MXPA99002996A - Methods for treating an ischemic disorder and improving stroke outcome - Google Patents

Abstract

The present invention provides for a method for treating an ischemic disorder in a subject with comprises administering to the subjet a pharmaceutically acceptable form of a selection antagonist in a sufficient amount over a sufficient time period to prevent white blood cell accumulation so as to treat the ischemic disorder in the subject. The invention further provides a method for treating an ischemic disorder in a subject which comprises administering to the subject carbon monoxide gas in a sufficient amount over a sufficient period of time thereby treating the ischemic disorder in the subject. The invention further provides a method for treating an ischemic disorder in a subject which comprises administering to the subject a pharmaceutically acceptable form of inactivated Factor IX in a sufficient amount over a sufficient period of time to inhibit coagulation so as to treat the ischemic disorder in the subject.

Description

METHODS TO DEAL WITH AN ISCHEMIC DISORDER AND IMPROVE THE RESULT CAUSED BY A SUDDEN ATTACK DESCRIPTION OF THE INVENTION The invention described herein has been made with government support under the National Institutes of Health, National Heart, Lung and Blood Institute grant HL55397 of the Department of Health and Human Services. Consequently, the government of the United States has certain rights over this invention. Through this application, reference is made to different publications. The descriptions of these publications in their entirety are incorporated herein by reference in this application for the purpose of more fully describing the state of the art as known to those familiar in the present to the date of the invention described and claimed in the art. I presented.

BACKGROUND OF THE INVENTION The treatment of ischemic disorders has been the focus of research for many years. The recent availability of transgenic mice has led to a flourishing number of reports describing the effects of specific gene products on the pathophysiology of the attack (sudden attack, infarction or crisis). Although models of focal cerebral ischemia in rat models have been well described, descriptions of a mouse model of occlusion of the middle cerebral artery are scarce, and sources of potential experimental variability remain undefined. Acute recruitment of neutrophils to cardiac tissue or postischemic pulmonary tissue has harmful effects in the early period of impact, but the mechanisms and effects of neutrophil inflow in the pathogenesis of the development of the attack remain controversial.

BRIEF DESCRIPTION OF THE INVENTION . The present invention provides a method for treating an ischemic disorder in a subject, which comprises administering to the subject a pharmaceutically acceptable form of a selectin antagonist in an amount sufficient for a sufficient period of time to prevent the accumulation of white blood cells in a manner in question the ischemic disorder in the subject. The invention further provides a method for treating an ischemic disorder in a subject which comprises administering to the subject gaseous carbon monoxide in a sufficient amount for a sufficient period of time in order to thereby treat the ischemic disorder in the subject. The invention further provides a method for treating an ischemic disorder in a subject, which comprises administering to the subject a pharmaceutically acceptable form of the inactivated factor IX in a sufficient amount for a period of time sufficient to inhibit coagulation so that the disorder is treated. Ischemic in the subject.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Neutrophil accumulation after focal cerebral ischemia and repercussion in mice. 45 minutes occlusion of the right middle cerebral artery was followed, followed by 23 hours of reperfusion in male C57B1 / J6 mice. One hour before occlusion of the middle cerebral artery, "3.3 x 105 neutrophils labeled with 1X1 In were injected into the tail vein. Ipsilateral (right hemisphere) and contralateral (left hemisphere) scores were obtained and normalized by g of tissue (n = 7, ** = p <0.01).

Figures 2A, 2B, 2C and 2D. Effect of preoperative neutrophil suppression on the indices of the attack outcome. C57B1 / J6 male mice were subjected to transient occlusion of the middle cerebral artery, as described above (wild type, n = 16) and compared with a similar procedure performed on mice in neutrophilic cuspids for 3 days before the day of treatment. surgery PMN-, n = 18). Figure 2A. Infarct volumes, calculated based on TTC serial stained brain slices, and expressed as% ipsilateral hemispheric volume. Figure 2B. Qualification of neurological deficiency, evaluated before anesthesia 24 hours after transient occlusion of the middle cerebral artery; the number 4 represents the most severe neurological deficiency. Figure 2C. Cerebral blood flow, measured by laser Doppler flow measurements 2 mm posterior to the bregma, expressed as% contralateral hemispheric blood flow. Figure 2D. Mortality 24 hours after transient occlusion of the middle cerebral artery. (* • = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 3. Expression of intercellular adhesion molecule-1 transcripts (ICAM-1) 24 hours after occlusion of the middle cerebral artery. RNA from the ipsilateral (infarct) and contralateral (non-infarct) hemispheres of the same mouse was prepared and agarose gel loaded with 20 μg of total RNA per band.

After overnight transfer to a nylon membrane, the Northern blot was probed with 32 p-labeled mouse ICAM-1 cDNA33. A / 3-actin probe was used for a control.

Figures 4A and 4B. Antigen expression for intracellular adhesion molecule-1 (ICAM-1) in the cerebral microvasculature 24 hours after occlusion of the middle cerebral artery. A coronal section of brain was obtained for immunostaining with ICAM-1, so that the non-infarcted and infarcted hemispheres of the same brain could be compared under identical staining conditions. The staining was performed using rat antibody against mouse ICAM-l, with primary antibody binding sites visualized by alkaline phosphatase. Figure 4A. Brain microvessels in the contralateral (non-infarcted) section of a brain obtained 24 hours after occlusion of the middle cerebral artery. Figure 4B. Brain microvessels of the ipsilateral (infarcted) hemisphere of the same brain section as shown in Figure 4A. The endothelial cells of the ipsilateral cerebral microvessels demonstrate increased expression of ICAM-1 (bright red staining). Amplification, 250X.

Figures 5A and 5B. Cerebrovascular anatomy in homozygous mice lacking ICAM-1 (FIG. 5B) and wild-type controls (FIG. 5A). Indian ink staining of the cerebrovascular anatomy with a lower view of the circle of Willis demonstrates that there are no gross anatomical differences in the vascular pattern of cerebral circulation, with posterior communicating arteries intact in both. Figures 6A and 6B. Sections in series stained with TTC at 24 hours from representative wild-type mice (Figure 6A) or homozygotes lacking ICAM-1 (Figure 6B) subjected to transient occlusion of the middle cerebral artery. The pale white area in the territory of the middle cerebral artery represents infarcted brain tissue, while the viable tissue is stained brick red. Figure 7A shows the quantification of infarct volumes by planimetry of the brain sections in series, in multiple experiments.

Figures 7A, 7B, 7C and 7D. The role of ICAM-l in the outcome of the attack. Transient occlusion of the middle cerebral artery was performed as described in ICAM-l + / + mice (wild type, n = 16) or ICAM-l - / - (n = 13), and the attack outcome indices were measured as described in figure 2. Figure 7A. Effect of ICAM-l on infarct volume, Figure 7B. Neurological deficiency score, Figure 7C. cerebral blood flow and Figure 7D. mortality (* = p < 0.05, ** = p < 0.01).

Figures 8A and 8B. Effect of hypoxia on the exocytosis of the Weibel-Palade body. Figure 8A. Human umbilical veins were exposed to hypoxia 4f > 02 15-20 Torr) or normoxia for the indicated durations, and the secretion of vonWillebrand factor (vWF) was quantified by ELISA. *** = P < 0.001 for hypoxia versus normoxia. Figure 8B. Similar experiments were performed for 8 h in the presence of 2 mm Ca ++ (Ca ++ 2 mM), 0 mM Ca ++ (free Ca ++), or Ca ++ O mM with 2 mM EGTA added to form chelates with residual extracellular Ca ++ (Ca ++ - free + EGTA ).

Figures 9A, 9B, 9C and 9D. Effect of endothelial hypoxia on the expression of P-selectin and adhesion of neutrophils. Figure 9A. Expression of P-selectin in HUVEC exposed to normoxia or hypoxia, determined by specific binding of radiolabeled monoclonal IgG against P-selectin (clone WPAS12.2). The data are expressed as a relative union compared to a normoxic time point of 4 h. Figure 9B. Effect of protein inhibitor synthesis on the hypoxia-induced expression of P-selectin. In a separate experiment, the effect of cycloheximide (10 μg / ml, + CHX) added at the beginning of the normoxic or hypoxic period of 4 hours, on the expression of P-selectin is shown. The comparison is made to simultaneous experiments performed in the absence of cycloheximide (-CHX), with data expressed as relative union compared with normoxic (-CHX) union, the means ± SEM (standard error mean) are shown; * = p < 0.05 versus normoxia (-CHX); t = p < 0.05 versus normoxia (+ CHX). Insertion: Effect of cyclohexy ida (10 μg / ml) on protein synthesis at 4 h, measured with proteins marked with 3SS, precipitable with trichloroacetic acid after administration of 35S-methionine and 3SS-cysteine. Figure 9C. Binding of neutrophils labeled with 1: L1Indium to endothelial monolayers of human umbilical normoxic (N) or hypoxic (H) umbilical vein at 4 h, in the presence of a blocking antibody against P-selectin (WAPS clone 12.2) or a non-blocking antibody against P-selectin (clone AC1.2). The means ± SEM are shown; ** = p < 0.01.

Figures IAA, 10B and 10C. Role of neutrophils and endothelial P-selectin in rodent cardiac preservation followed by heterotopic transplantation. Figure 10A. Cardiac preservation of rats. Hearts were transplanted immediately after harvest (Fresh, n = 8) or preserved for 16 h in lactated Ringer's solution at 4 ° C, followed by transplantation (Prsvd, n = 4). The effect of administering non-blocking antibody against P-selectin (AC1.2, n = 3), immunosuppressed neutrophil receptors before implantation of the donor heart (-PMN, n = 4) (, or when administering 250 μg of blocking IgG of mouse against P-selectin (n = 4) 10 minutes before the impact on the survival of the cardiac graft (bars with stripes) and leukostasis (myeloperoxidase activity, black bars) The means ± SEM are shown for graft survival , c versus a, p <0.0001; g versus c, p <0.05; i versus eoc, p <0.05, For infiltration of neutrophils in the graft, d versus b, p <0.01; h versus d, p < 0.05; j versus dof, p < 0.05 Figure 10B. Role of coronary endothelial P-selectin in cardiac preservation using donor hearts from mice lacking P-selectin (or wild-type control) that were subjected to washing free of blood before preservation. Graft survival was determined by the presence / absence of electrical / mechanical cardiac activity exactly ten minutes after the restoration of blood flow. Figure 10C: Quantification of neutrophils by measurement of myeloperoxidase activity (dABs 460 nm / min) as described15'18. (For the bars shown from left to right, n = 14, 8, 13 and 7, respectively, with the indicated values of P).

Figures HA and 11B. Weibel-Palade body release during human cardiac surgery in 32 patients. Figure HA. The coronary sinus blood (CSX) and the conclusion (CS2) of the ischemic period (transverse aortic subjection) were sampled. ELISA was performed for thrombomodulin (TM) and vWF. Figure 11B. Immunoelectrophoresis by vWF of a representative sample of CS-. and CS2 from the same patient's blood (diffusion factors are indicated). There is an increase in the high molecular weight multimers detected in the CS2 samples.

Figures 12A, 12B. 12C and 12D. General aspects of the operative installation for the mouse buccal cerebral ischemia model. Figure 12A. The diagram shows a retraction system based on suture. Figure 12B. View through the operating microscope. The large vascular stump represents the external carotid artery, which is located in the lower half in the field of operation. Figure 12C. Photograph of a cauterized occluded suture of the indicated caliber (5-0 [bottom part] or 6-0 nylon [top part]). Figure 12D. Schematic diagram of mouse cerebrovascular anatomy, with threading in the anterior cerebral artery, which occludes the middle cerebral artery at its point of origin.

Figure 13. Comparison of cerebrovascular anatomy between strains of mice. After anesthesia, the mice were given an intracardiac injection from India, followed by euthanasia without suffering. A circle of intact Willis can be observed in all strains, including the posterior and lateral communicating arteries, indicating that there are no gross differences related to staining in the cerebrovascular anatomy.

Figures 14A. 14B and 14C. Effects of the mouse strain on the attack result. Mice (males, 20-23 g) were subjected to occlusion for 45 minutes of MCA (using a 12 mm 6.0 occluding suture), followed by 24 hours of reproduction, and the attack outcome indices were determined. Figure 14A.

Effects of the strain on the volume of impact, determined as the percentage of ipsilateral hemispheric volume as described in the methods section. Figure 14B. Effects of the strain on the qualification of neurological deficiency, evaluated from no neurological deficiency (0) to severe neurological deficiency (4), with certain qualifications as described in the Method section. Figure 14C. Effects of the strain on the cerebral blood flow, measured by laser Doppler flowmetry as a relative flow over the infarcted territory, compared to the blood flow over the contralateral (non-infarcted) cortex. Strains include mice 129J (n = 9), CD1 (n = ll) and C57 / B16 (n = ll); * = p < 0.05 versus 129J mice.

Figures 15A. 15B and 15C. Effects of the size of the animal and waist diameter of the occlusion suture on the result of the attack. Male CD-1 mice of the indicated sizes were subjected to occlusion of the middle cerebral artery (45 minutes) followed by reperfusion (24 hours) as described in the section of Methods The size of the suture (caliber) is indicated on each panel. The small animals (n = ll) were those between 20-25 g (average 23 g), and large animals were between 28-35 g (mean 32 g, n = 14 for suture 6.0, n = 9 for suture 5.0). Figure 15A. Shows the effects of animal / suture size on infarct volume, Figure 15B. Neurological deficiency score, and Figure 15C. cerebral blood flow measured as described in figure 14. P values are shown.

Figures 16A, 16B and 16C. Effects of temperature on the result of the attack. Male C57 / B16 mice were subjected to 45 minutes of MCA occlusion (6.0 suture) followed by reperfusion. The core temperature at 37 ° C (normothermia, n = ll) was maintained for 90 minutes using an intrarectal probe with a thermocouple controlled heating device. In the second group (hypothermia, n = 12), the animals were placed in cages and left at room temperature after an initial 10 minutes of normothermia (mean core temperature, 31 ° C to 90 minutes). In both groups, after this observation period of 90 minutes, the animals returned to their boxes at room temperature maintained at 37 ° C for the duration of the observation. Twenty-four hours after the MCA occlusion, the indices of attack results were recorded; Figure 16A. infarct volume, Figure 16B. qualification of neurological deficiency, and Figure 16C. cerebral blood flow, measured as described in figure 3. The values of * = p < 0.05.

Figures 17A, 17B and 17C. Outcome comparisons between permanent focal cerebral ischemia and transient focal cerebral ischemia, followed by reperfusion. The MCA was permanently occluded (n = ll) or transiently (45 minutes, n = 17) with a 6.0-gauge suture in male C57 / B16 22-gram mice, as described in the methods section. Twenty-four hours after the occlusion of the MCA, the attack outcome indices were recorded; Figure 17A. infarct volume, Figure 17B. qualification of neurological deficiency, and Figure 17C. cerebral blood flow, measured as described in figure 14.

Figures 18A and 18B. Expression of P-selectin and accumulation of neutrophils (PMN) after occlusion of the middle cerebral artery (MCAO) in mice. Figure 18A. Expression of P-selectin followed by MCAO and reperfusion. Relative expression of the P-selectin antigen in the ipsilateral cerebral hemisphere after occlusion of the middle cerebral artery is demonstrated using either a monoclonal rat IgG against P-selectin IgG, labeled with 12SI or a non-immune rat IgG, marked with 12SI to control non-specific extravasation. The experiments were performed as described in the legend of Figure 18. The values are expressed against contralateral cps ipsilateral / cpm. n = 6 for each group, except for the control, of 30 minutes (n = 4); t = p < 0.001, 30 minutes of reperfusion versus immediate preocclusion; * = p < 0.025, change in the accumulation of P-selectin versus change in the control of IgG accumulation. Figure 18B. Course in PMN accumulation time followed by cerebral ischemia and reperfusion in the mouse. For these experiments, "3.3 x 105 PANS labeled with 1: L1In were intravenously injected, where they are injected intravenously into the wild type mouse (PS + / +) 15 minutes before occlusion of the middle cerebral artery (MCAO). The accumulation of PMN111 is measured immediately after sacrifice, as the proportion of ipsilateral / contralateral CPM under the following experimental conditions: before MCAO (Pre-O, n = 4), immediately after MCAO (Post-0, n = 6) ), and 10 minutes following MCAO but still before reperfusion (: 10 Post-O, n = 6). To establish the effect of reperfusion on PMN accumulation, reperfusion was initiated after 45 minutes of ischemia. The accumulation of PMN was measured following 30 minutes (n = 6), 300 minutes (n = 3), and 22 hours (n = 8) of reperfusion. Under identical conditions, accumulation of PMN is measured in mice lacking P-selectin (PS - / -) after 45 minutes of ischemia and 22 hours of reperfusion (n = 7, * = p <0.05 versus 45 minutes MCAO / 22 hours of reperfusion in PS + / + animals).

Figure 19. Role of P-selectin in no cerebrovascular reflux. Cerebral blood flow is measured in PS + / + (upper panel) and PS - / - (middle panel) of mice using a laser Doppler flow probe and expressed as the percentage of contralateral (nonischemic) hemispheric blood flow. Blood flow was measured at the following points in time: a, before MCAO (PS + / +, n = 16; PS - / -, n = 23); b, immediately after MCAO (PS + / +, n = 42; PS - / -, n = 40); c, 10 minutes after MCAO, but still before reperfusion (PS + / +, n = 36, PS - / -, n = 34); d, immediately after reperfusion (PS + / +, n = 36, PS - / -, n = 34); e, 30 minutes after reperfusion (PS + / +, n = 8, PS - / -, n = 5); and f, 22 hours after reperfusion (PS + / +, n = 15, PS - / -, n = 5). The lower panel represents an overlay of the two panels with error bars omitted for clarity.

Figure 20. Cerebrovascular anatomy in null homozygous mice in P-selectin, PS - / - (right) and wild-type controls, PS + / + (left). Indian ink / carbon black stain of the cerebrovascular anatomy with a lower view of the circle of Willis demonstrates that there are no gross anatomical differences in the vascular pattern of the vascular circulation, with the posterior intact communicating with the arteries in both.

Figure 21A, 21B and 21C. Effect of the gene for P-selectin on the results of the attack. Occlusion of the middle cerebral artery was performed for 45 minutes, followed by 22 hours of reperfusion in P-selectin at + / + (n = 10) or P-selectin - / - (n = 7) in mice. The effect of P-selectin on: Figure 21A. of the figure, infarct volume as evidenced by 2, 3, 5-triphenyl-2H-tetrazolium 2% chloride (TTC) as staining, and calculated as percent of the ipsilateral hemisphere; Figure 12B. qualification of neurological deficiency (1 = normal spontaneous movements, 2 = circles in the clockwise direction, 3 = clockwise rotation, 4 = lack of response to noxious stimuli); Ipsilateral figure. These data show that inhaled CO reduces infarct volumes after an attack. Figure 23B. Effect of inhalation of carbon monoxides on mortality after an attack. Experiments were performed as described above, mortality was shown at 24 hours. These data show that inhaled CO reduces mortality after the attack.

Figures 24A, 24B and 24C. Figure 24A. Dose-response curve of inhaled carbon monoxide on the attack result. The experiments are described in the above. CO was inhaled at the indicated doses. These data show that inhaled CO reduces infarct volume in a dose-dependent manner, with 0.1% providing optimal protection. Figures 24B and 24C. Role of heme oxygenase, the enzyme which produces CO, in the attack. The animals were either administered the vehicle alone (DMSO) or as a control of zinc protoporphyrin IX (ZnPP) or tin protoporphyrin IX (SnPP). In a final group, the mice were given biliverdin (Bili), a compound which is formed along CO during the heme degradation process by heme oxygenase. The left panel shows the violation volumes. The right panel shows mortality. These experiments show that the activity of activated heme oxygenase is blocked, the attack results are worse (greater infarcts and higher mortality). Because the administration of 21C. percent survival at the time of sacrifice. (* = p <0.05).

Figures 22A. 22B, 22C and 22D. Effect of blocking P-selectin on the attack results. PS + / + mice were administered either with rat IgG against mouse P-selectin, blocking (clone RB 40.34, 30 μg / mouse) or a similar dose of non-immune rat IgG immediately before surgery. Figure 22A. Cerebral blood flow at 30 minutes after reperfusion. After 22 hours of reperfusion, the volumes of Figure 22B of reperfusion infarction, the neurological deficiency ratings Figure 22C, and the mortality of the Figure 2D. are shown, (n = 7 for each group, * = p <0.05).

Figures 23A and 23B. Figure .23A. Effect of the inhalation of carbon monoxides on the volumes of cerebral infarction. The mice were placed in bell containers in which they were exposed to 0.1% CO for 12 hours. After this treatment, they were removed from the bell vessels and subjected to intraluminal occlusion of the middle cerebral artery. At 24 hours, the animals were sacrificed and infarct volumes were measured by staining with triphenyltetrazolium chloride (TTC) staining, as shown in Figure 25. The quantification of infarct volumes (mean ± SEM) is expressed As the percentage of infarction of the hemisphere biliverdin is not protective, these data suggest that the other product of heme oxygenase (CO) activity is protective.

Figure 25. TTC staining of serial brain slices for the animals of Figure 23. The infarcted tissue appears white, and the viable tissue appears brick-red.

Figures 26A-26F. Effect of focal cerebral ischemia on the induction of heme oxygenase I (HO-I). Figures 26A-26C show in situ hybridization of mRNA for HO-I in the attack (Figure 26B) and in the controls (Figures 26A and 26C). Figures 26D-26F show the immunohistochemistry of the HO-I protein. Figure 26E shows that the protein is expressed in blood vessels and astrocytes after the attack. Figures 26D and 26F show that the protein is not expressed in blood vessels and astrocytes in controls.

Figure 27. Effect of focal cerebral ischemia on the induction of heme oxygenase (HO-I) mRNA. The contralateral part indicates the side without attack of the brain. The ipsilateral part indicates the side of the brain that is under attack. In both animals, the side of the brain subjected to attack shows increased HO-I, but the side without attack does not present it.

Figure 28. Effect of hypoxia on the induction of heme oxygenase I (HO). Mice exposed to an epoxy environment for 12 hours (to simulate ischemia) show an increase in mRNA of heme oxygenase I, compared to normoxic controls. These data show a potential mechanism by which the epoxy preexposure may also contain protection against subsequent ischemic events, which have been found to be valid in mice subjected and hypoxia after attack.

Figure 29. Effect of hypoxia on the expression of the protein heme oxygenase I (HO-I) endothelial cells of the mouse brain.

Hypoxia causes the levels of HOI to increase in these endothelial cells derived from the brain.

Figure 30. Effect of hypoxia on the induction of heme oxygenase I (HO-I) mRNA in mouse brain endothelial cells. Hypoxia causes the levels of mRNA and HO-I in these endothelial cells derived from the brain to increase.

Figures 31A-31D. Expression of P-selectin and accumulation of neutrophils (PMN) after occlusion of the cerebral artery (MCAO) in mice. Figure 31A. Expression of P-selectin after MCAO and reperfusion. The relative expression of the antigen of P-selectin in the ipsilateral cerebral hemisphere after occlusion of the middle cerebral artery was demonstrated using rat monoclonal IgG against P-selectin labeled with 12SI or a non-immune rat IgG, labeled with 125I to control non-specific extravasation. The values are expressed as intralateral CPM, contralateral CPM. n = 6 for each group, except for the control that was 30 min (n = 4); t = p < 0.001, 30 minutes of reperfusion versus immediate preocclusion; * = p < 0.025, change in the accumulation of P-selectin versus change in the accumulation of control IgG. Figure 31B and 31C. Immunohistochemical localization of P-selectin expression in a brain section of a mouse subjected to 45 minutes of MCAO, followed by one hour of reperfusion. The ipsilateral and contralateral cerebral free cortical sections demonstrate the same mouse. The arrows point to a cerebral microvaso, with a dark brown color representing the expression of P-selectin as on the surface of endothelial cells. Figure 31D. Course in the time of PMN accumulation after focal cerebral ischemia and reperfusion in the mouse. For these experiments, "3.3 x 10 5 LN-labeled PMNs were intravenously injected into wild type mice (PS + / +) 15 minutes before occlusion of the middle cerebral artery (MCAO). The accumulation of 11: LIn-PMN is measured immediately after sacrifice, as the ratio of ipsilateral / contralateral CPM under the following experimental conditions: before MCAO (Pre-0, n = 4) immediately after MCAO (Post-O, n = 6), and 10 minutes after MCAO, but still before reperfusion (: 10 Post-O, n = 6). To establish the effect of reperfusion on PMN accumulation, reperfusion was initiated after 45 minutes of ischemia. PMN accumulation was measured following 30 minutes (n = 6), 300 minutes (n = 3), and 22 hours (n = 8) of reperfusion. Under identical conditions, accumulation of PMN in mice lacking P-selectin (PS - / -) is measured after 45 minutes of ischemia and 22 hours of reperfusion (n = 7, * = p <; 0.05 versus 45 minutes MCAO / 22 hours of reperfusion in animals PS + / +).

Figure 32A-32C. Role of P-selectin in the lack of cerebral vascular reflux. Cerebral blood flow is measured in PS + / + mice (upper panel) and PS - / - (middle panel) using a laser Doppler flow probe and expressed as the percentage of contralateral (nonischemic) hemispheric blood flow (± SEM) ). Blood flow is measured at the following points in time: a, before MCAO (PS + / +, n = 16, PS - / -, n = 23); b, immediately after MCAO (PS + / +, n = 42; PS - / -, n = 40); c, 10 minutes after MCAO but still before reperfusion (PS + / +, n = 36, PS - / -, n = 34); d, immediately after reperfusion (PS + / +, n = 36, PS - / -, n = 34); e, 30 minutes after reperfusion (PS + / +, n = 8, PS - / -, n = 5); and f, 22 hours after reperfusion (PS + / +, n = 15, PS - / -, n = 5). The lower panel represents an overlap of the two upper panels, where the error bars are omitted, for clarity.

Figures 33A-33B. Effect of the gene for P-selectin on the results of attacks. Occlusion of the middle cerebral artery was performed for 45 minutes, followed by 22 hours of reperfusion in P-selectin + / + (n = 10) or P-selectin - / - mice (n = 7). Effect of P-selectin on: Figure 33A. infarct volume, as evidenced by 2% staining with 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC), calculated as percent of ipsilateral hemisphere; Figure 33B. percent survival at the time of sacrifice. The means ± SEM are indicated, with the numbers of animals from which the indicated percentage was calculated above the survival bars (* = p <0.05).

Figure 34. Effect of blocking P-selectin on the results of attacks. PS + / + mice were administered either rat IgG against blocking P-selectin (clone RB 40.34, 30 μg / mouse) or a similar dose of non-immune rat IgG immediately before occlusion of the cerebral artery mean (Pre-MCAO, n = 7 for each group) or after occlusion of the middle cerebral artery (Post-MCAO, n = 9 for the control antibody, n = 6 for the blocking antibody against P-selectin functionally). In both cases, the intraluminal occlusion suture was extracted after an ischemic period of 45 minutes to simulate clinical reperfusion. After 22 hours of reperfusion, the infarct volumes (dark bar), the cerebral blood flow relative to 30 minutes after reperfusion (diagonal strips) and survival (bars with light shadows) are shown. The measures ± SEM are indicated, with the numbers of animals from which the survival percentages indicated above the survival bars were calculated. * = p < 0.05 versus control antibody.

Figures 35A-35B. Validation of quantitative spectrophotometric intracerebral hemorrhage assay, in the absence (Figure 35A) or presence (Figure 35B) of brain tissue. Figure 35A. Standard curve in which the known concentrations of hemoglobin were reduced to cyanomethaemoglobin after which D.O. at 550 nm. N = 5 determinations in each point, where the measurements + SEM are shown. The equation for the line of best fit and the value of r is shown. Figure 35B. Known concentrations of hemoglobin (using autologous blood diluted in saline) were added to fixed volumes of fresh brain tissue homogenate and the spectrophotometric hemoglobin assay was performed. The brains were divided into hemispheres, - for each animal, one hemisphere was immersed in physiological saline for 20 minutes (NS, solid line), and the other hemisphere was placed in triphenyltetrasolium chloride (TTC, dashed line) for 20 minutes ( similar to the procedure that would be done to measure the volume of cerebral infarction). For each concentration of aggregated hemoglobin, a spectrophotometric hemoglobin test was performed in 6 hemispheres. The measures ± SEM are displayed.

Figures 36A-36B. Quantitative spectrophotometric assay of hemoglobin. Figure 36A. Effects of infusion of collagenase and rt-PA on quantitative mouse ICH. He underwent stereotactic infusion with ICA-inducing agents in the right deep ganglia of the cortex / basal. The brains were collected 24 h later and the spectrophotometric hemoglobin test was performed to quantify ICH. Mice were subjected to: 1) zero treatment (Contro), 2) stereotactic infusion of 1 μm of normal saline (false), 3) stereotactic infusion of 0.024 μg of collagenase B in 1 μl of normal saline (Collagenase) , or 4) stereotactic infusion of collagenase B (as in the above) followed by intravenous tissue plasminogen activator (1 mg / kg in 0.2 μl of normal saline) by injection into the dorsal vein of the penis (Collagenase + rt-PA) . ** p < 0.001 versus false or control.

Figure 36B. Effect of rt-PA after focal ischemic attack in quantitative ICH in mouse. Mice were subjected to MCA occlusion for 45 minutes followed by reperfusion, and then: 1) 0.2 μm intravenous normal saline (attack + saline) or 2) intravenous tissue plasminogen activator (15 mg / kg in 0.2 μl normal saline) ) (Attack + rt-PA). The brains were harvested 24 h later and the spectrophotometric hemoglobin assay was performed to quantify ICH ** P < 0.05.

Figure 37. Demonstration of the rating system using for the visual determination of ICH after attack. Each cut, taken from different animals subjected to attack, represents the coronal cut of the brain which shows the maximum hemorrhagic diameter. The number corresponds to the visually determined hemorrhage score, defined in the Methods section.

Figures 38A-38B. Visual rating of ICH. Figure 38A. Effects of the infusion of collagenase and rt-PA on the visual qualification of ICH. The reactors were subjected to stereotactic infusion with ICH-inducing agents in the right deep crust / basal ganglia. Mice were subjected to: 1) lack of treatment (control), 2) stereotactic infusion of 1 μl of normal saline (false), 3) stereotactic infusion of 0.024 μg of collagenase B in 1 μl of normal saline (collagenase ), or 4) stereotactic infusion of collagenase B (as in the above), followed by intravenous tissue plasminogen activator (1 mg / kg in 0.2 μl of normal saline) by injection into the dorsal vein of the penis (Collagenase + rt-PA). Brains were collected 24 h later, sliced in 1 mm coronal slices and classified by an observer who does not know the protocol, as described in the methods section. * p < 0.05 versus collagenase, * p < 0.05 versus false or control. Figure 38B. Effect of rt-PA after focal ischemic attack on the visual qualification of ICH in mice. Mice were subjected to MCA occlusion for 45 minutes followed by reperfusion and then: 1) 0.2 μl intravenous normal saline (attack + saline) or 2) intravenous tissue plasminogen activator (15 mg / kg in 0.2 μl normal saline) ) (attack + rt-PA). The brains were harvested 24 h later, sliced in 1 mm coronal plates and classified by an observer who does not know the protocol, as described in the Methods section * p < 0.01. The individual values for the ICH visual ratings are shown, with the median value for each group indicated by a horizontal line.

Figure 39. Correlation between the visual qualification of ICH and the spectrophotometric hemoglobin assay. The optical density at 550 nm (ordinate) represents the results obtained from the spectrophotometric hemoglobin test in which the brain tissue was analyzed (from all the experiments). The corresponding visual ratings for ICH (as shown in Figure 38) are plotted along the abscissa. For each point, the measures ± SEM are displayed. Linear correlation is performed using Pearson's linear correlation, and the equation of the line and the value of r are shown.

Figures 40A-40F. Figure 40A. Effect of the attack and administration of factor IXai in attack on the accumulation of radiolabelled platelets. 11: Lindio-platelets were administered either with control animals without attack (n = 4), or in animals immediately before an attack with (n = 7) or without preoperative administration of factor IXai (300 μg / kg, n = 7 ). Platelet accumulation is expressed as ipsilateral CPM / contralateral CPM. The means + SEM are shown. * p < 0.05 versus no attack; ** p < 0.05 versus attack + vehicle. Figure 40B. Accumulation of fibrin in infarcted brain tissue. Twenty-two hours after focal cerebral ischaemia and reperfusion, a brain of a representative mouse was collected, which has been pretreated before surgery with either vehicle (two bands to the left) or factor IXai (300 μg / kg, two more bands to the right). The brains were divided into ipsilateral (R) and contralateral (L) hemispheres, and plasmin digestion was performed to solubilize accumulated fibrin. Immunoblotting was performed using a primary antibody directed against a neoepitope expressed on the cross-linked fibrin gamma-gamma chain dimer. Figure 40C-40F. Immunohistochemical identification of fibrin formation sites in the attack. Using the same antibody as described in Figure 2b to detect fibrin, brains were harvested from two mice after attack (upper and lower panels, each representing a mouse). The arrows identify cerebral microvessels. Note that in both ipsilateral hemispheres (right side panels), intravascular fibrin can be clearly identified by red staining, which is not seen in the non-ischemic contralateral hemispheres (left panels).

Figures 41A-41C. Figure 41A. Effect of factor IXai on relative CBF in a mouse attack model, measured by laser Doppler. CBF in animals treated with factor IXai (300 μg / kg, n = 48, dotted line) is significantly higher at 24 hours than vehicle-treated controls (n = 62). The means ± SEM are shown. * p < 0.05. Figure 41B. Effect of factor IXai on infarct volumes in a mouse attack model, measured by TTC staining of the coronal sections in series. The animals were administered vehicle (n = 62) or factor IXai (300 μg / kg, n = 48). It is shown in the means ± SEM. * p < 0.05. Figure 41C. Dose-response factor IXai in attack. Factor XXai was administered immediately before the onset of the attack, cerebral stroke volumes were determined as described in Figure 41B above. N = 62, 48, 6 and 6, for the vehicle, doses of 300 μg / kg, 600 μg / kg and 1200 μg / kg, respectively. The means ± SEM are shown. * p < 0.05 versus animals treated with vehicle.

Figures 42A-42B. Effect of factor IXai on intracerebral hemorrhage. Figure 42A. The hemoglobin spectrophotometric assay was performed as described in the Methods section. Side. at 550 nm it is linearly related to the hemoglobin content in the brain11'12. Figure 42B. ICH score, determined visually, by an observer who does not know the protocol, as described in the Methods section. The ICH score correlates with the hemoglobin content in the brain determined spectrophotometrically11'12. The means ± SEM are shown. * p < 0.05 versus animals treated with vehicle-.

Figure 4 Effect of synchronization of factor IXai administration on cerebral infarct volumes when administered after the onset of the attack. Mice were subjected to focal cerebral ischaemia and reperfusion, as described in the Methods section. The preocclusion administration data (2 bars to the left) are shown in Figure 42B. In additional experiments to determine the effects of factor IXai administered after the attack, immediately after suppression of the intraluminal occlusion suture, vehicle was administered intravenously (normal saline, n = 13) or factor IXai (300 μg / kg, n = 7). Cerebral infarct volumes were determined (based on serial sections stained with TTC, obtained at 10 pm). The means ± SEM are shown. * p < 0.05, ** p < 0.05 versus animals treated with vehicle.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating an ischemic disorder in a subject, which includes administering to the subject a pharmaceutically acceptable form of a selectin antagonist in an amount sufficient for a period of time sufficient to prevent the accumulation of white blood cells in a manner The ischemic disorder is treated in a subject. The selectin antagonist can be a mimetic peptide, a nucleic acid molecule, a ribozyme, a polypeptide, a small molecule, a carbohydrate molecule, a monosaccharide, an oligosaccharide or an antibody. The selectin may be a P-selectin, an E-selectin or an L-selectin. The antibody can be a P-selectin antibody. The antibody may further include a polyclonal antibody or a monoclonal antibody. The P-selectin antagonist may include a nitric oxide (NO) precursor such as L-arginine, a NO donor such as nitroglycerin or nitroprusside, a cyclic nucleotide analog such as cyclic GMP or cyclic AMP analog, or a phosphodiesterase inhibitor. The pharmaceutically acceptable form of the P-selectin antagonist may include a P-selectin antagonist and a pharmaceutically acceptable carrier. The carrier can include an aerosol, intravenous, oral or topical carrier. The white blood cell can be a neutrophil or a monocyte. The subject can be a mammal, the mammal can be a human, a cow, a pig, a sheep, a dog, a cat, a monkey, a domestic bird or any animal model of a disease or disorder of humans. Ischemic disorder may include, but is not limited to peripheral vascular disorders, venous thrombosis, pulmonary embolism, myocardial infarction, transient ischemic attack, unstable angina, reversible ischemic neurological deficit, falsiform cell anemia or an attack disorder. The subject may have experienced cardiac surgery, pulmonary surgery, spinal cord surgery, brain surgery, vascular surgery, abdominal surgery or organ transplant surgery, and organ transplant surgery may include heart, lung, pancreas, or transplant surgery. The present invention further provides a method for treating an ischemic disorder in a subject, which comprises administering to the subject gaseous carbon monoxide in an amount sufficient for a sufficient period of time to thereby treat the ischemic disorder in the subject. The administration of carbon monoxide can be via inhalation by the subject or via extracorporeal exposure to the blood or bodily fluids of the subject.

The amount of carbon monoxide which may be sufficient to treat the subject includes, but is not limited from about 0.0001% carbon monoxide in an inert gas, to about 2% carbon monoxide in an inert gas. The inert gas can be oxygen, nitrogen, argon or air. In one embodiment of the present invention, the amount of carbon monoxide administered may be 0.1% carbon monoxide in air. The period of time sufficient to administer carbon monoxide to a subject to treat an ischemic disorder includes, but is not limited to, from about 1 day before surgery to about 1 day after surgery. The period of time can be from approximately 12 hours before surgery to approximately 12 hours after surgery. The time period may also include from about 12 hours before surgery to about 1 hour after surgery. The time period may additionally include from about 1 hour before surgery to about 1 hour after surgery. The time period may also include from about 20 minutes before surgery to about 1 hour after surgery. The period of time sufficient to treat an ischemic disorder in a subject who does not undergo surgery may include before and during any physical manifestation of such disorder. The administration of carbon monoxide is preferable before manifestation in order to diminish such manifestation of an ischemic disorder. As described in the following, it has been shown that the administration of carbon monoxide is protective of ischemia in a subject, if administered before surgery. As used herein, the term "ischemic disorder" encompasses and is not limited to a peripheral vascular disorder, venous thrombosis, pulmonary embolism, myocardial infarction, a transient ischemic attack, pulmonary ischemia, unstable angina, reversible ischemic neurological deficit, adjunctive thrombolytic activity, excessive coagulation conditions, falsiform cell anemia or an attack disorder. The subject may undergo cardiac surgery, pulmonary surgery, spinal cord surgery, brain surgery, vascular surgery, abdominal surgery or organ transplant surgery. Organ transplant surgery may include heart, lung, pancreas or liver transplant surgery. Carbon monoxide can be administered in a direct manner. Instead of the subject inhaling directly or receiving carbon monoxide gas or a gas mixture, the subject may be administered compounds to stipulate the in vivo production of carbon monoxide. Such compounds may include, but are not limited to heme, ferritin, hematin, endogenous heme oxygenase precursors or heme oxygenase stimulators.

In addition, the subject may be exposed to an environment of low oxygen concentration compared to the normal atmosphere. Heme oxygenase is an endogenous enzyme which synthesizes carbon monoxide from the heme precursor group (it is part of the normal pathway in which the heme group is degraded and metabolized in the body). When mice are exposed to hypoxia or tissue ischemia, the concentrations of both messenger RNA which encodes the heme oxygenase protein and the protein itself are increased. In addition, the activity of the enzyme is increased, as indicated by carbon monoxide measurements in the tissue. Another embodiment of the present invention is a method for treating an ischemic disorder in a subject, which comprises administering to the subject a pharmaceutically acceptable form of inactivated factor IX, in an amount sufficient for a period of time sufficient to inhibit coagulation so that the ischemic disorder in the subject is treated. Sufficient amount may include, but is not limited to from about 75 μg / kg to about 550 μg / kg. The amount can be 300 μg / kg. The pharmaceutically acceptable form of inactivated factor IX includes inactivated factor IX and a pharmaceutically acceptable carrier. Factor IX can be inactivated by standard methods known to those familiar in the art, such as heat inactivation. Factor IX can be inactivated or the activity of factor IX can be inhibited by an antagonist. Such an antagonist can be a mimetic peptide, a nucleic acid molecule, a ribozyme, a polypeptide, a small molecule, a carbohydrate molecule, a monosaccharide, an oligosaccharide or an antibody. The present invention provides a method for identifying a compound that is capable of improving an ischemic disorder in a subject, which includes: a) administering the compound to an animal, which is an animal model of attack; b) measuring the result of the attack on the animal, and c) comparing the result of the attack in step (b) with that of the animal model of attack in the absence of the compound so as to identify a compound capable of improving an ischemic disorder in a subject. The animal model of attack includes a mouse model of focal cerebral ischemia and reperfusion. The result of the attack can be measured by physical examination, magnetic resonance imaging, laser Doppler flowmetry, triphenyltetrazolium chloride staining, chemical determination of neurological deficit, computed tomographic scan or cerebral cortical blood flow. The result of the attack on a human can also be measured by clinical measurements, quality of life scores and neuropsychometric tests. The compound may include a P-selectin antagonist, an E-selectin antagonist or an L-selectin antagonist.

The present invention further provides a method for identifying a compound that is capable of preventing the accumulation of white blood cells in a subject, which includes: a) administering the compound to an animal, which is an animal model of attack; b) measuring the result of the attack on the animal, and c) comparing the result of the attack in stage (b) with that of the animal model of attack, in the absence of the compound, so as to identify a compound capable of preventing an ischemic disorder in a subject. The aniamal attack model includes a mouse model of focal cerebral ischaemia and reperfusion. The result of the attack can be measured by physical examination, magnetic resonance imaging, laser Doppler flowmetry, triphenyltetrazolium chloride staining, chemical determination of neurological deficiency, computed tomography scan or cerebral cortical blood flow. The post-attack result in a human can also be measured by clinical measurements, quality of life scores and neuropsychometric tests. The compound may include a P-selectin antagonist, an E-selectin antagonist or an L-selectin antagonist. The present invention further provides a method for identifying a compound that is capable of preventing the accumulation of white blood cells in a subject which includes a) administering the compound to an animal, which is an animal model of attack; b) measuring the post-attack result in the animal and c) comparing the post-attack result in step (b) with that of the attack animal model in the absence of the compound so as to identify a compound capable of preventing the accumulation of white blood cells in the subject. White blood cells can be, but are not limited to, neutrophils, platelets or monocytes. The compound can be, but is not limited to, a selectin inhibitor, a monocyte inhibitor, a platelet inhibitor or a neutrophil inhibitor. The selectin inhibitor can be, but is not limited to, an inhibitor of P-selectin, E-selectin or L-selectin. Selectins are expressed on the surface of platelets and such inhibitors or selectin antagonists, as described herein, can prevent the expression of such selectins on the surface of the cell. The prevention of expression can be at transcriptional, translational or post-translational levels, or they can prevent the movement of such selectins through the cytosol and prevent delivery at the cell surface. The present invention provides treatment of ischemic disorders by inhibiting the ability of neutrophils, monocytes or other white blood cells to adhere properly. This can be done by removing the counter ligand, such as CD18. It has been shown, as discussed in the following, that "agénic" mice for CD18 (mice that do not have normal CD18 gene expression) are protected from adverse ischemic conditions. Endothelial cells on vessel surfaces in the subject may also be a target for treatment. In an attack mouse model, the administration of TPA as a thrombolytic agent causes some visible hemorrhage together with an improvement of the attack disorder. However, the administration of a P-selectin antagonist also improves the attack disorder in the animal model, but without the coincident hemorrhage. The present invention can be used in conjunction with a thrombolytic therapy to increase the efficacy of such therapy or to allow the subject to be dosed with lower doses of such therapy so as to reduce the side effects of thrombolytic therapy. As used herein, the term "suitable, pharmaceutically acceptable carrier" encompasses any of the pharmaceutically acceptable standard carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. An example of an acceptable triglyceride emulsion useful in the intravenous and intraperitoneal administration of the compounds is the triglyceride emulsion commercially known as Intralipid ™. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, fats or vegetable oils, gums, glycols or other known containers. Such carriers may also include flavor and color additives or other ingredients. This invention also provides pharmaceutical compositions that include therapeutically effective amounts of compositions and protein compounds capable of treating an attack disorder or of improving the post-attack outcome as the subject of the invention, together with diluents, preservatives, solubilizers, emulsifiers, suitable adjuvants and / or carriers, useful in the treatment of neuronal degradation due to aging, an inability to learn or a neurological disorder. Such compositions are liquid or leophilized or formulations dried in some other way and include diluents of a buffer content (for example Tris-HCl, acetate, phosphate), pH and variable ionic strength, additives such as albumin or gelatin to prevent absorption in surfaces, detergents (for example Tween 20, Tween 80, Pluronic F68, salts of bile acid), solubilizing agents (for example glycerol, polyethylene glycerol), antioxidants (for example ascorbic acid, sodium metabisulfite), preservatives (for example dimerosal, benzyl alcohol) , parabens), volume imparting substances or tonicity modifiers (for example lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the compound, formation of complexes with metal ions or incorporation of the compound into or on particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc. or on liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocytes without cytoplasm, or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate and in vivo clearance rate of the compound or composition. The choice of compositions will depend on the physical and chemical properties of the compound capable of relieving the symptoms of the attack disorder or of improving the post-attack outcome in the subject. Controlled or suspended release compositions include formulation in lipophilic deposits (eg, fatty acids, waxes, oils). Also included by the invention are particulate compositions coated with polymers (eg, poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms of protective coatings, protease inhibitors or permeation enhancers for various administration routes including parenteral, pulmonary, nasal and oral. The portions of the compound of the invention can be "labeled" by association with a detectable marker or indicator substance (eg, radiolabeled with 12SI or biotinylated) to provide reagents useful in the detection and quantification of the compound or its recipient carrier cells or their derivatives , in solid tissue and in fluid samples such as blood, cerebrospinal fluid or urine. When administered, the compounds are often rapidly cleared from circulation and therefore induce a relatively short duration of pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive compounds may be required to sustain therapeutic efficacy. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol and polypropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to show substantially longer half-lives in blood after intravenous injection compared to the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications increase the solubility of the compounds in aqueous solution, eliminate aggregation, improve the physical and chemical stability of the compound and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired biological activity can be obtained in vivo by the administration of such polymer-compound redoubts less frequently or with lower doses compared to the unmodified compound. The binding of polyethylene glycol (PEG) to compounds is particularly useful because PEG has a very low toxicity in animals (Carpenter et al., 1971). For example, a PEG adduct of adenosine deaminase has been approved in the United States for use in humans for the treatment of severe combined immunodeficiency syndrome. A second advantage provided by PEG conjugation is that it effectively reduces the immunogenicity and antigenicity of heterologous compounds. For example, a PEG adduct of a human protein may be useful for treatment of diseases in other mammalian species without the risk of activating a severe immune response. The compound of the present invention capable of alleviating the symptoms of a cognitive memory or learning disorder may be delivered in a microencapsulation device in a manner that reduces or prevents a host immune response against the compound or against the cells which produce the compound. The compound of the present invention can also be delivered microencapsulated in a membrane, such as a liposome. Polymers such as PEG can be conveniently linked to one or more reactive amino acid residues in a protein such as the alpha-amino group of the amino-terminal amino acid, the epsilon amino groups of the lysine side chains, the sulfhydryl groups of the cysteine side chains , the carboxy groups of the aspartyl and glutamyl side chains, the alpha-carboxyl group of the carboxy terminal amino acid, the tyrosine side chains or activated derivatives of the glycosyl chains linked to certain asparagine, serine or threonine residues. Many activated forms of PEG suitable for direct reaction with proteins have been described. PEG reagents useful for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or l-hydroxy-2-nitrobenzene. 4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of free sulfhydryl groups in the protein. Likewise, reagents containing aminohydrazine or hydrazine groups are useful for reaction with aldehydes generated by oxidation of periodate of carbohydrate groups in proteins. By means of well-known techniques such as titration and by taking into consideration the observed pharmacokinetic characteristics of the agent in the individual subject, a person skilled in the art can determine an appropriate dosage regimen. See, for example, Benet, et al., "Clinical Pharmacokinetics" in Chapter 1 (pp. 20-32) of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Edition, A.G. Gilman, et al. eds. (Pergamon, New York 1990). The present invention provides a pharmaceutical composition which comprises an agent capable of treating an attack disorder or of improving the post-attack outcome, and a pharmaceutically acceptable carrier. The carrier can include, but is not limited to, a diluent, an aerosol, a topical carrier, an aqueous solution, a non-aqueous solution or a solid carrier. This invention is illustrated in the Details section Experimental that follows. These sections are "set forth for the purpose of assisting in the understanding of the invention, but are not intended, and should not be construed as limiting the invention in any way, as set forth in the claims that follow thereafter.

EXPERIMENTAL DETAILS Abbreviations: EC, endothelial cell; PMN, polymorphic nuclear leukocyte, - WP, Weibel-Palade body, - vWF, von factor Willebrand; EGTA, ethylene glycol bis (aminoethyl ether) tetraacetic acid, - HBSS, balanced salt solution of Hank; CS, coronary sinus; IL, interleukin; PAF, platelet activating factor; ICAM-1, intercellular adhesion molecule-1; HUVEC, EC of the human umbilical vein; LR, lactated Ringer's solution; MCAO, occlusion of the middle cerebral artery, - rt-PA recombinant tissue plasminogen activator; HO-I or HOI or HO I, heme oxygenase I; ICH, intracerebral hemorrhage; OD, optical density, -MCA, middle cerebral artery; rt-PA, recombinant tissue-type plasminogen activator; AUNT, transient ischemic attack; TTC, triphenyltetrazolium chloride.

EXAMPLE 1: Brain protection in homozygous mice lacking ICAM-l after occlusion of the middle cerebral artery: role of adhesion of neutrophils in the pathogenesis of the attack To investigate whether polymorphonuclear leukocytes (PMN) contribute to adverse neurological sequelae and mortality after an attack, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of In an attack, a mouse model of transient focal cerebral ischemia was used and consisted of occlusion of the cerebral intraluminal middle artery (MCA) for 45 minutes followed by 22 hours of reperfusion. The accumulation of PMN monitored by deposition of LL-labeled PMN in postischemic brain tissue increased 2.5 times in the ipsilateral (infarcted) hemisphere compared to the contralateral (non-infarcted) hemisphere (p <0.01). Immunosuppressed neutrophilic mice before surgery demonstrated a 3.0-fold reduction in infarct volumes (p <0.001), based on triphenyltetrazolium chloride staining of serial brain slices, improved ipsilateral cortical cerebral blood flow ( measured by laser Doppler) and a reduced neurological deficit compared to controls. In wild-type mice subjected to 45 minutes of ischemia followed by 22 hours of reperfusion, the mRNA for ICAM-1 in the ipsilateral hemisphere was increased, with immunohistochemistry localizing increased expression of ICAM-1 in the cerebral microvascular endothelium. We investigated the role of ICAM-1 expression in the attack in homozygous mice lacking ICAM-1 (ICAM-1 - / -) compared to wild-type controls (ICAM-1 + / +). ICAM-1 - / - mice showed a 3.7-fold reduction in infarct volume (p <0.005) to a 35% increase in survival (p <0.05), and a reduced neurological deficit compared to ICAM-l + / + controls. Cerebral blood flow to the infarcted hemisphere was 3.1 times higher in ICAM-l - / - mice compared to the ICAM-l + / + controls (p <0.01) suggesting an important role for ICAM-l in genesis without Postischemic cerebral reflux. Because mice suppressed from PMN and deficient in ICAM-1 are relatively resistant to brain ischemia-reperfusion injury, these studies suggest an important role for PMN adhesion mediated by ICAM-1 in the pathophysiology of the developing attack.

INTRODUCTION: Neutrophils (PMN) are critically involved in the earliest stages of inflammation subsequent to tissue ischemia, initiating elimination functions which are ultimately related by macrophages. There is a darker side to the inflow of neutrophils, however, especially in post-ischemic tissues 1_7 where activated PMN can increase damage to the vascular and cellular elements of the parenchyma. The experimental evidence points to a pivotal role for endothelial cells in the establishment of PMN postischemic recruitment, in which the hypoxic / ischemic endothelial cells synthesize the proinflammatory cytosine IL-18 as well as the potent chemoattractant of neutrophils and activator, IL-89. The firm adhesion of PMN to activated endothelium in a postischemic vascular environment is promoted by the translocation of P-selectin to the cell surface10 as well as the increased production of platelet-activating factor and ICAM-11. Although strategies to block each of these neutrophil recruitment mechanisms are protective in various models of ischemia and reperfusion injury, their effectiveness in cerebral damage by ischemia / reperfusion remains controversial. There is considerable evidence that in the brain, as well as in other tissues, an influx of early PMN follows an ischemic episode12".17 Immunohistochemical studies describing the increased expression of PMN adhesion molecules to P-selectin have been described. and the intercellular adhesion molecule-1 (ICAM-1) in the postischemic cerebral vasculature 12'18"20. However, the pathogenic importance of the expression of the adhesion molecule in the brain remains controversial.; Data from a trial of a monoclonal antibody against ICAM-1 in a human attack is not yet available. In animal models, there is conflicting experimental evidence regarding the effectiveness of anti-adhesion molecule strategies in the experimental attack treatment.21"23 To determine if ICAM-1 participates in the pathogenesis of postischemic brain damage, the experiments reported here were designed in a mouse model of focal cerebral ischemia and reperfusion so that the role of a single critical PMN adhesion mediator (ICAM-1) could be determined.These studies demonstrated that increased expression of ICAM-1 and neutrophil inflow follows In addition, these studies showed that mice lacking neutrophil and transgenic deficient ICAM-1 are relatively resistant to cerebral infarction after ischemia and reperfusion, which provides strong evidence of an exacerbated role of ICAM-1. in the pathophysiology of attacks.

MATERIALS AND METHODS Mice: Experiments were carried out with ICAM-l deficient transgenic mice generated as previously reported24 by gene targeting in Jl embryonic pluripotent cells, injected in C57BL / 6 blastosistos to obtain germline transmission, and backcrossed to obtain homozygous mice lacking ICAM-l. All experiments were performed with ICAM-l - / - or wild type (ICAM-1 + / +) cousins of the fifth generation of backcrosses with C57BL / 6 mice. The animals were 7 to 9 weeks old and weighed between 25 and 36 grams at the time of the experiments.For certain experiments, neutrophil suppression was performed in C57BL / 6 mice by administering polyclonal rabbit antibody against neutrophils. of mouse25 (Accurate Scientific, Westbury NY) preabsorbed to remove red blood cells as a daily intraperitoneal injection (0.3 ml of a 1:12 solution) for 3 days.The experiments in these mice were carried out on the fourth day after confirm agranulocytosis by Wright-Giemsa staining of peripheral blood smears.

Transient occlusion of the middle cerebral artery26: Mice were anesthetized with an intraperitoneal injection of 0.3 ml of ketamine (10 mg / cc) and xylazine (0.5 mg / cc). The animals were placed in supine position on an operating surface controlled by rectal temperature (Yellow Spings Instruments, Inc., Yellow Springs, OH). It was maintained at the core temperature of the animal at 36-38 ° C intraoperatively and for 90 minutes postoperatively. The occlusion of the middle cerebral artery was performed as follows: an incision was made in the part of the midline of the neck to expose the coverage of the right carotid under the operating microscope (16-25X magnification zoom, Zeiss, Thornwood, NY). The common carotid artery was freed from its cover, and was isolated with 4-0 silk thread, and the occipital and pterygopalatine arteries were isolated and divided each. Total control of the internal carotid artery was obtained and the external carotid was cauterized and divided just proximal to its bifurcation in the lingual and maxillary divisions. Occlusion of the carotid transient was carried out by advancing a 13 mm suture of 5-0 blunt nylon with heat via the stump of the external carotid to the origin of the middle cerebral artery. After placement of the occlusal suture, the stump of the external carotid artery was cauterized to prevent bleeding through the arteriotomy and arterial flow was restored. In all cases, the duration of occlusion of the carotid was less than 2 minutes. After 45 minutes, the occlusal suture was removed to establish reperfusion. These procedures have been previously described in detail26.

Measurement of cerebral cortical blood flow: Transcranial measurements of cerebral blood flow were made using laser Doppler flow measurements (Perimed, Inc., Piscataway, NJ) after reflection of the cutaneous suprepary of calvary, as previously described27 (readings) transcranial were consistently the same as those performed after craniotomy in pilot studies, using a straight laser Doppler probe (model # PF303, Perimed, Piscataway, NJ) and previously published marks (2 mm posterior to breg, 6 mm on each side of the midline), relative cerebral blood flow measurements were performed as indicated, immediately after anesthesia, after occlusion of the middle cerebral artery, immediately after reperfusion and at 24 hours just before euthanasia. expressed as the proportion of the intensity of the Doppler signal of the ischemic hemisphere compared with the no Squamous Although this method does not quantify cerebral blood flow per gram of tissue, the use of laser Doppler flow measurements in precisely defined anatomically defined marks serves as a means to compare brain blood flows in the same animal in series, with respect to time. The surgical procedure / occlusion of the medial cerebral artery intraluminal and reperfusion are considered to be technically adequate if it is observed = 50% reduction in relation to the cerebral blood flow immediately after the placement of the intraluminal occlusion suture and an increase of = 33 % in the flow over the baseline occlusion observed immediately after removal of the occlusal suture. These methods have been used in previous studies26.

Preparation and administration of mouse neutrophils labeled with 11: LIn: citrated blood from wild-type mice was diluted 1: 1 with NaCl (0.9%) followed by gradient ultracentrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). After hypotonic lysis of residual erythrocytes (20-second exposure to distilled H20 followed by reconstitution with 1.8% NaCl NaCl), neutrophils were suspended in phosphate-buffered saline (PBS). 5-7.5 x 10 6 neutrophils were suspended in PBS with 100 μCi of xllIndioxine (Amersham Mediphysics, Port Washington, NY) for 15 minutes at 37 ° C. After washing with PBS, neutrophils were gently pelleted (450 g) and resuspended in PBS to a final concentration of 1.0 x 10 cells / ml. Immediately before surgery, 100 μl of radiolabeled PMN were mixed with physiological saline to a total volume of 0.3 ml ("3 x 106 cpm) and administered by injection into the penile vein. After euthanasia without suffering, the brains were obtained as described, and neutrophil deposition was quantified as cpm / g of each hemisphere.

Neurological examination: Twenty-four hours after occlusion and reperfusion of the middle cerebral artery, before providing anesthesia, the mice were examined for neurological deficit using a four-level evaluation system26: A rating of (1) is provided if the animal shows normal spontaneous movements; a rating of (2) is provided if the animal is seen to turn to the right (circles in a clockwise direction, when viewed from the top (ie, to the contralateral side), - is provided a rating of (3) if the animal is observed to rotate longitudinally (clockwise when viewed from the tail), - a rating of (4) is provided if the animal is crouching on all four legs , without response to harmful stimuli, this rating system has been previously described in mice26, and is based on similar qualification systems used in rats28,29 which are based on the contralateral movement of animals with attack, - after cerebral infarction, the contralateral side is "weak" and in this way the animal tends to turn towards the weakened side.Previous work on rats28 and mice26 shows that the largest cerebral infarcts are associated with with a greater degree of contralateral movement, to the point where the infarcts are too large so that the animals remain unresponsive.

Calculation of infarct volume: After neurological examination, mice are given 0.3 ml of ketamine (10 mg / ml) and xylazine (0.5 mg / ml), and final cerebral blood flow measurements are obtained. Human euthanasia is performed by decapitation under anesthesia, and the brains are removed and placed in a mouse brain matrix (Activational Systems Inc., Warren, MI) for 1 mm slices. Sections are submerged in 2,3,5, triphenyl, 2H-tetrazolium 2% chloride (TTC, Sigma Chemical Co., St. Louis, MO) in 0.9% phosphate buffered saline, incubated for 30 minutes at 37 ° C and placed in 10% formalin 26.30"32. The infarcted brains are visualized as an area of unstained tissue, in contrast to viable tissue, which dyes a brick-red color. infarction from planimetric serial cuts if they are expressed as the percentage of infarction in the ipsilateral hemisphere.

RNA extraction and Northern blot analysis: 24 hours after focal ischemia and reperfusion, the brains are obtained and divided into ipsilateral (with infarction) and contralateral (without infarction) brains. To detect the ICAM-1 transcripts, the total RNA of each hemisphere is extracted using an RNA isolation kit (Stratagene, La Jolla, CA). Equal amounts of RNA (20 μg / lane) are loaded on a 1.4% agarose gel containing 2.2 M formaldehyde for fractionation by size, and then transferred overnight to nylon membranes (Nytran) with 10X SSC buffer by pressure by capillary. A 33 cDNA probe for mouse ICAM-l (1.90 kb, ATCC, Rockville, MD) is labeled with 32P-c_-dCTP by random labeling of the primer or primer (Prime-A-Gene, Promega), which hybridizes at the sites at 42 ° C, followed by 3 washes with IX SSC / 0.5% SDS. Spots were developed with exposed X-Omat AR film with light sieves at -70 ° for 7 days. A β-actin probe (ATCC) was used to confirm loaded RNA.

Immunohistochemistry: Brains were removed at the indicated times after occlusion of the middle cerebral artery, fixed in 10% formalin, embedded in paraffin and cut for immunohistochemistry. Sections or sections were stained with rat antibody against mouse ICAM-l (1:50 dilution, Genzyme, Cambridge MA), and the primary antibody binding sites were visualized by a secondary antibody conjugated with alkaline phosphatase detected with FastRed ( TR / naphthol AS-MX, Sigma Chemical Co., St. Louis MO).

Data analysis: Cerebral blood flow, infarct volumes and neurological outcome scores were compared using the Student t test for unpaired variables. The deposition of neutrophils labeled with llxIndio was evaluated as paired data [when comparing the contralateral hemisphere (without infarction) with the ipsilateral (with infarction)], to control the variations in the injected accounts or the volume of distribution. The differences in survival between the groups were determined using continuity analysis with the Chi-square statistic. The values are expressed as means ± as SEM (standard error mean), with a p < 0.05 considered as statistically significant.

RESULTS: Accumulation of neutrophils in attack: Previous pathological studies have shown accumulation of neutrophils after cerebral infarction15"17.34" 36. To determine if neutrophils accumulate in the mouse model of focal cerebral ischemia and reperfusion, neutrophil accumulation after transient ischemia (45 minutes) and reperfusion was quantified (22 h), by measuring the deposition of lxlIn-labeled neutrophils administered to wild type mice before ischemic events. These experiments demonstrated a significantly higher accumulation of neutrophils (2.5-fold increase) in the ipsilateral hemispheres (with infarction) compared to the contralateral (non-infarcted) hemisphere (n = 7, p <0.01, Figure 1). Similar results were obtained when the inflow of neutrophils was monitored by myeloperoxidase assays, although low levels of activity were recorded in this latter assay (data not shown).

Effect of neutrophil suppression on the post-attack outcome: To determine the effect of neutrophil inflow on the post-attack outcome indices, neutrophilic mice were immunosuppressed beginning three days before surgery. When the surgery was performed on the fourth day, an almost complete agranulocytosis was evident in the peripheral blood smears. Neutropenic mice (n = 18) underwent ischemia for 45 minutes and 22 hours of reperfusion, and post-attack outcome indices were determined. Infarct volumes were 3 times smaller in neutropenic animals compared to wild-type controls (11.1 ± 1.6% versus 33.1 ± 6.4%, p <0.001, Figure 2A). The decrease in infarct volumes in neutropenic mice paralleled the ratings of reduced neurological deficit (Figure 2B), increased cerebral post-reperfusion cortical blood flows (Figure 2C) and a trend towards reduced nighttime mortality (22% mortality). in neutropenic mice versus 50% mortality in controls, Figure 2D).

Expression of ICAM-1 in attack in mice: To establish the effect of cerebral ischemia / reperfusion in the mouse model, mRNA concentrations for ICAM-1 after brain ischaemia and reperfusion in wild-type mice were evaluated. The ipsilateral (infarcted) cerebral hemisphere demonstrated mRNA for ICAM-l increased by Northern blot analysis, compared to RNA obtained from the contralateral (non-infarcted) hemisphere of the same animal (Figure 3). To evaluate the expression of the antigen for ICAM-1 in this mouse model, wild-type mice were subjected to ischemia for 45 minutes followed by 23 hours of reperfusion, and the cerebral microvasculature was examined by immunohistochemistry. The expression of the ICAM-1 antigen was not detectable in the cerebral microvasculature contralateral to the infarction (Figure 4A), but it increased markedly on the ipsilateral side, with a prominent ICAM-1 tension of the cerebral endothelial cells (Figure 4B) .

Role of ICAM-l in the attack: To explore the role of ICAM-l in the attack, transgenic mice were studied, which are homozygous deficient of ICAM-l24 in the mouse model of focal cerebral ischaemia and reperfusion. Because variations in cerebrovascular anatomy have been reported that result in differences in susceptibility to experimental attack in mice37, Indian ink staining was performed on the circle of Willis in lacking homozygous mice (ICAM-1 - / -) and ICAM-1 + / + These experiments (Figure 5) show that there are no general anatomical differences in the vascular pattern of cerebral circulation. To determine the role of the intercellular adhesion molecule-1 in the neutrophil inflow after focal cerebral ischemia and reperfusion, the accumulation of neutrophils was measured in homozygous mice lacking ICAM-1 (ICAM-1 - / -) ( n = 14) and in wild type control mice (n = 7) subjected to infusion with neutrophils labeled with U1ln. The relative accumulation of neutrophils (ipsilateral cpm / contralateral cpm decreased (39% reduction) in ICAM-1 - / - mice compared to the ICAM-1 + / + controls (1.70 ± 0.26 versus 2.9 ± 0.52, p, < 0.05) Subsequently, experiments were conducted to investigate if ICAM-l expression has a pathophysiological role in the post-attack outcome ICAM-l - / - mice (n = 13) were significantly protected from the effects of ischemia cerebral focal and reperfusion, based on a 3.7-fold reduction in infarct volume (p <0.01), compared with the ICAM-l + / + controls (Figures 6 and 7A) .This reduction in infarct volume it was accompanied by a reduced neurological deficit (Figure 7B) and increased cerebral cortical blood flow after reperfusion (Figure 7C). Given these results, it is not surprising that mortality was also significantly decreased in ICAM-1 mice. compared to the ICAM-l + / + controls (15% versus 50%, p < 0.05; Figure 7D).

DISCUSSION: Epidemiological evidence in humans suggests that neutrophils contribute both to the onset of attack38 as well as to brain tissue damage and poor clinical outcome39, with a potential role for neutrophils in postischemic hypoperfusion, neuronal dysfunction and scar formation40"44. Considerable experimental evidence suggests that neutrophils may exacerbate tissue damage after attack 13,45,48. Certain pieces of experimental data have been controversial since they do not find an association between agents that block neutrophil accumulation and subsequent outcome indices. attack. In a rat attack model, antibody-mediated suppression of neutrophils before attack significantly decreases water and brain content and infarct size13. However, leukocytopenia induced by cyclophosphamide in a model in gerbo49 or the administration of antibody against neutrophils to dogs50 does not show beneficial effects in the global models of cerebral ischemia. Experimental therapy that aims to interfere with neutrophil-endothelial interactions has also produced ambiguous results. In a phenyl model of transient focal cerebral ischemia, treatment with the antibody for CD18 (the common subunit of the ß2 integrins, which bind to the intercellular adhesion molecule l51) does not alter the recovery of cerebral blood flow, the return of the induced potentials or the volume of infarction23. However, other experiments have shown that microvascular patency after transient focal ischemia in primates is enhanced by antibodies to CD1814. In a similar model in rats, the anti-CDIIb / CD18 antibody has also been shown to reduce both neutrophil accumulation and neuronal damage related to ischemia52. The experiments reported here show that in a mouse model of focal cerebral ischaemia and reperfusion, neutrophils accumulate in "postischemic" brain tissue, a finding corroborated in other models which similarly show increased accumulation of granulocytes in areas of low cerebral blood flow in early periods during the post-ischemic period15'16'36.45 Not only neutrophils They accumulate during the post-ischemic period in mice, but their presence exacerbates the indices of post-attack outcome.When animals become neutropenic before the ischemic event, cerebral infarcts are smaller, with improved cerebral perfusion after the ischemic event. data are very similar to those reported in a rabbit model of thromboembolic attack, in which immunosuppression of neutrophils results in both reduced infarct volume and improved blood flow.35 Because neutrophils contribute to postischemic brain damage in mice, a strategy was designed to elucidate the role of ICAM-1 in the pathophysiology of the attack using mutant mice deleted from ICAM-124. Experiments indicate that homozygous mice lacking ICAM-1 are relatively resistant to the damaging effects of cerebral ischaemia and reperfusion. To demonstrate the role of both neutrophils and ICAM-1 in the pathogenesis of tissue damage in attack, the studies reported here used several methods to determine the post-attack outcome. Although numerous investigators have used TTC staining to quantify the volumes of cerebral infarction26'30"32,37,53-, there has been some controversy regarding the accuracy of this method, especially when it was evaluated for the first time after the ischemic event. In the TTC method, 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) reacts with intact oxidative enzymes on the mitochondrial crystals and is thus reduced to coloring.54 TTC staining is not reliable before two hours of ischemia have elapsed; After 36 hours, the cells are filtered in the infarcted tissue and can stain positively with TTC, thus obscuring a clear demarcation between the infarcted and non-infarcted tissues observed with the earliest staining31. Although infarct size delineated by TTC staining correlates well with infarct size delineated by haematoxylin and eosin staining30,32, direct morphometric measurements tend to overestimate infarct volumes due to cerebral edema, especially during first 3 days after the ischemic event32. However, given these limitations, the studies reported here incorporate three additional methods to define the role of neutrophils and ICAM-1 in the post-attack outcome, including the qualification of neurological deficit, the cerebral blood flow relative to the affected area and the mortality. These additional measures, which do not depend on the accuracy of TTC staining, strongly contribute to the identification of a pathogenic role for both neutrophils and ICAM-1 in the attack. There has been recent profusion of scientific studies that explore the mechanical basis for the recruitment of neutrophils to postischemic tissues. Endothelial cells appear to be the main regulators of neutrophil trafficking, regulating the process of chemoattraction, adhesion and emigration of neutrophils from the vasculature55. When exposed to an epoxy environment as a paradigm for tissue ischemia, endothelial cells synthesize the potent chemoattractant and activator of neutrophils interleukin-8 (IL-8) V blocking which appears to be beneficial in a lung model of ischemia and reperfusion6. In addition, the epithelial endothelial cells synthesize the proinflammatory cytokine interleukin-l8, which can up-regulate the endothelial expression of the neutrophil adhesion molecules of E-selectin and ICAM-l in an autocrine manner8,9,56. Other mechanisms of adhesion of neutrophils can also be activated in the brain after ischemia, such as the release of P-selectin from accumulated stored within the membranes of the body Weibel-Paladel10. In a primate model, the expression of P-selectin was increased rapidly and persistently after focal cerebral artery ischaemia and reperfusion18. Although P-selectin-dependent neutrophil recruitment appears to be harmful after cardiac ischaemia and reperfusion, 57 its pathophysiological importance in the establishment of the attack has not yet been determined. Although hypoxia induces de novo synthesis of bioactive lipid platelet activating factor (PAF) 11, in a reperfusion model of spinal cord ischemia, PAF antagonism does not offer an incremental benefit when administered simultaneously with antibody to CD11 / CD1848. The understanding of the role of ICAM-1 in the pathophysiology of the attack seems to be of particular relevance in humans for several reasons. An increased cerebrovascular expression of ICAM-1 has been demonstrated in primates at four hours of ischemia and reperfusion, particularly in the lenticulostriate microvasculature18. An autopsy study of recent cerebral infarcts in humans also demonstrates an increased expression of ICAM-I20. Since rats also express cerebral vascular ICAM-l in the following 24 hours both in the photochemically induced model of rat cerebral ischemia19 as well as in the occlusion model of the middle cerebral artery, 12 these data suggest the potential utility of deficient transgenic mice. in ICAM-l to elucidate the pathophysiological importance of the increased postischemic cerebral expression of ICAM-1. In particular, the expression time frame of ICAM-l (increased from 4 to 24 hours) in these models suggests that neutrophil-endothelial interactions mediated by ICAM-l may be the target in future pharmacological strategies to improve the subsequent outcome to attack in humans, because this time frame represents a realistic clinical interval for therapeutic intervention. Although neutropenic animals showed increased regional cerebral blood flow compared to controls, compared to neutropenic animals, mice deficient in ICAM-1 tend to have ipsilateral cerebral blood flows even greater than 24 hours. This observation can be related to the phenomenon of reflux deficiency, in which the blood flow does not return to the levels prior to the obstruction even after the release of a temporary vascular occlusion. A significant body of previous research has implicated neutrophils by plugging capillary microvascular beds in this process58, although in a global cerebral ischemia model, a reduction of 85% in circulating leukocyte counts does not decrease the incidence or severity of reflux49. The data suggest that non-neutrophil-dependent mechanisms, which nevertheless involve one, may contribute to the lack of post-ischemic cerebral vascular reflux. Since macrophages and lymphocytes express both LFA-1, which mediates an adhesive interaction with endothelial cells ICAM-I51, it is possible that mice with ICAM-I deficiency have a decreased recruitment of these mononuclear cells, a possibility which is currently the objective of additional research. This hypothesis is supported by multiple pathological observations that show the accumulation of macrophages and lymphocytes in 1-3 days after cerebral infarction12,17,19'34'59. Taken together, these studies indicate that in a mouse model of focal cerebral ischaemia and reperfusion, neutrophils accumulate in the infarcted hemisphere, which neutropenic animals demonstrate cerebral protection. Increased expression of ICAM-1 in cerebral endothelial cells appears to be an important mechanism that activates this recruitment of neutrophils, and mice which are not able to express ICAM-1 demonstrate improved postischemic blood flow, reduced infarct volumes and reduced mortality. These data suggest that pharmacological strategies aimed at interfering with neutrophil-endothelial interactions may improve post-attack outcome in humans.

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EXAMPLE 2: Hypoxia induced hypoxia of Weibel-Palade bodies of endothe cells: A mechanism for the rapid recruitment of neutrophils following cardiac preservation The period of hypoxia (H) is an important main event for vascular dysfunction which accompanies reperfusion, where endothe cells (EC) and neutrophils (PMN) play a central role. It has been hypothesized that the Weibel-Palade body exocytosis of EC (WP) during the hypoxic / ischemic period during organ preservation allows vigorous recruitment of PMNs in postischemic tissue, a process further amplified in a environment rich in oxidant. The exposure of human umbilical vein ECs to a hypoxic environment (p02 «20 torr) stimulates the release of von Willebrand factor (vWF), stored in EC bodies WP, as well as an increased expression of the adhesion molecule of PMN derived from the body WP, P-selectin on the surface of EC. The increased binding of L?: LN-labeled PMNs to the epoxy EC monolayers (as compared to normoxic controls) is blocked with a blocking antibody for P-selectin, but has no effect by a nonblocking control antibody. Although the increased expression of P-selectin and the release of vWF is also observed during reoxygenation, H alone (even in the presence of antioxidants) is sufficient to increase the exocytosis of the WP body. To determine the relevance of these observations in hypothermic cardiac preservation, during which the p02 within the cardiac tilt declines to similarly low levels, experiments were carried out in rodents (rat and mouse) of cardiac preservation / transplant model. Immunosuppression of recipient PMNs or administration of antibody to P-selectin blocker prior to transplantation results in reduced infiltration of neutrophils into the graft and improved graft survival, compared to identical preserved hearts transplanted to control receptors. To establish the important role of endothelial P-selectin expression on the donor vasculature, cardiac transplants were performed in mice using homozygous P-selectin-deficient donor hearts and washed wild-type controls free of blood / platelets prior to preservation. transplant. Hearts devoid of P-selectin transplanted into the wild type receptors demonstrated a remarkable (13-fold) reduction in neutrophil infiltration to the graft and increased survival to the graft compared to wild-type hearts transplanted into wild-type receptors. To determine if the exocytosis of the Weibel-Palade coronal endothelial body can occur during cardiac preservation in humans, the release of vWF in the coronary sinus was measured in 32 patients during open-heart surgery. The coronary sinus samples obtained at the beginning and at the end of the ischemic period showed an increase in the coronary sinus vWF antigen (by ELISA) consisting of multimers of predominantly high molecular weight (by immunoelectrophoresis). These data suggest that Weibel-Palade EC body exocytosis occurs during hypothermic cardiac preservation, by dissecting the vasculature to rapidly recruit PMNs during reperfusion.

Introducció: Endothelial cells (EC) adapt to hypoxia with a characteristic repertoire of responses (1), ranging from an increased expression of endothelin (2) to increased synthesis of basic fibroblast growth factor (3). Recent studies have indicated that many characteristics of CD responses to hypoxia are parallel to the characteristics of the inflammatory response; hypoxia selectively up-regulates EC expression of interleukin-1 (4), 6 (5), and 8 (6), platelet activating factor (PAF) (7,8), and ICAM-1 (4). ), which serve to promote the recruitment of neutrophils PMN, adhesion and activation in the ischemic site. Although these mechanisms may explain the later stages of reperfusion injury, the rapidity with which PMNs are recruited to the reperfused myocardium after a period of hypothermic preservation suggests that the mechanisms are involved which do not require the synthesis of de novo protein. In this regard, P-selectin may figure prominently in the earliest phases of PMN adhesion to the reperfused vasculature, and CDs may be rapidly expressed P-selectin pre-formed from sub-phalasmal storage sites in the membranes of the Weibel-body. Palade (9) in response to the abundance of free oxygen radicals generated in the reperfusion environment (10-12). In addition, recent data have pointed to a role of P-selectin-mediated leukocyte suppression in leukostasis and tissue damage associated with lung damage (13) and cardiac ischemia (14). Taken together, these findings lead to the hypothesis that the hypoxic / ischemic period associated with hypothermic myocardial preservation resembles the vasculature for its characteristic response during reperfusion by prominently displaying P-selectin on the EC surface before reperfusion, which serves as a spark which ignites and amplifies the subsequent inflammatory response. The experiments were designed to establish whether hypoxia per se (or hypothermic cardiac preservation, as occurs during cardiac surgery, in which p02 within the coronary bed declines at pO2 <20 Torr) (15) may result in exocytosis of WP bodies. In addition, experiments were designed to determine the role of P-selectin-dependent PMN adhesion in cardiac graft failure which characteristically follows a prolonged hypothermic preservation period. The results show that hypoxia is sufficient to induce the exocytosis of WP bodies of EC, even in the absence of reoxygenation (and the presence of antioxidants) and that the resulting expression of P-selectin causes ECs to bind to PMN in vitro. In rodents, the adverse consequences of P-selectin expression following hypothermic cardiac preservation can be completely abrogated either by neutrophil suppression, P-selectin blocking or by transplantation of hearts whose endothelial cells fail to express P-selectin. Because WP body exocytosis also occurs in patients undergoing open-heart surgery during the hypothermic cardiac preservation period, these data suggest that blocking P-selectin may represent an objective for pharmacological intervention to improve preservation. cardiac in humans.

Methods: Culture of endothelial cells and exposure of the cells to H or H / R. EC of human umbilical vein was prepared from umbilical cords and grown in culture by the method of Jaffe (16) modified by Thornton (17). The experiments used confluent EC (passages 1-4) that grew in 199 medium supplemented with fetal bovine serum (15%; Gemini, Calabasas, CA), human serum (5%; Gemini), endothelial growth supplement (Sigma, St. Louis, MO), heparin (90 μg / ml, Sigma) and antibiotics, as described (17). When the ECs reached confluence, experiments were performed placing cultures in an environmental chamber Coy Laboratory Products, Ann Arbor, MI) which was provided with temperature control (37 ° C) and atmosphere with the indicated amount of oxygen, carbon dioxide (5%) and the rest constituted of nitrogen. The use of this camera for cell culture experiments has been previously described (15,18). During the exposure of the CS to hypoxia (for a maximum of 16 hours), the oxygen tension in the culture medium was 14-18 torr and there was no change in the pH of the medium. Reoxygenation was performed by placing the EC in an ambient atmosphere containing carbon dioxide (5%) at 37 ° C.

Measurement of Weibel-Palade body exocytosis: ECs were plated in 24-well plates, 3 times Hank's balanced salt solution was soaked and exposed to hypoxia or normoxia for the indicated durations. For the experiments in which vWF was measured, the cells were maintained in serum-free medium. All other experiments with EC were performed in the EC growth medium as described above. For vWF measurement, 200 μl aliquots of culture supernatant were removed at the indicated times, and samples were taken in duplicate commercially available ELISA (American Diagnostica, Greenwich, CT), based on a polyclonal human vWF goat antibody, with a standard curve generated using purified human vWF antigen supplied by the same supplier. The expression of P-selectin in EC was determined by measuring the specific binding of a mouse monoclonal anti-human P-selectin antibody (WAPS 12.2 clone, Endogen, Cambridge, MA, this is an IgG1 which recognizes a calcium and block sensitive epitope P-selectin-dependent neutrophil adhesion The antibody was radiolabeled with 125 I by a lactoperoxidase method (19) using Enzymobeads (Bio-Rad, Hercules, CA), stored at 4 ° C and used in the following week of labeling The binding assays were performed in HUVEC seeded in 96-well plates, in which fresh M199 with 0.1% bovine serum albumin (Sigma, St. Louis, MO) was added immediately before each experiment. a humidified environment at 37 ° C and exposed to normoxia or H (in the presence or absence of 50 μM profucol as indicated, Sigma) for the indicated durations Cell monolayers were fixed for 15 minutes with paraformaldehyde % 10 (cells exposed to H were fixed while still within the hypoxic aminent), visually inspected to ensure that the monolayers remained intact, and washed twice with HBSS containing 0.5% bovine serum albumin (HBSS / A). The monlayers were then exposed to 105 cpm of antibody against 12SI-labeled P-selectin (WAPS 12.2) in the presence of 200 μg / ml unlabeled blocking antibody (WAPS 12.2) or non-blocking IgG against P-selectin of the same isotype ( anti-GMP-140, clone AC1.2, Becton-Dickinson, San Jose, CA) (20,21). After binding for 1 hour at 37 ° C, the monolayers were washed 4 times with HBSS / A, bound antibody was eluted with 1% triton X-100 in PBS (200 μl / well) and counted. For certain experiments, cycloheximide (10 μg / ml, Sigma) was added at the beginning of the normoxic or hypoxic period of 4 hours, as indicated. In separate experiments, designed to determine the degree of inhibition of protein synthesis by treatment with cycloheximide, the ECs were incubated with minimal essential medium methionine-poor or - cysteine (Gibco, Grand Island, NY) in the presence of 35S-methionine and 3SS-cysteine (either in the presence or in the absence of cycloheximide, 10 μg / ml) (3). After 4 hours of normoxic exposure, the precipitable material was collected and counted with trichloroacetic acid.

Preparation of human PMN and binding measurement: Briefly, citrated blood from healthy donors was diluted 1: 1 with NaCl (0.9%) followed by gradient ultracentrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Posterior to the hypotonic smooth of residual erythrocytes (exposure for 20 seconds to distilled H20 followed by reconstitution with 1.8% NaCl), PMNs were suspended in HBSS with 5 mg / ml human serum albumin (HBSS / HSA). 50-200 x 106 PMN were suspended in HBSS / HSA in the presence of 0.2-0.5 μCi of lxlIndium oxine (Amersham Mediphysics, Port Washington, NY) for 15 minutes at 37 ° C. After washing with HBSS / HSA, the PMN were gently sedimented (450 g) and resuspended in HBSS / HSA to a final concentration of 5.5 x 10 6 PMN / ml. After gentle agitation, 100 μl of radiolabeled PMN suspension was added to each well at the indicated time, incubated for 30 minutes at 37 ° C and then washed four times with HBSS / HSA. The monolayers were then treated with NaOH and the content of each well was extracted and counted.

Heterotropic model of cardiac transplantation in rat and mouse. Cardiac transplants were performed in the Ono-Lindsey heterotropic isograft model of cardiac transplantation (15,18,22).

Briefly, male Lewis rats (250-300 grams, Harran Sprague Dawley, Indianapolis, IN) were anesthetized, heparinized and the donor heart was rapidly removed after cardioplegic suppression with potassium, highly hypothermic. The hearts were preserved by washing the coronary arteries with lactated Ringer's solution (LR) at 4 ° C followed by heterotropic transplantation in paired receptors by gender / strain, with aortic anastomosis in the donor and recipient and pulmonary arterial anastomosis in the donor / in the inferior vena cava of the recipient performed sequentially. Graft survival was determined by the presence / absence of cardiac electrical / mechanical activity exactly 10 minutes after the restoration of blood flow, after which the grafts were excised and the neutrophil infiltrations were quantified by myeloperoxidase activity, measured as previously described (15,18). For certain experiments, neutrophil suppression of the recipient rats was carried out by administering a rabbit antibody against polyclonal rat neutrophils (23-25) (Accurate Scientific, Westbury NY) as a single intravenous injection 24 hours before the procedure of transplant. The suppression of neutrophils in these animals was confirmed and quantified by counting the remaining neutrophils, identified in Wright-Giemsa staining of peripheral blood. In other experiments, an igG anti-P-selectin blocker (250 μg / rat, Cytel, San Diego, CA) (13,14,26) was administered intravenously 10 minutes before the access of reperfusion. Heart transplants of mice were performed identically using homozygous male mice devoid of P-selectin or control, wild-type, with a C57BL / 6J background (27), with hearts excised immediately and released by native blood flow with 1.0 ml of LR at 4 ° C administered downwards through the transverse clamped aortic root followed by a hypothermic preservation period consisting of 3 hours of immersion in lactated Ringer's solution at 4 ° C.

Measurement of vWF in the coronary effluent of hypothermically preserved rat and human hearts: Human coronary sinus samples. After obtaining informed consent, coronary sinus blood was obtained at the beginning and at the conclusion of a normal cardiac surgery in a non-selected series of 32 patients, with simultaneous sampling of peripheral (arterial) blood in six. The coronary sinus samples were obtained from a retrograde perfusion catheter, which is usually placed in patients undergoing cardiopulmonary bypass. Palm samples were centrifuged for 5 minutes at 1500 x g to pellet the cellular elements, and aliquots were taken from the plasma and frozen at -70 ° C until the time of the assay. The ELISA test was performed to determine vWF (as described above) and thrombomodulin (Asserchron Thrombomodulin, Diagnostica Stago).

Immunoelectrophoresis of vWF: Multimeric composition of vWF in coronary sinus plasma samples and endothelial cell supernatants was evaluated when performing agarose gel immunoelectrophoresis. Samples were diluted 1:10, 1:20, and 1:30 (as indicated) and incubated for 30 minutes at 37 ° C in native sample buffer (Bio-Rad). The samples (20 μl) were then subjected to electrophoresis in 1.5% agarose gel (0.675 g of Low Mr agarose, Bio-Rad, 0.045 g of SDS, 45 ml of SDS Tris-tricine buffer [Bio-Rad]) . Molecular weight markers were run simultaneously on agarose gels and visualized by labeling and dividing the gel, with assigned molecular weight marker positions by Coomassie blue staining. The remaining half of the gel was washed in sodium borate (0.01 M) for 30 minutes followed by electrophoretic transfer overnight to a nitrocellulose membrane. The membrane was washed with wash buffer consisting of Tris buffered saline (pH 7.5) with 0.05% Tween-20 and then blocked for 1 hour with 50 ml of wash buffer containing 2.5 g of Carnation instant milk. After soaking with physiological saline, the membrane is immersed overnight in a wash buffer containing 1 g / dl of gelatin and a 1: 500 dilution of rabbit serum against human vWF (American Bioproducts, Parsippany, NJ). After washing 5 times with wash buffer, the membrane was immersed for 3 hours with gentle agitation in wash buffer containing 1 g / dl of gelatin and 16.6 μl of goat anti-rabbit IgG, conjugated with horseradish peroxidase (Bio -Rad) and was developed with 60 ml of HRP developer (30 mg HRP developer powder, Bio-Rad; 10 ml of methanol; 50 ml of saline buffered with Tris, - 50 μl of 30% hydrogen peroxide adjusted just before use).

Statistics. The analysis of variance was used to compare 3 or more conditions, with post-hoc comparisons tested using the Tukey procedure. The graft survival data were analyzed using contingency analysis with the Chi square statistic. Paired comparison of serial measurements (human CS and peripheral blood samples at baseline and at the end of cardiac surgery) were compared using the Stundent t test for paired variables. Values are expressed as means ± SEM (standard error mean), with a p < 0.05 considered as statistically significant.

Results: Exposure of cultured CDs to hypoxia resulting in the release of vWF and translocation of P-selectin to the cell surface. Previous studies have shown that the exposure of endothelial cells to hypoxia results in an elevation of intracellular calcium (28). In view of the association of increased cytosolic calcium with Weibel-Palade body exocytosis in CD, in response to thrombin or histamine (29,30), it was considered whether the exposure of CDs to hypoxia can initiate this process. The ECs placed in a hypoxic environment (p02 20 torr) released more vWF in the culture supernatants compared to their normoxic counterparts (Figure 8A, ELISA, confirmed by immunoelectrophoresis, data not shown). Although a trend towards increased concentrations of vWF was first observed at 1 hour of hypoxia, the differences between the normoxic hypoxic pWF concentrations are not statistically significant until 4 hours of exposure, and then increase steadily until 12 hours observational. To determine whether the increased vWF release observed by hypoxia at 4 hours of age, due to the release of preformed vWF, similar experiments were carried out in the presence of 10 μg / ml cycloheximide to inhibit protein synthesis. These experiments showed that the addition of cycloheximide at the beginning of the hypoxic period decreases the release of vWF decreased by hypoxia by 12.5%, suggesting that most of the vWF released by hypoxic exposure was preformed. Although these experiments were performed entirely within a hypoxic environment (ie, there was no reoxygenation), to further demonstrate that this H-mediated exocytosis of Weibel-Palade bodies is independent of the formation of reactive oxygen intermediates, it was added the antioxidant probucol (50 μM) to the EC at the beginning of H and it was found to have no effect (vWF, 4.7 ± 0.31 x 10"3 U / ml, at 6 hours of H.) The presence of probucol prevented the increase in the concentrations of vWF observed after reoxygenation in hypoxic ECs The calcium dependence of Weibel-Palade bodies exocytosis induced by hypoxia was demonstrated by experiments in which the ECs were placed in a calcium-free medium At the onset of hypoxic exposure, the absence of extracellular calcium attenuated the release of H-induced EC, and the addition of EGTA had an even more suppressive effect (basal endothelial release). e vWF also decreased by the reduction of extracellular calcium) (Figure 8B). To determine whether hypoxia also induces translocation of P-selectin to the plasmalemma surface of the ECs, the specific binding of IgG against P of selectin, labeled with 12SI to monolayer of normoxic or hypoxic EC was examined. The binding studies were performed on monolayers of fixed ECs with paraformaldehyde while they were still in a hypoxic environment, to eliminate the expression of P-selectin induced by oxygen free radicals during reoxygenation. These studies demonstrated an improved binding of IgG against labeled P-selectin against 125 I by hypoxic EC compared to normoxic EC (Figure 9A). This binding is blocked by the blocking IgG, against unlabeled P-selectin, but not by IgG against non-blocking P-selectin control of the same isotype. The surface expression of P-selectin was noted at the earliest points and time observed (60 minutes of H), and was observed at similar concentrations during the period of hypoxic exposure (up to 4 hours of observation). It is possible that hypoxia-induced endothelial P-selectin expression was detected at time points preceding a statistically significant increase in vWF release in similarly treated cells, because a portion of the vWF secreted initially binds firmly to the matrix subendothelial (31). To determine if protein synthesis was required for the hypoxia-induced expression of P-selectin, a separate experiment was performed in which cycloheximide was administered at the onset of normoxia or H, and binding of anti-P-selectin IgG was determined. , radio-labeled, at the time point of 4 hours. This experiment showed that even with >85% inhibition of protein sesis (Figure 9B, Insertion), hypoxia still increases the endothelial expression of P-selectin, albeit at reduced concentrations (Figure 9B). To establish that hypoxia-induced P-selectin on the cell surface can participate in neutrophil binding, radiolabeled human neutrophils were incubated with 111 indium oxin with hypoxic EC; increased binding to hypoxic monolayers was observed. The binding of PMN labeled with X11ln induced by hypoxia was blocked by the addition of a blocking anti-P-selectin IgG, but not by nonblocking anti-P-selectin IgG (Figure 9C) Role of P-selectin-dependent neutrophil adhesion in the preservation of hypothermic / ischemic myocardium. To establish the relevance of these observations to hypothermic myocardial preservation (in which the p02 of the preservation solution within the coronary basculature decreases below 20 Torr (15)), hearts of male Lewis rats were collected and subjected to Hypothermic preservation as described in the methods section. Because neutrophil mediated damage after cardiac ischemia has been well established (32-38), the potential pathophysiological role of P-selectin expression in an orthotopic model of rat heart transplantation in which subsequent reperfusion occurred was investigated. to a period of hypothermic preservation. These experiments showed excellent graft survival and poor neutrophil infiltration if the transplant was performed immediately after harvest (Figure 10A, fresh). However, when similar experiments were carried out with an intervention period (16 hours) of hypothermic preservation between transplant collection procedures, there was a high incidence (90%) of graft failure and marked leukostasis, confirmed histologically and by activity. determined myeloperoxidase (Figure 10A, Preserved). To demonstrate that neutrophil adhesion was responsible, at least in part for graft failure after prolonged preservation, transplants were performed after the suppression of neutrophils from recipient rats. Rabbit antibody against polyclonal rat PMN used (23-25) virtually eliminated all circulating PMN at the receptors (PMN count 1471 ± 56 versus 67 + 11 PMN / mm3 for control and immunosuppressed animals, respectively, p <0.001 ), with little effect on other types of cells. When reserved hearts were transplanted for 16 hours to suppressed neutrophil receptors to provide a neutrophil-free reperfusion environment, there was a significant reduction in myeloperoxidase activity in the graft and an increase in graft survival (Figure 10A, Preserved (- ) PMN). Normal recipient rats subjected to IgG infusion against blocking P-selectin 10 minutes before the restoration of blood flow demonstrated a reduction in both myeloperoxidase activity and an improvement in graft survival (Figure 10A, a-PS, blocked). a similar magnitude of neutrophil-suppressed receptors. This reduced infiltration of PMN and improved graft survival was observed despite 16 hours of hypothermic preservation of the donor heart. In conspicuous contrast, administration of a nonblocking control antibody (AC1.2) did not have a beneficial effect on graft leukostasis or graft survival (Figure 10A, a-PS, no blockage). Because in addition to the interactions between ECs and PMNs, platelets can also interact with PMNs via a P-selectin-dependent mechanism (39), an experiment was designed to isolate the construction of endothelial P-selectin to the leukostasis and graft failure which occurs after prolonged hypothermic cardiac preservation. For these experiments, the donor hearts of homozygous P-selectin-deficient mice were flushed and released from blood so that coronary endothelial cells lacking P-selectin can be transplanted to wild type receptors with platelets containing P-selectin. . Using a heterotrophic cardiac transplant model performed identically to the rat operation, donor hearts were obtained from both homozygous mice lacking P-selectin (27) and from wild-type controls; all hearts were transplanted in wild type receptors. These experiments demonstrated a significantly higher graft survival rate in transplants from mice lacking P-selectin to wild type, compared to transplants from the wild type to the wild type (Figure 10B). This improved graft survival in the first group parallels a marked reduction (13-fold) in graft leukostasis (Figure 10C). Because these hearts have been washed and released from blood at the beginning of preservation, these studies imply that the coronary endothelial P-selectin (instead of the platelet-derived one) in the poor preservation and deletion of leukocytes indicated after myocardial preservation hypothermic Exocytosis of the Weibel-Palade body during human cardiac surgery. To establish the relevance of these findings in humans, the following set of experiments was designed to demonstrate that coronary ECs release the contents of Weibel-Palade bodies during hypothermic cardiac preservation as occurs during regular cardiac surgery. Measurements of the release of vWF from the coronary vasculature were made during a well-defined period of cardiac ischemia, which occurs during the period of transverse aortic clamping. Blood samples were taken from the coronary sinus (drained from the heart) at the beginning (CS ^ and conclusion (CS2) of a cross-clamping aortic in 32 patients (this interval represents the ischemic period.) These patients (23 men, 9 women) presented clinical history of valvular heart disease (n = ll), or ischemic heart disease (n = 21) and experienced repair / replacement of the valve or coronary artery bypass graft, respectively Capture ELISAs performed for the thrombomodulin protein of integral membrane (40) showed no change in the concentrations between CS- and CS2 samples (4.35 ± 1.2 ng / ml versus 3.48 ± 0.8 ng / ml, p = NS), suggesting that ECs do not detach and that the integrity of the cell membrane is maintained during cardiac preservation Similar measurements performed for vWF showed that there is a consistent and significant increase in vWF that is secreted during the course of cardiac reserve (0.68 ± 0.06 U / ml versus 0.90 + 0.05 U / ml, CSX versus CS2, p < 0.01) (Figure HA). To demonstrate that this vWF is likely to be coronary endothelial rather than platelet-derived and therefore does not involve a consequence in cardiopulmonary bypass, peripheral blood samples were obtained spontaneously with samples and CS2, showed that vWF concentrations did not change (0.813 ± 0.52 U / ml versus 0.90 ± 0.41 U / ml, p = NS), suggesting that the mechanical disturbance of platelets during cardiopulmonary bypass is not causative. Because vWF is present in plasma as multimers with a range of Mr 's (41-44), with those vWF multimers of the accumulated stimulant (as opposed to the constitutively secreted) which is of higher molecular weight (45) , immunoelectrophoresis is performed on the CS samples. These gels demonstrated that in addition to a total increase in vWF in the CS2 / samples there appears to be an increase in high molecular weight multimers, suggesting release of a stimulable pool, as found in endothelial cells (Fibure 11B).

Discussion: The vasculature plays a critical role in maintaining the extracellular environment of organs undergoing ischemia and reperfusion, a role which is orchestrated mainly by the ECs found in the endovascular lumen. The EC responds to a period of oxygen deprivation by surprising phenotypic modulation, becoming prothrombotic (46) and proinflammatory (1,4,6). CDs exposed to hypoxia secrete proinflammatory cytokines IL-1 (4) and IL-8 (6) which can be used to direct leukocyte trafficking to areas of ischemia. Because these processes require de novo protein synthesis, they do not explain the immediate events that occur after a period of hypothermic preservation. Although the increased expression of ICAM-l and induction of E-selectin may contribute in periods after the suppression of leukocytes in cardiac grafts, this does not explain the rapid leukostasis observed after cold preservation, in which the synthesis is likely of protein decrease considerably. In this context, pretreatment with cycloheximide does not alter the early adhesion (90-120 minutes) of PMN observed after the hypoxic exposure of CDs (7), suggesting that de novo protein synthesis does not need to be involved in increases mediated by hypoxia in the PMN junction. Although platelet activation factor (PAF) can participate in PMN adhesion mediated by hypoxia (7, 47) and activation (48,49), PAF is not stored and must be synthesized, which may decrease its importance during the hypothermic period during myocardial preservation. It is for this reason that the rapid expression of P-selectin EC formed previously from subplasmalemal storage sites in the bodies of Weibel-Palade (9,50,51) may represent the most important mechanism for the early recruitment of later PMN to hypothermic preservation. The bodies of Weibel-Palade are found in abundance within the coronary microvasculature (52), which suggests their particular importance in cardiac preservation. The data show that Weibel-Palade exocytosis occurs both in response to hyp per se, as well as in human hearts during hypothermic preservation. Although it is difficult to accurately identify an endothelial origin for the vWFs observed in human coronary sinus samples, platelet studies after cardiopulmonary bypass show that there is no increase in surface expression of P-selectin or granule secretion or. (53.54). This suggests that the increase observed in vWF in the coronary sinus after aortic cross-clamping is not of platelet origin. Two aspects of the data also suggest that the vWF released after ischemia is of endothelial origin; (1) peripheral vWF concentrations remain unchanged while coronary sinus concentrations increase after myocardial ischemia, suggesting that elevated vWF arises from the heart, and not from the cardiopulmonary bypass device; (2) The donor hearts devoid of P-selectin, transgenic were released by washing donor blood at the start of preservation, so that when transplanted into wild-type recipient patients, coronary endothelial P-selectin was probably absent ( no platelet). These experiments demonstrate the important contribution of endothelial P-selectin to the recruitment of neutrophils which accompany reperfusion. It is not surprising that P-selectin is important after hypothermic myocardial preservation; Recent studies have shown that P-selectin is an important mediator of neutrophil-induced reperfusion damage following normothermic ischemia, as has been demonstrated in rabbit ears (26) and feline cardiac ischemia models (14). Because oxidants cause P-selectin expression on the EC surface (10), it is important in these studies to evaluate the role of the hypoxic period alone which can sebar the CIs to recruit the first wave of PMN, where recruitment Additional PMN amplified with the assault of the reactive oxygen intermediates produced in the reperfusion microenvironment. Although a report has suggested that hypoxia can induce the expression of P-selectin by CDs, these experiments (7) are currently performed after reoxygenation, a condition which is known to induce both its peroxide (18,55) and its adhesion. neutrophils to the cultivated CDs (56). In contrast, the experiments described herein were performed completely within a hypoxic environment to completely avoid the possibility of reoxygenation, and the antioxidants did not block hypoxia-induced expression of P-selectin, suggesting that the observations described in. the present reflect hypoxia hypoxia per se instead of reoxygenation. In addition, the cardiac protection demonstrated herein using a strategy wherein the preserved blood-free hearts of transgenic mice lacking P-selectin are transplanted into receptors with wild-type platelets demonstrate that the endothelial expression of P-selectin may be harmful subsequent to Hypothermic cardiac preservation. Because body exocytosis of Weibel-Palade bodies occurs during hypothermic cardiac preservation in humans, these studies suggest that myocardial preservation can be improved by therapeutic strategies designed to block the activity of P-selectin expressed on the endothelial surface.

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EXAMPLE 3: Procedure and strain-related variables that significantly affect the outcome in a mouse model of focal cerebral ischemia The recent availability of transgenic mice has led to an increasing number of reports describing the effects of specific gene products on the pathophysiology of attacks. Although focal cerebral ischemia models have been well described in rats, descriptions of a mouse model of medial cerebral artery occlusion are lacking, and sources of potential experimental variability remain undefined. It has been hypothesized that light technical modifications can result in highly discrepant results in a mouse model of attack and that control of surgical and procedural conditions can lead to post-procedural physiological and anatomical attack results. To test this hypothesis, a mouse model was established, which may allow permanent or transient focal cerebral ischemia due to intraluminal occlusion of the middle cerebral artery (MCA). This study provides a detailed description of the surgical technique and reveals important differences between the • strains commonly used in the production of transgenic mice. In addition to the differences related to the strain, the infarct volume, the neurological result and the cerebral blood flow seem to be significantly affected by the temperature during the ischemic and post-ischemic periods, the size of the mouse and the size of the suture. which obstructs the vascular lumen. When these variables are kept constant, there is a remarkable uniformity in the result after the attack. These data emphasize the protective effects of hypothermia in the attack, and can help to standardize techniques between different laboratories to provide a cohesive structure to evaluate the results of subsequent studies in transgenic animals.

Introduction: The recent advent of genetically altered mice provides a unique opportunity to evaluate the role of unique genetic products in the pathophysiology of attacks. Although there has been an increasing number of reports about the effect of cerebral ischemia in transgenic mice, to date, there is no detailed description of the mouse models involved, nor is there a detailed analysis of the potentially important procedural variables which they can alter the state after attack. Most descriptions of a mouse model (1,4,8,9,14,17-19,23,24) are developed descriptions of the widely used rat models of focal cerebral ischemia (22,26). Although there has been some attention given to the differences related to the strain with respect to the susceptibility of mice to cerebral ischemia (4), few technical considerations have been solved in published studies. Because the pilot data demonstrate that minor differences in the operative procedure or postoperative care result in greater differences in post-attack outcome, the present study was designed to systematically identify the important surgical, technical, and anatomical considerations necessary to obtain consistent results in a mouse model of focal cerebral ischemia.

When attacks are generated in a rigidly controlled manner, differences due to absence (or overexpression) of a single genetic process can be easily discerned. This study presents a detailed work on a reproducible mouse model of focal cerebral infarction based on modifications of the original rat model (26). This study identifies the procedural variables that are so intact in the result of the attack, which previously have not been reported in previous descriptions of mouse attack models. These variables include length and caliber d? the suture, methods of vascular control, regulation of temperature in mice and differences between strains commonly used in the proliferation of transgenic animals. As the model described turns itself into the study of permanent or transient focal cerebral ischemia, evidence is presented that with times of ischemia chosen with precision, a volume of infarction and mortality can be made in animals subjected to reperfusion that approaches those observed with permanent occlusion. Understanding the potential sources dependent on the variability model in the post-attack outcome can help clarify divergent results between different laboratories. The adoption of a standardized model which provides consistent results is an important first step towards the use of transgenic mice in the study of the pathophysiology of the attack.

Materials and methods : Acquired animals and anesthesia: Male mice were purchased from three different strains (C57 BlackJd, CD-1 and 129J) from Jackson Laboratories (Bar Harbor, ME). The animals were 8 to 10 weeks old and weighed between 18 and 37 grams (as indicated) at the time of the experiments. The mice were anesthetized with intraperitoneal injection of 0.3 ml of ketamine (10 mg / cc) and xylazine (0.5 mg / cc). An additional dose of 0.1 cc was administered prior to catheter removal in animals that experienced transient ischemia. On the day after surgery, anesthesia was repeated immediately before laser Doppler flow measurement and euthanasia without suffering. These procedures have been approved by the Institutional Committee for the care and use of animals at Columbia University and are in accordance with the AALAC guidelines for the care and human use of laboratory animals.

Surgical installation: The animals were placed in supine position on a calibrated pad which rests on a surface that operates at a controlled temperature (Yellow Springs Instruments, Inc. [YSI], Yellow Springs, OH). A rectal temperature probe (YSI) was inserted in order to regulate the temperature of the operating surface to maintain a constant temperature in the animal's core of 36-38 ° C. To facilitate exposure, the right hind leg and the left anterior leg were pasted to the operating surface, the right anterior leg was attached to the animal's chest and the tail was stuck to the rectal probe (Figure 12A). An incision was made in the midline of the neck by gently lifting the loose skin between the handlebar and the jaw, a skin circle of 1 cm2 was cut. The paired midline of the mandibular glands is directly underlying this area and was divided in a general way, with the left gland left in situ. The right gland was extracted cranially with a small straight Sugita aneurysm clip (Mizutto America, Inc., Beverly, MA) secured to the table by silk 4.0 and tape. The sternocleidomastoid muscle was identified and a 4.0 silk ligature was placed around its prominent part. This ligature was inferolaterally extracted and stuck to the table to expose the homoyoid muscle covering the carotid cover. The exhibition is shown in Figure 12B.

Operational approach: Once the carotid envelope was exposed, the mouse and temperature control surface were placed under an operating microscope (16-25X magnification zoom, Zeiss, Thornwood, NY), with a light source coaxial used to illuminate the field. Under the amplification, the homoyoid muscle was carefully divided with retractors. The common carotid artery (CCA) was carefully freed from its cover, taking care not to apply tension to the vagus nerve (which runs lateral to the CCA). Once released, the CCA is isolated with silk 4.0, sticks loosely to the operating table. Once proximal control of the CCA is obtained, the bifurcation of the carotid is placed in view. The occipital artery, which arises from the proximal external carotid artery and is directed posterolaterally through the proximal internal carotid artery (ICA) to enter the digastric muscle, is isolated at its origin and divided using a bipolar microcoagulator Malis ( Codman-Schurtleff, Randolph, MA). This allows a better visualization of the AAI as it is subsequently directed and is towards the lower cephalic part of the styloid muscle towards the base of the skull. Just before the ICA enters the skull, the pterygopalatine branch is divided, which is directed laterally and cranially. This branching is identified, isolated and divided at its origin, during which time the CCA-ICA axis becomes straight. A silk 4.0 suture is then placed around the internal carotid artery for distal control, the end of which sticks resolutely to the operating surface. Later, the external carotid artery is exposed. The cranio-medial course is contoured in its first branch, and the superior thyroid artery is cauterized and divided. The contouring is subsequently carried out distally by raising the yoid bone to expose the bifurcation of the artery in the lingual and maximal arteries. Just proximal to this bifurcation, the external carotid is cauterized and divided. Subsequently, sufficient tension is applied to the silk sutures surrounding the proximal and internal distal common carotid arteries to occlude the blood flow, taking care not to traumatize the wall of the artery. The tape of the occlusion sutures is re-adjusted to maintain occlusion.

Introduction and sewing of the intraluminal suture of occlusion: Immediately after the occlusion of the carotid, an arteriotomy is performed in the distal outer carotid wall just proximal to the cauterization area. Through this arteriotomy, a 5.0 or 6.0 nylon suture that has become blunted by heat is introduced (as indicated in the results section) (Figures 12C and 12D). As the suture advances to the level of the carotid bifurcation, the external stump gently retracts caudally, directing the tip of the suture to the proximal ICA. Once the occlusion suture enters the ICA, the tension on the proximal and distal control sutures is relaxed and the occlusion suture is advanced slowly up the ICA, towards the base of the skull under direct visualization (beyond from the level of the base of the skull, the view of the occlusion suture is lost).

The location of the distal tip of the occlusion suture through the origin of the middle cerebral artery (MCA) (proximal to the origin of the anterior cerebral artery) was determined by the length of the selected suture (12 mm or 13 mm). indicated by the results section, shown in Figure 12C) by laser Doppler flowmetry (see the section on ancient physiological procedures), and by post-sacral staining of the cerebral vasculature (see below). After the placement of the occlusion suture was completed, the stump of the external carotid artery was cauterized to prevent bleeding through the arteriotomy once the arterial flow was restored.

Completed surgical procedure: For all of the experiments shown, the occlusion of the carotid was less than two minutes. To close the incision, the sutures surrounding the proximal and distal CCA as well as the sternocleidomastoid muscle were cut and removed. The aneurysm clasp was removed from the submandibular gland and the gland was placed on the field of operation. The cutaneous edges were approached with a surgical staple and the animal was removed from the table.

Removal of the occlusion suture to establish transient cerebral ischaemia: Transient cerebral ischaemia experiments require the reexploration of the wound to remove the occlusion suture. For these experiments, an initial wound closure was performed with a temporary aneurysm clasp instead of a surgical staple to provide rapid access to the carotid. A proximal control with a 4-0 silk suture was restored before removal of the occlusion suture to minimize bleeding from the outer carotid stump. During the removal of the occlusion suture, cauterization of the stump of the external carotid artery was started early, before the distal suture had been completely cleared from the stump. Once the suture has been completely removed, the stump is cauterized more extensively. The restoration of flow in the extracranial internal carotid artery is visually confirmed and the wound closed as for the permanent focal ischemia described above. Confirmation of intracranial reperfusion is carried out with laser Doppler flowmetry (see section on ancient physiological procedures).

Calculation of the attack volume: Twenty-four hours after occlusion of the middle cerebral artery, the surviving mice were re-anesthetized with 0.3 cc of ketamine (10 mg / ml) and xylazine (0.5 mg / ml). After the final weight, temperature and cerebral blood flow readings (as described in the following), the animals were perfused with 5 ml of a 0.15% solution of methylene blue and saline to improve visualization of the cerebral arteries . Later the animals were decapitated and the brains were removed. The brains were then inspected to determine evidence of correct placement of the catheter, as evidenced by negative staining of the vascular territory irrigated by the MCA, and placed in the mouse brain matrix (Activational Systems Inc., Warren, MI) for 1 mm slices. The sections were immersed in 2%, 3, 5-triphenyltetrazolium chloride 2% (TTC) in 0.9% phosphate buffered saline, incubated for 30 minutes at 37 ° C and placed in 10% formalin (5) . After staining with TTC, the infarcted brain was visualized as an area of non-stained tissue (white) and a surrounding background of viable tissue (brick-red color). Serial sections were photographed and projected on plot paper to uniform amplification; all the sections in series were drawn, cut and the paper was weighed by a technician who did not know the protocol regarding the experimental conditions. Under these conditions, the infarct volumes are proportional to the summed weights of the roles that circumscribe the infarcted region and are expressed as a percentage of the right hemispheric volume. These methods have been validated in previous studies (3,12,15,16).

Ancient physiological studies: The ancient physiological studies were performed in each of three different strains used in the current experiments, immediately before and after the operative procedure. Systemic blood pressures were obtained by infrarenal abdominal aortic catheterization and measured using a Grass Model 7 polygraph (Grass Instrument Co., Quincy, MA). An arterial blood sample was obtained from this infrarenal aortic catheter; arterial pH, pCO, were measured. (mm Hg), pOa (mm Hg) and oxygen saturation and hemoglobin (%) using a blood gas analyzer and hemoglobinometer (Grass Instrument Co., Quincy, MA). Due to the need for arterial perforation and abdominal manipulation to measure these physiological parameters, the animals were designated only for these measurements (attack volume, neurological outcome and cerebral blood flow were not measured in these same animals). Transcranial measurements of cerebral blood flow were performed using laser Doppler flowmetry (Perimed, Inc., Piscataway, NJ) after reflection of the skin underlying the calvarium, as previously described (10) (the transcranial readings were consistently equal to those performed after craniectomy in pilot studies). To carry out these measurements, the animals were placed in a stereotactic head frame, after which they underwent cutaneous incision of the underlying midline from the nasion to the superior nuchal line. The skin was laterally separated, and a 0.7 mm straight laser Doppler probe (model # PF2B) was lowered onto the cortical surface, moistened with a small amount of physiological saline. The readings were obtained 2 mm posterior to the bregma, both 3 mm and 6 mm on each side of the midline using a stereotactic micromanipulator, maintaining the angle of the probe perpendicular to the cortical surface. Relative cerebral blood flow measurements were performed immediately after anesthesia, after occlusion of the MCA, and immediately before euthanasia, and were expressed as the proportion of the Doppler signal intensity of the ischemic hemisphere compared with the nonischemic one. For animals subjected to transient cerebral ischemia, additional measurements were taken just before and just after suture extraction, initiating reperfusion. Occlusion of the intraluminal surgical procedure / MCA was considered to be technically adequate if = 50% reduction in relative cerebral blood flow was observed immediately after placement of the intraluminal occlusion catheter (15 of 142 animals used in this study [10.6% ] were excluded due to an inadequate drop in blood flow at the time of occlusion). These exclusion criteria are shown in the preliminary studies to provide sufficient levels of ischemia to make infarct volumes consistent with TTC staining. It was considered that reperfusion was technically adequate if the cerebral blood flow at the time of catheter removal was at least twice the cerebral blood flow in the occlusion (13/17 animals in this study [76%]).

Temperature: The core temperature was carefully monitored during the peri-infarction period throughout the experimental period. Prior to surgery, rectal baseline temperature was recorded (YSI, model 74 of rectal probe Thermistemp, Yellow Springs Instruments, Inc., Yellow Springs, OH). Intraoperatively, the temperature was controlled using an operating surface controlled by thermocouple. After occlusion of MCA, the animals were placed for 90 minutes in an incubator, with a minimum temperature maintained at 37 ° C using the rectal probe connected via a thermocouple to a heating source in the incubator. The temperature was similarly controlled in those animals subjected to transient ischemia including a period of 45 minutes (ischemic) as well as a post-ischemic period of 90 minutes in the incubator. After placement of the core temperature in the incubator, the animals returned to their cages for the remaining duration of the observation before slaughter.

Neurological examination: Before administering anesthesia at the time of euthanasia, the mice were examined for obvious neurological deficits using the four-level grading system: (1) normal spontaneous movements, (2) the animal circles to the right, ( 3) the animal turns to the right, (4) the animal crouches on its four legs, without responding to harmful stimuli. It has been shown in preliminary studies that this system accurately predicts infarct size, and is based on systems developed for use in rats (6).

Data analysis: Attack volumes, neurological and brain blood flow scores, and arterial blood gas data were compared using a non-paired Student's t-test. The values are expressed as means ± SEM, with a p < 0.05 considered as statistically significant. Mortality data, when presented, were evaluated using the chi square analysis.

Results: Effects of the strain: Three commonly used, different strains of mice (CDl, C57 / B16, and 129J) were used to compare the variability in the post-attack outcome after permanent focal cerebral ischemia. To establish that there are no large anatomical differences in the collateralization of cerebral circulation, the circle of Willis was visualized using Indian ink in the three strains (Figure 13). These studies showed no large anatomical difference. Mice of similar sizes (20 ± 0.8 g, 23 ± 0.4 g, and 23 ± 0.5 g for 129J, CD1, and C57B1 mice, respectively) were then subjected to permanent focal ischemia under normothermic conditions using 12 mm long occlusion suture. nylon 6-0. Significant differences related to the strain were noted in the infarct volume, with infarcts in 129J mice significantly smaller than those observed in CDl and C57 / B16 despite identical experimental conditions (Figure 14A). The differences in infarct size were matched by neurological examination, with the highest scores (ie, most severe neurological damage) observed in the C57 / B16 and CDI mice (Figure 14B). To determine the relationship between infarct volume and cerebral blood flow to the core region, laser Doppler flowmetry was performed through the calvary of the thin mouse. No preoperative differences were observed related to the strain in the cerebral blood flow, which correspond to the lack of large anatomical differences in the vascular anatomy (Figure 13). The measurement of cerebral blood flow immediately after insertion of the occlusion catheter showed that similar degrees of flow reduction were generated by the procedure (the percentage of ipsilateral / contralateral flow immediately after insertion of the obstruction catheter was 23 ± 2 %, 19 ± 2%, 17 ± 3% for mice 129J, CDl and C57 / B16, respectively). It is not surprising that the blood flow to the core region measured at 24 hours just before euthanasia showed that the lowest blood flows are in those animals with the most severe neurological damage (Figure 14C).

Anatomical and physiological characteristics of the mice: baseline arterial blood pressures as well as pressures to bacterial lines after occlusion of the middle cerebral artery were almost identical for all the animals studied, and were not altered by strain or strain. size of the mouse (Table I). The analysis of arterial blood pH, pC02 and oxygen saturation by hemoglobin (%) revealed similarly that there are no significant differences (Table I).

Effect of animal size and perforation on the occlusion suture: To investigate the effects of mouse size on the result after the attack, mice of two different sizes (23 ± 0.4 g and 31 ± 0.7 g) were subjected to focal cerebral ischemia permanent. To eliminate other potential sources of variability in these experiments, the experiments were performed under normothermic conditions in mice of the same strain (CD1) using occlusion sutures of identical length and perforation (12 mm nylon 6-0). Under these conditions, small mice (23 ± 0.4 g) sustained consistently large infarct volumes (28 ± 9% of the ipsilateral hemisphere). Under identical experimental conditions, the large mice (31 + 0.7 g) showed much smaller infarcts (3.2 ± 3%, p = 0.02, Figure 15A), less morbidity in the neurological examination (Figure 15B) and a tendency to maintain a flow Cerebral blood ipsilateral greater after infarction compared to smaller animals (Figure 15C). Because it has been hypothesized that the reduction in infarct size affects these large animals and is related to poor diameter / length matching between the occlusion suture and the cerebral blood vessels, occlusion sutures were adapted. larger / thicker (13 mm, 5.0 nylon) for use in these larger mice. Large CDI mice (34 ± 0.8 g) which underwent permanent occlusion with these larger occlusion sutures sustained a marked increase in infarct volumes (50 ± 10% ipsilateral hemisphere, p <0.0001 compared to large mice infarcted with the smaller occlusion suture, Figure 15A). These larger mice were infarcted with larger occlusion sutures demonstrating higher scores for neurological deficiency (Figure 15B) and lower ipsilateral cerebral blood flows (Figure 15C) compared to similarly large mice infarcted with smaller occlusion sutures.

Effects of temperature: To establish the role of preoperative hypothermia in the attack volumes and neurological outcomes after occlusion of MCA, small mice C57 / B16 (22 ± 0.4 g) were subjected to permanent occlusion of MCA with a suture of 12-0 gauge 6-0 with normothermia maintained for two different durations; Group 1 ("Normothermia") was operated as described above, maintaining a temperature at 37 ° C from the preoperative period to 90 minutes postocclusion. Group 2 animals ("Hypothermia") were maintained at 37 ° C from the preoperative period until only 10 minutes after occlusion, as previously described (14). In the next 45 minutes after removal of the thermocouple controlled heating incubator, the core temperature of this second group of animals decreased to 33.1 ± 0.4 ° C (and further decreased to 31.3 + 0.2 ° C at 90 minutes). Animals operated under conditions of prolonged normothermia (group 1) showed larger infarct volumes (32 ± 9%) compared to hypothermic animals (group 2) (9.2 ± 5%, p = 0.03, Figure 16A). Differences in infarct volume were reflected by differences in neurological deficit (3.2 ± 0.4 versus 2.0 + 0.8, p = 0.02, Figure 16B), but were largely independent of cerebral blood flow (52 ± 5 versus 52 ± 7, p = NS, Figure 16C).

Effects of transient occlusion of ACM: Because reperfusion injury has been implicated as a major cause of neuronal damage following cerebrovascular occlusion (25) a subset of animals was subjected to a transient period (45 minutes) of ischemia followed by reperfusion as described in the above and comparisons were made with those animals which experienced permanent occlusion of CAM. The occlusion time was chosen based on preliminary studies (not shown) which demonstrate unacceptably high mortality rates (> 85%) with 180 minutes of ischemia and rare infarcts (<15%) with 15 minutes of ischemia. To minimize the confusing influence of other variables, the other experimental conditions were kept constant (small mice (22.5 ± 0.3 g) C57 / B16, the occlusion suture consisted of 12 mm of nylon 6-0 and the experiments were carried out under conditions normothermic). The initial decrease in CBF immediately post-occlusion was similar in both groups (16 ± 2% versus 17 ± 3% for groups of transient versus permanent occlusion, respectively, p = NS). Reperfusion was confirmed both by laser Doppler (increase of 2.3 see in the blood flow after removal of the occlusion suture at 66 ± 13%), and visually by injection of intracardiac methylene blue dye in representative animals. The infarct sizes (29 ± 0.5 versus 32 ± 9%), the neurological deficiency scores (2.5 ± 0.5 versus 3.2 ± 0.4) and the cerebral blood flow at sacrifice (46 ± 18% versus 53 ± 5%) were very similar among animals subjected to transient cerebral ischaemia and reperfusion, and those subjected to permanent focal cerebral ischemia (p = NS, for all groups) (Figures 17A-17C).

Discussion: The increasing availability of genetically altered mice has led to an increase in the use of mouse models of focal cerebral ischemia to allocate specific gene products in the pathogenesis of the attacks. Although recent publications describe the use of an intraluminal suture to occlude the middle cerebral artery to generate permanent.ey / transient cerebral ischemia in mice, there has been only a scant description of the necessary modifications of the original technical report in rats (8,14 , 17-19,24,26). The experiments described in this paper not only provide a detailed technical explanation of a mouse model suitable for permanent or transient focal middle cerebral artery ischemia, but also resolve potential sources of variability in the model.

Importance of the strain: One of the most important potential sources of variability in the model of cerebral ischemia in mice described herein is related to the animal strain used. The data suggest that, of the three strains tested, 129J mice are particularly resistant to neurological damage following occlusion of MCA. Although Barone similarly found differences in the attack volumes between 3 strains of mice (BDF, CFW and BALB / C), these differences were attributed to variations in the posterior communication arteries in these strains (4).

Since anatomical differences in cerebrovascular anatomy are not generally apparent in the study (Figure 13), the data suggest that non-anatomical differences related to the strain are also important in the outcome after occlusion of ACM. Since the result of the attack differs significantly between 2 strains of mice (129J and C57 / B16) commonly used to produce transgenic mice via homologous recombination in embryonic pluripotent cells (11), the data suggest a significant amount of experiments performed with transgenic mice. Because the initial founding progeny for the creation of transgenic animals with these strains have a mixed bottom 129J / C57 / B16, ideally the experiments were performed either with inbreeding controls or after a sufficient number of backcrosses to ensure purity of the strain Importance of size: Larger animals require a larger and thicker intraluminal suture to sustain infarct volumes which are consistent with those obtained in smaller animals with smaller occlusion sutures. The matching of the size of the animal and the suture seems to be important not only to produce a consistent cerebral infarct, but while a small suture leads to insufficient ischemia, too large a suture produces frequent intracerebral hemorrhage and vascular trauma (unpublished observation) . The use of animals of similar size is important not only to minimize the potential age-related variability in neuronal susceptibility to ischemic attack, but also to ensure that small differences in animal size do not obscure the comparison of data with meaning. In this example, it was shown that the difference in size of an amount as small as 9 grams can have a large impact on the volume of infarction and the neurological result after cerebral ischemia. Additional experiments using a larger perforation occlusion suture in older animals suggests that this increased propensity of small animals to present larger attacks is not due to the relative resistance of larger animals to ischemic neuronal damage, but rather to it owes to a small size of the suture used to occlude the MCA in large animals. Although these data were obtained using CDI mice, similar studies have been performed and it has been found that these results are also valid with other strains of mice, such as C57 / B16 (unpublished data). Reports previously published using mice of very different sizes (from 21 g to 35 g) as well as different suture diameters and lengths which have often not been reported (14,17). Studies indicate that the size of the animal and the suture are important methodological characteristics which must be solved in scientific reports.

Importance of temperature: It has long been recognized that hypothermia protects numerous organs from ischemic damage, including the brain. Studies in rats have shown that intrahischemic hypothermia up to 1 hour after occlusion of MCA is protective (2,15), reducing both mortality and infarct volumes with temperatures of 34.5 °. Although these results have been extrapolated to mouse models of cerebral ischemia in those studies, the maintenance of normothermia in animals is often described., the post-MCA occlusion temperature during monitoring periods has been extremely brief ("immediately after surgery" or "10 minutes after surgery") (4,14). The results indicate that the animals do not regulate their temperature beyond these brief durations, and they become severely hypothermic during the post-operative period and these temperature differences of up to 90 minutes after the occlusion of MCA can have a profound effect on the indices of post-seizure outcome that follows MCA occlusion (longer durations of normothermia have not been studied). Although others have claimed that normothermia using a feedback system based on rectal temperature similar to that described here, the duration of normothermia is often not specified (17). The results argue for a clear identification of methods to monitor and maintain the temperature, as well as the durations involved so that experimental results can be compared both within and between centers that study the pathophysiology of the attacks.

Transient versus permanent occlusion: The pathophysiology of certain aspects of permanent cerebral ischemia can be differentiated from cerebral ischemia followed by reperfusion, so it is important that a model is described which allows analysis in any condition. Although the differences between these two models are not extensively tested in the current series of experiments, under the conditions tested (45 minutes of ischemia followed by 23 hours of reperfusion), no significant differences were found in a post-attack outcome index. Variable durations of ischemia and reperfusion have been reported in other mouse models of transient cerebral ischemia, with ischemic times ranging from 10 minutes to 3 hours and reperfusion times ranging from 3 to 24 hours (17,24). Studies in rats have shown that brief periods of ischemia followed by reperfusion are associated with smaller infarcts than permanent occlusion (21,25). However, as the duration of ischemia increases beyond the critical threshold (between 120 and 180 minutes), reperfusion is associated with larger infarcts (7,21,26). For the current series of experiments, the durations of ischemia and reperfusion were chosen so that infarcts comparable to those observed after permanent occlusion of ACM were obtained, which is likely to explain the reason why the data do not show differences between the permanent and transient ischemia. These durations in the transient model were chosen after pilot experiments revealed that shorter ischemic durations (15 minutes) rarely lead to infarction, whereas 180 minutes of occlusion followed by reperfusion leads to massive infarction and almost 100% mortality in the following 4-6 hours in normothermic animals (observation not published). Although post-attack outcome indices can be measured earlier than 24 hours, a 24-hour observation time was chosen because observation at this time allows the study of delayed penumbral death, which is likely to be clinically relevant to the pathophysiology of human attack. In addition, it has been demonstrated in a rat model that 24 hours is sufficient for the maturation of a complete infarction (3,12,15,16).

Technical aspects of the mouse model: The technical aspects of the surgery necessary to create focal cerebral ischemia in mice differs in certain important aspects from that of rats. Self-retaining retractors, which have been mentioned in previous reports in rats (26), are undue unweildy in mice. Retraction based on suture secured with tape provides a superior alternative. In rats, closure occlusion of the proximal and distal carotid artery after the obligation of the external carotid artery has been reported (26), but generates more trauma to the carotid and hemorrhages in the mice. Without control of the distal internal carotid, which has not been previously described in mice, retrosternal retraction from the external carotid artery is consistently uncontrollable. By using the techniques described in this document, surgery can be completed virtually without blood loss, which is especially important given the small blood volume in mice. Unlike the rat model, the occlusion and transection of the branches of the external carotid artery and the pterygopalatine artery in the mouse model is obtained only with electrocautery. Previous reports of surgery in mice have not been clear as to whether the pterygopalatine artery has been taken or not (17,24). Other investigators have described a method with permanent occlusion of the common carotid artery and trans-carotid insertion of the suture without attention to the external carotid system or the pterygopalatine artery. Although it is effective for permanent occlusion, the latter method makes reperfusion studies impossible. The reperfusion method originally described in rats requires the removal of a blind catheter without anesthesia (26). When trying in pilot studies in mice, several animals presented hemorrhage. Therefore, a method of suture removal under direct visualization of the anesthetized animal was developed, which not only allows visual confirmation of the reperfusion of the extracranial carotid artery, but also allows meticulous hemostasis. In addition, the method allows immediate prior and subsequent reperfusion to laser Doppler flowmetry readings in the anesthetized animal. These laser Doppler flowmetry readings are similar to those described by Kamii et al. and Yang et al. where the readings are made intermittently and with the use of a stereotactic micromanipulator (17,24). However, the readings differ in that the coordinates used (2 mm posterior and 3 and 6 mm lateral to the bregma) are slightly more lateral and posterior than the previously published nucleus and the penumbral coordinates (1 mm posterior and 2 mm and 4.5 mm lateral to bregma). These coordinates, which were adopted based on the pilot studies, are the same as those used by Huang et al (14).

Conclusion: These studies demonstrate specific technical aspects of a mouse model of focal cerebral ischemia and reperfusion which allows the ability to reproduce measurements between different laboratories. In addition, these studies provide a basis for understanding important procedural variables which can have a large impact on the outcome after the attack, which can lead to a clear understanding of differences that are not related to the procedure under investigation. More importantly, this study indicates the need for careful control of the mouse strain, the size of the animal and the suture and temperature in the experimental animals as well as the controls. Conditions can be established so that the result after the attack is similar between models of permanent focal cerebral ischemia and transient focal cerebral ischemia, which can facilitate direct comparison and allow the study of reperfusion damage. The model described in this study may provide a cohesive basis for evaluating the results of subsequent studies in transgenic animals to facilitate understanding of the contribution of specific gene products in the pathophysiology of the attack.

Table I Pre-operative and postoperative physiological parameters. MAP, mean arterial pressure, - pC02, arterial C02 partial pressure (mm Hg); 02 Sat, saturation of 02 (%), - Hb, hemoglobin concentration (g / dl); preoperative, anesthetized animals before dissection of the carotid, in fake, anesthetized animals undergoing the surgery described in the text, immediately before the introduction of the occlusion suture; attack, anesthetized animals that underwent the surgery described in the text, immediately after the introduction of the occlusion suture p = NS for all comparisons between groups (data shown are for small mice of 22 grams C57 / B16).

PREOPERATIVE PARAMETER IN FALSE ATTACK MAP 102 ± 5.5 94 ± 1.9 88 ± 4.9 pH 7.27 ± 0.02 7.23 ± 0.04 7.28 ± 0.01 pC02 46 ± 1.3 44 ± 1.3 47 ± 3.5 02Sat 89 ± 1.6 91 ± 1.8 85 ± 2.2 Hb 14.6 + 0.42 14.3 + .12 14.2 ± 0.12 References 1. Backhaub C, Karkoutly C, Welsch M, Krleglstein J: A mouse model of focal brain ischemia for screening neuroprotective drug effects: J Pharmacol Methods 27: 27-32, 1992. 2. Baker CJ, Onesti ST, Solomon RA: Reduction by delayed hypothermia of cerebral infarction following middle cerebral artery occlusion in the rat: a time-course study. J Neurosurg 77: 438-444, 1992. 3. Baker CJ, Fiore AJ, Frazzini VI, Choudhri TF, Zubay GP, Solomon RA: Intraischemic hypothermia decreases the release of gluta ate in the cores of permanent focal cerebral infarcts. Neurosurgery 36: 1-9, 1995. 4. Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN: Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow M-tab 13: 683-692, 1993 ..

. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM: Evaluation of 2, 3, 5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17: 1304-1308, 1986. 6. Bederson JB, Pitts LH, Tsuji M: Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17: 472-476,] 9T6. 7. Buchan AM, Xue D, Slivka A: A new model of 0 temporary focal neocortical ischemia in the rat. Stroke 23: 273-279, 1992. 8. Chan PH, Kamii H, Yang G, Gafni J, Epstein CJ, Carlson E, Reola L: Brain infarction is not reduced in SOD-1 transgenic mice after permanent focal cerebral ischemia. 5 NeuroReport 5: 293-296, 1993. 9. Chiamulera C, Lump A, Reggiani A, Cristofori Q: Qualitative and quantitative analysis of the progressive cerebral damage after middle cerebral artery occlusion in mice. Brain Res 606: 251-258, 1993 .. 0 10. Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W: Continuous measurement of cerebral cortical blood flow by laser Doppler flowmetry in a mouse stroke model. J Cereb Blood Flow Metab 9: 589-596, 1989. 11. Donehower LA, Harvey M, Slagle BL, McArthur MJ, 5 Montgomery CA, Butel JS, Bradley A: Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356: 215-221, 1992. 12. Frazzini VI, Winfree CJ, Choudhri HF, Prestigia como CJ, Solomon RA: Mild hypothermia and MK-801 have similar but not additive degrees of brain protection in the rat permanent focal ischemia model. Neurosurgery 34: 1040-1046, 1994. 13. Ginsberg MD, Busto R: Rodent models of cerebral ischemia. Stroke 20: 1627-1642, 1989. 14. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA: Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265: 1883-1885, 1994. 15. Kader A, Brisman MH, Maraire N, Huh J-T, Solomon RA: The effect of mild hypothermia on permanent focal ischemia in the rat. Neurosurgery 31: 1056-1061, 1992. 16. Kader A, Frazzini VI, Solomon RA, Trifiletti RR: Nitric oxide production during focal cerebral ischemia in rats. Stroke 24: 1709-1716, 1993. 17. Kamii H, Kinouchi H, Sharp FR, Koistinaho J, Epstein CJ, Chan PH: Prolonged expression of hsp 70 mRNA following transient focal brain ischemia in transgenic mice overexpressing CuZn-superoxide dismutase. J Cereb Blood Flow Metab 14: 478-486, 1994. 18. Kinouchi H, Epstein CJ, Mizui T, Carlson R, Chen SF, Chan PH: Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Nati Acad Sci 88: 11158-11162, 1991. 19. Martinou JC, Dubois-Dauphin M, Staple JK, Rodriquez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C, Huarte J: Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental Ichemia. N-uron 13: 1017-1030, 1994. 20. Memezawa H, Smith ML, Siesjo BK: Penu bral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke 23: 552-559, 1992. 21. Menzies SA, Hoff JT, Betz AL: Middle Cerebral Artery Occlusion in Rats: A neurological and pathological evaluation of a reproducible model. Neurosurgery 31: 100-107, 1992. 22. Tamura A, Graham DI, McCullough J, Teasdale GM: Focal brain ischemia in the rat. 1: description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metabol 1 -. 53-60, 1981. 23. Welsh FA, Sakamoto T, McKee AE, Sims RE: Effect of lactacidosis on pyridine nucleotide stability during ischemia in mouse brain. J Neurochem 49: 846-851, 1987. 24. Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H: Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke 25: 165-170, 1994.

. Yang G-Y, Betz AL: Reperfusion- induced injury to the blood- brain barrier after middle cerebral artery occlusion in rats. Stroke 25: 1658-65, 1994. 26. Zea-Longa E, Weinstein PR, Carlson S, Cummin RW: Reversible middle cerebral artery occlusion without craniectomy in rat. Stroke 20: 84-91, 1989.

EXAMPLE 4: Exacerbation of brain damage in mice which express the gene for P-selectin: Identification of P-selectin blockade as a new target for attack treatment Currently there is a remarkable therapeutic gap for the treatment of an attack in development. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional significance of the expression of P-selectin in attack remains unexplored. In order to identify the pathophysiological consequences of the expression of P-selectin and to identify the blocking of P-selectin as a potential new approach for the treatment of attack, for the treatment of attack, experiments were performed using a mouse model of focal cerebral ischemia and reperfusion. Early expression of P-selectin in the postischemic cerebral cortex was demonstrated by the specific accumulation of IgG against mouse P-selectin, radio-labeled. In parallel experiments, the accumulation of neutrophils in the mouse ischemic cortex is that they express the gene for P-selectin (PS + / +) is significantly higher than that demonstrated in homozygous mice lacking P-selectin (PS - / -) . The reduced inflow of neutrophils was accompanied by higher postischemic brain reflux (measured by laser Doppler) in PS - / - mice. In addition, PS - / - mice demonstrated smaller infarct volumes (fivefold reduction, p <; 0.05) and improved survival compared to PS + / + mice (88% versus 44% p <0.05). Functional blocking of P-selectin in PS + / + mice using monoclonal antibodies directed against mouse P-selectin also improved early reflux and post-attack outcome, compared to controls. These data are the first to demonstrate a pathophysiological role of P-selectin in the attack, and suggest that blocking P-selectin may represent a new therapeutic target for attack treatment.

INTRODUCTION Ischemic attack is the third leading cause of death in the United States today1. Until very recently, there has not been a direct treatment to reduce brain tissue damage in the developing attack. Although the acute attack studies NINDS2 and ECASS3 rt-PA1 have suggested that there are potential therapeutic benefits of early reperfusion, 4 the increased mortality observed after treatment with streptokinase of acute ischemic attack5 clarifies the. It is evident that to date there is no clearly effective treatment for this developing attack. This gap in the current medical armament for the attack treatment has led to numerous innovative approaches6, although others different to rt-PA, none has reached the clinical objective. To identify a safe potential treatment and to avoid an attack in development, attention has been focused on the harmful role of recruited neutrophils. Recent work on a mouse model of reperfused attack has. demonstrated that the suppression of neutrophils (PMN) before the attack minimizes the damage to brain tissue and improves the functional result7, - the mice which lack the specific cell adhesion molecule, ICAM-1, are similarly protected7. P-selectin, a molecule that can be rapidly placed on the hypoxic endothelial surface from preformed storage sites8, is an important early mediator of neutrophil recruitment9, which facilitates the suppression of neutrophils mediated by ICAM-1. Although P-selectin is expressed in attack10 in primates, there are no published data which clarify the functional importance of P-selectin expression in any attack model whether reperfused or without reperfusion. To explore the pathophysiological role of P-selectin in the attack, a mouse model of focal cerebral ischaemia and reperfusion was used11 using both wild type mice and mice which were homozygous lacking the gene for P-selectin9 and a strategy of administration of an antibody against functionally blocking P-selectin. This study confirms not only that the expression of P-selectin after occlusion of the middle cerebral artery is associated with a reduced cerebral reflux after reperfusion worsens the result after attack, but that the blockade of P-selectin confers an important degree of cerebral protection post ica. These studies represent the first demonstration of the pathophysiological role of P-selectin expression in attack and suggest the exciting possibility that strategies against P-selectin may prove useful for the treatment of reperfused attack.

METHODS Mice: Experiments were carried out with transgenic mice deficient in P-selectin, produced as previously reported9, by means of genes in Jl embryonic pluripotent cells, injected in C57BL / 6 plastids to obtain germline transmission and backcrossed to obtain mice homozygous lacking P-selectin (PS - / -). The experiments were carried out with wild type PS - / - mice (PS + / +) of the third generation of backcrosses with C57BL / 6J mice. The animals were seven to twelve weeks old and weighed between 25 and 36 grams at the time of the experiments.

Transient occlusion of the middle cerebral artery: Mice were anesthetized (0.3 ce of 10 mg / cc of ketamine and 0.5 mg / cc of xylazine, ip), and placed supine on a surface operated with rectal temperature control (Yellow Springs Instruments, Inc., Yellow Springs, OH). The temperature of the animal was maintained at 37 ± 1 ° C intraoperatively and for 90 minutes postoperatively. An incision was made in the midline of the neck to expose the cover of the right carotid under the operating microscope (16-25X zoo magnification, Zeiss, Thornwood, NY). The common carotid artery was isolated with 4-0 silk and each of the occipital arteries, pterygopalatine and external carotid were isolated and divided. Occlusion of the middle cerebral artery (MCAO) was carried out by advancing a 5-0 blunt nylon suture by 13 mm heat via the stump of the external carotid. After placement of the occlusion suture, the stump of the external carotid artery was cauterized and the wound closed. After 45 minutes, the occlusion suture was removed to establish reperfusion. These procedures have been previously described in detail9.

Measurement of cerebral cortical blood flow: Cerebral blood flow measurements were performed using laser Doppler (Perimed, Inc., Piscataway, NJ), as previously described12. Using a 0.7 mm straight laser doppler probe (model # PF303, Perimed, Piscataway, NJ) and previously published marks (2 mm posterior to bregma, 6 mm on each side of the midline) 11,13, flow measurements were made relative cerebral blood as indicated; immediately after anesthesia, and 1 and 10 minutes after occlusion of the middle cerebral artery, as well as after 30 minutes, 300 minutes and 22 hours of reperfusion. The data are expressed as a proportion of the intensity of the Doppler signal of the ischemic hemisphere compared to the non-ischemic one. Although this method does not quantify cerebral blood flow per gram of tissue, the use of laser Doppler flow measurements in precisely defined anatomical landmarks serves as a means to compare brain blood flows in the same animal, serialized over time. The surgical procedure was considered technically adequate if a = 50% reduction in the relative cerebral blood flow was observed immediately after the placement of the intraluminal occlusion suture. These methods have been used in previous studies7,11.

Cerebrovascular anatomy was determined in representative animals in the following manner. Mice were anesthetized, and antemortem injection (0.1 ml) of India ink was administered: carbon black: methanol: physiological saline (1: 1: 1: 1, v: v: v: v) by left ventricular puncture. The brains were prepared by rapid decapitation followed by immersion in 10% formalin at 4 ° C for 2 days, after which the lower surfaces were photographed to demonstrate the vascular pattern of the circle of Willis.

Preparation and administration of 125 I-labeled proteins and mouse neutrophils labeled with lxlIn: Radioiodinated antibodies were prepared as follows. Rat IgG was radiolabeled against mouse P-selectin, monoclonal (Clone RB 40:34, Pharmingen Co., San Diego, CA) 14 and non-immune rat IgG (Sigma Chemical Co., St. Louis, MO) with 125 I by the lactoperoxidase method15 using Enzymobeads (Bio-Rad, Hercules, CA). Radiolabeled PMN were prepared in the following manner. Citrated blood from wild-type mice was diluted 1: 1 with NaCl (0.9%) followed by gradient ultracentrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). After hypotonic lysis of residual erythrocytes (20-second exposure to distilled H20 followed by reconstitution with 1.8% NaCl), the PMN were suspended in phosphate-buffered saline (PBS). Neutrophils (5-7.5 x 10s) were suspended in PBS with 100 μCi of lyoxane oxine (Amersham Mediphysics, Port Washington, NY), and subjected to gentle agitation for 15 minutes at 37 ° C. After washing with PBS, the PMN were gently pelleted (450 x g) and resuspended in PBS to a final concentration of 1.0 x 10 cells / ml.

Neurological examination: Before administering anesthesia, the mice were examined for neurological deficits 22 h after reperfusion using the four-level grading system11: a score of 1 was given if the animal demonstrated normal spontaneous movements; a rating of 2 was provided if the animal was observed to turn towards its ipsilateral side; a rating of 3 was provided if the animal was observed to rotate longitudinally (clockwise when viewed from the tail), - a rating of 4 is provided if the animal did not respond to noxious stimuli. This grading system has been previously described in mice7'11, and is based on similar grading systems used in rats16,17.

Calculation of infarct volumes: After neurological examination, the mice were anesthetized and final cerebral blood flow measurements were obtained. Euthanasia was performed without suffering by decapitation, and the brains were removed and placed in a mouse brain matrix (Activational Systems Inc., Warren, MI) for sectioned 1 mm. Sections or sections were immersed in 2%, 3, 5-triphenyl-2H-tetrazolium 2% chloride (TTC, Sigma Chemical Co., St. Louis, MO) in 0.9% phosphate-buffered saline, incubated for 30 minutes at 37 ° C and placed in 10% formalin lß. The infarcted brain was visualized as an area of unstained tissue. The infarct volumes were calculated from planimetric serial sections and were expressed as the percentage of infarction in the ipsilateral hemisphere. This method for calculating infarct volumes has been previously used7, 11,13, lß, and has been correlated with other functional indexes of post-attack outcome which have been described before.

Administration of unlabeled antibodies, radiolabeled PMNs and radiolabelled antibodies: For experiments in which unlabeled antibodies were administered, one of two different types of antibodies was used; either rat IgG against mouse P-selectin, blocking monoclonal (Clone RB 40.34, Pharmingen Co., San Diego, CA) 14'19'20 or non-immune rat IgG (Sigma Chemical Co., St. Louis, MO ). Antibodies were prepared as 30 μg in 0.2 ml of phosphate buffered saline containing 0.1% bovine serum albumin, which was subsequently administered into the penile vein 10 minutes before occlusion of the middle cerebral artery. In separate experiments, radiolabelled antibodies (0.15 ml, "2.6 x 10s cpm / μl) were intravenously injected 10 minutes before occlusion of the middle cerebral artery. In a third set of experiments, radiolabelled PMNs were administered intravenously 10 minutes before occlusion of the middle cerebral artery as a 100 μl injection (radiolabeled PMNs were mixed with physiological saline to a total volume of 0.15 ml, - «3 x 106 cpm / μl). For the experiments in which the unlabeled antibodies were administered, the time in which the measurements were made using the methods described above for determining cerebral blood flow, infarct volumes and mortality was indicated in the text. For those experiments which radiolabeled antibodies or radiolabeled PMNn were administered, the mice were sacrificed at the indicated time points and the brains were immediately removed and divided into ipsilateral (postischemic) and contralateral hemispheres. Deposition of radiolabeled antibodies or neutrophils was measured and expressed as ipsilateral / contralateral cpm.

Data analysis: Cerebral blood flow, infarct volume and deposition of PMN labeled with X11ln were compared using Student's t test for unpaired variables. The neurological deficiency scores were compared using the Mann-Whitney U test. Two-tailed ANOVA was performed to test for significant differences between baseline and final antibody deposition (30 minutes) between the two groups (experimental versus false). The Student t test was performed for unpaired variables in order to evaluate differences within the group (baseline versus time point at 30 minutes). Group survival differences were tested using contingency analysis with chi square statistics. The values were expressed as mean ± SEM with a value of p < 0.005 considered statistically significant.

RESULTS Expression of P-selectin in attack in mice: Because P-selectin mediates the initial phase of adhesion of leukocytes to activated endothelial cells 21, early cerebral expression of P-selectin was examined in a mouse model of reperfused attack. Mice were primed with mouse anti-mouse P-selectin IgG, monoclonal, labeled with 125 I before surgery and showed only 216% in antibody accumulation at 30 minutes of reperfusion compared to animals operated on false (p <0.001, Figure 18A). To demonstrate that this degree of antibody deposition in the reperfused hemisphere is due to P-selectin expression rather than non-specific accumulation, a comparison was made with animals treated identically to those that were administered non-immune rat IgG labeled with 12SI. These experiments demonstrated that there is a significantly higher accumulation of IgG against P-selectin compared to non-immune IgG (p <0.025, Figure 18A), suggesting that P-selectin is expressed in the brain in the next 30 minutes of reperfusion.

Accumulation of neutrophils in attack in mice: To delineate the course in time over which the PMN inflow occurs after the attack, the accumulation of PMN labeled with L? aTn was measured in wild type mice (PS + / +) before MCAO, immediately after and 10 minutes after MCAO, and at 30 minutes, 300 minutes and 22 h of reperfusion. In PS + / + mice, accumulation of PMN begins early after the onset of focal ischemia, and continues during the reperfusion period (Figure 18B). To establish the role for P-selectin in this postischemic neutrophil accumulation, experiments were performed using mice that are homozygous lacking for the gene for P-selectin (PS - / -). PS - / - mice showed significantly reduced PMN accumulation after occlusion of the middle cerebral artery and reperfusion (Figure 18B).

Role of PS in the lack of cerebrovascular reflux: To determine if the reduction in PMN accumulation in PS - / - mice results in improved cerebral blood flow after restoration of flow, serial measurements of relative CBF were obtained by laser Doppler in mice both PS + / + and PS - / -. Before the onset of ischemia (Figure 19, point a), the relative cerebral blood flows were almost identical between groups. Occlusion of the middle cerebral artery (Figure 19, point b) was associated with an almost identical decrease in cerebral blood flow in both groups. Immediately before the extraction of the intraluminal occlusion suture at 45 minutes of ischemia (Figure 19, point c), the cerebral blood flow increased slightly, although it remained significantly depressed in comparison with the baseline flows. Immediately after removal of the occlusion suture to initiate reperfusion (Figure 19, point d), cerebral blood flow in both groups increased to a comparable degree ("60% baseline in PS - / - mice and PS + / +.). The immediate failure of post-reperfusion cerebral blood flows to reach the levels prior to occlusion is characteristic of the lack of cerebrovascular reflux, 22 with subsequent decline in post-reperfusion cerebral blood flows, which represent late postischemic cerebral hypoperfusion23. At 30 minutes of reperfusion (Figure 19, point e), cerebral blood flow between the two groups of animals was separated, PS - / - animals show relatively higher cerebral blood flows significantly compared to PS + / + controls.

(Figure 19, point f). This discrepancy reflects significant differences in delayed postischemic cerebral hypoperfusion and persisted during the 22-hour observation period. Because variations in cerebrovascular anatomy have been reported to result in differences in susceptibility to experimental attack in mice24, Indian / carbon black staining was performed to visualize the vascular pattern of the illis circle in both mice PS - / - as PS + / +. These experiments demonstrated that there are no large anatomical differences in the vascular pattern of cerebral circulation (Figure 20).

Result after the attack-. The functional importance of P-selectin expression was tested by comparing the rates of post-attack outcome in PS - / - mice compared to PS + / + controls. PS - / - mice were significantly protected from the effects of focal cerebral ischemia and reperfusion, based on a 77% reduction in infarct volume (p <0.01) compared to the P-selectin + / + controls ( Figure 21A). This reduction in infarct volume was accompanied by a trend towards reduced neurological deficiency (p = 0.06, Figure 21B) and increased survival (p <0.05; Figure 21C) in PS - / - animals.

Blocking effect of P-selectin: After observing the functional role of the expression of P-selectin in the attack using rats by suppression, experiments were performed to determine if the pharmacological blockade of P-selectin can improve the result after attack in PS + / + mice. Using a strategy of administering 5 rat-blocking antibody, against mouse P-selectin, monoclonal (clone RB 40.34, 14.19.20) or non-immune control rat IgG immediately before surgery, it was observed that mice received blocking antibody have improved cerebral blood flow after reperfusion at 10 30 minutes (Figure 22A), as well as reduced neurological deficiencies (Figure 22B), reduced cerebral infarct volumes (Figure 22C) and a trend towards reduced mortality compared with the controls (Figure 22D).

DISCUSSION Despite the substantial advance in recent years in the primary prevention of attack1, the therapeutic options to treat an attack in development remain extremely limited *. Although the publication of two pivotal trials last fall demonstrates a reduced morbidity after treatment of ischemic stroke with rt-PA2,3, it was considered to introduce a new era of thrombolytic therapy in the attack treatment, 4 the excitement has been somewhat diminished by the hemorrhagic transformation and increased mortality observed in patients with ischemic attack treated with streptogenases. These divergent trials become more critical than ever that new and innocuous therapies are developed to treat an attack in development. Although restoring blood flow to a postischemic brain provides new opportunities for early therapeutic intervention, reperfusion is a double-edged sword. Given the cytotoxic potential of neutrophils, 25 it is not surprising that the influx of neutrophils into postischemic brain tissue can lead to additional damage and worsen the outcome after experimental attack.7,26"29 When using a mouse model of focal cerebral ischemia and reperfusion, an important contributory role of the cell adhesion molecule ICAM-l has recently been identified in the accumulation of neutrophils at 22 hours after the attack.7 However, the increased cerebrovascular endothelial expression of ICAM-1 requires the transcriptional and In the contrast, P-selectin, a membrane-spanning glycoprotein, which mediates the earliest phases of neutrophil adhesion, can be mobilized from its stored preformed pools to express itself rapidly in the surface of the ischemic endothelial cell.8'30 Since the clinical trials of the to thrombolytic for attack demonstrate a narrow time interval for potential benefit (in the following several hours of onset of attack) 2,3,5, this suggests that strategies designed to interfere with the earliest phases of PMN adhesion may be of theoretical benefit in human attack. These trials would result in higher numbers of patients presenting for earlier therapeutic intervention, which increases the need to solve the problem of reperfusion injury in medically revascularized territories. In addition, these trials underscore the increasing need to understand the contributions of individual adhesion molecules to the pathogenesis of the attack. Given the considerable body of literature describing the role of P-selectin in other models of ischaemia and reperfusion, 8,31"34 surprisingly little is known about the role of P-selectin in the attack." Knowledge of the specific role of P-selectin in the cerebral vasculature is important because the adhesion molecule requirements vary between vascular beds and the conditions under study.For example, in a model of intestinal transplantation35, antibodies against P-selectin do not reduce reperfusion damage, while that antibodies against CD11 / CD18 did, although blocking of P-selectin is not effective in reducing PMN adhesion and albumin leakage in rat mesenteric ischemia and in the reperfusion model, ICAM blockade l was effective.36 In an ischemia / reperfusion model of the hind limb of the rat, PMN adhesion selectin requirements differed between the pulmonary vascular beds and of crural muscle33.

The only published study that works with P-selectin in the ischemic brain is a histopathological description of primate attack, in which the expression of P-selectin in the microvasculature of the lenticulostriate was increased10. In addition, there are no data which clarify the functional importance of this expression of P-selectin. Studies are currently underway to study whether P-selectin expression contributes to cerebral post-ischemic accumulation of neutrophils, lack of reflux and tissue damage in a mouse model of reperfusion attack. Using a recently established model of focal cerebral ischaemia and reperfusion in mice11, expression of P-selectin was demonstrated by increased deposition of radiolabeled antibody in the ischemic territory. In this technique, the deposition of antibodies in the ischemic hemisphere was normalized so that in the non-ischemic hemisphere in each animal, not only are potential variations in the injection volume or volume of distribution minimized, but also that - allows comparison between animals given different antibodies. Due to the disruption of the function of the endothelial barrier in the ischemic cortex, the non-selective deposition of antibodies can be increased, and similar experiments were performed with rat IgG as a control. These data showed that the antibodies which bind P-selectin are deposited at an accelerated rate compared to the control antibody, which suggests that the local expression of P-selectin in the tissue subjected to reperfusion is increased. These data in the mouse model are parallel to those reported in the attack baboon model, 10 in which the expression of P-selectin was increased in the next hour after the ischemic event. The role of P-selectin expression in the recruitment of PMN in the post-ischemic zone was demonstrated using a strategy in which the accumulation of PMN labeled with ulIn was measured. Although previously it has been reported that at 22 hours it rises. accumulation of PMN in the ischemic hemisphere7, current data over time show that PMN accumulation begins shortly after ischemia access. The failure to express the gene for P-selectin was associated with a reduced accumulation of PMN, suggesting the participation of P-selectin in the cerebral postischemic recruitment of PMN. However, animals lacking P-selectin demonstrated a modest (though less than control) accumulation of neutrophils at 22 hours. These data indicate that P-selectin is not an exclusive effector mechanism responsible for the cerebral postischemic recruitment of PMN, and is consistent with the previous data that ICAM-1 also participates in the postischemic adhesion of PMN7. In addition, these data are not different from those in which intraabdominal instillation of thioglycollate in mice deficient in P-selectin causes delayed (but not absent) recruitment of PMN9.

Due to the critical need to identify the reasons for a failed reperfusion, current studies examined the role of P-selectin in delayed postischemic cerebral hypoperfusion22,23, the phenomenon in which blood flow declines during reperfusion, despite the restoration of adequate pressures of reperfusion. In cardiac models of ischemia, the lack of reflux worsens as the time elapses after reperfusion 37, which suggests an important role for the recruited effector mechanisms, such as progressive microcirculatory thrombosis, vasomotor dysfunction and recruitment of PMN. It has been demonstrated in other models that both P-selectin-dependent and ICAM-l38-dependent adhesion reactions as well as capillary tamponade by PMN39 participate in the lack of post-ischemic reflux. In the brain, PMN have been implicated in the lack of post-ischemic cerebral reflux40,41, but the role of P-selectin in this process has not yet been elucidated. The current study uses a relatively non-invasive technique (laser Doppler) to obtain serial measurements of the relative cerebral blood flow, in order to establish the existence, course in time and dependence of P-selectin on the lack of postischemic cerebrovascular reflux. In these experiments, animals lacking P-selectin and control were subjected to virtually identical degrees of ischemia, and the instantaneous recovery of blood flow after the removal of the intraluminal occlusion suture was the same in the two groups. However, cerebral blood flow declined in the period of time after reperfusion in the P-selectin + / + animals. In remarkable contrast, PS - / - animals showed only a slightly delayed postischemic cerebral hypoperfusion. This late (although limited) decline in cerebral blood flow by 22 hours is consistent with the modest recruitment of PMN observed in the animals PS - / - over the same period. This again suggests that other effector mechanisms (such as ICAM-1) may be responsible for the late decline in cerebral blood flow in PS - / - animals. The functional effects of P-selectin expression are evident from the current set of studies. Animals that fail to express the gene for P-selectin (or PS + / + animals treated with an antibody against functionally blocking P-selectin) show smaller infarcts, improved survival and survivors show improved neurological outcomes compared to controls. When these data are considered together with previously published data demonstrating a harmful role for the expression of ICAM-1 in an attack7, it becomes increasingly evident that there are multiple means to recruit PMNs to the postischemic cerebral cortex and that a blockade of each one represents a potential strategy to improve the result after attack in humans. Given the current recognition of the importance of reperfusion in time to stop the advance of the frontal wave of neuronal death after the attack, interfering with the adherence of PMN in its earliest stages seems to be an attractive option to reduce morbidity and mortality. In fact, the anti-adhesion strategies of the molecule can not only be beneficial on their own (that is, it includes patients not eligible for thrombosis), but it can expand the range of opportunities for thrombolytic intervention42. The current set of studies contributes to the understanding of the pathophysiological mechanisms that operate in a reperfusion attack. These studies suggest the need for clinical trials of therapies to develop attacks which optimize the reperfusion environment to reduce PMN accumulation.

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Springer TA: Adhesion rceptors of the immune system. Nature 1990; 346: 425-434 22. Ames There, Wright RL, Kowada M, Thurston JM, Majno G: Cerebral ischemia II: the no reflow-phenomenon. Am J Pathol 1968; 52: 437-447 23. Levy DE, Van Uitert RL, Pike CL: Delayed postischemic hypoperfusion-. a potentially damaging consequence of stroke. Neurology 1979; 29: 1245-1252 24. Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN: Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood _F_low Metab 1993, -13: 683-692 . Weiss SJ: Tissue destruction by neutrophils. N Engl J Med 1989; 320 (6): 365-376 26. Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP, Contreras TJ: Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke 1986; 17: 246-253 27. Kochanek PM, Hallenbeck JM: Polymorphonuclear leukocytes and monocytes / macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 1992; 23 (9): 1367-1379 28. Dutka AJ, Kochanek PM, Hallenbeck JM: Influence of granulocytopenia on canine cerebral ischemia induced by air embolism. Stroke 1989; 20: 390-395 29. Bednar MM, Ray ond S, McAuliffe T, Lodge PA, Gross CE: The role o-f- neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke 1991; 22 (1): 44-50 . Geng J-G, Bevilacqua MP, Moore KL, Mclntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA, McEver RP: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 1990; 343: 757-760 31. Weyrich AS, Ma X-L, DJ efer, Albertine KH, Lefer AM: In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest 1993; 91: 2620-2629 32. Winn RK, Liggltt D, Vedder NB, Paulson JC, Harian JM: Anti-P-selectin monoclonal antibody attenuates reperfusion injury in the rabbit ear. J Clin Invest 1993; 92: 2042-2047 33. Seekamp A, Till GO, Mulligan MS, Paulson JC, Anderson DC, Miyasaka M, Ward PA: Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am J Pathol 1994; 144: 592-598 34. Kubes P, Jutlla M, Payne D: Therapeutic potential of inhibiting leukocyt rolling in ischemia / reperfusion. J Clin Invest 1995; 95: 2510-2519 . Slocum MM, Granger DN: Early mucosal and microvascular changes in feline intestinal transplants. Gastroenterology 1993; 105: 1761-1768 36. Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, Rusche JR, Granger DN: Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage. Circ Res 1994; 74: 336-343 37. Kloner RA, Earned CE, Jennings RB: The "no-reflow phenomenon after temporary coronary occlusion in the dog. J Clin Invest 1974; 54: 1496-1508 38. Jerome SN, Dore M, Paulson JC, Smith CW, Korthuis RJ: P-selectin and ICAM-l -dependent adherence reactions: role in the genesis of postischemic no-reflow. Am _ J Physiol 1994; 266: H1316-H1321 5 39. Engler RL, Schmid-Schonbein GW, Pavelec RS: Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 1983; 111: 98-111 40. Mori E, Zoppo GJ, Chambers JD, Copeland BR, Arfors KE: Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons. Stroke 1992; 23: 712-718 41. Grogaard B, Schurer L, Gerdin B, Arfors KE: Delayed hypoperfusion after incomplete forebrain ischemia in the rat: the role of polymorphonuclear leukocytes. J Cereb Blood Flow Metab 1989; 9: 500-505 42. Bowes MP, Rothlein R, Fagan SC, Zivin JA: Monoclonal antibodies preventing leukocyte activation, reduces experimental neurological injury, and enhance efficacy of thrombolytic therapy. Neurolosy 1995; 45: 815-819.

Example 5 Homozygous mice lacking P-selectin are resistant to focal cerebral ischemia and reperfusion damage The role of neutrophils (PMN) in enhancing reperfusion damage in the face of focal ischemia in the central nervous system remains controversial. An important early stage in the capture of circulating PMN by the vasculature is measured by P-selectin expressed in the postischemic endothelium. Although the early and persistent expression of endothelial P-selectin has been described in the microvessels of the brain after occlusion of the middle cerebral artery in baboons, the consequences of the endothelial expression of P-selectin in the attack have not been determined. To define the role of P-selectin in the attack, a mouse model of focal cerebral ischaemia and reperfusion consisting of occlusion of the middle cerebral intraluminal artery (MCA) for 45 minutes followed by 22 hours of treatment was used in two groups of mice. reperfusion; transgenic mice that are homozygous lacking for P-selectin (PS - / -) and wild-type control cousins (PB + / +) were used. The volumes of cerebral infarction were calculated from sections or series sections planimetrated with triphenyltetrazolium chloride, and were expressed as the percentage of infarcted tissue in the ipsilateral hemisphere. The neurological result was based on that observed by a researcher who does not know the protocol (1: without deficit, 2: in circles, 3: turning, 4: immobile). The ipsilateral cortical cerebral blood flow (CBF) was determined by laser Doppler flowmetry and was expressed as a percentage of the contralateral cortical GBF. PS - / - mice showed a 3.8-fold reduction in infarct volumes compared to PS + / + controls (7.6 ± 4.4% vs. 29.2 ± 10.1%, p <0.05). This reduction in infarct volumes in mice lacking P-selectin was reflected by an improved survival (87% vs 42%, p <0.05) and a trend towards a reduced neurological deficit (1.9 ± 0.4 vs 2.5 ± 0.3 , p = NS) in the survivors. Because there was a trend for increased cerebral blood flow after cerebral ischaemia and reperfusion in the PS - / - cohort (65 ± 11% vs 46 ± 18% for controls, p = NS), these studies suggest that adherence P-selectin dependent can contribute to the lack of cerebral reflux. Taken together, these data imply an important role for the expression of P-selectin in the pathophysiology of the attack, and suggest novel pharmacological strategies to improve the post-attack outcome.

EXAMPLE 6; The absence of the gene for P-selectin reduces the cerebral postischemic accumulation of neutrophils, the lack of reflux and tissue damage in the mouse model of reperfusion attack Recent studies in humans indicate that the restoration of cerebral blood flow (CBF) during the early period after the onset of the attack reduces the neurological sequelae. It has been hypothesized that P-selectin (PS), an early-acting neutrophil adhesion molecule (PMN) expressed by the hyogenic endothelium, may have an important pathophysiological role in the development of a reperfusion attack. Studies were conducted to eliminate transient focal cerebral ischemia in a mouse model consisting of occlusion of the intraluminal middle cerebral artery for 45 minutes followed by 22 hours of reperfusion. In this model, mice which did not express the PS (PS - / -) gene have smaller infarct volumes, reduced neurological deficit ratings and improved survival compared to wild type controls (PS + / + ). Current studies were conducted to further define the mechanisms induced by PS of brain damage. PS + / + mice (n = 6) were given IgG against 125I-labeled PS before surgery and a 216% greater accumulation of antibody in the ipsilateral hemisphere was demonstrated by 30 minutes of reperfusion compared with animals operated on. false (n = 6, p <0.001) or with animals given non-immune IgG and underwent transient focal cerebral ischemia and 30 minutes of reperfusion (n = 4, p <0.03). In PS + / + mice, PMN accumulation begins early after the onset of focal ischemia, and continues through the reperfusion period (two-fold increase in ipsilateral / contralateral 113-In-PMN accumulation in 22 hours, n = 8, p <0.05). PS - / - mice show a 25% reduction in PMN accumulation in the ipsilateral hemisphere at 22 hours (n = 7, p <0.05). We investigated the effect of PS expression on the lack of postischemic cerebral reflux by measuring serial ipsilateral biphasic CBF during the evolution of the attack. Although baseline CBF, post-occlusion, and initial reperfusion were identical, CBF at 30 minutes of reperfusion were significantly greater in PS - / - mice (n = 5) compared to PS + / + mice (n = 8, 2.4 times higher, p <0.05). This difference is sustained during the rest of the reperfusion period of 22 h. These data support * an important early role for PS in the recruitment of PMNs, the lack of postischemic reflux and tissue damage in the development of the attack. This is the first demonstration of a pathophysiological role for PS in cerebral reperfusion injury, which suggests that PS block may represent a therapeutic target for reperfusion treatment.

EXAMPLE 7: Carbon monoxide and attack evolution Gaseous carbon monoxide, a toxic byproduct of heme catabolism, is involved in long-term potentiation and memory in the central nervous system. However, other physiological roles for the production of CO in the brain are unknown. Due to the fact that oxygenase is induced during inflammatory conditions, it was investigated whether the endogenous production of CO can confer a protective cerebral role in attack. In a mouse model of focal cerebral ischemia, heme oxygenase type I was induced in the mRNA (by Northern blot) and protein levels (by Western blot), located in the cerebral vascular endothelium in the ischemic hemisphere by in situ hybridization and immunohistochemistry. . Local CO production was observed by direct means in the ischemic area. In parallel experiments, mouse brain endothelial cells exposed to a hypoxic environment demonstrated a similar induction of mRNA for heme oxygenase, protein and CO generation. To determine whether CO production is incidental to the pathophysiology of the attack, CO production is blocked by administration of tin protoporphyrin (confirmed by direct measurement of reduced local concentrations of CO). These animals showed significantly larger infarct volumes, worse neurological outcomes and increased mortality, compared to untreated controls. In addition, the administration of CO before the attack confers significant brain protection. Since this protection was not observed in animals treated with biliverdin, the coincidental byproduct of heme catabolism, these data suggest that endogenous CO production per se has a protective role in the development of the attack.

Introduction There is a considerable body of literature and a common recognition of the toxic effects of exogenous carbon monoxide (CO), which binds avidly to the heme centers, inhibiting the transport of oxygen and poisoning cellular respiration. For many years, CO was considered an incidental byproduct of heme catabolism, but recent data in the brain suggest that CO produced - in neurons defined by heme oxygenase II - can modulate long-term potentiation. In rats, exposure to heat shock has been correlated with the expression of the 32 kDa heat shock protein (heme oxygenase I) in several organs including the. brain. The physiological significance of this HSP32 induction has been teleologically ascribed to the stoichiometric release of CO by heme oxygenase I (HOI). In most experimental studies, HOI induction serves only as an incidental marker of cellular oxidant stress. The recent identification of the anti-inflammatory role for HOI in a model of peritoneal inflammation has been ascribed to the production of natural antioxidant biliverdin during the process of heme catabolism. Current studies report for the first time that the post-ischemic brain generates huge amounts of CO. When using the mouse model of focal cerebral ischemia in which the middle cerebral artery was occluded by an intraluminal suture, the production of HOI in the ischemic hemisphere was significantly increased compared to the non-ischemic hemisphere. Due to immunohistochemistry and in situ hybridization localized at the source of HOI to the endothelial cells within the ischemic hemisphere, an in vitro model of cellular hypoxia was used to confirm the induction of the HOI message, the protein and the activity in the endothelial cells. mouse brain microvessels. The blockage of CO production using protoporphine IX tin or zinc was associated with an increase in the volume of cerebral infarction and mortality, while the exposure of the animals to CO immediately before ischemia conferred a significant brain protection, dependent on the dose, within a narrow therapeutic interval. The administration of biliverdin had no effect on this model. Taken together, these data indicate that ischemic brain tissue produces large amounts of CO, the production of which confers brain protection that limits the amount of tissue destroyed during an attack.

METHODS Preparation and administration of protoporphyrin. Initially tin protoporphyrin IX chloride (20 mg, Porphyrin Products, Logan, UT), zinc protoporphyrin IX (17 mg, Porphyrin Products, Logan, UT), or biliverdin (18 mg, Porphyrin Products, Logan, UT) were dissolved. . in dimethyl sulfoxide (2 ml). An aliquot of this solution (200 μl) is added to normal saline (9.8 ml) and this mixture is vortexed vigorously to produce a 2.7 x 10"4 M solution of the protoporphyrin. aluminum to prevent protoporphyrin photolysis and stored at 4 ° C until used.Smooth microosmotic pumps (# 1003D Alza Corp., Palo Alto, CA) are loaded with this protoporphyrin solution (91 μl / pump) and implanted subcutaneously In the anesthetized mouse via an incision in the dorsal midline of 1 cm 24 h before the start of surgery, these pumps administered drug solution at a rate of 0.95 + 0.02 μl / h. of the protoporphyrin solution (0.3 ml, iv) before insertion of the intraluminal occlusion catheter Each animal received the following total drug amounts (injection + pump) during the course of the study: protoporfir tin (0.070 mg), zinc protoporphyrin (0.059 mg) or biliverdin (0.061 mg).

EXAMPLE 8 Free radical hypoxia induces translocation of P-selectin (PS) to the surface of endothelial cells (EC), where it participates in the adhesion of neutrophils (PMN) during reperfusion. To explore a mechanism by which nitrovasodilators can attenuate the post-ischemic leukosclerosis, we tested whether the stimulation of the NO / cGMP pathway can attenuate surface expression of PS in human hypoxic umbilical vein ECs. The CDs were exposed to hypoxia (p02 <; 20 Torr for 4 hours), which demonstrated a 50% increase in the release of vWF (p <0.005) (vWF is packaged with PS), paralleled by an 80% increase in surface expression of PS (p. < 0.0001), as measured by specific binding of a radiolabeled antibody, against PS. Under similar conditions, the addition of the NO 3 donor -morpholino sidnonimine (SIN-1, 0.1 mM) or the cGMP analog 8-bromo-cGMP (cGMP, 10 nM) caused a reduction in the release of vWF; VWF control, 11 + 0.4 mU / ml; SIN-1, 9.1 + 0.3 mU / ml; CGMP, 9.7 ± 0.2 mU / ml; p < 0.005 for both SIN-1 or cGMP versus control. Compared with controls, SIN-1 or cGMP also reduces the surface expression of PS (40% and 48% decreases, respectively, of p <0.005 for each) using an immunofluorescent adhesion assay, both SIN-1 and cGMP. they reduced the unipon of HL60 to hypoxic HUVEC (53% and 86% decrease versus controls, p <0.05 for each). The fluorescence measurement of fura-2 showed that hypoxia increased the intracellular calcium concentration [Cai] and that the increased [Cai] can be blocked by cGMP. Neither SIN-1 nor cGMP can further reduce PS expression when ECs are placed in a calcium-free medium. These data suggest that the stimulation of the NO / cGMP pathway inhibits the expression of PS by inhibiting the flow of calcium in CDs, and identifies this inhibition as an important mechanism by which nitrovasosdilators can decrease the binding of PMN in postischemic tissues. .EXAMPLE 9: Factor IXai Factor IX is a coagulation factor which exists in humans and other mammals, and is an important part of the coagulation pathway. In the normal coagulation scheme, factor IX is activated either by factor Xla or by the tissue factor / VIIa complex to its active form, factor IXa. Then factor IXa can activate factor X, which drives the final part of the coagulation cascade, leading to thrombosis. Because factor X can be activated by one of two pathways, either extrinsic (via Vlla / tissue factor) or intrinsic pathway (via factor IXa), we hypothesized that inhibition of factor IXa can lead to decrease of some forms of hemostasis, but leaves hemostasis in response to damage in intact tissue. In other words, it can lead to blockage of some types of coagulation, but it may not lead to excessive or unwanted bleeding. Factor IXai is factor IX which has been chemically modified so that it still remembers factor IXa (and therefore can compete with native factor IXa), but which lacks its activity. This may "overtake" or cause a competitive inhibition of the normal coagulation-dependent factor IXa pathway. Because factor IXa binds to the endothelium and platelets and perhaps to other sites, blocking the activity of factor IXa can also be accomplished by administering agents which interfere with the binding of factor IXa (or by interference) with the activation of factor IX). In the attack and in other ischemic disorders, there may be a clinical benefit provided by lysing an existing thrombus, but there is also the potentially complicating complication of hemorrhage. In the current experiments, the mouse model of cerebral ischaemia and reperfusion was used (attack) . Mice received an intravenous bolus of 300 μg / kg factor factor IXai just before surgery. Attacks were generated by intraluminal occlusion of the right middle cerebral artery. When the results of the attack were measured 24 hours later, the animals that had received factor IXai had smaller infarct volumes, improved cerebral perfusion, fewer neurological deficits and reduced mortality compared to the controls underwent the same surgery but which did not receive factor IXai. It is also noted that animals with factor IXai were free of apparent intracerebral hemorrhage. In contrast, intracerebral hemorrhage was occasionally observed in the control animals who did not receive factor IXai. Table II Experimental Control Example 10: Exacerbation of brain damage in mice expressing the gene for P-selectin; Identification of the blockade of P-selectin as a new target for the treatment of attacks Abstract: Currently there is a remarkable therapeutic gap for the treatment of the development of attacks. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional importance of P-selectin expression in attacks remains unexplored. In order to identify the pathophysiological consequences of the expression of P-selectin and to identify the blocking of P-selectin as a potential new approach for the treatment of attacks, experiments were performed using a mouse model of focal ischemia and reperfusion. The early expression of P-selectin in the postischemic cerebral cortex was demonstrated by the specific accumulation of radiolabeled IgG against mouse P-selectin, with an increased expression of P-selectin localized in ipsilateral cerebral icrovascular endothelial cells by immunohistochemistry. In experiments designed to test the functional significance of the increased expression of P-selectin in the attack, it was shown that the accumulation of neutrophils in the ischemic cortex of mice expressing the gene for P-selectin (PS + / +) is significantly higher than in homozygous mice lacking P-selectin (PS - / -). The reduced inflow of neutrophils is accompanied by a higher postischemic cerebral reflux (measured by laser Doppler) in PS - / - mice. In addition, PS - / - mice demonstrated smaller infarct volumes (fivefold reduction, p <; 0.05) and improved survival compared to PS + / + mice (88% vs. 44%, p <0.05). Functional blocking of P-selectin in PS + / + mice using a monoclonal antibody directed against mouse P-selectin also improved early reflux and post-attack outcome, compared to controls, with reduced cerebral infarction volumes observed even when the blocking antibody was administered after occlusion of the middle cerebral artery. These data are the first to demonstrate a pathophysiological role for P-selectin in the attack, and suggest that blocking P-selectin may represent a new therapeutic target for the treatment of attacks.

INTRODUCTION: Ischemic attack is the third leading cause of death in the United States today1. Until very recently, there has been no direct treatment to reduce brain tissue damage in the development of an attack. Although the acute attack studies NINDS2 and ECASS3 and rt-PA2 + have suggested that there are potential therapeutic benefits of early reperfusion, 4 the increased mortality observed after treatment with acute ischemic attack streptokinase5 clarifies the evident fact that to date there is no Clearly effective treatment for an attack in development. This emptiness the current medical armament for the treatment of attack has led to innovative approaches6, although different from rt-PA, none has achieved clinical benefits. To identify a safe and effective potential treatment for a developing attack, we have focused on the damaging role of recruited neutrophils. In recent work in a model mouse reperfused attack has shown that the suppression of neutrophils (PMN) before the attack minimizes damage to brain tissue and improves functional outcome7, - mice which lack the specific cell adhesion molecule, ICAM-l are similarly protected7. P-selectin, a molecule which can be rapidly translocated to the hypoxic endothelial surface of previously formed storage sites8, is an important early mediator of neutrophil recruitment9, which facilitates the suppression of neutrophils mediated by ICAM-1. Although P-selectin is expressed in the attack on primates, 10 the functional significance of the expression of P-selectin in the attack is unknown. To explore the pathophysiological role of P-selectin in the attack, we used a mouse model of focal cerebral ischaemia and reperfusion11 using both wild type mice and mice which are homozygous lacking the gene for P-selectin9, and a strategy of administration of an antibody that functionally blocks P-selectin. In these studies, we confirm not only that the expression of P-selectin after occlusion of the middle cerebral artery is associated with reduced cerebral reflux after reperfusion and a worsened outcome after the attack, but that the P-selectin block confers a significant degree of postischemic brain protection. These studies represent the first demonstration of the pathophysiological role of the expression of P-selectin in the attack, and suggest the existing possibility that strategies against P-selectin may prove useful for the treatment of reperfused attack.

METHODS: Mice: Experiments were performed with P-selectin-deficient transgenic mice created as previously reported9 by gene targets in Jl embryonic pluripotent cells, injected into C57BL / 6 blastocysts to obtain a germ line of transmission, and backcrossed to obtain homozygous mice lacking P-selectin (PS - / -). The experiments were performed with PS - / - or wild type (PS + / +) prime mice of the third generation of backcrosses with C57BL / 6J mice. Animals that were 7 to 12 weeks old and that weighed between 25-36 grams at the time of the experiments. Because variations in cerebrovascular anatomy have been reported that result in differences in susceptibility to experimental attack in mice, 12 an Indian / carbon black ink stain was performed to visualize the vascular pattern of the Willis circle in both PS - / mice. - as in PS + / +. These experiments demonstrated that there are no large anatomical differences in the vascular pattern of cerebral circulation.

Transient occlusion of the middle cerebral artery: The mice were anesthetized (0.3 ce of 10 mg / cc of ketamine and 0.5 mg / cc of xylazine, i.p.) and placed supine on a rectal controlled operated surface (Yellow Springs Instruments, Inc., Yellow Springs, OH). The core temperature of the animal was maintained at 37 ± IoC intraoperatively and for 90 minutes postoperatively. An incision was made in the midline of the neck to expose the cover of the right carotid under the operating microscope (16-25X magnification zoom, Zeiss, Thornwood, NY). The common carotid artery was isolated with a 4-0 silk thread and each of the occipital, pterygopalatine and external carotid arteries were isolated and divided. The occlusion of the middle cerebral artery (MCAO) was performed by advancing a 13 mm nylon suture, blunt with 5-0 heat. ía- a stump of the external carotic. After placement of the occlusion suture, the stump of the external carotid artery was cauterized and the wound closed. After 45 minutes, the occlusion suture was removed to establish reperfusion. These procedures have been previously described in detail11.

Measurement of cerebral cortical blood flow: Transcranial measurements of cerebral blood flow were made using laser doppler (Perimed Inc., Piscataway, NJ), as previously described13. Using a 0.7 mm straight laser Doppler probe (model # PF303, Perimed, Piscataway, NJ) and previously published markings (2 mm post-bregma, 6 mm on each side of the midline), 11 were made as indicated by relative measurements of cerebral blood flow; immediately after anesthesia, 1 and 10 minutes after occlusion of the middle cerebral artery, as well as after 30 minutes, 300 minutes and 22 hours of reperfusion. The data were expressed as the proportion of the Doppler signal intensity of the ischemic part compared with the non-ischemic hemisphere. Although this method does not quantify cerebral blood flow per gram of tissue, the use of laser Doppler flow measurements at precisely defined anatomically indicated points serves as a means to compare in cerebral blood flows in the same animal serially with respect to weather. It was considered that the surgical procedure was technically adequate if _ > 50% reduction in relation to cerebral blood flow immediately after placement of the intraluminal occlusion suture. These methods have been used in previous studies7,11.

Preparation and administration of 125I-labeled proteins and mouse neutrophils labeled with X11ln: Radioiodinated antibodies were prepared as follows. Rat IgG was radiolabelled against mouse P-selectin, monoclonal (clone RB 40.34, Pharmingen Co., San Diego, CA) 14, and non-immune rat IgG (Sigma Chemical Co., St. Louis, MO) with 125 I by the lactoperoxidase method using Enzymobeads (BIO-Rad, Hercules, CA). Radiolabeled PMN were prepared in the following manner. Blood c treated from wild-type mice was diluted 1: 1 with NaCl (0.9%) followed by gradient ultracentrifugation in Ficoll-Hypaque (Pharmacia, Pisctaway, NJ). After hypotonic lysis of residual erythrocytes (20 seconds of exposure to distilled H20 followed by reconstitution with 1.8% NaCl), PMNs were suspended in phosphate-buffered saline (PBS). Neutrophils (5-7.5 x 106) were suspended in PBS with 100 μCi of indium oxina111 and subjected to gentle agitation for 15 minutes at 37 ° C. After washing with PBS, the PMN were gently pelleted (450 x g) and resuspended in PBS to a final concentration of 1.0 x 10 6 cells / ml.

Calculation of volumes < _1 = infarction; After the neurological examination, the mice were anesthetized and final cerebral blood flow measurements were obtained. Euthanasia was performed without suffering by decapitation, and the brains were removed and placed in a mouse brain matrix (Activational Systems Inc., Warren, MI) for 1 mm slices. The sections were immersed in 2, 3, 5-triphenyl-2H-tetrazolium 2% chloride in 0.9% phosphate-buffered saline, incubated for 30 minutes at 37 ° C and placed in 10% formalin 16. The infarcted brain was visualized as an area of unstained tissue. The infarct volumes were calculated from the planimetric serial sections and were expressed as the percentage of infarction in the ipsilateral hemisphere. This method of calculating infarct volumes has been previously used by our group7'11, and others16'17, and has been correlated with the other functional indexes of post-attack outcome, which are described above.

Administration of unlabeled antibodies. Radiolabelled PMN and Radiolabeled Antibodies: For experiments in which unlabelled antibodies were administered, one of two different types of antibodies was used; either rat IgG against monoclonal mouse P-selectin (clone RB40.34, Pharmingen Co., San Diego, CA) 14,18,19 or non-immune rat IgG (Sigma Chemical Co., St. Louis, MO). Antibodies were prepared as 30 μg in phosphate buffered saline containing 0.1% bovine serum albumin, which was then administered into the penile vein 10 minutes before occlusion of the middle cerebral artery. In separate experiments, radiolabelled antibodies (0.15 ml, ~ 6 c 105 cpm / μl) were intravenously injected 10 minutes before occlusion of the middle cerebral artery. In a third set of experiments, radiolabeled PMNs were administered intravenously 10 minutes before occlusion of the middle cerebral artery, such as 100 μl of injection (radiolabeled PMNs were mixed with physiological saline to a total volume of 0.15 ml; «3 x 106 cpm / μl). For experiments in which unlabeled antibodies were administered, the time in which measurements were made was indicated in the text, using the methods described above to determine cerebral blood flow, infarct volumes and mortality. For those experiments in which radiolabelled or radiolabeled PMN antibodies were administered, the mice were sacrificed at the indicated time points and the brains were immediately removed and divided into ipsilateral (postischemic) and contralateral hemispheres. Deposition of radiolabeled antibodies or neutrophils was measured and expressed as ipsilateral / contralateral cpm.

Immunohistochemistry: The brains were removed 1 hour after occlusion of the middle cerebral artery, fixed in 10% formalin, embedded in paraffin and sectioned for immunohistochemistry. Sections were stained with rabbit antibody against affinity-purified polyclonal human P-selectin (1:25 dilution, Pharmigen, San Diego, CA), and the primary antibody binding sites were visualized using goat anti-rabbit IgG, conjugated to biotin (1:20) detected with ExtrAvidin peroxidase (Sigma Chemical_ Co., St. Louis).

Data Analysis: Cerebral blood flow, infarct volume and deposition of 11: LIn-PMN were compared using the Student t test for unpaired variables. Two-tailed ANOVA (analysis of variance) was performed to test for significant differences between baseline and final antibody deposition (30 min) between the two groups (experimental versus false). Students t test for unpaired variables was performed to evaluate differences within the group (baseline versus point in time at 30 min). The differences in survival between groups was tested using contingency analysis with the Chi square statistic. The values were expressed as mean ±. SEM, with a value p < 0.05 considered as statistically significant.

RESULTS Expression of P-selectin in attack in mice: Because P-selectin mediates the initial phase of adhesion of leukocytes to activated endothelial cells, 20 we examined the early cerebral expression of P-selectin in a mouse model of reperfused attack. The mice were administered a monoclonal rat IgG against mouse P-selectin, labeled with 125 I before surgery, which showed a 216% increase in antibody accumulation at 30 minutes of reperfusion, compared to animals operated on. false (p <0.001, Figure 31A). To demonstrate that this degree of antibody deposition in the reperfused hemisphere is due to the expression of P-selectin instead of non-specific accumulation, comparison was made with animals treated identically to those administered non-immune rat IgG. marked with 125I. These experiments demonstrated that there is a significantly higher accumulation in IgG against P-selectin compared to non-immune IgG (p <0.025, Figure 31A), suggesting that P-selectin is expressed in the brain in the following 30 minutes of reperfusion. Examination of sections of brain tissue immunostained with P-selectin reveals that the expression of P-selectin is localized mainly in the microvascular endothelial cells in the ipsilateral cerebral cortex (Figure 31B).

Accumulation of neutrophils in attack in mice: To delineate the time course on the PMN inflow that occurs after an attack, the accumulation of PMN labeled 11: LIn in wild type mice (PS + / +) was measured before of MCAO, immediately after and 10 minutes after MCAO, and at 30 minutes, 300 minutes and 22 hours of reperfusion. In PS + / + mice, the accumulation of PMN begins early after the onset of focal ischemia, and continues during the reperfusion period (Figure 31C). To establish the role of P-selectin in this postischemic accumulation of neutrophils, experiments were performed using mice where they were homozygous lacking the gene for P-selectin (PS - / -). PS - / - mice showed significantly reduced accumulation of PMN after occlusion of the middle cerebral artery and reperfusion (Figure 31B).

Role of P-selectin in the lack of cerebrovascular reflux: To determine whether the reduction in PMN accumulation in PS - / - mice results in improved cerebral blood flow after restoration of flow, serial measurements of the relative CBF were obtained by laser doppler in mice both PS + / + and PS - / -. Before the onset of ischemia (Figure 32, point a), the relative cerebral blood flows were almost identical between groups. Occlusion of the middle cerebral artery (Figure 32, point b) was associated with an almost identical decrease in cerebral blood flow in both groups. Immediately before removal of the intraluminal occlusion suture at 45 minutes of ischemia (Figure 32, point c), cerebral blood flows have increased slightly, although they remain significantly depressed compared to baseline flows. Immediately after removal of the occlusion suture to initiate reperfusion (Figure 32, point d), cerebral blood flow in both groups increased to a comparable degree ("60% baseline in PS mice - / - and PS + / +). The immediate failure of cerebral blood flow after reperfusion to reach the levels prior to occlusion is characteristic of the lack of cerebrovascular reflux, 21 with subsequent decline in cerebral blood flow after reperfusion, representing delayed postischemic cerebral hypoperfusion22. For 30"minutes of reperfusion (Figure 32, point e), cerebral blood flows between the two groups of animals were separated, PS - / - animals showed significantly higher relative cerebral blood flows compared to PS + / + controls. (Figure 32, point f) This separation or divergence reflected significant differences in delayed postischemic hypoperfusion, and persisted during the 22-hour observation period.

Post-attack outcome: The functional significance of P-selectin expression was tested by comparing post-attack outcome rates in PS - / - mice with respect to PS + / + controls. PS - / - mice were significantly protected from the effects of focal cerebral ischemia and reperfusion, based on a 77% reduction in infarct volume (p <0.01) compared to P-selectin + / + controls (Figure 33A). This reduction in infarct volume was accompanied by increased survival in PS - / - animals (p z 0.05, Figure 33B).

Effect of blocking P-selectin: After observing the functional role of P-selectin expression in the attack using mutant mice by deletion, experiments were performed to determine if pharmacological blocking of P-selectin can improve the outcome after attack in PS + / + mice. By using a strategy of administration of a rat antibody against functionally blocking monoclonal mouse P-selectin (clone RB 40.3414'18,19 = or non-immune control rat IgG immediately before surgery, the animals that received the antibody The blocker immediately before occlusion of the middle cerebral artery was observed to obtain improved postreperfusion cerebral blood flows at 30 minutes, as well as reduced cerebral infarct volumes and a trend towards reduced mortality compared to controls (Figure 34, 6 bars more left) To increase the potential chemical relevance of a blocking strategy of P-selectin as a new treatment for attacks, additional experiments were performed in which the control or blocking antibody was administered after intraluminal occlusion of the artery. medium brain function (because most patients present after the beginning of the attack). In these studies, a significant reduction in infarct volumes was observed as well as a trend towards improved cerebral blood flow (Figure 34, 6 bars to the left).

DISCUSSION: Despite the substantial progress in recent years in the primary prevention of attacks1, the therapeutic options for dealing with an attack that is developing are extremely limited. Although the publication of two pivotal trials last fall demonstrates a reduced morbidity after treatment of ischemic attack with rt-PA2,3 which is considered a preamble to a new era of thrombolytic therapy in the treatment of attacks, 4 it has been enthusiastically tempered to some extent by the haemorrhagic transformation and the increased mortality observed in ischemic attack patients treated with streptokinase5. These divergent trials become more critical than ever to the extent that new safe therapies have been developed to treat an attack in development. Although restoring blood flow to the postischemic brain provides new opportunities for early therapeutic intervention, reperfusion is a double-edged sword. Given the cytotoxic potential of neutrophils, 23 it is not surprising that the inflow of neutrophils to postischemic brain tissue can generate more damage and worsen the outcome after experimental attack.7,24"27 Using a mouse model of focal cerebral ischaemia and reperfusion Recently, we have identified an important contributory role for the cell adhesion molecule ICAM-l in the accumulation of neutrophils at 22 hours after the attack.7 However, increased cerebrovascular endothelial ICAM expression requires transcriptional and translational events of In contrast, P-selectin, a glycoprotein distributed in the membrane which mediates the earlier phases of neutrophil adhesion, can be mobilized from its preformed storage pools to express rapidly on the surface of the ischemic endothelial cell8, 28. To the extent that clinical trials of thrombolytic therapy for the attack demonstrate a narrow time interval for potential benefit (in the first few hours after the onset of the attack) 2,3,5, this suggests that strategies designed to interfere with Earlier phases of PMN adhesion may be of theoretical benefit in an attack in humans. These trials may result in higher numbers of patients presenting for early therapeutic intervention, which increases the need to resolve the issue of reperfusion injury in immediately revascularized territories.

In addition, they underscore the growing need to understand the contributions of individual adhesion molecules to the pathogenesis of the attack. Given the considerable body of literature describing the role of P-selectin in other models of ischemia and reperfusion8'29"32, surprisingly little is known about the role of P-selectin in the attack.The knowledge of the specific role of P-selectin in the cerebral vasculature is important because the adhesion molecule requirements vary between vascular beds and conditions under study.For example, in a model of intestinal transplantation33, antibodies against P-selectin do not reduce reperfusion damage, whereas antibodies to CD11 / CD18 if they do so Although blockade of P-selectin is not effective in reducing PMN adhesion and albumin leakage in the model of mesenteric ischemia and reperfusion in rat, ICAM-I blockade was effective34. In the ischemia / reperfusion model of the hind limb in rat, PMN adhesion selectin requirements differ between pulmonary and crural muscle beds31. To our knowledge, the only published study that describes the increased expression of P-selectin in the ischemic brain is a histopathological description of the attack in primates, in which the expression of P-selectin in the lenticulostriate microvasculature was increased10. Current studies were carried out to study whether P-selectin expression contributes to postischemic brain neutrophil accumulation, reflux deficiency and tissue damage in a mouse model of reperfused attack. Using a recently established model of focal cerebral ischaemia and reperfusion in mice11, the expression of P-selectin was demonstrated by increased endothelial immunostaining and increased deposition of radiolabelled antibody in the ischemic territory. In this last technique, antibody deposition in the ischemic hemisphere was normalized, compared to the non-ischemic hemisphere in each animal, not only to minimize potential variations in injection volume or volume of distribution, but to allow comparison between animals given different bodies. Due to the interruption of the endothelial barrier in the ischemic cortex which can increase non-selective antibody deposition, similar experiments were performed with control rat IgG. These data show that the antibody which binds to P-selectin is deposited at an accelerated rate compared to the control antibody, suggesting that the local expression of P-selectin is increased in the reperfused tissue. These data in the mouse model are parallel to those reported in the attack baboon model, 10 in which the expression of P-selectin was increased in the next 1 hour after the ischemic event. The role of the expression P-selectin in the recruitment of PMN to the post-ischemic zone was demonstrated using a strategy in which the accumulation of PMN marked with?: L1In was measured. Although we have previously reported that at 22 hours, the accumulation of PMN is elevated in the ischemic hemisphere7, the current data of the course in time show that the accumulation of PMN begins shortly after the access of ischemia. The failure to express the gene for P-selectin was associated with a reduced accumulation of PMN, suggesting the participation of P-selectin in the recruitment of post-ischemic cerebral PMN. However, animals lacking P-selectin demonstrated a modest (although less than control) accumulation of neutrophils at 22 hours. These data indicate that P-selectin is not the exclusive reflector mechanism responsible for the recruitment of postischemic cerebral PMN, and it is consistent with our previous data that ICAM-l also participates in the postischemic adhesion of PMN7. In addition, these data are not different from those in which intraabdominal thioglycollate instillation in mice deficient in P-selectin causes delayed (but not absent) recruitment of PMN9. Due to the critical need to identify reasons for failed reperfusion, current studies examined the role of P-selectin in delayed postischemic cerebral hypoperfusion21,22, the phenomenon in which blood flow declines during reperfusion, despite the restoration of adequate perfusion pressures. In cardiac models of reflux in ischemia, it worsens as the time elapses after reperfusion, 35 suggesting an important role for the recruited effector mechanisms, such as progressive microcirculatory thrombosis, vasomotor dysfunction and PMN recruitment. In other models it has been shown that adhesion reactions dependent on both P-selectin and ICAM-l36 and capillary tamponade by PMN37 participate in the lack of postischemic reflux. In the brain, PMNs have been implicated in the lack of postischemic cerebral reflux38,39, but the role of P-selectin has not been previously elucidated. The current study uses a relatively non-invasive technique (laser Doppler) to obtain serial measurements of the relative cerebral blood flow, in order to establish the existence, course in time and dependence of P-selectin on the lack of post-ischemic cerebrovascular reflux. In order to demonstrate that the cooking procedure itself is not the cause of vascular damage and subsequent cerebral infarction, false ischemia experiments (n = 10) were performed in which the nylon suture was cooked inside of the internal carotid artery during a period without occlusion of 45 minutes. In these experiments, it was shown that cooking is not occlusive based on the lack of decline in perfusion by laser Doppler during the 45 minute period. Later, when the brains were collected and fined with TTC at 24 hours, none showed evidence of cerebral infarction. Therefore, we can conclude that the cooking procedure per se does not cause enough damage to affect our main outcome variables. When the relative cerebral blood flow was examined after occlusion of the frank middle cerebral artery in experimental animals, we observed that the animals lacking P-selectin and control subjected to virtually identical degrees of ischemia (there was an initial decrease -4.5 times in the relative cerebral blood flow after occlusion of the middle cerebral artery in both). However, there was a slight increase in relative cerebral blood flow in the first 10 minutes after occlusion, although the occlusion suture remained in place. This is an empirical observation that we have made consistently, for which there are probabilities of several possible explanations. There is a likelihood that some degree of collateral flow which opens in the ischemic territory. Another pertinent explanation is that there may be an initial vasospasm element in the region of the occlusal catheter tip, which is removed modestly in the following minutes. Although both explanations are possible, due to the small size of the mouse vasculature, we can not identify the mechanism with certainty in our model. However, to the extent that we have observed the same degree of flow recruitment in both control and experimental animals, these data do not alter our main conclusions that P-selectin is an important mediator of brain tissue damage in reperfused attack.

After the removal of the intraluminal occlusion suture, the instantaneous recovery of blood flow was the same in both P-selectin + / + and - / - animals. The fact that the flow levels never returned to the baseline (nor had there been an overshoot, as can be seen with reactive hyperemia) may be due to the severity and duration of the ischemic period, which is likely to recruit other mechanisms of lack of postischemic cerebrovascular reflux, such as thrombosis or neutrophil recruitment caused by mechanisms that do not depend on selectin. When, even, at later time points are examined (such as 30 minutes to 22 hours after removal of the occlusion suture), it is interesting to note that there is a slight decline in cerebral blood flow in the P-selectin animals. / -. This late (although limited) decline in cerebral blood flow at 22 hours is consistent with the modest recruitment of PMN observed in PS - / - animals during the same period, suggesting again the recruitment or activation of other effector mechanisms that limit the flow (such as ICAM-1) in PS - / - animals. The functional effects of P-selectin expression are evident from the current set of studies: animals which fail to express the gene for P-selectin (or PS + / + animals treated with an antibody against functionally blocking P-selectin ) show smaller infarcts and improved survival compared to controls. When these data are considered together with the previously published data demonstrating a harmful role for the expression of ICAM-1 in the attack7, it becomes increasingly evident that there are multiple means to recruit PMNs to the postischemic cerebral cortex and that the blocking each one represents a potential strategy to improve the outcome after attack in humans. Given our current recognition of the importance of reperfusion in time to stop the advance of the frontal wave of neuronal death after attack, interference with the adherence of PMN in its earliest stages seems to be an attractive option to reduce morbidity and mortality. In fact, strategies against the adhesion of molecules can not only be beneficial in themselves (that is, it includes patients ineligible for thrombolysis) but can extend the range of opportunities for thrombolytic intervention40. The current set of studies contributes to our understanding of the apsophysiological mechanisms that operate in the reperfused attack. These studies suggest the need for clinical trials of therapies for the attack in development which optimize the reperfusion environment to reduce the accumulation of PMN.

References: 1. Bronner LL, Kanter DS, Manson JE: Primary prevention of stroke. N Enql J Med 1995; 333 (21): 1392 -1400 2. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group: Tissue plasminogen activator for acute ischemic stroke. N Enql J Med 1995; 333: 1581-1587 3. Hacke, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH, Hennerici M, for the ECASS Study Group: Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke . J A M 1995; 274 (13): 1017-1025 4. del Zoppo GJ: Acute stroke - on the threshold of a therapy. H Enql J Med 1995; 333 (13): 1632-1633 . Hommel M, Cornu C, Boutitie F, Boissel JP, The MultiCenter Acute Stroke Trial - Europe Study Group: Thrombolytic therapy with streptokinase in acute ischemic stroke. N Ensl J Med 1996; 335: 145-150 6. Baringa M: Finding new drugs to treat stroke. Science 1996; 272: 664-666 7. Connolly ESJr, infree CJ, Springer TA, Naka Y, Liao H, Yan DS, Stern DM, Solomon RA, Gutierrez-Ramos JC, Pinsky DJ: Cerebral protection in homozygous nuil ICAM-l mice after middle cerebral artery occlusion: role of neutrophil adhesion in the pathogenesis of strokie. J Clin Invest 1996; 97: 209-216 8. Pinsky DJ, Naka Y, Liao H, Oz MC, Wagner DD, Mayada TN, Johnson RC, Hynes RO, Heath M, Lawson CL, Stern DM: Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies: a mechanism for rapid neutrophil recruitment after cardiac preservation. il Clin Invest 1996; 97: 493-500 9. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD: Leukocyte rolling and extravasation are severely compromised in P-selectin deficient mice. £ _al_L 1993; 74 (3): 541-554 . Okada Y, Copeland BR, Mori E, Tung MM, Thomas WS, Zoppo GJ: P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke 1994; 25: 202-211 11. Connolly ESJr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ: Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurg 1996; 38 (3): 523-532 12 Barone FC, Knudsen DJ, Nelson AH, Feuertstein GZ, Willette RN: Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J. Cereb Blood Flow Metab 1993, 13: 683-692 13. Dirnagl U, Kaplan B, Jacewicz M, Bulsinelli W: Continuous measurement of cerebral blood flow by laser-Doppler flowmetry in a mouse stroke model. J Cereb Blood Flow Metab 1989, -9: 589-596 14. Law K, Bullard DC, Arbones ML, Bosse R, Vestweber D, Tedder TF, Beaudet AL: Sequentlal contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 1995; 181: 669-675 . David GS, Reisfeld RA: Protein iodination with solid state lactoperoxidase. Biochem 1974; 13: 1014-1021 16. Bederson JB, Pitts LH, Tsuji M: Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472-476 17. Huang Z, Huang PL, Panahian N. Dalkara T, Fishman MC, Mopskowitz MA: Effects of cerebral ischemia in mice deficient in neuronal nitris oxide synthase. Science 1994; 265: 1883-1885 89. Bosse R, Vestweber D: Only simultaneous blocking of the L- and P-selectin inhibits completely neutrophil migration into mouse peritoneum. Eur J Immunol 1994; 24: 3019-3024 19. Kunkel EJ, Jung U, Bullard DC, Norman KE, Wolitzky BA, Vestweber D, Beaudet AL, Law K: Absence of trauma-induced leukocyte tolling in mice deficient in both P-selectin and ICAM-l. J Exp Med 1996; 183: 57-65 . Springer TA: Adhesion rceptors of the immune system. Nature 1990; 346: 425-434 21. Ames There, Wright RL, Kowada M, Thurston JM, Majno G: Cerebral ischemia II: the no reflow-phenomenon. Am J Pathol 1968; 52: 437-447 22. Levy DE, Van Uitert RL, Pike CL: Delayed postischemic hypoperfusion: a potentially damaging consequence of stroke. Neurology 1979; 29: 1245-1252 23. Weiss SJ: Tissue destruction by neutrophils. N Engl J Med 1989; 320 (6): 365-376 24. Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP, Contreras TJ: Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke 1986; 17: 246-253 . Kochanek PM, Hallenbeck JM: Polymorphonuclear leukocytes and monocytes / macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 1992; 23 (9): 1367-1379 26. Dutka AJ, Kochanek PM, Hallenbeck JM: Influence of granulocytopenia on canine cerebral ischemia induced by air embolism. Stroke 1989; 20: 390-395 27. Bednar MM, Raymond S, McAuliffe T, Lodge PA, Gross CE: The role of neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke 1991, -22 (1): 44-50 28. Geng J-G, Bevilacqua MP, Moore KL, Mclntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA, McEver RP: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 1990; 343: 757-760 29. Weyrich AS, Ma X-L, DJ efer, Albertine KH, Lefer AM: In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest 1993; 91: 2620-2629 30. Winn RK, Liggltt D, Vedder NB, Paulson JC, Harian JM: Anti-P-selectin monoclonal antibody attenuates reperfusion injury in the rabbit ear. J Clin Invest 1993; 92: 2042-2047 31. Seekamp A, Till GO, Mulligan MS, Paulson JC, Anderson DC, Miyasaka M, Ward PA: Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am J Pathol 1994; 144: 592-598 32. Kubes P, Jutlla M, Payne D: Therapeutic potential of inhibiting leukocyt rolling in ischemia / reperfusion. J Clin Invest 1995; 95: 2510-2519 33. Slacum MM, Granger DN: Early mucosal and microvascular changes 15 in feline intestinal transplants. Gastroenterology 1993; 105: 1761-1768 34. Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, Rusche JR, Granger DN: Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage. Circ Res 1994; 74: 336-343 . Kloner RA, Earned CE, Jennings RB: The "no-reflow phenomenon after temporary coronary occlusion in the dog. J Clin Invest 1974; 54: 1496-1508 36. Jerome SN, Dore M, Paulson JC, Smith CW, Korthuis RJ: P-selectin and ICAM-l-dependent adherence reactions: role in the genesis of postischemic no-reflow. Am J Physiol 1994; 266: H1316-H1321 5 37. Engler RL, Schmid-Schonbein GW, Pavelec RS: Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 1983; 111: 98-111 38. Mori E, Zoppo GJ, Chambers JD, Copeland BR, Arfors KE: Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons. Stroke 1992; 23: 712-718 39. Grogaard B, Schurer L, Gerdin B, Arfors KE: Delayed hypoperfusion after incomplete forebrain ischemia in the rat: the role of polymorphonuclear leukocytes. J Cereb Blood Flow Metab 1989; 9: 500-505 40. Bowes MP, Rothlein R, Fagan SC, Zivin JA: Monoclonal antibodies preventing leukocyte activation, reduces experimental neurological injury, and enhance efficacy of thrombolytic therapy. Neurology 1995; 45: 815-819.

Example 11: Use of carbon monoxide to treat an ischemic disorder-Ejection of protective effects of carbon monoxide in pulmonary ischemia In the initial patent application, we show data indicating that endogenous carbon monoxide production or the administration of exogenous carbon monoxide is beneficial to protect the brain against subsequent ischemic damage. As another example of the use of carbon monoxide in the treatment of ischemic disorders, we administer carbon monoxide to rats to test their effects on improved preservation of the lungs for transplantation (this is similar to an ischemic disorder, because the lungs of the When the donor is removed from the recipient, during the period in which the lungs are preserved and transferred from the donor to the recipient, there is an interruption in blood flow.

Methods to test the effect of carbon monoxide on lung preservation: Materials used to prepare preservation solution: For all experiments, the base preservation solution consisted of modified Euro Collins solution (EC) (Na * 10 mEq / 1, K + 115 mEq / 1, Cl "15 mEq / 1, H2P04" 85 mEq / 1, H2P04"15 mEq / 1, HC03"10 mEq / 1).

Lung collection, preservation and transplantation: Inbred male Lewis rats (250-300 g) were used for all experiments according to a protocol approved by the Institutional Animal Carea and Use Committee at Columbia University, in accordance with the guidelines established by the American Academic for Accreditation of Laboratory Animal Care (AAALAC). The lung transplant experiments were carried out in the following manner. Donor rats were given 500 units of heparin intravenously, and the pulmonary artery (PA) was drained with a volume of 30 ml of preservation solution at 4 ° C, at a constant pressure of 20 mmHg. When the lungs are preserved in this way, most of the infused lavage solution leaves the left ar- chaeal ventilation created by the donor lung, as well as the exit of the pulmonary veins posterior to the transection. The left lung is harvested or removed, a fist is placed in each vascular stump, a cylinder is inserted into the bronchus, and the lung was immersed for 6 hours in a preservation solution at 4 ° C which was identical to the PA wash solution. Paired rats in terms of genus / strain / size were anesthetized, incubated and ventilated with 02 to 100% using a rodent fan (Harvard Apparatus, South Natick, MA). An orthotopic left lung transplant was performed through a left thoracotomy using a fast cuff technique for complete anastomosis, with warm ischemic times maintained below 5 minutes. The cross-shaped staple was freed, blood flow and ventilation was restored to the transplanted lung. A loop was then passed through a surgical loop around the right PA, and Millar catheters were inserted (2F; Millar Instruments, Houston, TX) in the main PA and the left atrium (LA). A Doppler flow probe (Transonics, Ithaca, NY) was placed around the main PA.

Measurement of lung graft function Hemodynamic monitoring was carried out online using a MacLab and Macintosh Ilci computer. The hemodynamic parameters measured included LA and PA pressures (mmHg) and PA flow (ml / min). The oxygen arterial pressure (p02, mmHg) was measured during inspiration from 02 to 100% using a gas analyzer model ABL-2 (Radiometer, Copenhagen, Denmark). The PVRs were calculated as (mean pressure PA - pressure LA) / average flow PA, and were expressed as mmHg / ml / min. After the baseline measurements, the native right BP was ligated and serial measurements were taken every 5 minutes until euthanasia at 30 minutes (or until death of the recipient).

Carbon monoxide administration: At the indicated time before the surgery (4, 8 or 12 hours), the rats were placed in a bell cage, and carbon monoxide was administered at various concentrations (0.01%, 0.03% or 0.1%), with the rest of the gas mixture constituted of ambient air. (The gas was passed through a container of water prior to administration, in order to humidify for the comfort of the animal). At the indicated times after the start of the exposure, the rats were anesthetized and the lungs were collected as described above. These donor lungs were used in subsequent lung transplant experiments.

Results and Discussion: The results of these experiments indicated that compared to untreated controls, the inhalation of carbon monoxide before collection of the lungs provides significant protection for the lungs after transplantation. This protection is evidenced by: (1) improved arterial oxygenation of donor lung receptors pretreated with carbon monoxide; (2) increased pulmonary arterial blood flow (and reduced pulmonary vascular resistance) with the use of donor lungs pretreated with carbon monoxide; and (3) improved survival of donor lung receptors pretreated with carbon monoxide compared to controls. The beneficial effects of carbon monoxide were dose-dependent, that is, the best protection was observed at the 0.1% dose, with an intermediate level of protection observed at the 0.3% dose and the lowest protection was observed at the dose of 0.01%. The beneficial effects of carbon monoxide were also time dependent, where longer exposures seem to provide the greatest protection. Together, these data indicate that carbon monoxide may protect in another ischemic disorder (pulmonary ischemia) and suggest that the results may be generalizable to other ischemic disorders as well.

Example 12: Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice Abstract Background and Purpose There is a great interest in developing novel anticoagulant or thrombolytic strategies to treat ischemic attack. However, to date, there are limited means to accurately determine the hemorrhagic potential of these agents. Current studies were designed to develop and validate a method to accurately quantify the degree of intracerebral hemorrhage in mouse models. Methods In a mouse model, intracerebral hemorrhage (ICH) was induced by stereotactic intraparenchymal infusion of collagenase B alone (6 x 10"6 units, n = 5) or collagenase B followed by intravenous tissue plasminogen activator (rt-PA, 0.1 mg / kg, n = 6) The controls consisted of false surgery with stereotactic saline infusion (n = 5) or untreated animals (n = 5) .The ICH (1) was classified by a scale based on diameter maximum bleeding on coronal sections, and (2) was quantified by a spectrophotometric assay by measuring cyanometahemoglobin in chemically reduced extracts of homogenized mouse brain.This spectrophotometric assay was validated using known amounts of hemoglobin or autologous blood added to a separate cohort of homogenized brains. When using this trial, the degree of haemorrhage after occlusion / reperfusion of the focal mean bral artery was quantified in mice treated with high post-termination doses of rt-PA IV (10 mg / kg, n = 11) and the control mice were subjected to attack but were treated with physiological saline solution (n = 9). Results The known amounts of hemoglobin or autologous blood added to fresh whole brain tissue homogenate show a linear relationship between the aggregate amount and OD at the absorbance peak of cyanometahemoglobin (r = 1.00 and 0.98, respectively). When in vivo studies were performed to quantify experimentally induced ICH, the animals that received the intrabral infusion of collagenase B had significantly higher OD compared to the controls that were infused with saline solution (2.1-fold increase, p = 0.05). . In an occlusion and reperfusion attack model of the middle bral artery, the administration of rt-PA after reperfusion increased the OD by 1.8 times compared to the animals which received physiological saline (p <; 0.001). When the two methods of measuring ICH (visual grade and DO) were compared, there was a linear correlation (r = 0.88). Additional experiments demonstrated that staining with triphenyltetrazolium, which is commonly used to stain viable brain tissue, does not interfere with the spectrophotometric quantification of ICH. Conclusions These data demonstrate that the spectrophotometric assay accurately and reliably quantifies ICH in mice. This new method may aid in the objective determination of bleeding risks of novel anticoagulant or thrombolytic strategies to treat the attack and may facilitate the quantification of other forms of intracerebral hemorrhage.

Introduction The ischemic attack constitutes the great majority of the presents of acute attack. Therefore, there has been tremendous interest in designing strategies that can promptly and effectively restore blood flow to the ischemic region of the brain. Although heparin can be effective in an incipient attack (TIA) 3, its use during acute phases of attack can be associated with a high degree of morbidity and intracerebral hemorrhage.14 Similarly, at the beginning of the 1960s, the results discouraging trials of streptokinase for acute attack led to a reduction of physicians to trombalise acute attacks during the subsequent three decades.5,6 This reduction has been validated by recent trials in which the use of estrokinase has been associated with increased risk mortality and intracerebral hemorrhage.7 On the other hand, the use of recombinant cell-type plasminogen activator (rt-PA) to treat an attack in progress has shown to be more promising, 8 with a subset of patients with acute attack who are treated with rt -PA that show a reduced long-term morbidity if they are treated in the first 3 hours of the access of the symptoms9"11. Even so, other trials using the same agent (rt-PA) have shown no benefit or have had excessively high rates of ICH.9,12"15. This confusing imbroglio of clinical data underscores the urgent need to identify improved strategies for rapid reperfusion. For this purpose, it is imperative to identify an experimental model in which the potential benefits of reperfusion can be objectively weighted in time in an attack, against the risks of increased intracerebral hemorrhage, in most animal studies of thrombolytic therapy for clinical attack, the risks of intracerebral hemorrhage have been estimated instead of being measured quantitatively16"24. The current studies were designed to develop and validate a method to accurately quantify the degree of intracerebral hemorrhage in mouse models, in order to determine the potential risks of new anticoagulant or thrombolytic treatments for acute attack, Materials and methods Experimental animals In the present study, male C57BL / 6J mice were purchased from Jackson Laboratories (Bar Harbor, ME), and were used between 8 and 10 weeks of age (22-32 g). All procedures were performed in accordance with an institutionally approved protocol and are in accordance with the guidelines provided by the American Academy of Accreditation of Laboratory Animal Care (AAALAC).

Spectrophotometric assay for intracerebral hemorrhage The hemoglobin content of the brains subjected to the experimental procedures was quantified below using a spectrophotometric assay as follows. Complete brain tissue was obtained from control or experimental animals subjected to recent euthanasia, and each brain was treated individually as follows. Distilled water (250 μl) was added to each brain followed by homogenization for 30 seconds (Brinkman Instruments, Inc., Westbury, NY), sonication on ice with a pulse ultrasonicator, for 1 minute (SmithKline Corporation, Collegeville, PA), and centifugation at 13,000 rpm for 30 minutes (Baxter Scientific Products, Deerfield, IL). After collecting the supernatant containing hemoglobin, add 80 μl of Drabkin's reagent (purchased from Sigma Diagnostics, St. Louis, MO; K3Fe (CN) 6200 mg / l, JCN 50 mg / l, NaHCO3 lg / 1, pH 8.625) at a 20 ml aliquot and allowed to sit for 15 minutes. This reaction converts hemoglobin to cyanomethaemoglobin which has an absorbance peak at 540 nm, and whose concentration can be determined by the optical density of the solution at = 550 nm wavelength26. To validate the absorbance measured following these procedures that reflect the amount of hemoglobin, hemoglobin amounts of bovine erythrocytes were made (Sigma, St. Louis, MO) using similar procedures in conjunction with each brain tissue assay. As an additional measure, blood was obtained from control mice by cardiac puncture after anesthesia. Increasing aliquots of this blood were then added to fresh homogenate brain tissue obtained from untreated mice to generate a standard absorbance curve.

Intracerebral hemorrhage induced by collagenase The general procedures for inducing intracerebral hemorrhage in mice were adapted from a method which has previously been described in rats27. After anesthesia with an intraperitoneal injection of 0.35 ml of ketamine / 10 mg / ml) and xylazine (0.5 mg / ml), mice were placed in the prone position in a stereotactic head frame. Calvary was exposed by an incision in the midline of the skull from the nasion to the superior nuchal line and then the skin was laterally retracted. Using a variable speed drill (Dremel, Racine, Wl), a 1.0 mm hole was made at 2.0 mm posterior to the pregma and 2.1 mm to the right side of the midline. A single 22-gauge angiocatheter needle was inserted using a stereotactic guidewire into the right deep crustal / basal ganglia (coordinates: 2.0 mm posterior, 2.00 mm lateral). The needle was attached by plastic tubing to a microinfusion syringe and brain solutions were infused at a rate of 0.25 μl per minute for 4 minutes with an infusion pump (Bioanalytical Systems, West Lafayette, IN). The animals received: (1) 0.024 μg of collagenase B (Boehringer Mannheim, Mannheim, Germany) in 1 μl of normal saline (collagenase); 2) i μl of normal saline solution alone (false), - (3) without treatment (control); or (4) stereotactically guided infusion of collagenase B as above, but followed immediately by intravenous recombinant human tissue plasminogen activator (Genentech Inc., South San Francisco, CA, 1 mg / ml in 0.2 ml of normal saline (administered by injection into the dorsal penis vein (Collagenase + rt-PA) In the collagenase, false, and collagenase + rt-PA groups, the stereotactic needle was removed immediately after the infusion and the incision closed with surgical staples The brain tissue was collected immediately after rapid anesthetized decapitation.

Hemorrhagic conversion in a mouse focal cerebral ischemia model Focal cerebral ischemia occurred in animals due to transient occlusion of the right middle cerebral artery using a previously described method in detail28,29. Briefly, a 5-0 or 6-0 blunt-ended, 12-mm or 13-mm nylon suture is passed into the right internal carotid artery to the level of the middle cerebral artery. After 45 minutes, the occlusion suture is removed to restore reperfusion. Immediately after removal of the occlusion suture, the animals received intravenous tissue plasminogen activator (10 mg / kg in 0.2 ml of normal saline, attack + rt-PA) or normal saline (attack + saline) administered by injection into the dorsal vein of the penis. At 24 hours, brain tissue was immediately collected after rapid anesthetized decapitation. To evaluate the effect of 2, 3, 5-triphenyltetrazolium chloride (TTC), which is commonly used to differentiate infarcted non-infarcted brain tissue, 28,30 unmanipulated (control) brains were divided in half, and immersed in 2% TTC (Sigma Chemical Company, St. Louis, MO) or in 0.9% phosphate buffered saline, incubated for 30 minutes at 37 ° C, and then prepared as described above for the spectrophotometric hemoglobin assay. The other half of each brain was immersed in saline for an identical duration and then subjected to the procedures described in the foregoing for the spectrophotometric assay of hemoglobin.

Validation of quantitative intracerebral hemorrhage assay The ICH grade was first visually classified by an observer who does not know the protocol. For the visual qualification of the intracerebral hemorrhage in the mouse, the brains obtained from the mice that would have survived the time point of 24 hours after the procedure (collagenase-induced hemorrhage or MCA occlusion) were placed in a mouse brain matrix (Activational Systems, Inc., Warren, MI) to obtain coronal sections in 1 mm series. The sections were inspected by an observer who did not know the protocol and the brains were given an ICH score from a graded scale based on the maximum diameter of bleeding observed in any of the sections (ICH score 0, without bleeding).; 1, < 1 mm, 2, 1-2 mm, 3, > 2-3 mm; 4, > 3 mm]. The cuts of each brain were then accumulated, homogenized and then treated according to the procedures described above for the spectrophotometric assay of hemoglobin.

Statistics The correlations between the ICH scores visually determined and those of ICH spectrophotometric erminations were made using the Pearson linear correlation, indicating the correlation coefficients. To establish whether a given treatment (collagenase, false, attack, etc.) had a significant effect on either spectrophotometric or visually qualified ICH, comparisons were made on a non-paired double-tail T-test. For non-parametric data (visual ICH scores), nonparametric analysis was performed using the Mann-Whotney test. The values are expressed as means ± SEM, with p < 0.05 considered as statistically significant.

Results Hemoglobin spectrophotometric assay Initial studies were conducted to determine the reliability and reproducibility of the spectrophotometric hemoglobin assay. In the first set of experiments, known amounts of hemoglobin were converted to cyanomethaemoglobin according to previously published procedures and the OD was measured [Figure 35A) 26. In a second set of experiments, known amounts of autologous blood were added to fixed volumes of fresh brain tissue homogenate, with additional treatment of the samples as described above. These data show that the optical density of supernatants containing cyanometahemoglobin at 550 nm correlate linearly with the amount of blood added [Figure 35B]. These data show narrow linear correlations (r = 1.00 and 0.98 for Figures 35A and 35B, respectively), as well as excellent reproducibility as calibrated by relatively small standard errors of the mean. To establish that TTC (commonly used) to distinguish infarcted non-infarcted brain tissue28,30) does not affect the spectrophotometric assay of hemoglobin, unmanipulated brains (control) were divided in half, where each half underwent the standard staining procedure TTC and half was treated with saline as a control. These data (figure 35B, continuous and discontinuous lines) indicate that the previous treatment of TTC brain tissue does not affect the spectrophotometric hemoglobin assay. To determine if this method is capable of detecting ICH, an assay was performed on intracerebral mouse hemorrhage caused by two different procedures, intraparenchymal infusion of collagenase or occlusion / reperfusion of the middle cerebral artery. In the first procedure, collagenase B was applied as a local infusion through the perforation, in order to weaken the vascular wall to promote ICH (collagenase group). To further increase the propensity for, and the degree of ICH, a similar procedure was performed, with immediate administration of post-procedure rt-PA (collagenase group + rt-PA). Two control conditions were also included, a false operation which included drilling of the perforation, but with instillation of physiological saline solution (false), and an untreated group (control). These experiments demonstrated that the infusion of collagenase increases the amount of intracerebral blood detected by the spectrophotometric assay (especially with collagenase + rt-PA) compared to animals treated in false with normal controls [Figure 36A].

In the second method and perhaps the most clinically relevant method to induce ICH, an attack was generated by transient intraluminal occlusion of the middle cerebral artery followed by reperfusion. In addition, we try to increase the propensity for hemorrhagic conversion by administration of a thrombolytic agent. Two groups were studied, those who had received normal saline or those who received intravenous rt-PA immediately after the removal of the intraluminal occlusion suture. These data indicate that the addition of fibrinolytic agent after attack increases the amount of ICH which is detected by the spectrophotometric hemoglobin assay [Figure 36B]. It is interesting to note that the absorbance of the baseline is lower in animals subjected to attack compared to control / untreated animals [Figures 36A and 36B]. To further investigate the manner in which residual intravascular blood may affect the spectrophotometric hemoglobin assay, experiments were performed in which, immediately before the beheading of the animal for removal or harvesting of the brain, a cephalic perfusion of physiological saline solution (administered via the left cardiac ventricle) was performed. In control animals (n = 5) who received cardiac saline perfusion before brain harvesting, the optical density after tissue preparation and the spectrophotometric hemoglobin test was 0.25 + 0.3 (this is better than the optical density observed in non-cardiac perfused animals subjected or not to false surgery (n = 10, OD 0.34 ± 0.05, p = 0.05 versus cardiac perfused controls) On the other hand, after the attack, there were no differences in the OD either whether or not he has perfused cardiac saline (0.15 ± 0.04 for attack with cardiac saline solution n = 5; 0.15 ± 0.03 for infusion with cardiac saline solution, P = NS). When saline infused perfused animals were compared with animals perfused with saline but without attack, there was apparently a reduction in OD after the spectrophotometric hemoglobin test. -These data may suggest that animals with attack have less intracerebral blood detected, perhaps as a result of a reduction in the total amount of blood in the ipsilateral MCA region following ischemia.

Visual rating of ICH In order to further validate the spectrophotometric hemoglobin assay, we compare it with the morphometric determination of hemorrhage size, which has traditionally been used in the literature.31"35 We developed a visual qualification system (0-4) in which a Observer who does not know the protocol qualifies the degree of ICH in serial brain sections based on the maximum hemorrhage diameter.This visual determination was made in a photograph of the brain taken immediately before the spectrophotometric hemoglobin test [Figure 7] that the two techniques can be correlated in the same samples When compared to controls not subjected to any intervention, the animals that received false local infusion (ie, drilling + saline) show only a slight increase in the visual qualification of ICH [ Figure 38A.] However, when collagenase was added alone or collagenase + rt-PA to the infused material, visual ratings of ICH were significantly increased [Figure 38]. In the attack model, rt-PA similarly resulted in an increase in the visual rating of ICH [Figure 38B]. When the data is plotted to show the relationship between the visual qualification of ICH and the spectrophotometric technique to quantify ICH, a linear relationship (r = 0.88) was suggested, however, with smaller degrees of hemorrhage (ICH visual ratings of 0). or 1), this relationship was not maintained [Figure 39].

Discussion Recently, it has become evident that early intervention in the attack with certain intravenous thrombolytic agents (rt-PA) can be beneficial if it is instituted in the next 3 hours at the onset of symptoms, 9,10 however, the administration of thrombolytic agents outside of this narrow therapeutic range can cause an unacceptably high incidence of devastating ICH (streptokinase versus placebo, 10-day mortality 34.0% versus 18.2%, p = 0.002, mortality at 6 months, 73% versus 59%, p = 0.06 7) Therefore, it remains a clinical imperative to identify more adequate agents to restore perfusion, which are associated with a lower risk of hemorrhagic conversion. In order to study new agents which interfere with coagulation or fibrinolytic mechanisms, it is necessary to have an objective means to quantify the risk on the lower side of ICH. In the experimental literature, the quantification of ICH has been performed either by radiological imaging procedures32"37, or by visual estimation of the amount of hemorrhage in postmortem brain tissue31" 35. These procedures are of limited utility and depend on the conditions under study. For example, in addition to the logistical impediments imposed by the need for sophisticated equipment, most radiological imaging techniques are of limited use in mouse models, which may prevent their use in the evaluation of transgenic mice, a Potentially powerful tool to study fibrinolytic systems coagulation. The visual estimation of ICH is subjective in nature and, as our own data show, it may be relatively insensitive to detection of small ICH grades. In addition, none of the techniques, radiological or visual, allows the precise quantification of ICH when the hemorrhagic region is in the form of patches or is of multiple foci. Current studies were carried out to develop and validate an objective method to quantify ICH in experimental animals. The use of a spectrophotometric assay for the quantification of hemoglobin based on the conversion of hemoglobin to cyanometahemoglobin has been previously reported26,31. However, to the best of our knowledge, in the brain, it has only been used in rats to measure the size of a frank blood clot after its removal from adjacent brain tissue31. The spectrophotometric assay that we describe and validate can be used in animals as small as mice, which facilitates the use of many transgenic mouse strains now available (particularly those with alterations in thrombotic or fibrinolytic cascades). In addition, this spectrophotometric assay allows the quantification of ICH even when there are hemorrhages by patches or multiple foci, which would otherwise be to identify or isolate. Finally, in contrast to Lee's study, we have validated our study to determine its repeatability and reliability using known techniques of hemoglobin and autologous blood mixed with brain tissue31. Because the surgical procedure used in the attack experiments does not significantly alter blood hemoglobin concentrations (data not shown), the spectrophotometric hemoglobin assay can be used to extrapolate the volume of intracerebral hemorrhage when the concentration of hemoglobin at the time of bleeding is known. To develop and validate the spectrophotometric hemoglobin assay for situations that may be relevant for clinical ICH, we generated intracerebral hemorrhages by two different methods: (1) intracerebral injection of collagenase (to weaken the vascular wall, as it may present with an aneurysm or with trauma) and (2) in an attack model. In both cases, a group of animals also received rt-PA in order to validate the model at the high end of the ICH spectrum. Because there has not been a basic standard measurement for ICH in mice, our spectrophotometric measurements were compared to the ICH size independently determined by visual rating. Finally, to test the trial even more useful for experimental models of attack in which the brains are stained with triphenyltetrazolium chloride (TTC) to quantify the volume of cerebral infarction, the brains of animals subjected to occlusion / reperfusion by MCA were terminated with TTC before their accumulation and homogenization to establish that the TTC staining procedure itself does not interfere with the ability to quantify ICH by the spectrophotometric hemoglobin assay.

These data (Figure IB) indicate that there is no detectable cross-interference between the two procedures when used sequentially (first staining with TTC, followed by homogenization and the spectrophotometric assay of hemoglobin). In addition to its ability to detect ICH, current studies indicated that this technique can also provide an indication of the amount of residual intravascular blood after collection or removal of the brain. The perfusion procedure with cephalic saline solution does not alter the optical density for cyanoematohematoglobin in brains under attack, suggesting that the amount of intravascular blood is relatively fixed and not washed out by the procedure. However, in control animals which have not otherwise been treated, saline infusion treatment appears to decrease the optical density for cyanometahemoglobin by approximately 30%. Our experiments do not provide the reason for this difference, but it can be speculated that after the attack, there is an element of vasoconstriction / vasoocclusion in the infarct territory which makes the perfusion technique with less effective saline in the eliminated by washing additional residual intravascular blood. In addition, if there is actually an element of vasoconstriction after attack or experimentally induced intracerebral hemorrhage, this can reduce the accumulation of intravascular blood and therefore explain the total decrease in optical density when comparing the control and attack / ICH brains. (even if some extravascular blood is present in this last group). Several technical aspects of the spectrophotometric technique for measuring intracerebral hemorrhage also deserve mention. For the current experiments, although there is a broad absorbance peak for cyanometahemoglobin centered at approximately 450 nm, we measured the absorbance of cyanometahemoglobin at 550 mm. The reason for doing this is that many spectrophotometers have fixed wavelength capabilities that depend on pre-set filters, and 550 nm is a commonly used wavelength (especially in ELISA plate readers). Although the measurement of absorbance at 540 nm may provide slightly higher optical density measurements, the cyanometahemoglobin absorbance peak is large in this area, and therefore 550 nm can be used without the need to correct the absorbance of ferricyanide or ferrucianide (The extinction coefficients for cyanometahemoglobin at 551 nm and 540 nm are 11.5 and 11.1, respectively, compared with the extinction coefficient, 41 times lower than ferricyanide or ferrucianuro38). Studies using a continuous-wavelength spectrophotometer (which are used to measure OD at 540 nm) and ELISA-defined-spectrum plate readers (used to measure OD at 550 nm) gave similar results. Since this last technique is simpler, increases the performance of the procedure and allows us to minimize the sample volume, we chose to use this last technique for the studies shown in the Results section. There are some other potential technical considerations that should be considered when using the spectrophotometric assay. Although we have shown that the spectrophotometric procedure can be used in conjunction with TTC staining of sections or brain slices in series for analysis of infarct volume, the tissue should be homogenised subsequently and extracted, destroying tissue architecture and making it impossible characterization additional histological It is possible that this technique may overestimate the degree of ICH if extracerebral blood is unintentionally included during collection, or the. The technique can underestimate the degree of ICH if epidural, subdural or subarachnoid blood remains attached to the skull, which is discarded during the process of brain removal. Due to the nature of the measurement technique, in which light at a given wavelength is absorbed along a fixed length trajectory, any coas that cause turbidity of the homogenized brain supernatant can increase the OD reading . This may include lipids, abnormal plasma proteins and an eritocyte stroma. In fact, in preliminary experiments, we found that DOs were falsely raised when centrifugation was insufficient and part of the lipid layer was included in the assay. Free pyridines can alter the absorbance spectrum of cyanomethahemoglobin and there is the potential that other hemochromogens will also react with Drabkin's reagent39. However, as far as we know, these reactions do not interfere to a significant degree with blood determination / intracerebral hemorrhage. In summary, current data illustrate a simple and inexpensive way of spectrophotometric assays for hemoglobin which can provide a useful method for quantifying ICH. This technique may prove to be especially useful for evaluating the bleeding potential of newly developed thrombolytic or anticoagulant therapies for the attack treatment.

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Neurological Research 1993; 15: 344-349 18. Overgaard K, Sereghy T, Boysen G, Pedersen H, Diemer NH: Reduction of infarct volume by thrombolysis with rt-PA in an embolic rat stroke model. Scandinavian Journal of Clinical Laboratory Inves isa ion 1993; 53: 383-393 19. Benes V, Zabramski JM, Boston M, Puca A, Spetzler RF: Effect 5 of intra-arterial tissue plasminogen activator and urokinase on autologous arterial emboli in the cerebral circulation of rabbits. Stroke 1990; 21: 1594-1599 . Overgaard K, Sereghy T, Pedersen H, Boysen G: Effect of 0 delayed thrombolysis with rt-PA in a rat embolic stroke model.

Journal of Cerebral Blood Flow & Metabolism 1994; 14: 472-477 21. Lyden PD, Zivin JA, Clark WA, Madden K, Sasse KC, Mazzarella VA, Terry RD, Press GA: Tissue plasminogen activator-mediated 5 thrombolysis of cerebral em? Joli and its effect on hemorrhagic infarction in rabbits. Neurology 1989; 39: 703-708 22. Kochanek PM, Dutka AJ, Kumaroo KK, Hallenbeck JM: Effects of prostacyclin, indo ethacin, and heparin on cerebral blood flow and platelet adhesion after multifocal ischemia of canine brain. Stroke 1988; 19: 693-699 23. Slivka A, Pulsinelli W: Hemorrhagic causes of thrombolytic therapy in experimental stroke. Stroke 1987; 18: 1148-1156 24. Lyden PD, Zivin JA, Solí M, Sitzer M, Rothrock JF, Alksne J: Intracerebral hemorrhage after experimental embolic infarction. Anticoagulation. Arch Neurol 1987; 44: 848-850 25. Van Kampen EJ, Zijlstra WG: Standardization of hemoglobinometry. II. The hemiglobincyanide method. Clin Chim Acta 1961; 6: 538-544 26. Van Kampen EJ, Zijlstra WG: Standardization of 10 hemoglobinometry. II. The hemoglobincyanide method. Clin Chim Acta 1961; 6: 538 27. Rosenberg GA, Mun-Bryce S, Wesley M, Kornfield M: Collagenase-induced intracerebral hemorrhage in rats. Stroke 1990; 21: 801-807 28. Connolly ESJr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ: Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurs 1996; 38 (3): 523-532 29. Connolly ESJr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutiérrez-Ramos J-C, Pinsky DJ: Cerebral protection in homozygous nuil ICAM-l mice after middle cerebral artery occlusion. Role of neutrophil adhesion in th pathogenesis of stroke. J Clin Invest 1996; 97: 209-216 . Bederson JB, Pitts LH, Nishimura MC, Davis RL, Bartkowski HM: Evaluation of 2, 3, 5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 1986; 17: 1304-1308 31. Lee KR, Colon GP, Betz AL, Reep RF, Kim S, Hoff JT: Edema from intracerebral hemorrhage: the river of thrombin. J Neurosurg 1996; 84: 91-96 32. Del Bigio MR, Yan HJ, Buist R, Peeling J: Experimental intracerebral hemorrhage in rats: magnetic resonance imaging and histopathological correlates. Stroke 1996; 27: 2312-2320 33. Qian L, Nagaoka T, Ohno K, Tominaga B, Nariai T, Hirakawa K, Kuroiwa T, Takakuda K, Miyairi H: Magnetic resonance imaging and pathologic studies on lateral fluid percussion injury as a model of focal brain injury in rats. Bulletin of Tokyo Medical & Dental UOJversity 1996; 43: 53-66 34. Brown MS, Kornfeld M, Mun-Bryce S, Sibbitt RR, Rosenberg GA: Comparison of magnetic resonance imaging and histology in collagenase-induced hemorrhage in the rat. Journal of Neuroimagins 1995; 5: 23-33 . Thulborn KR, Sorensen AG, Kowall NW, McKee A, ai A, McKinstry RC, Moore J, Rosen BR, Brady TJ: The role of ferritin and hemosiderin in the MR appearance of cerebral hemorrhage: a histopathologic biochemical study in rats. American Journal of Euroradialogy 1990; 11: 291-297 36. Elger B, Seega J, Brendel R: Magnetic resonance imaging study on the effect of mildmopamil on the size- of intracerebral hemorrhage in rats. Stroke 1994; 25: 1836-1841 37. Weingarten K, Zimmerman RD, Deo-Narine V, Markisz J, Cahill PT, MD Deck: MR imaging of acute intracranial hemorrhage: findings on sequential spin-echo and gradient-echo images in a dog model. American Journal of Neuroradiolosy 1991; 12: 457-467 38. Drabkin DL, Austin JH: Spectrophotometric studies: II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 1935; 112: 51-65 39. Drabkin DL, Austin JH: Spectrophotometric studies. IV. Hemochromogens. J Biol Chem 1935; 112: 89-104 Example 13: Factor IXa blocked in active site limits microvascular thrombosis and brain damage in attack in mice, without increasing intracerebral hemorrhage Restore The clinical dilemma when performing the attack treatment is that the agents which restore vascular patency increase the risk of intracerebral hemorrhage. Factor IXa blocked in its active site (IXa), formed from factor IXa purified by dansylation of its active site, competes with the native factor IXa to inhibit the assembly of factor IXa in the intrinsic factor X activation complex. When pre-treated with factor IXai, mice subjected to focal cerebral ischemia and reperfusion demonstrate microvascular fibrin and reduced platelet deposition, increased cerebral perfusion and significantly smaller cerebral infarcts compared to vehicle-treated controls. Brain protection mediated by factor IXai is dose dependent, and is not associated with intracerebral hemorrhage at therapeutically effective doses, and can be observed even when factor IXai is administered after the onset of cerebral ischemia. The administration of factor IXai represents a new strategy to treat an evolving attack without increasing the risk of intracerebral hemorrhage.

Introduction Timely restoration of blood flow to the ischemic brain represents the paradigm of current treatment for an acute attack.13"Administration of a thrombolytic agent, even when delivered under optimal conditions, may not produce this desired clinical outcome. Frequency fails to return to preischemic levels (postischemic hyperfusion), which suggests that ischemic damage is not only caused by the original occlusion, but there is also an element of microcirculatory failure.Also, acute attack thrombolysis is associated with a risk increased intracerebral hemorrhage (ICH) 1"4, indicating that there remains a clear need to identify new agents which can promote reperfusion without increasing the risk of ICH. After an ischemic event, the vascular wall is modified from its resting state, anti-adhesive, antithrombotic, to one in which adhesion of leukocytes and thrombosis is promoted. In an acute attack, the active recruitment of leukocytes by adhesion receptors expressed in the ipsilateral microvasculature, such as ICA. -I5 and P-selectina6, potentiates post-ischemic hypoperfusion. However, in experiments with deletion mutant mice for each of these genes show that even in their absence, the postischemic cerebral blood flow (CBF) returns only partially to the baseline, suggesting the existence of additional mechanisms responsible for the lack of post-ischemic cerebrovascular reflux. To explore this possibility, a first set of experiments was designed to test the hypothesis that local thrombosis occurs at the level of the microvasculature (distal to the site of primary occlusion) in the attack. To determine the harmful consequences of microvascular thrombosis in the attack, the second set of experiments tested the hypothesis that selective blocking of the intrinsic pathway of coagulation could limit microvascular thrombosis, and thus protect the brain in attack. The strategy of selective inhibition of the intrinsic pathway of coagulation was chosen because it is the main responsible of intravascular thrombosis. Heparin, hirudin and fibrinolytic agents interfere with the final common pathway of coagulation to inhibit the formation or accelerate fibrin lysis, and therefore increase the propensity for ICH. We hypothesized that selective blockade of the activation complex assembly IXa / VIIIa / X may provide a novel mechanism to limit intravascular thrombosis while preserving extravascular hemostasis mechanisms via extrinsic clotting / tissue factor which may be critical in infarcted brain tissue or adjacent regions where small vessels are susceptible and undergo rupture. We used a novel strategy in which competitive factor IXa inhibitor (IXa blocked at the active site, or IXai) was administered to mice subjected to attack to test the hypothesis that the post-attack outcome can be improved without increasing ICH.

Methods Model d = attack an ra -_- Ón: Transient focal cerebral ischemia was induced in mice by intraluminal occlusion of the middle cerebral artery (45 minutes) and reperfusion (22 h) as previously reported7. Serial measurements of relative cerebral blood flow (CBF) were recorded via laser Doppler fluorometry7, and infarct volumes (ipsilateral hemisphere%) were determined by planimetric / volumetric analysis of serial brain sections stained with triphenyltetrazolium chloride (TTC). 7) Platelet studies as QQ indium: The platelet accumulation was determined using platelets labeled with xllindium, harvested and prepared as previously described8. Immediately prior to surgery, mice were administered 5 x 106 of platelets labeled with? X? Ln intravenously. Deposition was quantified after 24 hours by means of ipsilateral cpm / contralateral cpm.

Immunoblotting / Fibrin immunostaining: Post-slaughter fibrin accumulation (in fully heparinized animals) was measured using immunoblotting / immunostaining procedures which have been recently described and validated9. Because fibrin is extremely insoluble, extracts of brain tissue were prepared by digestion with plasmin, and then applied to a standard SDS-polyacrylamide gel for electrophoresis, followed by immunoblotting using rabbit anti-human or polyclonal antibody prepared to gamma-gamma chain dimers present in cross-linked fibrin which can detect mouse fibrin, with relatively small cross-activity with fibrinogen10. Fibrin accumulation was reported as an ipsilateral to contralateral ratio. In additional experiments, brains were embedded in paraffin, cut and immunostained using the same anti-fibrin antibody.

Spectrophotometric hemoglobin assay and visual rating of ICH; ICH was quantified by an assay based on spectrophotometry, which has been developed and validated11,12. Briefly, the mouse brains were homogenized, sonicated, centrifuged and the methaemoglobin in the supernatants was converted to cyanometahemoglobin (using the Drabkin reagent), whose concentration was determined by measuring the D.O. at 550 nm against a standard curve generated with known amounts of hemoglobin. A visual qualification of ICH was performed in coronal sections in series of 1 mm by an observer who does not know the protocol, based on the maximum diameter of hemorrhage observed in any of the sections [ICH score 0, without hemorrhage; 1, < 1 mm; 2, 1-2 mm; 3, > 2-3 mm; 4, > 3 mm].

Preparation of factor IXai13: Factor IXai was prepared by selectively modifying the active site histidine residue in factor IXa, using dansyl-glu-gly-arg-chloromethyl ketone. Proplex was applied to a preparative column containing monoclonal antibody for calcium-dependent factor IX, immobilized. The column was washed, eluted with buffer containing EDTA and then factor IX was activated in the eluate (confirmed as a single band in SDS-PAGE) when factor IXa was applied (incubated in the presence of CaCl 2) - The purified factor IXa was it reacted with a 100-fold molar excess of dansyl-glu-gly-arg-chloromethyl ketone and the mixture was dialyzed. The final product (IXai) lacking procoagulant activity, migrates identically to IXa in the SDS-PAGE analysis. This material (Factor IXai) is then used for subsequent experiments on filtration (0.2 μm) and chromatography on DeToxi-gel columns, to remove any traces of endotoxin contamination (in sample aliquots, there were no detectable lipopolysaccharides). Subsequently, IXai was frozen in aliquots at -80 ° C until its use. For those experiments in which IXai was used, it was administered as a single intravenous bolus at the indicated times and at the indicated doses.

Results To create an attack in a mouse model, a suture was introduced into the cerebral vasculature so that it occluded the right medial cerebral artery orifice, returning to irrigated ischemic territory. When the suture was removed after a period of 45 minutes of occlusion, a reperfused attack model was generated; the mice treated in this way demonstrated focal neurological deficits as well as clearly defined areas of cerebral infarction. Because the occlusion suture does not advance beyond the main vascular tributary (the middle cerebral artery), this model provides an excellent opportunity to investigate "downstream" events that occur within the cerebral microvasculature in response to the period of interrupted blood flow. Using this model, the role of microvascular thrombosis was investigated as follows. To demonstrate that platelet-rich thrombotic foci occur within the ischemic cerebral hemisphere, mice were administered X11ln labeled platelets immediately prior to the introduction of the intraluminal occlusion suture to monitor their deposition during the period after cerebral ischaemia and reperfusion. In animals not subjected to a surgical procedure to generate an attack, the presence of platelets is approximately equal between the right and left hemispheres, as expected [Figure 40A, left bar]. However, when animals are subjected to attack (and receive only vehicle for control of subsequent experiments), radiolabeled platelets accumulate preferentially in the ischemic hemisphere (ipsilateral) compared to a significantly lower deposition in the contralateral (nonischemic) hemisphere. [Figure 40A, middle bar] These data support the presentation of platelet-rich thrombi in the ischemic territory When factor IXai is administered to animals before the introduction of the intraluminal occlusion suture, there is a significant reduction in platelet accumulation radiolabeled in the ipsilateral hemisphere [Figure 40A, right bar] Another line of evidence also supports the presentation of microvascular thrombosis in the attack.This data comes from the immunodetection of fibrin, using an antibody directed against a neoepitope in the gamma-chain dimer -Grid of cross-linked fibrin. References demonstrate a band of increased intensity in the ipsilateral (right) hemisphere of vehicle treated animals subjected to focal cerebral ischaemia and reperfusion [Figure 40B, "vehicle"]. In animals treated with JXai factor (300 μg / kg) before the attack, there was no apparent increase in ipsilateral fibrin accumulation [Figure 40B "factor IXai"]. To demonstrate that fibrin accumulation is due to intravascular fibrin deposition (rather than due to non-specific permeability changes and exposure to the subendothelial matrix), fibrin immunostaining clearly localizes the increased fibrin in the lumen of intracerebral microvessels ipsilateral [Figure 40C]. To investigate whether factor IXai can limit intracerebral thrombosis and restore perfusion, IXai was administered to mice immediately before the attack (300 μg / kg). These experiments demonstrated a reduction in both the accumulation of X11ln platelets in the ipsilateral hemisphere [Figure 41A] as well as a decreased evidence of intravascular fibrin by immunostaining. In addition, there is a significant increase in CBF at 24 hours, suggesting the restoration of microvascular patency by factor IXai [Figure 41A]. The clinical relevance of this observation is underlined by the ability of factor IXai to reduce cerebral infarct volumes [Figure 4IB]. These beneficial effects of factor IXai are dose-dependent, and the optimal dose is 600 μg / kg [Figure 41C]. Because the development of ICH is a major concern with any anticoagulant strategy in the establishment of an attack, the effect of IXai on ICH was measured using our newly validated spectrophotometric method to quantify ICH11-12. These data indicate that at the lowest (and most effective) doses there is no significant increase in ICH [Figure 42A]. At the highest dose tested (1200 μg / kg) there is an increase in ICH, which was corroborated by a semiquantitative visual qualification method which we have also recently reported [Figure 42B] 11,12. Because therapies aimed at improving the after-outcome even acute attack must be delivered after clinical presentation, and because fibrin continues to form after an initial ischemic event in an attack, we test whether IXai can be effective when administered after the onset of cerebral ischemia. IXai administered after occlusion of the middle cerebral artery (after removal of the occlusion suture) provides significant brain protection judged by the ability to significantly reduce cerebral infarct volumes compared to vehicle-treated controls [Figure 43].

Discussion The data in these studies demonstrate clear evidence of intravascular thrombus formation (both platelets and fibrin) within the postischemic cerebral microvasculature. The pathophysiological importance of microvascular thrombosis in the attack is underlined by the ability of factor IXai to reduce microvascular thrombosis (the accumulation of both platelets and fibrin is reduced, with a concomitant increase in postischemic CBF) and the improvement in outcome after attack. These powerful antithrombotic actions of factor IXai are probably clinically important in the establishment of the attack, because factor IXai not only reduces infarct volumes in a dose-dependent manner, but also when administered after the onset of the attack . In addition, at clinically relevant doses, treatment with factor IXai does not cause an increase in ICH, performing a selective inhibition of the complex of factor IXa / VIIa / X activation complex with factor IXai as an attractive target for anti-seizure therapy in humans There are numerous reasons why targeted anticoagulant strategies may be an attractive alternative to the current use of thrombolytic agents in the management of acute attack, due to their success with ups and downs in clinical trials. Theoretically, an ideal treatment for acute attack would prevent the formation or induce the dissolution of the fibrin-platelet mesh that causes microvascular thrombosis in the ischemic area without increasing the risk of intracerebral hemorrhage. However, thrombolytic agents which have been studied in clinical trials of acute attack have consistently increased the risk of intracerebral hemorrhage.3 Streptokinase, administered in the first hours (<6) after the onset of the attack, was associated with an increased rate of hemorrhagic transformation (up to 67%); Although there was an increased early mortality, the surviving patients had less residual disability. The administration of tissue type plasminogen activator (tPA) in the following 7 hours (particularly in the next 3 hours) at the beginning of the attack results in increased early mortality and increased rates of hemorrhagic conversion (between 7-20%), although Survivors showed less residual disability. In order to develop improved anticoagulant or thrombolytic therapies, various animal models of attack have been examined. These models generally consist of the administration of coagulated blood in the internal carotid artery followed by administration of a thrombolytic agent. In rats, the administration of tPA in the next two hours to the attack improves cerebral blood flow and reduces the size of infarction up to 77% 14,1S. In a similar embolic attack model in rabbit, tPA was effective in restoring blood flow and reducing the size of the infarct, with occasional occurrence of intracerebral hemorrhage16'17. However, although there are advantages to the immediate dissolution of the clot, these studies (as well as the clinical trials of thrombolytic agents) indicate that there is a concomitant increased risk of intracerebral hemorrhage with this therapeutic approach.

Due to the usually precipitated onset of the ischemic attack, therapy has mainly focused on the lysis of the main fibrinous / atheroembolic residues which occlude a major vascular tributary to the brain. However, as shown by the current work, there is an important component of microvascular thrombosis which occurs downstream from the original occlusion site, which is likely to be of considerable pathophysiological significance for postischemic hypoperfusion (without reflux) and brain damage in a developing attack. These data agree in an excellent way with those that have been previously reported, in which microthrombi have been located topographically in the ischemic region in fresh cerebral infarcts18. The use of an agent which inhibits the assembly of the factor IXa / VIIIa / X activation complex represents a novel approach to limit the thrombosis that occurs within the vascular lumens, without damaging the extravascular hemostasis, the maintenance of which may be critical to avoid ICH. In current studies, treatment with factor IXai reduces the microvascular accumulation of platelets and fibrin, improves postischemic cerebral blood flow, and reduces cerebral stroke infarct volumes in the setting setting, without increasing ICH. The potency of factor IXai as an anticoagulant agent is based on the integral role of activated factor IX in the coagulation cascade. Not only does the strategy of blockade of factor IXa seem to be effective in establishing the attack, but it also seems to be effective to avoid progressive occlusion of the coronary artery induced after initial application of electric current to the left circumflex coronary artery in dogs13. As in those studies, in which factor IXai does not prolong the activated partial thromboplastin time (APTT (224), in the mouse model, the administration of factor IXai at the therapeutically effective dose of 300 μg / kg similarly does not significantly altered the protime or APTT [13.4 ± 0.7 and 7.9 ± 8.9 versus 12.1 ± 0.7 and 70.6 + 8.9 for PT and APTT of mice treated with IXai (n = 7) and treated with vehicles (n = 4), respectively, P = NS] The data which show that IXai administered after the onset of the attack is effective leads to another interesting hypothesis, that thrombus formation represents a dynamic equilibrium between the process of advancing thrombosis and fibrinolysis that is Even under normal (non-ischemic) establishments, it has been shown that this dynamic equilibrium occurs in man.19 The data in current studies, which show that factor IXai is effective even when After the access of the attack, the minister suggests that this strategy restores the dynamic equilibrium, which is displaced after cerebral ischemia in favor of thrombosis, and returns it to a vascular wall phenotype more at rest (antithrombotic).

As a final consideration, even if thrombolysis successfully removes major occlusion thrombi and / or anticoagulant strategies are effective in limiting progressive microcirculatory thrombosis, blood flow usually fails to return to pre-ischemic levels. This is exemplified by data in the current study, in which CBF is significantly improved by a factor IXai (which limits the accumulation of fibrin / platelets), CBF has not yet returned to pre-ischemic levels. These data support the existence of multiple effector mechanisms for postischemic cerebral hypoperfusion that include accumulation; postischemic neutrophils and consequent microvascular tamponade, with expression of P-selectin and ICAM-1 by the cerebral microvascular endothelial cells that are particularly relevant in this regard5,6. When observed from the perspective of the expression of leukocyte adhesion receptoreven when these adhesion receptors are absent, CBF levels are improved post-attack compared to controls but do not return to pre-ischemic levels. Taken together, these data suggest that microvascular thrombosis and leukocyte adhesion together contribute to postischemic cerebral hypoperfusion. In summary, the administration of a competitive inhibitor of factor IXa, factor IXa blocked in its active site, represents a novel therapy for the treatment of the attack. This therapy not only reduces microvascular thrombosis, improves postischemic cerebral blood flow and reduces damage to brain tissue after attack, but it can also do so if it is provided after the onset of cerebral ischemia and without increasing the risk of ICH. This combination of beneficial properties and relatively low downside risks of hemorrhagic transformation makes this approach extremely attractive for additional testing and potential clinical trials in human attack.

References 1. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group: Tissue plasminogen activator for acute ischemic stroke. New Ensl J Med 1995; 333: 1581-1587 2. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH, Hennerici M, for the ECASS Study Group: Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke J A M A. 1995; 274 (13): 1017-1025 3. del Zoppo GJ: Acute stroke - sn the threshold of a therapy. N Ensl J Med 1995; 333 (13): 1632 -1633 4. Hommel M, Cornu C, Boutitie F, Boissel JP, The MultiCenter Acute Stroke Trial - Europe Study Group: Thrombolytic therapy with streptokinase in acute ischemic stroke. N EnGl J Med 1996; 335: 145-150 5 5. Connolly ESJr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutiérrez-Ramos JC, Pinsky DJ: Cerebral protection in homozygous nuil ICAM-l mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis 0 of stroke. J Clin Invest 1996; 97: 209-216 6. Exacerbation of cerebral injury in mice which express the P-selectin gene: identification of P-selectin blockade as a new target for the treatment of stroke. Example 10 Hereinabove 5 7. Connolly ESJr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ: Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurg 1996; 38 (3): 523-532 0 8. Naka Y, Chowdhury NC, Liao H, Roy DK, Oz MC, Micheler RE, Pinsky DJ: Enhanced preservation of orthotopically transplanted rat lungs by nitroglycerin but not hydralazine: requirement for graft vascular homeostasis beyond harvest vasodilation. Circ Res 1995; 76: 900-906 9. Lawson CA, Yan SD, Yan SF, Liao H, Chen G, Sobel J, Kisiel W, Stern DM, Pinsky DJ: Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation . Journal of Clinical Investigation 1997; 99: 1729-1738 5 10. Lahiri B, Koehn JA, Canfield RE, Birken S, Lewis J: Development of an immunoassay for the COOH-terminal region of the gamma chains if human fibrin. Thromb Res 1981; 23: 103-112 0 11. Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice. Example 12 Hereinabove 12. Choudhri TF, Hoh BL, Solomon RA, Connolly ES, Pinsky DJ: Spectrophotometric hemoglobin assay: A new method to quantify; 5 experimental murine intracerebral hemorrhage and its potentiation by tissue plasminogen activator. Annual Meeting Joint Section on Cerebrovascular Surgery 1997 13. Benedict CR, Ryan J, Wolitzky B, Ramos R, Gerlach M, Tijburg 0 P, Stern D: Active site-blocked Factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting • extravascular coagulation in a canine thrombosis model. J Clin lH3 ££ St; 991; 88: 1760-1765 14. Papadopoulos SM, Chandler WF, Salamat MS, Topol EJ, Sackellares JC: Recombinant human tissue-type plasminogen activator therapy in acute thromboembolic stroke. J Neurosurg 1987; 67: 394-398 5 15. Overgaard K, Sereghy T, Pedersen H, Boysen G: Neuroprotection with NBQX and thrombolysis with rt-PA in rat embolic stroke. Neurol Res 1993; 15: 344-349 0 16. Crankcase LP, Guthkelch AN, Orozco J, Temeltas O- Influence of tissue plasminogen activator and heparin on cerebral ischemia in a rabbit model. Stroke 1992; 23: 883-888 17. Phillips DA, Fisher M, Davis MA, Smith TW, Pang RL: Delayed 5 treatment with a t-PA analogue and streptokinase in a rabbit embolic stroke model. Stroke 1990; 21: 602-605 18. Heye N, Paetzold C, Steinberg R, Cervos-Navarro J: The topography of m-crothrombi in ischemic brain infarct. Acta 0 Neuroloaica Scandinavica 1992; 86: 450-454 - 19. Nossel HL: Relative proteolysis of the fibrinogen B beta chain by thrombin and plasmin as a determinant of thrombosis. Nature 1981; 291: 155-167

Claims (45)

1. A method for treating an ischemic disorder in a subject, characterized in that it comprises administering to the subject a pharmaceutically acceptable form of a selectin antagonist in an amount sufficient for a period of time sufficient to prevent the accumulation of white blood cells so that the Ischemic disorder in the subject.

2. The method according to claim 1, characterized in that the selectin antagonist comprises a mimetic peptide, a nucleic acid molecule, a ribosome, a polypeptide, a small molecule. a carbohydrate molecule, a monosaccharide, an oligosaccharide or an antibody.

3. The method according to claim 2, characterized in that the antibody is a selectin antibody.

4. The method according to claim 3, characterized in that the antibody is a polyclonal antibody or a monoclonal antibody.

5. The method according to claim 1, characterized in that the selectin antagonist comprises nitroglycerin or an agent which stimulates the oxide-2'68-nitric pathway, 3 ', 5'-cyclic adenosine monophosphate, cyclic AMP or cyclic GPM. .

6. The method according to claim 1, characterized in that the pharmaceutically acceptable form comprises a selectin antagonist and a pharmaceutically acceptable carrier.

7. The method according to claim 6, characterized in that the carrier comprises an aerosol, intravenous, oral or topical carrier.

8. The method according to claim 1, characterized in that the white blood cell is a neutrophil, a monocyte or a platelet.

9. The method according to claim 1, characterized in that the subject is a mammal.

10. The method according to claim 9, characterized in that the mammal is a human.

11. The method according to claim 1, characterized in that the ischemic disorder comprises a peripheral vascular disorder, pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, unstable angina, reversible ischemic neurological deficit, falsiform cell anemia or a disorder of attack.

12. The method according to claim 1, characterized in that the subject undergoes cardiac surgery, pulmonary surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery or organ transplant surgery.

13. The method according to claim 12, characterized in that the organ transplant surgery comprises heart, lung, pancreas or liver transplant surgery.

14. The method according to claim 1, characterized in that the selectin is a P-selectin, an E-selectin or an L-selectin.

15. A method for treating an ischemic disorder in a subject, characterized in that it comprises administering to the subject gaseous carbon monoxide in an amount sufficient for a sufficient period of time to thereby treat the ischemic disorder in the subject.

16. The method according to claim 15, characterized in that the administration is via inhalation or extracorporeal exposure.

17. The method according to claim 15, characterized in that the amount comprises from about 0.001% carbon monoxide in the air to about 2% carbon monoxide in an inert gas.

18. The method according to claim 17, characterized in that the inert gas comprises air, oxygen, argon 0 nitrogen.

19. The method according to claim 15, characterized in that the amount comprises 0.1% carbon monoxide in air.

20. The method according to claim 15, characterized in that the period of time comprises from about 1 day before surgery to approximately 1 day after surgery

21. The method according to claim 15, characterized in that the period of time comprises from approximately 12 hours before surgery to approximately 12 hours after surgery.

22. The method according to claim 15, characterized in that the time period comprises from approximately 12 hours before surgery to approximately 1 hour after surgery.

23. The method according to claim 15, characterized in that the time period comprises from about 1 hour before surgery to about 1 hour after surgery.

24. The method according to claim 15, characterized in that the subject is a mammal.

25. The method according to claim 24, characterized in that the mammal is a human.

26. The method according to claim 15, characterized in that the ischemic disorder comprises a peripheral vascular disorder, pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, unstable angina, reversible ischemic neurological deficit, falsiform cell anemia or a disorder of attack.

27. The method according to claim 15, characterized in that the subject undergoes cardiac surgery, pulmonary surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery or organ transplant surgery.

28. The method according to claim 27, characterized in that the organ transplant surgery comprises heart, lung, pancreas or liver transplant surgery.

29. A method for treating an ischemic disorder in a subject, characterized in that it comprises administering to the subject a pharmaceutically acceptable form of inactivated factor IX in a sufficient amount for a period of time sufficient to inhibit coagulation so as to treat the ischemic disorder in the subject . -

30. The method according to claim 29, characterized in that the amount comprises from about 75 μg / kg to about 550 μg / kg.

31. The method according to claim 29, characterized in that the amount comprises 300 μg / kg.

32. The method according to claim 29, characterized in that the pharmaceutically acceptable form comprises inactivated factor IX and a pharmaceutically acceptable carrier.

33. The method according to claim 32, characterized in that the carrier comprises an aerosol, intravenous, oral or topical carrier.

34. The method according to claim 29, characterized in that the subject is a mammal.

35. The method according to claim 34, characterized in that the mammal is a human.

36. The method according to claim 29, characterized in that the ischemic disorder comprises a peripheral vascular disorder, pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, unstable angina, reversible ischemic neurological deficit, falsiform cell anemia or a disorder of attack.

37. The method according to claim 29, characterized in that the subject undergoes cardiac surgery, pulmonary surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery or organ transplant surgery.

38. The method according to claim 37, characterized in that the surgery of organ transplantation comprises heart, lung, pancreas or liver transplant surgery.

39. A method for identifying a compound that is capable of improving an ischemic disorder in a subject, characterized in that it comprises: a) administering the compound to an animal, which is an animal model of attack; b) measuring the post-attack result in the animal, and c) comparing the post-attack result in step (b) with that of the animal model of attack in the absence of the compound so as to identify a compound capable of improving an ischemic disorder in a subject.

40. The method according to claim 39, characterized in that the animal model of attack comprises a mouse model of focal cerebral ischaemia and reperfusion.

41. The method according to claim 39, characterized in that the result caused by a sudden attack is measured by physical examination, magnetic resonance imaging, laser Doppler flowmetry, trifeni-Ltetrazolium chloride staining, chemical determination of neurological deficiency, exploration of computed tomography or cerebral cortical blood flow.

42. The method according to claim 39, characterized in that the compound comprises a P-selectin antagonist.

43. A method for identifying a compound that is capable of preventing the accumulation of white blood cells in a subject, characterized in that it comprises: a) administering the compound to an animal, which is an animal model of attack; b) measure the attack result in the animal, and c) compare the result after the attack in stage __ (b) with that of the animal model of attack in the absence of the compound so as to identify a compound capable of improving an ischemic disorder in a subject.

44. The method according to claim 43, characterized in that the white blood cell is a neutrophil, a monocyte or a platelet.

45. The method according to claim 43, characterized in that the compound is an inhibitor of P-selectin, a monocyte inhibitor, a platelet inhibitor or a neutrophil inhibitor.