RU2642961C1 - Method for prevention of brain ischemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to experimental pharmacology and neurology, and can be used to prevent ischemic brain conditions. Method includes a preliminary, prior to modeling the ischemic state of the brain, administration of a preconditioning agent to the laboratory animal. As such an agent, tadalafil is used at a dose of 1 mg/kg, which is injected once intragastrically through the probe. After 60 minutes, a simulation of cerebral ischemia is performed by coagulation of two vertebral arteries and temporary occlusion of two common carotid arteries.

EFFECT: method provides a frank correction of cerebral ischemia in an animal during the experiment due to the induction of tadalafil mechanisms of metabolic adaptation and the realization of preconditioning effect.

1 cl, 4 dwg, 1 ex, 6 tbl

Description

The invention relates to medicine, in particular to experimental pharmacology and neurology.

According to well-known literature, ischemic stroke or cerebral infarction is the main pathology among severe forms of vascular brain damage. In Russia, this pathology takes first place as the cause of disability, and second place in the structure of general mortality (Melnikova E.V. Neuroprotection for cerebral ischemia [Text] / E.V. Melnikova, A.A. Shmonin // Farmateka. - 2012. - No. 9. - S. 36-42).

Prevention and correction of cerebral ischemia is theoretically possible using pharmacological preconditioning, the essence of which is the activation of endogenous defense mechanisms that reduce the degree of damage during a subsequent prolonged ischemic episode (Dolzhikova I.N. Distant and pharmacological preconditioning using erythropoietin and tadalafil in experimental renal ischemia : the dissertation of the candidate of biological sciences: 03/14/06 / IN Dolzhikova. - Belgorod). Promising for the study of the preconditioning effect of cerebral ischemia is a phosphodiesterase-5 inhibitor, tadalafil.

A technical solution is known "Means for the prevention or treatment of acute and chronic cerebrovascular disorders, the use and method of treatment" (RU No. 2468813, publ. 10.12.2012), which contains a pharmaceutical composition comprising methionyl-glutamyl-histidyl-phenylalanil as active components -prolyl-glycyl-proline and choline alfoscerate in the following ratio of components, in wt.%: methionyl-glutamyl-histidyl-phenylalanyl-prolyl-glycyl-proline 0.01-2.00; choline alfoscerate 98.00-99.99. Also disclosed is the use of said pharmaceutical composition for the manufacture of an agent and method of treatment.

The main disadvantage of this method is that the model of cerebral ischemia is not specific for many types of studies related to circulatory disorders of the brain. In addition, the use of this McGraw neurological deficit assessment scale is not always informative. Due to the wide variability of the anatomy of the arterial bed, pools, internal and external carotid arteries, it is necessary to exclude the possibility of collateral nutrition of the brain in the experiment.

Known technical solution "A method for the treatment of cerebral ischemia" (RU No. 2281765, publ. 08/20/2006). The essence of the invention lies in the fact that in a method of treating cerebral ischemia in its model in a mammal, the amounts of succinic bis [(2-hydroxyethyl) -N, N, N-trimethylaminium] are administered to the animal. The effect of reducing the death of brain neurons with the introduction of succinic bis [(2-hydroxyethyl) -N, N, N-trimethylaminium] was evaluated in a rat model with chronic cerebral ischemia caused by ligation of the carotid arteries. The nature and mechanism of damage to brain neurons in this model corresponds to that observed in ischemic stroke. The death of brain neurons was quantified by 1 H NMR in vivo by reducing the level of the neuronal marker N-acetyl aspartate (NAA) in the brain. NAA is a well-known non-invasive marker for neuronal integrity. NAA is found exclusively in neurons. A decrease in NAA or in the ratio of NAA to creatine (relative level of NAA) is strongly correlated with the loss of neurons when they are damaged or degenerate. (Demougeot C et al. J Neurochem. 2004 90 (4): 776-83. Roitberg B et al. Neurol Res. 2003 25 (1): 68-78. Schuhmann MU et al. J Neurotrauma. 2003 20 (8) : 725-43). Carotid ligation was performed in Wistar male rats 3 hours before the first injection. The rats were then injected with i.p. for seven days. injections physical R-RA (control), 1 mg / kg or 50 mg / kg of succinic bis [(2-hydroxyethyl) -N, N, N-trimethylaminium]. On day 15 from the time of ligation, the level of NAA in the brain was measured by 1 H NMR in vivo.

The main disadvantage of this method is that 1H NMR in vivo is a very expensive diagnostic method. It is not always possible to carry it out.

The objective of the invention is a method for the prevention of cerebral ischemia, including a phosphodiesterase-5 inhibitor, tadalafil, as a preconditioning agent.

The objective is achieved in that in a method comprising reproducing a four-vessel model of pathology and administering to a laboratory animal a dose of 1 mg / kg of tadalafil, which is administered once intragastrically through a probe as a preconditioning agent 60 minutes before the experiment, and after 60 minutes, brain ischemia is simulated by coagulation of two vertebral arteries and temporary occlusion of two common carotid arteries.

The mechanism of the cerebroprotective action of tadalafil, like IFDE-5, consists in influencing the pathway of NO-cGMP protein kinase G. When PK-G is activated, phosphorylation of K + channels occurs, the membrane is less sensitive to nerve impulses and its hyperpolarization. Then there is a decrease in the concentration of free Ca + 2 cytoplasm. These mechanisms are the basis of the vasodilating effects of nitric oxide. The main role in this mechanism is played by phosphodiesterase-5. It is it that reduces the accumulation of cGMP (Dolzhikova I. N. Distant and pharmacological preconditioning using erythropoietin and tadalafil in experimental kidney ischemia: the dissertation of the candidate of biological sciences: March 14, 06 / I. N. Dolzhikova. - Belgorod).

The main advantage of the proposed method is that:

- as a preconditioning agent, tadalafil is administered once at a dose of 1 kg / kg 60 minutes before the experiment intragastrically, due to this, 30 minutes (the first protective window) after administration, ATP-dependent K + channels open, which subsequently leads to an increase in resistance brain tissue, 12-48 hours after the administration of tadalafil, 2 protective windows open in which the preconditioning effect is realized, including through the action of nitric oxide (NO) and the further activation of metabolic adaptation mechanisms;

- the effectiveness of the drug is evaluated comprehensively: the neurological deficit of the animal, the behavioral status, the level of two specific markers of brain damage S100b and NSE are examined, histological and morphometric studies are carried out.

- using the electrophysiological method, a correctly executed model of pathology is monitored.

The method leads to a pronounced correction of cerebral ischemia, since tadalafil in this dose opens ATP-dependent K + channels and further induces metabolic adaptation mechanisms, due to which the preconditioning effect is realized.

 The method is carried out as follows.

The study was performed on 50 sexually mature Wistar male rats weighing 230-260 g. The experimental animals were divided into 5 groups: 1) intact (n = 10), 2) false-operated, 3) group with a two-vessel pathology (n = 10), 4 ) a group with four-vascular pathology (n = 10), 5) a group with four-vascular pathology and correction of tadalafil (1 mg / kg, ip), (n = 10). The animals were kept in standard vivarium conditions of the National Research University "BelSU" with free access to food and water. The keeping of animals and the setting up of the experiment was carried out in accordance with the requirements of orders No. 1179 of the Ministry of Health of the USSR of 10/11/1983 and No. 267 of the Russian Federation of 06/19/2003, as well as the international rules "Guide for the Care and of Laboratory Animals".

Group 3 animals were simulated with bovascular local ischemia: blood flow in the common carotid arteries was blocked for 4 minutes. Rats of group 4 were simulated with total four-vessel cerebral ischemia with an ischemic period of 4 minutes. Group 5 animals, 60 minutes before the simulation of total ischemia, were administered an intragastric phosphodiesterase-5 inhibitor, tadalafil, at a dose of 1 mg / kg. The animals were anesthetized with the preparation Zoletil 100 60 mg / ml and chloral hydrate 150 mg / ml. The control of the correctly executed technique was carried out by registering the animals with an electroencephalogram. Animals were taken out of the experiment 72 hours after it began, by overdose of anesthetic drugs.

The severity of the anti-ischemic effect was judged by the severity of neurological deficit, behavioral status, the level of specific markers of brain damage S100b and NSE, the analysis of the histological and morphometric state of brain sections.

When statistical data processing is calculated, the average value, the standard deviation. Differences are considered significant at p <0.05.

EXAMPLE OF SPECIFIC PERFORMANCE

The neurological deficit in animals was assessed on days 1, 3, 7, and 14 after modeling the pathology using a McGraw point scale in the modification of I.V. Gannushkina (1996) (according to the average score in the group) (Table 1), a group of intact animals was taken for control, which did not statistically differ from the false-operated ones:

Table 1

Dynamics of the severity of neurological disorders in the studied groups according to McGraw modified by I.V. Gannushkina (1996) (according to the average score in the group) (M ± m; n = 10).

Period Groups intact LO 2-sos. 4 minute igm 4-sos. 4 minute igm IFE-5 + IGM 1 day 0 0 0.2 ± 0.08 * 2.05 ± 0.49 * 0.8 ± 0.21 * 3 days 0 0 0 1 ± 0.42 * 0.75 ± 0.23 * 7 days 0 0 0 1 ± 0.44 * 0.4 ± 0.15 * 14 days 0 0 0 0.4 ± 0.44 * 0.2 ± 0.13 *

Note: here and everywhere further * - p <0.05, # - p> 0.05 in relation to the control group of rats.

In rats after 2-vascular cerebral ischemia lasting 4 minutes, a slight neurological deficit was observed on the first day. This was manifested in lethargy of limbs and slow movement and did not statistically differ from the group of falsely operated animals (p> 0.05). On the 3rd, 7th and 14th day, neurological symptoms were absent. On the first day after the simulation of IGM, the neurological deficit in rats with a 4-vascular model and an ischemic period of 4 minutes was of moderate severity (p <0.05): lethargy and slow movement were observed in 100% of the rats, and unilateral in 50% of the rats half-eye of the right eye, 10% bilateral half-heart disease, 20% of rats showed paralysis of the hind left paw, tremor. The lethargy and slow movement by 3 days after modeling the pathology disappeared. On days 3, 7, and 14, paralysis of the hind left limb and semiptosis of the right eye persisted. Neurological deficit, in a group of rats against the background of correction by pharmacological preconditioning of tadalafil, was with milder symptoms compared to a group of animals with IHM without drug administration (p <0.05): lethargy and slow movement were observed in 40% of rats, in 60% one-sided semiptosis of the right eye. The lethargy and slow movement in animals by 3 days after modeling the pathology disappeared. On the 3rd, 7th, 14th day, semiptosis of the right eye persisted. Compared with the control group, the animals of this group were significantly more active even on the first day after modeling the pathology.

The behavioral status of rats was assessed using an elevated cruciform maze test. Parameters such as the total time spent in the dark sleeve, the light one, and also the number of racks and hangings were evaluated (Table 2):

table 2

Assessment of the behavioral activity of animals in the test "Elevated cruciform labyrinth" (M ± m; n = 10).

Criterion Groups Intact
ny LO 2-sos. 4 minute igm 4-sos. 4 minute igm IFE-5 + IGM Dark sleeve, t 154.4 ± 4.5 156.2 ± 4 # 158.1 ± 4.1 # 170 ± 1.5 * 159 ± 2.3 * Light sleeve, t 26.6 ± 4.3 23.8 ± 4 # 22.9 ± 3.9 # 10 ± 1.5 * 20.7 ± 2.1 * Racks 10.5 ± 0.9 10.1 ± 0.8 # 7.9 ± 0.6 * 4.3 ± 0.4 * 7.7 ± 0.8 * Hanging pieces 4.8 ± 0.7 4.4 ± 0.7 # 3.3 ± 0.3 * 1.6 ± 0.3 * 3.6 ± 0.6 *

In the 2-vascular 4-minute model of pathology in animals, a slight decrease in horizontal activity, vertical activity was observed, which was manifested in a decrease in the number of racks and hangings. Orientation-research behavior decreased, statistically significant (p <0.05). In the group with 4-vascular 4-minute IHM, animals showed a significant decrease in horizontal activity, an increase in the time spent in the dark arms. The decrease in vertical activity was manifested in a decrease in racks, hangings by about 60%. The minimum approximate research behavior is retained (p <0.05). In the behavioral test of PKL, a group of rats with IHM and correction of tadalafil was more active in comparison with the control group. This was manifested in an increase in horizontal and vertical activity (Table 2), which was reflected in more racks and hangings (p <0.05). Indicatively - research behavior is reduced, but not significantly.

The motor activity of animals was assessed using an actimetry test with infrared monitoring of IR Actimeter activity (Table 3).

Table 3

Assessment of the behavioral activity of animals in the actimetry test (M ± m; n = 10).

Criteria Groups intact LO 2-sos. 4 minute igm 4-sos. 4 minute igm IFE-5 + IGM Total activity 939.9 ±
40.5 851 ± 49 # 595.7 ±
40.17 * 406.9 ±
59 * 553.3 ± 28.26 * Stereotypes of movement 77.5 ±
4.04 72.4 ± 5.34 # 42 ± 2.83 * 29.0 ±
2.05 * 35.7 ± 2.31 * Top speed 42.1 ±
2.41 36.6 ± 1.80 # 27.74 ±
1.78 * 22.01 ±
2.09 * 24.89 ± 1.85 * Total distance 2261.8 ±
104.55 1797.47 ±
103.20 # 1290.52 ±
93.09 * 772.3 ±
52.27 * 1190.61 ±
105.28 * Time relax 89.66 ±
7.54 111.71 ±
10.37 # 155.41 ±
8.87 * 196.39 ±
7.21 * 159.5 ± 8.61 *

When assessing the locomotor activity of animals in an actimetry test with infrared monitoring of IR Actimeter activity, it was found that the activity of false-operated rats decreased compared to intact rats, but not statistically significantly (p> 0.05). With an increase in the time of the ischemic period in the experiment, the activity of rats decreases: the total activity, the number of stereotypical movements, the maximum speed, and the total distance decrease. Rest time with increasing time of cerebral ischemia increases (Table 3). When comparing the motor activity of animals of the control group with the tadalafil group in the actimetry test on an IR Actimeter IR activity monitor, it was found that the activity of rats with preliminary administration of the drug is higher (p <0.05).

Next, assessed the level of specific markers of brain damage S100b and NSE in these groups (Table 4).

Table 4

The concentration of markers of brain damage in animal plasma on the 3rd day (M ± m; n = 10).

intact LO 2-sos. 4 minute igm 4-sos. 4 minute igm IFE-5 + IGM S100b Nse S100b Nse S100b Nse S100b Nse S100b Nse 0.77 ± 0.21 0.365 ± 0.36 0.95 ± 0.18 0.38 ± 0.23 1.498 ± 1.04 0.434 ± 0.29 2.03 ± 0.33 * 0.547 ± 0.22 * 0.585 ± 0.30 # 0.212 ± 0.31 #

When analyzing the levels of S100b and NSE in animals of all experimental groups, an increase in the concentration of markers in blood serum was observed. A statistically insignificant (p> 0.05) increase in the concentration of markers was observed in falsely operated animals (hypothetically associated with surgical intervention and the effect of general anesthesia on the body), with a two-vascular 4-minute IHM. A statistically significant (p <0.05) increase in concentration was in the group with four-vascular 4-minute and IHM. When analyzing the levels of S100b and NSE in animals of the group with IHM with preliminary correction of tadalafil, a decrease in the concentration of damage markers was observed even below the level of the control group (p> 0.05).

Under review microscopy in intact animals, the results obtained were consistent with descriptions of the cytoarchitectonic features of the frontal lobe and hippocampus. The neurons were predominantly pyramidal, rounded or polygonal in shape, with large rounded nuclei and a fine-grained cytoplasm. In many neurons, the basophilic substance of the cytoplasm had the appearance of large clumps located peripherally. In some neurons, one or two centrally located nucleoli were clearly defined (Fig. 1), where: A is the frontal lobe of the intact rat, X 100, okr. hematoxylin + eosin; B - hippocampus of an intact rat, X 400, okr. hematoxylin + eosin.

The frontal lobe region was characterized by a low location density, and the hippocampal region CA1 was of high density, medium-sized neurons. Morphometric changes in neurons in the two-vessel model of ischemia tended to increase and were characterized by an increase in the maximum and minimum diameters of pericarions, their perimeter and area. The diameter, area and perimeter of the nuclei of neurons also increased. The total number of hyperchromic neurons was 36.7%, and bipolar nuclei - 16.7% (Table 5). Changes in similar indicators in the CA1 region of the hippocampus were less significant, however, a general tendency to their increase was observed (Table 6). Qualitative changes were characterized by an increase in the number of hyperchromic neurons, chromatolysis, and moderate disorganization of layers in the CA1 region. The cell bodies lost their sharpness, deformed. Nuclear changes were polymorphic and were manifested both by swelling and pycnosis of individual nuclei. In some neurons, a displacement of the nucleus to the periphery of the pericarion was observed. An increase in the number of binucleate neurons in combination with a general increase in the area of the nucleus and pericarion is a morphological manifestation of regenerative processes and an increase in their functional activity arising in response to local cerebral ischemia (Fig. 2 - a two-vessel model of cerebral ischemia), where: A is the frontal lobe , okr. hematoxylin + eosin; B - CA1 region of the hippocampus, okr. Nissl thionine, X 400.

In the four-vascular model of ischemia, marked hyperchromia of neurons of the frontal lobe with perivascular and pericellular edema was noted. The shape of the cells was predominantly polygonal, elongated, and nuclei were not determined in many of them. The capillaries are paretically dilated, full-blooded. In the CA1 region of the hippocampus, disorganization of the neuronal layers, chromatolysis, swelling and pyknotic changes in the nuclei were observed, bipolar nucleons were practically not detected (Fig. 3 - four-vascular model of cerebral ischemia), where: A - frontal lobe, X 400, okr. hematoxylin + eosin; B - CA1 area of the hippocampus, X 400, okr. Nissl thionine.

With the introduction of tadalafil at a dose of 1 mg / kg an hour before the simulation of the four-vascular pathology, the morphological changes in neurons were mainly necrobiotic in nature. The number of dead neurons was significantly lower than without tadalafil correction. In the hippocampus, impaired stratification of the layers was moderate (Fig. 4 - four-vascular model of cerebral ischemia with correction of tadalafil), where: A - frontal lobe, X 100, okr. hematoxylin + eosin; B - hippocampus, X 400, okr. hematoxylin + eosin.

A statistical study found that ischemic damage to neurons, and the frontal lobe, and the hippocampus were significantly more pronounced in the group with a four-vessel model of brain ischemia without tadalafil correction. The minimum diameter, perimeter and area of neurons, diameter, perimeter and area of nuclei were significantly different (p <0.05). The number of bipolar nuclei is significantly higher when corrected by tadalafil (p <0.05), which reflects the functional state of the neurons, their greater activity and ability to regenerative and reparative processes. No significant differences were found when measuring the maximum diameter of the nuclei of neurons (p> 0.05).

Thus, in the two-vessel model of rat cerebral ischemia, moderate necrobiotic changes were observed with signs of activation of regenerative processes in both the frontal lobe and hippocampal neurons. In the four-vessel model of ischemia, the damage to most neurons was irreversible, without signs of activation of reparative processes. Correction of ischemic damage with tadalafil showed a statistically significant increase in the resistance of neurons of the frontal lobe and CA1 of the hippocampus to hypoxia and ischemia, a significant decrease in the number of dead neurons and a greater activity of reparative processes.

Table 5

Morphometric characteristic of neurons of the frontal lobes of the brain of rats (M ± m; n = 30).

Options intact 2-vascular IGM 4-vascular IGM IFDE-5 + IGM Max D 12.51 ± 0.29 13.23 ± 0.42 # 13.54 ± 0.63 # 8.57 ± 0.35 # Min d 8.46 ± 0.28 10.41 ± 0.40 * 6.58 ± 0.21 * 5.62 ± 1.51 * Cell perimeter 35.87 ± 0.90 41.58 ± 1.27 * 38.60 ± 1.38 * 23.83 ± 4.46 * S cells 83.33 ± 3.25 120.48 ± 5.81 * 75.45 ± 3.62 * 40.67 ± 1.28 * Core perimeter 21.98 ± 0.89 29.68 ± 1.01 * 23.58 ± 0.65 * 16.38 ± 1.89 * S core 35.08 ± 2.21 58.61 ± 4.21 * 38.90 ± 2.02 * 20.56 ± 0.39 * D Kernels 6.28 ± 0.24 9.15 ± 0.42 * 7.37 ± 0.24 * 5.64 ± 0.11 * D Nucleolus 1.88 ± 0.07 Hypochromic neurons- (46.7%) 2.65 ± 0.50
Hyperchromic- (36.7%)
Bipolar - (16.7%) - 2.40 ± 0.24 Hypochromic neurons- (10%) 1.67 ± 0.18
Hyperchromic- (90%)
Bipolar Hypochromic neurons- (43.4%) 1.63 ± 0.16
Hyperchromic- (43.4%)
Bipolar - (13.2%) 1.26 ± 0.10

Note: * - at (p <0.05), # - at (p> 0.05). For control took gr. intact animals. In the group "IFDE-5 + IGM" - column rats with four-vascular igm.

Table 6

Morphometric characteristics of rat hippocampal neurons (M ± m; n = 30).

Options intact 2-vascular IGM 4-vascular IGM IFDE-5 + IGM Max D 11.85 ± 0.29 13.64 ± 0.16 * 11.38 ± 0.16 # 8.49 ± 0.21 * Min d 8.03 ± 0.28 6.90 ± 0.20 * 6.34 ± 0.20 * 5.70 ± 0.78 * Cell perimeter 33.01 ± 0.80 37.22 ± 0.34 * 30.40 ± 0.34 * 24.30 ± 2.30 * S cells 68.90 ± 2.63 77.14 ± 2.63 * 57.71 ± 1.76 * 38.94 ± 0.81 * Core perimeter 22.66 ± 0.72 27.11 ± 0.47 * 20.86 ± 0.47 * 17.31 ± 1.28 * S core 38.06 ± 2.50 50.41 ± 1.25 * 29.96 ± 1.25 * 20.98 ± 0.26 * D Kernels 6.23 ± 0.26 6.47 ± 0.25 # 7.09 ± 0.25 * 5.56 ± 0.07 * D Nucleolus 2.38 ± 0.07 Hypochromic neurons -
Hyperchromic- (86.7%)
Bipolar - (13.3%) - 2.03 ± 0.11 Hypochromic
Hyperchromic- (90%)
Binucleolar - (10%) - 1.83 ± 0.23 Hypochromic neurons -
Hyperchromic- (90%)
Binucleolar - (10%) - 1.83 ± 0.23

Note: * - at (p <0.05), # - at (p> 0.05). For control took gr. intact animals. In the group "IFDE-5 + IGM" - column rats with four-vascular igm.

Thus, the obtained results indicate a pronounced correction of ischemic brain damage in the conditions of a total four-vascular model of rat brain ischemia IFDE-5, tadalafil at a dose of 1 mg / kg of animal body weight with a single intragastric administration.

Claims (2)

A method for the prevention of cerebral ischemia, including the reproduction of a four-vessel model of pathology and the administration of 1 mg / kg of tadalafil to a laboratory animal, which is administered once 60 minutes before the experiment, intragastrically through a probe as a preconditioning agent, and after 60 minutes, brain ischemia is modeled by coagulation two vertebral arteries and temporary occlusion of two common carotid arteries.

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