RU2572482C1 - Method for simulating cerebral vascular spasm in non-traumatic subarachnoidal haemorrhage in vivo - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves introducing fresh human venous blood into rat's occipital cistern. The introduction is preceded by puncturing or draining the occipital cistern with removing spinal fluid. After the first blood removal, the blood is re-introduced into the occipital cistern after a period of time providing continuous blood supply from the moment of the first introduction and required to develop cerebral vascular spasm.

EFFECT: method provides reproducing morphological changes of intracranial vessels as close to those in human individuals as possible, and ensures a possibility of studying vascular activity of preparations specific to human blood proteins.

9 cl, 4 dwg

Description

FIELD OF THE INVENTION

The invention relates to medicine, namely to neurosurgery, pathological anatomy, and can be used to study vascular spasm in non-traumatic subarachnoid hemorrhages in an experiment and conduct studies of the therapeutic efficacy of drugs for the prevention and treatment of vascular spasm.

State of the art

The prior art method for modeling non-traumatic subarachnoid hemorrhage in rats (Solomon RA, Antunes JL, Chen RY, Bland L., Chien S .: Decrease in cerebral blood flow in rat after experimental subarachnoid hemorrhage: a new animal model. Stroke 16:58 -64, 1985). With this technique, the authors performed a single puncture injection of fresh arterial autologous blood into the occipital cistern of the rat and carried out a subsequent assessment of cerebral perfusion up to 2 hours.

However, in the known method, a single injection of blood into the occipital cistern is performed, which makes it possible to observe a change in cerebral blood flow in the acute phase of the spasm. At the same time, with a single method of introducing changes in the deferred period of the experiment are practically absent. This model does not allow us to study the therapeutic efficacy of drugs specific for human blood proteins.

A method for modeling non-traumatic subarachnoid hemorrhage in rats is also known in the art (Bederson JB, Germano IM, Guarino L .: Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat. Stroke 26: 1086-1092, 1995 ) With this technique, the authors performed endovascular perforation of the internal carotid artery in rats and evaluated changes in cerebral blood flow in the first 24 hours from the experiment.

However, the known method also does not allow to observe delayed changes in vascular spasm and to study the therapeutic efficacy of drugs specific for human blood proteins.

One of the closest methods for modeling non-traumatic subarachnoid hemorrhage in rats is the double blood injection method (Meguro T., Clower BR, Carpenter R., Parent AD, Zhang JH Improved rat model for cerebral vasospasm studies. Neurol Res Oct 2001; 23 (7) : 761-6). With this technique, the authors performed a double injection of fresh arterial autologous blood into the occipital cistern of the rat with an interval of 48 hours between administrations and evaluated the morphological changes of intracranial vessels up to 7 days.

However, with all these methods, it is impossible to test the preventive and therapeutic effect of drugs that have an affinity for human blood proteins, for example, fibrinolytic drugs of the recombinant staphylokinase group, which are effective only in some animal and human species, but ineffective in rats (Species Reactivity to Staphylokinase. Exp Biol Med (Maywood) June 1949 71: 261-263, Lijnen HR, De Cock F., Matsuo O. & Collen D. Comparative fibrinolytic and fibrinogenolytic properties of staphylokinase and streptokinase in plasma of different species in vitro. Fibrinolysis 6, 33-37 (1992)).

Disclosure of invention

The objective of the invention is to create a convenient, affordable biological model for studying methods for the prevention and treatment of cerebral vascular spasm in non-traumatic subarachnoid hemorrhages using drugs specific for human blood proteins (for example, drugs of the recombinant staphylokinase group).

The technical result to which the claimed invention is directed is to reproduce the morphological changes of intracranial vessels during vascular spasm in laboratory white rats closest to those in humans, providing an adequate study of the therapeutic efficacy of drugs specific for human blood proteins.

The problem is solved in that the method of modeling cerebral vascular spasm in case of non-traumatic subarachnoid hemorrhage in an experiment involves the introduction of blood into the occipital cistern of the rat, while fresh human venous blood is introduced, and puncture or drainage of the occipital cistern with the removal of cerebrospinal fluid is performed before administration.

Puncture and blood injection are performed under the control of the frequency and depth of respiration of the animal. Excretion of cerebrospinal fluid from the subarachnoid space of the rat is carried out in a volume of from 0.05 to 0.2 ml for 3-7 minutes. The introduction of human blood into the occipital cistern is carried out in a volume of from 0.05 to 0.25 ml for 5-10 minutes.

In one embodiment of the invention, it is possible to reintroduce blood into the occipital cistern after a time, ensuring the constant presence of blood from the moment of the first injection, necessary for the development of cerebral vascular spasm. The indicated time is 24-48 hours.

After the introduction of blood, layered wound closure is performed, including closure of the occipital membrane.

In one embodiment, when blood is introduced into the occipital cistern, the animal is simultaneously sensitized by intravenous administration of human blood in a volume that does not lead to a violation of vital functions in the rat. The indicated volume is 0.5-2 ml.

Before the introduction of blood perform heparinization of the means of administration.

To exclude agglutination of human erythrocytes by rat agglutinins, before experiment, in 10 arbitrary animals, the group affiliation was assessed using the ABO system and the Rh factor using coliclones and tested for individual compatibility with all groups of human blood. In addition, before each introduction of human blood, an individual compatibility test was performed. No agglutination between rat serum and human erythrocytes was observed in any case, which suggests that human erythrocytes do not agglutinate under the influence of rat plasma.

With the comparative effectiveness of the intracisternal administration of recombinant staphylokinase preparations followed by external drainage of the cerebrospinal fluid in a model using fresh autologous blood in rats, we obtained data indicating the absence of fibrinolytic activity both locally (in the occipital cistern) and systemically (effect on hemostasis parameters).

According to preliminary data, the intracisternal use of recombinant staphylokinase preparations on the model of introducing human blood into the occipital cistern on the 3-4th day of the experiment with subsequent external drainage of cerebrospinal fluid in a volume that does not lead to a violation of the vital functions of the rat (0.3-1.5 ml of cerebrospinal fluid fluid per day), reduces the severity of morphological changes in vascular spasm both in the wall of intracranial vessels and in brain tissue.

Brief Description of the Drawings

The invention is illustrated by photographs of the image of the object of study in the experiment, where in FIG. Figure 1 shows the verification of NAO: A — brain of an intact animal; B - the brain was seized 30 minutes after the introduction of 0.25 ml of autologous blood into the occipital cistern; in FIG. 2 - the upper third of the main artery (magnification × 400), control, A - stained with hematoxylin and eosin, B - Gram-Weigert stained, the positions indicated in the images: 1 - endotheliocyte nuclei, 2 - VEM, 3 - MMC. Corresponds to the normal structure of the upper third of the rat’s main artery; in FIG. Figure 3 shows the upper third of the main artery on the 5th day after twice blood injection (magnification × 400), A - staining with hematoxylin and eosin, B - staining according to Lie, the positions indicated in the images: 1 - nuclei of endotheliocytes, 2 - VEM, 3 - MMC (there are signs of vacuolization of the cytoplasm). There is a sharp narrowing of the lumen of the vessel, a significant thickening of the artery wall, the convergence of the nuclei of endotheliocytes. VEM has pronouncedly convoluted contours; in the cytoplasm of MMC, it is a common hydropic dystrophy; in FIG. Figure 4 shows the image of the upper sections of the rat bridge (Lie staining, magnification × 1000), A - the 3rd day after the blood injection, B - the 7th day after the blood injection, the positions indicated on the images: 4 - capillaries with the saved blood flow ( in Fig. A - full-blooded, in Fig. B - with a declining lumen), 5 - areas of pronounced perivascular edema.

The implementation of the invention

Below is a detailed description of the implementation of the invention and the results of morphological studies of the resulting model of cerebral vascular spasm when twice introduced into the occipital cistern of the rat fresh human venous blood. However, this detailed description does not limit the claimed invention, because in the experiment, the claimed result was obtained with a single injection of blood.

1. Anesthesia

Anesthetization of animals was carried out according to the following method.

The rat was placed in a closed container, at the bottom of which hygroscopic cotton wool soaked in diethyl ether was laid. After 1-2 minutes, the rat fell asleep. After this, premedication was performed to potentiate the main anesthesia: the following drugs were injected intramuscularly into the thigh with an insulin syringe: droperidol - 0.3 mg / kg body weight (0.03 ml 0.25% solution), diphenhydramine 1.2 mg / kg body weight (0.03 ml of a 1% solution), atropine - 0.012 mg / kg body weight (0.03 ml of a 0.1% solution). 15 minutes after premedication, the following drugs were injected into the thigh area of another rat paw: “Zoletil 100” - 24 mg / kg body weight (0.06 ml of solution), Xylazine - 3.2 mg / kg body weight (0.04 ml 2 % solution). As a rule, the rat fell asleep within 5-15 minutes. The depth of anesthesia was assessed by the severity of the corneal reflex. If necessary, after 20 minutes 0.02-0.04 ml of Zoletil 100 was added.

2. Operation technique

The introduction of blood into the occipital cistern (performed on the 1st and 2nd day of the experiment with an interval between injections of 24 hours). The animal was fixed on the operating table and the area of operation was shaved. After treatment of the skin with antiseptic solutions, a midline incision was made from the occipital elevation to the spinous process of the VI-VII cervical vertebrae. To reduce bleeding and potentiate anesthesia, the neck muscles were infiltrated with the combined local anesthetic Ultracain DS 1: 200000 (articaine hydrochloride 40 mg / ml + epinephrine hydrochloride 6 mg / ml) diluted with saline 1: 5. Further, the muscles at the place of their attachment to the skull were cut off and skeletonized the scales of the occipital bone, the atlantooccipital membrane and the arch of the first cervical vertebra. A puncture of the occipital cistern was performed with the removal of 0.05-0.2 ml of cerebrospinal fluid for 3-7 minutes under the control of the frequency and depth of respiration of the animal. After that, 0.05-0.2 ml of fresh human venous blood was injected into the occipital cistern for 5-10 minutes under the control of the frequency and depth of respiration of the animal. In order for most of the blood to accumulate in the area of the basal cisterns, the animal was left for 20 min with the head end lowered by 20 ° -30 °. After this, the wound was sutured in layers. If necessary, compensation for blood loss was injected subcutaneously with a mixture of 5% glucose solution and 0.9% sodium chloride solution with a volume of up to 5-10 ml.

3. Assessment of neurological status

Assessment of neurological status was performed 1 time per day for 7 days after blood injection according to the method proposed by J.B. Bederson et al. (1986) [Bederson J.B., Pitts L.H., Tsuji M., Nishimura M.C., Davis R.L., Bartkowski H.: Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke 17: 472-476, 1986] on a 4-point scale:

0 points - absence of neurological disorders

1 point - the rat is inactive, sluggishly reacts to touch. No focal neurological symptoms

2 points - there is an isolated paresis of one limb (when lifting the animal by the tail, the activity of movements in one of the limbs is reduced)

3 points - there is unilateral hemiparesis (when pushing the animal to the side, it falls over on the affected side)

4 points - there is unilateral hemiplegia (the animal cannot actively move, when moving forward there is a pronounced deviation to the affected side - movement in a circle).

According to our data, changes in neurological status were observed during the first day after any intervention under anesthesia up to 1 point and were not associated with the manifestation of vascular spasm

4. Morphological study

Before the brain was removed, the animal was euthanized with a mixture of preparations “Zoletil 100” (0.4 ml), xylazine hydrochloride (0.3 ml 2% solution) and rocuronium bromide (0.1 ml 1% solution). According to the existing section, the wound was opened. Decapitation was performed at the level of III-IV of the cervical vertebrae, the skull was opened and, if possible, without damaging the hard and soft meninges, the brain preparation was removed along with the upper cervical spinal cord. Next, the drug was placed for 24 hours in 5% formalin solution and embedded in paraffin. Sections at the level of the upper third of the basilar artery were used for morphological studies.

Paraffin sections were stained in the following ways:

- Overview: hematoxylin and eosin,

- To assess the state of elastic fibers of the artery wall: according to Gram-Weigert (determination of the morphological type of the wall of the basilar artery),

- To assess the condition of the contractile apparatus of smooth muscle cells (myofibrils): by Lie.

Description of micropreparations was carried out using a Leica DM 1000 optical microscope with an increase of 400-1000 times, microphotography was performed on a Leica EC 3 digital camera.

Direct qualitative signs of cerebral vascular spasm were obtained in the form of characteristic morphological changes in intracranial vessels and ischemic changes in brain tissue.

In the study of the upper third of the basilar artery in rats after a double injection of autoblood into the occipital cistern, the following changes were revealed:

4th day - morphologically traced signs of moderate subsidence of the lumen of the vessel without significant thickening of the wall of the main artery and changes in the internal elastic membrane (VEM). When stained for Lie and MSB, signs of hypercontractual changes in smooth muscle cells (GMC) were detected.

5th day — thickening of the vessel wall, emphasized tortuosity of the elastic membrane with deepening of its folds, narrowing of the interfold gaps, the convergence of endotheliocytes with nuclei protruding into the lumen, oriented along the tops of the sweet VEM, were noted. During the histochemical reaction (Lie stain), signs of dystrophic changes from focal to widespread hydropic cytoplasmic dystrophy in the form of perinuclear and intracellular vacuolization, as well as signs of intracellular fuchsinophilia of the cytoplasm, indicating hypercontractual changes in contractile myofibrils appeared in the MMC cytoplasm.

6th and 7th days — a moderate decrease in the lumen of the vessel with widespread dystrophic changes in the MMC cytoplasm was noted as manifestations of residual signs of impaired contractility of myocytes with resolution of the spasm.

In addition, signs of impaired capillary blood flow in rat brain tissue were identified, which is a direct sign of cerebral ischemia at the microcirculatory level.

The proposed method can be used to test methods for the prevention and treatment of cerebral vascular spasm in an experiment, including the intrathecal administration of drugs and the use of drugs that have an affinity for human blood proteins.

Claims (9)

1. A method for simulating cerebral vascular spasm in non-traumatic subarachnoid hemorrhage in an experiment to study the vascular activity of drugs specific for human blood proteins, comprising introducing fresh blood into the occipital cistern of a rat, characterized in that fresh human venous blood is introduced, and puncture is performed before or drainage of the occipital cistern with the removal of cerebrospinal fluid, after the first injection of blood, reintroduction of blood into the occipital qi is carried out Terni after a time that ensures the constant presence of blood from the first administration, necessary for the development of cerebral vasospasm. 2. The method according to p. 1, characterized in that the puncture or drainage of the occipital cistern and the introduction of blood is performed under the control of the frequency and depth of respiration of the animal. 3. The method according to p. 1, characterized in that the removal of cerebrospinal fluid from the rat subarachnoid space is carried out in a volume of from 0.05 to 0.2 ml for 3-7 minutes. 4. The method according to p. 1, characterized in that the introduction of human blood into the occipital cistern is carried out in a volume of from 0.05 to 0.25 ml for 5-10 minutes 5. The method according to p. 1, characterized in that the repeated administration of blood is carried out 24-48 hours after the first injection. 6. The method according to p. 1, characterized in that after the introduction of blood perform layer-by-layer suturing of the wound, including suturing the occipital membrane. 7. The method according to p. 1, characterized in that when blood is introduced into the occipital cistern, the animal is simultaneously sensitized by intravenous administration of human blood in a volume that does not lead to impaired vital functions in the rat. 8. The method according to p. 7, characterized in that said volume is 0.5-2 ml. 9. The method according to p. 1, characterized in that before the introduction of blood perform heparinization of the means of administration.

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