Establishment method of permanent cerebral ischemia model of eastern vole

Abstract

The invention belongs to the technical field of animal model construction, and relates to a method for establishing an oriental vole permanent cerebral ischemia model. A nylon wire bolt with the head end diameter of 0.22 + / -0.02 mm is used, a mark is made at the position, 1.5 cm away from the head end, of the wire bolt, the wire bolt is inserted through a neck artery wound of the eastern vole and enters the middle artery of the vole brain through the internal carotid artery, and therefore the front end of the middle artery is blocked, and the vole permanent cerebral ischemia model is completed. The permanent cerebral ischemia model of the oriental vole is successfully established. The model has the characteristics of stable infarction volume, low death rate and uniform cerebral infarction range, and provides a more reliable and effective animal model for stroke related research.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction and relates to a method for establishing a permanent cerebral ischemia model of the fortified voles. Background Art

[0002] Stroke is a brain injury disease caused by rupture or blockage of brain blood vessels. The research on this disease has attracted increasing attention from doctors and researchers. Due to various limitations in clinical research, animal models of cerebral ischemia have become an indispensable tool for studying the injury mechanism and prevention and treatment measures of cerebrovascular diseases. Therefore, a rapid and effective rodent model of repeated cerebral ischemia-reperfusion injury is urgently needed by pharmaceutical research institutes for this type of disease.

[0003] The existing construction of rat cerebral ischemia model has the following defects: For example, when preparing the MCAO model, the operation after the nylon thread enters the skull is performed under non-direct vision, and the blood flow blockage situation cannot be judged; for example, the animal mortality rate of 4-VO (four-vessel occlusion method) is relatively high; 4-VO and 3-VO (three-vessel occlusion method) have surgical trauma problems and difficult postoperative recovery; 2-VO (two-vessel occlusion method) requires simultaneous ligation of bilateral common carotid arteries, and the animal's brain quickly develops low blood flow perfusion, which can easily lead to a high mortality rate; BCAS (bilateral common carotid artery stenosis) model surgery is more difficult, and it takes a long time to wait for experimental animals to develop cognitive defects after surgery.

[0004] Therefore, in order to construct a more suitable rat cerebral ischemia model, it is necessary to carefully consider the selection of the model, the accuracy of the surgical operation, and the similarity between the model and the clinical pathological process to improve the stability and reproducibility of the model and the reliability of the experimental results. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to construct a permanent cerebral ischemia model of the Fort voles with stable infarct volume, low mortality and uniform cerebral infarction range. The specific technical scheme is as follows: In order to achieve the above object, the present invention designs and discloses the following technical solutions: The first aspect of the present invention discloses a method for establishing a permanent cerebral ischemia model of a fortified vole, which comprises using a nylon thread plug with a head end diameter of 0.22±0.02 mm, with a mark made 1.5 cm away from the head end, inserting the thread plug through the incision of the fortified vole's carotid artery and entering the vole's middle cerebral artery through the internal carotid artery, thereby blocking the front end of the middle cerebral artery to complete the permanent cerebral ischemia model of the vole.

[0006] The specific steps of establishing the method are as follows: (1) Select Fortis voles and inject atropine 200 μL / mouse into the hind limb muscle of Fortis voles. 15 minutes after the injection, inject Zota 50 into the other hind limb muscle at a concentration of 40 mpk. (2) After satisfactory anesthesia, the skin of the Fortis vole's neck was prepared and the surgical field was disinfected; (3) The prepared vole is fixed in a supine position on the operating table with the tail facing the surgeon. A longitudinal incision is made in the middle of the neck from the edge of the lower lip in front to the clavicle in the back. The skin is covered with sterile gauze, and the submandibular and sublingual glands are separated. The neck muscles of the vole are exposed with a surgical retractor. (4) Blunt separation of the neck muscles to fully expose the left common carotid artery and the bifurcation of the internal and external carotid arteries, and place a microsurgical pad under the common carotid artery; (5) Ligate the proximal end of the common carotid artery with 8-0 suture, leave a ligature in the middle of the common carotid artery and tie a loose knot, and use a microvascular clamp to temporarily clamp the external carotid artery and the internal carotid artery; (6) Make a small incision in front of the ligature at the proximal end of the common carotid artery and insert the thread plug at the incision. At this time, remove the microvascular clamp and continue to insert the thread plug. When the thread plug mark reaches the bifurcation of the internal carotid artery and the external carotid artery, stop inserting and ligate the reserved ligature in the middle of the common carotid artery, minus the excess thread at the tail of the thread plug; (7) Remove the gauze and surgical retractor, return the submandibular gland to its original position, close the neck incision with 4-0 sutures, turn the vole to the supine position, and place it on a heating pad until it can resume autonomous activities; (8) After recovery, a permanent cerebral ischemia model was established in the Fort Microtus elegans.

[0007] The fort voles are preferably female, 6-8 weeks old, and weigh 60±10 g.

[0008] The second aspect of the present invention discloses a permanent cerebral ischemia model of the fort voles, which is established by the above method.

[0009] The third aspect of the present invention discloses the use of the above-mentioned permanent cerebral ischemia model of the fortified voles for developing drugs for preventing or treating cerebral ischemia diseases.

[0010] Compared with the prior art, the present invention has the following advantages: In terms of constructing an animal model of cerebral ischemia, it was found that the Oriental vole cerebral ischemia model is less difficult to construct than the mouse cerebral ischemia model, and surgical modeling can be performed without the use of a microscope. At the same time, compared with the rat model, the Oriental vole model also has significant advantages in construction cost. The cost of raising rats is relatively high, not only requiring more resources for daily maintenance, but also in the later stage of drug efficacy evaluation, due to the larger size of rats, the amount of drugs required also increases accordingly, which undoubtedly increases the cost and complexity of the experiment. In contrast, the Oriental vole model shows higher economy and practicality in these aspects, providing a new and powerful tool for stroke-related research.

[0011] The present invention fully exposes the left common carotid artery and the bifurcation of the internal carotid artery and the external carotid artery by blunt separation of the neck muscles, and places a microsurgical pad under the common carotid artery to improve the neck blood vessel exposure method, which can more accurately control the insertion of the suture, reduce surgical trauma, and improve the stability and repeatability of the model.

[0012] The invention makes a small incision in front of the ligature at the proximal end of the common carotid artery, inserts a wire plug at the incision, removes the microvascular clamp at this time, continues to insert the wire plug, stops inserting when the wire plug mark reaches the bifurcation of the internal carotid artery and the external carotid artery, ligates the ligature reserved in the middle of the common carotid artery, and removes the excess wire head at the tail of the wire plug; improves the wire plug entry technique, avoids the wire plug from damaging the blood vessel, and can improve the success rate of the model and reduce the mortality rate of the animal.

[0013] After comparing different types of suture plugs, the present invention selected 8-0 sutures to better simulate the blood flow blocking effect and reduce damage to blood vessels.

[0014] The present invention successfully established a permanent cerebral ischemia model of the Fortis voles, which has the characteristics of stable infarction volume, low mortality rate and uniform range of cerebral infarction, and provides a more reliable and effective animal model for stroke-related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the existing methods and experiments, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the behavioral scoring result of the Fort Microtus suis in Example 2.

[0017] Figure 2 This is the laser speckle imaging result in Example 3.

[0018] Figure 3 The TTC staining results in Example 4 DETAILED DESCRIPTION

[0019] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The experimental methods and techniques in the following embodiments without specifying specific conditions are usually carried out according to conventional conditions in the field or according to the conditions recommended by the manufacturer.

[0020] Example 1 In the process of establishing the permanent cerebral ischemia model of the fort voles, the technical scheme of the present invention is detailed and specific, and every detail from the selection of voles, anesthesia, surgical skin preparation, disinfection to the surgical steps is strictly regulated and operated. This not only ensures the success rate of model establishment, but also ensures the consistency and repeatability of the model. The specific establishment method is as follows: (1) Eight male and eight female voles aged 6-8 weeks and weighing 60±10 g were selected. Atropine 200 μL / vole was injected into the hind limb muscle of the voles to prevent increased tracheal secretions after anesthesia. Fifteen minutes after the injection, Zota 50 was injected into the hind limb muscle of the other side at a concentration of 40 mpk.

[0021] (2) After satisfactory anesthesia, prepare the skin on the vole's neck and disinfect the surgical field.

[0022] (3) The prepared vole is fixed on the operating table in a supine position, with the tail facing the surgeon. A longitudinal incision is made in the middle of the neck from the edge of the lower lip in front to the clavicle in the back, and covered with sterile gauze. The submandibular gland and sublingual gland are separated, and the neck muscles of the vole are exposed with a surgical retractor.

[0023] (4) Bluntly separate the neck muscles to fully expose the left common carotid artery and the bifurcation of the internal and external carotid arteries, and place a microsurgical pad under the common carotid artery.

[0024] (5) Ligate the proximal end of the common carotid artery with 8-0 suture, leave a ligature in the middle of the common carotid artery and tie a loose knot. Use microvascular clamps to temporarily clamp the external carotid artery and internal carotid artery.

[0025] (6) Make a small incision in front of the ligature at the proximal end of the common carotid artery and insert a wire plug at the incision. At this time, remove the microvascular clamp and continue to insert the wire plug. When the wire plug mark reaches the bifurcation of the internal carotid artery and the external carotid artery, stop inserting it and ligate the reserved ligature in the middle of the common carotid artery, minus the excess thread at the tail of the wire plug.

[0026] (7) Remove the gauze and surgical retractor, return the submandibular gland to its original position, and suture the neck incision with 4-0 sutures. Turn the vole to the supine position and place it on a heating pad until it can resume autonomous activities.

[0027] (8) After recovery, a permanent cerebral ischemia model was established in the Fort Microtus elegans.

[0028] In the surgical procedure, through the key steps of blunt separation of the neck muscles, full exposure of the left common carotid artery and the bifurcation of the internal carotid artery and the external carotid artery, ligation of the proximal end of the common carotid artery, insertion of the suture and ligation, the present invention successfully achieves the blockage of the middle cerebral artery of the Foriental vole, thereby completing the establishment of a permanent cerebral ischemia model.

[0029] In addition, during the operation, the present invention also focuses on monitoring and maintaining the physiological state of the vole, such as injecting atropine to prevent increased tracheal secretions after anesthesia and using a heating pad to maintain the body temperature of the vole, all of which provide strong guarantees for the successful establishment of the model.

[0030] Example 2 Behavioral scoring method: 24 hours after modeling, the behavior of the Fort voles permanent cerebral ischemia model in Example 1 was scored, and the scoring criteria were as follows: Score Performance and evaluation 0 Normal, no neurological impairment 1 The left (paralyzed) front paw cannot be fully extended, with mild neurological deficits 2 When walking, he turns in circles to the left side (the paralyzed side) and has moderate neurological deficits 3 When walking, he falls to the left side (the paralyzed side) and has severe neurological deficits 4 Unable to walk spontaneously, loss of consciousness 5 die The results are as follows Figure 1 Compared with male Fortis voles, female Fortis voles suffer less damage and are less likely to die after the establishment of a permanent cerebral ischemia model. Therefore, when establishing a permanent cerebral ischemia model in Fortis voles, it is more stable to use female voles to establish the model.

[0031] Example 3 Laser speckle imaging detection method: 24 hours after the modeling of the fort vole in Example 1, the fort vole was anesthetized by injection of 50,40 mpk of Zotazone at the hind limbs, the skin of the top of the head was prepared, and a longitudinal incision was made in the middle of the top of the head to expose the entire parietal bone. The fort vole was placed prone on an operating table with a heat preservation function, and its brain blood flow was monitored using a laser speckle imaging system, and the difference between the blood flow on the operated side and the control side was statistically analyzed. The results are as follows: Figure 2 As shown in the figure, the treated side ( Figure 2 The blood flow rate in the left side of the fortress was slow, while the blood flow in the control side was normal, which can prove that after the treatment of the technical solution of the present invention, a permanent cerebral ischemia model of the fortress vole was successfully established.

[0032] Example 4 TTC staining method: 24 hours after the model was constructed, the fortified voles were killed by cervical dislocation, and the entire brain of the fortified voles was quickly peeled out and frozen in a -20℃ refrigerator for 30 minutes.

[0033] After the brain tissue is completely frozen, use a blade to cut the frozen brain into slices of uniform thickness and immerse them in a culture dish containing 0.2% TTC staining solution.

[0034] Place the cut brain slices in a 37°C incubator for more than 30 minutes, and constantly observe the staining. When the staining is satisfactory, take out the culture dish, use a dropper to carefully remove the TTC staining solution, and pour formalin to fix the brain slices.

[0035] The fixed brain slices were spread on a glass slide and photographed. Figure 3 As shown in the figure, it can be seen that the brain of the Oriental vole has a white part after staining, and the infarction area is relatively stable, which proves that the method of the present invention can cause partial damage to the brain tissue due to ischemia, and successfully establish a permanent cerebral ischemia model of the Oriental vole, which has stable infarction volume, low mortality rate and uniform cerebral infarction range.

[0036] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing a permanent cerebral ischemia model in Fortis voles, characterized in that: A nylon suture with a head diameter of 0.22±0.02 mm was used. A mark was made 1.5 cm away from the head end of the suture. The suture was inserted through the incision of the carotid artery of the Eastern vole and passed through the internal carotid artery into the middle cerebral artery of the vole, thereby blocking the front end of the middle cerebral artery to complete the permanent cerebral ischemia model of the vole.

2. The method for establishing a permanent cerebral ischemia model of Fortis voles according to claim 1, characterized in that: The specific steps of the establishment method are as follows: (1) Select Fortis voles and inject atropine 200 μL / mouse into the hind limb muscle of Fortis voles. 15 minutes after the injection, inject Zota 50 into the other hind limb muscle at a concentration of 40 mpk. (2) After satisfactory anesthesia, the skin of the Fortis vole's neck was prepared and the surgical field was disinfected; (3) The prepared vole is fixed in a supine position on the operating table with the tail facing the surgeon. A longitudinal incision is made in the middle of the neck from the edge of the lower lip in front to the clavicle in the back. The skin is covered with sterile gauze, and the submandibular and sublingual glands are separated. The neck muscles of the vole are exposed with a surgical retractor. (4) Blunt separation of the neck muscles to fully expose the left common carotid artery and the bifurcation of the internal and external carotid arteries, and place a microsurgical pad under the common carotid artery; (5) Ligate the proximal end of the common carotid artery with 8-0 suture, leave a ligature in the middle of the common carotid artery and tie a loose knot, and use a microvascular clamp to temporarily clamp the external carotid artery and the internal carotid artery; (6) Make a small incision in front of the ligature at the proximal end of the common carotid artery and insert the thread plug at the incision. At this time, remove the microvascular clamp and continue to insert the thread plug. When the thread plug mark reaches the bifurcation of the internal carotid artery and the external carotid artery, stop inserting and ligate the reserved ligature in the middle of the common carotid artery, minus the excess thread at the tail of the thread plug; (7) Remove the gauze and surgical retractor, return the submandibular gland to its original position, close the neck incision with 4-0 sutures, turn the vole to the supine position, and place it on a heating pad until it can resume autonomous activities; (8) After recovery, a permanent cerebral ischemia model of the Fortis voles was established.

3. The method for establishing a permanent cerebral ischemia model of Fortis voles according to claim 1, characterized in that: The fort voles are preferably female, 6-8 weeks old, and weigh 60±10 g.

4. A permanent cerebral ischemia model of the fortified voles, characterized in that: The method is established by any one of claims 1 to 3.

5. Use of the Fortis musculus permanent cerebral ischemia model as claimed in claim 4 in developing drugs for preventing or treating cerebral ischemic diseases.

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