Method for establishing systemic hypoperfusion cerebral infarction model

Abstract

The invention relates to a method for establishing a systemic hypoperfusion cerebral infarction model. The model provided by the invention simulates the clinical common pathological process of cerebral infarction caused by cerebral blood flow hypoperfusion caused by too low blood volume. The technical method that conventional bilateral carotid artery ligation can only simulate chronic cerebral ischemia, ischemic infarction lesions are not formed, and hypoperfusion cerebral infarction cannot be simulated is broken through, and a composite method of bloodletting combined with improved MCAO is used for the first time to simulate clinically common hypoperfusion cerebral infarction caused by too low blood volume. The method provides a reliable animal model for studying the pathologic process of occurrence and development of the disease, pharmacodynamics of drugs, explaining the drug action mechanism and researching and developing new drugs.

Description

Technical Field

[0001] The invention relates to the technical field of medical models, and in particular to a method for establishing a systemic hypoperfusion cerebral infarction model. Background Art

[0002] According to the Chinese Ischemic Stroke Subtypes (CISS) classification, ischemic stroke can be divided into atherosclerotic type, cardioembolic type, perforating artery disease, other causes and unknown causes. Among them, large artery atherosclerosis type is divided into 4 types according to its pathogenesis: low perfusion / decreased embolic clearance, carrier artery (plaque or thrombus) blocking perforating artery, artery-to-artery embolism and mixed mechanism. Hypoperfusion-induced cerebral infarction is a more common type of atherosclerosis type, and its incidence accounts for 10% to 13% of the total number of acute strokes. The characteristic that distinguishes hypoperfusion stroke from other stroke subtypes is that there are often predisposing factors such as hypovolemia and hypotension before the onset of the disease, which leads to too low blood flow to some areas of the brain and insufficient perfusion of brain tissue. If it is lower than the threshold, it is easy to induce hypoperfusion cerebral infarction.

[0003] Cerebral blood circulation is part of the systemic circulation, but it has its own particularity. The brain mass accounts for about 2% to 3% of the body mass, but the cerebral blood volume accounts for 20% of the cardiac output. The normal function of the brain requires sufficient cerebral blood flow (CBF) to maintain, which is related to the effective brain perfusion pressure and vascular resistance. The whole brain CBF is about 50ml / (min·100g), and the local cerebral blood flow (r CBF) <23ml / (min·100g) is a low perfusion area. When acute hypoperfusion lasts for more than a certain period of time, the lesion progresses from the ischemic penumbra to acute cerebral infarction.

[0004] Hypoperfusion cerebral infarction is caused by insufficient blood supply to the brain, resulting in hypoxia of brain tissue, thus causing cerebral infarction. Common causes of acute hypoperfusion cerebral infarction are severe water loss or blood loss, excessive blood pressure reduction, shock, cardiac arrest, acute myocardial infarction, DIC and surgical anesthesia. Its pathogenesis is relatively complex, and it is currently believed to include hemodynamic disorders, stenosis or occlusion of blood supply arteries, impaired cerebrovascular reserve capacity, impaired blood purification function, and vascular tortuosity. Hypoperfusion cerebral infarction usually has two main manifestations: ① Local hypoperfusion cerebral infarction: When the large arteries in the neck or skull are severely narrowed or occluded, the local cerebral blood flow is reduced, causing low perfusion at the junction of adjacent cerebral vascular blood supply areas, which is called watershed infarction. ② Systemic hypoperfusion cerebral infarction: Due to severe water loss or blood loss, excessive blood pressure reduction, acute myocardial infarction, etc., the systemic perfusion pressure decreases, resulting in a decrease in blood flow to the entire brain. Ischemic brain damage is not limited to the watershed area, but may affect a wider range of brain tissue.

[0005] Local hypoperfusion cerebral infarction is generally produced by middle cerebral artery occlusion (MCAO). There is no mature model for simulating systemic hypoperfusion cerebral infarction. The bilateral carotid artery ligation model is a commonly used model for studying chronic hypoperfusion. This method simulates the state of chronic cerebral ischemia and is often used in the study of cognitive impairment. However, it does not form ischemic infarction lesions, so it cannot reflect the pathological process of cerebral infarction caused by hypoperfusion. The middle cerebral artery occlusion (MCAO) model is a classic animal model for studying acute cerebral infarction. It is achieved by inserting a suture from the internal jugular vein to the basilar artery circle of the brain to block the blood supply. After modeling, cerebral ischemic lesions can be seen. It is generally used to simulate local hypoperfusion cerebral infarction. Cerebral infarction caused by hypoperfusion is mostly caused by low blood volume, low blood pressure, and shock. The MCAO model fails to reflect the cause of hypoperfusion. Therefore, it is worth exploring how to simulate cerebral infarction caused by peripheral hypoperfusion and brain tissue hypoperfusion. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention discloses a method for establishing a systemic hypoperfusion cerebral infarction model, which uses an eyeball bloodletting combined with a modified middle cerebral artery occlusion (MCAO) rat model establishment method to simulate clinical hypoperfusion cerebral infarction caused by hypovolemia, comprising the following steps: Step 1: Bleed the rats' eyes with a blood volume of 0.5 ml / 100 g; Step 2: Use the exsanguinated rats to construct a modified middle cerebral artery occlusion (MCAO) rat model, specifically: Step 21, anesthetize the rat by intraperitoneal injection of 1% sodium pentobarbital, the injection dose is 40 mg / kg, and fix the anesthetized rat in supine position; Step 22, prepare the skin of the rat's neck, make a longitudinal incision of about 3 cm in the middle of the neck, bluntly peel off the subcutaneous tissue and muscle groups, and find the left common carotid artery (CCA) and the internal carotid artery (ICA) and the external carotid artery (ECA) at its bifurcation in the deep depression; Step 23, free the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), and ligate the proximal end of the left common carotid artery (CCA) with surgical sutures, about 0.5 cm away from the bifurcation of the left common carotid artery (CCA); ligate the internal carotid artery (ICA) and external carotid artery (ECA), and tie a slipknot with sutures; Step 24: Cut a V-shaped opening between the ligature and bifurcation of the left common carotid artery (CCA), adjust the curvature of the middle cerebral artery occlusion (MCAO) suture toward the midline of the trunk, insert the suture from the cut V-shaped opening, and slowly push it in along the direction of the internal carotid artery (ICA) entering the brain, referring to the bifurcation of the left common carotid artery (CCA). When the advancement length is 17-18 mm, resistance is felt and no further advancement is performed; Step 25: Ligate the left common carotid artery (CCA) firmly with surgical sutures, cut off the suture plug remaining outside the blood vessel to prevent it from being bitten and pulled out by the rat, and suture the skin; Step 3: Set up a sham control group: except that the thread plug described in step 24 is not inserted, other operations are carried out according to steps 1 and 2; Step 4: After the rats have been awake for 30 minutes, neurological function scores are performed.

[0007] As a preferred embodiment of the present invention, in step 24, the diameter of the thread plug is selected as follows: for rats weighing 280 g, a thread plug of 300 g is selected.

[0008] As a preferred embodiment of the present invention, after the skin is sutured in step 25, picric acid is applied to the sutured part to prevent the rats from biting each other.

[0009] As a preferred embodiment of the present invention, the indicators observed in the neurological function scoring performed in step 4 are: neurological function evaluation (mNSS), rotarod fatigue time, grip strength measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and histological staining.

[0010] Beneficial effects of the present invention: The model of the present invention simulates the pathological process of cerebral infarction caused by low blood volume, which is common in clinical practice. It breaks through the previous technical method that bilateral carotid artery ligation can only simulate chronic cerebral ischemia, does not form ischemic infarction lesions, and cannot simulate low perfusion cerebral infarction. For the first time, a composite method of bloodletting combined with modified MCAO is used to simulate low perfusion cerebral infarction caused by low blood volume, which is common in clinical practice. This method provides a reliable animal model for studying the pathological process of the occurrence and development of this disease, the pharmacodynamics and pharmacology of drugs, explaining the mechanism of drug action, and for the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The technical block diagram of the construction of the bloodletting MCAO rat model of the present invention;

[0012] Figure 2 This is a comparison chart of the neurological function scores of rats in each group of the present invention;

[0013] Figure 3 This is a comparison diagram of the rotarod fatigue time of each group of rats in the present invention;

[0014] Figure 4 This is a comparison chart of the gripping strength of rats in each group of the present invention;

[0015] Figure 5 This is a comparison diagram of the local cerebral blood flow (r CBF) of each group of rats with cerebral ischemia in the present invention;

[0016] Figure 6 Pseudo-color images of local cerebral blood flow in each group of rats with cerebral ischemia in the present invention;

[0017] Figure 7 This is a diagram of TTC staining results of each group of rats in the present invention;

[0018] Figure 8 This is a comparison diagram of HE staining of brain ischemic tissues of rats in each group of the present invention;

[0019] Fig. 9 It is a comparison diagram of Nissl staining of brain ischemic tissues of rats in each group of the present invention. DETAILED DESCRIPTION

[0020] Example 1

[0021] The method for establishing a systemic hypoperfusion cerebral infarction model of the present invention adopts an eyeball bleeding combined with a modified middle cerebral artery occlusion (MCAO) rat model establishment method to simulate clinical hypoperfusion cerebral infarction caused by hypovolemia, and comprises the following steps: Step 1: Bleed the rats' eyes with a blood volume of 0.5 ml / 100 g; Step 2: Use the exsanguinated rats to construct a modified middle cerebral artery occlusion (MCAO) rat model, specifically: Step 21, anesthetize the rat by intraperitoneal injection of 1% sodium pentobarbital, the injection dose is 40 mg / kg, and fix the anesthetized rat in supine position; Step 22, prepare the skin of the rat's neck, make a longitudinal incision of about 3 cm in the middle of the neck, bluntly peel off the subcutaneous tissue and muscle groups, and find the left common carotid artery (CCA) and the internal carotid artery (ICA) and the external carotid artery (ECA) at its bifurcation in the deep depression; Step 23, free the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), and ligate the proximal end of the left common carotid artery (CCA) with surgical sutures, about 0.5 cm away from the bifurcation of the left common carotid artery (CCA); ligate the internal carotid artery (ICA) and external carotid artery (ECA), and tie a slipknot with sutures; Step 24: Cut a V-shaped opening between the ligature and bifurcation of the left common carotid artery (CCA), adjust the curvature of the middle cerebral artery occlusion (MCAO) suture toward the midline of the trunk, insert the suture from the cut V-shaped opening, and slowly push it in along the direction of the internal carotid artery (ICA) entering the brain, referring to the bifurcation of the left common carotid artery (CCA). When the advancement length is 17-18 mm, resistance is felt and no further advancement is performed; Step 25: Ligate the left common carotid artery (CCA) firmly with surgical sutures, cut off the sutures remaining outside the blood vessel to prevent them from being bitten and pulled out by rats, suture the skin, and apply picric acid to the sutured area after suture of the skin to prevent rats from biting each other; Step 3: Set up a sham control group: except that the thread plug described in step 24 is not inserted, other operations are carried out according to steps 1 and 2; Step 4: After the rats were awake and active for 30 minutes, neurological function scores were performed. The observed indicators included neurological function evaluation (mNSS), rotarod fatigue time, grip strength measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and histological staining.

[0022] The choice of the thread plug diameter: for rats weighing 280g, choose the thread plug model of 300g.

[0023] Neurological function score results:

[0024] like Figure 2 As shown, the mNSS scores of each group were compared

[0025] Compared with the sham control group, the neurological deficit scores of rats in the MCAO group and the bloodletting + MCAO group on days 1, 3, 5, and 7 were higher than those in the sham control group (p<0.01).

[0026] like Figure 3 As shown, the comparison of the rotarod fatigue time of each group

[0027] Compared with the sham control group, the rotarod fatigue time of rats in the MCAO group and the exsanguination+MCAO group was significantly lower on the 3rd and 7th days than that in the sham control group (p<0.01). The exsanguination+MCAO group was significantly lower on the 7th day than that in the MCAO group (p<0.05).

[0028] like Figure 4 As shown, the upper limb grip strength test of each group is compared

[0029] Compared with the sham control group, the maximum upper limb grip strength of rats in the MCAO group and the bloodletting + MCAO group was lower on the 3rd and 7th days than that in the sham control group (p<0.01). The bloodletting + MCAO group was lower than the MCAO group on the 7th day, which was statistically significant (p<0.05).

[0030] like Figure 5 , Figure 6 As shown, the cerebral blood flow of each group was compared

[0031] Compared with the sham control group, the percentage of cerebral blood perfusion decreased in the MCAO group and the bloodletting + MCAO group on days 3 and 7 was significantly lower than that in the sham control group (p<0.01). The bloodletting + MCAO group was lower than the MCAO group on day 7, which was statistically significant (p<0.01).

[0032] like Figure 7 As shown, the percentage analysis of cerebral infarction area in each group of rats

[0033] After taking the brain, immediately place it in a -20 degree refrigerator to freeze for about 20 minutes; cut the brain tissue into 5-6 slices, one slice every 2mm. The first cut is at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut is at the optic chiasm; the third cut is at the infundibulum handle; the fourth cut is between the infundibulum handle and the caudal pole of the posterior lobe. Place the slices in 2% 2,3,5-triphenyltetrazolium chloride (TTC); cover with tin foil, and place in a 37°C incubator for 15-30 minutes, turning the brain slices from time to time to ensure even contact with the staining solution. The grayish white color is the infarct area.

[0034] There was no ischemic lesion in the sham control group. The ischemic lesions were obvious in the MCAO group and the bloodletting + MCAO group 3 and 7 days after modeling, and affected the parietal, temporal, frontal cortex, basal ganglia, internal capsule and other parts. The boundary between the lesion and the normal tissue was clear, and the ischemic area was obvious.

[0035] like Figure 8 , Fig. 9 As shown, pathological morphological analysis of brain ischemic tissues in rats of each group

[0036] The results of hematoxylin-eosin (HE) staining showed that the neuronal cells of the rats in the sham control group were arranged neatly and orderly, with intact structure, clear cell membrane, and nucleolus located in the center of the cell. Compared with the sham control group, the neuronal cells of the rats in the MCAO group and exsanguination+MCAO group were arranged irregularly, with condensed nuclei, increased separation, and neuronal cell degeneration.

[0037] Nissl staining results showed that the outlines of the neurons in the sham group were clear and the number was large. Compared with the sham group, the outlines of the neurons in the MCAO group and the exsanguination + MCAO group were unclear and the number was significantly reduced.

[0038] Conclusion: The neurological function score of the bloodletting combined with MCAO group is comparable to that of the MCAO group, and the rotarod fatigue time, grip strength, cerebral blood flow and other aspects are lower than those of the simple MCAO model. It simulates clinical low perfusion cerebral infarction caused by low blood volume.

[0039] The parts not described in detail herein are prior art. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer, and experimental steps not described in detail are referred to the prior art database.

[0040] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention, and modifications or deformations without creative labor are still within the protection scope of the present invention.

Claims

1. A method for establishing a systemic hypoperfusion cerebral infarction model, characterized in that: The eyeball bleeding combined with modified middle cerebral artery occlusion (MCAO) rat model was established to simulate the clinical low perfusion cerebral infarction caused by low blood volume, including the following steps: Step 1: Bleed the rats' eyes with a blood volume of 0.5 ml / 100 g; Step 2: Use the exsanguinated rats to construct a modified middle cerebral artery occlusion (MCAO) rat model, specifically: Step 21, anesthetize the rat by intraperitoneal injection of 1% sodium pentobarbital, the injection dose is 40 mg / kg, and fix the anesthetized rat in supine position; Step 22, prepare the skin of the rat's neck, make a longitudinal incision of about 3 cm in the middle of the neck, bluntly peel off the subcutaneous tissue and muscle groups, and find the left common carotid artery (CCA) and the internal carotid artery (ICA) and the external carotid artery (ECA) at its bifurcation in the deep depression; Step 23, free the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), and ligate the proximal end of the left common carotid artery (CCA) with surgical sutures, about 0.5 cm away from the bifurcation of the left common carotid artery (CCA); ligate the internal carotid artery (ICA) and external carotid artery (ECA), and tie a slipknot with sutures; Step 24: Cut a V-shaped opening between the ligature and bifurcation of the left common carotid artery (CCA), adjust the curvature of the middle cerebral artery occlusion (MCAO) suture toward the midline of the trunk, insert the suture from the cut V-shaped opening, and slowly push it in along the direction of the internal carotid artery (ICA) entering the brain, referring to the bifurcation of the left common carotid artery (CCA). When the advancement length is 17-18 mm, resistance is felt and no further advancement is performed; Step 25: Ligate the left common carotid artery (CCA) firmly with surgical sutures, cut off the suture plug remaining outside the blood vessel to prevent it from being bitten and pulled out by the rat, and suture the skin; Step 3: Set up a sham operation control experimental group: except that the thread plug described in step 24 is not inserted, other operations are carried out according to steps 1 and 2; Step 4: After the rats have been awake for 30 minutes, neurological function scores are performed.

2. The method for establishing a systemic hypoperfusion cerebral infarction model according to claim 1, characterized in that: In step 24, the diameter of the thread plug is selected: for rats weighing 280 g, a thread plug model with a weight of 300 g is selected.

3. The method for establishing a systemic hypoperfusion cerebral infarction model according to claim 1, characterized in that: After suturing the skin in step 25, apply picric acid to the sutured area to prevent the rats from biting each other.

4. The method for establishing a systemic hypoperfusion cerebral infarction model according to claim 1, characterized in that: The indicators observed in the neurological function score in step 4 are: neurological function evaluation (mNSS), rotarod fatigue time, grip strength measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and histological staining.

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