Method for establishing hypoperfusion cerebral infarction model

Abstract

The invention discloses a method for establishing a hypoperfusion cerebral infarction model, which is used for simulating a clinical cerebral infarction animal model caused by low blood pressure by adopting a method for establishing a model for combining hypotension with improved middle cerebral artery occlusion (MCAO) rats. Compared with the prior art, the technical method that existing 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, the composite method that blood pressure reduction is combined with MCAO improvement is used for the first time, and the clinical common pathological state of cerebral blood flow hypoperfusion cerebral infarction caused by hypotension is simulated. According to the invention, the pathologic process of occurrence and development of the disease and the pharmacology of the drug are researched, and the drug action mechanism is explained; a reliable animal model is provided for new drug research and development.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a method for establishing a low perfusion cerebral infarction model. Background Art

[0002] Stroke is a disease with high morbidity, high disability rate, high mortality rate, high recurrence rate and many complications. Stroke includes ischemic and hemorrhagic stroke, among which ischemic stroke is the most common type in clinical practice, accounting for about 70% of all strokes. Ischemic stroke can be divided into atherosclerotic type, small artery occlusive type, cardioembolic type, other causes and unknown causes according to etiology. Cerebral infarction caused by hypoperfusion is a more common type of atherosclerotic type, and its incidence accounts for 10% to 13% of the total number of acute strokes.

[0003] Cerebral perfusion refers to the process by which blood flows through the capillary network to deliver oxygen and nutrients to brain tissue for use. It is generally expressed as the cerebral blood flow (CBF) that passes through local brain tissue per unit time and is an important indicator of brain function. In normal people, the blood flow of gray matter structures such as the cerebral cortex, cerebellar cortex, basal ganglia, thalamus and brainstem is higher than that of white matter. The CBF of the whole brain is about 50ml / (min·100g). If the local cerebral blood flow (rCBF) is less than 23ml / (min·100g), it is a low perfusion area. Patients may experience certain clinical symptoms, which is helpful for the diagnosis of acute ischemic stroke.

[0004] Cerebral infarction caused by hypoperfusion refers to a type of disease in which the patient's intracranial perfusion pressure is lower than normal, resulting in decreased cerebral blood flow, ischemia of brain tissue, and thus symptoms of neurological deficits. There are two main types of cerebral infarction caused by hypoperfusion. One is severe stenosis or occlusion of the neck or intracranial arteries, which leads to local brain tissue hypoperfusion. Clinically, local cerebral ischemia is often seen at the boundary or watershed area of ​​adjacent cerebral vascular supply areas. The hypoperfusion of the junction area controlled by the artery is more obvious, which is called watershed infarction. The other is systemic hypoperfusion, that is, the decrease in systemic perfusion pressure leads to a decrease in brain tissue blood flow, so the ischemic focus is not limited to the watershed area. Common causes are hypotension and heart pump failure (myocardial infarction or severe arrhythmia).

[0005] There is a lack of animal models for basic research on low perfusion cerebral infarction. Most of the current studies use bilateral carotid artery ligation, which simulates the state of chronic cerebral ischemia and does not form ischemic infarction lesions. For research that is more suitable for cognitive impairment, it cannot reflect the pathological process of cerebral infarction caused by low perfusion. The MCAO animal model, which is currently used more, simulates the lesions of cerebral infarction, but fails to reflect the cause of low perfusion. Cerebral infarction caused by low perfusion is mostly caused by factors such as low blood pressure, blood loss, and shock, which lead to ischemic infarction of brain tissue. How to simulate peripheral low perfusion to cause low perfusion of brain tissue to cause cerebral infarction is worth exploring. The model of this patent is to imitate the pathological process of cerebral blood flow hypoperfusion caused by common clinical hypotension, which leads to cerebral infarction. The clinical treatment of cerebral infarction caused by low perfusion is different from that of other cerebral infarctions, and improving perfusion is the main means. Therefore, simulating clinical cerebral infarction caused by low perfusion is particularly important for studying the pathogenesis of this disease, observing the efficacy of drugs, and explaining the mechanism of action. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention discloses a method for establishing a hypoperfusion cerebral infarction model, which is characterized by comprising the following steps: Step 1, prepare compound reserpine suspension, grind 4 tablets of compound reserpine into powder, dissolve in 10 ml 0.9% sodium chloride solution, and store in a refrigerator at 4°C; Step 2, the compound reserpine suspension obtained in step 1 is gavaged at a dose of 0.016 mg / 2.5 ml / kg, once in the morning and once in the evening, and blood pressure is measured and recorded once a day; after continuous administration for 1 week, rats with systolic blood pressure ≤ 110 mmHg or 20% lower than the baseline systolic blood pressure are selected for modified MCAO modeling; Step 3, sodium pentobarbital was injected into the abdominal cavity at a dosage of 4 mg / kg. After anesthesia, the rats were fixed in a supine position, the neck skin was prepared and disinfected, a 3 cm longitudinal incision was made along the middle of the neck, the skin and anterior neck muscles on both sides were separated, the middle incision of the neck was made, the muscle groups were separated, and the right common carotid artery, external carotid artery and internal carotid artery were separated; the common artery and the proximal end of the external carotid artery were ligated respectively, and the internal carotid artery was temporarily clamped by an artery clamp; a small incision was made in the common carotid artery 5 mm away from the bifurcation with a 0.4 mm disposable sterile injection needle, and a silicone thread was inserted and pushed in the direction of the internal carotid artery entering the skull, the common carotid artery preparation line was tightened to prevent bleeding, the internal carotid artery artery clamp was loosened, and the thread plug was slowly inserted until the beginning of the middle cerebral artery; the common carotid artery was double-ligated and the thread was tightly wrapped, the surgical field was cleaned, the incision was sutured, and disinfection was performed, and the operation was completed; a small amount of picric acid solution was applied to the incision to prevent licking and biting of the wound, and a model group was obtained; Step 4: Select a new group of rats and repeat step 3, but do not give compound reserpine and do not insert silicone thread, thus obtaining the sham operation group; Step 5: After 30 minutes of awake activity, the model group and the sham operation group were scored for neurological function, including rotarod fatigue time, blood pressure measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and TTC staining.

[0007] As a preferred technical solution of the present invention, the length of the centerline plug in step three is 17-18 mm.

[0008] Beneficial effects of the present invention: The present invention 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 blood pressure reduction combined with improved MCAO is used to simulate the pathological state of cerebral infarction caused by cerebral blood flow hypoperfusion caused by clinically common hypotension. That is, the 7-day mortality rate of permanent cerebral ischemia MCAO reported in the past is about 50%. This method controls the depth of the ligation to 17-18mm, and the mortality rate is 25.5%, which is statistically different from the mortality rate of 48.5% at a depth of 20mm in the control group. The present invention is for studying the occurrence and development of the disease, the pharmacology of the drug, and the explanation of the drug's mechanism of action; it provides a reliable animal model for the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following will briefly introduce the drawings required for the specific implementation or the prior art description. In all the drawings, each element or part is not necessarily drawn according to the actual scale.

[0010] Figure 1 Comparison of neurological function scores of rats in each group;

[0011] Figure 2 Comparison of rotarod fatigue time of rats in each group;

[0012] Figure 3 Comparison of the gripping strength of rats in each group;

[0013] Figure 4 Comparison of regional cerebral blood flow (r CBF) in rats with cerebral ischemia in each group;

[0014] Figure 5 Comparison of pseudo-color images of regional cerebral blood flow in rats with cerebral ischemia in each group;

[0015] Figure 6 Comparison of blood pressure of rats in each group;

[0016] Figure 7 TTC staining results of rats in each group; DETAILED DESCRIPTION

[0017] Example 1 like Figures 1 to 7As shown, the present invention discloses a method for establishing a low perfusion cerebral infarction model, comprising the following steps: Step 1, prepare compound reserpine suspension, grind 4 tablets of compound reserpine into powder, dissolve in 10 ml 0.9% sodium chloride solution, and store in a refrigerator at 4°C; Step 2, the compound reserpine suspension obtained in step 1 is gavaged at a dose of 0.016 mg / 2.5 ml / kg, once in the morning and once in the evening, and blood pressure is measured and recorded once a day; after continuous administration for 1 week, rats with systolic blood pressure ≤ 110 mmHg or 20% lower than the baseline systolic blood pressure are selected for modified MCAO modeling; Step 3, sodium pentobarbital was injected into the abdominal cavity at a dosage of 4 mg / kg. After anesthesia, the rats were fixed in a supine position, the neck skin was prepared and disinfected, a 3 cm longitudinal incision was made along the middle of the neck, the skin and anterior neck muscles on both sides were separated, the middle incision of the neck was made, the muscle groups were separated, and the right common carotid artery, external carotid artery and internal carotid artery were separated; the common artery and the proximal end of the external carotid artery were ligated respectively, and the internal carotid artery was temporarily clamped by an artery clamp; a small incision was made in the common carotid artery 5 mm away from the bifurcation with a 0.4 mm disposable sterile injection needle, and a silicone thread was inserted and pushed 17 to 18 mm in the direction of the internal carotid artery entering the skull, the common carotid artery preparation line was tightened to prevent bleeding, the internal carotid artery artery clamp was loosened, and the thread plug was slowly inserted until the beginning of the middle cerebral artery; the common carotid artery was double-ligated and the thread was tightly wrapped, the surgical field was cleaned, the incision was sutured, and disinfection was performed, and the operation was completed; a small amount of picric acid solution was applied to the incision to prevent licking and biting of the wound, and a model group was obtained; Step 4: Select a new group of rats and repeat step 3, but do not give compound reserpine and do not insert silicone thread, thus obtaining the sham operation group; Step 5: After 30 minutes of awake activity, the model group and the sham operation group were scored for neurological function, including rotarod fatigue time, blood pressure measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and TTC staining.

[0018] The results are as follows: compared with the sham group, the mNSS scores of rats in the MCAO and hypotension+MCAO groups on days 1, 3, 5, and 7 were higher than those in the sham group, which was statistically significant (p<0.01).

[0019] Comparison of rotarod fatigue time among the groups showed that the rotarod fatigue time of rats in the MCAO and MCAO+hypotension groups was significantly lower than that in the sham group on days 3 and 7 (p<0.01). The rotarod fatigue time of rats in the MCAO+hypotension group was significantly lower than that in the MCAO group on day 7 (p<0.05).

[0020] Comparison of upper limb grip strength in each group showed that the maximum upper limb grip strength of rats in the MCAO and MCAO+hypotension groups on days 3 and 7 was lower than that in the sham group, which was statistically significant (p<0.01). The upper limb grip strength of the MCAO+hypotension group was lower than that of the MCAO group on day 7, which was statistically significant (p<0.05).

[0021] Comparison of cerebral blood flow among the groups showed that compared with the sham group, the percentage of cerebral blood flow perfusion of rats in the MCAO and MCAO+hypotension groups on days 3 and 7 was lower than that in the sham group, which was statistically significant (p<0.01). The percentage of cerebral blood flow perfusion in the MCAO+hypotension group was lower than that in the MCAO group on day 7, which was statistically significant (p<0.01).

[0022] Comparison of blood pressure among rats in each group showed that the blood pressure of the MCAO+hypotension group was significantly lower than that of the sham group and the MCAO group before modeling, and the difference was statistically significant (p<0.01). The blood pressure of the MCAO+hypotension group was lower than that of the MCAO group 1, 3, 5, and 7 days after modeling, and the difference was statistically significant (p<0.01).

[0023] The percentage of cerebral infarction area of ​​rats in each group was analyzed. After the brain was taken, it was immediately placed in a -20 degree refrigerator for quick freezing for about 20 minutes; the brain tissue was cut into 5-6 slices, and one slice was cut every 2 mm. The first cut was made at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut was made at the optic chiasm; the third cut was made at the infundibulum handle; and the fourth cut was made between the infundibulum handle and the caudal pole of the posterior lobe. The slices were placed in 2% 2,3,5-triphenyltetrazolium chloride (TTC); after covering with tin foil, they were placed in a 37°C incubator for 15-30 minutes, and the brain slices were turned over from time to time to make them evenly contact with the staining solution. The gray-white color is the infarction site. There was no ischemic lesion in the sham group. The ischemic lesions were obvious 3 days and 7 days after modeling in the MCAO group and the MCAO+hypotension group, and affected the parietal, temporal, frontal cortex, basal ganglia, internal capsule and other parts. The boundary between the lesion site and the normal tissue was clear, and the ischemic area was obvious.

[0024] The conclusion is as follows: the neurological function score of the hypotensive combined MCAO model is comparable to that of MCAO, and the cerebral blood flow, rotarod fatigue time, grip strength, and blood pressure are all lower than those of the simple MCAO model. The hypotension combined MCAO model has the most obvious decrease in rotarod fatigue time, blood pressure level, and cerebral blood flow. It simulates clinical low perfusion cerebral infarction caused by lowered blood pressure.

[0025] Components not described in detail herein are prior art.

[0026] 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 hypoperfusion cerebral infarction model, characterized in that: The steps include: Step 1, prepare compound reserpine suspension, grind 4 tablets of compound reserpine into powder, dissolve in 10 ml 0.9% sodium chloride solution, and store in a refrigerator at 4°C; Step 2, administer the compound reserpine suspension obtained in step 1 by gavage at a dose of 0.016 mg / 2.5 ml / kg, once in the morning and once in the evening, and measure and record blood pressure once a day; After 1 week of continuous administration, rats with systolic blood pressure ≤ 110 mmHg or 20% lower than the baseline systolic blood pressure were selected for modified MCAO modeling; Step 3, sodium pentobarbital is injected into the abdominal cavity at a dosage of 4 mg / kg. After anesthesia, the rat is fixed in a supine position, the neck skin is prepared and disinfected, a 3 cm longitudinal incision is made along the middle of the neck, the skin and anterior neck muscles on both sides are separated, the middle incision of the neck is made, the muscle groups are separated, and the right common carotid artery, external carotid artery and internal carotid artery are separated; The common artery and the proximal end of the external carotid artery were ligated respectively, and the internal carotid artery was temporarily clamped with an artery clamp; a small hole was punctured at the common carotid artery 5 mm away from the bifurcation with a 0.4 mm disposable sterile injection needle, and a silicone thread was inserted and pushed toward the direction of the internal carotid artery entering the skull, and the common carotid artery line was tightened to prevent bleeding, and the internal carotid artery artery clamp was loosened, and the line plug was slowly inserted until the beginning of the middle cerebral artery; the common carotid artery was double-ligated and the line was tightly wrapped, the surgical field was cleaned, the incision was sutured, and disinfection was performed, and the operation was completed; a small amount of picric acid solution was applied to the incision to prevent licking and biting of the wound, and the model group was obtained; Step 4: Select a new group of rats and repeat step 3, but do not give compound reserpine and do not insert silicone thread, thus obtaining the sham operation group; Step 5: After 30 minutes of awake activity, the model group and the sham operation group were scored for neurological function, including rotarod fatigue time, blood pressure measurement, laser speckle cerebral blood flow imaging, cerebral infarction area measurement, and TTC staining.

2. The method for establishing a hypoperfusion cerebral infarction model according to claim 1, characterized in that: The length of the centerline plug in step three is 17-18 mm.

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