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Effect of Buyanghuanwu Decoction on Neurological Function and Cytomorphology in Rats with Cerebral Ischemia
Effect of Buyanghuanwu Decoction on Neurological Function and Cytomorphology in Rats with Cerebral Ischemia

Cerebral ischemia belongs to the category of stroke, which is a common clinical disease with the characteristics of high incidence, high mortality and high disability rate, which seriously harms human health. So far, there is still a lack of effective chemotherapy drugs to treat cerebral ischemia and its sequelae at home and abroad. Evidence-based medicine shows that Chinese medicine has certain advantages in treating ischemic stroke. At present, a lot of research focuses on the protective effect of traditional Chinese medicine on acute cerebral ischemia injury. However, the functional recovery after cerebral ischemia is a long process, and there has been little research on it for a long time. Buyanghuanwu Decoction is a classic prescription for the treatment of ischemic stroke and sequela of stroke, which comes from Yilin Gaicao written by Wang Qingren in Qing Dynasty. This prescription is widely used in clinic and has definite curative effect, but its mechanism of promoting the recovery of neurological function needs to be further clarified. In this paper, the model of focal cerebral ischemia in rats was established by occlusion of middle cerebral artery, and the long-term protective effect of Buyanghuanwu decoction on its neurological function was discussed, in order to provide experimental basis for clinical medication of ischemic stroke.

1 material

1. 1 animal healthy male SD rat, 189, SPF grade, weighing 260 ~ 280 g, purchased from slack Jingda Experimental Animal Co., Ltd., Hunan Province, with the production license number SCXK (Hunan) 2009-0004 and the animal certificate number 04535.

1 prescription. 2 Buyanghuanwu Decoction comes from Wang Qingren's Lin Yi Gaicuo, which consists of Astragalus membranaceus 120 g and Paeonia lactiflora 4. 5 g, Ligusticum Chuanxiong 3 g, Guiwei 6 g, dried earthworm 3 g, safflower 3 g and peach kernel 3 g, all of which are in line with China Pharmacopoeia 2005 edition. Preparation of Buyanghuanwu decoction: soak that decoction pieces in clean wat for 30 min, adding medicinal materials and 5 times of double distil water for 60min in the first decoction, adding 5 times of double distilled water for 60 min in the second decoction, mixing the decoctions evenly, concentrating until the crude drug is 2g ml-1,and refrigerating for later use.

1.3 reagent 10% chloral hydrate (Yangzhou aoxin auxiliary factory), paraformaldehyde (Changsha Jinhua chemical co., ltd.), TTC (Shima red tetrazole company).

1.4 instrument Bx5 1 optical microscope and IPP5. 1 image analysis system (Olympus, Japan), FA electronic balance (Shanghai Taizhiheng Electronic Weighing Instrument Co., Ltd.), automatic double pure water distiller (Shanghai Glass Instrument Factory No.1), Finesse 325 slicer (Yamato, England).

Two methods

2. 1 Grouping and administration of rats were randomly divided into sham operation group, model group and Buyanghuanwu decoction group, with 63 rats in each group. Each group of animals was divided into three time points, namely 7, 14, 2 1 day after the first administration, and 2 1 animal at each time point. The animals in Buyanghuanwu decoction group began to take medicine 2 hours after operation, and the dosage was 5g kg-1 d-1,ig 1 time, daily1time. The model group and sham operation group were given equal volume distilled water.

2.2 Model Preparation and Evaluation References: Rat model of focal cerebral ischemia induced by middle cerebral artery occlusion: 10% chloral hydrate (3 3. 5 mL kg- 1) ip anesthesia. The median neck incision is about 3cm long, which is separated layer by layer. The external carotid artery is ligated, the internal carotid artery is clamped, and then the common carotid artery is ligated, about below the bifurcation of the common carotid artery. The sham operation group only cut the skin, separated the left common carotid artery and sutured it. After the operation, put the animals into a feeding box with clean stuffing, and they can drink and eat freely. After waking for 2 hours, animals were scored according to Longa and Bederson's five-point method, and those who scored 1 ~ 3 were enrolled in the group. 0: No symptoms of nerve injury; 1 min: the front paw of the opposite side cannot be fully extended; 2 points: Turn opposite; 3 points: dumping to the opposite side; 4 points: unable to walk autonomously and lose consciousness. The higher the score, the more serious the neurological deficit, and vice versa. Exclusion criteria: the score is lower than1; Subarachnoid hemorrhage; HE staining showed no pathological changes of cerebral ischemia; Die before time. When animals such as death are insufficient, they are randomly replaced.

2.3 Index detection

2.3. The cerebral infarction area ratio of 5 rats in each group at each time point was measured by 1 TTC staining method. After anesthesia with 10% chloral hydrate, the brain was decapitated and frozen at -20℃ for 20 min, and cut every 2 mm from the forehead 1 tablet. 1 the knife is at the midpoint of the line between the anterior pole of the brain and the optic chiasma; The second knife crosses in sight; The third knife is at the funnel handle; The fourth knife is between the funnel handle and the tail pole of the posterior lobe, with 5 pieces of ***A, B, C, D and E, stained with 2% red tetrazolium, and protected from light at 37℃ for 30 min. The normal tissue is dark red and the infarcted tissue is white. After taking pictures, the infarct area of the largest ischemic slice C was selected, and scanned and calculated by Image-Pro Express image analysis system, indicating the percentage of infarct area in the total brain area of slice C. In order to eliminate the error caused by cerebral edema in infarcted hemisphere, Swanson method was used to calculate the percentage of infarcted area on C slice after correction. Is = (s1-Sr)/2s/kloc-0 /×100% s1:the total area of the healthy side of the C piece; Sr: the area of non-infarcted area on the affected side of C film.

2.3.② Neurological function score: At each time point, rats in each group were scored 10, 2 hours after operation and 1 hour before the animals were killed.

2.3.3 Histopathological changes of hippocampus and cortex were observed by HE staining, with 6 rats in each group at each time point. Use the same method as 2. 3. 1, 4% paraformaldehyde was taken from the back of the brain and fixed for 24 h, dehydrated by gradient ethanol, embedded in conventional paraffin, and the brain was continuously sliced (with a thickness of 5 microns). The slices containing hippocampus were stuck on the glass slide, stained with he, and the morphological and pathological changes of the left hippocampus and cortical tissue cells in each group were observed and compared under the light microscope.

2.4 Statistical analysis

All data were expressed by X S, and the data were statistically analyzed by SPSS 19. 0. One-way ANOVA was used for comparison between groups, P

Three results

3.TTC staining compared the area of cerebral infarction in 1 group, and the sham operation group was evenly dyed deep red. 7, 14, 2 1 D model group and Buyanghuanwu decoction group showed uneven staining, and the local infarction area was white, and the infarction area was around the ischemic parietal cortex and hippocampus. The analysis of infarct area showed that the infarct area in the model group and Buyanghuanwu decoction group gradually decreased with time (P

3.2 The neurological function score of each group was 0 in sham operation group at each time point; The neurological function score of Buyanghuanwu decoction group was lower than that of model group, and the difference was statistically significant (P < 0). 05) 。

3.3 Morphological changes of hippocampus and cortex in each group

3.3. 1 Hippocampus sham-operation group has normal morphology, clear cell outline, uniform nuclear staining, orderly arrangement of cells and uniform interstitial staining. In 7, 14 and 2 1 d model groups, hippocampal neurons were shriveled, the nucleus was condensed, the nuclear margin was serious, vacuoles appeared in cells, the cell morphology changed from oval to spindle and triangle, the cell spacing increased, interstitial staining was sparse and there were holes. 7, 14, 2 1 d Compared with the model group, the number of vacuole-like cells in hippocampus was significantly reduced, the cell morphology was significantly improved, and the cell arrangement was more orderly. 7, 14 d Buyanghuanwu decoction group still has a few nuclei with deep staining and vacuolar changes, but the morphology of hippocampal cells in Buyanghuanwu decoction group is close to that in sham operation group.

3.3.2 The cortical cells in cortical sham-operation group were normal in morphology, with clear edges, neat arrangement and complete morphology, and the nuclei and intercellular substance were evenly stained. 7. In 14 and 2 1 d model groups, ischemic cells showed vacuolar necrosis, cell morphology lost, intercellular space increased, staining became shallow and showed reticular changes; The volume of remaining cells is reduced, the nucleus is deeply concentrated and stained, and the cell boundary is unclear. Compared with the model group in the same period, the number of vacuole-like cells in cortical cells in Buyanghuanwu decoction group was significantly reduced, the cell morphology was significantly improved, the cells were arranged neatly, the nucleus was clearer and the intercellular space staining was more uniform.

4 discussion

Focal cerebral ischemia, also known as ischemic stroke, is the most common clinical type of cerebrovascular disease. It is due to the disorder of blood supply to the brain, which leads to ischemia and hypoxia of brain tissue, necrosis of cerebral infarction in corresponding areas, loss of a large number of neurons, and corresponding symptoms and signs of neurological deficit. At present, the main methods of western medicine to treat cerebral ischemia are thrombolysis, anticoagulation, defibrasion, vasodilation, hyperbaric oxygen, interventional therapy, gene therapy, neural stem cell transplantation, etc., which are still in the animal experimental stage. Among these treatments, the effective rate of thrombolytic therapy is as high as 2 1% ~ 93% except ultra-early (within 3 hours of onset), and the effects of other treatments are uncertain. Moreover, due to its strict time window, thrombolytic therapy means that less than 5% patients have the opportunity to do this treatment. Therefore, about 80% patients with ischemic stroke will leave different degrees of sequelae-neurological deficit.

Typical ischemic stroke can be divided into three stages according to the course of disease. Acute phase 1, after stroke 1 month; The second recovery period, within 2 ~ 6 months after stroke; Stage 3 sequela, 6 months after stroke. Buyanghuanwu decoction is mainly used in the treatment of cerebral ischemia recovery period and sequelae period, and it is the first choice for the treatment of cerebral ischemia sequelae, with remarkable clinical effect. Therefore, in animal experiments, the longest observation time is 2 1 d after ischemia, which is equivalent to the sequelae of human cerebral ischemia. In this experimental study, the results of neurological function score show that Buyanghuanwu decoction can promote the recovery of neurological function in rats after cerebral ischemia. Its mechanism of action is considered by scholars to promote angiogenesis, dilate blood vessels, improve local microcirculation of cerebral ischemia by anticoagulation and thrombolysis, reduce brain edema and oxygen free radical damage, protect brain tissue, and thus promote the recovery of neurological function. Studies in our laboratory and other domestic laboratories show that Buyanghuanwu decoction can promote the proliferation, migration and differentiation of neural stem cells in the brain after ischemia, which may be one of its mechanisms to promote the recovery of neural function after ischemia. By comparing the ratio of cerebral infarction area of animals in each group, the results showed that the cerebral infarction area of Buyanghuanwu decoction group was significantly smaller than that of the model group at the same time. The possible reasons are as follows: 1. Buyanghuanwu decoction has a protective effect on the brain during acute cerebral ischemia, inhibiting the death and apoptosis of a large number of neurons and making the infarct area smaller; Secondly, Buyanghuanwu decoction promotes the regeneration, repair and compensation of ischemic brain tissue, resulting in a smaller infarct area than the corresponding model group; It is also possible to have both. In addition, by comparing the histological changes of hippocampus and cortex between Buyanghuanwu decoction group and model group after ischemia, the results show that Buyanghuanwu decoction can obviously improve the cell morphology and arrangement after cerebral ischemia injury and promote tissue repair.

In a word, this study confirmed that Buyanghuanwu decoction has the brain protection function of reducing the neurological function score of cerebral ischemia, reducing the area of cerebral infarction and improving the morphology and arrangement of brain tissue cells, which provided a strong experimental basis for the clinical treatment of ischemic stroke with this medicine.

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