Experimental Study On The Interventional Effects Of Tongxinluo On Angiogenesis And Compensatory Function After Focal Cerebral Ischemia

Objective: In this study, directed by collateral disease theory, using craniotomy ligation a side of the middle cerebral artery established the model of focal cerebral ischemia. To explore effectively therapeutic function on neurological recovery of ischemic stroke, we chose its representative prescription: Tongxinluo, and observed its interventional effects on behavioral scoring, morphology of brain tissue, microvascular density, diameter and volumes of blood perfusion, related angiogenesis factors and their expression. The results would be helpful for further providing experimental support for scientific value of collateral disease theory in ischemic cerebrovascular disease.Methods:1 The effects of Tongxinluo on infarction area and neurological behavior of focal cerebral ischemia ratsFocal cerebral ischemia model was developed with male SD rats of health SPF grade. The animals were randomly divided into six groups: the sham group, the model group, Tongxinluo high-dose (2.4g/kg?d) group, middle-dose (1.2g/kg?d) group, low-dose (0.6g/kg?d) group and the nimodipine (10mg/kg?d) group. Using craniotomy ligation a side of the middle cerebral artery established the model of focal cerebral ischemia. Sham group was only pushed aside meninges, not ligation vascular. All groups were irrigated, besides sham group and model group were gave isotonic Na chloride. Then each group was divided into three time points: 3d, 7d and 14d (n=6). Postural Reflex Test, Forelimb Muscle Strength Test and Beam Walking Test were used for neurological behavior grading. HE staining was used to detect the histomorphological changes of brain tissue and TTC staining was used to calculate the proportion of cerebral infarction area.2 The effects of Tongxinluo on microvascular density, diameter and volumes of blood perfusionGrouping and modeling idem. Microvascular density of cerebral cortex ischemia area was tested through immunohistochemical kit at 14d after heart perfusion fixation. Microvascular diameter, morphology and volumes of plasma perfusion of cerebral microvessels, through femoral vein being perfused with FITC-dextran, were analyzed with light microscope (40 times) at 14d after focal cerebral ischemia using laser scanning confocal microscopy (LSCM). The results could be obtained by image reconstruction, and volumes of blood perfusion were represented by percentage accounts of green fluorescent representative plasma capacity and scanning area total capacity.3 The effects of Tongxinluo on related angiogenesis factors and their expressionGrouping and modeling idem. The levels of NO, VEGF in serum were tested through specific kit at 14d after taking blood from carotid artery, taking brain by cutting head and separating serum conventionally. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the mRNA expression level of Ang-1 and Ang-2, and the immunohistochemical staining was used to detect the protein expression of VEGF, Ang-1 and Ang-2.Results:PartⅠ: The effects of Tongxinluo on infarction area and neurological behavior of focal cerebral ischemianeurological behavior scoring: High-dose Tongxinluo could effectively improve the behavior of focal cerebral ischemia model rats at 3d after ischemia (P<0.05), but middle-dose and low-dose had not obvious improving. Tongxinluo high-dose, middle-dose and Nim could improve the behavior of focal cerebral ischemia model rats at 7d after ischemia (P<0.05), but Tongxinluo middle-dose had not obvious improving. Tongxinluo high-dose, middle-dose, low-dose and Nim could improve the behavior of focal cerebral ischemia model rats at 7d after ischemia (P<0.05), and Tongxinluo high-dose group was better than low-dose group.Histomorphological changes: In model group, the ischemic cortex showed necrosis of neuron, swelling of cell, cytoplasm rarefaction, light dyeing and vacuolization, while in the sham group it showed no such pathological changes: the nucleolus was clear, round with intact karyolemma. Treatment groups to a certain extent were ameliorated. Tongxinluo high-dose significantly ameliorated the above-mentioned pathological changes, compared with the model groups, and necrosis cells obviously eliminated.The changes of infarction area: Tongxinluo high-dose and middle-dose groups could inhibit the infarction volume remarkabably at 3d after focal cerebral ischemia (P<0.01), and Tongxinluo high-dose group was better than middle-dose group (P<0.05). Tongxinluo high-dose, middle-dose groups and Nim could inhibit the infarction volume at 7d after focal cerebral ischemia (P<0.05). Tongxinluo high-dose and middle-dose groups could inhibit the infarction volume at 14d after focal cerebral ischemia (P<0.05, P<0.01).PartⅡ: The effects of Tongxinluo on microvascular density, diameter and volumes of blood perfusionHistochemical staining result was described as below: CD31 was mainly expressed in the treatment groups and model group of the brain cortex, and CD31 expression of Tongxinluo high-dose, middle-dose and Nim groups were intensive, while sham group was light dyeing of microvascular density. In treatment groups, Tongxinluo high-dose group was strongest dyeing.The results of microvascular diameter and morphology: Compared with sham group, the microvessel of model groups was irregular shape. The microvessel of treatment groups was significantly irregular shape, compared with model group, and Tongxinluo high-dose group was significantly irregular shape, numerous microvascular branch, and close connection between microvessels. The microvascular diameter of sham group was 6.80±0.93μm, and 35% of which was 6-7μm, but the rest of the paragraphs was less. The microvascular diameter of model group was 5.17±0.53μm, most (40%) of which was 5-6μm. The microvascular diameter of Tongxinluo high-dose group was 3.35±0.09μm, most (41%) of which was 2-3μm, but the contralateral brain microvascular diameter was 3.56±0.23, most (35%) of which was 3-4μm. The microvascular diameter of TXL-M group was 3.50±0.43μm, most (40%) of which was 3-4μm, but the contralateral brain microvascular diameter was 3.63±0.57, most (32%) of which was 3-4μm, but the rest of the paragraphs was less. The microvascular diameter of TXL-L group was 4.35±0.22μm, most (40%) of which was 4-5μm, but the contralateral brain microvascular diameter was 4.52±0.38, most (30%) of which was 5-6μm.The results of volumes of blood perfusion: Compared with model group, volumes of blood perfusion of Tongxinluo high-dose, middle-dose and Nim groups were significantly increased (P<0.05, P<0.01). In contralateral hemisphere, Tongxinluo high-dose group was significantly increased, compared with model group (P<0.05).PartⅢ: The effects of Tongxinluo on angiogenesis related factors and their expressionThe levels of NO, VEGF in serum: The levels of NO in serum of Tongxinluo high-dose and middle-dose group were significantly increased (P<0.05, P<0.01), and Tongxinluo high-dose group was more significant (P<0.05). The contents of VEGF in serum of Tongxinluo high-dose group was also significantly increased (P<0.05, P<0.01), compared with Tongxinluo low-dose and Nim group.The expressions of VEGF, Ang-1, and Ang-2 after focal ischemia: The expression of VEGF was slightly positive in sham group, but increased obviously in Tongxinluo high-dose group at 14d after focal ischemia, compared with model group. Other treatment groups had presented augmentative tendency, but there was no significant difference between these groups due to the extensive standard deviation inter-group. The expression of angiopoietin was little positive in sham group. The expression of Ang-1 in Tongxinluo high-dose and middle-dose group was intensified, compared with model group, but the expression of Ang-2 in Tongxinluo high-dose group was only. Other treatment groups had presented augmentative tendency, but there was no significant difference between these groups due to the extensive standard deviation inter-group.The expression of Ang-1 and Ang-2 mRNA: The expression of Tongxinluo high-dose and middle-dose group both increased remarkably, compared with model group (P<0.05), and Tongxinluo high-dose group increased more remarkably compared with Tongxinluo low-dose and Nim group (P < 0.05). Other treatment groups had presented augmentative tendency, but there was no significant difference compared with model group due to the extensive standard deviation intergroup.Conclusion:In conclusion, Tongxinluo could increase the levels of NO, VEGF in serum, obviously increase the expression of VEGF and angiopoietin protein, and obviously increase the expression of angiopoietin mRNA in cerebral cortex after focal cerebral ischemia. Tongxinluo could promote angiogenesis and compensatory increase volumes of blood perfusion of ischemia area. Thus, Tongxinluo could reduce neurons damage, decrease the cerebral infarction area, and improve neurological recovery, which could be one of the important mechanisms of effective therapy effect after focal cerebral ischemia.