| ObjectiveIschemic stroke has become an important cause of mortality and disability worldwide, and the risk of ischemic stroke in the diabetes patients is more significant than non-diabetic patients, and many studies have confirmed that diabetes can aggravate the condition of ischemic stroke and increased mortality rate. Endothelial progenitor cells (EPCs) is a kind of stem cells present in the adult bone marrow, released to the circulation, differentiating into mature endothelial cells, that counteract endothelial cells injury and replace dysfunctional endothelium and form blood vessels and augment neovascularization. However, level of circulating EPCs in the rats with focal cerebral ischemic reperfusion injury and with the risk factor diabetes is inconclusive when the situation changes; the associated with level of circulating EPCs and the setting of acute stroke has not been clarified; mechanism of level of circulating EPCs with stroke remains unclear. With the application of normal rats and experimental diabetic rats induced by 1% streptozotocin (STZ) solution, establishing focal cerebral ischemic reperfusion injury cases, observe level of circulating EPCs with brain tissue ischemic injury, and the expression of brain tissues' VEGF, bFGF and eNOS; to investigate changes of level of circulating EPCs cells in normal and diabetic rats with focal cerebral ischemia reperfusion injury and its significance and mechanisms for the prevention and treatment of ischemic stroke provides a theoretical basis.Methods(1) One hundred healthy adult male SD rats were randomly divided into normal control group (n=10), sham operation group (n=5), cerebral ischemia and reperfusion model group (CIR group, n=30), diabetes mellitus model group (n=5), diabetes sham operation group (n=5), and diabetes with CIR group (n=30). The models of experimental diabetes rats were established by a single intraperitoneal injection of STZ (60 mg/kg body weight). The ischemia stroke models establish the models of focal cerebral ischemia-reperfusion rats by Longa's technique.(2) The assessment standard of Longa' 5 scores was applied to assess the neural function of the rats after cerebral ischemia-reperfusion, as neurologic deficit score (NDS).(3) 24h after the reperfusion, some blood was taken in the ophthalmic vein from the rats. Level of circulating EPCs was examined using by flow cytometry.(4) 24h after the reperfusion, rat brains were removed successfully, fresh brain tissues were cut into 2mm thick slices and placed in 1% 2,3,5-triphenyl tetrazolium chloride (TTC) solution,and the volumes of staining in the infarct tissue were calculated.(5) The rats after 24h of the reperfusion, brains were removed, set in 4% paraformaldehyde solution and fixed, analyzed by immunohistochemistry in brain tissue VEGF, bFGF, and eNOS positive cells.(6) The data was analysed by SPSS 17.0 statistical software, expressed as mean±standard deviation (χ±s), and some groups were analyzed using single factor analysis of variance, and the two groups were compared by t test. Statistics dealing with multiple groups were analyzed using analysis of variance, two groups were compared by a number of q test, P<0.05 was considered as significant criterion.Results(1) The general situation of the normal rats and the diabetic rats The comparison of the quality and blood glucose between the diabetes rats and normal rats, diabetes rats appeared the diabetic symptoms such as drink more, eat more, urine more and weighting loss after the model made than the normal rats. The diabetic model group s' quality [(196.7±8.6) g] was significantly lower than control groups' [(250.8±17.7) g]; their blood glucose [(32.2±6.4) mmol/L] was significantly higher than the normal control groups' [(4.4±0.7) mmol/L] (all P<0.01).(2) The NDS of every group The normal control group, the sham operation group, the diabetic control group and the diabetic sham operation group had no significant nerve damage, the NDS was 0 point, the diabetic CIR group's NDS was (2.8±1.0),that was significantly higher than the CIR model group's (1.5±0.3) (P<0.05).(3) The TTC staining results of the rat's brain The volume of cerebral infarction in the diabetes CIR group was [(464.1±169.3) mm3], which was significantly greater than the CIR model group [(101.3±57.4) mm3] (all P<0.01).(4) The number of peripheral blood EPCs in every group The level of circulating EPCs in the other groups comparing with the normal control group and the normal sham operation control group, level in the CIR group significantly increased, and the level in the diabetic control group,the diabetic sham operation control group and the diabetic CIR group significantly reduced; comparing with the diabetic group and the diabetic sham operation group, the diabetic CIR group significantly reduced the level of circulating EPCs (all P<0.01).(5) The results of immunohistochemistry in brain tissue Comparing with the normal control group and the sham operation group, the expression of VEGF(expressed by neurons, glial cells,stellate cells), bFGF (expressed by endothelial cells) and eNOS (expressed by the endothelial cells) in the brain of the CIR group, was significantly increased;the diabetic control group and the diabetic sham operation group, the corresponding cells was no stained; the expression of VEGF, bFGF, and eNOS in the diabetic CIR group's brain tissues was slightly higher than the diabetic control group and the diabetic sham operation group (all P<0.01).Conclusion(1) The increasing level of circulating EPCs in the CIR group can help repair the impaired essential cells and protect brain tissue. (2) The level of circulating EPCs in the diabetic rats significantly reduced, and it in the diabetic CIR rats with decreased much more significantly.(3) Level of circulating EPCs with ischemic stroke may help predict the prognosis of disease.(4) The brain of normal rats with acute cerebral infarction, expressed a large number of VEGF, bFGF and eNOS, which mobilize the EPCs from the bone marrow to peripheral blood and angiogenesis in ischemic brain.(5) Level of circulating EPCs in the diabetes rats significantly reducing may be associated with the decreasing content of VEGF, bFGF, and eNOS in the brain tissue; the diabetic rats with focal cerebral ischemia reperfusion injury, although in the brain tissue the content of VEGF, bFGF, and eNOS increased slightly, but the number of peripheral blood EPCs reduced significantly, the mechanism was not entirely related with the damage of diabetes to the mobilization of EPCs. |
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