| 1.ObjectivePrevious study showed that Qi and Yin Deficiency and blood stasis were the most common Traditional Chinese Medicine (TCM) syndromes of diabetes-associated cerebral ischemia. Here, we establish animal models for the diabetes-associated cerebral ischemia due to Qi and Yin deficiency and blood stasis, and investigate the impact of diabetes on the spatial memory and nerve regeneration in hippocampus and BDNF expression in the cerebral ischemia rats. Furthermore, we evaluate the outcome of TCM of strengthening Qi, maintaining Yin, and promoting blood circulation in treatment of diabetes-associated cerebral ischemia model rats in vivo and in vitro and reveal the role of BDNF in neural regeneration.2.Methods2.1 We used continuous exhaustive swimming, gavage with thyroxine tablets suspension, and intraperitoneal injection of STZ composite preparations to establish the rat model for the diabetes-associated cerebral ischemia due to Qi and Yin deficiency and blood stasis. We explored the possibilities of the rat model for the diabetes-associated cerebral ischemia due to Qi and Yin deficiency and blood stasis from the aspects of glucose, nervous function, brain morphology, general state, tongue and the overall survival rates.2.2 Thirty rats were divided into 3 groups:1) sham,2) cerebral ischemia, and 3) diabetes-associated cerebral ischemia group (n=10 for each group). The BrdU incorporation assay was used to investigate the impact of diabetes on the proliferation, differentiation, and survival of neuron in the rat DG in response to cerebral ischemia. We also employed the immunohistochemistry to analyze the expression of BDNF and used the Morris water maze test to evaluate the effect of diabetes on the spatial learning and memory of cerebral ischemia rats.2.3 We set the following experimental groups:1) sham,2) diabetes-associated cerebral ischemia,3) diabetes-associated cerebral ischemia rats treated with TCM of benefiting Qi and maintaining Yin,4) diabetes-associated cerebral ischemia rats treated with TCM of benefiting Qi, maintaining Yin, and promoting blood circulation,5) diabetes-associated cerebral ischemia rats treated with TCM of benefiting Qi and maintaining Yin and K252a (an inhibitor of BDNF Trk receptor), and diabetes-associated cerebral ischemia rats treated with TCM of benefiting Qi and promoting blood circulation (n=10 for each group). We used Ginseng,Ligustrazine,Fragant solomonseal rhizome and Polygonatum Kingianum, which have been shown to benefit Qi, maintain Yin, and promote blood circulation. The BrdU incorporation assay was used to investigate the impact of diabetes on the proliferation, differentiation, and survival of neuron in the rat DG in response to cerebral ischemia. We also employed the immunohistochemistry to analyze the expression of BDNF and used the Morris water maze test to evaluate the effect of diabetes on the spatial learning and memory of cerebral ischemia rats. 2.4 The cultured neural stem cells were randomly divided into four groups:A) cultured in medium supplementing with normal rat sera, B) cultured in medium supplementing with sera from rats undergoing cerebral ischemia reperfusion, C) cultured in medium supplementing with high concentration glucose and sera from rats undergoing cerebral ischemia reperfusion, and D) cultured in medium supplementing with high concentration glucose and sera from rats undergoing cerebral ischemia reperfusion and treated with TCM for supplementing Qi, nourishing Yin, and promoting blood circulation. The proliferation of neural stem cells was measured using the 5-bro-modeoxyuridine (BrdU) incorporation assay. The activity of neural stem cells was determined using methyl thiazolyl tetrazolium colorimetry.3.Results3.1 Establishment of animal modelsWe used continuous exhaustive swimming, gavage with thyroxine tablets suspension, and intraperitoneal injection of STZ composite preparations to establish the rat model for the diabetes-associated cerebral ischemia due to Qi and Yin deficiency and blood stasis. The results showed that the overall successful rate for establishment of the disease model was 63%. The results showed that the model rats undergoing such treatments displayed phenotypes including tongue symptoms, neurological defects in brain morphology. The overall survival rate was consistent with the comprehensive evaluation index evaluation system for such animal models.3.2 The impact of diabetes on the spatial memory and nerve regeneration in hippocampus and BDNF expression in the cerebral infarction rats.Our current studies revealed that sustained long-term high blood sugar could increase damage of spatial learning and memory of the cerebral infarction rats in the Morris water maze test (P<0.05). We further investigated the impact of diabetes on neuron regeneration in cerebral infarction rats and found that diabetes significantly inhibited the survival and differentiation of newly proliferating neuron in the cerebral infarction rats(P<0.001), but it had little effect on facilitating neuron proliferation (P>0.05). Moreover, we found that the BDNF expression in the hippocampus of the diabetes-associated cerebral infarction rats was significantly lower than that in the hippocampus of the cerebral infarction without diabetes (P<0.001). Because BDNF expression is critical for neuron regeneration, our data suggested that diabetes might decrease the expression of BDNF, leading to inhibition of neuron differentiation and survival. This in turn affected the spatial learning and memory of cerebral isochemia patients.3.3Treatments of the diabetes-associated cerebral ischemia rats with TCM for benefiting Qi, maintaining Yin, and promoting blood circulation promote the recovery of spatial learning and memory via induction of BDNF-mediated nerve regeneration.Diabetes-associated cerebral ischemia increased the number of newly proliferating cells in the lower hippocampal dentate gyrus granule, but the treatments with these TCM did not increase the number of newly proliferating cells (P>0.05). It suggested that these TCM did not directly target the cellular proliferation pathway. On the other hand, we observed that the TCM for benefiting Qi, maintaining Yin, and promoting blood circulation or the TCM for benefiting Qi and promoting blood circulation considerably increased the survival rate and promoted the differentiation of newly proliferating neuron (P<0.05). We further found that both TCM significantly increased BDNF expression in the hippocampus of diabetes-associated cerebral ischemia mice(P<0.01) and improved their capability of spatial learning and memory (P<0.05-0.01).In addition, the beneficial effect of the TCM for benefiting Qi, maintaining Yin, and promoting blood circulation was considerably greater than the ones of the TCM for benefiting Qi and promoting blood circulation only (P<0.05) Furthermore, intracerebroventricular injection of the BDNF inhibitor K252a abolished the beneficial effects of the TCM on the survival and differentiation of newly proliferating cells (P<0.001) and on the spatial learning and memory (P<0.001). It suggested that induction of BDNF-mediated neural regeneration and differentiation to neuron plays a key role in the improvement of spatial learning and memory by the TCM for benefiting Qi, maintaining Yin, and promoting blood circulation.3.4 Sera containing medicine of supplementing Qi, nourishing Yin and promoting blood circulation increase cell proliferation and viability of neural stem cells of rats with high blood glucose and ischemia/reperfusion injuries.We found that sera containing medicine of supplementing Qi, nourishing Yin and promoting blood circulation could significantly increase proliferation (P<0.01) and viability of neural stem cells (P<0.01). MTT results showed that the viability of neural stem cells significantly decreased after reperfusion (P<0.001). Under high glucose tissue culture conditions, the viability of the cell after perfusion further decreased (P<0.05-0.01) compared to the low glucose control group. However, treatments of the neural stem cells with Sera contaning medicine of supplementing Qi, nourishing Yin and promoting blood circulation greatly increased the cellular viability and proliferation (P<0.01). These findings suggested that the joint use of Ginseng, Polygonatum, and Chuanxiong might promote the viability and proliferation of neural stem cells in rats with high blood glucose and ischemia/reperfusion injuries.4.Conclusion4.1 It is feasible to use continuous exhaustive swimming, gavage with thyroxine tablets suspension, and intraperitoneal injection of STZ composite preparations to establish the rat model for the diabetes-associated cerebral ischemia due to Qi and Yin deficiency and blood stasis4.2 Diabetes might inhibit BDNF expression, thus suppressing survival and differentiation of newly regenerated nerve cells in the hippocampus of the cerebral infarction rats and consequently affecting spatial learning and memory of cerebral infarction rats. 4.3 TCM for benefiting Qi, maintaining Yin, and promoting blood circulation promote the recovery of spatial learning and memory via induction of BDNF-mediated nerve regeneration.4.4 Chinese prescription of supplementing Qi, nourishing Yin and promoting blood circulation can promote the proliferation and the activities of neural stem cells cultured in high concentration glucose medium from rats undergoing cerebral ischemia reperfusion.
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