Effect Of Acute Hyperglycemia On Proliferation Of Neural Stem Cell And Phosphorylation Of CREB In Hippocampus Of Non-diabetes Rats With Acute Cerebral Infarction

Ischemic cerebrovascular diseases are a key object, because its high incidence, high morbidity and high mortality which serious harm to people’s health. Hyperglycemia after acute ischemic stroke is a common clinical phenomenon. And the blood glucose level is closely related to severity of acute ischemic stroke and prognosis. The blood glucose level is too high or too low will worsen the illness and affect prognosis. The influence of acute hyperglycemia to brain in acute cerebral infarction patients without diabetes is neurology controversial research for a long time. The relationship between acute hyperglycemia and degree of brain injury and its mechanism are inconclusive. So, we focused on acute hyperglycemia, discussed the effect of acute hyperglycemia on brain injury and proliferation of neural stem cell in non-diabetes rats after acute cerebral infarction,and tried to give a hand for setting up the best clinical blood glucose goals.Currently, thrombolytic therapy is the only internationally recognized treatment of acute cerebral ischemic stroke,it can make the embolism’s blood vessel recanalization. However, studies have shown that hyperglycemia increased risk of bleeding after thrombolysis in patients with acute cerebral ischemic stroke, increase mortality. In recent years, studies had found that hippocampus neurogenesis after acute cerebral ischemic stroke, endogenous repair mechanisms was activated, then reduced infarct volume and improved the prognosis. Glucose is the sole source of energy in the brain, which plays an important role in the processes. But the acute hyperglycemia in this research is still rare. Whether the effect of acute hyperglycemia in non-diabetes rats after acute cerebral infarction on brain injury was caused by weaken the proliferation capacity of neural stem cells in the hippocampus, that limit the repair ability of itself? Therefore, we simulated the status that acute hyperglycemia in non-diabetes rats after acute cerebral infarction, and used immunofluorescence and western blot method to study the effect of acute hyperglycemia after acute cerebral infarction on proliferation of hippocampal neural stem cells and phosphorylation of CREB. Trying to understand the pathogenesis of acute cerebral infarction in another angle and provide more reference on one hand for acute cerebral infarction patients to control the blood glucose levels.Part Ⅰ The relationship between levels of acute hyperglycemia in non-diabetes rats with acute cerebral infarction and degree of brain injuryObjectives:To establish a model of acute hyperglycemia in non-diabetes rats after acute cerebral infarction which is more convenient, fast and reliable. In addition, blood glucose levels is steady. And explore the relationship between acute hyperglycemia in non-diabetes rats after acute cerebral infarction and ischemic brain injury.Methods:1、The model of MCAO in rats:Rats were occluded the middle cerebral artery (MCAO) with intraluminal filament technique;2、The induced of acute hyperglycemia after MCAO:rats were rendered hyperglycemia with intraperitoneal (i.p.) injections of50%glucose at the indicated time points at5min before MCAO,45min and90min after MCAO. Blood glucose concentrations were detected at30min,75min and120min after MCAO by cutting the tail using glucose meter.3、The induced of various levels of acute hyperglycemia after MCAO and experimental group:SD rats were randomly divided into three groups---normoglycemia group (NG), two hyperglycemia groups(HG1and HG2). Dosage of each groups were the first2ml/Kg、2.5ml/Kg、8ml/Kg and follow two times were2ml/Kg. Blood glucose target of groups(NG、HG1and HG2) were4-5mmol/L、10mmol/L and20mmol/L, respectively. Normoglycemia group (NG) was given0.9%saline.4、Assessment criteria:The behavioral changes of the rats were evaluated using Zea Longa neurological functional scoring system and cerebral infarcti volumes was evaluated by TTC staining24h after cerebral ischemia.Results:1、Blood glucose levels were risen to target that the difference between groups were significant statistically (P<0.001) from the base that was not (P>0.05). Blood glucose levels (mmol/L) as follows, NG group:4.94±0.27-4.44±0.70, HG1group:5.66±0.87-10.05±1.38,HG2group:5.32±1.12-19.87±2.30. Within each group, at those time points----30min,75min and120min after MCAO,there was no significant statistically difference. That is, rats blood glucose levels were stable, and maintained to within120min after MCAO2/Zea Longa score (points) in rats24h after MCAO:NG group:1.4±0.55,HG1group:2.0±0.71, HG2group:2.8±0.45. HG2vs HG1, P=0.09; HG2vs NG, P=0.02; HG1vs NG,P=0.126.3、The percentage of cerebral infarct volumes in rats24h after MCAO (%): NG group:21.48±2.40; HG1group:29.85±6.87; HG2group:49.16±10.50. HG2vs HG1, P=0.030; HG2vs NG, P=0.009; HG1vs NG, P=0.126.4、Pearson correlation analysis: The correlation coefficient for acute hyperglycemia and cerebral infarction volume was0.857(P=0.000). The correlation coefficient for acute hyperglycemia and Zea Longa score was0.727(P=0.000). The correlation coefficient for cerebral infarction volume and Zea Longa score was0.815(P=0.000)Conclusion1、MCAO model combined with intraperitoneal injection of50%glucose solution can establish the model that acute hyperglycemia in non-diabetes rats after acute cerebral infarction.2/Mild hyperglycemic (10mmol/L) does not deteriorate ischemic brain injury in non-diabetes rats.3、Severe hyperglycemic (20mmol/L) obviously deteriorate ischemic brain injury in non-diabetes rats.4、The levels of acute hyperglycemia in non-diabetes rats after acute cerebral infarction were positively correlated with the degree of brain injury.Part Ⅱ The effects of acute hyperglycemia on proliferation in hippocampal neural stem cell of non-diabetes rats with acute cerebral infarction Objective:To discuss the effects of acute hyperglycemia on neural stem cells proliferation in hippocampal of non-diabetes rats with acute cerebral infarction.Methods:1、Using MCAO model combined with intraperitoneal injection of50%glucose solution establish the model that acute hyperglycemia in non-diabetes rats with acute cerebral infarction. And the behavioral changes of the rats were evaluated using Zea Longa neurological functional scoring system to evaluate succession of model.2、Proliferation cell markers:intraperitoneal injection of BrdU (300mg/Kg)3、Hippocampal proliferation cell detection:Using immunofluorescence double standard for BrdU and DCX, and counting cells number of BrdU+cells and BrdU+/DCX+cells in hippocampus. BrdU+cells number was used to assess the hippocampal cell proliferation after rat acute ischemic brain damage; BrdU+/DCX+was used to qualitative the proliferation cells, and be used to evaluate proliferation of neural stem cells in the hippocampus.Results:1、BrdU+cells in the hippocampus: After MCAO24h, BrdU+cells mainly distributed in the dentate gyrus of ipsilateral hippocampus in rats. The number of HG2group was minimal, the different with NG and HG1had statistical significance (p<0.05). There was no statistically significant difference between NG and HG1group(p>0.05)2、BrdU+/DCX+cells in the hippocampus: Distribution of BrdU+/DCX+cells as BrdU+. Comperented with NG and HG1, the number of HG2was reduced significantly, the different with NG and HG1had statistical significance (p<0.01) There was no statistically significant difference between NG and HG1group(p>0.05) Conclusion:1、At the acute after MCAO, mild hyperglycemia may be protection for proliferation of neural stem cells of cerebral ischemic ipsilateral dentate gyrus in non-diabetes rats;2、At the acute after MCAO, severely hyperglycemia reduce proliferation capacity of neural stem cells of cerebral ischemic ipsilateral dentate gyrus, and may aggravate ischemic brain injury in non-diabetes rats.Part III The effects of acute hyperglycemia on phosphorylation of CREB in hippocampus of non-diabetes rats with acute cerebral infarctionObjective:To discuss the effect of acute hyperglycemia on phosphorylation of CREB in hippocampus of non-diabetes rats with acute cerebral infarction.Methods:1、Using MCAO model combined with intraperitoneal injection of50%glucose solution establish the model that acute hyperglycemia in non-diabetes rats with acute cerebral infarction. And the behavioral changes of the rats were evaluated using Zea Longa neurological functional scoring system to evaluate succession of model.2、Proliferation cell markers:intraperitoneal injection of BrdU (300mg/Kg).3、The detection of p-CREB in hippocampal proliferation cell:Using immunofluorescence double standard for BrdU and p-CREB, and counting cells number of BrdU+/p-CREB+cells in hippocampal proliferative cells. BrdU+/p-CREB+cells number was used to assess the expression of p-CREB in hippocampal proliferative cells of non-diabetes rats with acute cerebral infarction. 4、The detection of phosphorylation of CREB protein in hippocampus:By Western blot (Western blot, WB) to detect levels of CREB and p-CREB protein in hippocampus.Results:1、BrdU+cells in the hippocampus:After MCAO24h, BrdU+cells mainly distributed in the dentate gyrus of ipsilateral hippocampus in rats. The number of HG2group was11.00±1.00, and reduced significantly, the different with NG and HG1had statistical significance (p<0.01). There was no statistically significant difference between NG and HG1group(p>0.05)2. BrdU+/p-CREB+cells in hippocampus:Distribution of BrdU+/p-CREB+cells as BrdU+. Comperented with NG and HG1, the number of HG2was3.00±1.00,and decrease than NG and HG1group.The different of HG2with NG and HG1had statistical significance (p<0.01). There was no statistically significant difference between NG and HG1group(p>0.05)3、CREB protein and p-CREB protein levels in cerebral ischemic ipsilateral hippocampus: In cerebral ischemic ipsilateral hippocampus,three groups all expressed CREB and p-CREB protein.The protein levels of each group had statistical significance difference(p<0.01). The ratio of p-CREB/CREB of HG2group was more low than NG and HG1group. The different of HG2with NG and HG1had statistical significance (p<0.01). There was no statistically significant difference between NG and HG1group(p>0.05)Conclusion:1、CREB signaling pathway play an important role in regulation of neural stem cells proliferation. This mainly is influenced by the ratio of p-CREB/CREB.2、The proliferation of neural stem cells in cerebral ischemic ipsilateral hippocampal dentate gyrus are closely related with CREB signaling pathways in non-diabetic rat with acute hyperglycemia after MCAO. This may be regulated by ratio of p-CREB/CREB.