Damage On The Neurons And Its Mechanism After Cerebral Ischemia/reperfusion In Rats With Hyperglycemia

Part I: The damage effects of hyperglycemia on the neurons after focal cerebral ischemia/reperfusion in rats. Objective: To study the damage effect of hyperglycemia on the neurons after focal cerebral ischemia/reperfusion in rats. Methods: A rat middle cerebral artery occlusion/reperfusion (MCAO) model was produced using the intraluminal filament method. The rats were divided into three groups: sham operation group( A ), hyperglycemia control group( B ) and non-hyperglycemia group( C ). After MCAO/reperfusion, the neurobehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenyltetrazolium chloride (TTC) staining. The H₂O% of the brain was calculated with the dry-wet method. The numbers and anatomic distribution of apoptotic cells were examined with terminal deoxynucleotidyl tranferase mediated dUTP-flourescein nick end-labeling (TUNEL) assay. Results: (1) After focal cerebral ischemia/reperfusion in rats, hyperglycemia can aggravate the deficits of the rats,increase the infarction area and increase the H₂O% of the brain of rats. (2) In the hyperglcemia group of rats, TUNEL-positive cells were preferentially located in the penumbral area and progressively increased up to and peaked at 24h after reperfusion, and then decreased at 2d, but still in high level. In the nonhyperglycemia group of rats, TUNEL-positive cells were also mainly in the penumbral area, but the number of the cells were significantly lower than that of the hyperglycemia group. The time-phase pattern of TUNEL-positive cells is similar to that of the hyperglycemia group. Conclusions: These results suggest that the neuronal apoptosis is a dynamic ongoing process. hyperglycemia might play an important role in the aggravation of the reperfusion injury after MACO/reperfusion. Part II: Effects of hyperglycemia on the expression of CytC mRNA and Caspase-3 mRNA after focal cerebral ischemia/ reperfusion in rats. Objective: To study the effects of hyperglycemia on the expression of CytC mRNA and Caspase-3 mRNA after focal cerebral ischemia/reperfusion in rats. Methods: A rat middle cerebral artery occlusion/reperfusion (MCAO) model was made using the intraluminal filament method. The rats were divided into three groups: sham operation group (A) , hyperglycemia group(B) and non-hyperglycemia control group(C). After MCAO/reperfusion, the neurobehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenyltetrazolium chloride (TTC) staining. The expression of CytC mRNA and Caspase-3 mRNA were determined by in site hybridization. Results (1) In the sham operation group and the hyperglycemia group, there was only a few CytC-positive cells were seen in the normal cerebral tissue. In the hyperglycemia group, the upregulation of CytC mRNA began 6h after ischemia/reperfusion, reached a maximum at 6h (cortex)～24h (striatum), then subsided gradually, but still in high level. In the non-hyperglycemia control group, CytC-positive cells were also mainly in cortex and striatum, but the number of the cells were significantly lower than that of the hyperglycemia group. The time-phase pattern of CytC mRNA is similar to the pattern of hyperglycemia group. (2) In the sham operation group and the hyperglycemia group, there is only a few Caspase-3-positive cells were seen in the normal cerebral tissue. In the hyperglycemia group, the upregulation of Caspase-3 mRNA began 6h after MCAO/reperfusion, reached a maximum at 24h and subsided at 48h, but still in high level. In the non-hyperglycemia group, Caspase-3-positive cells were also mainly in the penumbral area, but the number of the cells were significantly lower than that of the hyperglycemia group. The time-phase pattern of Caspase-3 mRNA is similar to that of the hyperglycemia group. Conclusions: The over-expression of CytC mRNA and Caspase-3 mRNA might play a key role in ischemic brain injury after MCAO/reperfusion. Hyperglycemia can increase the over-expression of CytC mRNA and Caspase-3 mRNA in the cerebral cortex, and might play an important role in the damage of ischemic neurons after MCAO/reperfusion. Part III: Effect of hyperglycemia on brain cell apoptosis of rats with focal cerebral ischemia and reperfusion injury Objective To explore the brain cell apoptotic mechanism of wistar rats with focal cerebral ischemia reperfusion injury under Hyperglycemia . Methods Hyperglycemia model was made by injection of streptozocin through abdomen in wistar rats; At the the same time , focal cerebral ischemia injury was made by occluding the middle cerebral artery with lynon line in streptocin-induced hyperglycemic rats and then infarct volume were estimated;The bax and bcl-2 was detected through immunohistochemistry ;At the same time ,the terminal deoxynucleotiolyl transfer erase-mediated dUTP-biotin nick end labeling DNA fragments in situ (TUNEL) were used to detect the change of TUNEL positive cells in every group . Resuls the infarct volume was higher siginificantly in hyperglycemic rats than which of nonhyperglycemic rats .The expression of bax enhanced greatly in hyperglycemic group compared to nonhyperglycemic group and sham-control group,the bcl-2 expression was on the contrary;The number of TUNEL positive cells in the hyperglycemic group was higher than that of nonhyperglycemic group and sham-control group . Conclusion Hyperglycemia aggravated the injury of focal ischmia-reperfusion in wistar rats and the enhanced expression of bax, bcl-2 may be take part in brain cell apoptotic mechanism of focal ischemia reperfusion injury under hyperglycemia. PartIV: Effects of hyperglycemia on the expression of MMP-2 and MMP-9 after focal cerebral ischemia/reperfusion in rats. Objective: To study the effects of hyperglycemia on the expression of MMP-2 and MMP-9 after focal cerebral ischemia/reperfusion in rats. Methods: A rat middle cerebral artery occlusion/reperfusion (MCAO) model was produced using the intraluminal filament method. The rats were divided into three groups: sham operation group, hyperglycemia group and non-hyperglycemia group. After MCAO/ reperfusion, the H₂O% of the brain was calculated with the dry-wet method. The EB of the brain was measured with the method of colorimetry. The expression of MMP-2 and MMP-9 were determined by immunohistochemical analyses. Results: (1) After focal cerebral ischemia/reperfusion in rats, hyperglycemia can increase the H₂O% of the brain. (2)The EB of the brain increased after MCAO/reperfusion, hyperglycemia can increase the EB of the brain. (3) In the sham operation group and the hyperglycemia group, there was only a few MMP-2-positive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of MMP-2 began 24h after ischemia/reperfusion, reached a maximum at 3d～7d, then subsided gradually, but was still in high level at 14d. In the non-hyperglycemia group, the expression of MMP-2 was significantly lower than that of the hyperglycemia group. The time-phase pattern of MMP-2 was similar to that of the hyperglycemia group. (4) In the sham operation group and the hyperglycemia group, there was only a few MMP-9-positive cells were seen in the normal cerebral tissue. In the hyperglycemia group, the upregulation of MMP-9 began 6h after ischemia/reperfusion, significantly increased at 12h, reached a maximum at 2d, then subsided gradually at 3d, however, was no longer observed at 14d. In the non-hyperglycemia group, the expression of MMP-9 was significantly lower than that of the hyperglycemia group. The time-phase pattern of MMP-9 was similar to that of the hyperglycemia group. Conclusions: The over-expression of MMP-2 and MMP-9 might play a key role in ischemic brain edema after MCAO/reperfusion. hyperglycemia can affect the expression of MMP-2 and MMP-9 in the ischemic brain, and might play an important role in the damage of neurons after ischemia/reperfusion. Part V: Effects of hyperglycemia on the expression of uPA and uPAR after focal cerebral ischemia/ reperfusion in rats.Objective: To study the effects of hyperglycemia on the expression of uPA and uPAR after focal cerebral ischemia/reperfusion in rats. Methods: A rat middle cerebral artery occlusion/ reperfusion (MCAO) model was produced using the intraluminal filament method. The rats were divided into three groups: sham operation group, hyperglycemia group and non-hyperglycemia group. After MCAO/reperfusion, the neurobehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenyltetrazolium chloride (TTC) staining. Inaddtion ,immunohistochemistry and RT-PCR were used to detect the expression level of uPA, uPAR in wistar rats. Results: the infarct volume and the scores of neurological deficit was higher siginificantly in hyperglycemic rats than which of nonhyperglycemic rats . the same things happened in the expression of uPA, uPAR in the groups of hyperglycemic rats and nonhyperglycemic rats. Conclusions: Hyperglycemia aggreviates the injury of focal ischmia-reperfusion in wistar rats and the higher expression of uPA, uPAR may be one of the principles in aggraviated focal ischemia reperfusion injury .