The Study On The Neuronal Injury And The Expression And Regulation Of Caspase-9 And Correlative Factors Following Focal Ischemia- Reperfusion In Rats

Ischemic cerebral vascular disorder (ICVD) is one of the most common leading causes of adult death and disability in many countries. The pathogenesis, pathophysiological mechanism of cell death and therapy of ICVD are the key points of the recent research. Cumulative evidences indicate that apoptosis play an important role in the delayed neuron death after reperfusion. In this research we analyze the mitochondria-related apoptosis pathway and other related factors in ischemic neuronal injury. The result of this study shed light on a new approach to the developing therapeutic strategies for stroke. The cells in the ischemic core die by necrosis, whereas the cells in the inner boundary of the ischemic lesion, "penumbral" regions, die more slowly by apoptosis. The mitochondria-mediated caspase-related apoptotic signal pathway might play an important role inischemic neuronal injury. Neuroprotective therapy plays an essential role in all the therapeutic strategies for ICVD. GM1 can protect against the neuronal injury caused by ischemic injury.Objective: The present study aimed to observe the phenomenon of neuron death and the distribution of injury cells following MCAo (middle cerebral artery occlusion) in rats and clarify the pathophysiological mechanism of ischemic neuronal injury. This study also aimed to examine caspase-9 and Apaf-1 mRNA expression; Bcl-2 and Bax immunoreactivity and caspase protein change in penumbra region and to analyze the spatial and temporal relation between gene expression and neuronal apoptosis so as to understand the molecular mechanism of mitochondria related apoptosis pathway. The other purpose of this study was to investigate the effect of GM1 on neurological outcome and neuron apoptosis following the ischemia-reperfusion injury in rats.Methods: Focal cerebral ischemia was induced by the transient left MCAo in Wistar rats. The behavior scales of neural function of rats was used as a general assessment of brain injury, morphology changes were observed by electron microscope and DNA fragmentation was analyzed by using TUNEL technique. The expression of caspase-9, Apaf-1, Bcl-2 and Bax mRNA and/or protein level were investigated by using semi-quantity RT-PCR, immunohistochemistry and western blottingtechniques respectively. The behavior scales of neural function of rats and the outcome of TUNEL stain were observed in order to evaluate the effects of GM1.Result: The model of transient focal cerebral ischemia-reperfusion in rat by intraluminal MCA blockade with a suture simulated the clinical manifestation and injury distribution of cerebrovascular occlusion diseases in human. Ultrastructure examination showed that there were many neurons characterized by morphological changes of apoptosis. TUNEL-positive cells were more apparent in the perifocal tissue and particularly in the inner border regions immediately adjacent to the ischemic core. During reperfusion after 2h of ischemia, the expression of caspase-9 mRNA changed with time in the ischemic cortex while the expression remained at high level after 48h of reperfusion with peak expression at 16-24h, while in base ganglia the expression returned to baseline after 6h following reperfusion. The 35-37kDa activated caspase-9 hardly could be seen until 2h after reperfusion and remained during the following 48h. The expression of Apaf-1 mRNA did not change significantly in the ischemic cortex in 48h following reperfusion. During reperfusion after 2h of ischemia, the expression of Bcl-2 was induced in the ischemic cortex with peak expression at 6h, and then decreased. The expression of Bax was observed markedly in the ischemic cortex at early stage of reperfusion,while the imraunoreactivity of Bax increased with time. The ratio of Bcl-2/ Bax increased at the beginning of reperfusion with peak at 6h, and than decreased in the following time. The number of apoptotic neurons decreased significantly in GMl-treated group.Conclusions: The focal cerebral ischemia and reperfusion model made by thread embolism of MCA in rat ge...