| BackgroundCerebrovascular disease(CVD) is one of the three main diseases which can make human dead. Its feature is acute neurological dysfunction, and its main manifestation is acute hemiplegia, aphasia, convulsion, disturbance of consciousness, increased intracranial pressure and so on. It can be divided into ischemia and hemorrhage on the basis of pathological characteristics and ischemia accounts for more percentage. Cerebral infarction, myocardial infarction, shock, neonatal asphyxia, neonatal hypoxic ischemic encephalopathy and brain trauma all can cause cerebral ischemia injury. There are perfect in vivo and in vitro cerebral ischemia models. The in vivo models include global cerebral ischemia model and focal cerebral ischemia model.The in vitro models include hypoxia reoxygenation model and oxygen-glucose deprivation model. But none of the models can imitate human complex cerebral ischemia pathological process. The research for ischemic cerebrovascular disease focuses on the effect of ischemia reperfusion to the final infarct volume at present. It is generally believed that apoptosis is the main factor that caused ischemia reperfusion injury. Researchers were very interested in neuron for the mechanisms of apoptosis, but payed little attention to the extensive and functional glial cells.Cysteinyl aspartate-specific protease-3is an apoptotic factor that people investigated more and had been very familiar with it. It is the most critical protease in the downstream of apoptotic cascade reaction of cells and the only way to apoptotic program stimulated by various factors. Various study has found astrocytes can express Caspase-3after brain injury, and the expression is proportional to the apoptosis index. This tells us that Caspase-3is also related to neuroglial cell apoptosis.As the transmembrane channel, gap junction (GJ) is made of two hemichannels, one in each of the apposed membranes. Connexins(Cxs) form hemichannels and gap junction channels.As the most abundant Cx family member, Cx43has been confirmed to exist in neuroglial cell of human and rats. It is believed that gap junction may have some effect on the diffusion or repairment of brain injury, because gap junction can transport venenous metabolites and energy. Blockers of gap junction had been found to lessen the injury caused by cerebral ischemia or ischemia reperfusion and help to reduce infarct size. But the cells or tissues may be susceptible to injury if inhibiting the formation of gap junction in gene. So there are controversies about the conservation effects of gap junction to brain injury, which may due to the little specificity of gap junction blockers. It has been found that the unpaired hemichannels can survive alone and open in some pathological and physiological conditions. The opening of hemichannels can aggravate cell apoptosis and brain injury. More and more research indicates that gap junction and hemichannels have close relationship to wound healing, myocardial infarction, arrhythmia, cerebral infarction, epilepsy, tumor, etc. Cx in mitochondria and nucleus may inhibit cell death and proliferation, but related mechanism is not clear.Since gap junction and hemichannels are related to brain injury, the structural protein Cx with both channels is related to cell death, and the survival of neuroglial cells determines the prognosis of brain injury, we may investigate the role of Cx, gap junction and hemichannels in neuroglial cell apoptosis and ischemia reperfusion. So we choosed global cerebral ischemia reperfusion model of rats to detect the changes of Caspase-3, Cx43and Cx43-hemichannel (Cx43-hcl)(There was little report about the detection of Cx43-hemichannel.) in reperfusion. We wish to reveal that Cx43and Cx43-hemichannel may aggravate cerebral ischemia reperfusion injury by inducing neuroglial cell apoptosis and to provide new theory to clinical therapia of ischemic cerebralvascular disease.ObjectiveTo investigate the expressions of Caspase-3, Connexin43(Cx43) and Cx43-hemichannel in globle cerebral ischemia reperfusion injury of rats, detect the role of Cx43and Cx43-hemichannel, and explore the new theoretical foundation and therapeutic strategy follow the cerebral ischemia.Methods56male adult Wistar rats (supplementing animals if the model was abandoned) were randomly divided into sham-operated group (Sham); global cerebral ischemia for10min, reperfusion for0.5h (IR/0.5h), global ischemia for lOmin, reperfusion for6h (IR/6h) and global ischemia for lOmin, reperfusion for24h group (IR/24h). The weight of rats was about270-320g. Each group had14animals of which8for Immunofluorescence histochemistry and6for Western blot. The global cerebral ischemia-reperfusion injury model was established by the method of improved4-vessel occlusion. All groups were sacrificed by decapitation at the predetermined time, the expression of Caspase-3, Cx43and Cx43-hemichannel in SVZ (sub ventricular zone) were detected by Immunofluorescence histochemistry and Western blot respectively.Results1Preparation of animal models In56male adult Wistar rats,47rats were successfully made of global cerebral ischemia reperfusion models (supplementing animals if the model was abandoned),and the ratio of success is84%.In the9unsuccessful models,4rats died(of which2for anesthesia accident,1for aspiration and1for hemorrhea),5rats did not meet the standard(of which3for no pupil greaten,2for convulsions).2Immunofluorescence histochemistry The expression of Caspase-3was little and the expressions of Cx43and Cx43-hemichannel were small in sham-operated group. Although their expressions started to increase in IR/0.5h group, there was no significant difference between Sham and IR/0.5h groups (P>0.05). Comparing IR/6h and IR/24h groups with Sham group, the expressions of Caspase-3, Cx43and Cx43-hemichannel were significantly increased (P<0.05).3Western blot The expressions of Caspase-3, Cx43and Cx43-hemichannel began to increase in IR/0.5h group, but there was no significant difference between Sham and IR/0.5h groups (P>0.05). Comparing IR/6h and IR/24h groups with Sham group, the expressions of Caspase-3, Cx43and Cx43-hemichannel were significantly increased (P<0.05).ConclusionCerebral ischemia reperfusion can induces neuroglial cell apoptosis, Cx43and Cx43-hemichannel promote apoptosis of astrocytes. This may be one of the mechanisms of ischemia reperfusion occurrence and development.SignificanceThis study was to preliminary explore the promoting effect of Cx43and Cx43-hemichannel to neuroglial cell apoptosis and this may be one of the mechanisms of ischemia reperfusion occurrence and development. Thus we provided one new method for the prevention and treatment of ischemic cerebrovascular disease. |
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