| Background and objectiveRecent years, accompanied with the improvement of the clinical emergency ability, the successful rate of rescuing patients undergoing cardiopulmonary resuscitation, myocardial infarction, asphyxia, shock and other critical conditions is increased gradually. The cerebral ischemia and ischemia-reperfusion injury caused by these diseases has become a hotspot. Among them, the main difficulty in clinical research lies in how to reduce neuronal death after cerebral ischemia and reperfusion. Through a series of researches on cerebral ischemia animal model, a large number of researchers confirmed that the apoptosis exist in cerebral ischemia-reperfusion injury in a morphology or a biochemistry way. How to inhibit apoptosis will be the key to reduce cerebral ischemia-reperfusion injury.The major ubiquitin ligase required for mitosis is the anaphase-promoting complex/cyclosome (APC/C). This unusually complex E3 ubiquitin ligase targets cell-cycle-related proteins such as cyclins and securin for degradation by the proteasome in mitosis and meiosis.The APC/C is regulated by phosphorylation, as well as by various activators and inhibitors that alter its substrate specificity at different phases of the cell cycle. Such tight control ensures appropriately timed degradation of key cell-cycle regulators.Cdhl-APC plays important roles in function and neuronal survival.,patterning synaptic development and the regulation of axonal growth.However there was few report about the feature of APC-Cdhl expression in central nervous system after cerebral ischemia reperfusion injury and the role of APC-Cdhl intervention in isc hemic cerebral injury.In our study, we first established a model of global cerebral ischemia and reperfusion injury, then and we investigated the relationship between the expression of APC-Cdhl, the neuronal apoptosis and the neurological functions changes. Later we used lentivius mediated RNAi vector to reduce the expression of APC-Cdhl and to investigate the effect of APC-Cdhl in ischemic brain injury and the possible mechanism. This study provided an experimental evidence for selecting the APC-Cdhl as a new strategy for brain protection.Methods and Results1. The changes of cognitive function and neural cell apoptosis in rats with cerebral ischemia reperfusion injuryMethods 80 male Sprague-Dawley rats were randomly divided into Sham-operated group (SH) and ischemia reperfusion group (IR). The rats of ischemia reperfusion group were induced by modified four-vessel occlusion (4-VO). In sham group, the vertebral arteries of the rats were permanently electrocauterized and the bilateral common carotid arteries were not electrocauterized. Both common arteries were occluded for seven minutes in IR group. According to the time points of ischemia reperfusion, the two groups were divided into 1d,3d,7d three subgroups respectively. At different time points, fresh frozen sections were prepared from brain tissue of the two group rats. Nissl dyeing was used to observe surviving hippocampus neurons. And neuronal apoptosis was detected by TUNEL staining. Neurons density and apoptotic index were calculated.Water maze was employed and analyzed by computer image analysis system. Seven days after the operation, learning and memory of the rats in two groups were tested by Morris water maze. Results 1). Nissl dyeing:In SH group, the hippocampal neurons were stained blue.The cell arranges were in neat rows. The forms were a complete hippocampus. CA1 area of nerve cells arranged in 4 to 5 layers and large round nucleus, clear nucleolus.Abundant Nissl bodies were showed by azomethine blue staining. In ischemia reperfusion group, the hippocampal neurons were apparent disorganized and irregular. The number of Nissl bodies were reduced or even disappeared. Nuclears became smaller and showed stained chromatin condensation.Compared with Sham group, the neuron density in ischemia reperfusion group was significantly lower (p<0.05).2). The results of TUNEL method showed that in Sham group, apoptosis of hippocampus neurons were occasionally in the CA1 area. But in ischemia reperfusion group, there were a large number of apoptosis hippocampus neurons in the CA1 area. Compared with Sham group, the apoptotic index of hippocampal neurons in ischemia reperfusion group was significantly higher (p<0.05).3). In Morris water maze tests, the incubation period of seeking the platform and the learning and memorizing ability in ischemia reperfusion group were significantly longer than sham-operated group(p<0.05).2. The feature of APC-Cdhl expression in central nervous system after cerebral ischemia reperfusion injury.Methods 60 male Sprague-Dawley rats were randomly divided into Sham-operated group (SH) and ischemia reperfusion group (IR). The rats in ischemia reperfusion group were induced by modified four-vessel occlusion (4VO). The methods were same to the first study. At different time points, fresh frozen sections were prepared from brain tissue of the two group rats.The expression of Cdhl was examined in the hippocampus by immunohistochemistry.At different times after the operations, the expression of APC-Cdhl of hippocampus was observed by quantitive real time PCR and Western Blot.Results The immuno-staining showed that Cdhl was highly expressed in cerebral cortex and hippocampus.Compared with SH group, the expression of Cdhl mRNA was significantly decreased at 1 day after injury and increased obviously at 3 days, but decreased again at 7 days. Compared with control group, the expression of Cdhl protein was significantly decreased at 1 day after injury and increased obviously at 3 days, but decreased again at 7 days(p<0.05).3. Expression and function of lentivirus-mediated Cdhl-SiRNA in cerebral ischemia-reperfusion injury in ratsMethods 150 male Sprague-Dawlcy rats were divided into group A(n=50), group B(n=50)and group C(n=50). The rats of 3 groups received injection with normal saline, lentivirus vector and recombinant lentivirus respectively. At 3 days after injection, all rats were implemented with global brain ischemia model by modified four-vessel occlusion (4-VO). The methods were same to the first study.Seven days after the surgery,the expression of Cdhl and Cyclin B were examined by quantitive real time PCR and Western Blot respectively. Apoptosis was examined using TUNEL staining method. The behavior was evaluated by Morris water maze.Results The expression of Cdhl mRNA in group C was significantly lower than those in group A and B (p<0.05), but the expression of Cyclin B in group C was higher (p<0.05). In addition,the results of TUNEL method showed that compared with group A and B, the apoptotic index of hippocampal neurons in group C was significantly increased(p<0.05). In Morris water maze tests, compared with group A and B the incubation period of seeking the platform and the learning and memorizing ability in group C was significantly longer(p<0.05).Conclusions1. The number of apoptotic neurons in hippocampus was increased and the cognitive ability was decreased after global cerebral ischemia reperfusion injury in rats 2. This result indicated that APC-Cdhl may participate in the pathophysiology of global cerebral ischemia-reperfusion injury and play an important role in the injury of central nervous system.3. APC-Cdhl may be mediated by Cyclin B accumulation in ischemic neuronal apoptosis.SummaryThis study shows that global cerebral ischemia-reperfusion injury model was successfully established by modified 4-VO. The hippocampus in rats suffered from delayed neuronal death and the apoptotic cells were increased. The learning and memory of rats were declined. This model could cause the hippocampus injury.Then we found the expression of APC-Cdhl in the rat hippocampus was increased after global cerebral ischemia-reperfusion injury. This observation suggests that APC-Cdhl may be involved in pathophysiology change of cerebral ischemia-reperfusion injury.Last Cdhl RNA interference lentiviral vector was injected to the rat hippocampus by Stereotactic injection.After three days,modified 4-VO cerebral ischemia-reperfusion injury model was established. Seven days later,we observed that the expression of Cdhl decreased and the expression of Cyclin B increased.The learning and memory of rats decreased.The results suggested that the cerebral ischemia-reperfusion injury could be aggratated by lentivirus-mediated Cdhl RNA interference.Our study exhibited the differential expression of Cdhl in global cerebral ischemia-reperfusion injury model and furtherly explored the effect of the APC-Cdhl intervention for the treatment of cerebral ischemia-reperfusion injury.lt could provided a new option and an experimental evidence for cerebral protection.
|
PDF Full Text Request