| Cerebrovascular diseases is usual diseases. The prevalence rate , death rate and disability factor is high. Accompany with the increasing of living standard, the incidence rate of Cerebrovascular diseases increased year by year. The cerebral hemorrhage is the 10-20% of Cerebrovascular diseases. The cerebral hemorrhage induced the intracranial edama immediatly. It will reach peak on the2~3day, and recovers gradually. Water transport across microvasculature is a main cause of brain edema, a phenomenon that was once believed to be the result of simple diffusion. A recently described family of water channel proteins (aquaporins, AQPs) facilitates the passage of water and other small solutes across membranes and through cells, including the vascular endothelium, so study of aquaporins may shed light on the treatment of brain edema (Denker etal., 1988). AQP4 belongs to the aquaporin family, which includes 11 subtypes (AQP0 to AQP10). Among these, AQP4 is unique because of its exceptionally high intrinsic water permeability and predominant location in brain (Yang and Verkman, 1997). AQP4 is expressed in astrocyte foot processes adjacent to vascular endothelial cells, and in the basolateral membrane of the ependymal cells but not in neurons (Nielsen et al., 1997; Rash et al., 1998; Saadoun et al., 2002). AQP4 is proposed to play an important role in water homeostasis in the brain and in formation of brain edema (Nielsen et al., 1997). Recently, AQP4 expression has been investigated to clarify its role in the development of brain injury. It has been reportedtraumatic brain injury (Sun et al., 2003), and is up-regulated following non-traumatic brain injury such as stroke or water intoxication in rodents (Taniguchi et al., 2000). In AQP4 gene knockout mice, there is less brain swelling 24 h after brain ischemia (Manley et al., 2000). In a human brain autopsy study, AQP4 immunoreactivity was found increased after cerebral infarction (Aoki et al., 2003). However, the expression characteristics of AQP4 in human traumatic brain injury are unknown. Furthermore, in human brains, AQP4 expression increases in edematous human astrocytomas, and there is significant correlation between up-regulation of AQP4 expression and breakdown of the blood-brain barrier (Aoki et al., 2003; Saadoun et al., 2002). AQP4 expression is up-regulated following a variety of diseases, such as infections, hyponatremia, ischemia and trauma. AQP4 may be the final common way of edama .cerebral edama is harmful to neurons and is a common cause of permanent brain damage. Whether blood or thrombin activate astrocytes and increase AQP4 expression remains unknown. The aim of this study was to clarify the properties of AQP4 expression in cerebral hemorrhage. This may help determine the mechanisms responsible for brain edema associated with cerebral hemorrhage. We first create the cerebral hemorrhage model on the rabbits. Then(1) detect the changes of water by dry-wet weight.(2) detect the changes of BBB by the means of Belayev.(3) detect the changes of mRNA of AQP4 by RT-PCR.(4)detect the changes of protein of AQP4 by western blot.(5)detect the change of protein of AQP4 by immunohistochemistry. We perform the experiment in order to research the involvement of edema and provide the reason of new mechanism of edema. Furthermore, we use aprotinin and hirudin to interfere and compare with the group of cerebral hemorrhage rabbit. Part I Establish the cerebral hemorrhage mode and study the properties of AQP4 expression in cerebral hemorrhage. We establish the cerebral hemorrhagemodel and evaluate it by the pathological analysis (microscope;electron microscpe).The results are as follows: we do the 42 models. In that, there are 6 models which are not satisfied.In the others,we evalulate by pathological analysis. Pathological changes:The changes of microscope:The blood is mainly locate the area of basal ganglia. In the group of 6h . A few neurons and glials swell. In the group of 48~72h, There are more triangle neurons which deeply stain in basal ganglia.we detect the widespread edema .There are more necrotic neurons and glials. In the group of 120h the edema decreases gradually.Electro microscepe:In the 48~72h group, the mitochondrion swells. The nuclear chromatin gathers to the edge. the changes of cerebral edema are detected by dry-wet weight. The results are as follows: It reach peak in the 24~48h group. Then, it decrease gradually. the changes of BBB are detected by the means of Belayev. The results are as follows: It reach peak in the 24h group. Then, it decrease gradually. The mRNA of AQP4 is detected by the RT-PCR.The results are as follows:In the sham operated group, there is the positive expression .In the 6h group, the expression increase obviously. It reach peak in the 48~72h group. Then, it decrease gradually. The protein of AQP4 is detected by western blot. The results are as follows:The expressions of AQP4 on the 30KD. There are weekly positive expressions in sham operated group.The ambulance inrease in the 6h group and reach the peak in the 24h group .Then decrease gradually. The protein of AQP4 is detected by the iminuno-histochemistery. The results are as follows: In the sham operated group, there is weakly positive expression in the cytochiema and membrane of astrocytes. There are a few positive cells in the 6h group, the experession and the positive cell increase reach the peak in th 24h group , then decrease gradually. Part â…¡: Influerce of hirudin in the alternation of AQP4 and cerebral edema after cerebral hemorrhage. the changes of cerebral edama are detected by dry-wet weight. The results are as follows: It reach peak in the 24~48h group. Then, it decrease gradually. There is obviously difference between the 12h,24h,48h,120hgroup and the control. the changes of BBB are detected by the means of Belayev. The results are as follows: It reach peak in the 24h group. There is obviously difference between the 12h, 24h group and the control. The mRNA of AQP4 is detected by RT-PCR: The ambulance of AQP4 is more weak than the cerebral hemorrhage. It reach peak in the 48~72h group. There is obviously difference between the 12h, 24h, 48h group and the control. The protein of AQP4 is detected by the western blot: the ambulance is more weak than the cerebral hemorrhage group. There is obviously difference between the 12h, 24h, 48h group and the control. The protein of AQP4 is detected by the imniumo-histochemistry in the groups interfered with hirudin: The expression is more weak than the cerebral hemorrhage and the positive cells are fewer than cerebral hemorrhage. There is obviously difference between the 12h, 24h, 72h group and the control. Part â…¢: Influerce of aprotinin in the alternation of AQP4 and cerebral edema after cerebral hemorrhage. the changes of cerebral edama are detected by dry-wet weight. There is obviously difference between the 12h,24h,48h group and the control. the changes of BBB are detected by the means of Belayev. There is obviously difference between the 12h group and the control. The mRNA of AQP4 is detected by RT-PCR: The ambulance of AQP4 is more weak than the cerebral hemorrhage. There is obviously difference between the 24h group and the control. The protein of AQP4 is detected by the western blot: The ambulance is more weak than the cerebral hemorrhage group. There is obviously difference between the 12h, 24h group and the control. The protein of AQP4 is detected by the imniumo-histochemistry: The expression is more weak than the cerebral hemorrhage and the positive cells are fewer than cerebral hemorrhage. There is obviously difference between the 24h, 72h group and the control. Conclusion: we duplicate the cerebral hemorrhage model successful. The success rate approach to 85.7%. Through and pathological analysis, we think the model can be used to research the cerebral hemorrhage. A number of mechanisms... |
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