| ObjectiveIntracerebral hemorrhage (ICH) is defined as non - traumatic cerebral pa-renchymal hemorrhage, which has high incidence and fatality rate. Post hemor-rage cerebral edema plays an important role in the deterioration of clinical condition. Studies on cerebral edema post hemorrage provided new approach for treatment of ICH in recent years. Much attention have been payed to the toxiticity of thrombin that is released in blood coagulation post ICH in recent studies. As a normal serum serine protease, thrombin acts in a number of receptor mediated reactions besides blood clotting cascade. The physiopathologic mechanism of cerebral edema induced by mass release of thrombin in ICH remains uncertain.Lately, studies have been focused on the effect of AQP4 in cerebral edema post ICH. AQPs is a membrane channel protein determined in recent ten years, it only allows water molecular and some micromolecular substances, and forms the major molecular basis of water transmembrane transportation. It has been revealed that AQP4 is the predominant form of AQPs in CNS, mainly expressed in the astrocytes and ependymocytes. AQP4 might be important for water metabolism.Our study was performed to investigate dynamic change of water content, permeability of the blood - brain barrier (BBB) and expression of AQP4mRNA in Intracerebral hemorragic rats and the role that thrombin plays in cerebral edema post hemorrage, and to evaluate the effect of hirudin in reducing cerebral e-dema.Materials and Methods1. Materials1. 1 Animal grouping: 75 normal male and female Wistar rats, with body weight of 250 ~ 300 g, were randomly assigned to control group, cerebral hemorrhage group and hirudin treated group. After the injection, every five rats were sacrificed at6h, Id, 3d, 5d, 7d respectively.1. 2 Main apparatus: Experimental cranial sterotaxic apparatus, PCR amplification system, electrophoresis apparatus, gel imaging analysis system, tissue homogenater, ultraviolet spectrometer, microsyringe, pyro - drying cabinet, electro - balance.1.3 Agents; Evens blue, formamide, hirudin, Trizol, RNA PCR Kit (AMV) , DNA MaKer , 6 x Loading Buffer , DEPC, agarose, chloroform, avantin.2. Animal model:Animal model was made by injecting self arterial blood into the caudate nucleus of rats as Rosenberg et al reported. Control group were injected with 78jxl of NS,75 (xl of self artery blood and additional 3ui of NS for ICH group ,75 ui of self artery blood and additional 3jxl of NS( contains 15U hirudin) for hirudin group.3. Index detection3. 1 Brain water content:: Anterior brain tissue of needle puncture was re-sectioned and determined by wet - dry weight, as [ (wet weight dry weight) /wet weight] 100%.3. 2 Permeability of the BBB: Posteromedial brain tissue of needle puncture was resectioned and assessed by Evens blue stain, as Blayevs method. Permeability of the BBB is assessed by EB content! OD/mg).3. 3 Expression of AQP4mRNA( RT-PCR) : Posterior lateral brain tissue of needle puncture was resectioned for RT - PCR with Trizol kit and RNA PCR kit (AMV). PCR amplification products were subjected to electrophoresis, spectrometry was analyzed by gel imaging analysis system. Finally, AQP4mRNAexpression was calculated by the ratio of AQP4 products ( OD) and J3 - actin products (OD).4. Statistical treatmentAll data were presented by x ± s, analyzed by SPSS10. 0 and Excel soft ware package. Statistical treatments were done with ANOVA and t - test. Significant difference was considered if P <0.05.ResultsBrain water content and expression of AQP4mRNA increased after 6 hours of ICH and reached its climax at the 3rd day, then decreased at the 5th to the 7 th day. , and there is obvious correlation between them (r=0.815, p<0.01). The permeability of the BBB increased at 6 hours after ICH, got to the peak at Id to 3d,then decreased at 5d to 7d but higher than control. The expression of AQP4mRNA is obviously correlated to the chang of the BBB§ permeability (r = 0.686 ,p <0.01). Administration of hirudin, inhibitor of thrombin could reduce the water content, BBB permeability and AQP4mRNA expression.DiscussionBrain water content x BBB permeability and expression of AQP4mRNA increased after 6 hours of cerebral hemorrhage and reached its climax at the 3rd day, then decreased at the 5th to the 7th day. ,and there is obvious correlation between them. Increased AQP4mRNA expression and BBB permeability might participate the pathophysilosical mechanisms in cerebral edema. Positive correlation between AQP4mRNA expression and BBB permeability suggested that increased AQP4mRNA expression might affect BBB permeability so that it could cause brain edema. Hirudin, the special inhibitor of thrombin, could down -regulate AQP4mRNA expression, BBB permeability and cerebral water content at all stages. It was indicated that thrombin released after ICH may promote cerebral edema by up - regulate AQP4mRNA expression and BBB permeability. The mechanism may include increased PKC activity and intracellular Ca + relea-... |
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