| ObjectiveIntracerebral hemorrhage (ICH) is defined as non - traumatic cerebral pa-renchymal hemorrhage, which has high incidence and fatality rate. Brain edema after ICH is one of the significant reasons making pathogenetic condition worse. Treatment options were limited to osmotic agents such as mannitol and surgical decompression, none of which correct the molecular - level mechanisms responsible for brain edema. Aquaporin 4 (AQP4) is the most widespread aquaporin in the central nervous system, and important in pathomechanism of brain edema that is caused by ischemia, hemorrhage, trauma, tumor and etc. This experiment investigates the changes of expression of AQP4 mRNA of perihemotoma tissue after ICH, permeability of blood — brain barrier (BBB) and brain water content in rats, compared with that in rats received nimotop. This experiment studys the effects of Ca2 + and AQP4 on brain edema and the effects nimotop on protective mechanism of nerve injury after ICH in order to provide the theoretical foundation of clinical therapy.Materials and Methods1. Materials1.1 Animal grouping:75 normal male and female Wistar rats, with body weight of 250 -300g,were randomly assigned to control group, experimental group and interventional group. Every group was divided into five sub -group according to 6h, 1d, 3d, 5d, 7d after treatment.1.2 Main apparatusExperimental cranial sterotaxic apparatus, PCR amplification system, elec-trophoresis apparatus, gel imaging analysis system, tissue homogenater, ultraviolet spectrometer, microsyringe, pyro - drying cabinet, electronic analytical balance.1.3 AgentsEvens blue, formamide, nimotop, Trizol, RNA PCR Kit( AMV) , DNA MaKer , 6 x Loading Buffer , DEPC, agarose, chloroform, avantin.1.4 Animal modelAnimal model was made by injecting self arterial blood into the caudate nucleus of rats as Rosenberg et al reported. Control group were injected with 78μl of NS, 78μl of self artery blood for experimental group , The interventional group received nimotop(0. 5mg/kg) by peritoneal injection before injecting self arterial blood into the caudate nucleus, per 24h injecting once.2. Index detection2.1 Brain water content;Anterior brain tissue of needle puncture was resectioned and determined by wet — dry weight, as [ ( wet weight — dry weight)/wet weight] x 100%. With brain water content as degree of brain edema.2.2 Permeability of the BBBPosterior lateral brain tissue of needle puncture was resectioned and assessed by Evens blue stain, as Belayevs method. Permeability of the BBB is assessed by EB content(OD/mg).2.3 Expression of AQP4mRNAPosterior medialis 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, AQP4mRNA expression was calculated by the ratio of AQP4 products (OD) and β - actin products (OD).3. Statistical treatmentAll data were presented by X ± SD, analyzed by SPSS10. 5 and Excel soft ware package. Statistical treatments were done with ANOVA and t - test.ResultThe brain water content and the expression of AQP4 mRNA were increased at 6 hour after ICH, reached peak at the 3rd day, and were still higher than normal level at the 7th day. The changes of the brain water content was positively correlated with the expression of AQP4 mRNA ( R = 0. 805 , P < 0. 001). The brain water content and AQP4mRNA were closed related. The permeability of BBB began to increase at 6 hour after ICH, and reached peak at the 1st and 3rd day. The permeability of BBB were also positively correlated with the expression of AQP4 mRNA(R = 0. 540, P < 0.01). After nimotop be given, the brain water content, permeability of BBB and expression of AQP4 mRNA were decreased.DiscussionOver the past decade a family of related water - transporting proteins, called aquaporins, has been identified. At least ten aquaporin water channels have been cloned from mammals. Data suggest that aquaporin provide a key route for water movement in brain edema. AQP4 is the most widespread aquaporin in the central nervous system, and important in pathomechanism of brain edema. In our experiment we can see that the brain water content increased at 6 hour after ICH, reached peak at the 3rd day, and were still higher than normal level at the 7th day. The changes of the brain water content were positively correlated with the expression of AQP4 mRNA. As the expression of AQP4 mRNA increased, the brain water content increased. The quantity of EB began to increase at 6 hour after ICH, and reached peak at the 1st and 3rd day, and then decreased gradually. The quantity of EB was positively correlated with the expression of AQP4 mRNA. As the expression of AQP4 mRNA was increased, the... |
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