Aqps In Brain Edema Formation And Its Expression Regulation

PART ONETHE EXPRESSION REGULATION OF AQPS AND THEIR RELATIONSHIP WITH ISCHEMIC CEREBRAL EDEMAObjective: To investigate the relationship between the formation of ischemic cerebral edema and the changes of AQPs expression during pathological course of ischemic cerebral edema in rats, and preliminarily to discuss the role of AQPs and the mechanism of AQPs expression regulation during ischemic cerebral edema.Methods: A total of 392 healthy male adult Wistar rats were randomly divided into two groups, the ischemic group and the sham operation control group. MCAO (middle cerebral artery occluded) models were established by occluding unilateral middle cerebral artery (MCA) of the rats with the suture method. The operation procedure of the control group was as same as the ischemic group but without occluding unilateral MCA. The brains of the animals were taken out at interval times of 1h,3h,6h,12h and 24h, respectively. Subsequently, the morphological changes were observed with HE staining and transmission electron microscope (TEM). Brain water content (BWC) was measured by using wet-dry weighing method. The expression of AQPs in the edema brain tissue was detected with immunofluorence (IFC) and immunohistochemistry (IHC), respectively. The changes of AQPs and their mRNA expression in the edema brain tissue were detected with Western Blot analysis and real time reverse transcription polymerase chain reaction (Real-time PCR). The changes of ERK expression in the edema brain tissue were measured by Western Blot.Results: After operation, the morphological structure, BWC, expression of AQPs and their mRNA, the content of ERK were not significantly changed in the control group. While in the ischemic group, HE staining and transmission electron microscope (TEM) showed that the brain tissue had ischemic changes. The space around the cells and capillaries became larger, some of the neurons showed necrosis, the nuclei were deeply stained or disappeared and the number of the neurogliocyte increased. The brain water content of ischemic brain tissue significantly increased from MCAO 1h, and gradually increased along with the ischemic time extendence, and reached its peak at 24h after MCAO. The results of IHC and IFC showed that the positive expression of AQPs was observed in the hippocampus, choroid plexes, thalamus, supraoptic nuclei, brain cortex and other regions. The expression of AQP4, AQP9 and their mRNA was up-regulated along with the ischemic time extendence and reached their peak at MCAO 24h. At the same time, the expression of AQP3, AQP5 and AQP8 increased immedially and reached their peaks at MCAO 6h, then decreased gradually, but still higher than the control group at MCAO 24h. The expression of AQPs protein was positively correlated with AQPs mRNA (P <0.05). The expression of AQP4 and AQP9 was correlated with BWC after MCAO (P<0.05). The contents of ERK and phosphorylated ERK were not significantly changed both in the control group and ischemic group.Conclusions: The expression enhancement of AQP4 and AQP9 was correlated with the brain edema aggravation after MCAO, suggesting that the expression changes of AQP4 and AQP9 would have a close relationship with the formation of brain edema after MCAO. The up-regulation of AQP3, AQP5 and AQP8 may play a role in the neurocyte edema (neuron and astrocyte) in the early stage and cooperate with AQP4, AQP9 for astrocyte edema in the later stage. The contents of ERK and phosphorylated ERK were not significantly changed, suggesting ERK-MAPK do not play a role in the regulation of AQPs after MCAO. PART TWO THE EFFECT OF OSMOLARITY CHANGES ON EXPRESSION OF AQUAPORINS IN ASTROCYTES AND THE PRELIMINARY STUDY OF ITS EXPRESSION REGULATIONObjective: To investigate the expression rules of aquaporins (AQPs) and the mechanism of their regulation in cultured rat astrocytes after exposure to different osmotic stress.Methods: Rat cerebral cortical astrocytes from less than 2 days newborn Wistar rats were undergone the primary culture. The cells were divided into control group,hypotonic groups and hypertonic groups randomly. The cells of the control group were cultured in normal culture medium, the model of cell edema in the hypotonic group was established by exposed to hypotonic medium, and cell dehydration in the hypertonic group was established by exposed to hypertonic medium. Each group was examined at interval times of 1h, 3h, 6h, 12h and 24h, respectively. HE staining and transmission electron microscope were used to observe the cell morphological changes. The viability of cells in hypotonic or hypertonic medium was measured by MTT colorimetric method, LDH assay and trypan blue staining. The alteration of AQPs was studied by the methods of immunofluorence, immunocytochemistry and imaging analysis. The contents of AQPs and their mRNA expression of astrocytes were detected with Western Blot Analysis and real time reverse transcription polymerase chain reaction (Real-time PCR). The content changes of ERK of cells were measured by Western Blot.Results: There were no significant change of the cell morphology, the cell viability, the expression of AQPs and their mRNA and the content changes of ERK in the control cells, which were cultured in normal culture medium. At the same time point, astrocytes cultured in hypotonic medium (268, 254, 240 mmol/L) showed typical features of cell edema, and the cell viability decreased, especially in lower osmolality group. The expression of AQPs and their mRNA were remarkably higher in hypotonic group than that in the control group(P<0.05), and increased by the osmolarity decreased. The expression of AQPs reached their peak at 24 except for AQP5 which reached its peak at 6~12h after astrocytes were exposed to hypotonic medium. The changes of AQPs expression showed a positive relationship with AQPs mRNA(P<0.05). The content changes of ERK were constant in astrocytes which exposed to hypotonic medium(P>0.05). Exposed to the hypertonic medium (320, 333, 345 mmol/L), cells shrinked, and the cell viability decreased with a time dependent manner. The expression of AQP4 and AQP9 were significantly changes compared with the cells in control group before 12h, and then decreased until 24h, at 24h time point it was mild higher than control cells(P<0.05), while the expression of AQP3, AQP5 and AQP8 reached their peak at 6h, then decreased. The content of ERK had no changes in higher osmolality group, but the phosphorylated ERK increased before 6h and then decreased until 24, at 24h time point it was mild higher than control cells. Preincubation of U0126, the inhibitor for ERK, could down-regulate the expression of ERK/ERK2, phosphorylate ERK/ERK2 and AQP3, AQP5 and AQP8.Conclusions: The expression of AQPs had a direct correlationship with osmotic changes. Hyponotic medium could induce cell edema, inhibit viability of astrocytes and up-regulate the expression of AQPs and their mRNA, which is thought to be a maladaptation reaction. Hypertotic medium could induce cell shrink, inhibit viability of astrocytes, up-regulate the expression of AQPs and their mRNA, which may play an important compensation for the hypertonic dehydration in the early stage. The results mentioned above imply that ERK-MAPK participate in the enhancement expression of AQP3, AQP5 and AQP8 in the hypertonic condition. The expression of AQP4, AQP9 in the hypertonic condition and AQPs in hypotonic condition could be regulated by other signal path.