The Internalization Of AQP4 After Intracerebral Hemorrhage And Its Mechanism

BackgroundPrimary intracerebral hemorrhage (ICH) has high rates of mortality and morbidity. During its complicated pathological process, the formation of brain edema is an important clinical complication in the secondary brain injury, which causes neurological deficits or even death. Many studies suggest the alteration of aquaporin-4 (AQP4) is involved in the whole course of edema formation after ICH and the expression of AQP4 is dynamically changed over the time.As a predominant water-selective channel in the brain, AQP4 is strongly expressed in those astrocyte membrane domains forming the interface between brain parenchyma and fluid-filled compartments. The expression of AQP4 is enriched in the perivascular glial endfeet, subpial membrane domains of brain astrocytes and ependymocytes, which enables the efflux and influx of water driven by the osmotic gradients and hydrostatic pressure. Brain water transport via AQP4 is bidirectional under different forms of brain edema induced by different pathological models, which means AQP4 is involved in the brain edema formation and its resolution. Therefore, AQP4 is a potentially therapeutic target for the pharmacological treatment of brain edema, both in the cytotoxic edema of prevention of edema formation and in the vasogenic edema of resolving the edema.It has been recognized that AQP4 is subject to regulation at the level of subcellular distribution. The membrane-bound proteins including AQP4 can be regulated by internalization which alters their subcellular distribution. This internalization may or may not be accompanied by subsequently lysosomal sorting and degradation. The internalization of AQP4 can influence its role in the brain volume homeostasis and water balance. Since no similar regulation has so far been reported for AQP4 following intracerebral hemorrhage, this study aims to clarify whether AQP4 is internalized and/or lysosomally degraded after ICH.Therefore, this research work has been designed to do both in vivo and in vitro. In vivo experiment we try to discover whether AQP4 is internalized and/or lysosomally degraded in the rat brain after ICH. In vitro experiment the ICH model is made in which astrocyte cultures receive thrombin treatment. The internalization of AQP4 and the anchoring protein a-syntrophin are examined. The correlation of AQP4 and a-syntrophin is explored. Further experiments are needed to elucidate the precise mechanisms of AQP4 internalization to facilitate our understanding of the potential of new treatments for brain edema.Objective1. To investigate whether AQP4 is internalized and/or lysosomally degraded in the rat brain after ICH, so as to explore the possible role of internalized AQP4 in the brain edema after ICH.2. To investigate the effects on AQP4 internalization in thrombin-treated astrocyte, so as to explore the correlation of AQP4 and anchoring protein a-syntrophin.Methods1.114 male adult SD (Sprague-Dawley) rats were randomized into two groups, the intracerebral hemorrhage group (n=75) and the sham-operated group (n=39). The ICH model was induced by administration of 2 u1 bacterial collagenase (0.25 U/μ1) into the right caudate nucleus. The following studies were carried out at 6 h,12 h,24 h, 48 h,72 h post-ICH. The hematoma was observed by HE staining, and the ultrastructure of vessels was demonstrated by trasmission electron microscopy (TEM) which identified the brain edema category. The wet-dry method was applied to measure the brain water content (BWC) at different time points post-ICH, and fluorescent tracer Evans blue (EB) was applied to examine the integrity of blood-brain barrier (BBB). Fluorescent q-PCR (quantitative polymerase chain reaction) and Western blot were applied to detect the levels of AQP4 mRNA and AQP4 protein. Double immunofluorescence labeling was applied to examine the co-expression of AQP4 with the marker of late endosome (mannose-6-phosphate receptor, MPR) and the marker of lysosome (lysosomal associated membrane protein-1, LAMP1) respectively. The ratio of co-expressed AQP4 with LAMP1 to total AQP4 was calculated by Image analysis software Image-Pro Plus 6.0.2. Rat cerebral cortical astrocytes from less than 2 days newborn SD rats were undergone the primary culture. The cells were divided into control group and thrombin-treated group randomly. The cells in control group were kept in tact, and the cells in thrombin-treated group were exposed to 100 U/ml thrombin for 24 h. The viability of astrocytes after thrombin treatment was evaluated by measuring the release of lactic acid dehydrogenase (LDH). The expression of AQP4 was examined by immunofluorescence staining. Double immunofluorescence labeling was applied to determine the co-expression of AQP4 with MPR, AQP4 with LAMP1, AQP4 with α-syntrophin respectively. Western blot was applied to detect the levels of AQP4 and a-syntrophin proteins.Results1. The internalization and lysosomal degradation of AQP4 after ICH① Transmission electron micrographs showed the alterations of vascular ultrastructure. At 1 d post-ICH, the perivascular endfeet was swollen and the swelling basement membrane was discontinuous, besides, the perivascular space was widen. ② A rise in brain water content started at 3 h and became significantly at 6 h post-ICH, then it continued to increase until to reach its peak at 48 h post-ICH. Evans blue extravasation was observed at 1 h post-ICH and continued to increase until to reach its peak at 6 h post-ICH compared with the sham group (P<0.01). ③ The expression of AQP4 mRNA was significantly increased at 24 h,48 h and 72 h post-ICH (P＜0.01). The upregulation of AQP4 mRNA climax occurred at 24 h post-ICH. The expression of AQP4 protein was increased significantly at 12 h (P<0.05) and reached the highest level at 24 h post-ICH. ④ At 6 h,12h, 24 h,48 h post-ICH, the co-expression of AQP4 with MPR, AQP4 with LAMP1 were determined in the choroid plexue, perihematomal region and cerebral cortex. The calculated ratios of AQP4 with LAMP1 demonstrated that the ratio was largely a function of time post-ICH. The ratio was increased significantly compared with the sham group (P<0.05).2. The effects on AQP4 by thrombin treatment in astrocyte cultures① 100 U/ml thrombin administration resulted in 30.17% ± 2.79% cell death in astrocyte cultures. ② AQP4 was mainly expressed on the astrocytic membrane domains in the control group. After the treatment of thrombin, a large proportion of AQP4 was expessed in the cytoplasm which was granule-shaped. The level of AQP4 protein was not changed significantly with thrombin treatment. ③ After the treatment of thrombin, the co-expression of AQP4 with MPR, AQP4 with LAMP1 were detected in astrocyte cultures. ④ The expression of a-syntrophin in astrocyte was decreased significantly and its protein level was downregulated significantly with thrombin treatment (P<0.01).Conclusions1. The levels of AQP4 mRNA and AQP4 protein were upregulated post-ICH, which was involved in brain edema formation. Furthermore, AQP4 was internalized and the lysosome was involved in the degradation of internalized AQP4 post-ICH.2. AQP4 was internalized and subsequently accompanied by lysosomal sorting and degradation in astrocyte cultures with thrombin treatment. The co-expression of AQP4 and a-syntrophin was hardly detectable and the a-syntrophin protein was decreased significantly with thrombin treatment. The down-regulated a-syntrophin could affect the anchoring of AQP4 to plasma membrane, which caused alteration of subcellular distribution.