| Intracerebral hemorrhage (ICH) is a common disease in the elder, with high mortality and morbidity. Brain edema is one of the most important complications of ICH, which is the major cause of death and severe neurological deficits, so its mechanism has been the focus of research in neuroscience. The key of brain edema is the imbalance of water in the cells of brain. The discovery of aquaporins (AQPs) provides a new opportunity to further clarify the balance of water in brain and its molecular mechanism.The AQPs are plasma membrane water-transporting proteins, which play an important role in the water balance of CNS. Aquaporin 4 (AQP4) is the principal member of AQPs family in the CNS, and it contributes to brain water homeostasis in perivascular and subpial membrane domains of astrocytes where they are concentrated in the astrocytic endfeet. AQP4 is abundantly concentrated in the astrocytic endfeet apposed to capillaries and the pia mater, which is called "polarized expression". The AQP4 at the astrocytic endfeet can form crystal-like orthogonal arrays of particles (OAPs), which is the structural basis of AQP4 to perform its efficient water transport function. Under physiological conditions, AQP4 is expressed at the astrocytic endfeet closely related to water transport, playing a crucial role in the balance of water. Whereas under pathological conditions, the polarized expression of AQP4 is lost, while OAPs is reduced significantly, causing water balance of blood-brain/blood-cerebrospinal disturbed.The polarized expression of AQP4 is associated with the dystrophin-dystroglycan complex (DDC), including dystroglycan (DG), syntrophin, and dystrobrevin. The key component of DDC is DG, which comprises α-and β-subunits. a-dystroglycan (a-DG) binds to extracellular matrix (ECM) components such as agrin, whereas β-dystroglycan (β-DG) is a membrane-spanning protein mediating between a-DG and the cytoskeleton and other components of the DDC such as a-syntrophin. AQP4 is tethered to astrocytic endfeet mediating by the a-syntrophin. In the brain of DG knockout mouse, the polarized expression of AQP4 was lost in perivascular endfeet.Previous studies reported on the AQP4 involvement in brain edema development and the changes of AQP4 expression following ICH. However, in these studies neither the change of AQP4 polarized distribution, nor the comparison of brain edema development and AQP4 polarity were established. To address this question, we investigated the effect of AQP4 polarized expression on brain edema following ICH, to provide experimental basis for the mechanism of brain edema and to provide new ideas for the treatment of brain edema.Objective:1. To observe the changes of aquaporin 4 (AQP4) polarized expression in the brain following ICH, and to explore its roles in the brain edema following ICH.2. To research the relationship of the polarized localization of AQP4 with β-DG in the brain tissue following ICH, and to explore the roles of β-DG on the change of AQP4 polarized localization.Methods:A total of 204 healthy adult male SD rats were randomly divided into two groups, the sham operation group and the ICH group (1 d,3 d,7 d post ICH), and autologous blood was injected into the right caudate nucleus to establish ICH model in rats. The hematoma was measured by magnetic resonance imaging (MRI), the brain water content (BWC) was measured by the wet and dry weight methods, Evans Blue (EB) was injected into vena jugularis externa to measure the integrity of blood-brain barrier (BBB), HE staining and transmission electron microscope (TEM) were used to observe the structure changes, Western blotting and real-time quantitative PCR (qPCR) were used to detect the changes of AQP4 protein and mRNA, and the polarized expression of AQP4 was detected by immunofluorescence (IFC). The co-expression of AQP4 and β-DG in the perihematomal tissue were detected by IFC, the P-DG protein was detected by Western blotting (WB), and β-DG mRNA was detected by qPCR.Results:1. The polarized expression of AQP4 was lost in the brain post ICH.MRI detection showed all model groups had obvious and clear hematoma, and the hematoma diameter of 1 d post ICH was biggest, these results showed that after injecting 50μl autologous bloods into the caudate nucleus, the ICH model was established successfully. The brain water content of 1 d,3 d post ICH were increased significantly, and compared with the sham group, the difference had statistical significance (P<0.05). The EB contents of 1 d,3 d post ICH were increased significantly, and compared with the sham group, the difference had statistical significance (P<0.05). HE staining and ultrastructure testing found, in the perihematomal tissue, the organization structure was destroyed obviously, especially in the tissue of 1 d post ICH, the endothelial cell of capillary was swollen and contained vesicles and vacuoles. At 1 d post ICH, AQP4 protein and mRNA in the perihematomal tissue were increased significantly, and compared with the sham group, the difference had statistical significance (P<0.05). At 3 d and 7 d post ICH, AQP4 protein and mRNA in the perihematomal tissue were not changed significantly, and compared with the sham group, the difference had no statistical significance (P>0.05). The results of IFC showed that, in the sham group, AQP4 was expressed on the astrocytes processes comprising the glial limitans externa beneath the pia mater, on the glial limitans interna beneath the ependymocytes and on the perivascular endfoot processes of caudate nucleus. However, in the brain tissue post ICH, AQP4 was gathered on the glial limitans externa, and AQP4 was expressed on the ependymocytes diffusely and multilayered, and AQP4 was expressed on the soma of astrocytic in the perihematomal tissue, which suggested that the polarized expression of AQP4 was lost in the brain following ICH.2. The decreased expression of P-DG was associated with the loss of AQP4 polarized expressionIn sham group, AQP4 and P-DG were co-expressed on the astrocytic processes comprising the glial limitans externa beneath the pia mater, on the astrocytic membranes of glial limitans interna beneath the ependymocytes, and on the perivascular endfeet processes in the caudate nucleus. However, in the ICH group, on the perihematomal tissue, ependymocyte cells, and the glial limitans externa, the polarized expression of AQP4 was lost, accompany with the expression of β-DG was decreased sharply, only little P-DG was expressed on the soma of astrocytes. AQP4 and p-DG were still co-expressed on the subfornical organ, supraoptic nucleus, hippocampi, and dentate convolution, where AQP4 was still polarized localization. On the perihematomal tissue at 1 d,3 d,7 d post ICH, the protein of β-DG was decreased sharply, and compared with the sham group, the difference had statistical significance (P<0.05). While the mRNA of P-DG was not changed, and compared with the sham group, the difference had no statistical significance (P>0.05)Conclusions:1. Water balance was disturbed in the brain following ICH, and the brain edema following ICH was vasogenic brain edema.2. The AQP4 in the perihematomal tissue was increased. While in the brain following ICH, the polarized expression of AQP4 was lost.3. In the brain following ICH, accompany with the loss of AQP4 polarity localization, the expression of β-DG was decreased sharply, suggesting that the decreased expression of P-DG was associated with the loss of AQP4 polarized expression.4. Loss of AQP4 polarized expression has important pathological and physiological significance. Accompany with decreased of β-DG, the AQP4 could not connect with the basement membrane via β-DG, resulting that AQP4 could not expressed on the astrocytic endfeet. In the perihematomal tissue, the brain edema was induced by the retraction of the clot, and by the disruption of the integrity of the blood-brain barrier. However, the polarized expression of AQP4 was decreased at the astrocytic endfeet of perivascular, and water in extracellular cannot eliminated by a transcellular AQP4-dependent route into vessel, delaying clearance of the edema. In other words, loss of AQP4 polarized expression may promote the development of brain edema following ICH. |
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