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1.
BACKGROUND:Previous studies have demonstrated that aquaporin-4 (AQP4) plays a key role in the formation and resolution of brain edema.However,the molecular mechanisms and role of AQP4 in hypoxia-ischemia-induced brain edema remain poorly understood.OBJECTIVE:To establish a newborn animal model of astrocytic oxygen-glucose deprivation and reintroduction,to observe the correlation between AQP4 and cellular volume,and to investigate the role of AQP4 in the development of brain edema following oxygen deprivation and reintroduction.DESIGN,TIME AND SETTING:A comparative experiment was performed at the Experimental Center of West China Second University Hospital between October 2007 and April 2009.MATERIALS:Astrocytes were derived from the neocortex of Sprague Dawley rats aged 3 days.METHODS:Astrocytes were incubated in glucose/serum-free Dulbecco's modified Eagle's medium,followed by 1% oxygen for 6 hours.Finally,oxygen-glucose deprivation and reintroduction models were successfully established.MAIN OUTCOME MEASURES:Real-time polymerase chain reaction and Western blot analysis were used to measure expression of AQP4 mRNA and protein in cultured rat astrocytes following oxygen-glucose deprivation and reintroduction.Astrocytic cellular volume,as determined by [3H]-3-O-methyl-D-glucose,was used to represent the extent of astrocytic swelling.RESULTS:During oxygen-glucose deprivation,AQP4 mRNA and protein expression gradually decreased in astrocytes,whereas cellular volume increased in a time-dependent manner (P< 0.01).Following oxygen-glucose reintroduction,AQP4 mRNAand protein expression was upregulated,peaked at day 7,and then gradually decreased,but still higher than normal levels (P < 0.05).However,cellular volume gradually decreased (P < 0.01),and then reached normal levels at day 7.CONCLUSION:AQP4 expression highly correlated with cellular volume changes,suggesting that AQP4 played an important role in modulating brain water transport in an astrocytic oxygen-glucose deprivation and reintroduction model.  相似文献   

2.
In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.  相似文献   

3.
Nicchia GP  Rossi A  Mola MG  Procino G  Frigeri A  Svelto M 《Glia》2008,56(16):1755-1766
Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.  相似文献   

4.
Cerebral edema contributes to morbidity and mortality in stroke. Aquaporins (AQPs)-1, -4, and -9 have been identified as the three main water channels in the brain. To clarify their role in water movement, we have compared their expression patterns with brain swelling after transient focal brain ischemia. There were two peaks of maximal hemispheric swelling at 1 hr and at 48 hr after ischemia, coinciding with two peaks of AQP4 expression. At 1 hr after occlusion, AQP4 expression was significantly increased on astrocyte endfeet in the core and in the border of the lesion. At 48 hr, AQP4 expression was increased in astrocytes in the border of the lesion over the whole cell. AQP9 showed a significant induction at 24 hr that increased gradually with time, without correlation with the swelling. The expression of AQP1 remained unchanged. These results suggest that AQP4, but not AQP1 or AQP9, may play an important role in water movement associated with the pathophysiology of edema after transient cerebral ischemia in the mouse.  相似文献   

5.
Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin‐4 (AQP4), an astrocyte water channel, or of Na+‐permeable channels, which mediate cellular osmolyte influx. However, the spatio‐temporal functional interactions between AQP4 and Na+‐permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na+‐permeable channels that are newly upregulated. Here, using co‐immunoprecipitation and Förster resonance energy transfer, we report that AQP4 physically co‐assembles with the sulfonylurea receptor 1—transient receptor potential melastatin 4 (SUR1‐TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell‐swelling studies using calcein fluorescence imaging of COS‐7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1‐TRPM4‐AQP4, was required for fast, high‐capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold‐injury to the cerebellum, we found that astrocytes newly upregulate SUR1‐TRPM4, that AQP4 co‐associates with SUR1‐TRPM4, and that genetic inactivation of the solute pore of the SUR1‐TRPM4‐AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1‐TRPM4‐AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4‐mediated pathological conditions.  相似文献   

6.
Brain edema is a serious complication of hepatic encephalopathy associated with fulminant hepatic failure (FHF). A major component of the edema seems to be cytotoxic, involving astrocyte swelling. Although the mechanism of brain edema in FHF is incompletely understood, it is generally believed that ammonia is involved critically in this process. Recent studies have shown that exposure of cultured astrocytes to ammonia results in the mitochondrial permeability transition (MPT), a phenomenon associated with mitochondrial failure and subsequent cellular dysfunction. The present study examined the potential role of the MPT in the astrocyte swelling associated with ammonia toxicity. Treatment of cultured astrocytes with ammonia (5 mM) caused a time-dependent increase in astrocyte cell volume (swelling), which was completely inhibited by the MPT inhibitor cyclosporin A (CsA). In this study, CsA also inhibited the ammonia-induced aquaporin 4 (AQP4) upregulation, which had been shown previously to be increased in cultured astrocytes by ammonia treatment. These findings suggest that the MPT plays a significant role in the ammonia-induced astrocyte swelling and may contribute to the brain edema associated with FHF.  相似文献   

7.
Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin‐4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β‐dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β‐DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia. © 2012 Wiley Periodicals, Inc.  相似文献   

8.
In multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), dysfunction of the blood–brain barrier (BBB) leads to edema formation within the central nervous system. The molecular mechanisms of edema formation in EAE/MS are poorly understood. We hypothesized that edema formation is due to imbalanced water transport across the BBB caused by a disturbed crosstalk between BBB endothelium and astrocytes. Here, we demonstrate at the light microscopic and ultrastructural level, the loss of polarized localization of the water channel protein aquaporin-4 (AQP4) in astrocytic endfeet surrounding microvessels during EAE. AQP4 was found to be redistributed over the entire astrocytic cell surface and lost its arrangement in orthogonal arrays of intramembranous particles as seen in the freeze-fracture replica. In addition, immunostaining for the astrocytic extracellular matrix receptor β-dystroglycan disappeared from astroglial membranes in the vicinity of inflammatory cuffs, whereas immunostaining for the dystroglycan ligands agrin and laminin in the perivascular basement membrane remained unchanged. Our data suggest that during EAE, loss of β-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic endfeet, and thus to edema formation in EAE. An erratum to this article can be found at  相似文献   

9.
Aquaporin-4 (AQP4) is the predominant water channel in brain and is selectively expressed in astrocytes. Astrocytic endfoot membranes exhibit tenfold higher densities of AQP4 than non-endfoot membranes, making AQP4 an excellent marker of astrocyte polarization. Loss of astrocyte polarization is known to compromise astrocytic function and to be associated with impaired water and K+ homeostasis. Here we investigate by a combination of light and electron microscopic immunocytochemistry whether amyloid deposition is associated with a loss of astrocyte polarization, using AQP4 as a marker. We used the tg-ArcSwe mouse model of Alzheimer's disease, as this model displays perivascular plaques as well as plaques confined to the neuropil. 3D reconstructions were done to establish the spatial relation between plaques and astrocytic endfeet, the latter known to contain the perivascular pool of AQP4. Changes in AQP4 expression emerge just after the appearance of the first plaques. Typically, there is a loss of AQP4 from endfoot membranes at sites of perivascular amyloid deposits, combined with an upregulation of AQP4 in the neuropil surrounding plaques. By electron microscopy it could be verified that the upregulation reflects an increased concentration of AQP4 in those delicate astrocytic processes that abound in synaptic regions. Thus, astrocytes exhibit a redistribution of AQP4 from endfoot membranes to non-endfoot membrane domains. The present data suggest that the development of amyloid deposits is associated with a loss of astrocyte polarization. The possible perturbation of water and K+ homeostasis could contribute to cognitive decline and seizure propensity in patients with Alzheimer's disease.  相似文献   

10.
Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin‐4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen‐activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal‐regulated kinase (ERK)1/2, C‐Jun amino‐terminal kinase (JNK)1/2/3, and p38‐MAPK. Inhibitors of ERK1/2 and p38‐MAPK (but not of JNK) blocked (40–60%) the manganese‐induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4‐related neurological complications associated with manganese toxicity. © 2010 Wiley‐Liss, Inc.  相似文献   

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