Novel variants in human Aquaporin-4 reduce cellular water permeability

Cerebral edema contributes significantly to morbidity and mortality after brain injury and stroke. Aquaporin-4 (AQP4), a water channel expressed in astrocytes, plays a key role in brain water homeostasis. Genetic variants in other aquaporin family members have been associated with disease phenotypes. However, in human AQP4, only one non-synonymous single-nucleotide polymorphism (nsSNP) has been reported, with no characterization of protein function or disease phenotype. We analyzed DNA from an ethnically diverse cohort of 188 individuals to identify novel AQP4 variants. AQP4 variants were constructed by site-directed mutagenesis and expressed in cells. Water permeability assays in the cells were used to measure protein function. We identified 24 variants in AQP4 including four novel nsSNPs (I128T, D184E, I205L and M224T). We did not observe the previously documented M278T in our sample. The nsSNPs found were rare ( approximately 1-2% allele frequency) and heterozygous. Computational analysis predicted reduced function mutations. Protein expression and membrane localization were similar for reference AQP4 and the five AQP4 mutants. Cellular assays confirmed that four variant AQP4 channels reduced normalized water permeability to between 26 and 48% of the reference (P < 0.001), while the M278T mutation increased normalized water permeability (P < 0.001). We identified multiple novel AQP4 SNPs and showed that four nsSNPs reduced water permeability. The previously reported M278T mutation resulted in gain of function. Our experiments provide insight into the function of the AQP4 protein. These nsSNPs may have clinical implications for patients with cerebral edema and related disorders.     

Lactic acid increases aquaporin 4 expression on the cell membrane of cultured rat astrocytes

The water channel protein aquaporin (AQP) may play roles in the homeostasis of water content in the brain and brain edema. One possible mechanism of brain edema is glial swelling due to lactic acidosis associated with ischemia. Here, we investigated the effect of lactic acid on the expression and cellular distribution of AQP 4 in cultured rat astrocytes. After 24h of incubation, the AQP4 expression level increased maximally with 35mM lactic acid. The AQP4 expression levels also increased with hydrochloric acid or acetic acid. In contrast, with sodium lactate, the AQP4 levels did not increase. The increase in AQP4 expression level occurred without a significant increase in AQP4 mRNA expression level by lactic acid. Under the conditions of de novo protein synthesis inhibition with cycloheximide, lactic acid increased the AQP4 expression level. Furthermore, lactic acid increased the AQP4 expression level on the cell surface of the astrocytes, as determined by a cell surface biotinylation assay and immunocytochemical examination. The increase in AQP4 expression level on the cell membrane of astrocytes induced by lactic acid may be a new regulation mechanism of AQP4 in the brain.     

胶质瘤细胞对血脑屏障水通道表达的影响

目的:探索胶质瘤细胞对血脑屏障水通道表达的影响及其病理生理意义。方法:通过内皮细胞系与胶质细胞体外共培养的方法建立体外血脑屏障模型。利用重水及高效液相分析的方法观察胶质瘤细胞作用对血脑屏障水转运的影响。运用半定量RT-PCR的方法分析胶质瘤细胞作用后血脑屏障内皮细胞及胶质细胞水通道1和水通道4的表达变化。结果:胶质瘤细胞可以诱导血脑屏障内皮细胞水通道1的异常表达并降低胶质细胞水通道4的表达水平。同时,胶质瘤细胞明显增强了体外血脑屏障模型对水由内皮细胞腔面至基底面的扩散。结论:胶质瘤性脑水肿不一定是血浆等大分子物质通透性增加的结果。血脑屏障中水通道的表达变化是胶质瘤性脑水肿发生的重要分子机制。     

Aquaglyceroporin 9 in brain pathologies

Aquaglyceroporins belong to the aquaporin family and are permeable to water and also to small solutes such as glycerol and urea. In this review, we will compare the expression of aquaporin 9 (AQP9), an aquaglyceroporin, with that of AQP4, a pure water channel, in pathological conditions. In astrocytes, AQP4 is mainly involved in water and ionic homeostasis. Its expression is highly modified in several brain disorders and it plays a key role in cerebral edema formation. AQP9 is expressed in astrocytes and in catecholaminergic neurons. The level of expression of brain AQP9 is under the control of blood insulin concentrations, and its expression is increased in diabetes, suggesting that AQP9 could be involved in brain energy metabolism. The induction of AQP9 in astrocytes is observed over time after stroke onset, suggesting participation in the clearance of excess lactate in the extracellular space. In some models, AQP9 is also induced in non-catecholaminergic neurons after global ischemia and in the periphery of gliomas, however functional roles are still unclear. The review of literature underlies that each AQP has several distinctive roles which depend on the AQP and cell types.     

New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice

Aquaporin-4 (AQP4) is the major water channel in the CNS. Its expression at fluid-tissue barriers (blood-brain and brain-cerebrospinal fluid barriers) throughout the brain and spinal cord suggests a role in water transport under normal and pathological conditions. Phenotype studies of transgenic mice lacking AQP4 have provided evidence for a role of AQP4 in cerebral water balance and neural signal transduction. Primary cultures of astrocytes from AQP4-null mice have greatly reduced osmotic water permeability compared with wild-type astrocytes, indicating that AQP4 is the principal water channel in these cells. AQP4-null mice have reduced brain swelling and improved neurological outcome following water intoxication and focal cerebral ischemia, establishing a role of AQP4 in the development of cytotoxic (cellular) cerebral edema. In contrast, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema caused by freeze-injury and brain tumor, probably due to impaired AQP4-dependent brain water clearance. AQP4-null mice also have markedly reduced acoustic brainstem response potentials and significantly increased seizure threshold in response to chemical convulsants, implicating AQP4 in modulation of neural signal transduction. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the CNS associated with altered brain water balance.     

Acute and chronic changes in aquaporin 4 expression after spinal cord injury

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.     

Effect of mild hypothermia on the expression of aquaporin family in cultured rat astrocytes under hypoxic condition

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In this study, we investigated the effect of mild hypothermia on the expression of AQP4, AQP5 and AQP9 in rat astrocytes cultured under hypoxic conditions. At 37 degrees C, a marked decrease in the expression of AQP4, AQP5 and AQP9 mRNAs was observed. However, at 32 degrees C (mild hypothermia), the expression of AQP5 mRNA was restored to its basal level. Interestingly, under mild hypothermia AQP4 mRNA expression transiently decreased and then increased about two-fold; while AQP9 mRNA expression decreased the same as at 37 degrees C. The changes in the expression of AQP4 and AQP9 proteins were confirmed by Western blot analysis. The restoration of the AQP4 and AQP5 expression at 32 degrees C from the hypoxia-induced decrease at 37 degrees C may play an important role in the reduction of brain edema under hypothermic conditions.     

Localization of Reversion-Induced LIM Protein (RIL) in the Rat Central Nervous System

Reversion-induced LIM protein (RIL) is a member of the ALP (actinin-associated LIM protein) subfamily of the PDZ/LIM protein family. RIL serves as an adaptor protein and seems to regulate cytoskeletons. Immunoblotting suggested that RIL is concentrated in the astrocytes in the central nervous system. We then examined the expression and localization of RIL in the rat central nervous system and compared it with that of water channel aquaporin 4 (AQP4). RIL was concentrated in the cells of ependyma lining the ventricles in the brain and the central canal in the spinal cord. In most parts of the central nervous system, RIL was expressed in the astrocytes that expressed AQP4. Double-labeling studies showed that RIL was concentrated in the cytoplasm of astrocytes where glial fibrillary acidic protein was enriched as well as in the AQP4-enriched regions such as the endfeet or glia limitans. RIL was also present in some neurons such as Purkinje cells in the cerebellum and some neurons in the brain stem. Differential expression of RIL suggests that it may be involved in the regulation of the central nervous system.     

Functional and molecular interactions between aquaporins and Na,K-ATPase

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Astrocytes play a key role in the clearance of both potassium (K⁺) and glutamate released during neuronal activity. Emerging evidence suggests that AQP4 facilitates K⁺ clearance by astrocytes and contributes to recovery of neuronal excitability. Here we report that AQP4 can assemble with its regulator metabotropic glutamate receptor 5 (mGluR5) and with Na,K-ATPase; the enzyme responsible for active K⁺ transport and for establishing the electrochemical gradient across the cell plasma membrane. We have, by use of pull down assays in rat brain tissue, identified the segment in the AQP4 NH₂-terminus containing the amino acid residues 23–32 as the site for interaction with Na,K-ATPase catalytic subunit and with mGluR5. Mutagenesis studies revealed that the AQP4 amino acids K27 and W30 are of key importance for interaction with both Na,K-ATPase and mGluR5. To confirm that interaction also occurs within intact cells, we have performed fluorescence resonance energy transfer (FRET) studies in primary astrocytes derived from rat striatum. The results indicate close proximity of wild type AQP4 and Na,K-ATPase in the plasma membrane of rat astrocytes. FRET efficiencies observed with the mutants AQP4 K27A and AQP4 W30A were significantly lower, highlighting the importance of these residues for the interaction between AQP4 and Na,K-ATPase. We conclude that AQP4/Na,K-ATPase/mGluR5 can form a macromolecular complex/transporting microdomain in astrocytes. This complex may be of functional importance for the regulation of water and K⁺ homeostasis in the brain, as well as for neuron-astrocyte metabolic crosstalk.     

Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice

Aquaporin-type water channels are expressed widely in mammalian tissues, particularly in the kidney, lung, eye and gastrointestinal tract. To define the role of aquaporins in organ physiology, we have generated and analysed transgenic mice lacking aquaporins (AQP) 1, 3, 4 and 5. Multiple phenotype abnormalities were found in the null mice. For example, in kidney, deletion of AQP1 or AQP3 produced marked polyuria whereas AQP4 deletion produced only a mild concentrating defect. Deletion of AQP5, the apical membrane water channel in the salivary gland, caused defective saliva production. Deletion of AQP1 or AQP5, water channels in lung endothelia and epithelia, resulted in a 90% decrease in airspace-capillary water permeability. In the brain, deletion of AQP4 conferred marked protection from brain swelling induced by acute water intoxication and ischaemic stroke. The general paradigm that has emerged from these phenotype studies is that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. However, we have found many examples in which the tissue-specific expression of an aquaporin is not associated with any apparent phenotypic abnormality. The physiological data on aquaporin null mice suggest the utility of aquaporin blockers and aquaporin gene replacement in selected human diseases.     

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K⁺–Cl⁻ cotransporter 1 (KCC1), believed to provide the pathways for Cl⁻ and K⁺ secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.     

Water transport becomes uncoupled from K+ siphoning in brain contusion, bacterial meningitis, and brain tumours: immunohistochemical case review

Specimens of normal human brain, contused brain, brain with bacterial meningitis, and brain tumours were immunolabelled for aquaporin 4 (AQP4) and Kir4.1. In normal brain tissue, AQP4 and Kir4.1 were detected around the microvessels. In pathological brain tissue, AQP4 was upregulated in astrocytes in oedematous regions and Kir4.1 was upregulated in astrocytes in damaged brain. Changes in alpha syntrophin expression paralleled those of AQP4 and Kir4.1. The following hypothesis is proposed: in astrocytes, under normal conditions, AQP4 couples water transport with Kir4.1 mediated K+ siphoning, but in pathological states, AQP4 facilitates the flow of brain oedema fluid, and Kir4.1 buffers increased extracellular K+.     

SIRT1对早期脑缺血大鼠水通道蛋白4表达的影响

目的:检测水通道蛋白4(AQP4)及沉默信息调节因子1(SIRT1)在脑缺血大鼠脑组织中的表达变化,探究SIRT1对AQP4的调节作用,明确二者在脑缺血及脑水肿发生发展中的作用。方法:雄性SD大鼠随机分成假手术组(sham组)和脑缺血模型组(MCAO组),其中MCAO组又分为6 h、12 h、24 h和48 h 4个时点组别。采用线栓法阻塞大脑中动脉建立局灶性脑缺血模型,于相应时点进行神经症状评分,Morris水迷宫检测大鼠学习认知功能,TTC染色观察脑梗死体积,干湿重法检测脑含水量的变化,HE染色观察大脑皮层周围组织神经细胞形态学变化,Western blot检测AQP4、SIRT1和基质金属蛋白酶9(MMP-9)的表达情况。结果:与sham组相比较,随着再灌注时间增加,MCAO组大鼠神经功能评分、脑梗死体积、脑组织通透性和脑组织含水量均持续增加,而大鼠学习认知功能则降低显著,HE染色显示脑缺血大脑皮层周围组织神经元细胞形态不规则,数目减少;Western blot实验结果显示AQP4表达水平呈增高趋势,SIRT1表达降低,MMP-9表达增高,MCAO-48 h组与sham组比较差异最明显(P0.01)。结论:脑缺血后,伴随脑组织损伤的加剧,SIRT1和MMP-9信号通路的表达激活影响了AQP4的表达活化,共同参与了脑水肿的形成。     

Expression of Aquaporin 1 and Aquaporin 4 in the Temporal Neocortex of Patients with Parkinson's Disease

The astrocytic water channel proteins aquaporin 1 (AQP1) and aquaporin 4 (AQP4) are known to be altered in brains affected by several neurodegenerative disorders, including Alzheimer's disease. However, AQP expression in brains affected by Parkinson's disease (PD) has not been described in detail. Recently, it has been reported that α‐synuclein (α‐syn)‐immunolabeled astrocytes show preferential distribution in several cerebral regions, including the neocortex, in patients with PD. Here, we investigated whether AQP expression is associated with α‐syn deposition in the temporal neocortex of PD patients. In accordance with the consensus criteria for dementia with Lewy bodies, the patients were classified into neocortical (PDneo), limbic (PDlim), and brain stem (PDbs) groups. Expressions of α‐syn, AQP1, and AQP4 in the temporal lobes of the individual PD patients were examined immunohistochemically. Immunohistochemical analysis demonstrated more numerous AQP4‐positive and AQP1‐positive astrocytes in the PDneo group than in the PDbs, PDlim, and control groups. However, in the PDneo cases, these astrocytes were not often observed in α‐syn‐rich areas, and semiquantitative analysis revealed that there was a significant negative correlation between the levels of AQP4 and α‐syn in layers V–VI, and between those of AQP1 and α‐syn in layers II–III. These findings suggest that a defined population of AQP4‐ and AQP1‐expressing reactive astrocytes may modify α‐syn deposition in the neocortex of patients with PD.     

Combustion smoke exposure induces up-regulated expression of vascular endothelial growth factor,aquaporin 4, nitric oxide synthases and vascular permeability in the retina of adult rats

Retinal cells respond to various experimental stimuli including hypoxia, yet it remains to be investigated whether they react to smoke inhalation. We show here that retinal cells in rats, notably the ganglion cells, Müller cells, astrocytes and blood vessels responded vigorously to a smoke challenge. The major changes included up-regulated expression of vascular endothelial growth factor (VEGF), aquaporin 4 (AQP4) and nitric oxide synthase (NOS). VEGF expression was localized in the ganglion cells, Müller cells, astrocytes and associated blood vessels. AQP4 was markedly enhanced in both astrocytes and Müller cells. Increase in vascular permeability after smoke exposure was evidenced by extravasation of serum derived rhodamine isothiocyanate which was internalized by Müller cells and ganglion cells. The tracer leakage was attenuated by aminoguanidine and N^G-nitro-l-arginine methyl ester (l-NAME) treatment which suppressed retinal tissue NOS and nitric oxide (NO) levels concomitantly. It is suggested that VEGF, AQP4 and NO are involved in increased vascular permeability following acute smoke exposure in which hypoxia was ultimately implicated as shown by blood gases analysis. NOS inhibitors effectively reduced the vascular leakage and hence may ameliorate possible retinal edema in smoke inhalation.     

Transcellular water transport and stability of expression in aquaporin 1-transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device

We investigated a portable bioartificial renal tubule device (BRTD) consisting of renal tubule cells and hollow fibers, to improve the quality of life of patients. It is necessary for a BRTD system to be compact. A compact portable BRTB requires transfection of an appropriate water channel or electrical pump genes in tubular epithelial cells, which should be based on physiological similarities to human kidney function. LLC-PK(1) cells, into which rat kidney aquaporin 1 (AQP1) cDNA was stably transfected, were evaluated for water transport ability. The expression and localization of water AQP1 were examined by Western blotting, RT-PCR, and immunofluorescence. To measure transcellular water permeation, a simple method was applied, using phenol red as a cell-impermeant marker of concentration. In contrast to wild-type LLC-PK(1) cells, rat AQP1-transfected cells had high transcellular osmotic water permeability. The expression of rat AQP1 mRNA (ratio of AQP1 to beta-actin mRNA) and protein bands (a 28-kDa band and a broad, 35- to 45-kDa band) was confirmed to be stably maintained until a population doubling level of 24. In AQP1-transfected LLCPK(1) cells, the protein was localized mainly to the basolateral side, but also the apical side, of the plasma membrane. Wild-type LLC-PK(1) cells were not stained at the plasma membrane. It is possible that enough AQP1-transfected tubule epithelial cells were supplied for a bioartificial renal tubule device.     

Aquaporin 4: a player in cerebral edema and neuroinflammation

Neuroinflammation is a common pathological event observed in many different brain diseases, frequently associated with blood brain barrier (BBB) dysfunction and followed by cerebral edema. Neuroinflammation is characterized with microglia activation and astrogliosis, which is a hypertrophy of the astrocytes. Astrocytes express aquaporin 4, the water channel protein, involved in water homeostasis and edema formation. Aside from its function in water homeostasis, recent studies started to show possible interrelations between aquaporin 4 and neuroinflammation. In this review the roles of aquaporin 4 in neuroinflammation associated with BBB disruption and cerebral edema will be discussed with recent studies in the field.     

Rapamycin alleviates brain edema after focal cerebral ischemia reperfusion in rats

Brain edema is a major consequence of cerebral ischemia reperfusion. However, few effective therapeutic options are available for retarding the brain edema progression after cerebral ischemia. Recently, rapamycin has been shown to produce neuroprotective effects in rats after cerebral ischemia reperfusion. Whether rapamycin could alleviate this brain edema injury is still unclear. In this study, the rat stroke model was induced by a 1-h left transient middle cerebral artery occlusion using an intraluminal filament, followed by 48?h of reperfusion. The effects of rapamycin (250?μg/kg body weight, intraperitoneal; i.p.) on brain edema progression were evaluated. The results showed that rapamycin treatment significantly reduced the infarct volume, the water content of the brain tissue and the Evans blue extravasation through the blood–brain barrier (BBB). Rapamycin treatment could improve histological appearance of the brain tissue, increased the capillary lumen space and maintain the integrity of BBB. Rapamycin also inhibited matrix metalloproteinase 9 (MMP9) and aquaporin 4 (AQP4) expression. These data imply that rapamycin could improve brain edema progression after reperfusion injury through maintaining BBB integrity and inhibiting MMP9 and AQP4 expression. The data of this study provide a new possible approach for improving brain edema after cerebral ischemia reperfusion by administration of rapamycin.     

Diabetes alters the localization of glial aquaporins in rat retina

Glial cells control the water homeostasis in the neural retina, in part via water transport through aquaporin (AQP) water channels. We investigated whether the immunolocalization of two water channels, AQP1 and AQP4, alters in the rat retina during experimental diabetes. Wistar rats were rendered diabetic by a single dose of streptozotocin, and retinal tissues were immunostained following 4 and 6 months. In control tissues, immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina and by distinct amacrine cells. In diabetic retinas, additional strong expression of AQP1 was found in glial cells located in the innermost retinal layers. The superficial retinal vessels were surrounded by AQP4 in control retinas, and by AQP1 in diabetic retinas. A similar alteration in the localization of AQP1 and AQP4 has been described in the rat retina after transient ischemia. The data suggest that the glial cell-mediated water transport in the retina of diabetic animals is altered especially at the superficial vessel plexus.     

Ischemia-reperfusion alters the immunolocalization of glial aquaporins in rat retina

Glial cells control the retinal osmohomeostasis, in part via mediation of water fluxes through aquaporin (AQP) water channels. By using immunohistochemical staining, we investigated whether ischemia-reperfusion of the rat retina causes alterations in the distribution of AQP1 and AQP4 proteins. Transient ischemia was induced in retinas of Long–Evans rats by elevation of the intraocular pressure for 60 min. In control retinas, immunoreactive AQP1 was expressed in the outer retina and by distinct amacrine cells, and AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina. After ischemia, retinal glial cells in the nerve fiber/ganglion cell layers strongly expressed AQP1. The perivascular staining around the superficial vessels altered from AQP4 in control retinas to AQP1 in postischemic retinas. The data suggest that the glial cell-mediated water transport in the retina is altered after ischemia especially at the superficial vessel plexus.