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Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE., (© 2024. The Author(s).)

Background: Ischemic stroke is one of the leading causes of human death and disability. Brain edema and peri-infarct astrocyte reactivity are crucial pathological changes, both involving aquaporin-4 (AQP4). Studies revealed that acute inhibition of AQP4 after stroke diminishes brain edema, however, its effect on peri-infarct astrocyte reactivity and the subacute outcome is unclear. And if diffusion-weighted imaging (DWI) could reflect the AQP4 expression patterns is uncertain., Methods: Rats were subjected to middle cerebral artery occlusion (MCAO) and allocated randomly to TGN 020-treated and control groups. One day after stroke, brain swelling and lesion volumes of the rats were checked using T2-weighted imaging (T2-WI). Fourteen days after stroke, the rats successively underwent neurological examination, T2-WI and DWI with standard b-values and ultra-high b-values, apparent diffusion coefficient (ADC) was calculated correspondingly. Finally, the rats' brains were acquired and used for glial fibrillary acidic protein (GFAP) and AQP4 immunoreactive analysis., Results: At 1 day after stroke, the TGN-020-treated animals exhibited reduced brain swelling and lesion volumes compared with those in the control group. At 14 days after stroke, the TGN-020-treated animals showed fewer neurological function deficits and smaller lesion volumes. In the peri-infarct region, the control group showed evident astrogliosis and AQP4 depolarization, which were reduced significantly in the TGN-020 group. In addition, the ultra-high b-values of ADC (ADCuh) in the peri-infarct region of the TGN-020 group was higher than that of the control group. Furthermore, correlation analysis revealed that peri-infarct AQP4 polarization correlated negatively with astrogliosis extent, and ADCuh correlated positively with AQP4 polarization., Conclusion: We found that acutely inhibiting AQP4 using TGN-020 promoted neurological recovery by diminishing brain edema at the early stage and attenuating peri-infarct astrogliosis and AQP4 depolarization at the subacute stage after stroke. Moreover, ADCuh could reflect the AQP4 polarization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Lin, Yin, Hao, Tian, Zhang, Ren, Li and Yang.)

The predominant form of edema that occurs during the early stage of ischemic stroke is cytotoxic, resulting in neuronal injury during brain ischemia and reperfusion. Intracellular calcium (Ca2+) is elevated following brain ischemia leading to increased cell membrane permeability. Ca2+/calmodulin-dependent protein kinase II (CaMK II), the downstream molecular signal of N-methyl-d-aspartate receptors (NMDARs), is sensitive to elevations in intracellular Ca2+. Aquaporin-4 (AQP4), which is expressed primarily in the brain, is a water-transport protein. However, it is unclear whether CaMK II regulates AQP4 expression to modulate cellular water permeability. We exposed cultured astrocytes to a hypoxic and glucose-free environment to mimic an ischemic environment in vitro. We investigated the effects of oxygen-glucose deprivation (OGD) on astrocytic viability and swelling, as well as CaMK II and AQP4 expression. We also studied the effects of CaMK II inhibition on cell swelling, viability and AQP4 expression. OGD increased astrocytic swelling and expression of CaMK II and AQP4, and it decreased astrocyte viability. Inhibition of CaMK II resulted in reduced astrocyte water permeability and AQP4 expression. We concluded that the upregulation of CaMK II promoted astrocyte swelling by increasing the expression of AQP4 after OGD., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 μL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

The present study was designed to determine the effect of different doses of oxytocin (OXT) on neuronal injury, spatial memory, blood-brain barrier (BBB) integrity and to explore possible underlying molecular mechanisms in the early stage of stroke in mice. Stroke model was generated by middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion in mice. OXT at doses of 1, 2, 4 and 8 IU/per mouse was administrated intranasally at the beginning of brain ischemia. Brain injury, BBB integrity, and spatial memory were evaluated by standard methods. Changes in the expression of nuclear factor-kappa B (NF-κB), and TUNEL positive cell were detected by immunohistochemistry. The levels of vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4) and brain-derived neurotrophic factor (BDNF) proteins were determined by western blotting and ELISA methods. OXT at doses of 4 and 8 IU/per mouse reduced the infarct size by 42% and 52%, respectively, and improved spatial memory function (p < 0.001). OXT (8 IU/per mouse) significantly reduced brain edema, BBB disruption and upregulated the AQP4 expression (p < 0.001). Finally, OXT significantly diminished the number of apoptotic, NF-κB positive cells and enhanced the expression of BDNF and VEGF proteins in the brain tissue (p < 0.001). These findings provide important evidences that OXT significantly suppresses neuronal damage in the early stage of stroke by inhibiting apoptotic and NF-κB signaling pathway, increasing the expression of VEGF, AQP4 and BDNF proteins and reducing the BBB leakage.

Background: Specific highly polarized aquaporin-4 (AQP4) expression is reported to play a crucial role in blood-brain barrier (BBB) integrity and brain water transport balance. The upregulation of polymerase δ-interacting protein 2 (Poldip2) was involved in aggravating BBB disruption following ischemic stroke. This study aimed to investigate whether Poldip2-mediated BBB disruption and cerebral edema formation in mouse bacterial meningitis (BM) model occur via induction of AQP4 polarity loss., Methods and Results: Mouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern. Recombinant human Poldip2 (rh-Poldip2) was administered intranasally at 1 hour after BM induction. Small interfering ribonucleic acid (siRNA) targeting Poldip2 was administered by intracerebroventricular (i.c.v) injection at 48 hours before BM induction. A specific inhibitor of matrix metalloproteinases (MMPs), UK383367, was administered intravenously at 0.5 hour before BM induction. Western blotting, immunofluorescence staining, quantitative real-time PCR, neurobehavioral test, brain water content test, Evans blue (EB) permeability assay, transmission electron microscopy (TEM), and gelatin zymography were carried out. The results showed that Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model. Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes, alleviated brain edema, preserved the integrity of BBB, and relieved the loss of AQP4 polarity in BM model. Rh-Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein (GFAP) and downregulated the expression of β-dystroglycan (β-DG), zonula occludens-1 (ZO-1), occludin, and claudin-5; whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP, and upregulated β-DG, ZO-1, occludin, and claudin-5. Similarly, UK383367 downregulated the expression of GFAP and upregulated the expression of β-DG, ZO-1, occludin, and claudin-5., Conclusion: Poldip2 inhibition alleviated brain edema and preserved the integrity of BBB partially by relieving the loss of AQP4 polarity via MMPs/β-DG pathway., (© 2020 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3-7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dexamethasone can alleviate the severity of bronchial and alveolar edema and therefore is widely applied in the treatment of various exudative diseases including pulmonary edema. However, the effectiveness of dexamethasone is still being questioned and its mechanism is not fully understood. Aquaporins (AQPs) are mainly responsible for the transmembrane transport of water, which is tightly associated with pulmonary edema. Small ubiquitin-like modifiers (SUMOs) are considered to play a protective role in some pathological conditions. In this study, we demonstrated that dexamethasone can upregulate the expression of AQPs in A549 cells by inducing SUMOylation. We found that a low dose of dexamethasone significantly upregulated the levels of SUMOylation and AQP expression in A549 cells, accompanied by a translocation of SUMOs from the cytoplasm to the nucleus. We also explored the possible relation between SUMOylation and AQPs. Knockdown of SUMO2/3 by RNA interference decreased the level of AQP4 in A549 cells after dexamethasone stimulation. Together, our findings demonstrated that AQP4 expression was upregulated in A549 cells exposed to dexamethasone, and SUMOylation may participate in the regulation of AQP4.

Objectives: To observe glymphatic transport and evaluate enlarged perivascular spaces (PVSs) in spontaneously hypertensive rats (SHRs)., Methods: SHRs were used as an animal model of cerebral small vessel disease, and Wistar Kyoto (WKY) rats were used as the control group. Histopathology was used to evaluate the enlargement of PVSs. A fluorescent tracer was infused into the cisterna magna of rats, and the proportion of the brain parenchyma area exposed to the fluorescent tracer was later quantified to evaluate the influx and efflux function of the glymphatic system. The global and polarized expression of aquaporin protein 4 (AQP4) was analyzed by immunofluorescence., Results: Compared with WKY rats, SHRs exhibited obviously enlarged PVSs and significantly decreased influx and efflux function of the glymphatic system. The results showed a significant decrease in AQP4 polarity in SHRs, but a difference in global AQP4 expression was not observed between SHRs and WKY rats., Conclusions: Impaired glymphatic transport may be involved in the pathogenesis of arteriolosclerotic cerebral small vessel disease, and enlarged PVSs may be a manifestation of the impaired glymphatic system., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: In the skeletal muscles, water metabolism is mainly regulated by water channel aquaporin 4 (AQP4). Although the expression level of AQP4 was reduced by long-term denervation, during denervation the relationship between muscle atrophy initiation and AQP4 expression decrease initiation remains unknown. The present study examined the relationship between the timing of muscle atrophy initiation and that of AQP4 expression decrease initiation, during the early stage of denervation., Methods: Female 344 rats (8 weeks of age) were randomly assigned to control (C), day 1 post-sciatic denervation (D1), day 4 post- sciatic denervation (D4) and day 7 post- sciatic denervation (D7) groups (n=6 per group). In the tibialis anterior (TA) muscles of each group, the expression levels of some target proteins were quantified by Western blot analysis., Results: The expression level of AQP4 significantly decreased on day 4 post-denervation (p<0.05). Moreover, the beginning of the decrease in AQP4 expression level was concurrent with the timing of muscle atrophy in the skeletal muscles during the early stage of denervation., Conclusions: The present study suggested that the progression of the decrease in the AQP4 expression level is partly related to the progression of muscle atrophy during the early stage of denervation., Competing Interests: The authors have no conflict of interest.

This study aimed to investigate the underlying pathological muscle damage in neuromyelitis optica spectrum disorder (NMOSD) patients without muscular symptoms. We prospectively enrolled 15 patients with aquaporin 4 (AQP4) antibody seropositive NMOSD and 16 patients with non-NMOSD diseases as a control group. Biceps biopsy samples from 18 patients were examined. Six NMOSD patients exhibited inflammatory lesions/edema in lower muscles on muscle MRI. On histopathological examination, NMOSD samples showed significantly decreased IgG-targeting AQP4 expression on sarcolemma compared with non-NMOSD samples in terms of the area of positive staining and integrated optical density. Muscle biopsy can support the differential diagnosis of NMOSD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Stroke is one of the leading causes of mortality and disability worldwide. Long noncoding RNAs (lncRNAs) including MALAT1 have been shown to have critical roles in cerebral ischemia reperfusion injury (CIRI). However, the underlying mechanism of MALAT1 in CIRI has not been elucidated. The present study aimed to investigate the function and potential regulatory mechanism of MALAT1 in cerebral ischemic reperfusion injury. We established the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reoxygenation (OGD/RX) model in vivo and in vitro, and then Cell Counting Kit-8 (CCK-8), RT-qPCR, flow cytometry analysis, lactate dehydrogenase (LDH) analysis, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to examine cell viability, MALAT1, aquaporin-4 (AQP4) expression, LDH release, and infarct volume, respectively. The level of AQP4 was remarkably upregulated in CIRI 24 h/48 h or OGD/RX 24 h/48 h compared with the sham group. Knockdown of AQP4 could alleviate OGD/RX-induced injury through enhancing cell viability and reducing LDH release and the rate of apoptotic cells. Furthermore, we found that MALAT1 was also increased in OGD/RX 24 h/48 h and silencing of MALAT1 could decrease AQP4. Inhibition of MALAT1 could also protect OGD/RX-induced injury, while the protective effect of MALAT1 siRNA on cerebral ischemic reperfusion was disappeared after transfection with AQP4 plasmid, indicating that MALAT1 may play a protective role in brain stroke through regulating AQP4. Taken together, our study provides evidence that MALAT1 is involved in ischemic stroke by inhibiting AQP4. Therefore, MALAT1 may serve as a potential target for therapeutic intervention in ischemic brain injury., (The Author(s). Published by S. Karger AG, Basel.)

Aquaporin (AQP4) could be associated with inflammation, common in central nervous system diseases. We investigated the effect of lipoxin A4 (LXA4) on the activation of astrocytes, AQP4 expression, and inflammatory response induced by lipopolysaccharide (LPS). Astrocytes were cultured in vitro and changes in transcript and protein levels of AQP4, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX-2), and protein levels of P38 and phospho-P38 were determined. The LPS group showed increased AQP4, IL-1β, TNF-α, and COX-2 levels, whereas they decreased in the LPS + LXA4 group, suggesting that LXA4 inhibits AQP4 expression. Furthermore, levels of phospho-P38 increased in the LPS group, but decreased in the LPS + LXA4 group. In conclusion, LXA4 alleviated the LPS-induced increase in AQP4 expression and inflammatory cytokine secretion by astrocytes, possibly by inhibiting P38 phosphorylation. For the first time, we found that LXA4 may inhibit the expression of inflammatory factors by regulating the expression of AQP4. AQP4 on astrocytes is likely to be the target of anti-inflammatory effect of LXA4., (Copyright © 2019. Published by Elsevier Inc.)

Background: Spinal cord ischemia-reperfusion injury (SCII) is a common complication of spinal surgery as well as thoracic and abdominal surgery. Acute cytotoxic edema is the key pathogenic alteration. Therefore, avoiding or decreasing cellular edema has become the major target for SCII treatment., Methods: The antiedema activity of ginsenoside Rb1 on aquaporin (AQP) 4, nerve growth factor (NGF), and brain-derived neurotrophic factor expression was detected by western blot and real-time polymerase chain reaction under conditions of oxygen-glucose deprivation/reoxygenation (OGD/R) in a rat astrocyte model in vitro. In addition, the cellular membrane permeability of AQP4 overexpressing cells or AQP4 small interfering RNA-transfected cells was detected., Results: Ginsenoside Rb1 significantly prevented OGD/R-induced AQP4 downregulation in rat astrocytes. In addition, ginsenoside Rb1 treatment or AQP4 overexpression in rat astrocytes significantly attenuated the OGD/R-induced increase of cellular membrane permeability. Moreover, ginsenoside Rb1 obviously prevented the OGD/R-induced decrease of NGF and BDNT expression in rat astrocytes., Conclusion: These findings demonstrate that ginsenoside Rb1 can relieve spinal cord edema and improve neurological function by increasing AQP4 expression.

Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC., (Copyright © 2019. Published by Elsevier Inc.)

Isoflurane is commonly used in pediatric population, but its mechanism of action in cognition is unclear. Aquaporin 4 (AQP4) regulates water content in blood, brain, and cerebrospinal fluid. Various studies have provided evidence for the role of AQP4 in synaptic plasticity and neurocognition. In this study, we aimed to determine whether a prolonged exposure to isoflurane in infant rats is associated with cognition and what effect this exposure has on AQP4 expression. Ten-day-old [postnatal day (P) 10] Wistar albino rats were randomly allocated to isoflurane group (n = 32; 1.5% isoflurane in 50% oxygen for 6 hours) or control group (n = 32; only 50% oxygen for 6 hours). Acute (P11) and long-term (P33) effects of 6-hour anesthetic isoflurane exposure on AQP4 expression were analyzed in whole brains of P11 and P33 rats by RT-qPCR and Western blot. Spatial learning and memory were assessed on P28 to P33 days by Morris Water Maze (MWM) test. The analysis revealed that isoflurane increased acutely both mRNA (~4.5 fold) and protein (~90%) levels of AQP4 in P11 rats compared with control group. The increasing levels of AQP4 in P11 were not observed in P33 rats. Also, no statistically significant change between isoflurane and control groups was observed in the latency to find the platform during MWM training and probe trial. Our results indicate that a single exposure to isoflurane anesthesia does not influence cognition in infant rats. In this case, acutely increased AQP4 after isoflurane anesthesia may have a protective role in neurocognition.

The blood-spinal cord barrier (BSCB) changes badly after spinal cord injury (SCI), and it is an important pathophysiological basis of SCI secondary damage. Aquaporin-4 (AQP4), one of the transmembrane proteins in spinal cord, has been shown to be closely related to the development of the BSCB and edema. We established a SCI model in rats using a free-falling weight drop device to subsequently investigate AQP4 expression. AQP4 messenger RNA (mRNA) and protein expression and immunoreactivity were detected in spinal cord tissue using reverse transcription-real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry and Western blot analysis. We found the water content and edema of the spinal cord were significantly higher than the control group after SCI, which was related to the growth of BSCB permeability; both reached their peak on the third day after injury. One, 3, 5, 7 days after injury, the immune response and protein expression in the model group increased from 1 to 3 days, with a plateau period from 3 to 5 days and a decline from 5 to 7 days, showing a significant difference compared with the sham group at each time point (P < 0.05), while the RT-qPCR results showed a decline of mRNA just after 3 days. In conclusion, after SCI, the water content of the spinal cord and the BSCB permeability increases, together with the excessive expression of AQP4, which reached a peak on the third day. AQP4 expression is closely relative to the permeability of BSCB and the water content of the spinal cord., (© 2019 Japanese Society of Neuropathology.)

Ischemic stroke occurs through embolic or thrombotic obliteration of an artery from cerebral circulation and represents over 80% of all stroke cases. One of the fiercest complications after stroke is edema, which results from imbalanced water diffusion around the blood vessels walls. Water diffusion around blood vessel walls occurs physiologically mainly through two protein-formed pores, namely aquaporins (AQPs) 1 and 4. Here, we compare for the first time the expression patterns and colocalization degrees of the two AQPs in control brain tissue and in peri-ischemic regions, on tissue obtained from eight patients with confirmed ischemic pathology and from five control cases. Our analysis showed that AQP4 is more abundant that AQP1, especially in the cortex and in the organized scar areas. The colocalization of the two markers was high, both located on the astrocytes membranes, but the colocalization degree decreased in the scar peri-ischemic regions. Colocalization with basement membranes was also lower for AQP1 compared to AQP4, in all regions analyzed.

Myocardial ischemia, hypoxia and reperfusion injury are induced by aortic occlusion, cardiac arrest and resuscitation during cardiopulmonary bypass (CPB), which can severely affect cardiac function. The aim of the present study was to investigate the effects of hydrogen‑rich solution (HRS) and aquaporin (AQP) on cardiopulmonary bypass (CPB)‑induced myocardial injury, and determine the mechanism of the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague Dawley rats were divided into a sham operation group, a CPB surgery group and a HRS group. A CPB model was established, and the hemodynamic parameters were determined at the termination of CPB. The myocardial tissues were observed by hematoxylin and eosin, and Masson staining. The levels of myocardial injury markers [adult cardiac troponin I (cTnI), lactate dehydrogenase (LDH), creatine kinase MB (CK‑MB) and brain natriuretic peptide (BNP)], inflammatory factors [interleukin (IL)‑1β, IL‑6 and tumor necrosis factor‑α (TNF‑α)] and oxidative stress indicators [superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO)] were determined by ELISA. Furthermore, H9C2 cells were treated with HRS following hypoxia/reoxygenation. Cell viability and cell apoptosis were investigated. The expression of apoptosis regulator Bcl‑2 (Bcl‑2), apoptosis regulator Bax (Bax), caspase 3, AQP‑1, AQP‑4, phosphorylated (p)‑Akt, heme oxygenase 1 (HO‑1) and nuclear factor erythroid 2‑related factor 2 (Nrf2) were investigated using western blotting and quantitative‑polymerase chain reaction of tissues and cells. Following CPB, myocardial cell arrangement was disordered, myocardial injury markers (cTnI, LDH, CK‑MB and BNP), inflammatory cytokines (IL‑1β, IL‑6 and TNF‑α) and MDA levels were significantly increased compared with the sham group; whereas the SOD levels were significantly downregulated following CPB compared with the sham group. HRS attenuated myocardial injury, reduced the expression levels of cTnI, LDH, CK‑MB, BNP, IL‑1β, IL‑6, TNF‑α, MDA and MPO, and increased SOD release. Levels of Bcl‑2, AQP‑1, AQP‑4, p‑Akt, HO‑1 and Nrf2 were significantly increased following HRS; whereas Bax and caspase‑3 expression levels were significantly reduced following CPB. HRS treatment significantly increased the viability of myocardial cells, reduced the rate of myocardial cell apoptosis and the release of MDA and LDH compared with the CPB group. A PI3K inhibitor (LY294002) was revealed to reverse the protective effect of HRS treatment. HRS was demonstrated to attenuate CPB‑induced myocardial injury, suppress AQP‑1 and AQP‑4 expression following CPB treatment and protect myocardial cells via the PI3K/Akt signaling pathway.

This study firstly used a rat traumatic brain injury model to compare the therapeutic effects of different intravenous infusion speed of 7.5% hypertonic saline (HS). Then the authors applied different delivery rate of 7.5% HS to two groups of patients to figure out the optimal infusion rates. A total of 100 rats were randomly divided into control group, group A (7.5% HS 6 mL/h), group B (7.5% HS 3 mL/h), and group C (7.5% HS 2 mL/h). All rats were established for the brain injury model. A total of 30 patients were selected and randomly divided into group A (250 mL/h) and group B (125 mL/h), with 15 cases in each group. Urine amount was recorded per hour; furthermore, blood was extracted from the patients to measure the levels of AQP4, NKCC1, tumour necrosis factor-α (TNF-a), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Compared with other groups, the expression levels of NKCC1 and AQP4 mRNA in group A was the lowest (P < 0.05). NKCC1 and AQP4 protein expression levels were the lowest in all the groups (P < 0.05). On the aspect of patients, group A displayed more significant difference compared with B group in terms of AQP4, NKCC1, TNF-a, IL-1β, and IL-6 (P < 0.05). In the two groups, a significant difference was noted in the urine amount at 4 h after administration (P < 0.05). In our study, infusion of hypertonic saline (250 mL/h) at the optimal rate of 7.5% HS decreased the intracranial pressure, brain tissue edema, and inflammatory cytokine expression; moreover, it can promote brain tissue protection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

The role of astrocytes in brain plasticity has not been extensively studied compared with that of neurons. Here we adopted integrative translational and reverse-translational approaches to explore the role of an astrocyte-specific major water channel in the brain, aquaporin-4 (AQP4), in brain plasticity and learning. We initially identified the most prevalent genetic variant of AQP4 (single nucleotide polymorphism of rs162008 with C or T variation, which has a minor allele frequency of 0.21) from a human database (n=60 706) and examined its functionality in modulating the expression level of AQP4 in an in vitro luciferase reporter assay. In the following experiments, AQP4 knock-down in mice not only impaired hippocampal volumetric plasticity after exposure to enriched environment but also caused loss of long-term potentiation after theta-burst stimulation. In humans, there was a cross-sectional association of rs162008 with gray matter (GM) volume variation in cortices, including the vicinity of the Perisylvian heteromodal language area (Sample 1, n=650). GM volume variation in these brain regions was positively associated with the semantic verbal fluency. In a prospective follow-up study (Sample 2, n=45), the effects of an intensive 5-week foreign language (English) learning experience on regional GM volume increase were modulated by this AQP4 variant, which was also associated with verbal learning capacity change. We then delineated in mice mechanisms that included AQP4-dependent transient astrocytic volume changes and astrocytic structural elaboration. We believe our study provides the first integrative evidence for a gliogenetic basis that involves AQP4, underlying language-associated brain plasticity.

Aquaporin-4 (AQP4) is a selective water channel, which expresses on the plasma membrane of myofibers and regulates the osmotic pressure, energy metabolism and morphological changes in myofibers by modulating water transport across sarcolemma in skeletal muscles. Although the physiological roles of AQP4 have been gradually clarified in skeletal muscles, the regulatory mechanisms of AQP4 expression have been poorly understood in skeletal muscles. Recently, it was reported that the expression of AQP4 decreased in atrophied skeletal muscles following sciatic nerve transection, but not tail-suspension. Therefore, expecting that the nerve supply to myofibers would be one of the major regulatory factors regulating AQP4 expression in skeletal muscles, we investigated whether the expression patterns of AQP4 were changed in skeletal muscles by denervation and subsequent reinnervation. As a result, while the APQ4 expression levels were significantly decreased by sciatic nerve freezing-induced denervation, subsequently the expression levels of AQP4 were fully restored during reinnervation in skeletal muscles (p < 0.05, respectively). On the other hand, the expression levels of α1-syntrophin and AQP1, which are respectively structural and functional related AQP4 factors, were stably maintained during the denervation and subsequent reinnervation. Therefore, the present study demonstrated that the expression of AQP4 may be regulated depending on the innervation to skeletal muscles. Moreover, AQP4 regulatory mechanisms may be fundamentally different to those of AQP1 in skeletal muscles.

Aim: Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury., Methods: Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining., Results: Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05)., Conclusion: Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Glioma is the most common and lethal central nervous system tumors. Temozolomide (TMZ) is an effective drug for malignant glioma, however, the intracellular and molecular mechanisms behind this anti-cancer effect have yet to be fully understood. The aim of the present study was to determine whether TMZ inhibits proliferation, invasion of glioma cells in vitro and whether these effects can be mediated through modulation of aquaporin 4 (AQP4) and phosphorylation of the MAPK pathway. The viability of U87 and U251 human glioma cells was evaluated using MTT assay. The cell cycle distribution was detected with flow cytometry. Migration ability and invasion ability were tested by scratch assays and transwell assays, respectively. The levels of AQP4 and MAPK were measured using immunoblot analyses. Our results showed that TMZ inhibited proliferation, migration and invasion, and induced G2/M arrest in U87 and U251 glioma cell lines. These changes were associated with a decrease in the levels of AQP4 expression as well as activation phosphorylated level of p38. Treatment with a p38 chemical activator (anisomycin) resulted in similar effects as TMZ treatment on glioma cells. And p38 chemical inhibitor (SB203580) could block these effects in glioma treated with TMZ, suggesting a direct up-regulation of the p38 signaling pathway. Therefore, we identified that TMZ might have therapeutic potential for controlling proliferation, invasion of malignant glioma by inhibiting AQP4 expression through activation of p38 signal transduction pathway. J. Cell. Biochem. 118: 4905-4913, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Spinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R)., Methods: The post-natal day 1-2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett's test., Results: The OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated-at least in part-via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes., Conclusions: HMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.

Background: Intramedullary ependymal cysts of the spinal cord are rare, benign, fluid-filled cysts usually situated along the ventral surface of the spinal cord. Only 32 cases have been reported since they were first described. Thus, owing to the rarity at which these cysts are encountered, their management and pathogenesis remain controversial. Whereas some investigators have advocated for cystosubarachnoid shunt placement for symptomatic ependymal cysts, others have argued for complete cyst resection or simple fenestration. Here we report the case of a 56-year-old female with a T11-T12 ependymal cyst that was successfully managed with cyst fenestration. We further investigated a potential pathological mechanism of cyst formation by performing immunohistochemistry to detect aquaporin expression in the cyst lining., Case Description: A 56-year-old female was found to harbor an enlarging cystic lesion of the conus that was discovered on workup of progressive paraparesis and urinary incontinence. She had lower extremity weakness and progressive myelopathy. Thoracic laminectomy with cyst fenestration arrested her neurologic deterioration. Pathological analysis revealed an intramedullary ependymal cyst. Immunohistochemistry was subsequently performed for expression of aquaporin-1 and aquaporin-4. There was dense staining of the underlying neuropil with concurrent membranous staining pattern of the cyst lining., Conclusions: Intramedullary ependymal cysts are rare, cystic lesions of the spinal cord. Early cyst fenestration decompresses the cyst and prevents neurologic deterioration. Here we report for the first time that aquaporins are expressed in the cyst wall, which is consistent with a passive, osmotic pathogenic mechanism of cyst formation., (Published by Elsevier Inc.)

Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. It is predominantly expressed in astrocytes at the blood-brain and blood-liquor interfaces. Although several AQP4 isoforms have been identified in the mammalian brain, two, AQP4a (M1) and AQP4c (M23), have been confirmed to cluster into plasma membrane supramolecular structures, termed orthogonal arrays of particles (OAPs) and to enhance water transport through the plasma membrane. However, the role of the newly described water-conductive mammalian isoform AQP4e is unknown. Here, the dynamics of AQP4e aggregation into OAPs and its role in the regulation of astrocyte water homeostasis have been studied. Using super-resolution structured illumination, atomic force, and confocal microscopies, the results revealed that, in female rat astrocytes, AQP4e isoform colocalizes with OAPs, affecting its structural dynamics. In hypoosmotic conditions, which elicit cell edema, OAP formation was considerably enhanced by overexpressed AQP4e. Moreover, the kinetics of the cell swelling and of the regulatory volume decrease was faster in astrocytes overexpressing AQP4e compared with untransfected controls. Furthermore, the increase in maximal cell volume elicited by hypoosmotic stimulation was significantly smaller in AQP4e-overexpressing astrocytes. For the first time, this study demonstrates an active role of AQP4e in the regulation of OAP structural dynamics and in water homeostasis. SIGNIFICANCE STATEMENT Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. To date, only AQP4a and AQP4c isoforms have been confirmed to enhance water transport through plasmalemma and to cluster into orthogonal arrays of particles (OAPs). We here studied the dynamics, aggregation, and role in the regulation of astrocyte water homeostasis of the newly described water-conductive mammalian isoform AQP4e. Our main findings are as follows: brain edema mimicking hypoosmotic conditions stimulates the formation of new OAPs with larger diameters, due to the incorporation of additional cytoplasmic AQP4 channels and the redistribution of AQP4 channels of the existing OAPs; and AQP4e affects the dynamics of cell swelling and regulatory volume decrease in astrocytes exposed to hypoosmotic conditions., (Copyright © 2017 the authors 0270-6474/17/3710748-09$15.00/0.)

To estimate the spatial and temporal expression of Aquaporin-4 (AQP-4) in a murine model of automated cerebral cryoinjury and correlate AQP-4 expression with development of brain oedema and neurological function. AQP-4 levels were determined quantitatively by Western blots at site of injury and at sites adjacent to and distant from injury in brains of cryoinjured (experimental) (n=18), sham injured (n=18) & normal mice at 24, 48, 72h post injury. AQP-4 expression was correlated with percentage water content of brain, Neurological Severity Score (NSS) and rotarod scores. We found a 1.4-fold increase in expression of AQP-4 at the site of injury and at sites distant from injury at 24h when compared to normal mice (p=0.05). The increase in expression of AQP-4 24h post injury was significantly higher in experimental group at the site of injury and at the site adjacent to the injury in the ipsilateral hemisphere when compared to the sham injured mice (p=0.05). At 24h post injury the median NSS score in the experimental group was 9 (interquartile range 7.25-10) and that in the sham group was 0.5 (interquartile range 0.0-1.0) (p<0.001). At 48 and 72h, AQP-4 expression remained elevated in the experimental group when compared to normal brain, but the levels were not significantly different from that in sham group. AQP-4 expression was significantly elevated in the ipsilateral hemisphere in the first 24h following cerebral cortical injury in mice and this could be correlated with worsening of neurological function. Over the next 48h, there was a trend towards decrease in AQP-4 expression that was associated with partial recovery of neurological function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of upper and lower motor neurons. The disease progression is associated with the astrocytic environment. Aquaporin-4 (AQP4) water channels are the most abundant AQPs expressed in astrocytes, exerting important influences on central nervous system homeostasis. The present study aimed to characterize the alterations in AQP4 expression and loca-lization in superoxide dismutase 1 (SOD1) G93A transgenic mice. SOD1G93A mice were sacrificed during the presymptomatic, disease onset and end stages and immunostaining was performed on spinal cord sections to investigate neuronal loss, glial activation and AQP4 expression in the spinal cord. It was observed that global AQP4 expression increased in the spinal cord of SOD1G93A mice as the disease progressed. However, AQP4 polarization decreased as the disease progressed, and AQP4 polarized localization at the endfeet of astrocytes was decreased in the spinal ventral horn of SOD1G93A mice at the disease onset and end stages. Meanwhile, motor neuron dege-neration and decreased glutamate transporter 1 expression in astrocytes in SOD1G93A mice were observed as the disease progressed. The results of the present study demonstrated that AQP4 depolarization is a widespread pathological condition and may contribute to motor neuron degeneration in ALS.

Purpose: Aquaporin 4 (AQP4), a water channel protein, is known to be expressed in retinal Müller cells. The purpose of this study was to determine the effects of VEGF and AQP4 channels on the volumetric changes in Müller cells., Methods: Retinas from diabetic rats and a cultured Müller cell line, TR-MUL5, were used in this study. Intravitreal injections of VEGF or PBS were performed on either streptozotocin (STZ)-induced diabetic or normoglycemic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. VEGF protein levels from collected retinas were determined by western blot analysis. Volumetric changes and nitric oxide (NO) levels in cultured Müller cells were determined using flow cytometry (FACS), in the presence or absence of VEGF and TGN-020, a selective AQP4 inhibitor., Results: In the diabetic rat retina, VEGF immunoreactivity was concentrated in the internal retinal layers, and AQP4 immunoreactivity was higher than controls. The expressions of AQP4 were colocalized with GFAP. Protein levels of VEGF in the hyperglycemic rat retina were significantly higher than controls. FACS analyses showed that exposure to VEGF enlarged Müller cells, while exposure to TGN-020 suppressed the enlargement. Intracellular levels of NO were increased after exposure to VEGF, which was suppressed following the addition of TGN-020., Conclusion: The observed Müller cell swelling is mediated by VEGF and AQP4.

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., (© 2016 International Society of Neuropathology.)

The discovery of AQP4-IgG (a pathogenic antibody that targets the astrocytic water channel aquaporin-4), as the first sensitive and specific biomarker for any inflammatory central nervous system demyelinating disease (IDD), has shifted emphasis from the oligodendrocyte and myelin to the astrocyte as a central immunopathogenic player. Neuromyelitis optica (NMO) spectrum disorders (SDs) represent an evolving spectrum of IDDs extending beyond the optic nerves and spinal cord to include the brain (especially in children) and, rarely, muscle. NMOSD typical brain lesions are located in areas that highly express the target antigen, AQP4, including the circumventricular organs (accounting for intractable nausea and vomiting) and the diencephalon (accounting for sleep disorders, endocrinopathies, and syndrome of inappropriate antidiuresis). Magnetic resonance imaging brain abnormalities fulfill Barkoff criteria for multiple sclerosis in up to 10% of patients. As the spectrum broadens, the importance of highly specific assays that detect pathogenic AQP4-IgG targeting extracellular epitopes of AQP4 cannot be overemphasized. The rapid evolution of our understanding of the immunobiology of AQP4 autoimmunity necessitates continuing revision of NMOSD diagnostic criteria. Here, we describe scientific advances that have occurred since the discovery of NMO-IgG in 2004 and review novel targeted immunotherapies. We also suggest that NMOSDs should now be considered under the umbrella term autoimmune aquaporin-4 channelopathy., (© 2015 New York Academy of Sciences.)

Aquaporins (AQPs) are integral membrane proteins which maintain cellular water and ion homeostasis. Alterations in AQP expression have been reported in rod-dominated rodent retinas exposed to light. In rodents and also in birds, light of moderate intensities (700-2000 lux) damages the retina, though detailed changes were not examined in birds. The aim of our study was to see if light affects cone dominated retinas, which would be reflected in expression levels of AQPs. We examined AQP1 and AQP4 expressions in chick retina exposed to 2000 lux under 12 h light:12 h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod) and 24L:0D (constant light). Additionally, morphological changes, apoptosis (by TUNEL) and levels of glutamate and GFAP (a marker of injury) in the retina were examined to correlate these with AQP expressions. Constant light caused damage in outer and inner nuclear layer (ONL, INL) and ganglion cell layer (GCL). Also, there were associated increases in GFAP and glutamate levels in retinal extracts. In normal photoperiod, AQP1 was expressed in GCL, outer part of INL and photoreceptor inner segments of. AQP4 was additionally expressed in nerve fiber layer. Immunohistochemistry and Western blotting revealed over all decreased AQP1 and AQP4 expression in constant light condition compared to those in other two groups. The elevated GFAP and glutamate levels might be involved in the reduction of AQPs in constant light group. Such decreases in AQP expressions are perhaps linked with retinal cell damage seen in constant light condition, while their relatively enhanced expression in two other conditions may help in maintaining a normal retinal architecture, indicating their neuroprotective potential.

Background: In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells., Results: Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema., Conclusions: We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood-brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients.

Background: We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior., Methods and Results: Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels., Conclusions: Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1.

Rotavirus (RV) is the primary cause of severe dehydrating gastroenteritis and acute diarrheal disease in infants and young children. Previous studies have revealed that genistein can inhibit the infectivity of enveloped or nonenveloped viruses. Although the biological properties of genistein are well studied, the mechanisms of action underlying their anti-rotavirus properties have not been fully elucidated. Here, we report that genistein significantly inhibits RV-Wa replication in vitro by repressing viral RNA transcripts, and possibly viral protein synthesis. Interestingly, we also found that aquaporin 4 (AQP4) mRNA and protein expression, which was downregulated in RV-infected Caco-2 cells, can be upregulated by genistein in a time- and dose-dependent manner. Further experiments confirmed that genistein triggers CREB phosphorylation through PKA activation and subsequently promotes AQP4 gene transcription. These findings suggest that the pathophysiological mechanism of RV infection involves decreased expression of AQP4 and that genistein may be a useful candidate for developing a new anti-RV strategy by inhibiting rotavirus replication and upregulating AQP4 expression via the cAMP/PKA/CREB signaling pathway. Further studies on the effect of genistein on RV-induced diarrhea are warranted.

Aquaporin-4 (AQP4) is the most popular water channel protein expressed in brain tissue and plays a very important role in regulating the water balance in and outside of brain parenchyma. To investigate the expression of aquaporin-4 in the rat brain tissue after dexamethasone therapy of meningitis induced by Streptococcus pneumonia, total 40 of 3-week old Sprague-Dawley rats were divided into infection group (n=30) and normal control group (n=10). The meningitis groups were infected with 1×10(7) cfu/ml of Streptococcus pneumoniae and then randomized into no treatment (untreated group, n=10), treatment with ceftriaxone (CTRX group, n=10) and treatment with dexamethasone combined ceftriaxone (CTRX+DEXA group, n=10). The normal control group was established by using saline. The rats were euthanized when they reached terminal illness or five days after infection, followed by detection of AQP4 through using immunohistochemistry and Western blot methods. Data has showed that expression of AQP4 in model group remained higher than the control and treatment group (P<0.05). AQP4 expression in CTRX+DEXA group was lower than that in CTRX group (P<0.05). There was no statistical difference between CTRX+DEXA group and the control group (P>0.05). These data suggested that Dexamethasone could down-regulate the expression of AQP4 in the brain tissue of rats with meningitis and provides evidence for the mechanism of protective effect of Dexamethasone on central neurosystem.

Objective: To understand how aquaporin4 (AQP4) and dystroglycan (DG) polarized distribution change and their roles in brain edema formation after traumatic brain injury (TBI)., Methods: Brain water content, Evans blue detection, real-time PCR, western blot, and immunofluorescence were used., Results: At an early stage of TBI, AQP4 and DG maintained vessel-like pattern in perivascular endfeet; M1, M23, and M1/M23 were increased in the core lesion. At a later stage of TBI, DG expression was lost in perivascular area, accompanied with similar but delayed change of AQP4 expression; expression of M1, M23, and DG and the ratio of M1/M2 were increased., Conclusion: At an early stage, AQP4 and DG maintained the polarized distribution. Upregulated M1 and M23 could retard the cytotoxic edema formation. At a later stage AQP4 and DG polarized expression were lost from perivascular endfeet and induced the worst cytotoxic brain edema. The alteration of DG expression could regulate that of AQP4 expression after TBI.

Acute lung injury (ALI) occurs frequently in patients with severe traumatic brain injury (TBI) and is associated with a poor clinical outcome. Aquaporins (AQPs), particularly AQP1 and AQP4, maintain water balances between the epithelial and microvascular domains of the lung. Since pulmonary edema (PE) usually occurs in the TBI-induced ALI patients, we investigated the effects of a thaliporphine derivative, TM-1, on the expression of AQPs and histological outcomes in the lung following TBI in rats. TM-1 administered (10 mg/kg, intraperitoneal injection) at 3 or 4 h after TBI significantly reduced the elevated mRNA expression and protein levels of AQP1 and AQP4 and diminished the wet/dry weight ratio, which reflects PE, in the lung at 8 and 24 h after TBI. Postinjury TM-1 administration also improved histopathological changes at 8 and 24 h after TBI. PE was accompanied with tissue pathological changes because a positive correlation between the lung injury score and the wet/dry weight ratio in the same animal was observed. Postinjury administration of TM-1 improved ALI and reduced PE at 8 and 24 h following TBI. The pulmonary-protective effect of TM-1 may be attributed to, at least in part, downregulation of AQP1 and AQP4 expression after TBI.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy., Approach and Results: Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression., Conclusions: An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system., (© 2014 American Heart Association, Inc.)

The regulation of water movement is of utmost importance for normal retinal function. Under physiological conditions, water is transported, dependent on the osmotic gradient, through the retinal pigment epithelial cell layer from the subretinal space to the choroid. The osmotic gradient has been found to be modified in eye diseases, thus leading to water accumulation in the subretinal space and the sensory retina, and subsequently contributing to the formation of macular oedema. Understanding the regulation of aquaporin expression is therefore crucial. In this study, we investigated the effects of hyperosmolarity on aquaporin-4 (AQP4) protein expression in the human retinal pigment epithelial cell line, ARPE‑19. AQP4 expression was examined by PCR, western blot analysis and immunofluorescence. Ubiquitinylation was examined by immunoprecipitation. The results revealed that hyperosmotic stress rapidly decreased AQP4 expression in the ARPE-19 cells. The effect remained unmodified by lysosomal or mitogen-activated protein kinase inhibitors, but was reversed by proteasome inhibitors. However, no ubiquitinylation of AQP4 was detected. Our results suggest that hyperosmotic stress markedly reduces AQP4 expression possibly through a proteasome ubiquitinylation-independent pathway. This may represent an adaptation to hyperosmotic stress. The results presented in this study contribute to our understanding of the formation of macular oedema.

Importance: Reports of neuromyelitis optica spectrum disorder (NMOSD) occurring in the setting of neoplasia suggest that aquaporin-4 autoimmunity may in some cases have a paraneoplastic basis., Observations: In this case report, we describe a patient with NMOSD whose test results were seropositive for aquaporin-4 IgG and who had a hepatic metastasis from a small-bowel neuroendocrine tumor. The tumor cells expressed aquaporin-4 immunoreactivity. She presented to the Neurology Department at Wayne State University with bilateral leg weakness, ascending paresthesias, and decreased sensation., Conclusions and Relevance: This case extends the context of NMOSD as a paraneoplastic disorder.

Neuromyelitis optica (NMO) is an autoimmune inflammatory, neurodestructive disease primarily targeting the optic nerve and spinal cord. An autoantibody against water channel protein aquaporin-4 (AQP4), which is expressed at endofeet of astrocytes has been implicated in the pathogenesis of NMO. We evaluated the impact of sera of seropositive patients with NMO spectrum disorders (NMOSDs) on the rodent optic nerve and retina. Serum was obtained either from patients with seropositive NMOSD (AQP4+), seronegative patient with idiopathic optic neuritis (AQP4-), and healthy volunteers (control). Anti-AQP4 antibody in a serum was measured by a previously established cell-based assay. The patients' sera were applied on the optic nerve after de-sheathed. Immunohistochemistry showed that at 7 days after the treatment, the area of the optic nerve exposed to the AQP4+ sera lost expression of both AQP4 and glial fibrillary acidic protein. Also, Human-IgG immunoreactivity and marked invasion of inflammation cells were observed in the optic nerve treated with AQP4+ serum. Immnoreactivity of neurofilament was reduced at 14 days after the treatment, not 7 days. Real-time polymerase chain reaction revealed the reduced gene expression of neurofilament in retina from the eye that was exposed to the AQP4+ sera at 14 days. Retrograde fluorogold-labeling on the retinal flatmount disclosed the significantly reduced number of retinal ganglion cells when the AQP4+ sera were applied. The present model has demonstrated that the sera from patients with seropositive NMOSDs led to the regional astrocytic degeneration and inflammatory cell invasion in the optic nerve, resulting in the ultimate loss of RGCs and their axons at areas beyond the injury site., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Stroke results in inflammation, brain edema, and neuronal death. However, effective neuroprotectants are not available. Recent studies have shown that high mobility group box-1 (HMGB1), a proinflammatory cytokine, contributes to ischemic brain injury. Aquaporin 4 (AQP4), a water channel protein, is considered to play a pivotal role in ischemia-induced brain edema. More recently, studies have shown that pannexin 1 channels are involved in cerebral ischemic injury and the cellular inflammatory response. Here, we examined whether the pannexin 1 channel inhibitor probenecid could reduce focal ischemic brain injury by inhibiting cerebral inflammation and edema. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion (MCAO) for 1 h. Infarct volume, neurological score and cerebral water content were evaluated 48 h after MCAO. Immunostaining, western blot analysis and ELISA were used to assess the effects of probenecid on the cellular inflammatory response, HMGB1 release and AQP4 expression. Administration of probenecid reduced infarct size, decreased cerebral water content, inhibited neuronal death, and reduced inflammation in the brain 48 h after stroke. In addition, HMGB1 release from neurons was significantly diminished and serum HMGB1 levels were substantially reduced following probenecid treatment. Moreover, AQP4 protein expression was downregulated in the cortical penumbra following post-stroke treatment with probenecid. These results suggest that probenecid, a powerful pannexin 1 channel inhibitor, protects against ischemic brain injury by inhibiting cerebral inflammation and edema.

Mesial temporal sclerosis (MTS) is the most frequent cause of drug resistant symptomatic partial epilepsy. The mechanism and genetic background of this unique pathology are not well understood. Aquaporins (AQP) are regulators of water homeostasis in the brain and are expressed in the human hippocampus. We explored the role of AQP genes in the pathogenetic mechanisms of MTS through an evaluation of gene expression in surgically removed human brain tissue. We analyzed AQP1 and 4 mRNA levels by quantitative real-time polymerase chain reaction and normalized to ABL and cyclophilin genes, followed by immunohistochemistry for AQP4. Relative expressions were calculated according to the delta Ct method and the results were compared using the Mann-Whitney U test. Brain specimens of 23 patients with epilepsy who had undergone surgery for MTS and seven control autopsy specimens were investigated. Clinical findings were concordant with previous studies and 61% of the patients were seizure-free in the postoperative period. AQP1 and 4 gene expression levels did not differ between MTS patients and control groups. Immunofluorescence analysis of AQP4 supported the expression results, showing no difference. Previous studies have reported contradictory results about the expression levels of AQP in MTS. To our knowledge, only one study has suggested upregulation whereas the other indicated downregulation of perivascular AQP4. Our study did not support these findings and may rule out the involvement of AQP in human MTS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although propofol has been reported to offer neuroprotection against cerebral ischemia injury, its impact on cerebral edema following ischemia is not clear. The objective of this investigation is to evaluate the effects of propofol post-treatment on blood-brain barrier (BBB) integrity and cerebral edema after transient cerebral ischemia and its mechanism of action, focusing on modulation of aquaporins (AQPs), matrix metalloproteinases (MMPs), and hypoxia inducible factor (HIF)-1α. Cerebral ischemia was induced in male Sprague-Dawley rats (n = 78) by occlusion of the right middle cerebral artery for 1 h. For post-treatment with propofol, 1 mg kg(-1) min(-1) of propofol was administered for 1 h from the start of reperfusion. Nineteen rats undergoing sham surgery were also included in the investigation. Edema and BBB integrity were assessed by quantification of cerebral water content and extravasation of Evans blue, respectively, following 24 h of reperfusion. In addition, the expression of AQP-1, AQP-4, MMP-2, and MMP-9 was determined 24 h after reperfusion and the expression of HIF-1α was determined 8 h after reperfusion. Propofol post-treatment significantly reduced cerebral edema (P < 0.05) and BBB disruption (P < 0.05) compared with the saline-treated control. The expression of AQP-1, AQP-4, MMP-2, and MMP-9 at 24 h and of HIF-1α at 8 h following ischemia/reperfusion was significantly suppressed in the propofol post-treatment group (P < 0.05). Propofol post-treatment attenuated cerebral edema after transient cerebral ischemia, in association with reduced expression of AQP-1, AQP-4, MMP-2, and MMP-9. The decreased expression of AQPs and MMPs after propofol post-treatment might result from suppression of HIF-1α expression.

Increased cranial pressure due to development of edema contributes significantly to the pathology of traumatic brain injury (TBI). Induction of an astrocytic water channel protein, Aquaporin 4 (AQP4), is known to predominantly contribute to cytotoxic edema following TBI. However, the mechanism for the increase in AQP4 following 24 h of TBI is poorly understood. Here we show that transcriptional activation of a ubiquitously expressed mammalian forkhead transcription factor, Foxo3a, induces cerebral edema by increasing the AQP4 level in the controlled cortical impact model of TBI in mice. TBI stimulates nuclear translocation of Foxo3a in astrocytes and subsequently augments its binding to AQP4 promoter in pericontusional cortex. Nuclear accumulation of Foxo3a is augmented by a decrease in phosphorylation at its Ser256 residue due to inactivation of Akt after TBI. Depletion of Foxo3a in mice rescues cytotoxic edema by preventing induction of AQP4 as well as attenuates memory impairment after TBI in mice.