Aquaporin-4 Expression In Astrocytes Research Articles

Exploring the reduction in aquaporin-4 and increased expression of ciliary neurotrophic factor with the frontal-striatal gliosis induced by chronic high-fat dietary stress.

High-fat diet (HFD)-induced obesity induces peripheral inflammation and hypothalamic pathogenesis linking the activation of astrocytes and microglia. Clinical evidence indicates a positive correlation between obesity and psychiatric disorders, such as depression. The connectivity of the frontal-striatal (FS) circuit, involving the caudate putamen (CPu) and anterior cingulate cortex (ACC) within the prefrontal cortex (PFC), is known for its role in stress-induced depression. Thus, there is a need for a thorough investigation into whether chronic obesity-induced gliosis, characterized by the activation of astrocytes and microglia, in these brain regions of individuals with chronic obesity. The results revealed increased S100β+ astrocytes and Iba1+ microglia in the CPu and ACC of male obese mice, along with immune cell accumulation in meningeal lymphatic drainage. Activated GFAP+ astrocytes and Iba1+ microglia were observed in the corpus callosum of obese mice. Gliosis in the CPu and ACC was linked to elevated cleaved caspase-3 levels, indicating potential neural cell death by chronic HFD feeding. There was a loss of myelin and adenomatous polyposis coli (APC)+ oligodendrocytes (OLs) in the corpus callosum, an area known to be linked with injury to the CPu. Additionally, reduced levels of aquaporin-4 (AQP4), a protein associated within the glymphatic systems, were noted in the CPu and ACC, while ciliary neurotrophic factor (CNTF) gene expression was upregulated in these brain regions of obese mice. The invitro study revealed thathigh-dose CNTF causing a trend of reduced astrocytic AQP4 expression, but it significantly impaired OL maturation. This pathological evidence highlights that prolonged HFD consumption induces persistent FS gliosis and demyelination in the corpus callosum. An elevated level of CNTF appears to act as a potential regulator, leading to AQP4 downregulation in the FS areas and demyelination in the corpus callosum. This cascade of events might contribute to neural cell damage within these regions and disrupt the glymphatic flow.

Continuous theta burst stimulation ameliorates cognitive deficits in microinfarcts mice via inhibiting glial activation and promoting paravascular CSF-ISF exchange

Microinfarcts are widespread in the elderly, accompanied by varying degrees of cognitive decline. Continuous theta burst stimulation (cTBS) has been demonstrated to be neuroprotective on cognitive dysfunction, but the underlying cellular mechanism has been still not clear. In the present study, we evaluated the effects of cTBS on cognitive function and brain pathological changes in mice model of microinfarcts. The spatial learning and memory was assessed by Morris water maze (MWM), Glymphatic clearance efficiency was evaluated using in vivo two-photon imaging. The loss of neurons, activation of astrocytes and microglia, the expression and polarity distribution of the astrocytic aquaporin-4 (AQP4) were assessed by immunofluorescence staining. Our results showed that cTBS treatment significantly improved the spatial learning and memory, accelerated the efficiency of glymphatic clearance, up-regulated the AQP4 expression and improved the polarity distribution of AQP4 in microinfarcts mice. Besides, cTBS treatment increased the number of surviving neurons, whereas decreased the activated astrocytes and microglia. Our study suggested that cTBS accelerated glymphatic clearance and inhibited the excessive gliogenesis, which ultimately exerted neuroprotective effects on microinfarcts mice.

Norepinephrine-Activated p38 MAPK Pathway Mediates Stress-Induced Cytotoxic Edema of Basolateral Amygdala Astrocytes.

The amygdala is a core region in the limbic system that is highly sensitive to stress. Astrocytes are key players in stress disorders such as anxiety and depression. However, the effects of stress on the morphology and function of amygdala astrocytes and its potential mechanisms remain largely unknown. Hence, we performed in vivo and in vitro experiments using a restraint stress (RS) rat model and stress-induced astrocyte culture, respectively. Our data show that norepinephrine (NE) content increased, cytotoxic edema occurred, and aquaporin-4 (AQP4) expression was up-regulated in the basolateral amygdala (BLA) obtained from RS rats. Additionally, the p38 mitogen-activated protein kinase (MAPK) pathway was also observed to be significantly activated in the BLA of rats subjected to RS. The administration of NE to in vitro astrocytes increased the AQP4 level and induced cell edema. Furthermore, p38 MAPK signaling was activated. The NE inhibitor alpha-methyl-p-tyrosine (AMPT) alleviated cytotoxic edema in astrocytes, inhibited AQP4 expression, and inactivated the p38 MAPK pathway in RS rats. Meanwhile, in the in vitro experiment, the p38 MAPK signaling inhibitor SB203580 reversed NE-induced cytotoxic edema and down-regulated the expression of AQP4 in astrocytes. Briefly, NE-induced activation of the p38 MAPK pathway mediated cytotoxic edema in BLA astrocytes from RS rats. Thus, our data provide novel evidence that NE-induced p38 MAPK pathway activation may be one of the mechanisms leading to cytotoxic edema in BLA under stress conditions, which also could enable the development of an effective therapeutic strategy against cytotoxic edema in BLA under stress and provide new ideas for the treatment of neuropsychiatric diseases.

Molecular mechanism of cerebral edema improvement via IL-1RA released from the stroke-unaffected hindlimb by treadmill exercise after cerebral infarction in rats

Cerebral edema following cerebral infarction can be severe and directly affect mortality and mobility. Exercise therapy after cerebral infarction is an effective therapeutic approach; however, the molecular mechanism remains unclear. Myokines such as interleukin-1 receptor antagonist (IL-1RA) are released during skeletal muscle contraction with effects on other organs. We hypothesized that myokine release during exercise might improve brain edema and confirmed the hypothesis using transient middle cerebral artery occlusion (tMCAO) model rats. Rats subjected to tMCAO were divided according to the severity of illness and further assigned to exercise and non-exercise groups. Treadmill exercises were performed at a speed of 2–8 m/min for 10 min from 1–6 days post-reperfusion after tMCAO. Exercise significantly reduced edema and neurological deficits in severely ill rats, with a reduction in aquaporin-4 (AQP4) expression in the ischemic core and increased blood IL-1RA release from the stroke-unaffected hindlimb muscle after tMCAO. Administration of IL-1RA into the lateral ventricles significantly reduced edema and AQP4 expression in the ischemic core. In conclusion, treadmill exercise performed in the early phase of stroke onset alleviated the decrease in blood IL-1RA following ischemic stroke. IL-1RA administration decreased astrocytic AQP4 expression in the ischemic core, suppressing brain edema.

Sulforaphane Upregulates Cultured Mouse Astrocytic Aquaporin-4 Expression through p38 MAPK Pathway.

Protein misfolding and/or aggregation are common pathological features associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson disease (PD). Abnormal protein aggregation may be caused by misfolding of the protein and/or dysfunction of the protein clearance system. Recent studies have demonstrated that the specific water channel protein, aquaporin-4 (AQP4), plays a role in the pathogenesis of neurodegenerative diseases involving protein clearance system. In this study, we aimed to investigate the role of sulforaphane (SFN) in the upregulation of AQP4 expression, along with its underlying mechanism using cultured mouse astrocytes as a model system. At low concentrations, SFN was found to increase cell proliferation and result in the activation of astrocytes. However, high SFN concentrations were found to suppress cell proliferation of astrocytes. In addition, our study found that a 1 μM concentration of SFN resulted in the upregulation of AQP4 expression and p38 MAPK phosphorylation in cultured mouse astrocytes. Moreover, we demonstrated that the upregulation of AQP4 expression was significantly attenuated when cells were pretreated with SB203580, a p38 MAPK inhibitor. In conclusion, our findings from this study revealed that SFN exerts hormesis effect on cultured mouse astrocytes and can upregulate astrocytic AQP4 expression by targeting the p38 MAPK pathway.

Differentially Aquaporin 5 Expression in Submandibular Glands and Cerebral Cortex in Alzheimer's Disease.

Impaired brain clearance mechanisms may result in the accumulation of aberrant proteins that define Alzheimer’s disease (AD). The water channel protein astrocytic aquaporin 4 (AQP4) is essential for brain amyloid-β clearance, but it is known to be abnormally expressed in AD brains. The expression of AQPs is differentially regulated during diverse brain injuries, but, whereas AQP4 expression and function have been studied in AD, less is known about AQP5. AQP5 functions include not only water transport but also cell migration mediated by cytoskeleton regulation. Moreover, AQP5 has been reported to be expressed in astrocytes, which are regulated after ischemic and traumatic injury. Additionally, AQP5 is particularly abundant in the salivary glands suggesting that it may be a crucial factor in gland dysfunction associated with AD. Herein, we aim to determine whether AQP5 expression in submandibular glands and the brain was altered in AD. First, we demonstrated impaired AQP5 expression in submandibular glands in APP/PS1 mice and AD patients. Subsequently, we observed that AQP5 expression was upregulated in APP/PS1 cerebral cortex and confirmed its expression both in astrocytes and neurons. Our findings propose AQP5 as a significant role player in AD pathology, in addition to AQP4, representing a potential target for the treatment of AD.

Canonical Bone Morphogenetic Protein Signaling Regulates Expression of Aquaporin-4 and Its Anchoring Complex in Mouse Astrocytes.

Aquaporin-4 (AQP4) is the predominant water channel in the brain; it is enriched in astrocytic foot processes abutting vessels where it is anchored through an interaction with the dystrophin-associated protein (DAP) complex. Enhanced expression with concomitant mislocalization of AQP4 along astrocyte plasma membranes is a hallmark of several neurological conditions. Thus, there is an urgent need to identify which signaling pathways dictate AQP4 microdistribution. Here we show that canonical bone morphogenetic proteins (BMPs), particularly BMP2 and 4, upregulate AQP4 expression in astrocytes and dysregulate the associated DAP complex by differentially affecting its individual members. We further demonstrate the presence of BMP receptors and Smad1/5/9 pathway activation in BMP treated astrocytes. Our analysis of adult mouse brain reveals BMP2 and 4 in neurons and in a subclass of endothelial cells and activated Smad1/5/9 in astrocytes. We conclude that the canonical BMP-signaling pathway might be responsible for regulating the expression of AQP4 and of DAP complex proteins that govern the subcellular compartmentation of this aquaporin.

Inhibition of CAMK II modulates water permeability by reducing AQP4 expression in astrocytes after oxygen-glucose deprivation

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.

Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

Defective aquaporin4 (AQP4)-mediated glymphatic drainage has been linked to tauopathy and amyloid plaque in Alzheimer’s disease. We now show that brain interleukin33 (IL33) is required for regulation of AQP4 expression in astrocytes, especially those at neuron-facing membrane domain (n-AQP4). First, IL33-deficient (Il33−/−) mice showed a loss of n-AQP4 after middle age, which coincided with a rapid accumulation of abnormal tau in neurons and a reduction in drainage of abnormal tau to peripheral tissues. Second, injection of recombinant IL33 induced robust expression of AQP4 at perivascular endfoot (p-AQP4) of astrocytes, but not n-AQP4, in Il33−/− brains. Although the increased p-AQP4 greatly accelerated drainage of intracerebroventricularly injected peptides, it did not substantially accelerate drainage of abnormal tau. These results suggest that p-AQP4 drives overall convective flow toward perivenous space, i.e., glymphatics, whereas n-AQP4 may generate an aqueous flow away from neurons to remove neuronal wastes, e.g., abnormal tau. We have previously shown the role of brain IL33 in DNA repair and autophagy in neurons with oxidative stress. Now, we show that IL33 deficiency also impairs glymphatic drainage. Defects in those mechanisms together may lead to chronic neurodegeneration and tauopathy at old age in IL33-deficient mice.

Activated microglia-derived macrophage-like cells exacerbate brain edema after ischemic stroke correlate with astrocytic expression of aquaporin-4 and interleukin-1 alpha release

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.

Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression.

Brain edema is a common and serious complication of ischemic stroke with limited effective treatment. We previously reported that methylene blue (MB) attenuated ischemic brain edema in rats, but the underlying mechanisms remained unknown. Aquaporin 4 (AQP4) in astrocytes plays a key role in brain edema. We also found that extracellular signal-regulated kinase 1/2 (ERK1/2) activation was involved in the regulation of AQP4 expression in astrocytes. In the present study, we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema. Rats were subjected to transient middle cerebral artery occlusion (tMCAO), MB (3 mg/kg, for 30 min) was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia (1.5 mg/kg, for 15 min). Brain edema was determined by MRI at 0.5, 2.5, and 48 h after tMCAO. The decreases of apparent diffusion coefficient (ADC) values on diffusion-weighted MRI indicated cytotoxic brain edema, whereas the increase of T2 MRI values reflected vasogenic brain edema. We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO. In addition, MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO. In a cell swelling model established in cultured rat astrocyte exposed to glutamate (1 mM), we consistently found that MB (10 μM) attenuated cell swelling, AQP4 increases and ERK1/2 activation. Moreover, the ERK1/2 inhibitor U0126 (10 μM) had the similar effects as MB. These results demonstrate that MB improves brain edema and astrocyte swelling, which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway, suggesting that MB may be a potential choice for the treatment of brain edema.

Silencing matrix metalloproteinase 9 exerts a protective effect on astrocytes after oxygen-glucose deprivation and is correlated with suppression of aquaporin-4

Cerebral ischemia, followed by brain edema, can be life-threatening. It has been widely reported that matrix metalloproteinase-9 (MMP-9) and aquaporin-4 (AQP4) have prominent roles in the development of brain edema. However, the exact mechanisms by which MMP-9 and AQP4 influence brain edema are not fully understood.In this study, astrocytes were subjected to oxygen-glucose deprivation (OGD) /reperfusion (OGD/R) injury, an in vitro model of Ischemia/reperfusion (I/R). Cell viability was evaluated through the measurement of LDH release. The expression of MMP-9 and AQP4 also were measured by qPCR and western blot. Subsequently, we knocked out the MMP-9 gene using MMP-9 siRNA. AQP4 and its gene expression, and the LDH release rate were measured using ELISA, Western blotting, and RT-PCR. We also assessed cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMK II) in MMP-9 knockout astrocytes. All measurements were performed with or without an OGD/R challenge.OGD/reperfusion enhanced LDH release levels, and also increased MMP-9 and AQP4 expression in astrocytes. Silencing the MMP-9 gene decreased LDH release levels, and also was associated with decreased AQP4 expression. The expression of PKC, but not PKA, PKG, or CaMK II, was decreased. This study revealed that OGD/reperfusion could cause cell damage in vitro. MMP-9 silencing protected astrocytes from hypoxic insult, and the protective effect may be enhanced by the downregulation of AQP4 expression. In conclusion, downregulating MMP-9 expression may be useful for the prevention and treatment of brain ischemia.

Dystroglycan is involved in the activation of ERK pathway inducing the change of AQP4 expression in scratch-injured astrocytes

We previously reported that aquaporin 4 (AQP4) played a critical role in formation of brain edema and the altered expression of dystroglycan (DG) could relate with AQP4 expression after traumatic brain injury (TBI). However the mechanisms of this process remain unclear. DG was showed could act as a scaffold involved in adhesion-mediated signaling in ERK/MAPK pathway. We hypothesize that after scratch, extracellular α-DG and transmembrane β-DG may act as the scaffold in scratch mechanical force activating ERK pathway which may regulate the expression of AQP4. Use ERK inhibitor and activator to confirm whether the expression of AQP4 is regulated by the activation of ERK pathway in scratched astrocytes. Use DG siRNA to confirm whether DG takes part in the process that the extracellular signal transduces into cell and activates the ERK pathway. The significant increase of AQP4 and DG expression induced by scratch could be abolished by blocking ERK signaling and enhanced by activating ERK signaling. Blockade of DG by siRNA led to no obvious effect of scratched-injury on the ERK signaling pathway. It demonstrated that DG may act as the scaffold in scratch mechanical force activating ERK pathway which can regulate the expression of AQP4 in astrocytes after scratch.

Effect of matrix metalloproteinase-9 on aquaporin-4 expression in astrocytes after hypoxia and glucose deprivation and its regulatory mechanism

Objective To investigate the effects and regulating mechanisms of matrix metalloproteinase 9 (MMP-9) gene silencing on aquaporin 4(AQP4) expression in astrocytes induced by oxygen-glucose deprivation. Methods Cerebral cortical astrocytes from 2 days newborn SD rats were undergone the primary culture. The ischemic cell model was established by oxygen-glucose deprivation. This experiment were divided into control group, negative control group and MMP-9 gene silencing group. The leakage rate of lactated dehydrogenase(LDH)was detected by chromatoptometry. The MMP-9 gene silencing was carried out by Lentivirus transfection. Real-time fluorescence quantitative PCR and Western blot were used to detect the expression levels of AQP4 and MMP-9. The expressions of PKA, PKC, PKG and CaMKⅡ were determined by Western blot. Results Compared with control group, LDH leakage rate was significantly higher in astrocytes induced by oxygen-glucose deprivation(t=13.35, P 0.05). Conclusion The permeability of astrocytes is increased by oxygen-glucose deprivation. Gene silencing MMP-9 could induce expression of AQP4 mRNA and protein decreased, and MMP-9 may regulate AQP4 expression by regulating PKC activity. Key words: Gene silencing; Matrix metalloproteinase 9; Aquaporin-4; Astrocyte; Oxygen-glucose deprivation

Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice.

Age is characterized by chronic inflammation, leading to synaptic dysfunction and dementia because the clearance of protein waste is reduced. The clearance of proteins depends partly on the permeation of the blood–brain barrier (BBB) or on the exchange of water and soluble contents between the cerebrospinal fluid (CSF) and the interstitial fluid (ISF). A wealth of evidence indicates that physical exercise improves memory and cognition in neurodegenerative diseases during aging, such as Alzheimer’s disease (AD), but the influence of physical training on glymphatic clearance, BBB permeability and neuroinflammation remains unclear. In this study, glymphatic clearance and BBB permeability were evaluated in aged mice using in vivo two-photon imaging. The mice performed voluntary wheel running exercise and their water-maze cognition was assessed; the expression of the astrocytic water channel aquaporin 4 (AQP4), astrocyte and microglial activation, and the accumulation of amyloid beta (Aβ) were evaluated with immunofluorescence or an enzyme-linked immunosorbent assay (ELISA); synaptic function was investigated with Thy1–green fluorescent protein (GFP) transgenic mice and immunofluorescent staining. Voluntary wheel running significantly improved water-maze cognition in the aged mice, accelerated the efficiency of glymphatic clearance, but which did not affect BBB permeability. The numbers of activated astrocytes and microglia decreased, AQP4 expression increased, and the distribution of astrocytic AQP4 was rearranged. Aβ accumulation decreased, whereas dendrites, dendritic spines and postsynaptic density protein (PSD95) increased. Our study suggests that voluntary wheel running accelerated glymphatic clearance but not BBB permeation, improved astrocytic AQP4 expression and polarization, attenuated the accumulation of amyloid plaques and neuroinflammation, and ultimately protected mice against synaptic dysfunction and a decline in spatial cognition. These data suggest possible mechanisms for exercise-induced neuroprotection in the aging brain.

Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague-Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin-4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation-mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31–8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation.

Astrocytes are one of the most abundant cell types in the mammalian central nervous system (CNS), and astrocyte swelling is the primary event associated with brain edema. Glutamate, the principal excitatory amino acid neurotransmitter in the CNS, is released at high levels after brain injury including cerebral ischemia. This leads to astrocyte swelling, which we previously demonstrated is related to metabotropic glutamate receptor (mGluR) activation. Aquaporin 4 (AQP4), the predominant water channel in the brain, is expressed in astrocyte endfeet and plays an important role in brain edema following ischemia. Studies recently showed that mGluR5 is also expressed on astrocytes. Therefore, it is worth investigating whether AQP4 mediates the glutamate-induced swelling of astrocytes via mGluR5. In the present study, we found that 1 mM glutamate induced astrocyte swelling, quantified by the cell perimeter, but it had no effect on astrocyte viability measured by the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays. Quantitative reverse transcription polymerase chain reaction analyses revealed that AQP4, among AQP1, 4, 5, 9 and 11, was the main molecular expressed in cultured astrocytes. Glutamate-induced cell swelling was accompanied by a concentration-dependent change in AQP4 expression. Furthermore, RNAi technology revealed that AQP4 gene silencing inhibited glutamate-induced astrocyte swelling. Moreover, we found that mGluR5 expression was greatest among the mGluRs in cultured astrocytes and was co-expressed with AQP4. Activation of mGluR5 in cultured astrocytes using (S)-3,5-dihydroxyphenylglycine (DHPG), an mGluR5 agonist, mimicked the effect of glutamate. This effect was abolished by co-incubation with the mGluR5 antagonist fenobam but was not influenced by DL-threo-β-benzyloxyaspartic acid (DL-TBOA), a glutamate transporter inhibitor. Finally, experiments in a rat model of transient middle cerebral artery occlusion (tMCAO) revealed that co-expression of mGluR5 and AQP4 was increased in astrocyte endfeet around capillaries in the penumbra, and this was accompanied by brain edema. Collectively, these results suggest that glutamate induces cell swelling and alters AQP4 expression in astrocytes via mGluR5 activation, which may provide a novel approach for the treatment of edema following brain injury.

Abstract TP452: Systemic Inflammation Reverses Stroke-induced Changes in Astrocytic Aquaporin-4 Expression

Introduction: Aquaporin-4 (AQP4) is a perivascular water channel that plays a key role in regulating the blood-brain barrier (BBB). Stroke acutely reduces expression and polarization of AQP4 on astrocytic endfeet, and these vascular changes might persist chronically and evolve with systemic inflammation. Reduced polarization of AQP4 after traumatic brain injury (TBI) leads to impaired perivascular clearance of debris and cognitive decline in mice, and if similar changes occur after stroke, then they may also contribute to post-stroke dementia. Hypothesis: Stroke will cause lasting changes in vascular organization, reducing astrocytic AQP4 expression and polarization in the area of injury. Methods: Thirty male Balb/cJ mice were subjected to distal middle cerebral artery ligation, allowed to survive for 28 days, and sacrificed 24 hours after injection with lipopolysaccharide (LPS, 0.33 mg/kg i.p.) or saline to model systemic inflammation. Three sequential coronal sections (20 μm) near the site of infarct (0.25 mm from Bregma) were stained with lectin, AQP4, and glial fibrillary acidic protein (GFAP) to evaluate BBB organization. Vessel density was evaluated with AngioTool. AQP4 expression was evaluated by Western blot. Results: Stroke increased vascular density in the injured hemisphere (11 ± 0.8% vs 7.6 ± 0.8% in sham mice, p < 0.05). Decreased AQP4 expression and polarization near the site of the infarct was observed, which were reversed with LPS (Figure 1). LPS increased AQP4 protein levels in stroke (2.5 ± 0.37 fold-increase, p < 0.05), but not sham mice. Conclusions: Stroke caused a chronic increase in vascular density, with peri-infarct vessels displaying decreased expression and changed polarity of AQP4. However, with systemic inflammation, this is reversed, potentially contributing to neuroinflammation and worsened cognitive outcomes in stroke patients who encounter infections via increased permeability of these abnormal vessels.

Aquaporin-4 antibodies, CNS acidosis and neuromyelitis optica: A potential link

BackgroundNeuromyelitis optica (NMO, Devic’s syndrome) is a severely disabling disorder of the central nervous system characterized by optic neuritis and longitudinally extensive myelitis. In around 80% of cases, NMO is caused by autoantibodies to astrocytic aquaporin-4 (AQP4), the most abundant water channel in the CNS. Acute NMO attacks are frequently accompanied by elevated levels of lactate in the cerebrospinal fluid (CSF). As a strongly dissociated anion (pK′=3.7) directly changing the strong ion difference, lactate causes a reduction in the dependent anion [HCO3-] and a rise in [H+], resulting in “metabolic” acidosis in the CSF. CSF acidosis also develops during respiratory failure due to brainstem or high cervical spinal cord lesions, the most common cause of death in NMO. However, lactic acid and more generally, a decrease in pH, has been shown to increase the membrane expression of AQP4 in astrocytes. An increase in AQP4 membrane expression during acute NMO attacks could potentially enhance the complement-mediated humoral immune reaction against AQP4-expressing astrocytes characteristic for NMO and, thus, result in more severe astrocytic damage. Moreover, lactate and acidosis have been shown to cause astrocytic swelling and to affect astrocytic viability, potentially rendering astrocytes more susceptible to AQP4-Ab-mediated damage. Finally, increased AQP4 expression could be an independent risk factor in NMO and other forms of CNS inflammation, as indicated by the finding of grossly attenuated experimental autoimmune encephalomyelitis in AQP4-null mice. Therefore, we hypothesize that CSF acidosis might play a role in the pathophysiology of AQP4-Ab-positive NMO and that alterations in CSF pH might possibly influence the outcome of acute attacks in this condition. In addition, we discuss potential clinical implications and make proposals on how to test the hypothesis. Finally, other factors that influence astrocytic AQP4 membrane expression and might play a role in NMO are discussed.

Temporal relationship between aquaporin-4 and glial fibrillary acidic protein in cerebellum of neonate and adult rats administered a BBB disrupting spider venom

Two astrocyte markers, the glial water channel aquaporin-4 (AQP4) and the glial fibrillary acidic protein (GFAP), have been implicated in several physiological and pathological conditions in the central nervous system (CNS) as well as in blood–brain barrier breakdown (BBBb). By color segmentation the immunoreactivity of both proteins, we demonstrate that the expression of AQP4 and GFAP was increased in the cerebellum of neonate (14-day-old, P14) and adult (8-week-old) rats administered Phoneutria nigriventer spider venom (PNV) known to cause perivascular edema, BBBb and convulsion. In the cerebellum's gray matter, PNV produced a major response, especially in the granular layer. Parallel increases in AQP4 and GFAP expression occurred 24 h after envenomation in the white matter of P14 and in the molecular layer of adults, as well as in the granular layer 2 h after envenomation. In the Purkinje layer there was a tendency of increased AQP4, for both, neonates (5 h), and adults (2 and 24 h). Moreover, PNV also provoked nonparallel upregulation of both markers with prevalence of upregulation of AQP 4 for P14 rats, and GFAP for adults. The major expression of both proteins was in the gray matter. The data indicates a venom effect in water/electrolyte balance in the cerebellum and the participation of AQP4 in these effects. Age-related and time-related regional differences probably reflect specificity in AQP4 distribution in different astrocytic membrane domains as well as its participation in K+ buffering and neural activity. This study is the first to associate astrocytic AQP4 expression and reactive gliosis in a model of BBB permeability promoted by P. nigriventer venom. Our data provide compelling evidence that AQP4 expression was increased in the cerebellum of rats administered PNV.