Downregulation Of Aquaporin-4 Expression Research Articles

Decreased kynurenine in cerebrospinal fluid and potential role in neuromyelitis optica spectrum disorder.

Tryptophan (Trp) metabolism has been implicated in neuroinflammatory and neurodegenerative disorders, but its relationship with neuromyelitis optica spectrum disorder (NMOSD) is unclear. In this pilot study, cerebrospinal fluid (CSF) was prospectively collected from 26 NMOSD patients in relapse and 16 controls with noninflammatory diseases and 6 neurometabolites in the tryptophan metabolic pathway, including 5-hydroxytryptamine (5-HT), kynurenine (KYN), melatonin (MLT), 5-hydroxyindoleacetic acid (5HIAA), 3-hydroxy-o-aminobenzoic acid (3-HAA), and kynurenic acid (KYA), were measured by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The association of Trp metabolites with NMOSD and its clinical parameters was evaluated. The role of KYN, which is a Trp metabolite involved in the binding of NMOSD-IgG antibody to aquaporin 4 (AQP4), was also evaluated in vitro. CSF KYN was significantly decreased in patients with relapsing NMOSD compared to controls, and CSF KYN was associated with CSF white blood cells in NMOSD. In vitro experiments showed that NMOSD-IgG specifically recognized KYN, which reversed the NMOSD-IgG-induced downregulation of AQP4 expression. Our results show that abnormal Trp metabolism occurs in NMOSD and that KYN might be a potential target for the treatment of AQP4-IgG-positive NMOSD patients.

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.

Hydrogen sulfide induces neuroprotection against experimental stroke in rats by down-regulation of AQP4 via activating PKC

Hydrogen sulfide (H2S) is now known as an important neuromodulator in the central nervous system. The aim of the current study was to investigate whether exogenous H2S gas can attenuate brain edema induced by experimental stroke and to clarify the potential mechanisms. Rats underwent 2-h middle cerebral artery occlusion (MCAO) and received 40ppm or 80ppm H2S inhalation for 3h at the beginning of reperfusion. The effects of H2S were investigated by evaluating neurological function, infarct size, brain edema volume, and aquaporin4 (AQP4) protein expression at 24h after reperfusion. Moreover, to explore the possible mechanisms for the neuroprotective effects of H2S, protein kinase C (PKC) activity was detected and a PKC inhibitor, Go6983, was used via intracerebral ventricular injection. Our results showed that 40ppm or 80ppm H2S inhalation significantly reduced neurological deficits, infarct size, and brain edema after MCAO. The expression of AQP4 in the peri-infarct area of brain was also inhibited after inhalation of H2S. PKC was activated by H2S treatment and the PKC inhibitor attenuated the neuroprotection of H2S with an increased AQP4 expression at the same time. In conclusion, H2S inhalation attenuates brain edema, reduces infarct volume, and improves neurologic function in a rat experimental stroke model. The therapeutic benefits of H2S inhalation are associated with down-regulation of AQP4 expression via activating PKC.

Neuroprotection of Sanhua Decoction against Focal Cerebral Ischemia/Reperfusion Injury in Rats through a Mechanism Targeting Aquaporin 4.

Sanhua decoction (SHD) is a famous classic Chinese herbal prescription for ischemic stroke, and aquaporin 4 (AQP4) is reported to play a key role in ischemic brain edema. This study aimed to investigate neuroprotection of SHD against focal cerebral ischemia/reperfusion (I/R) injury in rats and explore the hypothesis that AQP4 probably is the target of SHD neuroprotection against I/R rats. Lentiviral-mediated AQP4-siRNA was inducted into adult male Sprague-Dawley rats via intracerebroventricular injection. The focal cerebral ischemia/reperfusion model was established by occluding middle cerebral artery. Neurological examinations were performed according to Longa Scale. Brain water content, was determined by wet and dry weight measurement. Western blot was adopted to test the AQP4 expression in ipsilateral hippocampus. After the treatment, SHD alleviated neurological deficits, reduced brain water content and downregulated the expression of AQP4 at different time points following I/R injury. Furthermore, neurobehavioral function and brain edema after I/R were significantly attenuated via downregulation of AQP4 expression when combined with AQP4-siRNA technology. In conclusion, SHD exerted neuroprotection against focal cerebral I/R injury in rats mainly through a mechanism targeting AQP4.

Mesenchymal stem cells maintain blood-brain barrier integrity by inhibiting aquaporin-4 upregulation after cerebral ischemia.

Cerebral ischemia upregulates aquaporin-4 expression, increases blood-brain barrier (BBB) permeability, and induces brain edema. Mesenchymal stem cells (MSCs) can repress inflammatory cytokines and show great potential for ischemic stroke therapy. However, the effect of MSCs regarding the protection of ischemia-induced BBB break down is unknown. We test whether MSCs therapy protects BBB integrity and explore the molecular mechanisms of aquaporin-4 on BBB integrity. Two hundred and twenty-eight adult CD1 male mice underwent 90 minutes transient middle cerebral artery occlusion and received 2 × 10(5) MSCs intracranial transplantation. The neurological severity score was improved and both ischemia-induced brain edema and BBB leakage were reduced in MSC-treated mice. MSCs therapy reduced astrocyte apoptosis and inhibited ischemia-induced aquaporin-4 upregulation. In addition, small-interfering RNA knockdown of aquaporin-4 after cerebral ischemia effectively reduced aquaporin-4 expression, brain edema, BBB leakage, and astrocyte apoptosis. Conditional medium from lipopolysaccharide (LPS)-activated microglia enhanced aquaporin-4 expression, p38 and JNK phosphorylation, and apoptosis of cultured astrocytes. MSC treatment reduced the expression of inflammatory cytokines in LPS-activated microglia, and subsequently reduced aquaporin-4 expression and apoptosis of astrocytes. Knockdown of aquaporin-4 in cultured astrocytes also reduced apoptosis. Treatment with p38 and JNK inhibitors showed that p38, but not the JNK signaling pathway, was responsible for the aquaporin-4 upregulation. MSCs protected BBB integrity by reducing the apoptosis of astrocytes after ischemic attack, which was due to the attenuation of inflammatory response and downregulation of aquaporin-4 expression via p38 signaling pathway.

Aquaporin-4 inhibition mediates piroxicam-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rodents.

Aquaporin-4(AQP4) is an abundant water channel protein in brain that regulates water transport to maintain homeostasis. Cerebral edema resulting from AQP4 over expression is considered to be one of the major determinants for progressive neuronal insult during cerebral ischemia. Although, both upregulation and downregulation of AQP4 expression is associated with brain pathology, over expression of AQP4 is one of the chief contributors of water imbalance in brain during ischemic pathology. We have found that Piroxicam binds to AQP4 with optimal binding energy value. Thus, we hypothesized that Piroxicam is neuroprotective in the rodent cerebral ischemic model by mitigating cerebral edema via AQP4 regulation. Rats were treated with Piroxicam OR placebo at 30 min prior, 2 h post and 4 h post 60 minutes of MCAO followed by 24 hour reperfusion. Rats were evaluated for neurological deficits and motor function just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, RT-PCR and western blot experiments. Piroxicam pretreatment thirty minutes prior to ischemia and four hour post reperfusion afforded neuroprotection as evident through significant reduction in cerebral infarct volume, improvement in motor behavior, neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde were also found to be significantly reduced in ischemic brain regions in treated animals. This neuroprotection was found to be associated with inhibition of acid mediated rise in intracellular calcium levels and also downregulated AQP4 expression. Findings of the present study provide significant evidence that Piroxicam acts as a potent AQP4 regulator and renders neuroprotection in focal cerebral ischemia. Piroxicam could be clinically exploited for the treatment of brain stroke along with other anti-stroke therapeutics in future.

Effect of dexmedetomidine on permeability of blood-brain barrier in rats subjected to global cerbral ischemia-reperfusion

Objective To evaluate the effects of dexmedetomidine on the permeability of blood-brain barrier in rats subjected to global cerebral ischemia-reperfusion (I/R).Methods Thirty-six male Sprague-Dawley rats,weighing 250-300 g,were randomly divided into 3 groups (n =12 each):sham operation group (group S),global cerebral I/R group (group I/R) and dexmedetomidine group (group D).Global cerebral I/R was induced by occlusion of bilateral common carotid arteries combined with hypotension (MAP was maintained at 35-45 mm Hg) in anesthetized rats.In group D,dexmedetomidine was infused at a rate of 3μg· kg-1 · h-1 until 2 h of reperfusion after a loading dose of dexmedetomidine 3 μg/kg was injected intravenously immediately after onset of I/R.The rats were sacrificed at 24 h of reperfusion and their brains were immediately removed for microscopic examination of hippocampal CA1 region and for determination of the cell apoptosis,brain water content,Evans blue content and aquaporin 4 (AQP4) expression.Results The number of apoptotic cells was significantly larger,and brain water content,Evans blue content and AQP4 expression were higher in groups I/R and D than in group S (P ＜ 0.05 or 0.01).The number of apoptotic cells was significantly smaller,and brain water content,and Evans blue content and AQP4 expression were lower in group D than in group I/R (P ＜ 0.05 or 0.01).Global cerebral I/R-induced pathological changes were significantly attenuated in group D.Conclusion Dexmedetomidine can decrease the permeability of blood-brain barrier and attenuate global cerebral I/R injury in rats,and down-regulation of AQP4 expression may be involved in the mechanism. Key words: Dxmedetomidine ; Reperfusion injury; Cerebral ; Blood-brain barrier

Effect of ulinastatin on expression of aquaporin-4 during cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of ulinastatin on the expression of aquaporin-4 (AQP4) during focal cerebral ischemia-reperfusion (I/R) in rats.Methods One hundred and thirty-five male adult SpragueDawley rats,weighing 230-280 g,were randomly divided into 3 groups (n =45 each):sham operation group (S group),I/R group and ulinastatin group (group U).The rats were anesthetized with intraperitoneal 10％ chloral hydrate 3.5 ml/kg.Focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion followed by 24 repeffusion.In U group,ulinastatin 100000 U/kg was injected immediately after beginning of reperfusion,while the equal volume of normal saline was injected in S and I/R groups.The rats were sacrificed at 6,24 and 48 h of repeffusion and brains were removed for determination of infract volume (by TTC),brain water content and expression of AQP4 (by immunohistochemistry) in brain tissues.Results Compared with S group,the infarct volume and brain water content were significantly increased,and the expression of AQP4 was up-regulated at each time point in I/R and U groups (P ＜ 0.05).Compared with I/R group,the infarct volume and brain water content were significantly decreased,and the expression of AQP4 was down-regulated at each time point in U group (P ＜0.05).Conclusion The mechanism by which ulinastatin mitigates focal cerebral I/R injury is related to down-regulation of AQP4 expression in brain tissues. Key words: Trypsin inhibitors; Reperfusion injury; Brain; Aquaporin 4

ATP‐P2X7 receptor signaling controls basal and TNFα‐stimulated glial cell proliferation

Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal-entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X(7) receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2'-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X(7) receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X(7) receptors with the specific antagonists oxATP, BBG, and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X(7) signaling. Furthermore, TNFα activation of P2X(7) receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X(7) signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα.

Hypertonic saline ameliorates cerebral edema through downregulation of aquaporin-4 expression in the astrocytes

Osmotherapy with 10% hypertonic saline (HS) alleviates cerebral edema through osmotic force. Aquaporin-4 (AQP4) has been reported to be implicated in the pathogenesis of cerebral edema resulting from a variety of brain injury. This study aimed to determine if 10% hypertonic saline ameliorates cerebral edema through downregulation of AQP4 expression in the perivascular astrocytes in the ischemic cerebral edema. Adult male Sprague–Dawley (SD) rats were subjected to permanent right-sided middle cerebral artery occlusion (MCAO) and treated with a continuous i.v. infusion of 10% HS. Brain water content (BWC) analyzed by wet-to-dry ratios in the ischemic hemisphere of SD rats was attenuated after 10% HS treatment. This was coupled with the reduction of neuronal apoptosis in the peri-ischemic brain tissue. Concomitantly, downregulated expression of AQP4 in the perivascular astrocytes after 10% HS treatment was observed. Our results suggest that in addition to its osmotic force, 10% HS exerts anti-edema effects possibly through downregulation of AQP4 expression in the perivascular astrocytes. The reduction of brain edema after 10% HS administration can prevent ischemic brain damage.