Aquaporin-4-IgG-seropositive neuromyelitis optica spectrum disorders:progress of experimental models based on disease pathogenesis

Neuromyelitis optica spectrum disorders are neuroinflammatory demyelinating disorders that lead to permanent visual loss and motor dysfunction.To date,no effective treatment exists as the exact causative mechanism remains unknown.Therefore,experimental models of neuromyelitis optica spectrum disorders are essential for exploring its pathogenesis and in screening for therapeutic targets.Since most patients with neuromyelitis optica spectrum disorders are seropositive for IgG autoantibodies against aquaporin-4,which is highly expressed on the membrane of astrocyte endfeet,most current experimental models are based on aquaporin-4-IgG that initially targets astrocytes.These experimental models have successfully simulated many pathological features of neuromyelitis optica spectrum disorders,such as aquaporin-4 loss,astrocytopathy,granulocyte and macrophage infiltration,complement activation,demyelination,and neuronal loss;however,they do not fully capture the pathological process of human neuromyelitis optica spectrum disorders.In this review,we summarize the currently known pathogenic mechanisms and the development of associated experimental models in vitro,ex vivo,and in vivo for neuromyelitis optica spectrum disorders,suggest potential pathogenic mechanisms for further investigation,and provide guidance on experimental model choices.In addition,this review summarizes the latest information on pathologies and therapies for neuromyelitis optica spectrum disorders based on experimental models of aquaporin-4-IgG-seropositive neuromyelitis optica spectrum disorders,offering further therapeutic targets and a theoretical basis for clinical trials.

Anti-aquaporin-4 antibody(AQP4-IgG)and anti-myelin oligodendrocyte glycoprotein antibody(MOG-IgG)in the cerebrospinal fluid

In the last decade,a new neurological disease concept known as anti-myelin oligodendrocyte glycoprotein antibody(MOG-IgG)-associated disease(MOGAD)has emerged and is currently one of the most focused research areas in the field of neuroimmunology.MOG is a membrane protein mainly expressed on the surface of oligodendrocytes(Zhou et al.,2006).The exact pathogenic role of MOG-IgG in patients with MOGAD remains unclear;however,MOG-IgG has been suggested to cause tissue alterations and damage MOG-expressing cells(Zhou et al.,2006).The pathogenicity of MOG-IgG is further supported by the observation that only a few patients with acquired central nervous system(CNS)demyelinating syndromes exhibit both anti-aquaporin-4 antibody(AQP4-IgG)and MOG-IgG simultaneously,particularly with clear positivity levels of these antibodies as indicated by a cell-based assay result with a titer≥1:100(Sechi et al.,2021;Banwell et al.,2023).

Anti-glial fibrillary acidic protein antibody and anti-aquaporin-4 antibody double-positive neuromyelitis optica spectrum disorder:A case report

BACKGROUND A case of neuromyelitis optica spectrum disorder(NMOSD)with positive cerebrospinal fluid(CSF)anti-aquaporin-4 antibody(AQP4-IgG)and anti-glial fibrillary acidic protein IgG(GFAP-IgG)at the time of relapse was reported.The exact roles of GFAP-IgG in NMOSD are not fully understood and are the subject of ongoing research.This study revealed the possible connection between GFAPIgG and the occurrence or development of diseases.CASE SUMMARY A 19-year-old woman was admitted to the hospital due to a constellation of symptoms,including dizziness,nausea,and vomiting that commenced 1 year prior,reoccurred 2 mo ago,and were accompanied by visual blurring that also began 2 mo ago.Additionally,she presented with slurred speech and ptosis,both of which emerged 1 mo ago.Notably,her symptoms deteriorated 10 d prior to admission,leading to the onset of arm and leg weakness.During hospitalization,magnetic resonance imaging showed high T2-fluid attenuated inversion recovery signals,and slightly high and equal diffusion-weighted imaging signals.The serum antibody of AQP4-IgG tested positive at a dilution of 1:100.CSF antibody testing showed positive results for GFAP-IgG at a dilution of 1:10 and AQP4-IgG at a dilution of 1:32.Based on these findings,the patient was diagnosed with NMOSD.She received intravenous methylprednisolone at a daily dose of 500 mg for 5 d,followed by a tapering-off period.Afterward,the rate of reduction was gradually slowed down and the timely use of immunosuppressants was implemented.CONCLUSION The CFS was slightly GFAP-IgG-positive during the relapse period,which can aid in the diagnosis and treatment of the disease.

重组EGFP-aquaporin-4融合蛋白真核表达载体的构建及其在FRT细胞中的表达和定位

目的:构建以增强型绿色荧光蛋白(EGFP)为报告基因的重组真核表达载体pEGFP-aquaporin-4,以EGFP示踪aquaporin-4在Fisher大鼠甲状腺滤泡上皮细胞(FRT细胞)中的表达和定位,为进一步研究aquaporin-4提供实验依据。方法:应用RT-PCR方法获得aquaporin-4编码区基因,克隆入真核表达载体pEGFP-N1。经酶切和测序鉴定证实为aquaporin-4后,Lipofectamine 2000脂质体转染重组EGFP-aquaporin-4融合蛋白真核表达载体至FRT细胞中,倒置荧光显微镜下观察aquaporin-4在FRT细胞中的表达和分布,并应用Western blotting法检测aquaporin-4蛋白的表达。同时检测转入FRT细胞内钙黄绿素的相对荧光强度以判断FRT细胞水通透性的状况。结果:EcoRⅠ和KpnⅠ双酶切和测序重组载体结果证实目的基因aquaporin-4成功克隆到真核表达载体pEGFP-N1中。荧光显微镜下观察融合EGFP的aquaporin-4主要在FRT细胞膜上表达;Western blotting结果证实脂质体转染重组载体的FRT细胞表达aquaporin-4。转染重组EGFP-aquaporin-4融合蛋白真核表达载体的FRT细胞水通透性显著高于未转染的FRT细胞,其水通透性是未转染FRT细胞的1.65倍。结论:成功构建aquaporin-4真核表达载体,证实其可在FRT细胞中表达,并具有明显的膜蛋白特性和良好水通透性。

Aquaporin-4 in the formation of cerebral edema following severe burns What role do arginine vasopressin levels play?

BACKGROUND： Aquaporin-4 （AQP-4）, which is able to rapidly transport water within the brain, is highly expressed in brain tissue. It also plays an important role in the formation of cerebral edema following brain injury. However, the role of AQP-4 in the formation of cerebral edema following severe bums remains unknown. OBJECTIVE： To study changes in AQP-4 protein and mRNA expression during formation of cerebral edema following severe burns, and to explore the correlation between AQP-4 protein and mRNA expression with plasma levels of arginine vasopressin （AVP）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Research Center of Neuroscience, Chongqing Medical University from 2007 to 2008. MATERIALS： Biotin-labeled goat anti-rabbit antibody was provided by Beijing Zhongshan Biotechnology, China; in situ hybridization kit was provided by Wuhan Boster Biotechnology, China; rabbit anti-AQP-4 polyclonal antibody and horseradish peroxidase-labeled goat anti-rabbit IgG were provided by Chemicon, USA; AVP radioimmunoassay kit was provided by the Research Department of Neurobiology, the Second Military Medical University of Shanghai, China. METHODS： A total of 180 adult, healthy, Wistar rats were randomly assigned to control and burn groups with 30 rats in each group. The burn group was observed at five different time points： 2, 6, 12, 24, and 48 hours after burn. Hair on the mouse back was removed to expose skin on the back. After 1 day, skin with the hair removed was dipped into 100℃ water for 15 seconds to induce grade III bum injury that measures 30% of total bum surface area. MAIN OUTCOME MEASURES： Brain water content was measured using the dry-wet weight method. AQP-4 protein and mRNA expressions were detected using immunohistochemistry, in situ hybridization, Western blot, and reverse transcription-polymerase chain reaction; dynamic changes in plasma AVP were detected using radioimmunoassay. RESULTS： Brain water content gradually increased following severe burn injury. AQP-4 protein and mRNA expressions were upregulated in the supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, hippocampus, choroid plexus, and cerebral cortex. Plasma AVP levels increased following burn injury. AQP-4 protein and mRNA expressions positively correlated with brain water content and AVP levels during formation of cerebral edema （r= 0.870, 0.848, P 〈 0.01）. CONCLUSION： AQP-4 participated in the formation of cerebral edema following burn injury. Plasma AVP upregulated AQP-4 expression in brain tissue, thereby promoting formation of cerebral edema.

Increased expression of aquaporin-4 in human traumatic brain injury and brain tumors

Objective:To characterize the expression of aquaporin-4(AQP4),one of the aquaporins(AQPs),in human brainspecimens from patients with traumatic brain injury or brain tumors.Methods:Nineteen hnman brain specimens were obtahledfrom the patients with traumatic brain injury,brain tumors,benign meningioma or early stage hemorrhagic stroke.MRI or CTimaging was used to assess brain edema.Hematoxylin and eosm staining were used to evaluate cell damage,Immunohistochem-istry was used to detect the AQP4 expression.Results:AQP4 expression was increased from 15 h to at least 8 d after injury.AQP4immunoreactivity was strong around astrocytomas,ganglioglioma and metastatic adenocarcinoma.However,AQP4 immunore-activity was only found in the centers of astrocytomas and ganglioglioma,but not in metastatic adenocarcinoma derived from lung.Conclusion:AQP4 expression increases in human brains alter traumatic brain injury,within brain-derived tumors,and aroundbrain tumors.

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema Correlation with variation in apparent diffusion coefficient

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 pL shRNA- aquaporin-4 （control group） or siRNA- aquaporin-4 solution （1：800） （RNA interference group） into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25- 6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5 4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson＇s correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient （r = -0.806, P 〈 0.01）. These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracelfular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema.

Protective mechanisms of picroside Ⅱ on aquaporin-4 expression in a rat model of cerebral ischemia/reperfusion injury

BACKGROUND： Aquaporin-4 （AQP-4） over-expression following cerebral ischemia results in cerebral edema. Picroside Ⅱ has been shown to exhibit a neuroprotective effect on neuronal apoptosis. However, few reports have addressed the neuroprotective mechanisms and therapeutic times following cerebral ischemic reperfusion injury. OBJECTIVE： To explore the neuroprotective effects and ideal treatment window for picroside Ⅱ treatment of middle cerebral artery occlusion and reperfusion injury in rats. DESIGN, TIME AND SETTING; A randomized, controlled, animal experiment was performed at Institute of Cerebrovascular Diseases, Qingdao University Medical College from September 2008 to May 2009. MATERIALS： Picroside II was purchased from Tianjin Kuiqing Medical Technology, China. METHODS： A total of 165 adult, healthy, male, Wistar rats were randomly assigned to sham-surgery （n = 15）, model （n = 75）, and treatment groups （n = 75）. Rats in the model and treatment groups underwent middle cerebral artery occlusion and reperfusion through the use of an intraluminal monofilament suture on the left external-internal carotid artery, The treatment group was injected with 1.0% picroside Ⅱ （10 mg/kg） into the tail vein, and the model and sham-surgery groups were injected with 0.1 mol/L phosphate buffered saline （250 μL）. MAIN OUTCOME MEASURES： Neurological functional scores were evaluated using the Longa＇s method; cerebral infarction volume was detected through the use of tetrazolium chlodde staining; cellular apoptosis was determined through the use of the in situ end-labeling method; aquaporin-4 expression was measured using fluorescence labeling analysis and reverse transcription polymerase chain reaction technique. RESULTS： At 0.5 hour following cerebral ischemic injury, neurological functional scores were low, and a small infarction focus was detected in the ischemic cortex of the model group. Along with prolonged ischemia and an increased number of apoptosis-positive cells, AQP-4 mRNA and protein expression was increased. At 1-2 hours after ischemia, neurological scores and infarction sizes were significantly increased in the model group. Apoptotic-positive cells were widespread in the ipsilateral cortex and stdatum. In addition, AQP-4 mRNA and protein expression levels were increased. Picroside II treatment significantly decreased neurological scores and infarction volume, and reduced AQP-4 mRNA and protein expression levels compared with the model group （P 〈 0.05 or P 〈 0.01）. At 1 hour after ischemia, the therapeutic effect of picroside Ⅱ was notable （P 〈 0.01）. CONCLUSION： Picroside Ⅱ played a protective role in cerebral ischemic reperfusion injury by inhibiting apoptosis and regulating AQP-4 expression. The best therapeutic time window was 1 hour after cerebral ischemic reperfusion.

Effects of propofol on ammonium chloride-exposed astrocyte morphology and aquaporin-4 expression

Ammonia induces astrocyte swelling, which is strongly associated with overexpression of aquaporin-4. However, the mechanisms by which ammonia induces astrocyte swelling, and subsequently upregulating aquaporin-4 expression, remain unknown. In the present study, astrocytes were cultured in vitro and exposed to ammonium chloride （NH4CI）, followed by propofol protein kinase C agonist, or antagonist, respectively. Astrocyte morphology was observed by light microscopy, and aquaporin-4 expression was detected by western blot analysis. Results showed that propofol or protein kinase C agonist significantly attenuated the degree of NH4CI-induced astrocyte swelling and inhibited increased aquaporin-4 expression. Propofol treatment inhibited aquaporin-4 overexpression in cultured astrocyte induced by NH4CI; protein kinase C pathway activation is potentially involved.

Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cellular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffusion-weighted imaging（DWI）. We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and si RNA-aquaporin-4 was immediately injected via the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefficient value on T2-weighted imaging（T2WI） and DWI gradually increased within 0.5–6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2 WI and DWI reduced, relative apparent diffusion coefficient value was increased, and cellular edema was obviously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefficient value was similar between treatment and model groups, but angioedema was still obvious in the treatment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefficient value and the area of high signal intensity on T2 WI and DWI can reflect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

Effect of hypotonic medium on aquaporin-4 expression in cultured astrocytes

BACKGROUND： Aquaporin-4 （AQP4） is abundant in astrocytes, ependymal cells, and the choroid plexus, and is associated with cerebrospinal fluid formation and osmoregulation. AQP4 is speculated to be the hypothalamic osmoreceptor and regulator of water balance. OBJECTIVE： To examine AQP4 expression and its role in cultured rat astrocytes after exposure to hypotonic medium. DESIGN, TIME AND SETTING： Randomized control experiment. This experiment was carried out in the Research Room of Neurobiology, Chongqing University of Medical Science, China, between April and October 2003. MATERIALS： Two-day-old newborn Wistar rats （n =20）, weighing 10- 15 g, were purchased from the Experimental Animal Center of Chongqing University of Medical Science, China. METHODS： Purified rat cerebral cortical astrocytes were isolated fiom Wistar rats for in vitro cell culture experiments. The cells were randomly divided into control and hypotonic groups. The in vitro cell edema model was established by exposing astrocytes to hypotonic medium （268, 254, or 240 mmol/L）. Cells in the control group were cultured in normal culture medium. MAIN OUTCOME MEASURES： Morphological changes in astrocytes were observed under an inverted microscope and a transmission electron microscope after cells were cultured for 3, 6, 12, or 24 hours with hypotonic medium or normal culture medium. In each group, AQP4 protein and mRNA expression were assessed by immunocytochemistry, in situ hybridization, and reverse transcription polymerase chain reaction at the different time points. RESULTS： After astrocytes were cultured for 3, 6, 12, or 24 hours with hypotonic medium （268, 254, 240 mmol/L）, they showed typical features of cell edema. In the control group, no astrocytes developed pathological changes. There were no significant changes in the AQP4 mRNA and protein expression in the control group at any of the time points alter astrocytes were cultured with normal culture medium （P 〉 0.05）. Compared with the control group, AQP4 mRNA and protein expression in the hypotonic group were remarkably increased at all time points after astrocytes cultured with hypotonic medium （268, 254, 240 mmol/L; P 〈 0.05）. AQP4 mRNA and protein expression increased with increasing exposure time and with decreasing concentration of the hypotonic medium. CONCLUSION： Hypotonic medium induced cell edema and increased AQP4 mRNA and protein expression. Up-regulated expression of AQP4 was correlated with hypotonic medium concentration in a time dependent manner.

Aquaporin-4 and spinal cord injury

Edema formation is a major problem following traumatic spinal cord injury(SCI) that acts to exacerbate secondary damage.Severity of edema correlates with reduced neurological outcome in human patients.To date, there are no effective treatments to directly resolve edema within the spinal cord.The aquaporin-4(AQP4) water channel is found on membranes of astrocytic endfeet in direct contact with blood vessels, the glia limitans in contact with the cerebrospinal fluid and ependyma around the central canal.Being so locally expressed at the interface between fluid and tissue allow AQP4 channels to play an important role in the bidirectional regulation of water homeostasis under normal conditions and following trauma.With the need to better understand the pathophysiology underlying the devastating cellular events in SCI, animal models have become an integral part of exploration.Inevitably, several injury models have been developed(contusion, compression, transection) resulting in difficult interpretation between studies with conflicting results.This is true in the case of understanding the role of AQP4 in the progression and resolution of edema following SCI, whose role is still not completely understood and is highly dependent on the type of edema present(vasogenic vs cytotoxic).Here, we discuss regulation of AQP4 in varying injury models and the effects of potential therapeutic interventions on expression, edema formation and functional recovery.Better understanding of the precise role of AQP4 following a wide range of injuries will help to understand optimal treatment timing following human SCI for prime therapeutic benefit and enhanced neurological outcome.

Correlation between aquaporin-4 and brain edema in an animal model of astrocytic oxygen-glucose deprivation and reintroduction

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

Detection of anti-aquaporin-4 autoantibodies in the sera of Chinese neuromyelitis optica patients

In this study, we recruited 10 neuromyelitis optica patients, two multiple sclerosis patients and two myelitis patients. Chinese hamster lung fibroblast （V79） cells transfected with a human aquaporin-4-mCherry fusion protein gene were used to detect anti-aquaporin-4 antibody in neuromyelitis optica patient sera by immunofluorescence. Anti-aquaporin-4 autoantibody was stably detected by immunofluorescence in neuromyelitis optica patient sera exclusively. The sensitivity of the assay for neuromyelitis optica was 90% and the specificity for neuromyelitis optica was 100%. The anti-aquaporin-4 antibody titers in sera were tested with serial dilutions until the signal disappeared. A positive correlation was detected between Expanded Disability Status Scale scores and serum anti-aquaporin-4 antibody titers. The anti-aquaporin-4 antibody assay is highly sensitive and specific in the sera of Chinese neuromyelitis optica patients. Detection of aquaporin-4 autoantibody is important for the diagnosis and treatment of neuromyelitis optica.

Increased expression of aquaporin-4 in brain tissue of amygdala-kindled rats

Recurrent epileptic seizures can lead to brain edema, indicating that water regulation may be perturbed by seizures. We hypothesized that the expression of the brain water channel aquaporin-4 （AQP-4） may be upregulated in the epileptic brain. In the present study, we established the amygdala kindling model of epilepsy, and quantified AQP-4 protein and mRNA levels, using reverse transcription-PCR, immunohistochemistry and western blotting, in epileptic and control rats. We found that AQP-4 was overexpressed in the cerebral cortex of rats with epilepsy compared with controls. These findings show that AQP-4 is highly expressed in the brain of amygdala-kindled rats, suggesting that repeated seizures affect water homeostasis in the brain.

Altered Taurine Release in Hypothalamus of Aquaporin-4 Knockout Mice

Taurine is concentrated in glial cells in the hypothalamus and released in an osmo-dependent manner through volume-sensitive anion channels. Released tanrine acts on glycine receptors on vasopressin neurons to control vasopressin secretion. Water channel AQP4 is abundant in astrocytes in osmosensory areas such as the supraoptic nucleus of hypothalamus. An HPLC-based method was established to quantify taurine release from isolated hypothalamus tissues in wildtype and AQP4 knockout mice. Under the basal condition, there was no difference in taurine release from AQP4^＋/＋ and AQP4^-/- hypothalamuses. Taurine release from AQP4^-/- hypothalamus under hypoosmotic stimulation was significantly lower than that from AQP4^＋/＋ mice. AQP4 expression in the glial cells of the hypothalamus may play an important role in osmoregulation oftaurine release and subsequent vasopressin secretion.

Effects of propofol on neuronal apoptosis and aquaporin-4 expression in a rat model of traumatic brain injury

BACKGROUND： Several studies have demonstrated that propofol exhibits protective effects in the central nervous system. OBJECTIVE： To observe the effects of propofol on neuronal apoptosis and aquaporin-4 （AQP-4） expression in a rat model of traumatic brain injury and to further investigate the mechanisms of action. DESIGN, TIME AND SETTING： The present neuronal, pathomorphological experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical College between April 2007 and March 2008. MATERIALS： Traumatic brain injury was induced by free falling objects in 150 healthy, male, Wistar rats. Propofol was produced by AstraZeneca, China. Rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemistry kit, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling （TUNEL） kit were purchased from Wuhan Boster Bioengineering Co., Ltd., China. METHODS： All 150 rats were randomly and evenly divided into lesion-only and propofol-treated groups. One hour after traumatic brain injury, propofol-treated animals received 1% propofol （10 mg/kg） through the caudal vein, followed by a sustained perfusion of 30 mg/kg propofol per hour for 2 hours, while the lesion-only group received equal volumes of physiological saline in parallel. MAIN OUTCOME MEASURES： At 6, 12, 24, 48, and 72 hours after traumatic brain injury, morphological changes in the peritraumatic and adjacent brain areas were analyzed in all rats by hematoxylin-eosin （HE） staining. In addition, cellular apoptosis was detected by TUNEL assay and the number of AQP-4-positive cells was determined by immunohistochemistry techniques. Brain water content was calculated as the ratio of dry to wet tissue weight. RESULTS： HE staining results demonstrated that, in the lesion-only group, the peritraumatic area exhibited neuronal and glial cell necrosis and disintegration. The adjacent area displayed swollen neuronal perikarya and vascular endothelial ceils, cellular edema, and a small amount of proliferating glial ceils. In contrast, these pathological changes were noticeably alleviated in the peritraumatic and adjacent areas of propofol-treated animals. Compared with the lesion-only group, the number of apoptotic cells was significantly decreased in the propofol-treated group at each time point after traumatic brain injury, in particular at 24 and 48 hours （P 〈 0.05-0.01）. In the lesion-only group, AQP-4 expression began to increase at 6 hours after traumatic brain injury, reached a peak level at 24-48 hours, and began to decrease by 72 hours. In the propofol-treated group, the number of AQP-4 positive cells was significantly less than the lesion-only group. This was the case at all time points, in particular at 12 and 24 hours （P 〈 0.01-0.05）. CONCLUSION： Propofol can dowuregulate neuronal apoptosis and AQP-4 expression in rats following traumatic brain injury, in particular at 24-48 hours.

Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND： Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE： To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS： A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 （Santa Cruz, USA） and Evans blue （Sigma, USA） were used to analyze the tissue. METHODS： The rats were randomized into sham-operated （n = 16） and ischemia/reperfusion （n = 96） groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES： Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS： Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Evans blue content significantly increased on the ischemic side at 4 hours after ischemia/reperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group （P 〈 0.05）. Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion （r1 = 0.68, r2 = 0.81, P 〈 0.05）. CONCLUSION： Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.

Preventive administration of cromakalim reduces aquaporin-4 expression and blood-brain barrier permeability in a rat model of cerebral ischemia/reperfusion injury

Cromakalim,an adenosine triphosphate-sensitive potassium channel opener,exhibits protective effects on cerebral ischemia/reperfusion injury.However,there is controversy as to whether this effect is associated with aquaporin-4 and blood-brain barrier permeability.Immunohistochemistry results show that preventive administration of cromakalim decreased aquaporin-4 and IgG protein expression in rats with ischemia/reperfusion injury;it also reduced blood-brain barrier permeability,and alleviated brain edema,ultimately providing neuroprotection.

Effect of lidocaine on retinal aquaporin-4 expression after ischemia/reperfusion injury in the rat

BACKGROUND： Several studies have demonstrated that high doses of lidocaine can reduce edema in rats with brain injury by down-regulating aquaporin-4 （AQP4） expression. The hypothesis for the present study is that lidocaine could retinal edema that is associated with AQP4 expression. OBJECTIVE： This study was designed to investigate the interventional effects of lidocaine on retinal AQP4 expression and retinal edema following ischemia/reperfusion injury in the rat. DESIGN, TIME AND SETTING： This study, a randomized, controlled, animal experiment, was performed at the Basic Research Institute, Chongqing Medical University from September 2006 to May 2007. MATERIALS： Seventy-five, healthy, adult, female, Sprague-Dawley rats were included. A total of 50 rats were used to establish a retinal ischemia/reperfusion injury model using an anterior chamber enhancing perfusion unit. Rabbit anti-rat AQP4 antibody was purchased from Santa Cruz Biotechnology, USA. METHODS： All 75 rats were randomly divided into three groups, with 25 rats in each： control, model, and lidocaine. At each time point （1, 6, 12, 24, and 48 hours after modeling, five rats for each time point）, each rat in the lidocaine group was intraperitoneally administered lidocaine with an initial dose of 30 mg/kg, followed by subsequent doses of 15 mg/kg every six hours. The entire treatment process lasted three days for each rat. At each above-mentioned time point, rats in the model group were modeled, but not administered any substances. Rats in the control group received the same treatments as in the lidocaine group except that lidocaine was replaceld by physiological saline. MAIN OUTCOME MEASURES： Following hematoxylin-eosin staining, rat retinal tissue was observed to investigate retinal edema degree through the use of an optical microscope and transmission electron microscope. Retinal AQP4 expression was determined by immunohistochemistry. RESULTS： At each above-mentioned time point, AQP4 expression was significantly increased in the model group compared to the control group （P 〈 0.05）; this change was consistent with the degree of retinal edema. In the lidocaine group, retinal AQP4 expression was significantly decreased （P 〈 0.05）, and retinal edema was reduced, compared with the model group. CONCLUSION： Lidocaine inhibits rat retinal AQP4 expression following ischemia/reperfusion injury, leading to a reduction of retinal edema.