低糖对星形胶质细胞水通道蛋白4表达及分布的影响

目的探讨低糖体外培养对大鼠原代星形胶质细胞水通道蛋白4(aquaporin 4,AQP4)表达量及分布的影响。方法对体外培养的原代大鼠星形胶质细胞进行0mmol/L(无糖)和1mmol/L葡萄糖低糖培养,以5.5mmol/L葡萄糖作为正常糖浓度对照进行如下实验:(1)采用活细胞成像技术检测无糖培养后的星形胶质细胞的体积变化;(2)以Western blot检测无糖培养0h、3h、6h、12h和24h,以及0mmol/L、1mmol/L和5.5mmol/L葡萄糖培养24h后星形胶质细胞的AQP4表达量;(3)以免疫荧光染色检测0mmol/L、1mmol/L和5.5mmol/L葡萄糖培养6h后AQP4在星形胶质细胞上的分布。结果 (1)与0min时比较,无糖培养15min(1.14±0.03)和30min时(1.15±0.02)星形胶质细胞相对体积均明显增大(P0.01)。(2)无糖0mmol/L(含血清)培养实验中,与0h(AQP4相对表达量为1)比较,12h(1.55±0.21)、24h时(1.67±0.16)AQP4相对表达量均明显增加(P0.01);0mmol/L、1mmol/L和5.5mmol/L葡萄糖(含血清)培养24h后,与5.5mmol/L组(AQP4相对表达量为1)比较,0mmol/L组AQP4相对表达量(3.23±0.23)明显增加(P0.01)。(3)与0h时(AQP4相对表达量为1)比较,无糖(无血清)培养6h后AQP4蛋白相对表达量(1.32±0.09)明显上调(P0.05),24h时相对表达量(0.80±0.05)下降(P0.05);0mmol/L、1mmol/L和5.5mmol/L葡萄糖(无血清)培养24h后,与5.5mmol/L(AQP4相对表达量为1)组比较,0mmol/L组AQP4相对表达量(0.75±0.07)明显降低(P0.05)。无论含或不含血清,与5.5mmol/L葡萄糖组比较,0mmol/L和1mmol/L组AQP4在星形胶质细胞向膜上集中特异性分布更加明显。结论低糖处理可引起星形胶质细胞水肿,AQP4蛋白表达量发生变化和向细胞膜上集中分布。AQP4在低血糖性脑水肿中可能发挥重要作用。     

缺氧/复氧条件下星形胶质细胞水通道蛋白4和5表达变化的研究

目的：观察缺氧/复氧条件下星形胶质细胞水通道蛋白（AQP）4和5表达的变化,探讨脑缺血再灌注后脑水肿与AQP4和AQP5的关系。方法：取新生24h内的SD大鼠皮质新鲜脑组织,进行原代和传代培养。以95%N2和5%CO2造成细胞缺氧,用倒置相差显微镜对细胞进行形态学观察,用锥虫蓝染色法测定缺氧及复氧后不同时间点星形胶质细胞的死亡数以反映星形胶质细胞的存活能力,应用细胞免疫化学技术测定星形胶质细胞缺氧及复氧后各个时间点AQP4、AQP5表达的变化。结果：缺氧4及8h后细胞形态变化不明显,随着复氧时间的延长出现细胞损伤的表现。与对照组比较,缺氧后4及8h有少量细胞死亡（P〈0.05）,随着复氧时间延长细胞死亡数亦逐渐增多（P〈0.01）;缺氧4及8h后,AQP4、AQP5阳性表达细胞数均减少（P〈0.01）,而复氧后AQP4、AQP5阳性表达细胞数逐渐升高并随时间延长呈增高趋势（P〈0.01）。结论：星形胶质细胞对缺氧的耐受能力较强,但复氧时出现明显损伤;AQP4、AQP5表达的变化与缺血-再灌注损伤后脑水肿存在相关性。     

实验性脑出血后水通道蛋白-4的表达变化

目的　研究出血性脑水肿病理过程中水通道蛋白 4 (aquaporin 4 ,AQP4 )的表达与脑水肿形成之间的关系。方法　采用胶原酶制作大鼠脑出血模型 ,原位杂交和免疫组化法检测AQP4mRNA和蛋白质的表达变化 ,并用电镜技术和微血管灌注法观察脑水肿区的超微结构和微血管的变化。结果　与对照组相比 ,脑出血后 6h ,AQP4mRNA和蛋白质在脑水肿区表达增强 ,AQP4mRNA(吸光度值 ,A)由 0 2 9上升到 0 5 7(P <0 0 1) ,AQP4蛋白A值由 0 0 6上升到 0 15 (P <0 0 1) ,电镜下可见脑组织轻度水肿 ;至 72h ,AQP4mRNA和蛋白质的表达达到高峰 ,AQP4mRNA的A值为 0 88,AQP4蛋白A值为 0 2 5 ,此时脑组织严重水肿 ,神经元、胶质细胞和内皮细胞明显肿胀 ,毛细血管造影可见毛细血管开始增生 ;第 7天 ,AQP4的表达已减少 (尤以mRNA明显 ) ,但仍显著高于对照组 ,此时脑水肿已减轻。在整个脑水肿的形成过程中 ,AQP4mRNA和蛋白的表达呈高度正相关 (rs>0 82 ,Ps<0 0 1)。结论　脑出血后AQP4表达明显增强 ,提示AQP4参与了出血性脑水肿的发生发展过程 ,在出血性脑水肿的形成过程中起重要作用。     

星形胶质细胞的水通道蛋白4在神经系统疾病相关认知障碍中的作用

星形胶质细胞足突的水通道蛋白-4(AQP4)锚定在血管基底膜形成胶质血管联系,组成神经血管单元的部分结构,维持水和离子平衡,调控血脑屏障及脑血流量,调节突触可塑性和神经炎症,近年来星形胶质细胞水通道蛋白-4越来越被重视,然而在神经系统疾病相关认知障碍中的研究相对不足。本文综述了其在神经系统疾病相关认知障碍的发病机制中的研究,尤其是突触可塑性和神经炎症等方面的研究进展,为其防治提供新的策略。     

头孢曲松钠减轻水通道蛋白4抗体对星形胶质细胞的毒性作用

目的探讨头孢曲松钠在水通道蛋白4(AQP4)抗体诱导的星形胶质细胞损伤中的作用以及机制。方法常规体外培养新生SD大鼠大脑皮质细胞,将培养的细胞分为4组,分别加入健康人血清(对照组)、AQP4抗体阳性患者血清、头孢曲松钠+AQP4抗体阳性血清以及单纯头孢曲松钠。细胞培养24h后采用免疫组织化荧光染色观察不同组星形胶质细胞数目的变化,采用比色法测定上清液谷氨酸浓度以及免疫印迹分析谷氨酸转运体-1(GLT-1)蛋白表达水平。结果和对照组比较,AQP4抗体阳性血清组星形胶质细胞数目和谷氨酸转运体-1(GLT-1)蛋白表达明显减少,上清液谷氨酸浓度明显增高(均P0.01),而单纯头孢曲松钠组仅显著增加GLT-1蛋白表达(P0.01),并不影响星形胶质细胞数目和谷氨酸水平(P0.05);和AQP4抗体阳性组比较,头孢曲松钠+AQP4抗体阳性血清组星形胶质细胞数目和GLT-1蛋白表达增加,上清液谷氨酸浓度明显下降(P0.01)。结论头孢曲松钠可能通过上调星形胶质细胞GLT-1表达、减少细胞外液谷氨酸水平发挥减轻AQP4抗体对体外培养的大鼠脑皮质星形胶质细胞的毒性作用。     

缺氧与复氧对星形胶质细胞水通道蛋白-9表达的影响

目的探讨体外培养星形胶质细胞缺氧及复氧后水通道蛋白-9(AQP9)表达的变化特点及其所起的作用。方法取生后2d的Wistar大鼠大脑皮层进行星形胶质细胞纯培养,缺氧时间分别为:12、24、48h,并取缺氧24h后复氧12、24、48h的细胞进行存活率、乳酸脱氢酶活性的测定,采用免疫细胞化学方法检测细胞AQP9的表达。结果缺氧组及复氧组的星形胶质细胞死亡数与对照组相比无明显变化,缺氧和复氧后乳酸脱氢酶活性亦无明显改变(P(0.05),缺氧组细胞AQP9表达较对照组增高,并随缺氧时间延长而明显增高(P<0.05),复氧后24h内AQP9表达逐渐下降,24h后AQP9表达仍未恢复正常水平。结论星形胶质细胞对缺氧较为耐受,缺氧对星形胶质细胞存活能力无明显影响,缺氧引起AQP9的表达上调,而复氧使其表达明显下降。     

脑出血患者血肿周围脑组织水通道蛋白-4的表达变化

目的：探讨脑出血血肿周围脑组织水通道蛋白-4（AQP4）的表达规律及其与脑水肿形成的关系。方法：32例行颅内血肿清除术患者，按发病至手术时间不同分为〈6h、6～24h、24～72h和〉72h组。取术中获得的脑组织做病理标本，检测AQP4的表达变化，并观察其组织病理及超微结构变化。结果：〈6h组脑组织轻微水肿，AQP4表达呈弱阳性，6h后水肿逐渐加重，AQP4表达开始增强，至24～72h达高峰。此后，AQP4表达下降，水肿减轻。结论：脑出血后AQP4表达呈动态变化与脑组织水肿程度一致，提示AQP4在脑出血患者脑水肿的发生、发展中起着重要作用。     

凝血酶与实验性脑出血后水通道蛋白-4表达变化的研究

目的应用水蛭素拈抗凝血酶,探讨凝血酶与脑出血（intracerebral hemorrhage,ICH）后水通道蛋白-4（aquaporin-4,AQP4）表达的关系。方法健康大耳白兔,随机分为水蛭素干预组和对照组,每组出血后6h、12h、24h、2d、3d、5d各包括6只兔。RT-PCR法对AQP4 mRNA表达进行观察；Western blot法对AQP4蛋白表达进行观察。结果水蛭素干预组各时相AQP4 mRNA、AQP4蛋白表达均低于对照组。出血后12h、24h、2d与对照组相应时相比较有显著差异。结论凝血酶是脑出血早期导致AQP4 mRNA、AQP4蛋白表达增高的重要原因,其机制可能与低氧诱导因子（HIF-1）及凝血酶所致脑血肿周围细胞坏死有关。     

核转录因子κB涉及水通道蛋白-4抗体阳性血清对星形胶质细胞的毒性作用

目的探讨核转录因子κB(nuclear factor kappa B,NF-κB)在水通道蛋白4(aquaporin-4,AQP4)抗体诱导的星形胶质细胞损伤中的作用。方法纯化培养新生SD大鼠大脑皮质星形胶质细胞,按随机数字表法分为对照组、吡咯醛二硫氨基甲酸(PDTC)组、AQP4抗体阳性血清组(AQP4抗体组)和AQP4抗体+PDTC组,其中对照组加入10%(体积分数)健康人血清,PDTC组给予10μmol/L PDTC预处理1h后加入等量健康人血清;抗体组加入AQP4抗体阳性血清;AQP4抗体+PDTC组先用PDTC预处理1h后再加入AQP4抗体阳性血清。细胞培养12h后采用免疫细胞化学荧光法观察各组NF-κB p65入核情况,MTS比色法检测细胞存活度,免疫印迹法检测细胞内p65蛋白、磷酸化p65(p-p65)蛋白的表达。结果 AQP4抗体组可见明显NF-κB p65入核,其余三组均未见明显入核;AQP4抗体+PDTC组细胞存活度高于AQP4抗体组(吸光度值分别为0.4380±0.005、0.3897±0.045,F=133.355,P0.05);各组间NF-κB p65蛋白表达水平比较差异无统计学意义(P0.05);AQP4抗体+PDTC组p-p65蛋白表达水平较AQP4抗体组明显减少(分别为0.7027±0.020,0.8197±0.027,F=10.794,P0.05)。结论 AQP4抗体阳性血清可能通过激活NF-κB途径促进星形胶质细胞损伤,抑制NF-κB活性对星形胶质细胞具有保护作用。     

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     

Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytes

Agrin is a heparan sulfate proteoglycan of the extracellular matrix and is known for organizing the postsynaptic differentiation of the neuromuscular junction. Increasing evidence also suggests roles for agrin in the developing CNS, including the formation and maintenance of the blood-brain barrier. Here we describe effects of agrin on the expression and distribution of the water channel protein aquaporin-4 (AQP4) and on the swelling capacity of cultured astrocytes of newborn mice. If astrocytes were cultured on a substrate containing poly DL-ornithine, anti-AQP4 immunoreactivity was evenly and diffusely distributed. If, however, astrocytes were cultured in the presence of agrin-conditioned medium, we observed an increase in the intensity of AQP4-specific membrane-associated staining. Freeze-fracture studies revealed a clustering of orthogonal arrays of particles, representing a structural equivalent of AQP4, when exogenous agrin was present in the astrocyte cultures. Neuronal and non-neuronal agrin isoforms (agrin A0B0 and agrin A4B8, respectively) were able to induce membrane-associated AQP4 staining. Water transport capacity as well as the density of orthogonal arrays of intramembranous particles was increased in astrocytes cultured with the neuronal agrin isoform A4B8, but not with the endothelial and meningeal isoform A0B0. RT-PCR demonstrated that agrin A4B8 increased the level of the M23 splice variant of AQP4 and decreased the level of the M1 splice variant of AQP4. Implications for the regulation and maintenance of the blood-brain barrier including oedema formation under pathological conditions are discussed.     

精氨酸加压素对大鼠脑水肿形成及水孔蛋白-4表达的影响

目的 探讨精氨酸加压素(AVP)对大鼠脑水肿形成和水孔蛋白-4(AQP4)表达的影响.方法 90只SD大鼠随机分为正常对照组、假手术6 h、1 d、3 d、5 d组及AVP6 h、1 d、3 d、5 d组.于大鼠侧脑室注射AVP后,分别于相应时间点对脑室周围组织进行脑含水量测定,免疫组化技术检测脑室周围组织AQP4的表达.结果 假手术组与正常对照组脑组织含水量及AQP4表达未见增加.AVP组大鼠侧脑室注射AVP 6 h脑室周围脑组织含水量开始增加,1 d即达高峰,与正常对照组比较差异有统计学意义(均P＜0.01),5 d降到基础水平;脑室周围AQP4表达亦在注射AVP 6 h开始增加,1 d即达高峰,与正常对照组比较差异有统计学意义(均P＜0.01),第5 d恢复到基础水平,与脑水肿的变化相一致.结论 AVP可上调AQP4表达,引起大鼠脑含水量增加,导致脑水肿的发生.     

Negative and positive regulation of astrocyte DNA synthesis by ethanol

Ethanol suppression of astrocyte mitogenesis is well recognized but ethanol, under some conditions, has also been shown to stimulate astrocyte proliferation. This study addressed the role of protein kinase C and other mitogenic factors as mechanisms responsible for the bidirectional effects of ethanol on astrocyte DNA synthesis. Ethanol treatment inhibited astrocyte DNA synthesis both at 4 hr (short term) and 24 hr (long term) in serum free medium. In contrast, when the medium contained serum, ethanol was less effective in inhibiting DNA synthesis at 4 hr and treatment with ethanol for 24 hr increased DNA synthesis. Protein kinase C activity was increased in cells treated with ethanol for either 4 or 24 hr. Ethanol inhibition of DNA synthesis in serum free medium was not reversed by down regulating protein kinase C. In contrast, downregulating protein kinase C activity by continuous treatment with phorbol myristic acetate partially reversed the effect ethanol had on DNA synthesis. Also, directly inhibiting protein kinase C with H-7 in cells maintained and treated in the presence of serum abolished the stimulatory effect ethanol had on DNA synthesis. It appears that the negative regulation of astrocyte DNA synthesis by ethanol occurs by protein kinase C and serum independent mechanisms whereas adaptive or stimulatory effects of ethanol on astrocyte DNA synthesis requires the interaction of protein kinase C with other factors present in serum. J. Neurosci. Res. 50:1010–1017, 1997. © 1997 Wiley-Liss, Inc.     

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 Un...     

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.     

Loss of perivascular aquaporin-4 in idiopathic normal pressure hydrocephalus

Idiopathic normal pressure hydrocephalus (iNPH) is a subtype of dementia that may be successfully treated with cerebrospinal fluid (CSF) diversion. Recently, magnetic resonance imaging (MRI) using a MRI contrast agent as a CSF tracer revealed impaired clearance of the CSF tracer from various brain regions such as the entorhinal cortex of iNPH patients. Hampered clearance of waste solutes, for example, soluble amyloid-β, may underlie neurodegeneration and dementia in iNPH. The goal of the present study was to explore whether iNPH is associated with altered subcellular distribution of aquaporin-4 (AQP4) water channels, which is reported to facilitate CSF circulation and paravascular glymphatic drainage of metabolites from the brain parenchyma. Cortical brain biopsies of 30 iNPH patients and 12 reference individuals were subjected to AQP4 immunogold cytochemistry. Electron microscopy revealed significantly reduced density of AQP4 water channels in astrocytic endfoot membranes along cortical microvessels in patients with iNPH versus reference subjects. There was a significant positive correlation between density of AQP4 toward endothelial cells (perivascular) and toward parenchyma, but the reduced density of AQP4 toward parenchyma was not significant in iNPH. We conclude that perivascular AQP4 expression is attenuated in iNPH, potentially contributing to impaired glymphatic circulation, and waste clearance, and subsequent neurodegeneration. Hence, restoring normal perivascular AQP4 distribution may emerge as a novel treatment strategy for iNPH.     

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.