缺氧／复氧对星形胶质细胞AQP4表达的影响

目的 探讨缺氧/复氧对于体外培养星形胶质细胞表达AQP4的影响及缺血性脑血管病脑水肿的发病机制.方法 选取新生24 h Wistar大鼠脑组织行星形胶质细胞原代培养并建立缺氧/复氧模型,应用Western blot和免疫细胞化学法检测星形胶质细胞AQP4的表达变化.结果 与对照组比较,缺氧3 h和6 h星形胶质细胞AQP4蛋白表达减少(P<0.01),复氧后6 h和9 h其表达明显增高(P<0.01).结论 AQP4表达变化与细胞损害相关.     

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。     

新生动物星形胶质细胞体外缺糖缺氧/复糖复氧模型中水通道蛋白4与细胞水肿的关系

背景 脑缺氧缺血常常引起的脑水肿是新生儿神经发育障碍的主要原因。可是脑水肿的分子机制目前仍不清楚,所以近80年脑水肿的治疗方案改变甚少,10年前,水通道蛋白（AQP）的发现,提供了理解脑中水转运的分子基础,近期研究阐明了AQP4在脑水肿的形成和消退中起着重要作用,但是它在新生儿缺氧缺血性脑水肿的发生中的准确作用还不清楚,因此,我们研究AQP4在星形胶质细胞缺氧和复氧期水肿中的作用,以发现新生儿缺氧缺血性脑水肿的治疗新策略。 方法 我们采用实时荧光定量PCR和Western blot分别测定培养的星形胶质细胞在缺氧期和复氧期AQP4mRNA和蛋白的表达,用3H标记的甲基D葡萄糖测定细胞的体积,代表细胞的水肿程度。 结果 在缺氧缺血期,星形胶质细胞AQP4 mRNA和蛋白表达逐渐下降,而细胞体积按照时间依赖方式增加。复氧期,AQP4 mRNA和蛋白表达上调,而细胞体积逐渐下降,在复氧第7天恢复到正常对照组水平。 结论 我们通过星形胶质细胞缺氧缺血模型的研究,发现AQP4可能在脑组织水转运调控中起着重要作用,提示AQP4可能作为改善新生儿缺氧缺血性脑水肿的重要目标。     

亚低温对缺氧/复氧后星形胶质细胞AQP4表达的影响

目的 观察缺氧/复氧条件下大脑星形胶质细胞水通道蛋白4(AQP4)的表达变化以及亚低温对其表达的影响,探讨脑缺血再灌注脑水肿与AQP4的关系以及亚低温对脑缺血再灌注损伤的保护机制.方法 利用新生24 h内的SD大鼠,进行原代、传代培养,将星形胶质细胞分为对照组、常温组及亚低温组.用台盼蓝染色法测定37℃及32℃时,缺氧/复氧不同时间点星形胶质细胞的存活率,作为细胞受损指标,用倒置相差显微镜对细胞进行形态学观察,应用细胞免疫化学技术检测星形胶质细胞缺氧/复氧各个时间点AQP4的表达变化及亚低温的干预效果.结果 (1)缺氧4,8h时细胞形态变化不明显,随着复氧时间的延长,可见细化逐渐明显,而亚低温干预的细胞形态及细胞存活力变化均较相应的常温组明显减轻;(2)缺氧及复氧早期AQP4的表达降低,复氧后6h随着时间延长AQP4表达明显增多,在复氧≤8h,常温组及亚低温组的表达均低于对照组(均P＜0.05或0.01),而复氧后10,12h,AQP4蛋白表达均明显高于对照组(均P＜0.05或0.01);(3)在复氧后各时间点亚低温组AQP4的表达均明显低于常温组(均P＜0.05或0.01).结论 星形胶质细胞对缺氧的耐受能力较强,亚低温可以减轻缺氧/复氧后星形胶质细胞的损伤,通过降低AQP4的表达,可能是亚低温减轻缺血性脑水肿的作用机制之一.     

精氨酸加压素对星形胶质细胞水孔蛋白-4表达的调节及脑水肿形成影响的研究

目的研究精氨酸加压素(AVP)对星形胶质细胞水孔蛋白-4(AQP4)表达的调节,以及p38 MAPK信号通路在AQP4表达过程的作用,明确AVP及AQP4在脑水肿发生过程中的作用。方法大鼠大脑皮质分离星形胶质细胞,星形胶质细胞经分别用AVP、V1a受体(V1aR)拮抗剂和SB 203580进行处理,采用免疫组织化学技术及RT-PCR对AQP4 mRNA进行检测,Western blot检测p38 MAPK信号通路在AVP诱导AQP4表达中的活化程度。结果500nmol/L的AVP处理6h后,AQP4 mRNA表达开始升高(P<0.01),到12h达高峰(P<0.01),24h后仍维持在较高的水平(P<0.05)。加入p38 MAPK抑制剂SB 203580干预后,AQP4 mRNA表达水平与对照组比较差异不显著(P>0.05);AVP处理15min后p38 MAPK磷酸化水平开始增加,30min达高峰,持续到60min开始下降。V1aR拮抗剂处理后p38 MAPK磷酸化水平整个时间段均未出现明显变化。结论AVP通过激活V1aR引起p38MAPK信号通路活化从而诱导AQP4 mRNA高表达,从基因水平对AQP4进行调节,可能在脑水肿发生中,尤其是在星形胶质细胞水肿形成中起重要作用。V1aR拮抗剂及p38 MAPK抑制剂能抑制AQP4 mRNA的表达,避免星形胶质细胞肿胀。     

脑缺血大鼠水通道蛋白-9mRNA表达与钙离子浓度的改变及其关系

目的 研究脑缺血后脑组织水通道蛋白-9(AQP9)mRNA表达和Ca2 浓度的改变及其关系.方法 制作脑缺血大鼠模型,在脑缺血6 h、1 d、2 d,3 d、5 d,用半定量逆转录聚合酶链反应(RT-PCR)检测缺血脑组织AQP9 mRNA的表达水平;用Fura-2/AM荧光法测定水肿区脑细胞内Ca2 浓度;并与对照组比较.结果 与对照组相比,脑缺血后6 h,水肿区脑组织AQP9 mRNA表达水平和Ca2 浓度均开始增高,在脑缺血后第2 d、3 d表达最强(P<0.05～0.01);AQP9 mRNA表达水平与Ca2 浓度呈正相关(r=0.7654,P＜0.05).结论 脑缺血后AQP9表达增强,Ca2 浓度增高;提示AQP9和Ca2 可能共同参与缺血性脑水肿的形成.     

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。     

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。     

缺氧/复氧后星形胶质细胞AQP5表达的变化及亚低温的干预效应

目的观察缺氧/复氧条件下大脑星形胶质细胞水通道蛋白5(AQP5)的表达变化以及亚低温对其表达的影响。方法利用新生24 h内的SD大鼠,进行原代、传代培养,将星形胶质细胞分为对照组、常温组及亚低温组。用台盼蓝染色法测定37℃及32℃时,缺氧/复氧不同时间点星形胶质细胞的存活率,作为细胞受损指标,用倒置相差显微镜对细胞进行形态学观察,应用细胞免疫化学技术检测星形胶质细胞缺氧/复氧各个时间点AQP5的表达变化及亚低温的干预效果。结果 (1)缺氧4、8 h细胞形态变化不明显,随着复氧时间的延长,可见活化逐渐明显,而亚低温干预的细胞形态及细胞存活力变化均较相应的常温组明显减轻;(2)缺氧及复氧早期AQP5的表达水平降低,复氧后6 h随着时间延长AQP5表达明显增多,在复氧≤8 h常温组及亚低温组的表达水平均低于对照组(P<0.05或0.01),而复氧后10、12 h AQP5蛋白表达水平均明显高于对照组(P<0.05或0.01);(3)在复氧后各时间点亚低温组AQP5的表达水平均明显低于常温组(P<0.05或0.01)。结论亚低温可以减轻缺氧/复氧后星形胶质细胞的损伤,通过降低AQP5的表达水平,可能是亚低温减轻缺血性脑水肿的作用机制之一。     

水通道蛋白4在脑出血患者脑内皮层的表达

目的观察水通道蛋白4(aquaporin-4,AQP4)在脑出血继发脑水肿患者脑内皮层的表达。方法采用逆转录聚合酶链式反应(RT-PCR),免疫组织化学和Western-blot技术,分析比较10例脑出血患者脑组织与6例正常脑组织标本AQP4的mRNA和蛋白的表达情况。结果AQP4主要表达在皮层上的星形胶质细胞。脑出血继发脑水肿患者脑组织AQP4的mRNA和蛋白的表达均显著高于正常对照组(P<0.01)。结论AQP4参与了脑出血继发脑水肿的发生,可能是脑水肿产生的重要分子基础。     

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...     

The roles of aquaporin-4 in brain edema following neonatal hypoxia ischemia and reoxygenation in a cultured rat astrocyte model

Aquaporin-4 (AQP4), a water channel protein, is abundantly expressed in astrocytes and plays a key role in the development of brain edema. However, it is not clear whether AQP4 contributes to astrocytic swelling in hypoxia-ischemia (HI). To investigate the roles of AQP4 in astrocytic swelling during HI and reoxygenation, we measured AQP4 expression and astrocytic cellular volume in cultured rat astrocytes following HI and reoxygenation. RNA interference was used to knockdown AQP4 expression (AQP4(-/-)). Real-time polymerase chain reaction and Western blot analysis were used to detect the inhibitory efficiency of AQP4. We found that the maximal inhibition of AQP4 mRNA and protein in astrocytes after AQP4 siRNA transfection (AQP4(-/-)) was approximately 77 and 85%, respectively, compared to wild-type AQP4 (AQP4(+/+)) expression. Cellular volume in both AQP4(-/-) and AQP4(+/+) astrocytes was significantly increased during HI compared to cells cultured in normoxia (P<0.05). However, cellular volume during HI in AQP4(-/-) astrocytes was significantly less than that in AQP4(+/+) astrocytes (P<0.05). After reoxygenation, the cellular volume gradually decreased to control levels at 7 days in AQP4(-/-) but at 5 days in AQP4(+/+) astrocytes. The different roles of AQP4 during HI and reoxygenation suggest that AQP4 knockdown may protect against water influx in the formation of astrocyte swelling during HI, and may also delay water clearance in the resolution of astrocyte swelling during reoxygenation. In conclusion, AQP4 mediates bidirectional transport of water across astrocytes during HI and reoxygenation. AQP4 manipulation may serve as a novel therapeutic strategy during different periods of hypoxic-ischemic brain edema in neonates.     

Testosterone up-regulates aquaporin-4 expression in cultured astrocytes

Aquaporin-4 (AQP4) is located on astrocyte endfeet that face blood vessels in the brain and in the pia. It is thought to play a crucial role in the development of brain edema. To confirm the notion that sex steroids and dexamethasone influence brain edema through AQP4 regulation, we investigated the effects of 17beta-estradiol, testosterone, and dexamethasone on the expression of AQP4 in cultured astrocytes. Testosterone significantly up-regulated AQP4 at the level of both protein and mRNA. At a concentration of 100 nM, testosterone significantly increased AQP4 protein levels and ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Moreover, this effect was attenuated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, which can rapidly decrease AQP4 mRNA expression, indicating that the response was specific. These results indicate that AQP4 can alter the osmotic fragility of astrocytes and that testosterone can influence brain edema through AQP4 regulation, whereas 17beta-estradiol and dexamethasone cannot.     

Thrombin inhibits aquaporin 4 expression through protein kinase C-dependent pathway in cultured astrocytes

Aquaporin 4 (AQP4) is a key molecule for maintaining water balance in the central nervous system, and its dysfunction might cause brain edema. However, little is known about the regulation of AQP4 expression. Because thrombin has been implicated in brain edema formation, the purpose of this study is to determine whether thrombin affects expression of AQP4 in astrocytes. Here, the effect of thrombin on AQP4 expression in vitro was evaluated using Western blot analysis and RT-PCR. Meanwhile, we investigated whether the effect of thrombin on AQP4 expression was due to protease-activated receptor 1 (PAR-1). In addition, we examined the role of protein kinase C (PKC) in the effect of thrombin on AQP4 expression using Western blot analysis. We found that thrombin did not affect cell viability at concentrations of 0.05, 0.5, 5, or 50 nM but killed astrocytes at concentrations of 500 nM, with approx 72% of astrocytes surviving at 500 nM thrombin. Our data showed that AQP4 protein expression achieved only 28% of controls in 500 nM thrombin treatment, even if astrocytes survived approx 72% of controls at 500 nM thrombin. Thrombin significantly inhibited AQP4 in a time- and dose dependent manner in vitro (p<0.05). Cathepsin-G, a thrombin PAR-1 inhibitor, reversed significantly (p<0.05) the effect of thrombin on AQP4 mRNA and protein expression in astrocytes. We also observed that PKC inhibitor H-7 or prolonged pretreatment with TPA can rapidly increase AQP4 expression (p<0.05). Thrombin might inhibit AQP4 expression in rat astrocytes, and this effect is possibly mediated by the PKC pathway.     

An increase in intracellular free calcium ions by nicotinic acetylcholine receptors in a single cultured rat cortical astrocyte

Neuronal nicotinic acetylcholine receptors (nAChRs) are composed of an assembly between at least seven alpha (alpha2-alpha7, alpha9) and three beta (beta2-beta4) subunits in mammals. The addition of 50 mM KCl or 1 mM nicotine immediately increased the number of cells with high fluorescence intensity in rat cortical astrocytes on fluo-3 fluorescence measurement. Nicotine was effective at increasing the fluorescence intensity in astrocytes cultured for 2 days after replating, but not in those used 1 or 5 days after replating, without markedly affecting the cellular viability irrespective of the exposure period. Nicotine markedly increased the fluorescence intensity in a concentration-dependent manner at a concentration range of 10-100 microM in cultured astrocytes when analyzed on a responsive single cell. In these responsive single cells, the increase by nicotine was significantly prevented by the heteromeric alpha4/beta2 subtype antagonist dihydro-beta-erythroidine and the homomeric alpha7 subtype antagonist methyllycaconitine, as well as by nifedipine and EGTA but not thapsigargin. Methyllycaconitine failed to inhibit further the increase by nicotine in the presence of nifedipine, however, whereas the expression of mRNA was seen for all mammalian neuronal nAChR subunits in cultured rat cortical astrocytes as well as neurons. These results suggest that nicotine may increase intracellular free Ca2+ through the influx of extracellular Ca2+ across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to the alpha4/beta2 and/or alpha7 nAChR channels functionally expressed in cultured rat cortical astrocytes.     

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.