三七总皂苷预处理对急性内脏痛大鼠星形胶质细胞的影响

目的:探讨三七总皂苷预处理对急性内脏痛大鼠的影响,初步阐述三七总皂苷对急性内脏痛的影响机制。方法:成年雌性SD大鼠54只随机分为正常组(n=6),生理盐水预处理组(n=24),三七总皂苷预处理组(n=24)。正常组常规条件饲养,不做干预及建立急性内脏痛模型;生理盐水预处理组和三七总皂苷预处理组大鼠分别预先腹腔注射生理盐水(2.86 ml/kg)或7 mg/ml的三七总皂苷(2.86 ml/kg),每12 h一次,连续7 d,第8天腹腔注射1%乙酸(10 mg/kg),建立急性内脏痛模型,立即观测SD大鼠扭体反应。按(30、60、90、180 min)不同存活时间处死动物,免疫组化法观测脊髓背角GFAP的表达变化。结果:VPI评分显示,三七总皂苷能显著下调内脏痛模型VPI评分,减轻疼痛。免疫组化显示在相同时间点,三七总皂苷预处理组大鼠GFAP表达弱于生理盐水预处理组,尤其在30、60、90 min存活组。结论:对SD大鼠急性内脏痛模型预先腹腔注射三七总皂苷可以抑制脊髓胶质细胞激活,从而减轻急性内脏痛。     

甘珀酸对大鼠大脑中动脉缺血再灌注后星形胶质细胞增殖及活化的影响

目的探讨缝隙连接阻断剂甘珀酸对大鼠大脑中动脉(Middle Cerebral Artery,MCA)缺血再灌注模型半暗带区星形胶质细胞增殖及活化的影响。方法成年雄性SD大鼠80只,随机分为生理盐水组(n=35)、CBX干预组(n=35)和假手术组(n=10)。CBX干预组术前1h右侧侧脑室注射CBX,生理盐水组右侧侧脑室注射生理盐水,建立标准大脑中动脉梗死模型,缺血1h后再灌注6h、1d、3d、7d,免疫荧光及免疫印迹的方法观察GFAP,Ki67,PCNA的表达情况。结果与对照组比较,CBX干预组大鼠术后半暗带区GFAP的表达量减少,Ki67与GFAP双阳性细胞减少(P0.05),PCNA的表达量没有明显的变化。结论甘珀酸可以抑制缺血引起的星形胶质细胞的活化增殖。     

LPS诱导海马星形胶质细胞活化与活化的星形胶质细胞对海马神经元的影响

探讨脂多糖(Lipopolysaccharide,LPS)对长时间存活大鼠海马内星形胶质细胞的反应以及对神经元的影响。方法:本实验用10只健康成年雄性SD大鼠,海马CA3区注射LPS 10μ1．7和14d后,尼氏染色观察神经元的变化,免疫组织化学染色结合图像分析方法观察海马CA3区注射部位胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)、的表达变化。结果:脂多糖可促进海马星形胶质细胞的活化,但并不能引起海马区神经元的损伤。结论:星形胶质细胞在脑损伤后的脑内炎症反应起了一定的作用,但并不能引起神经元的损伤。     

甘珀酸干预对大鼠脑缺血再灌注损伤的影响

目的观察缝隙连接阻断剂甘珀酸对局灶性脑缺血/再灌注损伤的影响。方法采用大鼠大脑中动脉阻塞再灌流模型（MCAO）,将动物随机分为脑缺血60min再灌注（MCAO）组,脑缺血再灌注加甘珀酸干预（MCAO＋CBX）组和假手术组（sham）。采用尼氏染色显示脑梗死灶并计算梗死灶体积;应用免疫荧光与TUNEL染色法分别观察脑缺血后3d与7d不同时间点缺血边缘区胶质纤维酸性蛋白（GFAP）的表达和细胞凋亡情况。结果（1）缺血后3d、7d MCAO＋CBX组大鼠梗死体积小于MCAO组,3d、7d MCAO＋CBX组大鼠梗死体积较MCAO组分别缩小5%和4.6%;（2）缺血后3d、7d于缺血边缘区可见大量TUNEL阳性染色细胞,且MCAO组大鼠缺血边缘区细胞凋亡数目明显多于MCAO＋CBX大鼠（P〈0.001）;（3）缺血后3d和7d组缺血边缘区GFAP表达明显增强,3d的MCAO组与MCAO＋CBX组大鼠缺血边缘区GFAP的表达均较假手术组强（P〈0.05）,7d的MCAO＋CBX组大鼠缺血边缘区GFAP的表达较假手术组强（P〈0.001）,但明显弱于MCAO组大鼠（P〈0.01）;结论缝隙连接阻断剂甘珀酸可减少大鼠大脑中动脉阻塞后脑梗死体积,其机制可能与阻断缝隙连接后缺血边缘区神经元凋亡降低有关,星型胶质细胞的反应性变化参与了该过程。     

星形胶质细胞糖代谢对神经元的支持及保护作用

脑组织有着极其复杂的功能,这些功能的完成有赖于神经元细胞与胶质细胞之间的广泛合作。星形胶质细胞作为人脑内数量最多的细胞,其与神经元细胞之间的相互作用就显得十分重要。葡萄糖代谢途径包括糖酵解,有氧氧化及磷酸戊糖三条途径。其为脑组织维持其正常功能的前提。研究表明星形胶质细胞和神经元在糖代谢方面有着各自的特点,神经元在能量底物及抗氧化应激中对星形胶质细胞糖代谢途径存在一定的依赖性,干扰星形胶质细胞与神经元之间的代谢过程会导致疾病的发生。本综述主要从糖酵解及磷酸戊糖两条糖代谢途径阐述了星形胶质细胞与神经元的关系。这或许会对研究脑的代谢,脑疾病中神经元的损伤机制及如何保护神经元提供全新的视角,并可能为一些疾病的治疗开辟了新的途径。     

电针足三里穴对糖尿病胃轻瘫大鼠延髓多巴胺能神经元和 星形胶质细胞活性的影响

目的:研究电针足三里穴对糖尿病胃轻瘫大鼠延髓多巴胺能神经元内酪氨酸羟化酶(tyrosine hydroxylase,TH)和星形胶质细胞内胶质原纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)表达的影响。方法:32只实验大鼠分为空白对照(空白)组、糖尿病胃轻瘫模型(模型)组、模型组+电针足三里穴(足三里)组和模型组+电针非经非穴(非经非穴)组(每组8只)。模型制备采用腹腔注射5%四氧嘧啶和熟地灌胃诱导的方法。实验3周后取大鼠延髓进行抗TH和抗GFAP的单一和双重免疫组化染色,观察并记数TH和GFAP在延髓内的表达。结果:与空白组比较,各实验组TH阳性多巴胺能神经元和GFAP阳性星形胶质细胞集中表达于延髓迷走孤束复合体内,有明显的定位特点;高倍镜下观察到TH阳性神经元周围有大量GFAP阳性星形胶质细胞包绕。各组TH和GFAP表达以模型组最高;而足三里组TH阳性多巴胺能神经元数量明显减少(31.3±4.4→16.8±3.2),GFAP阳性产物表达明显降低(113.8±7.6→95.4±8.4),且它们之间有统计学意义(P<0.01);非经非穴组与模型组之间差异没有统计学意义。结论:针刺调节糖尿病胃运动功能障碍大鼠与其调控延髓多巴胺能神经元及其周围的星形胶质细胞功能活动有关。     

成年大鼠神经元和星形胶质细胞细胞周期蛋白表达的差异研究

目的比较研究成年大鼠细胞周期蛋白在神经元和星形胶质细胞的表达差异。方法应用免疫荧光和激光扫描共聚焦显微镜观察成年大鼠生理状态下大脑皮层或海马CA1区神经元和星形胶质细胞细胞周期素D1、E、A、B1、(CyclinD1、E、A、B1)的表达。结果成年大鼠海马CA1区和大脑皮层的神经元有Cyclin D1、E、A和B1的表达,细胞核和细胞浆均有表达,以胞核为主;星形胶质细胞也有上述细胞周期蛋白的表达但细胞数目较少,并且表达这些指标的星形胶质细胞多聚集在海马CA1区。结论成年大鼠大脑皮层和海马区的神经元和星形胶质细胞均表达细胞周期蛋白,而其在神经元的表达较星形胶质细胞更为普遍。     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

大鼠脑皮质星形胶质细胞的限制性细胞培养

介绍一种新的脑组织星形胶质细胞培养方法即限制性细胞培养（constraint cell culture）。常规分离纯化星形胶质细胞,将其低密度种植,维持在添中低量血清的化学成分限定的培养基中培养,并在长时期内不给予更换或补加培养液。利用波形蛋白(vimentin）和胶质纤维酸性蛋白（glial fibrary acidic protein）抗体的免疫荧光染色法鉴定观察不同培养时期的星形胶质细胞及其形态学变化。结果发现星形胶质细胞在最初的5天之内有一定程度的增殖,未出现过度增殖导致的细胞相互融合现象;接下来的3－5天内细胞形态明显分化,星形胶质细胞突起细长、胞体明显缩小、形态多样,最后细胞突起之间相互连接形成星形胶质细胞网络,并在相当长的时间内保持不变。实验结果显示在限制细胞种植密度和限制给予培养液的培养条件下星形质细胞的体外形态发育与在体的情形基本一致。提示该细胞培养方法可能有助于研究中枢神经系统中星形胶质细胞的生理功能。     

星形胶质细胞的生物学功能

人类大脑由两类细胞组成:一类是神经元,另一类是神经胶质细胞。神经胶质细胞的数量约为神经元的10倍,但其作用长期以来一直被认为仅限于在神经元之间充当填充物,填满大脑中的剩余空间,同时为神经元提供营养。但近年来认识到神经胶质细胞的主要成员星形胶质细胞能够感知外界刺激,它的反应选择性甚至高于相邻神经元。神经元的反应活动很多都要经过星形胶质细胞的介导才能完成。本文介绍了星形胶质细胞在神经调制、突触调节和神经血管系统偶联方面的一些新进展,以期在不久的将来对星形胶质细胞的功能有更深入的了解,并能应用于临床实践。     

Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture

Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high-density lipoprotein-like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE-mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low-density lipoprotein receptor-related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform-specific manner and that cell surface HSPGs and/or HSPG-LRP pathway may mediate this apoE-promoted lipid efflux.     

13C heteronuclear NMR studies of the interaction of cultured neurons and astrocytes and aluminum blockade of the preferential release of citrate from astrocytes

Citrate has been identified as a major tricarboxylic acid (TCA) cycle constituent preferentially released by astrocytes. We undertook the present study to examine further the nature of metabolic compartmentation in central nervous system tissues using ¹³C-labeled glucose and to provide new information on the influence of aluminum on the metabolic interaction between neurons and astrocytes. Metabolites released into the culture medium from astrocytes and neuron-astrocyte coculture, as well as the perchloric acid extracts of the cells were analyzed using 2D ¹H and ¹³C NMR spectroscopy. Astrocytes released citrate into the culture medium and the released citrate was consumed by neurons in coculture. Citrate release by astrocytes was blocked in the presence of aluminum, with progressive accumulation of citrate within the cells. We propose citrate supply is a more efficient energy source than lactate for neurons to produce ATP, especially in the hypoglycemic state on account of it being a direct component of the TCA cycle. Astrocytes may be the cellular compartment for aluminum accumulation as a citrate complex in the brain.     

Intra-amygdala microinfusion of neuropeptide S attenuates neuropathic pain and suppresses the response of spinal microglia and astrocytes after spinal nerve ligation in rats

The amygdala circuitry and neuropeptide S (NPS) have been shown to play an important role in the pain modulation. However, the alleviative effect of NPS in amygdala on neuropathic pain (NP) is not fully understood. Here, we demonstrate a possibility that the intra-amygdala microinfusion of NPS attenuates NP symptoms and suppresses the response of spinal microglia and astrocytes after spinal nerve injury. Spinal nerve ligation (SNL) in rats resulted in a striking decline in level of NPS and density of NPS-immunopositive cells in amygdala. SNL rats randomly received chronic bilateral microinjections of NPS (1, 10 and 100 pmol/side) or saline into the amygdala via cannulas on days 3, 6, 9, 12, 15 and 18 post-surgery. Chronic treatment with NPS increased thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) on day 11–21 post-SNL. The simultaneous treatment with SHA68 as non-peptide NPS receptor antagonist decreased the TWL and MWT, and reversed the inhibitory effects of NPS in SNL rats. NPS also significantly attenuated immunoreactivities of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein for microglia and astrocytes. Furthermore, the elevated levels of inflammatory mediators and expressions of nuclear factor κB p65 and CX3C chemokine receptor 1 due to SNL were significantly attenuated by NPS in amygdala. These effects of NPS were also counteracted by SHA 68. SHA 68 per se deteriorated the symptom of NP and the response of spinal microglia and astrocytes in SNL rats. Our study identified a protective role for NPS in amygdala against the development of NP, possibly attributing to its anti-inflammatory activity and inhibition of spinal microglia and astrocytes.     

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.     

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.     

应激和肠道菌群在肠易激综合征内脏疼痛中的机制研究

内脏痛是一种躯体内脏器官引起的疼痛,是功能性胃肠病（FGIDs）如肠易激综合征（irritable bowel syndrome,IBS）最常见的临床症状。慢性应激被认为是IBS的发病病因之一,应激可调节脑—肠轴的结构和功能,同时激活肠道免疫,破坏肠道黏膜屏障以及引起内脏感觉过敏。近年来,肠道微生物与脑—肠轴的双向交流及相互作用逐渐被认识,本研究就应激与肠道菌群在IBS内脏疼痛的机制研究作一综述。     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

Pituitary astrocytes from the neural lobe of rats

Summary Tissue culture preparations of adult and neonatal rat pituitary neural lobes were examined by use of cell-type specific immunohistochemical markers. Cultures obtained from explanted or dissociated adult tissue or explanted neonatal tissue produced cells immunoreactive for endothelial and fibroblast markers. In contrast, dissociated neonatal tissue produced, in addition, two distinct forms of astrocytic glial cells immunoreactive for glial fibrillary acidic protein, one of which was also immunoreactive for the ganglioside GD3.     

Effects of garcinol on free radical generation and NO production in embryonic rat cortical neurons and astrocytes

Garcinol (camboginol) is a polyisoprenylated benzophenone derivative isolated from fruit rind of Garcinia indica. This study was to elucidate the anti-oxidative and neuroprotective properties of garcinol in rat cortical neuron cultures. First, garcinol protects DNA from Fenton reaction-induced breakage in a dose-dependent manner, with an IC(50) value of 0.32 microM. Garcinol also inhibits xanthine oxidase activity with an IC(50) value of 52 microM and exhibits competitive inhibition. To further ascertain the neuroprotective effects of garcinol in inflammatory-mediated neurotoxicity, we utilized primary neuron/astrocyte co-cultures treated with LPS or cytokine. Our data implicate that treatment with garcinol (5 microM) for 7 days promotes neuronal attachment and neurite extension. The formation of nitric oxide (NO) by LPS in rat astrocytes has been suggested to correlate with the neurodegenerative process. In identifying the effect of neuroprotection, we found that garcinol prevented NO accumulation in LPS-treated astrocytes. Garcinol significantly reduced the expression of LPS-induced inflammatory mediators, such as iNOS and COX-2. Consequently, our results suggest that the neuroprotective effects of garcinol are associated with anti-oxidation and inhibition of iNOS induction in astrocytic cells. Garcinol may exert a similar anti-inflammatory effect and may be neuroprotective against brain injury.