da Cruz-Höfling Maria Alice, Rapôso Catarina, Verinaud Liana, Zago Gabriela Mariotoni
Department of Histology and Embryology, Institute of Biology, PO Box 6109, University of Campinas - UNICAMP, Zip Code 13083-970 Campinas, SP, Brazil.
Neurotoxicology. 2009 Jul;30(4):636-46. doi: 10.1016/j.neuro.2009.04.004. Epub 2009 Apr 22.
Phoneutria nigriventer spider venom (PNV) causes uneven BBB permeability throughout different cerebral regions. Little is known about cellular and molecular responses which course with the PNV-induced BBB opening. We investigate by immunohistochemistry (IHC) and Western blotting (WB), the GFAP, S100, IFN-gamma and TNF-alpha proteins expression in hippocampus and cerebellum after different time-points from venom or saline intravenous injection. All proteins variably altered its expression temporally and regionally. WB showed increased GFAP content at 15-45 min followed by a shift below the control level which was less pronounced in hippocampus. IHC showed reactive gliosis during all the trial period. In cerebellum, GFAP was mostly immunodetected in astrocytes of the molecular layer (Bergmann glia), as was S100 protein. The maximum S100 immunolabeling was achieved at 5h. IFN-gamma and TNF-alpha, expressed mostly by hippocampal neurons, increased along the trial period, suggesting a role in BBB permeability. In envenomed animals, closer contacts astrocyte-astrocyte, granule cells-granule cells and astrocytes-Purkinje cells were observed in cerebellum. Closer contacts between neurons-neurons-astrocytes-astrocytes were also seen in hippocampus. PNV contains serotonin, histamine, Ca(2+) channels-blocking toxins, some of which affect glutamate release. The hypothesis that such substances plus the cytokines generated, could have a role in BBB permeability, and that calcium homeostasis loss and disturbance of glutamate release are associated with the marked GFAP/S100 reaction in Bergmann glia is discussed. The existence of a CNS mechanism of defense modulated differentially for fast synthesis and turnover of GFAP, S100, IFN-gamma and TNF-alpha proteins was evident. A clear explanation for this differential modulation is unclear, but likely result from regional differences in astrocytic/neuronal populations, BBB tightness, and/or extent/distribution of microvasculature and/or ion channels density/distribution. Such differences would respond for transient characteristics of BBB disruption. This in vivo model is useful for studies on drug delivery throughout the CNS and experimental manipulation of the BBB.
黑腹捕鸟蛛毒液（PNV）会导致不同脑区的血脑屏障（BBB）通透性不均。关于PNV诱导血脑屏障开放过程中的细胞和分子反应，我们所知甚少。我们通过免疫组织化学（IHC）和蛋白质免疫印迹法（WB），研究了在静脉注射毒液或生理盐水后的不同时间点，海马体和小脑中胶质纤维酸性蛋白（GFAP）、S100、γ干扰素（IFN-γ）和肿瘤坏死因子-α（TNF-α）蛋白的表达情况。所有蛋白质的表达在时间和区域上均有不同程度的变化。蛋白质免疫印迹法显示，在15 - 45分钟时GFAP含量增加，随后下降至对照水平以下，在海马体中这种下降不太明显。免疫组织化学显示在整个试验期间均有反应性胶质增生。在小脑中，GFAP主要在分子层的星形胶质细胞（伯格曼胶质细胞）中被免疫检测到，S100蛋白也是如此。在5小时时达到最大的S100免疫标记。主要由海马神经元表达的IFN-γ和TNF-α在试验期间增加，表明它们在血脑屏障通透性中发挥作用。在中毒动物中，在小脑中观察到星形胶质细胞-星形胶质细胞、颗粒细胞-颗粒细胞以及星形胶质细胞-浦肯野细胞之间的接触更紧密。在海马体中也观察到神经元-神经元-星形胶质细胞-星形胶质细胞之间的接触更紧密。PNV含有5-羟色胺、组胺、钙通道阻断毒素，其中一些会影响谷氨酸的释放。本文讨论了这样一种假说，即这些物质加上产生的细胞因子可能在血脑屏障通透性中起作用，并且钙稳态的丧失和谷氨酸释放的紊乱与伯格曼胶质细胞中明显的GFAP/S100反应有关。很明显存在一种中枢神经系统防御机制，对GFAP、S100、IFN-γ和TNF-α蛋白的快速合成和周转进行差异调节。对于这种差异调节的明确解释尚不清楚，但可能是由于星形胶质细胞/神经元群体、血脑屏障紧密性以及/或者微血管的范围/分布和/或离子通道密度/分布的区域差异所致。这些差异将解释血脑屏障破坏的短暂特征。这种体内模型对于研究药物在整个中枢神经系统中的递送以及血脑屏障的实验操作很有用。
