Soares Edilene S, Stávale Leila M, Mendonça Monique C P, Coope Andressa, Cruz-Höfling Maria Alice da
Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas, Campinas, SP 13083-863, Brazil.
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, SP 13083-887, Brazil.
Int J Mol Sci. 2016 Nov 23;17(11):1462. doi: 10.3390/ijms17111462.
We have previously demonstrated that venom (PNV) causes blood-brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8-10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8-10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8-10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of .
我们之前已经证明，毒液（PNV）会导致大鼠血脑屏障（BBB）破坏、星形胶质细胞终足肿胀以及液体渗透到脑间质中。小窝和水通道通过共同参与剪切应力反应以及水肿形成/消退来应对血脑屏障的改变。在此，我们展示了腹腔注射PNV的大鼠海马中两种与血脑屏障相关的转运蛋白的产后发育相关变化：内皮小窝蛋白-1（Cav-1），它是小窝结构的主要支架蛋白；以及星形胶质细胞水通道蛋白-4（AQP4），它是在星形胶质细胞血管周围终足过程中表达的主要水通道蛋白。在产后第14天（P14）和8 - 10周龄大鼠的中毒关键时期，通过蛋白质免疫印迹法检测蛋白质水平、免疫组织化学（IHC）检测CA1、CA2和CA3亚区的蛋白质分布，并通过实时聚合酶链反应（qPCR）评估基因表达。中毒表现的强度和持续时间表明，P14新生大鼠比成年大鼠更容易受到PNV的影响。组织学上，用PNV处理的P14和8 - 10周龄大鼠的毛细血管显示血管周围水肿，而对照组则没有。P14中毒表现的强度随时间下降（2小时＞5小时＞24小时），而相反，AQP4和Cav-1的表达在24小时达到峰值，此时经PNV处理的动物在临床上与生理盐水对照组没有差异。AQP4的免疫组织化学显示，在中毒表现最明显的2小时，海马CA1的表达最少。亚区免疫组织化学定量分析显示，在P14大鼠中，Cav-1在中毒表现消失的24小时达到峰值，而在8 - 10周龄大鼠中，Cav-1在中毒症状最严重的2小时达到峰值，并逐渐减弱这种增加，直到24小时，但仍显著高于基线水平。考虑到星形胶质细胞与内皮细胞的物理和功能相互作用，我们推测AQP4和Cav-1的年龄相关调节可能与产后发育过程中星形胶质细胞功能特性的变化以及毒液诱导的血脑屏障破坏有关。
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