RhoA Signaling and Synaptic Damage Occur Within Hours in a Live Pig Model of CNS Injury, Retinal Detachment.
RhoA Signaling and Synaptic Damage Occur Within Hours in a Live Pig Model of CNS Injury, Retinal Detachment.
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DOI：
10.1167/iovs.16-19447
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发表时间：
2016-07-01
影响因子：
4.4
通讯作者：
Townes-Anderson E
中科院分区：
文献类型：
--
作者：
Wang J;Zarbin M;Sugino I;Whitehead I;Townes-Anderson E
视网膜损伤后，RhoA通路被激活。然而，在活体内，激活的时间和后果尚不清楚。在体外研究的基础上，我们重点研究了猪视网膜脱离后2小时的一段时间，猪的视网膜是全息血管的，含有视锥细胞。在麻醉下，视网膜脱离是通过在视网膜下注射平衡盐溶液造成的。2小时后处死动物，取核进行GTP酶活性测定、Western印迹定量分析和共聚焦显微镜分析。视网膜脱离组的RhoA活性是正常眼或仅接受玻璃体切割术的眼的1.5倍。RhoA效应器肌球蛋白轻链的磷酸化也增加。2小时后，视杆细胞已将其终末向胞体收缩，破坏了光感受器到双极突触，并在视网膜外核层产生了大量带有SV2免疫标记的球体。在脱离的眼睛中，仍然附着的远端视网膜也显示出RhoA活性和突触分离的显著增加。RAC1活性和胶质纤维酸性蛋白(GFAP)的增加并不是脱离所特有的，也没有看到两极树突的萌发，据报道是由于分离时间较长。RhoA激酶抑制剂Y27632显著减少视杆细胞的轴突回缩。RhoA通路的激活在损伤后迅速发生，并促进突触损伤，这可以通过抑制RhoA激酶来控制。我们建议视网膜脱离加入中枢神经系统损伤的名单，如中风和脊髓损伤，应考虑进行快速治疗干预。
The RhoA pathway is activated after retinal injury. However, the time of onset and consequences of activation are unknown in vivo. Based on in vitro studies we focused on a period 2 hours after retinal detachment, in pig, an animal whose retina is holangiotic and contains cones. Under anesthesia, retinal detachments were created by subretinal injection of a balanced salt solution. Two hours later, animals were sacrificed and enucleated for GTPase activity assays and quantitative Western blot and confocal microscopy analyses. RhoA activity with detachment was increased 1.5-fold compared to that in normal eyes or in eyes that had undergone vitrectomy only. Increased phosphorylation of myosin light chain, a RhoA effector, also occurred. By 2 hours, rod cells had retracted their terminals toward their cell bodies, disrupting the photoreceptor-to-bipolar synapse and producing significant numbers of spherules with SV2 immunolabel in the outer nuclear layer of the retina. In eyes with detachment, distant retina that remained attached also showed significant increases in RhoA activity and synaptic disjunction. Increases in RAC1 activity and glial fibrillary acidic protein (GFAP) were not specific for detachment, and sprouting of bipolar dendrites, reported for longer detachments, was not seen. The RhoA kinase inhibitor Y27632 significantly reduced axonal retraction by rod cells. Activation of the RhoA pathway occurs quickly after injury and promotes synaptic damage that can be controlled by RhoA kinase inhibition. We suggest that retinal detachment joins the list of central nervous system injuries, such as stroke and spinal cord injury, that should be considered for rapid therapeutic intervention.