Abstract
Purpose:
Visual recovery after a retinal detachment is often not complete with more than half of patients having visual acuity ≤20/50 after reattachment surgery (Ozgur and Esgin, 2007; Ross et al., 2000). We discovered that an increase in RhoA signaling, which promotes axonal retraction by photoreceptors and consequent synaptic breakage between rod and bipolar cells, is a key to detachment injury in vitro (Fontainhas and Townes-Anderson, 2008, 2011). In this study, we used a pig model of retinal detachment to determine whether RhoA signaling is involved in retinal detachment in vivo, as an initial step to develop a therapy to stabilize synaptic connections after detachment.
Methods:
Under general anesthesia, adult female Yorkshire pigs underwent pars plana vitrectomy; retinal detachments were created by injecting balanced salt solution subretinally. The animals were kept under anesthesia for a total of 2 hrs, and then sacrificed for enucleation. Neural retinal explants from detached and non-detached retinal areas were frozen and lysed for GTPase activity assays and western blot analysis. RhoA activation was determined by a Rhotekin binding assay, while RAC1 activation was determined by a p21 activated kinase 1 binding assay. Remaining tissue was fixed for morphology.
Results:
After 2 hrs, the ratio of RhoA-GTP/RhoA-Total was increased by 180% (p<0.05) in detached retina in the operated eye compared to the corresponding area in the fellow, unoperated eye. RhoA activity in the non-detached area in the operated eye was also elevated (137%, p < 0.05). Vitrectomy alone did not cause elevation of RhoA activity. The phosphorylation of myosin light chain II (MLCII), which is responsible for actomyosin contraction, and the activity of RAC1 increased significantly in the detached retina by 147% and 62%, respectively. Rod axonal retraction was observed after 2 hrs in both detached and non-detached areas of the operated eye.
Conclusions:
The activity of RhoA and the phosphorylation of MLC II, a downstream effector of RhoA signaling, are increased after 2 hrs of retinal detachment in vivo, concomitant with the axonal retraction by rod photoreceptors. Increases in RAC-1 activity may also contribute to the rod synaptic rearrangement. These findings suggest that blocking RhoA signaling may prevent the deleterious synaptic breakage between rod and bipolar cells after detachment in vivo.
Keywords: 697 retinal detachment •
648 photoreceptors •
650 plasticity