RhoA Signaling and Synaptic Damage Occur Within Hours in a Live Pig Model of CNS Injury, Retinal Detachment

Abstract

PurposeThe 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.MethodsUnder 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.ResultsRhoA 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.ConclusionsActivation 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.

Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment

“…15 Sections were immunolabeled for SV2 (Developmental Studies Hybridoma Bank, Iowa City, IA) with secondary antibodies conjugated to Alexa Fluor 488 (Life Technologies, Norwalk, CT), followed by propidium iodide (PI; 1 μg/mL; Sigma-Aldrich Corp., St. Louis, MO) to stain nuclei. Labeled sections were covered with Fluoromount-G medium (SouthernBiotech, Birmingham, AL) and preserved under coverslips sealed with nail polish.…”

ROCK inhibitors in ocular disease

“…One and 10 mM of Y27632 and 10 mM of fasudil achieved significant prevention of rod axonal remodeling in the detached area of the treated eye compared to the detached area of the fellow non-treated eye. In this model Y27632 also seemed to be more effective than fasudil: 10 mM Y27632 had significant effect on prevention of synaptic changes in both detached and non-detached retinal areas, compared to the control areas from the drug-free eye, whereas fasudil prevented synaptic retraction only in the detached area of retina [99], [unpublished data]. In vitro findings also suggest that other kinases in the RhoA/ROCK pathway, specifically Lim kinase (LIMK), play a role in structural remodeling of photoreceptors and could be future therapeutic targets after retinal detachment [98].…”

“…However, rods maintained their morphology and showed no axonal retraction with 100 µM Y27632 pretreatment [97] and in similar conditions with even lower concentrations (10, 30, 100 µM) in a dose-dependent manner [98]. An in vitro retinal detachment model in porcine eyes demonstrated that RhoA activity increased immediately after detachment [94,99]; recent experiments, therefore, have explored short time periods after the detachment injury. Porcine retinal explants treated with 1, 10 and 100 µM of Y27632 significantly reduced the number of the rod synaptic terminals which retracted into the outer nuclear layer, moreover six hour-delayed administrations of 100 µM Y27632 also reduced synaptic breakage [94].…”

Concise Review: Update on Retinal Pigment Epithelium Transplantation for Age-Related Macular Degeneration

“…In addition, Wang et al found that rod‐bipolar synaptic disjunction could be reduced with subretinal administration of a ROCK inhibitor (Fig. ) . Townes‐Anderson et al reported that subretinal injection of fasudil, a ROCK inhibitor that is approved for clinical use for a different indication, also reduces rod‐bipolar synaptic disjunction .…”

“…Finally, there was also a significant reduction in labeled pixels in YD compared with that in YC by 24.4% (**, p = .009; n = 48 retinal sections; 16 samples; four pigs). Reproduced with permission from Wang et al .…”