Abstract
PurposeRetinal detachment disrupts the rod-bipolar synapse in the outer plexiform layer by retraction of rod axons. We showed that breakage is due to RhoA activation whereas inhibition of Rho kinase (ROCK), using Y27632, reduces synaptic damage. We test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment.MethodsDetachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). In some animals, fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours.ResultsSubretinal injection of fasudil (10 mM) reduced retraction of rod spherules by 51.3% compared to control detachments (n = 3 pigs, P = 0.002). Intravitreal injection of 10 mM fasudil, a more clinically feasible route of administration, also reduced retraction (28.7%, n = 5, P < 0.05). Controls had no photoreceptor degeneration at 2 hours, but by 4 hours apoptosis was evident. Fasudil 10 mM reduced pyknotic nuclei by 55.7% (n = 4, P < 0.001). Phosphorylation of cofilin and myosin light chain, downstream effectors of ROCK, was decreased with 30 μM fasudil (n = 8–10 explants, P < 0.05).ConclusionsInhibition of ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment.Translational RelevanceThese results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.
Highlights
Detachment is well-known to affect synapses in the outer plexiform layer (OPL).[1,2] Synaptic injury begins with retraction of the rod presynaptic terminals towards their cell bodies
Examining first isolated salamander photoreceptors in culture, retinal explants from adult pigs, and iatrogenicallyproduced detachments in living pigs, we demonstrated that activation of the RhoA signaling pathway leads to axonal retraction by rod cells (Fig. 1A).[13,14,15]
ROCK inhibition with Y27632 reduced the amount of retraction observed in the outer nuclear layer (ONL) by rod photoreceptors in vitro and in vivo.[13,14,15]
Summary
Detachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). Fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours
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