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

Ellen Townes-Anderson, Jianfeng Wang, Éva Halász, Ilene Sugino, Amy Pitler, Ian Whitehead, Marco Zarbin, Ellen Townes-Anderson, Jianfeng Wang, Éva Halász, Ilene Sugino, Amy Pitler, Ian Whitehead, Marco Zarbin

Abstract

Purpose: Retinal 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.

Methods: Detachments 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.

Results: Subretinal 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).

Conclusions: Inhibition of ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment.

Translational relevance: These results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.

Keywords: ROCK inhibition; RhoA signaling; photoreceptor synapse; retinal detachment; synaptic plasticity.

Figures

Figure 1
Figure 1
(A) Proposed mechanism for fasudil inhibition of photoreceptor axonal retraction. Fasudil inhibits ROCK, which reduces LIMK activity and phosphorylation of cofilin and MLC. Phosphorylation of cofilin and MLC cause actin turnover and actomyosin contraction, which leads to axon retraction. (B, C) Location of iatrogenic retinal detachments and sample collection. Retinal detachments were created in the inferior nasal quadrant after vitrectomy. Samples from the yellow square areas were used for morphological anyasis. (B) Control eye. BD, detached area, detached with subretinal injection of BSS; BC, attached area. (C) Treated eye. FD, detached area, detached with subretinal injection of fasudil in BSS; FC, attached area; T, temporal; N, nasal; S, superior; I, inferior; OD, optic disc.
Figure 2
Figure 2
Axonal retraction by rod photoreceptors after 2 hours of detachment. (A) The normal retina, labeled for synaptic membrane protein (SV2, green) and nuclei (stained with PI, red). SV2 label is present in the OPL and IPL. (B) Two hours after detachment SV2-immunolabel appears in the ONL. This eye received a subretinal injection of 1 mM fasudil during creation of the detachment. SV2-labeled spots (white arrows) in the ONL indicate axonal retraction. Other retinal layers appear normal. INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 3
Figure 3
Effect of subretinal injection of 10 mM fasudil on axonal retraction by photoreceptors 2 hours after retinal detachment. (A) Representative images of retina labeled for SV2 (green) and nuclei (red). Control eye: attached (BC) and detached (BD) retinae. Treated eye: attached (FC) and detached (FD) retinae treated with 10 mM fasudil. SV2-labeled spots (white arrows) in the ONL indicate axonal retraction. (B) Binary images created from the green channel from images in (A). SV2-labeled spots are indicated with red arrows. (C) Comparison of SV2-labeled pixels/μm ONL length in different retinal areas. There was a significant reduction of SV2-labeled pixels, by 51.3%, in FD (29.2 ± 2.7 pixels/μm ONL) compared to BD (60.7 ± 2.7 pixels/μm ONL) indicating a reduction of axon retraction (**P = 0.002, n = 3 animals, 300 images, ± SD). There was no significant difference between BC and BD in SV2-labeled pixels.
Figure 4
Figure 4
Effect of intravitreal injection of fasudil on axonal retraction by photoreceptors 2 hours after retinal detachment. (A) Representative images of retina labeled for SV2 (green) and nuclei (red). Control eye, detachment made with BSS; treated eye, detachment made with BSS, fasudil injected immediately afterward intravitreally to achieve a 10 mM concentration. SV2-labeled spots (white arrows) in the ONL indicate axonal retraction. (B) Binary images created from the green channel from (A). SV2-labeled spots indicated with red arrows. (C) Comparison of the average of attached and detached areas between BSS control and fasudil-treated eye. The axonal retraction was significantly reduced by 28.7% in fasudil-treated eyes compared to BSS (*P < 0.05, n = 5 animals, 500 images, ± SD).
Figure 5
Figure 5
Levels of phosphorylated cofilin (p-cofilin) and MLC (pMLC) after 2 hours of treatment with fasudil (30 μM) in detached retinal explants. (A, B) Typical Western blots using antibodies against p-cofilin, cofilin, pMLC, MLC, and GAPDH. (C) The ratio of p-cofilin/cofilin-Total was reduced significantly with fasudil treatment by 25.8 ± 26.8% (*P < 0.05, n = 5 eyes, 10 explants, ± SD), while total cofilin (normalized by GAPDH) did not change. (D) The ratio of pMLC/MLC-Total was significantly reduced by 23.2 ± 8.2% with fasudil (**P < 0.05, n = 4 eyes, 8 explants, ± SD), while total MLC (normalized by GAPDH) did not change significantly.
Figure 6
Figure 6
Rod cell degeneration in the detached retina after a 4-hour detachment. Fasudil (10 mM) was injected intravitreally 2 hours after the detachment was created; samples were collected an additional 2 hours later. Nuclei were stained with PI (red); synaptic terminals were labeled for SV2 (green). (A, B) Representative sections of retina from untreated (BSS) and treated (Fasudil) eyes. Densely stained (pyknotic) nuclei are present in the ONL of the untreated detached retina (arrowheads) where rod cell bodies reside. (C) Treatment with fasudil significantly reduced the number of densely stained nuclei in the ONL by approximately 55.7% compared to controls (BSS, 14.1 ± 3.5; fasudil, 6.3 ± 3.1 nuclei/mm ONL; ***P < 0.001, n = 4 animals, 400 images, ± SD).

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