Oral Selumetinib Does Not Negatively Impact Photoreceptor Survival in Murine Experimental Retinal Detachment

Abstract

Purpose: Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling is neuroprotective in some retinal damage models but its role in neuronal survival during retinal detachment (RD) is unclear. In addition, serous RDs are a prevalent side effect of MEK inhibitors (MEKi), blocking MAPK/ERK signaling for treatment of certain cancers. We tested the hypothesis that MEKi treatment in experimental RD would increase photoreceptor death.

Methods: The MEKi selumetinib was delivered daily to C57BL/6 mice at a clinically relevant dose (10 mg/mL) starting 1 day prior to creating RD with subretinal hyaluronic acid injection. Photoreceptor TUNEL and outer nuclear layer (ONL) thickness were analyzed. Phospho-ERK1/2 (pERK) distribution, glial fibrillary acidic protein (GFAP) accumulation, and Iba-1 (microglia/macrophages) were evaluated with immunofluorescence.

Results: pERK accumulated in the Müller glia in detached retinas, but this was effectively blocked by selumetinib. Selumetinib did not induce serous RDs at day 1 and did not increase TUNEL positive photoreceptors or further decrease ONL thickness compared to controls. Retinal gliosis was not altered, but selumetinib did block the increase in intraretinal microglia/macrophage Iba-1 fluorescence intensity and acquisition of amoeboid morphology.

Conclusions: MAPK/ERK is neuroprotective in some retinal damage models; in RD, selumetinib blocked Müller pERK accumulation and changed the retinal microglia/macrophage phenotype but did not alter photoreceptor survival. This is consistent with the relatively good visual acuity seen in patients developing transient retinal detachments on MEK inhibitor therapy. Compensation by other neuroprotective pathways in the retina during retinal detachment may occur in the presence of MEK inhibition.

Figure 1

Selumetinib (AZD6244) jelly pellet fabrication. Identical shape and volume (100 μl) of strawberry flavored selumetinib jelly pellets were fabricated on the engraved parafilm sheet cast on a 1000 μl micropipette tip rack.

Figure 2

Selumetinib (AZD6244) blocks the increased accumulation of Müller pERK in RD. (A) Representative images of immunofluorescence for pERK1/2 (red) and Müller marker Sox2 (green) with DAPI counterstain for nuclei (blue) for day 3 (n = 6 mice/group) and day 7 (n = 5 mice/group) RD and fellow eye controls. pERK accumulates in the detached Müller endfeet in the GCL. The insert areas to the right highlight the colocalization of pERK1/2 and Sox2 accumulation in the INL; pERK was significantly increased after RD compared to control (P = 0.0960 day 3; P = 0.0025 day 7). pERK1/2 accumulation was blocked in Müller glia in RD eyes of selumetinib treated mice at day 3 (P = 0.0040) and day 7 (P = 0.0050) compared to vehicle treated eyes. Scale bar denotes 50 microns. (B) Quantitation of retinal pERK1/2 intensity *P < 0.05.

Figure 3

Selumetinib (AZD6244) does not increase cell death in detached photoreceptors. Representative day 3 RD images with TUNEL positive cells (red) counterstained with DAPI (blue) (A–D). There was no significant increase in TUNEL positive photoreceptors in RD eyes treated with selumetinib (D) compared to vehicle (B). Quantitation (E) showed no difference (P = 0.5192, n = 6 mice/group). (F) There was no difference in the ONL thickness ratio at day 14 between the selumetinib and vehicle treated RD groups (P = 0.450, n = 8 mice/group). Scale bar denotes 50 microns.

Figure 4

Oral selumetinib (AZD6244) does not significantly impact increased GFAP accumulation in RD. Immunofluorescence staining shows GFAP accumulation (green) after RD (B, D) compared with attached controls (A, C) at day 7 (n = 6/group). (E) Treatment with selumetinib does not significantly reduce GFAP staining in detached retina (P = 0.881). Scale bar denotes 50 microns.

Figure 5

Oral selumetinib (AZD6244) reduces Iba-1 intensity and amoeboid morphology of microglia RD. Confocal images of immunofluorescent staining for Iba-1 (green) in microglia/macrophages shows more amoeboid morphology in vehicle treated RD versus control mice (A, B). Selumetinib treatment (C, D) resulted in less amoeboid and more tamified microglia/macrophage morphology in RD compared with vehicle treatment (B). The intensity of fluorescence signal was measured resulting in a significant increase in vehicle treated RD eyes compared to control (P = 0.0222; E) and a significant decrease in AZD-treated RD eyes compared to vehicle-treated RD eyes (P = 0.0306; E). Iba-1 positive cell counts per mm² were significantly increased in RD eyes compared to controls in both selumetinib- and vehicle-treated eyes (P = 0.0291 and P = 0.00009, respectively; F) and higher in vehicle compared with selumetinib treated controls (P = 0.0276; F). Contingency table of microglia morphologic grade (G) showed a significant increase in amoeboid morphology (grade 3) in vehicle treated RD compared to selumetinib treated RD (P = 0.0086). Cell morphology was also determined using the form factor 1 (FF1) value, resulting in a significant decrease of amoeboid cells in selumetinib treated eyes (P = 0.0357; H). Scale bar denotes 50 microns.