A Spontaneous Nonhuman Primate Model of Myopic Foveoschisis

Abstract

Purpose: Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis.

Methods: We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene.

Results: Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected.

Conclusions: This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.

Figure 1.

Clinical examination and multimodal imaging of NHP foveoschisis. (A) Scatterplots of refractive errors and axial lengths of adult rhesus macaques at the California National Primate Research Center that underwent streak retinoscopy and A-scan biometry, separated by sex, including the affected animal (asterisk). (B) Boxplots of average vitreous length, anterior chamber (AC) depth, and lens thickness comparing the affected animal (asterisk) to other adult macaques. (C) External photographs, (D) color fundus photographs (FP), (E) infrared reflective (IR), (F) late-phase fluorescein angiography (FA), (G) blue-peak fundus autofluorescence (AF), and (H) spectral-domain optical coherence tomography (OCT) imaging of left and right eyes of the affected macaque at presentation at 27 years of age, and (I) repeat OCT imaging of the same animal 14 months later at time of necropsy. For comparison, representative OCT images from (J) a normal age-matched macaque, (K) a 7-year-old boy with X-linked retinoschisis, and (L) a 23-year-old woman with -15-diopters pathologic myopia and myopic traction maculopathy. Abbreviations: OD, right eye; OS, left eye. Scale bars, 200 µm to 1 mm.

Figure 2.

Retinal glial and photoreceptor morphology in NHP foveoschisis. Confocal fluorescence images of (A–F) glial fibrillar acidic protein (GFAP)-stained macroglia (green) and IBA-1+ microglia (red), or (G–L) rhodopsin+ rod (red) and M/L-opsin+ cone photoreceptors (green), along with DAPI (blue) to label cell nuclei, located at the foveal or parafoveal region A, D, G, and J, with magnified views of the dashed-box regions shown in B, E, H, and K, and peripheral retina C, F, I, and L in the rhesus macaque affected by foveoschisis A to C and G to I and representative age-matched control animal D to F and J to L. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; and RPE, retinal pigment epithelium. Scale bars, 100 to 500 µm.

Figure 3.

Single-nuclei RNA sequencing analysis of retinal tissues in NHP foveoschisis. (A) Identified cluster of single-nuclei expression profiles from the affected rhesus macaque and three control macaque samples. (B) Bar graphs comparing distribution and proportion of the seven cell types in the foveoschisis animal compared to three controls. (C) Volcano plots demonstrating significant DEGs comparing the affected to control macaques (adjusted P < 0.001 and fold change >2) with a bar graph summarizing the total number of DEGs for each retinal cell types. (D) Top GO term enriched by each DEG lists from the identified retinal cell types. Abbreviations: ACs, amacrine cells; BCs, bipolar cells; HCs, horizontal cells; MG, Müller glia; and RGCs, retinal ganglion cells.

Figure 4.

RS1 gene and expression in the retina of NHP with foveoschisis. (A) Table of gene variants identified in the introns and 3′ UTR of the RS1 gene from the rhesus macaque with foveoschisis. (B) Pedigree showing the affected animal (shaded) with its relatives (squares = males, circles = females, crossed lines = deceased, and question mark = not examined). (C) Violin plots from snRNA-seq comparing RS1 expression in the affected macaque and 3 control animals showing RS1 expression mostly in rods and cones. (D) Confocal fluorescence images of the eye with foveoschisis and a 23-year-old control animal stained with antibodies against RS1 (red) and DAPI (blue) to label the nuclei. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bars, 100 µm.