Repair of the degenerate retina by photoreceptor transplantation

Abstract

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6β(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

Fig. 1.

Photoreceptor integration is dependent upon recipient disease type. (A, Left axis, box plots) number of integrated rods 3 wk after transplantation into 6- to 8-wk-old models of retinal degeneration, compared with wild-type controls. n = number of eyes. Black bars: statistical significance (ANOVA with Tukey’s correction). Right axis (black dots), recipient ONL thickness at 6–8 wk (n = 3 per model). Black asterisks: statistical significance. (B) Representative images of integrated cells in each model. (Scale bar, 25 μm.) Dotted line denotes boundary of ONL/INL, dashed line denotes boundary of ONL. ONL, outer nuclear layer; INL, inner nuclear layer.

Fig. 2.

Morphology of integrated rods is influenced by recipient retinal environment. (A and B) Percentage of integrated rods that develop outer segments (A) and presynaptic-like structures (B) (n = 3 or more per model; ANOVA with Tukey’s correction). (C and D) Typical morphology, outer-segment length (C) and presynaptic-bouton formation (D) of integrated cells. (C) Integrated cells expressed the rod-specific transcription factor Nrl (green), rod α-transducin (C, ii), peripherin-2 (C, v), rhodopsin (C, vi), or β-PDE (C, vii) (red), as appropriate; such markers are absent in the respective endogenous photoreceptors. (D) Most (arrowheads) but not all colocalized with RIBEYE (red). Dotted line in vii denotes ONL/INL boundary. (Scale bar, 25 μm.)

Fig. 3.

Disease progression significantly but differentially affects photoreceptor transplantation efficacy according to disease type. (A) Black: impact of disease progression upon transplantation outcome, compared with wild-type. n = number of eyes examined. ANOVA with Tukey’s correction. Blue: linear regression denotes integration trend. Note that changes in Crb1^rd8/rd8 retinae were bimodal (shown as dashed line). (B) Gliosis in early and late degeneration, as assessed by CSPG (green) and GFAP (red) expression. (C) OLM integrity in early and late degeneration, as assessed by ZO-1 (red) expression. Disturbances in OLM integrity indicated by white arrows. (Scale bar, 50 µm.) (D) Trend correlations (nonquantitative) for integration (black), OLM integrity (red), and gliosis (green).

Fig. 4.

Manipulation of OLM and gliosis significantly increases integration and permits restoration of visual function. (A) impact of OLM disruption (using ZO-1 siRNA) and CSPG degradation (using ChABC), singularly and combined, on transplantation outcome in Rho^−/− and wild-type recipients. n ≥ 7 per condition; ANOVA with Tukey’s correction. (B) Contrast sensitivity against number of integrated rod-photoreceptors in subset (n = 7) of Rho^−/− recipients that underwent optomotor testing 3–4 wk after receiving combined treatment.