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. 2012 May 3;485(7396):99-103.
doi: 10.1038/nature10997.

Restoration of Vision After Transplantation of Photoreceptors

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Free PMC article

Restoration of Vision After Transplantation of Photoreceptors

R A Pearson et al. Nature. .
Free PMC article

Abstract

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Figures

Figure 1
Figure 1. Improved transplantation protocols significantly improve photoreceptor integration into the adult Gnat1−/− model of retinal dysfunction
a, Nrl-GFP+ rod-photoreceptor integration using new and previously published protocols; mean ± s.e.m., analysis of variance (ANOVA); n, number of eyes. b, Typical example of integrated Nrl-GFP+ rods (green). Scale bar, 50 µm. cf, Integrated Nrl-GFP+ rods expressed rod-α-transducin (c, d; red) and rod-arrestin (e, f; red) and demonstrated correct, counter-directional light-mediated translocation of these proteins (bottom panels in cf, respectively). gi, Integrated Nrl-GFP+ rods formed spherule synapses and expressed dystrophin (g), bassoon (h) and ribeye (i) (all red). Inserts, 3 µm projections of regions marked. IS, inner segments; OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Scale bar, 10 µm. j, Electron micrographs of low- (inset) and high-power views (consecutive sections), showing Nrl-GFP+ rod terminals (tT1, tT2). tT1 formed classic triad with horizontal axon terminals (H, orange) and bipolar dendritic terminal (B, red). Endogenous rod terminals (nT; blue) were DAB negative. Arrows, synaptic ribbons; scale bars, 500 nm. k, Schematic of rod-triad synapse, reproduced with permission from Webvision (H. Kolb et al., http://webvision.med.utah.edu/).
Figure 2
Figure 2. Transplanted Nrl-GFP+ rod photoreceptors are light-responsive and project light information to the visual cortex
a, Flash-response families from single dark-adapted wild type (black), bleached/regenerated wild type (red) and bleached/regenerated-Nrl-GFP+ (blue) rods. See Supplementary Information for flash intensities. b, Comparison of saturated response amplitude and dim-flash response parameters. Individual data points are shown (open circles) with mean ± s.e.m. (ANOVA, top three panels) or median ± range (Kruskal–Wallis, bottom panel); n, number of cells; s, half-saturating flash intensity. Not all parameters were obtainable for each cell. c, Representative retinal slice showing light-sensitive Nrl-GFP+ rod (red circle) and surrounding non-responsive GFP negative rods (white circles). d, Schematic of optical intrinsic imaging set-up (see Supplementary Information). Au, auditory cortex; S1, somatosensory cortex. e, Visual stimuli (Stim) (1) elicited optical signals in V1 only from Nrl-GFP+-treated Gnat1−/− (5) and wild type (2), but not untreated (3) or sham-injected (4), Gnat1−/− eyes. Far right, overlapping parts of the four stimuli were colour-coded (1). Only responses present for two overlapping stimuli were considered genuine sensory-evoked signals and represented with the corresponding colour (2–5). Scale bar, 1 mm.
Figure 3
Figure 3. Rescue of scotopic optokinetic head-tracking behaviour in Nrl-GFP-treated Gnat1−/− mice
a, The Optomotry™ set-up (see Supplementary Information). b, Measures of visual function include contrast sensitivity and visual acuity. c, d, Scotopic contrast sensitivity and visual acuity threshold measurements for Nrl-GFP-(green bars) or sham-(Gnat1−/−) treated Gnat1−/− eyes, and the averages of left and right eyes for Gnat1−/− mice receiving original protocol transplants or sham injections to both eyes, or untreated Gnat1−/− or wild type (white bars) controls. OR, optomotor response. Paired t-test. e, f, Scatter plots of contrast sensitivity and visual acuity against integrated Nrl-GFP+ rod number. g, h, Photopic contrast sensitivity and visual acuity for Nrl-GFP-(light grey) and sham-treated (dark grey) eyes before and after transplantation. Means ± s.e.m.; n, number of animals.
Figure 4
Figure 4. Nrl-GFP-treated Gnat1−/− mice can solve the visually guided water-maze task under scotopic conditions
a, Schematic of water-maze apparatus (adapted from ref. ; see Supplementary Information). Mice were trained to associate striped grating with escape from water by a hidden platform. An animal ‘passes’ a trial by crossing the red line (decision point) on the side of the divider with the striped grating. b, Pass rate of Nrl-GFP-treated (black), sham-injected (dark grey) and non-injected (mid grey) Gnat1−/− and non-injected wild-type (light grey) mice. Nrl-GFP-treated animals with a pass-rate of at least 70% are shown in green throughout. Mouse numbers in red refer to mice shown in Supplementary Movie. c, Average performance rate of all groups. d, Visual acuity and e, contrast sensitivity measurements for responders from Nrl-GFP-treated (green) and wild-type (light grey) groups. f, Swim-time latencies (time-to-platform) for all (light grey) and correct choice-only (dark grey) trials. g, Ability to solve water-maze task plotted against integrated Nrl-GFP photoreceptor number. h, Examples of integration in animals that successfully (top; Nrl-GFP-treated, number 6) or unsuccessfully (bottom; Nrl-GFP-treated, number 5) solved the task, as indicated in g (circled, red). Scale bar, 100 µm. ik, Pass rate (i), visual acuity (j) and contrast sensitivity (k) for Nrl-GFP-treated (light grey bars) and sham-injected (dark grey bars) Gnat1−/− mice before and after transplantation under photopic conditions. Means ± s.e.m.; ANOVA; n, number of animals.

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