Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors

Abstract

Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.

Fig. 1

The outer limiting membrane. (a) Semithin section of a wildtype mouse retina, stained with Toluidine Blue showing the location of the outer limiting membrane (OLM). Scale bar, 50 μm. Electron micrograph of the highlighted area shows the electron dense adherens junctions (black arrow head) that form the OLM (insert below). (b) Single confocal image of a retinal section from an Nrl.gfp (green) mouse, stained for zonula occludins-1 (ZO-1; red), an adherens junction protein. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 20 μm. (c) Schematic diagram illustrating the adherens junctions (red) that form the OLM, between the cells of the mammalian retina (green photoreceptors; grey Müller cells; blue nuclei). An enlargement of an adherens junction demonstrates the presence of the actin binding protein ZO-1 at the OLM (insert). (d) The chemical structure of alpha-aminoadipic acid (AAA) showing its similarity to glutamate. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium.

Fig. 2

The effect of AAA on adult wildtype retinal morphology. Light images of Haemotoxylin and Eosin (H&E) stained retinal sections from wildtype mice treated with 20 μg, 100 μg and 320 μg of AAA. Mice were sacrificed at 6 h, 24 h and 3 weeks post AAA intravitreal injection. (a, d, g) Vacuoles were present at 6 h post AAA administration in all doses (black arrow heads). (b, c) The retina recovered normal morphology by 24 h, post 20 μg AAA injection. (e, h) Displaced photoreceptor cell bodies suggested outer limiting membrane disruption at 24 h, post 100 μg and 320 μg AAA administration (white arrow heads). (f) Recovery of the retina was observed 3 weeks post 100 μg AAA injection, with a few remaining vacuoles between the outer segments (white arrow). (i) Disruption of the inner limiting membrane (black arrow), loss of retinal layers and degeneration of the photoreceptor inner/outer segments (white arrow), was present 3 weeks post 320 μg AAA injection. Highlighted sections are shown magnified to the right. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane; IS/OS, inner and outer segments; RPE, retinal pigment epithelium. Scale bars, 20 μm.

Fig. 3

Intravitreal administration of AAA causes transient disruption of the outer limiting membrane. Single confocal images of retinal sections from Nrl-GFP (green) (a) and wildtype (b, c) mice treated with AAA (100 μg) 24, 48, 72 h, 1 and 2 weeks prior to sacrifice. (a) Images show sequential displacement of photoreceptors towards the subretinal space (white arrowheads). The peak of disruption occurred at 72 h and ceased after 1 week post AAA intravitreal injection. (b) Sequential staining for ZO-1 (red), an adherens junction protein, demonstrates some outer limiting membrane disruption at 24 and 48 h, and maximal disruption at 72 h post AAA administration (white arrows). (c) Apoptotic cells, demonstrated by sequential TUNEL staining (red; white horizontal arrows). Nuclei were counterstained with Hoechst 33342 (blue). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OLM, outer limiting membrane; IS/OS, inner and outer segments. Scale bars, 20 μm.

Fig. 4

Ultrastructural recovery of the outer limiting membrane 1 week post AAA administration. Electron micrographs of retinal sections from PBS and AAA treated wildtype mice, sacrificed at 72 h (a, b) and 1 week (c) post intravitreal injection. (a) Normal retinal structure was observed in the PBS treated retina and continuous electron dense adherens junctions were present (insert). (b) Disrupted retinal lamination was observed at 72 h post AAA administration. Mislocalized photoreceptors (black arrow heads) and vacuoles (white arrow heads) were present in the segment layer, and a lack of adherens junctions was seen (black arrows and insert). (c) Relatively normal retinal structure was observed 1 week post AAA administration. A few vacuoles remained in the segment layer (white arrow heads) and almost continuous adherens junctions were present (insert). Contrast stained. ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Scale bars, 5 μm; insert scale bars, 2 μm.

Fig. 5

Integration of photoreceptor precursors into retinas with disrupted outer limiting membranes. (a) Number of integrated cells in retinas treated with PBS control or AAA, 72 h and 1 week, prior to cell transplantation (N = 9 for each timepoint, **P = 0.009 two-tailed paired t-test). (b) Normalized fold-difference in the number of integrated cells for the paired PBS versus AAA treated eyes, 72 h and 1 week prior to cell transplantation. (c,d) Confocal projection images of integrated photoreceptors (Nrl.gfp; green) in wildtype adult retinas, treated with PBS, AAA or a non-treated control, 72 h and 1 week, prior to cell transplantation. Nuclei were counterstained with Hoechst 33342 (blue). Nomarski images are also shown. INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 10 μm.