Rearrangement of the cone mosaic in the retina of the rat model of retinitis pigmentosa

Abstract

In retinitis pigmentosa (RP), the death of cones normally follows some time after the degeneration of rods. Recently, surviving cones in RP have been studied and reported in detail. These cones undergo extensive remodeling in their morphology. Here we report an extension of the remodeling study to consider possible modifications of spatial-distribution patterns. For this purpose we used S334ter-line-3 transgenic rats, a transgenic model developed to express a rhodopsin mutation causing RP. In this study, retinas were collected at postnatal (P) days P5-30, 90, 180, and P600. We then immunostained the retinas to examine the morphology and distribution of cones and to quantify the total cone numbers. Our results indicate that cones undergo extensive changes in their spatial distribution to give rise to a mosaic comprising an orderly array of rings. These rings first begin to appear at P15 at random regions of the retina and become ubiquitous throughout the entire tissue by P90. Such distribution pattern loses its clarity by P180 and mostly disappears at P600, at which time the cones are almost all dead. In contrast, the numbers of cones in RP and normal conditions do not show significant differences at stages as late as P180. Therefore, rings do not form by cell death at their centers, but by cone migration. We discuss its possible mechanisms and suggest a role for hot spots of rod death and the remodeling of Müller cell process into zones of low density of photoreceptors.

Figure 1

Confocal micrographs taken from vertical sections of retinas processed for M-opsin immunoreactivity. The micrographs are for P30 N (A), P15RP (B), P30RP (C), and P90 RP (D). In P30 N retinas, entire M-opsin-immunoreactive cones are labeled. In P15 RP retinas, the OS are distorted in orientation (arrow). In P30 RP retinas, M-opsin immunoreactive cones are shortened in length and show disorganized axon terminals (C). In P90 RP retinas, M-opsin-immunoreactive cones are positioned “flat” against the outer part of the INL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, Inner nuclear layer; OS, outer segment; N, normal; RP, retinitis pigmentosa. Scale bars = 20 μm.

Figure 2

Confocal micrographs taken from whole mounts processed for M-opsin and S-opsin immunoreactivities. Low-power micrographs illustrate the homogeneous distributions of M-opsin (A) and S-opsin (B) cones in P90 normal retina. Double exposure (C) demonstrates no colocalization of M-opsin and S-opsin immunoreactivity. Low-power micrographs show that M-opsin (D) and S-opsin (E) cones in P90 RP retinas exhibit spatial organizations in matrices of rings. Double exposures (F) demonstrates that both types of cones form rings at the same locations in the RP retinas. High-power micrographs of part of a ring marked with inset rectangles in D–F, are shown in G–I, respectively. The orientation of M-opsin (G) and S-opsin (H) immunoreactive cones in rings are shown. Double exposures (I) demonstrates the same orientation of M-opsin and S-opsin cones. Scale bars = 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Confocal micrograph taken from P30 (A–C) and P90 (D–F) whole mount RP retinas processed for M-opsin immunoreactivities showing rings in their distribution (A,D). Light micrograph taken at the same retinal location under DIC mode shows no retinal folds (B,E). Double exposure (C,F) confirms no retinal folds are associated with rings. Light micrographs taken from RP vertical retinas processed with hematoxylin staining (G–J). At P15, the thickness of the ONL is uniform (G). H: Higher-power micrograph of G is shown. At P30, the ONL show “grooves.” J: Higher-power micrograph of groove is shown. No nuclei are visible at the trough of the groove (arrow). Confocal micrograph taken from P30 whole mount RP retina processed with M-opsin antibody (red) and TOPRO-3 (blue). Nuclei at the center of the ring are not in the same focal plane as M-opsin-immunoreactive cones (K). L: Higher-power micrograph of K is shown. DIC, differential interference contrast; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 100 μm in A–J; 50 μm in K,L. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Confocal micrograph taken from P15 whole mount RP retinas processed for M-opsin (A), rhodopsin (B) immunoreactivities and for apoptotic cells (C). Triple exposure (A–C) indicates a cluster of apoptotic cells inside the ring. Where rings are not observed, apoptotic cells are scattered randomly. Scale bar = 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Confocal micrograph taken from whole mount RP retina processed for M-opsin (red) and S-opsin (green) immunoreactivities at P15 (A,B), 30 (C,D), 180 (E,F), and P600 (G,H). Double exposure shows a ring of M-opsin and S-opsin immunoreactive cone at P15 (A). A higher-power micrograph of a ring is shown (B). It illustrates the change of orientation of cones; starting to lie flat with their processes pointing toward the center of the ring. Many rings are visible by P30 (C). D: Higher-power micrograph of a ring is shown (C). All the COS and the cell bodies are near the rims of the rings, whereas the processes are pointing toward the center of the ring. At P180, rings start to lose their form (E). Higher-power micrograph of a part of what probably used to be a ring reveal M-opsin- and S-opsin-immunoreactive cones are no longer organized in the previously observed orientation (F). A lot of cones show growth of abnormal processes and loss of their OS. At P600, rings are no longer clear (G). Higher-power micrograph illustrates the cones' extensive growth of processes and their loss of OS (H). Scale bars = 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Composite image of confocal micrographs taken from the whole mount RP retinas processed for M-opsin immunoreactivities at P30 (A) and at P90 (B). At P30 there are comparatively more rings in the dorso-peripheral region of the retina. At P90 rings are seen throughout the entire retina. A graph of the mean total number of rings versus retinal regions of the P30 RP retinas (n = 3) suggest significantly greater number of rings in the dorsal region of the retinas compared to the ventral region (C). A graph of mean total number of rings versus postnatal age (n = 3; D) and a graph of mean diameter of rings (μm) versus postnatal age (n = 2; E) indicate rings grow both in their number and size from P30 to P90. Data presented as mean ± standard error. *P < 0.005 or better. Scale bars = 1 mm.

Figure 7

A graph of mean total number of immunoreactive cells versus postnatal age (A) shows no significant differences between the normal and the RP retinas at P30 (n = 2) and P180 (n = 3). Significant reduction is seen in P600 RP retinas (n = 3) for both M-opsin- and S-opsin-immunoreactive cone counts. A graph of RP retinal area (mm²) versus postnatal age indicates growth of retina in size with age (B: P30, n = 3; P180, n = 2; P600, n = 3). There are no significant differences in the retinal area between the normal and the RP retinas. Confocal micrographs taken from whole mount heterozygous (left) and homozygous (right) RP retinas processed for M-opsin immunoreactivities (C). Both show the same morphology, arrangement, and orientation of M-opsin-immunoreactive cones—the COS forming the rim of the ring and their processes in the inside of the ring. The densities of M-opsin-immunoreactive cones cell bodies in the dorsal wing of P180 heterozygous and homozygous RP retinas (n = 4 each) indicated no significant difference (D). Data presented as mean ± standard error. *P < 0.005 or better. Scale bar = 100 μm.