Development of a Post-vitrectomy Injection of N-methyl-N-nitrosourea as a Localized Retinal Degeneration Rabbit Model

Abstract

Since genetic models for retinal degeneration (RD) in animals larger than rodents have not been firmly established to date, we sought in the present study to develop a new rabbit model of drug-induced RD. First, intravitreal injection of N-methyl-N-nitrosourea (MNU) without vitrectomy in rabbits was performed with different doses. One month after injection, morphological changes in the retinas were identified with ultra-wide-field color fundus photography (FP) and fundus autofluorescence (AF) imaging as well as spectral-domain optical coherence tomography (OCT). Notably, the degree of RD was not consistently correlated with MNU dose. Then, to check the effects of vitrectomy on MNU-induced RD, the intravitreal injection of MNU after vitrectomy in rabbits was also performed with different doses. In OCT, while there were no significant changes in the retinas for injections up to 0.1 mg (i.e., sham, 0.05 mg, and 0.1 mg), outer retinal atrophy and retinal atrophy of the whole layer were observed with MNU injections of 0.3 mg and 0.5 mg, respectively. With this outcome, 0.2 mg MNU was chosen to be injected into rabbit eyes (n=10) at two weeks after vitrectomy for further study. Six weeks after injection, morphological identification with FP, AF, OCT, and histology clearly showed localized outer RD - clearly bordered non-degenerated and degenerated outer retinal area - in all rabbits. We suggest our post-vitrectomy MNU-induced RD rabbit model could be used as an interim animal model for visual prosthetics before the transition to larger animal models.

Keywords: Animal model; Intravitreal injection; N-methyl-N-nitrosourea; Optical coherence tomography; Retinal degeneration; Vitrectomy.

Fig. 1. Photograph of a rabbit during the vitrectomy procedure. Two-port 23-gauge core vitrectomy was performed with a direct biconcave lens (a, b). Light was provided from a surgical microscope and the vitreous was removed using a vitreous cutter (b).

Fig. 2. Ultra-wide-field color FP, AF, histology with H&E staining, and OCT images at one month after intravitreal injection of MNU without vitrectomy. One month after injection, no significant changes were observed in the FP, AF, histology, and OCT images of rabbit eyes with either 0.05 mg (a~d) or 0.1 mg (e~h) of injected MNU. Focal geographic hyper-AF areas (red arrows) were observed in the cases of 0.2 mg (j), 0.3 mg (n), and 0.4 mg (r) MNU injections. More severe retinal atrophy as seen in the photograph with H&E staining and OCT was induced via the 0.2 mg (k, l) and 0.4 mg (s, t) MNU injections versus with the 0.3 mg MNU injection (o, p). The green line on infrared FP shows the plane where the OCT image was harvested. Each scale bar on H&E staining (c, g, k, o, and s), infrared FP (left side image of d, h, l, p, and t), and OCT (right side image of d, h, l, p, and t) represents 100 µm, 800 µm, and 400 µm, respectively.

Fig. 3. Ultra-wide-field color FP and AF, spectral-domain OCT, and H&E staining images at one month after intravitreal MNU injection with pars plana vitrectomy. At one month after injection, no significant changes were observed in the FP, AF, OCT and H&E staining images of the rabbit eyes that received sham (a~d) or 0.05 mg (e~h) or 0.1 mg (i~l) of MNU injections after vitrectomy. Localized geographic hyper-AF areas with linear hyper-AF borders (red arrows) were observed with the 0.3 mg (n) and 0.5 mg (r) MNU injections after vitrectomy. Additionally, degenerative changes to the retina in OCT were observed in 0.3 mg (p) and 0.5 mg (t) MNU injections after vitrectomy. Loss of the photoreceptor layer and disruption of the layers of the retina were observed via H&E staining at the degeneration region with OCT (o, s). The green line on infrared FP shows the plane where the OCT image was harvested. Each scale bar on H&E staining (c, g, k, o, and s), infrared FP (left side image of d, h, l, p, and t), and OCT (right side image of d, h, l, p, and t) represents 100 µm, 800 µm, and 400 µm, respectively.

Fig. 4. Ultra-wide-field color FP, AF, spectral-domain OCT, and ERG images at six weeks after MNU (0.2 mg) injection with vitrectomy. Hyper-AF in the AF image (red arrow) and significant retinal changes in OCT at hyper-AF lesions were found (a~d). In the magnified OCT image (d, dashed-line box in Figure 4c), the borderline between the degenerated area and non-degenerated area was clearly delineated and the degeneration of the outer retina and retinal thinning were detected in the degenerated area (d, yellow dashed-line box). ERG responses were normal (e).

Fig. 5. The difference in histology with H&E staining and immunohistochemistry with PNA, Rhodopsin, PKCα, Brn3, GFAP, and TUNEL assay between the non-degenerated and degenerated areas at six weeks after intravitreal injection of 0.2 mg of MNU with vitrectomy. Histology with H&E staining showed not only the photoreceptor layer but also all layers of the retina appeared normal in the non-degenerated area (a), but loss of the photoreceptor layer was found in the degenerated area (b). In the non-degenerated area, cone and rod photoreceptor cells were normally expressed with PNA (c) and Rhodopsin staining (e), respectively. However, in the degenerated area, the cone photoreceptor cells were less expressed with PNA staining (d) and the rod photoreceptor cells were less expressed with Rhodopsin staining (f). Bipolar cells were well-expressed with PKCα staining (g, h) and ganglion cells were well-expressed with Brn3 staining (i, j) in both the non-degenerated (g, i) and degenerated areas (h, j) of the retina. Both the non-degenerated and degenerated areas showed an increase in GFAP staining (k, l), and there were no TUNEL-positive cells in either area (m, n). Next to all figures, three times enlarged figures (the part of dashed yellow box of a~n) are shown (a′~n′).

Fig. 6. Comparison of MNU injection between non-vitrectomy and vitrectomy group. Both without pars plana vitrectomy (PPV) group and with PPV group, 4 rabbits per each MNU dose were used. The exception was found with 0.2 mg MNU injection, 7 rabbits for each PPV group and non-PPV group, respectively. (a) Without vitrectomy group, 0.2 mg MNU induced significantly more retinal thinning than 0.1 mg (^***p<0.001) but there was no difference between 0.2 mg and 0.3 mg injection (not significant (n.s): p>0.05). With vitrectomy group, 0.2 mg MNU induced significantly different retinal thinning with all other doses (0.1 mg, 0.3 mg, and 0.5 mg). (b) Comparison of 0.2 mg MNU injection between non-PPV group and PPV group. In non-PPV group (n=7), significantly more retinal thinning was induced than that in PPV group (^**p<0.01). The standard deviation (S.D.) was smaller in vitrectomy group than non-vitrectomy group. (c) From the data of A, curve fitting was performed, and sigmoidal curve fit provides the best R² value (R²= 0.99) in vitrectomy group. Mean and S.D. was shown in each figure. Student-t test was performed for statistical analysis.

Fig. 7. Light-evoked RGC responses in non- and degenerated retinal areas. (a) ON RGC response of non-degenerated retinal area stimulated with full-field illumination (40 µW/cm² intensity), top: peri-event raster plot (ON: 1 s, OFF: 2 s, 30 trials), bottom: post-stimulus time histogram (PSTH) constructed from 30 trials (bin: 5 ms). (b) OFF RGC response of non-degenerated retinal area. (c) No light responses in degenerated retinal area.