A visually guided swim assay for mouse models of human retinal disease recapitulates the multi-luminance mobility test in humans.

PURPOSE: The purpose of this study was to develop a visually guided swim assay (VGSA) for measuring vision in mouse retinal disease models comparable to the multi-luminance mobility test (MLMT) utilized in human clinical trials. METHODS: Three mouse retinal disease models were studied: Bardet-Biedl syndrome type 1 (Bbs1M390R/M390R), n = 5; Bardet-Biedl syndrome type 10 (Bbs10-/-), n = 11; and X linked retinoschisis (retinoschisin knockout; Rs1-KO), n = 5. Controls were normally-sighted mice, n = 10. Eyeless Pax6Sey-Dey mice, n = 4, were used to determine the performance of animals without vision in VGSA. RESULTS: Eyeless Pax6Sey-Dey mice had a VGSA time-to-platform (TTP) 7X longer than normally-sighted controls (P < 0.0001). Controls demonstrated no difference in their TTP in both lighting conditions; the same was true for Pax6Sey-Dey. At 4-6 M, Rs1-KO and Bbs10-/- had longer TTP in the dark than controls (P = 0.0156 and P = 1.23 × 10-8, respectively). At 9-11 M, both BBS models had longer TTP than controls in light and dark with times similar to Pax6Sey-Dey (P < 0.0001), demonstrating progressive vision loss in BBS models, but not in controls nor in Rs1-KO. At 1 M, Bbs10-/- ERG light-adapted (cone) amplitudes were nonrecordable, resulting in a floor effect. VGSA did not reach a floor until 9-11 M. ERG combined rod/cone b-wave amplitudes were nonrecordable in all three mutant groups at 9-11 M, but VGSA still showed differences in visual function. ERG values correlate non-linearly with VGSA, and VGSA measured the continual decline of vision. CONCLUSION: ERG is no longer a useful endpoint once the nonrecordable level is reached. VGSA differentiates between different levels of vision, different ages, and different disease models even after ERG is nonrecordable, similar to the MLMT in humans.

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

OBJECTIVE: To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. METHODS: AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1µL) or subretinal (N = 21; 2µL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. RESULTS: The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. CONCLUSIONS: AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.

Progressive retinal degeneration of rods and cones in a Bardet-Biedl syndrome type 10 mouse model.

Bardet-Biedl syndrome (BBS) is a multi-organ autosomal-recessive disorder caused by mutations in at least 22 different genes. A constant feature is early-onset retinal degeneration leading to blindness. Among the most common forms is BBS type 10 (BBS10), which is caused by mutations in a gene encoding a chaperonin-like protein. To aid in developing treatments, we phenotyped a Bbs10 knockout (Bbs10-/-) mouse model. Analysis by optical coherence tomography (OCT), electroretinography (ERG) and a visually guided swim assay (VGSA) revealed a progressive degeneration (from P19 to 8 months of age) of the outer nuclear layer that is visible by OCT and histology. Cone ERG was absent from at least P30, at which time rod ERG was reduced to 74.4% of control levels; at 8 months, rod ERG was 2.3% of that of controls. VGSA demonstrated loss of functional vision at 9 months. These phenotypes progressed more rapidly than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. This study defines endpoints for preclinical trials that can be utilized to detect a treatment effect in the Bbs10-/- mouse and extrapolated to human clinical trials.

Correlation between electroretinography, foveal anatomy and visual acuity in aniridia due to PAX6 mutations.

BACKGROUND: Aniridia patients have poor visual acuity and iris malformation. The fovea in these patients is underdeveloped, but the relationship between structure and electrophysiologic function remains incompletely understood. This study correlates electrophysiology, visual acuity and optical coherence tomography (OCT) in patients with aniridia secondary to mutations in the PAX6 gene and compares with age-similar controls. METHODS: Patients were recruited from clinical practice. The mfERG protocol was a 4-min 103-hexagon protocol covering approximately 40° in diameter of central retina. Diagnosys full-field ERG (ffERG) and VERIS multifocal ERG (mfERG) were obtained using standard International Society for Clinical Electrophysiology of Vision protocols. OCT central thickness was recorded, and an OCT foveal score was calculated. Nonparametric permutation testing was utilized to determine the statistical significance. RESULTS: A total of 6 aniridia patients and 25 control patients were recruited. On mfERG, aniridia patients had significantly lower amplitudes in rings 1-3 (p = 0.0006, 0.0013, 0.0132), shorter latencies in ring 1 (p = 0.0312) and longer latencies in rings 5 and 6 (p = 0.0026, p = 0.0042) than controls. There was a significantly positive relationship in aniridia patients between logMAR visual acuity and mfERG amplitude in ring 4 (p = 0.0392) and ring 5 (p = 0.0489). On ffERG, there was no difference in amplitudes, though photopic 3.0 a- and b-wave latency, 30 Hz flicker latency and scotopic 0.01 b-wave latency were significantly longer in aniridia patients versus control (p = 0.0018, 0.0.0005, 1.00 x [Formula: see text], 0.0198). Thicker central macula on OCT correlated with lower mfERG amplitudes in rings 4-6 (p = 0.0369, 0.0292, 0.0255). There was no correlation between visual acuity and central macular thickness or foveal hypoplasia score as determined by OCT. CONCLUSIONS: Higher amplitude on mfERG correlated with poorer visual acuity in rings 4 and 5 in patients with PAX6 mutations. The slope of the change in amplitude from central to peripheral rings on the mfERG is significantly different in aniridia patients compared to controls, with a slower drop-off of amplitude from center to periphery. Additionally, mfERG in aniridia showed lower amplitudes than controls in rings 1-3. These changes along with the lack of correlation between visual acuity and central macular thickness/OCT score suggest that changes in electrical topography may be important to visual deficits in patients with PAX6 gene mutations.

Correlation between electroretinography, foveal anatomy and visual acuity in albinism.

BACKGROUND: Albinism patients have poor visual acuity in addition to hypopigmentation. Their foveal anatomy is abnormal, but correlation with function is incompletely understood. This study correlates retinal electrophysiology, visual acuity and optical coherence tomography (OCT) anatomy in albinism patients and compares with age-similar controls. METHODS: Institutional Review Board approval was obtained (IRB# 201408782). Patients were recruited from clinical practice. Inclusion criteria were at least three clinical features of albinism including iris transillumination, nystagmus, fundus hypopigmentation, or foveal hypoplasia on OCT and/or molecular genetic confirmation. Diagnosys (Lowell, Mass) full-field ERG (ffERG) and VERIS multifocal ERG (mfERG; Electro-Diagnostic Imaging, Milpitas, California) were obtained using standard International Society for Clinical Electrophysiology of Vision protocols. The mfERG protocol was a 4-min 103-hexagon protocol covering approximately 40° in diameter of central retina. Control subjects without albinism were recruited by in-hospital notices and invitations in clinic. OCT central thickness was recorded, and an OCT foveal score was calculated. Nonparametric permutation testing was utilized to determine the statistical significance. RESULTS: A total of 16 albinism patients and 19 age-similar controls were recruited. Four of 16 albinism patients had no nystagmus. Seventeen non-albinism controls had no ocular disorder other than refractive error. Two controls had infantile nystagmus with normal maculas on OCT. There was no statistically significant difference in mfERG amplitude or latency between albinism patients with or without nystagmus (lowest p = 0.68; 0.54, respectively). mfERG: 12 of 16 (75%) albinism patients had average ring 1 amplitudes within one standard deviation of controls despite having abnormal foveal anatomy on OCT. Patients averaged shorter latencies in rings 1 and 2 than controls (p = 0.005, p = 0.02). Patients averaged higher amplitudes than controls in rings 4, 5 and 6 (p = 0.03, p = 0.006, p = 0.004). There was no significant correlation between visual acuity and mfERG amplitudes in any ring (smallest p = 0.15). ffERG: Patients averaged higher amplitudes on 30 Hz flicker (p = 0.008). In all conditions, albinism patients had higher amplitude a-waves (p ≤ 0.03). B-waves were higher amplitude than controls in light-adapted 3.0 (p = 0.03). There was no statistical correlation between ffERG amplitudes and visual acuity (smallest p = 0.45). OCT: In albinism patients, thicker central macula on OCT correlated with lower mfERG amplitudes in all rings except for ring 1 (p < 0.05) and lower ffERG a-wave amplitudes on dark-adapted 0.01 (p = 0.003). Macular thickness on OCT did not correlate with visual acuity (p = 0.51); OCT foveal score did (p = 0.0004). CONCLUSIONS: Amplitude of mfERG does not correlate with visual acuity in any ring in patients with albinism. The slope of the change in amplitude from central to peripheral rings on the mferg is significantly different in albinism patients versus controls whether or not nystagmus is present. The decreased slope of change in amplitudes from center to periphery of the macula in albinism patients indicates changes in macular topography that are more important to visual deficits than the foveal depression.