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Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1$^{−/−}$) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1$^{−/−}$ retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1$^{−/−}$ mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

The absence of cyclic nucleotide-gated (CNG) channels in cone photoreceptor outer segments leads to achromatopsia, a severely disabling disease associated with the complete lack of cone photoreceptor function. In a common form, loss of the CNGA3 subunit disrupts visual transduction in cones and causes progressive degeneration. Here, we show that adeno-associated viral vector-mediated gene replacement therapy added the lacking sensual quality, cone-mediated vision, in the CNGA3$^{−/−}$ mouse model of the human disease. The functional rescue of cone vision was assessed at different sites along the visual pathway. In particular, we show electrophysiologically that treated CNGA3$^{−/−}$ mice became able to generate cone-mediated responses and to transfer these signals to bipolar and finally ganglion cells. In support, we found morphologically that expression of CNGA3 delayed cone cell death. Finally, we show in a behavioral test that treated mice acquired photopic vision suggesting that achromatopsia patients may as well benefit from gene replacement therapy.

Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Muller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

BACKGROUND: Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research. METHODOLOGY/PRINCIPAL FINDINGS: Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region. CONCLUSIONS/SIGNIFICANCE: The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.

BACKGROUND: Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. METHODOLOGY/PRINCIPAL FINDINGS: We achieved to adapt a commercial 3(rd) generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. CONCLUSIONS/SIGNIFICANCE: We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeut

PURPOSE: Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, we report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration. METHODS: C57BL/6 and BALB/c wild type mice and three different mouse models of hereditary retinal degeneration (Rho$^{-/-}$, rd1, RPE65$^{-/-}$) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data. RESULTS: In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRT$_{C57BL/6}$ = 237 $\pm$ 2microm and CRT$_{BALB/c}$ = 211 $\pm$ 10microm). RPE65$^{-/-}$ mice at 11 months of age showed a significant reduction of retinal thickness (CRT$_{RPE65}$ = 193 $\pm$ 2microm) with thinning of the outer nuclear layer. Rho$^{-/-}$ mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRT$_{Rho}$ = 193 $\pm$ 2microm). Examining rd1 animals before and after the onset of retinal degeneration allowed to monitor disease progression (CRT$_{rd1 P11}$ = 246 $\pm$ 4microm, CRT$_{rd1 P28}$ = 143 $\pm$ 4microm). Correlation of CRT assessed by histology and SD-OCT was high (r$^{2}$ = 0.897). CONCLUSION: We demonstrated cross sectional visualization of retinal structures in wild type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and facilitate characterization of disease dynamics and evaluation of putative therapeutic effects following experimental interventions.