Retinal safety evaluation of photoacoustic microscopy.

Photoacoustic microscopy (PAM) has significant potential as a promising diagnostic method for eye diseases and can provide anatomic and functional information of the retinal and choroidal vasculature. However, there are no FDA-approved PAM systems for ophthalmic imaging. In this study, a comprehensive safety evaluation was performed to evaluate the safety of PAM retinal imaging and whether PAM causes damage to retinal structure or function in rabbit eyes. 12 Dutch-Belted pigmented rabbits received photoacoustic imaging to 57% of the retinal surface area with a laser energy of 5% of the ANSI safety limit for five consecutive days and followed before imaging and 3 days, 1, 2, 3, and 4 weeks post imaging. Retinal morphologic analyses using slit lamp examination, fundus photography, red free, FA, FAF, ICGA, and OCT showed no retinal hemorrhage, edema, detachment, vascular abnormalities, or pigmentary abnormalities in the retina or choroid after PAM imaging. Full-field ERG analysis showed no significant difference in scotopic or photopic a- and b-wave amplitudes or implicit times between the control and experimental eyes over time (n = 6, P values > 0.05). Retinal ultrastructural evaluation using TEM showed normal structure of organelles and nuclei, and no significant loss of cells after PAM. TUNEL assay showed no evidence of cells apoptosis in retina. Retinal histopathology indicated that the architecture and thickness of the retinal layers was well preserved in all experimental eyes. A positive control at 500% of the ANSI limit demonstrated significant damage. The comprehensive retinal safety evaluation demonstrated no damage to retinal structure or function for 4 weeks after PAM imaging in rabbits.

Characterization of a canine model of autosomal recessive retinitis pigmentosa due to a PDE6A mutation.

PURPOSE: To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. METHODS: Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry, and assay for retinal PDE6 levels and enzymatic activity. RESULTS: The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments stopped, and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells, which appeared to be reduced in number relatively early in the disease process, other inner retinal cells were preserved in the early stages of the disease, although there was marked and early activation of Müller glia. Western blot analysis showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is essential for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. CONCLUSIONS: This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa, and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face of a rapid loss of rod cells.

Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors.

Neural developmental programs require a high level of coordination between the decision to exit cell cycle and acquisition of cell fate. The Maf-family transcription factor NRL is essential for rod photoreceptor specification in the mammalian retina as its loss of function converts rod precursors to functional cones. Ectopic expression of NRL or a photoreceptor-specific orphan nuclear receptor NR2E3 completely suppresses cone development while concurrently directing the post-mitotic photoreceptor precursors towards rod cell fate. Given that NRL and NR2E3 have overlapping functions and NR2E3 expression is abolished in the Nrl(-/-) retina, we wanted to clarify the distinct roles of NRL and NR2E3 during retinal differentiation. Here, we demonstrate that NRL binds to a sequence element in the Nr2e3 promoter and enhances its activity synergistically with the homeodomain protein CRX. Using transgenic mice, we show that NRL can only partially suppress cone development in the absence of NR2E3. Gene profiling of retinas from transgenic mice that ectopically express NR2E3 or NRL in cone precursors reveals overlapping and unique targets of these two transcription factors. Together with previous reports, our findings establish the hierarchy of transcriptional regulators in determining rod versus cone cell fate in photoreceptor precursors during the development of mammalian retina.

CNGB3 achromatopsia with progressive loss of residual cone function and impaired rod-mediated function.

PURPOSE: CNGB3 encodes the beta-subunits of cyclic nucleotide-gated channels in the photoreceptor plasma membrane. CNGB3 mutations cause a channelopathy that results in impaired cone function manifesting achromatopsia. The clinical physiology and phenotype of three affected sisters and three carriers were evaluated in a family with a homozygous CNGB3 mutation and an unrelated male harboring both CNGB3 and CNGA3 mutations. METHODS: Index patients were screened for mutations in CNGA3 and CNGB3 by DNA sequencing. Visual examination included acuity, color vision, Goldmann visual fields (GVF), dark-adapted absolute thresholds (DAT), electroretinography, and fundus photography. RESULTS: The three affected sisters were homozygous for a 1-bp deletion (c.1148delC) in CNGB3 that induces a frame shift after Thr383, whereas the carriers were heterozygous for this mutation. The unrelated male carried a heterozygous 8-bp deletion (c.819_826del8bp) in exon 6, as well as a heterozygous base substitution (c.1208G-->A) in exon 11 that causes an Arg403Gln exchange. All affected subjects had acuity ranging between 20/200 and 20/400, moderately constricted GVFs, normal DATs, reduced rod b-wave amplitudes, and extinguished photopic b-wave and flicker responses. Rod photoreceptor sensitivity and amplitude, calculated by fitting the rod a-waves by a model of activation of phototransduction were below normal mean. Carriers had mildly decreased acuity (20/25-20/40), normal rod and cone ERGs, and normal color vision. The fundi of the affected subjects showed macular atrophy by middle age, while the carriers showed peripheral RPE granularity in childhood and macular atrophy in late middle age. CONCLUSIONS: Foveomacular atrophy can occur in CNGB3-affected subjects, and even heterozygous carriers can exhibit maculopathy. Cone ERG responses in affected subjects are nearly extinguished, but some retain residual function into middle age and then progressively lose even this remnant. Rod responses are impaired in some CNGB3-affected subjects.