Age-Related RPE changes in Wildtype C57BL/6J Mice between 2 and 32 Months.

Purpose: This study provides a systematic evaluation of age-related changes in RPE cell structure and function using a morphometric approach. We aim to better capture nuanced predictive changes in cell heterogeneity that reflect loss of RPE integrity during normal aging. Using C57BL6/J mice ranging from P60-P730, we sought to evaluate how regional changes in RPE shape reflect incremental losses in RPE cell function with advancing age. We hypothesize that tracking global morphological changes in RPE is predictive of functional defects over time. Methods: We tested three groups of C57BL/6J mice (young: P60-180; Middle-aged: P365-729; aged: 730+) for function and structural defects using electroretinograms, immunofluorescence, and phagocytosis assays. Results: The largest changes in RPE morphology were evident between the young and aged groups, while the middle-aged group exhibited smaller but notable region-specific differences. We observed a 1.9-fold increase in cytoplasmic alpha-catenin expression specifically in the central-medial region of the eye between the young and aged group. There was an 8-fold increase in subretinal, IBA-1-positive immune cell recruitment and a significant decrease in visual function in aged mice compared to young mice. Functional defects in the RPE corroborated by changes in RPE phagocytotic capacity. Conclusions: The marked increase of cytoplasmic alpha-catenin expression and subretinal immune cell deposition, and decreased visual output coincide with regional changes in RPE cell morphometrics when stratified by age. These cumulative changes in the RPE morphology showed predictive regional patterns of stress associated with loss of RPE integrity.

Deletion of histone demethylase Lsd1 (Kdm1a) during retinal development leads to defects in retinal function and structure.

Purpose: The purpose of this study was to investigate the role of Lysine specific demethylase 1 (Lsd1) in murine retinal development. LSD1 is a histone demethylase that can demethylate mono- and di-methyl groups on H3K4 and H3K9. Using Chx10-Cre and Rho-iCre75 driver lines, we generated novel transgenic mouse lines to delete Lsd1 in most retinal progenitor cells or specifically in rod photoreceptors. We hypothesize that Lsd1 deletion will cause global morphological and functional defects due to its importance in neuronal development. Methods: We tested the retinal function of young adult mice by electroretinogram (ERG) and assessed retinal morphology by in vivo imaging by fundus photography and SD-OCT. Afterward, eyes were enucleated, fixed, and sectioned for subsequent hematoxylin and eosin (H&E) or immunofluorescence staining. Other eyes were plastic fixed and sectioned for electron microscopy. Results: In adult Chx10-Cre Lsd1fl/fl mice, we observed a marked reduction in a-, b-, and c-wave amplitudes in scotopic conditions compared to age-matched control mice. Photopic and flicker ERG waveforms were even more sharply reduced. Modest reductions in total retinal thickness and outer nuclear layer (ONL) thickness were observed in SD-OCT and H&E images. Lastly, electron microscopy revealed significantly shorter inner and outer segments and immunofluorescence showed modest reductions in specific cell type populations. We did not observe any obvious functional or morphological defects in the adult Rho-iCre75 Lsd1fl/fl animals. Conclusion: Lsd1 is necessary for neuronal development in the retina. Adult Chx10-Cre Lsd1fl/fl mice show impaired retinal function and morphology. These effects were fully manifested in young adults (P30), suggesting that Lsd1 affects early retinal development in mice.

Electrophysiologic and Morphologic Strain Differences in a Low-Dose NaIO3-Induced Retinal Pigment Epithelium Damage Model.

Purpose: We aimed to explore differences in the NaIO3-elicited responses of retinal pigment epithelium (RPE) and other retinal cells associated with mouse strains and dosing regimens. Methods: One dose of NaIO3 at 10 or 15 mg/kg was given intravenously to adult male C57BL/6J and 129/SV-E mice. Control animals were injected with PBS. Morphologic and functional changes were characterized by spectral domain optical coherence tomography, electroretinography, histologic, and immunofluorescence techniques. Results: Injection with 10 mg/kg of NaIO3 did not cause consistent RPE or retinal changes in either strain. Administration of 15 mg/kg of NaIO3 initially induced a large transient increase in scotopic electroretinography a-, b-, and c-wave amplitudes within 12 hours of injection, followed by progressive structural and functional degradation at 3 days after injection in C57BL/6J mice and at 1 week after injection in 129/SV-E mice. RPE cell loss occurred in a large posterior-central lesion with a ring-like transition zone of abnormally shaped cells starting 12 hours after NaIO3 treatment. Conclusions: NaIO3 effects depended on the timing, dosage, and mouse strain. The RPE in the periphery was spared from damage compared with the central RPE. The large transient increase in the electroretinography was remarkable. Translational Relevance: This study is a phase T1 translational research study focusing on the development and validation of a mouse model of RPE damage. It provides a detailed foundation for future research, informing choices of mouse strain, dosage, and time points to establish NaIO3-induced RPE damage.

Age-Related Retinal Changes in Wild-Type C57BL/6J Mice Between 2 and 32 Months.

Purpose: The purpose of this study was to extend our understanding of how aging affects normal retina function and morphology in wild-type C57BL/6J mice, by analyzing electrophysiological recordings and in vivo and post mortem anatomy. Methods: Electroretinograms (ERGs), spectral domain optical coherence tomography (SD-OCT), and confocal scanning laser ophthalmoscope (cSLO) in vivo images were obtained from mice between the ages of 2 and 32 months in four groups: group 1 (<0.5 years), group 2 (1.0-1.5 years), group 3 (1.5-2.0 years), and group 4 (>2.0 years). Afterward, mouse bodies and eyes were weighed. Eyes were stained with hematoxylin and eosin (H&E) and cell nuclei were quantified. Results: With aging, mice showed a significant reduction in both a- and b-wave ERG amplitudes in scotopic and photopic conditions. Additionally, total retina and outer nuclear layer (ONL) thickness, as measured by SD-OCT images, were significantly reduced in older groups. The cSLO images showed an increase in auto-fluorescence at the photoreceptor-RPE interface as age increases. H&E cell nuclei quantification showed significant reduction in the ONL in older ages, but no differences in the inner nuclear layer (INL) or ganglion cell layer (GCL). Conclusions: By using multiple age groups and extending the upper age limit of our animals to approximately 2.65 years (P970), we found that natural aging causes negative effects on retinal function and morphology in a gradual, rather than abrupt, process. Future studies should investigate the exact mechanisms that contribute to these gradual declines in order to discover pathways that could potentially serve as therapeutic targets.

Morphometric Analysis of Retinal Pigment Epithelial Cells From C57BL/6J Mice During Aging.

Purpose: To quantitatively evaluate the changes in orientation and morphometric features of mouse retinal pigment epithelial (RPE) cells in different regions of the eye during aging. Methods: We segmented individual RPE cells from whole RPE flatmount images of C57BL/6J mice (postnatal days 30 to 720) using a machine-learning method and evaluated changes in morphometric features, including our newly developed metric combining alignment and shape of RPE cells during aging. Results: Mainly, the anterior part of the RPE sheet grows during aging, while the posterior part remains constant. Changes in size and shape of the peripheral RPE cells are prominent with aging as cells become larger, elongated, and concave. Conversely, the central RPE cells maintain relatively constant size and numbers with aging. Cell count in the central area and the overall cell count (approximately 50,000) were relatively constant over different age groups. RPE cells also present a specific orientation concordance that matches the shape of the specific region of the eyeball. Those cells near the optic disc or equator have a circumferential orientation to cover the round shape of the eyeball, whereas those cells in the periphery have a radial orientation and corresponding radial elongation, the extent of which increases with aging and matches with axial elongation of the eyeball. Conclusions: These results suggest that the fluid RPE morphology reflects various growth rates of underlying eyeball, and RPE cells could be classified into four regional classes (near the optic disc, central, equatorial, and peripheral) according to their morphometric features.