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.

Spatial organization of the mouse retina at single cell resolution by MERFISH.

The visual signal processing in the retina requires the precise organization of diverse neuronal types working in concert. While single-cell omics studies have identified more than 120 different neuronal subtypes in the mouse retina, little is known about their spatial organization. Here, we generated the single-cell spatial atlas of the mouse retina using multiplexed error-robust fluorescence in situ hybridization (MERFISH). We profiled over 390,000 cells and identified all major cell types and nearly all subtypes through the integration with reference single-cell RNA sequencing (scRNA-seq) data. Our spatial atlas allowed simultaneous examination of nearly all cell subtypes in the retina, revealing 8 previously unknown displaced amacrine cell subtypes and establishing the connection between the molecular classification of many cell subtypes and their spatial arrangement. Furthermore, we identified spatially dependent differential gene expression between subtypes, suggesting the possibility of functional tuning of neuronal types based on location.

Gene augmentation therapy to rescue degenerative photoreceptors in a Cwc27 mutant mouse model.

Previous reports have demonstrated that defects in the spliceosome-associated protein CWC27 can lead to the degeneration of retinal cells in Cwc27 mutant mouse models. However, it is unknown whether gene replacement therapy can rescue this phenotype. The purpose of this study was to evaluate whether AAV based gene therapy could rescue the retinal degeneration observed in Cwc27 mutant mice. By 6 months of age, Cwc27 mutant mice show a retinal degenerative phenotype, including morphological and functional abnormalities, primarily driven by the death of photoreceptors. We hypothesize that subretinal injection of AAV8 to drive exogenous CWC27 protein expression will improve the retinal phenotype. We evaluated these improvements after gene therapy with electroretinography (ERG) and histology, either hematoxylin and eosin (H&E) or immunostaining. In this study, we demonstrated that subretinal injection of AAV8-GRK-Cwc27-FLAG in mutant mice can improve the functionality and morphology of the retina. Immunostaining analyses revealed a notable decrease in photoreceptor degeneration, including cone cell degeneration, in the AAV-injected eyes compared to the PBS-injected eyes. Based on these results, gene replacement therapy could be a promising method for treating retinal degeneration caused by mutations in Cwc27.

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.

Characterization of LSD1 Expression Within the Murine Eye.

Purpose: The purpose of this study was to extend the current understanding of endogenous lysine-specific demethylase 1 (LSD1) expression spatially and temporally in the retina. Toward that end, we determined the localization and levels of LSD1 and its substrates H3K4me1 and H3K4me2 (H3K4me1/2) within the murine eye. Methods: Immunofluorescent microscopy for LSD1, H3K4me1, and H3K4me2 was conducted on murine formalin-fixed paraffin-embedded eye sections across development in addition to Western immunoblotting to assess localization and protein levels. Results: Retinal LSD1 protein levels were highest at postnatal day 7 (P7), whereas its substrates H3K4me1 and H3K4me2 had equally high levels at P2 and P14. Concentrations of all three proteins gradually decreased over developmental time until reaching a basement level of ∼60% of maximum at P36. LSD1 and H3K4me1/2 were expressed uniformly in all retinal progenitor cells. By P36, there was variability in LSD1 expression in the ganglion cell layer, uniform expression in the inner nuclear layer, and dichotomous expression between photoreceptors in the outer nuclear layer. This contrasted with H3K4me1/2 expression, which remained uniform. Additionally, LSD1 was widely expressed in the lens, cornea, and retinal pigment epithelium. Conclusions: Consistent with its known role in neuronal differentiation, LSD1 is highly and uniformly expressed throughout all retinal progenitor cells. Variability in LSD1 expression, particularly in photoreceptors, may be indicative of their unique transcriptomes and epigenetic patterns of rods and cones. Murine rod nuclei exhibit LSD1 expression in a ring or shell, rather than throughout the nucleus, consistent with their unique inverted chromatin organization. LSD1 has substantial expression throughout adulthood, especially in cone nuclei. By providing insight into endogenous LSD1 expression, our current findings could directly inform future studies to determine the exact role of Lsd1 in the development and maintenance of specific structures and cell types within the eye.

Comparison of histologic findings in age-related macular degeneration with RPE flatmount images.

Purpose: To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD. Methods: Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images. Results: Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0­51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60­93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (<63 µm), medium (63.0­124 µm), and large ( ≥ 125 µm) drusen. The RPE was arranged in rosette-like structures overlying small drusen, attenuated overlying medium-sized drusen, and consisted of large multinucleated cells overlying large drusen. The RPE in the area of geographic atrophy was attenuated and depigmented. Conclusions: Confocal images of flatmounts from eyes with AMD showed RPE patterns overlying various types of drusen and geographic atrophy that correlated with histologic characteristics. We propose RPE repair mechanisms that may result in the patterns that we observed.

Set screw homogenization of murine ocular tissue, including the whole eye.

Purpose: To compare methods for homogenizing the mouse whole eye or retina for RNA extraction. Methods: We tested five homogenization techniques for the whole eye and the retina. Two established shearing techniques were a version of the Potter-Elvehjem homogenizer, which uses a plastic pellet pestle in a microfuge tube, and a Dounce homogenizer. Two modern bead-beating methods used commercially manufactured devices, the Next Advance Bullet Blender and the Qiagen TissueLyser LT. The last method involved vortex mixing multiple samples simultaneously in a buffer containing a stainless-steel set screw, a novel approach. RNA was extracted from the tissue after each technique was used. Degradation of RNA was measured with the RNA integrity number (RIN score) after electrophoresis on an Agilent BioAnalyzer RNA LabChip. Nucleic acid yields were measured with ultraviolet (UV) spectroscopy in a BioTek Synergy H1 Hybrid plate reader. The purity of the nucleic acids was assessed with the mean absorbance ratio (A260/A280). The preparation time per sample was measured with a digital stopwatch. Costs of necessary consumables were calculated per ten samples. Results: The RIN scores for all homogenization methods and both tissue types ranged from 7.75±0.64 to 8.78±0.18; none were statistically significantly different. The total RNA yield per whole eye from the bead-based methods ranged from 7,700 to 9,800 ng and from 3,000 to 4,600 ng for the pellet pestle and Dounce shearing methods, respectively. The total RNA yield per retina from the bead-based methods ranged from 4,600 to 8,400 ng and from 2,200 to 7,400 ng for the pellet pestle and Dounce shearing methods, respectively. Homogenization was faster using the bead-based methods (about 15 min for ten samples) because multiple samples could be run simultaneously compared to the shearing methods that require samples be homogenized individually (about 45-60 min per ten samples). The costs in consumables for the methods tested ranged from $2.60 to $14.70 per ten samples. The major differences in overall costs come in the form of one-time equipment purchases, which can range from one hundred to thousands of dollars. The bead-based methods required less technician involvement and had less potential for sample contamination than the shearing methods. Conclusions: The purity and quality of RNA were similar across all methods for both tissue types. The novel set screw method and the two bead-based methods (bullet blender and TissueLyser) outperformed the two shearing methods (the pellet pestle and Dounce techniques) in total RNA yields for the whole eye. Although the bullet blender, TissueLyser, and set screw methods produced comparable levels of RNA yield, purity, and quality, the set screw method was less expensive. Researchers seeking the efficiency of sophisticated bead homogenization equipment without the high equipment costs might consider this novel method.

Differential Exon Expression in a Large Family of Retinal Genes Is Regulated by a Single Trans Locus.

Transcription and RNA processing can generate many variant mRNAs (isoforms) from a given genomic locus. The more we learn about RNA processing the more we realize how complex it can be. Examining the expression profiles of individual exons, we observed that specific exons were differentially expressed across a large number of genes in mice. We found that each isoform or exon is independently expressed compared to other exons from the same gene and regulated separately in trans. Each trans locus was identified by mapping using linkage analysis in a large mouse recombinant inbred strain set. We present evidence for a limited number of these master regulatory loci in the retina. One major locus controls about half the expression of the individual exons and resides on Chromosome 4, between 133 and 136 Mb.