Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa.

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Pentosan Polysulfate Sodium Causes Diminished Function and Subtle Morphological Changes in Retina and RPE of Mice.

Purpose: There are numerous reports of a distinctive maculopathy in adults exposed to pentosan polysulfate sodium (PPS), a drug prescribed to treat bladder discomfort associated with interstitial cystitis. We tested whether PPS treatment of mice injures RPE or retina to provide insight into the etiology of the human condition. Methods: Mice were fed PPS-supplemented chow over 14 months. RPE and retinal function was assessed by electroretinography (ERG) regularly. Following euthanasia, one eye was used for sagittal sectioning and histology, the contralateral for RPE flatmounting. ZO-1 positive RPE cell borders were imaged using confocal microscopy and cell morphology was analyzed using CellProfiler. Results: After 10 months of PPS treatment, we observed diminution of mean scotopic c-wave amplitudes. By 11 months, we additionally observed diminutions of mean scotopic a- and b-wave amplitudes. Analysis of flatmounts revealed altered RPE cell morphology and morphometrics in PPS-treated mice, including increased mean en face cell area and geometric eccentricity, decreased RPE cell solidity and extent, and cytosolic translocation of alpha-catenin, all markers of RPE cell stress. Sex and regional differences were seen in RPE flatmount measures. Shortened photoreceptor outer segments were also observed. Conclusions: PPS treatment reduced RPE and later retina function as measured by ERG, consistent with a primary RPE injury. Post-mortem analysis revealed extensive RPE pleomorphism and polymegathism and modest photoreceptor changes. We conclude that PPS treatment of mice causes slowly progressing RPE and photoreceptor damage and thus may provide a useful model for some retinal pathologies.

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.

Self-supervised semantic segmentation of retinal pigment epithelium cells in flatmount fluorescent microscopy images.

MOTIVATION: Morphological analyses with flatmount fluorescent images are essential to retinal pigment epithelial (RPE) aging studies and thus require accurate RPE cell segmentation. Although rapid technology advances in deep learning semantic segmentation have achieved great success in many biomedical research, the performance of these supervised learning methods for RPE cell segmentation is still limited by inadequate training data with high-quality annotations. RESULTS: To address this problem, we develop a Self-Supervised Semantic Segmentation (S4) method that utilizes a self-supervised learning strategy to train a semantic segmentation network with an encoder-decoder architecture. We employ a reconstruction and a pairwise representation loss to make the encoder extract structural information, while we create a morphology loss to produce the segmentation map. In addition, we develop a novel image augmentation algorithm (AugCut) to produce multiple views for self-supervised learning and enhance the network training performance. To validate the efficacy of our method, we applied our developed S4 method for RPE cell segmentation to a large set of flatmount fluorescent microscopy images, we compare our developed method for RPE cell segmentation with other state-of-the-art deep learning approaches. Compared with other state-of-the-art deep learning approaches, our method demonstrates better performance in both qualitative and quantitative evaluations, suggesting its promising potential to support large-scale cell morphological analyses in RPE aging investigations. AVAILABILITY AND IMPLEMENTATION: The codes and the documentation are available at: https://github.com/jkonglab/S4_RPE.

Suprachoroidal Delivery in Rats and Guinea Pigs Using a High-Precision Microneedle Injector.

Purpose: Methods of injection into the suprachoroidal space (SCS) have been developed for larger animals and humans, but reliable administration to the SCS of rodents remains challenging given their substantially smaller eyes. Here, we developed microneedle (MN)-based injectors for SCS delivery in rats and guinea pigs. Methods: We optimized key design features, including MN size and tip characteristics, MN hub design, and eye stabilization, to maximize injection reliability. Performance of the injection technique was characterized in rats (n = 13) and guinea pigs (n = 3) in vivo using fundoscopy and histological examinations to validate targeted SCS delivery. Results: To enable SCS injection across the thin rodent sclera, the injector featured an ultrasmall, hollow MN measuring 160 µm in length for rats and 260 µm for guinea pigs. To control MN interaction with the scleral surface, we incorporated a three-dimensional (3D) printed needle hub to restrict scleral deformation at the injection site. A MN tip outer diameter of 110 µm and bevel angle of 55° optimized insertion without leakage. Additionally, a 3D printed probe was used to secure the eye by applying gentle vacuum. Injection by this technique took 1 minute to perform, was conducted without an operating microscope, and yielded a 100% success rate (19 of 19) of SCS delivery determined by fundoscopy and histology. A 7-day safety study revealed no notable adverse ocular effects. Conclusions: We conclude that this simple, targeted, and minimally invasive injection technique can enable SCS injection in rats and guinea pigs. Translational Relevance: This MN injector for rats and guinea pigs will expand and expedite preclinical investigations involving SCS delivery.

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.

Treadmill exercise promotes retinal astrocyte plasticity and protects against retinal degeneration in a mouse model of light-induced retinal degeneration.

Exercise is an effective neuroprotective intervention that preserves retinal function and structure in several animal models of retinal degeneration. However, the retinal cell types governing exercise-induced neuroprotection remain elusive. Previously, we found exercise-induced retinal neuroprotection was associated with increased levels of retinal brain-derived neurotrophic factor (BDNF) and required intact signal transduction with its high-affinity receptor, tropomyosin kinase B (TrkB). Brain studies have shown astrocytes express BDNF and TrkB and that decreased BDNF-TrkB signaling in astrocytes contributes to neurodegeneration. Additionally, exercise has been shown to alter astrocyte morphology. Using a light-induced retinal degeneration (LIRD) model, we investigated how exercise influences retinal astrocytes in adult male BALB/c mice. Treadmill exercise in dim control and LIRD groups had increased astrocyte density, GFAP labeling, branching, dendritic endpoints, and arborization. Meanwhile, inactive LIRD animals had significant reductions in all measured parameters. Additionally, exercised groups had increased astrocytic BDNF expression that was visualized using proximity ligase assay. Isolated retinal astrocytes from exercised LIRD groups had significantly increased expression of a specific isoform of TrkB associated with cell survival, TrkB.FL. Conversely, inactive LIRD isolated retinal astrocytes had significantly increased expression of TrkB.T1, which has been implicated in neuronal cell death. Our data indicate exercise not only alters retinal astrocyte morphology but also promotes specific BDNF-TrkB signaling associated with cell survival and protection during retinal degeneration. These findings provide novel insights into the effects of treadmill exercise on retinal astrocyte morphology and cellular expression, highlighting retinal astrocytes as a potential cell type involved in BDNF-TrkB signaling.

Systemic Treatment with Nicotinamide Riboside Is Protective in Two Mouse Models of Retinal Ganglion Cell Damage.

Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD+) metabolism occurs in neurodegenerative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD+ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern electroretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.

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.

Computational Model-Based Estimation of Mouse Eyeball Structure From Two-Dimensional Flatmount Microscopy Images.

Purpose: Retinal pigment epithelial (RPE) cells serve as a supporter for the metabolism and visual function of photoreceptors and a barrier for photoreceptor protection. Morphology dynamics, spatial organization, distribution density, and growth patterns of RPE cells are important for further research on these RPE main functions. To enable such investigations within the authentic eyeball structure, a new method for estimating the three-dimensional (3D) eyeball sphere from two-dimensional tissue flatmount microscopy images was investigated. Methods: An error-correction term was formulated to compensate for the reconstruction error as a result of tissue distortions. The effect of the tissue-distortion error was evaluated by excluding partial data points from the low- and high-latitude zones. The error-correction parameter was learned automatically using a set of samples with the ground truth eyeball diameters measured with noncontact light-emitting diode micrometry at submicron accuracy and precision. Results: The analysis showed that the error-correction term in the reconstruction model is a valid method for modeling tissue distortions in the tissue flatmount preparation steps. With the error-correction model, the average relative error of the estimated eyeball diameter was reduced from 14% to 5%, and the absolute error was reduced from 0.22 to 0.03 mm. Conclusions: A new method for enabling RPE morphometry analysis with respect to locations on an eyeball sphere was created, an important step in increasing RPE research and eye disease diagnosis. Translational Relevance: This method enables one to derive RPE cell information from the 3D eyeball surface and helps characterize eyeball volume growth patterns under diseased conditions.

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.

Systemic Treatment With Nicotinamide Riboside Is Protective in a Mouse Model of Light-Induced Retinal Degeneration.

Purpose: Maintaining levels of nicotinamide adenine dinucleotide (NAD+), a coenzyme critical for cellular energetics and biosynthetic pathways, may be therapeutic in retinal disease because retinal NAD+ levels decline during retinal damage and degeneration. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor that is orally deliverable and well-tolerated by humans, is protective in a mouse model of light-induced retinal degeneration. Methods: Mice were injected intraperitoneally with vehicle or NR the day before and the morning of exposure to degeneration-inducing levels of light. Retinal function was assessed by electroretinography and in vivo retinal morphology and inflammation was assessed by optical coherence tomography. Post mortem retina sections were assessed for morphology, TUNEL, and inflammatory markers Iba1 and GFAP. Retinal NAD+ levels were enzymatically assayed. Results: Exposure to degeneration-inducing levels of light suppressed retinal NAD+ levels. Mice undergoing light-induced retinal degeneration exhibited significantly suppressed retinal function, severely disrupted photoreceptor cell layers, and increased apoptosis and inflammation in the outer retina. Treatment with NR increased levels of NAD+ in retina and prevented these deleterious outcomes. Conclusions: This study is the first to report the protective effects of NR treatment in a mouse model of retinal degeneration. The positive outcomes, coupled with human tolerance to NR dosing, suggest that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.

Analysis of Damage and Wound Healing in the Retinal Pigmented Epithelium.

Previous studies of retinal pigment epithelium (RPE) morphology found cell-level and spatial patterning differences in many quantitative metrics in comparing normal and disease conditions. However, most of these studies examined eyes from deceased animals. Here we sought to compare noninvasively imaged RPE cells from live mice to histopathology. We describe changes to improve noninvasive imaging of RPE in the live mouse. In retinal diseases, there can be invasion by Iba1-positive cells, which can be detected by noninvasive imaging techniques. Here we can detect potential Iba1-positive cells at the level of the RPE noninvasively.

Initial Assessment of Lactate as Mediator of Exercise-Induced Retinal Protection.

Physical exercise is protective in rodent models of retinal injury and disease. Data suggest that this is in part mediated by brain-derived neurotrophic factor (BDNF) signal transduction. It has been hypothesized that exercised-induced neuroprotection may be mediated by increases in circulating lactate that in turn alter BDNF secretion. We therefore tested whether mice undergoing a treadmill running regimen previously shown to be protective in a mouse model of retinal degeneration (RD) have increased serum levels of lactate. Lactate levels in exercised and non-exercised mice were statistically indistinguishable. A role for circulating lactate in exercise-induced retinal protection is unsupported.

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.

Wheel running exercise protects against retinal degeneration in the I307N rhodopsin mouse model of inducible autosomal dominant retinitis pigmentosa.

Purpose: We previously reported that modest running exercise protects photoreceptors in mice undergoing light-induced retinal degeneration and in the rd10 mouse model of autosomal recessive retinitis pigmentosa (arRP). We hypothesized that exercise would protect against other types of retinal degeneration, specifically, in autosomal dominant inherited disease. We tested whether voluntary running wheel exercise is protective in a retinal degeneration mouse model of class B1 autosomal dominant RP (adRP). Methods: C57BL/6J mice heterozygous for the mutation in I307N rhodopsin (Rho) (also known as RHOTvrm4/+, or Tvrm4) are normal until exposed to brief but bright light, whereupon rod photoreceptor degeneration ensues. I307N Rho mice were given access to free spinning (active) or locked (inactive) running wheels. Five weeks later, half of each cohort was treated with 0.2% atropine eye drops and exposed to white LED light (6,000 lux) for 5 min, then returned to maintenance housing with wheels. At 1 week or 4 weeks after induction, retinal and visual function was assessed with electroretinogram (ERG) and optomotor response (OMR). In vivo retinal morphology was assessed with optical coherence tomography (OCT), and fundus blue autofluorescence assessed using a scanning laser ophthalmoscope. The mice were then euthanized, and the eyes fixed for paraffin sectioning or flatmounting. The paraffin sections were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to assess retina morphology and apoptosis. Half of the flatmounts were stained for ZO-1 and α-catenin to assess RPE cell structure and stress. (We previously reported that translocation of α-catenin from cell membranes into the cytosol indicates RPE cell stress.) The remaining flatmounts were stained for ZO-1 and Iba-1 to assess the RPE cell size and shape, and inflammatory responses. Results: In vivo measures revealed that induction of the I307N Rho degeneration decreased retinal and visual function, decreased the thickness of the retina and photoreceptor layers, and increased the number of blue autofluorescence spots at the level of the photoreceptor-RPE interface. Post-mortem analyses showed that induction caused loss of photoreceptors in the central retinal region, and increased TUNEL labeling in the outer nuclear layer (ONL). The RPE was disrupted 1 week after induction, with changes in cell size and shape accompanied by increased α-catenin translocation and Iba-1 staining. These outcomes were partially but statistically significantly prevented in the exercised mice. The exercised mice that underwent induced I307N Rho degeneration exhibited retinal function and visual function measures that were statistically indistinguishable from that of the uninduced mice, and compared to the unexercised induced mice, had thicker retina and photoreceptor layers, and decreased numbers of subretinal autofluorescent spots. Post-mortem, the retina sections from the exercised mice that had undergone induced I307N Rho degeneration exhibited numbers of photoreceptors that were statistically indistinguishable from those of uninduced mice. Similarly, exercise largely precluded a degeneration-induced increase in TUNEL-positive cells in the ONL. Finally, the RPE of the exercised mice appeared normal, with a regular cell shape and size, and little to no alpha-catenin translocation or Iba-1 immunosignal. Conclusions: Voluntary wheel running partially protected against retinal degeneration and inflammation, and RPE disruption in a model of inducible adRP. This is the first report of exercise protection in an adult adRP animal model. It is also the first report of an RPE phenotype in the I307N Rho mouse. These findings add to a growing literature reporting that modest whole-body exercise is protective across a wide range of models of retinal damage and disease, and further highlights the potential for this accessible and inexpensive therapeutic intervention in the ophthalmic clinic.

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.