Conditional Knockouts of Interphotoreceptor Retinoid Binding Protein Suggest Two Independent Mechanisms for Retinal Degeneration and Myopia.

Purpose: Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration. Methods: IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry. Results: Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs. Conclusions: Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.

POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells.

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON-OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2-/- mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

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.

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.

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.

A Tropomycin-Related Kinase B Receptor Activator for the Management of Ocular Blast-Induced Vision Loss.

Pressure waves from explosions or other traumatic events can damage the neurons of the eye and visual centers of the brain, leading to functional loss of vision. There are currently few treatments for such injuries that can be deployed rapidly to mitigate damage. Brain-derived neurotrophic factor (BDNF) and activation of its receptor tropomycin-related kinase B (TrkB) have neuroprotective effects in a number of degeneration models. Small molecule activators of TrkB, such as N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC), cross the blood-brain and blood-retina barriers after systemic administration. We characterize the effects of blast-induced ocular trauma on retinal and visual function. We show that systemic administration of HIOC, a potent small molecule activator of the BDNF/TrkB receptor, preserves visual function in mice exposed to ocular blast injury. The HIOC treatment for one week preserves visual function for at least four months. The HIOC treatment effectively protected vision when the initial dose was administered up to 3 h after blast, but not if the initial treatment was delayed for 24 h. We provide evidence that the therapeutic effect of HIOC is mediated by activation of BDNF/TrkB receptors. The results indicate that HIOC may be useful for managing ocular blast injury and other forms of traumatic optic neuropathy.

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.

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.

Ambient Light Regulates Retinal Dopamine Signaling and Myopia Susceptibility.

Purpose: Exposure to high-intensity or outdoor lighting has been shown to decrease the severity of myopia in both human epidemiological studies and animal models. Currently, it is not fully understood how light interacts with visual signaling to impact myopia. Previous work performed in the mouse retina has demonstrated that functional rod photoreceptors are needed to develop experimentally-induced myopia, alluding to an essential role for rod signaling in refractive development. Methods: To determine whether dim rod-dominated illuminance levels influence myopia susceptibility, we housed male C57BL/6J mice under 12:12 light/dark cycles with scotopic (1.6 × 10-3 candela/m2), mesopic (1.6 × 101 cd/m2), or photopic (4.7 × 103 cd/m2) lighting from post-natal day 23 (P23) to P38. Half the mice received monocular exposure to -10 diopter (D) lens defocus from P28-38. Molecular assays to measure expression and content of DA-related genes and protein were conducted to determine how illuminance and lens defocus alter dopamine (DA) synthesis, storage, uptake, and degradation and affect myopia susceptibility in mice. Results: We found that mice exposed to either scotopic or photopic lighting developed significantly less severe myopic refractive shifts (lens treated eye minus contralateral eye; -1.62 ± 0.37D and -1.74 ± 0.44D, respectively) than mice exposed to mesopic lighting (-3.61 ± 0.50D; P < 0.005). The 3,4-dihydroxyphenylacetic acid /DA ratio, indicating DA activity, was highest under photopic light regardless of lens defocus treatment (controls: 0.09 ± 0.011 pg/mg, lens defocus: 0.08 ± 0.008 pg/mg). Conclusions: Lens defocus interacted with ambient conditions to differentially alter myopia susceptibility and DA-related genes and proteins. Collectively, these results show that scotopic and photopic lighting protect against lens-induced myopia, potentially indicating that a broad range of light levels are important in refractive development.

Photoreceptor Degeneration in Homozygous Male Per2luc Mice During Aging.

The Per2luc mouse model developed by Takahashi laboratory is one of the most powerful models to study circadian rhythms in real time. In this study, we report that photoreceptors degenerate in male Per2luc mice during aging. Young (2.5- to 5-month-old) and aged (11- to 13.5-month-old) homozygous male Per2luc mice and C57BL/6J mice were used for this study. Retina structure and function were investigated via spectral domain optical coherence tomography (SD-OCT), fundus imaging, and electroretinography (ERG). Zonula occludens-1 (ZO-1) immunofluorescence was used to analyze the retinal pigment epithelium (RPE) morphology. Fundus examination revealed no difference between young Per2luc and wild-type (WT) mice. However, the fundus of aged Per2luc mice showed white deposits, suggestive of age-related drusen-like formation or microglia, which were absent in age-matched WT mice. No differences in retinal structure and function were observed between young Per2luc and WT mice. However, with age, Per2luc mice showed a significant reduction in total retinal thickness with respect to C57BL/6J mice. The reduction was mostly confined to the photoreceptor layer. Consistent with these results, we observed a significant decrease in the amplitude of a- and b-waves of the ERG in aged Per2luc mice. Analysis of the RPE morphology revealed that in aged Per2luc mice there was an increase in compactness and eccentricity with a decrease in solidity with respect to the values observed in WT, pointing toward signs of aging in the RPE of Per2luc mice. Our data demonstrate that homozygous Per2luc mice show photoreceptor degeneration during aging and a premature aging of the RPE.

Drug Tissue Distribution of TUDCA From a Biodegradable Suprachoroidal Implant versus Intravitreal or Systemic Delivery in the Pig Model.

Purpose: To determine local ocular tissue levels of the bile acid, tauroursodeoxycholic acid (TUDCA), in the pig model using oral, intravenous (IV), intravitreal injection (IVitI) and low- and high-dose suprachoroidal, sustained-release implants (SCI-L or SCI-H). Methods: Forty-six pigs (92 globes) were included in the study. TUDCA was delivered orally in 5 pigs, IV in 4, IVitI in 6, SCI-L in 17, and SCI-H in 14. Testing timeframes varied from the same day (within minutes) for IV; 1 to 6 days, oral; and 1 to 4 weeks, IVitI and SCI. Enucleated globes were dissected, specimens from specific tissues were separated, and TUDCA was extracted and quantified using mass spectrometry. Results: The highest TUDCA tissue levels occurred after IV delivery in the macula (252 ± 238 nM) and peripheral retina (196 ± 171 nM). Macular choroid and peripheral choroid levels were also high (1032 ± 1269 and 1219 ± 1486 nM, respectively). For IVitI delivery, macular levels at day 6 were low (0.5 ± 0.5 nM), whereas peripheral choroid was higher (15.3 ± 16.7 nM). Neither the SCI-L nor SCI-H implants delivered meaningful macular doses (≤1 nM); however, peripheral retina and choroid levels were significantly higher. Bile acid isoforms were found in the serum specimens. Conclusions: The highest TUDCA tissue levels in the pig model were obtained using IV delivery. Oral delivery was associated with reasonable tissue levels. Local delivery (IVitI and SCI) was able to achieve measurable local ocular tissue levels. Translational Relevance: Diffusional kinetics from the suprachoroidal space follow the choroidal blood flow, away from the macula and toward the periphery.

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.

Effects of Cone Connexin-36 Disruption on Light Adaptation and Circadian Regulation of the Photopic ERG.

Purpose: The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. Methods: Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. Results: HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. Conclusions: Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.

Dopamine 2 Receptor Signaling Controls the Daily Burst in Phagocytic Activity in the Mouse Retinal Pigment Epithelium.

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.

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.

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.

Low-Intensity Exercise in Mice Is Sufficient to Protect Retinal Function During Light-Induced Retinal Degeneration.

Purpose: We previously reported that a specific treadmill running exercise regimen protects against light-induced retinal degeneration (LIRD) in mice. We hypothesized that this protective effect varies with running intensity. To test this, mice undergoing LIRD were run at different treadmill speeds and retinal function was assessed. Methods: BALB/c mice were assigned to LIRD groups at varying treadmill speeds-0, 5, 10, or 20 m/min labeled inactive, low, medium, and high, respectively-and compared with naïve mice exposed to standard lighting (50 lux; naïve). Following 2 weeks of exercise, a subset of mice were exposed to toxic light (10,000 lux; LIRD) for 4 hours. After 5 additional days of exercise, retinal function was assessed by ERG. Corticosterone levels in serum and cathepsin B (CTSB) protein levels in muscle, brain, serum, and retina were measured. The retinal gene expression of complement factor 1qa (C1qa) and CTSB were measured. Results: The low+LIRD and medium+LIRD exercise groups had greater a- and b-wave ERG amplitudes when compared with the inactive+LIRD group (P < 0.02). The high+LIRD mice only differed from the inactive+LIRD mice in their dark-adapted b-waves. Serum corticosterone increased in the high+LIRD mice (P < 0.006). Retinal CTSB protein levels were higher in the low+LIRD versus high+LIRD mice (P < 0.004) but were otherwise unchanged. Exercise of any intensity decreased C1qa gene expression. Conclusions: Faster running did not additionally protect against LIRD, but it did increase serum corticosterone, suggesting stress-induced limits to exercise benefits. Unexpectedly, exercise did not increase CTSB proteins levels in muscle or serum, suggesting that it may not mediate exercise effects. Our results have implications for the use of low-intensity exercise as a vision loss treatment.

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.