A comparison of optophysiological biomarkers of photoreceptor stress and phototoxicity in BALB/cJ, B6 (Cg)-Tyrc-2J/J, and C57Bl/6J mouse strains.

Ophthalmic imaging instruments, including the confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography system, originally intended for revealing ocular microstructures in the human eye, have been deployed by vision researchers to evaluate the eyes of numerous small and large animal species for more than two decades. In this study, we have used these two instruments to obtain imaging data sequentially from the retinas of three prominent, widely used experimental mouse models to document changes induced by two contrasting vivarium lighting conditions. Mice studied include albino BALB/cJ and B6(Cg)-Tyrc-2J/J and pigmented C57Bl/6J. Mice were reared under dim light conditions until ~8 weeks of age where they underwent baseline imaging. Following, mice were returned to the dim vivarium or relocated to the top rack cage position in a standard vivarium. Mice were then followed for several months by ocular imaging to catalog the retinal dynamics as a function of long-term dim vs. elevated, standard vivarium lighting exposure levels. Upon exposure to elevated light levels, B6(Cg)-Tyrc-2J/J underwent similar changes as BALB/cJ in regard to photoreceptor outer segment shortening, photoreceptor layer proximal aspect hyperreflective changes, and the development of retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate. Noteworthy, however, is that infoldings and infiltrate occurred at a slower rate of progression in B6(Cg)-Tyrc-2J/J vs. BALB/cJ. The photoreceptor outer nuclear layer thickness of BALB/cJ degenerated steadily following elevated light onset. In contrast, B6(Cg)-Tyrc-2J/J degeneration was unremarkable for many weeks before experiencing a noticeable change in the rate of degeneration that was concomitant with a plateau and decreasing trend in number of retinal infoldings and monocyte infiltrate. Pathological changes in C57Bl/6J mice were unremarkable for all imaging biomarkers assessed with exception to autofluorescent sub-retinal inflammatory monocyte infiltrate, which showed significant accumulation in dim vs. elevated light exposed mice following ~1 year of observation. These data were evaluated using Spearman's correlation and Predictive Power Score matrices to determine the best imaging optophysiological biomarkers for indicating vivarium light stress and light-induced photoreceptor degeneration. This study suggests that changes in proximal aspect hyperreflectivity, outer segment shortening, retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate are excellent indicators of light stress and light-induced degeneration in albino B6(Cg)-Tyrc-2J/J and BALB/cJ mouse strains.

Cellular Changes in Retinas From Patients With BEST1 Mutations.

Best disease (BD), also known as vitelliform macular dystrophy, is an inherited disease of the central retina caused by more than 300 pathogenic variants in the BEST1 gene. The phenotype of BD is variable, and there are just a few reports on the histopathology of eyes from donors with BD. Here, we describe the histopathological comparison of donor's eyes from two patients with BD. Eyes obtained from 85-year-old (donor 1) and 65-year-old (donor 2) donors were fixed within 25 h postmortem. Perifoveal and peripheral retinal regions were processed for histology and immunocytochemistry using retinal-specific and retinal pigment epithelium (RPE)-specific antibodies. Three age-matched normal eyes were used as controls. DNA was obtained from donor blood samples. Sequence analysis of the entire BEST1 coding region was performed and identified a c.886A > C (p.Asn296His) variant in donor 1 and a c.602T > C (p.Ile201Thr) variant in donor 2; both mutations were heterozygous. Fundus examination showed that donor 1 displayed a macular lesion with considerable scarring while donor 2 displayed close to normal macular morphology. Our studies of histology and molecular pathology in the perifovea and periphery of these two BD donor eyes revealed panretinal abnormalities in both photoreceptors and RPE cellular levels in the periphery; donor 1 also displayed macular lesion. Our findings confirm the phenotypic variability of BD associated with BEST1 variants.

Evidence of complement dysregulation in outer retina of Stargardt disease donor eyes.

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4-/-. These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype.

Geographic Atrophy: Confocal Scanning Laser Ophthalmoscopy, Histology, and Inflammation in the Region of Expanding Lesions.

Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion. Methods: Enucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD. Results: Around the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells. Conclusions: Fluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Retinal Glial and Choroidal Vascular Pathology in Donors Clinically Diagnosed With Stargardt Disease.

Purpose: The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). Methods: Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis. Results: Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Müller cells formed pre- and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss. Conclusions: Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Müller cell membranes correlating with RPE loss. Müller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

Prolonged ocular exposure leads to retinal lesions in mice.

Retinal lesions in the posterior pole of laboratory mice occur due to native, developmental abnormalities or as a consequence of environmental or experimental conditions. In this study, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Following experimental preparation induction procedures (EPIP) involving general anesthesia, mydriasis/cycloplegia, and topical anesthesia to the cornea, two ocular recovery conditions (protected and unprotected) were tested within two different animal recovery chambers (open or closed). The anterior and posterior poles were evaluated for the development of retinal lesions using digital color photography, scanning laser ophthalmoscopy, and spectral-domain optical coherence during anesthesia recovery and up to 2.5 months thereafter. In some mice, electroretinograms, histological and immunohistological evaluations were performed to assess functional and structural changes that accompanied the retinal lesions detected by in vivo imaging. Our data suggests that prolonged ocular surface exposure to circulating ambient room air leads to significant anterior and posterior segment ocular complications. The most abundant, semi-reversible complication observed was the development of lesions in the outer retina, which had a 90% probability of occurring after 45 min of exposure. The lesions mostly resolved short-term, but functional and imaging evidence suggest that some perturbations to the outer retina may persist one or more months following initial development.

Correction to: Retinal Degenerative Diseases.

The title of the chapter is "Melatonin as the Possible Link Between Age-Related Retinal Degeneration and the Disrupted Circadian Rhythm in Elderly" but degeneration was incorrectly published as regeneration. Now this has been corrected to degeneration.

Retinal angiotensin II and angiotensin-(1-7) response to hyperglycemia and an intervention with captopril.

HYPOTHESIS: Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina. MATERIALS AND METHODS: The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of streptozotocin-induced diabetes in rats. RESULTS: In the nondiabetic eye, Ang II was localized to the endfeet of Müller cells, extending into the cellular processes of the inner plexiform layer and inner nuclear layer; Ang-(1-7) showed a wider distribution, extending from the foot plates of the Müller cells to the photoreceptor layer. Eyes from diabetic animals showed a higher intensity and extent of Ang II staining compared with nondiabetic eyes, but lower intensity with a reduced distribution of Ang-(1-7) immunoreactivity. Treatment of the diabetic animals with the angiotensin-converting enzyme inhibitor (ACEI) captopril showed a reduced intensity of Ang II staining, whereas increased intensity and distribution were evident with Ang-(1-7) staining. CONCLUSIONS: These studies reveal that pharmacological inhibition with ACEIs may provide a specific intervention for the management of the diabetes-induced decline in retinal function, reversing the profile of the endogenous angiotensin peptides closer to the normal condition.

Correction: DJ-1-Dependent Regulation of Oxidative Stress in the Retinal Pigment Epithelium (RPE).

[This corrects the article DOI: 10.1371/journal.pone.0067983.].

Absence of DJ-1 causes age-related retinal abnormalities in association with increased oxidative stress.

Oxidative stress alters physiological function in most biological tissues and can lead to cell death. In the retina, oxidative stress initiates a cascade of events leading to focal loss of RPE and photoreceptors, which is thought to be a major contributing factor to geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative stress under normal and pathological conditions remains largely unknown. A better understanding of the mechanisms involved in regulating RPE and photoreceptors oxidative stress response is greatly needed. To this end we evaluated photoreceptor and RPE changes in mice deficient in DJ-1, a protein that is thought to be important in protecting cells from oxidative stress. Young (3 months) and aged (18 months) DJ-1 knockout (DJ-1 KO) and age-matched wild-type mice were examined. In both group of aged mice, scanning laser ophthalmoscopy (SLO) showed the presence of a few autofluorescent foci. The 18 month-old DJ-1 KO retinas were also characterized by a noticeable increase in RPE fluorescence to wild-type. Optical coherence tomography (OCT) imaging demonstrated that all retinal layers were present in the eyes of both DJ-1 KO groups. ERG comparisons showed that older DJ-1 KO mice had reduced sensitivity under dark- and light-adapted conditions compared to age-matched control. Histologically, the RPE contained prominent vacuoles in young DJ-1 KO group with the appearance of enlarged irregularly shaped RPE cells in the older group. These were also evident in OCT and in whole mount RPE/choroid preparations labeled with phalloidin. Photoreceptors in the older DJ-1 KO mice displayed decreased immunoreactivity to rhodopsin and localized reduction in cone markers compared to the wild-type control group. Lower levels of activated Nrf2 were evident in retina/RPE lysates in both young and old DJ-1 KO mouse groups compared to wild-type control levels. Conversely, higher levels of protein carbonyl derivatives and iNOS immunoreactivity were detected in retina/RPE lysates from both young and old DJ-1 KO mice. These results demonstrate that DJ-1 KO mice display progressive signs of retinal/RPE degeneration in association with higher levels of oxidative stress markers. Collectively this analysis indicates that DJ-1 plays an important role in protecting photoreceptors and RPE from oxidative damage during aging.

Retinal Histopathology in Eyes from a Patient with Stargardt disease caused by Compound Heterozygous ABCA4 Mutations.

BACKGROUND: The goal of this study was to define the histopathology of the retina in donor eyes from a patient with Stargardt disease (STGD1) due to compound mutations in the ABCA4 gene. MATERIALS AND METHODS: Eyes were obtained from a 66-year-old female and fixed within 18 hours postmortem. The fundi of the posterior globes were evaluated with macroscopic, SLO and OCT imaging. The perifoveal and peripheral regions were processed for electron microscopy and immunocytochemistry using cell specific antibodies. Two age-similar normal eyes were used as controls. Prior ophthalmic examinations and genetic test results were also reviewed. RESULTS: All imaging modalities showed scattered bone spicules in the peripheral retina. Atrophy of the RPE was present around the optic nerve as evidenced by the absence of SLO autofluorescence. Histology analysis showed a severely degenerated fovea with little evidence of any retinal layering or remaining RPE. The fovea was severely degenerated, with little evidence of any retinal cell layer, including the RPE. In contrast, retinal nuclear layers were present in the periphery. The perifoveal region contained few cones labeled with cone-specific antibodies; some rhodopsin-labeled cells, reactive glia labeled with GFAP; and decreased autofluorescence of the RPE. The fovea was free of cone-specific labeling, contained a few disorganized rhodopsin-labeled cells and showed substantial GFAP labeling and no autofluorescent material in the retina. The periphery displayed stubby cells labeled with cone-specific antibodies, decreased rhodopsin-labeled cells, increased GFAP staining, and autofluorescent granules in the RPE. CONCLUSIONS: The histopathology of the retina in this patient with Stargardt disease displayed a highly degenerated fovea. In all retinal locations studied, cones were more severely affected than rods.

Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice.

DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis.

Retinal histopathology in eyes from patients with autosomal dominant retinitis pigmentosa caused by rhodopsin mutations.

PURPOSE: To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations. METHODS: Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells. RESULTS: DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea. CONCLUSIONS: Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina.

The BALB/c mouse: Effect of standard vivarium lighting on retinal pathology during aging.

BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse.

Light-Regulated Thyroid Hormone Signaling Is Required for Rod Photoreceptor Development in the Mouse Retina.

PURPOSE: Ambient light is both a stimulus for visual function and a regulator of photoreceptor physiology. However, it is not known if light can regulate any aspect of photoreceptor development. The purpose of this study was to investigate whether ambient light is required for the development of mouse rod photoreceptors. METHODS: Newborn mouse pups (C57BL/6) were reared in either cyclic light (LD) or constant dark (DD). Pups were collected at postnatal day (P)5, P10, P17, or P24. We performed retinal morphometric and cell death analysis at P5, P10, and P17. Rhodopsin expression was assessed using immunofluorescence, Western blot, and quantitative RT-PCR analysis. Electroretinograms were performed at P17 and P24. Radioimmunoassay and ELISA were used to follow changes in thyroid hormone levels in the serum and vitreous. RESULTS: In the DD pups, the outer nuclear layer was significantly thinner at P10 and there were higher numbers of apoptotic cells at P5 compared to the LD pups. Rhodopsin expression was lower at P10 and P17 in DD pups. Electroretinogram a-waves were reduced in amplitude at P17 in the DD pups. The DD animals had lower levels of circulating thyroid hormones at P10. Light-mediated changes in thyroid hormones occur as early as P5, as we detected lower levels of total triiodothyronine in the vitreous from the DD animals. Drug-induced developmental hypothyroidism resulted in lower rhodopsin expression at P10. CONCLUSIONS: Our data demonstrate that light exposure during postnatal development is required for rod photoreceptor development and that this effect could be mediated by thyroid hormone signaling.

Histopathological comparison of eyes from patients with autosomal recessive retinitis pigmentosa caused by novel EYS mutations.

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) imaging. Age-similar normal eyes and an eye donated by donor 1's asymptomatic mother (donor 4, family 1) were used as controls. The perifovea and peripheral retina were processed for microscopy and immunocytochemistry with markers for cone and rod photoreceptor cells. DNA analysis revealed EYS mutations c.2259 + 1G > A and c.2620C > T (p.Q874X) in family 1, and c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del in family 2. Imaging studies revealed the presence of bone spicule pigment in arRP donor retinas. Histology of all three affected donor eyes showed very thin retinas with little evidence of stratified nuclear layers in the periphery. In contrast, the perifovea displayed a prominent inner nuclear layer. Immunocytochemistry analysis demonstrated advanced retinal degenerative changes in all eyes, with near-total absence of rod photoreceptors. In addition, we found that the perifoveal cones were more preserved in retinas from the donor with the midsize genomic rearrangement (c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del) than in retinas from the donors with the truncating (c.2259 + 1G > A and c.2620C > T (p.Q874X) mutations. Advanced retinal degenerative changes with near-total absence of rods and preservation of some perifoveal cones are observed in arRP donor retinas with EYS mutations.