The retina is one of the most metabolically active tissues in the body and utilizes glucose to produce energy and intermediates required for daily renewal of photoreceptor cell outer segments. ...Glucose transporter 1 (GLUT1) facilitates glucose transport across outer blood retinal barrier (BRB) formed by the retinal pigment epithelium (RPE) and the inner BRB formed by the endothelium. We used conditional knockout mice to study the impact of reducing glucose transport across the RPE on photoreceptor and Müller glial cells. Transgenic mice expressing Cre recombinase under control of the Bestrophin1 ( Best1) promoter were bred with Glut1
mice to generate Tg-Best1-Cre:Glut1
mice ( RPEΔGlut1). The RPEΔGlut1 mice displayed a mosaic pattern of Cre expression within the RPE that allowed us to analyze mice with ~50% ( RPEΔGlut1
) recombination and mice with >70% ( RPEΔGlut1
) recombination separately. Deletion of GLUT1 from the RPE did not affect its carrier or barrier functions, indicating that the RPE utilizes other substrates to support its metabolic needs thereby sparing glucose for the outer retina. RPEΔGlut1
mice had normal retinal morphology, function, and no cell death; however, where GLUT1 was absent from a span of RPE greater than 100 µm, there was shortening of the photoreceptor cell outer segments. RPEΔGlut1
mice showed outer segment shortening, cell death of photoreceptors, and activation of Müller glial cells. The severe phenotype seen in RPEΔGlut1
mice indicates that glucose transport via the GLUT1 transporter in the RPE is required to meet the anabolic and catabolic requirements of photoreceptors and maintain Müller glial cells in a quiescent state.
High levels of oxidative radicals generated by daily light exposure and high metabolic rate suggest that the antioxidant machinery of the retina and retinal pigment epithelium (RPE) is crucial for ...their survival. DJ-1 is a redox-sensitive protein that has been shown to have neuroprotective function in the brain in Parkinson's disease and other neurodegenerative diseases. Here, we analyzed the role of DJ-1 in the retina during oxidative stress and aging. We induced low-level oxidative stress in young (3-month-old) and old (15-month-old) C57BL/6J (WT) and DJ-1 knockout (KO) mice and evaluated effects in the RPE and retina. Absence of DJ-1 resulted in increased retinal dysfunction in response to low levels of oxidative stress. Our findings suggest that loss of DJ-1 affects the RPE antioxidant machinery, rendering it unable to combat and neutralize low-level oxidative stress, irrespective of age. Moreover, they draw a parallel to the retinal degeneration observed in AMD, where the occurrence of genetic variants may leave the retina and RPE unable to fight sustained, low-levels of oxidative stress.
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•Antioxidants are upregulated in young DJ-1 KO RPE but downregulated in the retina.•DJ-1 KO retinas are degenerated under low-level oxidative stress, regardless of age.•Retinas of both young C57BL and DJ-1 KO were able to regulate antioxidant genes upon low-level oxidative stress.•Retinas of both aged C57BL and DJ-1 KO were unable to regulate antioxidant genes upon low-level oxidative stress.•RPE of aged C57BLl mice upregulated some antioxidant genes.
This study investigated the number of outdoor orientation programs--high-impact transition programs for college students--in the United States. Researchers used a census methodology to identify the ...number of outdoor orientation programs existing at accredited, residential, bachelor's degree-granting colleges and universities within the United States (N = 1,217). Researchers found 212 outdoor orientation programs (OOPs), representing 17% of the identified colleges, the largest number of OOPs in the history of these programs. A follow-up survey found OOPs were notably aligned in risk management procedures (medical training of leaders, tracking incidents, etc.) and programmatic similarities (use of name games, problem-solving and initiative activities). Programs diverged in the use of curricular activities (i.e., solo experiences) and the specifics of risk management (i.e., physician oversight). The COVID-19 pandemic led to 83% of programs being cancelled in 2021. A question remains as to how well these high-impact programs will recover after the pandemic.
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.
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.
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.
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.
ABSTRACT
The predominant function of the blood‐retinal barrier (BRB) is to maintain retinal homeostasis by regulating the influx and efflux between the blood and retina. Breakdown of the BRB occurs ...in a number of ocular diseases that result in vision loss. Understanding the molecular and cellular pathways involved in the development and maintenance of the BRB is critical to developing therapeutics for these conditions. To visualize the BRB in vivo, we used the transgenic Tg(l‐fabp:DBP‐EGFP:flk1:mCherry) zebrafish model that expresses vitamin D binding protein (a member of the albumin gene family) tagged to green fluorescent protein. Retinoic acid (RA) plays a number of important roles in vertebrate development and has been shown to play a protective role during inflammation‐induced blood‐brain barrier disruption. The role of RA in BRB development and maintenance remains unknown. To disrupt RA signaling, Tg(l‐fabp:DBP‐EGFP:flk1:mCherry) zebrafish were treated with N, N‐diethylaminobenzaldehyde and 4‐(1E)‐2‐5, 6‐dihydro‐5, 5‐dimethyl‐8‐(2‐phenylethynyl)‐2‐naphthalenylethenyl benzoic acid, which are antagonists of retinal dehydrogenase and the RA receptor, respectively. Treatment with either compound resulted in BRB disruption and reduced visual acuity, whereas cotreatment with all‐ trans RA effectively rescued BRB integrity. Additionally, transgenic overexpression of Cyp26a1, which catalyzes RA degradation, resulted in breakdown of the BRB. Our results demonstrate that RA signaling is critical for maintenance of the BRB and could play a role in diseases such as diabetic macular edema.—Pollock, L. M., Xie, J., Bell, B. A., Anand‐Apte, B. Retinoic acid signaling is essential for maintenance of the blood‐retinal barrier. FASEB J. 32, 5674–5684 (2018). www.fasebj.org
In the diabetic retina, cellular changes in the retinal pigment epithelium (RPE) and neurons occur before vision loss or diabetic retinopathy can be identified clinically. The precise etiologies of ...retinal pathology are poorly defined, and it remains unclear if the onset and progression of cellular dysfunction differ between type 1 and type 2 diabetes. Three mouse models were used to compare the time course of RPE involvement in type 1 and type 2 diabetes. C57BL/6J mice injected with streptozotocin (STZ mice) modeled type 1 diabetes, whereas Lepr(db/db) mice on both BKS and B6.BKS background strains modeled type 2 diabetes. Electroretinogram (ERG)-based techniques were used to measure light-evoked responses of the RPE (direct current-coupled ERG, dc-ERG) and the neural retina (a-wave, b-wave). Following onset of hyperglycemia, a-wave and b-wave amplitudes of STZ mice declined progressively and by equivalent degrees. Components of the dc-ERG were also altered, with the largest reduction seen in the c-wave. Lepr(db/db) mice on the BKS strain (BKS.Lepr) displayed sustained hyperglycemia and a small increase in insulin, whereas Lepr(db/db) mice on the B6.BKS background (B6.BKS.Lepr) were transiently hyperglycemic and displayed severe hyperinsulinemia. BKS.Lepr mice exhibited sustained reductions in the dc-ERG c-wave, fast oscillation, and off response that were not attributable to reduced photoreceptor activity; B6.BKS.Lepr mice displayed transient reductions in the c-wave and fast oscillation that correlated with hyperglycemia and magnitude of photoreceptor activity. In summary, all mouse models displayed altered RPE function concomitant with the onset of hyperglycemia. These results suggest that RPE function is directly reduced by elevated blood glucose levels. That RPE dysfunction was reversible and mitigated in hyperinsulinemic B6.BKS.Lepr mice provides insight into the underlying mechanism.
Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test ...whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.
•Tamoxifen provides structural and functional photoreceptor protection against NaIO3.•Tamoxifen results in more functional protection for cones than rods.•Estrogen-receptor modulation may be retina-protective.
Sodium iodate (NaIO3) is a commonly used model for age-related macular degeneration (AMD), but its rapid and severe induction of retinal pigment epithelial (RPE) and photoreceptor degeneration can ...lead to the premature dismissal of potentially effective therapeutics. Additionally, little is known about how sex and age affect the retinal response to NaIO3. This study aims to establish a less severe yet reproducible regimen by testing low doses of NaIO3 while considering age- and sex-related effects, enabling a broader range of therapeutic evaluations. In this study, young (3–5 months) and old (18–24 months) male and female C57Bl/6J mice were given an intraperitoneal (IP) injection of 15, 20, or 25 mg/kg NaIO3. Damage assessment one week post-injection included in vivo imaging, histological examination, and qRT-PCR analysis. The results revealed that young mice showed no damage at 15 mg/kg IP NaIO3, with varying degrees of damage observed at 20 mg/kg. At 25 mg/kg, most young mice displayed widespread retinal damage, with females exhibiting less retinal thinning than males. In contrast, older mice at 20 and 25 mg/kg displayed a more patchy degeneration pattern, outer retinal undulations, and greater variability in degeneration than the young mice. The most effective model for minimizing damage while maintaining consistency utilizes young female mice injected with 25 mg/kg NaIO3. The observed sex- and age-related differences underscore the importance of considering these variables in research, aligning with the National Institutes of Health's guidance. While the model does not fully replicate the complexity of AMD, these findings enhance its utility as a valuable tool for testing RPE/photoreceptor protective or replacement therapies.
•25 mg/kg intraperitoneal NaIO3 is the lowest dose that induces consistent damage.•Young male mice have more retinal thinning than females given 25 mg/kg IP NaIO3.•Old mice show different damage pattern, with males having less damage than females.
The neural retina metabolizes glucose through aerobic glycolysis generating large amounts of lactate. Lactate flux into and out of cells is regulated by proton‐coupled monocarboxylate transporters ...(MCTs), which are encoded by members of the Slc16a family. MCT1, MCT3, and MCT4 are expressed in the retina and require association with the accessory protein basigin, encoded by Bsg, for maturation and trafficking to the plasma membrane. Bsg−/− mice have severely reduced electroretinograms (ERGs) and progressive photoreceptor degeneration, which is presumed to be driven by metabolic dysfunction resulting from loss of MCTs. To understand the basis of the Bsg−/− phenotype, we generated mice with conditional deletion of Bsg in rods (RodΔBsg), cones (Cone∆Bsg), or retinal pigment epithelial cells (RPEΔBsg). RodΔBsg mice showed a progressive loss of photoreceptors, while ConeΔBsg mice did not display a degenerative phenotype. The RPEΔBsg mice developed a distinct phenotype characterized by severely reduced ERG responses as early as 4 weeks of age. The loss of lactate transporters from the RPE most closely resembled the phenotype of the Bsg−/− mouse, suggesting that the regulation of lactate levels in the RPE and the subretinal space is essential for the viability and function of photoreceptors.
Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces ...acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors.
Intravitreal injection of iron induces photoreceptor oxidative stress, resulting in increased autofluorescence, lipid peroxidation, and myeloid cell infiltration associated with retinal degeneration, geographic atrophy, and choroidal neovascularization.
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