Peripheral RPE Research Articles

A Single-Cell Transcriptome Atlas of the Human Retinal Pigment Epithelium.

Human retinal pigment epithelium cells are arranged in a monolayer that plays an important supporting role in the retina. Although the heterogeneity of specific retinal cells has been well studied, the diversity of hRPE cells has not been reported. Here, we performed a single-cell RNA sequencing on 9,302 hRPE cells from three donors and profiled a transcriptome atlas. Our results identified two subpopulations that exhibit substantial differences in gene expression patterns and functions. One of the clusters specifically expressed ID3, a macular retinal pigment epithelium marker. The other cluster highly expressed CRYAB, a peripheral RPE marker. Our results also showed that the genes associated with oxidative stress and endoplasmic reticulum stress were more enriched in the macular RPE. The genes related to light perception, oxidative stress and lipid metabolism were more enriched in the peripheral RPE. Additionally, we provided a map of disease-related genes in the hRPE and highlighted the importance of the macular RPE and peripheral RPE clusters P4 and P6 as potential therapeutic targets for retinal diseases. Our study provides a transcriptional landscape for the human retinal pigment epithelium that is critical to understanding retinal biology and disease.

A Case Report Discussing an Uncommon Peripheral Exudative Hemorrhagic Chorioretinopathy

Purpose: The purpose of this case report is to discuss an uncommon, unique hemorrhagic chorioretinopathy. Method: A 79-year-old-white female presented to clinic with complaints of difficulty reading at close distances and seeing faces at far distances in both eyes. An 85-year old white female presented with no visual complaints but stated redness in the nasal corner of her right eye. This is a case report of two patients that presented with geographic atrophy of the macula as well as midperipheral RPE alterations in both eyes, which resembled intraretinal hemorrhages. Results: The ocular findings associated with peripheral exudative hemorrhagic chorioretinopathy may include midperipheral intraretinal hemorrhages, peripheral RPE mottling, subretinal fluid or hemorrhages, subretinal exudation, pigment epithelial detachments, and/or vitreous hemorrhages. Conclusions: Although this condition is rare, primary eye care providers should be aware of this unique clinical entity, its relationship to age-related macular degeneration, and when these patients require a referral to a retinal specialist.

Sprint interval training versus moderate-intensity continuous training during inpatient rehabilitation after spinal cord injury: a randomized trial.

Randomized trial. To evaluate the effectiveness of a 5-week sprint interval training (SIT) protocol on an arm-crank ergometer in individuals with sub-acute spinal cord injury (SCI). Inpatient rehabilitation. Individuals with SCI (N = 20; 9 tetraplegia/11 paraplegia; time since injury, 14-182 days; age, 46 ± 16 years; 15 M/5 F) were randomized to SIT or moderate-intensity continuous training (MICT). SIT consisted of 3 × 20 s. 'all-out' cycle sprints (≥100% peak power output) interspersed with 2 min of active recovery (10% peak power output; total time commitment, 10 mins). MICT involved 20 min of cycling (45% peak power output; total time commitment, 25 mins). Both training interventions were delivered 3 times/week for 5 weeks. Heart rate and Borg's Rating of Perceived Exertion (RPE; 6-20) were monitored throughout training sessions. Maximal and sub-maximal power outputs were assessed on an arm-crank ergometer. Exercise enjoyment, exercise self-efficacy, and pain were assessed at the end of the intervention. During training sessions, heart rate (135 bpm vs. 119 bpm; p = 0.05), peripheral RPE (16 vs. 12; p = 0.000), and central RPE (15 vs. 11; p = 0.004) responses were higher in the SIT group, yet total work performed was greater in MICT. Peak power output increased significantly with training (36%), with no difference between groups (39% vs. 33%; p = 0.524). Similarly, improvements in sub-maximal power output were not different across groups. There were no between-group differences in exercise enjoyment (p = 0.385), exercise self-efficacy (p = 0.930), or pain (p = 0780). Five weeks of SIT improved physical capacity to the same extent as MICT in individuals with sub-acute SCI, despite a significantly lower time commitment with SIT.

Phenotypic Progression of Stargardt Disease in a Large Consanguineous Tunisian Family Harboring New ABCA4 Mutations.

To assess the progression of Stargardt (STGD) disease over nine years in two branches of a large consanguineous Tunisian family. Initially, different phenotypes were observed with clinical intra- and interfamilial variations. At presentation, four different retinal phenotypes were observed. In phenotype 1, bull's eye maculopathy and slight alteration of photopic responses in full-field electroretinography were observed in the youngest child. In phenotype 2, macular atrophy and yellow white were observed in two brothers. In phenotype 3, diffuse macular, peripapillary, and peripheral RPE atrophy and hyperfluorescent dots were observed in two sisters. In phenotype 4, Stargardt disease-fundus flavimaculatus phenotype was observed in two cousins with later age of onset. After a progression of 9 years, all seven patients displayed the same phenotype 3 with advanced stage STGD and diffuse atrophy. WES and MLPA identified two ABCA4 mutations M1: c.[(?_4635)_(5714+?)dup; (?_6148)_(6479_+?) del] and M2: c.[2041C>T], p.[R681∗]. In one branch, the three affected patients had M1/M1 causal mutations and in the other branch the two affected patients had M1/M2 causal mutations. After 9-year follow-up, all patients showed the same phenotypic evolution, confirming the progressive nature of the disease. Genetic variations in the two branches made no difference to similar end-stage disease.

Modeling Perceived Exertion during Graded Arm Cycling Exercise in Spinal Cord Injury.

RPE may be useful for exercise testing and prescription in individuals with spinal cord injury (SCI), although the roles of differentiated central and peripheral fatigue during exercise are not clear. We aimed to model differentiated RPE responses during graded arm cycling in individuals with SCI and to describe their relationship to cardiorespiratory outcomes. Thirty-six individuals with SCI (13 paraplegia and 23 tetraplegia) completed a maximal graded arm cycling exercise test to volitional exhaustion (5 W·min paraplegia; 10 W·min tetraplegia). Participants were asked to report central RPE (CRPE) and peripheral RPE (PRPE) every minute using the Borg category ratio (CR10) scale until termination of exercise. Heart rate and breath-by-breath respiratory outcomes were collected throughout the exercise test. Ventilatory threshold (VT) was assessed using the ventilatory equivalents method. Cardiorespiratory indices increased linearly during graded arm exercise. By contrast, both CRPE and PRPE responses were best fit to a quadratic model with positively accelerating growth in individuals with paraplegia (P < 0.01) and tetraplegia (P < 0.05). PRPE developed faster than CRPE in individuals with tetraplegia (P < 0.01). Individuals with paraplegia had accelerated CRPE (P < 0.05) and PRPE (P < 0.05) responses compared with tetraplegia, but not when considering only individuals who reached VT. PRPE was higher than CPRE only in the late stages (80%-100% test duration; P < 0.05) in both groups when only considering individuals who reached VT. PRPE develops faster than CRPE in individuals with tetraplegia in a nonlinear fashion, despite linear increases in cardiorespiratory responses during graded arm cycling. Although there is promise to use differentiated RPE for exercise testing and prescription within the SCI population, our results indicate that there are differences in how individuals with tetraplegia perceive peripheral versus central exertion.

Bilateral paraneoplastic optic neuropathy and unilateral retinal compromise in association with prostate cancer: a differential diagnostic challenge in a patient with unexplained visual loss

We report a 77-year-old Caucasian man with a 1-year complaint of unexplained visual loss and a 4-year history of prostate cancer. A complete ophthalmologic exam, Goldmann visual fields (GVFs), intravenous fluorescein angiography (IVFA), macular and disc optical coherence tomography (OCT), pattern-reversal visual evoked potentials (PVEPs), and flash electroretinograms (ERGs) were performed. On examination, visual acuity was reduced bilaterally. Fundus exam showed juxtapapillary changes (OS > OD) and, in OS, disc pallor, peripheral RPE dropout and whitish retinal discoloration along the arcades. OCTs were normal OU. Cancer-associated retinopathy (CAR) was suspected. A flash ERG was normal OD and markedly reduced and electronegative OS. An IVFA showed bilateral juxtapapillary staining and changes highly suggestive of sequelae of central retinal artery occlusion (CRAO) OS , in which a cilioretinal artery existed along the papillomacular bundle. GVFs showed bilateral blind spot enlargement and centrocecal scotomas, and PVEPs were delayed. These findings suggested cancer-associated optic neuropathy (CAON), confirmed by presence of anti-optic nerve autoantibodies (auto-Abs). No anti-retinal auto-Abs were found. CAON is a less common paraneoplastic manifestation than CAR and it is rarely observed in association with prostate cancer. A combination of visual function testing methods permitted the recognition, in this highly unusual case, of the concurrent presence of unilateral ERG changes most likely attributable to CRAO complications in OS, in all likelihood unrelated to CAON, and not to be confused with unilateral CAR. Auto-Ab testing in combination with visual function tests helps achieve a better understanding of the pathophysiology of vision loss in paraneoplastic visual syndromes.

Dye toxicity in the context of recurrent retinal detachment repair

Sir, We thank Dr Khan1 for his comments regarding our paper.2 In the context of retinal detachments (RD) where the break is not clinically detectable, the issue is balancing the risk of inevitable persistent RD and vision loss, against the use of a technique that will allow the break to be identified while causing minimal complications. There is no standard accepted technique at this time. Although Fourier domain OCT can show attenuation, alteration, and disruption of the junction line between photoreceptor outer segment/inner segment (OS/IS), however, the correlation of these findings with postoperative vision remains inconsistent and most reports continue to include examples of exceptions to statistical trends.3 The decision to use dye at our centre was made after the data on Trypan blue (at a higher concentration than used in our series) injection was available.4 The use of this dye was later reported in >45 patients.5 Before adopting the use of the dye at our centre, the senior author used either silicone oil or encirclement. Both techniques were associated with their own complications; the latter was only effective in 60% of cases in this group of patients and often associated with pain. Penha et al's6 investigation was in the context of macular holes. Toxicity is more relevant in the macular area and the dyes were shown to cause RPE atrophy in the areas that the subretinal bleb was created. Trypan blue caused the least amount of toxicity of the dyes investigated. In the context of identifying clinically undetectable breaks that are most likely to be peripheral, and possibly associated with atrophic retinal areas that have had previous cryotherapy, the risk of further peripheral RPE atrophy caused by dye toxicity needs to be balanced against the risk of persistent RD and visual loss if the break is not found. Regardless of the use of dye, the RD itself may cause disruption to the microanatomy (OS/IS junction).7, 8 Finally, with respect to the occurrence of retinal breaks near to areas of cryopexy, we simply chose to report our findings rather than attempt to draw any conclusions, as the number of cases were thankfully small.

Mitochondrial DNA Damage and Repair in RPE Associated with Aging and Age-Related Macular Degeneration

Mitochondrial DNA (mtDNA) damage may be associated with age-related diseases, such as age-related macular degeneration (AMD). The present study was designed to test whether the frequency of mtDNA damage, heteroplasmic mtDNA mutations, and repair capacity correlate with progression of AMD. Macular and peripheral RPE cells were isolated and cultured from human donor eyes with and without AMD. The stages of AMD were graded according to the Minnesota Grading System. Confluent primary RPE cells were used to test the frequency of endogenous mtDNA damage by quantitative PCR. Mutation detection kits were used to detect heteroplasmic mtDNA mutation. To test the mtDNA repair capacity, cultured RPE cells were allowed to recover for 3 and 6 hours after exposure to H(2)O(2), and repair was assessed by quantitative PCR. The levels of human OGG1 protein, which is associated with mtDNA repair, were analyzed by Western blot. This study showed that mtDNA damage increased with aging and that more lesions occurred in RPE cells from the macular region than the periphery. Furthermore, mtDNA repair capacity decreased with aging, with less mtDNA repair capacity in the macular region compared with the periphery in samples from aged subjects. Most interestingly, the mtDNA damage was positively correlated with the grading level of AMD, whereas repair capacity was negatively correlated. In addition, more mitochondrial heteroplasmic mutations were detected in eyes with AMD. These data show macula-specific increases in mtDNA damage, heteroplasmic mutations, and diminished repair that are associated with aging and AMD severity.

Longterm cultures of the aged human RPE do not maintain epithelial morphology and high transepithelial resistance

Dear Editor, Visual rehabilitation achieved with peripheral RPE autografts in exudative AMD is only modest. Moreover, complication rates using the popular RPE–choroid patch technique remain relatively high. Tissue-engineered transplants might improve the outcome of RPE transplantation. A culturing step could place cells on a substrate prior to transplantation, thereby optimizing delivery and long-term function [1]. This would require sufficient cellular plasticity (i.e., response to environmental stimuli) to achieve a well-differentiated, functional graft. While culture plasticity was confirmed for fetal cells or RPE cell lines [2, 3], only sparse information is available for aged RPE [4]. We studied the influence of culture conditions on select differentiation characteristics in long-term primary RPE cultures from ten human donors above age 55, and compared them to a 23-year-old and two fetal donors (20 and 21 weeks of gestation respectively). The main outcome measures at 6 weeks post-confluence (PC) were morphology and transepithelial resistance (TER), an electrophysiological method for assessing tight junctions build-up. Eyes were preserved within 6 hours of death. Adult primary RPE cultures (aRPE) were initiated from: (1) mechanically scraped RPE, or (2) collagenase pretreated (1 mg/ml for 40 minutes, CLS I, Worthington Biochemical Corp, Lakewood, NJ, USA) and mechanically scraped RPE, or (3) fetal (fRPE) and adult cultures started from floater passages [2]. Cells were grown on permeable polyester inserts (Transwells, Corning Inc., Corning, NY, USA) in modified Hu & Bok media [2], containing 2% calf serum, basic FGF (5 ng/ml), L-alanyl-L-glutamine (2 mM), Albumax (200 μg/ml), and antibiotics/antimycotics. RPE culture purity was confirmed by a uniform pancytokeratin staining. Experimental matrices analyzed the influence of above harvesting methods, seeding density, laminin coating (2 μg/cm), and media composition (2% vs 10% serum until confluence, or calcium switch at confluence, or 5 mM vs 25 mM glucose) on above outcome measures. Morphology was monitored weekly in all and transepithelial resistance (TER) in all conditions, except the laminin matrix (i.e., seven of ten aged donors). Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA, April 30–May 4 2006.

Expression of hypoxia-inducible factor−1α and −2α in human choroidal neovascular membranes

Up-regulation of pro-angiogenic cytokine expression occurring secondary to hypoxia in physiologic and pathophysiologic conditions is mediated by the family of transcription regulators know as hypoxia inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in human choroidal neovascularization (CNV) and the posterior segment of young and old eyes. Surgically excised CNV from patients with either age-related macular degeneration (AMD; n = 9), punctuate inner choroidopathy (PIC; n = 3) and young normal eyes were immunohistochemically probed with monoclonal antibodies against HIF-1alpha and -2alpha and compared to that for cell markers specific for vascular endothelial cells (CD34), macrophages (CD68), retinal pigment epithelial cells (RPE; panel cytokeratins/CK18) and VEGF. Following secondary antibody amplification, reactions were visualized with fast red chromogen. Cellular immunoreactivity of membranes for HIF-2alpha was strong in eight out of nine AMD specimens but it was only weakly positive for HIF-1alpha in five specimens. In contrast, two out of three PIC specimens were weakly positive for HIF-1alpha but demonstrated no staining for HIF-2alpha. Immunohistochemical analysis revealed areas within the CNV membranes that were predominantly immunopositive for CD68 and cytokeratin indicating the presence of RPE and/or macrophages and that these cells strongly co-localized with the presence of HIF and VEGF. No immunochemical co-localization was observed with HIF and the endothelial cell marker CD34 in any membranes studied. Normal globes also demonstrated HIF-2 positivity to be predominantly localized to the central RPE rather than peripheral RPE irrespective of age of donor. The localization of HIF expression supports the concept that hypoxia is a major stimulus for the development of submacular wound healing and within this context CNV is but one component of this process.

Effects of Music on Physiological and Perceived Exertion Responses to Varying Exercise Modes

Music is considered a motivational stimulator during exercise and a distraction from the discomfort and fatigue caused by physical activity. Previous studies have examined these effects, especially in terms of intensity, but few have compared a variation of exercise mode. PURPOSE: To investigate the effects of music on perceived exertion (RPE) and blood pressure (BP) responses during three different modes of aerobic exercise at a prescribed moderate intensity of exercise heart rate. METHODS: Eight relatively physically active students performed a submaximal Astrand cycle ergometer (BE) test and three moderate intensity (75% of predicted HRmax), submaximal steady state exercise sessions in a randomized fashion on a treadmill, rpm independent workload BE, and elliptical trainer (ET) with and without music. Heart rate (HR) and BP were measured before, twice during, and immediately following the activity while RPE (peripheral and overall) was assessed at three times during each exercise bout. To simulate realistic exercise conditions, visual and auditory distractions were not eliminated from the environment. Statistical analysis consisting of ANOVA and t-tests were performed with significance set at the .05 level. RESULTS: HR was kept within the desired target range for each condition and exercise session (151.3 ± 6.2 with music and 149.8 ± 7.1 without, mean ± SD). Overall RPE values with music (11.6 ± 1.9, 12 ± .7, and 12.7 ± 2.1 for the treadmill, ET, and BE, respectively) were similar (not significantly different) to those without music 12.8 ± 1.9, 12.5 ± 2.2, and 12.6 ± 2.1, in the same order). Peripheral RPE scores were similarly not significantly different for the treatment conditions. Systolic BP was higher with music during BE and ET but not significantly different (means = 145.1 vs. 139.8 and 137.8 vs 133.5 mmHg, respectively). CONCLUSIONS: These results support previous research studies (Copeland and Franks, 1991) that have not found an effect of music in lowering RPE in trained individuals (Matesic and Cromartie, 2002) during exercise. It is possible that the stimulatory and distractive qualities of music together negate each other's effect on RPE, especially in typical non-sterile surroundings. Musical stimulation of BP may vary according to mode and should be further investigated.

Topographic and age-dependent expression of heme oxygenase-1 and catalase in the human retinal pigment epithelium.

To investigate the hypothesis that there are topographic and age-related changes in the expression of heme oxygenase (HO)-1 and catalase in the RPE. Cryosections of the macula and periphery of human eyes (n = 18; aged 27-87 years) were subjected to a high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization protocol to determine the expression of HO-1 and catalase. The immunoreactivity of HO-1 and catalase were also investigated in the same sample set. Specimens were examined by light microscopy, and images were captured with a digital camera. The total number of RPE cells and HO-1- and catalase-labeled RPE cells was counted in each section, and the ratio of labeled RPE cells to total RPE cells was calculated in both the macular and the peripheral regions of each donor eye. There was a mosaic pattern of mRNA and protein expression of HO-1 and catalase in macular and peripheral RPE. Topographical differences in the expression of HO-1 at the mRNA level and catalase at both the mRNA and protein levels was also observed. The topographical differences between the expression of HO-1 in the macula and periphery protein were not statistically significant but showed similar trends. For HO-1, the only significant age-related decline in expression was observed in the macula and periphery. Expression of HO-1 at the protein level and that of catalase at both the mRNA and protein levels showed no significant decline with age. There is a possible age-related decline in HO-1 expression, whereas catalase expression remains unchanged with aging. Both exhibit mosaic patterns in the RPE monolayer.

The Effects of Wheelchair Camber on Physiological and Perceptual Responses in Younger and Older Men

This study examined the effects of 0, 4, and 8 degrees camber on the physiological and perceptual responses in younger (19-44 years) and older (45-74 years) sedentary, able-bodied men propelling a wheelchair at 2 kmh. Physiological and perceptual (rating of perceived exertion, RPE) responses were monitored using standardized procedures. Significant increases (p &lt; .05) in oxygen uptake, ventilation rate, and heart rate were observed with increasing camber angle. These values were not significantly (p &gt; .05) different between age groups. Central and peripheral RPE was unchanged as a result of camber angle in either group. Central RPE was significantly higher (p &lt; .05) for the older participants at a camber angle of 8 degrees. It was concluded that the physiological stress increases with camber angle during manual wheelchair propulsion at 2 kmh in younger and older men. The higher perceptual stress in older participants could be due to performance at a higher percentage of their maximum physiological capacity.

Respiratory, muscular, and overall perceptions of effort

Overall, respiratory and peripheral muscular perceptions of exercise have been examined in 16 subjects (eight men and eight women), each performing four types of exercise (two-leg, one-leg, arm + shoulder, and arm ergometry) under both normoxic and hypoxic (12% oxygen) conditions. Subjects could distinguish ratings for the three types of sensation. Both overall and peripheral muscular perceptions associated with a given oxygen intake or power output increased more than respiratory perceptions as the volume of active muscle was decreased. Hypoxia tended to increase both overall and respiratory perceptions for a given absolute oxygen consumption. Cross-modal comparisons suggested an average overall RPE of close to 13 units at 70% of a task and environment-specific peak oxygen intake, irrespective of exercise conditions, but the SD of individual responses varied by at least +/- 2 units about this average. Peripheral muscular sensations dominated small muscle tasks, but the peripheral muscular RPE became relatively consistent when related to external work rate per liter of active muscle. Respiratory perceptions provided some guide to the intensity of physical activity with a given mode of exercise and fixed environmental conditions. However, if respiratory RPE is used to "fine-tune" cross-modal exercise prescriptions, account must be taken of the distorting influence of active muscle volume and of hypoxia.

Congenital Hypertrophy of the Retinal Pigment Epithelium: Electron Microscopic and Morphometric Observations

Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is a well-circumscribed, flat, pigmented fundus lesion that is stable and generally nonprogressive. Light and electron microscopy and morphometric analysis was used to study a lesion with the clinical characteristics of CHRPE found in an eye enucleated for a posterior segment malignant melanoma. These studies showed that the lesion was composed of tall, maximally pigmented RPE cells that had a density 1.7 times greater than the density of the adjacent normal peripheral RPE. These observations suggested that cellular hyperplasia and hypertrophy may contribute to CHRPE. Ultraviolet fluorescence microscopy showed no autofluorescent granules of lipofuscin in the CHRPE, suggesting that the lesion's constituent cells lack the capacity to phagocytose and digest photoreceptor outer segments. Photoreceptor degeneration in the overlying retina consequent to this functional defect could be responsible for the localized visual field defects that typically occur in patients with CHRPE.