Oral minocycline for the treatment of retinitis pigmentosa-associated cystoid macular edema: results of a phase I/II clinical trial.

PURPOSE: The etiology of retinitis pigmentosa (RP)-associated cystoid macular edema (CME) has been related to retinal neuroinflammation and microglial activation. Minocycline, a drug FDA-approved for anti-microbial indications, also inhibits microglial activation and expression of inflammatory mediators. This study investigates the safety and efficacy of oral minocycline as primary treatment for RP-associated CME. METHODS: A single-center, prospective, open-label phase I/II clinical trial enrolled five participants with RP-associated CME. Participants had lead-in assessments prior to the initiation of oral minocycline 100 mg twice daily for 12 months. Main outcome variables included changes in best-corrected visual acuity (BCVA) and retinal central subfield thickness (CST) measured using spectral domain optical coherence tomography relative to mean of pre-treatment measurements. RESULTS: The study drug was well tolerated and not associated with any severe adverse events. No significant changes in mean BCVA from study baseline were noted in either the study eye (+ 0.7 ± 4.1 letters at 6 months, - 1.1 ± 1.7 letters at 12 months) or the qualifying fellow eye (- 0.3 ± 3.4 letters at 6 months, - 0.3 ± 4.6 letters at 12 months) (p > 0.05 for all comparisons). Mean percentage changes in CST from baseline however decreased progressively with treatment (decreases at 6 and 12 months: study eyes 3.9 and 9.8%; qualifying fellow eyes 1.4 and 7.7%). Considering all eyes (n = 10), mean percentage CST decrease at 6 and 12 months was 2.7 ± 9.5% (p = 0.39) and 8.7 ± 9.5% (p = 0.02) respectively. CONCLUSION: Oral minocycline administration over 12 months was associated with no significant changes in mean BCVA and a small but progressive decrease in mean CST. TRIAL REGISTRATION: NCT02140164 (05/2014).

Innate Immunity in Age-Related Macular Degeneration.

Multiple lines of investigation have demonstrated that inflammation plays significant roles in etiology of age-related macular degeneration (AMD). Although interventional trials in AMD therapy targeting inflammatory pathways have been conducted, they have not yet been successful and a detailed understanding as to why some have failed is still elusive. One limitation is the relative dearth of information on how immune cells interact with retinal cells to generate AMD phenotypes at each disease stage. Here, we summarize current research evidence and hypotheses regarding potential pathogenic roles of innate immune cells in the eye, which include resident retinal microglia, macrophages derived from infiltrating systemic monocytes, and macrophages resident in the choroid. We relate recent findings regarding the physiology, function, and cellular interactions involving innate immune cells in the retina and choroid to AMD-related processes, including: (1) drusen formation and regression, (2) the onset and spread of degeneration in late atrophic AMD, and (3) the initiation, growth, and exudation of neovascular vessels in late "wet" AMD. Understanding how innate immune cells contribute to specific AMD phenotypes can assist in generating a comprehensive view on the inflammatory etiology of AMD and aid in identifying anti-inflammatory therapeutic strategies and selecting appropriate clinical outcomes for the planned interventions.

Intravitreous treatment of severe ocular von Hippel-Lindau disease using a combination of the VEGF inhibitor, ranibizumab and PDGF inhibitor, E10030: Results from a phase 1/2 clinical trial.

BACKGROUND: Treatment options for severe ocular von Hippel-Lindau (VHL) disease are limited. This trial evaluated preliminary safety and potential efficacy of combination intravitreous injection with ranibizumab, a vascular endothelial growth factor (VEGF) inhibitor, and E10030, a PDGF inhibitor, for eyes with VHL disease-associated retinal hemangioblastoma (RH) not amenable or responsive to thermal laser photocoagulation. METHODS: This was a prospective, single-arm, open-label phase 1/2 study, comprised of three adults with VHL-associated RH and vision loss. Intravitreous injections of ranibizumab (0.5 mg) and E10030 (1.5 mg) were given unilaterally every 4 weeks in the study eye through 16 weeks, then every 8 weeks through 48 weeks. Supplementary standard care therapies were allowed without restriction after 40 weeks. The primary outcome was the ocular and systemic adverse effect profile at 52 weeks. Secondary outcomes included changes in best-corrected visual acuity (BCVA), RH size, exudation, epiretinal proliferation and retinal traction, and need for ablative treatment of RH or ocular surgery at week 52. RESULTS: Three participants each received nine injections prior to week 52 and were followed for 104 weeks. One participant manifested mild episodic ocular hypertension in the study eye. Change in BCVA in the study eye at week 52 for the three participants was -5, -12 and +2 letters. No reduction in RH size was measured at 52 weeks. Variable mild improvements in exudation in two participants at week 16 were not sustained through week 52. CONCLUSIONS: Combination intravitreous injection with ranibizumab and E10030 demonstrated a reasonable preliminary safety profile, but limited treatment effect.

Deep Learning Automated Detection of Reticular Pseudodrusen from Fundus Autofluorescence Images or Color Fundus Photographs in AREDS2.

PURPOSE: To develop deep learning models for detecting reticular pseudodrusen (RPD) using fundus autofluorescence (FAF) images or, alternatively, color fundus photographs (CFP) in the context of age-related macular degeneration (AMD). DESIGN: Application of deep learning models to the Age-Related Eye Disease Study 2 (AREDS2) dataset. PARTICIPANTS: FAF and CFP images (n = 11 535) from 2450 AREDS2 participants. Gold standard labels from reading center grading of the FAF images were transferred to the corresponding CFP images. METHODS: A deep learning model was trained to detect RPD in eyes with intermediate to late AMD using FAF images (FAF model). Using label transfer from FAF to CFP images, a deep learning model was trained to detect RPD from CFP (CFP model). Performance was compared with 4 ophthalmologists using a random subset from the full test set. MAIN OUTCOME MEASURES: Area under the receiver operating characteristic curve (AUC), κ value, accuracy, and F1 score. RESULTS: The FAF model had an AUC of 0.939 (95% confidence interval [CI], 0.927-0.950), a κ value of 0.718 (95% CI, 0.685-0.751), and accuracy of 0.899 (95% CI, 0.887-0.911). The CFP model showed equivalent values of 0.832 (95% CI, 0.812-0.851), 0.470 (95% CI, 0.426-0.511), and 0.809 (95% CI, 0.793-0.825), respectively. The FAF model demonstrated superior performance to 4 ophthalmologists, showing a higher κ value of 0.789 (95% CI, 0.675-0.875) versus a range of 0.367 to 0.756 and higher accuracy of 0.937 (95% CI, 0.907-0.963) versus a range of 0.696 to 0.933. The CFP model demonstrated substantially superior performance to 4 ophthalmologists, showing a higher κ value of 0.471 (95% CI, 0.330-0.606) versus a range of 0.105 to 0.180 and higher accuracy of 0.844 (95% CI, 0.798-0.886) versus a range of 0.717 to 0.814. CONCLUSIONS: Deep learning-enabled automated detection of RPD presence from FAF images achieved a high level of accuracy, equal or superior to that of ophthalmologists. Automated RPD detection using CFP achieved a lower accuracy that still surpassed that of ophthalmologists. Deep learning models can assist, and even augment, the detection of this clinically important AMD-associated lesion.

CHOROIDAL THICKNESS AND VASCULARITY VARY WITH DISEASE SEVERITY AND SUBRETINAL DRUSENOID DEPOSIT PRESENCE IN NONADVANCED AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To investigate how choroidal features vary with age-related macular degeneration (AMD) severity in early-intermediate disease. METHODS: One hundred fifty-one eyes of 151 participants >50 years with no to intermediate AMD were analyzed with enhanced depth imaging optical coherence tomography. Mean macular choroidal thickness (CT), choroidal vascular thickness (CV), and choroidal vascularity index (CVI) were determined, and statistical associations were calculated. RESULTS: Decreased CT and CV were associated with increased axial length (+30 and +14 µm/mm, respectively; P < 0.0001 each), whereas decreased CVI was associated with increased age (+0.1%/year; P = 0.004). Compared with eyes with no/early AMD (Group 0), eyes with large drusen without late AMD in the fellow eye (Group 1) showed increased CV and CVI (+22 µm, P = 0.03 and +2.2%, P = 0.02, respectively). However, eyes with large drusen and late AMD in the fellow eye (Group 2) resembled Group 0. Eyes with subretinal drusenoid deposits demonstrated lower mean CT/CV/CVI than Group 0 (-57 µm, P = 0.02; -31 µm, P = 0.02; -3.6%, P = 0.007). CONCLUSION: Early AMD progression seems associated with biphasic alterations in choroidal dimensions, increasing during early drusen formation but decreasing thereafter. Subretinal drusenoid deposits are independently associated with marked reductions in all choroidal parameters. Changes in choroidal vascular anatomy may drive or reflect the pathobiology of AMD progression.

A Deep Learning Approach for Automated Detection of Geographic Atrophy from Color Fundus Photographs.

PURPOSE: To assess the utility of deep learning in the detection of geographic atrophy (GA) from color fundus photographs and to explore potential utility in detecting central GA (CGA). DESIGN: A deep learning model was developed to detect the presence of GA in color fundus photographs, and 2 additional models were developed to detect CGA in different scenarios. PARTICIPANTS: A total of 59 812 color fundus photographs from longitudinal follow-up of 4582 participants in the Age-Related Eye Disease Study (AREDS) dataset. Gold standard labels were from human expert reading center graders using a standardized protocol. METHODS: A deep learning model was trained to use color fundus photographs to predict GA presence from a population of eyes with no AMD to advanced AMD. A second model was trained to predict CGA presence from the same population. A third model was trained to predict CGA presence from the subset of eyes with GA. For training and testing, 5-fold cross-validation was used. For comparison with human clinician performance, model performance was compared with that of 88 retinal specialists. MAIN OUTCOME MEASURES: Area under the curve (AUC), accuracy, sensitivity, specificity, and precision. RESULTS: The deep learning models (GA detection, CGA detection from all eyes, and centrality detection from GA eyes) had AUCs of 0.933-0.976, 0.939-0.976, and 0.827-0.888, respectively. The GA detection model had accuracy, sensitivity, specificity, and precision of 0.965 (95% confidence interval [CI], 0.959-0.971), 0.692 (0.560-0.825), 0.978 (0.970-0.985), and 0.584 (0.491-0.676), respectively, compared with 0.975 (0.971-0.980), 0.588 (0.468-0.707), 0.982 (0.978-0.985), and 0.368 (0.230-0.505) for the retinal specialists. The CGA detection model had values of 0.966 (0.957-0.975), 0.763 (0.641-0.885), 0.971 (0.960-0.982), and 0.394 (0.341-0.448). The centrality detection model had values of 0.762 (0.725-0.799), 0.782 (0.618-0.945), 0.729 (0.543-0.916), and 0.799 (0.710-0.888). CONCLUSIONS: A deep learning model demonstrated high accuracy for the automated detection of GA. The AUC was noninferior to that of human retinal specialists. Deep learning approaches may also be applied to the identification of CGA. The code and pretrained models are publicly available at https://github.com/ncbi-nlp/DeepSeeNet.

DeepSeeNet: A Deep Learning Model for Automated Classification of Patient-based Age-related Macular Degeneration Severity from Color Fundus Photographs.

PURPOSE: In assessing the severity of age-related macular degeneration (AMD), the Age-Related Eye Disease Study (AREDS) Simplified Severity Scale predicts the risk of progression to late AMD. However, its manual use requires the time-consuming participation of expert practitioners. Although several automated deep learning systems have been developed for classifying color fundus photographs (CFP) of individual eyes by AREDS severity score, none to date has used a patient-based scoring system that uses images from both eyes to assign a severity score. DESIGN: DeepSeeNet, a deep learning model, was developed to classify patients automatically by the AREDS Simplified Severity Scale (score 0-5) using bilateral CFP. PARTICIPANTS: DeepSeeNet was trained on 58 402 and tested on 900 images from the longitudinal follow-up of 4549 participants from AREDS. Gold standard labels were obtained using reading center grades. METHODS: DeepSeeNet simulates the human grading process by first detecting individual AMD risk factors (drusen size, pigmentary abnormalities) for each eye and then calculating a patient-based AMD severity score using the AREDS Simplified Severity Scale. MAIN OUTCOME MEASURES: Overall accuracy, specificity, sensitivity, Cohen's kappa, and area under the curve (AUC). The performance of DeepSeeNet was compared with that of retinal specialists. RESULTS: DeepSeeNet performed better on patient-based classification (accuracy = 0.671; kappa = 0.558) than retinal specialists (accuracy = 0.599; kappa = 0.467) with high AUC in the detection of large drusen (0.94), pigmentary abnormalities (0.93), and late AMD (0.97). DeepSeeNet also outperformed retinal specialists in the detection of large drusen (accuracy 0.742 vs. 0.696; kappa 0.601 vs. 0.517) and pigmentary abnormalities (accuracy 0.890 vs. 0.813; kappa 0.723 vs. 0.535) but showed lower performance in the detection of late AMD (accuracy 0.967 vs. 0.973; kappa 0.663 vs. 0.754). CONCLUSIONS: By simulating the human grading process, DeepSeeNet demonstrated high accuracy with increased transparency in the automated assignment of individual patients to AMD risk categories based on the AREDS Simplified Severity Scale. These results highlight the potential of deep learning to assist and enhance clinical decision-making in patients with AMD, such as early AMD detection and risk prediction for developing late AMD. DeepSeeNet is publicly available on https://github.com/ncbi-nlp/DeepSeeNet.

Longitudinal Study of Dark Adaptation as a Functional Outcome Measure for Age-Related Macular Degeneration.

PURPOSE: To investigate the natural history of dark adaptation (DA) function as measured by the change in rod intercept time (RIT) over 4 years and to correlate RIT change with age-related macular degeneration (AMD) severity. DESIGN: Longitudinal, single-center, observational study. PARTICIPANTS: A total of 77 participants aged ≥50 years with a range of AMD severities. METHODS: Participants each contributing a single study eye to the analysis were assigned into person-based AMD severity groups based on fundus characteristics (drusen, pigmentary changes, late AMD, and subretinal drusenoid deposits [SDDs]). The DA function was assessed in study eyes at baseline and 3, 6, 12, 18, 24, 36, and 48 months. Mean change in DA function over time was calculated using the slope of linear regression fits of longitudinal RIT data. Patient-reported responses on a Low Luminance Questionnaire (LLQ) were obtained at baseline and yearly. Nonparametric statistical testing was performed on all comparisons. MAIN OUTCOME MEASURE: The RIT, defined as the time taken after a photobleach for visual sensitivity to recover detection of a 5×10-3 cd/m2 stimulus (a decrease of 3 log units), was monitored in study eyes over 4 years, and the mean rate of change was computed. RESULTS: Longitudinal analysis of 65 study eyes followed on the standard testing protocol (mean age, 71±9.3 years; 49% were female) revealed that higher rates of RIT prolongation were correlated with AMD severity group assignment at baseline (P = 0.026) and with severity group assignments at year 4 (P = 0.0011). Study eyes that developed SDD during follow-up demonstrated higher rates of RIT prolongation relative to those that did not (P < 0.0001). Overall, higher rates of RIT prolongation were significantly correlated with greater 4-year decreases in LLQ scores (total mean score, P = 0.0032). CONCLUSIONS: Longitudinal decline in DA function, which correlated with patient-reported functional deficits, was accelerated in eyes with greater AMD severity and especially in eyes with SDD both at baseline and at 4 years. The RIT prolongation as a measure of changing DA function may be a functional outcome measure in AMD clinical studies.

LONGITUDINAL CHANGES IN EYES WITH HYDROXYCHLOROQUINE RETINAL TOXICITY.

PURPOSE: To characterize functional and structural changes in hydroxychloroquine (HCQ) retinal toxicity after drug cessation. METHODS: Twenty-two patients (91% female; mean age 58.7 ± 11.4 years; mean duration of HCQ treatment 161.1 ± 90 months; mean dose 5.9 ± 1.9 mg/kg) with detected HCQ retinopathy were monitored for 6 months to 82 months after HCQ cessation with multimodal imaging including spectral domain optical coherence tomography and fundus autofluorescence imaging at 488 nm (standard) and 787 nm (near-infrared autofluorescence). Tests of visual function including visual acuity, Humphrey visual field testing, and multifocal electroretinography (mfERG) were performed. Study eyes were categorized into four separate severity stages by qualitative grading of spectral domain optical coherence tomography macular scans taken at the time of HCQ cessation. Changes in outcome measures between drug cessation and last follow-up visit were computed and compared between eyes of different severity stages. RESULTS: Study eyes (n = 44) were categorized based on optical coherence tomography criteria into: Stage 1 (subtle changes confined to parafoveal region; n = 14), Stage 2 (clear localized changes in parafovea; n = 17), Stage 3 (extensive parafoveal changes; n = 7), and Stage 4 (foveal involvement, n = 6). Visual acuity measurements across follow-up were stable in Stage 1 and Stage 2 eyes but decreased significantly in Stage 3 and 4 eyes. Humphrey visual field measures were also stable in stages 1 and 2 but deteriorated in Stage 3 eyes. mfERG testing demonstrated significant improvement in the R1/R2 ratio after HCQ cessation in Stage 1 eyes (mean change = -0.86 ± 0.79, P = 0.03) but did not change significantly in eyes of higher stages. Decreases in macular thickness in ≥1 of 9 Early Treatment Diabetic Retinopathy Study subfields on spectral domain optical coherence tomography were found in eyes of all stages, with Stage 2 eyes demonstrating thinning in most subfields (eight of nine subfields). In eyes with a measurable central foveal ellipsoid zone band island (9 of 17 Stage 2 eyes and 7 of 7 Stage 3 eyes), progressive decrease in the foveal ellipsoid zone band length was observed in 6 of 9 (67%) Stage 2 eyes and 6 of 7 (86%) Stage 3 eyes. Changes indicative of progressing retinopathy were detected in 17% of Stage 1 eyes, 46% of Stage 2 eyes, and 43% of Stage 3 eyes on standard fundus autofluorescence imaging, and in 17% of Stage 1 eyes, 38% of Stage 2 eyes, and 14% of Stage 3 eyes on near-infrared autofluorescence imaging. CONCLUSION: Eyes with detected HCQ retinopathy do not demonstrate general stability in retinal structure and function after HCQ cessation but instead demonstrate a range of changes during follow-up whose magnitudes correlate with retinopathy severity at the time of cessation. After cessation, eyes with only subtle and localized retinopathy were mostly stable and may show some functional improvement, whereas more severely affected eyes continued to progress. These findings provide evidence that early detection and prompt cessation in HCQ retinopathy may be needed to arrest retinopathy progression and to optimize long-term outcomes.

Adaptive and Maladaptive Complement Activation in the Retina.

The complement system, commonly associated with innate immune responses to invading pathogens, has been found in the CNS to exert a host of noncanonical functions influential during development and disease. In the retina, local complement expression and activation have been detected in response to injury, and polymorphisms in complement genes have also been linked to the genetic risk for retinal disease. While knowledge regarding the functions, effects, and mechanisms underlying complement in the retina is incomplete, complement expression and activation have been intriguingly linked to both increases and decreases in retinal degeneration in separate contexts and model systems. Here we review the evidence for the varying adaptive and maladaptive contributions of complement and comment on the implications for therapeutic strategies at complement modulation in retinal pathologies.

Tamoxifen Provides Structural and Functional Rescue in Murine Models of Photoreceptor Degeneration.

Photoreceptor degeneration is a cause of irreversible vision loss in incurable blinding retinal diseases including retinitis pigmentosa (RP) and atrophic age-related macular degeneration. We found in two separate mouse models of photoreceptor degeneration that tamoxifen, a selective estrogen receptor modulator and a drug previously linked with retinal toxicity, paradoxically provided potent neuroprotective effects. In a light-induced degeneration model, tamoxifen prevented onset of photoreceptor apoptosis and atrophy and maintained near-normal levels of electroretinographic responses. Rescue effects were correlated with decreased microglial activation and inflammatory cytokine production in the retina in vivo and a reduction of microglia-mediated toxicity to photoreceptors in vitro, indicating a microglia-mediated mechanism of rescue. Tamoxifen also rescued degeneration in a genetic (Pde6brd10) model of RP, significantly improving retinal structure, electrophysiological responses, and visual behavior. These prominent neuroprotective effects warrant the consideration of tamoxifen as a drug suitable for being repurposed to treat photoreceptor degenerative disease.SIGNIFICANCE STATEMENT Photoreceptor degeneration is a cause of irreversible blindness in a number of retinal diseases such as retinitis pigmentosa (RP) and atrophic age-related macular degeneration. Tamoxifen, a selective estrogen receptor modulator approved for the treatment of breast cancer and previously linked to a low incidence of retinal toxicity, was unexpectedly found to exert marked protective effects against photoreceptor degeneration. Structural and functional protective effects were found for an acute model of light-induced photoreceptor injury and for a genetic model for RP. The mechanism of protection involved the modulation of microglial activation and the production of inflammatory cytokines, highlighting the role of inflammatory mechanisms in photoreceptor degeneration. Tamoxifen may be suitable for clinical study as a potential treatment for diseases involving photoreceptor degeneration.

Clostridium difficile toxin B induces autophagic cell death in colonocytes.

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3-II, formation of LC3+ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB-induced autophagy was also accompanied by the repression of phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy-related genes Beclin 1, Atg5 and Atg7 attenuated TcdB-induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro-death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.

Progression of Geographic Atrophy in Age-related Macular Degeneration: AREDS2 Report Number 16.

PURPOSE: To analyze the prevalence, incidence, and clinical characteristics of eyes with geographic atrophy (GA) in age-related macular degeneration (AMD), including clinical and genetic factors affecting enlargement. DESIGN: Prospective cohort study within a controlled clinical trial. PARTICIPANTS: Age-Related Eye Disease Study 2 (AREDS2) participants, aged 50-85 years. METHODS: Baseline and annual stereoscopic color fundus photographs were evaluated for GA presence and area. Analyses included GA prevalence and incidence rates, Kaplan-Meier rates, mixed-model regression, and multivariable analysis of the square root of GA, area adjusted for covariates, including clinical/imaging characteristics and genotype. MAIN OUTCOME MEASURES: (1) Presence or development of GA; (2) change in the square root of GA area over time. RESULTS: At baseline, 517 eyes (6.2%) of 411 participants (9.8%) had pre-existing GA (without neovascular AMD), with the following characteristics: 33% central, 67% noncentral; and the following configurations: 36% small, 26% solid/unifocal, 24% multifocal, 9% horseshoe/ring, and 6% indeterminate. Of the remaining 6530 eyes at risk, 1099 eyes (17.3%) of 883 participants developed incident GA without prior neovascular disease during mean follow-up of 4.4 years. The Kaplan-Meier rate of incident GA was 19% of eyes at 5 years. In eyes with incident GA, 4-year risk of subsequent neovascular AMD was 29%. In eyes with incident noncentral GA, 4-year risk of central involvement was 57%. GA enlargement rate (following square root transformation) was similar in eyes with pre-existing GA (0.29 mm/year; 95% confidence interval 0.27-0.30) and incident GA (0.28 mm/year; 0.27-0.30). In the combined group, GA enlargement was significantly faster with noncentrality, multifocality, intermediate baseline size, and bilateral GA (P < 0.0001 for interaction in each case) but not with AREDS2 treatment assignment (P = 0.33) or smoking status (P = 0.05). Enlargement was significantly faster with ARMS2 risk (P < 0.0001), C3 non-risk (P = 0.0002), and APOE non-risk (P = 0.001) genotypes. CONCLUSIONS: Analyses of AREDS2 data on natural history of GA provide representative data on GA evolution and enlargement. GA enlargement, which was influenced by lesion features, was relentless, resulting in rapid central vision loss. The genetic variants associated with faster enlargement were partially distinct from those associated with risk of incident GA. These findings are relevant to further investigations of GA pathogenesis and clinical trial planning.

Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.

Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration.

BACKGROUND: The bromodomain and extraterminal domain (BET) family proteins (BET2, BET3, and BET4) "read" (bind) histone acetylation marks via two distinct bromodomains (Brom1 and Brom2) facilitating transcriptional activation. These epigenetic "readers" play crucial roles in pathogenic processes such as inflammation. The role of BETs in influencing the degenerative process in the retina is however unknown. METHODS: We employed the rd10 mouse model (Pde6b rd10 mutation) of retinitis pigmentosa (RP) to examine the involvement of BET proteins in retinal neurodegeneration. RESULTS: Inhibition of BET activity by intravitreal delivery of JQ1, a BET-specific inhibitor binding both Brom1 and Brom2, ameliorated photoreceptor degeneration and improved electroretinographic function. Rescue effects of JQ1 were related to the suppression of retinal microglial activation in vivo, as determined by decreased immunostaining of activation markers (IBA1, CD68, TSPO) and messenger RNA (mRNA) levels of inflammatory cytokines in microglia purified from rd10 retinas. JQ1 pre-treatment also suppressed microglial activation in vitro, decreasing microglial proliferation, migration, and mRNA expression of inflammatory cytokines (TNFα, MCP-1, IL-1ß, IL-6, and RANTES). Expression of BET2, but not BET3 and BET4, was significantly elevated during photoreceptor degeneration at postnatal day (PN)24 in retinas of rd10 mice relative to age-matched wild-type controls. siRNA knockdown of BET2 but not BET4, and the inhibitor of Brom2 (RVX208) but not of Brom1 (Olinone), decreased microglial activation. CONCLUSIONS: These findings indicate that BET inhibition rescues photoreceptor degeneration likely via the suppression of microglial activation and implicates BET interference as a potential therapeutic strategy for the treatment of degenerative retinal diseases.

Decreased Visual Function Scores on a Low Luminance Questionnaire Is Associated with Impaired Dark Adaptation.

PURPOSE: We investigate whether responses on a Low Luminance Questionnaire (LLQ) in patients with a range of age-related macular degeneration (AMD) severity are associated with their performance on focal dark adaptation (DA) testing and with choroidal thickness. DESIGN: Cross-sectional, single-center, observational study. PARTICIPANTS: A total of 113 participants older than 50 years of age with a range of AMD severity. METHODS: Participants answered the LLQ on the same day they underwent DA testing using a focal dark adaptometer measuring rod intercept time (RIT). We performed univariable and multivariable analyses of the LLQ scores and age, RIT, AMD severity, subfoveal choroidal thickness [SFCT], phakic status, and best-corrected visual acuity. MAIN OUTCOME MEASURES: The primary outcome of this study was the score on the 32-question LLQ. Each item in the LLQ is designated to 1 of 6 subscales describing functional problems in low luminance: driving, emotional distress, mobility, extreme lighting, peripheral vision, and general dim lighting. Scores were computed for each subscale, in addition to a weighted total mean score. RESULTS: Responses from 113 participants (mean age, 76.2±9.3 years; 58.4% were female) and 113 study eyes were analyzed. Univariable analysis demonstrated that lower scores on all LLQ subscales were correlated with prolonged DA testing (longer RIT) and decreased choroidal thickness. All associations were statistically significant except for the association of choroidal thickness and "peripheral vision." The strongest association was the LLQ subscale of driving with RIT (r =-0.97, P < 0.001). Multivariable analysis for each of the LLQ subscale outcomes, adjusted for age, included RIT, with total LLQ score, "driving," "extreme lighting," and "mobility" also including choroidal thickness. In all multivariable analyses, RIT had a stronger association than choroidal thickness. CONCLUSIONS: This cross-sectional analysis demonstrates associations of patient-reported functional deficits, as assessed on the LLQ, with both reduced DA and reduced choroidal thickness, in a population of older adults with varying degrees of AMD severity and good visual acuity in at least 1 eye. These analyses suggest that local functional measurements of DA testing (RIT) and choroidal thickness are associated with patient-reported functional deficits.

Microglial phagocytosis and activation underlying photoreceptor degeneration is regulated by CX3CL1-CX3CR1 signaling in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation-bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well-defined. In this study, we investigate the role of CX3CR1, a microglia-specific receptor, in regulating microglia-mediated degeneration using the well-established rd10 mouse model of RP. We found that in CX3CR1-deficient (CX3CR1(GFP/GFP) ) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1-sufficient (CX3CR1(GFP/+) ) rd10 littermates. CX3CR1-deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at-risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1-CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1-CX3CR1 signaling is a molecular mechanism capable of modulating microglial-mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479-1491.

Optical Coherence Tomography Reflective Drusen Substructures Predict Progression to Geographic Atrophy in Age-related Macular Degeneration.

PURPOSE: Structural and compositional heterogeneity within drusen comprising lipids, carbohydrates, and proteins have been previously described. We sought to detect and define phenotypic patterns of drusen heterogeneity in the form of optical coherence tomography-reflective drusen substructures (ODS) and examine their associations with age-related macular degeneration (AMD)-related features and AMD progression. DESIGN: Retrospective analysis in a prospective study. PARTICIPANTS: Patients with intermediate AMD (n = 349) enrolled in the multicenter Age-Related Eye Disease Study 2 (AREDS2) ancillary spectral-domain optical coherence tomography (SD OCT) study. METHODS: Baseline SD OCT scans of 1 eye per patient were analyzed for the presence of ODS. Cross-sectional and longitudinal associations of ODS presence with AMD-related features visible on SD OCT and color photographs, including drusen volume, geographic atrophy (GA), and preatrophic features, were evaluated for the entire macular region. Similar associations were also made locally within a 0.5-mm-diameter region around individual ODS and corresponding control region without ODS in the same eye. MAIN OUTCOME MEASURES: Preatrophy SD OCT changes and GA, central GA, and choroidal neovascularization (CNV) from color photographs. RESULTS: Four phenotypic subtypes of ODS were defined: low reflective cores, high reflective cores, conical debris, and split drusen. Among the 349 participants, there were 307 eligible eyes and 74 (24%) had at least 1 ODS. The ODS at baseline were associated with (1) greater macular drusen volume at baseline (P < 0.001), (2) development of preatrophic changes at year 2 (P = 0.001-0.01), and (3) development of macular GA (P = 0.005) and preatrophic changes at year 3 (P = 0.002-0.008), but not development of CNV. The ODS at baseline in a local region were associated with (1) presence of preatrophy changes at baseline (P = 0.02-0.03) and (2) development of preatrophy changes at years 2 and 3 within the region (P = 0.008-0.05). CONCLUSIONS: Optical coherence tomography-reflective drusen substructures are optical coherence tomography-based biomarkers of progression to GA, but not to CNV, in eyes with intermediate AMD. Optical coherence tomography-reflective drusen substructures may be a clinical entity helpful in monitoring AMD progression and informing mechanisms in GA pathogenesis.

The involvement of regulatory non-coding RNAs in sepsis: a systematic review.

BACKGROUND: Sepsis coincides with altered gene expression in different tissues. Accumulating evidence has suggested that microRNAs, long non-coding RNAs, and circular RNAs are important molecules involved in the crosstalk with various pathways pertinent to innate immunity, mitochondrial functions, and apoptosis. METHODS: We searched articles indexed in PubMed (MEDLINE), EMBASE and Europe PubMed Central databases using the Medical Subject Heading (MeSH) or Title/Abstract words ("microRNA", "long non-coding RNA", "circular RNA", "sepsis" and/or "septic shock") from inception to Sep 2016. Studies investigating the role of host-derived microRNA, long non-coding RNA, and circular RNA in the pathogenesis of and as biomarkers or therapeutics in sepsis were included. Data were extracted in terms of the role of non-coding RNAs in pathogenesis, and their applicability for use as biomarkers or therapeutics in sepsis. Two independent researchers assessed the quality of studies using a modified guideline from the Systematic Review Center for Laboratory animal Experimentation (SYRCLE), a tool based on the Cochrane Collaboration Risk of Bias tool. RESULTS: Observational studies revealed dysregulation of non-coding RNAs in septic patients. Experimental studies confirmed their crosstalk with JNK/NF-κB and other cellular pathways pertinent to innate immunity, mitochondrial function, and apoptosis. Of the included studies, the SYRCLE scores ranged from 3 to 7 (average score of 4.55). This suggests a moderate risk of bias. Of the 10 articles investigating non-coding RNAs as biomarkers, none of them included a validation cohort. Selective reporting of sensitivity, specificity, and receiver operating curve was common. CONCLUSIONS: Although non-coding RNAs appear to be good candidates as biomarkers and therapeutics for sepsis, their differential expression across tissues complicated the process. Further investigation on organ-specific delivery of these regulatory molecules may be useful.

LONGITUDINAL STRUCTURAL CHANGES IN LATE-ONSET RETINAL DEGENERATION.

PURPOSE: To characterize longitudinal structural changes in early stages of late-onset retinal degeneration to investigate pathogenic mechanisms. METHODS: Two affected siblings, both with a S163R missense mutation in the causative gene C1QTNF5, were followed for 8+ years. Color fundus photos, fundus autofluorescence images, near-infrared reflectance fundus images, and spectral domain optical coherence tomography scans were acquired during follow-up. RESULTS: Both patients, aged 45 and 50 years, had good visual acuities (>20/20) in the context of prolonged dark adaptation. Baseline color fundus photography demonstrated yellow-white, punctate lesions in the temporal macula that correlated with a reticular pattern on fundus autofluorescence and near-infrared reflectance imaging. Baseline spectral domain optical coherence tomography imaging revealed subretinal deposits that resemble reticular pseudodrusen described in age-related macular degeneration. During follow-up, these affected areas developed confluent thickening of the retinal pigment epithelial layer and disruption of the ellipsoid zone of photoreceptors before progressing to overt retinal pigment epithelium and outer retinal atrophy. CONCLUSION: Structural changes in early stages of late-onset retinal degeneration, revealed by multimodal imaging, resemble those of reticular pseudodrusen observed in age-related macular degeneration and other retinal diseases. Longitudinal follow-up of these lesions helps elucidate their progression to frank atrophy and may lend insight into the pathogenic mechanisms underlying diverse retinal degenerations.