Glutaminase deficiency in rod photoreceptors disrupts nonessential amino acid levels to activate the integrated stress response and induce rapid degeneration.

The bioenergetic demand of photoreceptors rivals that of cancer cells, and numerous metabolic similarities exist between these cells. Glutamine (Gln) anaplerosis via the tricarboxylic acid (TCA) cycle provides biosynthetic intermediates and is a hallmark of cancer metabolism. In this process, Gln is first converted to glutamate via glutaminase (GLS), which is a crucial pathway in many cancer cells. To date, no study has been undertaken to examine the role of Gln metabolism in vivo in photoreceptors. Here, mice lacking GLS in rod photoreceptors were generated. Animals lacking GLS experienced rapid photoreceptor degeneration with concomitant functional loss. Gln has multiple roles in metabolism including redox balance, biosynthesis of nucleotides and amino acids, and supplementing the TCA cycle. Few alterations were noted in redox balance. Unlabeled targeted metabolomics demonstrated few changes in glycolytic and TCA cycle intermediates, which corresponded with a lack of significant changes in mitochondrial function. GLS deficiency in rod photoreceptors did decrease the fractional labelling of TCA cycle intermediates when provided uniformly labeled 13C-Gln in vivo. However, supplementation with alpha-ketoglutarate provided only marginal rescue of photoreceptor degeneration. Nonessential amino acids, glutamate and aspartate, were decreased in the retina of mice lacking GLS in rod photoreceptors. In accordance with this amino acid deprivation, the integrated stress response (ISR) was found to be activated with decreased global protein synthesis. Importantly, supplementation with asparagine delayed photoreceptor degeneration to a greater degree than alpha-ketoglutarate. These data show that GLS-mediated Gln catabolism is essential for rod photoreceptor amino acid biosynthesis, function, and survival.

Severe Spontaneous Tilt of Scleral-Fixated Intraocular Lenses.

PURPOSE: To report and evaluate a multicenter series of 18 cases of severe, spontaneous IOL tilt involving the flanged intrascleral haptic fixation technique (FISHF). DESIGN: Clinical study with historical controls. METHODS: We report a cross-sectional study of 46 FISHF cases using the CT Lucia 602 IOL at a single academic center over a period of 24 weeks to determine the incidence of severe rotisserie-style rotational tilt. These rates were then compared with the same time-frame the prior year to help determine if this is a new phenomenon. Additional cases of severe tilt were solicited from another 4 academic centers. RESULTS: Among 46 FISHF cases at a single center, 5 developed severe tilt. No clear pattern in surgical technique, ocular history, or ocular anatomy was evident in these cases compared with controls, although the involved IOLs clustered within a narrow diopter range, indicative of a batch effect. In the same 24-week interval the year before, 33 FISHF cases were performed, none of which exhibited severe rotational tilt. In our multicenter dataset, 18 cases of tilt were identified. Surgeons included fellow and early-career physicians as well as surgeons with multiple years of experience with the Yamane technique. A variety of surgical approaches for FISHF were represented. In at least 8 of the cases, haptic rotation and/or dehiscence at the optic-haptic junction were documented. CONCLUSIONS: The identification of haptic rotation and dehiscence intraoperatively in several cases may reflect a new stability issue involving the optic-haptic junction.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Increase in Retinal Detachment Repair Over a Ten-Year Period at an Academic Center Compared to National Trends.

BACKGROUND AND OBJECTIVE: This study aimed to determine whether cases of surgical retinal detachment (RD) repair at a tertiary care center from January 1, 2011 to December 31, 2020 increased proportionately to macular surgery cases as a control and to national trends. PATIENTS AND METHODS: Current Procedural Terminology codes were used to identify cases of primary RD repair (67107, 67108), complex RD repair (67113), pneumatic retinopexy (67110), and vitrectomy with membrane peeling (67041, 67042) at an academic center and in the Part B National Summary Data Files. Numbers of cases and mean case times at the academic center were determined. RESULTS: We identified 5,183 and 948,831 operative cases locally and nationally, respectively. Between 2011 and 2019, the total volume of RD repair at the academic center increased by 118.7%, compared to 23.3% for cases of membrane peeling. In contrast, surgical RD repairs and membrane peelings increased by 26.0% and 6.8% cases nationally. The ratio of RD repairs to membrane peelings from 2011 to 2019 increased from 1.5 to 2.6 locally compared to 0.6 to 0.7 nationally. Complex RD repairs increased more than primary RD repairs locally (129.3% vs 110.9% cases) and less than primary RD repairs nationally (20.6% versus 30.2% cases). CONCLUSION: Cases of surgical RD repair increased disproportionately compared to macular surgery at our institution and compared to RD repairs nationwide. [Ophthalmic Surg Lasers Imaging Retina 2023;54:505-511.].

Metabolic Alterations Caused by Simultaneous Loss of HK2 and PKM2 Leads to Photoreceptor Dysfunction and Degeneration.

HK2 and PKM2 are two main regulators of aerobic glycolysis. Photoreceptors (PRs) use aerobic glycolysis to produce the biomass necessary for the daily renewal of their outer segments. Previous work has shown that HK2 and PKM2 are important for the normal function and long-term survival of PRs but are dispensable for PR maturation, and their individual loss has opposing effects on PR survival during acute nutrient deprivation. We generated double conditional (dcKO) mice lacking HK2 and PKM2 expression in rod PRs. Western blotting, immunofluorescence, optical coherence tomography, and electroretinography were used to characterize the phenotype of dcKO animals. Targeted and stable isotope tracing metabolomics, qRT-PCR, and retinal oxygen consumption were performed. We show that dcKO animals displayed early shortening of PR inner/outer segments, followed by loss of PRs with aging, much more rapidly than either knockout alone without functional loss as measured by ERG. Significant alterations to central glucose metabolism were observed without any apparent changes to mitochondrial function, prior to PR degeneration. Finally, PR survival following experimental retinal detachment was unchanged in dcKO animals as compared to wild-type animals. These data suggest that HK2 and PKM2 have differing roles in promoting PR neuroprotection and identifying them has important implications for developing therapeutic options for combating PR loss during retinal disease.

Flow cytometric method for the detection and quantification of retinal cell death and oxidative stress.

Retinal cell death is the major cause of vision loss in many forms of blinding retinal disease. A plethora of research is focused on understanding the mechanisms of retinal cell death to identify potential neuroprotective strategies that prevent vision loss in these diseases. Traditionally, histological techniques have been used to determine the type and extent of cell death in the retina. These techniques, such as TUNEL labeling and immunohistochemistry, are laborious and time consuming, resulting in low throughput and variable results depending on the experimenter. To increase throughput and reduce variability, we developed several flow cytometry-based assays to detect and quantify retinal cell death. The methods and accompanying data presented demonstrate that flow cytometry can readily detect both retinal cell death and oxidative stress and importantly, the efficacy of neuroprotective agents. These methods will be of interest to investigators looking to increase throughput and efficiency without compromising sensitivity as the methods herein reduce analysis time from several months to less than a week. As such, the flow cytometry methods presented have the potential to expedite research efforts focused on developing novel strategies for retinal cell neuroprotection.

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2's efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

Rod photoreceptor-specific deletion of cytosolic aspartate aminotransferase, GOT1, causes retinal degeneration.

Photoreceptor cell death is the cause of vision loss in many forms of retinal disease. Metabolic dysfunction within the outer retina has been shown to be an underlying factor contributing to photoreceptor loss. Therefore, a comprehensive understanding of the metabolic pathways essential to photoreceptor health and function is key to identifying novel neuroprotective strategies. Glutamic-oxaloacetic transaminase 1 (Got1) encodes for a cytosolic aspartate aminotransferase that reversibly catalyzes the transfer of an amino group between glutamate and aspartate and is an important aspect of the malate-aspartate shuttle (MAS), which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Previous work has demonstrated that the activity of this enzyme is highest in photoreceptor inner segments. Furthermore, ex vivo studies have demonstrated that the retina relies on aspartate aminotransferase for amino acid metabolism. Importantly, aspartate aminotransferase has been suggested to be an early biomarker of retinal degeneration in retinitis pigmentosa and a possible target for neuroprotection. In the present study, we characterized the effect of Got1 deletion on photoreceptor metabolism, function, and survival in vivo by using a rod photoreceptor-specific, Got1 knockout mouse model. Loss of the GOT1 enzyme from rod photoreceptors resulted in age-related photoreceptor degeneration with an accumulation of retinal aspartate and NADH and alterations in the expression of genes involved in the MAS, the tricarboxylic acid (TCA) cycle, and redox balance. Hence, GOT1 is critical to in vivo photoreceptor metabolism, function, and survival.

Iatrogenic Paracentral Visual Field Defect After Pars Plana Vitrectomy for Macula-On Retinal Detachment Repair.

Perfluorocarbon liquid (PFCL) is an important adjunct in pars plana vitrectomy (PPV) for complex retinal detachment (RD). Complete removal of PFCL is critical to prevent retinal inflammation and cellular toxicity, but removal is not risk-free. We report a case of a new postoperative onset paracentral visual field defect after PPV with PFCL use for treatment of a macula-on RD. We present pre- and postoperative imaging that suggests a likely perioperative iatrogenic cause. [Ophthalmic Surg Lasers Imaging Retina 2022;53:644-646.].

A Caveat About Financial Incentives for Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Retinopathy.

PURPOSE: To highlight the financial incentive to the physician of choosing an intravitreal anti-vascular endothelial growth factor (VEGF)-based strategy for treating non-proliferative and proliferative diabetic retinopathy, and its possible risks to the patient and costs to the health care system. DESIGN: Perspective with retrospective cost and profit analysis. METHODS: Review and synthesis of selected literature on the treatment of diabetic retinopathy, with interpretation of activity-based and time-based costing of an intravitreal aflibercept strategy for diabetic retinopathy. Data from the DRCR Retina Network Protocols W and AB and PANORAMA trial were used to illustrate the potential financial incentive underlying such a treatment strategy. RESULTS: Physician treatment algorithms for diabetic vitreous hemorrhage and non-proliferative diabetic retinopathy may be influenced by the substantial financial incentives that intravitreal aflibercept strategies present, despite functional equivalence with alternative and less profitable strategies. For example, pursuing an intravitreal aflibercept-based strategy for diabetic vitreous hemorrhage presents a 76% increased profit over pars plana vitrectomy with laser, with equivalent functional outcomes. For non-proliferative diabetic retinopathy, preventative aflibercept injections represent a potential 414% increase in profit over observation and an increased cost of $12,164 to $17,542 over 2 years per patient, with no improvement in visual function. These findings demonstrate that there may be misaligned financial incentives in the management of diabetic retinopathy. CONCLUSIONS: While anti-VEGF therapy is a useful tool in the management of proliferative diabetic retinopathy and diabetic macular edema, it is believed that physicians should avoid overreliance on anti-VEGF injections in the treatment of diabetic retinopathy. Retinal specialists should be cognizant of the limitations, costs, and risks of anti-VEGF monotherapy and prophylactic therapy, and of the imperative to avoid bias towards financially remunerative practice patterns when equally effective alternatives exist.

Dark-reared rd10 mice experience rapid photoreceptor degeneration with short exposure to room-light during in vivo retinal imaging.

Inherited retinal diseases (IRDs) are a collection of rare genetic conditions, which can lead to complete blindness. A large number of causative genes have been identified for IRDs and while some success has been achieved with gene therapies, they are limited in scope to each individual gene and/or the specific mutation harbored by each patient with an IRD. Multiple studies are underway to elucidate common underlying mechanisms contributing to photoreceptor (PR) loss and to design gene-agnostic, pan-disease therapeutics. The rd10 mouse, which recapitulates slow degeneration of PRs, is an in vivo IRD model used commonly by vision researchers. Light deprivation by rearing animals in complete darkness significantly delays PR death in rd10 mice, subsequently increasing the time window for in vivo studies investigating neuroprotective strategies. Longitudinal in vivo retinal imaging following the same rd10 mice over time is a potential solution for reducing the number of animals required to complete a study. We describe a previously unreported phenotype in the dark-reared rd10 model that is characterized by dramatic PR degeneration following brief exposure to low-intensity light. This exquisite light sensitivity precludes the use of longitudinal studies employing in vivo imaging or other functional assessment requiring room light in rd10 mice and highlights the importance of closely following animal models of IRD to determine any deviations from the expected degeneration curve during routine experimentation.

SEROLOGY NEGATIVE BARTONELLA NEURORETINITIS IN AN IMMUNOCOMPROMISED PATIENT.

BACKGROUND/PURPOSE: To report a case of serology-negative severe disseminated Bartonella neuroretinitis in an immunocompromised patient in which diagnosis was made by detection of B. henselae DNA by universal polymerase chain reaction of brain tissue. METHODS: Case report. RESULTS: A 57-year-old man with immunoglobulin A vasculitis on immunosuppressive therapy presented with lethargy, weight loss, and bilateral decreased vision. Fundus examination revealed bilateral mild vitritis, marked optic disc edema, vascular sheathing, and numerous white inner retinal and preretinal lesions. Brain magnetic resonance imaging revealed multiple foci of restricted diffusion and a ring-enhancing focus in the left parietal lobe. Serologies, cerebrospinal fluid, and vitreous biopsies were all negative for Bartonella. A brain biopsy was performed and B. henselae DNA was detected by universal polymerase chain reaction of the specimen. The patient demonstrated resolution of fundus findings with antibiotic treatment. Repeat serological testing demonstrated seroconversion. CONCLUSION: In immunocompromised patients, infection by Bartonella henselae can present as severe disseminated disease. Establishing the diagnosis can be challenging as serologic testing is often unrevealing in the setting of a blunted immune response. Polymerase chain reaction has been used in select cases to establish the diagnosis.

Association of metformin and development of dry age-related macular degeneration in a U.S. insurance claims database.

PURPOSE: To assess whether metformin is associated with dry age-related macular degeneration (dAMD) development. METHODS: In this retrospective cohort study, patients enrolled in a nationwide U.S. medical insurance claims database from 2002 to 2016 were included if they had diabetes mellitus, were ⩾55 years old, and were enrolled for ⩾2 years without a prior AMD diagnosis. The primary exposure was metformin use analyzed as either active or prior use or cumulative metformin dosage over the study period. A time updating Cox proportional hazard regression was used to estimate the hazard ratio of dAMD incidence with metformin exposure. RESULTS: Among 1,007,226 diabetic enrollees, 53.3% were female and 66.4% were white with a mean hemoglobin A1c of 6.8%. Of eligible enrollees, 166,115 (16.5%) were taking metformin at the index date. Over the study period, 29,818 (3.0%) participants developed dAMD. In the active versus prior use of metformin model, active use conferred an increased hazard of developing dAMD (HR, 1.08; 95% CI, 1.04-1.12) while prior use had a decreased hazard (HR, 0.95; 95% CI 0.92-0.98). The cumulative metformin dosage model showed a significant trend toward increased hazard of dAMD incidence with increasing cumulative dosage (p < 0.001), with the lowest dosage quartile having decreased hazard of dAMD incidence (HR, 0.95; 95% CI, 0.91-0.99) and the highest having increased hazard (HR, 1.07; 95% CI, 1.01-1.13). CONCLUSIONS: Small, conflicting associations between metformin exposure and development of dAMD were observed depending on cumulative dosage and whether drug use was active, suggesting metformin did not substantially affect the development of dAMD.

Clinical Presentation and Outcomes of Rhegmatogenous Retinal Detachments During the COVID-19 Lockdown and Its Aftermath at a Tertiary Care Center in Michigan.

BACKGROUND AND OBJECTIVE: To investigate the effect of the coronavirus disease 2019 (COVID-19) lockdown on the presentation and management of acute, primary rhegmatogenous retinal detachment (RRD). PATIENTS AND METHODS: This was a single-center, consecutive case series with historic controls, examining patients during the COVID-19 "stay-at-home" order (March 24 to June 1, 2020), the subsequent reopening phase (June 1 to July 31, 2020), and corresponding preceding intervals (March 24 to July 31, 2016 to 2019). RESULTS: Despite a significant increase in patients presenting with macula-off RRD during the COVID-19 lockdown compared to the 2016 to 2019 timeframe (P = .03), the rate of single surgery anatomical success was similar between all groups (P = .66), as was final visual acuity (P = .61). No delays between presentation and surgical intervention were observed during the lockdown (P = .49). CONCLUSIONS: Despite the limitations of the COVID-19 lockdown, patients underwent surgery in a timely manner and achieved comparable visual outcomes to controls before COVID-19. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:593-600.].

Retinal Detachment After Endophthalmitis: Risk Factors and Outcomes.

PURPOSE: To analyze the risk factors, clinical course, and visual and anatomic outcomes of retinal detachment (RD) after endophthalmitis. PATIENTS AND METHODS: This retrospective study included 108 patients diagnosed with endophthalmitis between August 2014 and May 2019 at a single tertiary referral center. Sixteen patients developed RD after endophthalmitis. Retrospective analysis was performed to compare the cohort of endophthalmitis alone versus the cohort that developed RD after endophthalmitis, with analysis of potential risk factors for RD after endophthalmitis and treatment outcomes. RESULTS: The incidence of RD after endophthalmitis was 14.8% (N=16/108). The median time to develop RD after endophthalmitis was 27 days (range: 1-581 days, IQR: 25.3). Thirteen (81.3%) cases of RD occurred less than 2 months after the diagnosis of endophthalmitis. The incidence of aphakia (p=0.023) and posterior synechia (PS) (p=0.014) were significantly higher in the RD group. The mean initial and final visual acuity (VA) of the endophthalmitis alone group was 1.9±0.8 logMAR and 1.2±1.0 logMAR (p<0.0001), respectively, and 1.9±0.9 logMAR and 1.3±1.2 logMAR (p=0.07) in the RD group, respectively. Enucleation or evisceration occurred in 31.3% of cases with RD after endophthalmitis. The rate of final retinal re-apposition for the RD cohort was 56.3%. CONCLUSION: The anatomic and functional outcomes for RD after endophthalmitis remain poor, with significant risk for permanent vision loss. Aphakia and posterior synechiae were seen more often in cases with RD after endophthalmitis.

Surgical repair of primary non-complex rhegmatogenous retinal detachment in the modern era of small-gauge vitrectomy.

OBJECTIVE: To report anatomic and visual outcomes of pars plana vitrectomy (PPV), as well as scleral buckling (SB) and PPV/SB as surgical treatments for the management of primary, non-complex rhegmatogenous retinal detachment (RRD). METHODS AND ANALYSIS: Data from 751 eyes that underwent PPV, SB or combined PPV/SB as a surgical treatment for primary non-complex RRD with at least 3 months of follow-up were analysed to determine rates of single surgery anatomic success (SSAS) and final anatomic success (FAS). Patients or the public were not involved in the design, conduct or reporting of this research. RESULTS: PPV accounted for 89.0% (n=668), PPV/SB for 6.8% (n=51) and SB for 4.2% (n=32) cases. Overall SSAS (91.2% PPV, 84.3% PPV/SB, 93.8% SB; p=0.267) and FAS (96.7% PPV, 94.1% PPV/SB and 100.0% SB; p=0.221) were reported for the three surgical groups. SSAS and FAS were similar for lens status, macular detachment status and the presence or absence of inferior retinal breaks for each of the PPV, PPV/SB and SB groups. CONCLUSIONS: In this large, single institution, retrospective case series, we report surgical outcomes for patients with primary non-complex RRD managed with PPV, SB or PPV/SB in the modern era of small-gauge vitrectomy. We demonstrate that primary PPV without adjunct SB provides excellent anatomic and visual outcomes irrespective of lens status, macular involvement or pathology location.