Corneal Endothelium Viability Assay Using Trypan Blue Dye After Preloaded Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: The purpose of this study was to establish a validated method, consistent with Eye Bank Association of America medical standards, for evaluating endothelial cell loss (ECL) from an entire Descemet membrane endothelial keratoplasty (DMEK) graft using trypan blue dye as an alternative to specular microscopy. METHOD: Twenty-nine corneas were prepared for preloaded DMEK by a single technician, and the endothelium was stained with trypan blue dye for 30 seconds. The technician estimated total cell loss as a percentage of the graft and captured an image. Images were evaluated by a blinded technician using ImageJ software to determine ECL and compared with endothelial cell density from specular microscopy. Tissue processing intervals were analyzed for 4 months before and after implementation of this method. RESULTS: For the 29 grafts, there was no statistically significant difference ( t test, P = 0.285) between ECL estimated by a processor (mean = 5.8%) and ECL calculated using an ImageJ software (mean = 5.1%). The processor tended to estimate greater ECL than the actual ECL determined by ImageJ (paired t test, P = 0.022). Comparatively, postprocessing endothelial cell density measured by specular microscopy were higher compared with the preprocessing endothelial cell density (mean = 4.5% P = 0.0006). After implementation of this evaluation method, DMEK graft processing time intervals were reduced by 47.9% compared with specular microscopy evaluation ( P < 0.001). CONCLUSIONS: Our results show that visual ECL estimation using trypan blue staining by a DMEK graft processor is a reliable and efficient method for endothelial assessment. Unlike specular microscopy, this method achieves comprehensive visualization of the entire endothelium, reduces total time out of cold storage, and decreases total time required to prepare and evaluate DMEK grafts.

Development of Anterior Segment Focused Biologic Therapies to Regenerate Corneal Tissue for the Treatment of Disease: Drug Development Experience.

On February 24-27, 2021, the Association for Ocular Pharmacology and Therapeutics (AOPT) held its 15th biennial scientific meeting online. The meeting was organized by Dr. Sanjoy Bhattacharya of the University of Miami in conjunction with the board of trustees of the AOPT. The 3-day conference was attended by academic scientists, clinicians, and industry and regulatory professionals. The theme of the meeting was Restoring Vision through Regeneration and it was sponsored, in part, by the National Institutes of Health, Bright Focus, Regeneron, and Santen (USA). During the 3 days of the meeting, presentations from several sessions explored different aspects of regenerative medicine in ophthalmology, including optic nerve regeneration, drugs and devices in glaucoma, retinal neuroprotection and plasticity, visual perception, and degeneration of trabecular meshwork. This article summarizes the proceedings of the session on corneal regenerative medicine research and discusses emerging concepts in drug development for corneal epithelial and endothelial regeneration. Since the meeting in 2021, several of these concepts have advanced to clinical-stage therapies, but so far as of 2023, none has been approved by regional regulatory authorities in the United States. One form of corneal endothelial cell therapy has been approved in Japan and only for bullous keratopathy. Ongoing work is proceeding in the United States and other countries. Clinical Registration No: National Clinical Trials 04894110, 04812667; Japan Registry for Clinical Trials a031210199.

DMEK surgical training: An instructional guide on various wet-lab methods.

Descemet membrane endothelial keratoplasty (DMEK) is a partial-thickness corneal transplantation procedure that involves selective transplantation of the Descemet membrane and endothelium. DMEK offers significant advantages over other keratoplasty techniques, such as faster visual rehabilitation, better final visual acuity due to minimal optical interface effects, lower risk of allograft rejection, and less long-term dependence on topical steroids. Despite all its advantages, DMEK has been found to be more challenging than other corneal transplantation techniques, and its steep learning curve appears to be an obstacle to its widespread use and adoption by corneal surgeons worldwide. DMEK surgical training laboratories (wet labs) provide a window of opportunity for surgeons to learn, prepare, manipulate, and deliver these grafts in a risk-free environment. Wet labs are a significant learning tool, especially for those institutions that have limited tissue availability in their local centers. We provide a step-by-step guide for preparing DMEK grafts using different techniques on human and nonhuman models with instructional videos. This article should eventually help the trainees and the educators understand the requirements for performing DMEK and conducting a DMEK wet lab and develop their skills and interests from a wide variety of available techniques.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

Histological characterization of retinal degeneration in mucopolysaccharidosis type IIIC.

Heparan-α-glucosaminide N-acetyltransferase (HGSNAT) participates in lysosomal degradation of heparan sulfate. Mutations in the gene encoding this enzyme cause mucopolysaccharidosis IIIC (MPS IIIC) or Sanfilippo syndrome type C. MPS IIIC patients exhibit progressive neurodegeneration, leading to dementia and death in early adulthood. Currently there is no approved treatment for MPS IIIC. Incidences of non-syndromic retinitis pigmentosa and early signs of night blindness are reported in some MPS IIIC patients, however the majority of ocular phenotypes are not well characterized. The goal of this study was to investigate retinal degeneration phenotype in the Hgsnat knockout mouse model of MPS IIIC and a cadaveric human MPS IIIC eye. Cone and rod photoreceptors in the eyes of homozygous 6-month-old Hgsnat knockout mice and their wild-type counterparts were analyzed using cone arrestin, S-opsin, M-opsin and rhodopsin antibodies. Histological observation was performed on the eye from a 35-year-old MPS IIIC donor. We observed a nearly 50% reduction in the rod photoreceptors density in the Hgsnat knockout mice compared to the littermate wild-type controls. Cone photoreceptor density was unaltered at this age. Severe retinal degeneration was also observed in the MPS IIIC donor eye. To our knowledge, this is the first report characterizing ocular phenotypes arising from deleterious variants in the Hgsnat gene associated with MPS IIIC clinical phenotype. Our findings indicate retinal manifestations may be present even before behavioral manifestations. Thus, we speculate that ophthalmological evaluations could be used as diagnostic indicators of early disease, progression, and end-point evaluation for future MPS IIIC therapies.

COVID-19 and Eye Banking: Ongoing Impacts of the Pandemic.

PURPOSE: The purpose of this study was to assess the impact of ongoing waves of the COVID-19 pandemic and resulting guidelines on the corneal donor pool with resumption of clinical operations. METHODS: A retrospective analysis of donors deemed eligible for corneal transplantation at an eye bank from July 1, 2020, through December 31, 2021. Donors ineligible due to meeting Eye Bank Association of America (EBAA) COVID-19 guidelines or a positive postmortem COVID-19 testing were examined. The correlation between COVID-19 rule outs and state COVID positivity was calculated. The number of scheduled surgeries, suitable corneas, imports, and international exports was compared with a pre-COVID period. Postmortem testing was reduced for the final 5 months of the study, and numbers were compared before and after the policy change. RESULTS: 2.85% of referrals to the eye bank were ruled out because of EBAA guidelines. 3.2% of postmortem tests were positive or indeterminate resulting in an ineligible tissue donor (0.42% of referrals). Over the 18-month period, there was a 4.30% shortage of suitable corneas compared with transplantation procedures. There was a significant correlation between postmortem testing and state COVID-19 positivity (r = 0.37, P <0.01), but not with EBAA guidelines (r = 0.19, P = 0.07). When postmortem testing was reduced, significantly more corneas were exported internationally. CONCLUSIONS: Although corneal transplant procedures were back to normal levels, there was a shortage of suitable corneal tissue. The discontinuation of postmortem testing was associated with a significant increase in international exports of corneal donor tissue.

Effect of Increasing Povidone-Iodine Exposure on Corneal Epithelium and Impact on Donor Rim Cultures.

PURPOSE: The purpose of this study was to assess the effect of a second povidone-iodine (PVP-I) application at the time of donor tissue recovery on overall tissue quality and to analyze the rate of positive fungal and bacterial rim cultures before and after implementing increased PVP-I exposure. METHODS: The left cornea was recovered after a single application of PVP-I, while the right cornea was recovered after double PVP-I application in research-consented donors. The epithelial cell death rate was estimated using viability assay in corneal whole mounts under 10× objective (n = 5). Clinical characteristics of epithelium, stroma, and endothelium; positive rim culture rate; and incidences of infectious postoperative adverse reactions were compared for a period of 14 months before and after implementation of increased PVP-I protocol. RESULTS: The average epithelial cell death rate was unaltered between single and double PVP-I exposure groups. We observed a modest 10% increase in the number of tissues with mild edema after implementation of increased PVP-I exposure. Nonetheless, the percentage of tissues with moderate or severe edema was unaltered. The average positive rim culture rate decreased from 1.17% to 0.88% (P = 0.075) after implementation of the double PVP-I soak procedure. There has been only one report of infectious postoperative adverse reactions since this procedure change. By contrast, there were 5 reports for a period of 14 months before implementation of this protocol. CONCLUSIONS: These results indicate that new donor preparation methods with an additional 5 minutes of PVP-I exposure do not affect tissue quality, reduce positive rim cultures, and lead to lower incidence of postoperative infection.

Neuronal Bmal1 regulates retinal angiogenesis and neovascularization in mice.

Circadian clocks in the mammalian retina regulate a diverse range of retinal functions that allow the retina to adapt to the light-dark cycle. Emerging evidence suggests a link between the circadian clock and retinopathies though the causality has not been established. Here we report that clock genes are expressed in the mouse embryonic retina, and the embryonic retina requires light cues to maintain robust circadian expression of the core clock gene, Bmal1. Deletion of Bmal1 and Per2 from the retinal neurons results in retinal angiogenic defects similar to when animals are maintained under constant light conditions. Using two different models to assess pathological neovascularization, we show that neuronal Bmal1 deletion reduces neovascularization with reduced vascular leakage, suggesting that a dysregulated circadian clock primarily drives neovascularization. Chromatin immunoprecipitation sequencing analysis suggests that semaphorin signaling is the dominant pathway regulated by Bmal1. Our data indicate that therapeutic silencing of the retinal clock could be a common approach for the treatment of certain retinopathies like diabetic retinopathy and retinopathy of prematurity.

Aster proteins mediate carotenoid transport in mammalian cells.

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme ß-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues. We now provide evidence that Aster (GRAM-domain-containing) proteins, recently recognized for their role in nonvesicular cholesterol transport, engage in carotenoid metabolism. Our analyses revealed that the StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity. We further showed that carotenoids and cholesterol compete for the same binding site. We established a bacterial test system to demonstrate that the StART-like domains of mouse and human Aster proteins can extract carotenoids from biological membranes. Mice deficient for the carotenoid catabolizing enzyme BCO2 concentrated carotenoids in Aster-B protein-expressing tissues such as the adrenal glands. Remarkably, Aster-B was expressed in the human but not in the mouse retina. Within the retina, Aster-B and BCO2 showed opposite expression patterns in central versus peripheral parts. Together, our study unravels the biochemical basis for intracellular carotenoid transport and implicates Aster-B in the pathway for macula pigment concentration in the human retina.

Histopathological assessments reveal retinal vascular changes, inflammation, and gliosis in patients with lethal COVID-19.

PURPOSE: The purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. METHODS: Eyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for Epon embedding and immunocytochemistry. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the controls. OCT-based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes; however, the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggests that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Povidone-Iodine Attenuates Viral Replication in Ocular Cells: Implications for Ocular Transmission of RNA Viruses.

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1ß) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

Impact of the COVID-19 Pandemic on Keratoplasty and Corneal Eye Banking.

PURPOSE: The purpose of this study was to assess the impact of COVID-19 guidelines for corneal donor tissue screening and the utility of routine postmortem COVID-19 testing of donors intended for surgical use at a single eye bank. METHODS: A retrospective analysis of referrals to and eligible donors from an eye bank between March 1, 2020, and June 30, 2020, was performed, with the same time period in 2019 as a control. Referrals who were not procured because of Eye Bank Association of America COVID-19 guidelines and eye bank-specific restrictions were noted. The results of 1 month of routine postmortem testing performed by the eye bank were examined. Analysis of variance tests were performed to assess the change between donors from 2019 to 2020. RESULTS: There was a significant reduction in both the number of total referrals to the eye bank (P = 0.044) and donors eligible for surgical transplantation (P = 0.031). Eye Bank Association of America COVID-19 guidelines reduced the number of referrals over this period by 4% to 14%. Of the 266 surgically eligible donors who received postmortem COVID-19 testing in June by the eye bank, 13 resulted positive (4.9%). CONCLUSIONS: Despite a reduction in referrals and eligible corneal transplant donors at a single eye bank, there was a surplus of surgically suitable corneal tissue during the first wave of the COVID-19 pandemic. Eye banks should consider routine postmortem COVID-19 testing to identify asymptomatic infected donors although the risk of transmission of COVID-19 from infected donors is unknown.

Histopathological assessments reveal retinal vascular changes, inflammation and gliosis in patients with lethal COVID-19.

PURPOSE: To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. DESIGN: Comparative analysis of human eyes. SUBJECTS: Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed. METHODS: Globes were evaluated ex-vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for epon-embedding and immunocytochemistry. MAIN OUTCOME MEASURES: Retinal thickness and histopathology, detection of SARS-CoV-2 Spike protein, changes in vascular density, gliosis, and degree of inflammation. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the control. OCT based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes, however the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggested that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues.

BACKGROUND: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. METHODS: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in additional 10 research-intent COVID-19 positive donors. FINDINGS: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection. INTERPRETATIONS: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

Thyroid Activating Enzyme, Deiodinase II Is Required for Photoreceptor Function in the Mouse Model of Retinopathy of Prematurity.

Purpose: Retinopathy of prematurity (ROP) is a severe complication of premature infants, leading to vision loss when untreated. Presently, the molecular mechanisms underlying ROP are still far from being clearly understood. This study sought to investigate whether thyroid hormone (TH) signaling contributes to the neuropathology of ROP using the mouse model of ROP to evaluate longitudinal photoreceptor function. Methods: Animals were exposed to hyperoxia from P7 to P12 to induce retinopathy, thereafter the animals were returned to room air (normoxia). The thyroid-activating enzyme type 2 deiodinases (Dio2) knockout (KO) mice and the littermate controls that were exposed to hyperoxia or maintained in room air and were then analyzed. The retinal function was evaluated using electroretinograms (ERGs) at three and seven weeks followed by histologic assessments with neuronal markers to detect cellular changes in the retina. Rhodopsin protein levels were measured to validate the results obtained from the immunofluorescence analyses. Results: In the ROP group, the photoreceptor ERG responses are considerably lower both in the control and the Dio2 KO animals at P23 compared to the non-ROP group. In agreement with the ERG responses, loss of Dio2 results in mislocalized cone nuclei, and abnormal rod bipolar cell dendrites extending into the outer nuclear layer. The retinal function is compromised in the adult Dio2 KO animals, although the cellular changes are less severe. Despite the reduction in scotopic a-wave amplitudes, rhodopsin levels are similar in the adult mice, across all genotypes irrespective of exposure to hyperoxia. Conclusions: Using the mouse model of ROP, we show that loss of Dio2 exacerbates the effects of hyperoxia-induced retinal deficits that persist in the adults. Our data suggest that aberrant Dio2/TH signaling is an important factor in the pathophysiology of the visual dysfunction observed in the oxygen-induced retinopathy model of ROP.

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues.

BACKGROUND: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. METHODS: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors. FINDINGS: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection. INTERPRETATIONS: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

Prenatal alcohol exposure and maternal glutamine supplementation alter the mTOR signaling pathway in ovine fetal cerebellum and skeletal muscle.

Prenatal alcohol exposure causes fetal neurodevelopmental damage and growth restriction. Among regions of the brain, the cerebellum is the most vulnerable to developmental alcohol exposure. Despite vast research in the field, there is still a need to identify specific mechanisms by which alcohol causes this damage in order to design effective therapeutic interventions. The mammalian target of rapamycin (mTOR) is known to be associated with axonal regeneration, dendritic arborization, synaptic plasticity, cellular growth, autophagy, and many other cellular processes. Glutamine and glutamine-related amino acids play a key role in fetal development and are known to alter the mTOR pathway; recent research has shown that disturbances in their bioavailability and signaling pathways may mediate adverse effects of prenatal alcohol exposure. This study investigated the role of the mTOR signaling pathway in the fetal cerebellum and skeletal muscle after third trimester-equivalent prenatal alcohol exposure and maternal l-glutamine (GLN) supplementation using a sheep model. Fetal cerebella and skeletal muscles were sampled for Western blot analysis of mTOR and its downstream targets S6 kinase and eukaryotic initiation factor 4E-bindin protein (4E-BP1). The expression of cerebellar phosphorylated mTOR relative to the total mTOR was elevated in the alcohol+GLN group compared to the saline and GLN groups. Alcohol exposure increased the ratio of phosphorylated S6K to total S6K in fetal cerebellum, and no significant effect of GLN supplementation was observed. On contrary, maternal GLN supplementation reduced the activation of mTOR and S6K in fetal skeletal muscle, possibly to make GLN and other amino acids available for use by other organs. These findings suggest prenatal alcohol exposure and maternal GLN supplementation during the third trimester-equivalent alter the mTOR signaling cascade, which plays a possible key role in alcohol-induced developmental damage.

Maternal choline supplementation mitigates alcohol-induced fetal cranio-facial abnormalities detected using an ultrasonographic examination in a sheep model.

Early detection of prenatal alcohol exposure is critical for designing and testing effectiveness of interventional therapeutics. Choline supplementation during and after prenatal alcohol exposure has shown promising benefits in improving outcomes in rodent models and clinical studies. A sheep model of first trimester-equivalent binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Pregnant sheep were randomly assigned to six groups: Saline + Placebo control, Saline + Choline, binge Alcohol + Placebo (light binging), binge Alcohol + Choline, Heavy binge Alcohol + Placebo (heavy binging), and Heavy binge Alcohol + Choline. Ewes received intravenous alcohol or saline on three consecutive days per week from gestation day (GD) 4-41 to mimic a first trimester-equivalent weekend binge-drinking paradigm. Choline (10 mg/kg in the daily food ration) was administered from GD 4 until term. On GD 76, 11 fetal ultrasonographic measurements were collected transabdominally. Heavy binge alcohol exposure reduced fetal Frontothalamic Distance (FTD), Mean Orbital Diameter (MOD), and Mean Lens Diameter (MLD), and increased Interorbital Distance (IOD) and Thalamic Width (TW). Maternal choline supplementation mitigated most of these alcohol-induced effects. Maternal choline supplementation also improved overall fetal femur and humerus bone lengths, compared to their respective placebo groups. Taken together, these results indicate a potential dose-dependent effect that could impact the sensitivity of these ultrasonographic measures in predicting prenatal alcohol exposure. This is the first study in the sheep model to identify biomarkers of prenatal alcohol exposure in utero with ultrasound and co-administration of maternal choline supplementation.

The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.

A single pool of multipotent retinal progenitor cells give rise to the diverse cell types within the mammalian retina. Such cellular diversity is due to precise control of various cellular processes like cell specification, proliferation, differentiation, and maturation. Circadian clock genes can control the expression of key regulators of cell cycle progression and therefore can synchronize the cell cycle state of a heterogeneous population of cells. Here we show that the protein encoded by the circadian clock gene brain and muscle arnt-like protein-1 (Bmal1) is expressed in the embryonic retina and is required to regulate the timing of cell cycle exit. Accordingly, loss of Bmal1 during retinal neurogenesis results in increased S-phase entry and delayed cell cycle exit. Disruption in cell cycle kinetics affects the timely generation of the appropriate neuronal population thus leading to an overall decrease in the number of retinal ganglion cells, amacrine cells, and an increase in the number of the late-born type II cone bipolar cells as well as the Müller glia. Additionally, the mislocalized Müller cells are observed in the photoreceptor layer in the Bmal1 conditional mutants. These changes affect the functional integrity of the visual circuitry as we report a significant delay in visual evoked potential implicit time in the retina-specific Bmal1 null animals. Our results demonstrate that Bmal1 is required to maintain the balance between the neural and glial cells in the embryonic retina by coordinating the timing of cell cycle entry and exit. Thus, Bmal1 plays an essential role during retinal neurogenesis affecting both development and function of the mature retina.-Sawant, O. B., Jidigam, V. K., Fuller, R. D., Zucaro, O. F., Kpegba, C., Yu, M., Peachey, N. S., Rao, S. The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.

Maternal and fetal effects of dexmedetomidine infusion in pregnant ewes anesthetized with sevoflurane.

OBJECTIVE To characterize the maternal and fetal cardiopulmonary effects of a low-dose infusion of dexmedetomidine without a loading dose in pregnant ewes anesthetized with sevoflurane. ANIMALS 11 pregnant ewes. PROCEDURES Anesthesia was induced with propofol and maintained with sevoflurane. Ewes and fetuses were instrumented with arterial and venous catheters, and thermodilution-pulmonary arterial catheters were placed in the ewes. Baseline measurements were obtained at an end-tidal sevoflurane concentration of 3.4%, then dexmedetomidine (2 µg/kg/h, IV) was infused for 90 minutes without a loading dose. Cardiovascular and blood gas variables were measured at predetermined time points. RESULTS Dexmedetomidine infusion resulted in approximately 30% decreases in maternal systemic vascular resistance, blood pressure, and heart rate. Maternal cardiac index, oxygenation variables, and acid-base status remained unchanged, whereas pulmonary arterial pressure, pulmonary vascular resistance, and stroke volume increased, compared with baseline values. Uterine blood flow decreased by approximately 30% to 36%. Fetal heart rate and blood pressure remained unchanged, but significant increases in fetal plasma glucose and lactate concentrations were detected. CONCLUSIONS AND CLINICAL RELEVANCE Pregnant ewes receiving a combination of sevoflurane and an infusion of dexmedetomidine without a loading dose had cardiac index in acceptable ranges and maintained normoxia. This balanced anesthesia did not produce significant changes in fetal blood pressure or heart rate. However, the increase in fetal plasma lactate concentration and changes in maternal pulmonary vascular resistance and uterine blood flow require further investigation to better elucidate these effects.