Long-Term Outcomes of Vitrectomy for Idiopathic Epiretinal Membrane With Internal Limiting Membrane Removal in Patients With Good Preoperative Visual Acuity.

Purpose: To evaluate the long-term visual results of vitrectomy with epiretinal membrane (ERM) and internal limiting membrane (ILM) removal for idiopathic ERM in eyes with a preoperative visual acuity (VA) of 20/50 or better. Methods: This retrospective review of a consecutive case series comprised 337 patients. Of these, 36 eyes of 36 patients had ERM and ILM removal from 2017 to 2018. Inclusion criteria included a subjective decrease in VA, a preoperative VA of 20/50 or better, vitrectomy with ERM and ILM removal for ERM, and a minimum 6-month follow-up. Paired t tests were used to determine the statistical significance (P < .05) of VA changes postoperatively. Results: The mean (±SD) best-corrected logMAR VA improved to a maximum of 0.125 ± 0.09 (Snellen equivalent 20/26.4) at a mean of 11.1 months postoperatively (P < .001). The VA continued to significantly improve over the long term (mean, 41.6 months; range, 6-63; P < .001). Overall long-term data trended heavily toward VA improvement (25/36 patients [69.4%]) and stability (10/36 patients [27.7%)] after ERM and ILM removal, with only 1 patient (2.8%) having worse VA. There were no intraoperative or postoperative complications related to ERM and ILM removal. Conclusions: Surgery to remove idiopathic ERM and ILM for patients with significant symptoms and good preoperative VA may result in excellent long-term visual results.

Pembrolizumab-Induced Nodular Posterior Scleritis.

Purpose: To report a case of painless posterior scleritis presenting as a choroidal nodule in a patient with history of a tumor being treated with pembrolizumab. Methods: A case and its findings were analyzed, and a relevant literature review was performed. Results: A 20-year-old woman with a history of ependymoma presented with painless blurred vision in the right eye after being started on pembrolizumab for a tumor recurrence. Fundoscopy showed a solitary amelanotic choroidal lesion with surrounding subretinal fluid in the affected eye. Ultrasonography showed moderate internal reflectivity and fluid in Tenon capsule consistent with nodular posterior scleritis. After a course of systemic steroids and discontinuation of the pembrolizumab, the choroidal lesion completely resolved. Conclusions: Clinicians should be aware of posterior scleritis as an ocular complication of this class of medications.

Visual outcomes of macula-involving rhegmatogenous retinal detachment in patients with and without age-related macular degeneration.

BACKGROUND: The prognosis for patients with macula-off rhegmatogenous retinal detachment (RRD) and concomitant age-related macular degeneration (AMD) is not well known. The purpose of this study is to compare visual outcomes in macula-off RRD in eyes with AMD versus a group of comparison eyes without AMD. METHODS: This was a retrospective chart review of 1149 patients. A total of 191 eyes met study criteria, 162 non-AMD eyes (controls), and 29 AMD eyes. The main outcome measure was postoperative visual acuity following pars plana vitrectomy (PPV), scleral buckle (SB), or combined PPV/SB in control eyes versus AMD eyes. This was compared using Fisher's exact test. RESULTS: There was a statistically significant difference in postoperative visual acuity by AMD status, with those without AMD having a worse visual outcome overall (p = 0.0048). A similar percentage of AMD versus non-AMD eyes achieved vision better than 20/40. More patients in the non-AMD group achieved a final visual acuity between 20/40 and 20/200. Of patients with AMD, more had vision worse than 20/200 though 58% maintained functional vision (better than 20/200). Those without AMD had a higher frequency of Count Fingers (CF), Hand Motion (HM), Light Perception (LP), or No Light Perception (NLP) vision (p = 0.023). CONCLUSIONS: Though postoperative visual acuity was worse overall in the non-AMD group with a higher frequency of patients having final vision of CF, HM, LP, or NLP, this is likely a function of the difference in sample size and composition between the two groups. Importantly, this study suggests AMD patients can expect similar outcomes to non-AMD patients after RRD repair. We conclude that AMD patients can achieve functional vision after RRD surgery, similar to those without AMD.

Pax6 mutant cerebral organoids partially recapitulate phenotypes of Pax6 mutant mouse strains.

Cerebral organoids show great promise as tools to unravel the complex mechanisms by which the mammalian brain develops during embryogenesis. We generated mouse cerebral organoids harbouring constitutive or conditional mutations in Pax6, which encodes a transcription factor with multiple important roles in brain development. By comparing the phenotypes of mutant organoids with the well-described phenotypes of Pax6 mutant mouse embryos, we evaluated the extent to which cerebral organoids reproduce phenotypes previously described in vivo. Organoids lacking Pax6 showed multiple phenotypes associated with its activity in mice, including precocious neural differentiation, altered cell cycle and an increase in abventricular mitoses. Neural progenitors in both Pax6 mutant and wild type control organoids cycled more slowly than their in vivo counterparts, but nonetheless we were able to identify clear changes to cell cycle attributable to the absence of Pax6. Our findings support the value of cerebral organoids as tools to explore mechanisms of brain development, complementing the use of mouse models.

Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Using organoids to study human brain development and evolution.

Recent advances in methods for making cerebral organoids have opened a window of opportunity to directly study human brain development and disease, countering limitations inherent in non-human-based approaches. Whether freely patterned, guided into a region-specific fate or fused into assembloids, organoids have successfully recapitulated key features of in vivo neurodevelopment, allowing its examination from early to late stages. Although organoids have enormous potential, their effective use relies on understanding the extent of their limitations in accurately reproducing specific processes and components in the developing human brain. Here we review the potential of cerebral organoids to model and study human brain development and evolution and discuss the progress and current challenges in their use for reproducing specific human neurodevelopmental processes.

Cerebral organoids as tools to identify the developmental roots of autism.

Some autism spectrum disorders (ASD) likely arise as a result of abnormalities during early embryonic development of the brain. Studying human embryonic brain development directly is challenging, mainly due to ethical and practical constraints. However, the recent development of cerebral organoids provides a powerful tool for studying both normal human embryonic brain development and, potentially, the origins of neurodevelopmental disorders including ASD. Substantial evidence now indicates that cerebral organoids can mimic normal embryonic brain development and neural cells found in organoids closely resemble their in vivo counterparts. However, with prolonged culture, significant differences begin to arise. We suggest that cerebral organoids, in their current form, are most suitable to model earlier neurodevelopmental events and processes such as neurogenesis and cortical lamination. Processes implicated in ASDs which occur at later stages of development, such as synaptogenesis and neural circuit formation, may also be modeled using organoids. The accuracy of such models will benefit from continuous improvements to protocols for organoid differentiation.

The transcription factor E2A drives neural differentiation in pluripotent cells.

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

Fears, Depression, and Anxieties of Patients With Diabetic Retinopathy and Implications for Education and Treatment.

Objective: This study investigates undiagnosed depression and anxiety related to diabetes in patients with diabetic retinopathy and identifies commonly feared complications that these patients experience. Methods: The 74 consecutive individuals with diabetes were recruited for this investigation from the office of a retina specialist, and data were obtained from the participants through a self-report survey given to the patients before their appointment. Results: The most feared complication reported by patients surveyed was blindness (38.36%). When asked about depression and anxiety related to their diabetes, 20.27% of patients stated they have depression related to their diabetes, whereas 18.92% had anxiety related to their diabetes. Only 17.57% of the patients said they were being treated for their depression and/or anxiety at the time of the survey. Conclusions: This study demonstrates that many patients with diabetic retinopathy have coexisting fears and mental health concerns. Because most retina specialists treat a high number of patients with diabetes, it is crucial to understand the barriers and comorbidities related to this patient population. Retina specialists may play a role in identifying the hidden and underlying fears, depression, and anxieties in patients with diabetes so that these patients can get the necessary help and counseling they need.

Mouse vs man: Organoid models of brain development & disease.

Brain organoids have rapidly become established as promising tools for studying both the normal embryonic development of the brain and the mechanistic roots of neurodevelopmental disorders. Most recent studies are based on brain organoids derived from human pluripotent stem cells (PSCs), as these are likely to be the best way to understand normal human development and disease. However, brain organoids grown from mouse cells still have a role to play. We discuss recent work showing how mice and mouse organoids can be employed to complement studies using human organoids. Mouse stem cell-derived organoids are useful for the development of improved protocols to generate organoids, including brain region-specific organoids. Importantly, the wealth of existing in vivo data on mouse brain development together with detailed descriptions of mutant phenotypes provide invaluable points of comparison to validate organoids as tools to study the genetics of brain development. Further, organoids have significant potential to replace or reduce the numbers of animals used in studies of normal brain development.

Loss of Pax6 Causes Regional Changes in Dll1 Expression in Developing Cerebral Cortex.

The transcription factor Pax6 controls multiple aspects of forebrain development. Conditional deletion of Pax6 in embryonic mouse cortex causes increased proliferation of cortical progenitor cells and a concomitant decrease in neural differentiation. Notch signaling regulates the balance between proliferation and differentiation of cortical progenitor cells, suggesting a possible connection between Pax6 and Notch signaling. We investigated how expression of the Notch ligand delta-like 1 (Dll1) is altered by loss of Pax6. Acute cortex-specific deletion of Pax6 resulted in a widespread decrease in the density of Dll1+ cells at embryonic days 12.5 and 13.5 (E12.5 and E13.5). In constitutive loss-of-function mutants, decreases in the densities of Dll1+ cells were more limited both spatially and temporally. Controlled over-expression of Pax6 had no detectable effect on Dll1 expression. The proneural transcription factor Neurog2 is a target of Pax6 that can activate Dll1 expression and we found clear co-expression of Neurog2 and Dll1 in radial glial progenitors, suggesting that Pax6's effect on Dll1 could be mediated through Neurog2. However, we found no change in Dll1+ cells in Neurog2 -/- cortex suggesting either that Neurog2 is not directly involved, or that its loss of function in embryonic cortex can be compensated for.

Heparan Sulfate Sulfation by Hs2st Restricts Astroglial Precursor Somal Translocation in Developing Mouse Forebrain by a Non-Cell-Autonomous Mechanism.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st-/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st-/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS.SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.

Tissue-Specific Actions of Pax6 on Proliferation and Differentiation Balance in Developing Forebrain Are Foxg1 Dependent.

Differences in the growth and maturation of diverse forebrain tissues depend on region-specific transcriptional regulation. Individual transcription factors act simultaneously in multiple regions that develop very differently, raising questions about the extent to which their actions vary regionally. We found that the transcription factor Pax6 affects the transcriptomes and the balance between proliferation and differentiation in opposite directions in the diencephalon versus cerebral cortex. We tested several possible mechanisms to explain Pax6's tissue-specific actions and found that the presence of the transcription factor Foxg1 in the cortex but not in the diencephalon was most influential. We found that Foxg1 is responsible for many of the differences in cell cycle gene expression between the diencephalon and cortex and, in cortex lacking Foxg1, Pax6's action on the balance of proliferation versus differentiation becomes diencephalon like. Our findings reveal a mechanism for generating regional forebrain diversity in which one transcription factor completely reverses the actions of another.

Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors.

Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene Cdk6, which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6, other Pax6-regulated cell cycle genes are likely to be primary mediators of Pax6's actions on cortical progenitor cell cycles. Following acute cortex-specific deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6's actions. We then used flow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase.

Gli3 controls the onset of cortical neurogenesis by regulating the radial glial cell cycle through Cdk6 expression.

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.

The Role of Focal Laser in the Anti-Vascular Endothelial Growth Factor Era.

INTRODUCTION: To review important studies examining focal laser for diabetic macular edema (DME), to examine real-world data regarding actual treatments patients are receiving, to present long-term visual outcomes in real-world practice, and to suggest an evidence-based approach for the use of focal laser. METHODS: This study is a review of landmark studies evaluating focal laser and pharmacologic therapy for DME. In addition, the authors include a retrospective review of 102 consecutive eyes of 53 patients in our practice setting in rural Alabama. A chart review was performed, and patients were included if they were diagnosed with DME and were treated with both focal laser and bevacizumab. Bevacizumab and focal laser were given on a "as needed basis" at the discretion of one treating physician (J.O.M.). Worse visual acuity or worsening macular edema were indications for additional treatment. Statistical analysis was performed using frequencies and percentages. Best-corrected visual acuity (BCVA) was recorded at baseline and at the end of treatment (mean of 5 years) in the medical record. Primary outcome measures were BCVA, patients with better than 20/40 BCVA, patients with worse than 20/200 BCVA, and patients with stable BCVA. RESULTS: Anti-vascular endothelial growth factor (VEGF) therapies are the first-line treatment for DME, but real-world claims data suggest that diabetic patients cannot come in for monthly injections as in large clinical trials. In our series, after a mean of 5 lasers and 5.5 injections, 90% of eyes had stable or better BCVA, 65% were ≥20/40, and only 13% were ≤20/200. CONCLUSIONS: Laser treatment for DME remains an important adjunctive therapy.

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing.

BACKGROUND: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. RESULTS: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. CONCLUSIONS: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

Lateral cortical Cdca7 expression levels are regulated by Pax6 and influence the production of intermediate progenitors.

BACKGROUND: We studied whether regulation of Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6's cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6's effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6's expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis. METHODS: We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6 -/- embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells. RESULTS: We found that Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation. CONCLUSION: Our results suggest that Pax6 normally represses Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.

Vitrectomy and Vitrector Port Needle Biopsy of Choroidal Melanoma for Gene Expression Profile Testing Immediately before Brachytherapy.

PURPOSE: Transvitreal and transscleral needle biopsy can result in complications including vitreous hemorrhage and retinal detachment. This study evaluated a technique using 25-gauge vitrectomy as an adjunct to needle biopsy immediately before brachytherapy to minimize these complications and preserve good visual acuity. DESIGN: Retrospective, observational case series. PARTICIPANTS: Fifty-seven consecutive eyes of 57 patients with treatment-naïve medium choroidal melanomas without extraocular extension from July 2012 through September 2015. METHODS: Fifty-seven consecutive eyes of 57 patients with a clinically diagnosed choroidal melanoma underwent complete 25-gauge posterior vitrectomy followed by transvitrector port fine-needle aspiration biopsy of the tumor immediately before implantation of a radioactive iodine 125 plaque as treatment for the tumor. Cytopathologic analysis was not performed on the tumor aspirates in this study. MAIN OUTCOME MEASURES: Best-corrected postoperative visual acuity, postoperative complications of the reported technique, implantation tumor development, local tumor recurrence, presence of metastatic disease after surgery, and sufficiency of the tumor aspirates obtained by the reported technique for successful gene expression profile testing and prognostic classification. RESULTS: Mean preoperative and postoperative visual acuities were similar (20/60 vs. 20/80, respectively). Mean tumor thickness was 5.0 mm (range, 2.5-10 mm) and mean tumor basal diameter was 13.1 mm (range, 7-22 mm). Only 1 of 57 eyes (1.8%) showed a transient vitreous hemorrhage, biopsy yield was 100% for genetic analysis, and no patients showed recurrence or implantation tumor at the vitrector site. CONCLUSIONS: Combined 25-gauge posterior vitrectomy and 25-gauge trans-vitrector port needle aspiration biopsy immediately before brachytherapy is excellent for obtaining tumor aspirate for gene expression profiling while controlling for hemostasis, resulting in few complications.