Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed sFlt1 or sFlt4 were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied ex vivo to reduce the risk of failure in cases of "high-risk" corneal transplantation.

The human macula is a specialized, cone-rich region of the eye, critical for high-acuity vision, yet the pathways regulating its development remain poorly understood. RA-catabolizing enzyme CYP26A1 establishes the chick high-acuity area via upregulation of fibroblast growth factor 8 (FGF8). However, detailed analysis of this pathway and its functions has not been performed in early human fetal tissue. Fluorescent in situ hybridization revealed striking biphasic CYP26A1 expression but little FGF8 in the presumptive macula region between post-conception weeks (PCW) 6-17. Pharmacological retinoic acid (RA) signaling inhibition in human retinal organoids mimicking the two waves of CYP26A1 revealed early RA inhibition prompted early cell cycle exit and increased cone genesis, while late inhibition altered cone subtype specification. Conversely, recombinant FGF8 had no effect on photoreceptor fate. This work provides spatiotemporal examination of CYP26A1 across human macular development, as well as experimental evidence for the different roles of RA signaling inhibition in a human model of retinal development.

Photoreceptor degeneration is a leading cause of untreatable sight loss. Previously, we showed that human pluripotent stem cell-derived cone photoreceptors (hCones) can rescue retinal function in the Rd1 mouse model of rod-cone dystrophy. However, retinal degenerations display markedly different severities and concomitant remodeling of the remaining retina; for photoreceptor replacement therapy to be broadly effective, it must work for a variety of disease phenotypes. Here, we sought to rescue the Aipl1-/- model of Leber congenital amaurosis, a particularly fast, severe condition. After transplantation of hCones, host cone bipolar cells underwent extensive remodeling and formed nascent synaptic-like connections. Electrophysiological recordings showed robust rescue of light-evoked activity across visually relevant photopic intensities, and treated mice exhibited visually evoked optokinetic head-tracking behavior. Thus, human cone photoreceptor replacement therapy is feasible even in very severe cases of retinal dystrophy, offering promise as a disease-agnostic therapy in Leber congenital amaurosis (LCA) and in other advanced retinal degenerations.

PURPOSE. Little is known about the development of Bruch's membrane (BrM), the structure separating and supporting the retina and choroid, nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies, both in the development of accurate in vitro models, and effective RPE transplant strategies. Here, we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE. METHODS. The presence of laminin, elastin, fibronectin, and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR). RESULTS. In human fetal retina, laminin and fibronectin were present from 6 PCW, type IV collagen from 8 PCW, elastin from 12 PCW, type I collagen by 17 PCW, and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW, becoming distinct by 17 PCW. In hPSC-derived RPE cultures, basement membranes containing laminin and fibronectin were present from week 1, type IV collagen from week 2, and type I collagen from week 4. Type III collagen was present at all timepoints, although not localized as a basement membrane. Elastin was absent at all timepoints. CONCLUSIONS. BrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development, excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease, and inform cell therapy approaches, as de novo BrM-like membrane has the potential to support transplanted donor RPE.

107 Background: Apalutamide became available for mHSPC along with androgen deprivation treatment (ADT) in the UK in November 2021. We present here, to our best knowledge the first real world multicentre experience of Apalutamide use in mHSPC patients from three tertiary cancer centres from the UK. Our primary aim was to assess for toxicity and tolerability at this stage. Methods: We collected retrospective data from hospital prescription registries including all patients with mHSPC receiving Apalutamide between November 2021 and January 2023. Median period of follow up in our cohort was 14 months. Results: Two hundreds and ten patients received Apalutamide for mHSPC from three centres. The median age was 73 years. A total of 16.4% of the patients had recurrent mHSPC previously treated for localised PC and 83% were de novo metastatic. Of all patients, 43.3% had high-volume disease, and 56.7% had low-volume disease. The median presenting Prostate Specific Antigen (PSA) was 62 ng/l at diagnosis (range 0.87 to 5580). Undetectable PSA achieved in 43.3% of patient after starting Apalutamide and ADT. For those who have detectable PSA, median value was 0.22. Ninety percent of patients maintained their performance status (PS) at 0 or 1 on treatment. Most common side effects were fatigue (35.7%) and rash (11.4%). Cardiac adverse effects were recorded in 1.9% and thromboembolic events in 0.4%. Overall grade 3 and 4 toxicity was 5.2% across all symptoms assessed. To date, 83.3% patients continue on Apalutamide, 7.6 % discontinued due to toxicity and 9.1% had progressive disease (PD). Three deaths were recorded and all due to PD. Conclusions: In this multicentre retrospective analysis we found that Apalutamide and ADT is well tolerated in the real-world setting. Our patients are older and had lower volume metastasis compared with patients from the registry Trial (TITAN). Fatigue is more prevalent. Majority patients continue on Apalutamide with manageable side effects and good PS.

Up to 30% of youth with concussion experience persistent post-concussive symptoms (PPCS) impacting cognitive, social, mental, and physical health. Limited evidence-based therapies exist to treat PPCS. We present a case of an adolescent female with multiple sports-related concussions with persistent symptoms, including headache, nausea, dizziness, brain fog, and anxiety. After 4 weeks of treatment with an auricular percutaneous electrical nerve field stimulation (PENFS) device, she had near-complete resolution of her symptoms, allowing a return to her previous level of academic achievement, as well as physical activity. Emerging evidence suggests that various forms of neuromodulation, such as repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), can benefit patients with post-concussive symptoms. This adolescent female's improvement with PENFS contributes to this expanding field.

The human macula is a specialized, M/L cone-rich region of the eye, critical for high acuity vision, but little is known about the pathways regulating its development. Transcriptional regulation by Retinoic Acid (RA) is essential for many aspects of human eye development. Here, we report a striking biphasic expression of the RA-catabolizing enzyme, CYP26A1, in early human macular development between post-conception weeks 6-17. Early inhibition of RA signaling in human retinal organoids (hROs) prompts early cell cycle exit, and an increase in cone photoreceptors, while late inhibition alters cone subtype specification. Conversely, FGF8, which is negatively regulated by RA and vital for High Acuity Area specification in chick, is not expressed in the nascent human macula and had no effect on hRO photoreceptor fate. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. * CMZ : Ciliary marginal zone CS : Carnegie Stage CYP26 : cytochrome P450 family 26 d : Day FGF : Fibroblast growth factor GCL : Ganglion cell layer HAA : High acuity area hROs : Human stem cell derived retinal organoids INL : Inner nuclear layer LE : Lens epithelium mROs : Mouse stem cell derived retinal organoids NBL : Neuroblastic Layer ONH : Optic Nerve Head ONL : Outer nuclear layer PCW : Post-conception weeks PM : Presumptive Macula RA : Retinoic Acid RAR : Retinoic Acid Receptor RARE : Retinoic Acid Response Element RFZ : Rod-free zone RGC : Retinal ganglion cells RPCs : Retinal Progenitor Cells RXR : Retinoid X receptor [1]: pending:yes

While COVID-19 is no longer the “hot-topic” it was 2 years ago, its prevalence and impact are still significant. In 2022, the CDC estimated that over 90% of children from 6 months to 17 years old have had COVID-19 infection. While most children have limited and mild symptoms, a substantial subset experiences significant neurological manifestations and/or complications which may lead to long-lasting morbidity or even mortality. Such neurological manifestations of SARS-CoV-2 include acute encephalitis, seizures, central demyelinating disease, cerebrovascular events, peripheral neurological disorders, and chronic symptoms in the setting of long COVID, which may affect up to 25% of infected children and adolescents. Given the high prevalence of COVID-19 in the general and pediatric population, it is essential for clinicians to understand the full breadth of its potential effects. In this article, we review common neurological manifestations and sequelae of SARS-CoV-2 in the pediatric population and describe their prevalence, timing, and associated neuroimaging findings.

Stem cell-based therapy has gained importance over the past decades due to huge advances in science and technology behind the generation and directed differentiation of pluripotent cells from embryos and adult cells. Preclinical proof-of-concept studies have been followed by clinical trials showing efficacy and safety of transplantation of stem cell-based therapy, which are beginning to establish this as a modality of treatment. Disease candidates of interest are primarily conditions that may benefit from replacing dead or dying cells, including advanced inherited retinal dystrophies and age-related macular degeneration, and predominantly seek to transplant either RPE or photoreceptors, although neurotrophic approaches have also been trialed. Whilst a consensus has yet to be reached about the best stage/type of cells for transplantation (stem cells, progenitor cells, differentiated RPE and photoreceptors) and the methods of implantation (sheet, suspension), several CTs have shown safety. There remain potential concerns regarding tumorigenicity and immune rejection; however, with ongoing improvements in cell generation, selection, and delivery, these can be minimized. Earlier studies showed efficacy with immunosuppressive drugs to prevent rejection, and recent donor-matched transplants have avoided the need for immunosuppression. Retinal regenerative medicine is a challenging field and is in a nascent stage but holds tremendous promise. This narrative review delves into the current understanding of stem cells and the latest clinical trials of retinal cell transplantation.

Objective:Herpes simplex virus (HSV) encephalitis can be associated with many secondary neurological complications, but having multiple episodes of recurrent neurological complications is rare in an individual. Understanding the course of each complication can reduce time to diagnosis and adequate treatment. Additionally, we postulate the role of RNF213 mutation in HSV susceptibility. Methods:We describe a unique presentation of HSV-1 encephalitis in an infant with a pathogenic RNF213 mutation who went on to develop multiple rare neurological complications over the course of her illness. Results:Our patient was first diagnosed with neonatal HSV-1 encephalitis at age 2 weeks. She had recurrence of HSV encephalitis (HSE) with associated vasculopathy that led to right middle cerebral artery and posterior cerebral artery infarctions at 13 months, and then later developed post-HSE anti-N-methyl- d-aspartate receptor encephalitis. All of this occurred concomitant with RNF213 mutation. Interpretation:This patient demonstrates that, though rare, multiple neurological complications can occur in a single person, thus highlighting the importance of close surveillance of patients with a history of neonatal HSE and pursuing a broad differential in patients with subtle or recurrent symptoms. Furthermore, we propose a potential role of RNF213 mutation in the pathogenesis of our patient's multiple medical conditions.

Background: Although millions of children sustain concussions each year, a rapid and objective test for concussion has remained elusive. The aim of this study was to investigate quantitative pupillometry in pediatric patients in the acute, postinjury setting. Methods: This was a prospective case -control study of concussed patients presenting to the emergency department within 72 hours of injury. Pupillary measurements were gathered using NeurOptics' PLR 3000; evaluation included a symptom checklist and neurocognitive assessment. Data were analyzed using descriptive statistics and regression models. Results: A total of 126 participants were enrolled. One significant difference in pupillometry between concussed and control participants was found: left minimum pupil diameter in 12- to 18 year -olds (P = 0.02). Models demonstrating odds of a concussion revealed significant associations for time to 75% recovery (T75) of the left pupil in five- to 11 -year -olds and average dilation velocity of the left pupil in 12- to 18 -year -olds (P = 0.03 and 0.02 respectively). Models predicting symptom improvement showed one significant association: percent change of the right pupil in five -to -11 -year -olds (P = 0.02). Models predicting neurocognitive improvement in 12- to 18 -year -olds demonstrated significant association in T75 in the left pupil for visual memory, visual motor processing speed, and reaction time (P = 0.002, P = 0.04, P = 0.04). Conclusions: The limited statistically significant associations found in this study suggest that pupillometry may not be useful in pediatrics in the acute postinjury setting for either the diagnosis of concussion or to stratify risk for prolonged recovery. (c) 2024 Elsevier Inc. All rights reserved.

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation and nanotube formation. For complete details on the use and execution of this protocol, please refer to Kalargyrou et al. (2021).1.

The retina encompasses a network of neurons, glia and epithelial and vascular endothelia cells, all coordinating visual function. Traditionally, molecular information exchange in this tissue was thought to be orchestrated by synapses and gap junctions. Recent findings have revealed that many cell types are able to package and share molecular information via extracellular vesicles (EVs) and the technological advancements in visualisation and tracking of these delicate nanostructures has shown that the role of EVs in cell communication is pleiotropic. EVs are released under physiological conditions by many cells but they are also released during various disease stages, potentially reflecting the health status of the cells in their cargo. Little is known about the physiological role of EV release in the retina. However, administration of exogenous EVs in vivo after injury suggest a neurotrophic role, whilst photoreceptor transplantation in early stages of retina degeneration, EVs may facilitate interactions between photoreceptors and Müller glia cells. In this review, we consider some of the proposed roles for EVs in retinal physiology and discuss current evidence regarding their potential impact on ocular therapies via gene or cell replacement strategies and direct intraocular administration in the diseased eye.

Objective: Review the under-recognized risk for childhood stroke for unvaccinated children. Background: Approximately thirty five percent of pediatric strokes may be attributed to focal cerebral arteriopathy (FCA). FCA is a unifocal and unilateral stenosis of a large intracranial artery of the anterior circulation. Cases have been associated with varicella infection in the year prior to stroke. Acute ischemic strokes caused by FCA have a 25% recurrence risk in one year and therefore need to be monitored closely. Literature suggests that patients with FCA may benefit from treatment with steroids, in addition to anti-thrombotic therapy, making early diagnosis essential for optimizing outcomes. We present a case of FCA in the setting of recent varicella infection. Design/Methods: n/a Results: A 14-year-old unvaccinated female presented to the emergency department with left facial droop, left hemiparesis and speech disturbance. Symptoms started four days prior, worsening on day of arrival. NIH Stroke Scale on presentation was 2 for aphasia. CT head was normal. MRI brain showed restricted diffusion involving the right internal capsule and deep grey matter. MRA showed right internal carotid artery narrowing. Given delayed presentation, she was out of the window for acute interventions and was transferred for higher level of care. History was notable for varicella 7 months ago and COVID-19 5 months ago. Vessel wall imaging showed narrowing in the right internal carotid and A1, consistent with FCA. Patient was started on pulse dose steroids and aspirin. Conclusions: We present a case arterial ischemic stroke secondary to FCA in a patient with history of varicella infection. Wild type varicella infection has been associated with FCA up to 12 months post-infection and under-vaccination is associated with over 8-fold increase in risk of childhood stroke. Incidence of strokes related to varicella infection has decreased with the availability of vaccinations, highlighting the importance of childhood vaccines. Disclosure: Dr. Gupta has nothing to disclose. Dr. Pearson has nothing to disclose.

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1(-/-) and Pde6b(rd1/rd1), while Versican showed regional increases in the periphery of Rho(-/-) mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair.