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Myopia, a prevalent refractive error disorder worldwide, is characterized by the elongation of the eye, leading to visual abnormalities. Understanding the genetic factors involved in myopia is crucial for developing therapeutic and preventive measures. Unfortunately, only a limited number of genes with well-defined functionality have been associated with myopia. In this study, we found that the homozygous TGM2-deleted gene in mice protected against the development of myopia by slowing down the elongation of the eye. The effectiveness of gene knockdown was confirmed by achieving a 60 percent reduction in TGM-2 transcript levels through the use of TGM-2-specific small interfering RNA (siRNA) in human scleral fibroblasts (SFs). Furthermore, treating normal mouse SFs with various transglutaminase inhibitors led to the down-regulation of TGM-2 expression, with the most significant reduction observed with specific TGM-2 inhibitors. Additionally, the study found that the pharmacological blockade of muscarinic receptors also slowed the progression of myopia in mice, and this effect was accompanied by a decrease in TGM-2 enzyme expression. Specifically, mice with homozygous mAChR5, mAChR1, and/or mAChR4 and knockout mice exhibited higher levels of TGM-2 mRNA compared to mice with homozygous mAChR2 and three knockout mice (fold changes of 5.8, 2.9, 2.4, −2.2, and −4.7, respectively; p < 0.05). These findings strongly suggest that both TGM-2 and muscarinic receptors play central roles in the development of myopia, and blocking these factors could potentially be useful in interfering with the progression of this condition. In conclusion, targeting TGM-2 may have a beneficial effect regarding myopia, and this may also be at least partially be the mechanism of anti-muscarinic drugs in myopia. Further studies should investigate the interaction between TGM-2 and muscarinic receptors, as well as the changes in other extracellular matrix genes associated with growth during the development of myopia.

Current management of glaucomatous optic neuropathy is limited to intraocular pressure control. Neuroglobin (Ngb) is an endogenous neuroprotectant expressed in neurons and astrocytes. We recently showed that exogenous intravitreal Ngb reduced inflammatory cytokines and microglial activation in a rodent model of hypoxia. We thus hypothesised that IVT-Ngb may also be neuroprotective in experimental glaucoma (EG) by mitigating optic nerve (ON) astrogliosis and microgliosis as well as structural damage. In this study using a microbead-induced model of EG in six Cynomolgus primates, optical coherence imaging showed that Ngb-treated EG eyes had significantly less thinning of the peripapillary minimum rim width, retinal nerve fibre layer thickness, and ON head cupping than untreated EG eyes. Immunohistochemistry confirmed that ON astrocytes overexpressed Ngb following Ngb treatment. A reduction in complement 3 and cleaved-caspase 3 activated microglia and astrocytes was also noted. Our findings in higher-order primates recapitulate the effects of neuroprotection by Ngb treatment in rodent EG studies and suggest that Ngb may be a potential candidate for glaucoma neuroprotection in humans.

Contains fulltext : 292919.pdf (Publisher’s version ) (Open Access) BACKGROUND: High myopia (HM), defined as a spherical equivalent refractive error (SER) ≤ -6.00 diopters (D), is a leading cause of sight impairment, through myopic macular degeneration (MMD). We aimed to derive an improved polygenic score (PGS) for predicting children at risk of HM and to test if a PGS is predictive of MMD after accounting for SER. METHODS: The PGS was derived from genome-wide association studies in participants of UK Biobank, CREAM Consortium, and Genetic Epidemiology Research on Adult Health and Aging. MMD severity was quantified by a deep learning algorithm. Prediction of HM was quantified as the area under the receiver operating curve (AUROC). Prediction of severe MMD was assessed by logistic regression. FINDINGS: In independent samples of European, African, South Asian and East Asian ancestry, the PGS explained 19% (95% confidence interval 17-21%), 2% (1-3%), 8% (7-10%) and 6% (3-9%) of the variation in SER, respectively. The AUROC for HM in these samples was 0.78 (0.75-0.81), 0.58 (0.53-0.64), 0.71 (0.69-0.74) and 0.67 (0.62-0.72), respectively. The PGS was not associated with the risk of MMD after accounting for SER: OR = 1.07 (0.92-1.24). INTERPRETATION: Performance of the PGS approached the level required for clinical utility in Europeans but not in other ancestries. A PGS for refractive error was not predictive of MMD risk once SER was accounted for. FUNDING: Supported by the Welsh Government and Fight for Sight (24WG201).

The effectiveness of current antifungal therapies is hampered by the emergence of drug resistance strains, highlighting an urgent need for new alternatives such as adjuvant antifungal treatments. This study aims to examine the synergism between propranolol and antifungal drugs, based on the premise that propranolol is known to inhibit fungal hyphae. In vitro studies demonstrate that propranolol potentiates the antifungal activity of azoles and that the effect is more pronounced for propranolol–itraconazole combination. Using an in vivo murine systemic candidemia model, we show that propranolol–itraconazole combination treatment resulted in a lower rate of body weight loss, decreased kidney fungal bioburden and renal inflammation when compared to propranolol and azole treatment alone or untreated control. Altogether, our findings suggest that propranolol increases the efficacy of azoles against C. albicans, offering a new therapeutic strategy against invasive fungal infections.

Small animals, such as rodents, serve as an attractive option for investigating the intrinsic process of photoreceptor degeneration because of their wide availability and versatility in disease models and gene manipulations. However, there was a lack of an objective and quantitative approach to measure their outer retina dynamics while preserving spatial heterogeneity. Here, we demonstrate an automated, unbiased approach for functional retinal imaging in rodents based on unsupervised machine learning. Our method automatically searches for and classifies nanoscopic cellular dynamics from obscure speckle patterns captured by a low-cost, phase-sensitive optical coherence tomography. Using this approach, we revealed highly reproducible Type-I and Type-II signals in rodents related to different parts of the outer retina. The fast Type-I signal was not reported previously and we hypothesized that it originated from the movement of rod outer segments. We also characterized the light-induced response of the outer retina under scotopic and photopic conditions, and demonstrateden-facemapping of the outer retina function in an extended field of view (12°), analogous to multifocal electroretinograms, but only with a single shot and yielding much higher spatial resolution. Our approach can be widely applied to investigating tissue-specific retinal dynamics across animal models, as well as facilitating clinical translations for the early detection of neurodegenerative diseases.

PURPOSETo evaluate the duration-dependent and synergetic impact of high-intensity light (HL) and optical refocus (RF) on lens-induced myopia (LIM) development in chickens.METHODSMyopia was induced in one eye in chicks (10 groups, n=126) from day 1 post- hatching (D1) until D8 using -10D lenses. Fellow eyes remained uncovered as controls. Nine groups were exposed daily to continuous 2 hours (h), 4h, or 6h of either HL (15,000 lux); RF (removal of -10D lens); or both (HL+RF). One group served as the LIM group without any interventions. Ocular axial length (AL), refractive error, and choroidal thickness were measured on D1, D4, and D8. Outcome measures are expressed as inter-ocular difference (IOD= experimental - control eye) ±SEM.RESULTSBy D8, LIM increased AL (0.36±0.04 mm), myopic refraction (-9.02±0.37D), and choroidal thinning (-90.27±16.44 µm) in the LIM group (all, PCONCLUSIONDaily exposure to 2h, 4h, or 6h of HL, RF, or HL+RF reduced lens-induced myopic refraction in a duration-dependent manner in chickens. Only 6h of HL+RF completely stopped LIM development. The synergetic effect of HL and RF is dependent on the duration of the interventions.

To identify choroidal characteristics associated with susceptibility to development of naturally occurring and experimentally induced myopia.We compared choroidal properties between pigmented and albino guinea pig (GP) strains. Biometry, cycloplegic refractive error (RE), and eye wall sublayer thickness were measured from 171 GPs at postnatal day (P)6, 14, and 28. Forty-three P14 GPs underwent two-week monocular form-deprivation myopia (FDM). En face images of choroidal vasculature were obtained with a customized swept-source optical coherence tomography. Multivariate regression analyses were performed, with P28 RE as the outcome and P14 choroidal thickness (ChT) as the main predictor variable. Proteomic analysis was performed on choroidal tissue from P14 albino and pigmented GPs.At P14, RE was correlated with thickness of the choroid (β = 0.06), sclera (β = 0.12), and retina (β = 0.27; all P0.001). P14 ChT was correlated with P28 RE both with (β = 0.06, P = 0.0007) and without FDM (β = 0.05, P = 0.008). Multivariate regression analysis, taking into account FDM (versus physiological growth) and strain, revealed that for every 10-µm greater ChT at P14, P28 RE was 0.50D more positive (P = 0.005, n = 70). En face images of choroidal sublayers showed that albino choroids were relatively underdeveloped, with frequent avascular regions. Consistent with this finding, proteomic analysis suggested abnormalities of the nitric oxide system in the albino GP choroid.Current results are consistent with the notion that greater ChT could protect from or delay the onset of myopia, while lower ChT is associated with greater susceptibility to myopia development. The underlying mechanism could be related to dysfunction of the choroidal vascular system.

Phase-sensitive Fourier-domain optical coherence tomography (FD-OCT) enables label-free imaging of cellular movements in vivo with detection sensitivity down to the nanometer scale. Due to this high sensitivity, it is widely employed in various emerging functional imaging modalities such as optoretinography (ORG), optical coherence elastography (OCE), and optical coherence tomography angiography (OCTA). However, achieving shot-noise limited detection sensitivity remains a major challenge for in-vivo measurements where the sample is constantly affected by vascular pulsation, breathing, eye and head motion, and other involuntary movements. Here, we propose a phase-restoring subpixel motion correction (PRSMC) method for post-hoc image registration in FD-OCT. Based on a generalized FD-OCT model, this method enables translational shifts of OCT images by arbitrary displacements while accurately restoring physically meaningful phase components, both with subpixel precision. With the sample movements estimated from averaged Doppler shift or normalized cross-correlation, we reconstructed the OCT images by correcting the axial displacement in the spectrum (k) domain and the lateral displacement in the spatial frequency domain, respectively. We validated our method in simulations, phantom experiments, and in-vivo optoretinogram imaging, where the advantages over conventional approaches for both amplitude stability and phase accuracy were demonstrated. Our approach significantly reduces the motion-induced phase error (MIPE) when imaging moving samples, achieving systematic phase sensitivities close to the shot-noise regime.

Burn wounds are susceptible to microbial invasion from both resident and exogenous bacteria, which becomes a critical public health issue and causes substantial economic burden. There is a perceived demand to produce new antimicrobial wound dressings that hinder bacterial colonization while accelerating the healing process and hence would provide an improved standard of care for patients. Since ancient times, herbal extracts from medicinally important plants have extensively been used for treating burn injuries. This work reports the utility of electrospun nanofibers containing plant extracts and antibiotics combination as a multifunctional scaffold for treating second-degree burns. First, we determined the various components of plant extracts from Gymnema sylvestre by two different processing methods and their synergism with minocycline antibiotics. Then, we prepared core-shell nanofibrous dressings with poly-e-caprolactone/gelatin laden with minocycline hydrochloride as a shell and gelatin infused with G. sylvestre extracts (ultrasound-assisted extracts and cold macerated extracts) as the core using coaxial electrospinning. The electrospun nanofibers displayed a smooth, continuous, and bead-free morphology with adequate wettability. The presence of extract components in the core-shell nanofibers resulted in enhanced mechanical properties when compared to pristine mats. The core-shell structures resulted in sustained release of the bioactive components when compared to nanofiber blends. Core-shell nanofiber mats containing plant extracts and antibiotic combinations displayed potent antimicrobial and antibiofilm properties while promoting the spread and proliferation of skin cells when compared to pristine mats. In a porcine model of cutaneous second-degree burns, we showed that wounds treated with the antimicrobial dressing improved re-epithelialization and collagen organization in comparison to untreated wounds.

The retina is part of the central nerve system (CNS) and has various interneurons and sensory neurons such as photoreceptor cells. Retinitis pigmentosa (RP) is an inherited condition that is characterized by photoreceptor degeneration. Herein, we developed a fluorescent probe - NeuA - for detecting retinal neuronal cells and applied NeuA to discriminate between healthy and RP retinas. The staining pattern of NeuA in the retinas of healthy and RP mouse models was examined in vitro , ex vivo and in vivo using confocal microscopy, the fluorescent fundus microscopy and optical coherent tomography (OCT). NeuA strongly stained the outer segment layer of photoreceptor cells and some bipolar cells in the healthy retina, but there was only weak staining in the photoreceptor degenerated retinas. Therefore, NeuA probe can be used as the detecting RP tools in the preclinical conditions.

The purpose of this study is to characterize the retinal and choroidal vascular networks in some of the most common animal species using swept-source optical coherence tomography angiography (SS-OCTA). Retinal angiographic images were acquired from healthy, anesthetized animals of seven species (mouse, rat, pig, rabbit, guinea pig, chicken, and non-human primate). We generated the enface angiograms to visualize the different retinal vascular plexuses and the choroidal vascular plexus. Quantitative OCTA metrics, including perfusion density, vessel density, and fractal dimension, were compared amongst the different species. There was a noticeable difference in the OCTA enface maps of the distinct vascular layers amongst the various species. Specifically, the non-human primate retina has the highest level of perfusion density and vessel density, whereas the rabbit retina exhibited the lowest level of vessel density. The mouse and the rat retina shared similar vascular patterns, and there was no difference in the OCTA metrics. Using one specific SS-OCTA system for all experiments eliminated multiple instrument-dependent factors, but the lateral resolution was still affected by eye size. Effects of varying lateral resolution on vascular metrics were investigated via a simulation. Overall, we achieved a large field of view of the distinct retinal and choroidal vascular plexuses and quantified the vascular metrics in multiple species, which could serve as protocol guidance and atlas to study the retinal and choroidal vascular abnormalities and their roles in ocular diseases resembling in-vivo histology.

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, e-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with e-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three e-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by e-lysylation.

The development of antimicrobial nanofibre dressings that can protect the injured tissues from commensal pathogens while promoting tissue regeneration finds enormous potential in plastic and reconstructive surgery practices. To achieve this goal, we investigated the effect of chondroitin sulphate on the morphology, mechanical properties, wettability and biocompatibility of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium. To extend the durability of dressings, we prepared composite dressings containing polycaprolactone (PCL) and gelatin as blend or core-shell nanofibres. Nanofibre blends presented greater tensile strength and stretchability, while core-shell nanofibres displayed superior photoluminescent properties. In a porcine model of cutaneous burn injury, both the blend and core-shell nanofibre dressings displayed improved re-epithelialization, wound closure and clinical outcome in comparison to untreated burns. Histology of the biopsied tissues indicated smooth regeneration and collagen organization of the burns treated with core-shell nanostructures than untreated burns. This study compared the physico-chemical and biological properties of composite nanofibres that are capable of accelerating burn wound healing and possess antimicrobial properties, highlighting their potential as wound dressings and skin substitutes.

BackgroundTo evaluate the impact of full-spectrum light-emitting diodes (LEDs) mimicking sunlight on ocular axial elongation and refractive error development in a chicken model of myopia.MethodsA total of 39 chicks (Lohmann brown), 1 day-old, were randomly distributed into 3 groups. Animals were housed for 28 days in a temperature-controlled enclosure, under a 12/12h light/dark cycle of isoluminant (∼285 Lux) fluorescent [n = 18, (4000K, FL-4000)] or Sunlike-LED [n=12, (4000K, SL-4000); n = 9, (6500K, SL-6500)] white lights. Myopia was induced monocularly in all chicks by random occlusion of one eye with a frosted diffuser, from day 1 post-hatching (D1) until D14. On D14, diffusers were removed, and recovery from myopia was monitored under the same experimental light condition. Axial length (AL), refractive status, choroidal thickness and anterior chamber depth were recorded on days 1, 7, 14, 22 and 28.Ex vivoscleral collagen fibre thicknesses were measured from scanning electron microscopy images. Differences in outcome measures between eyes and groups were compared using 2-way repeated-measures ANOVA.ResultsThere was no significant difference between groups in the AL and refraction of form-deprived (FD) eyes during form-deprivation (D1 to D14). FD eyes of animals raised under SL-4000 and SL-6500 recovered more rapidly from excessive axial elongation than those of animals raised under FL-4000, by D22 and D28. Correspondingly, the refractive status of FD eyes exposed to SL-4000 and SL-6500 was close to that of control eyes by D28. The choroid became thicker during recovery in FD eyes compared to control eyes, in all groups. Choroidal thickness was significantly greater in FD eyes of chickens raised under SL-6500 than in animals raised under FL-4000 (P< 0.01). The diameter of scleral collagen fibrils was significantly greater in recovering FD eyes of chickens raised under SL-6500, than in those raised under FL-4000 (P= 0.04) and SL-4000 (P= 0.002).ConclusionsCompared to fluorescent light, moderate intensities of full-spectrum Sunlike-LEDs can accelerate recovery from form-deprivation myopia in chickens, potentially through choroid-mediated pathways increasing the diameter of scleral collagen fibrils. This study highlights an important implication of the spectral content of white light on ocular growth and emmetropization.

Corneal diseases, along with glaucoma and cataract, are the leading causes of blindness worldwide. While corneal surgery offers good success rates, early diagnosis and disease prevention is the preferred choice over expensive and complex surgical procedures. Non-contact, high-resolution imaging modalities for imaging the corneal structures are highly desirable for clinical examinations for early diagnosis of corneal diseases. The currently used clinical standard is the in vivo confocal laser scanning microscopy, which is a contact based method. In this context, an in-house developed, non-contact, Gaussian epi-illumination based imaging system was used to image corneal injury in preclinical animal models in vivo. New Zealand white rabbits and Wistar rats were used as the preclinical models for the investigations. The lateral resolution of the developed system is 1.1 μm. The images obtained with the developed system, as well as the dimensional information of the corneal structures, are in good agreement with the data previously reported using standard methods. The proposed system offers a simple, easy operable, real-time, non-contact imaging modality for the evaluation of corneal structures in high-resolution. Agency for Science, Technology and Research (A*STAR) Nanyang Technological University National Medical Research Council (NMRC) The authors acknowledge financial support received through A*STAR-MIG project (BMRC1619077002), SERI-NTU ADVANCED OCULAR ENGINEERING (STANCE) Program Fund, NMRC Singapore Translational Research Investigator Award (NMRC/STaR/0023/2014), NMRC Transition Award (NMRC/TA/0040/2015), and NMRC Transition Award (NMRC/TA/0057/2017) for pursuing parts of the contents presented in this paper, and the research manpower and facilities provided through funding at COLE, NTU.

Pancreatic islet transplantation into the anterior chamber of the eye (ACE) has been shown to improve glycemic control and metabolic parameters of diabetes in both murine and primate models. This novel transplantation site also allows the delivery of therapeutic agents, such as immunosuppressive drugs, locally to prevent islet graft rejection and circumvent unwanted systemic side effects. Local intravitreal administration of micronized dexamethasone implant was performed prior to allogeneic islet transplantation into the ACEs of non-human primates. Two study groups were observed namely allogeneic graft without immunosuppression (n = 4 eyes) and allogeneic graft with local immunosuppression (n = 8 eyes). Survival of islet grafts and dexamethasone concentration in the ACE were assessed in parallel for 24 weeks. Allogeneic islet grafts with local dexamethasone treatment showed significantly better survival than those with no immunosuppression (median survival time- 15 weeks vs 3 weeks, log-rank test p

Acquired retinal diseases such as age-related macular degeneration and diabetic retinopathy rank among the leading causes of blindness and visual loss worldwide. Effective treatments for these conditions are available, but often have a high treatment burden, and poor compliance can lead to disappointing real-world outcomes. Development of new treatment strategies that provide more durable treatment effects could help to address some of these unmet needs. Gene-based therapeutics, pioneered for the treatment of monogenic inherited retinal disease, are being actively investigated as new treatments for acquired retinal disease. There are significant advantages to the application of gene-based therapeutics in acquired retinal disease, including the presence of established therapeutic targets and common pathophysiologic pathways between diseases, the lack of genotype-specificity required, and the larger potential treatment population per therapy. Different gene-based therapeutic strategies have been attempted, including gene augmentation therapy to induce in vivo expression of therapeutic molecules, and gene editing to knock down genes encoding specific mediators in disease pathways. We highlight the opportunities and unmet clinical needs in acquired retinal disease, review the progress made thus far with current therapeutic strategies and surgical delivery techniques, and discuss limitations and future directions in the field.

One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes. Here, we demonstrate that a bio-functional polymer by itself is able to prevent retinal scarring in an experimental rabbit model of proliferative vitreoretinopathy. This is mediated primarily via clathrin-dependent internalisation of polymeric micelles, downstream suppression of canonical EMT transcription factors, reduction of RPE cell hyper-proliferation and migration. Nuclear factor erythroid 2–related factor 2 signalling pathway was identified in a genome-wide transcriptomic profiling as a key sensor and effector. This study highlights the potential of using synthetic bio-functional polymer to modulate RPE cellular behaviour and offers a potential therapy for retinal scarring prevention.

The traditional intravitreal injection delivery of anti-vascular endothelial growth factor (anti-VEGF) to the posterior segment of the eye for treatment of retinal diseases is invasive and associated with sight-threatening complications. To avoid such complications, there has been significant interest in developing polymers for topical drug delivery to the retina. This study reports a nanomicelle drug delivery system made of a co-polymer EPC (nEPCs), which is capable of delivering aflibercept to the posterior segment topically through corneal-scleral routes. EPC is comprised of polyethylene glycol (PEG), polypropylglycol (PPG) and polycaprolactone (PCL) segments. In this study, aflibercept-loaded nEPCs (nEPCs+A) is capable of penetrating the cornea in ex-vivo porcine eye models and deliver a clinically significant amount of aflibercept to the retina of laser-induced choroidal neovascularisation (CNV) murine models, causing CNV regression. nEPCs+A also demonstrates biocompatibility in-vitro and in-vivo. Interestingly, this study also suggests that nEPCs have intrinsic anti-angiogenic properties. The ability to deliver anti-VEGF drugs and the intrinsic anti-angiogenic properties of nEPCs may result in synergistic effects which can be harnessed for effective therapeutics. nEPCs may be a promising topical anti-VEGF delivery platform for the treatment of retinal diseases. This article is protected by copyright. All rights reserved.

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5–25.0 μg/ml (5.2–10.4 μM)] and S. epidermidis [MIC = 12.5 μg/ml (5.2 μM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 μg/ml (10.4–20.8 μM)]. CaD23 (at 25 μg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 μg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg—33% (20/60) and wild-type (WT)—7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.

Background Retinal regenerative therapies hold great promise for the treatment of inherited retinal degenerations (IRDs). Studies in preclinical lower mammal models of IRDs have suggested visual improvement following retinal photoreceptor precursors transplantation, but there is limited evidence on the ability of these transplants to rescue retinal damage in higher mammals. The purpose of this study was to evaluate the therapeutic potential of photoreceptor precursors derived from clinically compliant induced pluripotent stem cells (iPSCs). Methods Photoreceptor precursors were sub-retinally transplanted into non-human primates (Macaca fascicularis). The cells were transplanted both in naïve and cobalt chloride-induced retinal degeneration models who had been receiving systemic immunosuppression for one week prior to the procedure. Optical coherence tomography, fundus autofluorescence imaging, electroretinography, ex vivo histology and immunofluorescence staining were used to evaluate retinal structure, function and survival of transplanted cells. Results There were no adverse effects of iPSC-derived photoreceptor precursors on retinal structure or function in naïve NHP models, indicating good biocompatibility. In addition, photoreceptor precursors injected into cobalt chloride-induced retinal degeneration NHP models demonstrated an ability both to survive and to mature into cone photoreceptors at 3 months post-transplant. Optical coherence tomography showed restoration of retinal ellipsoid zone post-transplantation. Conclusions These findings demonstrate the safety and therapeutic potential of clinically compliant iPSC-derived photoreceptor precursors as a cell replacement source for future clinical trials.

To investigate the correlation between posterior pole choroidal blood flow evaluated with digital subtraction indocyanine green angiography and enface optical coherence tomography angiography (OCTA). Imaging in animal study. The anatomy of 2 cynomogulus monkeys was studied. Each monkey was given a 0.75 mg/kg injection of indocyanine green in the saphenous vein. The dynamic angiographic filling sequence was recorded at 15 frames per second using the Heidelberg Spectralis. After image registration, sequential frame subtraction was used to image the dye front moving through the choroid. The OCTA was obtained by frame averaging nine separate choriocapillaris slab flow images obtained from the Zeiss Plex Elite 9000. Posterior pole choriocapillaris filling pattern in relation to the choriocapillaris anatomy as imaged by OCTA. In the posterior pole, the choriocapillaris fills in the pattern of discrete units with variable sizes and shapes. The cycle of dye filling begins in the peripapillary area and progresses toward the periphery in a wavelike manner. This filling pattern repeats in a cyclical manner, consistent with the cardiac cycle. OCTA shows a uniform mesh of vessels. While OCTA shows a uniform meshwork appearance of the choriocapillaris, the dynamic dye angiography suggests an irregular configuration of functional units partitioned by pressure gradients as opposed to structural boundaries. Disturbance of local perfusion pressure within choroidal vasculature may result in abnormal flow patterns, which could be evaluated in the clinic using commercially available equipment.

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin–proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity, furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy. This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

Internal-tamponade agents are crucial surgical adjuncts in vitreoretinal surgery. Clinically used endotamponade agents act through buoyancy forces, yet can result in prolonged post-operative positioning, temporary loss of vision, raised intra-ocular pressure, cataract formation or the need for additional removal surgery. Here, we describe a thermogelling polymer that provides an internal tamponade effect through surface tension and swelling counter-forces. We tested the long-term biocompatibility of the polymer endotamponade in rabbit vitrectomy models, and its surgical efficacy and biocompatibility in a non-human primate retinal-detachment model. We also show that, while the thermogel biodegrades during the three months following surgery, it promotes the reformation of a vitreous-like body that mimics the biophysical properties of the natural vitreous. The thermogelling endotamponade might serve as a long-term vitreous substitute. A thermogelling polymer that acts as an internal tamponade can repair detached retinas and trigger the formation of a vitreous-like body, as shown in retinal-detachment rabbit and non-human-primate models.