Ocular Gene Therapy in a Patient with Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic disease caused by damaging variants in COL7A1, which encodes type VII collagen. Blistering and scarring of the ocular surface develop, potentially leading to blindness. Beremagene geperpavec (B-VEC) is a replication-deficient herpes simplex virus type 1-based gene therapy engineered to deliver functional human type VII collagen. Here, we report the case of a patient with cicatrizing conjunctivitis in both eyes caused by dystrophic epidermolysis bullosa who received ophthalmic administration of B-VEC, which was associated with improved visual acuity after surgery.

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.

PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Brief incubation of corneal grafts in activated platelet rich plasma enhances corneal endothelial cell survival and regeneration.

Corneal transplantation is the most frequent organ transplantation worldwide. Unfortunately, corneal graft failure is common and endothelial decompensation is considered the major cause. Corneal endothelial cells (CECs) lack the capacity to reproduce, and perioperative and postoperative endothelial cell loss remains a significant challenge associated with corneal graft viability. Therefore, strategies to preserve CEC density are critical to extend graft survival. Activated platelet rich plasma (aPRP), a product extracted from autologous blood, has both antioxidant and regenerative properties. aPRP eye drops have shown effectiveness in the treatment of corneal pathologies such as ulcers, dry eye, and burns. Our purpose is to determine the protective and regenerative effect of aPRP on corneal grafts by evaluating aPRP's effect on the survival and proliferation of human CECs. Human corneal grafts were incubated in aPRP for 15 min to assess the activation of the CEC pAkt survival pathway as measured by ELISA. Evaluation of the protective effect of aPRP was made using an apoptotic model, which simulated oxidative stress conditions. Expression of apoptotic markers was measured using ELISA and endothelial cell viability was determined by optical microscopy. The CEC proliferation rate was measured in vitro with Ki-67 staining. Corneal graft gross structure was evaluated by Hematoxylin & Eosin and Masson trichrome staining. Our results indicate that a short incubation of human corneal grafts in aPRP protects CECs from apoptosis by upregulating the pAkt survival pathway and promoting CEC proliferation. Additionally, aPRP incubation does not induce histological changes in the grafts. A brief pre-treatment of human corneal grafts in aPRP may be beneficial for transplant longevity, as it protects CECs from apoptosis by upregulating intracellular survival pathways and promoting proliferation. In addition, this approach appears to be safe and has the potential to improve surgical outcomes following corneal transplantation.

Tumor necrosis factor-alpha and interferon-gamma induce inflammasome-mediated corneal endothelial cell death.

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.

Use of autologous plasma rich in growth factors fibrin membrane in the surgical management of ocular surface diseases.

PURPOSE: To evaluate the safety and efficacy of the surgical use of autologous plasma rich in growth factors fibrin membrane (mPRGF) in improving corneal wound healing and regeneration in a variety of complex ocular surface defects. METHODS: Chart review on 15 eyes of 14 included patients undergoing ocular surface intervention using intraoperative mPRGF at the Bascom Palmer Eye Institute and at the Instituto Oftalmológico Fernández-Vega was performed. Patients were grouped based on type of intervention or condition (penetrating keratoplasty, superficial keratectomy, neurotrophic or persistent corneal ulcers, and corneal perforation). Patients were followed for an average of 11 ± 5 months. Main outcomes measured were mPRGF dissolving time, best-corrected visual acuity, and evidence of any persistent epithelial defects, rejections, or complications. RESULTS: All 15 eyes underwent successful placement of mPRGF. Average dissolving time for fibrin membrane was 21 ± 3 days. mPRGF resulted in total healing of the corneal defects in 13/15 (86.7%) of the treated eyes and partial healing in 2/15 (13.3%) eyes in which persistent epithelial defects were noted on follow-up. Visual acuity improvement was seen in 9/15 (60%) of the cases. CONCLUSION: The use of autologous mPRGF in the healing and regeneration of the ocular surface is a secure and efficacious surgical option. Our data demonstrate that PRGF fibrin membrane should be contemplated as an important tool to optimize ocular surface regeneration in complex cases.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

Amniotic membrane use for management of corneal limbal stem cell deficiency.

PURPOSE OF REVIEW: The current article reviews the most recent surgical techniques for management of corneal limbal stem cell deficiency (LSCD) using amniotic membrane tissue. RECENT FINDINGS: Early successes with amniotic membrane transplantation (AMT) for the treatment of ocular surface disorders have encouraged clinicians to investigate new applications. The use of AMT as a temporary patch in emergency cases in which LSCD may develop has considerably improved the prognosis of these patients. Amniotic membrane does not have stem cells of its own, but it supports regeneration of limbal epithelial stem cells (LESCs). Similarly, the combination of AMT with classic surgical techniques has enhanced the surgical success rates in most case series. Furthermore, based on its advantageous properties as a cell carrier, new applications to support in-vivo and ex-vivo cell expansion have been reported recently. SUMMARY: LSCD constitutes a general indication for AMT. Based on the clinical scenario, AMT may be performed alone to support regeneration of LESCs, in combination with other surgical techniques, or even supporting the in-vivo or ex-vivo expansion of LESCs.

Intraocular lens dislocation and tube shunt in the posterior chamber: a case report.

BACKGROUND: To describe management of a case of intraocular lens (IOL) and capsular bag (CB) dislocation in an eye with an Ahmed glaucoma valve in the posterior chamber. CASE PRESENTATION: A 75-year-old pseudophakic man with open-angle glaucoma and diabetic retinopathy developed neovascular glaucoma. After two intravitreous injections of bevacizumab and panretinal photocoagulation were administered, the new vessels regressed. However, goniosynechiae were observed over 360° of the angle. An Ahmed glaucoma valve model FP7 was implanted with the tube in the posterior chamber with adequate intraocular pressure control. Nineteen years after cataract surgery, when the IOL-CB complex became dislocated, they were sutured transclerally to the sulcus without Ahmed glaucoma valve modification. After a coughing episode, the vitreous pushed the IOL-CB complex forward and the tube was behind the IOL-CB complex. A 25-gauge posterior vitrectomy was performed, and the tube was returned to in front of the optic of the IOL using a forceps tip through a sclerotomy. CONCLUSION: This case suggested that management of IOL-CB dislocation can modify glaucoma shunt function. A complete pars plana vitrectomy may be required in order to reposition the dislocated IOL-CB complex in the presence of a posterior chamber drainage tube implant.

A Comprehensive Review of Donor Corneal Preservation Strategies.

ABSTRACT: Successful corneal transplantation relies on the viability of the corneal endothelium. Although various preservation systems have been developed in the field of eye banking, long-term storage of the corneal endothelium poses challenges and is costly. Optisol-GS in the past has been the most commonly used solution for intermediate-term corneal storage in the United States. However, disruptions in the availability of Optisol-GS, caused by rising costs and supply shortages, have necessitated alternative methods of corneal preservation. Previously described preservation methods include hypothermia (2-8°C) for short-term storage (7-14 days), organ culture (28-37°C) for intermediate storage (4-7 weeks), and cryopreservation for longer-term storage. In this review, we examine standard practice and alternative methods for corneal storage.

Novel treatments for dry eye syndrome.

Dry eye syndrome (DES) is a prevalent and multifactorial disease that leads to a self-perpetuating cycle of inflammation and damage to the ocular surface. This results in symptoms such as redness, burning, and blurred vision, which can negatively affect a patient's quality of life. While treatments are available to manage DES, they only temporarily relieve symptoms. Furthermore, long-term use of certain medications can cause harm to the ocular surface. Therefore, there is a need for safer and effective treatments for DES. This review highlights the latest advancements in DES therapy, providing valuable insights into ongoing efforts to improve patient outcomes.

Detection of Subclinical Neurotrophic Keratopathy by Noncontact Esthesiometry.

INTRODUCTION: This study aimed to analyze corneal sensitivity with a new noncontact and handheld esthesiometer (Brill Engines, Spain) in patients with dry eye disease (DED) and patients on hypotensive drops, and to compare it with healthy subjects. METHODS: A total of 31 patients (57 eyes) with DED, 23 patients (46 eyes) with glaucoma, and 21 healthy patients (33 eyes) were recruited. In all patients, corneal sensitivity was measured. Subsequently, a keratography test (Keratograph 5M, Oculus) was carried out to measure tear meniscus height (TMH), non-invasive breakup time (NIBUT), bulbar redness (Jenvis scale), and corneal staining (CS, Oxford scale). Both corneal sensitivity and ocular surface parameters were compared between DED, glaucoma, and healthy subjects. Linear mixed models were constructed to utilize data from both eyes of patients. An alpha level of 0.05 was considered statistically significant. RESULTS: The mean age was 56.1 ± 16.1 years in the DED group, 69.5 ± 11.7 years in the glaucoma group, and 37.190 ± 11.677 years in the control group. After adjustment for age and sex, corneal sensitivity was significantly reduced in DED and glaucoma vs control group (P = 0.02 and P = 0.009, respectively). NIBUT was lower in DED and glaucoma groups (P < 0.001 and P = 0.001, respectively). Redness and CS values were higher in the DED group (P = 0.04 and P = 0.001, respectively). TMH was lower in the glaucoma group (P = 0.03). CONCLUSIONS: Corneal sensitivity measured with a novel noncontact esthesiometer was reduced in DED and glaucoma groups compared to controls. In clinical practice, this esthesiometer could be an easy-to-use device to screen for patients with subclinical neurotrophic keratopathy.

Living with Dry Eye Disease and its Effects on Quality of Life: Patient, Optometrist, and Ophthalmologist Perspectives.

Dry eye disease is a very common condition, especially among aging women. People often think of it as a very mild and non-harmful issue, but the reality is that it has a huge deleterious effect on patients' quality of life. Most publications usually focus on the scientific aspects of this pathology: its epidemiology, diagnosis, or management. However, in this article we highlight the patient's perspective and the challenges of living with dry eye disease. With prior informed consent, we interviewed a patient whose life has drastically changed since she first got the diagnosis. We also asked healthcare professionals based in Miami who were involved in this patient's care for their opinions. We hope that the messages and commentaries resonate with patients and physicians involved in the care of dry eye disease worldwide.

The inflammasome pathway: A key player in ocular surface and anterior segment diseases.

Inflammasomes are multicomplex molecular regulators with an emerging importance in regulating ocular surface and anterior segment health and disease. Key components found in the eye include NF-κB, NLRP3, NLRC4, NLRP6, ASC, IL-1ß, IL-18, and caspase-1. The role of NLRP1, NLRC4, AIM2, and NLRP3 inflammasomes in the pathogenesis of infectious ulcers, DED, uveitis, glaucoma, corneal edema, and other diseases is being studied with many developments. Attenuation of these diseases has been explored by blocking various molecules along the inflammasome pathway with agents like NAC, polydatin, calcitriol, glyburide, YVAD, and disulfiram. We provide a background on the inflammasome pathway as it relates to the ocular surface and anterior segment of the eye, discuss the role of inflammasomes in the above diseases in animals and humans, investigate new therapeutic targets, and explore the efficacy of new anti-inflammasome therapies.

Neurotrophic Keratopathy After Slow Coagulation Transscleral Cyclophotocoagulation.

PURPOSE: Decreased corneal sensation and subsequent neurotrophic keratopathy (NK) is an uncommon complication after transscleral cyclophotocoagulation (TSCPC). Post-TSCPC NK has been rarely reported in the literature, predominantly after traditional, "pop technique" continuous-wave TSCPC or micropulse CPC. The authors report the first case series of NK after slow-coagulation TSCPC (SC-TSCPC). METHODS: This was a respective chart review of patients who developed NK after SC-TSCPC. The collected data included demographic data, type of glaucoma, risk factors for corneal anesthesia in addition to the number of laser spots, and the extent of the treated area. RESULTS: Four eyes experienced NK after SC-TSCPC. The median time for the development of NK was 4 weeks. At the final visit, 2 patients had a resolution of NK, 1 had a persistent corneal ulcer, and 1 had worsening NK and corneal perforation. CONCLUSIONS: NK is a rare but a vision-threatening complication that can develop after SC-TSCPC in patients with risk factors for decreased corneal sensation. Early diagnosis and proper management are crucial to reducing the risk of vision loss and improving the prognosis of these cases.

Comparison of Transcriptomic Analysis of the Conjunctiva in Glaucoma-Treated Eyes with Dry Eyes and Healthy Controls.

Ocular surface disease (OSD) associated with topical glaucoma drugs is a common issue impacting treatment adherence. We aimed to identify conjunctival transcriptomic changes in glaucoma and dry eye patients, comparing them to healthy controls. Bulbar conjunctival specimens were collected via impression cytology from 33 patients treated for glaucoma, 9 patients with dry eye, and 14 healthy controls. RNA extraction and bulk RNA sequencing were performed, followed by bioinformatics analysis to detect gene dysregulation. Ingenuity pathways analysis (IPA) identified pathways and biological processes associated with these transcriptomic changes. Sequencing analysis revealed 200 modified genes in glaucoma patients compared to healthy individuals, 233 differentially expressed genes in dry eye patients versus controls, and 650 genes in treated versus dry eye samples. In glaucoma patients, 79% of altered pathways were related to host defense, while dry eye patients showed a 39% involvement of host response, 15% in cellular proliferation and integrity, and 16% of mitochondrial dysfunction. These findings were validated through qRT-PCR. Glaucoma patients showed an intensified conjunctival immune response as a potential cause of OSD, whereas in dry eye patients, in addition to the immune response, other mechanisms such as mitochondrial dysfunction or reduced cellular proliferation were observed.

Ocular symptoms secondary to meningeal carcinomatosis in a patient with lung adenocarcinoma: a case report.

BACKGROUND: Meningeal carcinomatosis (MC) is a rare complication associated with hematologic and solid tumors. MC develops when malignant cells gain access to the leptomeningeal space, producing several clinical symptoms. Loss of vision and ocular motility deficit are the most frequent ocular symptoms reported. Fundus examination usually appears normal, although optic nerve alterations like optic atrophy or papilledema have been described. MC diagnosis is usually completed by magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis. Indicated treatment for MC usually involves intrathecal chemotherapy combined with radiotherapy, although survival rate is extremely low. CASE PRESENTATION: A 66-year old man with stage IV metastatic lung adenocarcinoma, presented to the Ophthalmology Department with a two-month history of double vision, soft headaches and dizziness episodes. The patient presented a best visual corrected acuity of 0.7 in his right eye and 0.8 in his left eye. Diplopia was corrected with 6-prism diopters base-out prism in right eye. Funduscopy showed a bilateral papilledema, juxtapapillary exudates and splinter hemorrhages. Brain MRI showed a diffuse leptomeningeal enhancement in cortical sulcus. Lumbar puncture was performed and cerebrospinal fluid (CSF) cytology revealed malignant cells compatible with a diagnosis of MC. Intrathecal chemotherapy was administered. CONCLUSION: MC is a serious complication of systemic cancer patients, involving a poor prognosis. Early diagnosis is extremely important, although treatment is frequently aimed to reduce the symptoms and extend survival. Eye symptoms may be the chief complaint, so MC should be considered in any patient with vision loss or diplopia accompanied by neurologic symptoms and in the absence of an intraocular cause, especially in the context of systemic cancer.

Risk and Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Infection on Corneal Transplantation: A Case-Control Study.

PURPOSE: The purpose of this study was to evaluate the risk of symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after corneal transplantation surgery, with cataract surgeries as controls, and the impact of the novel coronavirus disease pandemic in the clinical and surgical complications of corneal transplantation and cataract surgeries. METHODS: A retrospective matched case-control study of 480 consecutive individuals who underwent surgery at the Bascom Palmer Eye Institute between May 2020 and November 2020. A total of 240 patients who underwent corneal transplantation with tissue obtained from the Florida Lions Eye Bank were age, race, ethnicity, and sex matched with 240 patients who underwent cataract surgery during the same day and by the same surgical team. Only the first corneal transplant or cataract surgery during this period was considered for each individual. All donors and recipients were deemed SARS-CoV-2 negative by a nasopharyngeal polymerase chain reaction test before surgery. Postoperative SARS-CoV-2 infections were defined as previously SARS-CoV-2(-) individuals who developed symptoms or had a positive SARS-CoV-2 polymerase chain reaction test during the first postoperative month. RESULTS: Mean age, sex, race, and ethnicity were similar between groups. There were no differences between the corneal transplant and cataract groups in the rates of SARS-CoV-2 infection before (5.8% vs. 7.5%, P= 0.6) or after surgery (2.9% vs. 2.9%, P = 1). The rates of postoperative complications did not increase during the pandemic, compared with previously reported ranges. CONCLUSIONS: In this study, postoperative SARS-CoV-2 infection was similar for individuals undergoing corneal transplantation or cataract surgery. Further research is required to evaluate the transmission of SARS-CoV-2 through corneal tissue.

Effects of Mitomycin-C and 5-Fluorouracil on Ocular Adnexal Sebaceous Carcinoma Cells.

PURPOSE: To investigate the effects of mitomycin-C (MMC) and 5-fluorouracil (5-FU) on the viability, proliferation, and migratory capacity of cultured ocular adnexal sebaceous carcinoma (SC) cells. DESIGN: Laboratory investigation. METHODS: Human SC cell lines (Bascom Palmer 50 and 52 [BP50 and BP52]) and human limbal stem cells (LSCs) were treated with various concentrations of MMC and 5-FU. Cytotoxicity was assessed with the tetrazolium MTT colorimetric viability assay on normal corneal vs tumor cells. Growth curves and scratch assays were performed to characterize the effects of these chemotherapeutic agents on SC proliferation and migration, respectively. RESULTS: MMC decreased BP52 cell viability in a dose-dependent manner with a half-maximal effective dose (EC50) of 11.8 µM after 72 hours. SC viability decreased >50% at 80 mM 5-FU after 72 hours. MMC reduced LSC viability in a dose-dependent manner with an EC50 value of 3.24 µM, and 5-FU decreased LSC viability >50% at 160 µM. MMC decreased SC cell proliferation and migration in a dose-dependent manner. 5-FU displayed antiproliferative effects but did not affect cell migration at concentrations below 1000 µM. CONCLUSIONS: Our in vitro data corroborate clinical observations that MMC is efficacious for treating ocular adnexal SC, albeit at the expense of LSC viability. Our findings also demonstrate that topical 5-FU exhibits antiproliferative effects that supersede its cancer-killing and antimigratory effects on cultured SC cells.