Sustained Release Formulation of Hydroxypropyl-ß-cyclodextrin Eye Drops Using Xanthan Gum.

Bietti's crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-ß-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-ß-CyD. Our results suggest that HP-ß-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-ß-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-ß-CyD alone. Furthermore, the slow release of HP-ß-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-ß-CyD, and that HP-ß-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

Two novel non-coding single nucleotide variants in the DNase1 hypersensitivity site of PRDM13 causing North Carolina macular dystrophy in Korea.

Purpose: Pathogenic variants in North Carolina macular dystrophy (NCMD) have rarely been reported in the East Asian population. Herein, we reported novel variants of NCMD in 2 Korean families. Methods: The regions associated with NCMD were analyzed with genome sequencing, and variants were filtered based on the minor allele frequency (0.5%) and heterozygosity. Non-coding variants were functionally annotated using multiple computational tools. Results: We identified two rare novel variants, chr6:g.99,598,914T>C (hg38; V17) and chr6:g.99,598,926G>A (hg38; V18) upstream of PRDM13 in families A and B, respectively. In Family 1, Grade 2 NCMD and a best-corrected visual acuity of 20/25 and 20/200 in the right and left eyes, respectively, were observed. In Family B, all affected individuals had Grade 1 NCMD with characteristic confluent drusen at the fovea and a best-corrected visual acuity of 20/20 in both eyes. These two variants are 10-22 bp downstream of the reported V10 variant within the DNase1 hypersensitivity site. This site is associated with progressive bifocal chorioretinal atrophy and congenital posterior polar chorioretinal hypertrophy and lies in the putative enhancer site of PRDM13. Conclusion: We identified two novel NCMD variants in the Korean population and further validated the regulatory role of the DNase1 hypersensitivity site upstream of PRDM13.

Longitudinal Natural History Study of Visual Function in Bietti Crystalline Dystrophy: Implications for Early Intervention.

Purpose: To delineate the natural history of visual function parameters over time in individuals with Bietti crystalline dystrophy. Methods: This was a single-center retrospective longitudinal cohort study. Participants (n = 29) with a clinical diagnosis of Bietti crystalline dystrophy who harbored two alleles of disease-causing variants of the cytochrome P450 family 4 subfamily V member 2 gene (CYP4V2) were enrolled. Best-corrected visual acuity (BCVA), visual field (VF), and full-field ERG (ffERG) at baseline and their changes during the follow-up period were evaluated. Annual progression rates were calculated using three methods. Results: The mean age at the initial visit was 34.2 ± 7.5 years, with 5.9 ± 3.1 years follow-up. The annual progression rate from the longitudinal analysis using averaged individual progression rates was 0.079 logMAR units for BCVA, 1.14 dB for mean defect (MD) value of VF, and -18.06 µV and -5.45 µV for the b-wave amplitudes of scotopic 3.0 ERG and photopic 3.0 ERG, respectively. Mixed-model linear regression revealed annual progression rates of 0.068 logMAR units, 0.86 dB, -13.29 µV, and -3.75 µV, respectively. Cross-sectional progression rates from visual function versus age at baseline were 0.011 logMAR units, 0.47 dB, -1.85 µV, and -1.07 µV, respectively, which were significantly slower than those from the longitudinal data. Interocular symmetries for the MD values of VF and ffERG were good. Conclusions: Annual BCVA, VF, and ffERG progression rates were rapid, emphasizing the need for regular follow-up and early intervention. The progression rate cannot be inferred accurately from cross-sectional data from patients of different ages.

Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: an open-label, single-arm, exploratory trial.

Bietti crystalline corneoretinal dystrophy is an inherited retinal disease caused by mutations in CYP4V2, which results in blindness in the working-age population, and there is currently no available treatment. Here, we report the results of the first-in-human clinical trial (NCT04722107) of gene therapy for Bietti crystalline corneoretinal dystrophy, including 12 participants who were followed up for 180-365 days. This open-label, single-arm exploratory trial aimed to assess the safety and efficacy of a recombinant adeno-associated-virus-serotype-2/8 vector encoding the human CYP4V2 protein (rAAV2/8-hCYP4V2). Participants received a single unilateral subretinal injection of 7.5 × 1010 vector genomes of rAAV2/8-hCYP4V2. Overall, 73 treatment-emergent adverse events were reported, with the majority (98.6%) being of mild or moderate intensity and considered to be procedure- or corticosteroid-related; no treatment-related serious adverse events or local/systemic immune toxicities were observed. Compared with that measured at baseline, 77.8% of the treated eyes showed improvement in best-corrected visual acuity (BCVA) on day 180, with a mean ± standard deviation increase of 9.0 ± 10.8 letters in the 9 eyes analyzed (p = 0.021). By day 365, 80% of the treated eyes showed an increase in BCVA, with a mean increase of 11.0 ± 10.6 letters in the 5 eyes assessed (p = 0.125). Importantly, the patients' improvement observed using multifocal electroretinogram, microperimetry, and Visual Function Questionnaire-25 further supported the beneficial effects of the treatment. We conclude that the favorable safety profile and visual improvements identified in this trial encourage the continued development of rAAV2/8-hCYP4V2 (named ZVS101e).

Acute calcareous corneal degeneration in a patient with chronic graft-versus-host disease.

Objective: To describe acute calcareous corneal degeneration as a complication of chronic graft-versus-host disease. Materials and methods: Clinical case and review of the literature. Results: We presented a case of bilateral acute calcareous corneal degeneration in a patient with chronic graft-versus-host disease. Conclusions: Chronic graft-versus-host disease (cGVHD) occurs in 50-70% of bone marrow transplantation patients, the most frequent ocular complication being keratoconjunctivitis sicca (KCS). Calcareous corneal degeneration is a type of calcium deposition that can be secondary to chronic ocular inflammation or dry eye, but there are few cases reported of acute calcareous corneal degeneration and recurrent perforation in cGVHD. Abbreviations: GVHD = Chronic graft-versus-host disease, aGVHD = Acute graft-versus-host disease, cGVHD = Chronic graft-versus-host disease, KCS = Keratoconjunctivitis sicca, PKP = Penetrating keratoplasty, AMT = Amniotic membrane transplantation, PRGF = Plasma rich in growth factors, OD = Right eye, OS = Left eye.

Deep anterior lamellar keratoplasty and penetrating keratoplasty in macular corneal dystrophy: comparison of visual and topographic outcomes and complications.

PURPOSES: This study aims to assess and compare the postoperative visual and topographic outcomes, complications, and graft survival rates following deep anterior lamellar keratoplasty and penetrating keratoplasty in patients with macular corneal dystrophy. METHODS: In this study we enrolled 59 patients (23 male; and 36 female) with macular corneal dystrophy comprising 81 eyes. Out of these, 64 eyes underwent penetrating keratoplasty, while 17 eyes underwent deep anterior lamellar keratoplasty. The two groups were analyzed and compared based on best-corrected visual acuity, corneal tomography parameters, pachymetry, complication rates, and graft survival rates. RESULTS: After 12 months, 70.6% of the patients who underwent deep anterior lamellar keratoplasty (DALK) and 75% of those who had penetrating keratoplasty (PK) achieved a best-corrected visual acuity of 20/40 or better (p=0.712). Following surgery, DALK group showed lower front Kmean (p=0.037), and Q values (p<0.01) compared to the PK group. Postoperative interface opacity was observed in seven eyes (41.2%) in the DALK group. Other topography values and other complications (graft rejection, graft failure, cataract, glaucoma, microbial keratitis, optic atrophy) did not show significant differences between the two groups. The need for regrafting was 9.4% and 11.8% in the PK and DALK groups, respectively (p=0.769). Graft survival rates were 87.5% and 88.2% for PK and DALK; respectively (p=0.88 by Log-rank test). CONCLUSION: Both PK and DALK are equally effective in treating macular corneal dystrophy, showing similar visual, topographic, and survival outcomes. Although interface opacity occurs more frequently after DALK the visual results were comparable in both groups. Therefore, DALK emerges as a viable surgical choice for patients with macular corneal dystrophy without Descemet membrane involvement is absent.

Congenital stromal corneal dystrophy in a Spanish family: Clinical, genetic and histological analysis.

PURPOSE: To present the clinical, genetic, and histopathological features of the ninth family affected by congenital stromal corneal dystrophy (CSCD) to date. METHODS: Twelve cases of a Spanish family affected by CSCD were analyzed regarding history, visual acuity (VA, decimal scale), an ophthalmologic exam and specular microscopy. Five eyes were treated by deep anterior lamellar keratoplasty (DALK), and thirteen eyes by penetrating keratoplasty (PK). In the two last generations, a genetic study was performed. RESULTS: Most of the patients affected were born with opaque corneas except for three, whose corneas were clear at birth. Biomicroscopy showed a whitish diffuse stromal opacity with an unaltered epithelium, causing poor VA (from hand motions to 0.4). Patients treated with PK presented mean postoperative VA of 0.19±0.20 over a follow-up time of 235.3±101.4months with 38% recurrences. Patients who underwent DALK experienced VA improvement to 0.17±0.11 over a follow-up time of 10.8±2.6months without signs of recurrence. In the latter, the big bubble technique was not achieved, so a manual technique was performed. The genetic study showed heterozygosis for a 1-bp deletion at nucleotide 962 in exon 8 of the decorin gene. CONCLUSIONS: CSCD is a rare entity, which should be treated by DALK whenever possible, obtaining better results than PK. Close monitoring of children of affected individuals is important, because CSCD can progress during the early years of life.

Topical netarsudil for the treatment of primary corneal endothelial degeneration in dogs.

This study evaluated the tolerability and efficacy of the topical rho-kinase inhibitor netarsudil for canine primary corneal endothelial degeneration (PCED). Twenty-six eyes of 21 client-owned dogs with PCED were enrolled in a prospective, randomized, vehicle control clinical trial and received topical netarsudil 0.02% (Rhopressa®) or vehicle control twice daily (BID) for the first 4 months. Then, all patients received netarsudil for the next 4 or 8 months. Complete ophthalmic examination, ultrasonic pachymetry, Fourier-domain optical coherence tomography, and in vivo confocal microscopy were performed at baseline and 1, 2, 4, 6, 8 and 12 months. Effect of netarsudil on central corneal thickness (CCT), percentage of cornea with edema, and endothelial cell density (ECD) were evaluated by repeated measures ANOVA. Kaplan-Meier curves and log-rank test were used to compare corneal edema and clinical progression of eyes in netarsudil versus vehicle control groups. All dogs developed conjunctival hyperemia in at least one eye while receiving netarsudil. Unilateral transient reticulated intraepithelial bullae and stromal hemorrhage were observed respectively in 2 dogs in the netarsudil group. Two dogs showed persistently decreased tear production while receiving netarsudil, requiring topical immunomodulatory treatment. No significant differences in CCT, ECD, corneal edema or clinical progression were observed between netarsudil or vehicle treated eyes. When comparing efficacy of topical netarsudil BID and topical ripasudil 0.4% administered four times daily from our previous study, dogs receiving ripasudil had significantly less progression than those receiving netarsudil.

[Intracorneal selective stromal transplantation]. / Vnutrirogovichnaya transplantatsiya stromy.

Deep anterior lamellar keratoplasty or penetrating keratoplasty are currently considered the optimal methods of surgical treatment of stromal dystrophies and corneal degeneration. Despite certain advantages and benefits of these methods, they also have significant limitations: involvement of superficial corneal layers in the surgery, need for suturing, development of post-keratoplasty astigmatism etc. PURPOSE: This study aimed to test and describe the new method of closed sutureless keratoplasty (intracorneal selective stromal transplantation), which was indicated in isolated dystrophic and degenerative pathology of the stroma. MATERIAL AND METHODS: Intracorneal selective stromal transplantation was performed in a 62-year-old patient with stromal degeneration and intact corneal layers between the altered stroma and the Descemet's membrane posteriorly, and the Bowman's layer anteriorly. The patient also had immature senile cataract. Corneal stroma was removed and replaced with a graft in the optical center of the lens, while the endothelium, the Descemet's membrane and the Bowman's layer remained intact. RESULTS: The proposed technique of intracorneal selective stromal transplantation makes it possible to replace only the pathologically altered stroma through closed surgical approach, without affecting the anterior and posterior surfaces of the cornea. Best-corrected visual acuity has increased in the patient from 0.01 to 0.6, while mean endothelial cell density has not changed in the course of 24-months follow-up. CONCLUSION: The proposed keratoplasty method can be used in patients with dystrophy or degeneration of the corneal stroma and preserved endothelial cells, intact Descemet's membrane and Bowman layer. Since the superficial corneal layers are not involved during the surgery, intracorneal selective stromal transplantation combined the advantages of both deep anterior lamellar keratoplasty and endothelial keratoplasty. The biologically favorable result in this first clinical case allows a preliminary conclusion on the technical possibility and functional effectiveness of the proposed method, but further long-term observation and more clinical cases are required.

Familial primary calcific band-shaped keratopathy with late onset systemic disease: a case series and review of the literature.

BACKGROUND: Familial calcific band-shaped keratopathy (BSK) is a very rare disease, with no underlying cause. There is no underlying disease in this form of the disease. This article introduces a family with seven children, three of whom were diagnosed with familial primary calcific BSK. One of them developed a systemic disease 38 years after ocular manifestation. CASE PRESENTATION: In this case report, three Iranian siblings from a family with familial calcific band-shaped keratopathy (BSK) are introduced. Systemic and ocular examinations performed on these patients indicated the occurrence of chronic kidney disease in the older child, a 41-year-old woman, 38 years after ocular manifestation. The examinations conducted on the other two siblings revealed no pathological findings. The 41-year-old sister and 37-year-old brother underwent unilateral deep anterior lamellar keratoplasty (DALK), while the 33-year-old sister underwent bilateral superficial keratectomy (SK). CONCLUSION: Considering the late onset of systemic disease in one of the siblings diagnosed with familial calcific band-shaped keratopathy (BSK), it is crucial to emphasize the necessity of long-term follow-up for these patients and their families.

Unilateral Granular Type 2 Corneal Dystrophy With Exacerbation After LASIK.

PURPOSE: The aim of this study was to report a case of unilateral granular corneal dystrophy type 2 (GCD2) with exacerbation after bilateral laser in situ keratomileusis (LASIK). METHODS: Clinical evaluation, Scheimpflug imaging, anterior segment optical coherence tomography (AS-OCT), cytology, and genetic testing were used to confirm the diagnosis of unilateral GCD2 with exacerbation after bilateral LASIK. Detailed literature review for possible unilateral GCD2 presentations was performed. RESULTS: A 54-year-old White woman presented with blurred vision in her left eye and a history of bilateral LASIK performed 8 years before. Examination revealed dense opacities in the left cornea only, which were confirmed to be confined to the LASIK interface and adjacent corneal stromal tissue, as determined by AS-OCT. The patient underwent flap lift, interface debris removal, and stromal bed phototherapeutic keratectomy. Cytological analysis showed eosinophilic corneal stromal deposits that stained with trichrome stain and were congophilic on Congo red stain. Genetic testing was positive for heterozygous GCD2 transforming growth factor ß-induced gene ( TGFBI ), c.371G>A, p.R124H mutation. There were no opacities identifiable in the right eye on serial slit-lamp examination, Scheimpflug imaging, or OCT imaging at 4 or 8 years after bilateral LASIK. Literature review failed to identify any previous reports of unilateral GCD2. CONCLUSIONS: This is the first known reported case of unilateral granular corneal dystrophy type 2. LASIK is contraindicated in eyes with corneal stromal dystrophies related to mutations in TGFBI as both flap creation and laser ablation can exacerbate visually significant opacity formation. Scheimpflug and AS-OCT imaging are useful to identify opacities in GCD2.

Establishment of mouse model of neurotrophic keratopathy through TRPV1 neuronal ablation.

Neurotrophic keratopathy (NK) is a challenging disease with the reduced innervation to the cornea. To establish a genetic and stable mouse model of NK, we utilized the TRPV1-DTR mice with intraperitoneal injection of diphtheria toxin (DT) to selectively eliminate TRPV1 neurons. After DT administration, the mice exhibited robust ablation of TRPV1 neurons in the trigeminal ganglion, accompanied with reduced corneal sensation and nerve density, as well as the decreased calcitonin-gene-related peptide (CGRP) and substance P levels. According to disease progression of TRPV1 neuronal ablation, tear secretion was reduced from day 3, which followed by corneal epithelial punctate lesions from day 7. From day 11 to day 16, the mice exhibited persistent corneal epithelial defects and stromal edema. By day 21, corneal ulceration and stromal melting were observed with the abundant inflammatory cell infiltration, corneal neovascularization, and enhanced cell apoptosis. Moreover, subconjunctival injection of CGRP delayed the NK progression with the characteristics of reduced severe corneal epithelial lesions and corneal inflammation. In addition, the impairments of conjunctival goblet cells, lacrimal gland, and meibomian gland were identified by the diminished expression of MUC5AC, AQP5, and PPARγ, respectively. Therefore, these results suggest that the TRPV1-DTR mice may serve as a reliable animal model for the research of NK pathogenesis.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

IC3D Classification of Corneal Dystrophies-Edition 3.

PURPOSE: The International Committee for the Classification of Corneal Dystrophies (IC3D) was created in 2005 to develop a new classification system integrating current information on phenotype, histopathology, and genetic analysis. This update is the third edition of the IC3D nomenclature. METHODS: Peer-reviewed publications from 2014 to 2023 were evaluated. The new information was used to update the anatomic classification and each of the 22 standardized templates including the level of evidence for being a corneal dystrophy [from category 1 (most evidence) to category 4 (least evidence)]. RESULTS: Epithelial recurrent erosion dystrophies now include epithelial recurrent erosion dystrophy, category 1 ( COL17A1 mutations, chromosome 10). Signs and symptoms are similar to Franceschetti corneal dystrophy, dystrophia Smolandiensis, and dystrophia Helsinglandica, category 4. Lisch epithelial corneal dystrophy, previously reported as X-linked, has been discovered to be autosomal dominant ( MCOLN1 mutations, chromosome 19). Classic lattice corneal dystrophy (LCD) results from TGFBI R124C mutation. The LCD variant group has over 80 dystrophies with non-R124C TGFBI mutations, amyloid deposition, and often similar phenotypes to classic LCD. We propose a new nomenclature for specific LCD pathogenic variants by appending the mutation using 1-letter amino acid abbreviations to LCD. Pre-Descemet corneal dystrophies include category 1, autosomal dominant, punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD) ( PRDX3 mutations, chromosome 10). Typically asymptomatic, it can be distinguished phenotypically from pre-Descemet corneal dystrophy, category 4. We include a corneal dystrophy management table. CONCLUSIONS: The IC3D third edition provides a current summary of corneal dystrophy information. The article is available online at https://corneasociety.org/publications/ic3d .

Protocolo de tratamiento del edema macular quístico asociado a retinosis pigmentaria y otras distrofias hereditarias de la retina / Protocol for the treatment of cystoid macular edema secondary to retinitis pigmentosa and other inherited retinal dystrophies

Las distrofias hereditarias de la retina (DHR) son la causa principal de ceguera legal en la población laboral. El edema macular quístico (EMQ) es una de las causas tratables de pérdida visual afectando hasta un 50% de los pacientes. Se ha realizado una revisión bibliográfica combinando «inherited retinal dystrophy», «retinitis pigmentosa», «macular oedema» y un protocolo diagnóstico/terapéutico según los niveles de evidencia y recomendaciones de la «US Agency for Healthcare Research and Quality». Este protocolo se ha discutido en las reuniones mensuales del grupo XAREA DHR con la participación de más de 25 profesionales, creando un documento de consenso. La etiología del EMQ es multifactorial: disfunción de la barrera hematorretiniana, del epitelio pigmentario de la retina y de las células de Müller, inflamación y tracción vítrea. La OCT es la prueba de elección para el diagnóstico y seguimiento del EMQ asociado a las DHR. Los fármacos con mayor grado de evidencia científica son los inhibidores de la anhidrasa carbónica (IAC). Los corticoides, anti-VEGF intravítreos y vitrectomía con pelado de la membrana limitante interna no disponen de suficiente evidencia. Se propone un esquema de tratamiento en el EMQ en las DHR en adultos, otro para pacientes pediátricos y otra en las DHR y cirugía de catarata. Los IAC orales y tópicos son efectivos en el tratamiento del EMQ secundario a las DHR. El tratamiento con corticoides, anti-VEGF y vitrectomía son opciones de segunda línea. Se requieren ensayos clínicos aleatorizados para poder establecer la escala terapéutica en estos pacientes.(AU)

Inherited retinal dystrophies (IRD) are the leading cause of legal blindness in the working population. Cystic macular edema (CME) is one of the treatable causes of visual loss, affecting up to 50% of the patients. A bibliographic review has been carried out combining “inherited retinal dystrophy”, “retinitis pigmentosa”, “macular edema” and a diagnostic-therapeutic protocol according to the levels of evidence and recommendations of the “US Agency for Healthcare Research and Quality”. This protocol has been discussed in the monthly meetings of the XAREA DHR group with the participation of more than 25 experts, creating a consensus document. The etiology of CME is multifactorial: dysfunction of the blood-retinal barrier, retinal pigment epithelium, and Müller cells, inflammation, and vitreous traction.OCT is the test of choice for the diagnosis and follow-up of CME associated with IRD. The drugs with the highest degree of scientific evidence are carbonic anhydrase inhibitors (IAC). Intravitreal corticosteroids, anti-VEGF, and vitrectomy with peeling of the internal limiting membrane do not have sufficient evidence. A treatment scheme is proposed for the CME in IRD in adults, another for pediatric patients and an another for IRD and cataract surgery. Oral and topical IACs are effective in the treatment of CME secondary to IRD. Treatment with corticosteroids, anti-VEGF, and vitrectomy are second-line options. Randomized clinical trials are required to establish the therapeutic scale in these patients.(AU)

Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea.

PURPOSE: Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs. METHODS: The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits. RESULTS: We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age. CONCLUSION: This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.

Selective Bowman's layer transplantation in the treatment of neurotrophic corneal ulcers.

The aim of this paper is to present the surgical technique of selective Bowman's layer transplantation as well as the results of the surgery in patients with trophic corneal ulcers. The methodology of the study included three eyes from three patients with neurotrophic corneal ulcers grafting with corneal Bowman's layer. Pre- and postoperative analyses of the anterior segment of the eye were based on optical coherence tomography and slit-lamp assessment. Postoperatively a gradual corneal stroma restoration and fast corneal epithelialization were noticed. Based on our observations, the stiff and firm structure of Bowman's layer is supposed to strengthen the corneal surface and maintain its shape. It acts also as an antipathogen and antitoxin corneal barrier. Application of this acellular corneal layer transplantation to cover trophic corneal ulcers is an interesting, modern and crucial method of neurotrophic keratitis treatment.

Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.