Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation.

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Efficacy and safety of 0.05% micellar nano-particulate (MNP) cyclosporine ophthalmic emulsion in the treatment of moderate-to-severe keratoconjunctivitis sicca: a 12-week, multicenter, randomized, active-controlled trial.

BACKGROUND: Keratoconjunctivitis sicca or dry eye disease (DED) is a multifactorial disorder underpinned by a complex inflammatory cycle. Introduction of topical cyclosporine has been a significant advance in the management of DED. In recent years advancements in formulation technology have led to development of micellar nano-particulate (MNP) cyclosporine formulations that promise better penetration into ocular target tissues and potential for reduced ocular surface irritation. METHODS: We compared two dosing regimes of a proprietary MNP cyclosporine emulsion with the widely marketed topical cyclosporine formulation Restasis™ in a multicenter parallel-group randomised trial in patients with DED. Patients were randomised to one of 3 treatment groups with 90 patients eligible for the per protocol analysis: 30 in the higher dose test arm A; 32 in the lower dose test arm B; and 28 in the Restasis™ control arm C. All scored efficacy endpoints were tested for significance by comparing the mean change in scores from baseline in the test groups with that in the control group at 12 weeks, using the Student's t test. Wilcoxon's rank sum test was used to test individual symptom scores and clinician's global evaluation of treatment grades. RESULTS: Corneal fluorescein staining score, the primary efficacy endpoint, decreased by 6.8 ± 4.0, 5.7 ± 3.9, and 4.6 ± 3.6 points in the 3 groups respectively, indicating superior efficacy in test arm A in comparison to control arm C (p = 0.0026). Schirmer's tear test, conjunctival lissamine staining score, ocular surface disease index, and individual dry eye symptom scores also favoured higher dose MNP cyclosporine over Restasis™. The study failed to differentiate the treatment arms in terms of clinician's global evaluation of treatment, use of tear substitutes, best corrected visual acuity or safety and toleration. CONCLUSION: The results indicate that the dose of 1 drop of a 0.05% w/v ophthalmic emulsion of MNP cyclosporine administered topically twice daily yields better outcomes at 12 weeks than the lower dose tested in the study, and is more efficacious than an equivalent dose of Restasis™, the active control used in the study. TRIAL REGISTRATION: This trial was registered in the Clinical Trials Registry of India on 29/03/2019, and was assigned registration number CTRI/2019/03/018319.

Generating minicorneal organoids from human induced pluripotent stem cells.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Inflammation, vascularization and goblet cell differences in LSCD: Validating animal models of corneal alkali burns.

Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid-Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.

Cataract surgery in ocular surface diseases: clinical challenges and outcomes.

PURPOSE OF REVIEW: Cataracts are a significant cause of blindness and visual impairment worldwide. With the recent advances in cataract surgery techniques, outcomes have improved significantly in uncomplicated cases. This article discusses the challenges and outcomes of cataract surgery in complex cases targeting eyes with ocular surface diseases like Stevens-Johnson syndrome, ocular cicatricial pemphigoid, Mooren's ulcer, vernal keratoconjunctivitis and limbal stem cell deficiency. RECENT FINDINGS: Ocular surface diseases are commonly associated with corneal scarring and vascularization, conjunctival inflammation, symblepharon and forniceal shortening. Any surgical intervention in a hostile ocular surface environment may worsen the disease, which may result in visual deterioration. In recent past, with the use of immunosuppressants and with the introduction of different ocular surface reconstruction surgeries; stabilization of ocular surface can be achieved prior to planning cataract surgery. This kind of step-wise approach can considerably improve visual outcomes in such cases. SUMMARY: Cataract surgery in ocular surface diseases needs careful preoperative, intraoperative and postoperative planning. Performed in a staged manner under favorable ocular environment, it has good visual outcomes and can improve the quality of life in such patients.

Simple limbal epithelial transplantation.

PURPOSE OF REVIEW: Simple limbal epithelial transplant (SLET) is a technique for addressing limbal stem cell deficiency. Limbal tissue from a donor eye, typically the patient's fellow healthy eye, is transplanted onto an amniotic membrane attached to the surface of the diseased eye. SLET was developed to address limitations of other techniques, specifically the technical difficulty of ex-vivo expansion of cells required in some techniques and the larger amount of valuable limbal tissue harvested in techniques not relying on ex-vivo expansion. We described how the provision of this procedure adds to the armamentarium of techniques available to treat some of the many thousands of uniocular corneal blind around the world. RECENT FINDINGS: A total of 125 patients from a recent series from our centre and 68 from a multicentre study provide evidence for efficacy mainly in cases of unilateral corneal burn. Results were comparable to other stem cell techniques described in other papers. Numerous small case reports describe the use of SLET in other contexts including ocular surface squamous neoplasia and pterygium excision. SUMMARY: SLET offers a cheaper and perhaps safer alternative to other techniques. Further evaluation of clinical success against its most similar analogues of conjunctival limbal autograft and cultivated limbal autograft is required.

Simple Limbal Epithelial Transplantation: Long-Term Clinical Outcomes in 125 Cases of Unilateral Chronic Ocular Surface Burns.

PURPOSE: This study describes the long-term clinical outcomes of autologous simple limbal epithelial transplantation (SLET), a relatively new technique of limbal stem cell transplantation. DESIGN: This was a single-center prospective interventional cases series. PARTICIPANTS: This study included 125 patients, 65 adults and 60 children who developed unilateral limbal stem cell deficiency (LSCD) after suffering with ocular surface burns and underwent SLET between 2010 and 2014. METHODS: A 1-clock hour limbal biopsy sample was obtained from the unaffected eye. At the same sitting, the recipient eye was surgically prepared and the donor tissue was divided into small pieces and transplanted using an amniotic membrane scaffold with fibrin glue. MAIN OUTCOME MEASURES: The diagnosis and outcome in every case was validated by 5 independent masked assessors. The primary outcome measure was restoration of a completely epithelized, stable, and avascular corneal surface. The secondary outcome measure was improvement in visual acuity. Complications, risk factors for failure, and immunohistochemistry analysis of corneas that underwent SLET also were described. RESULTS: At a median postoperative follow-up of 1.5 years (range, 1-4 years), 95 of 125 eyes (76%; 95% confidence interval, 68.5%-83.5%) maintained a successful outcome. Kaplan-Meier analysis revealed a comparable survival probability at 1 year of 80% in adults and 72% in children (P = 0.304). Two-line improvement in visual acuity was seen in 75.2%, and 67% of successful cases attained 20/60 or better vision (P < 0.0001). Progressive conjunctivalization occurred in 18.4% of eyes. The clinical factors associated with failure were identified as acid injury, severe symblepharon, SLET combined with keratoplasty, and postoperative loss of transplants (P ≤ 0.0075). Success rates were comparable among faculty and trainees (P = 0.71). Immunohistochemistry revealed successful regeneration of normal corneal epithelium (CK3(+)/12(+)) without admixture of conjunctiva cells (Muc5AC(-)/CK19(-)) and replenishment of limbal stem cell (ΔNp63α(+)/ABCG2(+)) reserve. CONCLUSIONS: Autologous SLET is an effective, reliable and replicable technique for long-lasting corneal regeneration and vision restoration in unilateral chronic ocular surface burns. Simple limbal epithelial transplantation is probably preferable to other techniques of limbal stem cell transplantation, particularly where cell cultivation facilities are unavailable.

IKZF1, a new susceptibility gene for cold medicine-related Stevens-Johnson syndrome/toxic epidermal necrolysis with severe mucosal involvement.

BACKGROUND: Stevens-Johnson syndrome (SJS) and its severe form, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface, oral cavity, and genitals. These reactions are very rare but are often associated with inciting drugs, infectious agents, or both. OBJECTIVE: We sought to identify susceptibility loci for cold medicine-related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement (SMI). METHODS: A genome-wide association study was performed in 808 Japanese subjects (117 patients with CM-SJS/TEN with SMI and 691 healthy control subjects), and subsequent replication studies were performed in 204 other Japanese subjects (16 cases and 188 control subjects), 117 Korean subjects (27 cases and 90 control subjects), 76 Indian subjects (20 cases and 56 control subjects), and 174 Brazilian subjects (39 cases and 135 control subjects). RESULTS: In addition to the most significant susceptibility region, HLA-A, we identified IKZF1, which encodes Ikaros, as a novel susceptibility gene (meta-analysis, rs4917014 [G vs. T]; odds ratio, 0.5; P = 8.5 × 10(-11)). Furthermore, quantitative ratios of the IKZF1 alternative splicing isoforms Ik1 and Ik2 were significantly associated with rs4917014 genotypes. CONCLUSION: We identified IKZF1 as a susceptibility gene for CM-SJS/TEN with SMI not only in Japanese subjects but also in Korean and Indian subjects and showed that the Ik2/Ik1 ratio might be influenced by IKZF1 single nucleotide polymorphisms, which were significantly associated with susceptibility to CM-SJS/TEN with SMI.

Erratum to: Endophthalmitis in Boston keratoprosthesis: case series and review of literature.

To report the clinical and microbiological characteristics of infectious endophthalmitis after Boston type I keratoprosthesis (B-K-Pro) implantation. Retrospective analysis of 45 eyes that received a B-K-Pro type 1 between 2009 and 2012 was performed. Five eyes with a diagnosis of exogenous endophthalmitis after B-K-pro type 1 were identified and information about demographic data, indication for K-Pro, post-operative bandage contact lens use, post-operative prophylactic antibiotic use, timing and clinical presentation of endophthalmitis, gram stain and culture results of intraocular fluid and preoperative and post-operative visual acuity were collected. The incidence of endophthalmitis was 11.1 % (5 of 45 eyes) and average time to develop endophthalmitis was 5.62 months (range 2 days to 8 months). Mean patient age was 31.4 years (5 to 65 years). The surgical indications included corneal injury due to chemical burns (n = 2), multiple failed grafts secondary to microbial keratitis (n = 2) and congenital glaucoma with congenital herpetic keratitis (n = 1). Post-Boston K-Pro, the visual acuity ranged from light perception (LP) to 20/50. K-pro was explanted in 4 patients. There was bacterial and fungal growth in two patients each and one vitreous did not grow anything. All the eyes were phthisical at last visit. Infectious endophthalmitis after K-Pro implantation in our study had a higher incidence, early onset and extremely poor visual outcome compared with post-cataract surgery endophthalmitis, as reported in literature. Not only bacterial but also fungal infections are an important etiology for infectious endophthalmitis in these cases.

Endophthalmitis in Boston keratoprosthesis: case series and review of literature.

To report the clinical and microbiological characteristics of infectious endophthalmitis after Boston type I keratoprosthesis (B-K-Pro) implantation. Retrospective analysis of 136 eyes that received a B-K-Pro type 1 between 1999 and 2012 was performed. Five eyes with a diagnosis of exogenous endophthalmitis after B-K-pro type 1 were identified and information about demographic data, indication for K-Pro, post-operative bandage contact lens use, post-operative prophylactic antibiotic use, timing and clinical presentation of endophthalmitis, gram stain and culture results of intraocular fluid and preoperative and post-operative visual acuity were collected. The incidence of endophthalmitis was 3.67% (5 of 136 eyes) and average time to develop endophthalmitis was 5.62 months (range 2 days to 8 months). Mean patient age was 31.4 years (5 to 65 years). The surgical indications included corneal injury due to chemical burns (n = 2), multiple failed grafts secondary to microbial keratitis (n = 2) and congenital glaucoma with congenital herpetic keratitis (n = 1). Post-Boston K-Pro, the visual acuity ranged from light perception (LP) to 20/50. K-pro was explanted in 4 patients. There was bacterial and fungal growth in two patients each and one vitreous did not grow anything. All the eyes were phthisical at last visit. Infectious endophthalmitis after K-Pro implantation in our study had a higher incidence, early onset and extremely poor visual outcome compared with post-cataract surgery endophthalmitis, as reported in literature. Not only bacterial but also fungal infections are an important etiology for infectious endophthalmitis in these cases.

Measurement of consensual accommodation in vision-impaired eyes.

PURPOSE: To measure the accommodative response in unsighted or profoundly vision impaired (PVI) eyes when accommodation is elicited in the fellow, sighted eye. METHODS: Eighty-eight unilaterally PVI subjects (UPS) and 97 bilaterally sighted subjects (BSS) (10 to 45 years) were enrolled. Subjects had clear ocular media for auto-refraction and could steadily fixate targets with the sighted eye. For BSS, a long-pass filter was placed in front of one eye to simulate unilateral blindness. Both eyes were measured with a Shin-Nippon auto-refractor while fixating a 4/40 letter at 4 m and then an N8 letter at 40 cm and at 33 cm. Accommodation was calculated as the difference between distance and near refraction. RESULTS: Only subjects with repeatable alignment between measurements were included in the analyses (64 UPS, 95 BSS). Results were analyzed using t test and a generalized linear mixed model including age, sightedness, distance spherical equivalent, and accommodation as factors. The t test found no significant difference between eyes for UPS (p = 0.981 at 40 cm and p = 0.663 at 33 cm). For BSS, the sighting eye produced statistically significant but only slightly greater amounts of accommodation than the filtered eye (0.098 diopters [D], p = 0.002 at 40 cm and 0.189 D, p < 0.001 at 33 cm). The generalized linear mixed model found no difference between BSS and UPS in terms of difference in accommodation between eyes (p = 0.128 at 40 cm and p = 0.157 at 33 cm). CONCLUSIONS: The PVI eyes of unilaterally PVI individuals display similar accommodative response to their fellow, sighted eyes when accommodation is elicited by near target of up to 3 D to the fellow eye. However, the difference in accommodative response between PVI and fellow, sighted eye is related to the amount of accommodation elicited.

Chronic conjunctivitis due to Mycobacterium tuberculosis.

To report the clinical characteristics and treatment outcome in six patients with chronic conjunctivitis due to Mycobacterium tuberculosis. In this retrospective observational case series, all patients with a diagnosis of conjunctival tuberculosis seen in our clinics between January 2000 and January 2010 were reviewed. The clinical presentation, diagnostic investigations and response to medical therapy and outcomes were analyzed. Six patients (age range 15-47 years) were diagnosed with conjunctival tuberculosis. The mean duration from onset of symptoms to diagnosis was 6.5 months (range 1-12 months). Of the six patients, two had ulceration, one had a nodulo-ulcerative lesion, one had bilateral nodular epibulbar masses, and one had a hypertrophied papillary lesion. Systemic signs of tuberculosis were noted in two patients--pleural effusion in one and preauricular and submandibular lymph node involvement in the other. All patients had resolution of symptoms after treatment with four-drug anti-tuberculosis therapy (ATT). None had ocular or systemic recurrences after completion of ATT. Tuberculosis of the conjunctiva can have varied clinical presentation. Although a rare entity, it should be suspected in non-responding chronic conjunctivitis. A high index of suspicion and clinical examination aided by appropriate microbiological and histopathological testing can help in early diagnosis and management.

Outcome of tenonplasty in scleral ischemia secondary to acute ocular surface burn.

PURPOSE: To study the outcome of tenonplasty in eyes with perilimbal ischemia in acute ocular surface burns. METHODS: This was a single-center retrospective study that included all patients presenting between May 2012 and December 2022 with acute ocular surface burn (within 6 weeks from injury) with perilimbal scleral ischemia, without perforation or previous surgical intervention for the same. All these patients underwent single-stage tenonplasty with amniotic membrane transplantation and medical management. Successful outcome was defined as globe preservation at 6 months. RESULTS: Twenty-three eyes of 20 patients were included in our study. Out of these, alkali was the most common offending agent (10 eyes of 8 patients). The extent of scleral ischemia was <90° in seven eyes, 90°-180° in six eyes, and >180° in 10 eyes. The mean interval between the injury and tenonplasty was 16.5 days. Globe integrity in 20 of the 23 eyes was maintained 6 months post injury. Three eyes that developed hypotony at the end of 6 months had more than 180° of perilimbal scleral ischemia at presentation. The extent of scleral ischemia correlated with the development of phthisis bulbi, but the values were not statistically significant (P = 0.081). CONCLUSION: Tenonplasty in the acute stage acts as a globe-salvaging procedure in acute chemical burns with perilimbal scleral ischemia. Extensive scleral ischemia in an acute chemical burn remains a poor prognostic factor for globe integrity in the long term.

Bridging the gap: The promise of corneal bioengineering and regeneration.

In recent years, significant advances in tissue engineering and regenerative medicine have led to innovative approaches in addressing the various challenges associated with corneal transplants using bioengineered corneas. This mini-review aims to introduce the general ophthalmologist to the concept and technique of bioengineered cornea and provide an overview of the developments so far and an insight into the future direction. By summarizing the latest research and current limitations, we aim to highlight their potential for the future in ultimately contributing to vision restoration.

Visual outcome and postoperative complications of cataract surgery in patients with ocular surface disorders.

PURPOSE: To determine the visual outcome and postoperative complications of cataract surgery in patients with ocular surface disorders (OSDs). SETTING: Tertiary eyecare center in North India. DESIGN: Retrospective observational study. METHODS: Patients with various OSDs with stabilized ocular surfaces who underwent cataract surgery during this period and had a minimum postoperative follow-up of 6 weeks were included. The primary outcome measures were postoperative corrected distance visual acuity (CDVA) at 6 weeks, best CDVA achieved, and postoperative complications. RESULTS: The study included 20 men and 24 women. A total of 55 eyes were evaluated: Stevens-Johnson syndrome (SJS) 35 eyes, ocular cicatricial pemphigoid (OCP) 4 eyes, 8 eyes with dry eye disease (DED), 6 eyes with chemical injury and 2 eyes with vernal keratoconjunctivitis (VKC). The mean duration of OSD was 33.9 ± 52.17 months. The median preoperative CDVA was 2.0 (interquartile range [IQR], 1.45 to 2.0). The median CDVA ever achieved was 0.50 (IQR, 0.18 to 1.45) at 2 months and the median CDVA at 6 weeks was 0.6 (IQR, 0.3 to 1.5). Maximum improvement in CDVA was noted in patients with DED and SJS and the least in OCP. Phacoemulsification was performed in 47.27% eyes with intraoperative complications noted in 9% eyes. Postoperative surface complications occurred in 12 (21.82%) eyes. Other postoperative complications occurred in 9 (16%) eyes. CONCLUSIONS: Cataract surgery outcome can be visually rewarding in patients with OSDs provided ocular surface integrity is adequately maintained preoperatively and postoperatively.

Dealing With Pericylindrical Melts in Keratoprosthesis: Tenon Patch Graft to the Rescue.

PURPOSE: The aim of this study was to describe the outcomes of autologous Tenon patch graft in the management of Auro keratoprosthesis-related pericylindrical corneal melt. METHODS: We report 3 cases of sterile pericylindrical corneal melt in patients with Auro keratoprosthesis implantation after a mean duration of 5 years (1.5-8 years). Case 1 was a patient with severe graft-versus-host disease. Cases 2 and 3 were cases of chemical injury.All these cases of sterile pericylindrical corneal melt (4-6 mm) underwent autologous Tenon patch graft. The technique included freshening of the edges around the melt, followed by measuring the size of the defect. A Tenon graft harvested from the patient's own eye was used to seal the defect and act as a scaffold. The Tenon patch graft was spread over the melt and held in place by the application of fibrin glue and/or interrupted 10-0 nylon sutures. A bandage contact lens was then placed on the eye. RESULTS: Tenon patch graft was well taken in all patients. The mean duration of epithelial healing was 1 month. Globe integrity was well maintained with no postoperative complications at a mean follow-up duration of 12 months (6-18 months). CONCLUSIONS: Corneal melt is one of the most dreaded complications of KPro because its occurrence could threaten visual prognosis and globe integrity. Autologous Tenon patch is a simple yet innovative and effective option to steer such eyes away from potentially dreadful complications.

Corneal stromal changes following simple limbal epithelial transplantation on Scheimpflug densitometry: Early results.

CONTEXT: Chemical injury to eyes causes limbal stem cell deficiency (LSCD), which leads to conjunctival epithelium and underlying stromal changes. Simple limbal epithelial transplant helps to restore corneal epithelium and corneal stromal clarity. These changes are documented in this prospective eye study. PURPOSE: To report the changes in epithelial/stromal (E/S) reflectivity ratio and corneal stromal densitometry following simple limbal epithelial transplantation (SLET) in patients with unilateral chemical burn-induced LSCD. METHODS AND MATERIAL: It is a prospective imaging study of cornea before and after SLET. Corneal densitometry with Pentacam and epithelial and stromal reflectivity on anterior segment optical coherence tomography were analyzed for five patients (n = 5 eyes), who underwent autologous SLET for unilateral chemical burn-induced LSCD. STATISTICAL ANALYSIS USED: Mann-Whitney U-test and Pearson correlation. RESULTS: A significant improvement in E/S reflectivity was noted at 1 month (P < 0.05) after SLET, and this was maintained until the end of 6 months. The densitometry measurements decreased in the entire cornea at every level, that is, anterior, central, and posterior cornea. However, the values did not normalize till the last follow-up at 6 months. CONCLUSIONS: A trend of normal corneal epithelization exists in eyes undergoing SLET. Densitometry at all the levels, the anterior, central, and posterior cornea, also decreases after SLET. SLET improves visual outcomes in LSCD eyes not only by epithelization but also by reducing stromal scarring.

In vitro Assay to Examine the Function of Tears on Corneal Epithelial Cells.

Tears contain numerous secreted factors, enzymes, and proteins that help in maintaining the homeostatic condition of the eye and also protect it from the external environment. However, alterations to these enzymes and/or proteins during pathologies such as mechanical injury and viral or fungal infections can disrupt the normal ocular homeostasis, further contributing to disease development. Several tear film components have a significant role in curbing disease progression and promoting corneal regeneration. Additionally, several factors related to disease progression are secreted into the tear film, thereby serving as a valuable reservoir of biomarkers. Tears are readily available and can be collected via non-invasive techniques or simply from contact lenses. Tears can thus serve as a valuable and easy source for studying disease-specific biomarkers. Significant advancements have been made in recent years in the field of tear film proteomics, lipidomics, and transcriptomics to allow a better understanding of how tears can be utilized to gain insight into the etiology of diseases. These advancements have enabled us to study the pathophysiology of various disease states using tear samples. However, the mechanisms by which tears help to maintain corneal homeostasis and how they are able to form the first line of defense against pathogens remain poorly understood and warrant detailed in vitro studies. Herein, we have developed an in vitro assay to characterize the functional importance of patient isolated tears and their components on corneal epithelial cells. This novel approach closely mimics real physiological conditions and could help the researchers gain insight into the underlying mechanisms of ocular pathologies and develop new treatments. Key features • This method provides a new technique for analyzing the effect of tear components on human corneal epithelial cells. • The components of the tears that are altered in response to diseases can be used as a biomarker for detecting ocular complications. • This procedure can be further employed as an in vitro model for assessing the efficacy of drugs and discover potential therapeutic interventions.

Chemical (Alkali) Burn-Induced Neurotrophic Keratitis Model in New Zealand Rabbit Investigated Using Medical Clinical Readouts and In Vivo Confocal Microscopy (IVCM).

PURPOSE: Chemical eye injury is an acute emergency that can result in vision loss. Neurotrophic keratitis (NK) is the most common long-term manifestation of chemical injury. NK due to alkali burn affects ocular surface health and is one of its most common causes. Here, we established a rabbit model of corneal alkali burns to evaluate the severity of NK-associated changes. MATERIAL METHODS: Alkali burns were induced in NZ rabbits by treating the cornea with (i) a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (Mild NK) and (ii) trephination using a guarded trephine (5 mm diameter and 150-micron depth), followed by alkali burn, with a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (a severe form of NK). Immediately after, the cornea was rinsed with 10 mL of normal saline to remove traces of NaOH. Clinical features were evaluated on Day 0, Day 1, Day 7, Day 15, and Day 21 post-alkali burn using a slit lamp, Pentacam, and anterior segment optical coherence tomography (AS-OCT). NK-like changes in epithelium, sub-basal nerve plexus, and stroma were observed using in vivo confocal microscopy (IVCM), and corneal sensation were measured using an aesthesiometer post alkali injury. After 21 days, pro-inflammatory cytokines were evaluated for inflammation through ELISA. RESULTS: Trephination followed by alkali burn resulted in the loss of epithelial layers (manifested using fluorescein stain), extensive edema, and increased corneal thickness (550 µm compared to 380 µm thickness of control) evaluated through AS-OCT and increased opacity score in alkali-treated rabbit (80 compared to 16 controls). IVCM images showed complete loss of nerve fibers, which failed to regenerate over 30 days, and loss of corneal sensation-conditions associated with NK. Cytokines evaluation of IL6, VEGF, and MMP9 indicated an increased angiogenic and pro-inflammatory milieu compared to the milder form of NK and the control. DISCUSSION: Using clinical parameters, we demonstrated that the alkali-treated rabbit model depicts features of NK. Using IVCM in the NaOH burn animal model, we demonstrated a complete loss of nerve fibers with poor self-healing capability associated with sub-basal nerve degeneration and compromised corneal sensation. This pre-clinical rabbit model has implications for future pre-clinical research in neurotrophic keratitis.

Kuragel: A biomimetic hydrogel scaffold designed to promote corneal regeneration.

Cornea-related injuries are the most common cause of blindness worldwide. Transplantation remains the primary approach for addressing corneal blindness, though the demand for donor corneas outmatches the supply by millions. Tissue adhesives employed to seal corneal wounds have shown inefficient healing and incomplete vision restoration. We have developed a biodegradable hydrogel - Kuragel, with the ability to promote corneal regeneration. Functionalized gelatin and hyaluronic acid form photo-crosslinkable hydrogel with transparency and compressive modulus similar to healthy human cornea. Kuragel composition was tuned to achieve sufficient adhesive strength for sutureless integration to host tissue, with minimal swelling post-administration. Studies in the New Zealand rabbit mechanical injury model affecting corneal epithelium and stroma demonstrate that Kuragel efficiently promotes re-epithelialization within 1 month of administration, while stroma and sub-basal nerve plexus regenerate within 3 months. We propose Kuragel as a regenerative treatment for patients suffering from corneal defects including thinning, by restoration of transparency and thickness.