Dual-ROS-scavenging and dual-lingering nanozyme-based eye drops alleviate dry eye disease.

Efficiently removing excess reactive oxygen species (ROS) generated by various factors on the ocular surface is a promising strategy for preventing the development of dry eye disease (DED). The currently available eye drops for DED treatment are palliative, short-lived and frequently administered due to the short precorneal residence time. Here, we developed nanozyme-based eye drops for DED by exploiting borate-mediated dynamic covalent complexation between n-FeZIF-8 nanozymes (n-Z(Fe)) and poly(vinyl alcohol) (PVA) to overcome these problems. The resultant formulation (PBnZ), which has dual-ROS scavenging abilities and prolonged corneal retention can effectively reduce oxidative stress, thereby providing an excellent preventive effect to alleviate DED. In vitro and in vivo experiments revealed that PBnZ could eliminate excess ROS through both its multienzyme-like activity and the ROS-scavenging activity of borate bonds. The positively charged nanozyme-based eye drops displayed a longer precorneal residence time due to physical adhesion and the dynamic borate bonds between phenyboronic acid and PVA or o-diol with mucin. The in vivo results showed that eye drops could effectively alleviate DED. These dual-function PBnZ nanozyme-based eye drops can provide insights into the development of novel treatment strategies for DED and other ROS-mediated inflammatory diseases and a rationale for the application of nanomaterials in clinical settings.

Comprehensive dry eye therapy: overcoming ocular surface barrier and combating inflammation, oxidation, and mitochondrial damage.

BACKGROUND: Dry Eye Disease (DED) is a prevalent multifactorial ocular disease characterized by a vicious cycle of inflammation, oxidative stress, and mitochondrial dysfunction on the ocular surface, all of which lead to DED deterioration and impair the patients' quality of life and social functioning. Currently, anti-inflammatory drugs have shown promising efficacy in treating DED; however, such drugs are associated with side effects. The bioavailability of ocular drugs is less than 5% owing to factors such as rapid tear turnover and the presence of the corneal barrier. This calls for investigations to overcome these challenges associated with ocular drug administration. RESULTS: A novel hierarchical action liposome nanosystem (PHP-DPS@INS) was developed in this study. In terms of delivery, PHP-DPS@INS nanoparticles (NPs) overcame the ocular surface transport barrier by adopting the strategy of "ocular surface electrostatic adhesion-lysosomal site-directed escape". In terms of therapy, PHP-DPS@INS achieved mitochondrial targeting and antioxidant effects through SS-31 peptide, and exerted an anti-inflammatory effect by loading insulin to reduce mitochondrial inflammatory metabolites. Ultimately, the synergistic action of "anti-inflammation-antioxidation-mitochondrial function restoration" breaks the vicious cycle associated with DED. The PHP-DPS@INS demonstrated remarkable cellular uptake, lysosomal escape, and mitochondrial targeting in vitro. Targeted metabolomics analysis revealed that PHP-DPS@INS effectively normalized the elevated level of mitochondrial proinflammatory metabolite fumarate in an in vitro hypertonic model of DED, thereby reducing the levels of key inflammatory factors (IL-1ß, IL-6, and TNF-α). Additionally, PHP-DPS@INS strongly inhibited reactive oxygen species (ROS) production and facilitated mitochondrial structural repair. In vivo, the PHP-DPS@INS treatment significantly enhanced the adhesion duration and corneal permeability of the ocular surface in DED mice, thereby improving insulin bioavailability. It also restored tear secretion, suppressed ocular surface damage, and reduced inflammation in DED mice. Moreover, it demonstrated favorable safety profiles both in vitro and in vivo. CONCLUSION: In summary, this study successfully developed a comprehensive DED management nanosystem that overcame the ocular surface transmission barrier and disrupted the vicious cycle that lead to dry eye pathogenesis. Additionally, it pioneered the regulation of mitochondrial metabolites as an anti-inflammatory treatment for ocular conditions, presenting a safe, efficient, and innovative therapeutic strategy for DED and other inflammatory diseases.

Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

A single chain variable fragment antibody (Tn 64) cognate to fibronectin type III repeats promotes corneal wound healing by inhibiting fibrosis.

Corneal wound healing requires epithelial reorganization and stromal extracellular matrix (ECM) remodeling, with ECM proteins such as Tenascin C (TnC) regulating and maintaining corneal homeostasis. The N-terminal globular domain and C-terminal fibrinogen-related domains of TnC are separated by epidermal growth factor (EGF)-like repeats, and upto fifteen fibronectin type III domains (Tn fn). Overexpression of Tn fn 1-5 and its splice variants occurs in varied pathologies. We have previously used Tn64 (a single chain variable fragment antibody cognate to Tn fn 1-5) to establish roles of Tn fn 1-5 in fibrotic pathologies such as rheumatoid arthritis and posterior capsular opacification. Here, we show that Tn64 binds to Tn fn repeats 3-5 (which constitute the major site for binding of soluble fibronectin within TnC). Unlike other Tn fn domains, Tn fn 3-5 displays no inhibition of fibronectin matrix assembly. Rather, the Tn fn 3-5 construct is pro-fibrotic and elicits increased expression of fibronectin. We examined corneal epithelial as well as stromal wound healing through Tn64 binding to Tn fn 3-5, using a human corneal epithelial cell (HCEC) line, primary cultures of human corneal fibroblasts (HCFs), and an ex-vivo corneal organ culture model. Tn64 enhanced proliferation and adhesion of corneal epithelial cells, while inhibiting the migration of corneal fibroblasts and myofibroblasts. Tn64 appears to attenuate inflammation through downregulation of TNF-α, prevent corneal fibrosis by limiting fibronectin polymerization, and promote regeneration of corneal epithelia and stroma, suggesting that it could be developed as a therapeutic agent for effective anti-fibrotic corneal wound healing.

RiTe conjugate mediated corneal collagen crosslinking, a novel therapeutic intervention for keratoconus - in vitro and in vivo study.

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5'Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.

Nanomicelles empower natamycin in treating fungal keratitis: An in vitro, ex vivo and in vivo study.

Fungal infections of cornea are important causes of blindness especially in developing nations with tropical climate. However, the challenges associated with current treatments are responsible for poor outcome. Natamycin is the only FDA-approved antifungal drug to treat fungal keratitis, but unfortunately due to its poor water solubility, it is available as suspension. The marketed suspension (5% Natamycin) has rapid precorneal clearance, poor corneal permeability, a higher frequency of administration, and corneal irritation due to undissolved suspended drug particles. In our study, we developed clear and stable natamycin-loaded nanomicelles (1% Natcel) to overcome the above challenges. We demonstrated that 1% Natcel could permeate the cornea better than 5% suspension. The developed 1% Natcel was able to provide sustained release for up to 24 h. Further, it was found to be biocompatible and also improved the mean residence time (MRT) than 5% suspension in tears. Therefore, the developed 1% Natcel could be a potential alternative treatment for fungal keratitis.

Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium.

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.

Silk Fibroin Formed Bioadhesive Ophthalmic Gel for Dry Eye Syndrome Treatment.

PURPOSE: Dry eye syndrome (DES), arising from various etiologic factors, leads to tear film instability and ocular surface damage. Given its anti-inflammatory effects, cyclosporine A (CsA) has been widely used as a short-term treatment option for DES. However, poor bioavailability and solubility of CsA in aqueous phase make the development of a cyclosporine A-based eye drop for ocular topical application a huge challenge. METHODS: In this study, a novel strategy for preparing cyclosporine A-loaded silk fibroin nanoemulsion gel (CsA NBGs) was proposed to address these barriers. Additionally, the rheological properties, ocular irritation potential, tear elimination kinetics, and pharmacodynamics based on a rabbit dry eye model were investigated for the prepared CsA NBGs. Furthermore, the transcorneal mechanism across the ocular barrier was also investigated. RESULTS: The pharmacodynamics and pharmacokinetics of CsA NBGs exhibited superior performance compared to cyclosporine eye drops, leading to a significant enhancement in the bioavailability of CsA NBGs. Furthermore, our investigation into the transcorneal mechanism of CsA NBGs revealed their ability to be absorbed by corneal epithelial cells via the paracellular pathway. CONCLUSION: The CsA NBG formulation exhibits promising potential for intraocular drug delivery, enabling safe, effective, and controlled administration of hydrophobic drugs into the eye. Moreover, it enhances drug retention within the ocular tissues and improves systemic bioavailability, thereby demonstrating significant clinical translational prospects.

Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using 15N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO3-) obtained from the diet is converted to nitrite (NO2-) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite. To gain deeper insights into nitrate metabolism in porcine eyes, our current study involved feeding pigs either NaCl or Na15NO3 and assessing the levels of total and 15N-labeled NO3-/NO2- in various ocular tissues. Three hours after Na15NO3 ingestion, a marked increase in 15NO3- and 15NO2- was observed in all parts of the eye; in particular, the aqueous and vitreous humor showed a high 15NO3- enrichment (77.5 and 74.5%, respectively), similar to that of plasma (77.1%) and showed an even higher 15NO2- enrichment (39.9 and 35.3%, respectively) than that of plasma (19.8%). The total amounts of NO3- and NO2- exhibited patterns consistent with those observed in 15N analysis. Next, to investigate whether nitrate or nitrite accumulate proportionally after multiple nitrate treatments, we measured nitrate and nitrite contents after supplementing pigs with Na15NO3 for five consecutive days. In both 15N-labeled and total nitrate and nitrite analysis, we did not observe further accumulation of these ions after multiple treatments, compared to a single treatment. These findings suggest that dietary nitrate supplementation exerts a significant influence on nitrate and nitrite levels and potentially NO levels in the eye and opens up the possibility for the therapeutic use of dietary nitrate/nitrite to enhance or restore NO levels in ocular tissues.

Substance P promotes transforming growth factor-ß-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-ß on collagen type I synthesis without affecting that of IL-1ß on the expression of matrix metalloproteinase-1. This effect of SP on TGF-ß-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-ß-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-ß on collagen type I synthesis without affecting that of interleukin-1ß on the expression of matrix metalloproteinase-1.

Chlorine-Induced Toxicity on Murine Cornea: Exploring the Potential Therapeutic Role of Antioxidants.

Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.

Balanced activation of Nrf-2/ARE mediates the protective effect of sulforaphane on keratoconus in the cell mechanical microenvironment.

Keratoconus (KC) is a progressive degenerative disease that usually occurs bilaterally and is characterized by corneal thinning and apical protrusion of the cornea. Oxidative stress is an indicator of the accumulation of reactive oxygen species (ROS), and KC keratocytes exhibit increased ROS production compared with that of normal keratocytes. Therefore, oxidative stress in KC keratocytes may play a major role in the development and progression of KC. Here, we investigated the protective effect of sulforaphane (SF) antioxidants using a hydrogel-simulated model of the cell mechanical microenvironment of KC. The stiffness of the KC matrix microenvironment in vitro was 16.70 kPa and the stiffness of the normal matrix microenvironment was 34.88 kPa. Human keratocytes (HKs) were cultured for 24 h before observation or drug treatment with H2O2 in the presence or absence of SF. The levels of oxidative stress, nuclear factor E2-related factor 2 (Nrf-2) and antioxidant response element (ARE) were detected. The high-stress state of HKs in the mechanical microenvironment of KC cells compensates for the activation of the Nrf-2/ARE signaling pathway. H2O2 leads to increased oxidative stress and decreased levels of antioxidant proteins in KC. In summary, SF can reduce endogenous and exogenous oxidative stress and increase the antioxidant capacity of cells.

Role of Polyunsaturated Fatty Acids (PUFAs) and Eicosanoids on Dry Eye Symptoms and Signs.

Polyunsaturated fatty acids (PUFAs) generate pro- and anti-inflammatory eicosanoids via three different metabolic pathways. This study profiled tear PUFAs and their metabolites and examined the relationships with dry eye (DE) and meibomian gland dysfunction (MGD) symptoms and signs. A total of 40 individuals with normal eyelids and corneal anatomies were prospectively recruited. The symptoms and signs of DE and MGD were assessed, and tear samples (from the right eye) were analyzed by mass spectrometry. Mann-Whitney U tests assessed differences between medians; Spearman tests assessed correlations between continuous variables; and linear regression models assessed the impact of potential confounders. The median age was 63 years; 95% were male; 30% were White; and 85% were non-Hispanic. The symptoms of DE/MGD were not correlated with tear PUFAs and eicosanoids. DE signs (i.e., tear break-up time (TBUT) and Schirmer's) negatively correlated with anti-inflammatory eicosanoids (11,12-dihydroxyeicosatrienoic acid (11,12 DHET) and 14,15-dihydroxyicosatrienoic acid (14,15, DHET)). Corneal staining positively correlated with the anti-inflammatory PUFA, docosahexaenoic acid (DHA). MGD signs significantly associated with the pro-inflammatory eicosanoid 15-hydroxyeicosatetranoic acid (15-HETE) and DHA. Several relationships remained significant when potential confounders were considered. DE/MGD signs relate more to tear PUFAs and eicosanoids than symptoms. Understanding the impact of PUFA-related metabolic pathways in DE/MGD may provide targets for new therapeutic interventions.

UVB induced reactivation leads to HSV1 in the corneas of virtually all latently infected mice and requires STING to develop corneal disease.

Reactivation of latent herpes simplex type 1 results in virus returning to the cornea leading to recurrent herpetic stromal keratitis (rHSK). We compare two competing models to reactivate viruses from latency, UV-B irradiation and cyclophosphamide (CP). Results revealed that while both result in corneal recrudescence, only UV-B irradiation results in rHSK. To better understand the dynamics of reactivation, we analyzed corneas for both the presence of infectious viruses and the dynamics of exposure to multiple reactivations using UV-B. We noted that multiple reactivations result in progressively worse corneal disease. We also noted that expression of IFNα and STING, surragate markers for the presence of virus, are induced by the presence of reactivated virus. Studies to determine the importance of STING to the development of HSK revealed that in the absence of STING, mice do not develop significant HSK and the magnitude of the infiltrate of CD45+ cells in these corneas is significantly reduced. The resulting paucity of CD45+CD11b+GR-1+F4/80-neutrophils, and to a lesser extent CD45+CD11b+GR-1-F4/80+ macrophages in B6-STING KO mice following reactivation is likely the underlying cause for lack of rHSK as has been noted by ourselves and others. These results underscore the critical importance of STING's role in developing rHSK.

Baicalein glycymicelle ophthalmic solution: Preparation, in vitro antimicrobial activities, and antimicrobial mechanism evaluations.

The purpose of this study was to develop a novel baicalein (BAI) loaded glycymicelle ophthalmic solution with small molecule phytochemical glycyrrhizin as nanocarriers and to explore this solution's potential as an antimicrobial agent against ocular infections. The optimized BAI glycymicelles had a high encapsulation efficiency (98.76 ±â€¯1.25 %), a small particle size (54.38 ±â€¯2.41 nm), a uniform size distribution (polydispersity index = 0.293 ±â€¯0.083), and a zeta potential of -28.3 ±â€¯1.17 mV. The BAI glycymicelle ophthalmic solution exhibited an excellent short-term storage stability. BAI glycymicelles significantly increased the apparent solubility and in vitro release capability of BAI. The BAI glycymicelle ophthalmic solution exhibited no hen's egg-chorioallantoic membrane' irritation and strong in vivo ocular tolerance in rabbits. The BAI glycymicelles noticeably enhanced the in vivo corneal permeation. The BAI glycymicelles also precipitated increased in vitro antioxidant activity and significantly improved in vitro antipathogen activities. Various antimicrobial mechanisms, including the destruction of the bacterial cell wall, damage to the bacterial cell membranes, interruptions to the biofilm structure, and the apoptosis of bacteria, were inflicted on BAI glycymicelles. These findings provided useful knowledge regarding the development of a novel ophthalmic solution and formulation of BAI.

Revisiting rabbit models for keratoconus: A long-term study on collagenase-induced disease progression.

Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.

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.

A Comparative Pharmacokinetic Study for Cysteamine-Containing Eye Drops as an Orphan Topical Therapy in Cystinosis.

Cystinosis is a low-prevalence lysosomal storage disease. The pathomechanism involves abnormal functioning of the cystinosine lysosomal cystine transporter (CTNS), causing intraliposomal accumulation of the amino acid cysteine disulfide, which crystallizes and deposits in several parts of the body. The most common ophthalmic complication of cystinosis is the deposition of "gold dust" cystine crystals on the cornea, which already occurs in infancy and leads to severe photosensitivity and dry eyes as it gradually progresses with age. In the specific treatment of cystinosis, preparations containing cysteamine (CYA) are used. The availability of commercialized eyedrops for the targeted treatment is scarce, and only Cystadrops® are commercially available with strong limitations. Thus, magistral CYA-containing compounded eyedrops (CYA-CED) could have a key role in patient care; however, a rationally designed comprehensive study on the commercialized and magistral products is still missing. This work aims to build up a comprehensive study about commercialized and magistral CYA eye drops, involving pharmacokinetic and physicochemical characterization (applying mucoadhesivity, rheology test, investigation of drug release, and parallel artificial membrane permeability assays), as well as ex vivo tests, well supported by statistical analysis.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.