Subconjunctival injection of rapamycin-loaded polymeric microparticles for effective suppression of noninfectious uveitis in rats.

Noninfective uveitis is a major cause of vision impairment, and corticosteroid medication is a mainstay clinical strategy that causes severe side effects. Rapamycin (RAPA), a potent immunomodulator, is a promising treatment for noninfective uveitis. However, because high and frequent dosages are required, it is a great challenge to implement its clinical translation for noninfective uveitis therapy owing to its serious toxicity. In the present study, we engineered an injectable microparticulate drug delivery system based on biodegradable block polymers (i.e., polycaprolactone-poly (ethylene glycol)-polycaprolactone, PCEC) for efficient ocular delivery of RAPA via a subconjunctival injection route and investigated its therapeutic efficacy in an experimental autoimmune uveitis (EAU) rat model. RAPA-PCEC microparticles were fabricated using the emulsion-evaporation method and thoroughly characterized using scanning electron microscopy, fourier transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. The formed microparticles exhibited slow in vitro degradation over 28 days, and provided both in vitro and in vivo sustained release of RAPA over 4 weeks. Additionally, a single subconjunctival injection of PCEC microparticles resulted in high ocular tolerance. More importantly, subconjunctival injection of RAPA-PCEC microparticles significantly attenuated the clinical signs of EAU in a dose-dependent manner by reducing inflammatory cell infiltration (i.e., CD45+ cells and Th17 cells) and inhibiting microglial activation. Overall, this injectable microparticulate system may be promising vehicle for intraocular delivery of RAPA for the treatment of noninfective uveitis.

The new preservative-free ophthalmic formulation of bilastine 0.6% preserves the ocular surface epithelial integrity in a comparative in vitro study.

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5-10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface.

Biochemical and histopathological evaluation of systemic and ocular toxicity of favipiravir in rats.

Purpose: Favipiravir (FAV) used against COVID-19 is an antiviral drug that causes adverse reactions, such as hyperuricaemia, liver damage, and hematopoetic toxicity. The aim of the study was to investigate the systemic and ocular side-effects of FAV in rats, for the first time.Materials and methods: A total of 18 albino male Wistar rats were used in the study. The rats were divided into 3 groups as the healthy group (HG), the group given 50 mg/kg/day favipiravir (FAV50), and the group given 200 mg/kg/d favipiravir (FAV200). These doses were given to the experimental groups for one week. At the end of the experiment histopathological examinations were performed on the conjunctiva and sclera of the eye. In addition, malondialdehyde (MDA), total glutathione (tGSH), superoxide dismutase (SOD), interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-α) levels were measured in blood samples taken from rats. Results: Compared to HG, the MDA (1.37 ± 0.61 vs. 4.82 ± 1.40 µmol/mL), IL-1ß (2.52 ± 1.14 vs. 6.67 ± 1.99 pg/mL), and TNF-α levels (3.28 ± 1.42 vs. 8.53 ± 3.06 pg/mL) of the FAV200 group were higher. The levels of tGSH (7.58 ± 1.98 vs. 2.50 ± 0.98 nmol/mL) and SOD (13.63 ± 3.43 vs. 3.81 ± 1.43 U/mL) the FAV200 group were lower than the HG (p < 0.05, for all). The degree of damage to the cornea and sclera of the FAV200 group was quite high according to HG (p < 0.001). Conclusions: FAV can cause damage to rat conjunctiva and sclera by increasing oxidant stress and inflammation at high dose.

Effects of topical ketorolac tromethamine on tear parameters, meibography, goblet cell density, and conjunctival oxidative stress in healthy dogs.

OBJECTIVES: The objective of the study was to evaluate whether a twice-daily instillation of 0.45% preservative-free ketorolac tromethamine (FKT) or 0.4% benzalkonium chloride-preserved ketorolac tromethamine (BACKT), every 12 h for 30 days may affect tear film parameters and the meibography in healthy dogs. Additionally, we assessed whether the same treatments irritated the ocular surface, affected goblet cell density (GCD), and the levels of oxidative stress biomarkers (OSB) in the conjunctiva of the same dogs. PROCEDURES: Experimental and masked comparison study. In 11 healthy dogs baseline values of the lipid layer thickness, tear meniscus height, non-invasive tear breakup time (NI-TFBT), and the meibomian gland (MG) loss were assessed by OSAvet®. For each dog, one eye received 40 µL of BACKT, while the other received 40 µL FKT, every 12 h for 30 consecutive days. Tear parameters and meibography were repeated 15, 30, and 60 days post-treatments. Conjunctival hyperemia and blepharospasm were monitored at the same time points. At baseline and Day 30, a conjunctival biopsy was collected for GCD and OSB determination. RESULTS: Conjunctival hyperemia and blepharospasm were not observed. At Day 15, the MG loss increased only in FKT-treated eyes (p < .001). On Day 30, both treatment groups showed increased MG loss, shortened NI-TFBT, and reduced GCD and catalase (p < .05). At Day 30, BACKT-treated eyes showed lower levels of superoxide dismutase (SOD) (p = .006) and higher levels of malondialdehyde (MDA) (p = .02). Differences between treatments were not observed for any parameter at any time point (p > .05). 60 days after treatment, OSAvet® parameters tended to return to values assessed at baseline; however, significant differences remained for MG loss (p < .05). CONCLUSIONS: Twice-daily instillation of KT, containing or not BAC, for 30 consecutive days shortened NI-TFBT, decreased GCD, and increased the MG loss in healthy dogs. KT should be used with caution when prescribed for long periods, particularly in patients with tear film abnormalities. However, future controlled studies using KT, BAC, and other topical NSAIDs are indicated to further support this finding.

Comparison of cytotoxicity effects induced by four different types of nanoparticles in human corneal and conjunctival epithelial cells.

The impact of particulate matter (PM) on ocular surface health has attracted increased attention in recent years. Previous studies have reported that differences in the chemical composition of PM can affect the toxicological response. However, available information on the toxic effects of chemical components of PM on the ocular surface is insufficient. In this paper, we aimed to investigate the toxicity effects of chemical components of PM on the ocular surface, focusing on the effects of four different types of nanoparticles (NPs) in human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs), which include titanium dioxide (TiO2), carbon black (CB), zinc dioxide (ZnO), and silicon dioxide (SiO2). We found that the in vitro cytotoxic effects of CB, ZnO, and SiO2 NPs are dependent on particle properties and cell type as well as the exposure concentration and time. Here, the order of increasing toxicity was SiO2 → CB → ZnO, while TiO2 demonstrated no toxicity. Moreover, toxic effects appearing more severe in HCECs than HCjECs. Reactive oxygen species (ROS)-mediated oxidative stress plays a key role in the toxicity of these three NPs in HCECs and HCjECs, leading to apoptosis and mitochondrial damage, which are also important contributors to aging. Sirtuin1 (SIRT1) as an NAD+-dependent protein deacetylase that seems to play a potential protective role in this process. These findings implied that ROS and/or SIRT1 may become a potential target of clinical treatment of PM- or NP-related ocular surface diseases.

Simplified ex-vivo drug evaluation in ocular surface cells: Culture on cellulose filters of cells obtained by impression cytology.

Drug development, resource- and time-intensive, extensively employs cell-based assays to assess the efficacy and safety of candidate drugs. The widely used immortalized cell lines, experimentally convenient, have limited predictive value. In contrast, ex-vivo models more faithfully reproduce diseases but are technically challenging to establish. To address this need, we developed a simplified process for ex-vivo cell culture, demonstrating its feasibility in ocular surface cells. Conjunctival cells were harvested by impression cytology and grown on mixed cellulose ester membrane filters (MCFs). Human and rabbit conjunctival cells cultured on MCFs are 100% viable at 24 h, and 43% viable at 72 h. A gene expression study evaluating 84 genes involved in ocular inflammation demonstrated that ex-vivo culturing maintains intact the expression of two thirds of these genes in human cells. That these cells are suitable for the assessment of ocular drugs was demonstrated by studying the effect of phosphosulindac (PS), a small molecule under development for the treatment of dry eye disease, in both human and rabbit conjunctival cells. PS, for example, suppressed the expression of CXCL10, a cytokine participating in the pathogenesis of dry eye disease, in human and in rabbit conjunctival cells cultured ex-vivo by 32% and 70%, respectively. Conjunctival cells cultured ex-vivo can be transfected to evaluate mechanistic questions. We successfully transfected such cells with a plasmid expressing luciferase under the control of an IFN-γ-responsive promoter or its control plasmid. IFN-γ stimulated luciferase expression by 85% in cells with the responsive plasmid but not in controls; PS significantly suppressed this induction by 37% without affecting the control plasmid. These findings demonstrate that human and rabbit conjunctival cells cultured ex-vivo with our method are viable and maintain their biological integrity; respond to biological and pharmacological agents; and are transfectable with informative plasmids. The unique advantage of this method is to potentially accelerate the development of novel drugs for the treatment of ocular surface diseases, and to advance our understanding of ocular surface pathophysiology.

Effects of eye drops containing a mixture of 3% diquafosol sodium and tocopherol acetate (vitamin E) on the ocular surface of murine dry eye.

PURPOSE: To investigate the efficacy of topical application of 3% diquafosol sodium (DQS) and tocopherol (TCP) acetate mixtures in a mouse model of experimental dry eye (EDE). METHODS: After exposure to desiccating stress for 5 days, eye drops consisting of 3% DQS alone, 0.01% TCP alone, or 3% DQS and 0.005% or 0.01% TCP mixture were applied for the treatment of EDE. Tear volume, tear film break-up time (TBUT), corneal fluorescein staining scores (CFSS), and tear film lipid layer grades (TFLLG) were measured at 0, 5 and 10 days after treatment. The 2',7'-dichlorodihydrofluorescein diacetate assay (DCFDA) for reactive oxygen species (ROS) production, enzyme-linked immunosorbent assay (ELISA) for malondialdehyde (MDA), and flow cytometry for CD4 + interferon (IFN)-γ+ T cells were evaluated on the ocular surface at 10 days after treatment. In addition, levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and chemokine CC motif ligand 4 (CCL4) in the conjunctiva were measured using a multiplex immunobead assay, and conjunctival goblet cells were counted by periodic acid-Schiff staining at 10 days after treatment. RESULTS: Both the TCP mixture groups indicated a significant improvement in TBUT, ROS production, and MDA concentrations compared to those in the DQS alone group. Furthermore, the 0.01% TCP mixture group also showed higher tear film lipid layer grades and conjunctival goblet cell density and lower corneal fluorescein staining scores, number of CD4 + IFN-γ+ T cells, and levels of TNF-α, IL-1ß, and CCL4 than the DQS alone group (P < 0.05). CONCLUSIONS: Application of eye drops containing the mixture of DQS and TCP could stabilize the tear film lipid layer, improve TBUT and corneal epithelial damages, decrease ROS production, inflammatory molecules, and T cells, and increase conjunctival goblet cell density on the ocular surface. Topical DQS and TCP mixtures may have a greater therapeutic effect on clinical signs, oxidative damage, and inflammation of dry eye than DQS eye drops.

The Protective Effect of Oral Application of Corni Fructus on the Disorders of the Cornea, Conjunctiva, Lacrimal Gland and Retina by Topical Particulate Matter 2.5.

Particulate matter 2.5 (PM2.5) may aggravate dry eye disease (DED). Corni Fructus (CF), which is fruit of Cornus officinalis Sieb. et Zucc., has been reported to have various beneficial pharmacological effects, whereas the effect of CF on the eye is still unknown. Therefore, in this study, we investigated the effect of oral administration of water extract of CF (CFW) on the eye, hematology, and biochemistry in a DED model induced by topical exposure to PM2.5. Furthermore, the efficacy of CFW compared with cyclosporine (CsA), an anti-inflammatory agent, and lutein, the posterior eye-protective agent. Sprague-Dawley rats were topically administered 5 mg/mL PM2.5 in both eyes four times daily for 14 days. During the same period, CFW (200 mg/kg and 400 mg/kg) and lutein (4.1 mg/kg) were orally administered once a day. All eyes of rats in the 0.05% cyclosporine A (CsA)-treated group were topically exposed to 20 µL of CsA, twice daily for 14 days. Oral administration of CFW attenuated the PM2.5-induced reduction of tear secretion and corneal epithelial damage. In addition, CFW protected against goblet cell loss in conjunctiva and overexpression of inflammatory factors in the lacrimal gland following topical exposure to PM2.5. Furthermore, CFW markedly prevented PM2.5-induced ganglion cell loss and recovered the thickness of inner plexiform layer. Meanwhile, CFW treatment decreased the levels of total cholesterol and low-density lipoprotein cholesterol in serum induced by PM2.5. Importantly, the efficacy of CFW was superior or similar to that of CsA and lutein. Taken together, oral administration of CFW may have protective effects against PM2.5-induced DED symptoms via stabilization of the tear film and suppression of inflammation. Furthermore, CFW may in part contribute to improving retinal function and lipid metabolism disorder.

Inhibition of Aberrant α(1,2)-Fucosylation at Ocular Surface Ameliorates Dry Eye Disease.

Fucosylation is involved in a wide range of biological processes from cellular adhesion to immune regulation. Although the upregulation of fucosylated glycans was reported in diseased corneas, its implication in ocular surface disorders remains largely unknown. In this study, we analyzed the expression of a fucosylated glycan on the ocular surface in two mouse models of dry eye disease (DED), the NOD.B10.H2b mouse model and the environmental desiccating stress model. We furthermore investigated the effects of aberrant fucosylation inhibition on the ocular surface and DED. Results demonstrated that the level of type 2 H antigen, an α(1,2)-fucosylated glycan, was highly increased in the cornea and conjunctiva both in NOD.B10.H2b mice and in BALB/c mice subjected to desiccating stress. Inhibition of α(1,2)-fucosylation by 2-deoxy-D-galactose (2-D-gal) reduced corneal epithelial defects and increased tear production in both DED models. Moreover, 2-D-gal treatment suppressed the levels of inflammatory cytokines in the ocular surface and the percentages of IFN-γ+CD4+ cells in draining lymph nodes, whereas it did not affect the number of conjunctival goblet cells, the MUC5AC level or the meibomian gland area. Together, the findings indicate that aberrant fucosylation underlies the pathogenesis of DED and may be a novel target for DED therapy.

Insight into levofloxacin loaded biocompatible electrospun scaffolds for their potential as conjunctival substitutes.

The rehabilitation of visual acuity with severe conjunctival fibrosis depends on ocular reconstruction with suitable conjunctival substitutes. In this study, we have developed poly(lactic acid) (PLA) electrospun nanofibrous membranes (EFMs) surface coated by cellulose nanofibrils (CNF) and/or silk peptide (SP). The CNF coating improved the hydrophilicity and the SP coating proliferated conjunctival epithelial cells (CjECs). To prevent post-operative infections, the composite scaffolds were loaded with levofloxacin (LF), constantly exerting efficient bactericidal effects. In in vivo evaluations, the PLA EFMs presented excellent therapeutic effects by promoting structural and functional restoration of conjunctiva after transplant. Even with reduced topical administration of antibiotics, the coloboma treated with LF loaded scaffolds presented no infections. It could be deduced that the potent bacterial inhibition feature could save troubles for patients by minimizing the application of antibiotics post-surgery. Hence, the developed PLA EFMs loaded with LF could be promising conjunctival substitutes.

The effect of topical ocular moxifloxacin on conjunctival and nasal mucosal flora.

To determine the short-term effect of topically administered ocular moxifloxacin on conjunctival and nasal bacterial mucosal flora. The study included 20 patients with newly diagnosed age-related macular degeneration. Each patient's diseased eye was selected as the treatment eye and the fellow eye was selected as the control eye. All treatment eyes constituted the treatment group and all controls eyes constituted the control group. All patients received intravitreal injection of ranibizumab. Cultures were obtained from the inferior conjunctival fornix and the nostrils in all patients. Patients were instructed to administer moxifloxacin eye drops to the treatment eye 4 times daily for 1 week. The patients were instructed to come for a follow-up exam 1 week post intravitreal injection. The bacterial culture positivity rate and the bacteria isolated from the conjunctiva and nostrils were recorded in the 2 groups before and after use of topical ocular moxifloxacin. Mean age of the patients (12 female and 8 male) was 64.9 years. Before use of topical ocular moxifloxacin the conjunctival and nasal culture positivity rates in the treatment group were both 100%, versus 90% and 95%, respectively, in the control group. At the follow-up exam the conjunctival and nasal mucosa culture positivity rates in the treatment group decreased to 20% (4/20) and 30% (6/20), respectively (P < 0.001), versus 85% (17/20) and 80% (16/20), respectively, in the control group (P = 0.68 and P = 0.72 for conjunctival and nasal). This is the first study to show that moxifloxacin applied to the ocular surface topically has a significant effect on nasal flora. Daily administration of topical ocular moxifloxacin for 1 week significantly reduces the nasal bacterial flora in addition to conjunctival flora.

Characterization of the rabbit conjunctiva: Effects of sulfur mustard.

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5 ac producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures.

Astragalus â £ ameliorates the dry eye injury in rabbit model via MUC1-ErbB1 pathway.

The therapeutic effects and potential mechanisms of astragaloside IV on a rabbits dry eye model induced by benzalkonium chloride (BAC) was examined. In our study, a BAC-induced dry eye rabbit model was treated with eye drops containing astragaloside IV (5, 10 µM) or solvent four times a day. The clinical evaluations, such as tear break-up time (BUT) and Schirmer tear test (STT), were performed on days 0, 7, 14, 21, and 28. On day 28, the cornea and bulbar conjunctiva tissues (left eye and right eye) were collected with histology, and immunofluorescent staining conducted. The levels of MUC1 and ErbB1in the corneas were determined by real-time quantitative PCR (qRT-PCR) and the proteins levels of MUC1 and ErbB1 were detected by Western blot. It was demonstrated that both astragaloside IV (5, 10 µM) treatments resulted in an increased STT and BUT on days 7, 14, 21 and 28. Additionally, the astragaloside IV (5, 10 µM)-treated group showed increasing PAS-positive goblet cells than model group (0 µM). Moreover, the MUC1 in model group (0 µM) was decreased, while the expression of MUC1 in astragaloside IV (5, 10 µM) group was increased. Furthermore, astragaloside IV had a protective effect on BAC-induced rabbits' dry eye and demonstrated clinical improvements, which indicated that astragaloside IV served as a potential protective agent in the clinical treatment of dry eye.

A prospective multicentre study of intravitreal injections and ocular surface in 219 patients: IVIS study.

PURPOSE: To assess the impact of intravitreal injections (IVTI) on ocular surface of patients treated with multiple injections. METHODS: Prospective, tricentric study conducted in patients treated with unilateral IVTI. An asepsis protocol with povidone-iodine was used for all patients during IVTI. The primary endpoint was the difference between the pre-IVTI Ocular Surface Disease Index (OSDI 1) score and that measured on day one (D1) post-IVTI (OSDI 2). Secondary endpoints were the evaluation of predictive factors for OSDI scores, pain assessment on D1, and the Lacrydiag® analysis of tears from the injected eye versus contralateral eye before IVTI. RESULTS: Two hundred and nineteen patients with a mean age of 75.9 ± 10 years were included. The mean OSDI2-OSDI1 difference was 19.2 ± 20.6 (p < 0.001). The mean noninvasive tear break-up time was 6.41 ± 4.59 seconds in the injected eye versus 7.36 ± 4.36 seconds in the contralateral eye (p < 0.001). In the multivariate analysis, the factors significantly associated with the OSDI 2 score were the OSDI 1 score (p < 0.001), the pain score on D1 (p < 0.001) the number of instilled glaucoma eye drop (p = 0.01) and a centre effect (centres 2 and 3 versus centre 1, p < 0.001). CONCLUSION: Our results confirm the impairment of the ocular surface and quality of life immediately after an IVTI. These results suggest 3 levels of action to improve the immediate tolerance: improving the basal status of the ocular surface, reducing the contact time with povidone-iodine that might be toxic to the surface, and improving immediate post-IVTI treatment.

Rituximab as an Adjuvant Rescue Treatment for Ocular Cicatricial Pemphigoid.

PURPOSE: Ocular cicatricial pemphigoid (OCP) can lead to devastating ocular complications without prompt treatment. A number of immunomodulatory agents have been attempted with varying success. The objective of this study was to evaluate the use of rituximab as an adjuvant to immunomodulatory treatments (IMTs) in refractory OCP. METHODS: The clinical records of 14 patients with treatment refractory OCP treated with rituximab as monotherapy or in combination with IMTs were retrospectively reviewed, with a focus on demographics, treatments prerituximab and postrituximab, total number of rituximab infusions, response to treatment, and ocular outcomes including staging and best-corrected visual acuity. RESULTS: Thirteen patients (92.9%) achieved a complete response with rituximab over a mean period of 4.3 months. The average sustained complete response time in those without relapse was 29.7 months. Five patients relapsed over a mean period of 15 months, 2 of whom were able to regain control over a mean of 2.5 months with additional rituximab treatments. At the final evaluation, rituximab-based therapy improved ocular outcomes in OCP by stabilizing Foster staging and preventing the deterioration of best-corrected visual acuity in 72% of eyes. In total, 90% of eyes with Foster stages 3 or less did not progress. All patients were able to decrease IMT dosage and/or transition to less potent adjuvant treatments. CONCLUSIONS: The consideration of rituximab earlier in treatment of OCP as a rescue and/or maintenance therapy could result in an earlier arrest of disease progression, resulting in preservation of patients' vision, and enable tapering of adjuvant IMT.

Ocular surface response of two preservative-free cylcosporine A emulsion eye drops in a mouse model of dry eye.

PURPOSE/AIM: Dry eye (DE) disease is a multifactorial disease in which uncontrolled inflammation can lead to corneal epithelium lesions and symptoms of discomfort. The aim of the present study was to evaluate the efficacy of two cyclosporine emulsions in a mouse model of DE with corneal epithelium lesions. MATERIALS AND METHODS: Six- to 9-week-old female C57BL/6 N mice were housed in a controlled-environment room to induce DE. Following DE development, mice were instilled with: QD 0.1%CsA cationic emulsion (CaEm), BID 0.05%CsA anionic emulsion (AEm), or left untreated. Aqueous tear production and corneal epithelium lesions were assessed throughout the experiment. At the end of the treatment period, left eyes were sampled, fixed, and stained for histology, while the cornea, conjunctiva, and lacrimal gland of right eyes were sampled for transcriptomic analysis. RESULTS: Corneal lesion scores were reduced by 10.4%, 18.4%, and 10.9% at day 6, 10, and 14, respectively, with CaEm (QD), and by 2.6%, 3.0%, and 5.5% at day 6, 10, and 14, respectively, with AEm (BID). Histology demonstrated that 7 out of 10 DE mice presented moderate to severe ocular lesions, while only 2 and 5 out of 10 mice presented slight to moderate ocular lesions when treated with the CaEm (QD) and AEm (BID), respectively. The transcriptomic profile analysis suggests that a different set of inflammatory genes are modulated in the cornea, conjunctiva, and lacrimal gland upon DE development. In addition, the two emulsions distinctively modulate the gene expression profile. CONCLUSIONS: This study demonstrates that both emulsions were effective at reducing corneal lesions, with the CaEm (QD) being slightly better than the AEm (BID). Interestingly, this study suggests that ocular tissues may not respond similarly to a dry environment and that a different set of genes is modulated by the two formulations in the ocular tissues.

The action of subconjunctival cyclophosphamide in rabbits / Ação da ciclofosfamida subconjuntival em coelhos

ABSTRACT Objective: To observe clinically, in rabbits, the side effects of topical injection of subconjunctival cyclophosphamide, studying its role as an antifibrotic drug. Methods: Prospective study in 20 albino rabbits of New Zealand race. All rabbits were treated with cyclophosphamide, 10mg/ml in a volume of 0.3 ml, in the left eye through subconjunctival injection. They were evaluated for 1, 7, 30, and 60 days after the procedure. All the animals were examined for the detection of ocular reactions such as necrosis, hyperemia, chemosis, secretion, opacity, and iritis. Other side effects as changes in the behavior, in the feed, and the water consumption were also evaluated. Results: It was observed that from the 20 rabbits studied, three rabbits (15%) showed side effects only at the 24 hours analysis. One rabbit (5%) presented hyperemia, one rabbit (5%) had hyperemia associated with iritis, and one rabbit (5%) presented hyperemia associated with secretion. These reactions were not observed at 1, 7, 30, and 60 days. Conclusion: Cyclophosphamide subconjunctival injection induces minor side effects on the conjunctiva of rabbits such as hyperemia, associated with iritis and secretion.

RESUMO Objetivo: Observar clinicamente os efeitos colaterais de injeção subconjuntival de ciclofosfamida, pensando em sua ação como um agente antifibrótico. Métodos: Estudo prospectivo realizado com 20 coelhos albinos da raça Nova Zelândia. Todos os coelhos foram submetidos a 0,3 ml de injeção subconjuntival de ciclofosfamida 10mg/ml no olho esquerdo e foram avaliados de acordo com os efeitos locais no primeiro dia após a injeção, 7, 30 e 60 dias. Foram examinados para detecção de reações oculares como necrose, hiperemia, quemose, secreção, opacidade corneana, irite além de alterações comportamentais e variação no consumo de água e alimentação. Resultados: Dos 20 coelhos estudados, apenas 3 apresentaram reações oculares e somente na leitura de 24 horas. Um coelho (5%) apresentou hiperemia, 1 coelho (5%) apresentou hiperemia associada a presença de irite e 1 coelho (5%) apresentou hiperemia associada a presença de secreção. As reações não foram mais observadas durante os exames de 7, 30 e 60 dias. Conclusão: A ciclofosfamida subconjuntival causou poucos efeitos colaterais na conjuntiva dos coelhos. Os únicos efeitos encontrados foram hiperemia, irite e secreção.

A combination of CMC and α-MSH inhibited ROS activated NLRP3 inflammasome in hyperosmolarity stressed HCECs and scopolamine-induced dry eye rats.

An important mechanism involved in dry eye (DE) is the association between tear hyperosmolarity and inflammation severity. Inflammation in DE might be mediated by the NLRP3 inflammasome, which activated by exposure to reactive oxygen species (ROS). A combination of carboxymethylcellulose (CMC) and α-melanocyte stimulating hormone (α-MSH) may influence DE through this mechanism, thus avoiding defects of signal drug. In this study, we assessed whether treatment comprising CMC combined with α-MSH could ameliorate ocular surface function; we found that it promoted tear secretion, reduced the density of fluorescein sodium staining, enhanced the number of conjunctival goblet cells, and reduced the number of corneal apoptotic cells. Investigation of the underlying mechanism suggested that the synergistic effect of combined treatment alleviated DE inflammation through reduction of ROS level and inhibition of the NLRP3 inflammasome in human corneal epithelial cells. These findings indicate that combined CMC + α-MSH treatment could ameliorate lesions and restore ocular surface function in patients with DE through reduction of ROS level and inhibition of NLRP3 signalling.

Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature.

We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFß1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors' activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.