Rabbit Corneal Epithelial Cell Line Research Articles

Formulating Resveratrol and Melatonin Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) for Ocular Administration Using Design of Experiments.

Recent studies have demonstrated that Sirtuin-1 (SIRT-1)-activating molecules exert a protective role in degenerative ocular diseases. However, these molecules hardly reach the back of the eye due to poor solubility in aqueous environments and low bioavailability after topical application on the eye's surface. Such hindrances, combined with stability issues, call for the need for innovative delivery strategies. Within this context, the development of self-nanoemulsifying drug delivery systems (SNEDDS) for SIRT-1 delivery can represent a promising approach. The aim of the work was to design and optimize SNEDDS for the ocular delivery of two natural SIRT-1 agonists, resveratrol (RSV) and melatonin (MEL), with potential implications for treating diabetic retinopathy. Pre-formulation studies were performed by a Design of Experiment (DoE) approach to construct the ternary phase diagram. The optimization phase was carried out using Response Surface Methodology (RSM). Four types of SNEDDS consisting of different surfactants (Tween® 80, Tween® 20, Solutol® HS15, and Cremophor® EL) were optimized to achieve the best physico-chemical parameters for ocular application. Stability tests indicated that SNEDDS produced with Tween® 80 was the formulation that best preserved the stability of molecules, and so it was, therefore, selected for further technological studies. The optimized formulation was prepared with Capryol® PGMC, Tween® 80, and Transcutol® P and loaded with RSV or MEL. The SNEDDS were evaluated for other parameters, such as the mean size (found to be ˂50 nm), size homogeneity (PDI < 0.2), emulsion time (around 40 s), transparency, drug content (>90%), mucoadhesion strength, in vitro drug release, pH and osmolarity, stability to dilution, and cloud point. Finally, an in vitro evaluation was performed on a rabbit corneal epithelial cell line (SIRC) to assess their cytocompatibility. The overall results suggest that SNEDDS can be used as promising nanocarriers for the ocular drug delivery of RSV and MEL.

Transcriptional profiles along cell programming into corneal epithelial differentiation

Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4+, KLF4+, Myc+, ΔNp63α+, ABCG2+, Vimentin+, Zeb1+, VANGL1+, Krt3-, Krt12- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.

(-)-Epigallocatechin-3-gallate, reduces corneal damage secondary from experimental grade II alkali burns in mice

BackgroundSince recent reports have shown that (-)-Epigallocatechin-3-gallate (EGCG) could be used for treating proliferative and inflammatory disorders, we explored its use for the management of corneal chemical burns. Materials and methodsInitially, EGCG was assayed on the rabbit corneal epithelial cell line RCE1(5T5) to establish the best testing conditions, and to avoid unwanted outcomes in the experimental animals. Then, we studied its effects on cell proliferation, cell cycle progression and cell differentiation. Afterwards, we instilled EGCG in experimental grade II corneal alkali burns in mice, three times a day up to 21days, and evaluated by slit lamp examination and histological sections of corneal epithelial, corneal endothelial and stromal edema, as well as the presence of inflammatory cells and neovascularization. ResultsEGCG reduced cell growth and led to a decline in the proportion of proliferative cells in a concentration dependent manner. At 10μM, EGCG promoted cell differentiation, an effect not related with apoptosis or cytotoxicity. When 10μM EGCG was instilled in corneal alkali burns in mice three times a day up to 21days, EGCG significantly reduced corneal opacity and neovascularization. The improved clinical appearance of the cornea was associated to a controlled epithelial growth; epithelial morphology was similar to that observed in normal epithelium and contrasted with the hyperproliferative, desquamating epithelium observed in control burn wounds. EGCG reduced corneal, stromal and endothelial edema, and wound inflammation. ConclusionThis work constitutes the first evidence for the use of EGCG in the acute phase of a corneal alkali burn, representing a possible novel alternative to improve patient outcomes as an add-on therapy.

Palmitoyl Glycol Chitosan Micelles for Corneal Delivery of Cyclosporine.

Different substitution degrees of palmitoyl glycol chitosan (PGC), prepared according to the literature, were used to obtain polymeric micelles that have been assessed in comparison with Pluronic F127 micelles as possible carriers for poorly soluble drugs, such as cyclosporine A. Both PGC and Pluronic micelles were studied for their interactions with cell culture substrates. The least substituted and most hydrophilic derivative, PGC21 (approximately 5% substitution), showed a strong association with cyclosporine, more than tripling the colloidal concentration with respect to the saturated solution. It showed a greater ability to open Caco-2 tight junctions and to enhance the permeability of Caco-2 substrates with respect to micelles based on higher palmitoyl substitution, conceivably due to the lower modification of the chitosan chains. Permeation and penetration experiments were performed with PGC21 and Pluronic micelles on a rabbit corneal epithelial cell line (RCE) and on excised pig corneas. It was found that both PGC and Pluronic micelles could increase the permeation of the fluorescent probe rhodamine B through RCE cells by more than ten-fold. In RCE and in pig cornea, the micelles improved the penetration of both rhodamine and cyclosporine. For cyclosporine, the PGC21 micelles allowed penetration of approximately 1 μg/mg cyclosporine A in corneal tissue, demonstrating a potential for use in immunosuppression therapies.

Effect of sertraline on Ca²⁺ fluxes in rabbit corneal epithelial cells.

The effect of sertraline, a selective serotonin reuptake inhibitor (SSRI), on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) in a rabbit corneal epithelial cell line (SIRC) is unclear. This study explored whether sertraline changed basal [Ca²⁺](i) levels in suspended SIRC cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Sertraline at concentrations between 10-100 μM increased [Ca²⁺](i) in a concentration-dependent manner. The Ca²⁺ signal was reduced by 23% by removing extracellular Ca²⁺. Sertraline induced Mn²⁺ influx, leading to quench of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A₂ inhibitor aristolochic acid, but not by store-operated Ca²⁺ channel blockers and protein kinase C/A modulators. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone greatly inhibited sertraline-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 abolished sertraline-induced [Ca²⁺](i) rise. At concentrations of 5-50 μM, sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of 25 μM sertraline was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, sertraline induced [Ca²⁺](i) rises by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive Ca²⁺ channels. Sertraline-caused cytotoxicity was mediated by Ca²⁺-independent pathways.

Prednisolone-loaded nanocapsules as ocular drug delivery system: development, in vitro drug release and eye toxicity

Objective: To develop non-toxic aqueous ocular drug delivery systems containing prednisolone by means of its nanoencapsulation. Materials and methods: Nanocapsules were prepared by interfacial deposition of preformed polymer [poly(ε-caprolactone) or Eudragit® RS100]. Particle size distribution was determined by laser diffractometry, photon correlation spectroscopy and nanoparticle tracking analysis. Ocular irritation and cytotoxicity were evaluated in vitro on the chorioallantoic membrane (CAM) and rabbit corneal epithelial cell line, respectively. Results and discussion: Nanocapsules showed mean particle sizes between 100 and 300 nm and prednisolone encapsulation efficiency of around 50%. Controlled release of prednisolone occurred for 5 h for both formulations according to the biexponential model. Both formulations were found to be non-irritant in the CAM test and non-cytotoxic toward rabbit corneal epithelial cells. Conclusions: Encapsulation of prednisolone in nanocapsules was reported for the first time, being suitable for producing eye drops for the treatment of ocular inflammatory and no eye toxicity was indicated.

Physiological and elastic properties of highly porous hydroxyapatite potential for integrated eye implants: Effects of SIRC and L-929 cell lines

Hydroxyapatite (HAp)-based highly porous integrated orbital implants with a property of mimicking the movements of fellow eye have been developed recently. Before designing this kind of scaffolds, vascularization and angiogenesis in the constructs need to be considered. Moreover, eye cells expressed with time on these highly porous implants may exert some compressive load whose effect both mechanically and physiologically may indicate its long-term life in vivo. In the present investigation the effect of expression of eye cell lines [rabbit corneal epithelial cell line (SIRC)] on highly porous HAp scaffolds were compared with the normal fibroblastic cells on HAp in general, in terms of in vitro dissolution studies, cell culture cytotoxicity and cell adhesion properties. Elastic properties of macro-porous HAp with a wide range of porosities have also been estimated by ultrasonic non-destructive test methods and the results were compared with its compressive properties for potential application as integrated ocular implants. Cell viability of HAp in contact with SIRC is far better than the L-929 during the initial periods (48h) but cell adhesion behavior however showed better results in L-929 than the SIRC during the same initial time period. SIRC cells however eventually formed better adhesion properties on the surface of HAp as the days goes by than L-929. High concentration of both Ca and P in culture media might be another factor in cell growth modification. Both the ions had a counter effect on the L-929 and SIRC cells.

Molecular analysis of rubella virus in travelers suspected of measles infection in São Paulo, Brazil

To identify measles virus genotypes in three cases of travelers suspected of measles infection. Samples (blood and urine) were collected for serology, virus isolation, and genotyping. Sera were analyzed for IgM antibodies against measles virus and rubella virus by enzyme-linked immunosorbent assay (ELISA) (Siemens - Marburg, Germany). Clinical samples (lymphocytes and urine) were inoculated into Statens Serum Institute rabbit corneal epithelial cell line- ATCC CL 60 (SIRC) and Vero Slam cells. RNA was extracted from clinical samples and cell culture was inoculated and processed by polymerase chain reaction (PCR) with oligonucleotides specific for measles virus (MV) and rubella virus (RV). All patients showed IgM negative serology for MV and positive IgM for RV. RV belonging to genotypes 1B, 1C, and 1E were isolated from patients who came from Finland, Peru, and Germany, respectively. Genotype 1B has been found in Europe and on the East Coast of South America; 1C has been found in Peru and the West Coast of South America, and 1E, first identified in 1997, now appears to have worldwide distribution. Information about RV and MV genotypes circulating in São Paulo is essential for the control of measles, rubella, and congenital rubella syndrome (CRS) in Brazil.

Functional Characterization of Folate Transport Proteins in Staten’s Seruminstitut Rabbit Corneal Epithelial Cell Line

Purpose:The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten’s Seruminstitut rabbit corneal (SIRC) epithelial cell line.Methods: [3H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [3H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [3H]folic acid in the presence and absence of 1 mM cold folic acid.Results: Linear increase in [3H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent Km of 14.2 ± 0.2 nM, Vmax of (1.5 ± 0.1)*10−5 micro.moles/min/mg protein, and Kd of (2.1 ± 0.2)*10−6 min−1. The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [3H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca2+/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [3H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10−05 cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10−05 cm/sec.Conclusions: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.

Dictyostelium polycephalumInfection of Human Cornea

<i>Dictyostelium polycephalum</i>Infection of Human Cornea

Development of polyvinyl alcohol–gelatin membranes for antibiotic delivery in the eye

Aim: The aim of the present study was to develop biosynthetic hybrid polymer-based ocular insert for topical administration of antibiotics for treatment of ocular infections. Methods: Ciprofloxacin hydrochloride-loaded three different inserts were prepared by solution casting method by esterification of a biopolymer (gelatin) with a synthetic polymer (polyvinyl alcohol, PVA). Esterification between PVA and gelatin was confirmed by Fourier transform infrared spectroscopy. Results: Inserts were found to be wettable and swellable with simulated tear fluid and had a contact angle <50° with simulated tear fluid. Mechanical properties of PVA–gelatin (10:3 wt%) inserts included a maximal tensile strength of 8.6 ± 2 MPa and the inserts showed adequate mucoadhesion with reconstituted mucin. In vitro drug release of ciprofloxacin hydrochloride for up to 24 hours was observed from the inserts. Inserts were found to be biocompatible using SIRC rabbit corneal epithelial cell line by sulforhodamine B assay and by Draize test in albino rabbits. Further inserts showed higher ocular penetration of sodium fluorescein in goat eye as compared to eyedrop solution. Conclusions: In brief, the study suggests that PVA–gelatin polymeric blends are promising as ocular inserts for prolonged release of antibiotic in the eye as compared to eyedrops. Such inserts may also be therapeutically beneficial for treatment of corneal ulcers and external ocular infections.

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion byCandida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans. Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.

Assessment of Proliferation Induced in Fibroblasts and Rabbit Corneal Epithelial Cells by a Platelet Lysate Formulation: A Stability Study

Introduction. Platelets contain a mixture of growth factors (GF). These can be released from platelet lysate (PL) whose application to wounds is supposed to favour the healing process. The application of PL in a suitable formulation can improve therapeutic efficacy and patient compliance. A formulation of PL with a selected biocompatible vehicle has been developed for application in buccal mucosal damages (mucositis), that are the most common complications of the nonsurgical therapy of cancer. The same vehicle can be proposed also for ophthalmic formulations as several studies have shown that topical application of the growth factors to the injured cornea facilitates wound repair by rapid regrowth of the epithelial cells. Aim of the present work was to develop in vitro tests for a fast evaluation of PL activity in the formulation to evidence excipient compatibility, and the effect of formulative parameters on PL stability.The proliferative effect on two different models, fibroblasts and a corneal epithelial cell line, was evaluated. The stability of PL in the formulation has been assessed after two weeks at 4–8 °C storage.Methods. PL was mixed in a 1:1 ratio to the selected vehicle and stored at 4–8 °C. Immediately after preparation (time zero, T0) and after 2, 7, 10 and 15 days (T2–T15), aliquots were used to assess proliferative effect on cell cultures. Both fibroblast (primary human cell lines) and RCE (Rabbit corneal epithelial cell lines, ECACC) were seeded in 96-well plates with area of 0.34 cm2 at the concentrations of either 10000 or 5000 cells/well. After 24 hours the cells were added with either complete growth medium (Reference), minimal medium not supplemented with foetal calf serum and for REC cells without also EGF (Control), PL formulation diluted 1/20 (containing PL at 5%) and PL formulation diluted 1/40 (containing PL at 2.5%). After 24 hours, neutral red (NR) test was performed (Tox Kit 4, Sigma-Aldrich, Milano I). The NR solution absorbance was determined by means of a plate reader (Perkin Elmer, Milan, I) at wavelength of 490 nm. The absorbance read for each sample was compared with that of Reference, whose proliferation was assumed as 100%.Results. At time zero, the percentage of proliferation showed no significant differences with respect to the cells in complete growth medium (Reference) in the case of fibroblasts, both at 5000 and 10000 seeding level. The two cell models gave comparable results although slightly higher values of proliferation were obtained in fibroblasts. This can be attributed to the epithelial nature of the RCE and to the possible different percentage in the platelet derived mixture of FGF and EGF. In both cases it can be argued that the vehicle is compatible with cell growth. Both for fibroblasts and for RCE no decrease of activity with time was observed, and no statistical differences with respect to the control have been obtained until 15 days storage. (See figure 1).The results obtained suggest that the two cell culture models evaluated can be suitable to assess the activity of PL in easy and fast way. This will be useful to develop formulations intended for the treatment of mucositis and of damages of corneal epithelia. In particular, the formulation tested seems to be stable and to allow the release of GF from PL until 15 days of storage. [Display omitted]

Establishing and functional testing of a canine corneal construct

To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison. The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air-liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically. Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E(2) (PGE(2)) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry. A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE(2) production, which could be reduced by dexamethasone. The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.

Breakdown Evaluation of Corneal Epithelial Barrier Caused by Antiallergic Eyedrops Using an Electrophysiologic Method

The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.

Differential cytotoxicity of phospholipid analogues to pathogenic Acanthamoeba species and mammalian cells

Previous studies have reported the ability of several phospholipid analogues to successfully inhibit the growth of Acanthamoeba species in vitro. This study tests further phospholipid analogues, either as free drug or in liposomal formulations, and unlike previous studies, examines their comparative toxicities to mammalian cells. The relative cytotoxic activities of the phospholipid derivatives hexadecyl-PC, octadecyl-PC, elaidyl-PC, erucyl-PC and edelfosine, against Acanthamoeba castellanii, Acanthamoeba polyphaga and a rabbit corneal epithelial (RCE) cell line, was determined by the alamarBlue assay. Free and liposomal formulations were compared for hexadecyl-PC and elaidyl-PC. Both hexadecyl-PC and octadecyl-PC (IC50 values between 3.9 and 7.8 microM) demonstrated considerable activity against A. castellanii, as did elaidyl-PC (IC50 values between 15.6 and 31.25 microM). Both hexadecyl-PC and elaidyl-PC also proved effective against A. polyphaga (IC50 values between 15.6 and 31.25 and between 31.25 and 62.5 microM, respectively). In contrast, neither erucyl-PC nor edelfosine was inhibitory against either Acanthamoeba species. The growth of RCE cells was inhibited by octadecyl-PC, erucyl-PC and edelfosine (octadecyl-PC and erucyl-PC IC50 values between 7.8 and 15.6 microM and edelfosine IC50 values between 31.25 and 62.5 microM). Liposomal formulations of hexadecyl-PC and elaidyl-PC were less effective than free drug against both Acanthamoeba species. These results demonstrate that hexadecyl-PC has the highest therapeutic index and is the most promising for the treatment of acanthamoebiasis.

Transient receptor potential vanilloid 1 activation induces inflammatory cytokine release in corneal epithelium through MAPK signaling

In certain epithelial tissues, activation of transient receptor potential (TRP) vanilloid subtype 1 (TRPV1) by noxious stimuli induces pro-inflammatory cytokine release, which helps to mitigate the challenge. While the corneal epithelium elicits such responses to a variety of challenges, it remains unknown whether TRPV1 mediates pro-inflammatory cytokine secretion. Accordingly, we probed for TRPV1 expression and function in human (HCEC) and rabbit corneal epithelial cell (RCEC) lines, in their primary counterparts, and in human and mouse corneal epithelium in situ. Cell membrane and perinuclear TRPV1 expression was detected in all preparations and its identity verified by Western blot analysis. Capsaicin (CAP) (1-10 microM) increased nonselective cation channel whole cell currents (2.5-fold +/- 0.5-fold between -60 and 130 mV), resulting in calcium transients that were fully blocked by the TRPV1 antagonists capsazepine (CPZ) and ruthenium red, or removal of extracellular calcium. Another signaling event involved transient activation of global mitogen-activated protein kinase (MAPK) superfamily, which was followed by up to 3.3- and 9-fold increases in interleukins (IL)-6 and -8 release, respectively. Such increases in inflammatory mediators' release were suppressed by exposure to CPZ or MAPK inhibitors, or removal of Ca2+. Taken together, TRPV1 receptors may play a role in mediating corneal epithelial inflammatory mediator secretion and subsequent hyperalgesia.

Liposome-encapsulated mitomycin C for the reduction of corneal healing rate and ocular toxicity

Mitomycin-C (MMC), a potent antibiotic-antineoplastic drug, has recently proven to be useful as additional treatment in ocular surgery (pterygium, glaucoma filtering, photorefractive laser keratectomy, etc.) when inhibiting the epithelial wound healing response and reducing surgical scarring is beneficial to surgical success. However, the therapeutic benefits of MMC are frequently offset by ocular toxicity and undesirable side effects (retinal toxicity, corneal edema, corneal perforation, cataract, secondary glaucoma, iritis, scleral calcification, pain, etc.). The purpose of this study was to evaluate if a liposomal preparation containing MMC was capable of reducing the corneal healing rate and drug toxicity of a corneal lesion in a rabbit model. To do this, a liposomal formulation containing 0.2 mg/ml of MMC was prepared and tested against aqueous solutions and viscous formulations based on tamarind seeds polysaccharide (TSP), which were used as references. In vitro release of MMC from the vehicles through a dialysis membrane was characterized by a fast diffusion of MMC from the aqueous solution, while a more gradual release from the TSP and the liposomal formulations was observed. To evaluate the intraocular penetration of MMC, the drug amount in the aqueous and vitreous humor of the animals was determined 60 min after treatment. MMC toxicity was analyzed by monitoring the proliferation and viability of a rabbit corneal epithelial cell line (RCE) in the different formulations under study. These results showed reduced cytotoxicity for MMC in TSP viscous vehicles; however, only the liposomal MMC vehicle appeared to produce both lower toxicity for a rabbit corneal epithelial cell line (RCE) culture and a substantial reduction of the corneal healing rate in vivo.

Pax6Overexpression Suppresses Cell Proliferation and Retards the Cell Cycle in Corneal Epithelial Cells

The gene encoding transcription factor Pax6 resides at the top of a genetic hierarchy controlling development and morphogenesis of the eye. Pax6 continues to be expressed in the ocular surface epithelia of the postnatal eye. The goal of this study was to investigate a possible role for Pax6 in controlling dynamics of the ocular surface epithelia. Full-length mouse Pax6 (mPax) cDNA, or truncated mPax6Delta286 lacking the transcriptional activation domain was inserted into a tetracycline-inducible vector (Tet-on). A rabbit corneal epithelial cell line SIRC was used to establish stable transformants. Induction of Pax6 or truncated Pax6Delta286 proteins by doxycycline (DOX) was examined by Western blot and immunohistochemistry. The effects of Pax6 overexpression on cell cycle progression were assessed by the cell proliferation index, cell growth curve, and cell cycle assay. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. Recombinant adenovirus-carrying mPax6 or mPax6Delta286 transgenes were used for transient transduction of primary rabbit corneal epithelial cells, and the effect on cell cycle progression was assayed. The level of Pax6 or truncated Pax6 was tightly regulated by DOX. Overexpression of full-length Pax6 retarded the rate of cell proliferation, whereas the truncated form had no effect. Full-length Pax6 affected the rate at which individual cells traversed the cell cycle and induced caspase-3-independent apoptosis in a small percentage of cells. Transient transduction of cells with recombinant mPax6 adenovirus also inhibited cell proliferation. Inhibition of cell proliferation in Pax6-overexpressing corneal epithelial cell lines and primary cell culture is consistent with the notion that Pax6 plays a role in controlling corneal epithelial cell dynamics in vivo.

Herpes simplex virus-1–associated congenital cataract

Purpose To report association of herpes simplex virus-type 1 (HSV-1) in four cases of congenital cataract. Design Prospective interventional case series. Methods Four infants younger than 12 months, presenting with unilateral or bilateral congenital cataract, were included. The cases were clinically evaluated by the pediatric ophthalmologist. The lens aspirates collected at the time of cataract surgery were processed for HSV-1 culture in rabbit corneal epithelial (SIRC) cell line and for HSV-1 DNA by polymerase chain (PCR). The sera of the children and the mother were tested for HSV-1 immunoglobulin (Ig) M and IgG by enzyme linked immunosorbent assay (ELISA). Results HSV-1 was isolated in tube cultures in three of four lens aspirates, and all four lens aspirates were positive for HSV-1 DNA by PCR. Serum HSV-1 IgM was positive in all babies and in three cases HSV-1 IgM was positive in the mother's serum. Conclusion Based on a computerized literature search, we believe this may be the first report of HSV-1 associated congenital cataract.