Cornea Epithelial Cells Research Articles

Effect of Pseudomonas and Acanthamoeba infection upon inflammation produced by human cornea epithelial cells

Effect of <i>Pseudomonas</i> and <i>Acanthamoeba</i> infection upon inflammation produced by human cornea epithelial cells

Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation

Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.

Epistasis between Pax6Sey and genetic background reinforces the value of defined hybrid mouse models for therapeutic trials

The small eye (Sey) mouse is a model of PAX6-aniridia syndrome (aniridia). Aniridia, a congenital ocular disorder caused by heterozygous loss-of-function mutations in PAX6, needs new vision saving therapies. However, high phenotypic variability in Sey mice makes development of such therapies challenging. We hypothesize that genetic background is a major source of undesirable variability in Sey mice. Here we performed a systematic quantitative examination of anatomical, histological, and molecular phenotypes on the inbred C57BL/6J, hybrid B6129F1, and inbred 129S1/SvImJ backgrounds. The Sey allele significantly reduced eye weight, corneal thickness, PAX6 mRNA and protein levels, and elevated blood glucose levels. Surprisingly, Pax6Sey/Sey brains had significantly elevated Pax6 transcripts compared to Pax6+/+ embryos. Genetic background significantly influenced 12/24 measurements, with inbred strains introducing severe ocular and blood sugar phenotypes not observed in hybrid mice. Additionally, significant interactions (epistasis) between Pax6 genotype and genetic background were detected in measurements of eye weight, cornea epithelial thickness and cell count, retinal mRNA levels, and blood glucose levels. The number of epistatic interactions was reduced in hybrid mice. In conclusion, severe phenotypes in the unnatural inbred strains reinforce the value of more naturalistic F1 hybrid mice for the development of therapies for aniridia and other disorders.

Amyloid Beta Deposition Could Cause Corneal Epithelial Cell Degeneration Associated with Increasing Apoptosis in APPswePS1 Transgenic Mice

ABSTRACTObjective: To investigate the expression of amyloid precursor protein (APP) and amyloid beta (Aβ) in cornea and further explore the pathological and ultrastructural changes in corneal epithelium in APPswePS1 transgenic mice.Methods: Twelve wild type mice were grouped into control group and twelve TgAPPswePS1 mice at least 8 months old were grouped into the young experiment group (Tg-8M group), and another twelve transgenic mice at least 15 months old were selected into the aged experiment group (Tg-15M group). The pathological degeneration, ultrastructural changes, and the expression of APP, Aβ deposition, and the TUNEL reaction in corneal epithelial cells were observed. Western blot analysis was performed to determine expression levels of APP and Aβ with scraped epithelial debridement. All the results were quantified and analyzed.Results: In transgenic mice, the H&E-stained cornea sections demonstrated histopathological changes in corneal epithelial cells with irregular arrangement and the number of cell layers decreased, while normal structure observed in controls. In Tg-15M group, the corneal epithelial cell displaced a significant number of intracellular vacuoles with 1–2 cell layers left. Transmission electron microscopy (TEM) further confirmed the dramatic degeneration in corneal epithelium, the microvilli suffered degenerative changes and found with typical fingerpoint-like morphology in controls; however, microspike-like in Tg-15M group, and the number of microvilli decreased considerabely. An APP-positive immunoreaction was detected with a diffuse pattern in the corneal epithelial cells layer, about 3.122 ± 0.596 and 7.372 ± 0.936 fold changes in Tg-8M and Tg-15M groups, respectively, as compared with controls. On corneal flatmount, Aβ deposition found a diffuse pattern in the cytoplasm by fluorescence staining in TgAPPswePS1 with significantly increasing as compared with the controls, but no plaque was found. The apoptosis of TUNEL cells were observed in TgAPPswePS1 mice and increased 16.329 ± 3.542 fold changes in Tg-15M group as compared with controls.Conclusion: The APP expression and Aβ deposition might cause cornea epithelial cells degeneration in TgAPPswePS1 mice, associated with apoptosis in basal lamina cells.

Montmorillonite/chitosan nanoparticles as a novel controlled-release topical ophthalmic delivery system for the treatment of glaucoma.

BackgroundTo date, the rapid clearance from ocular surface has been a huge obstacle for using eye drops to treat glaucoma, since it has led to the short preocular residence time and low bioavailability.MethodsThe novel nanoparticles (NPs) were designed for topical ophthalmic controlled drug delivery system through intercalating the BH into the interlayer gallery of Na-montmorillonite (Na+Mt) and then further enchasing chitosan nanoparticles. The resulting nanoparticles had a positive charge (+29±0.18 mV) with an average diameter of 460±0.6 nm.ResultsIn vitro study of drug release profiles suggested controlled release pattern. The irritation experiment analysis on both human immortalized cornea epithelial cell (iHCEC) and chorioallantoic membrane-trypan blue staining (CAM-TBS) showed good tolerance for ocular tissues. It was interestingly found that the nanoparticles could enter into iHCEC from the result of cellular uptake experiment measured by confocal layer scan microscopy (CLSM). Meanwhile, multilayered iHCEC was used to simulate the barrier of corneal epithelial cells for in vivo preocular retention capacity study, which suggested that BH-Mt/CS NPs could prolong the retention time in comparison with BH solution. The ocular pharmacokinetics studied by microdialysis sampling technique showed that AUC0−t and MRT0−t of BH-Mt/CS NPs were 1.99-fold and 1.75-fold higher than those of BH solution, indicating higher bioavailability. Moreover, the study of blood drug concentration, few researchers have reported, showed that low level drug could enter into blood, suggesting lower systematic side effect. Importantly, pharmacodynamics studies suggested that BH-Mt/CS NPs could make a significant decreased intraocular pressure on glaucomatous rabbits.ConclusionInspired by these advance of montmorillonite/chitosan nanoparticles, we envision that the BH-Mt/CS NPs will be a potential carrier for BH, opening up the possible applications in glaucoma therapy.

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain.

Fermented &lt;i&gt;Cordyceps cicadae&lt;/i&gt; Mycelia Extracts Ameliorate Dry Eye Symptoms through Reduction of Cornea Epithelial Cell Apoptosis and Maintenance of Conjunctival Goblet Cells in a Mouse Dry Eye Model

Cordyceps cicadae (Cc), a traditional Chinese medicine, has been shown to possess immunomodulatory and anti-inflammatory activities, and is regarded as having effects in vision improvement, but with no reported evidence. This study investigated the effects of Cordyceps cicadae fermented mycelia extracts (Cc extracts) in a benzalkonium chloride (BAC)-induced mouse dry eye model. Female ICR mice aged 6 weeks were randomly divided into four groups: blank, BAC-damaged without Cc extracts, BAC-damaged with 10 mg/kg bodyweight of Cc extracts, BAC-damaged with 100 mg/kg bodyweight of Cc extracts. The results showed that tear volume, tear film breakup time, and cornea surface indexes, including smoothness, opacity, topography, and the extent of lissamine green staining, were all improved with intake of Cc extracts intake, when compared to the status of the BAC-damaged group without Cc extracts. Immunohistochemical assays showed moderate change of Ki-67+ and Np63+ epithelial cell populations, while apoptotic epithelial cells, as detected by TUNEL assay, were decreased. PAS stain showed that the conjunctival goblet cell number and total cell area were decreased in the BAC-damaged group with Cc extracts at 10 mg/kg bodyweight. This study demonstrated that Cc extracts effectively ameliorate BAC-induced dry eye symptoms through enhancement of cornea resilience against BAC-induced damages and maintenance of conjunctival goblet cells.

Expression, Regulation, and Effects of Interleukin-17f in the Human Ocular Surface

ABSTRACTPurpose: To evaluate the basal and possibly stimulated expression of interleukin (IL)-17 in the context of the ocular surface and potential downstream effects.Methods: Western blot and immunofluorescent staining were used to evaluate IL-17F expression in human cornea/conjunctival tissues and cornea epithelial cell line. Cytokines and matrix metalloproteinase (MMP) transcripts were quantified by qPCR. IL-17F effects on NF-κB were investigated by the secretary alkaline phosphatase assay.Results: IL-17F was expressed in the cytoplasm of human corneal and conjunctival epithelial tissues. In the corneal cell line, exogenous IL-17F did not increase the NF-κB activity, but Pam3CSK4 increased IL-17F transcripts. IL-17F stimulated MMP-9 activity, promoted IL-6, IL-8, tumor necrosis factor-α transcripts levels, and depressed monocyte chemoattractant protein-1 expression, but did not affect transforming growth factor beta-1 transcript levels.Conclusions: Normal corneal/conjunctival epithelial cells express IL-17F. Microbial agents may stimulate IL-17F via the NF-κB pathway. Matrix dissolution stimulated by IL-17F may have a role in the melting and necrosis of cornea in severe inflammation.

Human umbilical cord mesenchymal stem cells improve generation of spheres in human corneal epithelial cells and promotes cell migration

Event Abstract Back to Event Human umbilical cord mesenchymal stem cells improve generation of spheres in human corneal epithelial cells and promotes cell migration Siti M. Azmi1*, Nur Zawani M. Kamal1, Wai H. Ng1, Rafeezul Mohamed1, Jun J. Tan1 and Bakiah Shaharuddin1 1 Universiti Sains Malaysia, Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Malaysia Transplantation of cornea limbal stem cells is the contemporary cellular therapy for corneal surface regeneration. Cells in three-dimensional (3D) cultures mimic the microenvironment in vivo thus improve the engraftment and survival rates of cellular transplantation (Fennema, Rivron et al. 2013, Kawaguchi, Machida et al. 2013, Oltolina, Zamperone et al. 2015, Wan, Wang et al. 2015). Umbilical cord-derived mesenchymal stem cells (UCMSCs) possess regenerative capability and are rich with paracrine factors (Guo, Sun et al. 2015). The aim of this project was to study human cornea epithelial cells (hCEC) sphere formation under the influence of UCMSC, and its effect on cellular migration, and to investigate the stem cell markers expression in 3D co-cultures of hCEC-USMSC. In this study, spheres were generated by hanging drop technique from either hCEC alone, or combination with UCMSC using sphere induction medium (50% DMEM and 50% DKSFM supplemented with 10 ng/ml EGF and 20 ng/ml bFGF). Spontaneous sphere formation was performed on low cell-attachment plate and served as control. Spheres were harvested on day 4 and characterised. Cellular migration was evaluated by scratch migration assay in sphere cultures versus control. The results showed that hCEC sphere formation was improved in 3D by the hanging drop method compared to spontaneous generation on low adherent surface. hCEC sphere formation improved by the presence of UCMSC compared to hCEC alone (Fig 1). Immunofluorescence revealed that hCEC in spheres maintained their epithelial stem cell marker p63 without expression of differentiation marker cytokeratin 3 (Fig 2). Preliminary study on cell migration demonstrated capability for gap closure was enhanced in sphere cultures. In conclusion, UCMSC improves generation of sphere in HCECs. 3D sphere cultures showed a better condition for maintaining epithelial stem cell markers. Further functional studies to examine the effects of spherical hCEC-UCMSC co-culture for corneal regeneration will be investigated further. Figure 1 Figure 2 Acknowledgements This work was funded by Fundamental Research Grant Scheme (203.CIPPT.6711508) Ministry of Higher Education Malaysia. Keywords: 3D culture, Umbilical cord mesenchymal stem cell, Corneal epithelial, spherical culture, Cord Factors Conference: 6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016. Presentation Type: Poster Topic: Stem cells and Regenerative Medicine Citation: Azmi SM, Kamal NM, Ng WH, Mohamed R, Tan JJ and Shaharuddin B (2016). Human umbilical cord mesenchymal stem cells improve generation of spheres in human corneal epithelial cells and promotes cell migration. Front. Bioeng. Biotechnol. Conference Abstract: 6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting. doi: 10.3389/conf.FBIOE.2016.02.00019 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Dec 2016; Published Online: 19 Dec 2016. * Correspondence: Ms. Siti M Azmi, Universiti Sains Malaysia, Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Kepala Batas, Pulau Pinang, 13200, Malaysia, maisura@usm.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Siti M Azmi Nur Zawani M Kamal Wai H Ng Rafeezul Mohamed Jun J Tan Bakiah Shaharuddin Google Siti M Azmi Nur Zawani M Kamal Wai H Ng Rafeezul Mohamed Jun J Tan Bakiah Shaharuddin Google Scholar Siti M Azmi Nur Zawani M Kamal Wai H Ng Rafeezul Mohamed Jun J Tan Bakiah Shaharuddin PubMed Siti M Azmi Nur Zawani M Kamal Wai H Ng Rafeezul Mohamed Jun J Tan Bakiah Shaharuddin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Long‐term cultivation of corneal stem cells – possible applications from benchside to bedside

SummaryLong‐term cultures of corneal stem cells can be developed for future tissue engineering and clinical applications. Cornea limbal and stromal tissue explants can be cultivated and expanded for longer periods of time (months) under special conditions and without the use of scaffolds. Viable 3D cell outgrowth from the explants can be achieved within 4 weeks time. The outgrowing limbal epithelial stem cells (LESCs) revealed a unique fingerprint of markers specific for stemness, proliferation, limbal epithelial cells and differentiated cornea epithelial cells. Morphological and immunostaining analysis concluded that long‐term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization of collagen, which was similar to that formed by corneal stromal stem cells (CSSCs). Overall, modelling 3D long‐term structures of corneal stem cells can be used for generating highly pluripotent, long‐standing 3D cultures from limbal and stromal stem cells, which can be used for further research purposes and clinical transplantation.

Changes of Ocular Surface and the Inflammatory Response in a Rabbit Model of Short-Term Exposure Keratopathy.

PurposeTo evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy.MethodsShort term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis.ResultsSignificant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure.ConclusionsShort term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.

OCT/LCT monitoring of drug action on the structure of the human cornea in vivo

The effect of anti-glaucoma medicinal prepaprations Timolol-AKOS and Cosopt on the structure components of human eye cornea are studied. The eye drops Timolol-AKOS 0.5% and Cosopt were used as the object of study. 10 voluntary patients of the Eye Disease Clinic aged from 70 to 75 suffering from glaucoma took part in the studies. The study of cornea in vivo was carried out using the methods of laser scanning confocal tomography (LCT) and optical coherence tomography (OCT) before the application of drugs and at different time moments after the application. The eye cornea thickness values measured using the OCT at the initial moment of time and at different times after the application of the medicinal preparations are presented. From the LCT data the density and the mean diameter of the cornea epithelial cells was calculated. As a result of the complex studies carried out, it was shown that the Timolol-AKOS medication causes the swelling of the cornea within the limits of 1-5%. Under the use of Cosopt medication the cornea dehydration was observed.

TLR3/TRIF signalling pathway regulates IL-32 and IFN-β secretion through activation of RIP-1 and TRAF in the human cornea.

Toll-like receptor-3 (TLR3) and RNA helicase retinoic-acid-inducible protein-1 (RIG-I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG-I signalling pathway was stimulated by viral infection to produce interleukin (IL)-32-mediated pro-inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)-infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG-I that are responded to EBV-encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL-32-mediated pro-inflammatory cytokines and IFN-β through up-regulation of TRIF/TRAF family proteins or RIP-1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF-κB and IRFs to produce pro-inflammatory cytokines and IFN-β than RIG-I-siRNA transfection in HCECs/EBV. Blockade of RIP-1, which connects the TLR3 and RIG-I pathways, significantly blocked the TLR3/TRIF-mediated and RIG-I-mediated pro-inflammatory cytokines and IFN-β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF-dependent signalling pathway against viral RNA might be a main target to control inflammation and anti-viral responses in the ocular surface.

Hypercalcemia Leads to Delayed Corneal Wound Healing in Ovariectomized Rats.

Hypercalcemia is often observed in postmenopausal women as well as in patients with primary hyperparathyroidism or malignant tumors. In this study, we investigated the relationship between calcium ion (Ca(2+)) levels in lacrimal fluid and the rate of corneal wound healing in hypercalcemia using ovariectomized (OVX) rat debrided corneal epithelium. We also determined the effects of Ca(2+) levels on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T). The calcium content in bones of OVX rats decreased after ovariectomy. Moreover, the Ca(2+) content in the blood of OVX rats was increased 1 month after ovariectomy, and decreased. The Ca(2+) content in the lacrimal fluid of OVX rats was also increased after ovariectomy, and then decreased similarly as in blood. Corneal wound healing in OVX rats was delayed in comparison with Sham rats (control rats), and a close relationship was observed between the Ca(2+) levels in lacrimal fluid and the rate of corneal wound healing in Sham and OVX rats (y=-0.7863x+8.785, R=0.78, n=25). In addition, an enhancement in Ca(2+) levels caused a decrease in the viability in HCE-T cells. It is possible that enhanced Ca(2+) levels in lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in a delay in corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage of patients with hypercalcemia.

Decrease in Corneal Damage due to Benzalkonium Chloride by the Addition of Mannitol into Timolol Maleate Eye Drops.

We investigated the protective effects of mannitol on corneal damage caused by benzalkonium chloride (BAC), which is used as a preservative in commercially available timolol maleate eye drops, using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constant (kH), as well as cell viability, were higher following treatment with 0.005% BAC solution containing 0.5% mannitol than in the case BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without mannitol. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.5% mannitol was significantly higher than that for eyes instilled with timolol maleate eye drops without mannitol, and the addition of mannitol did not affect the corneal penetration or IOP reducing effect of the timolol maleate eye drops. A preservative system comprising BAC and mannitol may provide effective therapy for glaucoma patients requiring long-term treatment with anti-glaucoma agents.

A new approach of delivering siRNA to the cornea and its application for inhibiting herpes simplex keratitis.

Small interfering RNA (siRNA) is a potential agent for the treatment of ocular surface diseases. Previous studies delivered siRNA by directly injecting siRNA into cornea or conjunctiva. In the present study we sought to explore an alternative approach to deliver siRNA into mouse cornea via eye drops that contains cy3-labeled siRNA (cy3-siRNA) and different cationic complexing agents and to evaluate the effects of siRNA targeting HSV-1 ICP4 gene (ICP4-siRNA) on mouse herpes simplex keratitis (HSK). Cy3-siRNA was mixed with Lipofectamine 2000, Entranster(TM)-in vivo, polyethyleneimine (PEI) or PEO-PPO-PEO polymers at different ratios. The efficacy of delivery was analyzed after topical application of the complexes to normal, EDTA treated, and epithelial scraped cornea of BALB/c mouse eyes. Compared to the other delivery agents and schedules, PEI at 0.75 mg/ml with 20 μM cy3-siRNA complex delivered eight times daily for two days was the most efficient as revealed by its production of the greatest fluorescence in cornea epithelial cells. In mouse HSK, the application of ICP4-siRNA+PEI eye drops reduced the damage to the corneal epithelia and decreased viral VP16 expression in the corneal tissue. These results proved the idea that siRNA can be formulated into eye drops with carriers for effective delivery into the cornea and that the formulated eye drops containing ICP4-siRNA can inhibit HSV-1 replication in the mouse corneas.

ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration.

Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R-2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing.