Effects of Ripasudil Hydrochloride on Epithelial Repair in a Mouse Cornea.

PURPOSE: Effect of topical administration of a Rho kinase inhibitor, ripasudil, on epithelial wound healing in a mouse cornea was investigated. Effects of treatment of cultured human corneal epithelial cell (HCEC) line and organ-cultured corneal epithelium with ripasudil on expression of p-ERK was also examined. METHODS: Epithelial defects with a diameter of 2.0 mm were prepared in the central corneas of C57BL/6 mice with or without 1-week travoprost pre-treatment, to which ripasudil or PBS as a control was instilled every 6 h immediately after preparation. The mice eyes were cultured with or without travoprost for 24-hrs. The expression levels of p-ERK in epithelium of mice eyes were compared by immunostaining after further 24-hrs culture with or without ripasudil for 24-hrs. HCEC were cultured with or without ripasudil and processed for examination for proliferation activity and protein expression of p-ERK by either immunostaining or Western blotting. The cells were also treated with or without travoprost for 24-hrs, and were further cultured with or without ripasudil. Expression levels of p-ERK were examined by Western blotting. RESULTS: Ripasudil treatment suppressed post-debridement epithelial healing in association with reduced proliferation activity in peripheral (limbal) epithelium in cornea with or without pre-treatment with travoprost. Ripasudil treatment accelerated p-ERK expression. Ripasudil supplementation upregulated proliferation with increased p-ERK in HCEC. CONCLUSION: Ripasudil treatment promotes wound healing of the mouse corneal epithelium by enhancing cell proliferation on peripheral (limbal) epithelium.

Characteristics of daily foraging activity of Camponotus japonicus via time series analysis.

Social insects often share tasks among individuals. In this study, we analyzed the foraging activity of ants (Camponotus japonicus) and recorded the daily passage event counts of individual workers between a nest chamber and a foraging arena in five monodomous colonies. We proposed two hypotheses on the time series of foraging frequency by individual worker ants as follows: (i) for the time series of foraging frequency by individual worker ants, the foraging frequency on a certain day could be expressed by the product of the foraging frequency on the previous day and the exponential of a random number. (ii) The random numbers are correlated between some pairs of worker ants. The results for the five tested ant colonies showed that the probability of total daily passage counts (the sum of an individual's passage count) followed a log-normal distribution. The worker ants behaved differently in terms of active days and foraging frequency. However, for > 54% of the worker ants, the probability of the daily passage count was characterized by a log-normal distribution, and these worker ants performed > 72% of the tasks in each colony. Furthermore, for > 73% of the worker ants, the time development of the passage count was mathematically modeled; the logarithmic first difference between the passage counts on a certain day and those on the previous day was a random normal variable. These results support hypothesis (i). Additionally, the random numbers that were equivalent to the logarithmic first difference were correlated for some pairs of worker ants. These results support hypothesis (ii).

Delayed regression of laser-induced choroidal neovascularization in TNFα-null mice.

We investigated the effects of lacking TNFα on the development and regression of Argon-laser-induced choroidal neovascularization (CNV) in mice. We lasered ocular fundus for induction of CNV in both wild-type (WT) and TNFα-null (KO) mice. Fluorescence angiography was performed to examine the size of CNV lesions. Gene expression pattern of wound healing-related components was examined. The effects of exogenous TNFα on apoptosis of human retinal microvascular endothelial cells (HRMECs) and on the tube-like structure of the cells were investigated in vitro. The results showed that Argon-laser irradiation-induced CNV was significantly larger in KO mice than WT mice on Day 21, but not at other timepoints. Lacking TNFα increased neutrophil population in the lesion. The distribution of cleaved caspase3-labelled apoptotic cells was more frequently observed in the laser-irradiated tissue in a WT mouse as compared with a KO mouse. Exogenous TNFα induced apoptosis of HRMECs and accelerated regression of tube-like structure of HRMECs in cell culture. Taken together, TNFα gene knockout delays the regression of laser-induced CNV in mice. The mechanism underlying the phenotype might include the augmentation of neutrophil population in the treated tissue and attenuation of vascular endothelial cell apoptosis.

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line.

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 µM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 µM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell-cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell-cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.

Verification of mathematical models of response threshold through statistical characterisation of the foraging activity in ant societies.

The concept of response threshold (RT) has been developed to explain task allocation in social insect colonies, wherein individual workers engage in tasks depending on their responsiveness to the task-related stimulus. Moreover, a mathematical model of RT has been proposed to explain data obtained from task allocation experiments; however, its applicability range warrants clarification through adequate quantitative analysis. Hence, we used an automatic measuring system to count passage events between a nest chamber and a foraging arena in five colonies of ants, Camponotus japonicus. The events were measured using radio-frequency identification tags attached to all workers of each colony. Here, we examined the detailed forms of i) labour distribution during foraging among workers in each colony and ii) the persistence of rank-order of foraging among workers. We found that labour distribution was characterized by a generalized gamma-distribution, indicating that only few workers carried out a large part of the workload. The rank-order of foraging activity among workers in each colony was maintained for a month and collapsed within a few months. We compared the obtained data with testable predictions of the RT model. The comparison indicated that proper evaluation of the mathematical model is required based on the obtained data.

Impaired healing of cornea incision injury in a TRPV1-deficient mouse.

The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor ß1 (TGFß1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFß1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFß-induced granulation tissue formation.

UMATracker: an intuitive image-based tracking platform.

Image-based tracking software are regarded as valuable tools in collective animal behaviour studies. For such operations, image preprocessing is a prerequisite, and the users are required to build an appropriate image-processing pipeline for extracting the shape of animals. Even if the users successfully design an image-processing pipeline, unexpected noise in the video frame may significantly reduce the tracking accuracy in the tracking step. To address these issues, we propose UMATracker (Useful Multiple Animal Tracker), which supports flexible image preprocessing by visual programming, multiple tracking algorithms and a manual tracking error-correction system. UMATracker employs a visual programming user interface, wherein the user can intuitively design an image-processing pipeline. Moreover, the software also enables the user to visualize the effect of image processing. We implement four different tracking algorithms to enable the users to choose the most suitable algorithm. In addition, UMATracker provides a manual correction tool for identifying and correcting tracking errors.

Lack of plakoglobin impairs integrity and wound healing in corneal epithelium in mice.

We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (JupCEΔ/CEΔ) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jupf/f) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.g., brushing and wound healing. Immunohistochemistry analysis demonstrated that, although Jup ablation did not affect BrdU incorporation, basal cell-like cells labeled for keratin 14 were ectopically present in the supra-basal layer in mutant corneal epithelium, suggestive of altered cell differentiation. Plakoglobin-deficient epithelium exhibits increased fragility against mechanical intervention when compared to wild-type controls under identical treatment. Closure of an epithelial defect was significantly delayed in JupCEΔ/CEΔ epithelium. Our findings indicate that the lack of plakoglobin significantly affects corneal epithelium differentiation, as well as its structural integrity. Plakoglobin is essential to the maintenance of the structure of the corneal epithelium and its wound healing.

Ocular surface alkali injury damages meibomian glands in mice.

PURPOSE: To examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice. METHODS: Three µL of 1 N NaOH were applied under general anesthesia to the right eye of 10-week-old BALB/c (n = 54) mice to produce a total ocular surface alkali burn. The meibomian gland morphology was examined at days 1, 2, 5, 10, and 20 by stereomicroscopy and non-contact infrared meibography. Mice were then sacrificed and eyelids processed for histology with hematoxylin-eosin and immunohistochemistry for ELOVL4, PPARγ, myeloperoxidase (a neutrophil marker) and F4/80 macrophage antigen, as well as TUNEL staining. Another set of specimens was processed for cryosectioning and Oil red O staining. RESULTS: Alkali injury to the ocular surface produced cellular apoptosis, infiltration of neutrophils and macrophages, degeneration of the meibomian gland, and ductal dilation. Inflammation in and destruction of acunal stricture seemed more prominent in the lower eyelid, while duct dilation was more frequently observed in the upper eyelid during healing. Surviving acinar cells were labeled for ELOVL4 and PPARγ. Oil red O staining showed that the substance in the dilated duct contained predominantly neutral lipid. CONCLUSIONS: Alkali injury to the ocular surface results in damage and destruction of the eyelid meibomian glands. The pattern of the tissue damage differs between glands of the upper and lower eyelids.

Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas.

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor ß, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.

Selection of the Rotation Direction for a Camphor Disk Resulting from Chiral Asymmetry of a Water Chamber.

Self-motion of a camphor disk rotating inside a water chamber composed of two half-disks was investigated. The half-disks were joined along their diameter segments, and the distance between their midpoints (ds) was considered as the control parameter. Various types of camphor disk motions were observed depending on ds. When ds = 0, the chamber had a circular shape, so it was symmetric. A camphor disk showed either a clockwise (CW) or counterclockwise (CCW) rotation with the direction determined by its initial state. The symmetry of the chamber was broken for ds > 0. For moderate distances between the midpoints, a unidirectional orbital motion of the disk was observed. The preferred rotation direction was determined by the shape of the chamber, and it did not depend on the initial rotation direction. For yet larger ds, the unidirectional circular motion was no longer observed and the trajectory became irregular. A mathematical model coupling the camphor disk motion with the dynamics of the developed camphor molecular layer on water was constructed, and the numerical results were compared with the experimental results. The selection of motion type can be explained by considering the influence of camphor concentration on the disk trajectory through the surface tension gradient.

Effects of epiplakin-knockdown in cultured corneal epithelial cells.

BACKGROUND: To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. METHODS: Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and proliferation were examined by using scratch assay and Alamar blue assay, respectively. RESULTS: Scratch assay and Alamar blue assay showed migration and proliferation of the cells was accelerated by epiplakin knockdown. siRNA-knockdown of epiplakin suppressed protein expression of E-cadherin, keratin 6 and vimentin. CONCLUSIONS: Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.

Inhibition of development of laser-induced choroidal neovascularization with suppression of infiltration of macrophages in Smad3-null mice.

We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-ß1 (TGFß1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFß1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFß1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFß1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFß1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.

Modulation of Smad signaling by non-TGFß components in myofibroblast generation during wound healing in corneal stroma.

Corneal scarring/fibrosis disturbs normal transparency and curvature of the tissue and thus impairs vision. The lesion is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix. Inflammatory/fibrogenic growth factors or cytokines expressed in inflammatory cells that infiltrate into injured tissues play a pivotal role in fibrotic tissue formation. In this article the pathogenesis of fibrosis/scarring in the corneal stroma is reviewed focusing on the roles of myofibroblast, the key player in corneal stromal wound healing and fibrosis, and cytoplasmic signals activated by the fibrogenic cytokine, transforming growth factor ß (TGFß). Although it is established that TGFß/Smad signal is essential to the process of keratocyte-myofibroblast transformation in a healing corneal stroma post-injury. This article emphasizes the involvement of non-TGFß molecular mechanisms in modulating Smad signal. We focus on the roles of matricellular proteins, i.e., osteopontin and tenascin C, and as cellular components, the roles of transient receptor potential (TRP) cation channel receptors are discussed. Our intent is to draw attention to the possibility of signal transduction cascade modulation (e.g., Smad signal and mitogen-activated protein kinases, by gene transfer and other related technology) as being beneficial in a clinical setting to reduce or even prevent corneal stromal tissue fibrosis/scarring and inflammation.

Suppression of In Vivo Neovascularization by the Loss of TRPV1 in Mouse Cornea.

To investigate the effects of loss of transient receptor potential vanilloid receptor 1 (TRPV1) on the development of neovascularization in corneal stroma in mice. Blocking TRPV1 receptor did not affect VEGF-dependent neovascularization in cell culture. Lacking TRPV1 inhibited neovascularization in corneal stroma following cauterization. Immunohistochemistry showed that immunoreactivity for active form of TGFß1 and VEGF was detected in subepithelial stroma at the site of cauterization in both genotypes of mice, but the immunoreactivity seemed less marked in mice lacking TRPV1. mRNA expression of VEGF and TGFß1 in a mouse cornea was suppressed by the loss of TRPV1. TRPV1 gene ablation did not affect invasion of neutrophils and macrophage in a cauterized mouse cornea. Blocking TRPV1 signal does not affect angiogenic effects by HUVECs in vitro. TRPV1 signal is, however, involved in expression of angiogenic growth factors in a cauterized mouse cornea and is required for neovascularization in the corneal stroma in vivo.

Transient Receptor Potential Channels and Corneal Stromal Inflammation.

Corneal transparency is dependent on the maintenance of the structural integrity and functional activity of its epithelial and endothelial limiting layers and the stroma. Different transient receptor potential (TRP) channel subtypes are expressed in cells and on corneal sensory nerve endings. They serve as sensors and transducers of environmental stimuli that can reduce tissue transparency. These nonselective cation channels are members of a superfamily sharing TRP box protein sequence homology having 6 membrane spanning domains with a pore between the fifth and sixth segments. TRP channels are composed of 4 monomeric subunits that oligomerize in homomeric or heteromeric configurations derived from different TRP subtypes belonging to the same or any of 6 different subfamilies. TRP subfamily members identified in the cornea include those belonging to the canonical, vanilloid, ankyrin, or melastatin subfamilies. In this review, we specifically focus on the functional roles of TRPV1 and TRPA1 expression in the cornea as their activation provides adaptive nociceptive and immune responses to noxious environmental stresses such as irritating ligands, temperature fluctuations, rises in ambient osmolarity, mechanical stretch, decline in pH, and tissue injury. Our previous studies have indicated that TRPV1 and TRPA1 subtypes are potential drug targets for improving corneal wound healing after alkali burns, because injury-induced fibrosis, neovascularization, and inflammation in either TRPV1 or TRPA1 gene-silenced mice were all significantly reduced.

Disruption of eyelid and cornea morphogenesis by epithelial ß-catenin gain-of-function.

PURPOSE: To examine the developmental pathobiology of the eyelid and the cornea caused by epithelial ß-catenin gain-of-function (gof) during mouse embryogenesis. METHODS: Compound mutant mice (Ctnnb1(GOFOSE) , gof of ß-catenin in the epidermis and the ocular surface epithelium) were generated by time-mating keratin 5-promoter-Cre recombinase (Krt5-Cre) and Ctnnb1(fE3/WT) (floxed exon 3 of Ctnnb1) mice. Eyes obtained from wild-type (WT) and mutant embryos at various gestation stages until E18.5 were examined with histology and immunohistochemistry. The ultrastructure of the ocular tissues of the E18.5 embryos was also examined. RESULTS: Expression of the gof-ß-catenin mutant protein in the epidermis severely impaired eyelid morphogenesis at E15.5, E17.5, and E18.5. The mutant stroma exhibited impaired keratocyte differentiation with accelerated cell proliferation and reduction in the accumulation of collagen type I. The mutant embryos also showed hyperproliferative nodules in the ocular surface epithelia with anomaly of cornea-type epithelial differentiation and the absence of the epithelial basement membrane. CONCLUSIONS: Expression of the gof-ß-catenin mutant protein in basal epithelial cells disrupts eyelid and cornea morphogenesis during mouse embryonic development due to the perturbation of cell proliferation and differentiation of the epithelium and the neural crest-derived mesenchyme.

Pathobiology of wound healing after glaucoma filtration surgery.

Conjunctival and subconjunctival fibrogenesis and inflammation are sight compromising side effects that can occur subsequent to glaucoma filtration surgery. Despite initial declines in intraocular pressure resulting from increasing aqueous outflow, one of the activated responses includes marshalling of proinflammatory and pro-fibrogenic cytokine mediator entrance into the aqueous through a sclerostomy window and their release by local cells, as well as infiltrating activated immune cells. These changes induce dysregulated inflammation, edema and extracellular matrix remodeling, which occlude outflow facility. A number of therapeutic approaches are being taken to offset declines in outflow facility since the current procedure of inhibiting fibrosis with either mitomycin C (MMC) or 5-fluorouracil (5-FU) injection is nonselective. One of them entails developing a new strategy for reducing fibrosis induced by wound healing responses including myofibroblast transdifferentiation and extracellular matrix remodeling in tissue surrounding surgically created shunts. The success of this endeavor is predicated on having a good understanding of conjunctival wound healing pathobiology. In this review, we discuss the roles of inappropriately activated growth factor and cytokine receptor linked signaling cascades inducing conjunctival fibrosis/scarring during post-glaucoma surgery wound healing. Such insight may identify drug targets for blocking fibrogenic signaling and excessive fibrosis which reduces rises in outflow facility resulting from glaucoma filtration surgery.

Histological analysis of a cornea following experimental femtosecond laser ablation.

Corneal photorefractive surgery is currently performed by ablation of corneal stroma under the stromal flap. A stromal flap is created using a femtosecond (FS) laser or mechanical microkeratome, although the FS laser procedure is considered safer and more accurate. This review assesses and compares the use of FS laser versus mechanical microkeratome ablation for corneal stromal characteristics mainly examined by histology and cellular biological responses. Supporting data from our studies, using corneas of enucleated porcine eye globes, are included in this review. Histological analysis and experimental studies of cellular/tissue responses to FS laser irradiation should be further investigated, and the equipment used to perform these techniques should be improved.

Effects of the loss of conjunctival Muc16 on corneal epithelium and stroma in mice.

PURPOSE: To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. METHODS: We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. RESULTS: The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. CONCLUSIONS: The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.