[Show abstract][Hide abstract] ABSTRACT: Beauveria bassiana is an entomopathogenic fungus and is a rare cause of keratitis. We present a case of fungal keratitis caused by B. bassiana that was diagnosed by in vivo confocal microscopy and in vitro corneal cultures. In addition, we determined the temperature- and drug-sensitivities of the isolated strain of B. bassiana.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study is to elucidate the clinical manifestations and the current treatment status of cytomegalovirus (CMV) endotheliitis via a large case series obtained from a national survey conducted in Japan.
[Show abstract][Hide abstract] ABSTRACT: To report lid-wiper epitheliopathy (LWE)-like staining at the lower eyelid margin (lower-LWE) and to determine the prevalence of LWE (upper-LWE) and lower-LWE in contact lens (CL) wearers and non-CL wearers.
[Show abstract][Hide abstract] ABSTRACT: PurposeTo investigate the changes in the tear flow velocities caused by ageing.Methods
Ninety-nine subjects (41 men, mean age 48.3 ± 20.7 years) were recruited from the Department of Ophthalmology of the Ehime University Hospital. None of the subjects had serious abnormalities of the external surface of the eye. The Krehbiel flow of tears was determined by 40-μm polymethylmethacrylate (PMMA) beads suspended in a fluorescein sodium solution (PPF). The movement of the beads was video recorded through a slit-lamp during normal blinking. The flow of the beads was determined with a Motion analyzer®software (KEYENCE Co., Osaka, Japan). The velocity of the beads in young age, 20–40 years, middle age, 41–60 years and old age, ≥61 years, groups was determined.ResultsThe equation describing the velocity (mm/second) of the PMMA particles as a function of age in the lower tear meniscus measured in the direction of the lacrimal punctum was Y = 2.49−0.04X, where Y = velocity and X = age (r2 = 0.214; p < 0.0001). For the upper meniscus, the equation was Y = 4.83−0.05X (r2 = 0.195, p < 0.0001). The average velocity was 0.70 ± 1.66 mm/second in the lower and 2.16 ± 1.93 mm/second in the upper tear meniscus (p < 0.0001). The particle velocity decreased significantly with increasing age, but no significant difference between the male and female groups except for the lower tear meniscus when all subjects were analysed.Conclusion
The PPF technique is a simple method of examining Krehbiel flow of tears and may be used for evaluating functional nasolacrimal duct obstruction quantitatively.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE. To investigate the differing characteristics of limbal niche cells (LNCs) and limbal stromal cells (LSCs) in the maintenance of limbal epithelial stem/progenitor cells in the cornea. METHODS. LNCs were obtained from direct dissection of the human corneal limbus, and LSCs were obtained from the explant cultures of limbal stromal tissues under the same culture conditions. The resulting cultures were examined for their ability to support the growth of limbal stem/progenitor cells in colony-forming capacity, stratified epithelial cell sheet formation, maintenance of limbal epithelial stem/progenitor cell characteristics, and gene expression levels of factors that supported the limbal epithelial stem/progenitor cells. RESULTS. The colony-forming efficiency of limbal epithelial stem/progenitor cells in LNCs group (6.57±1.54%) was significantly higher than that in the LSCs group (1.43±0.47%). The epithelial cell sheets in the LNCs group stratified into 4-5 layers compared with 2-3 stratified layers in the LSCs group. Both the staining of the colonies and epithelial cell sheets in the LNCs group showed a higher intensity of the limbal stem cell marker ΔNp63, and a lower intensity of the differentiated corneal epithelial cell marker keratin 3 than that in the LSCs group. Moreover, reverse transcription polymerase chain reaction analysis revealed that compared with the common expression of EGF etc., the LNCs showed a higher expression level of E-cadherin and a lower expression level of NT3 than that of the LSCs. CONCLUSIONS. LNCs had the distinct ability to support limbal epithelial stem/progenitor cells compared with LSCs, suggesting their different roles in maintaining the ocular surface.
[Show abstract][Hide abstract] ABSTRACT: To develop a side-view imaging technique for observing the dynamic behavior of posterior chamber structures (PCSs) in porcine eyes which mimics closed-eye cataract surgery in humans.
Enucleated porcine eyes were placed into liquid nitrogen for 5 seconds and immediately bisected at about a 45-degree angle to the equatorial plane. The anterior portion was attached firmly to a glass slide with superglue and sprinkled with wheat flour. Phacoemulsification and aspiration (PEA) was performed as in humans on 10 consecutive porcine eyes. The movements of the PCSs were monitored through the glass slide with a high-resolution video camera set below the cut surface of the eye. The intraocular pressure (IOP) was monitored during the surgery. The highest IOP, operation time, and volume of irrigation fluid of 10 whole eyes were compared to that obtained from the bisected eyes glued to a glass slide. In a second set of experiments, the strength of the seal between the bisected eye and the glass slide was tested in three sets of eyes: 1) frozen eye fixed with superglue with wheat flour for 3 min; 2) frozen eye fixed with superglue for 3 min; and 3) non-frozen eye fixed with superglue for 30 min. The highest IOP that led to a disruption of the seal was compared among the three groups.
PEA was successfully performed on 9 of 10 (90%) eyes with the movements of the PCSs clearly observed. The average maximum intraocular pressure of the 9 bisected eyes was 55.8 +/- 4.7 mmHg and that for the 10 unbisected eyes was 55.3 +/- 5.0 mmHg (P = 0.650). The frozen eye fixed with superglue in combination with wheat flour (Group 1) had the strongest sealing strength with an average IOP at the breaking point of 117.3 +/- 36.2 mmHg.
Our side-view imaging technique can be used to evaluate the changes of the PCSs during intraocular surgery and for surgical training of new residents.
[Show abstract][Hide abstract] ABSTRACT: To describe a new method of measuring early phase tear clearance by anterior segment optical coherence tomography (AS-OCT).
Sixty normal subjects were divided into a young group (30 subjects; 29.6 ± 7.2 years) and an elder group (30 subjects; 71.4 ± 10.8 years). AS-OCT (CASIA SS-1000, Tomey, Japan) with customized software was used to record the tear meniscus at the centre of the lower eyelid. Five microlitres of lukewarm saline solution was dropped into the lower conjunctival sac, and an image of the tear meniscus was obtained immediately and again 30 seconds after natural blinking. The tear meniscus height (TMH) and tear meniscus area (TMA) were measured in the AS-OCT images, and the percentage decrease in the TMH and TMA was used as a measure of the tear clearance. Correlations between tear clearance and clinical features including degree of conjunctivochalasis, degree of protrusion of inferior lacrimal punctum, distance of lacrimal punctum from the Marx line and fluorescein clearance rates were also determined in another healthy population consisting of 30 subjects.
The OCT tear clearance rate was 35.2 ± 11% for TMH and 28.1 ± 12.4% for TMA in the young group, and 12.4 ± 7.3% and 6.2 ± 9.1%, respectively in the elder group. The differences were significant for both the TMH (p = 0.017) and the TMA (p = 0.024). The OCT-determined tear clearance was positively correlated with the fluorescein clearance rate, and negatively correlated with the distance between the lacrimal punctum and Marx line, degree of conjunctivochalasis and degree of lacrimal punctum protrusion.
AS-OCT can be used as a rapid, non-invasive and quantitative method of determining the early phase tear clearance rate in a normal healthy population.
[Show abstract][Hide abstract] ABSTRACT: We report a patient who, based on the clinical manifestations, was originally diagnosed as having Chandler's syndrome and later developed varicella-zoster virus (VZV) DNA-positive anterior uveitis.
The patient with Chandler's syndrome who manifested anterior uveitis underwent a complete ophthalmologic examination. Polymerase chain reaction (PCR) was used to amplify the viral DNA in the aqueous humor to determine the cause of the intraocular inflammation.
Slit-lamp biomicroscopy showed focal iris atrophy and peripheral anterior synechiae (PAS); specular microscopy of the corneal endothelium disclosed the hammered-silver appearance. Based on these clinical findings, we diagnosed this patient as having Chandler's syndrome. During the follow-up period, however, the inflammatory cells suddenly appeared in the anterior chamber with formation of keratic precipitates and an increased intraocular pressure (IOP). VZV DNA was displayed in the aqueous humor by PCR. Based upon the diagnosis of VZV anterior uveitis, corticosteroids and acyclovir were given topically and systemically. The inflammation subsided with these medications; however, trabeculectomy was finally needed to control the IOP due to PAS progression.
The coincidence of VZV anterior uveitis with Chandler's syndrome may constitute an implication for the possible viral etiology of iridocorneal endothelial syndrome.
Case reports in ophthalmology. 09/2013; 4(3):274-8.
[Show abstract][Hide abstract] ABSTRACT: To compare 3 ocular lubricants containing sodium hyaluronate (SH), carboxymethylcellulose (CMC), and hydroxypropyl methylcellulose (HPMC) for their ability to enhance water retention and to protect human corneal epithelial cells (HCECs) from dehydration.
Experiments were performed using 0.1% and 0.3% solutions of the 3 lubricants diluted in Milli-Q water for the water retention assays and in DMEM/F12 culture medium for the cell viability assays. Five milliliters of each of the lubricants was dropped onto a filter paper, and the paper was kept in an open container at 25°C and a humidity of 36% to 38%. The weight of the paper was measured hourly for 4 hours. In the second set of experiment, cultured HCECs were exposed to the test lubricants for 60 minutes, and the lubricants were removed. Cells were then exposed to room air for up to 60 minutes. Cells were then incubated with a vital dye, and the absorption of the reduced form of the dye was measured. The cell survival rate was compared for the 3 lubricants.
Filter papers moistened with both 0.1% and 0.3% SH were significantly heavier than those moistened with CMC and HPMC at all time points. The survival rate of HCECs was significantly higher at most times with 0.1% and 0.3% SH than with CMC and HPMC solutions. The effects of CMC were not significantly different from those of HPMC.
These findings indicate that SH is significantly better for water retention and protection of HCECs from dehydration than HPMC and CMC.
[Show abstract][Hide abstract] ABSTRACT: Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β(2) (TGF-β(2)) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β(2) on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β(2) on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β(2) had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of recombinant TGF-β(2) (0.1-10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β(2) inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β(2) (10 ng/ml) affect HCEC migration. Exposure to TGF-β(2) increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex(TM) suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β(2) and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β(2) and/or FGF-2 induced cellular migration was determined by the Boyden chamber assay. Both TGF-β(2) and FGF-2 induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β(2) and FGF-2-induced migration of HCECs. These results indicate that TGF-β(2) reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β(2) and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.
Experimental Eye Research 12/2012; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose. To evaluate the disinfectant effect of methylene blue (MB)-mediated photodynamic therapy (PDT) on a pathogenic strain of Acanthamoeba. Methods. Acanthamoeba castellanii (ATCC 50370) used in this study were treated under one of four experimental conditions: light irradiation and incubation in MB (L+M+), light irradiation and incubation in physiologic solution (L+M-), incubation in MB only (L-M+), and incubation in physiologic solution (L-M-). M+ trophozoites were incubated in either 0.25 or 0.5 mM MB for 10 minutes. L+ organisms were irradiated for 30 minutes following incubation in solution. A halogen lamp (660 ± 10 nm) with a maximum output of 6 mW/cm(2) was used as the PDT light source. After treatment, antiacanthamoeba activity was evaluated by checking the respiratory activity of the amoeba with 5-cyano-2,3-tetrazolium chloride (CTC) staining. We also determined whether the effect of PDT with MB had been retained or augmented when it was performed in combination with conventional antiamoebic agents. Results. MB-PDT suppressed the respiratory activity of trophozoites in an MB-concentration-dependent manner at total light doses of 10.8 J/cm(2). The respiratory activity of each group as a percentage of that of L-M- is as follows: L+M+ 11.6% (0.5 mM), 60.9% (0.25 mM); L-M+ 116.5% (0.5 mM), 105.5% (0.25 mM); L+M- 107.6%; and L-M- 106.3%. (L+M+ versus L-M- P < 0.05). MB-PDT had a synergistic effect when used in combination with polyhexamethylene biguanide (PHMB) or amphotericin B, but not with voriconazole. Conclusions. MB-PDT is effective against Acanthamoeba in vitro and has synergistic effects with PHMB and amphotericin B.
[Show abstract][Hide abstract] ABSTRACT: To assess a newly developed eyelid pressure measurement system called a blepharo-tensiometer that uses a tactile pressure sensor.
The tactile sensor was 10 mm in diameter and approximately 0.4 mm thick. The sensor was covered with silicon rubber and was placed between a soft contact lens on the cornea and the inner surface of the eyelid. Under these conditions, the sensor measured the pressure of the eyelid on the ocular surface. The pressure of the upper and lower eyelids were measured separately while the eyelids were closed for at least 5 seconds in 34 eyes of 34 normal volunteers. To determine the reliability of the blepharo-tensiometer, the pressures of the upper and lower eyelids were measured on 3 separate days in both eyes of 12 normal volunteers. The correlation between the age and the eyelid pressure was calculated.
The intraclass correlation coefficients for the 3 measurements ranged from 0.675 to 0.911 for the upper eyelid and 0.663 to 0.925 for the lower eyelid. The mean eyelid pressure was 16.95±6.08 mm Hg for the upper lid and 16.11±7.27 mm Hg for the lower lid. The eyelid pressure decreased with increasing age, and both the upper and lower eyelid pressures were significantly and negatively correlated with age (upper eyelid pressures, P<0.0001; lower eyelid pressures, P=0.000432). No complication was detected after the measurements in all of the subjects.
Our blepahro-tensiometer can obtain repeatable measurements of the eyelid pressure and can be used to evaluate the pressure of the eyelids on the ocular surface in normal and diseased eyes.
[Show abstract][Hide abstract] ABSTRACT: To determine whether polymorphisms in the Toll-like receptor 4 (TLR4) gene are associated with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma (XFG) in Japanese individuals.
Genetic association study.
Setting: Multicenter study. Study population: One hundred eighty-four unrelated Japanese patients with POAG, 365 unrelated patients with NTG, and 109 unrelated patients with XFG from 5 hospitals. Procedures: Genomic DNA was extracted from leukocytes of the peripheral blood, and 8 polymorphisms in the TLR4 genes were amplified by polymerase chain reaction (PCR) and directly sequenced. Allele and genotype frequencies and the inferred haplotypes were estimated. Main outcome measures: Differences in allele and genotype frequencies and haplotypes between subjects with POAG, NTG, and XFG.
The allele frequency of rs2149356 of the TLR4 gene in the POAG, NTG, and XFG groups was the most significantly different from that of the control group (minor allele frequency 0.446, 0.395, 0.404, vs 0.308; P = .000058, P = .0030, and P = .015). The allele frequencies of the 5 TLR4 SNPs were higher in all of the glaucoma groups than that in the control group. The statistics of genotypes of TLR4 were approximately the same for all allele frequencies. The haplotypic frequencies with Tag SNPs studied earlier showed that only POAG was statistically significant. Other haplotypes, such as rs10759930, rs1927914, rs1927911, and rs2149356, had higher statistical significance (overall P = .00078 in POAG, overall P = .018 in NTG, and overall P = .014 in XFG).
This study demonstrated that TLR4 polymorphisms are associated with NTG in the Japanese, and they also play a role in the pathogenesis of POAG and XFG.
American Journal of Ophthalmology 07/2012; 154(5):825-832.e1. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Bullous keratopathy (BK), a severe sight-threatening disorder can have a variety of etiologiessuch as prophylactic laser iridotomy, intraocular surgery, trauma, and other ocular disorders.However, there are cases of unknown origins, among which a unique clinical entity namelypseudoexfoliation syndrome (PEX) is having increased importance. CASE PRESENTATION: In this case note, we report the clinical features and in vivo confocal microscopic andpathological findings of two BK cases of unknown cause. CONCLUSIONS: Our findings suggest that the BK was caused by the corneal endotheliopathy of PEX, acommon disease that could affect up to 30% of people over 60 years old and is moreprevalent than we have believed.
[Show abstract][Hide abstract] ABSTRACT: To investigate the role played by epiregulin in corneal epithelial wound healing in vivo in epiregulin-knockout (KO) mice and cultured mouse corneal epithelial cells (MCECs).
A 2-mm diameter central epithelial wound was created in epiregulin-KO and wild-type (WT) mouse corneas. The size of the unhealed area and the epithelial cell proliferation and migration were examined. Myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating corneal stroma. Real-time PCR was used to determine expression of the mRNA of inflammatory cytokines in the corneal epithelial cells. Expression of chemokine (C-X-C motif) ligand 2 (CXCL2) response to IL-1β was examined in MCECs with or without recombinant mouse epiregulin. Repetitive injuries were created to determine the effect of inflammation in healing in epiregulin-KO mice.
After a single injury, corneal epithelial wound healing and cell migration and proliferation were unimpaired. However, corneal opacities and a larger number of infiltrating PMN cells were observed in epiregulin-KO mice. Expression levels of IL-1β, IL-6, CXCL1, and CXCL2 were higher in epiregulin-KO than in WT corneal epithelia cells. The addition of epiregulin significantly reduced the expression of CXCL2 in response to IL-1β in MCECs. In response to repetitive injuries, a significant delay in healing and more severe opacities were observed in epiregulin-KO mice than in WT mice.
Our results indicate that during wound healing, epiregulin may regulate the expression of cytokines and chemokines to reduce an excessive accumulation of PMN cells, which will cause corneal opacity and persistent epithelial defects.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK.
Retrospective, cross-sectional study.
A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests.
Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis.
Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis.
The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness.
Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome.
The author(s) have no proprietary or commercial interest in any materials discussed in this article.
[Show abstract][Hide abstract] ABSTRACT: The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r = 0.83 and P < 0.01 for trophozoites; r = 0.60 and P < 0.01 for cysts; Spearman rank correlation test). The respiratory activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r = 0.70 and P < 0.01 for trophozoites; r = 0.64 and P < 0.01 for cysts; Spearman rank correlation test). The significant correlation of the results indicated that the CTC biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.
Journal of clinical microbiology 02/2012; 50(5):1606-12. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the contactin-associated protein-like 2 (CNTNAP2) gene for single-nucleotide polymorphisms (SNPs) in Japanese patients with the exfoliation syndrome (XFS).
One hundred and eight unrelated Japanese patients with the XFS, and 199 normal controls were studied. Genomic DNA was extracted from the leukocytes of the peripheral blood, and 8 SNPs, rs826802, rs1404699, rs7803992, rs700308, rs4725736, rs2107856, rs2141388, and rs6970064, were amplified by polymerase chain reaction (PCR), directly sequenced, and genotyped.
The allele frequencies of rs1404699 (p=8.57XE-3, odds ratio (OR)=1.59, 95% confidential intervals (CI); 1,12-2.24) and rs7803992 (p=5.43XE-4, OR=1.86, 95% CI; 1.31-2.65) were statistically significantly different between XFS and controls. In addition, there were significant differences in these genotype frequencies (p=0.0197 and 1.75XE-3). The allele and the genotype frequencies of rs2107856 and rs2141388, which were statistically significant SNPs in an earlier study, were not significantly different.
The variants, rs1404699 and rs7803992, of CNTNAP2 should be associated with XFS in the Japanese population.
[Show abstract][Hide abstract] ABSTRACT: To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells.
A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1(+/+), Sl/Sl(d), and W/W(v) mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix.
The speed of corneal wound healing was slower in Sl/Sl(d) and W/W(v) mice than in controls (p<0.01) and the speed of healing in Sl/Sl(d) mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/W(v) mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1(+/+) and 252% in Sl/Sl(d) MCECs by recombinant mouse SCF; however, no significant difference was found in W/W(v) MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes.
These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.