Reepithelialization of the human cornea is regulated by endogenous opioids

PURPOSE: To determine the influence of endogenous opioid modulation on reepithelialization of the human cornea. METHODS: Eight-millimeter-diameter epithelial defects were created with a trephine and mechanical scraping in the center of human corneas. Resurfacing was studied in organ culture. The size of the defect, the number of specimens with complete reepithelialization, and rate of closure were evaluated using topical fluorescein and morphometric analysis. The influence of opioid receptor blockade was studied using the potent and long-acting opioid antagonist, naltrexone (NTX; 10(-6) M), and the effects of excess (10(-6) M) opioid growth factor (OGF), [Met5]enkephalin, also were determined. The modulatory activity of NTX and OGF on DNA synthesis was evaluated by monitoring the labeling index (LI) using radioactive thymidine. The presence and location of OGF and its receptor (OGFr) were ascertained by immunocytochemistry 1 hour and 24 hours after abrasion. RESULTS: NTX accelerated the wound-healing process, with 21% to 89% less defect than controls observed from 24 to 96 hours. At 72 hours, 62% of the subjects in the NTX group had complete closure of the corneal defects, in contrast to only 19% of the control specimens. All epithelial abrasions were resurfaced in the NTX group between 96 and 120 hours, whereas all controls were not closed until 168 hours. The rate of healing in the NTX group was 1.06 mm2/h compared to a rate of 0.68 mm2/h in the control group. OGF delayed corneal wound healing, with 24% to 260% more defect recorded than in control specimens at day 7. The healing rate of the OGF group was 0.42 mm2/h compared to 0.82 mm2/h for control subjects. The corneal epithelium adjacent to the wound had an LI that was 152% greater than control specimens, whereas OGF decreased the LI of this region by 75%. OGF and OGFr were detected in the epithelium bordering the damaged region at 1 hour, and both peptide and receptor were noted in the regenerating epithelium at 24 hours. CONCLUSIONS: These results indicate that an endogenous opioid is present and functions as a tonically active, receptor-mediated, negative growth factor during reepithelialization of the abraded human cornea.     

Kinetics of corneal epithelium turnover in vivo. Studies of lovastatin

The authors developed a direct chemical approach for estimating the rate of turnover of the corneal epithelium in vivo. The method was used to examine the effects of lovastatin, a potent inhibitor of cholesterol biosynthesis, on proliferation and turnover of the epithelium. Corneal DNA was labeled by pulse injection (IP) of the rat with 3H-thymidine, and 3H-labeled DNA was recovered from peripheral and central corneas over the next 15 days. Only the epithelium became labeled, and the loss of label by cell desquamation began 3 days after injection. The loss of 3H-DNA from the cornea (peripheral plus central region) followed first-order kinetics. The half-life of the disappearance was about 3 days. The peripheral cornea became more highly labeled than the central cornea and began to lose 3H-DNA before the central cornea. These observations support the possibility of a higher mitotic rate in the peripheral region and the centripetal movement of a population of peripheral epithelial cells in the normal cornea. The half-lives of the disappearance of 3H-DNA from peripheral and central corneas measured between days 5 and 15 postinjection were identical, both at 3 days. Complete turnover of the corneal epithelium would, therefore, require about 2 weeks (4-5 half-lives). Treatment of the rat with lovastatin had no obvious effects upon the proliferation or turnover of the corneal epithelium. Although lovastatin inhibited corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthesis, the cornea compensated by induction of this enzyme so that there was no net inhibition of cholesterol synthesis in the cornea.     

The phenotype of limbal epithelial stem cells

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

In vivo investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM)

BACKGROUND: The confocal tandem scanning microscope was first used in 1985 by Lemp et al for in vitro and in 1990 by Cavanagh et al for in vivo investigation of human eyes. The aim of this study was to investigate the cells of the central and the peripheral portions of the corneal epithelium and to measure corneal epithelium thickness and the total thickness of the corneas of our volunteers with the new Rostock Laser Scanning Microscope. MATERIAL AND METHODS: A Heidelberg Retina Tomograph (HRT II) was used in combination with a water contact microscope lens (Zeiss, x63, 0.95), the Rostock cornea module (RCM) developed at our institute for the in vivo examination of the cornea. In this study, 92 eyes of 68 subjects between the ages of 15 and 88 years were examined. RESULTS: At the superficial cell layer, the average cell density in the central cornea was 840 +/- 295 cells/mm2, and in the periphery it was 833 +/- 223 cells/mm2. At the wing cell layer, the average cell density rises to 5070 +/- 1150 cells/mm2 in the central and to 5582 +/- 829 cells/mm2 in the peripheral cornea. At the basal cell layer, the cell density rises further to 8996 +/- 1532 cells/mm2 in the central and 10,139 +/- 1,479 cells/mm2 in the peripheral corneal epithelium. The average corneal thickness in the central region was found to be 545 +/- 25 microm, and 652 +/- 75 microm in the periphery. The average epithelium thickness was determined centrally to be 54 +/- 7 microm, and peripherally 61 +/- 5 micrim. CONCLUSIONS: The Rostock Scanning Laser Microscope offers a standardized, reproducible, safe, and fast diagnostic procedure for the evaluation of the corneal epithelium. This technology allows better image quality compared with confocal-slit scanning microscopes and produces a precise depth measurement.     

Involvement of p27(KIP1) degradation by Skp2 in the regulation of proliferation in response to wounding of corneal epithelium

PURPOSE: To examine the expression of the p27(KIP1)in the normal and epithelial-scraped cornea and whether degradation of p27(KIP1)by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium. METHODS: C57Bl6, p27(KIP1-/-), Skp2(-/-), and Skp2(-/-)/p27(KIP1-/-) double-knockout mice were examined. Normal and epithelial-scraped corneas were analyzed by immunocytochemistry using anti-p27(KIP1) antibody. Cells in the S phase of DNA synthesis were analyzed by immunocytochemistry using anti-bromodeoxyuridine (BrdU) antibody. RESULTS: The p27(KIP1) was expressed in basal cells of the central and peripheral region of the cornea and limbus. This expression was not detected 24 hours after the epithelial scraping, when there were many cells in the S phase of DNA synthesis in the corneal epithelium. There were no obvious differences in the thickness and anti-BrdU staining in the corneal epithelium of p27(KIP1-/-) mice from that of control animals. Twenty-four hours after epithelial scraping in the Skp2(-/-) mice, the corneal epithelium was thinner than in wild-type mice and had many p27(KIP1)-positive cells and few BrdU-positive cells. In contrast, 24 hours after epithelial scraping in the Skp2(-/-)/p27(KIP1)(-/-) double-knockout mice, the corneal epithelium was as thick as in wild-type mice and had many BrdU-positive cells. CONCLUSIONS: These results suggest that degradation of p27(KIP1) by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium.     

Superoxide dismutase isoenzymes in the normal and diseased human cornea

PURPOSE: The human cornea, a tissue much exposed to oxidative stress, is rich in extracellular superoxide dismutase (SOD). In this study, the contents and distributions of the SOD isoenzymes in the normal human cornea were compared with those in corneas affected by keratoconus and bullous keratopathy. METHODS: The central and peripheral parts of normal human corneas were analyzed separately. Central corneal buttons were obtained from patients with keratoconus and bullous keratopathy who were undergoing primary keratoplasty or retransplantation. SOD enzymatic activities were determined by a direct spectrophotometric method, and extracellular SOD and the cytosolic Cu- and Zn-containing SOD (CuZn-SOD) proteins were determined with ELISA and studied with immunohistochemistry. RESULTS: The total SOD content, and particularly the extracellular SOD content, was lower in the central than in the peripheral normal cornea. CuZn-SOD and extracellular SOD were demonstrated in all three corneal layers. CuZn-SOD was found in cells, whereas extracellular SOD appeared to be localized on cell surfaces, in basal membranes, and in the stroma. In keratoconus, corneal levels of extracellular SOD were half those in the control corneas, whereas CuZn-SOD and the mitochondrial Mn-containing SOD levels were normal. In bullous keratopathy, apart from edematous dilution, SOD isoenzyme levels were essentially normal. In a remarkable finding, the same pattern in SOD isoenzyme levels as in the original disease was also found at retransplantation. CONCLUSIONS: Extracellular SOD and CuZn-SOD show markedly different distribution patterns within the human cornea. Extracellular SOD activity in the central cornea is halved in keratoconus, compared with that in normal control corneas. The finding of a similar reduction at retransplantation in keratoconus suggests reduced corneal extracellular SOD synthesis in cells of the host as a cause of the low enzyme levels.     

Metabolic analysis of reepithelializing rabbit cornea using phosphorus-31 nuclear magnetic resonance spectroscopy

To investigate metabolic differences between the central and peripheral cornea the latter including the limbal area, corneas were dissected and examined using phosphorus-31 (31P) nuclear magnetic resonance spectroscopy. Since most 31P signals originate from the epithelium, 31P spectra of the cornea primarily represent the metabolic state of the epithelium. The spectra of the peripheral cornea showed all phosphorus resonances detected in the whole cornea; in contrast, the central cornea showed no phosphocreatine and glycerophosrylethanolamine, and only low levels of ATP. These results indicate that there is a higher metabolic activity in the peripheral epithelium, especially in the limbal area, than in the central epithelium. To evaluate the metabolic state of corneal epithelium during regeneration, we also examined corneas reepithelializing after 7 mm of central epithelial tissue had been removed by mechanical scraping. Rabbits were killed 24 and 48 h after scraping. The reepithelializing corneas clearly showed an increase in ATP, phosphocreatine, and sugar phosphates with time, although phosphorylcholine remained depressed. These findings suggest that the reepithelializing cornea has an elevated level of energy production and that it may have reached a higher steady state, thereby indicating accelerated metabolism of the epithelium during regeneration.     

Adaptation of homeostatic ocular surface epithelium to chronic treatment with the opioid antagonist naltrexone

PURPOSE: To determine how ocular surface epithelium adjusts to an increase in cell replication after treatment with the opioid antagonist naltrexone (NTX). METHODS: Adult male rats were given twice daily injections of 30 mg/kg NTX or vehicle for 7 days. Outcomes of NTX administration included DNA synthesis (monitored with BrdU), mitosis (assayed using colchicine), number of cell layers and cell diameter, apoptosis and necrosis, and packing density for the peripheral corneal epithelium, limbus, and conjunctiva. Also, transit time from basal to surface epithelial layers in the peripheral cornea was assessed with [H]thymidine as a marker. RESULTS: DNA synthesis and mitosis in the basal layer of the peripheral corneal epithelium of NTX-treated rats were increased 69% and 85%, respectively, from control levels; no changes in either parameter were recorded in the limbal or conjunctival epithelium (stem cell region). Epithelial thicknesses in the NTX group were increased by 8% to 38% from control subjects, without more cell layers. Packing density in NTX-treated rats was increased from control values by 26% in the basal layer of the limbus and by 12% to 28% in the suprabasal layers of the corneal epithelium, limbus, and conjunctiva. Epithelial cell diameters from corneas of NTX-exposed rats were subnormal in the basal and suprabasal cells of the limbus and conjunctiva. Apoptosis and necrosis were negligible in the epithelium of NTX-treated and control rats. Transit times of peripheral corneal epithelial cells of animals in the NTX group were shortened by 63% from control levels. CONCLUSIONS: These data show that a 1-week treatment with NTX does not induce proliferative pathology or toxicity in ocular surface epithelium, has a minimal effect on stem cell proliferation, and accelerates normal homeostatic processes. Topical application of NTX for stimulation of corneal epithelial wound healing results in no adverse sequelae, thereby supporting the therapeutic role for this drug in the treatment of ocular surface abnormalities.     

Localization of corneal epithelial stem cells in the developing rat.

A monoclonal antibody, 4G10.3, was developed that preferentially binds limbal basal cells in adult rat, rabbit, and human corneas. These cells were hypothesized to be the stem cells for the corneal epithelium. The antibody 4G10.3 was localized by immunofluorescence microscopy in rats 1 d and 1, 1.5, 2, 3, 4, and 6 wk of age. Until 1.5 wk, 4G10.3 bound intensely to all basal cells in the cornea and the limbus. At 2 wks, the basal cells at the central cornea abruptly changed their shape from flattened or ovoid to large and cuboidal and bound 4G10.3 with greatly reduced intensity. Increased stratification of epithelium also was seen. Cells binding 4G10.3 gradually became sequestered to the limbal area after 2 wk, concomitant with increased stratification. At 4 and 6 wk, 4G10.3 binding was identical to that in adult corneas with only limbal basal cells showing positive binding. Basal cells in the limbal epithelium did not decrease their intense binding of 4G10.3 or change their ovoid cellular shape from 1 d through adult life. These results suggest that, during development, stem or stem-like cells are localized throughout the basal layer of the corneal and limbal epithelium. As the cornea matures, these cells are sequestered in the limbus at the same time that stratification of the epithelium and shape changes occur in the basal cells.     

Inhibition of corneal neovascularization by vascular endothelia growth inhibitor gene

AIM: To evaluate the effect of EffecteneTM lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV). METHODS: Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to induce CNV and divided into 4 random teams, ten per each team: team A: transfected by pcDNA4-VEGI gene mediated by EffecteneTM lipofectine transfection; team B: by plasmid pcDNA4; team C: by EffecteneTM, and team D: by normal saline. Length and area of CNV were observed under slit lamp every day after tran- sfection. Immunohistochemistry was performed to detect the expression of VEGI protein in corneas at day 3, 7, 14 and 21. RESULTS: 1) Average occurrence of CNV was 6.3 days in team A, 3.1 days in team B, 3.2 days in team C, and 3.2 days in team D. Difference was significant between A and other teams (P<0.01); 2) Length and average area of CNV in each period in team A was significantly different from those in team B, C and D (P<0.01); 3) VEGI expressions were observed in epithelium, stroma, endothelium and the cliff of CNV in team A at 3 days after transfection by immunohistochemical staining. None VEGI positive cells were found in the control teams (team B, C and D) all the time. CONCLUSION: EffecteneTM lipofectine transfection technique can effectively transfect pcDNA4-VEGI gene into rabbit cornea and the length and CNV areas can be inhibited by VEGI gene.     

Expression of tissue inhibitor of metalloproteinase-4 in normal human corneal cells and experimental corneal neovascularization

PURPOSE: To study the expression of TIMP-4 in cultured corneal cells and in corneal neovascularization. METHODS: Human limbo-corneal epithelial cells, fibroblasts, and endothelial cells were cultured in serum-free, PMA- or basic fibroblast growth factor (bFGF)-treated condition. Neovascularization in rat cornea was induced by suturing. The expression of TIMP-4 was examined by immunohistochemistry, Western blot and RT-PCR. RESULTS: TIMP-4 was constitutively expressed in cultured human corneal cells. The expression was only mildly enhanced after mitogen treatment. TIMP-4 immunoreactivity was predominantly expressed in normal rat corneal epithelium, and also in ingrowing blood vessels following suturing, which persisted up to day 28. Increased staining in corneal epithelium and blood vessels were also noted in vascularized human corneas. CONCLUSIONS: TIMP-4 is expressed in the cornea, which may play a role in modulating extracellular matrix remodeling associated with corneal wound healing and angiogenesis.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.     

Interaction between corneal invasion of polymorphonuclear leukocytes and corneal epithelium

The interaction between corneal invasion of polymorphonuclear leukocytes (PMNs) and corneal epithelium was investigated following different types of corneal injury. Histological examination of centrally denuded corneas demonstrated that it took 18-24 hours for PMNs to infiltrate into the center of the denuded stroma, approximately synchronous with the reepithelialization. On the other hand, in the centrally denuded corneas immediately covered by glue to prevent the reepithelialization, the time required for the appearance of PMNs at the central portion of the corneas was more than 96 hours. Chemotactic activities of PMNs in the conditioned media of normal cornea, completely denuded cornea and reepithelializing cornea were examined using a Boyden chamber. The highest chemotactic activity was defected in the conditioned medium of the cornea with reepithelialization. These data suggest the possibility that the corneal epithelium, especially the re-covering epithelium, stimulates infiltration of the stroma by PMNs.     

In vivo confocal microscopic evaluation of langerhans cell density and distribution in the corneal epithelium of healthy volunteers and contact lens wearers

PURPOSE: To examine and compare the density and distribution of Langerhans cells (LCs) in the corneal epithelium of healthy volunteers and contact lens wearers. METHODS: A total of 225 eyes of 130 healthy volunteers (age, 17-81 years) without history of ocular inflammation, trauma, or surgery and 98 eyes of 55 contact lens wearers (age, 13-76 years) were examined in vivo with the combination of the Heidelberg Retina Tomograph II and in-house-invented Rostock Cornea Module. RESULTS: In healthy volunteers, in vivo confocal microscopy revealed LCs in 31% of all volunteers, with 37 of these 43 volunteers presenting LCs both in the center and the periphery of the cornea with densities of 34 +/- 3 and 98 +/- 8 cells/mm, respectively. In the group of contact lens wearers, 55% of all corneas presented with LCs, and 11 of these 33 corneas revealed LCs at central and peripheral locations. Although LC densities were markedly higher in both the central (78 +/- 25 cells/mm) and the peripheral cornea (210 +/- 24 cells/mm) of contact lens wearers, the gradient of LC density from peripheral to central cornea was found almost identical in both groups. In the central cornea, LC density decreased with duration of contact lens wear. LCs were located at the depth of 35 to 60 microm (ie, the level of lower intermediate cells, basal cells, and subepithelial nervous plexus). LCs presented as either large cells bearing long processes or smaller cells lacking cell dendrites, most supposedly indicating mature and immature phenotype, respectively. CONCLUSIONS: In vivo confocal microscopy enables evaluation of LC density and distribution in corneal epithelium. LCs were found present both in the center and the periphery of the cornea without difference in distribution between healthy volunteers and contact lens wearers. However, contact lens wearers revealed almost twofold higher LC densities in both locations, implying chronic mechanical irritation of the cornea in response to the contact lens as foreign body. Taken together, analysis of LC using in vivo confocal microscopy provides helpful information for a better understanding of contact lens-disturbed ocular homeostasis.