Moesin as a key cytoskeleton regulator in corneal fibrosis.

PURPOSE: : Corneal fibrosis is the third leading cause of blindness worldwide. α-Smooth muscle actin (SMA), a marker of fibrosis, is closely regulated through an intermediate group of submembrane molecules - cytoskeleton regulators. The purpose of this study was to elucidate the role of specific cytoskeleton regulators in a mouse model of corneal fibrosis. METHODS: : A mouse model of corneal fibrosis was developed using anterior keratectomy (AK) and the topical application of transforming growth factor (TGF)-ß1 (1 µg/ml). The RT² Profiler™ PCR Array for cytoskeleton regulators was used to assay changes in levels of specific members of this class of proteins. Moesin siRNA was delivered into the corneal stroma by iontophoresis in vivo. Transformation of the corneal keratocyte-to-myofibroblast in corneal fibrosis, as defined by the expression of α-SMA, was determined by Western blot. RESULTS: : After AK and topical application of TGF-ß1, moesin was the most highly upregulated gene among 84 cytoskeleton regulator genes; iontophoresing moesin siRNA into the corneal stroma reduced the expression of α-SMA to 0.22-, 0.52-, and 0.31-fold of control at postoperative (PO) day 1, 3, and 5, respectively; also, upregulation of phospho-Smad 2 induced by TGF-ß1 was reduced by moesin siRNA to 0.59-, 0.56-, and 0.31-fold of control and expression of phospho-Smad 3 was reduced to 0.58-, 0.53-, and 0.47-fold of control at the same PO days. CONCLUSIONS: : Moesin may be a potential drug target for inhibiting corneal fibrosis, and the details of moesin-related signaling pathways would be critical for understanding corneal fibrosis.

The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region.

Understanding the molecular mechanisms of antimicrobial peptide-membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3-5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)(2)KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3-9.7 microM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 microg/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5-10 mmicroM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 microM. The concentration of 5-10 microM for Y2 from FCS correlated with the concentration of 5 microM (6.25 microg/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5-5 microM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.

Epithelial microfilament regulators show regional distribution in mouse conjunctiva.

PURPOSE: The conjunctival epithelium is a continuous sheet of cells with regional characteristics that appear to be similar. This study was designed to investigate the distribution and levels of expression of a subset of microfilament regulators in the forniceal, palpebral, and bulbar conjunctival epithelia. METHODS: Balb/C mice were used. The localizations of paxillin, focal adhesion kinase, vinculin, talin1, cofilin, profilin, gelsolin, integrin ß1, and integrin α6 were studied with the use of cross-sectional immunofluorescent staining. For a detailed cellular analysis, positioning and ablation with the laser microbeam (PALM) Combi System was used to obtain forniceal, bulbar, and palpebral conjunctival epithelia for expression comparison with the use of western blot analysis and quantitative real-time polymerase chain reaction. RESULTS: Immunostaining showed that focal adhesion kinase, cofilin, profilin, gelsolin, talin1, and vinculin were expressed in all layers of the forniceal, palpebral, and bulbar conjunctival epithelia. Paxillin, integrin ß1, and α6 was found to be located in the basal cell layer in all three of these areas. Quantitative real-time polymerase chain reaction showed that the transcript levels of these microfilament regulators in the forniceal conjunctivae were higher than those levels found in the bulbar and palpebral conjunctivae. Western blot analysis confirmed the differential expression levels of these microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae. CONCLUSIONS: Differences in the levels of microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae suggest different modes of interaction with their microenvironment and within cell layers.

Multivalent Antimicrobial Peptides as Therapeutics: Design Principles and Structural Diversities.

This review highlights the design principles, progress and advantages attributed to the structural diversity associated with both natural and synthetic multivalent antimicrobial peptides (AMPs). Natural homo- or hetero-dimers of AMPs linked by intermolecular disulfide bonds existed in the animal kingdom, but the multivalency strategy has been adopted to create synthetic branched or polymeric AMPs that do not exist in nature. The multivalent strategy for the design of multivalent AMPs provides advantages to overcome the challenges faced in clinical applications of AMPs, such as: stability, efficiency, toxicity, maintenance of activity in high salt concentrations and under physiological conditions, and importantly overcoming bacterial resistance which is currently a leading health problem in the world. The multivalency strategy is valuable for moving multivalent AMPs toward clinical applications.

Effect of dispase denudation on amniotic membrane.

PURPOSE: To describe the cellular components, biochemical composition, and membrane surface characteristics of denuded human amniotic membrane (DHAM) treated with Dispase II. METHODS: DHAM was incubated with Dispase II (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed by gentle scraping to remove any remaining epithelial cells using a cell scraper. Histology, immunohistochemistry for extracellular matrix molecules and growth factors, and transmission (TEM) and scanning electron microscopy (SEM) were performed to assess the effects of increasing durations of incubation on DHAM structure. RESULTS: Dispase II treatment was associated with the digestion of several ECM molecules, particularly those in the basement membrane including collagen VI, fibronectin, and laminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, but TGF-alpha, TGF-beta1, TGF-beta 2R, PDGF-A, VEGF, and EGFR expression were all reduced by Dispase II incubation. TEM confirmed the disruption of DHAM ultrastructure with increasing duration of Dispase II incubation, beginning with disruption of the basal lamina and progressing to loosening of the stromal collagen network as well. CONCLUSIONS: The use of Dispase II in the preparation of DHAM causes significant changes to the ultrastructure of the membrane, particularly the BM. Prolonged incubation with dispase may cause significant disruption in DHAM structure which may affect cell growth in cultured explants.

Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation.

PURPOSE: To determine the expression of muscarinic receptor subtypes (mAChRs) in human and mouse scleral fibroblasts (SFs), to investigate the mechanism that mediate the role mAChRs play in cell proliferation, and to explore the underlying intracellular signaling pathways involved in mouse SFs with treatment of muscarinic agents. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of mAChRs in the human and mouse sclera. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cultured SFs. An immunohistochemical study was used to further detect the presence of mAChR proteins in frozen scleral sections. BrdU (5-bromo-2-deoxyuridine ) cell proliferation assay was performed to measure DNA synthesis. Enzyme linked immunosorbent assay (ELISA) was used to measure in vitro kinase activity for epidermal growth factor receptor (EGF-R), fibroblast growth factor (FGF-2), transforming growth factor (TGF)-beta1, and extracellular signal-regulated kinase (ERK)1/2. Expressions of epidermal growth factor-receptor (EGF-R); protein kinase C (PKC); Proline-rich tyrosine kinase 2 (Pyk-2), v-raf murine sarcoma viral oncogene homolog B1 (B-Raf), Rat Sarcoma (Ras), c-Jun N-terminal kinases (JNK1/2), and ERK1/2 were detected by immunoblot. RESULTS: mAChR for subtypes M(1)-M(5) were detected in both mouse and human SFs by protein, cellular, and mRNA analysis. EGF-R, PKC, Pyk-2, B-Raf, Ras, JNK1/2, and ERK1/2 were activated after treatment by agonists and antagonists, indicated by changes in phosphorylation of these proteins. Atropine abolished the carbachol-induced activation of SF cell proliferation in a concentration-dependent manner. Carbachol also activated p42/44 mitogen-activated protein kinase (MAPK) and Ras in a time-dependent manner. Muscarinic agents also modulated fibroblast growth factor expression in these cells. CONCLUSIONS: This study confirms the presence and functional role of all five mAChRs in human and mouse SFs. These results show that proliferative responses of SFs to muscarinic receptor stimulation are mediated via the activation of the classical MEK-ERK-MAPK cascade.

The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells.

AIM: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE). METHODS: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. RESULTS: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. CONCLUSIONS: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

Proteomic analysis of human tears: defensin expression after ocular surface surgery.

Human tear protein profiles were monitored by surface enhanced laser desorption/ionization-time-of-flight mass spectrometry ProteinChip technology (SELDI-TOF ProteinChip) and liquid chromatography-mass spectrometry (LC-MS). Tears were collected from 21 patients scheduled for surgery to remove an ocular surface neoplasm prior to surgery (day 0) and on days 1, 3, and 30 postoperatively. Using this proteomic approach, we verified that three human alpha-defensins (HNP-1, HNP-2, and HNP-3) were significantly up-regulated in their expression after surgery and that their levels decreased to approximately normal by day 30 by which time healing was complete. Further confirmation of the identity of the alpha-defensins in human tears was made by LC purification, trypsin digestion, and ESI-MS/MS analysis of their tryptic digests. The concentrations of HNP-1 and HNP-2 were determined and shown to be markedly increased after ocular surface surgery. The results of the study suggest that human alpha-defensins HNP-1, HNP-2, and HNP-3 are up-regulated after surgery, and may in addition to their antimicrobial properties have an important role in wound healing.

Confocal microscopy of Aspergillus fumigatus keratitis.

AIM: To use a confocal microscope to characterise the treated and untreated courses of fungal keratitis. METHODS: In the first experiment, Aspergillus fumigatus stromal keratitis was produced in both eyes of seven New Zealand white rabbits. In the second experiment, keratitis was induced in right eyes of 20 rabbits. Group 1 rabbits were treated with topical fluconazole, group 2 rabbits received oral fluconazole, and group 3 rabbits were used as controls. The rabbits were examined with a slit lamp and confocal microscope 2, 6, 10, 14, and 20 days after inoculation. The corneal cultures were taken on days 2, 14, and 20 and biopsies were taken on days 2 and 22. RESULTS: On days 14 and 22 confocal microscopy was more sensitive than culture technique in both treated and untreated animals, since not all cases of fungal keratitis can be cultured. CONCLUSION: This study indicates that confocal microscopy is a rapid and sensitive diagnostic tool for both the early diagnosis and non-invasive follow up of fungal keratitis

Dimensional growth of the rabbit eye.

The rabbit eye has been used experimentally in research in vision and ophthalmology. However, there is little information about postnatal growth. The purpose of this study was to determine growth patterns of the rabbit globe from birth to young adult status focusing in particular on the fibrous outer tunic of the globe, the sclera. Eyes of both sexes of New Zealand White rabbits were examined on postnatal days 1, 7, 14, 28, 42 and 56. Axial length, equatorial diameter and corneal diameter were measured using a digital caliper. Plastic-embedded tissue was used to quantify scleral thickness and cellular density at anterior, equatorial and posterior sites. Axial length increased 230% between postnatal days 1 and 56. The rate of growth was not linear and revealed two peaks. The first peak occurred between postnatal days 1-7, and the second between days 14-28. Eye opening occurred on days 9-11. Sex differences were seen, with the male eye being larger (p = 0.00001). Surface area measurements of sclera and cornea revealed that the sclera occupies approximately 5/6 of the outer surface of the globe and the cornea 1/6. Scleral thickness increased from day 1 to day 42 (70-155 microm at the equator) while fibroblast density was found to decrease (0.32 to 0.05 cells/microm(2)). The results suggested that eye opening precedes a unique increase in the rate of eye growth and visual processes may be playing a role in modulating the growth pattern between days 14 and 28.

Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry.

BACKGROUND: The axolemmal distribution and density of voltage-gated sodium channels largely determines the electrical excitability of sprouting neurites. Recent evidence suggests that accumulation of sodium channels at injured axonal tips may be responsible for ectopic axonal hyperexcitability and the resulting abnormal sensory phenomena of pain and paresthesias. For future improvement in pain management it is necessary to identify structurally significant generators of autorhythmicity. A first step in this regard will be to determine the predominant types of sodium channels in injured axons. The opportunity to test human specimens from painful and non-painful neuroma is of great value. METHODS: We employed immunocytochemical methods to investigate if two types of highly specific voltage-gated sodium channel subtypes could be detected in sections of human neuroma. FINDINGS: Both subtypes of sodium channels PN1 and PN3 accumulated abnormally in human neuromas. The immunoreactive pattern was more pronounced in painful neuromas. This is in contrast to previous reports that focused either on PN1 or PN3 as main generators of hyperexcitability induced pain. INTERPRETATION: Both, PN1 and PN3 seem to be involved in hyperexcitability induced pain. It can be expected that a variety of other highly specific voltage gated sodium channel subtypes will be detected in regenerating peripheral nerve in the near future, which contribute to the development of neuropathic pain states. Thus, in order to therapeutically control hyperexcitability induced neuropathic pain, it might be worthwhile to develop pharmaceuticals that can selectively block different sodium channel subtypes and subunits.A review of the role of sodium channels in neuropathic pain is implemented in the discussion.

Differential expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin and hypoxanthine phosphoribosyltransferase (HPRT) in postnatal rabbit sclera.

PURPOSE: GAPDH, beta-actin, HPRT and 18S rRNA are constitutively expressed in all mammalian cells. In accordance with the nature of invariant control, these genes have been used to standardize genes of interest in expression studies. Recent studies have suggested that GAPDH, beta-actin and HPRT in special situations may come under temporary regulatory control, but that 18S rRNA may be more likely to remain constitutive. However, little is known about the quantitative expression of these genes in fibroblasts and in particular during early postnatal development, a time of rapid changes in cell metabolism. In this study we have examined the differential expression of these genes in association with scleral development from an early postnatal age up to young adult status. METHODS: GAPDH, beta-actin, HPRT, and 18S rRNA gene expression were analyzed in the rabbit sclera from 1 day to 8 weeks postnatally by real-time, comparative PCR. RESULTS: Real-time PCR analysis showed that the expression levels of GAPDH, beta-actin, and HPRT were higher in the first postnatal week and then declined. However, from 2 to 8 weeks, the mRNA levels of these three genes underwent significant variations (P < 0.01) in their levels of expression. In contrast, the expression level of 18S rRNA showed no significant variation (P >or= 0.5) over this time period. Conclusions. The present study shows that GAPDH, beta actin and HPRT gene were differentially expressed in early postnatal scleral development. It also suggests that these gene products could be implicated in the developmental process and have a crucial role in the early postnatal period. This study demonstrates that 18S rRNA may be preferable to normalize genes of interest in studies of early development.

Model for avulsion injury in the rat brachial plexus using passive acceleration.

We have developed an experimental model for brachial plexus injuries in the rat that closely simulates the characteristics of human injury. This model produces avulsion injuries in a noninvasive manner. A prototype apparatus was designed that allowed a force to be transmitted to a restrained limb by passive acceleration. Reproducible results were obtained in 32 rats. A significant correlation was found between the test weight and the number of roots avulsed (r = 0.92; P < 0.05). The amount of force also correlated to the pattern of avulsion injury: a 230-g weight produced either C6 (54%), C7 (15%), or C6 and C7 (31%) avulsions; a 330-g weight produced C6 (18%), C7 (9%), or C6 and C7 (73%) avulsions; a 530-g weight produced C5 through C8 (75%) or C6 through T1 (25%) avulsions. This model of brachial plexus injury may be useful to further our understanding of the cellular response to this incapacitating injury and to develop therapeutic strategies with behavioral correlates.

Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys.

OBJECTIVE: To study the effects of lacrimal gland removal on basal and reflex tear production and on the ocular surface in the squirrel monkey. METHODS: Unilateral main lacrimal gland removal in 6 squirrel monkeys was followed by Schirmer testing, slit-lamp examination with fluorescein, and collection of basal and reflex (stimulated) tears for analysis of tear protein spectra between 0 and 20 kd, as well as histological evaluation. RESULTS: Schirmer test results showed an 80% decrease in basal tears and a 90% decrease in reflex tears during week 1, and a 32.2% and 33.3% decrease, respectively, at week 20 after surgery, compared with the contralateral control side. However, no gross abnormalities or fluorescein staining were seen in 5 of the 6 monkeys, and the conjunctival surfaces remained normal. The main and accessory lacrimal glands appeared to secrete similar types of proteins. No histological changes were seen in corneal, conjunctival, or eyelid tissues 20 weeks after surgery. CONCLUSIONS: Tears from accessory lacrimal glands were sufficient to maintain a stable tear layer on the cornea, suggesting that so-called basal tear flow is made up of fluid from both main and accessory lacrimal glands and that decreased tear production by the main lacrimal gland is not a causative factor in keratoconjunctivitis sicca. CLINICAL RELEVANCE: This study shows that total removal of the main lacrimal gland does not in itself lead to keratoconjunctivitis sicca. However, the nature of neural control of the accessory glands is not yet clear.

[Comparative study of tears protein composition in patients after photorefractive keratectomy and laser specialized keratomileusis]. / Sravnitel'noe issledovanie belkovogo sostava slezy patsientov posle fotorefraktsionnoi keratoéktomii i lazernogo spetsializirovannogo keratomileza.

Changes in the protein spectrum of the lacrimal fluid, resultant from refraction ocular surgery (photorefraction keratectomy, PRK, and laser specialized keratomileusis, LASIK) were evaluated. Lacrimal fluid was collected before operation and in various terms after it in 28 patients (48 eyes) subjected to PRK and in 34 patients (59 eyes) subjected to LASIK. Biochemical analysis of the lacrimal fluid was performed on a mass spectrometer. After PRK the greatest changes in the protein spectrum occurred immediately after the operation before reepithelialization. Changes after LASIK were negligible. Refraction laser operations cause changes in the protein composition of tears, LASIK being more physiological than PRK from viewpoint of intactness of the protein composition of tears.

Characterization of immortalized rabbit lacrimal gland epithelial cells.

To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm2 plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 microM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.

Denervation of rabbit lacrimal gland increases levels of transferrin and unidentified tear proteins of 44 and 36 kDa.

PURPOSE: The main lacrimal gland secretes proteins and fluid that make up the aqueous component of the tears. Previous reports indicate that the parasympathetic innervation of the gland influences the secretion of protein from the lacrimal gland. We investigated the effect of lacrimal nerve transection on the levels of individual proteins and overall protein concentration in the tear fluid. METHODS: The main lacrimal gland was unilaterally denervated in adult rabbits at the site of nerve entry to the gland. The contralateral gland (sham-operated) had identical surgical manipulations, excluding nerve transection. Tears were collected daily from both eyes for up to 9 days, after which lacrimal glands were collected. SDS-PAGE, densitometric and image analysis, and Western blot were performed. RESULTS: Consistently measurable tear protein bands ranged from 6 kDa to 85 kDa, using densitometric analysis. Lacrimal gland denervation produced a sustained increase in proteins of 85, 44, and 36 kDa in tears and lacrimal gland tissue from the denervated side, compared with the sham-operated side (0.025 > p > 0.001). The band at 85 kDa was identified as transferrin by Western blot. Tears from the denervated glands also showed transient decreases in low molecular weight tear proteins (18, 12/10, and 6 kDa), as well as a decrease in overall protein concentration, compared with tears from sham-operated glands and non-operated glands (p < 0.001). CONCLUSIONS: These results demonstrate that, in rabbit tears, the quantities of transferrin and two unidentified tear proteins, as well as overall protein concentration, are influenced by the sensory and/or autonomic innervation to the lacrimal gland. The decrease in overall tear protein concentration after lacrimal gland denervation may be related to a loss of nerve-regulated secretagogue-induced protein secretion.

Characterization of epithelial downgrowth by confocal microscopy.

A 43-year-old white woman with a history of multiple ocular surgeries, including 4 penetrating keratoplasties, developed a concentric retrocorneal membrane at the graft periphery in the right eye. A white-light, tandem, scanning confocal microscope using a 24x/0.60 contact objective was used to examine the right eye in vivo. At the endothelial layer, confocal microscopic images similar to corneal epithelial cells were detected at the graft periphery. Unlike normal endothelial cells, the imaged cells demonstrated easily recognizable nuclei.