Ocular cicatricial pemphigoid associated with hyperproliferation of the conjunctival epithelium

We determined the mitotic rate, measured by evaluating uptake of tritiated thymidine autoradiographically, and the frequency of goblet cells in conjunctival epithelial biopsy specimens from nine normal subjects and from 11 patients (seven women and four men ranging in age from 50 to 80 years) with ocular cicatricial pemphigoid. The mitotic rate of patients with the disease was significantly higher than that of normal subjects, 7.2 +/- 2.2 vs 1.6 +/- 0.2 labeled cells per 100 basal epithelial cells (P less than .01). The goblet cell frequency, however, was significantly less in patients than in normal subjects. This suggests that ocular cicatricial pemphigoid is associated with hyperproliferation of the conjunctival epithelium, with a concurrent failure of normal conjunctival differentiation.     

Biocompatibility of nanocomposites used for artificial conjunctiva: in vivo experiments

PURPOSE: To evaluate the biocompatibility of nanocomposites used for artificial conjunctiva. METHODS: Fifty New Zealand white rabbits were used for the experiments. Nanocomposites of poly -caprolactone (PCL) and of PCL coated with polyvinyl alcohol (PCL+PVA), polyvinyl pyrrolidone (PCL+PVP), or chitosan (PCL+C), and amniotic membrane (AM) as a control, were cut into small disks with a diameter of 3.5 mm. The disks were inserted underneath the conjunctiva to measure their inflammation-inducing properties. To investigate epithelial adhesion and goblet cell differentiation, the disks were transplanted after round conjunctival excision. Cultivated conjunctival epithelial cells on nanocomposite were then transplanted onto the abdomen of Balb/c athymic mice. The number of inflammatory cells and the density of goblet cells were measured using hematoxylin and eosin, periodic-acid-Schiff, and immunohistochemistry after 2 weeks. RESULTS: The number of inflammatory cells found inside of the inserts was as follows: 21 +/- 4.9 for controls, 21 +/- 15.1 for PCL, 49.6 +/- 26.0 for PCL+PVP, 40.2 +/- 17.1 for PCL+C, and 13.8 +/- 3.9 for PCL+PVA. In PCL+PVA, the accumulation of inflammatory cells was significantly lower than in the controls (p < 0.01, Mann-Whitney U). The reepithelialization of conjunctival cells was accomplished in more than 75% of all disks except for the PCL+C. In addition, we found the differentiation of goblet cells in the following order from greatest to least: amniotic membrane, PCL, and PCL+PVP. CONCLUSIONS: Nanocomposites of PCL were biocompatible in rabbit conjunctiva, suggesting that PCL may be considered as a candidate for use in the development of artificial conjunctiva.     

Effect of conjunctival structure and inflammatory cell counts on intraocular pressure after trabeculectomy

Purpose: To evaluate the conjunctival structure and inflammatory cell counts and to determine the predictive value of these histological parameters for postoperative intraocular pressure (IOP) levels after trabeculectomy. Methods: A clinical and histological study was performed on consecutive patients. Postoperative (mean 32. 8 +/- 18.4 months; range 6-60 months) conjunctival biopsies of 36 eyes of 28 primary open-angle glaucoma patients who had trabeculectomy between 1992 and 1995 were included in the study. According to postoperative pressure control, patients with < or = 16 mm Hg and those with >16 mm Hg were taken as groups 1 and 2, respectively. The control group (group 3) consisted of 15 age-matched patients without glaucoma, who had received no topical therapy. We compared the conjunctival structure and cell counts within these groups. Goblet cells, acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness, vascular density, mucopolysaccharides and collagen compositions were determined in groups 1, 2 and 3. Results: The number of goblet cells was significantly higher in group 1 (6.74 +/- 7.23) than group 2 (3. 09 +/- 2.77; p = 0.017). No statistical difference was observed in the number of acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness, vascular density, mucopolysaccharide or collagen compositions between groups 1 and 2 (p > 0.05). In addition, when groups 1 and 2 were compared with the control group, there was a significant decrease in goblet cells (p < 0.001), an increase in acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness and vascular density (p < 0.001), but there was no significant difference in mucopolysaccharide and collagen compositions (p > 0.05). Conclusions: This study suggests that at the time of surgery a high number of goblet cells may be a predictor of lowered IOP (< or = 16 mm Hg) following trabeculectomy without antimetabolite. Copyright Copyright 1999 S.Karger AG, Basel     

Isolation and characterization of cultured human conjunctival goblet cells

PURPOSE: To isolate and characterize goblet cells from normal human conjunctival tissue to determine whether epidermal growth factor (EGF) receptors are present and whether EGF can influence goblet cell proliferation. METHODS: Goblet cells were isolated from explant cultures established from normal conjunctival tissue harvested from patients during periocular surgery. The cells were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy, molecular biology, and biochemistry. Proliferation was determined with a MTT proliferation assay after exposing goblet cells, which had been serum deprived for 48 hours, to increasing concentrations of epidermal growth factor (EGF; 0-80 ng/mL) for 24 hours. RESULTS: Goblet cells were isolated from conjunctival explants by scraping nongoblet cells from the culture dish. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff (PAS) reagent, goblet cell-specific cytokeratin-7, HPA lectin, and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-4. The mRNA for MUC5AC was detected using RT-PCR. The presence of the EGF receptors EGFR, ErbB2, and ErbB3 was confirmed through Western blot analysis of cell lysates. EGF elicited a concentration-dependent increase in goblet cell proliferation of 160% +/- 0.5%, 188% +/- 0.45%, 293% +/- 1.3%, and 220% +/- 0.5% of control values with 10, 20, 40, and 80 ng/mL EGF, respectively. CONCLUSIONS: Human goblet cells that retain characteristics of goblet cells in vivo can be cultured. EGF receptors are present in human goblet cells, and EGF stimulates their proliferation.     

The in vitro and in vivo proliferative capacity of serum-free cultivated human conjunctival epithelial cells

PURPOSE: To investigate the in vitro and in vivo proliferative capacity of human conjunctival epithelial cells cultured in serum-free media, and to compare this with current methods that utilize serum-containing media and 3T3 feeder layers. METHODS: Human conjunctival epithelial cells were cultivated in serum-free media alone, serum-free media with a 3T3 feeder layer, and serum-containing media with a 3T3 feeder layer. The areas of outgrowth, colony-forming efficiencies and number of population doublings were compared. The in vivo proliferative potential was assessed by analyzing the number of cells generated by the implantation of cultured cells into athymic mice. Cultured cells were evaluated for the expression of cytokeratins K3, K4, K12, K19, as well as the gel-forming goblet cell mucin, MUC5AC. RESULTS: Cells cultivated in serum-free media, serum-free media and feeder cells, and serum-containing media and feeder cells achieved colony-forming efficiencies of 14.5 +/- 4.1%, 10.1 +/- 3.1%, and 20.4 +/- 6.7%, respectively, and number of population doublings of 24.8 +/- 4.3, 14.8 +/- 3.6, and 30.0 +/- 5.0, respectively. Nine-day old athymic mice conjunctival cysts derived from serum-free cultures comprised 1.29 x 10(6) +/- 0.46 x 10(6) cells, while cysts derived from serum-containing cultures comprised 1.30 x 10(6) +/- 0.53 x 10(6) cells. The degree of epithelial stratification was similar in both conditions. Serum-free cultivated conjunctival cells retained their in vivo characteristics and expressed K4, K19 and MUC5AC. The presence of MUC5AC mRNA in these cells was confirmed by RT-PCR. CONCLUSIONS: Conjunctival epithelial cells propagated in serum-free media demonstrated a similar in vivo proliferative capability, as compared to serum-containing media with 3T3 feeder cells. This has important clinical implications, as the serum-free ex vivo expansion of cells for clinical transplantation overcomes the problems associated with the use of animal serum and cells.     

Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system

PURPOSE: To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells. METHODS: Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin. RESULTS: The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RT-PCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes. CONCLUSIONS: This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.     

Comparison of goblet cell density after femtosecond laser and mechanical microkeratome in LASIK

PURPOSE: To study the effect of the LASIK procedure performed with a femtosecond laser and a manual microkeratome on the conjunctival goblet cell and epithelial cell populations. METHODS: In this prospective, nonrandomized, masked study, 64 eyes undergoing LASIK were included: 30 with the Moria M2 (M2) microkeratome and 34 with the IntraLase femtosecond laser (IL). The preoperative spherical equivalent was -2.0 +/- 3.8 D in the M2 group and -3.1+/- 3.1 D in the IL group. The time that the suction ring was applied on the eye was registered, and goblet cell density (GCD), epithelial cell morphology, and inflammatory cells were evaluated by conjunctival impression cytology, before and after the surgery. RESULTS: All the patients in both groups showed a decrease in GCD after LASIK (P < 0.001) that recovered after 6 months. At 1 week, 1 month, and 3 months, GCD was lower with IL than with M2 (P < 0.019, P < 0.001, and P < 0.024, respectively). The mean period that the suction ring was applied was longer in the IL than in the M2 group (P < 0.001). There was a high correlation between the decrease in GCD and the suction time (R = 0.8), and the preoperative spherical equivalent (R congruent with 0.4). CONCLUSIONS: Impression cytology showed a greater reduction in goblet cell populations after IL than after M2, probably because of the length of time that the suction ring exerted pressure on the conjunctiva. These changes in the goblet cells may contribute to the development of the ocular surface syndrome after LASIK procedures.     

Response of ocular surface epithelium to corneal wounding in retinol-deficient rabbits

The purpose of this study was to explore the nature and time course of the functional effects of early retinol deficiency on the ocular surface epithelium. To this end, the conjunctival epithelial healing rate, mitotic rate, and goblet cell frequency were determined following experimental ocular surface epithelial wounding in 15 rabbits sustained on a retinol-deficient diet and in 12 pair-fed controls. The animals were sacrificed at 2, 7, and 14 days after wound closure. The mean serum retinol level (+/- SEM) prior to wounding was 6.0 +/- 0.9 microgram/dl for the group fed the deficient diet. The mean serum and liver retinol levels for this group following defect closure were 4.0 +/- 0.5 micrograms/dl and 0 micrograms/g, respectively. These values are all significantly less than the analogous respective control values of 83.2 +/- 2.4 micrograms/dl, 77.6 +/- 2.3 micrograms/dl and 35.1 +/- 3.0 micrograms/g (P less than 0.001 for each pair, student t-test). In the retinol-deficient animals, the unwounded eyes had an abnormally high rate of conjunctival epithelial cell mitosis, the earliest ocular surface cellular abnormality detected. Hypermitosis of the unwounded corneal epithelium was also noted, though somewhat later than in the conjunctiva. Epithelial wound healing was delayed considerably in the retinol-deficient group, with only 33% of eyes in this group healed within the same time period as the controls (P less than 0.05, Chi-square). Normal numbers of goblet cells were noted in the conjunctiva of retinol-deficient animals, despite at least 5, and up to 8 weeks of profoundly depleted retinol stores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Density and distribution of canine conjunctival goblet cells

Conjunctival goblet cells (GCs) were quantitated to establish baseline values for density and distribution of these cells in healthy canine eyes. From each of 18 sites, tissue was collected, sectioned at 2 micron, and stained with periodic acid Schiff stain. Within each sampling site, 500 epithelial cells (GCs, squamous, polygonal, and basal epithelial cells) were counted and the ratio of GCs to total epithelial cells was computed as an index of goblet cell density or goblet cell index (GCI). A heterogenous distribution of canine conjunctival goblets cells was demonstrated. Lower nasal fornix (LNf) and adjacent sites, lower middle fornix (LMf) and lower nasal tarsal (LNt), had the highest mean densities of goblet cells. In contrast, GCs were essentially absent from the upper and lower bulbar areas. Remaining sites had intermediate GCIs. Sex differences in GCIs were noted for LNf and LNt sites. Mean tear film breakup times (BUTs) were determined, and, for normal beagle dogs, were 19.38 (+/- 4.80 secs) OS and 19.96 (+/- 5.01 secs) OD. The similarities between canine and human conjunctival goblet cell distributions support the use of the dog for studying the conjunctival mucous system.     

Expansion of conjunctival epithelial progenitor cells on amniotic membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells. Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture. After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase. Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.     

Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion

PURPOSE: To determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctival goblet cells and whether cholinergic agonists and VIP stimulate goblet cell secretion. METHODS: Immunofluorescence studies were performed using antibodies against the m1, m2, and m3 muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva were placed on filter paper and incubated for 15 to 120 minutes, with or without increasing concentrations of the cholinergic agonist carbachol or VIP. The muscarinic antagonist atropine and the muscarinic receptor-subtype-selective antagonists pirenzepine (M1), gallamine (M2), and 4-4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M3) were incubated with carbachol to determine specificity of receptor activation. RESULTS: Immunoreactivity to M2 and M3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M1 receptor was not on goblet cells but was on the stratitfied squamous cells. Immunoreactivity to VIPR2 was found on goblet cells with a localization similar to that of the M2 and M3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol, at 10(-4) M, induced a threefold increase in glycoconjugate secretion, which was completely inhibited by atropine (10(-5) M). Carbachol-induced secretion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by gallamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS: Cholinergic agonists, through M2 and/or M3 muscarinic receptors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, suggesting that goblet cell secretion in vivo is under the control of parasympathetic nerves.     

Tear function and ocular surface findings in premature and term babies

OBJECTIVE: To describe the ocular surface and tear function findings in premature and term babies. DESIGN: Prospective, case-control study. PARTICIPANTS: Forty-eight eyes of 24 premature babies seen at the Department of Ophthalmology of Uludag University School of Medicine, Bursa, Turkey, from March 2002 through September 2002 and 50 eyes of 25 healthy term babies were studied. INTERVENTION: The subjects underwent routine ophthalmic examinations; corneal sensitivity measurements; Schirmer test with anesthesia, with and without nasal stimulation; primary Jones test; fluorescein staining of the ocular surface; and conjunctival impression cytology. MAIN OUTCOME MEASURES: Premature and term babies were compared for corneal sensitivity, lacrimal drainage system patency, tear function and ocular surface staining parameters, goblet cell density, and squamous metaplasia grade. The relation of these parameters to the status of the ocular surface was also investigated. RESULTS: Mean corneal sensitivity scores were 45+/-5.0 mm and 55+/-4.5 mm in the premature and term babies, respectively (P<0.001). Premature babies had a mean corneal fluorescein staining score of 1.5+/-0.25 points, compared with 0.22+/-0.28 points in the term babies (P<0.001). The mean Schirmer test scores without and with stimulation were 1.5+/-2.5 mm and 4.15+/-2.5 mm in the premature babies, respectively, compared with 15+/-3.5 mm and 18.75+/-4.5 mm in the term babies. The intragroup and intergroup Schirmer test scores were statistically significant (P<0.001). The primary Jones test was positive in 20.8% of the eyes in the premature babies, whereas it was positive in 84% of eyes in the term babies. The premature babies with positive primary Jones test results all had corneal epithelial defects or severe superficial punctuate keratopathy. Mean conjunctival impression cytology squamous metaplasia scores were 1.86+/-1.2 in the premature babies and 0.86+/-0.47 in the term babies (P<0.001). Mean goblet cell densities were 393+/-484 cells/mm(2) and 739+/-503 cells/mm(2) in the premature and term babies, respectively (P<0.001). CONCLUSION: Decreased corneal sensitivity, reduced tearing, and lacrimal drainage patency are important determinants of ocular surface disease in premature infants. Premature newborns with low Schirmer test scores and a patent lacrimal system may experience corneal and conjunctival epithelial problems and should be carefully checked for the presence of dry eye complications.     

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus

PURPOSE: To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. METHODS: Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. RESULTS: Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. CONCLUSIONS: This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.     

VEGF-dependent conjunctivalization of the corneal surface

PURPOSE: To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency. METHODS: The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization. RESULTS: Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001). CONCLUSIONS: Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.     

Long-term effects of mitomycin C in pterygium surgery on scleral thickness and the conjunctival epithelium

PURPOSE: To evaluate the long-term effects of intraoperative application of mitomycin C on the scleral thickness and the conjunctival epithelium at the surgical site of pterygium excision. DESIGN: Prospective observational case series. PARTICIPANTS: Twenty-four patients who underwent excision of primary pterygium with intraoperative mitomycin C in our department during the year 1996. METHODS: Patients were evaluated by slit-lamp biomicroscopy, impression cytology, and high-frequency ultrasonography. Impression cytology was performed by applying a small nitrocellulose filter paper for a few seconds at the excision area and for a few seconds at the opposite perilimbal area, and subjecting the specimens to the periodic acid-Schiff-Gill modified Papanicolaou staining protocol. The morphology of the conjunctival epithelium and goblet cell density (GCD) were recorded. High-frequency ultrasound was performed at the same sites, and the scleral thickness was measured at a distance of 1 mm from the limbus. MAIN OUTCOME MEASURES: Goblet cell density, conjunctival epithelial morphology, and the scleral thickness at the operated and nonoperated sites. RESULTS: All patients had successful pterygium removal with no corneal recurrence after a mean follow-up of 77.2+/-3.9 months (range, 72-84). Impression cytology revealed normal nongoblet conjunctival epithelial cells at the excision area, with a 4-fold decrease in the GCD at the excision area when compared with the contralateral nonoperated site (296+/-120 cells/mm(2) and 1183+/-310 cells/mm(2), respectively; P = 0.0036). No differences were noted between the scleral thicknesses at the operated site (750+/-70 microm) and the opposite site (740+/-80 microm) (P = 0.84). CONCLUSIONS: A single application of mitomycin C after pterygium excision is not associated with reduction in scleral thickness more than 6 years postoperatively. The conjunctival epithelium retains its normal phenotype, with a marked reduction of the GCD.     

Upregulation of ICAM-1 expression in the conjunctiva of patients with chronic graft-versus-host disease

PURPOSE: To correlate conjunctival intercellular adhesion molecule 1 (ICAM-1) expression with cytologic and clinical findings of chronic graft-versus-host disease (GVHD). METHODS: Seven patients with chronic GVHD-related keratoconjunctivitis and five age-matched normal controls were recruited for the study. Clinical examination included medical history, visual acuity, evaluation of ocular signs and symptoms (scored from 0 to 3), corneal fluorescein staining (scored from 0 to 5 on the basis of the number of corneal sectors involved), Schirmer test type I, and break-up time (BUT). Impression cytology samples were collected from the nasal and inferior bulbar conjunctiva of patients and controls. Goblet cells were counted in three randomly selected fields and averaged. Immunofluorescent staining for ICAM-1 was carried out and the percentage of cells expressing the marker was evaluated. RESULTS: All patients showed signs and symptoms of keratoconjunctivitis sicca. Schirmer test type I and BUT were reduced (4.8+/-6.7 mm/5 min and 3.9+/-2.7 seconds, respectively). Goblet cells were significantly reduced in GVHD eyes with respect to normal eyes (65+/-30.5 and 192+/-16.9 cells/field respectively; p<0.001). Goblet cell number was directly related to Schirmer test values (p<0.01, rho=0.817) and inversely related to total sign score (p<0.01, rho=-0.939). ICAM-1 expression was increased in GVHD eyes with respect to normal controls, in which no staining was observed. ICAM-1 expression showed an inverse relation to goblet cell number (p<0.01, rho=-0.852) and Schirmer test values (p<0.01, rho=-0.926), and was directly correlated to total sign score (p<0.01, rho=0.982). CONCLUSIONS: Conjunctival ICAM-1 expression is increased in GVHD patients. The severity of the disease is associated with tear parameters, goblet cell decrease, and inflammatory markers, such as ICAM-1.     

Goblet cells of the normal human bulbar conjunctiva and their assessment by impression cytology sampling

Goblet cells of the conjunctiva are the main source of mucus for the ocular surface. The objectives of this review are to consider the goblet cells as assessed by various histological, cytological and electron microscopy methods, and to assess the consistency of published reports (over more than 25 years) of goblet cell density (GCD) from impression cytology specimens from nominally healthy human subjects. Reported GCD values have been notably variable, with a range from 24 to 2226 cells/mm2 for average values. Data analysis suggests that a high density of goblet cells should be expected for the healthy human conjunctiva, with a tendency toward higher values in samples taken from normally covered locations (inferior and superior bulbar conjunctiva) of the open eye (at 973 +/- 789 cells/ mm2) than in samples taken from exposed (interpalpebral) locations (at 427 +/- 376 cells/mm2). No obvious change in GCD was found with respect to age, perhaps because the variability of the data did not allow detection of any age-related decline in GCD. Analyses of published data from 33 other sources indicated a trend for GCD to be lower than normal across a spectrum of ocular surface diseases.     

Conjunctival epithelial wound healing

In vivo conjunctival epithelial healing in albino rabbits was investigated by light microscopy following both n-heptanol and trephined conjunctival wounding. Reepithelialization occurred faster following n-heptanol treatment (3 days) versus trephination (6-7 days). No goblet cells were present in the migrating epithelium during reepithelialization. After 1 day of wounding, goblet cells disappeared several millimeters peripheral to the wound margin in both types of wounds. Goblet cells first reappeared peripherally 1 week after wounding before they appeared in the central wound area. These observations indicate that a large area of conjunctival epithelium surrounding a wound is involved with repair of that wound. Since the goblet cell content of conjunctival epithelium appears to change as a result of the stresses of epithelial repair, the goblet cell population may reflect the presence of reparative or proliferative processes in the ocular surface.     

Protective effect of uridine on cornea in a rabbit dry eye model

PURPOSE: To investigate the effect of uridine on cultured human corneal epithelial cells and keratocytes in vitro and to evaluate whether the application of uridine-containing eye drops could improve ocular surface health in an in vivo dry eye model. METHODS: Uridine was added to cultured epithelial cells (3 x 10(4) cells/well) and keratocytes (1 x 10(4) cells/well) at various concentrations (0.5-50 microM). Cytotoxicity was tested with the use of MTT assay, and the cells were assessed for apoptosis with the use of flow cytometry. Expressions of hyaluronic acid (HA), glycosaminoglycan (GAG), nitric oxide (NO), and matrix metalloproteinase (MMP)-9 were measured. In vivo, the degree of reepithelialization was assessed after topical application of uridine (100 microM) in a rabbit corneal wound model. Changes in tear production and conjunctival goblet cell counts were investigated after instillation of various concentrations of uridine-containing eye drops in a rabbit dry eye model. RESULTS: In vitro, uridine showed no cellular toxicity. It increased the biosynthesis of HA and GAG and reduced MMP-9 levels in cultured corneal epithelial cells and keratocytes. In vivo, uridine enhanced corneal wound healing and significantly increased the number of conjunctival goblet cells in rabbits. CONCLUSIONS: Uridine can restore the health of the ocular surface in a rabbit corneal wound and dry eye model.     

An in vivo confocal masked study on corneal epithelium and subbasal nerves in patients with dry eye

PURPOSE: The objective of the present study was to determine whether dry eye (DE) associated with primary Sj?gren's syndrome (PSDE) and DE not associated with Sj?gren's syndrome (NSDE) are related to an alteration of corneal innervation. METHODS: Eleven healthy volunteers younger than 60 years (normal [N] < 60 group), 10 healthy volunteers 60 years of age or older (N > or = 60 group), 11 patients with PSDE, and 10 patients with NSDE were studied. Epithelial and stromal density and subbasal and stromal nerves were investigated by confocal microscopy. RESULTS: The density of the superficial epithelial cells was 741 +/- 306 cells/mm2 in the PSDE group; 1022 +/- 331 cells/mm2 in the NSDE group; 1523 +/- 294 cells/mm2 in the N > or = 60 group, and 1529 +/- 341 cells/mm2 in the N < 60 group (P < 0.0001, ANOVA). The number of subbasal nerves was 2.8 +/- 1.2 in the PSDE group, 3.3 +/- 0.7 in the NSDE group, 3.1 +/- 0.9 in the N > or = 60 group, and 4.6 +/- 0.8 in the N < 60 group (P < 0.0001, ANOVA). The number of beadlike formations observed in the different groups was 387 +/- 62/mm in the PSDE group, 323 +/- 64/mm in the NSDE group, 182 +/- 63/mm in the N > or = 60 group, and 198 +/- 66/mm in the N < 60 group (P < 0.0001, ANOVA). A correlation was found between the number of subbasal nerves and age (P < 0.01) and between the number of subbasal nerves and Schirmer's test (P < 0.001, Spearman rho). CONCLUSIONS: Patients with DE show alteration in the corneal innervations. The demonstration of such alterations introduces new strategies for treatment of this frequent disease.