5-Fluorouracil toxicity to the ocular surface epithelium

The antimetabolite, 5-fluorouracil (5-FU), has been used to control proliferation of retinal pigment epithelial cells and fibrocytes, and is currently the subject of a multicenter clinical trial of its value in the control of scarring after glaucoma operations. To evaluate possible ocular surface toxicity, the effect of 5-FU on the mitotic rate and differentiation of the ocular surface epithelium in rabbits was measured. 5-FU was instilled into eyes with 10-mm diameter central epithelial wounds and into nonwounded eyes at a dose of 9 mg per day for 4 days. Saline treated control wounded eyes healed within 4 days (n = 5) while 40% (4 of 10) of the 5-FU treated wounded eyes had defects at 4 days. The normal mitotic rate of the corneal epithelium was 1.0 +/- 0.3 (n = 4) tritiated thymidine labeled cells per 100 basal corneal epithelial cells after 2.5 hr incubation. Saline treated control wounded eyes had an increased mitotic rate, 7.1 +/- 1.3 (n = 5) labeled cells per 100 basal corneal epithelial cells after 2.5 hr incubation. Topical 5-FU decreased both of those rates to about 1% of normal. The normal conjunctival epithelial mitotic rate was 1.8 +/- 0.4 (n = 4) labeled cells per 60 basal cells after per 2.5 hr incubation. This rate was the same in wounded eyes, but was decreased in eyes treated with 5-FU. Thus, 5-FU (9 mg/day topically) has serious toxic effects to ocular surface epithelium which must be carefully considered if this drug is to be used clinically.     

Characteristics of the human ocular surface epithelium

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.     

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)     

Impairment of ocular surface epithelium barrier function in patients with atopic dermatitis

AIMS—To assess the integrity of the ocular surface epithelium in patients with atopic dermatitis from the viewpoint of its barrier function.
METHODS—49 patients with atopic dermatitis with blepharoconjunctivitis (ABC group), 27 age matched patients with seasonal or perennial allergic conjunctivitis (AC group), and 20 volunteers with normal healthy eyes (NH group) were assigned to this study. Ocular surface epithelium barrier function was evaluated by the fluorophotometric method using a slit lamp fluorophotometer. 3 µl of 0.5% sodium fluorescein was instilled into the conjunctival sac of the right eye and fluorescein uptake (ng/ml) 30 minutes later (20 minutes after eye washing) was measured in the central cornea and the temporal bulbar conjunctiva. Fluorophoto metric measurements performed were analysed in each group and compared between the groups.
RESULTS—The ABC group showed significantly higher fluorescein uptake (mean 28.2 (SEM 3.3) ng/ml) in the cornea than the AC (11.4 (2.2), p=0.001) and NH groups (9.3 (2.1), p=0.001). There was no significant difference between the AC and NH groups (p=0.930). The ABC group also showed significantly higher fluorescein uptake in the bulbar conjunctiva (393.4 (54.0)) than the AC (182.9 (24.6), p=0.011) and NH groups (169.3 (29.1), p=0.012). There was also no significant difference in fluorescein uptake between the AC group and the NH group (p=0.987).
CONCLUSION—This study suggested that ocular surface epithelium barrier function is impaired in patients with atopic dermatitis with blepharoconjunctivitis.

Keywords: atopic blepharoconjunctivitis; ocular surface epithelium; barrier function; fluorophotometry     

Differential regulation of membrane-associated mucins in the human ocular surface epithelium

PURPOSE: Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. METHODS: A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. RESULTS: MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. CONCLUSIONS: The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.     

连续配戴软性角膜接触镜对眼表上皮的影响

目的:探讨软性角膜接触镜(soft contact lens,SCL)连续配戴对眼表上皮的影响以及可能机制。方法:将SCL配戴者依据连续戴镜时间分为<12h组、12~24h组和>24h组,共3组。对正常对照组(35例70眼)与病例组(99例198眼)进行用角膜荧光素染色(fluores-cein vital staining,FLS)、结膜印迹细胞学检查(conjunctivalimpression cytology,CIC),对比各组间各观察指标差异。结果:SCL戴镜各组FLS记分、杯状细胞密度与结膜上皮鳞状化分级与正常对照组间差异有统计学意义(P<0.01);<12h组与12~24h组之间,<12h组与>24h组之间FLS、杯状细胞密度与结膜上皮鳞状化分级的差异有统计学意义(P<0.05);12~24h组与>24h组之间FLS、杯状细胞密度与结膜上皮鳞状化分级的差异无统计学意义。结论:连续配戴软性角膜接触镜会导致眼表上皮损伤和结膜杯状细胞减少,结膜上皮角化。且持续戴镜时间越长,变化越明显。     

Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders

PURPOSE: To study the short-term clinical results of transplanting of cultivated corneal/limbal epithelial cells on human amniotic membrane (AM) for limbal deficiency. DESIGN: Noncomparative, retrospective interventional case series. PARTICIPANTS: Thirteen eyes of 13 patients with severe limbal deficiency (Stevens-Johnson syndrome in eight eyes, ocular cicatricial pemphigoid in three eyes, and chemical burns in two eyes) were treated at the department of Ophthalmology, Tokyo Dental College, Japan. INTERVENTION: Cultivated allo-limbal epithelium was transplanted onto the ocular surface of patients with severe limbal deficiency. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, changes in visual acuity, and postoperative complications were studied. Histologic examinations were also performed on cultivated epithelium. RESULTS: Cultivated corneal epithelium on AM formed two to three layers with the formation of basement membrane-like structures. After the surgery, the epithelium regenerated and covered the ocular surface in eight eyes (61.5%). However, three of the eight eyes developed partial conjunctival invasion, and two eyes later developed epithelial defects. At last examination, corneal epithelialization was achieved in six eyes (46.2%). Five eyes had conjunctivalization, one eye had dermal epithelialization, and one eye was not epithelialized. Complications were corneal perforation in four eyes and infectious keratitis in two eyes. CONCLUSIONS: This study demonstrates that the success rate for transplanting cultivated allo-limbal epithelium on the AM is not different from the conventional limbal and AM transplantation for the treatment of severe limbal stem cell dysfunction.     

Topical cyclosporine to control ocular surface disease in patients with chronic glaucoma after long-term usage of topical ocular hypotensive medications

Purpose

To evaluate changes in ocular surface and central corneal sub-basal nerve fiber layer (SBNFL) after topical cyclosporin therapy in chronic glaucoma patients on long-term topical antiglaucoma therapy.

Methods

A prospective comparative study of ocular surface evaluation of chronic glaucoma patients on long-term topical therapy treated concurrently with a topical cyclosporine 0.05% twice daily for 6 months and controls was done. The study parameters evaluated at recruitment and at the 6-month follow-up included details of topical antiglaucoma medications, visual acuity, intraocular pressure, ocular surface evaluation parameters (TBUT, Schirmers I, ocular surface staining scores and ocular surface disease (OSD) index score (OSDI)), central corneal sensation (Cochet Bonnett aesthesiometer), and central confocal microscopy to study the SBNFL density (SBNFLD).

Results

Thirty-two eyes of 16 patients with chronic glaucoma and 30 eyes of 15 normal subjects as controls were studied. Mean TBUT, pre/post CsA treatment was 8.67±3.01/12.24±1.83 s (P=0.007). Mean conjunctival/corneal staining scores pre/post CsA treatment were 3.38±1.93/1.50±0.718 (P=0.00) /5.19±1.82/1.81±0.78 (P=0.098), respectively. Mean OSDI pre/post CsA treatment scores were 30.63±14.61/14.76±6.06 (P=0.007). Mean corneal sensations scores pre/post CsA treatment were 4.64±0.46/4.94±0.39 (P=0.002). Central corneal SBNFLD pre and post CsA treatment was 8811.35±2985.29/10335.13±4092.064 μm/mm² (P=0.0001).

Conclusions

Schirmer''s test, ocular surface staining scores, OSDI, corneal sensations, and corneal SBNFLD showed a statistically significant improvement following a 6-month concurrent topical CsA therapy.     

Change of paracellular permeability of ocular surface epithelium by vitamin A deficiency

Dietary vitamin A deficiency in young rabbits caused advanced squamous metaplasia with keratinization of conjunctival epithelium and concomitant reduced paracellular permeability to 3H-mannitol. Both morphologic and permeability changes were reversed with systemic administration of vitamin A. In adult rabbits, vitamin A deficiency caused milder changes of goblet cell loss and increased cellular stratification in conjunction with reduced permeability in the conjunctiva-like epithelium that covers the vascularized cornea after chemical injury with n-heptanol. Topically applied retinoid (tretinoin 0.1%) did not affect the morphology and permeability of the normal corneal or conjunctival epithelium of rabbits that were not vitamin A deficient. These studies showed that altered permeability is associated with the epithelial abnormality during vitamin A deficiency and helped clarify the physiologic function of retinoids in the ocular surface epithelia in the nondeficient state.     

Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model

PURPOSE: To report in vivo reconstruction of the ocular surface using amniotic membrane and heterologous transplants of epithelial limbal cells in rabbits with chemical burns. METHODS: After severe damage to the ocular surface with n-heptanol and keratectomy, 15 rabbits developed total limbal deficiency with conjunctival epithelialization, vascularization, and chronic inflammation. One month later, a complete keratectomy was performed in all eyes: 12 received additional transplantation of human amniotic membrane and heterologous limbal epithelial cells in a double amniotic membrane layer, 2 received amniotic membrane only, and 1 control eye received no procedure. RESULTS: After 1 month of follow-up, corneas in eight of the operated eyes presented minimal vascularization, without signs of rejection. Corneal surface reconstruction was demonstrated with the growth of new corneal-like epithelial phenotype and integration of amniotic membrane to the basal corneal surface. A superficial amniotic membrane (with the amnion side up as a dressing) peeled off after 7 to 10 days. The epithelialization with heterologous limbal epithelial cells was evident underneath. The other four operated eyes were followed for 6 months; the ocular surface was also stable with a corneal-like epithelial phenotype. CONCLUSION: Simultaneous transplantation of amniotic membrane and heterologous limbal epithelial cells in severe ocular surface disorders could restore ocular surface and may be useful in patients with severe bilateral limbal epithelial loss, giving new perspectives for the treatment of severe ocular surface disorders.     

Transdifferentiation of conjunctival epithelium after ocular surface rehabilitation through deep lamellar sclerokeratoplasty

PURPOSE: The quality of the postoperative corneal epithelia of patients with severe ocular surface disorders, whose ocular surface had been reconstructed through deep lamellar sclerokeratoplasty (DLSKP) using allografts, was examined. PATIENTS AND METHODS: Six eyes with ocular surface disorders in 6 patients who had received DLSKP were observed for periods of 2 years or longer (average: 3 years and 10 months). The rehabilitated corneal epithelium cells of some of these patients were analyzed with impression cytology and fluorescence in situ hybridization (FISH) analysis methods. RESULTS: All 3 cases analyzed using the impression cytology method showed normal corneal epithelium cell forms. In the 2 cases analyzed also with the FISH analysis method, in which the hosts and donors were of the opposite gender, the cells were host-derived (99.3% and 98.8%). CONCLUSION: It is considered that rehabilitation of severe ocular surface disorders using allograft epithelial transplantation procedures, including DL-SKP, is eventually concluded by transdifferentiation of the conjunctival epithelium cells derived from the host.     

Topical ciclosporin in the treatment of ocular surface disorders

Mounting evidence suggests that inflammation is the key factor in the pathogenesis of various ocular surface diseases, with a complex interplay of genetic, environmental, and psychosocial factors. Management of these conditions is often challenging. Topical corticosteroids, with their associated side effects, are the mainstay of current treatments for patients with vision threatening disease. Ciclosporin A is an immunomodulator that specifically inhibits T lymphocyte proliferation. Recently, a topical ciclosporin preparation was approved by the US Food and Drug Administration and became available for use in ophthalmology. Given the increasing use of ciclosporin eye drops, the goal of this article is to provide the reader with an overview of the well established uses of ciclosporin and to help refine the questions that should be addressed by future investigations.     

小梁切除术后远期泪膜和眼表上皮改变及其影响因素研究

目的 探讨青光眼患者小梁切除术后远期泪膜和眼表上皮的情况及其影响因素.方法 对103例(139只眼)小梁切除术后6个月以上的患者进行眼表泪膜检查和结膜印迹细胞学检查,分析术后远期薄壁滤过泡的发生率与术中使用丝裂霉素C(MMC)的关系,分析术后远期泪膜和眼表上皮改变与术中使用MMC及术后滤过泡形态的相关关系.结果 小梁切除术后患眼与正常对照组相比,泪膜破裂时间缩短,眼表活体染色增多,结膜上皮鳞状化生改变明显.高浓度MMC组术后薄壁滤过泡的发生率明显高于低浓度MMC组和空白对照组.术后远期眼表上皮杯状细胞密度(GCD)与术中MMC的使用及术后滤过泡的形态呈负相关.结论 小梁切除术后远期患眼存在干眼表现.术中使用高浓度MMC可增加术后远期薄壁滤过泡的发生率.术后远期结膜上皮杯状细胞密度的降低与MMC的使用及隆起于眼表的滤过泡有关.