Pharmacokinetic features of difluprednate ophthalmic emulsion in rabbits as determined by glucocorticoid receptor-binding bioassay.

PURPOSE: Difluprednate (6α,9-difluoro-11ß,17,21-trihydroxy-1,4-pregnadiene-3,20-dione 21-acetate 17-butyrate, DFBA) has long been used as an anti-inflammatory dermatological agent. The main objectives of the current study were to evaluate the pharmacokinetic and pharmacodynamic features of DFBA when used as an ophthalmic agent, and to compare these features with those of other common ophthalmic agents, to determine which has the highest activity. METHODS: A glucocorticoid (GC) receptor-binding test was performed to evaluate GC receptor-binding activity (GCRBA, the index of pharmacological effect). Using this information, we calculated dose-response curves, IC(50) values, and K(d) values to evaluate each drug's K(i) value. Finally, we performed studies in live rabbits to compare the activity of 4 formulations [0.002%, 0.01%, or 0.05% DFBA, or an ophthalmic solution of 0.1% betamethasone sodium phosphate (BMP)] at 4 time points (0.5, 1, 2, 4 h). At each time point, blood and eye samples were taken so that C(max) (the maximum equivalent concentration of the active DFBA metabolite, DFB), T(max) (the time at which C(max) was measured), and the area under the concentration-time curve could be compared across the 4 formulations. RESULTS: BMP had the highest K(i) value (8.4 × 10(-8) nmol/L), whereas DFB had the lowest (6.1 × 10(-11) nmol/L). The GCRBA of DFBA was intermediate to these 2 values (7.8 × 10(-10) nmol/L). Instillation of the DFBA ophthalmic emulsion in the eyes of rabbits led to dose-dependent increases in GCRBA, which was mostly attributable to the activity of DFB. The 0.05% DFBA ophthalmic emulsion elicited the greatest response in both aqueous humor and iris/ciliary body tissues, though there were no significant dose-dependent differences in GCRBA in plasma samples. CONCLUSIONS: The 0.05% DFBA ophthalmic emulsion appears to be an effective and safe anti-inflammatory treatment in ocular tissues. It is comparable, and possibly even superior, to the 0.1% BMP solution, and may be particularly useful in cases of severe disease where treatment with BMP solution alone is insufficient.

Tight junction-related protein expression and distribution in human corneal epithelium.

PURPOSE: To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium. METHODS: Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium. RESULTS: In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified. CONCLUSION: Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PURPOSE: To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS: Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS: The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS: The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

New surgical treatment for superior limbic keratoconjunctivitis and its association with conjunctivochalasis.

PURPOSE: To introduce a new surgical procedure for treating superior limbic keratoconjunctivitis (SLK) and to suggest the association of SLK with conjunctivochalasis. DESIGN: Interventional case series. METHODS: Six eyes of five patients with long-standing severe ocular irritation unresponsive to treatment with topical steroid and artificial tears were operated on using our new procedure. This technique consists of four steps as follows: (1) Rose bengal (RB) staining is used to localize the abnormal conjunctival area; (2) an arc-like conjunctival incision is placed from the 2 to the 10 o'clock position adjacent and distal to the RB-stained area; (3) the conjunctiva is resected to form a crescent using the arc-like incision as the base; the size of the resection is determined by conjunctival redundancy after removal of the subconjunctival connective tissue; and (4) the crescent conjunctival opening is closed with interrupted sutures. In two eyes, the new surgical procedure was performed together with surgery for inferior bulbar conjunctivochalasis. RESULTS: In all operated eyes, RB staining had disappeared by the end of the second postoperative week; recovery from symptoms and loss of inflammation were recorded by 1 month after treatment. In the case with the longest follow-up (14 months), there was cytologic evidence of goblet cell recovery at 3 months after the operation. In another, there was normalization of the nucleo/cytoplasmic ratio of conjunctival cells without the appearance of goblet cells. CONCLUSIONS: Our treatment very effectively resolved symptoms associated with SLK, even in eyes unresponsive to conventional therapy with eye drops. Considering that we did not address the diseased part of the conjunctiva but rather the adjacent conjunctival redundancy, we propose that superior bulbar conjunctivochalasis is involved in the pathogenesis of SLK.

The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane.

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.

Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease.

PURPOSE: Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction. METHODS: The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM. RESULTS: At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects. CONCLUSIONS: We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.

An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture.

PURPOSE: A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants. In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium. The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium. The new cell-suspension technique was compared with the existing explant method. METHODS: Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM. Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures. The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12). RESULTS: Both cell-suspension and explant culture methods produced a healthy epithelial cell layer. The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells. In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells. Both types of cultivated epithelium showed positive expression of K3 and K12 keratins. In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells. CONCLUSIONS: Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system. The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium. Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation.