Ray Jui-Fang Tsai

HUMAN ALLOGRAFT LIMBAL TRANSPLANTATION FOR CORNEAL SURFACE RECONSTRUCTION

Limbal allograft transplantation was performed consecutively in 16 eyes with thermal or chemical burns (n=5), Terriens degeneration (n=2), congenital sclerocornea (n=1), Stevens-Johnson syndrome (n=1), and chronic keratoconjunctivitis (n=7), by transplanting randomly selected cadaver limbocorneal grafts to the recipient eye that had received superficial lamellar keratectomy to remove fibrovascular pannus. Oral cyclosporine A was administered immediately for 2.9 ± 1.3 months. During 18.5 ± 5.4 months of follow-up, the results showed improved visual acuity in 13 eyes (81.3%) and rapid (within 1 week) surface healing in 10 eyes (62.5%). Donor limbal tissue developed engorged and tortuous blood vessels in 12 eyes within 1-2 months, but these regressed within 3 months after surgery. No acute graft failure or allograft rejection could be identified. Twelve eyes (75%) showed total regression of vascularization and four eyes had decreased vascularization. These preliminary results indicate that limbal allograft transplantation may be able to reconstruct a corneal surface that has undergone bilateral diffuse destruction, including the loss of limbal epithelial stem cells.

Amniotic membrane graft for primary pterygium: comparison with conjunctival autograft and topical mitomycin C treatment

AIM To study the efficacy and safety of amniotic membrane graft as an adjunctive therapy after removal of primary pterygium, and to compare the clinical outcome with conjunctival autograft and topical mitomycin C. METHODS 80 eyes of 71 patients with primary pterygia were treated with excision followed by amniotic membrane graft. The result was compared retrospectively with 56 eyes of 50 patients receiving conjunctival autograft, and 54 eyes of 46 patients receiving topical mitomycin C. Patients were followed for at least 6 months, and the averaged follow up periods for the three groups were 13.8, 22.8, and 18.4 months, respectively. RESULTS There were three recurrences (3.8%) in the amniotic membrane graft group, three recurrences (5.4%) in the conjunctival autograft group, and two recurrences (3.7%) in the topical mitomycin C group. There was no significant difference in recurrence rate among the three groups (p = 0.879). No major complications occurred in the amniotic membrane graft group or the conjunctival autograft group. One case of infectious scleritis due to scleral ischaemia occurred in the topical mitomycin C group. CONCLUSION This study showed that amniotic membrane graft was as effective as conjunctival autograft and mitomycin C in preventing pterygium recurrence, and can be considered as a preferred grafting procedure for primary pterygium.

Effect of Stromal Inflammation on the Outcome of Limbal Transplantation for Corneal Surface Reconstruction

Limbal transplantation (LT) is reportedly better than conjunctival transplantation in restoring rabbit corneal surfaces when performed 1–2 months (early stage) after severe damage. The outcome remains unclear if surgery is done at a later stage, and it is also unclear whether lamellar keratectomy should routinely be performed. Using the same rabbit model, LT was done at 3–4 months (intermediate limbal transplantation[ILT], n = 7) or 9–11 months (delayed limbal transplantation[DLT], n = 8) later. Lamellar keratectomy was also conducted with ILT in another group (keratectomy in intermediate limbal trans-plantation[IKLT], n = 7). External eye photography and fluorescein angiography were used to document corneal surface and stromal changes. The resultant epithelial phenotype was studied with AE-5 (cornea specific) and APSM-l/AM-3 (conjunctiva specific) monoclonal antibodies. As in previous studies of early limbal transplantation (ELT, performed at 1–2 months), ILT also had a high (eight of eight) success rate of restored corneal phenotype. In contrast, DLT yielded varying results: three of eight successes for corneal, three of eight for mixed, and two of eight for conjunctival phenotypes (p < 0.01, χ2 trend). IKLT yielded four of seven corneal, two of seven mixed, and one of seven conjunctival phenotype successes. These results indicate that intense stromal inflammation associated with disease chronicity or additional stromal damage by lamellar keratectomy can interfere with the capability of limbal grafts to attain normal corneal epithelial proliferation and differentiation. Future studies of how limbal stem cells are regulated by the stromal environment are crucial to enhancing other clinical applications.

Effects of aging on anterior and posterior corneal astigmatism.

Purpose: To evaluate age-related changes in astigmatism of both corneal surfaces and the whole cornea. Methods: The right eyes of 370 subjects were measured with a rotating Scheimpflug camera (Pentacam). Astigmatisms of the anterior and posterior corneal surfaces were determined. The total corneal astigmatism was derived using power vector summation and vergence tracing. Age-related changes to corneal astigmatism were evaluated using polar value analysis (both in diopter and millimeter). Results: For the anterior and total cornea, the proportion of with-the-rule astigmatisms decreased and those of oblique and against-the-rule astigmatisms increased with age. For the posterior cornea, most eyes displayed against-the-rule astigmatisms in all age groups. There was a significant trend toward against-the-rule astigmatism associated with increasing age for both anterior and total corneal astigmatisms (mean changes of −0.18 and −0.16 diopters/5 years, respectively), and toward with the rule in posterior corneal astigmatism (a mean change of 0.022 diopters/5 years). Regarding shape changes, a “flat meridian toward a more vertical orientation” trend with increasing age for both the anterior and posterior corneal surfaces was observed (mean changes of 0.0295 and 0.0224 mm/5 years, respectively). The posterior corneal surface compensated for the astigmatism arising from the anterior corneal surface in 91.4% and 47.7% of eyes in the 21-30 and ≥71 years groups, respectively. Conclusions: There were age-related shifts toward against-the-rule and with-the-rule astigmatisms for the anterior and posterior corneal surfaces, respectively. The compensating effects of the posterior corneal surface on anterior corneal astigmatism decreased with advancing age.

Fibroblasts isolated from human pterygia exhibit transformed cell characteristics

SummaryPterygium is a degenerative corneal limbal process and UV irradiation has been suggested as being a major environmental predisposing factor. The invasive nature of the fibroblasts associated with pterygia raises the question as to whether these cells are transformed. To test this hypothesis, we established fibroblast strains from autologous and heterologous pterygial and conjunctival specimens, respectively, from subjects between 40 to 50 yr of age, and compared their growth characteristics in culture. All pterygial fibroblast strains exhibited a reduced dependence on serum and exogenous growth factors for growth and reached a saturation population density that was threefold higher than conjunctival fibroblasts cultured under the same conditions. In addition, all pterygial fibroblast strains were able to form colonies in soft agar in 5% fetal bovine serum at a 6.0 to 7.5% efficiency. Under the same experimental conditions, none of the conjunctival fibroblast strains were able to grow. The results presented support the conclusion that pterygial fibroblasts have acquired many of the properties of the transformed phenotype.

Expression of Matrix Metalloproteinases 2 and 9 and Tissue Inhibitors of Metalloproteinase 1 and 2 in Inflammation-Induced Corneal Neovascularization

Purpose: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. Methods: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. Results: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. Conclusion: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.

Expression of Tissue Inhibitor of Metalloproteinase-4 in Normal Human Corneal Cells and Experimental Corneal Neovascularization

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

Conjunctival epithelial cells in culture-growth and globlet cell differentiation

Abstract Conjunctival epithelial cell growth and differentiation were studied by cultivating cells on tissue culture plastic surface and on natural substrata such as collagen gel and matrigel R . Well-differentiated goblet cells were unable to attach in plastic cultures and could only be preserved in collagen gel- or matrigel R - based cultures. Percoll density fractionation experimental suggested that, in the primary conjunctival epithelial cells, there were precursor cells for goblet and non-goblet epithelial cells. The goblet cell phenotypic expression of these precursor cells was influenced by the surface to which they attach and by the serum factors. The PAS and AM-1 positive cells could also be induced when the precursor cells are cultured on collagen gels in serum-free define medium supplemented with retinoic acid. To study how the goblet cell precursors are differentiated and from what stem cells they are derived, it is necessary to develop a culture system with a better mimicry of the in vivo conjunctival tissue. In this regard, wedeveloped an in vitro ‘conjunctival equivalent’, in which the epithelial cells were cultured on fibroblast-contracted collagen lattice to allow continued cross-interactions of the epithelial and mesenchymal cells. This experimental model should allow experimental inquiries that are difficult, if not impossible, in conventional cell cultures.

Ultrasound biomicroscopy in pigmented conjunctival cystic nevi

Purpose To report the use of ultrasound biomicroscopy in the clinical diagnosis and management of pigmented conjunctival cystic nevi. Method Two patients, aged 11 and 18 years, with rapidly growing raised conjunctival melanocytic lesions suspected to be inflamed juvenile conjunctival nevus underwent ultrasound biomicroscopic and histopathologic examinations. Results Ultrasound biomicroscopic examination of the lesions revealed multiple areas of cystic tissue, which is compatible with pathologic finding of compound nevus with epithelial inclusion cysts formation. Furthermore, clear interface was found between the mass and the underlying sclera Conclusion Pigmented conjunctival nevi may obscure cysts under slit-lamp examination. Ultrasound biomicroscopy is a useful diagnostic adjunct to distinguish cysts in conjunctival lesions. Additionally, this technique may be helpful in delineating the extent of lesions prior to excision biopsy.

Management of Infectious Scleritis with Combined Medical and Surgical Interventions

Purpose: Infectious scleritis is one of the most devastating external eye diseases. Previously, with topical medication as the only treatment, the clinical outcome was usually poor, and most cases required enucleation or evisceration. We retrospectively analyzed the outcome of patients with infectious scleritis who received surgical interventions with systemic and topical antibiotics. Patients and methods: Eight patients (M:F = 1:7, mean age = 66.0 ± 12.6 y/o) with culture-proven infectious scleritis were treated with standardized combined systemic antibiotic, surgical debridement (conjunctival recession with cryotherapy) in addition to topical antibiotic treatment. Low dose oral steroid was used in later stage to reduce inflammation. Moreover, amniotic membrane (AM) graft was performed in four of them with persist scleral melting to promote epithelial wound healing. Results: All the eyes were salvaged, and maintained useful vision that was 20/400 or better, and these patients receiving AM grafts showed a more rapid re-epithe- lialization and reduction of inflammation after grafting. Conclusion: Surgical debridement in combination with systemic and topical antibiotics can treat infectious scleritis efficiently. In conditions with extensive stromal melting, inflammation, and impending perforation, AM graft may be considered an adjunctive therapy to promote wound healing once sufficient antibiotic therapy has been administered.