Ray Jui-Fang Tsai

Studies on stem cells (SC) show that SC functions are determined by the extracellular microenvironment, known as the “niche”, and by intrinsic genetic programs in the SCs; both are involved in regulating the delicate balance of self-renewal and differentiation. We have identified an animal model of limbal SC (LSC) deficiency and transplantation of SC-containing limbal tissue to treat the LSC deficiency, which could not only replace LSCs by providing new healthy corneal epithelial cells but also restore the lost niche of the limbal stromal layer, causing the regression of vessels and rearrangement of the corneal stromal lamellae. The purpose of the ex-vivo expansion technique is to develop a method that will enable culture of a small number of SCs which could than be expanded in a defined cultured system while preserving the original characteristics and properties of the SCs. In addition, SC characteristics will continue to be maintained when the cultured cells are transplanted back into the host. Bromodeoxyuridine-retaining, ΔNp63, ABCG2, p120, and N-cadherin immunoreactive studies of LSC cultured on an amniotic membrane have been performed. Pathological studies have been conducted for cases with preexisting central corneal stromal opacity treated by transplantation of LSCs followed by penetrating keratoplasty. The results indicate that the amniotic membrane can provide the niche environment for cultured LSCs and maintain the limbal-like environment for the transplanted area of cornea.

To examine the corneal epithelial phenotype in an altered basement membrane. Corneas from 9 patients with symptoms of continuous unstable corneal curvature (CUCC) were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with transactivating and N-terminally truncated pP63 transcript (ΔNp63), cytokeratin 3 (Krt3), ATP-binding cassette sub-family G member 2 (ABCG2), connexin 43 (CX43), p38 mitogen-activated protein kinases (p38MAPK), activating protein 2 (TFAP2), and extracellular signal-regulated kinase (Erk1/2) monoclonal antibodies. Positive immunostaining with ABCG2, p38MAPK, and TFAP2 monoclonal antibodies was observed in the basal epithelial cells of CUCC patients, and CX43 and ΔNp63 were detected in the full-thickness epithelial cells of CUCC patients. Our results indicate that alteration of the corneal basement membrane induces a de-differentiation-like phenotype in corneal basal epithelial cells.

To identify the stem-cell property of the ex vivo expansion of limbal stem cells (LSCs) on amniotic membrane (AM) in culture system and after clinical transplantation. Four key factors have to be performed in the defined culture system: (1) the label-retaining cells have to be identified; (2) the cells can be serially expanded and passaged in vitro; (3) the expanded cells can be labeled by tissue-specific keratin or markers, and (3) their stem cells cannot be labeled by those keratin or markers. The ex vivo-expanded LSCs on AM were positive for p63 and ABCG2 and BrdU label-retaining studies on flat mount preparation. When the ex vivo-expanded LSCs with AM were transplanted into a subcutaneous layer of nude mice, they formed multiple layers of cells. Only the basal layer of cells was positive for p63 and BrdU. The cells over the suprabasal layers were positive for K12/K3. The pathologic studies of corneal specimen of successful LSC transplantation after penetrating keratoplasty demonstrated that P63-positive cells were noticed all over the basal layer of central cornea and AM could be identified at 10 months after LSC transplantation. These results indicate that the AM provided the niche function for cultured LSCs and maintained the limbal-like environment for the transplanted area of cornea. The survival of cases depends on the severity of the disease entity, culture technique, and maintenance of the niche environment for LSCs in the culture and after clinical transplantation.

Research from JAMA Ophthalmology — Reduction of Indocyanine Green–Associated Photosensitizing Toxicityin Retinal Pigment Epithelium by Sodium Elimination

Backgrounds/aims: Staining of internal limiting membrane with indocyanine green (ICG) has been reported to be associated with postoperative atrophic retinal pigment epithelium (RPE) change. Here the authors examined whether removing sodium from the solvent reduces ICG induced RPE cytotoxicity. Methods: Human RPE cells were exposed to ICG (0.25 and 0.025 mg/ml) reconstituted with balanced salt solution (BSS) or Na⁺ free BSS. Light microscopy, trypan blue dye exclusion, acridine orange/ethidium bromide staining, and DNA electrophoresis were used to evaluate the cytotoxic effects of ICG. ICG uptake was measured by optical absorption at 790 nm. Results: Sodium removal reduced the ICG induced changes in cell morphology and improved the RPE cell viability. When RPE cells were incubated for 4 hours in 0.25 mg/ml ICG dissolved in BSS and sodium free BSS, 86.3% (SD 6.7%) and 2.4% (1.1%) of the cells were stained with trypan blue, respectively. After ICG treatment, RPE dies mainly through a necrotic mechanism. ICG uptake by RPE was also reduced with sodium removal. Conclusions: ICG induced cytotoxicity in cultured human RPE was reduced with removal of sodium from the solvent. This reconstitution method may provide a safer intravitreal use of ICG in macular hole surgery.

To derive a unique database of intraocular lens (IOL) power for Taiwanese, an ethnic group with a strikingly high prevalence of myopia. A retrospective series of 3068 cases visiting Chang Gung Memorial Hospital, Linkou for cataract removal and posterior chamber IOL implantation between July 1999 and June 2000 was reviewed. The distribution of IOL powers and a possible age-correspondence was analyzed by one-way analysis of variance (ANOVA) test and multiple regression. Using the SRK/II linear regression formula, with an "A" constant of 118.5, the mean predicted IOL power required for emmetropia was 20.0 +/- 5.1 diopter (D). The mean IOL power for males was 19.8 +/- 5.1 D. The mean IOL power for females was 20.1 +/- 5.1 D. Moreover, ANOVA results documented a statistically significant tendency of age-dependence for IOL power distribution in the 3 groups (male, female, and male and female; F=24.53, p<0.05; F=16.39, p<0.05; F=40.54; p<0.05, respectively). In particular, it statistically significantly differed among decades over 40 years indicating that IOL power increased with age. However, the implanted IOL power decreased with age in patients younger than 40 years old. Multiple regression analysis showed that age, but not gender, was statistically significantly correlated to the IOL power distribution (p<0.05). We provide a unique database of IOL power for cataract surgeries in Taiwan. An age-related correspondence of the database of IOL powers was also documented in this study, which can therefore be regarded further cross-sectional evidence for the age-dependence prevalence of myopia.

RECENTLY, SEVERAL investigators have reported that peeling of the internal limiting membrane (ILM) to eliminate tangential traction force improves the anatomic closure rate and functional outcomes, ^1-4 although other investigators hold a different opinion.^5-7 However, if ILM removal is attempted during macular hole surgery, its visualization may be difficult. The staining of the ILM with indocyanine green (ICG) to enhance its visibility has recently been proposed.^8-12 The procedure involves direct application of ICG to the inner surface of the retina in the macular area. No adverse effects of ICG were reported in these studies. The concentration of ICG used in these reports ranged from 0.6 to 5 mg/mL, and ICG was left in the vitreous cavity from 30 seconds to 5 minutes.

Advanced Coats' disease is a threat to vision. Management of advanced Coats' disease has long been a challenge to ophthalmologists. Some people have attempted to use pars plana vitrectomy and intraocular diathermy on diseased vessels followed by intraocular gas or silicone oil injection. However, internal drainage is technically difficult. We present a case of advanced Coats' disease for which, after an encircling buckle and pars plana vitrectomy, intravitreal injection of perfluorodecaline was used to displace the subretinal fluid to the peripheral subretinal space, and transscleral external drainage was achieved. Finally panretinal laser photocoagulation, cryotherapy and endodiathermy were performed on diseased vessels. Visual improvement and reattachment of the posterior pole were achieved. So we think a high-density vitreous substitute can be a useful adjunct in the management of advanced Coats' disease. It minimizes the disadvantages of the internal or external approaches, while maintaining most of the advantages of both.

The Stevens–Johnson syndrome, ocular pemphigoid, and thermal or chemical burns can cause scarring and opacification of the cornea and loss of vision. Transplantation of epithelial cells from the limbus of the contralateral cornea can restore useful vision. However, this procedure requires a large limbal graft from the healthy eye and is not possible in patients who have bilateral lesions.