Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology.

This study demonstrated that growth and differentiation of rabbit conjunctival epithelial cells could be promoted by such substrata as collagen gel, matrigel or various mixtures of collagen and matrigel in a defined culture system. On conventional plastic or glass culture, the conjunctival epithelial cells adopted a monolayer of small epithelioid cells in primary cultures. They became enlarged, squamoid and exhibited notable senescence upon subcultures. On collagen gel, cells formed an organized monolayer sheet with cuboid shape and cell polarity. On matrigel, cells formed globules with stratified appearance including the basal layer of the outer part of globule and the squamoid cells of the central part of globule. The epithelial origin of these cultures was verified by the positive immunostaining of anti-keratin monoclonal antibodies. Expression of mucin antigen was lost on plastic or glass culture, but promoted on collagen gel or matrigel, as demonstrated by staining with periodic acid Schiff's reagent and anti-mucin monoclonal antibody stainings. These results indicate that both collagen gel and matrigel can provide a permissive substrate environment for goblet cell differentiation. Furthermore, this unique phenotypic expression may be possessed only by a selective cell subpopulation. This culture system will allow us to further explore the mechanism by which the goblet cell differentiation is controlled.

The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent.

PURPOSE: To study the effects of collagen matrix and fibroblasts on the growth and development of human bulbar conjunctival epithelial cells. METHOD: Human bulbar conjunctival epithelial cells were cultured on three-dimensional collagen gels containing either normal human conjunctival fibroblasts (HCF), Swiss 3T3 cells, or no cells. After 1 week of culturing, half of the cultures were raised to the air-liquid interface and the rest of the cultures remained submerged. On day 14, cultures were fixed and sectioned for light and electron microscopic studies. RESULTS: Conjunctival epithelial cells cultured on fibroblast-contracted collagen lattice developed into a multicell-layer epithelium with characteristic epithelial structural features including microvilli, desmosomes, early hemidesmosomes, and basement membrane-like structures. Formation of all or some of the above features appeared to be influenced by the type of fibroblasts in the collagen lattices. Structures such as hemidesmosomes and basement membrane were only observed in epithelium developed on 3T3- but not on conjunctival fibroblast-condensed collagen lattices. In contrast, goblet cell differentiation was only observed in epithelia developed on normal HCF-supported collagen matrix. Epithelial cells cultured on acellular collagen gels did not develop into multicell-layer epithelium, and no differentiated characteristics were observed. CONCLUSIONS: These results indicate that the type of fibroblasts dispersed in the collagen matrix plays an important role in the development and differentiation of conjunctival epithelial cells. Normal HCF-dispersed collagen matrix was less growth stimulating to epithelial cells and allowed them to undergo goblet cell differentiation. In contrast, 3T3-dispersed collagen matrix was more growth stimulating, resulting in thicker epithelium with a higher degree of stratification.

Inhibition of vascular endothelial cell morphogenesis in cultures by limbal epithelial cells.

PURPOSE: To study the in vitro angiogenic activity of human conjunctival and limbal epithelial cells and conjunctival, limbal, and corneal fibroblasts in a three-cell-type coculture model. METHODS: Human umbilical vein endothelial cells (EC) were cocultured with epithelial cells, fibroblasts, or epithelial cells and fibroblasts to test their effect on EC morphogenesis. Neutralizing antibodies to some known angiogenic factors were added to the culture to see whether the EC morphogenesis may be blocked by a particular antibody. RESULTS: Conjunctival and limbal epithelial cells exhibited very little or no stimulatory effect on EC tube formation when examined in an EC- epithelial cell coculture system. In contrast, conjunctival, limbal, and corneal fibroblasts all promoted EC morphogenesis when examined under the same culture conditions. Fibroblast-induced EC morphogenesis was inhibited by addition of anti-vascular endothelial growth factor (VEGF) and/or anti-basic fibroblast growth factor (bFGF) antibodies to the culture medium. In the three-cell-type coculture system consisting of ECs, fibroblasts, and epithelial cells, limbal epithelial cells (but not conjunctival epithelial cells) exhibited a strong inhibitory effect on fibroblast-induced EC tube formation. CONCLUSIONS: The proangiogenic activity of ocular surface fibroblasts is probably mediated through a paracrine mechanism by VEGF and bFGF. Limbal epithelial cells, but not conjunctival epithelial cells, inhibit fibroblast-stimulated angiogenesis.

Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium.

Previous studies have shown that using n-heptanol to create a total corneal epithelial defect beyond the limbus results in two different healing patterns with an unpredictable incidence. Between 14-68% of these wounded rabbit corneas (n = 287, combining various reports) showed extensive vascularization and conjunctivalization, whereas the remaining were not vascularized and had conjunctival transdifferentiation with a cornea-like epithelium. To investigate the role of the limbal epithelium in these two healing patterns, the authors treated rabbit eyes for various durations with n-heptanol and additional scraping. Histology showed that treatment for up to 120 seconds removed both the corneal and conjunctival epithelia but left the limbal basal cells intact. To prove viability, they cultured the treated limbal explants on collagen gel. After 14 days of culture, increased stratification of the limbal epithelium and an epithelial outgrowth onto the corneal stroma was observed. The latter was proven to be of corneal origin (positive to AE-5 but negative to AM-3 monoclonal antibody staining). The authors then surgically removed the entire limbal zone including 2 mm of peripheral cornea and 3 mm of adjacent conjunctiva in addition to n-heptanol debridement of the entire corneal epithelium in 54 rabbit eyes and observed a high incidence (96%) of corneal vascularization and conjunctivalization of the resultant epithelial phenotype (positive to AM-3, but negative to AE-5 monoclonal antibody staining). These results support the hypothesis that corneal epithelial stem cells are located in the limbus and indicate that an incomplete removal of the basal limbal epithelium by n-heptanol leads to unvascularized corneas with conjunctival transdifferentiation. Conversely, complete removal of such cells results in corneal vascularization and conjunctivalization.

Suppression of choroidal neovascularization by adeno-associated virus vector expressing angiostatin.

PURPOSE: To test the efficacy of a recombinant adeno-associated virus (rAAV) vector that expresses mouse angiostatin in suppressing experimental choroidal neovascularization (CNV) in a rat model. METHODS: An rAAV vector, rAAV-angiostatin, was constructed to deliver the mouse angiostatin gene. rAAV-angiostatin and a control virus, rAAV-lacZ, were delivered in vivo by subretinal injection in Brown Norway rats, and the delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). For a CNV suppression experiment, CNV was generated by fundus krypton laser photocoagulation 7 days after the viral vector injection and was evaluated by fluorescein angiography (FA) and histology. Apoptosis in retina was analyzed using the TUNEL assay. Inflammation in the retina was investigated by immunohistochemistry, using antibodies that recognize lymphocytes. RESULTS: rAAV-angiostatin injection led to sustained expression of the angiostatin gene in chorioretinal tissue for up to150 days. FA analysis revealed significant reduction of the average sizes of CNV lesions in rAAV-angiostatin-injected eyes when compared with rAAV-lacZ-injected eyes at both 14 (P = 0.019) and 150 (P = 0.010) days after injection. Moreover, histologic analysis of CNV lesions also revealed significantly smaller lesions in rAAV-angiostatin-injected eyes (P = 0.004). As for adverse effects, rAAV-angiostatin injection did not cause inflammation or apoptosis of cells in retina and choroid. CONCLUSIONS: This is the first report that subretinal injection of rAAV-angiostatin can significantly reduce the sizes of CNV lesions. This and the absence of apoptosis and inflammation in chorioretinal tissue indicate the feasibility of a gene therapy approach for treatment of CNV disease.

Laser in situ keratomileusis for myopia of -2 to -25 diopters.

PURPOSE: To evaluate excimer laser in situ keratomileusis in the treatment of low, high, and excessively myopic eyes in Taiwan, 77 patients (104 eyes) were treated with Nidek EC-5000 excimer laser and divided into four groups. METHODS: In situ keratomileusis was performed with a Chiron or SCMD microkeratome. The Nidek EC-5000 excimer laser was used, with multiple passes and a multiple zone ablation technique was performed for myopia higher than -10.00 D. RESULTS: In eyes with a preoperative spherical power equal to or less than -7.00 D (29 eyes of 18 patients), the preoperative cycloplegic mean spherical equivalent refraction was -5.44 +/- 1.36 D. The mean postoperative subjective spherical equivalent refraction was 0.25 +/- 0.64 D at 1 month, 0.056 +/- 0.55 D at 3 months, -0.069 +/- 0.415 D at 6 months, and 0.022 +/- 0.496 D at 9 months. The predictability of postoperative 9 month results demonstrated that 86.21% of eyes were within +/-0.50 D, 96.55% were within +/-1.00 D, and 100% were within +/-2.00 D of emmetropia. In eyes with a preoperative spherical power equal to or less than -10.00 D and higher than -7.00 D, 26 eyes of 17 patients had a preoperative cycloplegic mean spherical equivalent refraction of -8.41 +/- 0.84 D. The mean postoperative subjective spherical equivalent refraction was -0.043 +/- 1.15 D at 1 month, -0.12 +/- 0.92 D at 3 months, -0.21 +/- 1.05 D at 6 months, and -0.37 +/- 1.04 D at 9 months. The predictability of postoperative 9 month results demonstrated that 76.92% of eyes were within +/-0.50 D, 80.77% were within +/-1.00 D, and 96.5% were within +/-2.00 D of emmetropia. In eyes with a preoperative spherical power equal to or less than -15.00 D and more than than -10.00 D, 40 eyes of 27 patients had a preoperative cycloplegic mean spherical equivalent refraction of -12.65 +/- 1.51 D. The mean postoperative subjective spherical equivalent refraction was 0.275 +/- 1.72 D at 1 month, -0.30 +/- 1.34 D at 3 months, -0.47 +/- 1.23 D at 6 months, and -0.62 +/- 1.23 D at 9 months. The predictability of postoperative 9 month results demonstrated that 62.5% of eyes were within +/-0.50 D, 75% were within +/-1.00 D, and 87.5% were within +/-2.00 D of emmetropia. For 19 eyes of 15 patients with a preoperative spherical power greater than -15.00 D, the preoperative cycloplegic mean spherical equivalent refraction was -19.53 +/- 2.61 D. The mean postoperative subjective spherical equivalent refraction was -0.40 +/- 1.22 D at 1 month, -0.69 +/- 1.34 D at 3 months, -0.83 +/- 1.40 D at 6 months, and -0.65 +/- 2.99 D at 9 months. The predictability of postoperative 9 month results demonstrated that 31.5% of eyes were within +/-0.50 D, 52.63% were within +/-1.00 D, and 63.16% were within +/-2.00 D. CONCLUSION: LASIK is a safe and effective technique for the treatment of low, high, and excessive myopia.

Removal of sodium from the solvent reduces retinal pigment epithelium toxicity caused by indocyanine green: implications for macular hole surgery.

BACKGROUND: s/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.

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.

Intrascleral dissemination of infectious scleritis following pterygium excision.

AIMS: To assess the clinical pictures, possible pathogenesis, management, and therapy of patients with infectious scleritis associated with multifocal scleral abscesses following pterygium excision. METHODS: The records of patients with infectious scleritis after pterygium excision who developed multifocal scleral abscesses presenting from 1988 to the end of 1995 were reviewed. Early culture of abscesses was performed, and topical, systemic antimicrobials, or both were given to all patients. Fourteen eyes were operated on in addition to antimicrobial treatment. RESULTS: The initial culture reports of scleral ulcers identified Pseudomonas species in 12 of these 18 patients, Aspergillus in one, Mycobacterium fortuitum in one, and mixed organisms in four. Subsequent abscess cultures were taken from 15 of the infected eyes, and revealed the same organism as the initial culture in 12. Associated complications included four serous retinal detachments, three choroidal detachments, two double detachments, five complicated cataracts, and four recurrences of the initial infection. Four eyes required eventual enucleation and 11 eyes regained useful vision. CONCLUSIONS: With subsequent abscess cultures proving to be the same organism as found in the initial ulcer, the abscess formation appears to represent intrascleral dissemination. Early diagnosis and appropriate, prolonged topical plus systemic antimicrobial treatment are essential to halt the progression of such severe infections.

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 transplantation[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-1/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, chi 2 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.

Human allograft limbal transplantation for corneal surface reconstruction.

Limbal allograft transplantation was performed consecutively in 16 eyes with thermal or chemical burns (n = 5), Terrien's 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.

Fibroblasts isolated from human pterygia exhibit transformed cell characteristics.

Pterygium 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.

Trends and outcomes of treatment for primary rhegmatogenous retinal detachment: a 9-year nationwide population-based study.

PURPOSE: To determine the trends and outcomes for treating primary rhegmatogenous retinal detachment (RRD) in a nationwide population-based study in Taiwan. METHODS: We collected admission data during the period of 1997-2005, from the Taiwan National Health Insurance Research Database, a source that covers over 96% of Taiwan's 23 million citizens. Totally 28 911 patients with a first-time admission diagnosis of RRD (ICD-9-CM codes 361 to 361.07) and undergoing surgical treatment (scleral buckling (SB), pars plana vitrectomy (PPV), or their combination) were identified. The utilized operation type, 180-day readmission rate for recurrent retinal detachment, length of hospital stay, and admission charge were obtained. Contingency table/chi (2) test and t-test were employed for the statistical analysis. RESULTS: Primary PPV (with or without SB) was a primary procedure in 47.3% of cases in 1997. This rate rose significantly to 61.2% in 2005. A significant decrease in the total 180-day readmission rate occurred from 18.95% in 1997 to 13.81% in 2005. These rates also significantly decreased for each surgical modality (from 16.30 to 11.38% for SB, from 21.29 to 14.69% for PPV, and from 22.99 to 16.55% for PPV+SB). The length of hospital stay decreased for each surgical modality between 1997 and 2005. CONCLUSIONS: There was a significant trend towards more frequently employing primary PPV (with or without SB) for the management of primary RRD. In addition, significant improvements in the primary success rates were shown for each surgical modality group and for total samples between 1997 and 2005.

Limbal transplantation for ocular surface reconstruction--a review.

Based on the new concept that the limbal epithelium contains the stem cell population for corneal epithelial cellular proliferation and differentiation, a surgical procedure, limbal transplantation, has been devised. This paper reviews the background information regarding how this procedure was originally developed and the encouraging clinical as well as experimental results of corneal surface reconstruction using this procedure. The clinical aspects are also discussed concerning the surgical indications, procedural details, expected results, and potential problems. We also include the recent results using a rabbit model to compare the efficacy of surface reconstruction between limbal transplantation and original conjunctival transplantation. We hope, through this up-to-date review, that our capacities to manage these and other ocular surface disorders will be enhanced by understanding the new important concept of the limbal stem cell.

Recurrent herpetic keratitis induced by laser iridectomy: case report.

The mechanism for herpetic keratitis reactivation remains unclear. When observed clinically, the reactivation may be associated with a variety of endogenous and exogenous stimuli, such as strong sunlight, fever, menstruation, and psychiatric disturbances. In experimental studies, most methods of inducing recurrence have involved some degree of corneal trauma, inflammation, neuronal stimulation, or damage to the nerves that innervate the cornea. Although corneal damage after laser iridectomy (LI) is well documented, recurrent herpetic keratitis induced by LI has never been reported. Here we present an unusual case of recurrent herpetic keratitis induced by LI. The location of the bullous keratopathy was strongly correlated to the site of laser iridectomy. Clinical findings as well as the dramatic response to antiviral treatment supported the diagnosis. Although the energy for laser iridectomy is relatively safe for most circumstances, the possibility of inducing herpetic keratitis cannot be ignored. Therefore it is important for clinicians to beware of this potential complication.

Comparison of limbal and conjunctival autograft transplantation in corneal surface reconstruction in rabbits.

Destruction of corneal surface was created in one eye of 24 rabbits by n-heptanol corneal epithelial debridement and surgical removal of limbal zone. One month later, the animals were equally subdivided into three groups of eight for limbal transplantation, conjunctival transplantation, and control without transplantation. During a 6-month postoperative follow-up, all corneas in the control group showed progressive vascularization and conjunctivalization. All corneas with limbal transplantation showed progressive decrease of vascularity, verified by fluorescein angiography. In contrast, all but one of the eight corneas of conjunctival transplantation showed progressive vascularization (P = 0.01). More important, the resultant epithelia showed corneal phenotype in limbal transplantation, but remained conjunctival in conjunctival transplantation, as verified by monoclonal antibodies AM-3, APSM-1, and AE-5. These results support the concept of the limbal location of corneal epithelial stem cells, and indicate that complete destruction of the limbal zone resulted in corneal vascularization and conjunctivalization, and that limbal transplantation has a better efficiency than conjunctival transplantation in restoring such destroyed corneal surface.

Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells.

BACKGROUND: 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. METHODS: We took specimens of limbal epithelial cells from the healthy contralateral eyes of six patients with severe unilateral corneal disease. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane, together with the sheet of limbal epithelial cells, was transplanted to the denuded corneal surface of the damaged eye after superficial keratectomy to remove fibrovascular ingrowth. The mean (+/-SD) follow-up period was 15+/-2 months. RESULTS: Complete reepithelialization of the corneal surface occurred within two to four days of transplantation in all six eyes receiving transplants. By one month, the ocular surface was covered with corneal epithelium, and the clarity of the cornea was improved. In five of the six eyes receiving transplants (83 percent), the mean visual acuity improved from 20/112 to 20/45. In one patient with a chemical burn who had total opacification of the cornea, the acuity improved from the ability to count fingers at 40 cm to 20/200. No patient had recurrent neovascularization or inflammation in the transplanted area during the follow-up period. CONCLUSIONS: Transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral deficiency of limbal epithelial cells.

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms.

The contraction of collagen lattices made with foreskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracellular cyclic AMP-raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-beta (TGF-beta). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-beta, but not by aFGF and insulin, when added simultaneously. Thus, TGF-beta may attenuate CT's inhibition on collagen lattice contraction by attenuating CT-induced intracellular cyclic AMP increase, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMP-independent mechanism.

Topical ocular anesthetic abuse: case report.

Topical ocular anesthetic abuse is uncommon in our clinical experience. The complications associated with topical ocular anesthetic abuse included persistent corneal epithelial defect, ring-shaped stromal infiltrate, and anterior segment inflammation. This disorder can masquerade as Acanthamoeba keratitis or other infectious keratitis. We report a suspicious case of infectious keratitis unresponsive to antibiotics. The patient had an irritable manner, low pain-control threshold, and an analgesic drug abuse history. This information, along with finding a topical ocular anesthetic bottle at bedside helped alert us to possibility of drug abuse. After discontinuing use of the topical anesthetic and using lubricants, topical steroid, and a therapeutic soft contact lens, the condition improved.

Endothelial cell loss induced by phacoemulsification occurs through apoptosis.

BACKGROUND: Cornea endothelial cells are nondividing cells containing pumping function which is crucial for cornea clarity and integrity. Endothelial cell loss occurs after cataract surgical procedures such as phacoemulsification. The authors hypothesize that endothelium damage occurs through apoptosis. METHODS: Ultrasound was achieved by placing a phacoemulsification probe in the anterior chamber and delivering 0% or 50% of maximum power for 2.5 min. The corneal tissue was harvested immediately, and at 1 and 7 days after the operation. Corneal tissue was stained by hemotoxylin and eosin (H&E) and evaluated by light microscopy. Endothelium apoptosis was monitored using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay to detect DNA fragmentation. RESULTS: In corneas which underwent phacoemulsification with 0% energy, no cell loss or apoptosis was identified immediately, 1 or 7 days after the operation. Likewise, in corneas exposed to 50% energy, no cell loss or apoptosis was detected immediately after phacoemulsification. However, minimal amount of cell loss but prominent apoptosis was detected with the TUNEL assay 1 day after the operation, whereas significant cell loss but no apoptosis was detected 7 days after the operation by H&E stain. CONCLUSIONS: These results demonstrate that corneal endothelial cell loss induced by ultrasound damage occurs through apoptosis.