Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

Flap technique-assisted surgeries for advanced retinitis pigmentosa complicated with macular hole: a case report and literature review.

BACKGROUND: Full-thickness macular hole (FTMH) is a rare complication in retinitis pigmentosa (RP) patients and may increase intraoperative challenges. Furthermore, lens capsular flap transplantation and inverted internal limiting membrane (ILM) flap were reported to close complicated FTMH successfully. Here, we present a case of bilateral advanced RP complicated by a FTMH treated with a novel lens capsular flap transplantation and inverted internal limiting membrane flap. CASE PRESENTATION: A 46-year-old presented to our hospital with a complaint of progressively blurred vision and metamorphopsia in both eyes. Spectral-domain optical coherence tomography revealed a FTMH with retinoschisis in the right eye and another FTMH in the left eye. ILM peeling with inverted ILM flap technique was performed on the right eye and ILM peeling with anterior lens capsular flap technique was performed on the left eye. Post-operative follow-up showed successful closure of the FTMH and improved vision in both eyes. CONCLUSIONS: In our present case, flap-assisted techniques for retinitis pigmentosa with macular hole result in excellent visual and anatomic outcomes.

Artificial Intelligence-Assisted Early Detection of Retinitis Pigmentosa - the Most Common Inherited Retinal Degeneration.

The purpose of this study was to detect the presence of retinitis pigmentosa (RP) based on color fundus photographs using a deep learning model. A total of 1670 color fundus photographs from the Taiwan inherited retinal degeneration project and National Taiwan University Hospital were acquired and preprocessed. The fundus photographs were labeled RP or normal and divided into training and validation datasets (n = 1284) and a test dataset (n = 386). Three transfer learning models based on pre-trained Inception V3, Inception Resnet V2, and Xception deep learning architectures, respectively, were developed to classify the presence of RP on fundus images. The model sensitivity, specificity, and area under the receiver operating characteristic (AUROC) curve were compared. The results from the best transfer learning model were compared with the reading results of two general ophthalmologists, one retinal specialist, and one specialist in retina and inherited retinal degenerations. A total of 935 RP and 324 normal images were used to train the models. The test dataset consisted of 193 RP and 193 normal images. Among the three transfer learning models evaluated, the Xception model had the best performance, achieving an AUROC of 96.74%. Gradient-weighted class activation mapping indicated that the contrast between the periphery and the macula on fundus photographs was an important feature in detecting RP. False-positive results were mostly obtained in cases of high myopia with highly tessellated retina, and false-negative results were mostly obtained in cases of unclear media, such as cataract, that led to a decrease in the contrast between the peripheral retina and the macula. Our model demonstrated the highest accuracy of 96.00%, which was comparable with the average results of 81.50%, of the other four ophthalmologists. Moreover, the accuracy was obtained at the same level of sensitivity (95.71%), as compared to an inherited retinal disease specialist. RP is an important disease, but its early and precise diagnosis is challenging. We developed and evaluated a transfer-learning-based model to detect RP from color fundus photographs. The results of this study validate the utility of deep learning in automating the identification of RP from fundus photographs.

A New Perspective on Dry Eye Classification: Proposal by the Asia Dry Eye Society.

The 2017 consensus report of the Asia Dry Eye Society (ADES) on the definition and diagnosis of dry eyes described dry eye disease as "Dry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment, potentially accompanied by ocular surface damage." The report emphasized the instability of tear film and the importance of visual dysfunction in association with dry eyes, highlighting the importance of the evaluation of tear film stability. This report also discussed the concept of tear film-oriented therapy, which stemmed from the definition, and which is centered on provision of insufficient components in each tear film layer and ocular surface epithelium. The current ADES report proposes a simple classification of dry eyes based on the concept of tear film-oriented diagnosis and suggests that there are three types of dry eye: aqueous-deficient, decreased wettability, and increased evaporation. It is suggested that these three types respectively coincide with the problems of each layer: aqueous, membrane-associated mucins, and lipid/secretory mucin. Although each component cannot be quantitatively evaluated with the current technology, a practical diagnosis based on the patterns of fluorescein breakup is recommended. The Asia Dry Eye Society classification report suggests that for a practical use of the definition, diagnostic criteria and classification system should be integrated and be simple to use. The classification system proposed by ADES is a straightforward tool and simple to use, only through use of fluorescein, which is available even to non-dry eye specialists, and which is believed to contribute to an effective diagnosis and treatment of dry eyes.

Human antigen R protein modulates vascular endothelial growth factor expression in human corneal epithelial cells under hypoxia.

PURPOSE: Corneal avascularity is critical for corneal transparency; therefore, a tailored process has been presumed to minimize corneal neovascularization (NV). In most cell types, the transcription of vascular endothelial growth factor (VEGF) is up-regulated, and the stability of VEGF mRNA is sustained by human antigen R (HuR) during hypoxia; however, whether such response applies to corneal epithelial cells is unclear. METHODS: Human corneal epithelial cells (HCECs) and MCF-7 cells that serves as the control were incubated under 0.5% oxygen, and the levels of VEGF and HuR were examined time-dependently. The alteration of HuR was also examined in vivo using the closed-eye contact lens-induced corneal neovascularization rabbit model and immunohistochemistry. Additionally, the expression of HuR was modulated by transfection of plasmids encoding HuR or siRNA targeting HuR to validate the role of HuR in VEGF expression. RESULTS: We found that, unlike in control cells, the level of VEGF was not up-regulated, and the HuR expression was declined in HCECs following hypoxia. The HuR immunostaining intensities were decreased in corneal epithelial cells of rabbits wearing contact lenses. In addition, HuR overexpression restored the ability of HCECs to up-regulate VEGF under hypoxia; however, knockdown of HuR suppressed hypoxia-induced VEGF in control cells but did not further decrease VEGF in HCECs. These findings suggest that HCECs may modulate HuR to suppress hypoxia-mediated up-regulation of VEGF. CONCLUSION: Our study revealed a distinct regulation of VEGF via HuR in HCECs following hypoxia, which likely contributes to minimizing corneal NV and/or maintenance of corneal avascularity.

Therapeutic penetrating keratoplasty for microbial keratitis in Taiwan from 2001 to 2014.

PURPOSE: To study the surgical outcome of therapeutic penetrating keratoplasty (TPK) for medically uncontrolled microbial keratitis at a tertiary hospital in Taiwan over a 14-year period. METHODS: This is a retrospective case series study. Patients who underwent TPK at National Taiwan University Hospital in 2001-2014 were included. Patients were divided into 3 diagnostic groups: bacterial keratitis, fungal keratitis, and acanthamoeba keratitis. Each of the following criteria was evaluated: graft clarity at 1 month and 1 year postoperatively, cure of the disease, and anatomical success rate. RESULTS: A total of 107 TPKs were included. TPK eradicated the infection in 57/62 (91.9%) of bacterial keratitis, 33/41 (80.5%) of fungal keratitis, and 9/10 (90.0%) of acanthamoeba keratitis. 22/57 grafts (38.6%) of bacterial keratitis, 22/38 grafts (57.9%) of fungal keratitis, and 5/10 grafts (50.0%) of acanthamoeba keratitis remained clear at 1 year postoperatively. The 1-year graft survival rate did not significantly differ among these 3 groups. The leading causes of graft failure were late endothelial decompensation and graft reinfection. A higher percentage of graft clarity was achieved in grafts <8.5 mm in diameter than in larger grafts (55.4% vs. 31.4%, P = 0.027). Of the 14 patients with endophthalmitis at the time of TPK, the infection was successfully treated in 13 patients except 1 patient required evisceration. CONCLUSION: TPK is valuable in the management of medically uncontrolled microbial keratitis, although the 1-year graft survival rate is unsatisfactory. TPK combined with intravitreal antibiotics and vitrectomy is also a beneficial treatment strategy for patients with endophthalmitis secondary to microbial keratitis.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period.

PURPOSE: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. METHODS: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. RESULTS: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). CONCLUSION: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

Dermatologic tacrolimus ointment on the eyelids for steroid-refractory vernal keratoconjunctivitis.

PURPOSE: The study aims to evaluate the therapeutic effects of dermatologic tacrolimus ointment on eyelids to treat refractory vernal keratoconjunctivitis (VKC). METHOD: This institutional study examined the effects of steroid treatment for 10 patients diagnosed with severe VKC refractory to steroid treatment. Patients received 0.1% dermatologic topical tacrolimus treatment on their eyelids once or twice daily for concomitant atopic dermatitis. The therapeutic outcomes were evaluated according to change in severity of clinical findings recorded with serial external ocular photography and change in requirement for steroid treatment. RESULTS: Clinical signs and symptoms improved substantially after tacrolimus treatment. Significant reduction in size of papillae, decrease of discharge, improvement in hyperemia, and shield ulcer healing with re-epithelization were observed in all patients. Six out of 10 (60%) patients did not receive steroid treatment. Long-term maintenance of tacrolimus was required to prevent episodic exacerbation. Patients' only treatment-related complaints were of mild burning sensations during medication application to eyelids, and this sensation disappeared a few days after treatment. CONCLUSIONS: Application of 0.1% dermatologic tacrolimus ointment to eyelids is effective and safe in the treatment of refractory VKC in patients with concomitant atopic dermatitis. This treatment may serve as a substitute for or decrease the requirement of steroid treatment.

Clinicopathologic correlation of ocular surface squamous neoplasia from a university hospital in North Taiwan 1994 to 2014.

BACKGROUND/PURPOSE: To describe the clinical and histologic characteristics of ocular surface squamous neoplasia (OSSN) and evaluate factors significant in recurrence at a university hospital in North Taiwan. METHODS: Patient charts, clinical features, and pathology records were retrospectively reviewed in patients with pathology-proved OSSN from January, 1994 to December, 2014. Clinicopathologic correlation was analyzed. RESULTS: Thirty-six patients were recruited. Mean age was 63.4 ± 13.0 (ranging from 23 to 87) years old. OSSN was predominant in men (21/36). Clinical appearances included papilliform in 17 eyes, gelatinous in 11 eyes, leukoplakic in 3 eyes, and 5 eyes in corneal intraepithelial neoplasia (CIN). Of 31 conjunctival OSSN, there were 4 in CIN I, 11 in CIN II, 13 in CIN III, and 3 in squamous cell carcinoma. Superior location was associated with higher-grade OSSN. Although statistical analysis was not significant, papilliform and multifocal lesions showed a trend of high-grade OSSN. The stages of tumor were 4, 5, 26, and 1 eye(s) in T1 to T4, respectively. Recurrence of disease occurred in 9 cases (25%) with mean recurrence time of 20.6 (range: 4 to 65) months. Multifocal lesion has a higher tendency for recurrence. CONCLUSION: Superior location was associated with high-grade OSSN, and papilliform OSSN might have a tendency of severe and invasive lesions. Multifocal lesions might be associated with higher-grade OSSN and higher recurrence rates.

In Vivo Confocal Microscopic Study of Hard Contact Lens-Induced Lipid Keratopathy Secondary to Corneal Neovascularization in a Rabbit Hypercholesterolemic Model.

OBJECTIVES: In vivo confocal microscopy was used to observe the morphological presentations and anatomical correlations between corneal neovascularization (NV) and intracorneal lipid deposition in a rabbit model of contact lens (CL)-induced lipid keratopathy secondary to corneal NV. METHODS: Rabbits were divided into 3 groups: (1) 8-week normal diet, (2) 8-week high-cholesterol diet, and (3) 4-week normal diet followed by 4-week high-cholesterol diet. Corneal NV was induced by closed-eye CL. The formation and maturation of corneal NV were shown by immunohistochemical staining against CD31 and high-molecular-weight melanoma-associated antigen. In vivo confocal microscopy identified corneal NV and lipid deposition. Acquired images for each eye were arranged and mapped into subconfluent montages. RESULTS: In group 1, corneal NV sprouting formed from the peripheral to the central cornea by the end of week 4. Pericytes around vessels were shown after 2 weeks of CL wear. In group 2, lipid deposition started from the peripheral cornea and progressively covered the whole cornea. In group 3, lipid deposition was found first in the central cornea after 2 weeks of high-cholesterol diet and progressed to cover the peripheral cornea. In vivo confocal microscopy demonstrated four different patterns of intracorneal lipid deposition: spindle shapes arranged randomly or in parallel, amorphous shapes, multiangular shapes, and mixed types. Intracorneal lipid deposition was distributed from basal corneal epithelium to deep stroma. CONCLUSIONS: Intracorneal lipids tend to accumulate around newly formed corneal NV but can extend to the area covered with mature NV. In vivo confocal microscopy can demonstrate various shapes and depths of intracorneal lipid deposition.

Mini-Scleral Lenses for Correction of Refractive Errors After Radial Keratotomy.

OBJECTIVE: To evaluate the factors affecting clinical outcomes of correcting cumulative refractive errors or irregular astigmatism after radial keratotomy (RK) using mini-scleral lenses. METHODS: We retrospectively analyzed 36 eyes receiving mini-scleral lenses after RK from July 2011 to June 2016. Analyses included age, refractive errors, best spectacle-corrected visual acuity (BSCVA), best contact lens-corrected visual acuity (BCLCVA), topographic indices, RK treatment zone diameter (TZD), power difference (PrD) within the RK treatment zone, and decentration distance (DD) of the RK treatment zone center. An increase of three lines or more of Snellen visual acuity was considered a successful fitting. RESULTS: Of the eyes fitted with mini-scleral lenses, the BCLCVA (logarithm of the minimum angle of resolution [logMAR] 0.19±0.22) was significantly better than the BSCVA (logMAR 0.47±0.30). Twenty-three patients (64%) had a successful fitting. There was no statistically significant difference between successful and unsuccessful fittings with regard to age, prefitting refractive error, BSCVA, corneal astigmatism, simulated steep/flat K values, surface regularity index, TZD, or PrD. However, successful fittings had better centration of treatment zones with an average DD of 0.54±0.44 mm compared with a DD of 0.92±0.53 mm in the unsuccessfully fitted eyes. In addition, our case series showed that there was a negative impact on fitting outcomes in the presence of small central clear zones resulting from central incisions or scars. CONCLUSIONS: Although decentration of the RK treatment zone and small central clear zones were related to less optimal fitting results, mini-scleral lenses are a feasible option to correct cumulative refractive errors and irregular astigmatism in post-RK patients.

Analysis of SmartPlug Insertion-Related Complications.

PURPOSE: To evaluate the complications and the treatment results of SmartPlug-related complications. METHODS: Retrospective review of all patients from a single medical hospital who received SmartPlug (Medennium, Inc., Irvine, CA) insertions from October 2007 to February 2014. All patients who developed SmartPlug-related canaliculitis and pyogenic granuloma were analyzed. Lacrimal irrigation with antibiotics was performed in most patients. RESULTS: Six hundred thirty-one eyes received SmartPlug insertion and 18 eyes developed SmartPlug-related complications (14 canaliculitis and 4 pyogenic granuloma). The mean time interval from insertion to development of the complications is 3.0 (0.5-6.9) years. Sixteen eyes received lacrimal irrigation with antibiotics, and all the eyes showed improvement without recurrence. The other two eyes had recurrence of granuloma pyogenica only after surgical excision. CONCLUSIONS: SmartPlug-related complications, including canaliculitis and granuloma pyogenica, required long-term follow-up. Most of the complications can be cured by lacrimal irrigation of antibiotics.

Outcomes of pediatric cataract surgery with triamcinolone-assisted vitrectomy.

BACKGROUND/PURPOSE: To evaluate outcomes in pediatric patients undergoing lensectomy, posterior capsulotomy, and triamcinolone-assisted vitrectomy for congenital cataract. METHODS: This retrospective study included 34 patients younger than 72 months who underwent lensectomy, posterior capsulotomy, and triamcinolone-assisted vitrectomy with or without intraocular lens (IOL) implantation for cataract at the National Taiwan University Hospital from July 2006 to December 2012. RESULTS: Fifty-one eyes from 34 patients with cataract (unilateral in 17 patients, bilateral in 17 patients) were included. The mean age at surgery was 26.74 months (range: 2-72 months). The mean postoperative follow-up was 27.8 months (range: 6-72 months). Primary IOL implantation was performed in 25 eyes, 21 of which had the IOL implanted in the capsular bag. Fifty eyes had a central round pupil. The median logarithm of the minimum angle of resolution visual acuity was 0.3 in patients with unilateral cataract and 0.1 in those with bilateral cataract. Three eyes (5.9%) developed visual axis opacification (VAO) and required further surgery. Univariate analysis using Fisher's exact test indicated that surgery in the first 12 months of life was significantly associated with development of VAO (p=0.047). The incidence of postoperative VAO was approximately 15.8% in this age group. CONCLUSION: Triamcinolone-assisted vitrectomy can be used in pediatric cataract surgery without serious long-term adverse effects. While the incidence of VAO is low, it appears unavoidable in approximately one-sixth of patients who undergo surgery before 12 months of age.

Surgical result of pterygium extended removal followed by fibrin glue-assisted amniotic membrane transplantation.

BACKGROUND/PURPOSE: To report the recurrence rate and cosmetic results of conjunctival wound edge and caruncle, and complications after pterygium extended removal followed by fibrin glue-assisted amniotic membrane transplantation. METHODS: A prospective interventional cohort study enrolled 57 (58 eyes) patients undergoing pterygium extended removal followed by fibrin glue-assisted amniotic membrane transplantation. All patients received postoperative follow-up for at least 12 months. Recurrence rate was graded from 1 to 4, and cosmetic results of conjunctival edge and caruncle were graded from 1 to 5. RESULTS: The cohort included 48 eyes with nasal pterygium, 5 eyes with temporal pterygium, and 5 eyes with double pterygium. There were 81.0% (n=47), 0% (n=0), 12% (n=7), and 7% (n=4) of eyes with Grades 1-4 recurrence, respectively. The cosmetic results of conjunctival wound edge and caruncle in cases with nasal pterygium showed 59.3% (n=32), 14.8% (n=8), 9.3% (n=5), 16.6% (n=9), and 0% (n=0) of eyes with Grades 1-5 morphology, respectively. Overall, 5.1% (n=3), 3.4% (n=2), 3.4% (n=2), 3.4% (n=2), 1.7% (n=1), 6.9% (n=4), and 1.7% (n=1) of patients suffered from postoperative pyogenic granuloma, transient diplopia, permanent motility restriction, steroid glaucoma, fat prolapse, subamniotic membrane hemorrhage, and early detachment of amniotic membrane, respectively. CONCLUSION: Pterygium extended removal followed by fibrin glue-assisted amniotic membrane transplantation results in low recurrence, satisfactory cosmetic results and a low incidence of additional complications.

Inhibition of matrix metalloproteinase activity reverses corneal endothelial-mesenchymal transition.

Ex vivo culture or regeneration of corneal endothelial cells often is subjected to gradual endothelial-mesenchymal transition and loss of function. Here, we found that during ex vivo culture, bovine corneal endothelial cells underwent endothelial-mesenchymal transition and had an up-regulated expression and activity of matrix metalloproteinases. Inhibition of matrix metalloproteinase activity in confluent bovine corneal endothelial cells decreased the level of endothelial-mesenchymal transition regulators: snail and slug. The phosphorylation and degradation of the key Wnt signaling pathway modulator active ß-catenin also were accelerated with the broad-spectrum matrix metalloproteinase inhibitor Marimastat, which may result from decreased N-cadherin shedding and increased intact N-cadherin molecules on the cell membrane. Intracameral injection of Marimastat also suppressed basic fibroblast growth factor-induced endothelial-mesenchymal transition in a rat corneal endothelium cryo-injury model and significantly diminished the corneal edema. Our study indicated that inhibition of matrix metalloproteinase activity can reverse endothelial-mesenchymal transition and preserve the function of corneal endothelial cells both during ex vivo culture and in vivo. This may offer a potential therapeutic target in regenerative medicine for the treatment of corneal endothelial dysfunctions.

History and development of ophthalmology in Taiwan.

Western medicine was first introduced to Taiwan by medical missionaries in the mid-19th century. Modernization of medicine was systematically transplanted to Taiwan in the Japanese colonial period, and ophthalmology was established third among hospital departments, following internal medicine and surgery. Dr Hidetaka Yamaguchi, an ophthalmologist, was the first head of the Taihoku Hospital, later known as National Taiwan University Hospital (NTUH; Taipei, Taiwan). Ophthalmologists during the colonial period conducted studies on tropical and infectious eye diseases. After World War II, ophthalmologists at NTUH played an important role in medical education, residency training, studies, and teaching. Dr Yan-Fei Yang established the Taiwan Ophthalmological Society in 1960 and instituted its official journal in 1962. Dr Ho-Ming Lin established the Department of Ophthalmology at the Tri-Service General Hospital in the 1950s and the Veterans General Hospital in the 1960s. Taiwan ophthalmologists eradicated trachoma by 1971. Cataract surgery and penetrating keratoplasty were initially performed in the 1960s. Currently, there are about 1600 ophthalmologists in Taiwan conducting an estimated 120,000 cataract surgeries and 600 corneal transplantations annually. Many subspecialty societies have been established recently that serve to educate Taiwanese ophthalmologists and to connect with international ophthalmic societies. Taiwan ophthalmologists continue to contribute to the advancement of ophthalmic knowledge globally.

Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective Rho kinase inhibitor, Y-27632.

PURPOSE: To optimize isolation of viable bovine corneal endothelial cells (BCECs), we evaluated the effectiveness of various preparation protocols. This entailed comparing the effects of collagenase A and trypsin in the presence and absence of a Rho kinase inhibitor, Y-27632, on proliferation and tight junctional and cytoskeletal integrity during their expansion. METHODS: 5-bromo-2'-deoxyuridine (BrdU) incorporation evaluated cell proliferation. Western blot analysis evaluated F-actin, zonule occludin, and ZO-1 associated nucleic acid binding protein (ZONAB) and RhoA expression. Rho A pulldown assay evaluated Rho A activity. RESULTS: In the trypsin (TrypLE)-prepared BCECs, BrdU incorporation decreased whereas nuclear ZONAB expression increased and became stable from day 3 to 7. In contrast, in the collagenase-A-prepared BCECs, we observed preserved ZO-1 integrity, invariant nuclear ZONAB expression, and dense cortical F-actin expression, and BrdU incorporation was invariant from days 1 to 7. Y-27632 did not increase BrdU incorporation and nuclear ZONAB expression in the TrypLE-prepared and the collagenase-A-prepared BCECs. Moreover, Y-27632 increased irregular cellular morphology and downregulated the expression of ZO-1 in the collagenase-A-prepared BCECs from days 1 to 7. Y-27632 inhibited RhoA activation irrespective of whether the cells were isolated with trypsin or collagenase A. CONCLUSIONS: It is preferable to isolate BCECs with collagenase A and expand them without Y-27632. With this protocol, proliferative activity and tight junctional and cytoskeletal integrity are better preserved than if trypsin is used in the presence or absence of Y-27632.

Genes in dizygote twins with Bowman layer corneal dystrophy.

PURPOSE: To report a de novo R124C mutation of transforming growth factor ß-induced (TGFBI) gene in one of dizygotic twins with corneal dystrophy of the Bowman layer. CASE REPORT: An 11-year-old boy was one of dizygotic twins and had a history of bilateral blurred vision and recurrent corneal erosion. Examination of the visual acuity demonstrated 20/40 in his each eye. Biomicroscopy showed bilaterally central corneal subepithelial opacities and some dot opacities in the superficial stroma. Anterior segment optical coherence tomography demonstrated most hyperreflective opacities at the subepithelial layer with some interspersed opacities in the superficial stroma. Confocal microscopy revealed irregular hyperreflective materials in the Bowman layer and the superficial stroma. Several rough filaments were found in the middle layer of stroma, corresponding to a feature of amyloid lattice. The corneas of his parents and dizygotic sister were clear. Sequencing of the TGFBI gene revealed an R124C mutation in the affected boy but not in his three healthy family members. CONCLUSIONS: A de novo R124C mutation of the TGFBI gene may occur in one of dizygotic twins. Patients with an R124C mutation may have clinical features like corneal dystrophy of the Bowman layer. Confocal microscopy can be used to detect subclinical lattice lines, thereby facilitating differential diagnosis.

In vivo confocal microscopy of bulbar conjunctiva in patients with Graves' ophthalmopathy.

BACKGROUND/PURPOSE: To investigate the bulbar conjunctival changes in patients with Graves' ophthalmopathy (GO) using in vivo confocal microscopy. METHODS: A total of 15 GO patients and 15 control patients were recruited. Images of the superior site and temporal bulbar conjunctivas were taken by the Heidelberg Retina Tomograph/Rostock Corneal Module. The conjunctival thickness and cell density of superior epithelium, basal epithelium, Langerhans cells, and goblet cells were analyzed. Conjunctival impression cytology was performed to assess the grading of squamous metaplasia of the conjunctival epithelium. RESULTS: The superficial epithelial cell density in the superior bulbar conjunctiva of the GO group was 856.93 ± 461.68 cells/mm(2), which was significantly lower than that in the control group (1581.13 ± 556.34 cells/mm(2); p = 0.002). However, the difference in superficial epithelial cell density in the temporal bulbar conjunctiva between the two groups was not statistically significant. No significant differences in conjunctival thickness and the basal epithelial cell density were noted between the two groups. Increased Langerhans cell density and reduced goblet cell density were noted in the GO group. Impression cytology of the conjunctival epithelium revealed significantly higher degree of squamous metaplasia in the GO group. The superficial epithelial cell density in the upper bulbar conjunctiva showed negative correlation with marginal reflex distance in the GO group. CONCLUSION: GO patients suffered from more severe bulbar conjunctival damage and inflammation with the superior site than the temporal site. In vivo confocal microscopy can be a rapid and noninvasive tool for the quantitative evaluation of ocular surface changes in patients with GO.