I-Jong Wang

Control of cell attachment on pH-responsive chitosan surface by precise adjustment of medium pH.

The purpose of this study is to demonstrate pH-responsive chitosan is able to control cell behavior in response to small changes in environmental pH, which is at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. HeLa cells attached and spread well on chitosan at pH 6.99 and 7.20. When the pH was increased to 7.65, over 90% of cells would rapidly detached from chitosan surface within 1 h. Similarly, fibronectin adsorbed on chitosan at pH 7.20 also rapidly desorbed after increasing the medium pH. Most importantly and interestingly, medium pH adjustment could be facilitated by altering environment pCO(2). It was found over 80% of HeLa cells could be recovered from chitosan surface within 1 h and the viability of detached cells was more than 95% by transferring the culture plate from incubator to atmospheric condition. Additionally, chitosan substrate could effectively control attachment/detachment of various types of cells including cell lines HaCaT, H1299, NIH-3T3, and primary corneal fibroblasts, indicating the technology described here is easily reproducible and should be promising for controlling rapid fibronectin adsorption/desorption and cell attachment/detachment for tissue engineering applications.

Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development

Goblet cell metaplasia and mucus overproduction contribute to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notch signaling regulates cell fate decisions and is crucial in controlling goblet cell differentiation in the gut epithelium. Little is known, however, about how endogenous Notch signaling influences the goblet cell differentiation program that takes place in the postnatal lung. Using a combination of genetic and in vitro approaches here we provide evidence of a novel role for Notch in restricting goblet cell differentiation in the airway epithelium during the postnatal period. Conditional inactivation of the essential Notch pathway component Pofut1 (protein O-fucosyltransferase1) in Tgfb3-Cre-expressing mice resulted in an aberrant postnatal airway phenotype characterized by marked goblet cell metaplasia, decreased Clara cell number and increase in ciliated cells. The presence of the same phenotype in mice in which the Notch transcriptional effector Rbpjk was deleted indicated the involvement of the canonical Notch pathway. Lineage study in vivo suggested that goblet cells originated from a subpopulation of Clara cells largely present in proximal airways in which Notch was disrupted. The phenotype was confirmed by a panel of goblet cell markers, showed no changes in cell proliferation or altered expression of proinflammatory cytokines and was associated with significant downregulation of the bHLH transcriptional repressor Hes5. Luciferase reporter analysis suggested that Notch directly repressed MUC5AC transcription in lung epithelial cells. The data suggested that during postnatal life Notch is required to prevent Clara cells from differentiating into goblet cells.

Comorbidities of dry eye disease: a nationwide population‐based study

Purpose: To investigate the comorbidities of dry eye disease in a nationwide population‐based data in Taiwan.

Overnight orthokeratology-associated microbial keratitis.

Purpose: This study was designed to report the clinical aspects, microbiologic findings, and treatment outcomes of overnight orthokeratology-associated microbial keratitis. Methods: Medical records of patients with overnight orthokeratology-associated microbial keratitis at National Taiwan University Hospital from August 2000 to October 2001were reviewed. The clinical and microbiologic characteristics and treatment outcomes were investigated. Results: Nine patients (in total 10 eyes) from aged 8 to 17 (mean, 12.3 ± 2.9) years were included in this study. Eight patients had a unilateral infection and one had a bilateral infection. The initial best corrected visual acuities ranged from hand motion to 20/20. The lesions were located at the central cornea in nine eyes (90%). Smears and cultures from corneal scrapings were obtained from all patients. Four eyes were culture-positive, which included nonfermentative Gram-negative bacillus, Pseudomonas aeruginosa and Acanthamoeba. Positive smears from another two eyes revealed Gram-negative bacilli and double-walled cyst. All patients were cured using antimicrobial medications with complete re-epithelization and disappearance of corneal infiltrates. Four eyes had a final best corrected visual acuity of 20/30 or worse after a mean follow-up of 9.4 months, including one eye that had visual acuity of hand motion only. Complications included corneal opacity in all eyes, glaucoma in one eye, and cataract in one eye. Conclusions: Overnight orthokeratology is an important risk factor of microbial keratitis, especially in school children. Acanthamoeba and Gram-negative bacilli, especially Pseudomonas aeruginosa, are the most common pathogens in our series. The risk of microbial keratitis after overnight orthokeratology should not be overlooked.

In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi- THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.

Formation of keratocyte spheroids on chitosan-coated surface can maintain keratocyte phenotypes.

Corneal keratocytes have been reported to be able to form spheroids that can preserve their phenotypes after being seeded back onto tissue culture plate in specific culture media. In this study, we found that keratocytes could also form spheroids on a bioengineered material, chitosan-coated surface, with 10% horse serum and Dulbeccos modified Eagles medium. Under scanning electron microscopy observation, the cells in the spheroids were found to adhere each other tightly, and the cellular boundary could not be distinguished. They could return to a dendritic (keratocyte) morphology and proliferate after they were seeded back onto tissue culture plate. Immunocytochemistry was used to characterize these cells. Reverse transcription-polymerase chain reaction revealed that keratocytes in the spheroids were not from the PAX-6-positive progenitor cells. Further, the results of the seeding density and the number of spheroids formation, cell viability (MTT) assays, negative staining of Ki-67, and Live/Dead assay suggested that the spheroids were from cell aggregation instead of cell proliferation. Cells in the spheroids maintained phenotypes and functions characteristic of keratocytes, as seen by reverse transcription-polymerase chain reaction, collagen gel contraction assay, and challenges of keratocytes with transforming growth factor-beta1. Our results showed that corneal keratocytes could form spheroids on a chitosan-coated surface and maintain a keratocyte phenotype. However, such keratocyte spheroids do not proliferate and cannot withstand transforming growth factor-beta from myofibroblast differentiation.

Ethambutol-induced optic neuropathy: a nationwide population-based study from Taiwan

Aim To investigate the risk factors and comorbidities associated with ethambutol-induced optic neuropathy (EON). Method Using the Taiwan Longitudinal Health Insurance Database, we conducted a study within a nationwide representative cohort of patients treated with EMB. We identified 231 patients newly diagnosed with EON between 2000 and 2008, and 924 control subjects. Adjusted OR by estimating the risk of EON in relation to comorbidities and EMB prescription protocol was determined. Results Compared with the control group, EON patients were at risk with older age, hypertension (adjusted OR=1.62, 95% CI 1.16 to 2.26) and renal diseases (without end-stage renal diseases (ESRD), adjusted OR=2.11, 95% CI 1.02 to 4.35; with ESRD, adjusted OR=3.73, 95% CI 1.79 to 7.74). Patients with an EMB prescription duration longer than 3 months were not at elevated risk compared with those whose prescription less than 3 months (OR=1.35, 95% CI 0.99 to 1.83, adjusted for age, sex, hypertension and renal diseases). Patients whose average daily dose was greater than 1200 mg, compared with the other two groups (800∼1199 mg, less than 800 mg) were not at increased risk for EON. Conclusions Age, hypertension and renal diseases are risk factors for EON in the Taiwanese population.

Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats

The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans ∼4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5′-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5′-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

The phenotype of bovine corneal epithelial cells on chitosan membrane

Corneal wound healing is one of the major issues in ocular surface reconstruction and ocular surface diseases. Amniotic membrane (AM) transplantation is an excellent treatment modality to promote corneal wound healing and treat corneal diseases. It is interesting and valuable to search for another synthetic and biocompatible substitute for the study of mechanism of AM and the treatment of ocular surface disorders. Chitosan, the second-most abundant polymer in nature, has many biological advantages such as biocompatibility, biodegradability, hemostatic activity, and wound-healing property to be used as biomedical applications. The purpose of this project is to evaluate the phenotype of cultured corneal epithelial cells in vitro on synthetic chitosan membrane (CM). We cultivated bovine corneal epithelial cells on CM and AM, and then evaluated their phenotypes. The viability of the respective cell cultures was investigated using the 3-[4,5-dimethylrhiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cytotoxicity of CM and AM to corneal epithelial cells was evaluated by lactate dehydrogenase (LDH) assay. The morphology of cultivated corneal epithelial cells on CM and AM was observed by scanning electron microscopy. Additionally, immunocytochemical stainings were used to confirm the phenotype of corneal epithelial cells. In MTT and LDH assays we found that the CM can support the growth of cultured corneal epithelial cells in good condition with minimal toxicity. The SEM and immunohistocytochemistry showed that the phenotype of corneal epithelial cells is compatible with that of AM. We conclude that the CM has the potential to be a suitable biomaterial for treating ocular surface disorders.

Outcomes of penetrating keratoplasty with imported donor corneas.

PURPOSE To analyze factors influencing the surgical success of penetrating keratoplasty and long-term graft survival when using imported donor corneas. METHODS Sixty-three donor corneas imported to Taipei from the Cincinnati Eye Bank from July 1992-June 1993 were used for penetrating keratoplasty. The corneal endothelium was examined using specular microscopy on arrival in Taiwan. The endothelial morphology and endothelial cell density (ECD) were compared with the photograph of the same cornea taken in the United States. The relationships of the surgical success rate with donor age, death to enucleation time, death to surgery time, and ECD were analyzed. The long-term graft survival and ECD of clear grafts were analyzed 4 years after surgery. RESULTS On specular microscopic examination. the imported corneas showed diminished endothelial reflection, blurred cellular borders, and increased dark areas, which were markedly different from the pictures of the corneal endothelium taken in the United States. The average ECD before transportation was 2,525+/-267/mm2 and decreased to 1,934+/-250/mm2 after transportation (p < 0.001), with an average endothelial cell loss of 590+/-247/mm2. The overall surgical success rate was 89% and did not correlate with any of the donor factors tested except death to surgery time. The surgical success rate decreased when the time from death to surgery was >7 days (p = 0.05), mainly because of poor reepithelialization. Four years after surgery, 24 grafts remained clear. The ECD had decreased by 72+/-5% in the clear grafts. CONCLUSIONS Our findings show that endothelial changes in imported donor corneas do occur after transportation, but the surgical success rate may not be influenced significantly if the penetrating keratoplasty is performed within 7 days after donor death. However, the ECD in the clear grafts 4 years after surgery is low.