The cornea: stasis and dynamics

The physiological roles of the cornea are to conduct external light into the eye, focus it, together with the lens, onto the retina, and to provide rigidity to the entire eyeball. Good vision thus requires maintenance of the transparency and proper refractive shape of the cornea. Although the cornea appears to be a relatively static structure, dynamic processes operate within and around the cornea at the tissue, cell, and molecular level. In this article, I review the mechanisms responsible for maintenance of corneal homeostasis as well as the development of new modes of treatment for various corneal diseases. I. The static cornea: structure and physiological functions. The cornea is derived from ectoderm, so that it can be considered as transparent skin. It is devoid of blood vessels and manifests the highest sensitivity in the entire body. The surface of the cornea is covered by tear fluid, which serves both as a lubricant and as a conduit for regulatory molecules. The cornea is also supplied with oxygen and various nutrients by the aqueous humor and a loop vascular system in addition to tear fluid. The cornea interacts with its surrounding tissues directly as well as indirectly through tear fluid or aqueous humor, with such interactions playing an important role in the regulation of corneal structure and functions. The resident cells of the cornea-epithelial cells, fibroblasts (keratocytes), and endothelial cells--also engage in mutual interactions through network systems. These interactions as well as those with infiltrated cells and regulation by nerves contribute to the maintenance of the normal structure and functions of the cornea as well as to the repair of corneal injuries. II. The dynamic cornea: maintenance of structure and functions by network systems. Developments in laser and computer technology have allowed observation of the cells and collagen fibers within the cornea. Furthermore, progress in cell and molecular biology has allowed characterization of dynamic network systems-including cell-cell and cell-extracellular matrix interactions as well as cytokines and neural factors-that contribute to the maintenance of corneal transparency and shape. III. Disruption of network systems: persistent corneal epithelial defects and corneal ulcer. Selection of the appropriate treatment for pathologic lesions of the cornea and the accompanying decrease in visual acuity requires localization of the lesion with regard to the epithelium, stroma, or endothelium of the cornea. In certain instances, however, it is not possible to determine the cause of the problem within the cornea. In such cases, the cause of the pathologic lesion and the target for treatment may lie in the surrounding tissues or environment. For example, corneal epithelial wound healing may be delayed, leading to the development of persistent epithelial defects, as a result of disruption of intercellular junctions between epithelial cells, an abnormality of the corneal basement membrane, altered concentrations of various cytokines in tear fluid, a lowered corneal sensation, or allergic reactions in the lid conjunctiva. Loss of corneal epithelial barrier function can further allow inflammatory cytokines present in tear fluid, together with infiltrated cells, to activate keratocytes and elicit excessive degradation of collagen in the stroma, thereby giving rise to corneal ulcer. IV. Development of new drugs for corneal diseases. We have attempted to apply the results of basic scientific research to the development of new drugs for corneal diseases that remain difficult to treat. The process of authorization for new drugs from the Ministry of Health, Labor, and Welfare takes more than two decades, however. The path from the bench to clinical practice is thus a long one. 1. Development of eyedrops for treatment of persistent corneal epithelial defects. We demonstrated the clinical efficacy of fibronectin eyedrops for the treatment of persistent epithelial defects of the cornea. However, the possibility of blood-borne infections has interfered with the development of serum-derived fibronectin as a drug. An automated machine for the preparation of autologous fibronectin eyedrops has therefore recently been developed. Furthermore, in seeking an alternative to fibronectin eyedrops, we are investigating the effects of a peptide corresponding to the second cell-binding domain of fibronectin on corneal epithelial wound healing. Considering that urokinase-type plasminogen activator may be expressed at the site of corneal epithelial defects and facilitates epithelial migration, the potential clinical application of annexin V, which stimulates the secretion of urokinase-type plasminogen activator for the treatment of persistent corneal epithelial defects is also now under investigation in Japan. 2. Development of eyedrops for treatment of neurotrophic keratopathy. Substance P, a neurotransmitter, stimulates corneal epithelial migration in a synergistic manner with insulin-like growth factor (IGF)--1. We have shown that eyedrops containing both the substance P-derived peptide FGLM-amide and the IGF-1--derived peptide SSSR are effective for the treatment of persistent corneal epithelial defects in individuals with diabetic keratopathy or neurotrophic keratopathy, both of which are associated with a reduction in corneal sensation. 3. Development of drugs for corneal ulcer. Treatment of corneal infection with antibiotics does not necessarily halt the process of corneal ulceration, which is characterized by excessive degradation of stromal collagen, or resolve persistent corneal epithelial defects. In addition to eyedrops for the treatment of persistent corneal epithelial defects, we have therefore also been working on the development of new drugs for the treatment of corneal ulcer. To this end, we have established an experimental system in which corneal fibroblasts are cultured in a three-dimensional collagen gel. With this system, we have shown that triptolide and steroids inhibit collagen degradation by corneal fibroblasts. Triptolide or its derivatives are thus potential drugs for the treatment of corneal ulcer and would work by acting directly on corneal fibroblasts rather than by inhibiting the secreted enzymes(matrix metalloproteinases) responsible for collagen degradation.     

Successful treatment of cystoid macular edema with etanercept in a patient with rheumatoid arthritis associated panuveitis

BACKGROUND: The anti-tumor necrosis factor (TNF)-alpha agent has been shown to be effective for the treatment of various inflammatory diseases, including rheumatoid arthritis and inflammatory disorders of the colon. It also appears to be effective for treatment of macular edema secondary to uveitis. CASE: A 57-year-old woman complained of blurred vision in both eyes. Inflammatory cells in the anterior chamber, vitreous opacity, and cystoid macular edema (CME) were detected in both eyes. Her corrected visual acuity was 0.1 OD and 0.3 OS. Macular thickness measured by optical coherence tomography was 784 and 714 microm for the right and left eyes, respectively. She had active rheumatoid arthritis, and treatment with etanercept was started. The extent of CME decreased gradually after the treatment was begun, with the macular thickness having decreased to 204 and 190 microm in the right and left eyes, respectively, and visual acuity having improved to 0.9 OD and 1.2 OS by 4 months after the beginning of treatment. CONCLUSION: These results suggest that etanercept may be effective for the treatment of CME secondary to uveitis, and they implicate TNF-a in the pathogenesis of uveitic CME.     

A case of bilateral corneal epithelial dysplasia characterized by laser confocal biomicroscopy and cytokeratin immunofluorescence

PURPOSE: To describe a case of bilateral corneal epithelial dysplasia in which each lesion was characterized by both laser confocal biomicroscopy and cytokeratin immunofluorescence. METHODS: A 52-year-old Japanese woman with bilateral corneal epithelial dysplasia was treated by corneal epithelial debridement. We observed the affected area with laser confocal biomicroscopy before and after treatment and examined the immunofluorescence of cytokeratins to examine the characteristics of the abnormal epithelial cells. RESULTS: Laser confocal biomicroscopy revealed the atypical epithelial cells in all layers of the corneal epithelium as well as the reconstituted normal structure of the corneal epithelium after epithelial debridement. Immunofluorescence of cytokeratin 12 (K12) and K4 revealed the presence of four types of cells (those positive for one, both or neither of these cytokeratins) in each lesion. CONCLUSION: Cells expressing both K12 and K4 probably represented dysplastic cells that had invaded the cornea via the limbus and adopted characteristics of corneal epithelial cells. Cells lacking both K12 and K4 were probably either undifferentiated cells or epithelial cells in which cytokeratin expression had not been initiated.     

Metagenomic diagnosis of bacterial infections

To test the ability of high-throughput DNA sequencing to detect bacterial pathogens, we used it on DNA from a patient's feces during and after diarrheal illness. Sequences showing best matches for Campylobacter jejuni were detected only in the illness sample. Various bacteria may be detectable with this metagenomic approach.     

Pulmonary Artery Perforation Repair During Thrombectomy Using Microcoil Embolization

A distal pulmonary artery perforation was successfully occluded by percutaneous microcoil embolization via a microcatheter. Microcoil embolization is a reasonable alternative therapeutic approach for this rare complication of pulmonary interventional procedures.     

Determinants of the quality of life in Alzheimer's disease patients as assessed by the Japanese version of the Quality of Life-Alzheimer's disease scale

BACKGROUND: Although QOL is an important indicator to assess multiple facets of life, the QOL of Alzheimer's disease (AD) subjects with impaired cognitive ability due to dementia has not yet been fully investigated. In this study, we developed the Japanese version of the Quality of Life-Alzheimer's disease (QOL-AD) scale by means of back-translation, and ascertained its reliability and validity for evaluating the quality of life in AD subjects. We also hypothesized that the presence of neuropsychiatric symptoms may determine the characteristics and determinants of both the patients' and the caregivers' responses to the patients' QOL questionnaire. METHODS: We administered the QOL-AD questionnaire to subjects with mild or moderate AD (n = 140). The test-retest reliability was evaluated by the same interviewer after a month's interval. Data from the following tests were also collected to ascertain the validity of the questionnaire: Short Memory Questionnaire (SMQ), Neuropsychiatry Inventory (NPI), Hyogo Activities of Daily Living Scale (HADL) and Mini-Mental State Examination (MMSE). RESULTS: The Japanese version of the QOL-AD questionnaire demonstrated good internal reliability for both the patients' (Cronbach's alpha = 0.84) and the caregivers' responses (Cronbach's alpha = 0.82) and good test-retest reliability for both the patients' (intraclass correlation coefficient = 0.84) and caregivers' reports (intraclass correlation coefficient = 0.91). The concordance between the patients' self-report and the caregivers' observation was moderate (Pearson correlation coefficient = 0.60). The score for the 'mood factor' (apathy, depression/dysphoria) in NPI predicted the overall QOL score as determined from both the patients' and the caregivers' responses for subjects with mild (MMSE>or=21, n = 88) and moderate (MMSE< 21, n = 52) AD. The score for the 'psychosis factor' (delusions, hallucinations, anxiety, agitation, disinhibition, irritability, aberrant motor activity) in NPI predicted the total QOL score as determined by the patients and the caregivers among subjects with moderate AD only. CONCLUSIONS: As hypothesized, the presence of neuropsychiatric symptoms may be an important predictor of both the patients' and caregivers' responses to the patients' QOL questionnaire. QOL-AD appears to be a promising measure of the QOL of subjects with mild to moderate AD in Japan.     

Preparation of thermoresponsive polymer brush surfaces and their interaction with cells

Poly(N-isopropylacrylamide) (PIPAAm) brush surfaces with different layer thickness on polystyrene substrates were prepared by surface-initiated atom transfer radical polymerization (ATRP). Surface characteristics of PIPAAm brushes and their influence on adhesion and detachment of bovine carotid artery endothelial cells (ECs) were controlled by PIPAAm layer thickness. Surface hydrophilicity increased with PIPAAm layer thickness at 37 degrees C because PIPAAm brush surfaces with higher thickness provide more extended chain conformations with relatively high chain mobility, and accompanying polymer chain hydration. These surface property alterations lead to negligible cell adhesion through minimal matrix protein adsorption and also modified surface modulus. By adjusting polymerization reaction conditions and time, polymer layers supporting confluent cultures of ECs were possible. Confluent EC monolayers spontaneously detached as contiguous cell sheets from PIPAAm brush surfaces at reduced temperatures. Thermoresponsive cell adhesion and detachment behavior were analyzed from the standpoint of surface physicochemical characteristics. Thermoresponsive surfaces prepared by surface-initiated ATRP techniques allow surface selection in preparing cell sheets from attachment-dependent cells having relatively strong adhesive property for tissue engineering applications.     

A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization

The proper function of many tissues depends critically on the structural organization of the cells and matrix of which they are comprised. Therefore, in order to engineer functional tissue equivalents that closely mimic the unique properties of native tissues it is necessary to develop strategies for reproducing the complex, highly organized structure of these tissues. To this end, we sought to develop a simple method for generating cell sheets that have defined ECM/cell organization using microtextured, thermoresponsive polystyrene substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges (50 microm wide, 5 microm deep). Vascular smooth muscle cells cultured on these substrates produced intact sheets consisting of cells that exhibited strong alignment in the direction of the micropattern. These sheets could be readily transferred from patterned substrates to non-patterned substrates without the loss of tissue organization. Ultimately, such sheets will be layered to form larger tissues with defined ECM/cell organization that spans multiple length scales.     

Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture dishes

The oral mucosa is an attractive cell source for autologous transplantation in human patients who require regenerative therapies of various epithelia. However, the time-course of cellular changes in transplanted oral mucosal epithelia at ectopic sites remains poorly understood. By applying a rat model, we analyzed phenotypic changes in oral mucosal epithelial cell sheets after harvest from temperature-responsive culture dishes and subsequent autologous subcutaneous transplantation. We used monoclonal antibodies to identify epithelial-specific cytokeratins 4, 10, 13, and 14, the stem/progenitor cell marker p63, and proliferating cell nuclear antigen, within the regenerated tissues. Transplanted oral mucosal epithelial cell sheets proliferated during the first week after grafting in conjunction with host inflammation, but then began to degenerate afterward with complete disappearance after 3 weeks. Our findings suggest that host subcutaneous tissues support proliferation and differentiation of the oral mucosal epithelial cell sheets, but are unable to promote maintenance of stem and progenitor cells and therefore cannot produce long-term survivability.