Identification and Analysis of Primary Cilia in the Corneal Endothelial Cells of Patients with Bullous Keratopathy.

PURPOSE: To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK). METHODS: This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68-90 years) with BK who underwent Descemet's stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin. RESULTS: Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients. CONCLUSION: The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK.

Membrane structures and functional correlates in the bi-segmented eye lens of the cephalopod.

The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.

P2X7 receptor activation may be involved in neuronal loss in the retinal ganglion cell layer after acute elevation of intraocular pressure in rats.

PURPOSE: To investigate whether the P2X7 receptor is involved in retinal ganglion cell (RGC) death after the intraocular pressure (IOP) is elevated in rats. METHODS: After the IOP was elevated to 90 mmHg for 1 h, the rats were subsequently administered oxidized adenosine triphosphate (OxATP) and brilliant blue G (BBG) as P2X7 antagonists. The rats were euthanized 7 days after IOP elevation for histologic evaluation and at 1, 3, and 7 days after IOP elevation to immunostain for the P2X7 receptor and neuron-specific class III ß-tubulin in the retina. Changes in P2X7 receptor expression were measured in total retina extracts using western blot analysis. Quantitative real-time PCR was also performed using the entire retina to determine whether the P2X7 receptor is involved in upregulating tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 at 1, 2, and 3 days after the IOP was elevated. RESULTS: RGC density and the inner plexiform layer thickness significantly decreased 7 days after IOP elevation, but were dose-dependently preserved when treated with OxATP or BBG. P2X7 immunoreactivity in the RGCs increased after IOP elevation, with the peak occurring from day 1 through day 3. Protein levels of P2X7 receptor were significantly increased 1, 2, and 3 days after IOP elevation. The messenger ribonucleic acid expression of the P2X7 receptor, TNF-α, IL-1ß, and IL-6 was significantly upregulated in the retina after IOP elevation, and was suppressed by treatment with OxATP. CONCLUSIONS: These results suggest the expression of the P2X7 receptor is upregulated in the retina after IOP elevation, leading to RGC death. Upregulation of TNF-α, IL-1ß, and IL-6 might be involved in this mechanism of RGC death. Furthermore, P2X7 antagonists may prevent RGC death after IOP elevation.

Ocular toxicity of benzalkonium chloride homologs compared with their mixtures.

This study was performed to assess the in vivo ocular toxicity of benzalkonium chloride (BAK) homologs compared with commercially available BAK (BAK mixture) and to assess the ocular toxicity of BAK homolog after repeated ocular application. Rabbit eyes were examined by ophthalmology and scanning electron microscopy (SEM) after 10 applications of BAK homologs with C12 (C12-BAK) and C14 (C14-BAK) alkyl chain lengths and a BAK mixture at concentrations of 0.001% (w/v), 0.003% (w/v), 0.005% (w/v), 0.01% (w/v) and 0.03% (w/v). The ocular toxicity of C12-BAK to rabbit eyes was examined by ophthalmology and histopathology after repeated ocular application for 39 weeks. In addition, the antimicrobial activities of C12-BAK and C14-BAK against A. niger, S. aureus and P. aeruginosa were assessed. Ocular toxicity of C12-BAK was less than those of the BAK mixture and C14-BAK. No ocular toxicity was noted after ocular application of 0.01% C12-BAK to rabbits for 39 weeks. C12-BAK showed antimicrobial activities at a concentration of 0.003%. These results suggest that the use of C12-BAK to replace BAK mixture as a preservative in ophthalmic solutions should be considered in order to reduce the incidence of the corneal epithelial cell injury induced clinically by BAK.

Temperature effects on the disappearance and reappearance of corneal-endothelium primary cilia.

INTRODUCTION: To elucidate the specific functions of the primary cilia in corneal endothelial cells (CECs) by investigating the histological changes of corneal endothelium exposed at low temperature. STUDY DESIGN: Experimental study. METHODS: This study involved corneas freshly obtained from Japanese white rabbits preserved in Optisol™-GS (Bausch & Lomb) corneal storage medium at 4 °C for 0, 1, and 7 days. Corneas preserved for 7 days were also incubated at 37 °C in culture media for an additional 2 days. A rabbit CEC line was also preserved in Optisol™-GS at 4 °C for 0 and 1 day. The corneal endothelium specimens and CECs were then assessed by immunostaining and scanning electron-microscopy (SEM). RESULTS: Immediately post isolation, the CECs of the specimens showed positive immunostaining for primary cilia (i.e., approximately 20%) via anti-acetylated alpha Tubulin antibody and SEM observation. Primary cilia were found to have attenuated/disappeared on the corneal endothelium specimens preserved for 1 or 7 days at 4 °C. After an additional 2-day incubation at 37 °C, primary cilia reappeared on the corneal endothelium specimens (approximately 20%). The disappearance of cilia during the preservation period was also observed in the immortalized CECs. CONCLUSION: The findings in this study using rabbit corneas indicate that the primary cilia of corneal endothelium preserved at low temperature disappeared, then reappeared after returning to body temperature, suggesting that temperature has a direct effect on the primary cilia of corneal endothelium.

Toxicity of Amphotericin B in Rabbit Corneal Epithelial Cells Stored in Optisol™-GS: Corneal Epithelial Cell Morphology and Migration.

PURPOSE: To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. METHODS: Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6-8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. RESULTS: At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. CONCLUSIONS: The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery.

Tumor-associated calcium signal transducer 2 is required for the proper subcellular localization of claudin 1 and 7: implications in the pathogenesis of gelatinous drop-like corneal dystrophy.

Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

[A case presenting peculiar staining with fluorescein on the unilateral cornea].

BACKGROUND: Corneal staining with fluorescein resulting from a cause distinct from that of general epithelial barrier disturbance. CASE: A 66-year-old woman saw her doctor complaining of blurred vision and foreign-body sensation in her left eye. Superficial punctate keratopathy (SPK) was detected on her left cornea. Following instillation of some eye drops, the patient developed corneal staining with fluorescein. Assuming that the staining was due to drug toxicity, eye-drop instillation was discontinued but the staining did not improve and her doctor then referred her to us. Although we tried glucocorticosteroid and artificial tear eye drops, the staining spread until it covered the patient's cornea almost completely. On a following day, we attempted to observe the patient's cornea by confocal microscopy, yet her corneal epithelium could only partially be seen due to what can best be described as a "black-cloud-like" formation on her cornea. However, immediately following that observation the corneal staining diminished without epithelial disturbance and the patient's symptoms were improved. Suspecting that some foreign materials may be stuck to her cornea, we performed impression cytology and detected the existence of MUC16. CONCLUSION: The patient's corneal staining may have resulted from her cornea being covered by materials related to MUC16.

A thermoreversible hydrogel as a biosynthetic bandage for corneal wound repair.

Ocular trauma and disorders that lead to corneal blindness account for over 2 million new cases of monocular blindness every year. A popular ocular surface reconstruction therapy, amniotic membrane transplantation, has been shown to aid corneal wound repair. However, the success rates of the procedure are variable. Here, we proposed to bioengineer a novel synthetic material that would serve as a biomimetic corneal bandage. The PLGA-PEG-PLGA triblock copolymer was synthesised via ring-opening polymerisation. Thermoreversible gelation behaviour was investigated at different polymer concentrations (23%, 30%, 35%, 40%, 45%, w/v) at temperatures ranging between 5 and 60 degrees C. Viscoelastic properties were studied in dynamic mechanical analysis with 1 degrees C/min temperature ramp. Cryo-SEM revealed a porous hydrogel with interconnecting networks. No adverse cytotoxicity was observed with an in vitro scratch-wound assay and in in vivo biocompatibility tests. We have demonstrated that the PLGA-PEG-PLGA hydrogel possessed a suitable gelling profile and, for the first time, the biocompatibility properties for this application as a potential bandage for corneal wound repair.

Diagnosis of multiple myeloma in a patient with atypical corneal findings.

PURPOSE: To report the case of a 67-year-old woman with a diagnosis of multiple myeloma (MM) and atypical corneal opacity as the first appearance. METHODS: The patient consulted an ophthalmologist regarding blurred vision that progressed gradually without any systemic symptoms. The corneal epithelium and anterior stroma of both eyes harbored diffuse gray-white deposits of unknown etiology. She underwent a general checkup that included molecular genetic analysis. At cataract surgery, performed 5 months after the diagnosis, the corneal epithelium was removed with an epikeratome. The epithelial sheet was analyzed histologically. RESULTS: Her serum immunoglobulin G (IgG) level was increased; serum immunoelectrophoresis showed a monoclonal gamma-globulin spike of the IgG kappa-type. The presence of 30.4% pleomorphic plasma cells in the bone marrow confirmed the diagnosis of MM. Her visual acuity improved after combined cataract surgery and superficial keratectomy. The surgically removed epithelial sheet showed IgG deposits in the corneal epithelium; electron-dense intracellular deposits were observed in corneal epithelial cells. CONCLUSIONS: In some individuals, unusual corneal deposits may constitute the first sign of MM. Superficial keratectomy with an epikeratome is a minimally invasive treatment of MM with corneal opacification.

TNF-alpha is crucial for the development of autoimmune arthritis in IL-1 receptor antagonist-deficient mice.

IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice spontaneously develop autoimmune arthritis. We demonstrate here that T cells are required for the induction of arthritis; T cell-deficient IL-1Ra(-/-) mice did not develop arthritis, and transfer of IL-1Ra(-/-) T cells induced arthritis in nu/nu mice. Development of arthritis was also markedly suppressed by TNF-alpha deficiency. We found that TNF-alpha induced OX40 expression on T cells and blocking the interaction between either CD40 and its ligand or OX40 and its ligand suppressed development of arthritis. These findings suggest that IL-1 receptor antagonist deficiency in T cells disrupts homeostasis of the immune system and that TNF-alpha plays an important role in activating T cells through induction of OX40.

Cultivated corneal endothelial cell sheet transplantation in a primate model.

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

Assessment of epithelial integrity and cell viability in epithelial flaps prepared with the epi-LASIK procedure.

PURPOSE: To assess the histological integrity and cell viability in epithelial flaps prepared with epikeratomes. SETTING: Department of Ophthalmology, Kyoto Prefectural University of Medicine and The Baptist Eye Clinic, Kyoto, Japan. METHODS: Epithelial flaps were prepared by epi-LASIK surgery. After immediate fixation, they were examined by light and electron microscopy. To assess cell viability in fresh epithelial flaps, biostaining experiments were performed using propidium iodide (PI), calcein-AM, and Hoechst 33342 dye. In addition, some epithelial flaps were organ-cultured for 24 hours. RESULTS: Light and electron microscopy showed that most of the inspected areas showed nuclei and cytoplasm at significantly reduced density and discontinuity of the basement membrane. Biostaining experiments showed that approximately 90% of the basal cells in epithelial flaps were PI-positive dead cells; organ cultures showed detachment of basal cells from the epithelial flap after 24 hours of incubation. CONCLUSION: Most basal cells in epithelial flaps prepared with different epikeratome devices were dead.

The existence of dead cells in donor corneal endothelium preserved with storage media.

AIM: To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination. METHODS: Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue. RESULTS: Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%-10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells. CONCLUSION: Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.

Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium.

PURPOSE: The ocular surface is covered by two biologically distinct epithelia: corneal and conjunctival. The expression of keratin12 (K12) is currently considered a hallmark of cornea-type differentiation. In the current study, the biological features of K12-positive cells in human bulbar conjunctival epithelium were examined. METHODS: Human conjunctival tissues were subjected to investigate the K12-positive cells in conjunctiva by immunostaining, in situ hybridization, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was performed with introduced amplified-fragment length polymorphism (iAFLP). To determine the presence of stem- or progenitor cells, immunostaining and colony-forming assays were performed. RESULTS: Western blot analysis, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12-positive cells reside mainly in clusters in conjunctival epithelium. FACS analysis showed that 0.2% to 1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were K12 positive. iAFLP analysis revealed that the gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the K12-positive cells. Some colony-forming cells expressed K12. CONCLUSIONS: The K12-positive cells appear to be ectopically residing, self-maintaining corneal epithelial cells in the conjunctival epithelium.

Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation.

PURPOSE: The corneal epithelium is essential for maintaining corneal transparency, and efforts have been made to develop improved techniques for corneal epithelial transplantation in patients with total limbal failure. We evaluated the suitability of transplanted cultivated human conjunctival epithelium (HCjE) as a corneal epithelium replacement in rabbits with total corneal and limbal deficiency. METHODS: HCjE cells, cultivated on human amniotic membrane (AM) to confluence and exposed to an air-liquid interface (air-lifted), were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The cultivated HCjE sheet and the engrafted epithelium were analyzed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The transplanted HCjE remained transparent, smooth, and without epithelial defects during the follow-up period. Both the cultivated HCjE cells and the engrafted epithelium manifested five to six layers of stratified squamous epithelium similar in morphology to normal corneal epithelium. The basal cells expressed the putative stem cell markers (ABCG2 and P63) and hemidesmosome and desmosome component proteins. The cytokeratins (CK4, CK13, CK3, and CK12) and MUC4 were found in the engrafted epithelium. However, MUC5AC was not expressed. The results indicate that HCjE cultivated on AM has the potential to be used as an alternative corneal epithelium. CONCLUSIONS: The transplantation of cultivated HCjE sheets is a promising technique for the treatment of eyes with limbal failure.

Clinical impact of conjunctivochalasis on the ocular surface.

PURPOSE: To report the clinical impact of conjunctivochalasis on the ocular surface by evaluating the effect of a new concept of conjunctivochalasis operation on the resolution of patients' symptoms and corneal epithelial damage. Also, the association of inflammation with conjunctivochalasis is examined. PATIENTS AND METHODS: A total of 168 eyes of 131 conjunctivochalasis patients with (50 eyes) or without (118 eyes) dry eye who received the newly designed conjunctivochalasis operation were enrolled. All patients had prominent conjunctivochalasis at the lower tear meniscus and their ocular symptoms were not sufficiently controlled by the usual eyedrop therapy. Subjective symptoms of patients were assessed before and after the operation by questionnaires. Scores of corneal fluorescein staining were evaluated before and after the operation in patients with dry eye. Four samples of the lower part of conjunctiva from non-dry eye conjunctivochalasis patients were investigated by immunostaining and compared with samples from 4 normal conjunctiva and 3 conjunctiva showing inflammation due to Mooren ulcer, ocular cicatricial pemphigoid, and alkali burn. RESULTS: The most frequent chief subjective symptoms before the operation were irritation (51.7%) and lacrimation (31.4%) in conjunctivochalasis patients without dry eye and irritation (80.0%) in those with dry eye. Improvement of the chief symptoms was obtained in 88.2% and 78.0% of these 2 patient groups, respectively. Furthermore, in patients with dry eye, corneal fluorescein staining scores (mean+/-SD) were significantly improved after the operation compared with before the operation: A (area), 0.6+/-0.7 and D (density), 0.8+/-0.9 versus A, 1.3+/-0.5 and D, 1.9+/-0.9; P<0.0001. Based on the immunostaining study, conjunctival samples from eyes with conjunctivochalasis and normal eyes showed negligible inflammation compared with those from inflamed conjunctiva. CONCLUSIONS: This study suggests that conjunctivochalasis has a great clinical impact on the ocular surface, and the newly developed operation is very effective in resolving patient complaints and also ocular surface damage in conjunctivochalasis with dry eye. It may also be suggested that the conjunctivochalasis has a negligible association with conjunctival inflammation.

Expression of the P2Y2 receptor on the rat ocular surface during a 1-year rearing period.

PURPOSE: P2Y2 receptors are expressed on ocular surface tissues. Diquafosol ophthalmic solution (DIQUAS(®) ophthalmic solution 3 %; Santen Pharmaceutical Co., Ltd.) acts on these receptors and promotes the secretion of water and mucin. It has been shown to be an efficient dry eye treatment. If P2Y2 receptor expression on the ocular surface decreases with age, the effect of diquafosol may be reduced in elderly persons. In this study, we investigated the changes in P2Y2 receptor expression on the rat ocular surface over an extended period of time. METHODS: P2Y2 receptor expression in the conjunctiva, cornea, meibomian gland and lacrimal glands of male and female Sprague-Dawley rats was examined from 5 weeks until 53 weeks of age using immunostaining and quantitative-PCR. RESULTS: In the immunohistological examinations, P2Y2 receptor expression was observed in the conjunctival epithelium containing goblet cells, corneal epithelium, meibomian gland ductal epithelium and lacrimal gland ductal epithelium. However, its expression was not significantly different between each age group or between sexes. Regarding P2Y2 receptor mRNA expression, there was an age-related increase in the bulbar conjunctiva. In particular, a significant increase was observed in the 53-week-old age group as compared to the 5-week-old female age group. However, age-related changes in expression were not observed in the cornea or meibomian gland in males or females. CONCLUSIONS: We observed no significant age-related decrease was observed for P2Y2 receptor protein and mRNA expression on rat ocular surface tissues.

A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea.

This study investigated changes in collagen fibril architecture and the sulphation status of keratan sulphate (KS) glycosaminoglycan (GAG) epitopes from central to peripheral corneal regions. Freshly excised adult bovine corneal tissue was examined as a function of radial position from the centre of the cornea outwards. Corneal thickness, tissue hydration, hydroxyproline content, and the total amount of sulphated GAG were all measured. High and low-sulphated epitopes of keratan sulphate were studied by immunohistochemistry and quantified by ELISA. Chondroitin sulphate (CS) and dermatan sulphate (DS) distributions were observed by immunohistochemistry following specific enzyme digestions. Electron microscopy and X-ray fibre diffraction were used to ascertain collagen fibril architecture. The bovine cornea was 1021±5.42 µm thick at its outer periphery, defined as 9-12 mm from the corneal centre, compared to 844±8.10 µm at the centre. The outer periphery of the cornea was marginally, but not significantly, more hydrated than the centre (H=4.3 vs. H=3.7), and was more abundant in hydroxyproline (0.12 vs. 0.06 mg/mg dry weight of cornea). DMMB assays indicated no change in the total amount of sulphated GAG across the cornea. Immunohistochemistry revealed the presence of both high- and low-sulphated epitopes of KS, as well as DS, throughout the cornea, and CS only in the peripheral cornea before the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS remained constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also higher at mid-depth in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12 mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the distribution of low-sulphated KS.