Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips.

PURPOSE: Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA. METHODS: MUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined. RESULTS: MUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%. CONCLUSION: We developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients.

A Novel Model of Meibomian Gland Dysfunction Induced with Complete Freund's Adjuvant in Rabbits.

A novel meibomian gland dysfunction (MGD) model induced by the injection of complete Freund's adjuvant (CFA) in rabbits was developed to facilitate the understanding of the pathophysiology of MGD with meibomitis. In addition, we sought to evaluate treatment with steroid eye drops in this model. Male Japanese white rabbits were subcutaneously injected with CFA into the upper eyelid margin. The eyelid margins of the rabbits were chronologically observed through slit lamp examination. The development of meibomitis was assessed through histopathology. We evaluated the effects of topically applied tobramycin/dexamethasone (Tob/Dex) eye drops on the plugged orifices and telangiectasia. After the injection of CFA, slit lamp examination revealed markedly plugged orifices, telangiectasia around the orifices and a toothpaste-like meibum, as compared with the normal eyelids. Histopathology revealed granulation tissue with infiltration of inflammatory cells, hyperkeratinization of the ductal epithelium, and cystic dilatation of ducts in the meibomian gland. The orifices were plugged with a proteinaceous substance. Tob/Dex eye drops significantly suppressed the plugging and telangiectasia around the orifices. Through the injection of CFA, we successfully established a novel rabbit MGD that mimics the symptoms observed in humans meibomitis. This model should be useful in the evaluation of the efficacy of drug candidates.

Tear volume estimation using a modified Schirmer test: a randomized, multicenter, double-blind trial comparing 3% diquafosol ophthalmic solution and artificial tears in dry eye patients.

PURPOSE: We aimed to evaluate the feasibility of using a modified Schirmer test to determine the increase in tear volume after administration of 3% diquafosol ophthalmic solution (diquafosol 3%) in dry eye patients. PATIENTS AND METHODS: A randomized, multicenter, prospective, double-blind clinical study recruited 50 qualified subjects. They received diquafosol 3% in one eye and artificial tears in the other eye. The study protocol comprised a screening and treatment procedure completed within 1 day. The Schirmer test was performed on closed eyes three times a day. The primary efficacy end points were the second Schirmer test scores 10 minutes after the single dose. Secondary end points were the third Schirmer test scores 3 hours and 40 minutes after the single dose and the symptom scores prior to the second and third Schirmer tests. RESULTS: According to the Schirmer test, 10 minutes after administration, diquafosol 3% significantly increased tear volume compared to artificial tears. Diquafosol 3% and artificial tears both showed significant improvements in the symptom scores compared to baseline. However, there was no significant difference in the symptoms score between diquafosol 3% and artificial tears. CONCLUSION: The modified Schirmer test can detect a minute change in tear volume in dry eye patients. These findings will be useful in the diagnosis of dry eye, assessment of treatment benefits in daily clinical practice, and the development of possible tear-secreting compounds for dry eye.

Meibomian Gland Dysfunction Model in Hairless Mice Fed a Special Diet With Limited Lipid Content.

PURPOSE: A novel meibomian gland dysfunction (MGD) model was developed to facilitate understanding of the pathophysiology of MGD and to evaluate treatment with azithromycin ophthalmic solution (azithromycin). MGD was induced in HR-1 hairless mice by feeding them a special diet with limited lipid content (HR-AD). METHODS: Male HR-1 hairless mice were fed an HR-AD diet for 16 weeks. Development of MGD was assessed by histopathology at 4-week intervals. The lid margin was observed by slit-lamp examination. After cessation of the HR-AD diet, the mice were fed a normal diet to restore normal eye conditions. Expression of cytokeratin 6 was determined by immunostaining. We evaluated the effects of topically applied azithromycin on the plugged orifice in this model. RESULTS: After mice were fed the HR-AD diet, histopathology analysis showed hyperkeratinization of the ductal epithelium in the meibomian gland. Ductal hyperkeratinization resulted in the loss of acini, followed by atrophy of the gland. Slit-lamp examination revealed a markedly plugged orifice, telangiectasia, and a toothpaste-like meibum compared with that of a normal eyelid. Cessation of feeding with HR-AD ameliorated both the MGD signs and the expression of cytokeratin 6, restoring the tissue to a histologically normal state. Azithromycin treatment significantly decreased the number of plugged orifices and ameliorated atrophy, as revealed by histopathologic analysis. CONCLUSIONS: We developed a novel model that mimics human MGD signs in HR-1 hairless mice fed an HR-AD diet. Azithromycin treatment led to therapeutic improvement in this model. This MGD model could be useful for the evaluation of drug candidates for MGD.

Apafant, a potent platelet-activating factor antagonist, blocks eosinophil activation and is effective in the chronic phase of experimental allergic conjunctivitis in guinea pigs.

Platelet-activating factor (PAF) plays important roles in allergic reactions. In particular, there are many concerns about PAF, eosinophils, and the chronicity of allergic diseases. The purpose of the present studies is to elucidate the role of PAF in eosinophil activation at conjunctiva and to confirm the efficacy of Apafant (a potent PAF antagonist) ophthalmic solution in chronic experimental allergic conjunctivitis. Guinea pigs were actively immunized and allergic conjunctivitis was induced by repetitive instillation of 2.5% ovalbumin. PAF solution was topically applied and eosinophil activation was assessed by measuring the eosinophil peroxidase (EPO) activity in the tear fluid. Itch-scratching episodes and clinical symptoms scores were evaluated in the repetitive challenge conjunctivitis. From the instillation of PAF solution into guinea pig eyes, which were in a state of chronic allergic conjunctivitis, a significant increase in EPO activity was observed, and this increase was inhibited by pre-treatment with Apafant. In the repetitive challenge model, the animals treated with Apafant ophthalmic solution showed a significant reduction of clinical symptoms and the itch-scratch response in both the first and the second challenges. PAF has an activity, that induces mediator release from eosinophils in the conjunctival tissues and may be involved in the chronic phase of allergic conjunctivitis.