A New Dry Eye Disease Model: Evaluation Of A Murine Model Of Dry Eye Disease Induced By Topical Hyperosmolar Saline

Background and purposeDry eye disease, also known as keratoconjunctivitis sicca or tear dysfunction syndrome is caused by abnormal quality and quantity or unusual kinetics of tear which lead to tear film unstability causing ocular surface tissue disease. Dry eye disease is the most common ocular surface disease.Pathogenesis of dry eye disease is not clear so far. Many risk factors can lead to dry eye disease. National Eye Institute/Industry workshop suggested that dry eye disease should be divided into two categories,including deficient aqueous production (including Sj?gren's syndrome and non-Sj?gren's syndrome) and over evaporation.Researchers have proposed a variety of animal models of dry eye disease, however, no one animal model can accurately mimic characteristics of the disease,which include frequent occurance, gradual deterioration, and complex mechanism. Although many risk factors can induce dry eye disease and the pathogenesis of dry eye disease is complicated, the disease shares common final pathway that is the unstablity of tear film followed by the elevating electrolyte concentrations in tear, which can lead to increased tear osmolarity. The high tear osmolarity is a key factor in the pathogenesis of dry eye disease.Normal human tear osmolarity is 302±9.7mOsm / L according to the statistical analysis of the literatures. tear osmotic pressure in patients with dry eye disease was 326.9±22.1 mOsm / L. Taken 316mOsm / L as a diagnostic indicator of dry eye disease, the overall accuracy is better than any other indicators of diagnosis of dry eye disease.In this study, we use topical hypertonic saline to produce high osmotic pressure of tears to make a dry eye disease model and to observe whether the model mimics the signs and histopathological lesions of dry eye disease. Method1. The selection and grouping of animalsSixty female BALB / c mice of 6 to 8 week old were divided into blank group (20 mice), control group (20 mice), experimental group (20 mice) by random number table, and both eyes were used. The control group was given topical 308mOsmol / L sodium chloride solution, 5 times a day. The experimental group was given topical 500 mOsmol / L sodium chloride solution, 5 times a day. Mice in the blank group were not given any treatment.2. SchirmerⅠThe phenol red thread was placed in the eye canthus of BALB / c mice with a micro-tweezer under a slit lamp microscope. The phenol red thread was removed, the wetting length of the phenol red thread was measured after 60s.3. Corneal fluorescein staining and scoringInstillation of 1μl of 5% fluorescein sodium into the conjunctiva of BALB / c mice were made, photographic records and scoring were made with cobalt blue light under the microscope after 3 minutes.4. Rose bengal stainingRose bengal test strip was soaked using saline, the wet part of rose bengal test strip was gently touched the cornea of the BALB / c mouse. Photographic records were made.5. Tear fernsTear in fornix of BALB / c mice was collected with a capillary tube, the tear was coated in a clean slide, dried at room temperature. Image analysis of fern crystal and photographic records were done under the microscope within 48 hours.6. HE staining and PAS stainingThe morphology of corneal epithelia was observed by HE staining. The thickness of the central corneal epithelium was measured by image analysis software.The top fornix of the conjunctival fornix was stained using PAS staining.The number of fornix goblet cells was calculated under each visual field.7. Scanning electron microscopeThe morphology of corneal surface.was observed by scanning electron microscope.8. Statistical MethodsScores of corneal fluorescein were compared by Kruskal-Wallis H test among 3 groups at the same time points. Scores of corneal fluorescein staining were compared with Wilcoxon rank sum test at different time points in the same group. The thickness of the corneal epithelium, tear secrection, the number of goblet cells were compared by ANOVA among three groups at the same time points. The thickness of the corneal epithelium, tear secrection, the number of goblet cells were compared by T test in the same group at different time points.Result1. SchirmerⅠexperimentAt day 0, wetting length of phenol red thread of the mice in the blank group, the control group, the experimental group was not significantly statistically different (P> 0.05). From the 7th day, wetting length of phenol red thread of the mice in the experimental group was significantly deceased than that at day 0 (P <0.01) .The wetting length of phenol red thread at day 7 was decreased 28% compared with that at day 0. The wetting length of phenol red thread at day 42 was decreased 66% compared with that at day 0.2. Corneal fluorescein staining and scoringAt day 0, in the cornea of BALB / c mice in the blank group, the experimental group and the control group, little corneal fluorescein staining was found. Corneal score in each group showed no significant difference (P> 0.05). Punctate or sheet staining gradually emerged in the cornea of BALB / c mice in the experimental group. Corneal fluorescein staining score at day 7 was increased 75% compared with that at day 0 in the experimental group.3. Rose bengal stainingOcular surface rose bengal staining of BALB / c mouse in the blank group and the control group showed a small amount of punctate staining. At day 7, the staining changed from punctate to patches on central and nasal cornea.4. Tear fernsImage analysis of fern crystal and photographic records were done under the microscope. Tear ferns is well formed in the blank or the control group. The fern in tear of BALB / c mice in the experimental group diminished from the 7th day, connections between fern materials interrupted. Only small fern materials or even no fern material were observed from the 28th days under the microscope.5. HE staining Corneal epithelial thickness was measured and corneal epithelium was observed. In the blank group and the control group, corneal epithelial layer of BALB / c mice clearly showed 4 to 5 layers by HE staining.The columnar cells were arranged along the basis of epithelial layer. Cells became squamous when closed to corneal surface. The cornea of mice in the experimental group were found abnormal arrangement of the basal cells, decreased thickness of epithelial layer(P<0.05), and presentation of vacuole.6. PAS stainingClear goblet cell morphology and distribution in the epithelial cells, mainly in the fornix, were observed in the blank group and the control group. Goblet cell density was significantly decreased at day 14(P<0.01)in the experimental group.Conjunctival epithelial cells of BALB / c mice were irregularly arranged in the experimental group.7. Scanning electron microscopyEpithelial cells of corneal surface of BALB / c mice in the three groups were observed by scanning electron microscopy at 42th day. Epithelial cells in the blank group and the control group were found intact, polygonal types and close connection. Microvilli were found on the cell surface. In the experimental group, loss of microvilli and destruction of corneal surface epithelial cells were found on the corneal surface of BALB / c mice.Conclusion1, Signs and histopathological examinations of BALB/c murine model of dry eye disease induced by topical hyperosmolar saline mimic that of human with dry eye disease.2. BALB/c murine model of dry eye disease induced by topical hyperosmolar saline is easy-made, stable and repeatable.The modle can be used for the study of mechanism and treatment of dry eye disease.3, High osmotic pressure in tear may play an important role in the pathogenesis of dry eye disease.