Non-obese diabetic (NOD) mice are a commonly used murine model for the study of Sjögren's syndrome. However, variations in susceptibility to the disease among the mice has often yielded less stable results. Based on the correlation between the pathological changes and the tear tests, we attempt to establish a simple screening procedure to assure the validity of experimental results by excluding those mice with poor susceptibility to dry eyes.
Seventy male NOD mice were recruited. The tear film break-up test (BUT) and the phenol red cotton thread test (CTT) were implemented while the mice were under anesthesia. The mice were divided into four groups (grades 1 to 4) based on their BUT readings, and four similar groups based on CTT measurements. Tear samples in each grade were collected for IL-1beta detection with ELISA. The lacrimal glands and conjunctiva of the mice were used to detect the levels of leucocyte common antigen (LCA). LCA-Positive staining was considered as the "gold standard" in the receiver operating characteristic curve (ROC curve) analysis. C57BL/6 mice were used as wild-type controls.
There were 13 (18.57%), 43 (61.43%), 10 (14.29%) and 4 (5.71%) mice in grades 1, 2, 3 and 4 by BUT test, and 34 (48.57%), 15 (21.43%), 14 (20.00%) and 7 (10.00%) in grades 1, 2, 3 and 4 by CTT test respectively. Fifty-one out of the 70 mice (72.86%) were detected LCA-positive, and they were mainly in grades 1 and 2 of both the BUT and CCT grading systems. ELISA showed significant variances of IL-beta levels among the four groups (p < 0.01), with much lower IL-beta levels in group 3 and 4 when both BUT and CTT were used for grouping. The tear IL-beta level in the wild-type mice was similar to those of the grade 4 mice, using either BUT or CTT for grouping. The ROC curve analysis provided optimal cutting lines, which were 2 seconds in BUT readings and 4 mm/min in CTT measurements respectively.
BUT and CTT tests are useful methods in screening high susceptible NOD mice. Cutting lines at BUT < or = 2 seconds and CTT < or = 4 mm/min provide a good balance between the assurance of susceptibility and the maximization of use of NOD mice for the study of Sjögren's syndrome.