After animals had been anesthetized by ketamine/xylazine, tear production was determined by using a round piece of absorbent paper of 6 mm diameter, which was folded in the middle, taken with a pair of tweezers and immersed into the tear meniscus on the lower conjunctival fornix for 20 sec (left and right eye). Next, the paper was photographed under a microscope and the moistened area determined with Image J (NIH, http://rsb.info.nih.gov/ij/ (accessed on 11 March 2019)). The measured uptake of tear fluid in mm2 was compared to a standard curve prepared using absorbent paper to uptake known volumes of a stock basic solution (0.9% NaCl) (Figure 8).

Standard curve of fluid uptake using absorbent paper and known volumes of a basic solution (0.9% NaCl). The resulting uptake areas were plotted against volume. This curve was then used to convert actual mouse tear uptake areas into tear volume.

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