Use of linseed oil to treat experimentally induced keratoconjunctivitis sicca in rabbits

Twenty male New Zealand white rabbits (Oryctolagus cuniculus) were housed in individual metal cages with water and food ad libitum. All of the procedures were approved by the Ethics Committee on the Use of Animals of UNOESTE under protocol no. 014/09. The induction model of KCS in rabbits was based on a previously published study [11], which described the removal of the lacrimal glands and nictitating membranes and the administration of atropine sulfate 1% eye drops TID until the confirmation of KCS diagnosis (≤5 mm/min Schirmer's tear test (STT) and/or Rose Bengal test (RBT) positive) and then throughout the treatment period (12 weeks) for KCS maintenance. The rabbits were evaluated at M0 (prior to the KCS induction surgery), M1 (1 week after the induction of KCS and the beginning of treatment), and M2 to M12 (i.e., assessments as weekly intervals beginning 1 week after the initiation of treatment). Of the 20 rabbits, 15 were induced using the KCS protocol described above and 5 were allocated to the control group without KCS induction. A week after the induction of KCS, the groups of animals were treated for 12 weeks as follows: group C (n = 5, control, one drop of placebo, 0.9% NaCl solution, topically twice a day (BID) in both eyes), group OLO (n = 5, 1 g/day of liquid LO made by Laboratory Ophthalmos-SP (São Paulo, Brazil) orally), group TLO (n = 5, one drop of LO eye drops made by Laboratory Ophthalmos-SP BID in both eyes), and group OTLO (n = 5, 1 g/day of liquid LO orally with one drop of LO eye drops BID in both eyes). STT was performed without anesthetic eye drops and was considered positive if the KCS values were ≤5 mm/min. The fluorescein test (FT) was performed to observe ulcers colored with 1% fluorescein strips moistened with a small amount of normal saline; they were scored 1 (negative for a corneal ulcer) or 2 (positive for a corneal ulcer). RBT was performed on stained cells devitalized by KCS; after instilling the anesthetic eye drops, we used one drop of 0.5% Rose Bengal, and they were scored 1 (the absence of stained cells devitalized by KCS) or 2 (the presence of stained cells devitalized by KCS). For the histopathological analysis at the end of the experiment (M12), the rabbits were euthanized using 2.5% IV sodium thiopental (200 mg/kg); after transpalpebral enucleation, the eyeball was placed in a solution of 10% formaldehyde for 24 to 48 h. The eyes were stored in 70% alcohol and were processed according to the technique for inclusion in paraffin; 5-μm-thick cuts of the cornea and conjunctiva were obtained, which were colored with hematoxylin and eosin (HE) as well as periodic acid-Schiff (PAS). For STT variables and the density of caliciform cells, we used the variance analysis test for paired samples in contrast to Tukey's method. For FT, RBT, and histopathology, a non-parametric Friedman test was used to compare the various times, and the Kruskal-Wallis test with Dunn's method was used for between-group comparisons. We adopted a significance level of P < 0.05.