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

Background The objective of this study was to evaluate the effectiveness of various linseed oil (LO) preparations (oral, topical, oral and topical combined) in treating experimentally induced keratoconjunctivitis sicca (KCS) in rabbits. Twenty male New Zealand white rabbits were divided into four groups: group C (control), group OLO (oral LO), group TLO (topical LO), and group OTLO (oral and topical LO). The animals were evaluated weekly using Schirmer's tear test (STT), fluorescein test (FT), and Rose Bengal test (RBT) and were euthanized at the end of the experiment for histopathological analysis. Findings There were significant improvements in the parameters analyzed (STT, FT, and RBT) and in the histopathological finding in all of the groups using LO. Conclusions The analyzed results demonstrate that LO, administered orally or topically, was effective in treating experimentally induced KCS in rabbits, although combined oral and topical LO did not show additional benefits greater than those with a single route of administration.

The ω-3 and ω-6 EFAs are found in many foods, such as nuts, cold-water fish, soybeans, rapeseed oil, and linseed oil. Linseed, Linum usitatissimum, is composed of 57% ω-3, 16% ω-6, 28% monounsaturated fatty acids, and only 9% unsaturated fatty acids, and the ω-3-to-ω-6 ratio of 1:3 is considered close to ideal. Linseed is considered to be a natural anti-inflammatory agent due its potential in synthesizing non-inflammatory mediators, such as PGE1 and TXA1 [8][9][10]. The objective of this study was to evaluate the effectiveness of various linseed oil (LO) preparations (oral, topical, oral and topical combined), which are rich in ω-3 and ω-6, in treating experimentally induced KCS in rabbits.

Materials and methods
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   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 nonparametric 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.

Results
The STT, FT, and RBT results are shown in Figure 1. An interesting clinical finding was from rabbit no. 4 of group OTLO (oral and topical LO), which presented a melting ulcer at M1, and there was a partial resolution of the process until the end of the experiment (Figure 2). The histopathology results of the cornea and conjunctiva in group C showed no changes ( Figure 3A). Upon histopathology of the cornea, there was slight edema in group OLO ( Figure 3B) compared to group TLO, which presented moderate edema ( Figure 3C,D,E). In group TLO, the presence of inflammatory infiltrates with exocytosis of neutrophils was observed ( Figure 3F,G). In the histopathological results of the conjunctiva, the OLO ( Figure 3B) and OTLO ( Figure 3H) groups had mild and moderate edema, respectively, whereas group TLO had severe edema. The OLO and OTLO groups ( Figure 3I) showed mild necrosis, whereas group TLO showed moderate necrosis. There was no statistical significance (P > 0.05) in the difference in the caliciform or goblet cell density in the conjunctiva among the groups, and the values were 9.8 ± 1.9 (C), 9.5 ± 2.3 (OLO), 10.7 ± 2.7 (TLO), and 9.0 ± 3.8 (OTLO).

Discussion and Conclusions
The group treated with oral LO (group OLO) had normal tear production in the second week after starting treatment and a significant increase above the control group after 3 weeks. This finding is consistent with several studies, especially in patients with Sjögren's syndrome, in whom oral ω-3 and ω-6 supplementation has been recommended to control KCS symptoms [2][3][4]12]. In this study, topical LO was more effective in the early treatment of KCS, increased production of tears, and corneal ulcer resolution. The lipid mediators play an important role in the resolution and repair of corneal injuries [7,12,13].
The LO mechanism of action might be anti-inflammatory; the inhibitory response of the arachidonic acid inflammatory cascade has been proven to synthesize the noninflammatory mediators PGE1 and TXA1 from ω-6 series by the action of COX1 on DGLA, which likely reduces the inflammation on the cornea, conjunctiva, and lacrimal glands; increases the production of tears; and improves the activity of caliciform cells, consequently causing an increase in the production of mucin. This increased mucin provides an improvement in the PTF quality, which leads to reduced tear evaporation in the aqueous portion of the eye [2,4,6,7,12]. In this study, LO topical instillation did not induce any ocular side effects, such as irritation, hyperemia, or discomfort. In addition, the concomitant use of LO orally and topically did not show benefits beyond a single route of administration. A hypothesis for this result is that an excess of ω-3 and ω-6 results in an improper ratio of these two EFAs. More pharmacokinetic and pharmacodynamic studies of oral LO and of LO in ocular topical formulations should be conducted to clarify these results. Therefore, we conclude that LO, orally and topically, was effective in treating experimentally induced KCS in rabbits, although combined oral and topical LO did not demonstrate additional benefits beyond those from a single route of administration.