Comparison of 1 % cyclosporine eye drops in olive oil and in linseed oil to treat experimentally-induced keratoconjunctivitis sicca in rabbits

PURPOSE
To evaluate the effectiveness of topical 1% cyclosporine eye drops diluted in either of the two vehicles-olive and linseed oil-and that of the oils themselves in treating experimentally-induced keratoconjunctivitis sicca (KCS) in rabbits.


METHODS
KCS was induced in 25 New Zealand rabbits using 1% atropine sulfate eye drops for 7 days before treatment and throughout the treatment period (12 weeks). The rabbits were divided into five groups: one control (C) group without KCS induction and four treatment groups in which KCS was induced and treated topically with olive oil (O), linseed oil (L), cyclosporine in olive oil (CO), and cyclosporine in linseed oil (CL). The animals were evaluated using Schirmer tear test 1 (STT), the fluorescein test (FT), tear-film break-up time (TBUT), the rose bengal test (RBT), and histopathological analysis.


RESULTS
Values of STT and TBUT significantly decreased 1 week post-induction (p<0.05) and were similar to initial values after the 4th week of treatment, in all groups. After KCS induction, there was significantly less corneal damage in group L than in group CL, as assessed FT and RBT. Histopathology demonstrated that Groups L and CL presented less edema and corneal congestion. There was no significant difference in the goblet cell density (cells/mm2) between the groups (p=0.147).


CONCLUSION
Cyclosporine diluted in olive oil or linseed oil was effective in the treatment of KCS, although it had better efficacy when diluted in linseed oil. Linseed oil presented better effectiveness, whether associated or not, than olive oil. These results may contribute to the creation of novel topical ophthalmic formulations for KCS treatment in future.

The objective of this study was to evaluate the effectiveness of cyclosporine, a potent immunosuppressor used in the treatment of KCS, as 1% eye drops diluted in olive oil (already used as a vehicle for cyclosporine) or linseed oil, in addition to the use of each oil separately (without KCS), in the treatment of experimentally induced KCS in rabbits.

AnimAls
This experiment was approved by the Ethics Committee on the Use of Animals of UNOESTE (protocol n o 720/11).Twenty-five male New Zealand white rabbits (Oryctolagus cuniculus) of a mean age of 198 days and a mean weight of 3,600 g were selected from the Central Bioterium of UNOESTE and kept in individual metal cages with access to water and food ad libitum.

The inducTion of Kcs
The induction model of KCS in rabbits was based on previously published studies that used 1% atropine sulfate eye drops three times daily (15,16,(20)(21)(22) .Atropine is a competitive antagonist of the muscarinic acetylcholine receptors.As the ocular surface and main lacrimal gland are innervated by parasympathetic fibers of the seventh cranial nerve; the use of atropine is known to cause decreased tear secretion and con sequent inflammation in a similar way to dry eye syndrome (20)(21)(22) .Eye drops were administered until the diagnosis of KCS (7 days to induce KCS in all rabbits) was confirmed by a Schirmer tear test 1 (STT; Teste de Schirmer®; Ophthalmos Laboratory) result of ≤5 mm/min and/or ≤10 seconds tear-film break-up time (TBUT) (23,24) and throughout the treat ment period (12 weeks) for KCS maintenance.

Groups
The data from both eyes were pooled from the 25 rabbits.Of these rabbits, 20 were induced using the KCS protocol, and 5 were allocated to the non-induced control group.The timing of the 1% atropine eye drop instillation was 6:00 am, 2:00 pm, and 10:00 pm, and the timing of the treatment eye drop instillation was 8:00 am and 8:00 pm.Beginning 1 week after KCS induction, the animals were treated for 12 weeks as follows: Group C (control; n=5) (one drop of placebo, 0.9% NaCl solution [Fresenius Kabi, Campinas, São Paulo, Brazil], topically applied twice a day [BID] in both eyes); Group CO (one drop of 1% cyclosporine in olive oil [Ophthalmos®, São Paulo, Brazil], topically applied BID in both eyes); Group CL (one drop of 1% cyclosporine in linseed oil [Ophthalmos®], topically applied BID in both eyes); Group O (one drop of olive oil [Ophthalmos Laboratory], topically applied BID in both eyes); and Group L (one drop of linseed oil [Ophthalmos Laboratory], topically applied BID in both eyes).The rationale behind using 1% CsA was that it is a manipulated concentration marketed by Ophthalmos Laboratory in Brazil.
STT was performed without anesthetic eye drops, and was used to determine the quantitative tear production.
TBUT for evaluation of the quality of the tears was performed twice, successively, and the mean was calculated.After placing a drop of 1% fluorescein eye drops (Fluoresceína®; Allergan, São Paulo, Brazil) into the lower fornix, a slit-lamp, set on a bright light setting with a cobalt blue filter, was used to measure the time between the last blink and the first appearance of a dark spot on the cornea (formation of a dry area).

hisTopATholoGicAl AnAlysis
For the histopathological analysis, the rabbits were euthanized at the end of the experiment (T12) using 2.5% sodium thiopental (200 mg/kg) administered intravenously.After transpalpebral enuclea tion, the eyeball was placed in a 10% formaldehyde solution for 24-48 hours.The eyes were stored in 70% alcohol, and then routine ly processed and embedded in paraffin.Three serial sections of 5 μm from the cornea and conjunctiva were obtained and stained with he matoxylin and eosin (HE) and periodic acid-Schiff (PAS).The cor nea and conjunctiva were assessed for the following: edema, cellular degeneration, necrosis, inflammatory infiltrate (neutrophilic, mononu clear, and mixed), and goblet cell count (bulbar conjunctiva).Edema, cellular degeneration, and necrosis were scored from 0 to 3: (0) absence, (1) mild, (2) moderate, and (3) severe.Inflammatory infiltrate was scored from 0 to 3: (0) absent, (1) mild (1-4 cells per field), (2) moderate (5-9 cells per field), and (3) severe (>10 cells per field).Goblet cell density (cells/mm 2 ) was counted in five high-power fields (40× objective) on each slide, corresponding to an area of around 0.5 mm 2 .One blinded observer evaluated the histopathological exams.

sTATisTicAl AnAlysis
The STT and TBUT values and goblet cell density were compared between the groups and time points using analysis of variance (ANOVA), in addition to Tukey's method.FT and RBT values, were analyzed with the Friedman and Kruskal-Wallis tests, for comparisons within groups over time and among different groups, respectively.A statistical signi ficance level of p<0.05 was adopted.The software used for the statistical analysis was Biostat 5.3.
The FT results are presented in figure 2 A. At T1, only group O showed an FT score of 2 (two stained quadrants), while the other groups: L, CO, and CL, showed an FT score of 1 (one stained quadrant).At T2 and T4, all treatment groups presented an FT score of 1, and at T8 and T12, no eyes showed positive staining.The RBT results are shown in figure 2 B. From T1 to T4, all treatment groups presented a score of 2 (only corneal staining).At T8, groups O, L, and CO presented scores of 2, and at T12, no eyes were RBT positive.
The histopathological images of the cornea and conjunctiva are presented in figure 3. A normal cornea from group C is shown in figure 3 A. Slight edema and conjunctival congestion were found in groups O (Figure 3 B) and CO.Regarding the goblet cell density (Figures 3 C  and D), there was no statistically significant difference between the groups (p=0.147),although group CL appeared to have higher values (11.2 ± 3.5) compared to groups C (8.0 ± 2.1), O (7.6 ± 3.7), L (9.0 ± 3.4), and CO (8.4 ± 3.2).The histopathological results of all the conjunctival samples from group CO (Figure 3 E) and O (Figure 3 F) showed more chronic inflammation than the other treatment groups.

DISCUSSION
When comparing the two different oils, linseed oil showed a more favorable and rapid effect than olive oil in terms of the STT and TBUT *P<0.05 (Tukey's test, compared to control group).results at T2, FT results at T4, and in the histopathological results.In general, Group O showed the worst results in the treatment of KCS.These variations could be explained by the difference in composition for each oil.Olive oil contains a larger quantity of MUFA (70-80% oleic acid) and only a small amount of PUFA (5-10% of ω-3, ω-6, and ω-9) (10,11) .Linseed oil is comprised of 16% ω-6 and 57% ω-3, which are natural anti-inflammatory agents due to their ability to synthesize noninflammatory mediators (12,25) .The ω-6 origin of PGE1 and TXA1 via cyclooxygenase 1 (COX1) and the effects of the ω-3 fatty acids eicosapentaenoic acid and docosapentaenoic acid could explain the clinical improvements in the animals that received the linseed oil treatment (18,19) .
These results are in agreement with a previous study that used a topical formulation containing an emulsion of alpha-linolenic acid and linoleic acid on mice with experimentally induced KCS; the inflammatory symptoms of KCS improved in the mice (17) .A recent study compared almond and linseed oil as diluents of tacrolimus, another immunosuppressor, in the treatment of KCS in rabbits and showed that linseed oil either as a diluent or in isolation was more effective than almond oil (16) .Another study evaluated the effectiveness of various linseed oil preparations (oral, topical, and an oral/topical combination) in treating experimentally induced KCS in rabbits, and concluded that orally or topically administered linseed oil was effective in the treatment of KCS, although the combined oral/topical treatment did not show additional benefits (15) .When the CsA groups were compared with the oil groups, groups CL and CO were found to present similar TBUT and FT results at the end of the experiment.Additionally, there was more rapid negative RBT staining for group CL, which also had higher, although non-statistically significant, goblet cell density.The better results in group CL for these parameters might be due to the combination of the anti-inflammatory properties of the linseed oil and the immunosuppressive effects of the cyclosporine.This result is in agreement with the findings from the study that used tacrolimus, another immunosuppressor used in KCS treatment; the group that was administered tacrolimus diluted with linseed oil showed better improvement in KCS control in rabbits, with increased goblet cell density in the conjunctiva (16) .In rabbits with KCS induced by autoimmune dacryoadenitis, the use of 0.05% CsA in an ophthalmic emulsion (Restasis®) promoted significantly increased tear production (26) .These CsA effects are due to the immunosuppressive action that minimizes the inflammatory da mage induced by KCS in the cornea, conjunctiva, and goblet cells (2)(3)(4)(5) .The increased density of goblet cells in KCS is very important because decreased goblet cell density can induce mucin deficiency in the precorneal tear film and may result in ulcerated corneas with irregular surfaces (4,27) .
The histopathological results also demonstrate a stronger inflammatory process in the O and CO groups (Figures 4 E and F), as assessed by the presence of mild edema in the cornea and moderate edema and congestion in the conjunctiva.Additionally, only group O demonstrated squamous metaplasia, which was most likely due to chronic ocular inflammation (1,2,5) .The goblet cell density was not significantly increased in group CL when compared to the other groups, but this difference could potentially become statistically significant in a larger population.
We conclude that cyclosporine diluted in olive oil and linseed oil is effective for the treatment of experimentally induced KCS in rabbits.However, linseed oil, when used alone or as a diluent for cyclosporine, is more effective at treating KCS than olive oil.This study reveals that both the immunosuppressive effect of cyclosporine and the properties of the oil medium, particularly the anti-inflammatory properties of linseed oil, are responsible for the improvement of dry eye symptoms in rabbits.These results may contribute to the development of novel topical ophthalmic formulations for the treatment of KCS in future.

Figure 1 .
Figure 1.Mean ± SD for (A) Schirmer's tear test I (STT I) a in mm/min and (B) tear-film break-up time (TBUT) b in seconds, in rabbits with experimentally induced keratoconjunctivitis sicca (KCS) that were treated with various topical formulations: placebo (group C), olive oil (group O), linseed oil (group L), 1% cyclosporine in olive oil (group CO), and 1% cyclosporine in linseed oil (group CL).

Figure 2 .Figure 3 .
Figure 2. Median results for (A) fluorescein test (FT) a and (B) rose bengal test (RBT) b in rabbits with experimentally induced keratoconjunctivitis sicca (KCS) that were treated with various topical formulations: placebo (group C), olive oil (group O), linseed oil (group L), 1% cyclosporine in olive oil (group CO), and 1% cyclosporine in linseed oil (group CL).