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Laboratory Sciences |

Topical Omega-3 and Omega-6 Fatty Acids for Treatment of Dry Eye FREE

Saadia Rashid, MD; Yiping Jin, MD, PhD; Tatiana Ecoiffier, MSc; Stefano Barabino, MD, PhD; Debra A. Schaumberg, ScD, MPH; M. Reza Dana, MD, MSc, MPH
[+] Author Affiliations

Author Affiliations: Schepens Eye Research Institute, Boston, Massachusetts (Drs Rashid, Jin, Barabino, Schaumberg, and Dana and Mss Ecoiffier); Harvard Medical School, Boston (Drs Rashid, Jin, Barabino, Schaumberg, and Dana and Ms Ecoiffier); Department of Neurosciences, Ophthalmology, and Genetics, University of Genoa, Genoa, Italy (Dr Barabino); Division of Preventive Medicine, Brigham and Women's Hospital, Boston (Dr Schaumberg); and Cornea Service, Massachusetts Eye and Ear Infirmary, Boston (Dr Dana).


Arch Ophthalmol. 2008;126(2):219-225. doi:10.1001/archophthalmol.2007.61.
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Published online

Objective  To study the efficacy of topical application of alpha-linolenic acid (ALA) and linoleic acid (LA) for dry eye treatment.

Methods  Formulations containing ALA, LA, combined ALA and LA, or vehicle alone, were applied to dry eyes induced in mice. Corneal fluorescein staining and the number and maturation of corneal CD11b+ cells were determined by a masked observer in the different treatment groups. Real-time polymerase chain reaction was used to quantify expression of inflammatory cytokines in the cornea and conjunctiva.

Results  Dry eye induction significantly increased corneal fluorescein staining; CD11b+ cell number and major histocompatibility complex Class II expression; corneal IL-1α and tumor necrosis factor α (TNF-α) expression; and conjunctival IL-1α, TNF-α, interferon γ, IL-2, IL-6, and IL-10 expression. Treatment with ALA significantly decreased corneal fluorescein staining compared with both vehicle and untreated controls. Additionally, ALA treatment was associated with a significant decrease in CD11b+ cell number, expression of corneal IL-1α and TNF-α, and conjunctival TNF-α.

Conclusions  Topical ALA treatment led to a significant decrease in dry eye signs and inflammatory changes at both cellular and molecular levels.

Clinical Relevance  Topical application of ALA omega-3 fatty acid may be a novel therapy to treat the clinical signs and inflammatory changes accompanying dry eye syndrome.

Figures in this Article

Dry eye syndrome (DES) is a highly prevalent health problem that affects more than 10 million people, primarily women, in the United States alone.1,2 It is a frequent cause of office visits due to ocular discomfort and commonly leads to problems with sustained visual activities such as reading and driving.3 Inflammation has been recognized as an important component of DES.4 The recently introduced topical cyclosporin A (Restasis; Allergen, Irvine, California) has been shown to decrease ocular surface inflammation, stimulate tear production, and improve signs and symptoms of dry eye,5 further signifying the role of inflammation and anti-inflammatory agents for dry eye treatment.

Naturally occurring essential polyunsaturated fatty acids (PUFA) of omega-3 (n-3) and omega-6 (n-6) series are promising natural anti-inflammatory agents shown to have beneficial effects in many inflammatory conditions such as rheumatoid arthritis and ulcerative colitis.6 The n-3 FAs include alpha-linolenic acid (18:3n-3; ALA) and its elongation and desaturation products, stearidonic acid (18:4n-3), eicosapentaenoic acid (20:5n-3; EPA), and docosahexaenoic acid (22:5n-3; DHA). The n-6 FAs include linoleic acid (18:2n-6; LA) and its products, gammalinolenic acid (18:3n-6; GLA), dihomogammalinoleic acid (20:3n-6; DGLA), and arachidonic acid (20:4n-6; AA). Both ALA and LA are called “essential” FAs because they cannot be synthesized by mammals and must be supplied in diet.

Recent studies have shown beneficial effects of dietary supplementation of FAs in DES.79 In a cross-sectional study of 32 470 women, women with a higher n-3 FA intake (more than 5-6 tuna servings per week as opposed to less than 1) were found to have 68% lower prevalence of DES.9 In 2 randomized clinical trials, oral supplementation with LA and GLA ameliorated the signs and symptoms of dry eye.7,8 It is postulated that when the n-6 to n-3 ratio is approximately 4:1 or lower, the conversion of DGLA to AA undergoes competitive inhibition with enhanced metabolism of DGLA to prostaglandin E1 (PGE1) series,10 an eicosanoid with anti-inflammatory properties. In aggregate, these data indicate that n-3 and n-6 FAs may play a role in the pathogenesis and treatment of DES. However, several important issues remain unresolved, in particular whether FAs can be provided topically, thereby bypassing excess caloric intake and gastrointestinal adverse effects associated with their oral supplementation. The purpose of this study was to evaluate the efficacy of topical n-3 and n-6 FAs using the controlled environmental chamber murine model of dry eye.11

INDUCTION OF DRY EYE

All animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Schepens Eye Research Institute Animal Care and Use Committee. The details of the controlled environmental chamber and dry eye end points have been published previously.11 Dry eye was induced in 6- to 10-week-old C57BL/6 mice (Taconic Farms, Germantown, New York) for variable periods ranging from 2 to 10 days. Mice were placed in the controlled environmental chamber (relative humidity <30%, airflow 15 L/min, temperature 21-23°C),11 modified with subcutaneous scopolamine administration for maximal ocular dryness.11 Scopolamine (Sigma-Aldrich, St Louis, Missouri) was injected in dorsal skin of mice (0.5 mg per 0.2 mL at 9 AM, 12 PM, and 3 PM; 0.75 mg per 0.3 mL at 6 PM). Controls were age-matched mice (relative humidity >80%, no airflow, temperature 21-23°C, no scopolamine).

TOPICAL FA FORMULATIONS AND TREATMENT REGIMEN

Formulations tested included 0.2% ALA, 0.2% LA, and 0.1% ALA to 0.1% LA (1:1 ratio of n-3/n-6, total FA amount equal to the individual FA formulations). The FAs are water insoluble and hence require emulsification with compatible surfactants. The vehicle used consisted of the surfactants and emulsifiers Tween-80 (2.6%) and Glucam E-20 (2.6%), vitamin E as an antioxidant, mixed with a packing solution (water, boric acid, sodium borate, sodium chloride, and ethylenediaminetetraacetic acid) and prepared in an emulsion (Johnson and Johnson Vision Care, Inc, Jacksonville, Florida).

Forty-eight hours after dry eye induction, each eye was randomized to receive one of the formulations or the vehicle. One microliter eye drop was applied topically to the eye of unanesthetized mouse once daily from 48 hours to day 4 (total 3 doses) or day 9 (total 8 doses) depending on the time point studied. The untreated group received no eye drops. Signs of dry eye were measured 24 hours after the last dose (day 5 or day 10). Mice were then killed for cellular and molecular studies.

MEASUREMENT OF CORNEAL FLUORESCEIN STAINING

Corneal fluorescein staining was performed at baseline (day 0), 48 hours (before administration of the first eye drop dose), day 5, and day 10. One microliter of 5% fluorescein was applied into the inferior conjunctival sac as previously described.12 Eyes were flushed with phosphate-buffered saline (PBS) to remove excess fluorescein at 3 minutes and examined with slitlamp biomicroscope in cobalt blue light. Punctate staining was recorded in a masked fashion using a standardized National Eye Institute grading system of 0 to 3 for each of the 5 areas of the cornea.13 Kruskal-Wallis and Mann-Whitney tests (unpaired data set) and Wilcoxon test (paired data set) were used for statistical analysis.

IMMUNOHISTOCHEMICAL STAINING

The following primary antibodies (BD Pharmingen, San Diego, California) were used for immunohistochemical staining: FITC-conjugated rat antimouse CD11b (monocyte/macrophage marker, catalog No. 557396; isotype FITC-conjugated rat antimouse IgG2bk, catalog No. 553988), purified hamster antimouse CD3e (T-cell marker, catalog No. 553057; isotype purified hamster IgG1, catalog No. 553969), biotin-conjugated rat antimouse GR-1 (neutrophil marker, catalog No. 553124, isotype biotin-conjugated rat IgG2b, catalog No. 553987), biotin-conjugated rat antimouse Iab (C57BL/6 major histocompatibility complex [MHC] Class II marker, catalog No. 553546; isotype biotin-conjugated mouse IgG2bk, catalog No. 559531). The secondary antibodies (Jackson Laboratories, Bar Harbor, Maine) included Cy3-conjugated goat anti-Armenian hamster (code No. 127165-160) and Cy3-conjugated Strepavidin antibodies (code No. 016-160-084). For whole-mount immunofluorescence corneal staining, freshly excised corneas were washed in PBS and acetone fixed for 15 minutes. Nonspecific staining was blocked with anti-FcR CD16/CD32 antibody (BD Pharmingen, catalog No. 553142), and Strepavidin and Biotin blocking solutions (Vector Laboratories, Burlingame, California). Next, the specimens were immunostained with primary or isotype antibodies for 2 hours, washed with PBS, incubated with secondary antibodies, and mounted using Vector Shield mounting medium (Vector Laboratories). Whole-mount corneal images were taken using confocal microscope (Leica TCS 4D; Lasertechnik, Heidelberg, Germany). Cells were counted in 8 to 10 areas each in the periphery (0.5-μm area from the limbus) and the center (central 2-μm area) of the cornea in a masked fashion using an epifluorescence microscope (model E800; Nikon, Melville, New York) at ×40 magnification. The mean number of cells were obtained by averaging the cell number in the 8 to 10 areas studied. Cell number was compared using 1-way analysis of variance (ANOVA), followed by pairwise comparisons adjusted for multiple comparisons by the least significant difference method. P values less than .05 were deemed statistically significant.

RNA ISOLATION, REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION, AND REAL-TIME POLYMERASE CHAIN REACTION

Total RNA was isolated from the cornea and conjunctiva (2 pooled corneas per group and 6 pooled conjunctivae per group) using Trizol (Invitrogen, Carlsbad, California, catalog No. 15596-026) for tissue homogenization and 70% ethanol for RNA precipitation, followed by extraction and purification using RNeasy Microkit (Qiagen, Valencia, California, catalog No. 74004). RNA was stored at −80°C until further use.

The first strand complementary DNA (cDNA) was synthesized from 300 ng of total RNA using SuperScript III Reverse Transcriptase (Invitrogen, catalog No. 18080) per manufacturer's protocol. Real-time polymerase chain reaction (PCR) was performed with FAM-MGB dye-labeled predesigned primers (Applied Biosystems, Foster City, California) for IL-1α (catalog No. 4329586), tumor necrosis factor α (TNF-α) (Assay ID Mm99999068_m1), GAPDH (Mm99999915_g1), IL-2 (Mm00801778_m1), IL-4 (Mm00445259_m1), IL-6 (Mm00446190_m1), IL-10 (Mm00439616_m1), and interferon γ (IFN-γ) (Mm00801778_m1) per manufacturer's protocol. One microliter of cDNA was loaded in each well and assays were performed in duplicates. A nontemplate control was included to evaluate DNA contamination of isolated RNA and reagents. The results of quantitative real-time PCR were analyzed by the comparative threshold cycle (CT) method and normalized by GAPDH as an internal control. The relative expression of cytokines at different time points was compared using 1-way ANOVA, post hoc test least significant difference with P < .05 deemed significant.

CLINICAL AND MOLECULAR SIGNS OF DRY EYE

Compared with day 0 (mean [SD] score, 1.3 [1.5]), corneal fluorescein staining scores were significantly higher at day 2 (5.4 [2.13]), day 5 (8.4 [1.2]), and day 10 (7.9 [3.6]) (Wilcoxon test, P = .01, n = 8). No significant difference was found between groups at day 2, day 5, or day 10. Thus, dry eye induction led to a significant increase in staining that remained steadily elevated through day 10.

The normal cornea has a resident population of bone marrow–derived immature (MHC Class II−CD80−CD86−) CD11b+ antigen presenting cells (APCs) that acquire MHC Class II in response to inflammation.14,15 Induction of dry eye for 10 days increased the CD11b+ cell number in the periphery by 44% (mean [SEM], 240 [23.2] vs 345.8 [15], P = .02, n = 3) and the center by 45% (183.4 [20.2] vs 265.5 [27.8], P = .09, n = 3). MHC Class II expression by CD11b+ cells, an important marker for the cells' maturation and T-cell stimulatory capacity, was increased by 104% in the periphery (75.1 [8.2] vs 152.9 [33.7], P = .07, n = 3) and 146% in the center (30.4 [6.5] vs 74.8 [13.6], P = .04, n = 3) of the dry eye cornea.

Corneal and conjunctival expression of proinflammatory cytokines IL-1α and TNF-α, was increased in dry eye relative to the normal eye (Figure 1 and Figure 2). However, expression of TH1 (IL-2 and IFN-γ) and TH2 cytokines (IL-4, IL-6, and IL-10) was not detected in the cornea. On the contrary, the conjunctiva showed increased expression of IL-6 (14.5-fold), IL-2 (4.9-fold), IFN-γ (16.3-fold), and IL-10 (97.6-fold) (Figure 2). IL-4 expression was not increased.

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Figure 1.

Real-time polymerase chain reaction results showing increased relative expression of IL-1α and tumor necrosis factor α (TNF-α) transcripts in dry eye corneas compared with normal corneas. Asterisk indicates P < .001. Data are presented as mean and standard error (error bars); n = 6 for day 0, n = 4 for day 2, n = 8 for day 5, and n = 4 for day 10.

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Figure 2.

Real-time polymerase chain reaction results showing increased relative expression of various cytokine transcripts in dry eye conjunctiva (day 10) compared with normal conjunctiva. Data are presented as mean and standard error (error bars); n = 18 for IL-1α and tumor necrosis factor α (TNF-α), and n = 6 for remaining cytokines. IFN-γ indicates interferon γ.

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CORNEAL FLUORESCEIN STAINING IN DRY EYE TREATED WITH FA FORMULATIONS

Two days after dry eye induction, eyes were randomized to receive 1 μL of ALA, LA, combined ALA and LA, or vehicle or no eye drops. Corneal fluorescein staining scores were measured at days 5 and 10. Only ALA-treated eyes showed a sustained and significant decrease in staining compared with the untreated and vehicle control groups at both days 5 and 10 (Figure 3). At day 5, all the treatment groups showed a significant decrease in staining compared with the untreated group. However, only ALA-treated eyes showed a significant decrease compared with the vehicle (45%, P = .04); no significant difference was noted between the eyes treated with LA, combined ALA and LA, and vehicle. At day 10, only the ALA-treated eyes showed a significant decrease in the corneal fluorescein staining compared with the vehicle (62%, P = .02) and untreated controls (71%, P = .007). No difference was seen between the eyes treated with vehicle, LA, and combined ALA and LA and untreated eyes at day 10 (Figure 3).

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Figure 3.

Topical alpha-linolenic acid (ALA) treatment produces a sustained decrease in corneal fluorescein staining compared with the vehicle-treated and untreated controls at days 5 and 10. Asterisk indicates P = .001 vs untreated and P = .04 vs vehicle; dagger, P = .007 vs untreated and P = .02 vs vehicle. Data are presented as mean and standard error (error bars); n = 8 for the untreated, vehicle, ALA, and linoleic acid (LA) groups and n = 6 for the combined ALA and LA treatment group.

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ENUMERATION OF CD11b+ MONOCYTES IN EYES TREATED WITH FA FORMULATIONS

The number of CD11b+ cells was found to be significantly decreased (P = .03) in ALA-treated eyes in the center of the cornea as compared with the untreated group and the vehicle, LA, and combined ALA and LA groups (Figure 4 and Figure 5). In the periphery, there was no significant difference between vehicle and ALA groups, although ALA treatment showed a significant decrease compared with untreated eyes (P = .001) (Figure 4). Treatment with ALA decreased the cell number by 37% (periphery) and 42% (center) compared with the untreated group and 21% (periphery) and 37% (center) compared with the vehicle. None of the other groups showed a significant difference in corneal cell number compared with the vehicle.

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Figure 4.

Alpha-linolenic acid (ALA) treatment decreases the number of CD11b+ cells in the periphery and center of dry eye corneas. Asterisk indicates P = .03 vs untreated; dagger, P = .001 vs untreated and P = .07 vs vehicle; and double dagger, P = .01 vs untreated and P = .03 vs vehicle. Data are presented as mean and standard error (error bars) and n = 3. LA indicates linoleic acid.

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Figure 5.

Representative confocal images of center of whole-mount corneas showing CD11b+ cells (green). Images show normal eyes (A); untreated eyes (B); and eyes treated with vehicle (C), alpha-linolenic acid (ALA) (D), linoleic acid (LA) (E), and combined ALA and LA (F). The number of CD11b+ cells is comparable with the normal (nondry) cornea only in the ALA-treated group.

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CORNEAL AND CONJUNCTIVAL EXPRESSION OF IL-1α AND TNF-α IN EYES TREATED WITH FA FORMULATIONS

Among-group comparisons showed that only ALA treatment persistently decreased corneal and conjunctival expression of IL-1α and TNF-α at days 5 and 10 compared with untreated eyes and eyes treated with vehicle, LA, and combined ALA and LA (Figure 6 and Figure 7). The conjunctival expression was only studied at day 10 since higher level of these cytokines was seen in the cornea at day 10. Only ALA-treated eyes showed significant decrease in IL-1α expression compared with untreated corneas at day 10 (P = .04), significant decrease in TNF-α expression compared with untreated (P = .02) and vehicle-treated corneas (P = .04) at day 5, and a decreased trend at day 10 (P = .07). Expression of TNF-α in ALA-treated conjunctivae was significantly decreased at day 10 compared with untreated eyes (P = .004) and a downward trend was also seen in IL-1α expression. No significant difference was seen between the untreated eyes and eyes treated with vehicle, LA, or combined ALA and LA in either IL-1α or TNF-α expression.

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Figure 6.

Real-time polymerase chain reaction results showing that corneas treated with alpha-linolenic acid (ALA) have significantly decreased relative expression of (A) IL-1α (asterisk indicates P = .04 vs untreated) and (B) tumor necrosis factor α (TNF-α) transcripts (asterisk indicates P = .02 vs untreated and P = .04 vs vehicle). Data are presented as mean and standard error (error bars); n = 6 for day 5 and n = 4 for day 10. LA indicates linoleic acid; mRNA, messenger RNA.

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Figure 7.

Real-time polymerase chain reaction results showing that conjunctiva treated with alpha-linolenic acid (ALA) has decreased relative expression of IL-1α and tumor necrosis factor α (TNF-α) transcripts. Asterisk indicates P = .004 vs untreated. Data are presented as mean and standard error (error bars) and n = 18. LA indicates linoleic acid; mRNA, messenger RNA.

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The preponderance of evidence suggests that inflammation, whether a cause or effect or both, frequently accompanies DES in rodents16 and humans.17 Artificial tears, the most common therapy for DES, often provide temporary symptomatic relief but do not address the underlying pathogenic mechanisms that lead to DES. The current study demonstrates for the first time a beneficial effect of topical application of the n-3 FA ALA in treating the ocular signs and reversing the inflammatory changes of dry eye at both molecular and cellular levels.

The ALA-treated eyes showed a significant reversal in corneal epithelial damage, manifested by decreased fluorescein staining as compared with the untreated eyes and eyes treated with vehicle, LA, or combined ALA and LA. The exact mechanism of corneal epithelial repair in the ALA-treated eyes is unknown but could theoretically be mediated directly by ALA or its metabolites, EPA and DHA. In healthy individuals, nearly 5% to 10% of dietary ALA is converted sequentially to EPA and DHA18,19 by delta-5 and delta-6 desaturase enzymes. Human corneal epithelial cells express the enzyme 15-lipoxygenase (ALOX15),20,21 and the endogenous formation of neuroprotectin D1 (NPD1), a novel DHA-derived ALOX15 product, has been reported in the murine cornea.22 Topical NPD1 application increases the re-epithelization rate in a mouse corneal wound model.22 Thus, endogenous production of NPD1 from topically administered ALA may be one of the mechanisms for reversing corneal epitheliopathy in DES.

At the molecular level, dry eye induction leads to a persistent increase in corneal expression of IL-1α and TNF-α. These cytokines are important mediators of inflammation implicated in the pathogenesis of corneal ulceration, uveitis, and corneal transplant rejection.2326 Produced constitutively in the corneal epithelium and on release by injury or death,27,28 IL-1α can up-regulate TNF-α release and its own autocrine production.29 Tumor necrosis factor α has been implicated as an important mediator of pathogenesis in DES.30 Elevated gene expression of IL-1, IL-6, IL-8, and TNF-α in the conjunctival epithelium31 and a higher tear concentration of IL-1 has been reported in patients with DES.32

Of the formulations tested, only ALA treatment was effective in decreasing the corneal and conjunctival expression of IL-1α and TNF-α. Because these cytokines are released early in response to epithelial cell damage and are also released by activated macrophages, the epithelial repair and decreased macrophages infiltration in the ALA-treated cornea may account for the decreased cytokine expression. Dietary ALA has been shown to decrease endotoxin-induced macrophage production of TNF-α.33 Healthy humans, when fed an ALA-rich diet, have shown to suppress IL-1β and TNF-α production by 30%.34,35 However, the precise mechanism of this suppression is not yet understood.

Our study showed a nearly 100-fold increased expression of IL-10 in dry eye conjunctiva. Interleukin 10 is produced by activated macrophages and some lymphocytes. The 2 major activities of IL-10 are to inhibit IL-1 and TNF production by macrophages and to inhibit the accessory function of macrophages in T-cell activation through reduced expression of MHC Class II.36 Consequently, IL-10 inhibits both innate and T-cell–mediated immunity. The enhanced IL-10 expression may represent a regulatory mechanism in the ocular surface to promote quiescence and maintain normal homeostasis.

Our study showed the novel finding of corneal infiltration by mature MHC Class II–expressing APCs in dry eye. The normal central cornea has a resident population of CD45+ bone marrow–derived cells that are uniformly of a highly immature (MHC Class II−CD80−CD86−) phenotype14,37 and acquire high expression of maturation markers in response to inflammation, thereby enhancing their capacity to stimulate T-cell–mediated responses.14,15 The vast majority of these resident cells are CD11b+ and of a monocyte/macrophage lineage.38 Up-regulation of HLA-DR in the conjunctiva of patients with DES has been reported,39 but to our knowledge, this is the first study to report corneal leukocytic infiltration and activation in dry eye. Interestingly, topical ALA application showed a reduction in the corneal leukocytic infiltration. This may be partly accounted for by the decreased cytokine expression, especially TNF-α. Both IL-1α and TNF-α can induce corneal APC mobilization and IL-1 induction of these cells' migration is largely mediated by TNF receptor function.40 These changes are important in disease pathogenesis and severity since the corneal APC mobilization is known to have a significant effect on the degree and rapidity of corneal immune reactions, including graft rejection41 and herpes keratitis.42,43

Previous studies have shown variable effects of LA and GLA oral intake in dry eye.7,8,44 In 2 clinical trials, oral LA and GLA intake was shown to ameliorate dry eye associated ocular discomfort,7,8 corneal epitheliopathy,7 and lissamine green staining of the conjunctiva8 and increase the tear PGE1 content.7 However, another randomized trial comparing GLA and placebo in 90 patients with Sjögren syndrome found no significant difference in dry eye signs and symptoms between the treatment and placebo groups.44 Another cross-sectional study showed no independent relationship between dietary LA intake and dry eye.9

In our study, we found no beneficial effect of combined LA and ALA formulation. The lack of beneficial effect in this combination may in part be dose-dependent. For example, it may be possible that a beneficial effect would be seen at higher doses of LA and ALA or with a different n-3 to n-6 ratio. However, the consistent failure of LA to demonstrate any positive effect clinically and cellularly, and its reversal of ALA therapeutic efficacy when a combination is used, suggests that at least for topical therapy, ALA (n-3 FA) is preferred. Recent data have emerged showing polymorphic variations in the desaturase enzymes, delta-5 and delta-6 desaturase, that can affect the FA composition in phospholipids.45 These insights suggest that common genetic variations in these and other genes might influence LA metabolism toward greater production of the more inflammatory eicosanoid products of AA rather than the relatively less inflammatory products of DGLA.45 Therefore, the combination of an n-3 FA (eg, ALA) with one of n-6 FA conversion products, such as DGLA, may very well demonstrate efficacy levels beyond what our current data suggest. Our present study does not compare the changes seen with topical ALA treatment with other available topical anti-inflammatory agents, such as steroids; such direct comparisons may be worthwhile future studies.

In summary, our study shows the beneficial effect of ALA omega-3 FA topical application in reversing the signs and the underlying inflammatory changes seen in dry eye. The use of these fatty acids in topical formulations to treat dry eye and potentially other inflammatory ocular surface conditions, would allow more flexibility in dosing without the accompanying systemic, in particular gastrointestinal, adverse effects that can be seen with oral intake of these fatty acids. Further studies are clearly indicated to optimize dosing and formulations that are maximally effective.

Correspondence: Reza Dana, MD, MSc, MPH, Schepens Eye Research Institute, 20 Staniford St, Boston, MA 02114 (reza.dana@schepens.harvard.edu).

Submitted for Publication: April 26, 2007; final revision received June 27, 2007; accepted July 4, 2007.

Financial Disclosure: Dr Schaumberg has served as a consultant to Vistakon and Johnson and Johnson. Dr Dana has served as a consultant to and received research support from Vistakon and Johnson and Johnson. Drs Rashid, Schaumberg, and Dana are listed as coinventors on the patent application for “compositions and methods for treating eye disorders and conditions.”

Funding/Support: This study was supported in part by Johnson and Johnson Vision Care, Inc, and the Sjögren's Syndrome Foundation.

Additional Information: Sandra Michaud, PhD, assisted with real-time PCR, Qiang Zhang, MD, assisted with immunohistochemical staining, and Don Pottle assisted with confocal microscopy.

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Brash  ARBoeglin  WEChang  MS Discovery of a second 15S-lipoxygenase in humans. Proc Natl Acad Sci U S A 1997;94 (12) 6148- 6152
PubMed Link to Article
Liminga  MOliw  E cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium. Biochim Biophys Acta 1999;1437 (2) 124- 135
PubMed Link to Article
Gronert  KMaheswari  NKhan  NHassan  IRDunn  MLaniado Schwartzman  M A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense. J Biol Chem 2005;280 (15) 15267- 15278
PubMed Link to Article
Dana  MRYamada  JStreilein  JW Topical interleukin 1 receptor antagonist promotes corneal transplant survival. Transplantation 1997;63 (10) 1501- 1507
PubMed Link to Article
Brito  BEO’Rourke  LMPan  YAnglin  JPlanck  SRRosenbaum  JT IL-1 and TNF receptor-deficient mice show decreased inflammation in an immune complex model of uveitis. Invest Ophthalmol Vis Sci 1999;40 (11) 2583- 2589
PubMed
Rosenbaum  JTPlanck  STHuang  XNRick  LAnsel  JC Detection of mRNA for the cytokines, interleukin-1 alpha and interleukin-8, in corneas from patients with pseudophakic bullous keratopathy. Invest Ophthalmol Vis Sci 1995;36 (10) 2151- 2155
PubMed
Fabre  EJBureau  JPouliquen  YLorans  G Binding sites for human interleukin 1 alpha, gamma interferon and tumor necrosis factor on cultured fibroblasts of normal cornea and keratoconus. Curr Eye Res 1991;10 (7) 585- 592
PubMed Link to Article
Wilson  SEMohan  RRMohan  RRAmbrosio  R  JrHong  JLee  J The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog Retin Eye Res 2001;20 (5) 625- 637
PubMed Link to Article
Wilson  SEHe  YGWeng  J  et al.  Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996;62 (4) 325- 327
PubMed Link to Article
West-Mays  JAStrissel  KJSadow  PMFini  ME Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop. Proc Natl Acad Sci U S A 1995;92 (15) 6768- 6772
PubMed Link to Article
Trousdale  MDZhu  ZStevenson  DSchechter  JERitter  TMircheff  AK Expression of TNF inhibitor gene in the lacrimal gland promotes recovery of tear production and tear stability and reduced immunopathology in rabbits with induced autoimmune dacryoadenitis. J Autoimmune Dis 2005;26
PubMed Link to Article
Pflugfelder  SCJones  DJi  ZAfonso  AMonroy  D Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjogren's syndrome keratoconjunctivitis sicca. Curr Eye Res 1999;19 (3) 201- 211
PubMed Link to Article
Solomon  ADursun  DLiu  ZXie  YMacri  APflugfelder  SC Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 2001;42 (10) 2283- 2292
PubMed
Morris  DDHenry  MMMoore  JNFischer  JK Effect of dietary alpha-linolenic acid on endotoxin-induced production of tumor necrosis factor by peritoneal macrophages in horses. Am J Vet Res 1991;52 (4) 528- 532
PubMed
James  MJGibson  RACleland  LG Dietary polyunsaturated fatty acids and inflammatory mediator production. Am J Clin Nutr 2000;71 (1) ((suppl)) 343S- 348S
PubMed
Endres  SGhorbani  RKelley  VE  et al.  The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 1989;320 (5) 265- 271
PubMed Link to Article
Abbas  AKLichtman  AHPober  JS Cytokines: Cellular and Molecular Immunology.  Philadelphia, PA WB Saunders Co1997;250- 276
Hamrah  PLiu  YZhang  QDana  MR The corneal stroma is endowed with a significant number of resident dendritic cells. Invest Ophthalmol Vis Sci 2003;44 (2) 581- 589
PubMed Link to Article
Hamrah  PHuq  SLiu  YZhang  QDana  MR Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003;74 (2) 172- 178
PubMed Link to Article
Stern  MEGao  JSchwalb  TA  et al.  Conjunctival T-cell subpopulations in Sjogren's and non-Sjogren's patients with dry eye. Invest Ophthalmol Vis Sci 2002;43 (8) 2609- 2614
PubMed
Dekaris  IZhu  SDana  MR TNF-alpha regulates corneal Langerhans cell migration. J Immunol 1999;162 (7) 4235- 4239
PubMed
Callanan  DPeeler  JNiederkorn  JY Characteristics of rejection of orthotopic corneal allografts in the rat. Transplantation 1988;45 (2) 437- 443
PubMed Link to Article
McLeish  WRubsamen  PAtherton  SSStreilein  JW Immunobiology of Langerhans cells on the ocular surface: II, role of central corneal Langerhans cells in stromal keratitis following experimental HSV-1 infection in mice. Reg Immunol 1989;2 (4) 236- 243
PubMed
Jager  MJBradley  DAtherton  SStreilein  JW Presence of Langerhans cells in the central cornea linked to the development of ocular herpes in mice. Exp Eye Res 1992;54 (6) 835- 841
PubMed Link to Article
Theander  EHorrobin  DFJacobsson  LTManthrope  R Gammalinolenic acid treatment of fatigue associated with primary Sjogren's syndrome. Scand J Rheumatol 2002;31 (2) 72- 79
PubMed Link to Article
Schaeffer  LGohlke  HMuller  M  et al.  Common genetic variants of the FADS1 FADS2 gene cluster and their reconstructed haplotypes are associated with the fatty acid composition in phospholipids. Hum Mol Genet 2006;15 (11) 1745- 1756
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Real-time polymerase chain reaction results showing increased relative expression of IL-1α and tumor necrosis factor α (TNF-α) transcripts in dry eye corneas compared with normal corneas. Asterisk indicates P < .001. Data are presented as mean and standard error (error bars); n = 6 for day 0, n = 4 for day 2, n = 8 for day 5, and n = 4 for day 10.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Real-time polymerase chain reaction results showing increased relative expression of various cytokine transcripts in dry eye conjunctiva (day 10) compared with normal conjunctiva. Data are presented as mean and standard error (error bars); n = 18 for IL-1α and tumor necrosis factor α (TNF-α), and n = 6 for remaining cytokines. IFN-γ indicates interferon γ.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Topical alpha-linolenic acid (ALA) treatment produces a sustained decrease in corneal fluorescein staining compared with the vehicle-treated and untreated controls at days 5 and 10. Asterisk indicates P = .001 vs untreated and P = .04 vs vehicle; dagger, P = .007 vs untreated and P = .02 vs vehicle. Data are presented as mean and standard error (error bars); n = 8 for the untreated, vehicle, ALA, and linoleic acid (LA) groups and n = 6 for the combined ALA and LA treatment group.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Alpha-linolenic acid (ALA) treatment decreases the number of CD11b+ cells in the periphery and center of dry eye corneas. Asterisk indicates P = .03 vs untreated; dagger, P = .001 vs untreated and P = .07 vs vehicle; and double dagger, P = .01 vs untreated and P = .03 vs vehicle. Data are presented as mean and standard error (error bars) and n = 3. LA indicates linoleic acid.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Representative confocal images of center of whole-mount corneas showing CD11b+ cells (green). Images show normal eyes (A); untreated eyes (B); and eyes treated with vehicle (C), alpha-linolenic acid (ALA) (D), linoleic acid (LA) (E), and combined ALA and LA (F). The number of CD11b+ cells is comparable with the normal (nondry) cornea only in the ALA-treated group.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Real-time polymerase chain reaction results showing that corneas treated with alpha-linolenic acid (ALA) have significantly decreased relative expression of (A) IL-1α (asterisk indicates P = .04 vs untreated) and (B) tumor necrosis factor α (TNF-α) transcripts (asterisk indicates P = .02 vs untreated and P = .04 vs vehicle). Data are presented as mean and standard error (error bars); n = 6 for day 5 and n = 4 for day 10. LA indicates linoleic acid; mRNA, messenger RNA.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 7.

Real-time polymerase chain reaction results showing that conjunctiva treated with alpha-linolenic acid (ALA) has decreased relative expression of IL-1α and tumor necrosis factor α (TNF-α) transcripts. Asterisk indicates P = .004 vs untreated. Data are presented as mean and standard error (error bars) and n = 18. LA indicates linoleic acid; mRNA, messenger RNA.

Graphic Jump Location

Tables

References

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PubMed Link to Article
Schein  ODHochberg  MCMunoz  B  et al.  Dry eye and dry mouth in the elderly: a population-based assessment. Arch Intern Med 1999;159 (12) 1359- 1363
PubMed Link to Article
Miljanovic  BMDana  RSullivan  DASchaumberg  DA Impact of dry eye syndrome on vision-related quality of life among women. Am J Ophthalmol 2007;143 (3) 409- 415
PubMed Link to Article
Stern  MEPflugfelder  SC Inflammation in dry eye. Ocul Surf 2004;2 (2) 124- 130
PubMed Link to Article
Pflugfelder  SC Antiinflammatory therapy for dry eye. Am J Ophthalmol 2004;137 (2) 337- 342
PubMed Link to Article
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PubMed Link to Article
Aragona  PBucolo  CSpinella  RGiuffrida  SFerreri  G Systemic omega-6 essential fatty acid treatment and pge1 tear content in Sjogren's syndrome patients. Invest Ophthalmol Vis Sci 2005;46 (12) 4474- 4479
PubMed Link to Article
Barabino  SRolando  MCamicione  P  et al.  Systemic linoleic and gamma-linolenic acid therapy in dry eye syndrome with an inflammatory component. Cornea 2003;22 (2) 97- 101
PubMed Link to Article
Miljanovic  BTrivedi  KADana  MRGilbard  JPBuring  JESchaumberg  DA Relation between dietary n-3 and n-6 fatty acids and clinically diagnosed dry eye syndrome in women. Am J Clin Nutr 2005;82 (4) 887- 893
PubMed
Simopoulos  AP The importance of ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmocother 2002;56 (8) 365- 379
PubMed Link to Article
Barabino  SShen  LChen  LRashid  SRolandao  MDana  MR The controlled-environment chamber: a new mouse model of dry eye. Invest Ophthalmol Vis Sci 2005;46 (8) 2766- 2771
PubMed Link to Article
Barabino  SChen  WDana  MR Tear film and ocular surface tests in animal models of dry eye: uses and limitations. Exp Eye Res 2004;79 (5) 613- 621
PubMed Link to Article
Lemp  MA Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21 (4) 221- 232
PubMed
Hamrah  PZhang  QLiu  YDana  MR Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells. Invest Ophthalmol Vis Sci 2002;43 (3) 639- 646
PubMed
Hamrah  PLiu  YZhang  QDana  MR Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol 2003;121 (8) 1132- 1140
PubMed Link to Article
Luo  LLi  DQDoshi  AFarley  WCorrales  RMPflugfelder  SC Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 2004;45 (12) 4293- 4301
PubMed Link to Article
Gulati  ASacchetti  MBonini  SDana  R Chemokine receptor CCR5 expression in conjunctival epithelium of patients with dry eye syndrome. Arch Ophthalmol 2006;124 (5) 710- 716
PubMed Link to Article
Emken  EAAdlof  ROGulley  RM Dietary linoleic acid influences desaturation and acylation of deuterium-labeled linoleic and linolenic acids in young adult males. Biochim Biophys Acta 1994;1213 (3) 277- 288
PubMed Link to Article
Gerster  H Can adults adequately convert alpha-linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)? Int J Vitam Nutr Res 1998;68 (3) 159- 173
PubMed
Brash  ARBoeglin  WEChang  MS Discovery of a second 15S-lipoxygenase in humans. Proc Natl Acad Sci U S A 1997;94 (12) 6148- 6152
PubMed Link to Article
Liminga  MOliw  E cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium. Biochim Biophys Acta 1999;1437 (2) 124- 135
PubMed Link to Article
Gronert  KMaheswari  NKhan  NHassan  IRDunn  MLaniado Schwartzman  M A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense. J Biol Chem 2005;280 (15) 15267- 15278
PubMed Link to Article
Dana  MRYamada  JStreilein  JW Topical interleukin 1 receptor antagonist promotes corneal transplant survival. Transplantation 1997;63 (10) 1501- 1507
PubMed Link to Article
Brito  BEO’Rourke  LMPan  YAnglin  JPlanck  SRRosenbaum  JT IL-1 and TNF receptor-deficient mice show decreased inflammation in an immune complex model of uveitis. Invest Ophthalmol Vis Sci 1999;40 (11) 2583- 2589
PubMed
Rosenbaum  JTPlanck  STHuang  XNRick  LAnsel  JC Detection of mRNA for the cytokines, interleukin-1 alpha and interleukin-8, in corneas from patients with pseudophakic bullous keratopathy. Invest Ophthalmol Vis Sci 1995;36 (10) 2151- 2155
PubMed
Fabre  EJBureau  JPouliquen  YLorans  G Binding sites for human interleukin 1 alpha, gamma interferon and tumor necrosis factor on cultured fibroblasts of normal cornea and keratoconus. Curr Eye Res 1991;10 (7) 585- 592
PubMed Link to Article
Wilson  SEMohan  RRMohan  RRAmbrosio  R  JrHong  JLee  J The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog Retin Eye Res 2001;20 (5) 625- 637
PubMed Link to Article
Wilson  SEHe  YGWeng  J  et al.  Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996;62 (4) 325- 327
PubMed Link to Article
West-Mays  JAStrissel  KJSadow  PMFini  ME Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop. Proc Natl Acad Sci U S A 1995;92 (15) 6768- 6772
PubMed Link to Article
Trousdale  MDZhu  ZStevenson  DSchechter  JERitter  TMircheff  AK Expression of TNF inhibitor gene in the lacrimal gland promotes recovery of tear production and tear stability and reduced immunopathology in rabbits with induced autoimmune dacryoadenitis. J Autoimmune Dis 2005;26
PubMed Link to Article
Pflugfelder  SCJones  DJi  ZAfonso  AMonroy  D Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjogren's syndrome keratoconjunctivitis sicca. Curr Eye Res 1999;19 (3) 201- 211
PubMed Link to Article
Solomon  ADursun  DLiu  ZXie  YMacri  APflugfelder  SC Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 2001;42 (10) 2283- 2292
PubMed
Morris  DDHenry  MMMoore  JNFischer  JK Effect of dietary alpha-linolenic acid on endotoxin-induced production of tumor necrosis factor by peritoneal macrophages in horses. Am J Vet Res 1991;52 (4) 528- 532
PubMed
James  MJGibson  RACleland  LG Dietary polyunsaturated fatty acids and inflammatory mediator production. Am J Clin Nutr 2000;71 (1) ((suppl)) 343S- 348S
PubMed
Endres  SGhorbani  RKelley  VE  et al.  The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 1989;320 (5) 265- 271
PubMed Link to Article
Abbas  AKLichtman  AHPober  JS Cytokines: Cellular and Molecular Immunology.  Philadelphia, PA WB Saunders Co1997;250- 276
Hamrah  PLiu  YZhang  QDana  MR The corneal stroma is endowed with a significant number of resident dendritic cells. Invest Ophthalmol Vis Sci 2003;44 (2) 581- 589
PubMed Link to Article
Hamrah  PHuq  SLiu  YZhang  QDana  MR Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003;74 (2) 172- 178
PubMed Link to Article
Stern  MEGao  JSchwalb  TA  et al.  Conjunctival T-cell subpopulations in Sjogren's and non-Sjogren's patients with dry eye. Invest Ophthalmol Vis Sci 2002;43 (8) 2609- 2614
PubMed
Dekaris  IZhu  SDana  MR TNF-alpha regulates corneal Langerhans cell migration. J Immunol 1999;162 (7) 4235- 4239
PubMed
Callanan  DPeeler  JNiederkorn  JY Characteristics of rejection of orthotopic corneal allografts in the rat. Transplantation 1988;45 (2) 437- 443
PubMed Link to Article
McLeish  WRubsamen  PAtherton  SSStreilein  JW Immunobiology of Langerhans cells on the ocular surface: II, role of central corneal Langerhans cells in stromal keratitis following experimental HSV-1 infection in mice. Reg Immunol 1989;2 (4) 236- 243
PubMed
Jager  MJBradley  DAtherton  SStreilein  JW Presence of Langerhans cells in the central cornea linked to the development of ocular herpes in mice. Exp Eye Res 1992;54 (6) 835- 841
PubMed Link to Article
Theander  EHorrobin  DFJacobsson  LTManthrope  R Gammalinolenic acid treatment of fatigue associated with primary Sjogren's syndrome. Scand J Rheumatol 2002;31 (2) 72- 79
PubMed Link to Article
Schaeffer  LGohlke  HMuller  M  et al.  Common genetic variants of the FADS1 FADS2 gene cluster and their reconstructed haplotypes are associated with the fatty acid composition in phospholipids. Hum Mol Genet 2006;15 (11) 1745- 1756
PubMed Link to Article

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