Systemic Omega-6 Essential Fatty Acid Treatment and PGE₁ Tear Content in Sjögren’s Syndrome Patients

Abstract

purpose. To determine the effect of oral omega-6 essential fatty acids on PGE₁ tear content and signs and symptoms of ocular discomfort in patients with Sjögren’s syndrome (SS).

methods. This randomized, double-masked, controlled, clinical trial involved 40 patients with primary SS, divided into two groups: group 1: 20 patients (18 women, 2 men; mean age, 36.9 ± 7.9 years [SD]) treated for 1 month with linoleic acid (LA; 112 mg), and γ-linolenic acid (GLA; 15 mg) administered twice daily; group 2: 20 patients (19 women, 1 man; mean age, 36.3 ± 5.5 years) treated twice daily with placebo. Patients underwent three examinations: at baseline (T0), after 1 month of treatment (T1), and 15 days after suspension of treatment (T2). At each examination, the following tests were performed: tear sampling (2 μL) from the inferior meniscus, tear break-up time (BUT), fluorescein stain of the ocular surface, and tear basal secretion. A symptom score was also obtained at each examination. PGE₁ was evaluated by enzyme immunoassay. The primary efficacy variable was PGE₁ content of tears.

results. The tear PGE₁ levels were significantly increased in group 1 at T1 versus T0 (PGE₁ level: T0, 44 ± 5.4 ng/mL; T1, 58.3 ± 5.5 ng/mL; P < 0.01 versus T0 and group 2 at T1). At examination T2, a statistically significant reduction of PGE₁ levels toward baseline was observed (45.7 ± 5.2 ng/mL; P < 0.01 versus T1). A statistically significant reduction of symptom score was observed in group 1 at examination T1 (P < 0.01 versus T0 and group 2 score). At examination T2, the symptom score was significantly higher than T1 but remained lower than T0. The corneal fluorescein stain in group 1 showed a statistically significant improvement at examination T1 versus T0 and group 2 (P < 0.01). This improvement was also present at T2 (P < 0.02). No statistically significant differences were found for the other tests. No statistically significant changes were observed in the patients in group 2 at all examination time points.

conclusions. Omega-6 administration increases the PGE₁ levels in tears of patients with SS and improves ocular surface signs and symptoms of ocular discomfort.

Sjögren’s syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of exocrine glands and mucosae. ^{1 2 3} The ocular surface shows, in both conjunctiva and lacrimal glands, T-cell infiltration and upregulation of expression in markers of immune activation. ^{4 5}  

It has been shown that topical anti-inflammatory treatment of patients with dry eye produces a significant reduction of activated lymphocyte in the conjunctiva, ⁶ thus demonstrating the potential usefulness of an anti-inflammatory treatment of dry eyes. 

γ-Linolenic acid (GLA) and its precursor linoleic acid (LA) are essential fatty acids found in certain plant seed oils. Their administration has been reported to have beneficial effects in the treatment of chronic inflammatory disorders, such as rheumatoid arthritis (RA), determining significant improvements in several clinical cases. ⁷  

GLA is metabolized to dihomo-γ-linolenic acid (DGLA), the immediate precursor of PGE₁, an eicosanoid with known anti-inflammatory properties. ^{8 9 10 11} In addition, both GLA and DGLA modulate the immune responses in an eicosanoid-independent manner by acting directly on T lymphocytes. ^{12 13 14} Administration of GLA reduces joint pain and swelling, as well as the need for nonsteroidal anti-inflammatory drugs and corticosteroids in patients with rheumatoid arthritis (RA) and active synovitis. ^{15 16 17} The addition of GLA to cells in vitro or the administration of GLA in vivo reduces secretion of IL-1β from lipopolysaccharide (LPS)-stimulated human mononuclear cells. ¹⁸ In fact, it has been shown that GLA reduces the autoinduction of IL-1β by inducing a protein that reduces pro-IL-1β mRNA stability. IL-1β is important in host defense, but the autoinduced amplification mechanism may be excessive in genetically predisposed patients. Thus, reduction of IL-1β autoinduction may be protective in some patients with diseases characterized by chronic inflammation. ¹⁹  

A possible role of GLA in the therapy of Sjögren’s syndrome and other immunologic disorders has been indicated. ²⁰ In patients with SS, the plasma and erythrocyte membrane levels of fatty acids of both the omega-6 and -3 series of fatty acids correlates inversely with levels of IgM rheumatoid factor and anti SSA/Ro antibodies. ²¹ It has also been reported that treatment with a mixture of LA and GLA improves the objective ocular status of patients with primary SS significantly and increases significantly the plasma levels of the GLA metabolite and PGE₁ precursor DGLA, although no correlation has been found between objective ocular status and DGLA levels in plasma and erythrocytes. ²² These data indicate that omega-6 treatment may play a role in the treatment of SS, whereas no data are available, as far as we know, about the possible effect of omega-3 fatty acids. 

Treatment with systemic LA and GLA has also been shown to reduce the expression of the inflammatory marker HLA-DR in conjunctival cells of subjects with dry eye. ²³  

However, there are no existing data showing a modification of tear content of PGE₁ after systemic therapy with essential fatty acids. 

The purpose of the present study was to evaluate the effects of treatment with systemic LA and GLA on the ocular surface and lacrimal secretion of patients with SS and to see whether this treatment could induce changes in the PGE₁ content of tears. 

Methods

Patient Population

The present research was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the University of Messina. 

All patients were aged more than 18 years and provided informed consent before being recruited into the study and after receiving a full explanation of all procedures and possible consequences. 

Forty patients (37 women and 3 men; average age, 36.6 ± 6.7 years) affected by primary SS that was diagnosed according to the classification criteria for Sjögren’s syndrome proposed by the American-European Consensus Group, ²⁴ were enrolled for this randomized, double-masked, controlled, clinical trial at the Ocular Surface Unit of the Department of Surgical Specialties of the University of Messina. All patients in both groups were instructed to follow their usual diets. A diary to register major deviations from the usual diet and from the study protocol was given to each patient at the beginning of the study and was returned at the follow-up visit. 

Inclusion criteria were ability and willingness to participate in the study; stable disease and general therapy for at least 1 month; no local treatment other than preservative-free cellulose-derivative eye drops; moderate-to-severe dry eye, as defined by tear basal secretion less than 5 mm/5 minutes, break-up time (BUT) less than 7 seconds, and signs of corneal damage demonstrated by corneal fluorescein staining (at least 2+ according to Lemp). ²⁵ During the course of the study, the patients were allowed to continue their general and local therapy. 

Exclusion criteria were as follows: any systemic disease different from SS; menopausal status; presence of systemic treatment with drugs such as β-blocking agents, benzodiazepines, hormones, or antihistamine agents that could interfere with tear production; and use of nonsteroidal anti-inflammatory drugs (NSAIDs) or steroids. 

Study Treatment

Once recruited, patients were randomly divided into two groups, according to a table of random numbers, and assigned, in a double-masked manner, to the following treatments: group 1, 20 patients (18 women, 2 men; mean age, 36.9 ± 7.9 years [SD]) assigned to oral treatment twice daily for 1 month with sachets containing LA (112 mg) and GLA (15 mg), and group 2, 20 patients (19 women, 1 man; mean age, 36.3 ± 5.5 years) treated twice daily with placebo sachets containing the excipients of the study drug (Table 1) . Patients were instructed to take the sachets diluted in water with a 12-hour interval between administrations. 

Tests Performed

Patients underwent three examinations in the course of the study: at baseline (T0), after 1 month of treatment (T1), and 15 days after suspension of treatment (T2). At each examination the tests were performed in the following order. 

Grading of Subjective Symptoms.

Self-reported symptoms were graded by means of a questionnaire administered at the beginning of each examination by a different observer (RS) from the one who performed the examinations (PA). The following symptoms were evaluated according to a scoring system from 0 (absent) to 3 (severe): burning, itching, foreign body sensation, dryness, mucous discharge, and photophobia. A global score, obtained by summing the scores of each symptom, was considered for the evaluation of ocular discomfort. 

Tear Sampling.

Samples were obtained from the inferior tear meniscus with glass capillary tubes (Behring Diagnostics, GmbH, Marburg, Germany). Care was taken to illuminate only the region of the lower tear meniscus where collection took place, to limit light stimulation. Furthermore, the procedure was performed taking care to avoid touching the lid margin and conjunctiva, to minimize stimulated tear production. Tear collection was immediately suspended if flow was seen or reported by the patients. 

Two microliters of tears were collected from each patient. The collected fluid was transferred into a 1-mL Eppendorf tube and 98 μL of assay buffer containing indomethacin (10 μg/mL) was added. Tear samples were stored at −20°C until analysis. 

Tear BUT.

A fluorescein break-up time (BUT) test for the evaluation of tear film stability was performed. The procedure suggested by Lemp ²⁵ was followed. A fluorescein strip (Haag-Streit AG, Köniz, Switzerland) was dampened with a drop of nonpreserved saline, and the strip was touched to the inferior palpebral conjunctiva. Patients were asked to blink several times to mix the fluorescein with the tear film. They were asked to open their eyes, and the time between the opening of the eyes and the appearance of the first dry spot was measured three times. The median time was recorded as representative of the patient’s BUT. 

Assessment of the Corneal Epithelium.

Fluorescein vital staining to evaluate the corneal epithelium, as suggested by Lemp, ²⁵ was performed after evaluation of BUT. The corneal fluorescein stain was evaluated 3 minutes after fluorescein instillation, by observing the cornea through a cobalt blue light. Five corneal areas were considered. The area of positive corneal staining was scored from 0 (absent) to 3 (diffuse loss of epithelium). 

Tear Basal Secretion.

This test was performed after anesthesia of the ocular surface was performed by the abundant instillation on of 0.4% oxybuprocaine (Novesina; Novartis Pharma S.p.A., Origgio, Italy). At least 10 to 15 drops of anesthetic were administered to the conjunctiva and both lid margins, to obtain the anesthesia of all the ocular structures that would come in contact with Schirmer’s strips (Alfa Intes; Industria Terapeutica Splendore, Casoria, Italy). The instillation was repeated twice with a 2-minute interval between each instillation. Schirmer’s strips were placed after 7 to 10 minutes from the last instillation, when the patients mentioned not feeling a wet sensation after instillation of the eye drops. ^{26 27}  

PGE₁ Assay

The tear content of PGE₁ was evaluated in a masked manner by enzyme immunoassay (EIA), using a commercial kit (Assay Designs Inc., Ann Arbor, MI). This assay used a polyclonal antibody to PGE₁ to bind, in a competitive manner, the PGE₁ in the sample or an alkaline phosphatase molecule that has PGE₁ covalently attached to it. After a simultaneous incubation at room temperature, the excess reagents were washed away, and the substrate was added. After 45 minutes at room temperature without shaking, the enzyme reaction was stopped, and the yellow color generated was read on a microplate reader at 405 nm. The intensity of the bound yellow color was inversely proportional to the concentration of PGE₁ in both standards and samples. The measured optical density was used to calculate the concentration of PGE₁. 

Statistical Analysis

The PGE₁ content of tears was a primary efficacy variable. The statistical analysis of the results was performed in a masked manner (SAS software, ver. 8.1; SAS, Cary, NC). Only right eye results were analyzed. Student’s t-test and the Mann-Whitney test were used, as appropriate. P ≤ 0.05 was considered statistically significant. 

Results

Neither deviation from the protocol nor adverse events were observed during the study. The results of the tests performed and of symptoms score are summarized in Tables 2 and 3 . The PGE₁ content of tears at baseline (T0) was 44 ± 5.4 ng/mL in group 1 and 44.1 ± 5.1 ng/mL in group 2. After 1 month of treatment (T1) there was a statistically significant increase in PGE₁ in group 1 (58.3 ± 5.5 ng/mL; P < 0.01 versus T0 and group 2 at same time of observation); no statistically significant differences were found in group 2. Fifteen days after the suspension of treatment (T2) a statistically significant reduction of PGE₁ levels toward baseline (45.7 ± 5.2 ng/mL; P < 0.01 versus T1) was observed in group 1; no significant changes were found in group 2 (Fig. 1) . 

A statistically significant improvement in symptom scores was observed at T1 in group 1 versus both T0 of the same group and versus T0 and T1 of group 2 (P < 0.01). At T2 in group 1, the symptom score was significantly higher than at T1 but remained significantly lower than at T0 and than all scores at the different times of observation in group 2 (Fig. 2) . When the individual symptoms were considered, it was observed that group 1 showed a statistically significant reduction of burning, foreign body sensation, and dryness at T1. A significant reduction in these symptoms was still present at T2. 

As for the other tests performed, we noted that in group 1 the corneal fluorescein stain showed a statistically significant improvement after 1 month of treatment versus both T0 and group 2 at the same time point (P < 0.01). This improvement was also present at T2 versus both group 1 T0 and group 2 at all observation times (P < 0.02; Fig. 3 ). 

No statistically significant differences were found throughout the study for BUT and basal tear secretion (Figs. 4 5 , respectively). No particular changes in the subjects’ diet were recorded. 

Discussion

The results of the present study show that the oral administration of a solution containing a mixture of omega-6 essential fatty acids induced a statistically significant increase in the PGE₁ content of tears and a statistically significant amelioration of symptoms of ocular discomfort and signs of corneal epithelial defects. No changes were found for both BUT and basal tear secretion. 

Several papers have shown that PGE₁ possesses anti-inflammatory properties and originates from omega-6 fatty acids. ^{28 29 30 31 32 33 34 35} The synthesis of PGE₁ is dependent on the availability of DGLA, which derives from the essential fatty acids LA and GLA by means of the enzymatic activity of desaturase and elongase. The enrichment of cellular lipids with DGLA is essential to increase the endogenous production of PGE₁. DGLA can be converted by cyclooxygenase into PGE₁ but can also be converted by a δ-5 desaturase in arachidonic acid. For this reason, the administration of omega-3 fatty acids has been suggested, together with omega-6, to increase the production of eicosanoids with less inflammatory properties than those derived from arachidonate. However, it has been shown that administration of GLA is able to reduce the production of AA-derived eicosanoids such as the leukotrienes B4 and C4, and platelet activating factor synthesis, while increasing DGLA, PGE₁, and 15-(S)-hydroxy-8,11,13-eicosatrienoic acid synthesis, all substances with anti-inflammatory properties. ^{10 17 28 29 30} PGE₁ has been shown to exert a powerful anti-inflammatory effect in the rat adjuvant arthritis model ^{31 32} and in the mouse lupus model. ³³ It has also been found to be a potent suppressor of synoviocyte proliferation in the mouse ^{9 34} and to inhibit collagenase activity in rabbits. ³⁵  

Several studies in humans proved that GLA supplementation produced a clear anti-inflammatory effect. In vitro studies showed a decreased synthesis of LTB₄ in neutrophils derived from healthy humans. ²⁸ GLA was able to reduce the autoinduction of IL-1β in peripheral blood monocytes derived from both normal volunteers and patients with rheumatoid arthritis, this being a protective activity in diseases characterized by chronic inflammation. ¹⁹ DGLA, the GLA derivative and immediate precursor of PGE₁, added to cells in vitro and administered orally in vivo was able to induce a reduced secretion of interleukin-1β and tumor necrosis factor-α from activated human peripheral blood monocytes in patients with rheumatoid arthritis. ¹⁸  

The biological effects of PGE₁ was demonstrated in vivo by feeding its precursor dietary fatty acids. There is substantial evidence that treatment with omega-6 fatty acids induces anti-inflammatory effects. Randomized, placebo-controlled trials have shown that patients with rheumatoid arthritis and active synovitis, treated for up to 1 year with omega-6, have progressive improvement, suggesting that omega-6 may function as a disease-modifying anti-rheumatic drug. ^{16 17} Omega-6 has also been shown to increase PGE₁ synthesis and cAMP levels in cultures of human synovial cells. ¹⁰  

It has been suggested that the incorporation of select diet-derived polyunsaturated fatty acids (PUFAs) may alter the dynamic lipid environment that regulates lateral protein diffusion of anchored receptors, thereby modulating their function. Omega-6 fatty acids have been shown to suppress T-cell activation by interfering with early events in the TcR/CD3-receptor-mediated signal transduction cascade. ^{36 37}  

It was recently shown that inflammation is one of the main mechanisms common to all forms of dry eye. ³⁸ Therefore, the therapeutic approach to dry eye should take into consideration this aspect, particularly in patients with SS, an autoimmune disease with a direct involvement of the ocular surface and tear-producing apparatus. ^{3 4 5}  

There have been several trials of the effect of anti-inflammatory treatment in dry eye. ^{39 40 41 42 43 44 45 46} The topical administration of unpreserved methylprednisolone induced a rapid and dramatic improvement of ocular irritation symptoms and signs of keratoconjunctivitis sicca in moderate to severe SS dry eye. ³⁹ Because SS is a chronic disease and long-term treatment with steroids is often accompanied by complications, the use of topical steroids for short-term “pulse” treatments in the course of exacerbation of keratoconjunctivitis sicca has been suggested. 

Cyclosporine is an immunomodulatory drug that exerts its action by inhibiting the release of proinflammatory cytokines such as IL-2 and IFN-γ by T cells. Used at concentrations of 0.05% or 0.1%, it has been shown to have a beneficial effect in patients with SS, who subsequently showed less need for additional palliative treatment. ^{40 41}  

The short-term use of NSAIDs has been indicated to induce a rapid improvement of subjective symptoms or amelioration of filamentary keratitis in patients with secondary SS. ⁴² In contrast, it has been demonstrated that treatment with NSAIDs is accompanied in patients with SS by decreased corneal sensitivity, with potential harm for the corneal epithelium. Therefore, these drugs should be used with caution and under close monitoring, and the treatment should be promptly discontinued if corneal epithelial defects develop or worsen during the treatment. ⁴⁷  

Interferon-α2, another drug with an immunomodulatory mechanism of action, has also been shown to have a favorable effect, improving tear and salivary function in patients with SS ⁴³ ; however, this drug is administered systemically and can have several side effects. Tetracycline has been shown to inhibit the expression of matrix metalloproteinase-9, which is overexpressed in the course of corneal ulcer and dry eye. ^{44 45}  

In a controlled study in patients with dry eye, in which equal doses of GLA and a quantity of LA four times lower than that used in the present study were used, it was shown that patients treated with omega-6 had a statistically significant reduction of the percentage of conjunctival cells expressing HLA-DR, a marker of inflammation. In the treated group, the percentage of HLA-DR-positive cells varied from 58.5% ± 14.1% at baseline to 41.3% ± 18.9% (P < 0.05), whereas in the placebo-treated control group a non–statistically significant expression of HLA-DR was observed in conjunctival epithelial cells. Moreover a statistically significant difference was observed at the end of the treatment between the two groups. ²³ The decreased expression of conjunctival HLA-DR in the treated group was also accompanied by a statistically significant decrease of the symptom scores and lissamine green staining of the conjunctiva. Another study showed that omega-6 fatty acids increased the rates of tear and saliva production in patients with SS. ⁴⁶  

In our study, we were not able to demonstrate an increase in basal tear secretion, perhaps because of the relatively short period of treatment for patients with moderate to severe lacrimal gland impairment, demonstrated by reduced tear basal secretion in a test. Another study also failed to show an increased tear production in patients with SS who had fatigue symptoms. ⁴⁸ These data may indicate a lack of a direct action of omega-6 fatty acids on tear production. 

The anti-inflammatory activity of omega-6 led us to undertake the present study about the effect of omega-6 administration in patients with SS, in which a chronic inflammation of the ocular surface was demonstrated, also considering that no data are available showing the effect of omega-3 treatment in this disease. 

From the results of our study, it appears that omega-6 fatty acids could be of help in controlling the evolution of signs and symptoms of dry eye. Their use, together with other anti-inflammatory agents, systemic or local, could contribute to obtain a good efficacy with lower dosage of more aggressive drugs, thus reducing the risk of the ocular and systemic side effects that often accompany the use of anti-inflammatory agents. 

Further studies of the effects of the combined action of both omega-6 and -3 fatty acids on inflammatory ocular disorders would be beneficial. 

 Supported by grants from the National Research Council and Bausch & Lomb.

 Submitted for publication November 29, 2004; revised May 10 and July 7, 2005; accepted October 17, 2005.

 Disclosure: P. Aragona, None; C. Bucolo, Bausch & Lomb (E, F); R. Spinella, None; S. Giuffrida, Bausch & Lomb (E, F); G. Ferreri, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Composition of the Study Drug and the Comparative Placebo

Study Drug	Placebo
Linoleic acid (112 mg)	Fructose (2383.3 mg)
Gamma-linolenic acid (15 mg)	Monohydrate citric acid (50 mg)
Fructose (2383.3 mg)	Aspartame (12.5 mg)
Monohydrate citric acid (50 mg)	Silicon dioxide (6 mg)
Aspartame (12.5 mg)	Bigrade aroma (45 mg)
Silicon dioxide (6 mg)	Citrus aroma (131 mg)
Bigrade aroma (45 mg)
Citrus aroma (131 mg)

Table 2.

 

Total Symptom Score and Study Test Results at the Three Time Points

	Symptoms (Total Score)	BUT (s)	Corneal Stain Score	Tear Basal Secretion (mm/5 min)	PGE1 (ng/mL)
Group 1
T0	5 ± 1.4	2.3 ± 1.1	4.4 ± 1.5	2.3 ± 1.7	44 ± 5.4
T1	2 ± 0.9*	2.7 ± 1.1	2 ± 1.3*	2.4 ± 1.7	58.3 ± 5.5, †
T2	3.5 ± 1.1, ‡	2.1 ± 1.1	2.6 ± 1.5, §	2.5 ± 1.4	45.7 ± 5.2
Group 2
T0	5.7 ± 1.1	2.4 ± 1	4.6 ± 1.4	2.4 ± 1.6	44.1 ± 5.1
T1	5.1 ± 1.2	2.5 ± 1.1	4.5 ± 1.5	2.7 ± 1.3	44.2 ± 4.7
T2	5.2 ± 1.2	2.1 ± 0.9	4.4 ± 1	2.5 ± 1	44.6 ± 4.6

*   P < 0.01 versus baseline and T2 in the same group and all examinations of group 2.

†   P < 0.01 versus baseline and T2 of group 1 and versus all examinations of group 2.

‡   P < 0.01 versus T1 and T0 of group 1 and all examinations of group 2.

§   P < 0.02 versus T0 of group 1 and all examinations of group 2.

Table 3.

 

Symptom Score and Total symptom Scores at the Three Time Points

	Burning	Itching	Foreign Body	Dryness	Mucus	Photophobia	Total Score
Group 1
T0	2 ± 0.6	0.1 ± 0.4	2.1 ± 0.5	0.6 ± 0.7	0.1 ± 0.3	0.5 ± 0.2	5 ± 1.4
T1	0.9 ± 0.5*	0 ± 0	1 ± 0.6, ‡	0.1 ± 0.3, ∥	0 ± 0.2, ¶	0 ± 0	2 ± 0.9, #
T2	1.5 ± 0.5, †	0.1 ± 0.5	1.4 ± 0.6, §	0.2 ± 0.4	0 ± 0.3, ¶	0 ± 0	3.5 ± 1.1, **
Group 2
T0	2.1 ± 0.7	0.4 ± 0.5	2.2 ± 0.6	0.4 ± 0.5	0.3 ± 0.5	0.1 ± 0.3	5.7 ± 1.1
T1	2.1 ± 0.6	0.2 ± 0.4	1.9 ± 0.7	0.4 ± 0.5	0.4 ± 0.5	0.1 ± 0.3	5.1 ± 1.2
T2	2.1 ± 0.6	0.1 ± 0.4	1.9 ± 0.6	0.4 ± 0.6	0.5 ± 0.5	0 ± 0.2	5.2 ± 1.2

*   P < 0.01 versus T0 and T2 in the same group and all examinations in group 2.

†   P < 0.01 versus T0 and T1 in the same group and all examinations in group 2.

‡   P < 0.03 versus T0 and T2 in the same group and P < 0.02 versus all examinations in group 2.

§   P = 0.03 versus T0 and T2 in the same group and P < 0.02 versus all examinations in group 2.

∥   P < 0.01 versus T0 in the same group and versus all examinations in group 2.

¶   P < 0.01 versus all examinations in group 2.

#   P < 0.01 versus T0 and T2 in the same group and all examinations in group 2.

**   P < 0.01 versus T1 and T0 in group 1 and all examinations in group 2.

Figure 1.

 

PGE₁ content of tears at the different examinations. *P < 0.01 versus baseline and T2 of group 1 and versus group 2 at all observation times.

Figure 2.

 

Symptom score at the different examinations. *P < 0.01 versus T0 of group 1 and all examinations in group 2. §P < 0.01 versus T1 and T0 in group 1 and versus all examinations in group 2.

Figure 3.

 

Corneal fluorescein stain score. *P < 0.01 versus group 1 T0 and group 2 T0 and T1. §P < 0.02 versus group 1 T0 and group 2 in all examinations.

Figure 4.

 

Tear film BUT. No statistically significant differences were found in either group.

Figure 5.

 

Tear basal secretion. No statistically significant differences were found in either group.