Tacrolimus and Hyaluronate Therapy Enhance Tear Film Stability in Canine Evaporative Dry Eye Disease

ABSTRACT

Objective

Evaluate the efficacy of a therapeutic approach combining tacrolimus and hyaluronate‐based lubricant for the management of evaporative dry eye disease (EDED) in dogs, compared to hyaluronate monotherapy.

Procedures

Fifty‐four client‐owned dogs with EDED were randomly assigned to three groups (n = 18 each): Group 1 received 0.03% tacrolimus and 0.3% hyaluronate twice daily; Group 2 received 0.3% hyaluronate four times daily; and Group 3 received 0.3% hyaluronate twice daily. Blink rate, clinical scoring, corneal esthesiometry, Schirmer tear test, tear film breakup time (TFBUT), punctate fluorescein staining, lissamine green staining, and owner‐reported symptoms were assessed at baseline, 15, and 45 days after initiating therapy.

Results

Group 1 showed the most significant improvements, with TFBUT increasing by +57% by Day 15 and + 93% by Day 45, accompanied by notable reductions in ocular discharge and conjunctival hyperemia. Most ocular changes and owner‐reported symptoms improved more rapidly and with greater amplitude in Group 1. Although Groups 2 and 3 also improved, changes were less pronounced despite the higher dosing frequency in Group 2. Neither corneal sensitivity nor corneal changes (fibrosis, neovascularization, pigmentation, and edema) showed significant variation in any group.

Conclusions

Dogs receiving combined tacrolimus and hyaluronate treatment showed faster and more pronounced improvements in both objective and subjective assessments of ocular health. These findings underscore the importance of addressing both the immunologic aspects and tear film instability associated with EDED, particularly in cases where inflammation plays a significant role. Future studies should directly evaluate tacrolimus monotherapy and explore different tacrolimus formulations and vehicles.

1. Introduction

Dry eye disease (DED) is a multifactorial condition characterized by disruption of the tear film homeostasis, leading to chronic inflammation of the ocular surface. DED is primary classified into two forms: quantitative tear film deficiency, or aqueous‐deficient dry eye (ADDE), which is marked by insufficient aqueous tear production, and qualitative tear film deficiency, also known as evaporative dry eye (EDED), which results from abnormalities in the lipid and/or mucin components of the tear film [1, 2, 3]. Although ADDE is well documented in companion animals [4, 5, 6], many animals with normal aqueous tear production, as evidenced by a normal Schirmer tear test, may still exhibit clinical signs consistent with DED, such as ocular discharge, conjunctivitis, and keratitis. Further diagnostic testing often reveals that these cases are attributable to EDED.

EDED is increasingly recognized in dogs [2, 7, 8], particularly in brachycephalic breeds such as Shih Tzus [9, 10, 11, 12], Pugs [12, 13, 14, 15], and French Bulldogs [12, 16], where the characteristic features of brachycephalic ocular syndrome—such as macropalpebral fissure, lagophthalmos, nasal fold or caruncular trichiasis, and corneal hypoesthesia—significantly compromise tear film stability [17]. A recent study by Sebbag et al. demonstrated that most Shih Tzu dogs exhibited a markedly reduced tear film breakup time (TFBUT) of 5.3 s on average, which likely contributes to the high prevalence of ocular surface disorders in this breed [9]. EDED can also occur from primary meibomian gland dysfunction [2, 18, 19, 20, 21], conjunctivitis [22, 23, 24, 25], surgical procedures such as distichiasis removal [26, 27], and ADDE resulting in mixed DED [19, 28].

Despite the increasing recognition of EDED in veterinary medicine, scientific literature on treatment strategies remains scarce. Tacrolimus, a macrolide immunomodulatory drug that has demonstrated efficacy in ADDE by promoting aqueous tear secretion [29, 30, 31, 32, 33], may also be beneficial in managing EDED by reducing cytokine‐mediated inflammation [34], and potentially supporting ocular surface nerve health, as suggested by findings with the closely related drug cyclosporine [35]. This study aims to evaluate the efficacy of a treatment approach combining topical tacrolimus and hyaluronate‐based lubricant in the management of EDED in dogs. By comparing this regimen with hyaluronate monotherapy, administered either twice or four times daily, we sought to determine whether the addition of tacrolimus offers superior benefits in terms of clinical outcomes and owner‐reported symptoms. We hypothesized that the combination of immunomodulatory and lubricative therapies would provide more rapid and sustained improvements in tear film stability and ocular surface health compared to lubrication alone.

2. Materials and Methods

2.1. Animals

Fifty‐four client‐owned dogs were enrolled in the study, with n = 18 dogs randomly assigned into one of three treatment groups (see Section 3). The study's sample size, calculated via one‐way ANOVA with a power of 80% and alpha 0.05 (SigmaPlot 15.0; Systat Software Inc., San Jose, CA, USA), was based on the assumption that TFBUT would increase by at least 50% (baseline 5.3 s, standard deviation 2.4 s) [9] when using topical hyaluron‐based lubricant and/or topical immunomodulatory drug [36]. Each dog underwent a complete physical examination and ophthalmic examination involving neuro‐ophthalmic examination (menace response, dazzle reflex, pupillary light reflexes, and palpebral reflexes), slit lamp biomicroscopy (SL‐17, Kowa, Tokyo, Japan) of the adnexa and anterior segment, indirect ophthalmoscopy (Eyetec, São Carlos, Brazil) of the fundus, intraocular pressure (IOP) measurement with rebound tonometry (TonoVet Plus, Icare, Vantaa, Finland), Schirmer tear test‐1 (STT‐1), and TFBUT. Dogs were included in the study if one or both eyes had low TFBUT (< 5 s) [2, 7], adequate STT‐1 values (≥ 15 mm/min) [37], and ≥ 1 clinical sign previously reported in canine patients with qualitative tear film deficiency (e.g., blepharospasm, ocular discharge, meibomian gland obstruction, eyelid redness, conjunctival redness and loss of corneal transparency) [2, 7, 8, 9, 11, 20]. The exclusion criteria were as follows: (i) Previous ophthalmic medications or ocular surgery in the past 6 months; (ii) Systemic illness confirmed via bloodwork and physical examination that could affect ocular parameters (e.g., endocrinopathy, and neuropathy), and (iii) Presence of corneal ulceration, prolapsed nictitans gland, or moderate entropion in one of both eyelids (i.e., entropion more severe than the typical mild medial lower eyelid entropion observed in brachycephalic dogs). Given the high prevalence of cilia abnormalities in breeds commonly affected by EDED (i.e., brachycephalic breeds), the presence of a few aberrant eyelashes was not considered a criterion for exclusion in this study. This included fine distichiae and ectopic cilia that remained covered by the palpebral conjunctiva, without protrusion or irritation to the ocular surface. Informed consent was obtained from all owners, and the study was approved by the Ethics Committee on Animal Experimentation of the Federal University of Bahia (protocol # 12/2022). Data collected from owners were limited to nonsensitive information related to the animals' clinical history, care, and treatments, obtained in the context of routine anamnesis. All responses were anonymized and no personally identifiable information was collected.

2.2. Exams and Diagnostic Tests

The study involved three separate visits, namely Day 0 (i.e., baseline, before any therapy), Day 15 (i.e., 15 days following initiation of therapy), and Day 45 (i.e., 45 days following initiation of therapy). All owners were asked to fill a detailed questionnaire at each visit. The questionnaire assessed outcomes related to the appearance of the dogs' eyes (i.e., ocular discharge, ocular redness, and ocular pruritus), each subjectively graded by the owners from 0 (none) to 10 (very pronounced) (Appendix S1). At each visit, all ophthalmic examinations and diagnostic tests were performed in the morning hours (8–12) by two examiners (SB, AO) in the same examination room, with ambient temperature and humidity recorded daily. The following procedures were performed in both eyes of each dog in the specific order described below [37], ensuring a minimum of 5–10 min interval between successive tests to allow the tear fluid to replenish [38]. Further, all exams and tests were performed at least 1 h after the administration of the morning dose of the assigned topical treatment on recheck days (Day 15 and Day 45), to assess therapeutic effects under routine treatment conditions.

• Blink rate: Eyes were observed from a distance by two evaluators, each consistently assigned to either the right or left eye throughout the study, ensuring consistency. Observations were conducted while the dog was in a “resting state” (i.e., no head manipulation, no external stimuli, in a quiet environment). The number of complete and incomplete blinks were counted separately over 2 min in each eye, a value divided by two to record complete blink rate and incomplete blink rate in blinks/min.

• Clinical scoring: In each eye, a grade between 0 and 3 (0 = absent, 1 = mild, 2 = moderate, and 3 = severe) was given to each of the following observation/lesion [39, 40]: Ocular discharge of any type (i.e., serous, seromucoid, mucopurulent), conjunctival hyperemia, corneal neovascularization, corneal pigmentation, corneal edema, corneal fibrosis.

• Corneal tactile sensation (CTS): A Cochet–Bonnet aesthesiometer (Luneau Ophtalmologie, Chartres, France) with a 0.12‐mm diameter monofilament was used to evaluate the corneal sensitivity in each eye. Starting at a filament length of 6 cm, the nylon fiber was held perpendicular to the ocular surface and advanced toward the central cornea until a slight bend in the fiber was noted. The filament was shortened in increments of 0.5 cm, and CTS was recorded as the length (in cm) that elicited a consistent blink reflex in at least three out of five attempts [9, 41].

• Schirmer tear test‐1 (STT‐1): A standard Schirmer strip (Tear Flo ophthalmic strips, Oasis Medical, Glendora, CA, USA) was placed in the lateral lower conjunctival fornix of each eye. The wet portion of the strip was measured after 1 min and recorded in mm/min.

• Tear film breakup time (TFBUT): In both eyes, 3 μL of 1% fluorescein was instilled on the ocular surface using a pipette (Eppendorf Reference 2, Hamburg, Germany). After three manual blinks, the eyelids were kept open and the dorsotemporal corneal surface was observed at 16× magnification with a slit lamp and cobalt blue filter. The TFBUT was recorded with a stopwatch (to the nearest tenth of a second) as the time from eyelid opening to the appearance of ≥ 1 dark spot(s) within the fluorescent green tear film. The average of two measurements per eye was used for data analysis.

• Punctate fluorescein staining (PFS): After flushing excess fluorescein from the ocular surface with sterile eyewash, the corneal surface was observed at 10× magnification with a cobalt blue filter (SL‐17). The PFS was graded as previously described [9]: 0 = no stain uptake, 1 = discrete stain uptake (< 1/3 of cornea), 2 = moderate stain uptake (1/3 to 2/3 of cornea), and 3 = diffuse stain uptake (> 2/3 of cornea).

• Lissamine green: A lissamine green ophthalmic dye strip (Green Touch Strips; Madhu Instruments, New Delhi, India) was wetted with a sterile eyewash and touched once to the ventral bulbar conjunctiva of each eye. After 3 manual blinks, the temporal bulbar conjunctiva was examined at 10× magnification. Grading was performed as previously described [42]: 0 = little to no stain retention, 1 = mild stain retention, 2 = moderate stain retention, and 3 = diffuse stain retention.

2.3. Treatment Protocols

Dogs were randomly assigned at baseline (Day 0) to one of three treatment groups using the permuted block method via Excel software (Microsoft Excel 2016 for Windows):

1. Group 1: Tacrolimus 0.03% (compounded in medical‐grade coconut oil vehicle with medium chain triglycerides; Drogavet compounding pharmacy, Brazil) administered twice daily in each eye, then (5–10 min later) preservative‐free 0.3% hyaluronate (I‐Drop Vet Gel; I‐Med Pharma Inc., Montreal, Canada) twice daily in each eye.

2. Group 2: Preservative‐free 0.3% hyaluronate (I‐Drop Vet Gel) administered four times daily in each eye, spaced evenly throughout waking hours (e.g., around 07:00 a.m., 12:00 p.m., 05:00 p.m., and 10:00 p.m.).

3. Group 3: Preservative‐free 0.3% hyaluronate (I‐Drop Vet Gel) administered two times daily in each eye.

Owners were asked to administer the eye drops on a daily basis as prescribed, including the morning of the recheck visits (Day 15 and Day 45).

2.4. Data Analysis

Normality of the data was assessed with the Shapiro–Wilk test; data were normally distributed (p ≥ 0.095), therefore results are presented as mean ± standard deviation (range). Results from right and left eyes were compared by means of paired t‐tests; since no significant differences were noted between eyes for any diagnostic test (p ≥ 0.152), the average of the measurements of the right and left eyes of each dog were used for further analysis. The percent complete blink was calculated in each dog by dividing the number of complete blinks by the number of total blinks (complete and incomplete).

Subjects characteristics at baseline were compared among the three experimental groups using one‐way ANOVA with post hoc Holm–Sidak test for continuous data (i.e., age, body weight, STT‐1, IOP, and scores for ocular discharge, conjunctival hyperemia, corneal vascularization, corneal pigmentation, and corneal fibrosis), or chi‐square tests for categorical data (i.e., skull conformation, sex, neuter status, Jones test, lagophthalmos, entropion, trichiasis, impacted meibomian glands, distichia, and ectopic cilia). One‐way ANOVA with post hoc Holm–Sidak tests were also used to compare each outcome among the three groups at Day 15 and at Day 45, and to compare the three groups for the percent change of each outcome between Day 15 and Day 0, and between Day 45 and Day 0. Last, survey results (owners' questionnaires) were compared at baseline then among time points with one‐way ANOVA and post hoc Holm‐Sidak tests for continuous data (i.e., discharge, redness, and pruritus) as well as chi‐square tests for categorical data (i.e., local irritation, omission, transient interruption, and intent to continue). Statistical analyses were performed with SigmaPlot 15.0 (Systat Software Inc., San Jose, CA, USA) and values p < 0.05 were considered statistically significant.

3. Results

The study population comprised 27 males (14 castrated, 13 intact) and 27 females (16 spayed, 11 intact), aged between 6 months and 11 years (4.7 ± 2.9 years) and weighing between 4 kg and 40 kg (13.1 ± 11.8 kg). Representative images of canine eyes with EDED are depicted in Figure 1.

FIGURE 1.

Representative photographs of dogs with clinical signs consistent with qualitative tear film deficiency: (A) 9‐year‐old male Yorkshire Terrier, (B) 5‐year‐old female Shih Tzu, and (C) 11‐year‐old male Shih Tzu. Clinical signs include ocular discharge, marginal blepharitis, meibomianitis, conjunctivitis, and keratitis.

At baseline (Day 0), no significant differences were noted among the three experimental groups for any outcome (p ≥ 0.072), except for the degree of conjunctival hyperemia (p = 0.011) being significantly higher at baseline between Groups 1 and 2 (p = 0.019) and between Groups 1 and 3 (p = 0.024) (Table 1). Throughout the entire study duration (6 months), ambient temperature and humidity varied from 19°C–27°C and 37%–72%, respectively; however, average differences in temperature and humidity from one visit day to another (for the same patients) were only 1.6°C and 4.3%, respectively.

TABLE 1.

Characteristics of canine patients with qualitative tear film deficiency at baseline. Continuous data are presented as mean standard deviation (range) and compared among groups using one‐way ANOVA with post hoc Holm–Sidak test, while categorical data are presented as percentages and compared among groups using chi‐square tests.

	Group 1	Group 2	Group 3	p
Age (years)	4.7 ± 3.1 (0.8–11)	4.7 ± 3.3 (0.5–9)	4.7 ± 2.2 (0.7–10)	1.000
Body weight (kg)	8.0 ± 4.8 (3.3–24)	7.1 ± 5.3 (2.7–26.7)	12.3 ± 11.8 (3.2–42.5)	0.118
Skull conformation (% brachycephalic)	61.1	66.7	55.6	0.750
Sex (% female)	55.6	61.1	44.4	0.199
Neuter status (% neutered)	55.6	55.6	55.6	1.000
STT‐1 (mm/min)	20.7 ± 3.9 (15–25)	19.0 ± 2.7 (15–24)	21.0 ± 3.4 (16.5–30)	0.172
IOP (mmHg)	20.1 ± 3.0 (14.5–25)	18.9 ± 3.2 (11–25)	19.8 ± 2.5 (15–24)	0.416
Jones test (% eyes)	28	17	50	0.250
Lagophthalmos (% eyes)	61	61	67	0.199
Entropion (% eyes)	86	72	58	0.220
Trichiasis (% eyes)	47	44	28	0.272
Impacted meibomian glands (% eyes)	28	36	3	0.083
Distichiasis (% eyes)	6	8	3	0.850
Ectopic cilium (% eyes)	6	3	0	0.802
Ocular discharge	1.7 ± 0.6 (0.5–3.0)	1.3 ± 0.7 (0–3.0)	1.2 ± 0.6 (0–2.0)	0.072
Conjunctival hyperemia	1.3 ± 0.7 (0–2.5)	0.6 ± 0.6 (0–2.0)	0.7 ± 0.8 (0–2.0)	0.011
Corneal vascularization	0.3 ± 0.5 (0–2.0)	0.2 ± 0.4 (0–1.0)	0.1 ± 0.3 (0–1.0)	0.273
Corneal pigmentation	0.4 ± 0.7 (0–2.5)	0.2 ± 0.5 (0–2.0)	0.1 ± 0.3 (0–1.0)	0.298
Corneal edema	0 ± 0 (0–0)	0 ± 0 (0–0)	0 ± 0 (0–0)	1.000
Corneal fibrosis	0.3 ± 0.4 (0–1.0)	0.1 ± 0.3 (0–1.0)	0.2 ± 0.4 (0–1.0)	0.197

3.1. Comparison Day 0 Versus Day 15 Versus Day 45

The following outcomes significantly differed between time points (Day 0 vs. Day 15 vs. Day 45) in at least one experimental group (Figure 2, Appendix S2): TFBUT (Figure 2A) for Group 1 (p < 0.001) and Group 2 (p = 0.025); PFS (Figure 2B) for Group 1 (p = 0.040); ocular discharge (Figure 2C) for Group 1 (p = 0.005); and conjunctival hyperemia (Figure 2D) for Group 1 (p = 0.002) and Group 2 (p = 0.004). In contrast, no significant differences were noted for the following outcomes in any experimental group: STT‐1 (p ≥ 0.219), lissamine green staining (p = 1.000), complete blink rate (p ≥ 0.982), percent complete blinks (p ≥ 0.253), corneal vascularization (p ≥ 0.678), corneal pigmentation (p ≥ 0.890), corneal edema (p = 1.000), and corneal fibrosis (p ≥ 0.529).

FIGURE 2.

Bar charts displaying the mean + standard deviation for various clinical outcomes at baseline (white bars), day 15 (gray bars), and day 45 (black bars) of therapy in canine patients with qualitative tear film deficiency. Patients received either 0.03% tacrolimus and 0.3% hyaluronate twice daily (Group 1), 0.3% hyaluronate four times daily (Group 2), or 0.3% hyaluronate twice daily (Group 3). Outcomes measured include tear film breakup time (A), punctate fluorescein staining (B), ocular discharge (C), and conjunctival hyperemia (D).

3.2. Comparison Percent Change (Recheck vs. Baseline)

When compared to baseline (Day 0), the percent change did not significantly differ among experimental groups for any outcome at Day 15 (p ≥ 0.122) and at Day 45 (p ≥ 0.144). However, greater percent changes were observed in Group 1 for TFBUT (Figure 3A), PFS (Figure 3B) and ocular discharge (Figure 3C), and in Group 2 for conjunctival hyperemia (Figure 3D). Further, ocular discharge appeared to improve earlier in Group 1, being noticeable at Day 15 versus Day 45 for Group 2 and Group 3 (Figure 3C).

FIGURE 3.

Scatter plots illustrating the percent change in various clinical outcomes from baseline to day 15 (white circles) and day 45 (black circles) of therapy in canine patients with qualitative tear film deficiency. Horizontal bars indicate the median percent change. For treatment details and outcome measures, refer to the legend of Figure 2.

3.3. Owners' Questionnaire

Owners of dogs from Group 1 reported significantly reduced ocular discharge (p < 0.001), ocular redness (p = 0.019), and ocular pruritus (p = 0.015) over time (Figure 4). Although reductions in ocular discharge, ocular redness, and ocular pruritus were also described for dogs from Group 2 and Group 3, these findings were not statistically significant (p ≥ 0.086).

FIGURE 4.

Bar charts displaying the mean + standard deviation for various owners‐reported symptoms at baseline (white bars), day 15 (gray bars), and day 45 (black bars) of therapy in canine patients with qualitative tear film deficiency. Outcomes measured include ocular discharge (A), ocular redness (B), and ocular pruritus (C).

4. Discussion

The present study demonstrates that the combination of immunomodulatory and lacrimomimetic eyedrops is effective for managing EDED in dogs, similar to the standard therapeutic approach for ADED in companion animals [43, 44]. Dogs in Group 1 (treated with tacrolimus and hyaluronate twice daily) exhibited a faster and more comprehensive improvement in ocular signs compared to those in Group 2 (hyaluronate four times daily) and Group 3 (hyaluronate twice daily). This was evident both through objective clinical assessments and subjective owner‐reported symptoms. In cases where tacrolimus therapy (or closely related cyclosporine) is unavailable due to supply issues, adverse effects, cost, or other factors, administering topical lubrication four times daily may be more effective than twice daily in dogs with EDED, though compliance could be a challenge.

In dogs, early clinical signs indicative of EDED may include ocular redness, excessive serous discharge (epiphora), inspissation of the meibomian gland orifices, and marginal blepharitis [2, 7, 8, 9, 11, 20]. However, these subtle signs often go unnoticed by owners, resulting in dogs typically presenting at more advanced stages of the disease, with pronounced clinical symptoms such as mucopurulent discharge, conjunctival hyperemia and chemosis, corneal fibrosis, neovascularization, and pigmentation. EDED is increasingly recognized in veterinary patients, underscoring the importance for veterinary ophthalmologists and general practitioners to conduct additional diagnostic tests beyond the STT to assess tear quality, as highlighted in a recent review article [37]. Here, all canine patients underwent STT as well as a series of ocular tests including TFBUT, PFS, lissamine green staining, and blinking assessment.

Tear film breakup time is a measure of precorneal tear film stability, determined by the time it takes for fluorescein dye, and consequently the tear film, to evaporate from the corneal surface. TFBUT serves as an indirect indicator of the mucin and/or lipid component(s) of the precorneal tear film [37], with reduced values commonly observed in companion animals with mucin or lipid deficiencies [7, 9, 21, 22]. In the present study, all three groups demonstrated improvement in TFBUT compared to baseline; however, the improvement was more pronounced and rapid in Group 1. By Day 15, TFBUT had increased significantly from baseline (+57%) and continued to rise through Day 45 of the experiment (+93%).

Punctate fluorescein staining is an important diagnostic marker of dry eye disease, reflecting disruption of corneal epithelial tight junctions, increased permeability, or epithelial cell damage [37]. The characteristic micro‐punctate staining results from fluorescein dye adhering to areas of epithelial cell loss or compromised glycocalyx, thereby revealing corneal surface damage [45]. In this study, all treatment groups demonstrated improvement in PFS; however, only patients in Group 1 exhibited a sustained and significant reduction in PFS scores by Day 45, with a 31% decrease compared to baseline.

Lissamine green is a diagnostic dye used to evaluate tear film deficiency by staining devitalized corneal and conjunctival epithelial cells without causing ocular irritation [42]. In our canine patients diagnosed with EDED, no lissamine staining was observed at any time point. This result contrasts with the findings from Smith et al., who reported a significant association between increased lissamine green staining and reduced TFBUT in dogs with ADED or mixed dry eye disease [42], rather than EDED. The degree of lissamine green staining has been shown to increase with repeated dye administration at 1‐min interval [46]; however, our protocol employed only a single drop application.

A complete blink is essential for promoting meibomian lipid secretion, distributing tear film components across the ocular surface, and ensuring proper tear drainage through the nasolacrimal duct (i.e., the lacrimal pump) [37]. Therefore, evaluating the frequency and extent of incomplete blinking is important in dogs, especially brachycephalic breeds, which have a high prevalence of lagophthalmos [37]. In our study, blink rates and the percentage of incomplete blinks remained unchanged over time across all treatment groups, suggesting that these therapies are insufficient to correct impaired blinking in canines with EDED. Alternative approaches, such as medial canthoplasty, may be necessary for selected patients with macropalpebral fissure and lagophthalmia [9, 17, 47], complementing the therapeutic benefits of topical eyedrops.

Ocular discharge and redness decreased in all dogs, with the most pronounced and significant improvement noted in Group 1. Objective clinical scoring and subjective owner surveys indicated that ocular discharge of Group 1 was significantly reduced by Day 15 (−32% and −52%, respectively) and continued to decline by Day 45 (−33% and −73%, respectively). Ocular redness in Group 1 also showed significant reduction by Day 15 (−41% and −76%, respectively), with further improvement by Day 45 (−63% and −74%, respectively). Although all treatment groups demonstrated improvement over time, it is important to note that baseline conjunctival hyperemia was higher in Group 1 compared to the other groups. This difference likely influenced the magnitude of improvement observed in this parameter, making it uncertain whether similar statistical significance would have been achieved had all groups started with comparable baseline hyperemia. As for the other exam findings, no significant changes were observed in corneal abnormalities, including edema, neovascularization, fibrosis, or pigmentation.

In clinical practice, artificial tears are typically recommended alongside immunomodulatory therapy as primary treatment options for dry eye disease [43, 44]. This approach is well established for ADED, and our study's findings suggest it is likely beneficial for canines with EDED as well.

Tacrolimus is a macrolide antibiotic with potent immunomodulatory properties, reportedly 10–100 times more effective than cyclosporine [30]. Immunomodulatory drugs like tacrolimus, cyclosporine, and pimecrolimus are routinely prescribed for canine patients with ADED, improving aqueous tear production by inhibiting T‐lymphocyte proliferation and reducing immune‐mediated dacryoadenitis [29, 30, 31, 32, 33, 43, 44, 48, 49]. Beyond their lacrimostimulant effect, these drugs offer additional benefits that could improve ocular surface homeostasis in dogs with EDED, such as reducing ocular surface inflammation and pro‐inflammatory cytokines [34] and supporting corneal nerve health, as reported for cyclosporine [35]. Studies have documented significant reductions in corneal inflammation in canine patients with ADED [29, 48, 50], though this effect was not observed in our study of EDED, likely due to the mild baseline corneal changes. Additionally, several studies have shown reduced conjunctival inflammation, with cytological and histological evidence supporting improved goblet cell health and mucin secretion [7]. This anti‐inflammatory action may also benefit meibomian gland health and lipid secretion, as demonstrated by Perry et al. [51], potentially through the reduction of extracellular traps that obstruct meibomian gland orifices [52].

Tear replacement therapy (i.e., lacrimomimetic) has long been considered a cornerstone in the management of dry eye disease, irrespective of its etiology [43, 44]. Artificial tear products are designed to mimic and replace one or more components of the tear film; however, they are rarely sufficient as standalone therapy [43, 44]. In this study, preservative‐free 0.3% hyaluronate (I‐Drop Vet Gel) was administered to dogs, based on the established benefits of preservative‐free eyedrops for eyes with ocular surface irritation, as well as the well‐documented advantages of sodium hyaluronate in stabilizing the tear film and supporting ocular surface health (e.g., mucoadhesive properties and promotion of wound healing) [36, 53]. Additionally, hyaluronate‐based lubricants have been widely reported to alleviate dry eye symptoms in canine patients [40, 54, 55, 56, 57].

Importantly, while the present study demonstrated the additive benefits of combining tacrolimus with hyaluronate, the individual contribution of tacrolimus to the observed improvements remains to be determined. Future research should specifically investigate tacrolimus monotherapy for canine EDED to clarify its standalone efficacy, as well as explore the impact of different tacrolimus formulations, concentrations, and vehicles on therapeutic outcomes and tolerability.

This study presents several limitations. First, we did not differentiate between mucin‐ or lipid‐deficient EDED. This approach reflects the practical constraints of many veterinary clinics, where fluorescein is readily available for TFBUT assessment, but more advanced diagnostics are not yet widely accessible. However, TFBUT alone has limitations, as it is a subjective test and may be influenced by factors such as age, corneal surface irregularities, or fluorescein concentrations in the tear film [58]. More comprehensive diagnostic tools, such as interferometry, meibography, noninvasive breakup time, or goblet cell quantification through impression cytology or conjunctival histology, could provide greater insights into therapeutic interventions for EDED in dogs. Second, owner‐reported symptoms were based on individual perception, as owners were not formally trained with standardized grading images, which may introduce variability. Despite this, subjective assessments remain valuable in evaluating real‐world clinical impact in veterinary medicine. Third, while an oily vehicle control or tacrolimus‐only group would have provided valuable insight, we prioritized statistical power and clinical relevance by focusing on three treatment groups based on personal experience; future studies should investigate the standalone effects of these components. Fourth, we used a single artificial lubricant, linear hyaluronate 0.3%, without comparison to alternative formulations such as cross‐linked hyaluronate [40, 57, 59] or lubricants designed to address lipid deficiencies [60]. Comparative studies could further clarify the therapeutic efficacy of different products [61]. Finally, while 45 days was deemed sufficient to evaluate tear film stability and acute ocular responses, chronic corneal changes such as neovascularization, fibrosis, and pigmentation may require a longer follow‐up period for comprehensive assessment.

In conclusion, this study demonstrated that the combination of tacrolimus and preservative‐free 0.3% hyaluronate was more effective in treating qualitative tear film deficiency in dogs compared to the use of hyaluronate alone. Dogs receiving the combined treatment showed faster and more pronounced improvements in both objective and subjective assessments of ocular health. Although TFBUT and PFS improved across all groups, the greatest improvements were observed in dogs treated with the combined regimen. These findings underscore the importance of addressing both the immunologic aspects and tear film instability associated with EDED, particularly in cases where inflammation plays a significant role. Future studies should directly assess tacrolimus monotherapy and investigate different tacrolimus formulations and vehicles, as well as explore the comparative efficacy of different hyaluronate‐based lubricants and advanced diagnostic tools for better differentiation of tear film deficiencies.

Author Contributions

Lionel Sebbag: conceptualization, writing – original draft, methodology, writing – review and editing, formal analysis, supervision, data curation. Sirlene F. Barbosa: investigation, methodology, writing – review and editing, data curation. Arianne P. Oriá: conceptualization, investigation, writing – review and editing, supervision, data curation.

Ethics Statement

Informed consent was obtained from all owners, and the study was approved by the Ethics Committee on Animal Experimentation of the Federal University of Bahia (protocol # 12/2022). Data collected from owners were limited to nonsensitive information related to the animals' clinical history, care, and treatments, obtained in the context of routine anamnesis. All responses were anonymized, and no personally identifiable information was collected.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Appendix S1.

Appendix S2.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.