The use of shotgun label-free quantitative proteomic mass spectrometry to evaluate the inflammatory response in aqueous humor from horses with uveitis compared to ophthalmologically healthy horses.

OBJECTIVE: The objective of this study was to use shotgun label-free tandem mass spectrometry (LF-MS/MS) to evaluate aqueous humor (AH) from horses with uveitis (UH) compared to ophthalmologically healthy horses (HH). ANIMALS STUDIED: Twelve horses diagnosed with uveitis based on ophthalmic examination and six ophthalmologically healthy horses (postmortem) purchased for teaching purposes. PROCEDURES: All horses received a complete ophthalmic examination and physical exam. Aqueous paracentesis was performed on all horses and AH total protein concentrations were measured with nanodrop (TPn) and refractometry (TPr). AH samples were analyzed with shotgun LF-MS/MS and proteomic data were compared between groups using Wilcoxon rank-sum test. RESULTS: A total of 147 proteins were detected, 11 proteins had higher abundance in UH, and 38 proteins had lower abundance in UH. Proteins with higher abundance included apolipoprotein E, alpha-2-macroglobulin (A2M), alpha-2-HS-glycoprotein, prothrombin, fibrinogen, complement component 4 (C4), joining chain for IgA and IgM, afamin, and amine oxidase. There were positive correlations between TPn (p = .003) and TPr (p = .0001) compared to flare scores. CONCLUSION: Differential abundance of A2M, prothrombin, fibrinogen, and C4 indicate upregulation of the complement and coagulation cascade in equine uveitis. Proinflammatory cytokines and the complement cascade have potential as therapeutic targets for equine uveitis.

Retrospective evaluation of the incidence of gastrointestinal bleeding in dogs receiving ophthalmic nonsteroidal anti-inflammatory drugs.

OBJECTIVES: To report the incidence of gastrointestinal (GI) bleeding and associated risk factors in a population of dogs receiving ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs). ANIMAL STUDIED: Medical records of dogs prescribed ophthalmic NSAIDs (cases), dogs receiving systemic NSAIDs alone and dogs receiving systemic prednisone alone (controls). PROCEDURES: Data were collected retrospectively from the medical records of 204 dogs prescribed ophthalmic NSAIDs (diclofenac, ketorolac, or flurbiprofen), which were subdivided based on if they received any concurrent systemic NSAIDs or glucocorticoids, 136 dogs receiving a systemic NSAID (carprofen or meloxicam) alone, and 151 dogs receiving a systemic glucocorticoid (prednisone) alone at a referral hospital from 2015 to 2019. RESULTS: Gastrointestinal bleeds developed in 8/79 (10.1%) of topical NSAID-only cases, 10/136 (7.4%) of systemic NSAID controls, and 14/151 (9.3%) of systemic glucocorticoid controls, with no significant difference between the three groups (p = .6103). There were no significant differences in GI bleed rates between cases treated with ketorolac, diclofenac, or flurbiprofen (p = .160), although severe GI bleeding was only seen in ketorolac-treated dogs. Presence of a known concurrent risk factor for GI bleeding was significantly associated with the development of GI bleed in dogs on ophthalmic NSAIDs (p = .032). CONCLUSIONS: Dogs treated with ophthalmic NSAIDs developed GI bleeding at a frequency comparable to dogs receiving systemic NSAIDs or systemic glucocorticoids alone, suggesting that dogs receiving ophthalmic NSAIDs may be at increased risk of GI bleeding.

Comparison of the TONOVET Plus®, TonoVet®, and Tono-Pen Vet™ tonometers in normal cats and cats with glaucoma.

OBJECTIVES: To determine the accuracy, precision, and clinical applicability of the ICare® TONOVET Plus (TVP) in cats. ANIMALS AND PROCEDURES: IOP readings obtained with the TVP were compared to values obtained concurrently with the original TONOVET (TV01) and Tono-Pen Vet™ (TP) in 12 normal cats (24 eyes) and 8 glaucomatous LTBP2-mutant cats (13 eyes) in vivo. Reproducibility of TVP readings was also assessed for three observers in the above cats. The anterior chambers of five different normal cat eyes were cannulated ex vivo. IOP was measured with the TVP, TV01, and TP at manometric IOPs ranging from 5 to 70 mmHg. Data were analyzed by linear regression, ANOVA and Bland-Altman plots. ANOVA was used to assess reproducibility of TVP readings obtained by different observers and an ANCOVA model controlled for variation of individual cats. p < .05 was considered significant. RESULTS: TVP values strongly correlated with TV01 values (y = 1.045x + 1.443, R2 = .9667). The TP significantly underestimated IOP relative to the TVP and TV01, particularly at high IOP. IOP values obtained by 1 observer were significantly higher (~1 mmHg average) compared to the other 2 observers via ANCOVA analysis (p = .0006479 and p = .0203). Relative to manometry, the TVP and TV01 were significantly more accurate (p < .0001) and precise (p < .0070) than the TP in ex vivo eyes. CONCLUSIONS: IOP readings obtained with the TVP and TV01 are broadly interchangeable between models and between observers, but subtle differences may be important in a research context. TP readings vastly underestimate high IOP in feline glaucoma.

Ocular penetration of oral acetaminophen in horses.

BACKGROUND: Acetaminophen (paracetamol) is increasingly used to treat painful conditions in horses but its ocular penetration has not been studied. OBJECTIVES: To determine whether orally administered acetaminophen penetrates the aqueous humour of the normal equine eye and report an aqueous humour:serum acetaminophen concentration ratio in horses. STUDY DESIGN: In vivo experiment. METHODS: Six privately owned horses with normal ophthalmic examinations weighing 568 ± 65 kg (mean ± standard deviation) and aged 11 ± 4 years were given 20 mg/kg acetaminophen orally every 12 h for a total of six doses. Physical exam parameters were recorded prior to, during, and after the dosing period. One hour after the final dose, horses were sedated and simultaneous aqueous humour and serum samples were collected and analysed for acetaminophen concentrations and selected eicosanoids. An aqueous humour:serum acetaminophen concentration ratio was calculated. A second aqueous humour sample was taken and analysed for eicosanoid concentrations 3 months after acetaminophen dosing. Physical exam data were compared between time points using a mixed model analysis (significance p < 0.05). RESULTS: Acetaminophen was detected in both serum and aqueous humour of all horses and mean ± standard deviation aqueous humour:serum acetaminophen concentration ratio was 44.9 ± 15.9%. No significant changes in physical exam parameters occurred during or after dosing. Eicosanoids were not detected in aqueous humour at any sampling point. MAIN LIMITATIONS: Presence of acetaminophen in the aqueous humour may not relate to clinical effect. A therapeutic level of acetaminophen has not been determined in horses, and the absence of ocular inflammation does not reflect conditions in which acetaminophen may be used. CONCLUSIONS: Acetaminophen readily penetrates the aqueous humour of the normal equine eye after consecutive oral dosing. Further study is required to determine whether acetaminophen is useful in the treatment of ocular pain and inflammation.

Pro-inflammatory cytokines in aqueous humor from dogs with anterior uveitis and post-operative ocular hypertension following phacoemulsification, primary glaucoma, and normal healthy eyes.

BACKGROUND: The aim of this study was to evaluate the levels of pro-inflammatory cytokines in aqueous humor (AH) from dogs with anterior uveitis and post-operative ocular hypertension (POH) following phacoemulsification, in AH from dogs with primary glaucoma, and in normal healthy eyes with no signs of anterior uveitis or other ocular diseases. METHODS: An exploratory study including 21 samples of AH collected from 15 dogs; post-phacoemulsification with anterior uveitis and POH ('POH group', n = 10 samples), primary glaucoma ('glaucoma group', n = 6 samples), and normal ('normal group', n = 5 samples). Target mass spectrometry via multiple reaction monitoring (MRM-MS) with the Canine Cytokine SpikeMix™ as internal standard was used to measure the pro-inflammatory cytokine levels. RESULTS: The MRM-MS method measured 15 pro-inflammatory cytokines. Tumor-necrosis-factor-alpha (TNFα) and interleukin-18 (IL-18) levels in AH were different between all three groups (glaucoma>POH>normal) (p = .05, p = .02, respectively). Additionally, IL-6 was higher in the 'POH group' compared to the 'glaucoma group' (p = .04) and IL-4 was higher in the 'POH group' compared to the 'normal group' (p = .04). Intraocular pressure (IOP) was positively associated with increased AH levels of IL-18 (Spearman correlation = .64, p = .03). CONCLUSIONS: MRM-MS using the Canine Cytokine SpikeMix™ as an internal standard was established as a method to detect pro-inflammatory cytokine levels in canine AH. The study demonstrated increased levels of IL-4, IL-6, IL-18, and TNFα in AH from canines with POH following phacoemulsification. Primary glaucomatous eyes had the highest levels of IL-18 and TNFα which may indicate that inflammation plays a role in the pathogenesis of primary glaucoma in dogs.

Serum albumin and total protein concentration in the tear film of horses with healthy or diseased eyes.

OBJECTIVE: To determine total protein content (TPC) and serum albumin levels in the tears of horses with healthy or diseased eyes. ANIMALS STUDIED: Forty-two horses with healthy eyes and 11 horses with unilateral (n = 10) or bilateral (n = 1) ocular disease. PROCEDURE: Each eye underwent an ophthalmic examination including detailed conjunctivitis scoring and tear collection with Schirmer strips. TPC and serum albumin levels were quantified in tear samples and compared among healthy eyes, affected eyes, and contralateral unaffected eyes. The impact of the following variables on lacrimal protein levels were assessed: age, breed, and sex (healthy eyes), as well as conjunctivitis score (diseased eyes). RESULTS: Lacrimal TPC ranged from 7.0 to 19.5 mg/mL in healthy eyes, while serum albumin ranged from 71.1 to 711.3 µg/mL (~1.6% of TPC) and was higher in tears of aged and female horses (P ≤ .033). Eyes with ocular disease had significantly greater (P ≤ .001) serum albumin in tears (median 679.6 µg/mL) compared to contralateral unaffected eyes (130.0 µg/mL) and eyes of the reference population (200.7 µg/mL). However, lacrimal TPC did not differ significantly among the 3 groups. Scoring of palpebral conjunctival hyperemia trended toward a positive association with serum albumin in tears (r = 0.49, P = .062). CONCLUSIONS: The protein profile in equine tears differs in health and disease. Serum albumin in tears increases with ocular disease and, similar to other species, might serve as a biomarker for ocular insult in horses. Future studies could investigate the protein levels in horses with specific ocular conditions and help determine the biological importance of albumin on the equine ocular surface.

Contribution of Habitual Activity to Cruciate Ligament Rupture in Labrador Retrievers.

OBJECTIVE: The aim of this study was to describe the contribution of signalment and habitual activity in the development of cruciate ligament rupture (CR) in Labrador Retrievers. STUDY DESIGN: Four hundred and twelve client-owned purebred Labrador Retrievers were recruited. Dogs were assigned either as affected with CR or as controls based on signalment, physical examination and radiographic evidence of disease. Clients were asked to complete a questionnaire related to signalment, concurrent disease and a questionnaire pertaining to their dog's activity before development of CR or general activity during the dog's most active years. RESULTS: Habitual activity was not significantly different between dogs affected with CR and controls. There was no significant difference in neuter status or body weight between CR affected dogs and controls. Labrador Retrievers with a yellow coat, and Labradors that did not maintain an optimal body weight in the opinion of their veterinarian were at increased risk of developing CR. CONCLUSIONS: Habitual activity level is not a risk factor for development of CR in Labrador Retrievers. Our study did not show neuter status, sex or body weight to be risk factors for CR. However, coat colour and not sustaining optimal body condition are significant risk factors for CR.