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.

Detection of pro-inflammatory cytokines in healthy canine tears using Canine Cytokine SpikeMix™ mass spectrometry via multiple reaction monitoring.

PURPOSE: To evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles. METHODS: A complete ophthalmic examination was performed on 15 healthy research Beagles to verify no ophthalmic diseases. Tears were collected OU by placing a Weck-Cel® cellulose spear in the ventral conjunctival fornix for 1 min. The Weck-Cel® spear was placed in a 2.0 mL tube with a centrifuge filter forcing tears to flow through the filter into the bottom of the tube. The tears were analyzed using the Canine Cytokine SpikeMix™ and MRM-MS. Descriptive data from this study was reported as the normalized total peak area (nTPA) and median (range) using data imported from the online MRM-MS Skyline program. RESULTS: The level of 16 pro-inflammatory cytokines was successfully detected in all 15 dogs. The four cytokines with the highest median amounts in the samples were IL-2 = 0.1243 (0.019-6.7289), IL-6 = 0.964 (0.0036-16.9365), TNFα = 0.1644 (0.0096-0.7138), and CSF-2 = 0.4022 (0.1475-2.6208). CONCLUSIONS: This study revealed that 16 pro-inflammatory cytokines in canine tears from healthy dogs can be detected with Canine Cytokine SpikeMix™ and MRM-MS.

The use of a "horizontal centrifugation protocol" to prepare autologous platelet-rich fibrin membranes for corneal reconstruction surgery in dogs with complicated corneal ulcerations: A case series.

OBJECTIVE: The purpose of this case series was to describe the effect of autologous PRF membrane for corneal reconstruction surgery in dogs. PRF membranes made from two healthy dogs unrelated to the current case series were used for PRF histologic analyses. ANIMALS: Seven dogs with complicated corneal ulcerations. PROCEDURE: A complete ophthalmic examination, hematology, and fibrinogen analysis were performed pre-surgery. A PRF clot was made from autologous blood in a serum tube after centrifugation in a horizontal Bio-PRF® Centrifuge at 700 × g for 8 min. The PRF clot was processed in a PRF-Box® into a PRF membrane. The PRF membrane was sutured to the corneal ulcer bed. Each dog had a follow-up at days 5-7, 12-14, and 30-40 post-surgery. A final long-term follow-up was performed as well. RESULTS: A positive outcome with healing and a "good" quality PRF membrane was seen in six out of seven dogs. One dog had a fibrinogen level below normal range and the PRF membrane was of "poor" quality. This dog developed a descemetocele 13 days post-surgery and needed rescue surgery. Mean healing time for all dogs was 9 ± 5.5 days. Minimal scarring, corneal pigmentation, and vascularization were observed at the final long-term follow-up 288 ± 44 days post-surgery. CONCLUSION: PRF membrane was successful as graft material for corneal ulceration reconstruction surgery. Low fibrinogen appeared to have negative effect on the quality of the PRF membrane, showing the importance for the surgeon to evaluate the quality of the PRF membrane prior to surgery.

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.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

A genetic investigation of equine recurrent uveitis in the Icelandic horse breed.

Equine recurrent uveitis (ERU) is an autoimmune disease defined by inflammation of the uveal tract of the eye. The cause of ERU is thought to be complex, involving both genetic and environmental factors. The purpose of this study was to investigate potential genetic risk factors for ERU in the Icelandic horse. Fifty-six Icelandic horses (11 affected with ERU and 45 controls) living in Denmark and the USA, eight years or older, were included in the study. A case-control GWAS was performed using the GGP Equine 80K array on the Illumina Infinium HD Beadchip using 40 horses. A mixed linear model analysis identified a single SNP on ECA 11 (BIEC2_141650; NC_009154.3:g.3817009A>G) that reached genome-wide significance (p = 1.79 × 10-7 ). This variant was within an intron of tissue inhibitor of metalloproteinase 2 (TIMP2), a gene previously implicated in ERU. Sanger sequencing identified a single coding variant in this gene; however it was a synonymous mutation (NC_009154.3:g.3858193C>T) and was not perfectly concordant with ERU phenotype (p = 0.68). Further investigation of TIMP2 is warranted. Additional horses and markers are needed to identify other potential loci worthy of further investigation as contributors to ERU risk in Icelandic horses.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Acidic chemical corneal ulceration secondary to nail polish spill in a puppy.

PURPOSE: To describe the treatment of an acidic chemical corneal ulceration in a puppy secondary to nail polish spilling on and adhering to the cornea. CASE SUMMARY: A 12-week-old neutered male Australian Shepherd mix dog was presented to Colorado State University Veterinary Teaching Hospital's (CSU-VTH) Emergency and Urgent Care service acutely after exposure to nail polish spilling on the left eye (OS). Initial ophthalmic examination revealed nail polish adhered to approximately 80% of the cornea with moderate diffuse corneal edema and chemosis, and green nail polish adhered to the skin and fur of the periocular area. Copious flushing with eye wash was performed, but no areas of reduced nail polish adherence were noted. The patient was treated with 1 drop of topical ofloxacin 0.3% ophthalmic solution OS and hospitalized overnight, receiving hyaluronic acid ophthalmic lubrication every hour OS. The treatment plan was carried out for 6 h until time of transfer. In the morning, CSU-VTH's Ophthalmology service removed a nonadhered nail polish plaque in 1 piece using Bishop-Harmon tissue forceps. Following removal, a superficial ulcer secondary to an acidic chemical burn covering 100% of the corneal surface was noted, as well as moderate diffuse corneal edema. Following topical treatment for 8 days, the superficial ulcer healed completely, with no significant long-term consequences. UNIQUE INFORMATION PROVIDED: This is the first report of management of an acidic chemical corneal ulceration secondary to nail polish spill in a puppy. Early assessment and vigilance in treatment was essential for a good prognosis and outcome.

Ocular abnormalities in the Icelandic horse with a focus on equine recurrent uveitis: 112 Icelandic horses living in Denmark and 26 Icelandic horses living in the United States.

PURPOSE: To describe the most common ocular abnormalities in the Icelandic horse with focus on equine recurrent uveitis (ERU) and association between ocular abnormalities and summer eczema and coat colors. METHODS: A descriptive cross-sectional study. A complete physical and ophthalmic examination as well as measurement of serum titers for Leptospira serovariants was performed on Icelandic horses from Denmark (DK) and the United States (USA). RESULTS: One hundred and twelve Icelandic horses living in DK and 26 Icelandic horses living in the United States were included in this study (total of 138 horses, 274 eyes). The three most common ocular abnormalities were follicular conjunctivitis (55.8%, 153/274 eyes), cataracts (27.4%, 75/274 eyes), and multifocal chorioretinopathy ("bullet holes"; 19%, 52/274 eyes). The prevalence for ERU among Icelandic horses aged "eight years and older" was 8% (6/75 horses). The "insidious ERU" type was found in 83% of the ERU cohort (5/6 horses), while "classic ERU" was found in 17% (1/6 horses). A correlation between ERU and cataracts was found in the population (p < .01). Leptospiral serology results were not associated with ERU in either DK or US-Icelandic horses (p = 1.00, p = 1.00, respectively). ERU was not associated with summer eczema (p = .49), and no coat colors were associated with the ocular abnormalities found in the Icelandic horse (all p-values > .05). CONCLUSION: Follicular conjunctivitis, cataracts, and multifocal chorioretinopathy were the three most common ocular abnormalities findings. Icelandic horses who were 8 years or older had an 8% prevalence for ERU. Summer eczema and coat color were not associated with evidence of ERU or other ocular abnormalities.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Cataracts and phacoemulsification in the Siberian Husky: A retrospective and multicentric study (2008-2018).

PURPOSE: To compare cataract characteristics and complications related to cataracts and phacoemulsification in the Siberian Husky (Huskies) versus other canine breeds (non-Huskies). ANIMALS: A total of 50 Huskies (92 eyes) and 96 non-Huskies (182 eyes) were evaluated. METHODS: Medical records of Huskies (at four university veterinary hospitals, 2008-2018) and non-Huskies (Colorado State University, 2017-2018) diagnosed with cataracts were reviewed. Age of dog, cataract stage at presentation, and pre- and post-operative complications were recorded and analyzed. RESULTS: Mean (±standard deviation) age at presentation was significantly lower in Huskies (3.5 ± 3.3 years) compared to non-Huskies (9.5 ± 2.9 years) (p < .0001). Huskies more commonly presented with hereditary cataracts than non-Huskies (84% versus 52%) and a significantly higher percentage of non-Huskies presented with diabetic cataracts than Huskies (48% versus 16%; p = .0001). Cataract stage at presentation did not differ between Huskies and non-Huskies. Phacoemulsification was performed in 40% (20 out of 50 dogs, 39 out of 92 eyes) of Huskies and 42% (40 out of 96 dogs, 74 out of 182 eyes) of non-Huskies. Pre-operative and post-operative retinal detachment were more common in Huskies than non-Huskies (13% versus 2% and 10% versus 1%, respectively) but the difference was not significant. Other post-operative complications occurred with similar frequency in both groups (p ≥ .17). CONCLUSIONS: Huskies evaluated for cataracts were younger and less likely to present with diabetic cataracts than other canine breeds and, although not statistically significant, had a clinically important increased risk of retinal detachment pre- and post-phacoemulsification.

Clinical ophthalmological diagnostic description of 10 healthy sugar gliders (Petaurus breviceps) and prevalence of ocular-related presentations in a larger hospital population.

OBJECTIVE: To provide reference values for ocular examination and diagnostics in ophthalmologically normal sugar gliders (Petaurus breviceps). To retrospectively determine the prevalence of ocular diseases in sugar gliders presenting to a single institution. ANIMALS: Ten client owned and 106 previously evaluated sugar gliders. PROCEDURE: A descriptive study evaluated sugar gliders presented to Colorado State University's Avian, Exotics, and Zoological Medicine Service (CSU-AEZ) from August-2019 to January-2020. A complete ophthalmic examination including Schirmer tear test II (STT II), phenol red threat test (PRTT), intraocular pressure (IOP) via rebound tonometry, fluorescein, and rose bengal stain was performed under anesthesia. Conjunctival aerobic culture swabs and cytology were collected prior to ophthalmic evaluation. A retrospective review of medical records of sugar gliders presented to CSU-AEZ from 2008 to 2018 for ocular disease was performed. RESULTS: Mean values ± standard deviation for selected diagnostics included the following: STT II: 2.2 ± 6.7 mm/min; PRTT: 0 ± 0 mm/15 s; IOP: 12 ± 2.6 mm Hg. Fluorescein and rose bengal staining highlighted corneal abrasions secondary to tear testing. The three most common conjunctival bacterial isolates cultured were Staphylococcus spp. (3/20, 15%), Coryneform spp. (3/20, 15%), and unidentified Gram-positive cocci (3/20, 15%). Retrospective analysis revealed ocular diseases to be the third most common abnormality resulting in sugar glider presentations (13/106, 12.3%). CONCLUSION: This descriptive study gives reference values for IOP, conjunctival microbiology, and cytology for sugar gliders. STT II and PRTT provide little clinical value in sugar gliders. The retrospective study revealed that ocular abnormalities, often secondary to dental disease, are a common reason for presentation.

Effect of topical administration of 0.1% diclofenac sodium ophthalmic solution at four frequencies on intraocular pressure in healthy Beagles.

OBJECTIVE: To evaluate effects of topical ophthalmic administration of diclofenac on intraocular pressure (IOP) when applied at 4 frequencies to eyes of Beagles. ANIMALS: 8 ophthalmologically normal Beagles. PROCEDURES: The study involved four 5-day experimental periods each separated by a 16-day washout period. During each period, 1 drop of 0.1% diclofenac sodium ophthalmic solution was administered to the right eye at 4 treatment frequencies (1, 2, 3, or 4 times/d); 1 drop of eyewash was administered to the left eye as a control treatment. A complete ophthalmic examination was performed on days 0 (day before first treatment) and 5 of each experimental period. Gonioscopy was performed on day 0 of the first period. The IOPs were measured at 7 am and 7 pm on days 1 through 5. RESULTS: No abnormalities were detected during neuro-ophthalmic and ophthalmic examinations on day 0 of each experimental period. No adverse reactions to administration of diclofenac or eyewash were observed at any time point. No abnormalities were detected during ophthalmic examinations performed on day 5, and IOPs remained < 25 mm Hg in all 4 periods. No significant differences were identified between the treated and control eyes or among the 4 treatment frequencies. CONCLUSIONS AND CLINICAL RELEVANCE: Topical ophthalmic administration of diclofenac up to 4 times/d in dogs with no ophthalmic abnormalities did not significantly increase the IOP. Additional research is needed to evaluate the effect of topical ophthalmic administration of diclofenac on IOP in dogs with anterior uveitis.

Bilateral Anterior Uveitis in a Northern Saw-whet Owl (Aegolius acadicus) With a Metastatic Pectoral Malignant Mesenchymoma.

A captive, adult, male northern saw-whet owl (Aegolius acadicus) was examined for blepharospasm of the left eye. The owl was diagnosed with bilateral anterior uveitis and a corneal ulceration in the left eye. It was treated with oral and topical nonsteroidal anti-inflammatory medications and a topical antibiotic. Multiple recheck examinations and medication adjustments were performed over the next 4 months, at the end of which time the bilateral anterior uveitis was controlled with a topical nonsteroidal anti-inflammatory applied 3 times per week to both eyes. The owl was re-examined 2 months later after 2 suspected neurologic episodes. On physical examination, the owl was quiet and had difficulty standing and ambulating. Five firm multilobular and immobile masses were identified overlying the pectoral muscle and sternum. Fine-needle aspiration from 1 mass revealed neoplastic cells consistent with a sarcoma. The owl was euthanatized. On the basis of results of histopathologic examination, the mass was diagnosed as a pleomorphic spindle cell sarcoma with features of rhabdomyosarcoma, liposarcoma, and osteosarcoma. Numerous tumor cells were immunopositive for myoglobin and desmin, indicating striated muscle origin. Although a metastatic lesion was present in 1 adrenal gland, lesions of inflammation or neoplasia were absent in either eye on histopathologic examination. This report describes an apparent ocular manifestation of systemic disease in an avian species with clinically diagnosed recurrent anterior uveitis.

Retrospective evaluation of canine palpebral masses treated with debulking and cryotherapy: 46 cases.

PURPOSE: To evaluate the recurrence frequency and the time to recurrence of canine palpebral masses treated with debulking and cryotherapy. METHODS: Medical records of dogs that underwent debulking and cryotherapy for palpebral masses from December 2010 to November 2015 at the University of Minnesota's Veterinary Medical Center were reviewed. A telephone survey with owners was used to determine recurrence and time to recurrence. RESULTS: Forty-six masses (31 dogs) were included in this retrospective study. Included palpebral masses were as follows: adenomas 17/46 (37%), epitheliomas 8/46 (17.4%), papillomas 6/46 (13%), inflammatory masses 6/46 (13%), mixed adenoma-papillomas 6/46 (13%), mixed epithelioma-papillomas 2/46 (4.4%), and a melanocytoma 1/46 (2.2%). Of the seven masses that were noted to recur (7/46, 15.2%), three were mixed adenoma-papillomas (three of six adenoma-papillomas, 50%), two were epitheliomas (two of eight epitheliomas, 25%), one was a papilloma (one of six papillomas, 16.7%), and one was a melanocytoma (one of one melanocytoma, 100%). Average recurrence time was 367.9 days [range: 30-904 days]. A significant association between tumor type and tumor recurrence was found (P = .009). Adenomas were significantly less likely to recur (P = .017), and mixed adenoma-papillomas were significantly more likely to recur (P = .001). CONCLUSIONS: Debulking and cryotherapy is an effective method of treatment for eyelid masses in dogs with a low study recurrence rate of 15.2% and average recurrence time of 367.9 days. Results also suggest that mixed adenoma-papilloma masses have a significantly higher recurrence rate than other eyelid masses, whereas adenomas had no recurrence in our study population.

Glaucoma with Descemet's membrane detachment in five horses.

PURPOSE: To describe the clinical and histopathologic features of glaucoma associated with Descemet's membrane (DM) detachment in five horses without prior history of intraocular surgery. ANIMALS STUDIED: Three Appaloosa horses and two Thoroughbreds were included in this study. The affected horses ranged in age from 16 to 27 years and presented with severe diffuse corneal edema. PROCEDURE: Five eyes were enucleated due to intraocular hypertension and/or chronic corneal ulceration. The enucleated globes were evaluated by the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Each globe was routinely processed for histopathology and analyzed by light microscopy. A histologic diagnosis of glaucoma was reached by demonstrating a loss of optic nerve axonal tissue by measuring neurofilament-immunopositive axons with automated image analysis software. RESULTS: All five horses presented with unilateral severe diffuse corneal edema that had developed between 2 and 16 weeks prior to enucleation. Intraocular pressures for the affected eyes were between 9 and 87 mmHg prior to enucleation. Descemet's membrane detachment was identified histopathologically in all five globes (5/5, 100%). All five eyes had an avascular spindle cell proliferation filling the space between the displaced peripheral DM and the corneal stroma. Neurofilament immunostaining revealed axonal loss consistent with glaucoma. CONCLUSION: Equine glaucoma may be associated with Descemet's membrane detachment. This detachment and glaucoma is a possible differential diagnosis for severe equine corneal edema. In this case series, an eye with a DM detachment had a poor prognosis for retention.

Modified Kuhnt-Szymanowski surgical procedure for secondary cicatricial ectropion in a horse.

A 1-year-old Dutch Warmblood gelding was presented to the University of Florida's Large Animal Hospital (UF-LAH) for correction of ectropion of the right lower eyelid. The ectropion was the result of a lower eyelid laceration. A primary repair was performed by the referring veterinarian; however, the horse prematurely removed the sutures and the wound healed with inversion of the eyelid margin. Surgical correction of the entropion, with removal of tissue from the lower eyelid, resulted in cicatricial ectropion. During the initial evaluation at UF-LAH, a corneal ulcer was noted in the right eye because of exposure from the anatomically nonfunctional lower eyelid. A modified Kuhnt-Szymanowski procedure was performed to correct the ectropion and repair the eyelid margin. The surgical site healed appropriately with an acceptable cosmetic and functional result. No recurrences of corneal ulcers in the right eye were reported in the 18 months following surgical correction of the ectropion.