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ABSTRACT NO.: 01

The expression of connective tissue growth factor (CTGF) and transforming growth factor-α (TGF-α) following glaucoma filtering surgery in a rabbit model

D. W. Esson,* M. B. Sherwood,* G. S. Schultz† and T. Blalock†

*Department of Ophthalmology, Institute for Wound Healing, University of Florida, Gainesville, FL, USA

Purpose: Glaucoma filtering surgery (GFS) may be indicated when medication fails to adequately control intraocular pressure (IOP). Excessive subconjunctival scarring following GFS is generally responsible for failure of the surgery. The processes involved in the scarring response appear to be heavily influenced by growth factors. We used a rabbit model of GFS, in which surgery rapidly fails secondary to aggressive scarring, to investigate the expression of CTGF and TGF-α following GFS, as well as the effect on the scarring response following the addition of these factors to the surgical site. Methods: In order to measure CTGF and TGF-α expression, GFS was performed in one eye of two sets of eight New Zealand White (NZW) rabbits by creating a limbal-based conjunctival flap and inserting a 22G IV cannula through a scleral tunnel into the anterior chamber to create a filtering bleb. Animals were sacrificed and tissues harvested for ELISA analysis at fixed time-points following surgery. CTGF and TGF-α were also immuno-localized in the conjunctival ‘bleb’ tissue. In order to evaluate the effect of exogenous CTGF and TGF-α, GFS was performed on one eye of six NZW rabbits as described above but the filtering sites were treated with a 5-min intraoperative application of 0.4 mg/mL Mitomycin-C (MMC) to increase bleb survival. Eight days following surgery, two randomly selected blebs were injected with 0.2 mL of balanced salt solution (BSS) containing 5 µg of recombinant CTGF, two with 0.2 mL of BSS containing 5 µg recombinant TGF-α, and two with BSS alone. Bleb survival was assessed for 21 days postoperatively by measuring IOP, bleb width, depth and height. Results: A peak in the levels of CTGF and TGF-α expression occurred at day 5 and days 5–7, respectively, following GFS, with reversion to baseline levels by days 7–10. MMC-treated blebs, receiving an injection of 0.2 mL BSS on day 8 following GFS, contracted to 50% of their original area by an average of 13 days after injection. In contrast, blebs treated with CTGF failed more rapidly, reaching 50% of their original size by an average of 6 days after injection and failed completely by an average of 16 days after injection, while all the BSS-treated blebs continued to survive at 21 days post injection. Similar MMC-treated blebs injected with TGF-α also failed completely by day 16 but decreased to 50% of their original area even earlier, by day 3. Conclusions: CTGF and TGF-α are induced in the bleb following GFS and exogenous CTGF and TGF-α significantly accelerate bleb failure in a rabbit model. Growth factors and their activation genes may provide targets for preventing the failure of GFS. Financial support: Supported in part by an unrestricted departmental grant and NIH grant EY05587. Commercial interest: None.

ABSTRACT No.: 02

Prevalence of the breed-related primary glaucomas in purebred dogs in North America

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K. N. Gelatt

Department of Small Animal Clinical Sciences and Gwathmey-Adams Laboratory of Vision Science, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610–0126, USA

Objective: To determine the prevalence of the breed-related glaucomas in purebred dogs presented to the veterinary medical teaching hospitals in North America that participate in the veterinary medical database (VMDB). Material and Methods: In this retrospective study, age at first diagnosis, breed and gender data for all breeds of dogs were collected from the VMDB with the clinical diagnosis of primary glaucoma (glaucoma-nos) at 5- to 10-year intervals from 1964 to 2002. The per cent prevalence for each breed (affected dogs compared to all dogs of each breed), any changes over the 38 years, and any gender differences for the these glaucomas were determined. Results: The prevalence of the primary breed-related glaucomas has gradually increased from 0.29% (1964–1973); 0.46% (1974–1983); 0.76% (1984–1993); to 0.89% (1994–2002). Breeds with the glaucomas consistently among the top 10 breeds from four different periods (1964 to 2002) included American Cocker Spaniel, Basset Hound, Wire Fox Terrier, and Boston Terrier. During the last observation period (1994–2002), 22 different breeds had 1% or higher prevalence of the glaucomas. The highest prevalence of glaucomas in 1994–2002 by breed included: American cocker spaniel (5.52%); Basset Hound (5.44%); Chow Chow (4.70%); Shar-Pei (4.40%); Boston Terrier (2.88%); Wire Fox Terrier (2.28%); Norwegian Elkhound (1.98%); Siberian Husky (1.88%); Cairn Terrier (1.82%); and Miniature Poodle (1.68%). A predominance of females with glaucoma occurred in the American Cocker Spaniel, Basset Hound, Cairn Terrier, Chow Chow, English Cocker Spaniel, Samoyed, and perhaps the Siberian Husky, and a predominance of males in the Australian Cattle dog and St Bernard. Age affects the time for first presentation of the glaucomas in the purebred dog. In the majority of breeds the glaucomas are presented for initial diagnosis in dogs between 4 and 10 years old. Conclusion: The breed-related glaucomas in purebred dogs are frequently presented to the veterinary medical teaching hospitals in North America. The prevalence of the breed-related glaucomas in the dog appears higher than any other animal species except humans, and in some breeds exceeds that in humans. In many breeds the high prevalence of the glaucomas suggests a genetic basis. Acknowledgment: The authors thank Ms Yun Shen, Operations Manager, Veterinary Medical Data Base/Canine Eye Registry Foundation, Purdue University; Ms Norma Pablo, Medical Records, Veterinary Medical Teaching Hospital, University of Florida; and the veterinary medical teaching hospitals which provided this information. Commercial interest: None.

ABSTRACT No.: 03

Lufenuron: determination of antifungal activity in vitro and measurement of blood concentrations following oral administration in horses

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N. C. Scotty,* P. J. Johnson,* E. A. Giuliano,* T. J. Evans,† G. Rottinghaus,† A. W. Fothergill‡ and T. J. Cutler§

*Departments of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Department of Pathology, University of Texas Health Science Center, §Animal Eye Specialty Clinic, West Palm Beach, FL, USA

Purpose: Lufenuron, a chitin synthetase inhibitor, is commonly advocated for the treatment of mycotic infections in horses. Experiment 1: to demonstrate the efficacy of lufenuron against Aspergillus sp. growth in vitro. Experiment 2: to demonstrate that lufenuron gains access to the circulation and achieves blood concentrations in a dose-dependent manner following oral administration in horses. Methods: Experiment 1. A colony of Aspergillus sp. isolated from diseased equine cornea was tested in vitro against technical grade lufenuron in a series of dilutions (in 100% DMSO) ranging from 0.125 µg/mL to 64 µg/mL, and compared to untreated control cultures. Experiment 2. Fifteen healthy adult horses were randomly divided into two treatment groups. Group 1 consisted of nine horses treated with lufenuron orally at a dose of 5 mg/kg, q 24 h for 3 days. Group 2 consisted of six horses treated with lufenuron orally at a dose of 20 mg/kg, q 24 h for 3 days. Blood samples were collected at 0, 1, 2, 3, 6, 12, 24, 48, 72 and 96 h following the first dose of lufenuron from all horses, and also at 5–14 weeks following the first dose of lufenuron from available horses. Whole blood lufenuron concentrations were determined using high performance liquid chromatography. Data were analyzed as a repeated measurement split-plot in time. Mean differences were determined using Fischer's least significant difference. Statistical significance was set at P < 0.05. Complete ophthalmic examinations consisting of slit-lamp biomicroscopy and indirect ophthalmoscopy were performed on all horses prior to the administration of lufenuron, and were repeated on available horses following the final dose of lufenuron. Results: Experiment 1. Lufenuron did not affect the rate of growth of Aspergillus sp. at the concentrations tested. Experiment 2. Group 1: mean lufenuron peak concentration was 19 p.p.b. (range 0–42 p.p.b.) at 72 h. Group 2: mean lufenuron peak concentration was 104 p.p.b. (range 41–176 p.p.b.) at 72 h (based on four horses, data pending). Conclusions: Lufenuron was absorbed into the circulation in a dose-dependent manner following oral administration in adult horses. Based on preliminary data, the highest mean blood concentration was attained using a dose of 20 mg/kg of body weight, and was 104 p.p.b. (0.104 µg/mL). Lufenuron failed to inhibit growth of an equine corneal Aspergillus isolate in vitro at concentrations ranging up to 64 µg/mL. Further studies are needed to determine whether lufenuron is effective against filamentous fungi, and to further characterize lufenuron's pharmacokinetic profile in horses. Financial support: Funded by the National Society of Phi Zeta, University of Missouri College of Veterinary Medicine. Commercial interest:  None.

ABSTRACT No.: 04

Changes in intraocular pressure in dogs wearing a collar vs. a harness

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A. M. Pauli, E. Bentley, K. A. Diehl and P. E. Miller

Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA

Purpose: It is frequently recommended that dogs with glaucoma, a weak or thin cornea, or those who have recently undergone intraocular surgery wear a harness rather than a collar. This recommendation is based on the assumption that a harness will result in less pressure on the neck, and therefore less of an increase in intraocular pressure (IOP) during exercise on a leash. Many harnesses, however, apply pressure at the thoracic inlet, and a harness allows the dog to pull against the leash to a greater degree. These factors may negate the usefulness of the harness and may actually result in a greater IOP during exercise. The purpose of this study was to evaluate the theory that application of neck pressure via a harness results in less of an increase in IOP than a collar. Methods: Fifty-one eyes from 26 healthy sled dogs were included in the study. Breeds represented were Malamute (n = 12), Siberian Husky (n = 8), Pit Bull (n = 4), Cocker Spaniel (n = 1), and Chinook (n = 1). Complete ophthalmic exams including slit-lamp biomicroscopy and binocular indirect ophthalmoscopy were performed on all dogs. One drop of 0.5% proparacaine was administered topically to both eyes, and the resting IOP was measured with applanation tonometry (Tono-Pen XL Mentor, Norwell, MA, USA). The tension each dog generated against a nylon leash was measured in kilograms using an Imada digital force gauge. Each dog pulled on the leash, first wearing a collar and then wearing a harness. Each dog was then restrained on the ground in a standing position and the previously measured force of pull was applied to the neck by pulling on the leash. After 10 s of applied force, the IOP of both eyes was measured and the application of the force was stopped. The IOP was measured again 1 min later. After at least 5 min to allow IOP to return to baseline this procedure was repeated with the harness for each dog. Significance was assessed with anova, and pair-wise comparisons performed with Student's t-test with the level of significance set at P < 0.05. Results: IOP was significantly increased over baseline values under force when the dog was wearing a collar (51.6%, up to a 35-mmHg increase) but not with the harness (15.8%, up to a 14.5-mmHg increase), and a collar significantly increased IOP compared to the harness (P < 0.05). IOP returned to baseline after 1 min. There was no correlation between the increase in IOP and the amount of tension applied to the neck (inline image= 0.1014, inline image= 0.0113), or with the tension generated per kilogram body weight by the dog (inline image= 0.0009, inline image= 0.1187). Conclusions: Dogs with weak or thin corneas, glaucoma or any condition for which an increase in IOP could be harmful would benefit from wearing a harness instead of a collar when exercising on a leash. Once the dog begins pulling against a leash while wearing a collar the force with which it pulls does not correlate to the increase in IOP it experiences. Commercial interest: None.

ABSTRACT No.: 05

Enrofloxacin and the feline retina: histologic effects

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R. R. Dubiezig,* M. M. Ford† and K. Narfstrom†

*School of Veterinary Medicine, University of Wisconsin, Madison, WI, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA

Purpose: The goal of this work is to characterize the morphology of retinal degeneration in the first few days following exposure to toxic levels of enrofloxacin administered orally to healthy cats. Methods: Twelve male and 12 female cats were given 50 mg/kg/day of enrofloxacin as a single dose orally. The status of the retina was monitored via funduscopy and electroretinography, and the cats were sacrificed 3, 5 and 7 days after the initial dose. Control cats were given sham medication and were monitored in exactly the same manner. Results: Cats sacrificed 3 days after the first dose had dramatic swelling and cytoplasmic vacuolation limited to the photoreceptors. On day 5, the number of rod photoreceptors was greatly diminished and nuclear pyknosis replaced cytoplasmic vacuolation as the most prominent change. By day 7 there were very few rod photoreceptor nuclei remaining. Cone nuclei were still detectable and at no time were changes in the inner retina detectable. Conclusions: Enrofloxacin, given at a rate of 50 mg/kg/day to healthy adult cats is acutely toxic to the rod photoreceptors leading to cytoplasmic vacuolation detectable by day 3 and profound atrophy of the outer retina by day 7. Rod photoreceptors appear to be more sensitive than cones, and the inner retina remains morphologically unaffected. Financial support: Supported by Bayer Animal Health. Commercial interest: None.

ABSTRACT No.: 06

Feline periocular peripheral nerve sheath tumor: a case series

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T. Blocker,* A. Hoffman,* R. R. Dubielzig† and E. J. Ehrhart‡

*Eye Care for Animals, Tustin, CA, College of Veterinary Medicine, University of Wisconsin, Madison, WI, College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA

Purpose: To describe signalment, clinical presentation, surgical treatment and outcome, survival, and histopathologic and immunohistochemical characteristics of periocular peripheral nerve sheath tumor in a series of cats. Methods: Six cats with a histologic diagnosis of peripheral nerve sheath tumor (PNST) of the eyelid or conjunctiva were identified from 3997 feline ocular histopathologic submissions between 1976 and 2002. Medical records were reviewed and data collected for signalment, eye affected, location, number of recurrences, surgical description and outcome, survival, and histopathologic description. Results of ancillary diagnostic tests were recorded when available. An immunohistochemistry (IHC) panel was performed on the tissue. Tumors from five cases were stained for vimentin, S-100, glial fibrillary acid protein (GFAP), laminin, actin, and neurofilament protein (NFP). Tumor from one case was stained for S-100, neuron specific enolase (NSE) and Melan A. Results: Mean age was 8 years (range: 5–15 years). There was an even gender distribution. Breeds represented were three DSH, two DLH and one Manx. The right eye was affected in four cats and the left eye in two cats. The upper eyelid was affected in the majority of the cats (5/6). Tumor regrowth occurred in all cases an average of three times. Five of six cats underwent wide excision with enucleation or exenteration as the final surgery. Tumor regrowth occurred in two of these cats. Histomorphologic evaluation of the tumors revealed them all to be of spindle cell type showing features of Antoni type B pattern in 5/6 tumors and Antoni type A pattern in 1/6 tumors typical of PNST. Bielchowski silver stain for axons was negative in 4/5 of the tumors. IHC revealed a strong reactivity for vimentin in 5/5 tumors; S100 in 5/6 tumors, GFAP in 0/5 tumors, laminin in 4/5 tumors, actin in 0/5 tumors, NFP in 1/5 tumors, and NSE in 1/1 tumors. Conclusions: Definitive histologic diagnosis of spindle cell tumors can be difficult. The presence of Antoni A and B histologic patterns, as well as immunohistochemical affinity for vimentin, S100, laminin and NFP, supports a diagnosis of PNST. The results of this study characterize periocular PNST as a rare tumor in cats that has a high rate of local recurrence. Wide excision that involved sacrificing a visual eye was necessary in the majority of the cases. Distant metastasis was not confirmed pre or postmortem. Commercial interest: None.

ABSTRACT No.: 07

Equine amniotic membrane transplantation for keratomalacia in three horses

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M. E. Lassaline, A. M. Komaromy, F. J. Ollivier, M. E. Kallberg, D. E. Brooks and K. N. Gelatt

College of Veterinary Medicine, University of Florida, Gainesville, FL USA

Purpose: Amniotic membrane has antifibrotic, antiangiogenic and antiprotease properties. This retrospective study was designed to investigate the use of equine amniotic membrane transplants (AMT) in preserving vision, maintaining the structural integrity of the globe and maximizing cosmesis in equine eyes with severe keratomalacia. Method: Equine amnion had previously been aseptically harvested from a 12-year-old Thoroughbred mare during an elective C-section. Sections of amnion were stored at −80 °C on a nitrocellulose membrane in DMEM with penicillin, streptomycin, neomycin and amphotericin B, and thawed as needed. Records of equine cases at the University of Florida with keratomalacia that received an amniotic membrane transplant without adjunctive conjunctival grafting were examined. Clinical description, details of medical and surgical treatment, and outcome were documented. Etiologies were determined by cytology, culture or histology. Results: Three horses with severe keratomalacia received an AMT without conjunctival graft between December 2002 and April 2003. Two were Thoroughbred racehorses, and one a Hanoverian stallion. All three had potentially career-limiting melting ulcers in the left eye. Pseudomonas spp. were cultured from all three eyes, with evidence of a concurrent fungal infection in two eyes (i.e. from cytology and culture of Aspergillus in one eye, and histology in the second). The ulcers involved 50% of corneal diameter in one case, and greater than 75% of corneal diameter in two cases, and were deemed poor candidates for conjunctival grafting due to the size of the subsequent scar and degree of visual impairment. All three ulcers worsened with aggressive medical therapy of antibiotics and antifungals (both systemic and topical), antiproteases, mydriatic/cycloplegics, and anti-inflammatories. Amnion was used without conjunctival graft in all three cases. One AMT was covered with an Acell membrane. In all three cases the AMT sloughed over a 4–6-week period. At last follow-up, all three eyes receiving AMT were comfortable and on no medication. The three eyes had light perception (indicated by positive dazzle and consensual papillary light reflex) and an inconsistent location-dependent menace response. All three horses returned to their prior work. Conclusions: Results of a small number of equine amniotic membrane transplants suggest that amnion can be used successfully to preserve both globe structure and limited vision, as well as optimize cosmesis, in cases with severe keratomalacia. Commercial interest: None.

ABSTRACT No.: 08

A retrospective, epidemiologic review of Horner's syndrome in the dog: 169 cases (1992–2003)

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K. L. Abrams

Veterinary Ophthalmology Services, Inc., Warwick, Rhode Island, USA

Purpose: To review the epidemiologic factors in 169 cases of Horner's Syndrome (HS) in the dog. Methods: The medical records of 169 cases of Horner's Syndrome in the dog were extracted from the practice's computerized patient files using the search feature of the Veterinary Specialists System's (VSS) software. Chi Square analysis was performed on a Palm m500 hand-held device using the tStat Chi Square software. Results: A total of 169 cases of Horner's Syndrome were reviewed out of 13 736 dogs examined at the practice. There was no significant difference between left and right eyes. Purebred dogs represented 132 cases and 49 breeds; 37 cases were mixed breed. The most commonly represented purebreds included the: Golden Retriever (17.8%), Labrador Retriever (15.4%), American and English Cocker Spaniels (10%), Collie (7%), Shetland Sheepdog (4.7%), Siberian Husky (3.6%), Weimaraner, Rottweiler, Samoyed, Bassett Hound (2.4% each), Doberman (1.8%), and Akita (1.2%). Compared with the general practice population, Golden Retrievers, Labrador Retrievers, Collies, Shelties, Weimaraners and Dobermans were over-represented. When all HS males and females were compared to the general practice population, there were significantly more males with Horner's Syndrome (P < 0.01). However, if gender is examined only for Golden Retrievers with HS compared with the general practice population of Golden Retrievers, there is no significant difference between all males and all females. Finally, there were 22 cases of HS with diabetic cataracts (13%), 17 of which had phacoemulsification, and five of which did not have surgery, and there was one case of nondiabetic cataracts. Compared with the general practice population of diabetic cataracts, there was a very significant over-representation of diabetic cataract patients who developed HS. Phenylephrine testing was performed on 122 patients with an average time to resolution of clinical signs of 9.9 min (± 5.3). Conclusions: The significantly high incidence of certain breeds with Horner's Syndrome in this review series is contrary to earlier reports in the U.S. (Morgan 1989, Kern 1989), but the highest breed representation of Golden Retrievers is consistent with two reports from the United Kingdom (Boydell 1995, 2000). Boydell reported that 110 dogs out of 155 Horner's Syndrome cases were Golden Retrievers but there was no conclusive comparison to the overall general population of canine patients. Of the 110 Goldens with HS, 95 were males, and in the current series males were over-represented. However, in the current series, when gender was examined by Golden Retrievers only, there was no significant difference. Finally, the existence of diabetes rather than cataract formation or phacoemulsification of the cataract appeared to be a significant risk factor for developing Horner's Syndrome. Commercial interest: None.

ABSTRACT No.: 09

Electroretinography in the Hispanolian Amazon parrot (Amazona ventralis)

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D. V. H. Hendrix* and M. H. Sims†

*University of Tennessee, Department of Small Animal Clinical Sciences, University of Tennessee, Department of Comparative Medicine, Knoxville, TN, USA

Purpose: This study was undertaken to characterize the normal latencies, amplitudes and presence of oscillatory potentials in the electroretinogram of Hispanolian Amazon parrots (Amazona ventralis). Methods: Six healthy 3-year-old Hispanolian Amazon parrots with clinically normal eyes were anesthetized using isoflurane. A contact lens electrode was placed on the cornea of the test eye, a subdermal needle electrode was placed 1 cm lateral to the lateral canthus, and a subcutaneous electrode (ground) was placed over the occipital protuberance of the skull. Photic stimuli were delivered from a stroboscopic flash placed 20 cm from the test eye. The birds were dark adapted for 4 min. The average ERG after 6 flashes of white light in the light-adapted state and the white, red or blue light in the dark-adapted birds was recorded. Each discharge of the strobe triggered two amplifiers of a four-channel electrodiagnostic system to record activity at the cornea for 225 ms. One channel amplifier was used for recording the ERG and the other for recording the OP. For recording the ERG, the channel amplifiers were set at a bandpass of 1–3000 Hz, while the bandpass for the OP was 100–500 Hz. Input to the amplifier was such that positive activity at the cornea caused an upward deflection in the recording. Positive peaks of the OP were labeled consecutively as 01 to 04. Positive peak latencies and amplitudes were measured using a cursor on the computer monitor and were recorded to the nearest 0.01 ms and 0.01 µV, respectively. Latency was measured from the onset of the stimulus to the positive peak, and the amplitude was measured from the positive peak to the preceding nadir. Results: The mean latency for the light-adapted a-wave was 11.35 (± 1.54) ms and the mean latency for the light-adapted b-wave was 24.4 (± 6.17) ms. The mean amplitude of the light-adapted a-wave was 20.4 (± 5.45) µV and the mean amplitude of the light-adapted b-wave was 57.34 (± 27.82) µV. The mean latency for the dark-adapted a-wave was 12.15 (± 1.55) ms and the mean latency for the dark-adapted b-wave was 29.65 (± 3.98) ms. The mean amplitude of the dark-adapted a-wave was 38.61 (± 13.44) µV and the mean amplitude of the dark-adapted b-wave was 129.47 OP (± 26.12) µV. The oscillatory potentials were composed of three or four peaks. Variability in latency increased from 01 to 04 and three eyes did not have recordable 04. Conclusions: This study showed that ERG results in 3-year-old Hispanolian Amazon parrots are more similar to the ERG of dogs seen for cataract evaluation in our hospital than to the ERG of raptors. A bird's diurnal or nocturnal habits and feeding habits should be taken into consideration when interpreting ERG results.

ABSTRACT No.: 10

Endothelin-1 and nitric oxide in the normal and glaucomatous dog eye

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M. E. Kallberg,* D. E. Brooks,* K. N. Gelatt,* W. W. Dawson,† A. T. Timmers,† D. A. Samuelson,* N. J. Szabo* and G. N. Lambrou‡

*College of Veterinary Medicine, College of Neuroscience, University of Florida, Gainesville, FL, USA, Novartis Pharma, Basal, Switzerland

Purpose: To document any differences in the levels of the endothelin-1 (ET-1) peptide and nitric oxide (NO) in aqueous humor and vitreous in the glaucomatous dog eye compared to the normal dog eye. Methods: Comparisons were made between normal (n = 30) and glaucomatous (n = 14) samples for levels of NO and ET-1, measured by enzyme immunoassay in aqueous humor and vitreous. The numerical data were analyzed using an anova 2*2 factorial analysis. The two factors of interest were the disease, with two levels (glaucomatous and normal), and breed, with two levels (Cocker Spaniel and non Cocker Spaniel). Results: The ranges for aqueous humor and vitreal ET-1 levels for all normal samples were 0.87–6.3 and 0.23–6.83 pg/mL, respectively. The ET-1 aqueous humor and vitreal ranges for all glaucomatous samples were 1.91–14.56 and 1.13–5.96 pg/mL, respectively. The mean (± SD) canine ET-1 aqueous levels for all breeds increased significantly (P = 0.0004) from 2.8 ± 1.52 pg/mL for the normal eyes to 6.86 ± 3.46 pg/mL for the glaucomatous eyes. The increase was significant for the Cocker Spaniel group (P = 0.0003) while the increase for the non Cocker Spaniel group was not significant. The mean (± SD) canine ET-1 vitreous levels for all breeds increased significantly (P = 0.0023) from 1.56 ± 1.86 pg/mL for the normal eyes to 3.47 ± 1.57 pg/mL for the glaucomatous eyes. The increase was significant for the Cocker Spaniel group (P = 0.0005), while the increase for the non Cocker Spaniel group was not significant. The mean vitreal ET-1 level for the normal Cocker Spaniel group was significantly lower (P = 0.0099), than for the normal non Cocker Spaniel group. The ranges for all normal aqueous humor and vitreal NO levels were 1.41–11.46 and 0.24–16.47 µm, respectively. The NO aqueous humor and vitreal ranges for all glaucomatous dogs were 2.25–37.94 and 1.07–46.98 µm, respectively. The mean (± SD) canine NO aqueous levels for all breeds increased significantly (P = 0.0176) from 3.9 ± 1.93 pg/mL for the normal eyes to 12.48 ± 13.46 pg/mL for the glaucomatous eyes. The increase was larger for the non Cocker Spaniel group although there was no interaction. The mean (± SD) canine NO vitreous levels for all breeds increased significantly (P = 0.0085) from 4.47 ± 2.92 pg/mL for the normal eyes to 15.33 ± 16.22 pg/mL for the glaucomatous eyes. The increase was significant for the non Cocker Spaniel group (P = 0.0148), while the increase for the Cocker Spaniel group was not significant. Conclusion: The increases in endothelin-1 in glaucomatous aqueous humor and vitreous were larger for the Cocker Spaniel group compared to the non Cocker Spaniel group, while the increases in NO were larger for the non Cocker Spaniel group. The distinct results for the two groups might imply a unique response of NO and ET-1 in narrow-angle glaucoma in Cocker Spaniels. Financial support: Supported by Novartis Pharma. Commercial interest: None.

ABSTRACT No.: 11

Inherited retinal dysplasia in Miniature Schnauzer dogs

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Bruce H. Grahn,* Eric S. Storey* and Catherine McMillan†

*Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4, Box 579, Delisle, Saskatchewan, S0L 0P0 Canada

Purpose: To define the clinical syndrome and determine the etiology of retinal dysplasia and persistent primary vitreous in miniature Schnauzer dogs. Methods: We examined 106 miniature Schnauzers with a biomicroscope and an indirect ophthalmoscope. Several enucleated eyes were fixed, sectioned and stained routinely. They were examined with routine light microscopy, and transmitting and scanning electron microscopy. A pedigree was constructed and related dogs were test-bred to define the mode of inheritance of this syndrome. Results: Congenital retinal dysplasia was confirmed in 24 of 106 related Miniature Schnauzer dogs. Physical and postmortem examinations revealed that congenital abnormalities were limited to the eyes. Biomicroscopic and indirect ophthalmoscopic and neuro-ophthalmic examinations confirmed that many of these dogs were blind secondary to bilateral retinal dysplasia and detachment (nonattachment) (n = 13), and the remainder had focal areas of retinal dysplasia (n = 11). Several of these dogs were also diagnosed with unilateral (n = 9) or bilateral (n = 6) persistent hyperplastic primary vitreous. These examinations and the pedigree and test-bred litters revealed that this condition is inherited. Conclusions: This study confirms that retinal dysplasia and persistent hyperplastic primary vitreous is a congenital syndrome that it is inherited as an autosomal recessive condition in Miniature Schnauzers. Financial support: Supported by Companion Animal Health Research Grant 702615. Commercial interest: None.

ABSTRACT No.: 12

Bilateral immune-mediated ulcerative keratitis with collagenolysis in two dogs

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Eric S. Storey and Bruce H. Grahn

Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada

Purpose: To describe clinical manifestations and laboratory findings of idiopathic bilateral circumferential collagenolytic steroid-responsive corneal ulcers in two dogs. Methods: We completed routine physical and ophthalmic examinations in two dogs presented with bilateral circumferential deep corneal ulcers. We also examined corneal and conjunctival biopsies with light microscopy and immunhistochemistry because these ulcers were nonresponsive to topical antibiotic and anticollagenolytic therapy. In addition, a quantitative immunoglobulin assessment was completed in one dog. Results: Clinical signs common to both dogs included progressive bilateral peripheral circumferential collagenolysis. Collagenolysis was progressive despite medical management including autologous serum and broad-spectrum antibiotics. Aerobic and anaerobic cultures and light microscopy of tissues failed to reveal any microorganisms. Conjunctival and corneal biopsies confirmed a lymphoplasmocytic inflammatory cell infiltrate and a paucity of pyogenic inflammation. Immunohistochemistry confirmed that the inflammatory cells were predominantly CD8 positive T-lymphocytes. Quantitative immunoglobulin analysis was normal except for a significant increase in IgA. The ulcerative keratitis responded favorably to topical immunosuppression with corticosteroids. Conclusions: The light microscopic, bacteriologic and immunohistochemical findings in both dogs supported an immune-mediated etiology of the corneal ulceration. These findings are similar to immune-mediated ulcerative keratitis in humans. Commercial interest: None.

ABSTRACT No.: 13

Photodynamic therapy for the treatment of periocular squamous cell carcinoma in horses

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E. A. Giuliano,* D. L. McCaw,* I. MacDonald,† P. J. Johnson,* G. Klauss,* M. M. Ford,* N. C. Scotty,* L. E. Galle* and C. P. Moore*

*Veterinary Medicine and Surgery, University of Missouri-Columbia, Columbia, MO, Photodynamic Therapy Center, Roswell Park Cancer Institute, Buffalo, NY, USA

Purpose: Photodynamic therapy (PDT) is an evolving new treatment modality. A pilot study seeking to demonstrate proof of principle and safety is being conducted. We hypothesize that surgical excision with adjunctive local PDT is an effective and safe treatment for equine periocular squamous cell carcinoma (PSCC). Methods: Three horses with naturally occurring PSCC were identified. Tumors were characterized by complete ophthalmic examination, 3-dimensional tumor measurement, extraocular photography, and histopathology. With patients under general anesthesia, PSCC tumors were treated with surgical debulking and infiltration of the resulting wound bed with pyropheophorbide-a-hexyl-ether (HPPH, PhotochlorTM) in dimethylsulfoxide (final concentration: 2 mg/mL; treatment dose = 1 mg/cm2 of tumor bed). Surgical beds were subsequently irradiated using a 665-nm wavelength diode laser (incident light dose = 100 J/cm2; fluence rate = 100 mW/cm2). Duration of treatment for each 3 cm-diameter treatment area was 16 min 40 s. Post-treatment progress of horses was regularly evaluated by complete ophthalmic examination and repeated biopsy. Results: Refractory PSCC had been unsuccessfully treated (using surgical excision with cryotherapy and/or intralesional cisplatin) in two of three horses prior to PDT. Partial surgical excision and PDT has subsequently yielded disease-free intervals of 14, 10 and 9 months in our study horses. These results were obtained following a single treatment in two horses and two treatments in one horse. In one horse, carcinoma in situ developed 2 months after partial surgical excision and PDT, requiring local excision under standing sedation. Other minor postoperative complications included mild-to-moderate eyelid swelling and regional tissue necrosis, typical of PDT. Conclusions: Preliminary results suggest that surgical excision and adjunctive PDT may represent a safe and useful novel treatment modality for PSCC in horses. Providing these initial favorable results are supported by results of on-going studies, additional advantages of PDT in the treatment of equine PSCC compared with current methods include improved nonrecurrence rates, shorter hospital periods, reduced overall cost, good cosmesis and excellent preservation of eyelid function. Future studies are underway to further evaluate the cellular and biochemical effects of PDT on equine PSCC. Commercial interest: None.

ABSTRACT No.: 14

Vascular reactivity and retinal histology in Abyssinian cats with progressive rod cone degeneration

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E. A. Giuliano,* R. A. Linsenmeier† and K. Narfstrom*

*Veterinary Medicine and Surgery, University of Missouri – Columbia, Columbia, MO, Biomedical Engineering and Neurobiology & Physiology, Northwestern University, Evanston, IL, USA

Purpose: Retinal vessel blood flow regulation at varying stages of photoreceptor degeneration in Abyssinian cats was evaluated in response to changes in blood oxygen levels. Photoreceptor loss and retinal thicknesses at central and peripheral locations were correlated to blood vessel changes. Methods: Eight Abyssinian cats (1–6.5 years old) with hereditary retinal degeneration, stages 1 (early) to 4 (late), were examined. Cats were placed under general anesthesia and color fundus photographs were taken while the animals were breathing room air, 100% oxygen, and 10% oxygen, sequentially. Measurements of vessel diameter (largest and smaller artery and vein) were made from digitized photographs. At the conclusion of general anesthesia, the retinas were fixed in 2% glutaraldehyde in 0.1 m cacodylate buffer. Selected retinal sections were routinely processed and 2-micron-thick sections were stained with toluidine blue for light microscopy. Total and outer retinal thickness (at ×400) were measured and photoreceptor nuclei were counted (average of three sections at ×1000) in the area centralis and far periphery. Results: Diameters of retinal arteries and veins decreased as the degeneration progressed, with stage 1 animals being indistinguishable from normal cats, and a stage 4 cat having vessels that were barely detectable. Artery and vein diameters increased in hypoxemia and decreased in hyperoxia. Outer nuclear photoreceptor counts correlated well with total retinal thickness and outer retinal thickness across animals and stages of disease. As the disease progresses, decreases in photoreceptor nuclei occur earlier in the periphery than in the area centralis. Additionally, there was an earlier loss of photoreceptor nuclei in the inferior retina than in the superior retina. Inner retinal thickness was well preserved in all stages of retinal degeneration. Conclusions: Retinal vessels become attenuated in photoreceptor degeneration, but regulate normally in response to hypoxia and hyperoxia. These results, in addition to earlier studies, support the conclusion that changes in vessels precede the structural loss of photoreceptors, and are more closely related to the loss of photoreceptor function. Financial support: NIH EY05034 (RAL); NCI (KN); Foundation Fighting Blindness (KN). Commercial interest: None.

ABSTRACT No.: 15

Utilizing b- and a-wave ERG amplitudes in a graphical representation and b/a-wave ratios in the diagnosis of feline hereditary rod cone degeneration

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J. Hyman* and K. Narfström†

*Eye Care for Animals, Kansas City, KS, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA

Purpose: To utilize a graphical representation of b- and a-wave amplitudes and b/a-wave ratios to diagnose hereditary retinal rod cone degeneration in funduscopically normal cats from heterozygous or homozygous rod cone degeneration parents. Methods: Electroretinograms (ERGs) were performed on 14 F2 cats (from parents heterozygous or homozygous for recessively inherited rod cone degeneration), aged 8–18 months, on two to three different occasions, 3–6 months apart. Results from 24 unrelated age-matched normal cats and six known heterozygous (F1 cats) were compared to the F2 ERGs. Following 2 h of dark adaptation, the cats’ pupils were dilated and anesthetized with medetomidine and ketamine IM. A computerized ERG stimulating and recording system was utilized (ERG system Tor, Global Eye Program, Reymyre, Sweden) with bilateral full-field light stimulation. ERGs using a scotopic white light intensity series in 0.5 log unit stops, from − 6.0 cd s/m2−0.6 cd s/m2, were performed. After 10 min of light adaptation (background 30 cd/m2) photopic stimulation was elicited at 5, 30 and 50 Hz at 0.0 cd s/m2. B- and a-wave amplitudes were calculated and fitted into a graph utilizing the median and the 1st and 99th percentiles as limits of normality. B/a-wave amplitude ratios were also obtained and compared to those of control cats. Results: Eight of the 14 F2 cats showed low amplitudes for the a-wave, close to or below the lower limits of normality for the control cats in this age group using our system. Similar differences were not as obvious at this age range for the b-wave for the same eight cats. Further, in these eight cats b/a-wave ratios were markedly increased when compared to the normal, F1 cats, and other F2 cats in the study. Conclusions: Homozygous animals were differentiated from heterozygotes mainly through evaluation using a graphical representation of ERG a- and b-wave amplitude results and b/a-wave ratios. The increase in b/a-wave ratios was apparently caused by a marked decrease in a-wave amplitudes in early disease. Financial support: Supported by the Foundation Fighting Blindness and the National Cancer Institute. Commercial interest: None.

ABSTRACT No.: 16

Deep lamellar endothelial keratoplasty (DLEK) in six horses

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D. E. Brooks, M. E. Lassaline, M. E. Kallberg, A. M. Komaromy, K. N. Gelatt and F. J. Ollivier

College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

Purpose: A technique for surgical correction of deep stromal abscesses (DSA) is described in six horses. Methods: Retrospective medical records study. The DLEK procedure involves an 18–25-mm limbal incision to a depth just anterior to Descemet's membrane, lamellar dissection to expose and remove the DSA, sutureless placement of a split-thickness, frozen, endothelial graft, and closure of the limbal incision with 7–0 vicryl. An 8-year-old Quarterhorse gelding, 1-year-old Thoroughbred colt, 17-year-old Paint mare, 5-year-old Tennessee Walking Horse mare, 8-year-old Thoroughbred mare, and a 4-year-old Quarterhorse stallion received the DLEK procedure. Results: The abscesses ranged from 5 to 8 mm in diameter. Healing was achieved in 14–28 days in four cases. Intralesional hyphae (n = 3) and hyphae/eosinophils were present (n = 1) in histologic specimens of the SA. Complications included wound dehiscence (n = 1), suture line infection with beta Streptococcus (n = 1), and sandwich keratitis (n = 1). One horse died of intestinal problems and a second from septic pneumonia and colitis. Conclusions: This procedure is relatively simple and has a high rate of tectonic success in limbal DSA of horses. Commercial interest: None.

ABSTRACT No.: 17

MMP production by microbial isolates from equine corneal ulcers

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D. E. Brooks, F. J. Ollivier, M. E. Lassaline, M. E. Kallberg, G. S. Schultz and K. N. Gelatt

College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

Purpose: Tear film matrix metalloprotease (MMP) activity is elevated in horses with corneal ulcers. There are several possible sources of the MMP. We wanted to determine if microbes isolated from corneal ulcers in horses are responsible for some of the elevation in MMP. Methods: Cultures of Staphylococcus areus, β-hemolytic Streptococcus, Pseudomonas aeruginosa, Aspergillus sp. and Fusarium sp. were obtained from horses with ulcerative keratitis presented to the University of Florida Veterinary Ophthalmology Service. Organisms were grown on Mueller Hinton medium at 37 °C, and 5% O2 and 95% CO2. Samples of culture medium were then collected from each organism at 24, 48 and 72 h, filtered, and then stored at −80 °C. Gelatin zymography and measurement of optical density were used to evaluate the proteolytic activity in the culture media. Results:Staphylococcus areus and β-hemolytic Streptococcus did not produce any detectable MMP. Pseudomonas aeruginosa produced alkaline protease and Pseudomonas elastase. Aspergillus produced a high molecular weight alkaline protease at 72 h. Conclusions: Some bacteria and fungi are directly responsible for a small portion of the elevation in tear film MMP activity in horses with corneal ulcers. Other microbes may induce disease by producing other growth factors or cytokines that induce production of MMP by the cornea and other sources. Commercial interest: None.

ABSTRACT No.: 18

Corneoconjunctival transpositional grafts − an inferior approach

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M. A. Mineo and C. A. Fischer

Animal Eye Clinics of Florida, Clearwater, FL, USA

Purpose: To compare the overall cosmetic appearance of the cornea when corneoconjunctival transpositional grafts (CCT) are performed using transposed tissue harvested inferior to the corneal lesion vs. a superior approach. The ophthalmologic reasons to use this surgical procedure, and the steps to perform it, are reviewed. Methods: Clinical observations and comparisons were made on patients who had undergone a CCT procedure for various conditions including deep infectious corneal ulcers, some with descemetocele formation, degenerative/calcific keratopathy and its complications or feline corneal sequestra. In some of these eyes the tissue was surgically harvested and advanced ventrally from a position superior to the lesion. In other eyes, the corneoconjunctival tissue was advanced dorsally from a position inferior to the lesion. The steps of this surgical procedure were performed in the same manner for all eyes involved. Postoperative medications and treatment regimes were similar in most cases; however, some were individualized to the specific needs of the patient. Results: In all cases, this surgical procedure successfully remedied the original reason for the surgery. After an appropriate amount of healing time, the only residual opacity visible to the untrained or unaided eye was the area of the transposed limbus. In the animals where the grafted tissue was harvested superior to the lesion there was significantly more visual residual corneal opacification than those where tissue was transposed from the inferior position. Conclusion: CCTs can be used to correct defects, lend support and facilitate healing for a multitude of corneal conditions. A far greater cosmetic appearance of the cornea can be achieved if the graft is surgically taken from the cornea and conjunctiva inferior to, rather than superior to, the lesion or defect which is to be corrected. In all but the most dorsal of lesions, the area of transposed limbus remains hidden underneath the third eyelid or lower lid margin. Commercial interest: None.

ABSTRACT No.: 19

Characterization of the pupil light reflex, electroretinogram and tonometric parameters in healthy mouse eyes

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S. D. Grozdanic,*,† R. A. Allbaugh,† D. M. Betts,* D. S. Sakaguchi,†,‡ Y. H. Kwon,§ R. H. Kardon§ and I. M. Sonea†

*Departments of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, Department of Zoology and Genetics, Iowa State University, Ames, IA 50011, §Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA

Purpose: To characterize the pupil light reflex (PLR), electroretinographic (ERG) and tonometric parameters which might be of importance for the in vivo characterization of mouse models of chronic ocular hypertension. Methods: C57/BL6 mice were used for experiments. The PLR was evaluated with a computerized pupillometer (n = 14), ERGs were recorded simultaneously from both eyes (n = 23) and IOP was measured with a modified Goldmann tonometer (n = 23). Results: The analysis of the PLR parameters confirmed that the consensual PLR did not have significantly different amplitude (P > 0.1) and latency time (P > 0.1) compared to the direct PLR. However, PLR velocity (P = 0.004) was significantly lower in the consensual PLR. Electroretinography revealed an a-wave amplitude of 168.3 ± 9.6 µV with a latency of 27.5 ± 0.6 ms and a b-wave amplitued of 403 ± 28.8 µV with a latency of 22.7 ± 0.6 ms. The flicker ERG recording revealed amplitudes of 20.6 ± 2.4 µV. Tonometry experiments revealed that modified Goldmann tonometer measurements correlated well with invasive manometry (r2 = 0.89). The mean IOP of the mouse was 15.3 ± 0.6 mmHg. Conclusions: Consensual PLR in mice is relatively slower than the direct PLR, but retains the same degree of constriction as the direct PLR. A modified Goldmann tonometer seems to be a reliable noninvasive tool for IOP measurements in mice. Comercial interest: None.

ABSTRACT No.: 20

Temporary recovery of the inner retina and optic nerve function in the rat model of chronic ocular hypertension

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S. D. Grozdanic,*,† S. N. Hildreth,* D. M. Betts,* D. S. Sakaguchi,†,‡ Y. H. Kwon,§ R. H. Kardon§ and I. M. Sonea†

*Departments of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, Department of Zoology and Genetics, Iowa State University, Ames, IA 50011, §Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA

Purpose: To functionally characterize the rat retina and optic nerve after chronic elevation of intraocular pressure (IOP) using electroretinography (ERG) and computerized pupillometry. Methods: Chronic elevation of IOP was induced in Brown Norway rats by combined injection of indocyanine green dye (ICG) into the anterior chamber and diode laser treatment, followed by ERG and pupil light reflex (PLR) monitoring. Results: Laser treatment induced significant elevation of IOP in operated eyes. Preoperative values for the PLRratio (ratio = indirect/direct PLR) were 68.5 ± 4% (mean ± SEM; %). Three days postoperatively, the PLRratio decreased to 60.3 ± 10.3%, but was not significantly different compared to preoperative values (P > 0.05, Kruskal–Wallis nonparametric test with Dunn's post-test). However, 7 days postoperatively the PLR function dramatically decreased to 14.6 + 8.6% and was significantly smaller compared to preoperative values (P < 0.01). Fourteen days postoperatively, the PLR function further decreased to 11.5 ± 6.7% (P < 0.01) and 21 days postoperatively the PLRratio was 12.6 ± 4% (P < 0.05). At day 28 the PLR significantly recovered and was not significantly different compared to preoperative values (PLRratio= 38.5 ± 8.6%, P> 0.05, Kruskal–Wallis nonparametric test with Dunn's post-test). However 35 days after surgery the PLR started to decrease once again in the operated eyes (PLRratio= 17.2 ± 7.4%) and was significantly smaller again compared to preoperative values (P< 0.05). The PLRratio continued to decrease and was 9.5 ± 7.3% (42 days postoperatively; P< 0.01) and 4.2 ± 4.2% at the last recording time point (60 days postoperatively, P< 0.001). ERG analysis of operated eyes revealed significantly decreased amplitudes of a-(P < 0.001) and b-waves (P < 0.001) 10 days postoperatively, while oscillatory potentials (OPs) and flicker ERG (flERG) amplitudes were not detectable. However, 28 days postoperatively OPs significantly recovered (OP ratio = 7.6 ± 2.2%, P < 0.001), while a-wave, b-wave and flERG amplitudes did not significantly change compared to values observed 10 days postoperatively. The ERG analysis of the operated eyes revealed dramatically reduced a-wave (ratiooperated/control= 8.9 ± 2.7%), and b-wave amplitudes (7.8 ± 2.5%), while OPs and flERG did not have recordable amplitudes 60 days postoperatively. Histologic analysis revealed degeneration of all retina layers and optic nerve axons. Conclusions: Chronic hypertensive rat eyes have capability to partially recover inner retina and optic nerve function observed by electroretinography and computerized pupillometry. Chronic ocular hypertension in rats dramatically damaged all retinal layers and optic nerves observed by morphologic and functional methods.

ABSTRACT No.: 21

Efficacy of topical antibiotic-chondroitin sulfate solutions in the therapy of canine recurrent corneal erosions

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E. C. Ledbetter,* R. J. Munger† and R. D. Ring†

*College of Veterinary Medicine, Cornell University, Ithaca, NY, †Animal Ophthalmology Clinic, Dallas, TX, USA

Purpose: To determine the efficacy of two topical antibiotic-chondroitin sulfate ophthalmic solutions in the therapy of recurrent corneal erosions in dogs. Methods: All dogs diagnosed with recurrent corneal erosion (based on the presence of a superficial epithelial defect that retained fluorescein, nonadherent epithelium adjacent to the lesion periphery, and minimal stromal involvement) were considered eligible for the study. Under topical anesthesia, nonadherent epithelium was removed manually with cotton-tipped swabs at the initiation of treatment. Eyes were treated topically 3–4 times daily with one of two products chosen randomly for each patient: 100 mg/mL chondroitin sulfate and 3 mg/mL tobramycin ophthalmic solution (Tobramax®; Labyes S.A., Buenos Aires, Argentina) or 200 mg/mL chondroitin sulfate and 3 mg/mL ciprofloxacin ophthalmic solution (Ciprovet®; Labyes S.A.). Patients were re-evaluated at 2-week intervals from the start of treatment, for a total of 4 weeks. Erosions were considered healed when the cornea did not retain fluorescein, the epithelium appeared firmly attached with no folds or breaks evident by biomicroscopy, and there was no reoccurrence of the erosion for the subsequent 4 weeks. Cotton-tipped applicators were used to confirm corneal healing by gently brushing the epithelium of all questionably healed cases at the final recheck. Results: Eighty-four erosions from 80 dogs were included in the study. Erosions were present unilaterally in 76 dogs and bilaterally in four dogs. Fifty-four erosions were treated with Tobramax® and 30 erosions were treated with Ciprovet®. The mean age of treated dogs was 9.1 years (range: 2.3–14.8) with 41 spayed females, 23 castrated males, nine intact males, and seven intact females. Twenty-six breeds were represented, with the most common being Boxer (n = 20), mixed breed (n = 13), Labrador Retriever (n = 5) and Cocker Spaniel (n = 5). After 2 weeks of treatment, 53.6% (30/54 eyes treated with Tobramax® and 15/30 eyes treated with Ciprovet®) of erosions had healed. After 4 weeks of treatment, 81.0% (44/54 eyes treated with Tobramax® and 24/30 eyes treated with Ciprovet®) of erosions had healed. The 16 erosions that failed to heal after 4 weeks of therapy were treated with a soft contact lens and either a multiple punctuate keratotomy (n = 14) or a grid keratotomy (n = 2). Following this treatment, 100% (16/16) erosions healed within 14 days. Conclusions: Topical therapy with an antibiotic-chondroitin sulfate ophthalmic solution compares favorably with other published medical and surgical therapies for recurrent corneal erosions. A longer treatment period may be required than with other therapeutic options. Acknowledgment: The authors gratefully acknowledge the contribution of the medication by Labyes S.A. of Buenos Aires. Commercial interest: None.

ABSTRACT No.: 22

Ocular biomicroscopy (UBM) in Yacare Caiman (Caiman yacare)

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F. B. N. Maia, J. A. T. Pigatto, P. M. Guedes and P. S. M. Barros

Ophthalmology Section, School of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil

Purpose: The UBM allows microscopic resolution imaging of anterior ocular structures. There is little information available on the anterior ocular segment in reptiles. The purpose of this study was to evaluate the anterior segment structures in eyes of Yacare Caiman. Methods: Twelve eyes from six Yacare Caiman with a mean age of 3 years were used. The eyes were obtained from a Brazilian commercial company that breeds wild animals (Pro-Fauna Commercial Ltd). Immediately after death the eyes were enucleated and transported to the laboratory in a moist chamber at 4 °C. The exam was performed within 6 h of enucleation. An Ultrasound Biomicroscope P40 50 MHz (Paradigm Medical, Inc.) with a mean resolution of 50 µm and a penetration depth of 5 mm was used. The eyes were immersed in 2% methylcellulose and axial and peripheral images were obtained. Parameters evaluated were central corneal thickness, anterior chamber depth, lens thickness and anterior segment abnormality. Results: Mean corneal central thickness was 688 µm and mean anterior chamber depth was 2.73 mm. The measurements of antero-posterior lens were higher than 5 mm. Two eyes showed anterior synechia. Conclusion: Ultrasound biomicroscopy can be used to accurately evaluate and detect abnormalities of the anterior ocular segment in exotic animals. Similarity was observed when comparing Yacare Caiman and domestic species eyes. Acknowledgment: We would like to thank Pro-Fauna commercial Ltd for supply eyes used in this research. Commercial interest: None.

ABSTRACT No.: 23

Evaluation of corneal endothelial loss following conventional extracapsular lens extraction and phacoemulsification in dogs

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J. A. T. Pigatto,* J. L. Laus,† J. M. Santos,‡ G. N. Rodrigues,† I. C. Talieri,† A. T. Jorge† and M. L. Rezende†

*School of Veterinary Medicine, University of Sao Paulo, Brazil School of Veterinary Medicine, Sao Paulo State University, Brazil,School of Agronomy, Sao Paulo State University, Brazil

Purpose: To compare corneal endothelial damage after conventional extracapsular lens extraction and phacoemulsification in dogs. Methods: Twelve Mongrel adult dogs free of ocular abnormalities were used. All procedures were performed in compliance with the ARVO statement for use of animals in ophthalmic and vision research. One eye underwent conventional extracapsular lens extraction and phacoemulsification was performed in the fellow eye. Six dogs were euthanized at postoperative day 1 and six dogs were euthanized 60 days postoperatively. Both eyes were obtained immediately following euthanasia and the corneas were processed and examined with a scanning electron microscope (JEOL-JSM 5410). Photomicrographs were taken of the central, inferior and superior corneal endothelium. The percentage of cell loss was estimated at postoperative day 1. At 60 postoperative days the cell density was calculated. Statistical data were analyzed using Tukey's test. Results: In both procedures the cell loss was greater in the superior, and lower in the inferior cornea when compared to that in the central cornea. Sixty days after surgery the endothelial cell densities were lower after conventional extracapsular lens extraction than phacoemulsification. Significant differences in postoperative loss of endothelial cells were found between the two procedures. Conclusion: The results indicate that phacoemulsification induced significantly less corneal endothelial damage than conventional extracapsular lens extraction. Acknowledgments: FAPESP (98/031153–0), Alcon SA, Schering Plough and Intervet. Commercial interest: None.

ABSTRACT No.: 24

Scanning electron microscope observations of the corneal endothelium in tropical freshwater fish (Prochilodus scrofa)

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J. A. T. Pigatto,* M. F. Tucunduva,* J. L. Laus,† J. M. Santos‡ and P. S. M. Barros*

*School of Veterinary Medicine, University of Sao Paulo, School of Veterinary Medicine, Sao Paulo State University, School of Agronomy, Sao Paulo State University, Brazil

Purpose: Although there have been many studies of the corneal endothelium of humans and some mammals, there have been few in other vertebrates. The aim of this study was to examine the surface morphology and cell density of the corneal endothelium in tropical freshwater fish. Methods: Twelve corneas from six 3-year-old Curimbata (Prochilodus scrofa) were used. The specimens were collected from a Brazilian company that breeds fishes. All procedures were performed in compliance with the ARVO statement for use of animals in ophthalmic and vision research. Eyes with evidence of ocular disease were excluded. Each cornea was prepared immediately postmortem for scanning electron microscopy. Three photomicrographs of the central corneal endothelium at ×750 magnification were obtained from each cornea using a scanning electron microscope (JEOL-JSM 5410). The morphometric analysis was performed with regard to polygonality, mean cell area and cell density. Statistical analysis was conducted using Tukey's test. Values of P < 0.05 were considered significant. Results: Scanning electron microscopy revealed corneal endothelium characterized by a continuous layer composed of polygonal cells uniform in size and shape. The predominant number of cells was hexagonal (75%) in shape, with pentagonal (12.5%) and heptagonal cells (12.5%) constituting the greater portion of the remaining corneal endothelium. The mean cell area was 265 ± 48 µm2 and the cell density was 3774 ± 645 cells per mm2. Statistical analysis showed that the comparison between right and left eye from the same fish did not exhibit differences in the variables determined. Conclusion: The structure of corneal endothelium of Curimbata is similar to those described for other vertebrates. Commercial interest: None.

ABSTRACT No.: 25

Use of small intestinal submucosa (SIS) as a graft for corneal perforations and abscesses in horses, dogs and cats

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S. G. Krohne, M. Bussieres, J. Stiles and W. M. Townsend

Purdue University School of Veterinary Medicine, West Lafayette, IN, USA

Purpose: To retrospectively evaluate the efficacy of small intestinal submucosa (SIS) grafts with conjunctival flaps for corneal and corneo-limbal repair in dogs, cats and horses. Methods: Patients that received SIS surgical grafts for corneal or corneo-limbal repair at the Purdue University Veterinary Teaching Hospital between August 1999 and February 2003 were included in the study. The clinical outcome of this surgical procedure in 15 animals including six dogs, two cats and seven horses was evaluated. Results: SIS was used as structural graft material in severely diseased corneas or at the limbus (one case) to replace cornea or sclera that was diseased and surgically debrided or excised in 15 patients. All but one of these cases had a full-thickness defect (from disease or surgery) at the time the graft was sutured in place. Nine/fourteen corneas had infections confirmed by culture results or biopsy. A conjunctival flap was sutured over the graft in all patients, and then trimmed several weeks later. All patients regained structural integrity of the cornea; infections, abscesses, ulcerations or neoplasia were resolved, and all patients were comfortable at the final evaluation 5 months after surgery. Five canine patients that were visual before surgery remained visual after surgery and trimming. One dog was not visual before or after surgery. The two feline and seven equine patients were visual at the final evaluation. All patients had a fairly cosmetic appearance to the surgical eye at the end of the study. Common complications of the procedure included aqueous humor leakage for a limited time, conjunctival flap partial dehiscence, synechiae, cataracts, and transient anterior chamber fibrin following surgery. Conclusions: This study provides additional cases to the literature documenting the successful use of SIS grafts for corneal repair in dogs and cats, when success is defined as vision and preserving structural integrity. SIS grafts were also found to be successful as a structural material to repair infected equine corneas. These successful repairs included large full-thickness corneal defects. Financial support: Supported by Purdue University VCS Internal Grants. Commercial interest: None.

ABSTRACT No.: 26

The effect of topical 0.02% tacrolimus aqueous suspension on tear production in dogs with keratoconjunctivitis sicca

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E. A. Berdoulay,*,† R. V. English,* B. Nadelstein† and A. K. Weigt‡

*Animal Eye Care Assoc. Inc., Cary, NC, Chesapeake, VA, Annapolis, MD, USA

Purpose: To investigate the effect of 0.02% tacrolimus in aqueous suspension on tear production in dogs with keratoconjunctivitis sicca (KCS). Methods: Topical 0.02% tacrolimus in aqueous suspension was administered twice daily in 107 dogs (209 eyes) diagnosed with KCS (STT ≤ 10 mm/min and clinical signs of dry eye). In addition, dogs with mildly decreased tear levels (9 ≤ 14 mm/min) but with clinical signs of KCS were also evaluated. Dogs were separated into four categories: (1) KCS dogs naïve to tear stimulator therapy: tacrolimus administration was initiated in 46 dogs (88 eyes) with KCS that received no prior topical cyclosporine A (CsA) or tacrolimus therapy. (2) KCS dogs responsive to CsA therapy: tacrolimus administration was substituted for CsA in 17 dogs (34 eyes) with KCS that were responsive to 1.0% or 2.0% CsA in mineral or canola oil. (3) KCS dogs nonresponsive to CsA therapy: tacrolimus administration was substituted for CsA in 24 dogs (47 eyes) with KCS that were not responsive to 1.0% or 2.0% CsA in mineral or canola oil. (4) Double blinded controlled study: tacrolimus or just the aqueous carrier alone was administered to 20 dogs (40 eyes) with KCS that received no prior topical tear stimulator therapy. STT and clinical signs were scored for all dogs in each category prior to and 6–8 weeks after initiating tacrolimus therapy. Paired samples t-test was used to evaluate differences in STT and Friedman two-way anova was used to evaluate differences in clinical scores prior to and after tacrolimus or placebo administration. Results:Category 1: tear production increased ≥ 5 mm/min in 86.7% of eyes with initial STT ≤ 10 mm/min, 88.0% of eyes with initial STT ≤ 2 mm/min, and 22.5% of eyes with initial STT ≥ 11 after tacrolimus administration. Category 2: mean tear production increased significantly (2.8 mm/min, P = 0.006) after tacrolimus administration. Category 3: tear production increased ≥ 5 mm/min in 38.7% of eyes with initial STT ≤ 10 after tacrolimus administration. Category 4: no significant change was noted in STT or clinical signs in dogs treated with the aqueous carrier alone. Clinical signs improved significantly among dogs within category 1 and 3, regardless of change in STT. Ocular irritation was noted in 1/107 dogs upon administration. Conclusions: Twice daily administration of 0.02% tacrolimus in aqueous suspension effectively increases tear production in dogs with KCS (initial STT ≤ 10). A trend towards increased responsiveness was noted among dogs with lower initial STT values. Short-term evaluation of efficacy revealed that CsA can be safely replaced with tacrolimus to maintain control of tear production in dogs with KCS. Dogs that fail to respond positively (= 5 mm/min) to topical CsA administration, may respond positively to topical 0.02% tacrolimus in aqueous suspension. Financial interest in product.

ABSTRACT No.: 27

Keratoconjunctival effects of diabetes mellitus in dogs

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C. L. Cullen,* S. L. Ihle* and A. A. Webb†

*Department of Companion Animals, Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Canada

Purpose: To compare Schirmer tear test (STT) values, corneal sensitivity, tear break-up times (BUTs), and tear glucose values in relation to conjunctival microflora between diabetic cataractous (D), nondiabetic cataractous (C) and nondiabetic noncataractous (N) dogs. Methods: Fifteen each of D, C and N dogs were subject to complete ocular examinations including neuro-ophthalmic examination, aerobic, anaerobic, and fungal cultures from the ventral conjunctival sacs, corneal touch threshold (CTT), STT, tear glucose, tear BUT, fluorescein stain, applanation tonometry, conjunctival swabs for cytology, and slit-lamp biomicroscopy. Dogs with recent/current ocular (other than cataracts for D and C groups) or systemic abnormalities (other than diabetes for D group), or receiving topical ocular medications were excluded. In particular, CTT was assessed from the central region of the cornea using a Cochet–Bonnet aesthesiometer. The aesthesiometer monofilament length was recorded (mm filament length) when three of five attempts to elicit a blink reflex were successful. The tear glucose was measured (mm) using the glucose section from a qualitative colorimetric urine dipstick. Tear BUTs were estimated by timed biomicroscopic cobalt blue filtered examination of fluorescein-stained tear film. The degree of cataract and uveitis were critically evaluated (ranked 0–4; none–hypermature or marked, respectively). Glycemic control was estimated via serum fructosamine and glycosylated hemoglobin values. Results: STTs were significantly lower in D dogs (15.7 mm/min (SD ± 6.5) in the right eye (OD); 15.5 mm/min (SD ± 6.4) in the left eye (OS)) compared to N dogs (21.3 mm/min (SD ± 5.9) OD; 21.6 mm/min (SD ± 5.5) OS). The length of filament required to elicit a blink reflex in D dogs was significantly shorter (29.1 mm (SD ± 15.8) OD; 26.2 mm (SD ± 15.1) OS) than that required for N dogs (43.1 mm (SD ± 9.5) OD; 44.3 mm (SD ± 9.2) OS). Tear BUTs were significantly less in D dogs (6.6 s (SD ± 3.8) OD; 6.5 s (SD ± 6.4) OS) compared to both N (18.4 s (SD ± 5.2) OD; 18.9 s (SD ± 7.1) OS) and C (13.2 s (SD ± 8.4) OD; 14.7 s (SD ± 7.5) OS) dogs. Tear glucose values were significantly higher in D dogs (median = 6 mm) compared to both N and C dogs (medians = 0 mm). The conjunctival microbial isolates did not appear different across groups. There were no significant differences in the degrees of cataract or uveitis between the D and C groups. Conclusions: D dogs have significantly reduced aqueous tears and corneal sensitivity compared to N dogs. D dogs have accelerated tear BUTs compared to N and C dogs. Increased tear glucose in D dogs does not appear to be associated with altered conjunctival microflora. The degree of glycemic control does not significantly affect these ocular parameters. Financial support: Supported by the Sir James Dunn Animal Welfare Center. Commercial interest: None.

ABSTRACT No.: 28

Preliminary findings from the multicenter clinical study group evaluating the Cullen canine frontal sinus valved glaucoma shunt

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C. L. Cullen,* K. Corcoran,† J. T. Bartoe,‡ A. H. Brightman,‡ P. D. da Costa,§ D. Hacker,¶ S. Keil,** J. S. Sapienza,†† J. Wolfer,‡‡ and W. L. Yakely§§

*Atlantic Veterinary College, University of Prince Edward Island, PE Canada, Veterinary Vision of Virginia, Springfield, VA, Department of Clinical Sciences, Kansas State University, KS, §Eye Center for Animals, New Orleans, LA, Animal Eye Specialists, El Cerrito, CA, **Mission MedVet, Mission, KS, ††Long Island Veterinary Specialists, Plainview, NY, ‡‡Animal Eye Clinic, Toronto, ON, §§Animal Eye Clinic of Spokane, Spokane, WA, USA

Purpose: To evaluate the efficacy of the Cullen frontal sinus valved glaucoma (FSVG) shunt (E. Benson Hood Laboratories, Pembroke, MA; patent pending) in maintaining normal intraocular pressure (IOP) in dogs with glaucoma. Methods: Selection criteria included dogs with medically uncontrolled glaucoma with no active uveitis. Glaucoma was confirmed by IOP greater than 30 mmHg and reduced or absent vision. The shunt was implanted in affected eyes of: (1) dogs with primary glaucoma (PG) (n = 11 eyes; 7/11 with vision); and (2) dogs with secondary glaucoma (SG) following phacoemulsification ± intraocular lens implantation (n = seven eyes; 3/7 with vision) or melanocytic glaucoma (n = one eye with vision). Antiglaucoma medications were discontinued in 14/19 shunted eyes immediately postoperatively. Ophthalmic examinations, including maze testing for visual eyes, applanation tonometry, and assessment of intracameral (IC) shunt position and patency, were completed daily to every other day for 2–9 days postoperatively, and at variable intervals thereafter. Results: Mean IOPs following shunt implantation (note: globes receiving postoperative antiglaucoma medications have been excluded) included: 10.5, 12.3 and 18.0 mmHg (n = 14 eyes); 10.3 mmHg (n = 11 eyes); 8.9 mmHg (n= eight eyes); 13.5 and 15.0 mmHg (n= two eyes); and 23 mmHg (n= one eye) for days 0 and 1 and weeks 1, 2, 3–5, 12–16, 20–24 and 36, respectively. Vision was maintained in 11/11 sighted eyes and one blind dog regained vision following shunt implantation. The duration of vision postoperatively ranged from 2 days to 36 weeks (mean = 8.4 weeks). Complications included IC fibrin with or without blood (n = 15 eyes); IC fibrinous shunt occlusion (n = seven eyes); IC shunt motion and/or retraction (n= three eyes); ulcerative keratitis (n= two eyes); endophthalmitis (n= one eye); and peri-sinus/–ocular swelling (n= one eye). Five of 19 shunted eyes required enucleation. Conclusions: The Cullen FSVG shunt appears to be an effective surgical therapy for canine glaucoma in the absence of active uveitis. Mean IOPs of shunted eyes not receiving antiglaucoma medications ranged from 8.9 to 23 mmHg. IC fibrin formation, with or without shunt occlusion, is the most common complication. Financial support: Supported by the Sir James Dunn Animal Welfare Center. Financial interest in product.

ABSTRACT No.: 29

Glaucoma associated with pectinate ligament dysplasia and narrowing of the iridocorneal angle of the Shiba Inu in Japan

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K. Kato, S. Matsunaga, M. Mochizuki, R. Nishimura, N. Sasaki and H. Ogawa

The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Purpose: Primary glaucoma in the Shiba Inu has been diagnosed frequently in Japan; however, there have been no reports of intraocular changes in affected eyes. The purpose of this study was to identify the shape of the pectinate ligaments (PLs) and the width of the iridocorneal angle (ICA) in glaucomatous eyes vs. nonglaucomatous eyes in Shiba Inu dogs. Methods: One hundred and fourteen Shiba Inu dogs that were presented to our institution between June 1998 and June 2003 were included in the study. Forty-four of these were found to have glaucoma. The other 70 dogs presented for routine vaccinations or heart worm testing. All of the dogs received complete ophthalmic examinations, including indirect ophthalmoscopy, tonometory (Tonopen) and gonioscopy. Photographs taken through the goniolens were used to evaluate both PL and ICA. Results: Of the 44 dogs with glaucoma, 15 (34%) were affected bilaterally and 29 (66%) were affected unilaterally. Of the 29 unilaterally glaucomatous dogs, the ICA of the nonglaucomatous eye was closed in 15 eyes (52%), narrow in 12 eyes (41%), and slightly narrow in two eyes (7%). The dogs with bilateral glaucoma were divided into three groups based upon the length of time between the onset of glaucoma in the first eye and the onset of glaucoma in the second eye. Of the eight dogs (53%) in whom the interval was less than 1 year, the ICA was closed in five dogs, narrow in two and slightly narrow in one. Of the six dogs (40%) with interval of onset between the two eyes of 1 to 3 years, the ICA was closed in four dogs, narrow in one and slightly narrow in one. In the dog with an interval of over 5 years, the ICA was narrow. Within the normotensive control group, the ICA was open in 14 dogs, slightly narrow in 28, narrow in 24, and closed in four dogs. In the dogs with narrow and slightly narrow ICAs, the PLs were thickened and in some cases formed a solid sheet. Conclusions: In this study the majority of dogs with glaucoma had changes in both the ICA and PL. However, there was also a tendency for the ICA to be narrow in the normotensive control dogs. This study suggests that a Shiba Inu with a narrow ICA in conjunction with thickened PL may be predisposed to developing glaucoma. Commercial interest: None.

ABSTRACT No.: 30

Ocular blastomycosis with no apparent systemic disease: review of the literature and a case presentation

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K. A. Caruso and S. A. Koch

The Animal Eye Clinic, Silver Spring, MD, USA

Purpose: A review of blastomycosis with a presentation of chronic uveitis caused by an atypical infection with Blastomyces dermatitidis. Methods: A case report of a 2-year-old, spayed female Labrador Retriever mix presenting with uveitis complicated with glaucoma secondary to Blastomyces dermatitidis infection OD. The dog was followed for 1.5 years post diagnosis and continues to have no systemic illness, and no systemic therapy was pursued. Results: A full systemic medical work-up was performed, which included CBC, chemistry profile, acute and convalescent fungal and tick titers, and chest and skull films, all of which were negative for systemic disease. Enucleation was elected due to glaucoma, and Blastomyces dermatitidis was diagnosed histologically. Further support of the histologic diagnosis was made with polymerase chain reaction and immunohistochemical tests. Conclusion: (1) We propose an alternate route of infection from the usual cutaneous or pulmonary seeding of the fungus. (2) All tests for systemic disease, including serology for B. dermatitidis were negative, indicating that B. dermatitidis may be a differential for uveitis despite negative serology and lack of systemic illness. (3) Blastomycosis can be regarded solely as an ocular disease, without systemic illness. Commercial interest: None.

ABSTRACT No.: 31

The effects of dexamethasome on growth and apoptosis of bovine corneal endothelial cells and scleral fibroblasts

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W. L. Chen,* F. R. Hu,* Y. B. Chou,† A. Fei† and C. T. Lin†

*Department of Ophthalmology, College of Medicine, National Taiwan University,Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Purpose: To investigate whether bovine corneal endothelial cells and scleral fibroblasts express glucocorticoid receptor (GR) and to assess the effects of dexamethasone (DEX) on growth and death of these cells. Methods: Primary cultures of bovine corneal endothelial cells and scleral fibroblasts were established from fresh tissues. GR mRNA and protein were analyzed in both kinds of cells by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. The cells were treated with various concentrations of DEX (ranging from 10−11 to 10−3 M). Growth rate and apoptosis analysis was performed at 0, 2, 4 and 6 days after DEX treatment of the cells. Growth rate of DEX-treated cells was analyzed by MTS assay and cell counting. Apoptosis and necrosis of DEX-treated cells were investigated by flow cytometry of cells stained with a fluorescein conjugate of Annexin V/propidium iodide and TUNEL assay. Results: RT-PCR, Western blotting and immunohistochemistry showed the expression of GR mRNA and protein in both corneal endothelial cells and scleral fibroblasts. An increased cell growth rate of both kinds of cells was observed following DEX treatment at concentrations ranging from 10−10 to 10−5 M, with maximum effects at 10−5 M (P < 0.05). DEX caused significant necrosis and apoptosis in both kinds of cells. Necrosis level of the cells was increased with increasing DEX concentrations treated. Apoptosis was observed at low concentrations (10−7 and 10−10 M) in DEX-treated corneal endothelium, but at a higher concentration (10−4 M) in scleral fibroblasts. Conclusions: Both bovine corneal endothelial cells and scleral fibroblasts express GR mRNA and protein. DEX possesses dual pro-apoptotic and pro-proliferative activities at respective specific concentrations in corneal endothelial cells and scleral fibroblasts. Commercial interest: None.

ABSTRACT No.: 32

Recovery of functional cones following intraocular gene therapy in RPE65 null mutation dogs

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K. Narfström* and M. Katz†

*Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, MO, Department of Ophthalmology, School of Medicine, University of Missouri, Columbia, MO, USA

Purpose: To investigate the long-term functional and structural effects following gene therapy in a group of dogs using a recombinant adeno-associated virus (AAV) RPE65 construct. Methods: Five RPE6S-/- dogs were treated with similar amounts of a rAAV.RPEG5 construct subretinally into one eye and with a construct containing rAAV-Green Fluorescent Protein (GFP) into the other eye in four of these dogs. The contralateral eye of the fifth dog was left untreated. A sixth RPE65-/- dog was treated in the same way as the first four dogs except that the RPE65 construct was injected intravitreally instead of subretinally. Bilateral, simultaneously recorded electroretinograms (ERGs) were performed at regular intervals throughout the study using previously published methods. The morphologic effects of rAAV.RPE65 and control treatment were also analyzed using fluorescence, light and electron microscopy and immunocytochemistry. Results: In the first five dogs rod recovery reached a maximum 3 months post surgery in the RPE65 treated eye but then declined, while cone recovery showed a slow increase up to 18 months after the gene transfer. There was also a delayed effect on the contralateral eye: an increase in photopic amplitude responses in the first five dogs starting 6 months postoperatively. In the intravitreally treated dog, a bilateral increase in photopic responses was observed 15 months after the gene transfer. The morphologic studies corroborated the ERG findings in that there was an abolishment in the abnormal RPE lipid inclusions of affected dogs in the gene transfer-treated area and less in other areas, compared to untreated affected dogs from earlier studies. Photoreceptor outer segment structures were more orderly and robust in the eye that received gene transfer than in the contralateral eye at 10 months post surgery. Conclusions: After an initial improvement there is a successive decline in rod function, starting 3 months after gene therapy, and a failure to transfer the effect to rods of the contralateral eye. Cone recovery is slower following unilateral gene transfer but increases after 6 months bilaterally up to at least 18 months. Also, unilateral intravitreal injection of the RPE65 construct may affect bilateral cone function positively on a long-term basis. Financial support: Supported by the Foundation Fighting Blindness and Research to Prevent Blindness, Inc. Commercial interest: None.

ABSTRACT No.: 33

Glutathione levels in blood and aqueous humor of normal and cataractous dogs

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C. T. Lin and S. M. Wang

Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Purpose: To investigate levels of the antioxidant glutathione in blood and aqueous humor from different age groups of dogs with or without cataracts. Methods: One hundred and fifty-three dogs were used in this study. All 153 dogs were analyzed for blood glutathione (GSH) levels; one hundred and three had normal lenses and 50 were cataract patients. Thirty-two dogs were assayed for aqueous GSH level. Ten of these were dogs with normal lenses and 22 were cataract patients. Dogs were divided into three age groups: < 5 years, > 5 years, and > 7 years. A modified chemical analysis protocol was established using reagents of a GSH detection system (BioChem). All data were compared between the same age groups and were analyzed statistically by the SAS program. Results: A reliable GSH detection system for body fluid analysis was established. The sensitivity of the system was 2.38 µm. The recovery rate for high, medium and low level of GSH was 96.62%, 91.02% and 76.4%, respectively. The intra- and inter-assay coefficients of variation revealed that the detection system has high sensitivity and accuracy for detecting high and medium concentrations of canine GSH samples. The average blood GSH level from 103 normal dogs was 1338.0 ± 487.5 µm (mean ± SD), while a level of 931.8 ± 511.1 µm was found in 50 cataractous dogs. The blood GSH levels from cataractous dogs were significantly lower than that in normal dogs (P < 0.05). No difference in blood GSH level was observed in terms of sex, neutering status or maturity of cataracts. The average aqueous GSH level from 10 normal young dogs was 90.3 ± 70.9 µm, while a level of 92.5 ± 80.0 µm was detected in 22 cataractous dogs. There was no significant difference between normal and cataract groups in aqueous GSH levels. Conclusions: The established GSH detection system is an easy and sensitive system. The cataractous dogs have lower blood GSH level than dogs without cataracts, especially in the > 7-years age group. The GSH may have a protective role for dogs against oxidant injuries, including cataractogenesis. Lower levels of GSH were found in the aqueous of young dogs; however, the aqueous GSH data were inconclusive due to difficulty in obtaining aqueous samples from the same age group of normal eyes as the cataract group (aged). Commercial interest: None.

ABSTRACT No.: 34

Qualitative tear film and goblet cell assessment in cats with corneal sequestrums

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S. G. Sisler,* E. S. Storey† and B. H. Grahn†

*Eye Care For Animals, Chicago, IL, Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4 Canada

Purpose: To evaluate the tear film qualitatively and goblet cells quantitatively in cat eyes with corneal sequestrums and compare them to cat eyes with no ocular abnormalities. Methods: Eleven cat eyes with sequestrums (one cat was affected bilaterally) and 14 cat eyes with no clinical ophthalmic disease (seven normal eyes were contralateral to eyes with sequestrums) were examined physically and then with complete ophthalmic examinations (neuro-ophthalmic, Schirmer tear tests, applanation tonometry, fluorescein stain, biomicroscopic and indirect ophthalmoscopic examinations). Cats ranged in age from 1.5 to 9 years and were of the Himalayan, Persian or Burmese breed. We assessed their tear film break-up times and submitted biopsies of the dorsal and ventral nasal, and dorsal and ventral temporal palpebral conjunctiva near the fornix on these cats. The conjunctival and sequestrum biopsies were fixed routinely in 10% buffered formaldehyde, processed and stained routinely with hematoxylin and eosin and periodic acid Schiff, and examined with a light microscope. Goblet cell numbers were quantified and compared to the number of basal epithelial cells in each quadrant and eye. The goblet/epithelial cell ratios were compared to the tear film break-up times of normal and diseased eyes statistically with paired t-tests. Samples of the sequestrums were submitted for polyermase chain reaction evaluation of Herpes felis, Chlamydia, and mycoplasm felis. Results: In normal cat eyes mean tear film break-up time was 21 (± 12) s. Normal cat eyes had an average goblet/epithelial cell ratio in the dorsal nasal palpebral conjunctiva of 0.66 (± 0.1), in the nasal ventral palpebral conjunctiva, 0.68 (± 0.2), in the temporal dorsal palpebral conjunctiva, 0.63 (± 0.2), and in the the temporal ventral palpebral conjunctiva, 0.55 (± 0.3). In cat eyes with corneal sequestrums mean tear film break-up time was 14 (± 13) s. The average goblet/epithelial cell ratio in respective areas of the palpebral conjunctiva was 0.56 (± 0.3), 0.57 (± 0.3), 0.48 (± 0.3), and 0.49 (± 0.3). PCR results were not available at the time of publication of the abstract. Conclusions: As a group there is no significant correlation between goblet cell numbers and the presence of a corneal sequestrum. There are multiple cats in which goblet cell numbers varied significantly from the controls. Further exploration of these cats is being undertaken. Commercial interest: None.

ABSTRACT No.: 35

Evaluation of a new hand-held ERG for screening of ophthalmic blinding disease

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S. Sisler,* L. Galle,† E. Giuliano,† M. M. Ford,† G. Klauss,† C. Moore,† B.-E. Andersson‡ and K. Narfström†

*Eye Care For Animals, Chicago, IL, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA, Global Eye Program, Rejmyre, Sweden

Purpose: To determine the ease of use and clinical efficacy of a new hand-held and portable electroretinograph (ERG) (Mjölner) in the evaluation of ophthalmic blinding disorders in small animal practice. Methods: High brightness white light emitting diodes (LEDs) were incorporated into a compact hand-held ERG unit, including miniature full-field stimulator and screen. Recordings from anesthetized normal and visually impaired small animals were performed, using jet lenses and platinum electrodes. The light intensity range during scotopic conditions was 2.0 cd s/m2; −3.0 to +1.2 log units in 0.3 log unit steps. Photopic recordings were performed with animals light adapted (background: 30 cd/m2), and single flash and 30 Hz flicker recordings were conducted using 2.0 cd s/m2 light stimuli. Up to 100 ERG recordings were stored in the ERG instrument from each recording session and later transported to a computer for further analysis. Results from the hand-held ERG were compared to those obtained from two commercially available ERG systems (Retinographics and ERG system Tor). Results: Isolated and reproducible rod and cone responses, mixed rod and cone responses, and intensity-response series were successfully recorded from normal cats and dogs. The waveforms obtained using the hand-held ERG were not identical to those of normal animals using the other two ERG recording systems. B-wave amplitudes in diseased retinas as low as 8 microvolts were reliably recorded using Mjölner. Conclusions: The new hand-held ERG, both compact and simple to use, yields stable recordings. The waveforms obtained using the hand-held ERG were not similar to those recorded using the ERG systems Retinographics and Tor, mainly due to inherent variations in the different photostimulators. The new hand-held ERG may prove useful in the diagnosis of ophthalmic blinding diseases. Additional studies are needed to establish reference ranges using this unit in veterinary clinical practice. Financial interest in product.

ABSTRACT No.: 36

The prevalence of posterior capsule disruption during phacoemulsification and associated postoperative complication rates in dogs: 244 eyes (1995–2002)

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N. S. Johnstone and D. A. Ward

Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USA

Purpose: The objective of this retrospective study was to report the prevalence of posterior capsule disruption during routine phacoemulsification and to document the postoperative outcomes and complications in eyes with disruption of the posterior capsule compared to eyes in which the posterior capsule remained intact. Methods: Records of 143 dogs (244 eyes) were reviewed. Data collected included whether the posterior capsule was disrupted, whether the disruption was planned or accidental, whether an intraocular lens (IOL) was implanted, and visual outcome. Records were further reviewed for postoperative complications, including ocular hypertension, anterior uveitis, retinal detachment, bacterial endophthalmitis, and poor positioning of IOLs. IOL implantation rates, complication rates, and visual outcomes were compared between intact and disrupted posterior capsule groups using Chi square analyzes. Results: The posterior capsule was disrupted in 33/244 eyes (14%). Planned capsulotomies accounted for 36% of the disruptions. IOLs were implanted in 76% of eyes without a disruption of the posterior capsule and in 31% of eyes with a posterior capsule disruption. IOLs were more likely to be implanted in eyes with a planned disruption of the posterior capsule (7/12; 58%) than in eyes with an accidental disruption (3/20; 15%). Ocular hypertension was the most common complication both in eyes with intact and ruptured posterior capsules, with similar rates in both groups (33% and 39%, respectively). Other complications were rare in both groups. There were no significant differences in any of the postoperative complications assessed between eyes with posterior capsule disruption and those without. Final postoperative visual outcome was not different in eyes with posterior capsule disruption compared to those with intact capsules. Conclusions: The most significant complication of posterior capsule disruption during phacoemulsification is the inability to implant an IOL. IOLs are more likely to be placed in eyes with intentional disruptions of the posterior capsule compared to those with accidental ruptures. No difference was noted between eyes with posterior capsule disruption and those without in regards to the development of postoperative ocular hypertension, anterior uveitis, retinal detachment, bacterial endophthalmitis, or poor positioning of the IOL. Commercial interest: None.

ABSTRACT No.: 37

Effects of systemic flunixin meglumine, topical oxytetracycline, and topical prednisolone on tear film proteinases in normal horses

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M. E. Rainbow,* F. Ollivier,† J. P. Pickett,* I. P. Herring* and D. E. Brooks†

*VA-MD Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, VA, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

Purpose: To determine effects of three medical treatments, topical tetracycline, topical prednisolone and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. Methods: Twelve ophthalmologically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (500 mg IM BID), topical 1% prednisolone acetate (0.2 mL TID), or topical oxytetracycline (0.2 mL TID), followed by a 9-day washout period. All topical medications were applied to the left eye, and the right eye was treated with a placebo. Tears were collected before the first treatment on day 1 and the morning following the last treatment on day 5. Tear film proteinase activity was measured using gelatin zymography and measurements of optical density. Statistical analysis of the difference between the treated and untreated eyes and the eyes before and after treatment was performed using mixed effects model for anova. Results: There was no significant difference between treated and placebo eyes for MMP-2 or MMP-9 for any of the treatments. Other than the decreased MMP-2 activity in the left eye of horses treated with flunixin meglumine (P = 0.0259), there was no significant difference after treatment compared to before treatment in either eye for any treatment for MMP-2 and MMP-9. Conclusions: Systemic flunixin meglumine, topical 1% prednisolone acetate, or topical oxytetracycline do not significantly change MMP-2 or MMP-9 activity in normal equine tear film. Financial support: Supported by Patricia Bonsall Stuart Award for Equine Research. Commercial interest: None.

ABSTRACT No.: 38

The effects of propofol on electroretinogram in Havanese dogs

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C. H. Mar, R. E. Merideth and R. L. Sigler

Eye Care for Animals, Phoenix, AZ, USA

Purpose: To determine whether the nonbarbiturate intravenous anesthetic agent, propofol, alters the electroretinogram (ERG) response in dogs when compared to the ERG response from dogs receiving topical anesthesia only. Methods: Fourteen (four males and 10 females between 1 and 6 years old), clinically normal, adult Havanese dogs were involved in the study. The dogs were dark adapted for 10 min and pupils were dilated with 1% tropicamide prior to the ERG. After topical instillation of 0.5% proparacaine, corneal electroretinograms of the right eye were recorded in each dog with Retinographics® using a white stimulus. Electroretinograms were repeated after intravenous administration of propofol. Propofol was given to produce the effects of general anesthesia (5.8–16.6 mg/kg). Dogs were intubated and provided 100% oxygen for the duration of anesthesia. Results: The mean a-wave amplitude and latency from dogs receiving topical proparacaine were 40.3 ± 16.6 µV and 11.6 ± 1.3 ms, while the mean b-wave amplitude and latency were 135.1 ± 41.0 µV and 18.2 ± 2.3 ms, respectively. With propofol induction, the a-wave amplitude and latency were 23.2 ± 10.6 µV and 12.1 ± 1.7 ms, respectively. The mean b-wave amplitude and latency after propofol induction were 103.1 ± 43.3 µV and 19.6 ± 4.1 ms, respectively. The a- and b-wave amplitudes between the topical anesthesia results and the propofol anesthesia results were significantly different (P < 0.01, paired t-test). The a- and b-wave latency between the topical anesthesia results and the propofol anesthesia results were not significantly different (P > 0.01, paired t-test). Conclusions: Propofol significantly lowers the amplitude response of the electroretinogram but does not alter the latency time in Havanese dogs. Commercial interest: None.

ABSTRACT No.: 39

The use of CO2 laser for the removal of canine eyelid meibomian gland adenomas

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M. Bussières, S. G. Krohne, J. Stiles and W. Townsend

Purdue University School of Veterinary Medicine, West Lafayette, IN 47907, USA

Purpose: To clinically evaluate the effectiveness of the CO2 laser for the removal of canine eyelid meibomian gland adenomas. Methods: A total of 12 meibomian adenomas (12 dogs) of at least 3 mm in diameter were ablated using the CO2 laser. The laser site was not sutured. Owners were asked to administer triple antibiotic ointment to the laser site three times daily for 1 week. Patients were re-evaluated at 7 days, 21 days and 6 months after lasering. The average age of the dogs included in this study was 9.4 years. Size of tumors and laser defects following ablation of the mass were measured. Assessment of pain, conjunctival hyperemia, eyelid hyperemia and cosmetic appearance were noted at each recheck. Results: Ten masses were found on the lower eyelid and two on the upper eyelid. The mean width and length of the masses was 5.5 × 5 mm. Seven of the masses involved the eyelid margin. The mean width and length of the laser site following ablation was 4 × 4 mm. Contracture of the tissue from the laser energy was responsible for the difference in size between the tumor and the laser defect. The laser site width had contracted to 3.5 mm at the 7 day recheck (n = 12). In five dogs a 1-mm defect remained at 21 days, while in five dogs the surgical site had completely closed. One dog was lost to follow-up after 7 days and one dog returned well after 21 days. At the 6-month recheck all patients had a 1-mm visible scar and slight lid margin irregularity. None of the patients developed corneal disease secondary to the laser procedure and no recurrence of tumor was noted. Conclusion: The use of the CO2 laser is an effective technique for the removal of meibomian gland adenomas in dogs, and results in good cosmetic appearance. Commercial interest: None.

ABSTRACT No.: 40

Diabetic retinopathy in dogs following lensectomy by phacoemulsification: 52 cases (1993–2003)

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M. P. Landry, I. P. Herring and D. L. Panciera

Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

Purpose: To determine the prevalence of diabetic retinopathy in diabetic dogs following lensectomy by phacoemulsification and to identify potential risk factors. Methods: Medical records of 52 dogs with diabetes mellitus and 179 nondiabetic dogs that underwent phacoemulsification from 1993 to 2003 at VMRCVM were reviewed and information was recorded on signalment, history, physical examination findings, CBC, serum chemistry, urinalysis, ophthalmic examination findings, ancillary diagnostic procedures, surgical information, and postsurgical ophthalmic examination findings. To allow categorization of glycemic control, a scoring system was developed based upon fasting blood glucose, perioperative body weight change, daily insulin dosage, and presence of glucosuria or ketonuria. Data from the diabetic and nondiabetic groups were analyzed to determine the prevalence of, and risk factors for development of diabetic retinopathy following phacoemulsification. Results: Of 52 dogs with diabetes mellitus, 11 (21.1%) developed ophthalmoscopic signs of diabetic retinopathy following lensectomy by phacoemulsification. Median time from diabetes diagnosis to appearance of retinopathy was 1.4 years (range: 0.5–3.2 years). The retinopathy was characterized by retinal hemorrhage/microaneurysms in the tapetal retina that were generally multifocal and pinpoint in appearance. Retinal hemorrhage was identified in only one (0.6%) nondiabetic dog following lensectomy. No risk factors for development of retinopathy were identified. Conclusions: Diabetic retinopathy appears to be much more prevalent and may occur earlier in the dog than previously thought. This may impact on future treatment, as diabetic dogs survive longer with improved glycemic control. Commercial interest: None.

ABSTRACT No.: 41

Activity of topically and orally applied Interferon omega in cats indicated by Mx-protein expression in conjunctival and white blood cells

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T. Bräcklein,* S. Speier,* A. Metzler,† B. Spiess and M. Richter*

*Ophthalmology Service, Department of Small Animals, University of Zurich, †Institute of Virology. Veterinary Faculty, University of Zurich, Switzerland

Purpose: The purpose of this study is to investigate the activity of feline Interferon (IFN) omega in cats after topical and oral application, by estimation of Mx protein expression. Mx proteins are specific biochemical markers for type II FN activity. Methods: In multiple single-dose drug studies 20 µL containing 50 U, 100 U, 1000 U, 10 000 U IFN omega, respectively, were applied topically in both eyes of 10 SPF cats. In a second study, the cats received 200 U IFN omega orally. The same cats served as control receiving 20 µL 0.9% NaCl topically or 100 µL 0.9% NaCl orally after a 2-week wash out period (cross-over design study). Conjunctival cells (CCs) and white blood cells (WBCs) were collected prior to application of IFN or NaCl (day 0) on day 1 and thereafter on every third day until all samples became negative. Samples were examined for Mx protein expression by immunostaining and Western blot immunoassay, respectively. Results: CC and WBC samples of cats inoculated with a single dose of 10 000 U IFN omega stained positively for Mx protein for 7 and 31 days, respectively, in 10/10 cats. CC samples stained positively for 21 days in 3/10 cats and WBC samples were positive for 45 days in 6/10 cats. WBC samples of cats inoculated with a single dose of 1000 U IFN omega were positive for at least 1 day in 10/10 cats and for 3 days in 6/10 cats. CC samples did not show Mx protein staining at any time in any cat inoculated with 1000 U IFN omega. Inoculation of 50 U and 100 U did not induce Mx protein expression either in CCs or in WBCs in any cat at any time. Following oral administration of a single dose of 200 U IFN omega, Mx protein was found in WBCs of 9/10 cats for at least 1 day, but disappeared in all cats at the third day after administration. CC did not reveal Mx protein at any time in any cat. Conclusions: Induction of Mx protein and therefore activity of IFN omega in the cat is dose dependent. Our findings indicate that IFN omega has a long-lasting biologic effect when administered topically in a concentration of 10 000 U/20 µL (= 500 000 U/mL). Funding sources: Research grant from the University of Zurich, Switzerland. Commercial interest: None.

ABSTRACT No.: 42

An investigation of the safety and efficacy of topical ophthalmic application of tacrolimus in dogs

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E. A. Adkins, D. V. H. Hendrix, J. L. Stuffle, D. A. Ward and B. J. Skorobohach

Department of Small Animal Clinical Sciences, University of Tennessee, Knoxville, TN, USA

Purpose: To determine if topical tacrolimus (TACRO) is a safe and effective treatment for keratoconjunctivitis sicca (KCS) in dogs. Our hypotheses are that twice daily use of 0.03% topical TACRO is safe in normal dogs and twice daily use of 0.03% topical TACRO is effective in the treatment of dogs with KCS. Methods: This study was a two-phase, randomized, controlled, masked clinical trial. Phase 1 determined if TACRO was safe in normal dogs. Six dogs received topical olive oil BID and seven dogs received 0.03% topical TACRO diluted in olive oil BID for 14 days. Schirmer tear tests (STTs), fluorescein staining and slit-lamp biomicroscopy were performed daily. Serum biochemistry panels and TACRO serum levels were performed on day 0 and day 15 to monitor for systemic toxicity. Phase 2 determined if TACRO was effective in the treatment of up to 40 client-owned dogs with KCS. One half of the dogs enrolled received 2% cyclosporine A in olive oil and the other dogs received 0.03% TACRO. Complete ophthalmic examinations and photography were carried out on day 0, then at weeks 4, 8 and 12. In phase 1 the two groups’ daily scores for conjunctival hyperemia, blepharospasm and epiphora were compared by Student's t-test. The daily STT of the two groups were compared by repeated measures anova. In phase 2 the control and treatment groups’ STTs were compared by repeated measures anova. Numerical scores were assigned to clinical signs. Scores from day 1 and week 12 were compared using the Student's t-test. Results: Phase 1: there was no significant difference in ocular signs between the two groups. Student's t-test evaluation of the STT revealed a trend for increased tear production in the treatment group on days 5, 11 and 15. In three dogs the TACRO serum levels were higher before treatment than after treatment. Phase 2: twenty-two dogs have enrolled in the study to date. Three dogs died during the study and two were dropped from the study due to corneal ulceration. Nine dogs have completed the study. There was no significant difference in STT comparing the two groups at any time point. There was no significant difference in the clinical signs based on the clinical scores. Conclusions: Twice daily topical TACRO is safe in normal dogs. A larger sample size is needed to determine if TACRO is effective in the treatment of KCS. Financial support: Supported by the American College of Veterinary Ophthalmology, University of Tennessee Center for Excellence, Department of Small Animal Clinical Sciences and Companion Animal Fund and the Rasmussen Charitable Gift Fund. Commercial interest: None.

ABSTRACT No.: 43

Blood and ocular tissue levels of topical dorzolamide in dogs

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D. A. Ward,* M. A. Cawrse,* S. K. Cox† and D. V. H. Hendrix*

*Department of Small Animal Clinical Sciences, Department of Comparative Medicine, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USA

Purpose: To evaluate levels of dorzolamide HCl in whole blood, aqueous humor, cornea and ciliary body following topical application of 2% dorzolamide HCl eyedrops in normal dogs. Methods: Blood concentrations were measured following a single dose and after chronic dosing. In the single dose study, 2% dorzolamide HCl eyedrops (Trusopt®) were applied to both eyes of 10 normal Beagles. Artificial tear eyedrops were applied bilaterally to an additional five Beagles. Blood samples were obtained at 30 min, 2 h, 6 h and 10 h following eyedrop application. The following day dorzolamide or artificial tear application was continued every 8 h for 6 additional days. On the 7th day, dorzolamide or artificial tears were applied at 7 a.m., and blood samples were collected 30 min, 2 h, 6 h and 10 h later. The next day 2% dorzolamide eyedrops were commenced every 8 h to both eyes of the five dogs that had received artificial tears. After 3 days of dosing these dogs were euthanized and enucleated. Aqueous humor, cornea and ciliary body were then dissected and submitted for analysis of dorzolamide concentrations. Whole blood was analyzed following treatment to disrupt RBC membranes. Tissue supernatants were analyzed following homogenization of pooled samples and centrifugation. Dorzolamide concentrations were measured by HPLC using a mobile phase of 80%A (0.085% H3PO4 and 1.2 g octane sulfonic acid/L) and 20%B (acetonitrile), with UV detection at 252 nm. Results: Baseline blood samples obtained prior to commencement of dorzolamide eyedrops did not contain measurable dorzolamide in any dog. None of the dogs receiving artificial tears had measurable dorzolamide concentrations following either single dose or chronic dosing. Mean blood concentrations (± SD) (expressed in ng/mL) following a single dose were 312.56 ± 117.85, 30 min after dosing, 435.70 ± 106.46, 2 h after dosing, 471.70 ± 124.43, 6 h after dosing, and 538.56 ± 136.54, 10 h after dosing. Mean blood concentrations following chronic dosing were 3914.80 ± 614.74, 30 min after the last dose, 3878.10 ± 576.64, 2 h after the last dose, 3719.90 ± 569.88, 6 h after the last dose, and 3566.50 ± 567.74, 10 h after the last dose. Ocular tissue concentrations following 3 days of dosing were 330.00 ± 81.30 ng/mL in aqueous humor, 1767.60 ± 589.85 ng/g in cornea, and 2324.70 ± 1333.36) ng/g in ciliary body. Conclusions: Dorzolamide reaches measurable concentrations in canine blood within 30 min following topical administration, and increases 10-fold following chronic administration. The highest ocular tissue levels obtained were in ciliary body homogenates, with demonstrable levels also present in cornea and aqueous humor. Commercial interest: None.

ABSTRACT No.: 44

Clinical features, etiologies and outcomes of hyphema in dogs: a retrospective study (1999–2002)

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T. A. Paul and D. A. Ward

Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USA

Purpose: To identify the etiologies, common clinical features, and outcomes of hyphema in dogs. Methods: Medical records from the University of Tennessee College of Veterinary Medicine with a diagnosis of hyphema between 1999 and 2002 were identified and reviewed. Results: Thirty-eight cases of hyphema were identified over the period studied. Eighteen different breeds and mixed breeds were represented, with no apparent breed predilection. Seventeen dogs were male and 21 were female. The left eye was affected in 21 dogs; the right eye in 16 dogs, and both eyes in one dog. Eight potential etiologies were identified: retinal detachment (19 cases), coagulopathies (four cases), systemic hypertension (four cases), perforating corneal trauma (three cases), blunt trauma (three cases), uveitis (three cases), complication of phacoemulsification (three cases), and intraocular tumor (one case). An etiology could not be identified in three cases, and five cases had more than one potential etiology. The approximate volume of hyphema was recorded in 25 cases: 14 cases had approximately 25% of the anterior chamber filled with blood; one case had 50%; two cases had 75%, and eight cases were total hyphemas. Follow-up periods ranged from no follow-up to 11 months. Two cases developed ocular hypertension secondary to the hyphema, and six developed glaucoma. At the close of follow-up, 29 eyes were nonvisual, and nine were visual. Most vision loss was not as a direct result of the hyphema, but rather associated with the cause of the hyphema (e.g. retinal detachment). Of the nine visual eyes, three were due to coagulopathies, two were due to uveitis, two were complications of phacoemulsification, and one each were due to perforating corneal trauma and idiopathic. Hyphema volume was recorded for seven of the visual eyes: 6/7 had 25% hyphemas, and 1/7 had a 50% hyphema. Most cases received topical corticosteroids and atropine, and numerous other therapies were applied depending on specific clinical situations. Conclusions: Retinal detachment is the most common cause of hyphema in dogs. The prognosis for vision is generally poor, but is better in cases of small hyphema volumes not associated with retinal detachment, hypertension or trauma. Commercial interest: None.

ABSTRACT No.: 45

A study of the viscosity of marine mammal tears

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A. H. Brightman,* S. Gerds,* T. W. Campbell† and M. R. Fedde*

*College of Veterinary Medicine, Kansas State University, Manhattan KS, College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA

Purpose: To attempt to quantify the clinical observation that marine mammals have extremely viscous tear films. Methods: Five species of marine mammals were identified as subjects for the study. Ocular examinations were performed and the animals were trained over a 6-month period to allow for the collection of tears while breached from the water or restrained by handlers. Depending on the species, 1.0–10.0 mL of tears were collected in a syringe. The samples were run, pool side. A 0.7-mL aliquot was measured with a cone-plate viscometer (Model LVTDV-II, Brookfield Engineering Laboratory, Stoughton, MA, USA). The plate temperature was set at 38 °C with an external water bath. Shear rates of 12 and 25/s were used and the apparent viscosity was recorded in cP. A viscosity range rather than a standard deviation was recorded. Two per cent hydroxyproplyl methylcellulose was used as control. Results: Tear film viscosity was measured for one Killer Whale, two Lesser Killer Whales, two Bottlenose Dolphins, three sea turtles and four Manatees. The control substance had a viscosity of 29–31(thousands) cP. The remaining results were Killer Whale: 18.7–21 cP, Lesser Killer Whale: 58–62 cP, Bottlenose Dolphin: 39.5–43 cP, sea turtle: 49–51 cP, and Manatee: 78.5–81 cP. Conclusions: The viscoelastic nature of the marine mammal tear film, as well as the mammals themselves, provides unique challenges in quantifying tear film viscosity. The marine mammals studied all had tear film viscosities in excess of the control substance, except the Killer Whale. The disparity was attributed to sample handling. The Manatee had the most viscous of the tear films studied, and of the animals studied is the only one that lives in both salt and fresh water. Various biochemical and environmental factors may have played a role in the development of the extremely viscous tear film in marine mammals. Financial support: Supported by the Ophthalmology fund, KSU Foundation. Commercial interest: None.

ABSTRACT No.: 46

Ocular histoplasmosis with cross reaction to Bartonellosis in four cats

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D. T. Paglia,* K. L. Ketring,† S. Sisler,‡ E. Howerth,§ W. Hardy¶ and R. R. Dubielzig**

*Veterinary Medical Teaching Hospital, University of California, Davis, CA, All Animal Eye Clinic, Cincinnati, OH, Eye Care for Animals, Wheeling, IL, §Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA, National Veterinary Laboratory, Franklin Lakes, NJ, **Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA

Purpose: To evaluate Bartonella-positive cats with uveitis and histologically confirmed Histoplasma and demonstrate that antibodies specific for histoplasmosis can cross react with bartonellosis. Methods: Eyes of four cats with a previous diagnosis of intraocular inflammation and positive Western blot test for Bartonella (results were scaled by the laboratory from +1 to +4) were enucleated and histologically examined. Based on the above results, immunohistochemistry (IHC) for bartonellosis as well as histoplasmosis was performed. All cats had most or all of the following diagnostics performed: complete blood count, serum chemistry panel, T4 levels, FeLV, FIV, feline coronavirus, and toxoplasmosis titers, and vitreal aspirates. Results: As per the selection criteria, all four cats had clinical signs of intraocular inflammation, a high (+3 or +4) titer for bartonellosis, and granulomatous or pyogranulomatous inflammation with demonstrable Histoplasma organisms in the eye. IHC for histoplasmosis demonstrated a strongly positive reactive staining, not only of the individual organisms, but also of granular protein debris in phagocytes. Anti-Bartonella antibodies labeled the same Histoplasma organisms but did not stain the granular debris. Two of the cats had numerous Histoplasma organisms while the remaining two cats had smaller numbers of organisms based on silver stain and IHC for histoplasmosis. Other diagnostic procedures failed to provide results that contributed to the diagnosis. Conclusions: Feline uveitis has several known etiologies; however, most cases remain undetermined. The role of Bartonella henselae in feline uveitis is unclear. A +3 or +4 titer for Bartonella is considered significant (National Veterinary Laboratory); however, histology and IHC support the diagnosis of histoplasmosis as the cause of granulomatous/pyogranulomatous uveitis in these cats. Furthermore, in this group, there is weak but specific cross-reactivity on IHC using anti-Bartonella antibodies. These results may be explained by: (1) a coexisting Histoplasma and Bartonella infection, (2) an immunologic cross reaction based on shared antigenicity, or, (3) a clinically quiescent exposure to Bartonella with an amplification of the immune response due to a coexisting inflammatory disease rather than specific cross reactivity. Commercial interest: None.

ABSTRACT No.: 47

Hemangiosarcoma and squamous cell carcinoma in the third eyelid of a horse

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P. M. Gearhart, B. A. Steficek and S. M. Petersen-Jones

College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA

Purpose: To describe the clinical presentation, histopathologic diagnosis and outcome of a third eyelid mass composed of both hemangiosarcoma and squamous cell carcinoma in a horse. Methods: This is a presentation of a clinical case. Results: A 12-year-old Belgian draft horse gelding presented for evaluation of red-tinged serous ocular discharge with a duration of approximately 3 months. The condition had been treated with triple antibiotic and hydrocortisone ointment once daily with no improvement. On ophthalmic examination, there was a red-tinged serous discharge from the right eye and nostril. A large, red, lobulated, friable mass with a granular surface was present on the palpebral surface of the right third eyelid. The remainder of the ophthalmic examination of the right eye was unremarkable. The left eye was normal except for a small raised plaque-like mass on the palpebral surface of the nictitans. The right nictitans was surgically removed in its entirety, including margins around the gross borders of the mass. The plaque-like mass was removed in an excisional biopsy from the left nictitans. Histopathology of the excised right nictitans mass was characterized by two infiltrative neoplastic processes. There was extensive vasoformative proliferation of neoplastic endothelial cells, consistent with a hemangiosarcoma. The presence of positive staining for Factor VIII-related antigen confirmed this diagnosis. There was also a locally extensive zone of neoplastic squamous epithelial cell proliferation which expanded the conjunctival epithelium and infiltrated the underlying submucosal stroma. This second lesion was consistent with a squamous cell carcinoma. Histologic examination of the plaque-like mass in the left nictitans showed that it was also a squamous cell carcinoma. Due to monetary constraints, the owners declined further treatment to the left nictitans and follow-up treatment for the right eye. The appearance of bloody ocular discharge from the right eye approximately 16 months postoperatively prompted the owners to request re-examination. At that time, there was clotted blood in and around the medial canthus of the right eye. Both the upper and lower eyelids were thickened and firm, suggesting tumor regrowth. The globe was unable to be visualized due to the thickening of the lids. The left nictitans was normal in appearance. Conclusions: There are only a few reports describing ocular tumors of vascular origin in the horse. These tumors have been described as arising from the limbal, perilimbal or palpebral conjunctiva, and less frequently from the nictitans. This case was unusual because there were two neoplasms present in the same mass that could not be differentiated grossly. Commercial interest: None.

ABSTRACT No.: 48

Transforming growth factor beta (TGF-β) in the equine tear film

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T. Miller-Michau, J. Salmon, J. Davis, M. G. Davidson and B. C. Gilger

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

Purpose: The equine cornea is susceptible to a wide range of injury and infection and frequently responds with significant fibrosis. TGF-β is a cytokine associated with increased extracellular matrix deposition, chemotaxis, angiogenesis, and immunosuppression. TGF-β has been identified in human and rabbit tear film, and TGF-β1 may be responsible for corneal fibrosis. The purpose of this study was to determine if TGF-β1 and TGF-β2 were present in the equine tear film and to detect changes in TGF-β with disease and following surgery. Methods: Tear fluid was collected with methylcellulose sponges from 32 normal horses (n = 32 eyes), 21 unaffected contralateral eyes of affected horses, and 40 affected horses (n = 41 eyes). TGF-β1 (total) and TGF-β2 (total and active) levels were determined by a human enzyme immunoassay (ELISA). Total TGF-β was determined following sample acid activation. Results: Total TGF-β2 was detected in 18/33 (54.5%) normal eyes, 16/21 (76.2%) unaffected contralateral eyes of affected horses, and 37/38 (97.4%) affected eyes, with the mean TGF-β2 levels (± SEM) being 939 ± 262, 1484 ± 449 and 5329 ± 1261 pg/mL, respectively. Total TGF-β1 was detected in 14/14 (100%) normal eyes, 10/10 (100%) unaffected eyes of affected horses, and 21/21 (100%) affected eyes with the mean TGF-β1 levels (± SEM) being 67 ± 14, 83 ± 36, and 34 ± 9 pg/mL, respectively. Active TGF-β2 was detected in 4/4 (100%) of normal horses and 16/16 (100%) of affected horses with the mean TGF-β2 levels (± SEM) being 72.6 ± 1.9 and 28.9 + 1.1 pg/mL, respectively. Total TGF-β2 and TGF-β1 were present in 4/4 (100%) postoperative samples (within 24 h) with the mean (± SEM) being 20 217 ± 9589 and 74 ± 35 pg/mL, respectively. TGF-β2 was significantly increased in stromal abscesses, and indolent and infected ulcers. It was not significantly increased in traumatic ulcers, SCC and uveitis. Conclusions: This study demonstrates that TGF-β1 and TGF-β2 are present in the equine tear film. All normal horses had detectable levels of total TGF-β1, although in much lower concentrations than TGF-β2; however, TGF-β2 was found in only half of normal horses. Latent TGF-β2 release increased in affected eyes, in the face of a dilution effect with increased tearing, while TGF-β1 decreased. Levels were most elevated in stromal abscesses and indolent ulcers. TGF-β1 levels remained the same and TGF-β2 levels increased within 24 h after corneal surgery indicating a release of TGF-β2 and alsoTGF-β1. TGF-β1 may be responsible for increased fibrosis following surgery. The presence of TGF-β may also play a role in the pathophysiology of stromal abscesses and possibly indolent ulcers in the horse. Support: American College of Veterinary Ophthalmologists. Commercial interest: None.

ABSTRACT No.: 49

Surgical and clinical comparative study between foldable acrylic hydrophilic and rigid PMMA lenses in canine cataract patients

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S. Pizzirani, F. Maggio, T. Miller-Michau, B. G. Gilger and M. G. Davidson

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

Purpose: To evaluate intraoperative and postoperative results of cataract surgery in dogs using an acrylic foldable intraocular lens, and to compare results with use of a rigid PMMA lens. Methods: Dogs undergoing bilateral cataract surgery had one eye that was randomly chosen to be implanted with a 5-mm biconvex optic (41D), triplate haptic, hydrophilic acrylic, 12-mm foldable intraocular lens (CANI/JAG, I-Medical, Pharma Inc.), and the fellow eye implanted with a 7-mm optic (41D) one piece, 17-mm rigid PMMA (Model K-9, 717, I-Medical Pharma Inc.), after routine phacoemulsification and aspiration of cataractous lenses. Operative parameters (time of phacoemulsification and surgery) and follow-up, including postoperative inflammation, overall degree of capsular opacification, and refractive state were compared between the two eyes. A score (0 = poor, 1 = fair, 2 = good, 3 = excellent) was also assigned to the clinical outcome for cases where follow-up was longer than 3 months. Results: Fifteen dogs were included: seven were diabetic and four eyes in each group had clinical signs of lens-induced uveitis. There were no significant differences in stage of cataract, phacoemulsification and/or surgery times between the two groups. Inflammation (aqueous flare and anterior chamber fibrin) in the first 3 weeks after surgery, overall capsular opacity and surgical outcome at time of last follow-up (mean 8.5 months, median 8, range 3–13 months) were not significantly different between the PMMA and the foldable lens groups. There was a tendency for the foldable lens to score lower rates of opacity, but the differences were not statistically significant. Averages of refraction in nine patients with PMMA and 11 patients with foldable IOLs resulted in undercorrection of +1.36D and +2.30D, respectively. Conclusions: Results of the study suggest comparable postoperative results can be obtained with PMMA and the CANI/JAG foldable IOL, including postoperative inflammation, short/medium-term capsular opacification, refraction and surgical outcome. There appeared to be a tendency for foldable IOL to produce lower scores of overall opacities and to maintain a clearer visual axis. Support: Foldable intraocular lenses were supplied by I-Medical Pharma Inc. Commercial interest: None.

ABSTRACT No.: 50

Calcific keratopathy in geriatric patients: a report of six cases

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S. Pizzirani, F. Maggio, T. Miller-Michau, M. G. Davidson and B. G. Gilger

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

Purpose: To describe clinical and histologic findings in six dogs with calcific keratopathy. Methods: Clinical records of canine patients with corneal ulceration and whose histopathologic results after lamellar keratectomy were consistent with stromal calcium deposition were reviewed. Von Kossa and Alizarine red stains were used to determine the mineral composition of the refractive stromal bodies. Results: Six dogs were included (three male castrated and three female spayed) with a median age of 15 years (10–18). There were three mixed-breed dogs, one WHWT, one Poodle and one Shetland sheepdog. Ocular findings consisted of a central corneal opacity, represented by multifocal stromal crystalline needle-shaped deposits. There was deep stromal corneal ulceration, 2–6 mm wide, in the center of the opacities. In four dogs these opacities were bilateral and asymmetric. In two dogs they were unilateral secondary to exophthalmos because of orbital neoplasia. Corneal ulceration had been present for an average of 16.5 days. Neovascularization was absent in the central cornea and very minimal when present at the limbus. All dogs were successfully treated with a keratectomy and conjunctival pedicle graft. In two dogs with bilateral involvement, the fellow eye underwent corneal ulceration 2 and 7 weeks later, respectively. One case was treated surgically and one medically due to the owner's choice. Conservative treatment resulted in persistence of corneal ulceration for 6 months, after which the patient was lost to follow-up. No recurrence has been recorded in the eyes treated surgically. Corneal cytology and histopathology did not reveal any signs of infection or inflammation. CBC and chemistry profiles of all patients were unremarkable and calcium levels were normal in all cases. CaxP product was less than 70 in all patients and ACTH stimulation test was negative in three and patients and positive in one. Low dose dexamethasone suppression test in the latter patient was normal. Conclusions: Calcific keratopathy manifests as a corneal degeneration with mineral deposition within the subepithelial and anterior stroma. Geriatric patients appear to be predisposed to the disease. Age-related collagen degeneration/senescence, decreased corneal sensitivity, blinking defects, tear film abnormalities and corneal exposure because of exophthalmos could singularly or as a whole theoretically explain why older patients and central cornea are predisposed. Ulcerative lesions have a decreased tendency to heal spontaneously or with medical therapy alone when the cornea is infiltrated with calcium. Hyperadrenocorticism, primary or secondary hypercalcemia or chronic renal failure with increased CaxP also do not appear to be related. Superficial keratectomy and conjunctival grafting allowed healing of the corneal ulcers and cleared the surrounding corneas in all treated eyes. Commercial interest: None.

ABSTRACT No.: 51

Elevations in adrenal sex hormones in canine sudden acquired retinal degeneration (SARDS)

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R. T. Carter,* E. Bentley,* J. W. Oliver,† P. E. Miller* and I. P. Herring‡

*School of Veterinary Medicine, University of Wisconsin-Madison, College of Veterinary Medicine, University of Tennessee, Knoxville, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA

Purpose: Dogs with SARDS often have persistent systemic clinical signs consistent with excessive glucocorticoid production, but often serum cortisol values are normal. Many dogs are ultimately euthanized after diagnosis because the owners feel the dogs have a poor quality of life due to the persistent clinical signs. Recently, it has been recognized that sex hormones also have glucocorticoid-like activity, which prompted us to investigate these hormones in dogs with SARDS. Methods: Ten dogs met the inclusion criteria of a history of sudden blindness, a normal fundic examination or funduscopic changes that were not consistent with the degree of vision loss, and an extinguished bright-flash electroretinogram. Seven were spayed females and three were castrated males. Breeds included Brittany Spaniel (3), Miniature Schnauzer (2), West Highland White Terrier (1), Dachshund (1), Golden Retriever (1), Pomeranian (1) and mixed breed (1). Ages ranged from 7 to 10 years with a mean of 8.7 years. Laboratory testing included analysis of sex hormones (androstenedione, estradiol, progesterone, 17-OH progesterone and testosterone; 10/10) and cortisol (10/10) both pre and post ACTH stimulation, serum chemistry panels (9/10), CBC (8/10), and resting ACTH levels (4/10). Results: Systemic clinical signs of glucocorticoid excess were present in 9/10 dogs. Nine/ten dogs had elevations in one or more sex hormone values. These elevations included 17-OH progesterone and progesterone (7/10), androstenedione (6/10) and estradiol (3/10). Eight/nine dogs with systemic clinical signs had elevations of one or more sex hormone values. One dog had no clinical signs, but had elevations in hormone values. Cortisol levels were normal in 5/10 dogs. Five/ten dogs had elevated cortisol post-ACTH stimulation. Endogenous ACTH levels were inappropriate (normal to low normal) in 3/3 dogs with elevated sex hormones, and normal in one dog with normal sex hormone values. The most frequent abnormality on CBC was an elevated total protein (4/8). The most frequent abnormalities on serum chemistry profiles included elevations in ALP (5/9) and ALT (4/9). Conclusions: In this preliminary study, most dogs with SARDS had elevations in sex hormone levels but not all dogs had elevated cortisol. The presence of a variety of increased sex hormone levels indicates increased adrenal activity. Endogenous ACTH levels in the dogs with elevated sex hormones are inappropriately high given the level of adrenal activity, which suggests increased activity at the level of the pituitary. For dogs with persistently elevated hormone levels, possible treatment options could be explored to alleviate their systemic clinical signs, thereby improving quality of life and possibly preventing euthanasia in some cases. Commercial interest: None.

ABSTRACT No.: 52

Nanoscale topography modulates corneal epithelial cell migration

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N. K. A. Diehl,* J. D. Foley,* P. F. Nealey† and C. J. Murphy*

*Department of Surgical Sciences, School of Veterinary Medicine, Department of Chemical Engineering, School of Engineering, ‡University of Wisconsin-Madison, Madison, WI, USA

Purpose: To engineer surfaces with nanoscale topographic features that mimic the corneal epithelial basement membrane and to study the effect of these features on corneal epithelial cell migration. Methods:Surface fabrication: Using electron-beam lithography and reaction ion etching, silicon wafers were patterned and etched with grooves and ridges of nano- and microscale dimensions (pitch range from 400 to 4000 nm) and coated with silicon oxide to achieve a chemically uniform surface. Additionally, polyurethane patterned surfaces were created through replication molding techniques. Migration of individual cells: SV-40 transformed human corneal epithelial cells in SHEM containing 10% fetal bovine serum (FBS) were sparsely seeded onto patterned and smooth control surfaces and allowed to adhere for 12–20 h. Sequential photographs of cells on surfaces with features of differing dimensions, as well as smooth control surfaces, were obtained every 30 min over 24 h to record orientation, with respect to the underlying surface pattern, and migration of individual cells. Cell dispersion assay: SV-40 cells in SHEM containing 10% FBS and pretreated with Mitomycin-C to prevent proliferation, were seeded onto patterned and smooth control surfaces in constrained, 500-µm diameter, circular areas, using custom-fabricated cell manifolds. Once cells were adherent (16–24 h), the manifolds were removed and outward migration of groups of cells on each surface monitored, recording the cell colony shape, dimensions and orientation, daily for 7 days. Results: Individual cells frequently aligned with respect to the underlying surface pattern and migrated preferentially parallel to grooves and ridges of nano- and microscale dimensions of all pitches assayed. Direction of migration of individual cells on smooth surfaces was random. Groups of cells migrated out from the initial circular seeded zone predominantly along grooves and ridges, elongating to elliptical colonies paralleling surface grooves and ridges of differing dimensions. On smooth surfaces, groups of cells migrated radially and equally in all directions, maintaining a circular colony shape. Conclusions: Biologic length scale substratum features resembling the basement membrane modulate corneal epithelial cell migration. These findings have relevance to the maintenance of corneal homeostasis and wound healing, and the conduction of in vitro studies of cell migration, as well as to tissue engineering and the development of corneal prostheses. Financial support: Supported by NIH grant NEI 12253–05 and NSF grant MRSEC CTS 9703207. Commercial interest: None.

ABSTRACT No.: 53

High resolution ultrasound imaging of the anterior segment of dogs with primary glaucoma prior to and following the topical application of 0.005% latanoprost

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P. E. Miller, E. Bentley, K. A. Diehl and R. Carter

University of Wisconsin-Madison, School of Veterinary Medicine, Madison, WI, USA

Purpose: The mechanisms triggering primary glaucoma in dogs are poorly understood. Similarly, it is unclear how 0.005% latanoprost can rapidly lower IOP in acute glaucoma if it acts solely by improving uveoscleral outflow. We used high-resolution ultrasound (HRUS) to try to elucidate the anatomic mechanisms by which primary glaucoma develops in dogs, and to investigate how latanoprost may affect these mechanisms. Methods: Longitudinal, 20 MHz, HRUS images were collected at the 12 o’clock position from nine dogs (14 eyes) with various stages of primary angle closure glaucoma (PACG), and from two dogs (four eyes) with primary open angle glaucoma (POAG) prior to, and 1–2 h following topical 0.005% latanoprost. IOP and resting pupil diameter (PD) were also recorded. Results: At the time of imaging, eight dogs had acute PACG (mean IOP = 56 mmHg; range 35–80 mmHg; PD = 7–11 mm) and one had intermittent episodes of PACG (IOP = 12 mmHg; PD = 13 mm). In five dogs with acute PACG the normotensive, but at-risk, fellow eyes (PACG suspects) also were present and imaged. Dogs with acute PACG appeared to have a form of reverse pupillary block with a sigmoidally shaped iris. The pupillary border of the iris was flattened and in close apposition to the anterior lens capsule, the mid-peripheral iris was concave, and the peripheral iris was convex and rotated anteriorly. This configuration was associated with gonioscopic angle closure, a relatively shallow posterior chamber, and ciliary cleft collapse. Similar, but less dramatic alterations were present in the eye with intermittent PACG, and in PACG suspects. In four out of five PACG suspects, however, the ciliary cleft was open even though the angle was gonioscopically narrow to closed. In POAG (mean IOP = 41 mmHg; PD = 8–14.5 mm), the iris had a planar configuration with collapse of the ciliary cleft. Latanoprost normalized IOP in five dogs with acute PACG, in all four eyes with POAG, and reduced IOP to 6 mmHg in the eye with intermittent PACG. In each of these eyes marked miosis occurred (1–4.5 mm). In PACG eyes post latanoprost the iris assumed a planar to uniformly convex configuration, the posterior chamber deepened, and the anterior chamber shallowed. The ciliary cleft was minimally altered. In the three dogs with acute PACG in which IOP remained elevated post latanoprost, miosis did not occur and the original configuration persisted. In POAG eyes the iris thinned, the ciliary cleft opened, and the posterior chamber deepened. Conclusions: Reverse pupillary block is one mechanism leading to PACG in dogs. Despite similar rises in IOP, POAG had a different anterior segment configuration and was associated with ciliary cleft collapse. Latanoprost may rapidly lower IOP in PACG by inducing miosis, which breaks the pupillary block, and by opening the ciliary cleft in eyes with POAG. Commercial interest: None.

ABSTRACT No.: 54

Prevalence of equine herpesvirus DNA from conjunctival swabs in Missouri

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L. E. Galle,* S. B. Kleiboecker,† P. J. Johnson,* E. A. Giuliano* and N. C. Scotty*

*Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Department of Pathobiology, College of Veterinary Medicine,University of Missouri-Columbia, MO, USA

Purpose: Equine herpesviruses 1, 2 and 4 have been associated with equine ocular disease. Of these, equine herpesvirus 2, a member of the gammaherpesvirinae subfamily, has been specifically implicated as a primary cause of keratitis. The prevalence of detectable equine herpesvirus in normal eyes, however, is not known. A pilot study to determine the prevalence of detectable herpesviruses from the ocular surface, nasopharynx and buffy coat of healthy horses lacking ocular disease was undertaken. Methods: Samples were collected from 10 healthy horses. All animals were free of corneal and conjunctival abnormalities at the time of sample collection. The following samples were collected from each animal: (1) 10 mLs whole blood; (2) nasopharyngeal swab; (3) pooled conjunctival swabs from both eyes. Swabs were vortexed in virus isolation media prior to storage, and the buffy coat was collected into virus isolation media after separation from other blood components. Samples were stored at −80 °C until assayed. All samples were subjected to a consensus primer PCR capable of detecting herpesvirus from all three Herpesviridae subfamilies, a PCR capable of detecting species of the gammaherpesvirinae subfamily, and virus isolation. Results: All animals had herpesvirus detected from at least one of the samples collected using consensus primer PCR. Seven of 10 animals had herpesvirus DNA detectable in conjunctival swabs; 5/10 conjunctival samples were positive for gammaherpesvirus DNA. Nine of 10 nasopharyngeal swabs were positive for herpes by consensus primer herpes PCR, and 8/10 were positive for gammaherpesvirus DNA. Three of 10 buffy coat samples were positive for herpesvirus DNA, and 2/10 buffy coat samples were positive for gammaherpesvirus DNA. All samples positive for gammaherpesvirus were also positive by the general herpesvirus assay. Extraction controls and no-template controls were all negative. Conclusions: Herpesvirus DNA was detectable in 7/10 conjunctival swabs in this study, with 6/10 also being gammaherpesvirus positive. Further investigation of the prevalence of equine herpesviruses, detectable from the ocular surface of horses lacking ocular disease, is needed prior to developing standardized diagnostic criteria for equine herpetic keratitis. Studies including a larger population sample from multiple horse herds and species-specific PCR with virus isolation are underway. Commercial interest: None.

ABSTRACT No.: 55

Effects of general anesthesia on Schirmer I tear tests and corneal touch threshold of dogs and cats

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K. V. Cutter-Schatzberg,* B. G. Mangan,* D. V. Nydam,† P. Maza* and T. J. Kern*

*Department of Clinical Sciences, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Purpose: Dogs and cats occasionally develop superficial, central corneal ulceration within 24 h of general anesthesia (GA). Schirmer I tear test (STT) values have been shown to decrease transiently in dogs after GA but STT has not been evaluated critically in cats post GA. Corneal touch threshold (CTT) has not been investigated in dogs or cats following GA. We hypothesize that STT values and central CTT (cCTT) of both dogs and cats decrease transiently following GA. Methods: Twenty-three adult dogs and 13 adult cats presented to Cornell University for routine neutering and received complete ophthalmologic examinations (exam a) including Cochet–Bonnet corneal aesthesiometry, STT, flourescein staining, slit-lamp biomicroscopy, indirect ophthalmoscopy, and applanation tonometry prior to GA. Male cats were anesthetized with ketamine, diazepam, hydromorphone, acepromazine, glycopyrrolate. Dogs and female cats were anesthetized with glycopyrrolate, thiopental or diazepam/ketamine, acepromazine, butorphanol, isoflurane. Ophthalmologic examinations were repeated within 12–24 h (exam b) and 24–48 h post GA (exam c). STTs were conducted using a commercial tear test strip. Tonometry was performed with a Tonopen XL II and 1 drop of topical 0.5% proparacaine per eye. The paired Student's t-test was utilized for statistical analysis and P< 0.05 was considered statistically significant. Results: The mean values for canine cCCT OS were 1.92 cm (a), 1.92 cm (b) and 2.1 cm (c), and the mean canine cCTT OD values were 1.98 cm (a), 1.85 cm (b) and 2.03 cm (c). The mean canine STTs OS were 20 mm/min (a), 14 mm/min (b) and 18 mm/min (c), and the mean canine STTs OD were 17 mm/min (a), 14 mm/min (b) and 22 mm/min (c). The mean values for feline cCTT OS were 3.89 cm (a), 2.87 cm (b) and 2.98 cm (c), and the mean feline cCTT OD values were 3.82 cm (a), 2.68 cm (b), 2.93 cm (c). The mean feline STT OS values were 14 mm/min (a), 11 mm/min (b) and 14 mm/min (c) and the mean feline STT OD values were 11 mm/min (a), 10 mm/min (b) and 11 mm/min (c). No significant differences in cCCT were calculated in dogs post GA (P-values: 0.40–1.00), whereas feline cCTT was decreased significantly at both post GA time points (P-values: 0.00007–0.0047). A significant decrease in canine STT was calculated within 12–24 h post GA for both eyes (OS: P= 0.0009, OD: P = 0.023) as shown previously. At 24–48 h post GA only the OS had a significant reduction in STT (OS: P= 0.024, OD: P= 0.061). In contrast, feline STT was not decreased significantly post GA (P-values: 0.15–0.85). Conclusion: This pilot study shows that cCTT was reduced in cats but not dogs for at least 48 h post GA and neutering. STT values decreased transiently in dogs but not in cats post GA and neutering. Commercial interest: None.

ABSTRACT No.: 56

The use of an Ahmed anterior chamber shunt and transscleral diode laser cyclophotocoagulation for controlling glaucoma in a horse

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K. V. Cutter-Schatzberg, N. I. Irby and R. McGrath

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Purpose: Glaucoma is diagnosed uncommonly in horses compared to dogs and cats. Equine glaucoma typically occurs secondary to equine recurrent uveitis (ERU), although primary equine glaucoma has been reported sporadically. A 14-year-old Quarter horse mare presented for a cloudy left (OS) cornea and was diagnosed with ERU OU and secondary glaucoma OS via applanation tonometry (intraocular pressure (IOP): 42 mmHg OS). Aggressive medical management using a full spectrum of both topical and oral antiglaucoma medications failed to control IOP. Therefore, surgical placement of an anterior chamber shunt combined with transscleral diode laser cyclophotocoagulation (CPC) was performed as a salvage procedure to reduce IOP. CPC was repeated 7 weeks after surgery as a standing procedure. Methods: An anterior chamber Ahmed shunt was placed and transscleral diode laser CPC was performed under general anesthesia. In brief, the dorsal bulbar conjunctiva was undermined and an Ahmed anterior chamber shunt was sutured to the sclera, medial to the dorsal rectus fascia using 7–0 nylon. The area was pretreated for 5 min with mitomycin-C prior to shunt placement. The shunt tubing was cut to the desired length with iris scissors. A 20-gauge needle was tunneled through the dorsal scleral shelf and into the anterior chamber at a 45° angle. The needle was then removed and the tubing was inserted quickly through the tract within the scleral shelf and into the anterior chamber. The dorsal bulbar conjunctiva was closed with 6–0 polygalactin. Transscleral CPC was performed subsequently OS using 60 hits at 1500 mSec and 1700 mWatts, divided evenly into specific quadrants of the dorsonasal, dorsotemporal, and ventrotemporal sclera, 4–5 mm caudal to the limbus as described previously. IOP remained normal postoperatively for 6 weeks. However, the horse presented 7 weeks after surgery for increased corneal edema OS, with an IOP of 30 mmHg OS. Transscleral diode laser CPC was repeated as a standing procedure using topical 0.5% proparacaine and intravenous (IV) butorphanol and detomidine hydrochloride. Results: After 8 months of follow-up, the IOPs OU have remained normal, ranging from 12 to 18 mmHg OS and 10 to 18 mmHg OD. The horse remains visual OU. Unfortunately, the long-term prognosis for vision is poor given the typical, devastating sequelae of ERU. Conclusion: This case report demonstrates that the use of an anterior chamber shunt device, such as the Ahmed valve, can aid in the control of equine glaucoma in select cases when combined with transscleral laser CPC. Transscleral diode CPC may be performed as a standing procedure in the horse using a combination of IV butorphanol and detomidine, obviating the need for general anesthesia. Commercial interest: None.

ABSTRACT No.: 57

Juvenile ocular abnormalities in a ferret

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K. V. Cutter-Schatzberg and R. R. Riis

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Purpose: Ferrets (Mustela putorius furo) are popular household pets and laboratory animals, particularly in the area of developmental retinal research. To accompany their popularity comes increased attention to the ocular diseases of ferrets. The purpose of this study was to determine the frequency of ocular disease within a large population of ferrets. Methods: Indirect ophthalmoscopy using both 28 and 40 diopter lenses was performed on 3257 intact, male (Hobbs) ferrets between 6 and 12 months of age. All ferrets were bred and housed at the same facility. Ocular examinations were performed by a single ACVO diplomate. All ferrets received 1 drop of 1% topical tropicamide for pupillary dilation 10–20 min prior to examination. Results of the ocular examinations were recorded. Histopathologic examinations also were performed on 87 globes of ferrets with ocular abnormalities within this population. Globes were fixed either in Bouin's solution or 10% formalin and were embedded subsequently in paraffin blocks. Paraffin embedded globes were sectioned and stained with hematoxylin and eosin (H&E). Results: Review of the data from 3257 ferret examinations revealed eight different categories: 1. normal (n = 1551); 2. bilateral cataracts (n = 66); 3. unilateral cataracts (n = 5); 4. bilateral retinal degeneration (n = 61); 5. unilateral retinal degeneration (n = 9); 6. bilateral optic nerve head hypoplasia (n = 1291); 7. unilateral optic nerve head hypoplasia (n = 253); 8. persistent hyaloid remnant (n = 21). The frequency of ocular disease within the examined population was 47.62% normal; 2.03% bilateral cataracts; 0.15% unilateral cataract; 1.87% bilateral retinal degeneration; 0.28% unilateral retinal degeneration; 39.64% bilateral optic optic nerve head hypoplasia; 7.77% unilateral optic nerve head hypoplasia, and 0.64% persistent hyaloid vascular remnant. Histopathology confirmed the presence of cataracts, retinal degeneration, retinal dysplasia and optic nerve head hypoplasia in the 87 globes evaluated. The retrobulbar optic nerve appeared normal in all histopathology samples examined, including cases of optic nerve head hypoplasia. Conclusion: To our knowledge, this is the first report of optic nerve head hypoplasia in ferrets. While some of these ocular abnormalities such as cataracts and severe retinal degeneration have significant visual consequences, the visual significance of optic nerve head hypoplasia and retinal dysplasia is unknown. The true incidence of ocular disease in the ferret cannot be determined from this study given the single population of ferrets studied. However, ocular examination is recommended for all ferrets being considered for breeding, research, or as pets. Commercial interest: None.

ABSTRACT No.: 58

Enrofloxacin and the feline retina: ophthalmoscopic and electroretinographic effects

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M. M. Ford, K. Narfström, E. A. Giuliano and C. P. Moore

College of Veterinary Medicine, University of Missouri, Columbia, MO, USA

Purpose: To characterize the ophthalmoscopic and electroretinographic (ERG) effects of oral enrofloxacin in healthy cats. Methods: Twenty-four (12 male, 12 female), pathogen-free, Domestic Short-haired cats were equally randomized by gender into one of two study groups (experimental, control). Groups were further divided into three subgroups according to duration of enrofloxacin administration (3, 5 or 7 days). Enrofloxacin was administered to experimental study group animals once daily at 10 times the recommended dose (50 mg/kg/day, PO) between day 1 and the day of humane euthanasia (3, 5 or 7 doses). Daily funduscopic examinations were performed by an investigator blinded to the treatment groups (KN). ERG studies were performed with cats under general anesthesia prior to, and every 2–3 days after onset of enrofloxacin administration. Cats received intramuscular (IM) injections of medetomidine (0.09 mg/kg) followed 15 min later with ketamine (5 mg/kg, IM), immediately prior to ERG testing. A computerized ERG (ERG System Tor, Global Eye Program, Rejmyre, Sweden) with full-field white light stimulation was used. After 2 h of dark adaptation a scotopic light intensity series from −6.0 cd s/m2 in 0.5 log unit steps was performed, as well as a high-intensity series with flashes up to 0.6 log cd s/m2. After 10 min of light adaptation, photopic stimulation was elicited at 5, 30 and 50 Hz using 0.0 cd s/m2 light intensity. Control cats underwent identical dosing regimens with placebo, clinical examinations, and testing procedures as experimental cats. Four animals per study group were humanely euthanized on days 3, 5 and 7 and eyes were collected for light and electron microscopy. Results: Six of eight cats that received enrofloxacin had absent menace responses by day 4. Neurologic signs (seizures, ptyalism, vestibular signs) were identified in two animals after 5 days of treatment. Funduscopically, gray discoloration with tapetal hyporeflectivity, first evident in the area centralis, and a generalized attenuation of retinal blood vessels were detected within 72 h in all treated cats. No clinical or funduscopic abnormalities were noted in any control animal. Within 24 h of initial dosing, b-wave amplitudes were decreased and were nonrecordable by day 3 for all stimuli used. A-wave amplitudes were nonrecordable by day 5. These changes were reflected by a progressive decrease in the b-/a-wave ratio. No significant changes in ERG recordings were noted in the control group. Conclusions: Enrofloxacin administered at a dose of 50 mg/kg/day PO to healthy adult cats is acutely toxic to rod and cone photoreceptors leading to a significant reduction in ERG a- and b-wave amplitudes by day 3 following treatment. Decreases in b-wave ERG amplitudes precede funduscopic changes. Financial support: Supported by a grant from Bayer. Commercial interest: None.

ABSTRACT No.: 59

Canine keratoconjunctivitis sicca associated with etodolac administration

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G. Klauss,* E. A. Giuliano,* C. P. Moore,* C. M. Stuhr,† S. L. Martin,‡ J. W. Tyler,* K. E. Fitzgerald† and D. A. Crawford‡

*College of Veterinary Medicine, University of Missouri, Animal Eye Clinic, Wilton, CT, Fort Dodge Animal Health (FDAH), Overland Park, KS, USA

Purpose: Etodolac is an oral nonsteroidal anti-inflammatory drug that has been implicated in the development of canine keratoconjunctivitis sicca (KCS). A retrospective study was undertaken to characterize the features of KCS in dogs administered oral etodolac and response to treatment. Methods: Cases studied were obtained from two sources. Group 1 (1) consisted of 65 cases obtained from an independent survey of veterinary ophthalmologists. Group 2 (2) contained 146 cases reported to FDAH. Schirmer tear test (STT) values, reported in mm of wetting/min, were used to categorize severity of KCS as follows: mild (10), moderate (5–9), and severe (< 5). Descriptive statistics were performed on both groups. Forward stepwise logistic regression models were developed predicting probability of complete remission or clinical improvement as a function of several variables. Independent variables considered are reported in results. Results from groups 1 and 2 were analyzed separately. Results: Bilateral KCS was present in 62/65 (1) and 96/146 (2) dogs. Average age was 10.1 ± 2.8 years in both groups. Breeds represented in the highest frequency were German Shepherds (14/65 (1); 32/146 (2)) and Labrador Retrievers (9/65 (1); 22/146 (2)). Average body weight was 32.5 ± 13.5 kg (1) and 30.0 ± 12.5 kg (2). Duration of administration prior to diagnosis of KCS was 9.1 ± 6.7 months (1) and 7.8 ± 7.7 months (2). Mild KCS was present in 11 eyes of eight dogs (1) and 11 eyes of eight dogs (2). Moderate KCS was present in 32 eyes of 22 dogs (1) and in 27 eyes of 19 dogs (2). Severe KCS was present in 84 eyes of 50 dogs (1) and in 111 eyes of 62 dogs (2). Treatment for KCS was reported in 48 (1) and 116 (2) cases, and 46/48 (1) and 99/116 (2) were treated with topical cyclosporine. KCS resolved in 7/65 (1) and 23/146 (2) dogs. KCS improved in 30/65 (1) and 39/146 (2) dogs. No response to treatment was observed in 26/65 (1) and 27/146 (2) dogs. Fifty-one (1) and 52 (2) dogs had records which were sufficiently complete to permit use in models used to predict complete remission and clinical improvement. In the group 1 analysis, none of the independent variables were significantly associated with the probability of remission or clinical improvement. In group 2, dogs with treatment intervals < 6 months prior to the onset of KCS were 4.2 times more likely to experience remission (P = 0.049) than were dogs with treatment intervals of 6 months. Conclusions: In one of these populations of dogs, shorter duration of etodolac administration (< 6 months) was associated with improved outcome. Careful monitoring of tear production should be performed prior to and during administration of etodolac in dogs. Financial support: Supported by a grant from Fort Dodge Animal Health. Commercial interest: Financial interest in product; investor; and employee.

ABSTRACT No.: 60

Feline malignant glaucoma/aqueous misdirection: 16 cases

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N. La Croix,* A. van der Woerdt,‡ R. H. Silverman,§ T. Blocker† and A. Hoffman†

*Eye Care for Animals, UT, Eye Care for Animals, CA, The Animal Medical Center, NY, §Weill Medical College of Cornell University, New York, NY, USA

Purpose: To describe signalment and response to medical and/or surgical therapy of cats with malignant glaucoma/aqueous misdirection. Methods: Medical records of cats presenting to the Eye Care for Animals for the past 3 years, and to the Animal Medical Center for the past 9 years, were reviewed. Cats with malignant glaucoma/aqueous misdirection were included if they were followed for at least 3 months after initial diagnosis. High frequency ultrasound biomicroscopy and histopathologic examination were described. Results: Sixteen cats were included in the study. The mean age was 12 years (range: 4–16). Thirteen cats were spayed females, and three were castrated males. All cats were Domestic Short-hairs, except for one Persian. The right eye was affected in 10 cats, the left eye in four cats, and both eyes in two cats. A total of 18 eyes were affected, with an average intraocular pressure (IOP) of 27 mmHg (range: 14–40) at initial presentation. Fourteen eyes were visual, three eyes had decreased vision, and one eye was blind at initial examination. Fifteen affected eyes were treated initially with the following: 2% dorzolamide q 12 h (four eyes); 1% pilocarpine q 12 h (three eyes); 0.5% pilocarpine q 12 h (one eye); or 0.5% timolol malate q 12 h (seven eyes). Initially, no therapy was prescribed for two eyes. Phacofragmentation with anterior vitrectomy was performed in one eye with an IOP of 40 mmHg. After 2–3 weeks, the average decrease in IOP was 8 mmHg (range: 3–15) with 0.5 or 1% pilocarpine. Average decrease in IOP was 8.5 mmHg (range of 0–11) with 2% dorzolamide. During the same time period, IOP of eyes treated with 0.5% timolol malate increased by 4 and 6 mmHg in two eyes, and decreased by an average of 8 mmHg (range: 3–13) in five eyes. The IOP increased by an average of 6 mmHg in the two eyes that were not treated. Phacofragmentation was performed on one eye that had been treated initially with 0.5% timolol maleate. This eye remained visual for the following 3 years without medication, maintaining an average IOP of 20 mmHg. The eye in which phacofragmentation had been performed initially was enucleated when its IOP returned to 40 mmHg 6 weeks postoperatively. Of visual eyes that were treated medically, five lost vision after an average of 10 months (range: 3–24), and 10 remained visual after an average of 13 months (range: 3–36). Histopathology of one of these blind eyes revealed anterior displacement of the lens and iris, occluding the iridocorneal angle. High frequency ultrasound biomicroscopy of one affected eye revealed a thicker lens, a deeper vitreous chamber, iris bowing, and anterior rotation of the ciliary processes when compared to a normal control. Conclusions: Malignant glaucoma/aqueous misdirection is most commonly seen in older, domestic Short-hairs. Response to medical management can be unpredictable. Surgical management controlled the IOP in one cat, but failed to control IOP in another. Commercial interest: None.

ABSTRACT No.: 61

Suppression of EAU using a plasmid encoding the ocular neuropeptide alpha-melanocyte stimulating hormone

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D. J. Biros and A. W. Taylor

The Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA, USA

Purpose: The ocular neuropeptide alpha-melanocyte stimulating hormone (α-MSH) suppresses inflammation on both innate and adaptive immunity. It suppresses endotoxin-mediated APC activation of inflammation and activation of IFN-g by Th1 cells; moreover, it induces activation of TGF-β-producing CD25+ and CD4+ regulatory T-cells (Treg). Intravenous injections of α-MSH into mice immunized for experimental autoimmune uveitis (EAU) suppress the incidence and severity of EAU. Also, an adoptive transfer of α-MSH-induced Treg cells specific to ocular autoantigen suppressed EAU. It is not known, however, if local delivery of α-MSH to the inflamed ocular environment can also suppress EAU. Therefore, we examined the effect of subconjunctival delivery of a plasmid encoding α-MSH on EAU. Methods: EAU was induced by subcutaneous immunization in B10.RIII, C57Bl/6, or melanocortin 5-receptor knockout (MC5r(–/–)) mice with human interphotoreceptor retinoid binding protein peptide (hIRBPp) mixed in complete Freund's adjuvant. Injected into the subconjunctival space of both eyes was 25 µg of either α-MSH plasmid or empty plasmid on days 6 and 9 for B10.RIII mice, or days 11 and 18 for C57Bl/6 and MC5r(–/–) mice. The ocular fundus was monitored every 3 days via direct ophthalmoscopy. Uveoretinal inflammation was scored on a range from 0 to 5. Mean maximum EAU scores in each treatment group were analyzed using the Mann–Whitney U-test. When the retinal inflammation resolved, aqueous humor was collected and assayed by an α-MSH ELISA. Also, the eyes were collected for histopathologic examination. Results: There was significant suppression (P < 0.0003) in the severity of EAU when B10.RIII mice were injected with the α-MSH plasmid by contrast to the empty plasmid (1.7 ± 0.2 and 3.1 ± 0.2, respectively). In C57Bl/6 mice a similar significant (P < 0.05) suppression of EAU was seen (1.5 ± 0.4 α-MSH plasmid and 2.4 ± 0.2 empty plasmid). However, injections of the α-MSH plasmid into eyes of MC5r(–/–) mice had no significant effect on the severity of EAU. The incidence of EAU was suppressed by 50% in α-MSH plasmid-treated eyes. Average time to clinical recovery was 33 days for B10.RIII mice receiving the empty plasmid and 17 days for the α-MSH plasmid. Similar results were seen in C57Bl/6 mice. Histologic examination of the retinas showed minimal damage in α-MSH plasmid-treated eyes. After recovery from EAU, treated eyes had a 3–4-fold increase in aqueous humor concentration of α-MSH. Conclusions: The reintroduction of the ocular immunosuppressive cytokine α-MSH during EAU suppresses the incidence and severity of the autoimmune disease. Financial support: Supported by PHS grant EY13913 and C Zycos, Inc. Commercial interest: None.

ABSTRACT No.: 62

Acoustic velocity of porcine, canine and rabbit lens and vitreous body at 10 MHz

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C. Görig,* T. J. Varghese,† T. Stiles,† J. Zagzebski† and C. J. Murphy*

*Department of Surgical Sciences, Department of Medical Physics, University of Wisconsin, Madison, WI, USA

Purpose: The goal of this study was to determine the acoustic velocities of porcine, canine and rabbit lens and vitreous body tissues. The influence of age, temperature and time after enucleation on acoustic velocity was also assessed. Methods: The eyes of research dogs, rabbits and slaughtered pigs were enucleated within 1 h after death and then immediately transferred into BSS and stored at 4 °C. All measurements were carried out within 12 h after death. A narrow band substitution technique was used, with distilled degassed water as the measurement medium. Ultrasound propagation speed was determined at temperatures ranging from 32 to 40 °C in 2 °C increments. The time-shift of a 10-MHz tone burst pulse at a zero crossing was measured when the lens or the vitreous body was introduced into the path of the ultrasound beam. Three consecutive estimates were recorded. The mean values and standard deviations were calculated. To test for a linear relationship between mean velocities and temperature, velocity and age, or time after enucleation, a regression analysis was done. An analysis of covariance was conducted to compare the slopes of the regression curves: cvitreous vs. temperature. Results:Table 1 lists the velocity values of the porcine, canine and rabbit lens and vitreous body at 36 °C and 10 MHz. Lens tissue exhibited a significant age effect, but no age effect could be demonstrated for vitreous body tissue. Temperature did not impact on acoustic velocity through lens tissue of any species in a systematic fashion. In contrast, the velocity through vitreous humor increased linearly with temperature in all species. Time from enucleation did not significantly alter the velocities measured. Conclusions: Sound velocity through lens tissue differs significantly between the three species examined and also differs from accepted values for the human lens. Ratios of measured velocities at 36 °C to the velocity in human lens are 1.024, 1.041 and 1.053 for porcine, canine and rabbit lens, respectively. The age of an animal examined has also to be taken into account when conducting biometry measurements of the eye. No significant difference among species was found for acoustic velocities through the vitreous body. Commercial interest: None.

Table 1. Acoustic velocities of porcine, canime and rabbit lens and vitreous body tissues
Speciescvitreous (m/s)clens (m/s)
Porcine1532 ± 0.8 (n = 16)1681 ± 6.3 (n = 9)
Canine1532 ± 0.7 (n = 17)1708 ± 20 (n = 40)
Rabbit1531 ± 1.1 (n = 23)1728 ± 20 (n = 20)

ABSTRACT No.: 63

Agryophilic nucleolar organizer regions (AgNOR) as a diagnostic indicator of ocular squamous epithelial tumors in the horse

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C. B. Mosunic,* P. A. Moore,† U. Dietrich,* A. Vidyashankar,* A. Cheng,‡ E. Howerth‡ and K. P. Carmichael†

*College of Veterinary Medicine, Department of Small Animal Medicine, Department of Pathology, Department of Statistics, University of Georgia, Athens, GA, USA

Purpose: To evaluate the correlation between the histologic diagnoses of ocular squamous epithelial tumors and AgNOR scores. Methods: Archived tissue sections and paraffin-embedded tissue blocks from ocular squamous epithelial tumors (squamous papilloma, squamous plaque, squamous cell carcinoma in situ, and invasive squamous cell carcinoma) were retrieved. The H&E tissue sections were reviewed for accuracy of histologic diagnosis. The tissue sections were deparrafinized and rehydrated in water. The tissue sections were stained in a 2 : 1 50% silver nitrate : gelatin solution and then subsequently washed in water and mounted. An agryophilic nucleolar organizer region (AgNOR) score was determined by counting the number of dark black/brown staining dots in the nucleus of 20 cells in five different quadrants, resulting in a total of one hundred cells counted. The total count of staining dots was divided by one hundred to achieve an average per cell AgNOR score. These counts were repeated two more times to assess intraobserver reliability. The three counts were then averaged to obtain a final AgNOR score for the tumor. A one-way analysis of variance was used to perform a pairwise comparison of variances between histologic types. All analyzes were performed at a significance level of P < 0.05. Results: AgNOR staining was performed on 11 plaques, nine squamous cell carcinoma in situ and 22 invasive squamous cell carcinomas. The average AgNOR score for invasive squamous cell carcinoma was 3.01 (n = 22), for squamous cell carcinoma in situ was 1.98 (n = 9), and for squamous plaque was 1.71 (n = 11). There was a statistically significant difference between the AgNOR score of invasive squamous cell carcinoma and squamous cell carcinoma in situ (P < 0.0001) and invasive squamous cell carcinoma and squamous plaque (P < 0.0001). There was not a significant difference between squamous cell carcinoma in situ and squamous plaque (P = 0.0898). Conclusion: AgNOR score can be a potentially helpful tool used in the diagnosis of ocular epithelial neoplasms of horses. Financial support: Supported by the University of Georgia Companion Animal fund. Commercial interest: None.

ABSTRACT No.: 64

Observation of tear overflow and evaluation of effect of the modified medial canthoplasty (MMC) on epiphora eyes of dogs

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A. Saito,* K. Takehana,† A. Tuduki* and T. Kotani‡

*Triangle Animal Hospital, Department of Veterinary Anatomy, School of Veterinary Medicine, Rakuno Gakuen University, Department of Veterinary Surgery, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan

Purpose: Behavior of tear fluid of epiphora eyes was observed and recorded on DVD. The lacrimal flow observed is described in this abstract. Further, to evaluate the effect of MMC, the profile of the tear overflow observed here was compared with that of the case of an epiphora dog treated by MMC. Materials and Methods: Two dogs with an abnormal medial canthus that was considered to cause epiphora were included in the study. A 3-year-old, female Miniature Dachshund with severe epiphora was used for the tear overflow recording. A slit-lamp microscope (BQ, Haag Streit) was employed for the observation of tear flow and a video image was recorded on a digital filing system (VK-2, Kowa) and DVD recorder (DMR-E30, Panasonic). After observation of the ocular surface and medial canthus, 1 µL of fluorescein/Rose bengal staining solution was instilled and lacrimal flow was observed and recorded under free blinking. A 5-month-old, female Bolognese that had suffered from severe epiphora from 3 months of age was given MMC treatment. The shape of the medial canthus, tear meniscus and facial hair staining were observed by slit-lamp microscopy pre- and post MMC. An open lacrimal punctum was observed in both eyes in both cases. The lacrimal levels of the two dogs were as follows: Miniature Dachshund, OD: PRT: 36 mm/15 s, STT-1: 23 mm/60 s, OS: PRT: 31 mm/15 s, STT-1: 24 mm/60 s; Bolognese, OD: PRT: 28 mm/15 s, STT-1: 21 mm/60 s, OS: PRT: 30 mm/15 s, STT-1: 25 mm/60 s. Results: Severe tear overflow from the medial canthus was observed in the Miniature Dachshund with epiphora. Tear overflow induced by the hair around the medial canthus of the lower eyelid was observed under higher magnification. Observation of the video image showed the behavior of tear overflow. In the recorded image, tear fluid was moved to the medial canthus and flowed out by blinking. Tear meniscus was observed clearly at the center of the lower eyelid, while it was not observed around the medial canthus. However, preoperative observation of the dog treated by MMC revealed tear overflow from the medial canthus and hair-mediated overflow at the medial canthus of the lower eyelid. Tear meniscus was observed clearly at the center of the lower eyelid, while it was not observed around the medial canthus in a similar manner as that of the Miniature Dachshund. After MMC, abnormal shape of the medial canthus was corrected and the hair that had induced the tear overflow was not observed. Tear meniscus was observed over the whole lower eyelid. Four months after the treatment of MMC a remarkable improvement of hair staining was observed. Discussion: Recorded images of the eyes of a Miniature Dachshund showed the mode of tear overflow by blinking. In the case of the Bolognese, the abnormal shape of the medial canthus was corrected by MMC treatment and epiphora was remarkably improved. Similar abnormality of the medial canthus was observed in both cases and it was suggested that such a characteristic abnormality may exist in dogs with epiphora. MMC was found to be an effective treatment as a corrective method for the treatment of epiphora. However, a variety of causes of epiphora are suggested, and the close observation of the medial canthus by slit-lamp microscopy and of tear behavior by tear staining is therefore important to determine whether MMC is applicable to each case. Commercial interest: None.

ABSTRACT No.: 65

Topical effect of cyclosporine A, acetylcysteine, ciprofloxacin, EDTA, doxycycline, polysulfated glycoaminoglycan, autoserum and artifical tears on matrix metalloproteinases 2 and 9 activity in the tear film of the normal dog

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S. Couture, M. Carrier, M. Doucet and M. Moreau

Department of Small Animal Clinical Sciences, Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada

Purpose: The normal tear film contains proteolytic enzymes better known as matrix metalloproteinases (MMPs). These are mainly produced by keratocytes, inflammatory cells and infectious organisms and are essential to the cellular remodelling process. However, in some cases of ulcerative keratitis, the physiologic mechanism of feedback control is affected. A marked increase in enzymes that cause excessive degradation of the macromolecules of the corneal extracellular matrix is then observed, finally creating a lesion commonly called a melting ulcer. In such cases, an effective reduction of enzyme activity is essential to optimize corneal healing. The gelatinases (MMP2 and MMP9) are particularly important in the progression of melting corneal ulcer because they are the only MMPs able to digest the basal membrane of the corneal epithelium, thus exposing the corneal stroma to other collagenase. Antiproteolytic agents have thus been highly recommended in these cases, but little information is available on the efficacy of medication to control this enzyme activity in vivo. The goal of this study was to evaluate the effect of various agents topically administered on the gelatinase activity present in the tear film of normal dogs. Methods: Following a complete ophthalmic examination eight Beagles were randomly selected. Each animal alternatively received one of the following agents: cyclosporine A 1%, artificial tears (control), acetylcysteine 10%, ciprofloxacin 0.3%, EDTA 1%, doxycycline 10 µg/mL, and polysulfated glycoaminoglycan 5% and autoserum during a 48-h period. The study was performed over a period of 8 weeks to allow each dog to receive each medication. Tear samples were collected with micro capillary pipettes following corneal surface irrigation of each eye with sterile saline at four different times (24 h before first treatment, 24 h after the treatment started, 1 h after the last treatment, and 6 h after the last treatment). The samples of both eyes were pooled for each animal and each collection time and placed in PCR tubes to be stored at −70 °C for further evaluation. An average of 85 µL was obtained from each eye. Gelatinase activity was evaluated by optical density obtained from the Gelatinase Activity Assay Kit (Chemicon International). Optical density (measured with a spectrophotometer) of each sample was compared to the optical density of a positive MMP2 control. From this ratio a semiquantitative measure of gelatinase activity was obtained. Results: Levels of total gelatinase activity were significantly lower than the control level when the eyes were treated with cyclosporine A, EDTA or ciprofloxaxin. However, this reduction was not significant compared to the reduction observed with the artificial tears. Conclusions: Though most of the agents employed in this study are known to inhibit MMP activity in vitro, in our study none of the agents significantly reduced the gelatinase activity compared to the artificial tears. Local application of an ophthalmic agent creates a dilution of the MMPs. In our study, this effect seemed to cause the reduction of activity more than the antiproteolytic action of each agent. Commercial interest: None.

ABSTRACT No.: 66

Diagnosis of equine fungal keratitis using polymerase chain reaction

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A. Neary,* P. A. Moore,† M. Chandler,† C. B. Mosunic,† K. P. Carmichael,‡ Ursula Dietrich† and Susan Sanchez*,§

*College of Veterinary Medicine, University of Georgia, Athens, GA, Athens Diagnostic Laboratory, Department of Small Animal Medicine and Surgery, §Department of Pathology, Department of Medical Microbiology, Athens, GA, USA

Purpose: To evaluate polymerase chain reaction (PCR) in the diagnosis of equine fungal keratitis. Methods: Corneal samples for PCR were obtained from equine cases evaluated for fungal keratitis, ulcerative keratitis and stromal abscess formation. Corneal samples were frozen in 1 mL of saline at −70 °C. Standard PCR was carried out using universal fungal primers and gel electrophoresis. The PCR results were compared to cytology, fungal culture and histopathology for the presence of fungal organisms. Results:Candida albicans serial dilutions determined 10-genome sensitivity for both universal fungal and C. albicans specific primers. Fungal PCR (n = 22), corneal cytology (n = 22), fungal cultures (n = 22) and histopathology (n = 16) were performed in 22 cases of equine keratitis. PCR results were positive for universal fungal primers in 50% of cases (n = 11/22). Corneal cytology was positive for fungal hyphae in 59.1% (n = 13/22) and fungal cultures were positive in 50% (n = 11/22) of cases. Histopathology confirmed the presence of fungi in 43.75% (n = 7/16) of cases. Of the 14 samples positive for fungal organisms by cytology, fungal culture or histopathology, 42.9% (n = 6/14) were positive by PCR. Of the eight samples negative for fungal organisms by cytology, fungal culture and histopathology, 62.5% (n = 5/8) were positive by PCR. Of these five cases, four were clinically agreeable with fungal keratitis (stromal abscess (n = 3) and deep progressive corneal ulceration (n = 1)). Conclusion: PCR is a fast and sensitive diagnostic tool to aid in the clinical diagnosis of equine fungal keratitis. Financial support: Supported by the Veterinary Medical Experiment Station, The University of Georgia. Commercial interest: None.

ABSTRACT No.: 67

Use of Acell Vet® xenograft in feline corneal sequestrum

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Michael M. S. Zigler

Eyevet Consulting Services, Oakville, Ontario, Canada

Purpose: To evaluate ACell Vet® porcine bladder derived extracellular matrix (ECM) in the surgical treatment of feline corneal sequestrum. Methods: Four cats with corneal sequestrum were evaluated and treated surgically to remove deep corneal sequestra. Following deep keratectomy to remove the corneal sequestrum, the ACell Vet® xenograft material was rehydrated, cut to size and sutured into the resulting surgical defect with 7–0 Vicryl. In each case, postoperative management included topical antibiotic and a viscous lubricant. Patients were rechecked at frequent intervals to follow and evaluate the healing response. Results: The material was easily handled and well tolerated, and the resulting postoperative clarity of the cornea was deemed to be better than conjunctival pedicle grafting or grafting with BioSIS-T®. Conclusions: ACell Vet® produced superior results in the treatment of feline corneal sequestrum compared to other methods of grafting. Commercial interest: None.

ABSTRACT No.: 68

Ocular canine ehrlichiosis:. a 3-year course

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M. Leiva, T. Peña and C. Naranjo

Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, UAB, Barcelona, Spain

Purpose: Ocular lesions are a well-recognized complication of systemic ehrlichiosis in the dog and have been reported experimentally as 50%. The purpose of this study was to characterize ehrlichiosis ocular signs, to determine the prevalence of those signs, and to describe concurrent diseases, response to treatment and ocular prognosis. Methods: Canine medical records of the Universitat Autònoma de Barcelona Veterinary Teaching Hospital, dating from January 2000 to December 2002, were retrospectively reviewed for suitable cases. Criteria for inclusion in the study were: positive serodiagnostic (ELISA) for Ehrlichia canis and available information about the clinical outcome. Results: Fifty-six cases of dogs with positive serologic titles for Ehrlichia canis were identified during the study period. Forty-six were included in the study based on the above criteria. Eighteen dog breeds were represented: 20 large breeds (43.4%), 23 (50%) medium breeds and 3 (6.6%) small breeds, of these 18 (39.1%) were female and 28 (60.9%) male. Systemic clinical signs included; epistaxis or other bleeding tendencies in 11 cases (23.9%), recurrent lameness in nine cases (16%), neurologic signs in four cases (7.2%) and dermatologic alterations in two cases (4.3%). Other clinical signs were depression, anorexia, fever, lymphadenopathy, vomiting and diarrhea. Seventeen cases (30.5%) were referred for bilateral ocular signs, of which 11 patients (65%) presented only ophthalmic signs. Panuveitis with exudative retinal detachment was present in 11 cases (64.7%), exudative anterior uveitis was diagnosed in five cases (29.4%), and optic neuritis in one case (9.09%). Of the patients with ocular involvement, five (29.4%) showed hyphema and/or retinal hemorrhages. All the patients with ocular signs and serology titer (ELISA) higher than 1 : 320 had panuveitis, while lower titers were associated with anterior uveitis. The most frequent concurrent disease was leishmaniosis in seven cases (15.2%), of which only one had ocular signs. Oral doxycycline (5 mg/kg twice a day for 21 days) was used for systemic treatment. Imidocarb dipropionate or a longer doxycycline treatment was used for treating severe, chronic or refractory cases. Systemic oral prednisone (0.5 mg/kg twice a day) was added in 11 cases of ocular ehrlichiosis. Ocular outcome was poor for five eyes (n = 34); major complications were retinal atrophy and phthisis bulbi. Conclusions: In this study, ocular signs were present in 30.5% of canine ehrlichiosis cases. The most frequent ocular sign was bilateral panuveitis. Higher serology titers were related to panuveitis and retinal detachment. Systemic prednisone added to doxycycline seems to be a good therapeutic choice for ocular ehrlichiosis. The most frequent concurrent disease was leishmaniosis. An accurate and rapid diagnosis and treatment determine a better visual prognosis. Commercial interest: None.

ABSTRACT No.: 69

Application of argon laser iridotomy (LI) and gonioplasty (LG) in dogs

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O. Igarashi,* M. Iijima,* K. Hase,* S. Sakamoto,* T. Chuma,* R. Igarashi* and T. Kotani†

*Kushiro Animal Hospital,Kushiro, Hokkaido, Japan, Department of Veterinary Surgery, ‡School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan

Purpose: Surgical procedures such as valve implantation and trabeculectomy, as well as other methods, have been applied for glaucoma in dogs that still have vision. In human ophthalmology, the first choice of treatment for mild to moderate elevation of intraocular pressure (IOP) in general is LI and LG. However, postoperative long-term endothelial cell disturbance after surgery has been reported. In this abstract we report on LI and LG performed in three canine cases. Methods: Three dogs (five eyes) were diagnosed with closed angle glaucoma with pupillary block, and normal vision was confirmed by ERG. Average age was 7.2 ± 2.3 years (mean ± S.D), body weight was 8.9 ± 3.6 kg, and IOP was 33.0 ± 3.2 mmHg. Both 2% dorzolamide twice a day and 0.005% latanoprost once a day were administered. One hour before laser therapy, topical 2% pilocarpine and 1% apraclonidine were given. Under general anesthesia, laser irradiation was performed using Pc-EDO (HGM Co. Ltd., 450–530 nm, central wave length: 514.5 nm), by slit-lamp method applying Abraham lenses. Laser settings were divided in two steps; spot size, laser power, laser duration and laser frequency were 200–500 and 50 µm, 200 mW and 1000 mW, 0.2 and 0.02 s, 4–8 and 150–240 times, respectively. Iris hole and excision were confirmed by slit-lamp. Laser gonioplasty (peripheral iridoplasty) was also applied for two cases. Spot size was 200 µm, laser power was 300 mW, duration was 0.5 s and the laser was used to irradiate 14–16 spots on the nasal half circumference. Results: LI mean duration required was 17.5 ± 5.6 min, and mean elevation of mean IOP of 5.2 ± 4.1 mmHg was observed after 12 h, but in 2 days it reduced below preoperative pressure. Mean IOP was reduced to 9.7 ± 4.8 mmHg 5 days postoperatively. Overall, mean reduction of IOP was 20.3 ± 5.6 mmHg. Complication of endothelial disturbance and any other condition were not observed. Conclusions: Although reports about long-term endothelial disturbance after LI and LG operations by argon and YAG laser are limited, its cause is not yet proved. Mild reversible hyperocular pressure was observed, but decreased and any other complications such as iritis, posterior synechia and cataract were not observed. Given these results, LI and LG by argon laser may be effective treatments for glaucoma in dogs. Commercial interest: None.

ABSTRACT No.: 70

Ocular histopathology of canine leishmaniosis

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M. T. Peña,* G. Klauss,† D. Fondevila,* M. Leiva,* C. Naranjo,* X. Roura,* M. Davidson‡ and R. R. Dubielzig§

*Departament de Medicina i Cirurgia Animals & Hospital Clinic Veterinari. Facultat de Veterinaria, Universitat Autónoma de Barcelona, Spain, Veterinary Medical Teaching Hospital, University of Missouri, MO, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, NC, §School of Veterinary Medicine, University of Wisconsin, WI, USA

Purpose: Relative prevalence of ocular signs in dogs with systemic leishmaniosis has been reported in four clinical studies to vary between 16 and 80%. In a retrospective study we found ocular and/or periocular lesions in 24.4% of cases. Previous histopathology reports are from individual cases or series of less than nine cases. The purpose of this study was to characterize and localize the type of ocular lesions and the frequency of lesions in dogs with leishmaniosis. Methods: Dogs that had died or were euthanized and had a diagnosis of leishmaniosis were included in the study. The diagnosis had to be confirmed by at least two of three means: cytologic or histopathologic identification of the organism, serologic results and/or PCR testing. All dogs were patients of the Teaching Veterinary Hospital at the UAB. Both eyes were enucleated and harvested in formalin. Slides stained with hematoxylin and eosin, and a Leishmania-specific immunoperoxidase test where evaluated for each eye. Seventy eyes from 35 dogs were included in the study over a 2-year period. Results: Twenty of 36 dogs had renal failure and ocular signs were only present in three animals. Six of 36 dogs had received treatment with allopurinol, 17 received allopurinol and glucantime, 13 of which also received systemic corticosteroids. Only 13 dogs did not receive antileishmania treatment. Treatment was given for a mean of 13.3 months (5 days−3 years). Perivascular lymphoplasmacytic infiltrate with some macrophages was found in 34/72 eyes (47.2%). Of these, Leishmania organisms were identified directly in five cases (10 eyes, 13.8%) by H&E, and in 23 eyes by immunoperoxidase (IP) test (31.9%). Leishmania parasites were also identified by IP test in three other eyes without inflammation (4.2%). Inflammation was present without parasite identification in nine eyes (12.8%). Unilateral lesions were present in seven (34% of affected) cases; of those, the parasite was identified in three eyes. Only five of the 13 dogs receiving systemic corticosteroids had cellular infiltrate and in four of them parasite identification was possible. Parasites have been found in the conjunctiva, sclera, cornea, ciliary body and choroid. Fifteen cases and 35 eyes had no inflammation and no parasites (41.6% of cases, 48.6% of eyes). Discussion: The presence of the parasite is higher than the number of eyes with clinical signs (3/15). Leishmania organism can be found at different ocular structures with and without inflammation. Degree of inflammation and the presence or absence of the parasite can vary depending on the disease's course and can also be modified by the disease's treatment. Commercial interest: None.

ABSTRACT No.: 71

Necrotizing scleritis due to leishmania: case report

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C. Naranjo, M. Leiva and T. Peña

Departament de Medicina i Cirurgia Animals, Hospital Clínic Veterinari, Facultat de Veterinària, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain

Purpose: To describe a case of ocular leishmaniosis in which a scleral granuloma evolved to necrotizing scleritis. Methods: A 5-year-old, female English Cocker Spaniel was referred to the UAB Veterinary Teaching Hospital for evaluation of a scleral mass in the left eye, present for 4 months. The referring veterinarian treated the condition with topical and systemic corticosteroids and topical antibiotic but the lesion reappeared when the treatment was discontinued. The general physical examination was unremarkable. Ophthalmic examination revealed a deforming pinkish mass in the dorso-temporal sclera of the left eye (OS) with mild stromal corneal edema. Results: Scleral biopsy revealed granulomatous inflammation and Leishmania infantum organisms evidenced by immunoperoxidase staining. Serum titers against Leishmania spp. were negative. CBC showed mild thrombocytopenia and biochemical parameters were within reference range. Treatment with oral allopurinol (10 mg/kg BID) and topical dexamethasone was started. Three months later the dog presented with a mass deforming the contour of the left globe. Anterior synechia and a blood clot at the 3 o’clock position were also detected. Oral prednisone (0.5 mg/kg BID) was added and the nodule reduced in size within 2 weeks. Despite this improvement, 2 weeks later severe scleral thinning was present. A sclerectomy was performed and a frozen canine scleral graft was placed. Postoperative complications included hyphema and ocular hypertension that were resolved with an anterior chamber lavage and administration of 10% mannitol (1 g/kg, IV), respectively. The animal was discharged with topical dexamethasone, tropicamide, chloramphenicol, timolol and dorzolamide, and systemic prednisone (0.5 mg/kg BID) and cephalexin (20 mg/kg BID). Five days later antiglaucoma treatment was discontinued due to normalization of intraocular pressure. Histopathology of the sclera showed necrotizing mononuclear scleritis but no organisms were detected with immunoperoxidase staining. Serum titers against L.infantum were negative. The medication was slowly tapered as clinical signs resolved. Conclusions: Granuloma formation is a typical sign of leishmaniasis. Within ocular structures they have been reported in eyelids, conjunctiva, nictitating membrane, cornea, limbus and iris, but to the authors’ knowledge they have never been described in the sclera. The differential diagnoses for the initial lesion in this case include nodular granulomatous episcleritis, nodular fasciitis, neoplasia, or granuloma caused by parasites. In the Mediterranean basin leishmaniosis must also be included. In this particular case, in which systemic signs were absent, histopathology and immunohistochemistry were essential to correctly diagnose and treat the animal. Some authors postulate these localized lesions may correspond with the inoculation site. Commercial interest: None.

ABSTRACT No.: 72

A PCR-based assay for the diagnosis of equine fungal keratitis

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V. J. Kuonen, C. M. H. Colitz, D. A. Wilkie, A. J. Gemensky Metzler, I. D. Bras and H. Koyama

Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, USA

Purpose: To develop a rapid and accurate method of diagnosing equine fungal keratitis by using polymerase chain reaction (PCR). Methods: Corneal scrapings or biopsies were collected from 17 horses presented for corneal disease from July 2002 to June 2003. Routine phenol-chloroform DNA extraction was performed and quantitated by spectrophotometry. PCR was performed using universal fungal, aspergillus-specific, and fusarium-specific primers. The equine housekeeping gene, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), was amplified to control for the DNA extraction process. All PCR experiments included positive controls using DNA extracted from aspergillus and fusarium cultures. A negative control without DNA was included in all PCR experiments. Other negative controls included DNA extracted from four lamellar keratectomies and seven corneal scrapings from euthanized horses with normal corneas. The horses were euthanized for reasons other than those of this study. The PCR products were visualized on 1.2% agarose gels stained with ethidium bromide. PCR products were purified using the QIAquick PCR purification kit and sequenced. Sequences were compared with published sequences in GenBank. Corneal cytology (n = 16), fungal culture (n = 14), and histopathology (n = 6) were also performed. Sensitivity, specificity, positive predictive value and negative predictive value of corneal cytology, fungal culture and histopathology were evaluated by use of results of PCR assay as true positives and true negatives. Results: Six samples (35.3%) tested positive using the PCR assay for fungal keratitis, five were positive for aspergillus (83.3%) and three were positive for fusarium (50%) (two samples were positive for both). Sequencing confirmed the PCR results. Five (31.3%) had hyphae on cytology. Three (21.4%) were positive for fungal culture. Two (33.3%) had hyphae on histopathology. All samples with a positive fungal culture or a positive histopathologic examination had a positive PCR assay confirmed by sequencing. Compared with PCR assay, corneal cytology had a sensitivity of 50%, specificity of 90%, positive predictive value of 75%, and negative predictive value of 75%. Fungal culture had a sensitivity of 50%, specificity of 100%, positive predictive value of 100% and negative predictive value of 72.7%. All values for histopathology were 100%. Conclusions: Results of the present study suggest that this PCR assay is a rapid and effective method for diagnosing aspergillus- or fusarium-associated fungal keratitis. Financial support: Supported by Ohio Animal Health Grant. Commercial interest: None.

ABSTRACT No.: 73

Electroretinographic and demographic findings of dogs presented for cataract surgery (1998–2003)

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A. J. Gemensky Metzler, K. A. Norris, D. A. Wilkie, C. M. H. Colitz, I. D. Bras and R. Elliott

College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Purpose: To establish normal values, by breed, for dogs with cataracts undergoing electroretinography and to determine whether differences exist between diabetic and nondiabetic dogs. Methods: An electroretinogram (ERG) was performed on 306 dogs between 1998 and 2003. Ocular ultrasonography was performed prior to ERG in all cataract patients. Dogs without cataracts or diagnosed with PRA (n = 25) and eyes without recordable b-waves were excluded. The pupils were pharmacologically dilated prior to the ERG. The ERG was performed under general anesthesia using a Cadwell Ganzfeld amplifier and Nihon Kohden Evoked Potential Measuring System. An eyelid speculum and conjunctival stay suture were placed to ensure forward globe placement. ERG Jets were applied to the cornea, and platinum reference electrodes were placed subdermally posterior to the lateral canthal ligament and the ground electrode over the occipital crest. For each dog, a b-wave value for each eye was recorded under photopic conditions followed by 5 min of dark adaptation. Using a neutral density filter, the scotopic ERG was recorded. Dogs were divided into diabetic and nondiabetic groups for statistical comparison and were separated by breed to establish mean photopic and scotopic values. The mean photopic and scotopic values for OD and OS for each group were averaged together when not significantly different (Prism software). Results: An ERG was recorded for 50 breeds and 281 dogs for which the average age was 7.69 years (range: 0.75–18 years) and the mean photopic values were 19.5 millivolts (mv) OS, 18.1 mv OD and mean scotopic values were 104.3 mv OS and 98.72 mv OD. Diabetes mellitus (DM) was the cause of cataracts in 108 dogs (38.4%) and 173 dogs were not diabetic. The average age, mean photopic and scotopic values for diabetic and nondiabetic dogs, respectively, were 8.5 years, 18.1 mv, 104.2 mv and 8.1 years, 19.3 mv, 97.4 mv. The most common breeds presented for cataract surgery, number evaluated, number with DM, average age, and mean photopic and scotopic values, respectively, were as follows: mixed breed (54; 23; 9.2 years; 18.6, 109), Miniature Schnauzer (26; 16; 7.3 years; 17, 76), Labrador Retriever (26; 12; 6 years; 21.5, 126.8), Miniature and Toy Poodle (25; 6; 9.2 years; 15.1, 66.6), Bichon Frise (14; 2; 5.3 years; 17.8, 85.4), Yorkshire Terrier (12; 6; 8.9 years; 14.7, 67.2), Siberian Husky (10; 2; 4.1 years; 34.2, 195), Lhasa Apso (9; 5; 10 years; 20.3, 88.2), Boston Terrier (6; 0; 8.5 years; 26.9, 127.7), Golden Retriever (6; 0; 6.8 years; 17.8, 75.6), Shih Tzu (6; 1; 9.5 years; 19.7, 130), Cairn Terrier (5; 5; 9.4 years; 18.7, 145.8), Miniature Pinscher (5; 4; 7.8 years; 13.6, 49). Conclusions: The ERG values were not significantly different between diabetic and nondiabetic dogs. Diabetic cataracts were most common in the mixed breed, Miniature Schnauzer, Cairn and Yorkshire Terriers, Miniature Pinscher and Labrador Retriever. Commercial interest: None.

ABSTRACT No.: 74

Evaluation of advanced glycation end-products in diabetic and inherited canine cataracts

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I. D. Bras, D. A. Wilkie, A. J. Gemensky, V. J. Kuonen, H. Koyama and C. M. H. Colitz

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Purpose: To immunolocalize cell-associated receptors of advanced glycation end-products (RAGE), and to determine whether there is a correlation between RAGE, DNA damage and cell proliferation in canine diabetic and inherited cataracts. Methods: Prior to phacoemulsification 32 anterior lens capsules from 22 dogs were collected, and either frozen at −70 °C or formalin fixed and paraffin embedded. Eleven dogs were diabetic (17 eyes) and 11 dogs had inherited cataracts (16 eyes). Standard avidin-biotin complex immunohistochemical staining was performed using antibodies against RAGE, gadd45 and PCNA. Negative control slides omitted the primary antibody. Standard Western blot analysis was performed on six anterior lens capsules (three diabetic and three inherited) using the same antibodies used in the immunohistochemical staining. Results: All capsules demonstrated up-regulation of RAGE and gadd45. Stain intensity varied among different populations of lens epithelial cells (LEC). The LEC monolayer adjacent to the anterior capsule revealed greater stain uptake than newer proliferating areas associated with pseudometaplasia and fibrotic plaque formation. Up-regulation of RAGE was most evident in the cytoplasm of the LEC. Up-regulation of gadd45 was mainly perinuclear and variably intranuclear. No difference was observed between diabetic and inherited specimens, stage or duration of cataract, or presence or absence of lens-induced uveitis at the time of sample collection. PCNA was up-regulated in diabetic capsules and all specimens from mature and hypermature inherited cataracts with variable stain characteristics. Stain uptake was observed mainly in the nucleus and perinuclear area of the proliferating LECs that surrounded fibrotic plaques. PCNA was down-regulated in 42% of the inherited cataracts, but only in specimens from immature cataracts. Western blot analysis was consistent with these findings. Conclusion: Up-regulation of RAGE indicates the presence of AGE products in canine inherited and diabetic cataracts. There was no correlation between expression of RAGE in inherited and diabetic cataracts in either the Western blot or immunohistochemistry. LEC exposed to AGEs up-regulate RAGE and transdifferentiate into the pseudofibroblastic phenotype seen in cataract and after-cataract. Staining characteristics for RAGE and gadd45 suggest that up-regulation of RAGE in LECs exposed to AGEs may contribute to pseudo-fibroblastic changes and altered cellular growth, establishing a correlation between oxidative stress, RAGE and DNA damage. Proliferation of LEC in diabetic cataracts and advanced stages of inherited cataracts are supported by PCNA up-regulation. Financial support: Supported by ACVO Resident's Research Grant. Commercial interest: None.

ABSTRACT No.: 75

Estrogen receptor-alpha expression in canine cataracts

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C. M. H. Colitz and C. A. Barden

College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Purpose: Estrogen receptor alpha (ES) has been previously immunolocalized to the human lens epithelium (LEC) and estradiol hormones have been shown to protect human LEC from H2O2 oxidative stress through unknown mechanisms. We were interested in evaluating ES and its potential role in canine LEC and comparing it to cataractous LEC as preliminary work in our laboratory has shown that there is an increased interaction of telomerase (TERT) with estrogen response element (ERE) in cataracts when compared to normal LEC, regardless of the sex of the dog. ERE is a DNA sequence in the promoter region of various genes, including TERT, that binds ER. Methods: Transcription factor array and Western blot analysis were performed. Briefly, the TranSignal™ TF–TF Interaction Array I (Panomics) was used to screen for 54 TF–protein interactions. Nuclear protein was extracted from two normal (one young, one old) and three cataractous (one diabetic, two inherited) canine anterior lens capsules and quantitated by standard Bradford assay. Each sample was incubated with the probes provided, allowing the TF cis-elements to bind to the protein. Immunoprecipitation was performed with anti TERT antibody in order to pull out the TF cis-elements interacting with TERT. This complex was eluted and hybridized to the TranSignal Array membrane (one membrane per sample). Direct comparisons of spot intensity were made between the five sample blots to identify differences in TF interactions using Gel-Pro software. Standard Western blot analysis was performed using 2.5 µg protein per sample (same samples as in array) with anti-ER antibody (Santa Cruz, 1 : 2000). Results: The transcription factor array confirmed an increased interaction between ERE and TERT in diabetic and inherited cataractous LEC, decreased interaction in the young normal LEC and no interaction in the old normal LEC. We also found increased expression of ER in the LEC of both diabetic and inherited canine cataracts when compared to normal old canine LEC. This difference was not sex specific. Conclusions: It is known that postmenopausal women have an increased incidence of cataracts, and a transgenic mouse model that had repressed ER had inducible cataracts in both males and females. Estrogen hormone can also directly increase TERT transcription and telomerase activity possibly leading to protection of the LEC from oxidative stress. Based on this and the data presented in this abstract, we hypothesize that ER may play a direct or indirect role in cataractogenesis, regardless of sex, possibly via telomerase. Financial support: Supported by NIH NEI 00414–05. Commercial interest: None.

ABSTRACT No.: 76

Use of hydroxyapatite orbital implants with cosmetic corneoscleral prostheses after enucleation in eight horses

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B. C. Gilger,* T. Miller Michau,* S. Pizzirani,* E. Smith,* D. A. Wilkie† and L. Johnston‡

*Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, Department of Clinical Sciences, The Ohio State University, Columbus, OH, E. R. Johnston and Associates, Hillsborough, NC, USA

Purpose: Use of hydroxyapatite (HA) implants in humans after enucleation has been shown to have less long-term complications and allow a more cosmetic result. However, the most common complication of HA implants in humans is infection of the porous implant and orbit prior to vascularization and fibrous ingrowth into the implant. The purpose of this study was to review the complications and outcome of the use of HA orbital implants after enucleation in eight horses. Methods: Horses that received an HA implant after enucleation for the purpose of supporting a corneo-scleral cosmetic prosthesis were included in this study. Seven horses had surgery at NCSU and one had surgery performed at OSU. All horses received a 40-mm HA orbital sphere implanted in a homologous cadaver scleral shell. After the enucleation, the extraocular muscles were sutured into the scleral shell and the Tenon's capsule and conjunctiva were closed over the implant. A temporary conformer was inserted into the conjunctival fornix under the eyelids to prevent contracture during healing and prior to final cosmetic shell fitting. Topical and systemic antibiotics were used for 2 weeks after surgery and oral NSAIDS were used as needed to reduce swelling and discomfort. Approximately 30 days after surgery, the ocularist (LJ) performed an orbital impression and the final corneo-scleral implant was made and fitted. Results: Enucleations were performed because of chronic uveitis (3), acute or chronic corneal perforations (3), endophthalmitis following repair of corneal laceration (1), and for chronic phthisis bulbi (1). Complications occurred in four of eight horses, including conjunctival epithelial dehiscence (3), upper eyelid entropion (2), and orbital infection (2). Complications were managed successfully in six of eight horses, but two horses with an orbital infection required implant removal. Complications were more common in eyes with ocular surface inflammation or infection (i.e. fungal keratitis, perforation, chronic phthisis bulbi with secondary orbital infection). Discussion: Despite a high rate of complications (50%), excellent cosmetic results were obtained in six of eight horses. Complications may develop from poorly fitted conformers, uncontrolled ocular surface infection or inflammation, or from inability to adequately manage and clean the conformer and orbit after surgery. The most severe complication was implant infection that occurred in two horses. Despite frequent orbital irrigation and systemic antibiotics, both required implant removal. Careful patient selection (i.e. temperament), owner education, control of infections prior to surgery, and frequent follow-up will help minimize complications. Commercial interest: None.

ABSTRACT No.: 77

Transplantation of cultivated canine corneal epithelium using temperature-responsive culture dishes

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M. Endo,* I. Endo,† K. Nishida,‡ M. Yamato,§ A. Masuda,¶ Y. Tano‡ and T. Okano§

*Kunitachi Veterinary Ophthalmic Service, Kunitachi Animal Hospital, Department of Ophthalmology, Osaka University Medical School, §Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, CellSeed Inc., Japan

Purpose:  In order to reconstruct the ocular surfaces with tissue engineering, autologous canine corneal epithelial cell sheets were prepared on temperature-responsive culture dishes. Harvested cell sheets were examined histologically and subjected to autotransplantation. Methods: Damaged ocular surfaces were created with n-heptanol and conjunctival excision including the limbus. The limbal stem cell deficiency (LSCD) model presented conjunctival epithelialization with vascularization during the initial 3 weeks. Autologous corneal epithelial cell sheets were prepared from limbal stem cells cocultured with 3T3 fibroblasts on temperature-responsive culture dishes. Cell sheets were harvested by reducing temperature without proteolytic treatment, examined histologically and subjected to autotransplantation. Results: Under a transmission electron microscopy as well as a light microscope, harvested corneal epithelial cell sheets showed multiple cell layers and the apical and basal structures were similar in appearance to those of normal corneal epithelium. Positive expression of keratin 3 and 12, and p63 was observed. Damaged corneal surfaces of LSCD were successfully treated with autotransplantation of autologous cultivated corneal epithelial cell sheets without suture. Treated corneas became transparent and repelled fluorescein dye even immediately after the surgery, although they showed minimal inflammation. Even after a 6-month follow-up, regenerated corneas maintained transparency. Conclusions: Reconstructed corneal surfaces of canine LSCD models were clear and smooth without signs of rejection. Immunohistochemical observation suggested that harvested cell sheets contained corneal epithelial stem cells. The present method utilizing temperature-responsive culture dishes should be proved for reconstruction of severe ocular surface disorders. Commercial and financial interest in product: None.

ABSTRACT No.: 78

Cultivation of canine corneal limbal epithelial cells on thermo-responsive culture dishes without murine 3T3 feeder cells

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A. Masuda,* S. Oguri,* M. Endo,† I. Endo,‡ K. Nishida,§ M. Yamato,¶ Y. Tano§ and T. Okano¶

*Kunitachi Veterinary Ophthalmic Service, Kunitachi Animal Hospital, Department of Ophthalmology, Osaka University Medical School, §Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, CellSeed Inc, Japan

Purpose: In order to reduce the risk of unknown infectious problems caused by exogenous biomaterials, canine corneal limbal epithelial cell cultures were performed in the absence of murine 3T3 feeder cells. Methods: Fragments of corneal limbal tissue were dissected from canine eyes. The epithelial cell layers were separated from stroma with dispase, dissociated into single cells with trypsin, and inoculated on the thermo-responsive culture dishes with or without feeder cells. Growing appearance, cell morphology, expression of differentiation markers, presence of colony forming cells, and ability of thermo-responsive detachment of the epithelial cell sheets were examined. Results: We successfully obtained confluent corneal epithelial cell sheets from 1 × 104 corneal limbal epithelial cells without any help of feeder cells, though the cell growth was a little slower than that with feeder cells. The cell morphology was quite similar to that with feeder cells. The cell sheets contained colony forming cells at a similar amount to those with feeder cells, and were positively stained with an anticytokeratin 3 antibody, which is specific to corneal epithelial cells, and antip63 antibody, which stains basal limbal epithelial cells. All the cultured cells were negative to an anticytokeratin 19 antibody, which stains conjunctival epithelia but not corneal epithelia. The sheets were strong enough to be detached from the culture surfaces after incubation at 20 °C for 30 min. Conclusions: We have successfully obtained regenerated corneal epithelial cell sheets from small corneal limbal fragments without the help of feeder cells. As the present culture method does not contain any cells of xenogeneic origin, the obtained cell sheets should be utilized more safely for regeneration therapy for canine corneal disorders. Commercial and financial interest in product: None.

ABSTRACT No.: 79

Evaluation of cidofovir on FHV-1 infected and noninfected feline corneal epithelial cells

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L. S. Sandmeyer, C. B. Keller and D. Beinzle

Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Purpose: To evaluate the safety and efficacy of cidofovir against FHV-1 infection of cultured feline corneal epithelial cells at concentrations similar to that which may be used clinically as topical antiviral therapy. Methods: Feline corneal epithelial (FCE) cells were cultured in supplemented hormonal epithelial medium (SHEM). To evaluate for cidofovir cytotoxicity, FCE cells were incubated with cidofovir concentrations ranging from 0.5% to 0.00005% (n= 8 cultures per concentration). FCE cell morphology was examined using phase-contrast microscopy at 24 and 48 h. At 48 h, viable cell counts and per cent viabilities were calculated using trypan blue exclusion. In a separate assay, FHV-1-infected and noninfected FCE cell cultures were incubated in the presence or absence of 0.2% and 0.5% cidofovir for 72 h (n = 36 cultures per group). Viral cytopathic effect (CPE) was evaluated semiquantitatively using phase-contrast microscopy every 24 h. Viral titres of culture supernatant were determined by the TCID50 method on CRFK cells for all infected cell cultures at 72 h. Viable cell counts and per cent viabilities of the noninfected FCE cell cultures were calculated using trypan blue exclusion at 72 h. Results: Cidofovir concentrations of 0.5%, 0.05% and 0.005% reduced mean viable cell counts compared to controls and to lower concentrations of the drug (P = 0.013). Concentrations of 0.5% and 0.2% cidofovir reduced the mean viable cell count by 1.71 and 1.65 times, respectively, compared to controls (P < 0.0001). Cidofovir at 0.5% slightly reduced the per cent viability of FCE cells compared to controls (P = 0.004). Cytopathic changes caused by FHV-1 infection were not observed at any time following incubation with 0.2% or 0.5% cidofovir. Viral titres of controls were 1014 TCID50/mL. Due to cytostatic effects of cidofovir-containing medium, definitive viral titres could not be calculated for cidofovir-treated groups; however, they were estimated to be 103.5 TCID50/mL. Conclusions: Cidofovir was highly effective against in vitro feline corneal herpesvirus infection, but had cytostatic effects on FCE and CRFK cells. Financial support: Funding provided by the Pet Trust Foundation. Commercial interest: None.