Abstracts: 35th Annual Meeting of the American College of Veterinary Ophthalmologists, Washington, DC, USA. October 21–23, 2004


ABSTRACT NO.: 01

Microarray analysis of the failure of glaucoma filtering ‘blebs’ in a rat model

D. W. Esson,* M. B. Sherwood,† M. P. Popp‡ and G. S. Schultz§

*Eye Care for Animals, Tustin California,Dept Ophthalmology, University of Florida, Gainesville, FL,UFSCC/ICBR Microarray Core, University of Florida, Gainesville, FL,§Institute for Wound Research, University of Florida, Gainesville, FL, USA

Purpose:  The failure of glaucoma filtering surgery (GFS) is generally caused by excessive conjunctival scarring and contraction of the ‘bleb’ site. Previous studies have demonstrated that Connective Tissue Growth Factor (CTGF) and Transforming Growth Factor-Beta (TGFβ) are important in mediating this process. The purpose of this study was to provide a broad overview of the factors which may play a role in clinical bleb failure following GFS, using microarray technology. Methods: GFS was performed in one eye of 27 adult Sprague-Dawley rats, by creating a limbal-based conjunctival flap, inserting a 30G, cannula through a scleral tunnel into the anterior chamber and suturing the conjunctiva closed to create a filtering bleb. Nine blebs were harvested at days 2, 5 and 12 respectively following surgery Blebs had failed by day 12. Three blebs from each treatment were pooled, to yield 3 replicates. In a similar manner, conjunctival and Tenons’ tissue were harvested from 9 further normal (unoperated) rats and pooled to yield 3 control replicates. For each of the pooled samples, total RNA was extracted, labeled and used to hybridize Affymetrix 230A rat genome microarrays. Results: Based on anova, 1542 genes were differentially expressed (P = 0.01) across bleb failure. The greatest number of changes in gene expression occurred between day 0 and day 2. Many of these genes had returned to control levels by day 12. The differentially expressed genes were sorted by Gene Ontology number and placed into ‘GenMapp’ pathways. Approximately 750 genes had no functional annotation. The most heavily populated functional categories included growth factors (notably CTGF, TGFβs 1, 2 & 3, Fibroblast Growth Factor and Insulin-like Growth Factor) structural proteins (notably procollagens I, II, X & XII, collagens I, III, V & XVIII, vimentin, intergrin & fibronectin) and matrix metalloproteinases (notably MMPs II, III & IX). Conclusion: This study represents the first large-scale gene expression analysis of GFS bleb failure. A greater understanding of the mechanisms of bleb failure may facilitate improved treatment strategies and may lead to the development of new and specific therapies which prolong bleb survival following GFS. Funded in part by an unrestricted grant from Research to Prevent Blindness. Commercial interest: None.

ABSTRACT NO.: 02

Application of a new subretinal injection device in the dog

A. M. Komáromy,* S. E. Varner,† E. de Juan,† G. M. Acland‡ and G. D. Aguirre*

*Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA;School of Medicine, Doheny Eye Institute, University of Southern California, Los Angeles, CA;Baker Institute, Cornell University, Ithaca, NY, USA

Purpose: To evaluate a new subretinal injection device (INNO JECT) for use in dogs. Methods: Young, adult normal dogs of various ages were used. The pupils were pharmacologically dilated, and the animals were anesthetized. Proper positioning of the globe was ensured by retrobulbar injection of saline and a scleral stay suture. In one group the temporal sclera was surgically exposed, cauterized, and the globe penetrated through the sclera with a 25G needle 6 mm from the limbus. The needle was then removed. A 30G Knolle anterior chamber irrigating cannula was inserted through the same site and brought into tangential apposition with the retinal surface for the subsequent subretinal injection of 150 µL saline or India ink with a conventional injection device. The fundus was visualized through a Machemer vitrectomy lens. The cannula was withdrawn and the conjunctiva over the sclerotomy site closed in standard fashion. In the second group the INNO JECT was used to penetrate the conjunctiva and the sclera 6 mm from the limbus. Once the tip of the INNO JECT was close to the retinal surface, the 39G capillary fiber was extended and brought into apposition with the retina for the subsequent subretinal injection. No closure of the conjunctiva was required. Postoperative management was identical in the two groups. The animals were monitored for 1 week after surgery. The eyes were then harvested for routine histological analysis. Results: Both approaches provided access to the subretinal space and were equally effective for subretinal injections. The INNO JECT was easier to use and required less manipulation than the conventional injection device. This resulted in a shorter surgery time and less postoperative conjunctival swelling. The INNO JECT created a smaller retinotomy with less leakage of reagent into the vitreous. Histologically, no difference could be observed between the two techniques. Conclusions: The INNO JECT is a new instrument that is useful for subretinal injections as used in gene therapy or cell transplantation in experimental studies in the dog model. Supported by NIH grants EY06855, EY13729, EY13132. Disclosure: AM Komáromy, None; SE Varner, InnoRx Inc. P; E de Juan, InnoRx Inc. I, P; GM Acland, None; GD Aguirre. Commercial interest: None.

ABSTRACT NO.: 03

Successful treatment of keratoconjunctivitis sicca (kcs) in dogs with pimecrolimus drops: a comparison with cyclosporine a (cya) ointment

R. Ofri,* I. Allgoewer,† U. Graenitz,‡ T. Pena,§ B. M. Spiess,¶ G. N. Lambrou** and E. Latour**

*Hebrew University of Jerusalem, Israel;Veterinary Eye Clinic, Berlin, Germany;Veterinary Eye Clinic, Chemnitz, Germany;§Autonomous University of Barcelona, Spain;University of Zurich, Switzerland; **Novartis Institutes for BioMedical Research, Basel, Switzerland

Purpose: The aim of this study was to conduct a clinical trial, testing the efficacy of topical pimecrolimus (Novartis Pharma, Basel, Switzerland) in alleviating clinical signs of KCS in dogs and compare it with a veterinary commercial form of CyA (Optimmune®, Schering Plough Animal Health, Union, NJ). Methods: We conducted an open-label, multicenter, randomized, 8-week study enrolling 44 dogs (77 eyes) previously untreated with CyA. KCS was diagnosed based on a Schirmer Tear Test I (STT) value ≤ 10 mm/min and an aggregate score ≥ 4 in 9 clinical signs of corneal and/or conjunctival inflammation (each sign scored from 0 = none to 4 = severe). Dogs were randomly assigned to a treatment group, and medicated twice daily with 1% pimecrolimus oil-based drops or 0.2% CyA ointment. Rechecks were conducted at 2, 4 and 8 weeks. Results: Baseline mean STT values in the pimecrolimus and CyA groups were 3.8 ± 0.7 mm/min (mean+-sem; n = 20) and 4.6 ± 0.7 mm/min (n = 24), respectively. Significant improvements were observed within both groups (P ≤ 0.001) within 2 weeks. After 8 weeks, mean increases of 9.2 and 5.8 mm/min were observed in the pimecrolimus and CyA groups, respectively. The difference was marginally significant (P = 0.085). Both drugs also caused a significant improvement in clinical signs of inflammation (P ≤ 0.001) within 2 weeks. Baseline mean total scores in the pimecrolimus and the CyA groups were 16.0 ± 1.1 and 13.9 ± 1.4, respectively. After 8 weeks, there was a significantly larger reduction in the total score in eyes treated with pimecrolimus (mean decrease 10.3) compared to eyes treated with CyA (mean decrease 7.6; P = 0.024). Two dogs from the pimecrolimus group were discontinued due to severe local irritation. Conclusions: The results show that 1% pimecrolimus drops are more effective than commercial 0.2% CyA ointment in controlling KCS in dogs. The findings confirm the interest to develop pimecrolimus for treatment of dry eye in humans. Support: Funded by Novartis Pharma AG. Commercial interests: Compensation (RO, IA, UG, TP, BS), and employee (EL, GL).

ABSTRACT NO.: 04

Congenital orbital varix in a dog

E. A. Adkins,* D. A. Ward,* P. T. Wooten,† S. Smith-Foley† and G. B. Daniel*

*Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine;Department of Radiology, University of Tennessee Medical Center, Knoxville TN, USA

Purpose: To describe the clinical presentation, diagnosis and outcome of a congenital orbital varix in a dog. Methods: This is a presentation of a clinical case. Results: A 10-week-old female Labrador retriever presented for evaluation of exophthalmos and exotropia of 8 weeks duration OS. The exophthalmos became more pronounced on compression of the left jugular vein. Diagnostic tests included orbital ultrasonography, computed tomography, angiography and venography. Ultrasonography with Doppler color flow demonstrated a vascular lesion with unidirectional blood flow. Computed tomography revealed a contrast-enhancing orbital soft tissue mass. An arteriovenous malformation was ruled out by angiography. Venography demonstrated a bi-lobed sacculated structure draining via the angular and dorsal external ophthalmic vein providing the definitive diagnosis of a large orbital varix OS. Treatment of the lesion was by embolization with stainless steel coils. All clinical signs resolved two weeks postoperatively. There has been no recurrence of the lesion 15 months postoperatively. Conclusions: There are few reports of orbital vascular anomalies in the dog. To the authors knowledge this is the first report of treatment of an ocular disease by coil embolization in veterinary medicine. Commercial interest: None.

ABSTRACT NO.: 05

Ocular toxicity of subconjunctival sustained release cyclosporine delivery devices in dogs

A. B. Beale,* J. Salmon,* M. R. Robinson† and B. C. Gilger*

*Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC;National Eye Institute, National Institutes of Health, Bethesda, MD, USA

Purpose: To evaluate potential toxicity of a subconjunctival sustained release cyclosporinee A (CsA) device in dogs. The implant is designed to provide an alternative to daily medication for the treatment of chronic keratoconjunctivitis sicca (KCS) or other chronic immune-mediated keratopathies in dogs. Methods: Six young female beagles were used in the study. All dogs were normal on initial ocular exam including Schirmer tear test I, tonography, slit lamp biomicroscopy, indirect ophthalmoscopy, ocular fundus photography and electroretinogram (ERG). All dogs had a normal complete blood count and serum chemistry profile. Each dog was sedated and each eye was prepared for sterile surgery. A pocket was created under the dorsal bulbar conjunctiva parallel to and approximately 8 mm posterior to the limbus. Two 0.75 inch 10% matrix CsA implants were inserted and the conjunctiva closed with 6–0 polyglatin 910. The other eye of each dog received 2 identical silicone implants without CsA. Triple antibiotic ointment was applied twice daily for 7 days. Ocular exams were repeated at Day 7 and then monthly for the duration of the study. ERGs and bloodwork were performed monthly. Two dogs were euthanized at 1, 3, and 6 months. The implants were removed, placed in a dessicator for 48 h, and then weighed to estimate the amount of drug delivered. The eyes were enucleated, fixed in 10% NBF and processed for routine histopathology. Results: Both the CsA and the silicone-only implants were well tolerated. STT values in both CsA implanted and silicone-only eyes rose at days 7 and 30 but then returned to baseline for the remainder of the study. No changes were noted on ocular exams, systemic bloodwork or ERGs. Reduction in implant weight suggested a rate of delivery of 25 µg/day with highest release rate over the first month. Due to implant extrusion in one dog (1 extrusion out of 24 implants) and damage to the implant on removal in another dog, it was not possible to determine accurate weight data for the 6 month time point. Histologic abnormalities were limited to a mild inflammatory response surrounding CsA and silicone-only implants. No evidence of pathology was noted in the cornea, uvea, sclera, lens or retina. Conclusions: Double implantation of 10% CsA silicone device did not caused signs of toxicity over a 6-month period in normal Beagle dogs based on clinical findings, ERG and histopathology. Further studies involving pharmacokinetics and clinical trials are underway to evaluate the efficacy of the sustained release CsA devices. Funding provided by the National Eye Institute. Commercial interest: Compensation (BCG).

ABSTRACT NO.: 06

Identification of individual dogs from retinal images: do retinal vessels change substantially over time?

J. R. Gionfriddo,* C. Lee,* E. Bridgeford,* K. K. Marren,* T. A. Precht† and C. C. Powell*

*College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO; Technology Driven Products, Loveland, CO, USA

Purpose: Positive identification of individual dogs is important for humane societies, breeders and dog owners. Most currently used identification methods, such as microchips and tattoos, may be altered, forged or lost. A pet product company developed a method of identifying individual dogs by retinal scans. As part of a feasibility study for this product, we were asked to determine if the retina could change sufficiently as the dog ages to make him/her unidentifiable by this method later in life. Methods: Fundic photographs (35-mm slides) of both eyes of 15 beagle dogs were analyzed. These photographs had been taken with a Kowa fundus camera by Dr Claire Lee as part of very large study on radiation effects on dogs and were taken yearly (starting at one year of age) throughout the life of each dog. We selected slides of dogs of both genders and used the photographs of their fundi at 3 different ages (1 years, 5 or 7, and 9 years). Each slide was analyzed to determine the positions of the major blood vessels using a software program developed by Technology Driven Products. In this program, a ring with a width of 0.38 pixels was superimposed on the image of the fundus. The ring was positioned to surround the optic disc by placing a dot in the middle of the ring, over the optic pit. The ring was divided into degrees of a circle with the 0 (360) degree position toward the 3 : 00 position, thus making 90 degrees at the 12 : 00 position and 180 degrees at the 9 : 00 position. The number of pixels occurring within the boundaries of the ring at each degree position was measured by the program. A value for the number of pixels in the background of the fundus was established and compared with the numbers of pixels present at each prominent blood vessel as it crossed the ring. Thus a plot of the ‘density’ of each vessel vs. its position around the ring was generated for each eye. The plots of each eye of each dog at the different ages were compared for all dogs. Results: After adjusting for variations in light intensities and the angles at which the photographs were taken, the plots of the fundic blood vessels were virtually identical in both eyes of all dogs at the three ages. Conclusions: The sizes and positions of the retinal blood vessels do not change sufficiently over the course of a dog's lifetime to preclude their use to positively identify an individual dog during it's lifetime. Supported by a grant from Pet Safe®, Knoxville, TN. C. Comercial interest: None.

ABSTRACT NO.: 07

The effect of elective phacofragmentation on central corneal thickness in the dog

G. L. Lynch and J. L. Brinkis

Eye Care For Animals, Tustin, CA, USA

Purpose: To characterize the short- and intermediate-term effects of elective phacofragmentation on central corneal thickness (CCT) in the dog. Methods: Forty-three dogs undergoing elective cataract surgery in a total of 66 eyes over a 7-month period at a single private ophthalmology referral clinic were enrolled in the study. CCT was measured via ultrasonic pachymetry using the Tomey SP-3000 unit just prior to surgery, the day following surgery, one week postsurgery, one month postoperatively, and greater than 2 months following phacofragmentation. Statistical comparisons were made using descriptive and inferential statistical methods with a level of significance set at P < 0.05. Results: The initial mean CCT of 611 µm was increased dramatically by one day postphacofragmentation to 741 µm. CCT remained slightly elevated (666 µm) at 1 week postoperatively, but became indistinguishable from preoperative measurements by 1 month postsurgery (626 µm) and remained so at the > 2 month time period (618 µm). The change over time and the trends remained remarkably similar, even when adjusted separately for age, gender, surgeon status, cataract type, and total surgery time (all P < 0.0001). Corneas of diabetic patients were thicker than those of nondiabetics on average and at all time periods. It also appears that there was a sharper increase from the preoperative to one day postoperative CCT in the diabetic group compared to the nondiabetic group. Overall, it appears that the CCT of the pseudophakic group took longer to return to baseline than the aphakic group. Interestingly, it appears that the mean CCT of the foldable IOL group was slower to return to baseline than either the rigid IOL or aphakic groups. Dogs with an in-hospital postoperative IOP spike (> 25 mmHg) developed a greater 1-day postsurgical increase in CCT. It appears that there was a sharper decrease in mean CCT during a ‘catch-up’ period from one month to greater than 2 months postoperatively in the postoperative hypertension group. Conclusions: Elective phacofragmentation surgery results in an increase in CCT in the dog, but this increase is transient, and as such should not deter the performance of such surgery. No benefit of the small incision cataract surgeries made possible by the use of foldable intraocular lenses could be demonstrated. Commercial interest: None.

ABSTRACT NO.: 08

Histochemical and immunohistochemical evaluation of 75 feline intraocular neoplasms

B. H. Grahn,* R. L. Peiffer,‡ C. L. Cullen§ and D. M. Haines†

*Department of Small Animal Clinical Sciences,Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4;Merck and Co. West Point, Pennsylvania,§Department of Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada

Purpose: To investigate the immunohistochemical (IHC) and histochemical differentiation of ocular neoplasms of cats, with the ultimate objective of developing an algorithm of histochemical and IHC staining to facilitate the diagnosis of poorly differentiated intraocular neoplasms in cats. Methods: Sections of fixed tissues from 75 eyes from cats with intraocular neoplasms were examined with light microscopy. The neoplasms were morphologically categorized according to the World Health Organization International Histological Classification. These included ocular sarcomas (32), diffuse iris melanomas (17), lymphomas (15), ciliary adenomas (3), metastatic carcinomas (1), and undifferentiated intraocular neoplasms (7). Sections of these globes were stained with hematoxylin and eosin and periodic acid-Schiff (histochemical stains), and IHC stains to identify expression of vimentin, actin, cytokeratin, S-100, tyrosinase, and HMSA-5 cellular markers. Neoplasms diagnosed as intraocular sarcomas and ciliary epithelial neoplasms were also stained with neurospecific enolase. Tumors diagnosed as diffuse iris melanomas and lymphosarcomas were also stained with LY5 and CD3 stains. Results: Histochemical and IHC classification based on detection of differentiation antigens revealed discordance from the morphologic diagnoses in 10 of 75 neoplasms. These included neoplasms classified based on IHC and histochemical staining as five lymphosarcomas, four intraocular sarcomas and one ciliary adenocarcinoma. IHC staining also revealed a diagnosis in 6/7 undifferentiated intraocular neoplasms. IHC, histochemical staining, and morphology provided a diagnosis for all but one poorly differentiated feline intraocular neoplasm in this study. Conclusions: While the diagnosis of the majority of feline intraocular neoplasms can be based on morphological criteria, adjunct histochemical and IHC stains are useful in determining the cell of origin in anaplastic feline ocular neoplasms. Funding sources: Companion Animal Health Fund, Western College of Veterinary Medicine. Commercial interest: None.

ABSTRACT NO.: 09

Retrobulbar injection technique and anesthesia in dogs

P. J. Accola,* E. Bentley,† L. J. Smith,† L. J. Forrest,† C. Baumel† and C. J. Murphy†

*Veterinary Emergency Service, Middleton, WI;School of Veterinary Medicine, University of Wisconsin–Madison, Madison WI, USA

Purpose: The use of systemic nondepolarizing neuromuscular blocking agents (NMBA, e.g. atracurium) to inhibit extraocular muscle activity is common during corneal and intraocular surgery in veterinary ophthalmology. Intensive monitoring is essential during administration of NMBAs to achieve a safe and therapeutic response, and positive pressure ventilation is recommended. A common complication of these drugs is hypoventilation, particularly when manual ventilation is used without capnometry. Respiratory acidosis occurs even when low doses that do not appear to affect respiration are used. Retrobulbar anesthesia can produce extraocular muscle akinesis and eliminate the need for systemic neuromuscular blockade. Additionally, retrobulbar anesthesia can provide additional analgesia for enucleation. The purpose of this study was to develop and evaluate the efficacy of various retrobulbar injection techniques in the dog. Methods: Three retrobulbar injection techniques were evaluated: a combined superior and inferior peribulbar, inferior-temporal palpebral, and peri-mandibular approach. The first study involved the retrobulbar injection of latex on multiple canine cadaver heads followed by orbitotomy and evaluation of latex distribution (n = 4, n = 4, n = 2, respectively). The second study used magnetic resonance imaging (MRI) to evaluate the distribution of a gadolinium solution injected in the retrobulbar space in dogs euthanized for unrelated studies (n = 4). Finally, a gadolinium/lidocaine (4 mg/kg) mixture was injected in anesthetized beagles (n = 2) and distribution was evaluated with MRI. The effect of the local anesthetic was evaluated by assessing ocular position, pupil diameter, and ease of manipulation of the globe. Results: Latex injections were localized to the orbital cone with leakage to adjacent retrobulbar structures using both the inferior-temporal palpebral and peri-mandibular techniques. The combined superior and inferior peribulbar technique resulted in adequate but less predictable coverage, thus this technique was not evaluated further. In MRI studies on cadaver dogs, the inferior-temporal palpebral technique resulted in more consistent retrobulbar coverage than the peri-mandibular technique. Finally, the MRI of live dogs given the gadolinium/lidocaine mixture via the inferior-temporal palpebral technique demonstrated appropriate distribution in the retrobulbar space with no distribution to the central nervous system, rapid onset of action, pupillary dilation, central eye position, and facilitation of globe manipulation. Conclusions: The inferior-temporal palpebral technique was the preferred approach for retrobulbar injection of anesthesia in dogs. This is a potential alternative to the use of systemic nondepolarizing neuromuscular blocking agents during ophthalmic surgery, and can provide additional local analgesia. Further studies are underway to evaluate its efficacy in the clinical setting. Commercial interest: None.

ABSTRACT NO.: 10

The effect of trophic factor supplementation on the viability of corneal endothelial cells after simple cold storage

E. Bentley, G. A. McKie, J. F. McAnulty and C. J. Murphy

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

Purpose: Preservation of endothelial cell viability for longer periods during storage of corneal tissue increases the availability of healthy tissue for transplantation and may decrease rejection of transplants, a major barrier to stable, optically clear corneal transplantation in dogs. Supplementation of storage media with various growth factors has been shown to improve storage times in corneas and other organs. The current study was designed to determine if endothelial cell viability in canine corneas can be improved by supplementing storage media with selected trophic factors. Methods: 24 pairs of eyes were collected from normal research dogs euthanized for reasons unrelated to this study. 22 dogs were between 5 and 7 years of age, and 2 were approximately one year old. Corneas were sterilely removed, bisected, and one half vital stained with alizarin red and trypan blue for baseline endothelial cell counts, and the other half placed in either Optisol GS® (OGS; Bausch & Lomb, St. Louis, MO) or Optisol GS® supplemented with trophic factors (OGS + TFS). Trophic factors used were bovine neutrophil peptide-1 (1 µg/mL), substance P (2.5 µg/mL), insulin-like growth factor-1 (10 ng/mL), and nerve growth factor (20 ng/mL). Right and left eyes from each dog were randomly assigned to either OGS or OGS + TFS. After storage for 7 days, 14 days, 21 days, and 28 days (n = 6 for each time point), corneas were removed and vital stained for endothelial cell counts. Endothelial cell counts were obtained by averaging cell counts from images obtained from 3 different areas of the paracentral cornea. A paired t-test was used to compare the change from baseline cell counts during the storage times between the two groups and to compare baseline endothelial cell counts to endothelial cell counts after storage times in samples stored in OGS and OGS + TFS. Results: Significantly less endothelial cell loss occurred in the OGS + TFS group than in the OGS group in 14 days of storage (P < 0.0223; 687 ± 361 cells/mm2 lost OGS + TFS, 1755 ± 704 cells/mm2 lost OGS). There was no significant difference in cell loss between the two groups at 7 days, 21 days, or 28 days. There was no significant change in endothelial cell counts from baseline in either the OGS or the OGS + TFS group at 7 days, but there was significant cell loss at all successive time points within both groups. Conclusions: Supplementation of storage media with multiple trophic factors significantly decreases endothelial cell loss compared to OGS alone after 14 days of storage, but has no significant effect during longer periods of storage. Significant endothelial cell loss still occurred from baseline to day 14 and later in both groups, however, trophic factor supplementation appears to be an effective strategy for extending corneal storage times. Supported by a grant from the ACVO. Commercial interest: None.

ABSTRACT NO.: 11

Detecting early functional deficits in rat models of acute and chronically elevated intraocular pressure

G. Ben-Shlomo,* S. Bakalash,† M. Schwartz† and R. Ofri*

*School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot, Israel;Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

Purpose: To use the pattern electroretinogram (PERG) to detect retinal function deficits in rat models of acute and chronically elevated intraocular pressure (IOP). Methods: Six male Lewis rats were used in each model. Elevated IOP was induced in one eye, with the contralateral eye serving as a normotensive control. Argon laser photocoagulation of the episcleral veins and limbal plexus was used to induce chronic elevation of IOP. An acute increase in IOP was achieved by insertion of a 30-gauge needle connected to a polyethylene tube and a bag of normal saline (0.9%) infusion. The infusion bag was placed 1 m above the rat's head, creating a closed loop circulation, for 1 h. Scotopic PERG responses were recorded prior to IOP elevation, and 2 weeks (in the acute model) or 3 weeks (in the chronic model) later. Stimulus was a series of 5 shifting (6.1 Hz) checkerboard patterns of decreasing spatial frequency (0.368–0.023 cycles per degree, cpd), projected directly on the animal's fundus from a distance of 7 cm using a specially modified direct ophthalmoscope. Results: Responses from the normotensive and hypertensive eyes of both models showed typical spatial frequency dependence. P1N2 amplitude increased with the size of the stimulus, peaking in response to a 0.046 cpd pattern. Chronic model: In the hypertensive eyes, signal amplitudes were reduced in response to all patterns, with significantly (P < 0.05) lower signals recorded in response to wide stimuli. Mean responses to 0.046 and 0.023 cpd patterns were reduced by 40% and 30%, respectively. Acute model: In the experimental eyes, signal amplitudes were significantly reduced in response to all patterns (P < 0.001). Mean responses to gratings of 0.368, 0.184, 0.092, 0.046 and 0.023 cpd, decreased by 79%, 86%, 90%, 88% and 88%, respectively. Conclusions: The PERG of rat eyes with chronically elevated IOP shows an amplitude reduction of 30–40% in response to wide gratings. These deficits provide an indication of selective functional damage in magnocellular-like pathways, which have been shown to be preferentially damaged in nonhuman primate and rat models of glaucoma. Acute elevation of IOP caused a reduction of 79–90% in responses to all gratings, indicating nonselective, massive functional deficits in inner retina activity. We propose that the PERG may be used for early detection, and to monitor progression, of glaucomatous damage in the inner retina of the rat model. Commercial interest: None.

ABSTRACT NO.: 12

Canine multifocal acquired chorioretinopathy: clinical and pathological characterization of a nonhereditary progressive retinal degeneration

G. M. Acland,*,† G. D. Aguirre† and S. E. Pearce-Kelling*

*Baker Institute, Cornell University;School of Veterinary Medicine, University of Pennsylvania, Philadelphia PA, USA

Purpose: Dogs that are heavily involved in sport or work can develop a (multi)focal chorioretinopathy, previously ascribed to various putative etiologies, including larval migrans, distemper and heredity. This study defines the clinical course, pathogenesis, and retinal pathology of this disease. Methods: Prospective studies were undertaken in 2 populations at risk. Borzois in a large breeding kennel were examined at approximately 6 month intervals over several years. Subsequently, selected dogs were monitored at less regular intervals. In a second study, over 1000 Border Collies competing at a high level in national herding trials were examined over several years, many on several occasions. Additional studies were undertaken of dogs in other affected or at risk populations. Eyes from selected dogs from each of these populations were obtained for retinal morphologic examination. Results: The initiating ophthalmoscopic lesion was a ‘jet’ microhemorrhage, estimated at approximately 5 µL, arising from the subretinal vasculature, and bursting through the overlying retina into the vitreous. Over several days to weeks the initial lesion resolved leaving a characteristic focal ‘scar’. Morphologically, the primary lesion appeared as a break in the normal laminar separation of the retina from underlying tissues, with focal retinal degeneration. In eyes with only 1 or very few such lesions, the uninvolved retina was normal. In some affected dogs multiple such lesions developed over several years. In such eyes the focal lesions often coalesced into larger degenerate areas and, in some cases, formerly uninvolved retinal regions exhibited a generalized degeneration. The incidence in male dogs was consistently higher than in females in all populations. Lesions were usually asymmetrical between eyes with left eyes, usually, more frequently and worse affected than right. Although shared environment is seen as a risk, no evidence for heritability was observed, either from pedigree analysis, or from limited experimental breedings. No evidence of parasitic (larval migans) involvement was detected. Conclusions: In the early stages of this disease the clinical and retinal morphologic appearrance is characteristic, but eventually can be virtually indistinguishable from other generalized retinal degenerations. The etiology remains unknown, but appears to involve microvascular accidents arising from athletic stress. Support: NIH Grants EY06855, EY13729, EY13132, The Foundation Fighting Blindness, American Border Collie Association/U.S.B.C.H.A. Commercial interest: None.

ABSTRACT NO.: 13

Canine goniodysgenesis-related glaucoma: a morphological review of 100 cases looking at acute inflammation and pigment dispersion

C. M. Reilly, R. Morris and R. R. Dubielzig

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

Purpose: To investigate the role of pigment dispersion and acute inflammation in the pathogenesis of breed-related goniodysgenesis associated glaucoma. Methods: Records from 2905 pathology submissions from dogs with glaucoma were reviewed. Cases with a diagnosis of goniodysgenesis were selected only when the duration of the disease was specified. Cases were subdivided into breeds, and only breeds having more than three samples fit for inclusion were part of the study. The total number of cases included was 100. Slides were evaluated for the following: Presence of pigment cells in the trabecular meshwork; the ability to predict the dependent side of the eye based on the distribution of pigment; the absence of pigment epithelial cells from the posterior iris; evidence of stripping of pigmented cells from the posterior iris epithelium; and free pigmented cells in the iridocorneal angle. Globes were evaluated further for the presence or absence of inflammatory cell infiltration in the trabecular meshwork. Neutrophilic and lymphoplasmacytic infiltration was quantified independently. Changes were compared at different intervals from the clinical onset of disease. Results: Of 100 cases evaluated, there were 62 Cocker Spaniels, 14 Bassett Hounds, 10 Labrador Retrievers, 7 Chow Chows and 7 Huskies. Sex distribution was approximately 2 females to 1 male. 14 were 0–3d in duration, 20 were 4–7d and 66 were > 7d. The mean duration of clinical signs was 53 days. 93% of 1–3d glaucomas and 95% of 4–7d glaucomas had free pigment. 79% of > 7d cases had free pigment. 43% 1–3d, 75% 4–7d and 55% of chronic glaucomas had pigment epithelium missing from the posterior iris surface. 21% 1–3d, 45% 4–7d, and 21% of > 7d cases had evidence of cell stripping from the posterior iris surface. 64% 1–3d, 95% 4–7d, and 50% of > 7d had pigmented cells in the iridocorneal angle. 86% of 1–3d, 50% 4–7d, and 14% of > 7d glaucomas had neutrophils. 64% of 1–3d, 60% 4–7d, and 48% of > 7d cases had lymphoplasmacytic inflammation. 57% 1–3d, 35% 4–7d and 12% > 7d cases had evidence of both types. In all cases, the degree of inflammation tended to be mild. Conclusions: Eyes enucleated for goniodysgenesis related glaucoma are twice as likely to be in the chronic phase of disease (> 7d). Acute inflammation is more likely to be seen within the first three days from the onset of clinical disease. Lymphoplasmacytic inflammation is seen with similar frequency at all time points. Pigment dispersion is a prominent finding at all time points, but is more prominent during the first 7 days. The finding of iris pigment epithelial loss or stripping away of the iris epithelium supports the theory that pupillary block associated with touching of the parapupillary iris to the lens is important in the pathogenesis of canine glaucoma associated with goniodysgenesis. Comercial interest: None.

ABSTRACT NO.: 14

Reduction in matrix metalloproteinase activity in the equine tear film during corneal healing in 10 horses with ulcerative keratitis

F. J. Ollivier,* D. E. Brooks,* M. E. Kallberg,* K. N. Gelatt,* S. E. Andrew,† S. Schultz‡ and G. Van Setten§

*Small and Large Animal Clinical Sciences,Obstetrics and Gynecology,University of Florida, Gainesville, Florida; Georgia Veterinary Specialists, Atlanta, Georgia;§Karolinska Institute, St Eriks Eye Clinic, Stockholm, Sweden

Purpose: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear films during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. We analyzed tear protease activity during healing of corneal ulcers in horses to test this hypothesis. Methods: From 10 horses with ulcerative keratitis, samples of tear fluid were obtained from both eyes on the day of admission (day 1) at the hospital and then at various time points until the complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases, the medical treatment included topical applications of equine serum, antibiotics, atropine, and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in five cases on day 2 in addition to the medical treatment. Results: The mean total MMP activity (± SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 ± 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 ± 0.68) (P = 0.006) on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (−82.4%) between the first day of admission and the day when the ulcer was completely healed (P = 0.0002). The activity level in the healed eye (0.43 ± 0.17) was not significantly different to the one in the contralateral eye (0.36 ± 0.18) at the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 ± 0.68–0.36 ± 0.18 but this 56.0% of decrease was not significant (P = 0.069). Conclusions: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity which decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In the horses, successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis. Supported by the office of Graduate Studies of the University of Florida College of Veterinary Medicine (Consolidated Research Development Award). Commercial interest: None.

ABSTRACT NO.: 15

Differences in location of matrix metalloproteinases 2 and 9 in normal and ulcerated canine corneas

M. E. Kallberg, D. E. Brooks, F. J. Ollivier, M. Nelligan, P. A. Lewis and K. N. Gelatt

Departments of Small and Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville FL, USA

Purpose: To examine whether ulcerated corneas differ from normal corneas with respect to MMP2 and MMP9 location, and whether MMP2 and MMP9 staining differ between ulcers with keratomalacia and traumatic ulcers with subsequent eye rupture. Methods: Unstained corneal paraffin sections of 10 ulcerated canine corneas were obtained from the Surgical Pathology service at the University of Florida Veterinary Teaching Hospital. Sections were from eyes enucleated secondary to corneal ulceration with keratomalacia, or traumatically ruptured eyes. Six normal canine eyes obtained from dogs euthanized in the senior terminal surgery elective were used as controls. The sections were immunoenzymatically stained using the avidin-biotin complex method. The slides were then subjectively evaluated. The specificity of the antigen-antibody response was tested using Western Blotting comparing canine corneas with horse corneas and a human control. Results: The Western Blot analysis showed good correspondence protein bands of horse MMPs and a human control. The corneal epithelium of all normal eyes stained distinctly for MMP2. Four of the six normal dog corneas did not stain for MMP9 in the epithelium. Normal dog corneas did not stain for either MMP in the stroma. Traumatically ruptured corneas stained faintly for both MMPs in the epithelium and stroma. Corneas with melting ulcers stained intensely for MMP2 in epithelium and stroma. The stain intensity in both the epithelium and stroma for MMP9 varied from none (3 dogs) to intense (3 dogs) for the eyes with keratomalacia. No obvious correlation was seen between intensity of stain for MMP9 and duration of disease or presence of bacteria. Conclusions: The MMP staining of the normal canine eyes was inconsistent with previous reported data on rat and bovine corneas but corresponded with equine studies. The MMP2 staining characteristics of ulcerated canine eyes were consistent with known data on the staining of ulcerated rat, mouse, rabbit, horse and human corneas. The increase of MMPs in diseased canine corneas supports the use of anti-MMP medication in the treatment of canine corneal ulcers. Further studies are needed to fully understand the role of MMPs in canine corneal disease. Funded by a CVM Resident Intramural Grant, University of Florida. Commercial interest: None.

ABSTRACT NO.: 16

Canine ocular onchocerciasis in the United States: two new cases and a review of the literature

M. K. Zarfoss,* R. R. Dubielzig,† M. L. Eberhard‡ and K. S. Schmidt§

*University of Missouri-Columbia, University of Wisconsin-Madison,Division of Parasitic Diseases,Centers for Disease Control and Prevention,§Veterinary Ophthalmology Services, Oxnard, CA, USA

Purpose: Since 1991, 53 cases of canine ocular onchocerciasis have been reported in the literature worldwide, 43 of these from Greece, five from Hungary, and five from the western United States. Information on the histopathologic features of canine ocular onchocerciasis is limited. The present report describes the histopathologic features of canine ocular onchocerciasis in two dogs from California that presented clinically with firm episcleral nodules and uveitis unilaterally. Pertinent literature and pathogenesis are reviewed; recognizable clinical features and treatment are discussed. Methods: The cases presented were diagnosed via histopathology of the enucleated globes and episcleral granulomas at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Positive identification of adult Onchocerca within episcleral granulomas was made based on light microscopic features. Results: Histopathologic examination of both globes revealed episcleral parasites surrounded by granulomas containing few to moderate numbers of eosinophils. Other sequelae in both cases included lymphoplasmacytic uveitis, preiridal fibrovascular membranes, peripheral anterior synechiae, retinal atrophy, and optic cupping. Both male and female worms were present, as were in utero microfilariae in both cases. Worms in both cases were tentatively identified as O. lienalis. Conclusions: Ocular onchocerciasis should be considered as a differential diagnosis in cases of canine conjunctival nodules or periorbital swelling, particularly in dogs from the western United States. Commercial interest: None.

ABSTRACT NO.: 17

The effect of topical bimatoprost and unoprostone isopropyl on the intraocular pressure of normal cats

J. T. Bartoe,* H. J. Davidson,* M. T. Horton,* Y. Jung† and A. H. Brightman*

*Department of Clinical Sciences, College of Veterinary Medicine,Department of Statistics, College of Arts and Sciences, Kansas State University, Manhattan KS, USA

Purpose: To evaluate the effect of bimatoprost and unoprostone isopropyl on the intraocular pressure, pupillary diameter, and parameters associated with ocular inflammation and pain over time. Methods: For an initial 5 days, intraocular pressure (IOP) bi-daily, pupillary diameter (PD), blepharospasm score (BS), conjunctival injection score (CS), and flare score (FS) were determined for each of 12 cats to establish baseline values. Following this period, 6 randomly selected cats were administered 30 µL topically of bimatoprost (Lumigan®, Allergan, Inc., Irvine CA) to a randomly selected eye once daily. The remaining 6 cats were administered 30 µL topically of buffered saline solution (BSS®, Alcon Laboratories, Inc., Fort Worth TX) to a randomly selected eye once daily. The contralateral eye of each cat remained untreated at all time points. The treatment period consisted of 10 days in which IOP bi-daily, PD, BS, CS, and FS were determined for each cat daily. This period was followed by 5 days without treatment in all cats with the aforementioned clinical parameters determined daily. Subsequently, 6 randomly selected cats were administered 30 µL topically of unoprostone isopropyl (Rescula®, Novartis Ophthalmics, Duluth GA) to a randomly selected eye twice daily. The remaining 6 cats were administered 30 µL topically of BSS to a randomly selected eye twice daily. The contralateral eye of each cat remained untreated at all time points. The treatment period consisted of 10 days in which IOP bi-daily, PD, BS, CS, and FS were determined for each cat daily. This period was followed by 5 days without treatment in all cats with the aforementioned clinical parameters determined daily. Results: There was no statistically significant difference (P < 0.05) between the IOP of bimatoprost and BSS treated cats at either the AM (p-0.178) or PM (p-0.348) assay points over the 10 day treatment period. Similarly, significance difference in IOP was not observed between unoprostone isopropyl and BSS treated eyes for the AM (p-0.103) and PM (p-0.457) assay points, respectively. Although transient miosis (< 2 h) was subjectively noted in the treated eye of all cats dosed with either bimatoprost or unoprostone isopropyl, a statistically significant difference in PD could not be detected between cats treated with bimatoprost vs. BSS or cats treated with unoprostone isopropyl vs. BSS at the time points assayed. BS, CS, and FS remained at a value of 0 (no evidence of blepharospasm, conjunctival injection, or flare) for all cats at all time points. Conclusions: Bimatoprost dosed topically once daily and unoprostone isopropyl dosed topically twice daily do not appear to significantly lower the intraocular pressure of normal cats. Supported by KSU Ross Interdepartmental Research Funds. Commercial interest: None.

ABSTRACT NO.: 18

Observations of a corneal endothelium in tropical freshwater fish (Prochilodus lineatus) using alizarin red and trypan blue

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

*College of Veterinary, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil,College of Agricultural and Veterinarian Sciences, São Paulo State University, Campus of Jaboticabal, SP, Brazil,Scholl of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil

Purpose: Although there have been many studies of the corneal endothelium of human and some mammals, there have been few in other vertebrates. The purpose of this study was to examine the endothelial surface morphology and to perform a morphometric analysis of the corneal endothelial cells of a tropical freshwater fish (Prochilodus lineatus). Methods: Twelve normal eyes from six freshwater fish with three months age and weighing 220 g each were used. These 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. Immediately after death the eyes were removed and the endothelium stained with trypan blue and alizarin red and examined using a light mcroscope. Five photomicrographs were taken of each cornea and analyzed with a digital imaging system (Sigma Scan). The morphometric analysis was performed with regard to polygonality, mean cell area and cell density. Statistical analysis was conducted using the Tukey test. Values of P < 0.05 were considered significant. Results: Normal freshwater fish (Prochilodus lineatus) corneal endothelium was characterized by a continuous layer of polygonal cells of uniform size and shape. The predominant number of cells was hexagonal (75%), with pentagonal (12.5%) and heptagonal (12.5%) cells constituting the greater portion of the remaining corneal endothelium. The mean cell area of corneal endothelium was 295 ± 67 µm2 and the endothelial cell density was 3379 ± 634 cells/mm2. The parameters evaluated did not differ significantly between the right and the left eye from the same fish. Conclusions: The combined staining with the vital stain trypan blue and the intercellular stain alizarin red provides a simple, quick technique for visualization of corneal endothelium. The structure of endothelial cells is similar to those described for other vertebrates. Acknowledgments: FAPESP (98/031153–0) and CAPES. Commercial interest: None.

ABSTRACT NO.: 19

Morphological analysis of the corneal endothelial cells of dogs using specular and scanning electron microscopy

J. A. T. Pigatto,* C. D. Freire,* F. C. Abib,† P. S. M. Barros,‡ R. Quinze,‡ J. P. D. Ortiz,§ J. M. Santos§ and J. L. Laus§

*College of Veterinary, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil,College of Medicine, Federal University of Parana, Curitiba, PR, Brazil,School of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil,§College of Agricultural and Veterinarian Sciences, São Paulo State University, Campus of Jaboticabal, SP, Brazil

Purpose: To compare the corneal endothelial morphology in normal eyes of dogs obtained by specular microscopy with those obtained after processing for scanning electron microscopy. Methods: Twenty normal eyes from 10 adult mixed-breed dogs weighing 10–12 kg were used. The animals were euthanatized for reasons unrelated to this study and treated in accordance with the Association for Research in Vision and Ophthalmology statement for use of animals in ophthalmic and vision research. Eyes with evidence of ocular disease were excluded. Immediately after enucleation the corneal endothelium were examined and photographed using a contact specular microscope (Bio-Optics LSM-2100C®). Three images of central endothelium with at least 100 endothelial cells per image were obtained from each cornea. Computer-assisted analysis provided the pleomorphism and the polymegathism. After the specular microscopic evaluation, the corneas were excised and processed for scanning electron microscopy. Three images of the central endothelium were obtained with at least 100 endothelial cells per image from each cornea. The pleomorphism and the polymegathism were determined through image analyzer software. Statistical analysis was conducted using the Tukey test and values of P < 0.05 were considered significant. Results: The predominant number of cells was hexagonal in shape, with pentagonal and heptagonal cells constituting the greater portion of the remaining corneal endothelium. An index of pleomorphism was 0.22. Pleomorphism and polymegathism did not differ significantly between specular and scanning electron microscopy. Statistical analysis showed that the parameters evaluated did not exhibit significant differences between the right and the left eyes from the same dog. Conclusion: Regarding morphological parameters direct comparisons of values obtained by specular microscopy can be made from those determined by scanning electron microscopy. Acknowledgments: FAPESP (98/031153–0) and CAPES. Commercial interest: None.

ABSTRACT NO.: 20

Cell size shape relationships in corneal endothelium of dogs

J. A. T. Pigatto,* C. D. Freire,* P. S. M. Barros,† R. Quinze,† J. P. D. Ortiz,‡ J. M. Santos‡ and J. L. Laus‡

*College of Veterinary, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil;School of Veterinary Medicine, University of São Paulo, São Paulo, SP;College of Agricultural and Veterinarian Sciences, São Paulo State University, Campus of Jaboticabal, SP, Brazil

Purpose: To investigate the relationship between cell shape and endothelial morphology in corneal endothelium of dogs using cell size as a parameter. Methods: Twenty normal eyes from adult mixed-breed dogs weighing around 12 kg were used. The animals were euthanatized for reasons unrelated to this study and treated in accordance with the ARVO statement for use of animals in ophthalmic and vision research. Eyes with evidence of ocular disease were excluded. Immediately after enucleation the corneas were excised and processed for scanning electron microscopy. The corneal endothelium was examined and photographed using a scanning electron microscopy. Three images of the central endothelium were obtained with at least 100 endothelial cells per image from each cornea. The pleomorphism and the polymegathism were determined through image analyzer software. Statistical analysis was conducted using the Tukey test and values of P < 0.05 were considered significant. Results: Regarding the polygonality of the corneal endothelium, the predominant number of cells was hexagonal in shape, with pentagonal, and heptagonal cells constituting the greater portion of the remaining corneal endothelium. An index of polymegathism was 0.22. Statistical analysis showed that the parameters evaluated did not exhibit significant differences between the right and the left eyes from the same dog. Conclusion: Corneal endothelium of dogs with a greater number of hexagonal cells demonstrated minimal variation in cell size. Acknowledgments: FAPESP (98/031153–0) and CAPES. Commercial interest: None.

ABSTRACT NO.: 21

Origins of the photopic negative response in rat retinal degeneration models

D. Raz-Prag,* S. Machida,† R. N. Fariss,‡ C. Vijayasarathy,* R. A. Bush* and P. A. Sieving*

*National Institute of Deafness and Communication Disorders, NIH;School of Medicine, Iwate Medical University,National Eye Institute, NIH, Bethsda MD, USA

Purpose: The light adapted electroretinogram in primates has a negative deflection (PhNR) following the b-wave that is associated with ganglion cells and axons. We explored the retinal origins of the PhNR in the rat during retinal degeneration. Methods: Photopic ERG's were recorded at different stages of retinal degeneration in RCS rats and p23 h rhodopsin transgenic rats. Eyes were removed for immunolabeling of KIR4.1, the dominant potassium channel on Mueller glial cells responsible for currents generating the ERG in the inner retina, and for biochemical analysis. Results: The PhNR increased in RCS as retinal degeneration progressed, but gradually declined in p23 h, whereas the b-wave decreased in both forms of degeneration. Immunolabeling revealed a slight KIR4.1 increase in the inner plexiform and ganglion cell layers in the RCS rats with age, and Western blot showed a shift of KIR4.1 from the natural tetramer form of the channel to the monomer form. No changes of KIR4.1 were observed in p23 h rats. Conclusions: Though both forms of retinal degeneration are characterized by photoreceptor death, the photopic ERG's and the PhNR demonstrate that they involve a different set of events. The PhNR may originate in potassium currents in the inner retina. Commercial interest: None.

ABSTRACT NO.: 22

Cyclooxygenase-1, -2, and -3 expression in clinically normal and glaucomatous canine eyes

C. L. Cullen,* D. E. Sims,† A. Singh,* C. McCarville,* B. P. Wilcock‡ and D. L. Simmons§

*Department of Companion Animals, and Department of Biomedical Sciences,Atlantic Veterinary College, University of Prince Edward Island, Canada;Histovet Surgical Pathology, Guelph, Ontario, Canada; §Department of Chemistry and Biochemistry, Brigham Young University, UT, USA

Purpose: (1) To determine the localization and expression of cyclooxygenase (COX)-1, -2, and -3 in normal canine eyes and canine eyes diagnosed with primary glaucoma; and (2) to enhance our present understanding of the pathogenesis of canine primary glaucoma. Methods: Twenty normal eyes, routinely fixed (Bouin's (n = 10 eyes) or 10% formalin (n = 10 eyes)) and paraffin-embedded, and 30 archived glaucomatous globes diagnosed histologically with primary glaucoma were analyzed for COX-1, -2, and -3 expression using immunohistochemistry. The degree of COX immunohistochemical staining was ranked from 0 to 3 (0 = none, 1 = weak, 2 = moderate, and 3 = marked expression) for each ocular region. Results: In the normal and glaucomatous eyes, moderate COX-1 immunoreactivity was observed in the conjunctival and corneal epithelia. In normal eyes, COX-1 expression was moderate in the nonpigmented ciliary epithelium (NPCE) (19/20 eyes), and weak in the iris stroma (10/20 eyes) and trabecular meshwork (TM; 16/20 eyes) while its expression in these same regions was weak in fewer glaucomatous eyes (17/30, 1/30, 0/30 eyes, respectively). Moderate COX-1 expression was observed in the nonpigmented retinal pigment epithelium (RPE) and tapetum of the majority of glaucomatous eyes compared to weak COX-1 immunoreactivity in these regions in fewer of the normal eyes. In contrast, there was no COX-2 expression in any of the normal eyes. Weak to moderate COX-2 immunoreactivity was observed in the corneal epithelium and stroma in those glaucomatous eyes with keratitis and/or corneal scarring. Both the normal and glaucomatous eyes had weak to moderate expression of COX-3 in the conjunctival epithelium, all layers of the cornea, and the NPCE. COX-3 immunoreactivity in the corneal endothelium was more frequently encountered in diseased eyes. Weak to moderate COX-3 expression was further observed in the nerve fiber layer (NFL), RPE and tapetum of glaucomatous eyes only. Conclusions: Reduced COX-1 immunoreactivity in the NPCE, iris stroma, and TM of glaucomatous eyes and increased expression of COX-1 in the RPE and tapetum suggest that alterations in COX-1-derived prostaglandins (PGs), those necessary for homeostatic physiologic processes, may play a role in the pathogenesis of canine primary glaucoma. Immunoreactivity of COX-3 in the NFL, RPE and tapetum of glaucomatous eyes suggests that COX-3-derived PGs may also play a role in the pathogenesis of primary glaucoma in dogs. Supported by the Sir James Dunn Animal Welfare Center. Commercial interest: None.

ABSTRACT NO.: 23

Cyclooxygenase-1, -2, and -3 expression in clinically normal and glaucomatous feline eyes

C. L. Cullen,* D. E. Sims,† C. McCarville,* A. Singh,* B. P. Wilcock‡ and D. L. Simmons§

*Department of Companion Animals, and Department of Biomedical Sciences,Atlantic Veterinary College, University of Prince Edward Island, Canada;Histovet Surgical Pathology, Guelph, Ontario, Canada; §Department of Chemistry and Biochemistry, Brigham Young University, UT, USA

Purpose: (1) To determine the localization and expression of cyclooxygenase (COX)-1, -2, and -3 in normal feline eyes and feline eyes diagnosed with glaucoma secondary to lymphonodular uveitis; and (2) to enhance our present understanding of the pathogenesis of feline secondary glaucoma. Methods: Twenty normal eyes, routinely fixed (Bouin's (n = 10 eyes) or 10% formalin (n = 10 eyes)) and paraffin-embedded, and 30 archived glaucomatous globes diagnosed histologically with glaucoma secondary to lymphonodular uveitis were analyzed for COX-1, -2, and -3 (n = 10 normal and 10 glaucomatous eyes for COX-3) expression using immunohistochemistry. The degree of COX immunohistochemical staining was ranked from 0 to 3 (0 = none, 1 = weak, 2 = moderate, and 3 = marked expression) for each ocular region. Results: In the normal and glaucomatous eyes moderate to marked COX-1 immunoreactivity was observed in the conjunctival epithelium, corneal epithelium and nonpigmented ciliary epithelium (NPCE) with weak to moderate COX-1 expression noted in the photoreceptors, Müller cells, and tapetum. In glaucomatous eyes, COX-1 expression was further observed in the nerve fiber layer (NFL) of the retina while its immunoreactivity was reduced, when compared to the normal eyes, in the ganglions cells (GCs) and the trabecular meshwork. In comparison, weak to moderate COX-2 expression was observed in the limbal-based conjunctival and corneal epithelia of normal eyes while only weak COX-2 immunoreactivity was noted in fewer diseased eyes in these regions. Weak COX-2 expression was also noted multifocally in the photoreceptors (4/20 eyes) and inner limiting membrane (ILM; 3/20 eyes) of only the normal eyes. All normal and glaucomatous globes exhibited weak to moderate COX-3 immunoreactivity in the conjunctival and corneal epithelia, Müller cells, and ILM. In the normal eyes, weak to moderate COX-3 expression was observed in the NPCE (9/10 eyes) with only weak (n = 2/10 eyes) to no COX-3 expression noted in the NPCE of the glaucomatous eyes. Conclusions: Reduced COX-1 immunoreactivity in the GCs and trabecular meshwork of glaucomatous eyes and increased expression in the NFL suggest that alterations in COX-1-derived prostaglandins (PGs), those necessary for homeostatic physiologic processes, may play a role in the pathogenesis of feline glaucoma. Reduced expression of COX-3 in the NPCE in glaucomatous eyes suggests that COX-3-derived PGs may also play a role in the pathogenesis of feline secondary glaucoma. Supported by the Sir James Dunn Animal Welfare Center. Commercial interest: None.

ABSTRACT NO.: 24

Ultrastructural findings in six feline corneal sequestra

C. L. Cullen,* D. W. Wadowska,† A. Singh‡ and Y. Melekhovets§

Department of Companion Animals,*Graduate Studies and Research,and Department of Biomedical Sciences,Atlantic Veterinary College, University of Prince Edward Island, Canada;§Laboratory Director, HealthGene Corporation, Molecular Diagnostic and Research Center, Toronto, Ontario, Canada

Purpose: (1) To describe the ultrastructural features of corneal sequestra in cats; and (2) to enhance our understanding regarding the pathogenesis of feline corneal sequestration. Methods: Six corneal sequestra were harvested via keratectomy from globes of six cats. Corneal sequestra were routinely fixed then postfixed for high resolution light and transmission electron microscopy (HR-LM and TEM, respectively). The tissues were embedded in Epon/Araldite. Sections 0.5 µm thick were cut and stained with 1% toluidine blue in 1% sodium tetraborate solution for HR-LM. Ultrathin sections were collected on copper grids and stained with uranyl acetate and Sato's lead stain for TEM. Ultrathin sections were examined and the images were captured on an Advantage HR CCD camera (AMT, USA) using a Hitachi 7500 electron microscope operated at 80 kV. Two healthy corneas from two cats were harvested immediately following euthanasia. These corneal tissues (control samples) were processed in the same manner as the corneal sequestra for HR-LM and TEM. A portion of each corneal sequestrum was also submitted for polymerase chain reaction (PCR) testing for infectious agents including feline herpesvirus-1 (FHV-1) and Toxoplasma gondii (T. gondii). Results: Ultrastructure of healthy corneal tissues revealed basal corneal epithelial cells aligned adjacent to a thin acellular layer similar to Bowman's layer with underlying tightly packed, regularly arranged collagen fibrils oriented in different planes. Keratocytes were elongated and had long and irregularly shaped nuclei, and cytoplasm contained rough endoplasmic reticulum and abundant membrane-bound vesicles. In contrast, corneal sequestra contained varying amounts of an amorphous, electron dense substance, continuous with intact basal epithelial basement membranes peripherally, and overlying corneal ulceration and loosely packed collagen fibrils. Remnants of necrotic keratocytes were seen in spaces between disarranged collagen layers. In all samples, occasional keratocytes exhibited morphology indicative of apoptosis including clumping and margination of chromatin, and shrunken cytoplasm. Varying degrees of inflammation were noted on HR-LM and TEM of affected corneas including peri- and intralesional neutrophils, lymphocytes, plasma cells, and macrophages. Corneal sequestra were FHV-1-positive (n = 2), FHV-1 and T. gondii-positive (n = 1), T. gondii-positive (n = 1), or negative for DNA of these infectious agents (n = 2) using PCR. Conclusions: Apoptosis may play a role in the pathogenesis of feline corneal sequestration independent of the presence or absence of DNA of these infectious organisms. Comercial interest: Employee (YM).

ABSTRACT NO.: 25

Tear film breakup times in young healthy cats

C. L. Cullen,* C. Lim† and J. Sykes†

Department of Companion Animals; *Veterinary Teaching Hospital,Atlantic Veterinary College, University of Prince Edward Island, Canada

Purpose: (1) To determine tear film breakup times (BUTs) in young healthy cats; (2) to determine tear BUTs in feline eyes within 8–20 h following general anesthesia; (3) to determine if tear BUT vary significantly preoperatively when compared to values obtained 8–20 h postoperatively; (4) to determine if Schirmer tear test (STT) values correlate with tear BUTs in young healthy cats; and (5) to determine if the isolation of particular etiologic agents from conjunctival swabs of healthy cats affects tear BUTs. Methods: Eighteen healthy domestic shorthair (n = 14) and domestic longhair (n = 4) cats, with normal ocular examinations, ranging in age from 0.5–3 years. Complete ophthalmic examinations, including tear BUTs, were performed on all cats. Conjunctival swabs from each eye of all cats and blood samples from all cats were collected and submitted for polymerase chain reaction (PCR) screening for FHV-1, Chlamydophila felis, Mycoplasma spp., and calicivirus. In 10 of 18 cats, STT values and tear BUTs were measured before general anesthesia was administered and again within 8–20 h following the end of anesthesia. Results: Mean (± SD) preanesthesia tear BUTs for all 18 cats were 17.4 (± 4.6) seconds for the right eye (OD) and 16.0 (± 4.5) seconds for the left eye (OS). Mean postanesthesia tear BUT results were 12.5 (± 4.3) seconds and 13.1 (± 4.0) seconds for OD and OS, respectively. Post-anesthesia tear BUTs were significantly more rapid than those measured before anesthesia OD only. There was also a positive correlation, both before and after anesthesia, between STT values in both eyes (OU) and tear BUTs OU. The isolation or lack of isolation of conjunctival microorganisms using PCR did not significantly affect tear BUTs. Conclusions: Mean tear BUT in young healthy domestic cats is 16.7 (± 4.5) seconds. Tear BUT is positively correlated with STT values. Although mean tear BUTs OD at 8–20 h following anesthesia were more rapid than preanesthesia values, this difference did not appear clinically relevant. Supported by the Companion Animal Trust Fund, Atlantic Veterinary College, University of Prince Edward Island. Commercial interest: None.

ABSTRACT NO.: 26

Canine ocular peripheral nerve sheath tumors: a report of 3 cases with clinical and histologic features and a review of the literature

K. Caruso,* S. A. Koch,† R. Marrion,‡ D. G. Dunn,§ D. Belote¶ and G. Saturday**

*,The Animal Eye Clinic, Silver Spring, MD;Essex County Veterinary Specialists, North Andover, MA;§,,**Armed Forces Institute of Pathology, Washington DC, USA

Purpose: To describe a unique orbital neoplasm in dogs with emphasis on clinical presentation, treatment and outcome, as well as histopathologic characteristics. A brief review of the literature will precede the cases. Methods: Three dogs with the histologic diagnosis of peripheral nerve sheath tumors (PNST) of the eyelid, palpebral conjunctiva, and retrobulbar area are described. The immunohistochemical stains for S-100 protein, glial fibrillary acidic protein (GFAP), and Melan-A were used to elucidate the diagnosis of PNST. Results: Mean age of the three dogs was 11-years-old. Gender distribution consisted of a spayed female, castrated male, and an intact male. Breeds represented were Labrador Retriever mix, Labrador Retriever, and a Schipperke. The right eye was affected in 2 dogs, and the left eye in one dog. The lower eyelid was affected in two dogs, and the retrobulbar area in one dog. One dog had tumor re-growth after eyelid reconstruction requiring exenteration; another dog was exenterated after initial presentation, and one dog is clinically free of re-growth after eyelid repair using a lip-to-lid reconstruction. Both dogs requiring exenteration have had subsequent re-growth in and around the orbit. Histomorphologic evaluation of the tumors revealed whorls of neoplastic spindle cells with mitotic figures ranging from 3 to 8 per high power field. Both dogs requiring exenteration with subsequent re-growth had neoplastic cells extending to surgical margins suggesting incomplete excision; whereas the dog that remains clinically free of re-growth had a well delineated mass that did not extend to any margin. Immunohistochemical evaluation of the neoplastic cells showed some to be strongly positive for S-100 protein, while others were negative. Immunohistochemical stains for GFAP and Melan-A were diffusely negative. Conclusions: Definitive diagnosis of peripheral nerve sheath tumors in dogs is difficult. No consistently sensitive and specific antigenic markers for these neoplasms exist; therefore immunohistochemistry may, at best, lend support to a diagnosis. The quagmire of controversy among pathologists is distinguishing low-grade stromal sarcomas and spindle cell melanomas from PNST. Nerve sheath tumors tend to be infiltrative, locally aggressive with a high rate of recurrence, but rarely metastasize. Location of the neoplasm and the ability to excise well outside the neoplastic margins may dictate recurrence. Although ocular PNST's are rare in animals, the same findings were substantiated recently in the feline. Commercial interest: None.

ABSTRACT NO.: 27

Electroretinographic evaluation of supplementation including lutein for canine progressive retinal atrophy

Y. Umeda, S. Maehara, S. Wakaiki, K. Tsuzuki, K. Yamashita, Y. Izumisawa and N. Itoh

Rakuno Gakuen University, School of Veterinary Medicine, Department of Companion Animal Clinical Sciences, Ebetsu, Japan

Purpose: Lutein belongs to the large class of plant pigments referred to as carotenoids, and its antioxidative activity in the retina has been reported. In human ophthalmology, lutein is administrated for cataract and age-related maculopathy. In this study we evaluated a supplement including lutein by electroretinography on canine progressive retinal atrophy (PRA). Methods: The subjects were 11 dogs diagnosed as having PRA by history, funduscopy and electroretinography. The breeds included 3 miniature dachshund, 2 golden retriever and one miniature schnauzer, Akita, English cocker spaniel, Welsh corgi, Yorkshire terrier and American cocker spaniel. The dogs was prescribed an oral supplement containing 3.0 mg lutein, 3.0 mg lycopene, 1.5 mg beta-carotene, 0.7 mg alfa-carotene and 0.2 mg zeaxanthin, daily for 120 days. Electroretinography was performed for all dogs before and after 120 days of supplementation. Furthermore, electroretinography was performed in 2 of 11 dogs 120 days after termination of supplementation. Four types of electroretinography included rod response, standard flash combined rod and cone response, single flash cone response and 30 Hz flicker cone response were performed. Results: Electroretinograms recorded before supplementation were reduced in 7 dogs and were nonrecordablein in 4 dogs. In the rod response and combined rod and cone response post supplementation, amplitude was increased in 5 of 7 dogs (P < 0.05, paired t-test). In 2 of 5 dogs that showed recovery of amplitude with supplementation, electroretinography was performed 120 days after terminate supplementation, and decreased amplitudes was observed in every response (P < 0.05, paired t-test) in both dogs. In 4 dogs that showed a nonrecordable electroretinogram before supplementation, no recovery in the electroretinogram was recorded after supplementation. Conclusion: Recovery of retinal electrophysiological function in dog with PRA was demonstrated after treatment with a supplementation including lutein. Further investigation is needed in many clinical cases; however, our findings suggest that the progression of canine PRA is inhibited by antioxidant supplementation including lutein. Commercial interest: None.

ABSTRACT NO.: 28

Retinal pigment epithelial degeneration observed in experimental hyperlipidemia model in dog

S. Wakaiki,* E. Uchida,* T. Sako,† H. Taniyama,† S. Maehara,* K. Tsuzuki,* K. Yamashita,* Y. Izumisawa* and N. Itoh*

*Rakuno Gakuen University, School of Veterinary Medicine, Department of Companion Animal Clinical Sciences,Rakuno Gakuen University, School of Veterinary Medicine, Department of Veterinary Pathology, Ebetsu, Japan

Purpose: Retinal pigment epithelial degeneration (RPED) is a condition characterized by accumulation of irregularly shaped spots with various size in the tapetal fundus. In this case series, we examined the results of ophthalmologic examinations, biochemical analyzes of blood samples, and histopathologic findings in three similar cases of RPED observed in an experimental hyperlipidemia model in the dog. Methods: Three male beagles were fed a high-calorie diet from two to six years of age to induce hyperlipidemia. Ophthalmologic evaluation including menace reflex, pupillary light reflex, intraocular pressure, ophthalmoscopic examination and electroretinography (ERG) was conducted in all cases. Fluorescein angiography (FAG) was performed in two dogs. Blood lipid (total cholesterol and triglyceride) and carbohydrate (glucose and fructosamine) levels were measured monthly for four years. Tissue samples were obtained when a retinal lesion was discovered, and were embedded in paraffin and stained with hematoxylin and eosin. Results: In all three cases, blood total cholesterol level was markedly high. However, triglyceride, glucose and fructosamine were within normal limits. Ophthalmoscopic examinations in all three cases revealed hyperreflective and irregularly shaped spots of various sizes scattered throughout the tapetal fundus. The nontapetal fundus showed depigmentation of the retinal pigment epithelium (RPE). ERG responses were reduced in both eyes, showing a marked reduction of the standard flash b-wave in all three dogs. FAG analysis in two dogs showed numerous spots, which altered from hypofluorescent in the choroidal phase to hyperfluorescent in the retinal venous phase. Histopathologic examination revealed hypertrophy of RPE cells forming nests. Discussion: The fundi of all three dogs showed characteristic features of RPED. The marked reduction of b-wave amplitude in ERG, existence of spots which changed from hypofluorescent to hyperfluorescent, and histopathologic findings were also consistent with RPED. Long-term hyperlipidemia is thus suggested to be one of the factors inducing RPED. Commercial interest: None.

ABSTRACT NO.: 29

Cone dysfunction with digoxin in dogs detected by multi color electroretinography using contact lens electrode with built in light source

S. Maehara, A. Ohsawa, S. Wakaiki, K. Tsuzuki, K. Yamashita, Y. Izumisawa and N. Itoh

Rakuno Gakuen University, School of Veterinary Medicine, Department of Companion Animal Clinical Sciences, Ebetsu, Japan

Purpose: It is difficult to functionally evaluate different color pathways with present conventional electroretinogram (ERG). In this study, we have developed contact lens electrodes with built in color: red (644 nm), green (525 nm), and blue (470 nm) light source (color LED-electrode). We further studied the effect of experimental digoxin intoxication in dogs using this system. Methods: Color ERG recordings using the newly developed color LED-electrodes were conducted in 17 normal beagle dogs to evaluate their properties. We used these electrodes to evaluate the effect of digoxin toxicity in 7 beagle dogs. Digoxin was administrated orally twice a day as a single dose of 0.0125 mg/kg for 14 days. Color ERG recordings and measurement of plasma concentration of digoxin were performed before and after administration and after termination. We recorded rod response, standard flash combined rod and cone response, single flash cone response and 30 Hz flicker cone response using the new electrodes. Results: ERG recording in normal dogs revealed that responses obtained by red electrode were significantly smaller than those with blue or green electrode (P < 0.01, student's t-test) and showed variable results: According to these observations, the red electrode was not used further. In the digoxin intoxication experiment, dogs showed a high plasma concentration of digoxin (2.4 ng/mL) on the 14th day after initiation of administration, and a decreased concentration of digoxin 14 day after termination (< 0.1 ng/mL). In rod response, no abnormalities were detected by ERG with each electrode. In combined rod and cone response, decreased amplitude in a-wave was detected by ERG with the white, blue and green electrodes (P < 0.01, paired t-test). In single flash cone response, latency was detected by color ERG with the blue and green electrodes (P < 0.01, paired t-test). In flicker cone response, decreased amplitude was only detected by color ERG with the blue electrode (P < 0.01, paired t-test). Decreased amplitude and extended latency recovered after termination of administration. Conclusion: Color ERG recordings using the blue or green LED-electrode effectively detected cone dysfunction induced by digoxin in dogs. ERG recordings with color stimuli may be more sensitive ways of evaluating cone function than those with white light stimuli. Commercial interest: None.

ABSTRACT NO.: 30

Evaluation of effect of the modified medial canthoplasty (mmc) on corneal epitheliopathy of a shih-tzu dog by the lacrimal flow observation

A. Saito and A. Tuduki

Triangle Animal Hospital, Toyko, Japan

Purpose: The fact that the dog of brachycephalic breeds with euryblepharon were commonly affected by corneal epitheliopathy has been known. Modified medial canthoplasty (MMC) is our recommended therapy for the treatment of these cases (2002 ACVO). The present report evaluates the effect of MMC during the observation of affected areas of corneal epithelium and the change of lacrimal flow behavior of prepost MMC treatment. Methods: The MMC was operated on a Shih Tzu dog (8 months old, male) with epiphora and corneal epitheliopathy. Observation of corneal epitheliopathy and the behavior of tear flow was conducted on pre- and post operation of MMC. A slit-lamp microscope (BQ, Haag Streit), digital filing system (VK-2, Kowa), DVD recorder (DMR-E30, Panasonic) fluorescein/Rose Bengal staining solution (instilled 1 µL/oneeye) were used for observation of tear flow and ocular surface. Result: Before the MMC treatment, the corneal surface was not covered with tear fluid by blinking. However, it became apparent that the cornea in contact with residual tear fluid around lower eyelid gave no sign of corneal epitheliopathy by the observation before MMC. And the corneal surface was not covered with tear fluid by blinking. After the MMC treatment, tear fluid was spread over the ocular surface and corneal epitheliopathy was remarkably improved. Discussion: In this case, the affected area of corneal epitheliopathy related to the condition of the tear fluid covering on ocular surface by blinking. It is thought that the uncompleted blinking is the cause of corneal epitheliopathy. In this study, however, residual tear fluid and its covering condition on the ocular surface was considered to be affective factor. The MMC was found to be an effective treatment for corneal epitheliopathy of brachycephalic breeds with euryblepharon because it improved the tear flow behavior. Commercial interest: None.

ABSTRACT NO.: 31

Evaluation of the tear formation during the neonatal period in small breed dogs, using the trimmed Schirmer tear test: STT1 & STT2

E. Garcia Da Silva* and P. D. Galera†

*Clínica Veterinária Lago Norte, Brasília/Brazil;College of Veterinary Medicine, University of Brasilia/Brazil

Purpose: The ability of newborns to produce tears has been debated in human literature over many decades. In the past there had been a general belief that lacrimal production is diminished or even absent in preterm and term infants. This prospective study intends to evaluate whether newborn dogs have a substantial aqueous tear production as well as to compare the results to the human ophthalmology data. The trimmed Schirmer tear test (TSTT) is also evaluated as a method for measuring reflex and basal tear secretion in canine neonates. Methods: Thirty healthy puppies were evaluated from six littermates, with each one having five animals randomly selected. Lhasa Apso, Poodle, West Highland White Terrier, Yorkshire Terrier, Miniature Pinscher and Shi-Tsu were the breeds studied. The age chosen to be evaluated was the forth week after birth, since the canine palpebral fissure opens between 10 and 15 days. Puppies on which an excessive restraint was required, as well as crying or being recently fed, were promptly excluded. The TSTT strip was obtained with the use of a n.64 beaver blade, to accurately transect a 5 mm-width commercial tear test strip in two 2.5 mm-width strips. Its anterior portion, which was bent at the notch to place in the inferior fornix, was remodeled in order to be identical in both strips. The TSTT1 was measured after one minute in both eyes. A topical anesthetic was used afterwards to conduct the TSTT2, under the following order: one minute after instilling the drop, the surface of the eye was wiped off with soft cotton tips, having the measurement being done after one additional minute. Both strips always touched the corneal surface. Mean values (± SD) were calculated. Results: The age of the puppies ranged from 22 to 28 days. The mean TSTT1s OD were 13.63 ± 3.45 mm/min, the mean TSTT1s OS were 13.56 ± 2.68 mm/min, being the final average of the TSTT1 13.6 ± 3.07 mm/min. The mean TSTT2s OD were 3.73 ± 1.76 mm/min, the mean TSTT2s OS were 3.63 ± 1.77 mm/min, being the final average of the TSTT2 3.68 ± 1.75 mm/min. Conclusions: Recent studies have established that the majority of term infants secrets tears normally and preterm infants have reduced basal and reflex tears. The TSTT which has been advocated in birds because of their small palpebral fissure, can successfully measure basal and reflex tear production in canine neonates. We concluded that small breed dogs do produce tears during the neonatal period, although they have significantly reduced reflex and basal tear secretion. Therefore the tear production in normal canine neonates could be potentially similar to that in human preterm infants. To the author's knowledge, there have been no previous articles evaluating the development of tearing during the neonatal period in the small animal medicine. Commercial interest: None.

ABSTRACT NO.: 32

Age related changes in electroretinogram in Beagle dogs

Y. Itoh,* S. Maehara,† A. Osawa,† N. Takiyama,* O. Igarashi,‡ A. Saito§ and N. Mizuki*

*Department of Ophthalmology & Visual Science, Graduate School of Medicine, Yokohama City University;Department of Companion Animal Clinical Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University;Kushiro Animal Hospital;§Triangle Animal Hospital, Toyko, Japan

Purpose: Single flash electroretinogram (ERG) has been reported to show the maximum amplitude in the young adult levels (15–24 years) and declines with age in humans. This investigation was designed to evaluate the effect of age on ERG in normal beagle dogs. Methods: Forty-six healthy beagle dogs (38 males and 8 females aged 8 months to 8 years, weight: 10.53 ± 0.7 kg) with normal vision and normal ocular fundi underwent the ERG using a contact lens electrode with a built-in Light-Emitting Diodes. ERG was performed with a TOMEY Corporation Medical LE-2000. Each response (Rod, Standard Flash (SF), Cone and 30-Hz Flicker (Flicker)) were recorded in both eyes following the standard protocol of International Society for clinical Electrophysiology of Vision and reporting of Kotani (Kotani et al. ACVO 2001) under sedation. Pupils were dilated with an ophthalmic solution consisting of 0.5% tropicamide and 0.5% phenylephrine. The ERG was recorded with the dogs in ventral decubitus position under sedation following 30 min of dark adaptation. We performed linear regression analysis and compared averages of each ERG response against age. Results: We found significant age-dependent reduction in the amplitude of Rod (Spearman, r = −0.23; P = 0.22), SF-a (r = −0.32; P = 0.001), SF-b (r =−0.47; P < 0.001), cone (r = −0.31; P = 0.003) and Flicker (r = −0.23; P = 0.03) responses, and significant decrease in the b/a ratio of the SF response (r = −0.39; P < 0.001), but no significant age correlations between the implicit times for all responses on those scatter grams. On average comparison, there was a tendency that all amplitudes began reduction at 6–7 years of age and no tendency aging correlation on each implicit time. Conclusions: Our results would provide evidence that age related changes in ERG responses in beagle dogs begin in the young adult levels (about 1 year), similar to the report in humans. Furthermore, an increased tendency of the b/a ratio between 7 years and 8 years is caused by higher reduction of SF-a. Commercial interest: None.

ABSTRACT NO.: 33

Experimental vogt-koyanagi-harada disease model in Akita dogs

N. Takiyama,* K. Yamaki,† S. Maehara,‡ S. Wakaiki,‡ N. Mizuki* and N. Itoh*

*Department of Ophthalmology & Visual Science, Graduate school of Medicine, Yokohama City University;Department of Ophthalmology, School of Medicine, Akita University,Department of Companion Animal Clinical Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan

Purpose: To establish an experimental model of Vogt-Koyanagi-Harada (VKH) disease in dog. Methods: Two Akita dogs were used in this study (1 year-old male and 2 years-old female). Both dogs were immunized with peptide mixtures of human Tyrosinase Related Protein (TRP) 1–1 to TRP 1–22 (1 mg of each peptide/dog). The male dog was immunized once and sacrificed about 20 months after immunization. At 15 months after the immunization, the female dog was immunized again and sacrificed 3 weeks later. The ophthalmological examination included funduscopy, slit-lamp biomicroscopy, and fluorescein angiography (FA) was performed. The histopathological examination was performed for eyes, skin, and brain. Results: Both dogs developed bilateral uveitis and exudative retinal detachment 2 weeks after the immunization. FA at 3 weeks after immunization showed multifocal leakages from the choroids. This uveitis gradually remitted. Three months after immunization, depigmentation of funds and skin were observed and gradually enlarged. Histopathologically, the exudative retinal detachment, thickness of choroid with infiltration of inflammatory cells, depigmentation, pigment dispersion and Dalen-Fuchs nodules were seen in all eyes. Depigmentation, pigment dispersion, and infiltration of inflammatory cells were seen around hair follicles and vessels of skin. In the subarachnoid space, inflammatory cells were infiltrated. Conclusions: The clinical course and histopathological changes were very similar to those seen in Akita dogs and human with spontaneously occurring VKH disease. We conclude that VKH disease has been successfully induced in Akita dogs. This model would help to study the mechanisms of human and dog VKH disease. Financial support: Supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (No 08672006, no. 09671784). Commercial interest: None.

ABSTRACT NO.: 34

Ectopic cilium in seven horses

S. D. Hurn,* A. G. Turner* and C. McCowan†

*Melbourne Veterinary Referral Center 70 Blackburn Road, Glen Waverley Victoria, Australia, 3150,The University of Melbourne, Veterinary Clinical Center, 250 Princes Highway Werribee, Victoria, Australia, 3030

Purpose: To describe the clinical presentation, clinical diagnosis, histopathologic diagnosis and surgical treatment of ectopic cilium in seven horse. Methods: This is a case series presentation. Ectopic cilium was diagnosed by slit lamp biomicroscopy and treated surgically by transconjunctival excision in seven adult horses. Histopathology was performed in two cases. Results: All cases presented with a common history of blepharospasm, ocular discharge and keratitis. Ophthalmic examination revealed a single translucent cilium in the upper central eyelid palpebral conjunctiva, emerging approximately 5 mm from the eyelid margin. Cilia emerged within a small discrete region of conjunctival pallor that was surrounded by conjunctival hyperemia. An area of wedge-shaped superficial vascularization of the adjacent cornea was observed directly below and adjacent the ectopic cilium in all cases. Corneal ulceration was not observed in any horse. All horses responded well to transconjunctival surgical excision and histopathology confirmed a diagnosis of ectopic cilium in two horses. Conclusions: There was recurrence of ectopic cilium following surgery in one horse. This is the first publication of ectopic cilia in the horse and emphasizes their importance as etiology for epiphora, blepharospasm and keratitis. Commercial interest: None.

ABSTRACT NO.: 35

Spectroscopic determination of photofrin® following periocular subcutaneous injection in horses

E. A. Giuliano,* E. J. Morgan,† P. J. Johnson* and S. A. Tucker†

*Veterinary Medicine and Surgery, University of Missouri-Columbia, Columbia, MO; Department of Chemistry, University of Missouri-Columbia, Columbia, MO, USA

Purpose: Local photodynamic therapy (PDT) represents a novel delivery method and treatment modality for equine periocular squamous cell carcinoma with favorable preliminary results: Retention time and tissue dissipation rate of photosensitizer diluted in dimethylsulfoxide (DMSO) after local injection into the tumor bed is currently unknown. A spectroscopic method for determining the concentration of Photofrin® (porfimer sodium) in equine periocular skin is described. Methods: Spectroscopic analysis was performed on samples from multiple equine subjects, under a variety of experimental conditions. Photofrin® was reconstituted in either 5% dextrose/water (D5W) or medical grade DMSO. Periocular equine skin overlying the ventral orbital rim was collected from recently deceased horses using 4, 6, and 8 mm punch biopsies. For initial studies, 1 mL samples were prepared in glass vials by dissolving fresh skin samples from each horse in the Solvable® tissue solubilizer, containing ∼1 µg/mL of Photofrin®. Samples were heated overnight (= 12 h at 50–55 °C) to dissolve the tissue. Experimental controls were treated in the same manner and included (1) solvent blanks – neat D5W, DMSO and Solvable®; (2) Photofrin® dissolved in D5W, DMSO or Solvable®; and (3) equine tissue dissolved in Solvable®. Same-size skin samples from each horse were also immediately frozen in liquid nitrogen, stored at −80 °C, and processed identically to fresh tissue samples. Given these results, Photofrin® dissolved in DMSO was injected subcutaneously and 6-mm punch biopsy samples were immediately collected, frozen in liquid nitrogen, and processed as above. Absorption (scan rate of 120 nm/min, a slit width of 1 nm and temperature of 25 °C) and fluorescence (slit widths of 16/8 nm, scan interval of 1 nm and internal average of three) spectra were measured on a Hitachi U-3000 spectrophotometer and a SLM 48000 DSCF/MHF spectrofluorometer, respectively. Results: Comparison of all Photofrin® spectra showed no difference between fresh vs. frozen tissue samples, tissue samples obtained from different punch sizes, or samples from different test subjects. Using the fluorescence emission signal (∼627 nm – excitation of 403 nm) from a series of Photofrin®: tissue standards, a calibration plot was generated to quantitatively determine of the concentration of Photofrin® after subcutaneous injection in treated horses. All fluorescence spectra were absorption and blank corrected. Conclusions: Photofrin® can be quantified in equine skin following local injection using spectroscopic analysis. Unlike other detection modalities, our experimental results were not affected by native equine skin fluorescence. Further studies are underway to evaluate the tissue dissipation rate of Photofrin® following subcutaneous injection in live horses. Commercial interest: None.

ABSTRACT NO.: 36

Treatment of equine periocular squamous cell carcinoma with photodynamic therapy: 5 cases

G. Klauss,* E. A. Giuliano,* D. L. McCaw,* I. MacDonald,† P. J. Johnson,* 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: To report the successful treatment of 5 cases of equine periocular squamous cell carcinoma (PCSS) with surgical excision and photodynamic therapy (PDT), a novel treatment modality, using pyropheophorbide-a-hexyl-ether (HPPH, Photochlor™). Methods: Five horses with naturally occurring PSCC confined to the eyelid with no evidence of orbital invasion or distant metastasis were included in this study. Horses underwent complete ophthalmic examination, extraocular photography, 3-dimensional tumor measurement, and biopsy of tumor to confirm diagnosis of SCC. Horses were placed under general anesthesia and tumors were surgically debulked. The resultant wound bed was infiltrated with pyropheophorbide-a-hexyl-ether (HPPH, Photochlor™) in dimethylsulfoxide (final concentration: 2 mg/mL; treatment dose = 1 mg/cm2 of tumor bed). Surgical beds were irradiated using a 665-nm wavelength diodelaser (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. Horses were regularly evaluated postoperatively with complete ophthalmic examinations and repeated biopsy. Results: Two of 5 horses with refractory PSCC had been unsuccessfully treated with surgical excision and cryotherapy with or without intralesional cisplatin prior to PDT. Surgical excision and PDT has yielded disease-free intervals of 27, 21.5, 20, 2.5 and 2 months. Four horses received a single treatment, and 1 horse required 2 treatments for apparent cure of tumor. In 1 horse, carcinoma in situ developed 2.5 months after surgical excision and PDT, requiring local excision under standing sedation with no subsequent tumor regrowth. Minor complications in the first 2 weeks postoperatively included eyelid swelling and regional tissue necrosis, typical of PDT. In 2 horses with PSCC involving the medial canthus, the inferior and superior nasolacrimal punctae were sacrificed intraoperatively and subsequent mild chronic ocular discharge or epiphora occurred. These 2 horses also experienced mild-moderate narrowing of the palpebral fissure, necessitating a medial canthoplasty to successfully widen the palpebral fissure in 1 horse. Conclusions: Surgical excision and local PDT with HPPH represents a novel, safe, and successful treatment modality for equine PSCC. Fewer treatment episodes, shorter hospitalization periods, reduced overall cost, good cosmesis and excellent preservation of eyelid function are additional advantages of PDT in the treatment of equine PSCC compared to other current treatment modalities. Study results have implications for the treatment of periocular SCC in other species. Supported by a grant from the ACVO Vision for Animals Foundation. Commercial interest: None.

ABSTRACT NO.: 37

Retrograde axonal transport obstruction of brain derived neurotrophic factor and its receptors TrkB and p75 in retinal and optic nerve in dogs with spontaneous glaucomatous eyes

S. Iwabe,* G. A. Garcia-Sanchez* and N. Moreno-Mendoza†

*Veterinary Medicine College, UNAM, Mexico,Biomedical Research Institute, UNAM, Mexico, Mexico City, Mexico

Purpose: To demonstrate that a retrograde axonal transport obstruction in optic nerve head at lamina cribosa level produce Brain-Derive Neurotrophic Factor delivery deprivation leading to apoptosis and RGC death in dogs presenting high intraocular pressure. Methods:  Immunocytochemistry analysis was performed to evaluate brain-derived neurotrophic factor (BDNF) and its receptors TrkB and p75 proteins expression in retina from normal and glaucomatous dogs. Immunofluorescent staining of retinal sections with fluorescence secondary antibodies was also performed to confirm protein for the neurotrophin and its receptors localization, as well as, a double-labeling to colocalize both receptor types. Results: In those normal eyes BDNF, TrkB and p75 immunoreactivity were detected in retinal ganglion cell layer (RGC), nerve fiber layer (NFL), photoreceptor layer, lamina cribosa cells, optic nerve head (ONH) astrocytes and optic nerve head tissue. BDNF was also observed in outer nuclear layer (ONL) and outer plexiform layer (OPL), as well as, TrkB and p75 receptors were detected in inner nuclear layer (INL) and inner plexiform layer (IPL). A more intensive labeling of TrkB was observed at lamina cribosa level and ONH. Also a specific expression of BDNF and TrkB was present in neurons body and dendrites along their axis. However none of these proteins were seen in retinal pigment epithelium (RPE). Considering an axonal transport obstruction due to an IOP elevation, in glaucomatous dog eyes the blockage was evident next to optic nerve head at lamina cribosa level where BDNF had a more intense labeling. TrkB and p75 labeling were more intense in glaucomatous than in normal retina at NFL and GCL level, as well as a more intensive pattern at (ONH) level at glial cells and in the prelaminar portions. Conclusion: Interruption of BDNF retrograde transport is substantially inhibited by intraocular pressure elevation. TrkB and p75 accumulation at the ONH in glaucoma suggests a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma, as well as, a paracrine and/or autocrine signaling within the lamina cribosa. Neurotrophin signaling may regulate more than neuronal development, survival and differentiation. BDNF neurotrophin and its receptors expression by lamina cribosa cells and ONH astrocytes in glaucomatous eyes may help to determine the role between of these cells as a paracrine source and these proteins in terms of retinal ganglion cell survival. Commercial interest: None.

ABSTRACT NO.: 38

Immunohistochemical localization of cancer related protein kinases in normal monkey retina with comparison to human retina

C. Giudice,* R. Ferrari,† Anna Maria Giusti† and A. Terron†

*DIPAV: Section of Pathology, University of Milan, Italy,Nerviano Medical Science. Preclinical Development. Pathology Department, Milan, Italy

Purpose: Kinase inhibitors are among the most advanced compounds in development using the new drug discovery paradigm of developing small-molecules drugs against molecular targets in cancer. The cyclin-dependent kinases (CDKs) are intimately involved in the pathway of controlling cell proliferation and disruption of this pathway is a recognized molecular event leading to cancer. However, CDKs, and structurally closely related kinases, such as GSK3, are also expressed in long-term life span cells, thus understanding their morphological localization using immunohistochemical detection would represent an important tool for the evaluation of unexpected toxicity during drug development. We evaluated the normal immunohistochemical localization of different kinases in Cynomologus monkey retina and normal human retina. Methods: Eyes from three Cynomologus monkey were removed immediately after death, fixed overnight in Davidson solution and routinely processed for histology. Additional samples from these monkey and from two human eyes were frozen immediately after removal. Immunohistochemistry (Envision and TSA amplification) was performed on 3 monkeys and 2 human eyes with a panel of mAb and pAB against CDK2; CDK4; CDK5; GSK3β; and their phosphorilated forms. Results: Immunohistochemically CDKs expression was similar in human and monkey retina: CDK2 stained always negative, CDK4 stained weakly to moderately positive in photoreceptor (PR), Inner Nuclear Layer (INL) and Ganglion Cell Layer (GCL), GSKβ and pGSKβ stained weakly to moderately positive in PR, Outer and Inner Nuclear Layer (OPL, IPL); CDK5 stained moderately to intensely positive in PR, INL and GCL. Conclusions: Cyclin-dependent kinases are intimately involved in the pathway controlling cell proliferation as well as in the control of sensitivity to cell death in several tissues, including the retina. In addition, CDK5 is also associated with neuronal differentiation and both CDK5 and GSK3β are involved in regulation of cytoskeleton components. Immunohistochemical localization of the different CDKs and GSK3β was indicative of their presence within the retina. Particularly CDK4 and 5 were consistently expressed in the PR layer. CDKs immunohistochemical expression was similar in human and monkey retina. These data indicate that inhibition of cancer targeted kinases could eventually lead to inhibition of additional cellular events unrelated to cell proliferation. Data also suggest that the monkey would represent an appropriate animal model to evaluate kinase inhibitor in the preclinical setting. Commercial interest: None.

ABSTRACT NO.: 39

Retinal pathology in natural cases of ovine scrapie

A. Regnier,* O. Andreoletti,† D. Cayez,* F. Schelcher† and P. L. Toutain*

UMR INRA/ENVT 181 Pharmacologie et Toxicologie Expérimentales, *UMR INRA/ENVT 1225 Interactions Hôte Agent Pathogène,Ecole Nationale Vétérinaire, 31076 Toulouse, France

Purpose: To evaluate ophthalmoscopic, electroretinographic and retinal histologic changes, and determine retinal distribution of pathologic prion protein (PrPSc) in scrapie-affected sheep. Methods: Seventeen scrapie-affected red face Manech ewes at various stages of disease progression and aged between 1 and 3 years were included in this study. Six clinically normal age-matched red face Manech served as controls. Funduscopic examination was done prior to performing electroretinography under general anesthesia. Six specific electroretinogram (ERG) responses were recorded using a computerized ERG system with full-field white light stimulation. Within one to 10 days after the ERG procedure, the animals were humanely euthanized and one randomly selected eye was collected for morphologic analysis of the retina and immunohistochemical detection of PrPSc in the retina. In both groups, diagnosis and scrapie status were eventually confirmed by postmortem histopathological and immunohistochemical examination of brain tissue. Results: No fundoscopic abnormalities were identified in any of scrapie-affected and control animals. In scrapie-affected sheep, the light- and dark-adapted ERG responses were significantly lower (P < 0.05) than those observed in control animals. There was evidence of retinal disease in all scrapie-affected animals on histology. Variable degrees of photoreceptor outer segment degeneration and decrease in the cellular density of the outer nuclear layer were observed in the outer retinal layers. In most cases, a multifocal accumulation of eosinophilic amorphous material was found between outer segments and the retinal pigment epithelium. There was also a decline in the number of cells contained within the inner nuclear layer. Immunostaining for PrPSc was not identified in any of the control retinas, but was observed in the retina from all scrapie-affected animals. PrPSc deposits were commonly found in the ganglion cell layer and inner plexiform layer, and occasionally detected in photoreceptors. In some cases, PrPSc accumulations were more marked in the central area of the retina. Conclusions: Although no appreciable fundoscopic lesions were observed in this sample population of scrapie-affected ewes, retinal dysfunction and morphologic changes were identified in these animals. In addition, detection of PrPSc with immunocytochemistry demonstrated that the disease-specific prion protein accumulates in the retina of sheep with naturally occurring scrapie. Commercial interest: None.

ABSTRACT NO.: 40

A re-examination of the mode of inheritance of posterior cortical cataracts in Labrador and Golden Retrievers

G. Aguirre,* D. Holle,† A. Yu-Speight,‡ C. Sarna† and E. Hall†

*University of Pennsylvania, Philadelphia, PA,The Seeing Eye, Inc., Morristown, NJ,Central Texas Veterinary Ophthalmology, Austin, TX, USA

Purpose: Inherited cataracts in the Labrador and Golden retrievers occur with high frequency, and affect approximately 5–8% of dogs in each breed. In the majority of cases, cataracts appear between 1.5 and 2 years, are bilaterally symmetrical, show minimal progression, and do not impair vision. In a small number of dogs, however, the cataracts are unilateral, or appear after 4–5 years of age. It is not clear whether these atypical cases are sporadic phenocopies, or represent a variable expression of the mutant gene. Efforts to reduce the number of affected dogs have been based on the published genetics of the disorder that considers the cataracts to be inherited as dominant with incomplete penetrance. Unfortunately, such control measures have been unsuccessful in reducing the frequency of the disease in the two breeds. In this study, we re-examine the inheritance of the posterior triangular subcapsular cataracts. Methods: A normal German shepherd male dog was bred to 2 female dogs affected with posterior, subcapsular, axial cataracts that were unilateral (Labrador/Golden retriever backcross) or bilateral (Golden retriever). Each female, respectively, produced 2 litters with a total number of 14 and 21 dogs. All dogs were examined using binocular indirect ophthalmoscopy and biomicroscopy after mydriasis with 1% tropicamide. Results: 12 of the 14 pups produced by the Labrador/Golden backcross female were normal when examined between 2 and 2.3 years of age; the remaining 2 dogs were euthanatized for unrelated medical problems at 1.2 and 1.7 years of age, and were normal at that age. Of the 21 pups produced by the Golden retriever with bilateral cataracts, 11 were normal at 2–2.4 years age. The remaining 10 dogs are not old enough to be past the age of risk, but, at ages ranging from 0.75 to 1.2 years, significant lenticular abnormalities have not been observed. Conclusions: Although this study is still in progress, the preliminary findings would suggest that the posterior subcapsular triangular cataracts present in these breeds are not inherited as dominant with incomplete penetrance. This conclusion would require that all dogs be examined past the age of risk, and, moreover, that the older dogs examined thus far continue to remain free of the cataracts that are characteristic of the breeds. Based on these findings, and the results of pedigree analysis from a larger set of the breeding population, an autosomal recessive mode of inheritance would be more likely. This will have implications, not only for the control of the disorder in these breeds, but also will require revision of the advice given to breeders when animals with these cataracts are identified. Supported by the Van Sloun Fund for Canine Genetic Research, and the Morris Animal Foundation. Commercial interest: None.

ABSTRACT NO.: 41

Transdifferentiation of the gerbil retina pigment epithelium by a copper depletion regimen

J. M. Clinton,* S. A. Baranowitz† and R. A. Eckhardt‡

*Animal Eye Clinic,EpiTek, Inc.,Brooklyn College, Brookyn NY, USA

Purpose: To determine whether a copper depletion regimen would cause transdifferentiation of the Retina Pigment Epithelium into lens in the Mongolian Gerbil, Meriones unguiculatus. Methods: Transdifferentiation, the conversion of one differentiated cell type into another, has been proven in mammals only in the rat pancreas-to-hepatocyte experimental system. The use of a copper chelator, along with a copper-depleted diet, was the method of transdifferention in that system. A similar regimen was tested in gerbils by feeding them with a specialized diet, and comparing this group to a control group fed on a standard laboratory diet. Lentectomy was performed on day 27, the animals were then then sacrificed on day 61 for histological and immunohistochemical analysis. Results: In 45% of the surviving experimental animals, and none of the control animals, regenerated lens were seen. The histologic and immunohistochemical data suggested that the new lenses had derived from transdifferentiated retina pigment epithelium, as occurs naturally in the lens regeneration system. Conclusions: A copper depletion regimen was effective in inducing transdifferentiation of the retina pigment epithelium into lens in the Mongolian Gerbil. Supported by Epitek, Inc. Commercial interest: S. A. Baranowitz (employee); R. A. Eckhardt, None.

ABSTRACT NO.: 42

Effects of ketoprofen, meloxicam and flunixin meglumine in reducing intraocular inflammation

G. Kennard* and M. Gilmour†

*Eye Care For Animals, Phoenix, Arizona,College of Veterinary Medicine, Oklahoma State University, Stillwater OK, USA

Purpose: (i) To determine in dogs the intraocular anti-inflammatory effects of ketoprofen, meloxicam and flunixin meglumine. (ii) To compare the relative intraocular anti-inflammatory effects of ketoprofen, meloxicam and flunixin meglumine. Methods: Twenty-four normal dogs undergoing terminal surgery were studied. All dogs were examined one day before the study to verify no pre-existing intraocular inflammation. The dogs were divided into four groups of six: a control group, which received intravenous (IV) saline; and three treatment groups, the ketoprofen, meloxicam and flunixin groups, which received ketoprofen IV, meloxicam IV and flunixin meglumine IV, respectively. All treatments were administered at general anesthetic induction. Thirty minutes following saline or NSAID administration, a limbal paracentesis of both eyes was performed using a 27-guage needle, and 200 µL of aqueous humor (AH) was aspirated. A second paracentesis was performed one hour after the first, using the same procedure. AH samples were stored at −80 °C for later protein assay. AH protein levels were measured using a commercial chemistry analyzer utilizing the biuret reaction and colormetric detection at 550 nm wavelength and 600 nm wavelength. Results: AH protein levels in the first collection sample were not significantly different between the four groups. Average protein level measured (mean ± SD) 0.114 mg/mL ± 0.052 mg/mL. Protein levels in the second collection sample measured 18.327 mg/mL ± 10.646 mg/mL in the control group and 12.445 mg/mL ± 7.446 mg/mL, 18.584 mg/mL ± 10.316 mg/mL and 16.930 mg/mL ± 8.696 mg/mL for the ketoprofen, meloxicam and flunixin groups, respectively. The difference between protein levels in the second and first collection samples from all four groups were statistically significant. The difference in protein levels between all four groups in the second collection sample was not statistically significant. Conclusions: (i) Preoperative intravenous administration of ketoprofen, meloxicam or flunixin meglumine does not seem to cause a significant reduction in intraocular inflammation in normal dogs. (ii) AH paracentesis is a reliable method for inducing anterior uveitis in dogs. Supported by Eye Care for Animals, Phoenix Arizona and by the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University. Commercial interest: None.

ABSTRACT NO.: 43

Early glutamate redistribution in the retina of dba/2j mice with glaucoma

H. C. Low,* J. R. Gionfriddo,* C. C. Powell* and J. E. Madl†

*Department of Clinical Sciences, Colorado State University,Department of Biomedical Sciences, Colorado State University, Fort Collins CO, USA

Purpose: Ischemia and glutamate release may contribute to retinal damage in glaucoma. We examined if ischemia-like glutamate redistribution occurs in the DBA/2 J (D2) mouse model of glaucoma. Methods: Eyes from female control C57BL6 and D2 mice were rapidly dissected and fixed by immersion. Adjacent 0.5 µm plastic sections were stained with toluidine blue for morphometry or immunogold stained to measure glutamate or glial fibrillary acidic protein (GFAP) levels in retinal cells. Results: In control mice, putative Muller cells contained very low levels of glutamate (55 + 3 gray scale) and GFAP while neurons of the ganglion cell layer (GCL) contained higher levels of glutamate (90–170 gray scale). Similar immunostaining was seen in D2 mice at eight months of age. In retinas of older D2 mice (10–12 months), signs of retinal damage were seen including decreased GCL cell densities, high levels of GFAP in Muller cells, and neuronal swelling. Some regions contained Muller cells with significantly more glutamate than controls (90 + 2 gray scale; P < 0.01 by t-test) while neurons maintained normal glutamate levels. In regions with neuronal loss, neurons frequently had reduced glutamate levels (< 75 gray scale) while Muller cells contained increased levels of glutamate compared to controls. Conclusions: Retinal damage in D2 mice varies in intensity in different areas. Less damaged regions with normal neuronal density contained stressed, GFAP-positive Muller cells that accumulated glutamate. In regions with decreased numbers of neurons, glutamate levels decreased in many neurons and increased in Muller cell. Changes of glutamate distribution consistent with ischemia may occur in early stages of glaucomatous retinal damage and contribute to neurotoxicity in D2 mice. Financial support: Funding for these studies was provided by grants from the College Research Council of Colorado State University. Commercial interest: None.

ABSTRACT NO.: 44

Effects of diode laser cyclophotocoagulation on corneal nerve morphology in the dog

N. C. La Croix,* M. A. Gilmour,† R. R. Dubeilzig‡ and C. F. Marfurt§

*Eye Care For Animals; Department of Veterinary Clinical Sciences, Oklahoma State University College of Veterinary Medicine;School of Veterinary Medicine, University of Wisconsin-Madison;§Indiana University School of Medicine, Indianpolis IN, USA

Purpose: To determine if corneal nerve damage (changes in sensitivity and/or morphology) follows diode laser cyclophotocoagulation in the dog. Methods: Twenty-four hours prior to cyclophotocoagulation 12 dogs were subjected to ophthalmic examination that included: slit-lamp biomicroscopy, indirect ophthalmoscopy, Schirmer tear testing (STT), fluorescein staining, intraocular pressure (IOP) testing, and corneal touch threshold (CTT) sensitivity testing in central corneal quadrants. Diode laser cyclophotocoagulation was performed under propofol (Schering-Plough Animal Health, Union, NJ) anesthesia 54 h prior to euthanasia. The direct contact cyclophotocoagulation tip contacted the sclera 5 mm caudal to the limbus, over the approximate location of the pars plicata of the ciliary body. A total of 100 J of laser energy was delivered to the ciliary body of the left eye of each dog. Energy was delivered as 1000 mWatt × 3000 ms (or 3 J per site) over 33 sites. The dogs’ right eyes served as untreated controls. Ophthalmic examinations were repeated 24 and 48 h after treatment. Six hours after the last exam, dogs were euthanized, corneas were resected from each globe, and in vivo corneal positioning marked with colored sutures. Eight pairs of treated and contralateral control corneas were fixed and sectioned. Corneal nerves were visualized in tangential and perpendicular sections by immunohistochemical staining with an antibody directed against neuronal class III beta-tubulin. Nerve bundles were counted for 7 pairs of corneas, by one observer who did not know which cornea had been treated. Twelve treated globes without corneas were processed for histopathology. Results: Corneal ulcerations did not occur, and there were no statistically significant changes in STT, IOP, and CTT sensitivity values. Dyscoria was found in 50% of treated eyes (0% control) 24 h post cyclophotocoagulation. Flare was present in 67% of treated eyes (0% control) at this time point. Anisocoria (treated pupil larger then control) developed in all 12 animals 48 h post treatment. One treated eye (< 10%) presented a retinal detachment. Nearly identical numbers of major stromal nerve bundles (average of 17.3 treated, 17.4 control) were found in the 7 pairs of corneas. Stromal nerve bundles in 50% of treated eyes (0% control), contained small numbers of degenerating axons. Histopathology revealed areas of ciliary nerve necrosis in 33% of treated eyes. Conclusions: Diode laser cyclophotocoagulation causes limited corneal nerve fiber damage. Prominent nerve fiber bundle dropout, previously reported for Nd:YAG cyclophotocoagulation, was not observed for the diode laser. Supported by a grant from Eye Care for Animals. Commercial interest: None.

ABSTRACT NO.: 45

A retrospective, morphological study to establish evisceration case selection criteria

N. S. Lester, R. R. Dubielzig and A. Hunkler

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

Purpose: To survey current attitudes regarding clinical indications for evisceration vs. enucleation in dogs and cats and to retrospectively determine the rate of and the underlying cause for enucleation subsequent to evisceration. The intention was to establish criteria for appropriate evisceration case selection, thereby, help reduce scleral shell failure. Methods: An E-mail: survey was conducted using the ACVO list-serve regarding evisceration case selection – 17 ophthalmologists responded. Archived morphologic specimens from the Comparative Ocular Pathology Laboratory of Wisconsin were also evaluated. Evisceration samples from dogs and cats were reviewed as well as scleral shells submitted after failure. The written record and the histologic material were reviewed when needed. Results: 17/17 of the survey respondents indicated that intrascleral prosthesis implantation was appropriate for primary glaucoma. 16/17 respondents thought the procedure was appropriate in the face of uveitis or trauma. 14/15 did not think that evisceration was appropriate in the face of infectious disease and 16/16 respondents did not think that the procedure was appropriate in the face of neoplasia. 55/754 canine evisceration samples had tumors, 43 of which were benign and 12 of which were malignant. 57/754 canine evisceration samples had supprative/granulomatous inflammation, almost always idiopathic. 5/45 feline eviscerations had tumors, all of which were malignant. Corneal disease leading to perforation or severe inflammation (12/32) and recurrent neoplasia (12/32) were the two main reasons for scleral shell failure in this study. The ratio of eviscerations with tumors to scleral shells with tumors were 6.8/1 (34/1 overall) in dogs and 1.25/1 (7.5/1 overall) in cats. Conclusions: The survey revealed that most of the respondents did not find it appropriate to eviscerate when infection or neoplasia was involved but found it acceptable to perform an evisceration in the face of uveitis. Data from the retrospective study showed that for every 6.8 canine eviscerations with a tumor, there is 1 scleral shell failure due to tumor. Both the overall rate of failure and the failure in the face of tumor is much worse in cats since 1/7.5 eviscerations in cats failed. No specific recommendation can be made regarding corneal disease despite a high failure rate due to corneal disease because it could not be determined what the status of the cornea was prior to evisceration. However, the presence of significant corneal disease or conditions which predispose to corneal disease (e.g. KCS, lagophthalmos) should be carefully considered prior to recommending evisceration surgery. Commercial interest: None.

ABSTRACT NO.: 46

Role of tetracyclines in healing of canine refractory corneal ulcers

H. L. Chandler, C. M. H. Colitz and D. F. Kusewitt

The Ohio State University, College of Veterinary Medicine, Department of Veterinary Biosciences, Columbus, Ohio, USA

Purpose: Epithelial defects in the cornea usually heal rapidly and without scarring, however, in refractory corneal ulcers, re-epithelialization does not occur normally. Normal corneal re-epithelialization occurs by the process of epithelial-mesenchymal transition. (EMT), the transformation of anchored epithelial cells into migrating fibroblast-like cells. EMT occurs during wound healing, where it is initiated by growth factors such as transforming growth factor-β (TGF-β) that stimulate production of Slug and Snail, members of the Snail family of transcription factors. These transcription factors modulate the changes in gene expression leading to EMT. Tetracyclines are of therapeutic benefit in treating refractory corneal ulcers. It is known that tetracyclines enhance production and activation of TGF-β and suppress protease production. Based on these observations, we hypothesize that treatment with tetracyclines stimulate increased TGF-β activity, leading to increased expression of Snail family transcription factors and, ultimately, the EMT required for successful corneal wound healing. Methods: Clinical samples of debrided ulcer edges and keratectomies of corneal ulcers were examined to determine if there was a failure to induce Slug and other contractile elements at the margins of refractory corneal ulcers; in the absence of such induction, EMT cannot occur normally. Routine immunohistochemistry was used to examine the expression of E-cadherin, β-catenin, β-actin, α-smooth muscle actin (α-SMA), tropomyosin and desmoplakin. Western blot and quantitative real-time PCR was used to examine the expression of Slug protein and mRNA in keratectomy samples compared to normal corneas. Corneal epithelial cells were wounded in vitro, then treated with 100 or 1 µg/mL of oxytetracycline. Wound closure was monitored over 72 h. Results: Slug was present in basal cells of the normal cornea. There was decreased expression of Slug protein and mRNA in refractory ulcers compared to normal corneas. E-cadherin, β-catenin, β-actin and desmoplakin expression was localized to the cell membranes while α-SMA and tropomyosin were not expressed in refractory ulcers. 100 µg/mL of oxytetracycline increased the rate of wound closure in corneal epithelial cells compared to untreated controls. Conclusions: During normal re-epithelialization, E-cadherin, β-catenin, and β-actin are relocated from the cell membrane to the cytoplasm and α-SMA and tropomyosin are expressed in cells actively migrating across the wound. In refractory ulcers, the presence and location of these elements indicates that proper EMT is not occurring. As well, during normal wound healing, Slug expression increases in cell undergoing EMT. Refractory ulcers have Slug expression lower than normal basal levels, indicating that abnormal cell migration is occurring. In vitro treatment of wounded corneal epithelial cells with oxytetracycline showed an increase rate of cell migration. Treatment of refractory ulcers with tetracycline may increase the expression of Slug and necessary contractile elements to normal levels, and allow normal re-epithelialization of the wound to occur. Commercial interest: None.

ABSTRACT NO.: 47

Phosphorylation of telomerase and estrogen receptor-alpha by akt in cataracts

C. M. H. Colitz, C. A. Barden, H. L. Chandler, P. Lu, A. G. Metzler, D. A. Wilkie, I. D. Bras and V. J. Kuonen

Department Vet. Clin. Sci., College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Purpose: Increased telomerase activity is a consistent finding in all cataracts (and PCO) that have lens epithelial cells (LEC) undergoing epithelial-mesenchymal transformation (EMT). EMT is a nonspecific change of LEC during cataractogenesis causing the cells to undergo pseudometaplasia and migration. Telomerase (TERT) and estrogen receptor alpha (ERα) interact in cataractous LEC but not in normal LEC and both require phosphorylation for activation. Our purpose was to characterize the kinase(s) responsible for the phosphorylation and activation of TERT and ERα in LEC and to determine if there is a difference between normal and cataractous LEC. Methods: Protein was extracted from normal and cataractous (diabetic and inherited) canine anterior lens capsules and quantified by standard Bradford assay. Routine Western blot analysis was performed to evaluate normal and cataractous LEC for ERα and phosphorylated Akt (pAkt). Then, coimmunoprecipitation was performed to evaluate whether pAkt was responsible for phosphorylating TERT and/or ERα. Results: ERα and pAkt were up-regulated in cataractous LEC while there was negligible expression in normal LEC. TERT and ERα do not coprecipitate with pAkt in normal LEC; further supporting that pAkt is not the kinase involved in normal LEC's telomerase activity. However, TERT and ERα coprecipitate with pAkt in cataractous LEC, implying that pAkt is responsible for the up-regulation of TERT and ERα in cataractogenesis. Conclusions: These results support the role of pAkt in activation of telomerase activity in cataractous LEC. Furthermore, pAkt also activates ERα in cataractous LEC, which may be involved in regulating TERT at the transcriptional level prior to or during EMT. A better understanding of the molecular modifications occurring during EMT of LEC may lead to genetic management of cataractogenesis or PCO. Commercial interest: None.

ABSTRACT NO.: 48

PCO in diabetic and nondiabetic patients following cataract surgery

I. D. Bras, A. J. Gemensky-Metzler, D. A. Wilkie, W. Saville, V. J. Kuonen, T. Robbin, R. Elliot and C. M. H. Colitz

Department Vet. Clin. Sci., College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Purpose: The purpose of this study was to quantify PCO in the canine patient, to determine if there was a difference in the development of PCO between inherited and diabetic cataracts, and to determine if age, breed, stage of cataract at the time of surgery, or presence of intraocular inflammation before and/or after surgery influence PCO development. Methods: One hundred 47 dogs (265 eyes) with inherited (69 dogs, 121 eyes) or diabetic cataracts (78 dogs, 144 eyes) underwent phacoemulsification and PMMA intraocular lens implantation and were prospectively evaluated for the development of PCO at 2 weeks, 6 weeks, 2–4 months, 6 months, and 1 year postoperatively. PCO scores were assigned using a scale of 0–4+ and presence or absence of intraocular inflammation was recorded. Pre-existing capsular plaques noted on ophthalmic exam immediately after surgery were not included in the PCO grading scale. Dogs were grouped by age and breed size and a categorical value assigned. PCO formation in the inherited and diabetic groups with and without inflammation were compared. The gender and right eye vs. left eye were also compared. Statistical analysis was performed using SAS software. Results: No difference in PCO development between diabetic and nondiabetic patients was present at any time interval. The average PCO score 1 year postoperatively was 1.2657 ± 0.79098 for dogs with inherited cataracts and 1.1818 ± 0.56580 for dogs with diabetic cataracts. There was no difference in PCO formation between the right and the left eye. A significant (P < 0.0001) increase in PCO formation was observed at each evaluation period in the inherited, diabetic, and the overall population. The age and gender of the animal did not significantly influence PCO formation. Small and medium breeds developed significantly more PCO in comparison to the large/giant breeds at 2 weeks and 2–4 months postoperatively. Eyes with early immature cataracts, up to 4 months postoperatively, had significantly increased PCO formation. There was no difference in the degree of PCO formation in eyes with inherited cataracts (P = 0.153), diabetic cataracts (P = 0.593), or the overall population (P = 0.238) with or without inflammation prior to and/or after surgery. Conclusion: PCO formation occurs in all canine patients undergoing phacoemulsification with IOL with 100% incidence 1 year postoperatively. PCO formation is a multifactorial process where age, breed size, and stage of cataract at the time of surgery influence PCO formation. The development of PCO increases over time in diabetic and nondiabetic patients and inflammatory episodes do not contribute to increased PCO formation. Future studies should focus on the development of a new IOL as well as the use of a quantitative method of evaluation. Commercial interest: None.

ABSTRACT NO.: 49

Intraocular pressure, tear production, and conjunctival bacteriologic culture results from a captive flock of humboldt penguins (Spheniscus humboldti)

R. L. Swinger,* J. N. Langan† and R. E. Hamor‡

*Animal Eye Speciality Clinic, Deerfield Beach, FL;Brookfield Zoo, Brookfield, IL;University of Illinois, College of Veterinary Medicine, Department of Clinical Sciences, Champaign-Urbana IL, USA

Purpose: To determine common ocular abnormalities, as well as normal intraocular pressure (IOP), tear production, and normal bacterial flora of the cornea and conjunctiva in captive Humboldt penguins. In addition, penguins housed in both salt and fresh water living environments were compared to provide input into proper husbandry techniques. Methods: Twenty-eight penguins from a captive flock housed at Brookfield Zoo (Chicago, IL) were studied. An equal number of penguins had been living in salt and fresh water environments. All penguins were manually restrained without sedation. Both eyes were examined with a slit lamp and direct ophthalmoscope, removing birds from the study with evidence of ocular disease or previous trauma. Sixteen penguins (32 eyes) were selected randomly for ocular culturing, eight from each living environment. A sterile schirmer tear test (STT) paper was used to measure and record the average tear production per minute. A topical anesthetic was applied to the cornea, followed by applanation tonometry using a Tono-Pen XL (Tono-Pen XL, Mentor, Norwell, MA 02061, USA). Results: The mean IOP was 20.36 ± 4.1 mmHg. The mean rate of tear production was 6.45 ± 2.9 mm/minute. The most prevalent bacteriologic organisms found were Corynebacterium spp. and Staphylococcus spp. Conclusions: The Humboldt penguin, Spheniscus humboldti, whose aquatic lifestyle requires excellent vision, has become a highly endangered species over the past decade. While we believe our IOP findings accurately establish a reference value for this species; tear production and ocular flora may be influenced by the species’ habitat. Commercial interest: None.

ABSTRACT NO.: 50

Prevalence of breed-related cataracts in the dog in North America

Kirk N. Gelatt and Edward O. MacKay

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

Purpose: To determine the prevalence of cataracts in dogs presented to veterinary medical teaching hospitals, Colleges of Veterinary Medical, in North American from 1964 to 2003. Methods: A retrospective study of all dogs presented with cataracts to veterinary medical teaching hospitals in North America from 1964 to 2003 was conducted to determine cataract prevalence. The different decades, breeds, gender, and age of presentation with cataract were compared. Results: The prevalence of dogs presented with cataract varied by decade and ranged from 0.95% (1964–197), 1.88% (1974–83), 3.5% (1984–93), and 2.8% (1994–2003). The total number of dogs presented with cataracts was 39 229. Approximately 60 breeds of dogs were affected with cataracts above the baseline prevalence of 1.61% in the mixed breed-hybrids dogs. From 1964 to 2003 the prevalence of cataract formation in this patient population increased by about 295%. The breeds most frequently diagnosed during 1964–2003 with cataract included: Smooth Fox Terrier (11.70%), Havanese (11.57%), Bichon Frise (11.45%), Boston Terrier (11.11%), Miniature Poodle (10.79%), Silky Terrier (10.29%) and Toy Poodle (10.21%). The breeds with the highest number of cataractous dogs during the entire four decades were the Boston Terrier (11.11%), Miniature Poodle (10.79%), American Cocker Spaniel (8.77%), Standard Poodle (7.00%), and Miniature Schnauzer (4.98%). Difference in gender ratios of cataractous dogs seemed to affect only limited number of breeds. Age of presentation with cataract diagnosis varied among several breeds. Conclusion: Cataract formation is one of the most prevalent eye diseases in the dog population, and in about 60 breeds of dogs the prevalence of cataract exceeds the baseline mixed-breed hybrid group. The prevalence of cataract is also influenced by age in most purebred dogs and affects 17.4% of the 7–15 + year-old mixed breed-hybrid dog population. The total and age related cataract prevalence in dogs seems very similar to that in man. Acknowledge: The authors thank Ms Yun Shen, Veterinary Medical Data Base/Canine Eye Registry Foundation, Purdue University and Ms Norma Pablo, Medical Records, University of Florida, and the veterinary medical teaching hospitals which provided this information. Support: The Jaqua Foundation, and Gwathmey-Adams Laboratory for Vision Science. Commercial interest: None.

ABSTRACT NO.: 51

Effects of systemic and topical administration of 0.5% tropicamide on intraocular pressure, pupillary diameter, blood pressure, and heart rate in normal cats

K. S. Schmidt,* D. V. Hacker,† P. Kass‡ and A. L. Barkhoodarian*

*Veterinary Ophthalmology Services;Animal Eye Specialists;University of California, Davis, Davis CA, USA

Purpose: The objective of the study was to determine the effects of systemic and topical 0.5% tropicamide on intraocular pressure (IOP), pupillary diameter (PD), blood pressure, and heart rate (HR) in normal felines with normotensive eyes. Methods: IOP, PD, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and HR were measured in 18 clinically healthy cats. Changes in SBP, DBP, MBP, and HR were evaluated using one-way repeated measures analysis of variance, with time as the repeated factor. IOP and PD were evaluated using two-way repeated measures analysis of variance, with time and side (OD vs. OS) as the repeated factors. P-values less or equal to 0.05 wereconsidered statistically significant. Results: After lingual tropicamide administration, the mean PD at T60 was 3.53 mm OD and 3.53 mm OS. The mean PD at T90 was 6.36 mm OD and 6.31 mm OS. The mean PD at T120 was 8.25 mm OD and 8.19 mm OS. This change in PD from T60, T90, and T120 was statistically significant, demonstrating a linear increase in PD over time after tropicamide application on the tongue (P < 0.0001). There was no statistically significant difference in PD when comparing the right to the left pupils (P = 0.10). The mean IOP at T60 was 14 mmHg OD and 12.94 mmHg OS. The mean IOP at T90 was 14.5 mmHg OD and 14.23 mmHg OS. The mean IOP at T120 was 14.94 mmHg OD and 14.89 mmHg OS. This change in IOP from T60, T90, and T120 was statistically significant, demonstrating a linear increase in IOP over time after tropicamide application on the tongue (P = 0.034). There was no statistically significant difference in IOP when comparing the right eye to the left eye (P = 0.28). After application of one drop of tropicamide ophthalmic solution in the right eye and one drop of lubricant eye drops was placed in the left eye, there was a statistically significant increase in PD in the right side, with a mean of 11.8 mm at T150 and mean of 12.0 mm at T180, compared to the left side, with a mean of 10.2 mm at T150 and mean of 10.4 mm at T180 (P = 0.0001). There were no significant changes in IOP over time from T150 to T180 (P = 0.42). However, the mean IOP at T150 was 16.28 mmHg OD and 15.44 mmHG OS. The mean IOP at T180 was 16.72 mmHg OD and 15.89 mmHg OS. This change reflects an increase in IOP in the tropicamide-treated eye in comparison to the control eye (P = 0.05). There were no statistically significant differences in SBP, DBP, MBP, and HR values over time for the duration of the study. Conclusions: We conclude that although lingual application of tropicamide appears to result in systemic absorption, causing significant pupillary dilation and elevations in IOP, systemic effects on SBP, DBP, MBP, and HR are not observed. Commercial interest: None.

ABSTRACT NO.: 52

Congenital glaucoma in the siamese cat – a new spontaneously occurring animal model for glaucoma research

G. J. McLellan, D. Betts, K. Sigle and S. Grozdanic

Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA

Purpose: The glaucomas constitute a leading cause of blindness in humans in developed countries. Although primary glaucomas are recognized infrequently in cats, we have diagnosed congenital, open-angle glaucoma in 11 closely related, purebred Siamese cats. The purpose of this study was to characterize the clinical features of this disease and to evaluate its potential suitability as a spontaneously occurring animal model for human glaucoma. Methods: Following the diagnosis of primary glaucoma in an 8-month-old, Siamese cat in 2002, a prospective study was initiated to evaluate related cats for clinical evidence of primary glaucoma. All cats included in the study were subjected to thorough ocular examination that included slit-lamp biomicroscopy, gonioscopy, applanation tonometry, and indirect and direct ophthalmoscopy. Results: A total of 11 closely related cats were available for clinical examination. The cats available for study comprised the proband, its’ parents, and 8 of the 13 surviving full siblings from 3 separate litters. Six male and 5 female cats were examined. Age at first examination ranged from 3 weeks to 3 years. All cats were found to have bilateral mild to moderate buphthalmos and moderate elevation in intraocular pressure (IOP). Mean IOPs at initial presentation were 31.6 mmHg (range 18–54 mmHg) and 29.7 mmHg (range 18–50 mmHg) in the right and left eyes, respectively. Additional clinical features that were commonly noted included prominent, thick, elongated ciliary processes and apparent microphakia/spherophakia that were observed in all 7 cats > 4 months of age at initial examination, but were not seen in kittens examined at 3 weeks of age. Haab's striae were noted in 5 eyes and lens subluxation was evident in 8 eyes. Neither episcleral injection nor signs of ocular pain were apparent and there was no evidence of intraocular inflammation. All eyes appeared visual at the time of initial examination and optic disc cupping was evident in only 2 eyes. Mild increase in latency of the pupillary light reflex was noted. Gonioscopy revealed open or slightly narrowed iridocorneal angles, with pectinate ligament dysplasia and prominent iris processes (10/10 eyes). One 3 week-old kitten demonstrated bilateral persistent pupillary membranes. Serologic testing for FeLV/FIV was negative in all cats. Medical therapy was instituted with topical dorzolamide in 4 cats, with subsequent addition of timolol and pilocarpine. Unilateral extraction of an anteriorly displaced lens was carried out in the proband and Ahmed valved anterior chamber shunts were implanted bilaterally, but did not lead to sustained reduction in IOP. Conclusions: Congenital glaucoma has been identified in 11 closely related Siamese cats. Clinical features identified in affected cats, including subtle anterior segment malformations, showed remarkable similarities to those seen in human infants with primary congenital and pediatric glaucoma. The early onset of clinical disease, combined with relatively modest elevation in intraocular pressure and slow progression of vision loss indicate that this inherited feline disease shows considerable promise as a spontaneously occurring animal model of human congenital open-angle glaucoma. A breeding colony has now been established in order to facilitate further clinical, pathological and genetic characterization of the disease. Supported by a Faculty Development Award, from the College of Veterinary Medicine, Iowa State University. Commercial interest: None.

ABSTRACT NO.: 53

Glutamate transporter activity and localization does not correspond to the temporary functional recovery and late degeneration after acute ocular hypertension

Daniel M. Betts,* Nigel L. Barnettl† and Sinisa D. Grozdanic*

*Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011;Vision, Touch & Hearing Research Center, School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Australia

Purpose: To determine whether the localization and activity of retinal glutamate transporters are affected by acute ocular hypertension and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration after induction of the acute ocular hypertension. Methods: Acute ocular hypertension was induced in rats by increasing the intraocular pressure to 110 mmHg for 60 min. Functional evaluation was performed by monitoring the pupil light reflex (PLR) and electroretinogram (flash, flicker ERG and oscillatory potentials) for 60 days postoperatively. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult. d-aspartate accumulation wasaffected during acute hypertensive episode, which was suggestive of impaired glutamate transporter activity. However, despite severe retinal degeneration d-aspartate was normally accumulated in the ischemic retinas at all postoperative time points. 10d postoperatively the PLRratio (ratio = indirect/direct PLR = 34 + 7.5%) was significantly less than the preoperative value (preop = 76.7 + 2.6%, P < 0.05). However, 25 and 35 days postoperatively PLR ratios significantly recovered and did not differ significantly from preoperative values (44.4 + 6.9% and 53.8 + 9.6%, P > 0.05). Forty-five and 60 days postoperatively the PLRratio declined again and was significantly lower than the preoperative value (33.8 + 8.7% and 26.2 + 8.9%, P < 0.05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days postoperatively when compared to the first time point recorded postoperatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or activity observed by d-aspartate (glutamate analog) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ocular hypertension. Mechanisms observed in the rat model of acute ocular hypertension might somewhat mimic changes observed in dogs with acute uncontrolled glaucoma. The pharamacological manipulation of glutamate transporter activity might be promising treatment during early onset of the acute ocular hypertension. Commercial interest: None.

ABSTRACT NO.: 54

Morphometric analysis of glaucomatous dog retinas – tapetal sparing of peripheral, but not central retina

K. S. Sigle,* J. Ostojic,*,† G. J. McLellan,* D. M. Betts,* D. S. Sakaguchi† and S. D. Grozdanic*

*Department of Veterinary Clinical Sciences, College of Veterinary Medicine;Neuroscience Program and Department Genetics, Development and Cell Biology-Iowa State University, Ames IO, USA

Purpose: To determine whether morphological preservation of the tapetal retina occurs in glaucomatous dog eyes. Methods: Morphometric analysis was performed in 9 glaucomatous dog eyes (Cocker Spaniel (n = 1; closed angle glaucoma), Brittany spaniel (n = 1, closed angle glaucoma), Chow-Chow (n = 2, closed angle glaucoma), Shar-Pei (n = 1, closed angle glaucoma), Labrador Retriever (n = 1, closed angle glaucoma), Basset Hound (n = 3, open angle glaucoma with pectinate ligament dysplasia)) and 9 control (healthy) dog eyes (Beagle; n = 9). The exclusion criteria for the study were: retinal detachment, nonglaucomatous retinal degeneration, retinal dysplasia, history of cryoablation or cyclophotocoagulation and posterior uveitis. Results: The central tapetal retina (CTR, 152 + 9 µm) in glaucomatous eyes was significantly thicker from the central nontapetal retina (CNTR, 127 + 14 µm; P = 0.012, Paired t-test). However, morphometric analysis of the healthy retinas revealed that the CTR (180 + 5 µm) is significantly thicker compared to the CNTR (155 + 2 µm; P = 0.002, Paired t-test). When data were presented as ratio between CTR and CNTR thickness, there was no statistical difference between glaucomatous eyes (126 + 9%) and control eyes (117 + 3%; P = 0.45, Student's t-test). Statistical analysis showed that CTR (P = 0.02, Student's t-test) but not CNTR (P = 0.07, Student's t-test) were significantly thinner in glaucomatous dogs compared to the healthy (control) eyes. The peripheral tapetal retina (PTR, 72 + 5 µm) in glaucomatous eyes was significantly thicker from the peripheral nontapetal retina (PNTR, 59 + 6 µm; P = 0.03, Paired t-test), which was not the case for healthy retinas (PTR = 97 + 3 µm; PNTR = 99 + 3 µm; P = 0.5, Paired t-test). When data were presented as ratio between PTR and PNTR thickness, there was statistical difference between glaucomatous (126 + 9%) and control eyes (98 + 3%; P = 0.01, Student's t-test). Statistical analysis showed that PTR (P = 0.003, Student's t-test) and PNTR (P = 0.002, Student's t-test) were significantly thinner in glaucomatous eyes compared to the healthy (control) eyes. Semiquantitative analysis revealed statistically significant increase in glial fibrillary acidic protein (GFAP) immunoreactivity in glaucomatous eyes when compared to the healthy (control) eyes (P = 0.01, Student's t-test). However there was no statistically significant difference in the GFAP immunoreactivity between the tapetal and nontapetal retina of glaucomatous eyes (P = 0.4, Paired t-test). Glaucomatous optic nerve heads (ONH) had significantly more cupping (ONH cup depth = 434 + 166 µm) compared to healthy control eyes (120 + 20 µm; P = 0.002 Student's t-test). Conclusions: Detailed morphometric analysis showed decreased retinal thickness, increased GFAP immunoreactivity and cupping in glaucomatous dog eyes. Peripheral tapetal retinas seemed to be more spared compared to the nontapetal retinas, however, no evidence of tapetal sparring was present in central retinal regions of glaucomatous dog eyes. Commercial interest: None.

ABSTRACT NO.: 55

Early loss of the 03 component of oscillatory potentials in a rat model glaucoma

S. A. Hildreth, T. Lazic, D. M. Betts and S. D. Grozdanic

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames IO, USA

Purpose: To determine which ERG component is the most dramatically affected in glaucomatous eyes. Methods: The glaucoma was induced in 9 adult Brown Norway rats by combining injection of indocyanine green dye (ICG) into the anterior chamber, followed by diode laser treatment (30 spots) of the episcleral veins enriched with the dye. Electroretinograms (scotopic flash ERG and OPs, photopic flicker ERG) were recorded from the operated and control eyes preoperatively and 10 days postoperatively. Results: Laser treatment induced significant elevation of the IOP in operated eyes 7 days after surgery: control = 23.5 + 0.7 mmHg, operated = 30.2 + 0.7. ERG analysis of operated eyes revealed significantly decreased amplitudes of all tested components 10 days after surgery: a-wavectrl = 175 + 10 µV, a-waveoper = 127.7 + 11 µV (P < 0.01, Paired t-test); b-wavectrl = 340 + 15 µV, a-waveoper = 264 + 2 µV (P < 0.04, Paired t-test); flickerctrl = 44.8 + 2.5 µV, flickeroper = 27.8 + 3.3 µV (P < 0.01, Paired t-test); OPcrtl = 311 + 44.5 µV, OPoper = 200 + 20.2 µV (P < 0.02, Paired t-test). Detailed analysis of the OP components (O1, O2, O3, O4) revealed that O3 component ratio was significantly smaller (P < 0.01, Repeated measures anova) when compared to other oscillatory potential components: O1ratio(operated/control) = 72.2 + 6.9%; O2ratio = 83.7 + 16.6%; O3ratio = 31.3 + 9.3%; O4ratio = 63.9 + 16.2%. Conclusions: The function of the retina decreases significantly in a rat model of glaucoma. The O3 component of oscillatory potentials seems to be the most dramatically affected. Careful analysis of different oscillatory potential components might be a useful strategy for the early detection of glaucomatous functional changes in animals. Supported by The Glaucoma Foundation, NY; and Carver Trust – ISU Biotechnology Foundation. Commercial interest: None.

ABSTRACT NO.: 56

Protection of optic nerve function following acute elevation of intraocular pressure by sustained intravitreal delivery of neurotrophic growth factors incorporated into biodegradable microspheres

S. D. Grozdanic,* S. N. Hildreth,‡ T. Lazic,* J. Orasky,† E. Lavik,§ D. M. Betts,* M. H. Kuehn,¶ Y. H. Kwon,¶ R. H. Kardon¶ and D. S. Sakaguchi*,

*Dept. Vet. Clinical Sci. – College of Veterinary Medicine,Dept. of Genetics, Development and Cell Biology,Iowa State University, Ames, IA;§Dept. Biomedical Engineering, Yale University, New Haven, CT;Dept. of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA

Purpose: To perform molecular analysis of the ischemic retinas after acute elevation of intraocular pressure (IOP) and determine if release of neurotrophic growth factors from intravitreally injected biodegradable microspheres can improve functional and morphological outcome of the damaged retina and/or optic nerve. Methods: Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 min). Microarray analysis was performed with Affymetrix GeneChip arrays. Microspheres for slow release of neurotrophic factors were made using a spontaneous emulsification technique forming poly lactic-coglycolic acid(PLGA) spheres. Microspheres (BDNF, GDNF, CNTF or blank/control) were injected into the vitreous 1 day after elevation of IOP. Pupil light reflexes (PLR) were recorded preoperatively and at days 10, 20, 30, 40, 50 and 60 postoperatively. Electroretinograms (flash and flicker ERG, oscillatory potentials) were recorded preoperatively and 60 days postoperatively. Results: Analysis of the PLR amplitudes showed significantly better functional outcome in acute ischemic rat eyes which received BDNF or GDNF, but not CNTF microspheres compared to rats which received empty/blank or no microspheres: PLRratio(BDNF) = 54.5 + 1.5% (P < 0.001), PLRratio(GDNF)= 48 + 3% (P < 0.05), PLRratio(CNTF) = 44.3 + 1.6% (P > 0.05), PLRratio(blank) = 39.1.5 + 1.8%, PLRratio(no microspheres) = 36.4 + 4.4%. Rats which received GDNF microspheres showed no functional recovery in the first 10 days postoperatively which correlates with down regulation of the GDNF receptor (observed by microarray analysis). CNTF microspheres produced no significant effect, probably due to spontaneous up-regulation of CNTF (observed by microarray analysis) in ischemic retinas which might be responsible for the temporary recovery of PLR function in rats which did not receive any treatment. ERG and histology data analysis revealed no statistically significant difference between all groups. Conclusions: Sustained release of BDNF and GDNF from biodegradable microspheres may protect the inner retina and optic nerve function. Molecular profiling of specific disease conditions is the most important step in order to precisely determine selection and timing for specific neuroprotective factor application. Microspheres engineered to release neurotrophic factors may be a promising strategy for the treatment of optic nerve ischemic disease and glaucoma in humans and animals. Commercial interest: None.

ABSTRACT NO.: 57

Neuroglobin and cytoglobin – new respiratory proteins in mammalian eyes

S. D. Grozdanic,* J. Ostojic,*,† N. A. Syed,§ M. S. Hargrove,‡ J. T. 3rd Trent,‡ D. M. Betts,* Y. H. Kwon,§ M. H. Kuehn,§ R. H. Kardon§ and D. S. Sakaguchi*,

*Department of Veterinary Clinical Sciences – College of Veterinary Medicine,Department of Genetics, Development and Cell Biology,Department of Biochemistry, Biophysics and Molecular Biology-Iowa State University;§Department of Ophthalmology and Visual Sciences-University of Iowa, Iowa City, IO, USA

Purpose: To determine the distribution of neuroglobin and cytoglobin in human and dog retinas and to examine whether mRNA and protein expression are altered in glaucomatous rodent and dog eyes. Methods: Specific polyclonal antibodies to neuroglobin and cytoglobin were generated and used to determine expression and localization by immunofluorescence microscopy in human and dog retinal sections. The Real Time PCR and microarray analysis were used to examine change in the mRNA expression in acute and chronic hypertensive rodent eyes. Results: Neuroglobin and cytoglobin are two recently discovered members of the globin family of oxygen binding molecules. Neuroglobin immunoreactivity appeared to be present in the photoreceptor layer (predominantly in the inner segments of the photoreceptors), in some cells of the inner nuclear layer and was very abundant in the retinal ganglion cell layer. Cytoglobin immunoreactivity appeared to be predominantly localized to the ganglion cell layer and inner nuclear layer. Some immunoreactivity was present in the outer plexiform layer. Comparative analysis of healthy and glaucomatous dog eyes revealed increased expression of neuroglobin in glaucomatous dog retinas. Molecular analysis showed significant up-regulation of neuroglobin in acute hypertensive rodent eyes (P < 0.05, control vs. acute hypertensive) and cytoglobin in chronic hypertensive rodent eyes (P < 0.05, control vs. chronic hypertensive). Conclusions: Neuroglobin and cytoglobin are widely distributed globins in mammalian retinas, including humans and dogs. The specific kinetics of these hexacoordinate globins supports their role as temporary oxygen reservoirs, which might provide a minimal, but continuous supply of intracellular oxygen during ischemic and anoxic conditions. Specific localization of neuroglobin and cytoglobin is suggestive of a potentially important role in oxygen homeostasis and ischemia-induced cell signaling in retinas and optic nerves affected by ischemia and/or glaucoma. Commercial interest: None.

ABSTRACT NO.: 58

Contact lenses in an aphakic dog

E. O. Ropstad,* T. M. Seland† and E. Bjerkås*

*Norvegian School of Veterinary Science, Oslo, Norway;Seland Optikk, Sandnes, Norway

Purpose: To describe the behavioral presentation, with and without corneal contact lenses of a dog that had undergone bilateral lensectomy following primary lens luxation. Methods: A 5 years old intact female Sealyham terrier was presented with blepharospasm and redness of the right eye (OD). On examination, congested scleral and conjunctival vessels were diagnosed and the lens was found luxated into the anterior chamber. Intra-ocular pressure (IOP) was 50 mmHg (Tono-pen®). Funduscopic findings were normal. The lens of the left eye (OS), was subluxated posteriorly with iridodonesis and a deep anterior chamber. IOP was 18 mmHg. The fundus of OS was not evaluated in detail, as the pupil was not dilated for fear of provoking a complete lens luxation in this eye as well. The lens of OD was removed through an intracapsular extraction as an emergency. Six months later the dog was presented for similar ocular signs OS. At this point, there was a complete anterior lens luxation, also in this eye. IOP was 37 mmHg (Tono-pen®). The lens was removed following the same surgical procedure as in OD. Results: The wound of OD healed without problems, except from some corneal opacity due to scar formation. The fundus was unaltered postoperatively. The surgery of OS resulted in slightly more corneal opacities, as well as some keratoconus. After the first surgery the dog became inactive and insecure in new surroundings. In humans lens subluxation is known to cause poor visual acuity, head ache and double vision. The dog was referred to an optometrist, who measured a refraction of +14 diopters (D) in OD, whereafter the dog was provided with a temporary +8 D soft contact lens (Vasurfilcon A, Precision UV®). The lens was applied to the eye using a lubricant (Lubrithal®, Leo Pharma, Denmark). Wearing the new contact lens, the dog's behavior changed completely, the dog becoming more active and apparently happier. The dog seemed comfortable wearing the contact lens. The lens stayed on the eyes until changed by the owner once a month, as recommended by the manufacturer. In order to achieve emmotropia in both eyes after the surgery of OS, +14 D lenses (MMA/VP, Omniflex®) were applied. These lenses were of a harder consistency than the former ones, and thus more likely to fall out, especially in OS. The post operative keratoconus in this eye may be a contributing factor. The same behavioral changes occurred wearing the new type of contact lenses, however. Conclusions: Refractive errors can be important disabling conditions in dogs. By correcting refractive errors in aphakic dogs with the use of contact lenses, the quality of life can be significantly improved. Soft contact lenses seem more suitable for dogs than harder ones. Commercial interest: None.

ABSTRACT NO.: 59

Evaluation of retinal phenotype in Tibetan terrier ceroid-lipofuscinosis

K. Narfström* and M. Katz†

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

Purpose: Studies were performed to characterize storage body accumulation in the retina of Tibetan terrier Ceroid-Lipofuscinosis (CL) and to determine whether the disease in this breed is accompanied by impaired retinal function and degeneration. Methods: Three clinically affected Tibetan Terriers, aged 7–10 years, and one 5-year-old normal dog of the same breed were studied by routine ophthalmic evaluations, including bilateral full-field electroretinography (ERG), utilizing a protocol for differentiation of rod and cone function (Narfstrom et al. Doc Ophthalmol, 105: 83–92, 2002). Fluorescence, light- and electron microscopy were performed on the retinae of the affected dogs as recently described (Katz et al. In Press, Am J of Vet Res, 2004). Results: Pupillary light reflexes were abnormal in 2 of the 3 CL-affected dogs. Fundus appearances varied from normal to moderately advanced retinal atrophy. ERGs indicated moderately to severely depressed rod function while cone function was normal to only moderately impaired. Morphologically there was an accumulation of disease-specific storage bodies in retinal neurons, particularly in retinal ganglion and Muller cells. There was a disruption of outer segment morphology and a variable loss of photoreceptors cells; the latter from slight to moderate. Conclusions: Impairment of retinal function appears to be relatively mild in affected dogs, well beyond 7 years of age, even when morphology shows a substantial amount of intracellular storage-bodies, predominantly in the inner retina, and a certain loss of photoreceptor cells. The similarities between Tibetan Terrier CL and some forms in humans suggest that the canine disease may have a genetic and biochemical basis similar to that of one of the human CL disorders. Supported by the American Kennel Club, Canine Health Foundation; Research to Prevent Blindness, Inc.; and NIH grant NS 38987 (MK). Commercial interest: None.

ABSTRACT NO.: 60

A clinical and ultrasonographic examination of the eyes of Bouvier des Flandres dog with an emphasis on identifying risk factors for primary angle-closure glaucoma

P. E. Miller, E. Bentley, K. A. Diehl and C. J. Murphy

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

Purpose: Although primary angle-closure glaucoma (PACG) in association with pectinate ligament dyplasia (PLD) is common in Bouviers, PLD alone is a nonspecific marker for PACG since the vast majority of dogs with PLD do not develop PACG. An improved ability to identify dogs at risk of PACG would allow earlier initiation of glaucoma preventative therapy and more useful breeding advice. This study sought to evaluate the ability of a variety of ophthalmic parameters to improve the identification of dogs at risk for overt PACG. Methods: 98 eyes of 50 Bouviers (17 males, 33 females) underwent streak retinoscopy, gonioscopy, applanation tonometry pre- and postpupil dilation, indirect ophthalmoscopy, slit-lamp biomicroscopy, and A-, B-, and 20 MHz high resolution ultrasonography (HRUS). Drainage angles were graded as described by Ekesten et al. Results: Mean age was 63.3 month (range 10–140 month). Two dogs were monocular from previous PACG. Forty-nine of 98 eyes were = 0.5D hyperopic and the overall mean refractive error was 0.38D. Although 12 dogs (24%) did not have PLD on gonioscopy, only 5 dogs (10%) had an angle that was ‘open’ or ‘wide open’ its entire circumference. In 2 sibling females without PLD the trabecular meshwork was filled with pigment. The ciliary cleft was also closed in the more severely affected dog. Mean (s.d.) IOP predilation was 13.3 ± 3.2 mmHg and 14.0 ± 3.2 mmHg postdilation. No dog exceeded 25 mmHg postdilation. The mean (s.d.) axial length was 21.96 ± 0.53 mm; anterior chamber depth was 4.22 ± 0.46 mm; and axial lens thickness was 7.09 ± 0.31. On HRUS the ciliary cleft was closed in 5 eyes of 3 dogs: a 50-mo-old male with severe PLD, narrow angles and overt PACG in the fellow eye, a 35-mo-old male with severe PLD and 2.5D hyperopia OU, and a 74-mo-old female without PLD but extensive pigment deposition in the trabecular meshwork and a shallow anterior chamber (3.3–3.4 mm) OU. The remaining eye of the other dog with overt PACG in the fellow eye had a narrowed ciliary cleft and shallow anterior chamber (3.21 mm). Conclusions: Although PLD is common in Bouviers, other angle malformations that may lead to glaucoma such as angle narrowing and pigment deposition within the trabecular meshwork also occur. Ciliary cleft narrowing or closure on HRUS may improve the identification of dogs at risk of overt PACG, as may the presence of a shallow anterior chamber or hyperopia. Support: Bouvier Des Flandres Health Foundation. Commercial interest: None.

ABSTRACT NO.: 61

Expression of matrix metalloproteinases 2 and 9 in experimentally wounded canine corneas and spontaneous chronic corneal epithelial defects (SCCEDs)

R. T. Carter,* R. Kambampati,* J. D. Foley,* M. Mineo,† C. J. Murphy* and E. Bentley*

*School of Veterinary Medicine, University of Wisconsin, Madison, WI,Animal Eye Clinics of Florida, Clearwater, FL, USA

Purpose: Spontaneous chronic corneal epithelial defects (SCCEDs) in dogs and humans are characterized by the persistence of an epithelial defect with nonadherent epithelium surrounding the denuded stroma. Samples obtained from canine patients demonstrate sheets of poorly adherent epithelial cells, absence of basement membrane components, and the presence of a stromal superficial hyalinized acellular zone. Matrix metalloproteinases (MMPs) remodel the extracellular matrix and are reported to be altered during normal wound healing and in various pathological processes, including recurrent erosions in humans. Here, we report MMP activity to be modulated during normal wound healing in dogs and in canine patients with SCCEDs. Methods: Right eyes of 22 normal dogs underwent mechanical epithelial debridement. 24 h (n = 8), 48 h (n = 5) or one week (n = 9) postwounding the dogs were euthanized for reasons unrelated to this study and corneas collected. Superficial keratectomies were performed on canine SCCED patients for therapeutic reasons, and samples were collected for analysis (n = 17). Samples were homogenized and supernatants collected. Zymography was performed as previously described. Gels were imaged and analyzed using Kodak imaging software. Densitometry values were evaluated relative to normal (T-test, significant P  0.05). Results: Wounded corneas showed significant up-regulation of MMP-9 at all time points. Maximal values were seen at 48 h. At 7 days post wounding values had diminished markedly but remained elevated above unwounded controls. There was no significant difference in values for MMP-2 in acutely wounded corneas vs. controls at any time point. SCCED samples demonstrated a significant increase in both MMP-9 and MMP-2 compared to normal, unwounded controls but were less than in acutely wounded corneas. Conclusions: Wounded corneas show a significant increase in MMP-9 during corneal epithelial wound healing with decreasing expression as healing occurs. SCCED samples showed a significant increase in both MMP-9 and MMP-2. It is unclear what role elevations of MMP-9 and MMP-2 may have in the pathogenesis of SCCED. Further work is being conducted in chronically wounded dogs to determine if the altered MMP values are simply associated with the presence of a chronic corneal epithelial defect or if the elevations are uniquely associated with SCCED. Supported by a grant from the ACVO. Commercial interest: None.

ABSTRACT NO.: 62

Combination plasmid delivery of ocular immunosupressive factors enhances suppression of experimental autoimmune uveitis

D. J. Biros and A. W. Taylor

Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Harvard MA, USA

Purpose: We have recently demonstrated that periocular delivery of a plasmid encoding the ocular immunosuppressive cytokine alpha-melanocyte stimulating hormone (α-MSH) suppresses the incidence and severity of Experimental Autoimmune Uveitis (EAU) in B10.RIII mice by 50% while significantly preserving the retinal structure and increasing levels of α-MSH in the aqueous humor. Since we have previously shown that the ocular immunosuppressive cytokine transforming growth factor-beta 2 (TGF-β2) enhances the α-MSH-mediated suppression of IFN-γ in primed activated CD3+ T cells in vitro, we asked if the subsequent delivery of a plasmid encoding the gene for TGF-β2 in EAU eyes that have already received a single α-MSH plasmid would further enhance the beneficial effects of α-MSH plasmid-mediated EAU suppression. Methods: Six week-old B10.RIII mice were immunized to human interphotoreceptor retinoid binding protein peptide (161–180). 25 µg subconjunctival plasmid injections of either empty plasmid, a plasmid encoding the gene for α-MSH or a plasmid encoding the gene for TGF-β2 was given to both eyes on day 6 and 9 postimmunization. Uveoretinal inflammation is monitored by direct ophthalmoscopy and scored on a scale of 0–5 over 30 days, and the mean maximum scores for each group were analyzed using the Mann–Whitney U-test. Eyes representing the mean maximum score from each group were collected for histopathology examination. Results: Mice that received the α-MSH plasmid on day 6 followed by the TGF-β2 plasmid on day 9 had a 42% reduction in EAU incidence and severity (P < 0.05). The results are similar to previous work where mice received multiple α-MSH plasmid injections. When mice received either a single injection of α-MSH plasmid or a single injection of TGF-β2 plasmid there was no significant suppression of EAU when compared to mice receiving the empty plasmid. Histopathology from each group correlated with the results of the clinical observations. Conclusions: The delivery of the α-MSH plasmid alone or in combination with TGF-β2 plasmid significantly suppressed the incidence and severity of EAU. Our results imply that although multiple injections of α-MSH plasmid alone are sufficient to suppress ocular autoimmune disease, the inclusion of injecting TGF-β2 plasmid may help further to restore the ocular immunosuppressive microenvironment by replacing a missing factor in uveitic eyes. Supported by NIH grants EY 10752 and EY 13913 C. Zycos, Inc., Genzyme, Inc. Commercial interest: None.

ABSTRACT NO.: 63

Analysis of dogs with sudden acquired retinal degeneration syndrome (SARDS)

M. A. Gilmour,* M. R. Cardenas,† M. Blaik,* R. Bahr* and J. F. McGinnis†,

*Department of Veterinary Clinical Sciences, Oklahoma State University;Department of Ophthalmology, University of Oklahoma Health Sciences Center; Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City OK, USA

Purpose: (1) To evaluate dogs with SARDS for evidence of pituitary gland, adrenal gland, and pulmonary neoplasia. (2) To evaluate dogs with SARDS for evidence of antiretinal antibodies. (3) To evaluate dogs with cancer for antiretinal antibodies. Methods: Sixteen dogs with SARDS, confirmed by ophthalmic examination and electroretinography, were evaluated in the study. Thoracic radiographs (3-view), ultrasonography, and contrast computed tomography were used to evaluate the lungs, adrenal glands, and pituitary gland, respectively, in 15 of the SARDS dogs. Western blot analysis was used to evaluate the sera of all 16 SARDS dogs, 20 dogs with cancer, and 20 normal dogs (free of known ophthalmic and systemic disease) for negative controls. Electrophoresis was performed using canine retinal homogenate on 10% SDS-polyacrylamide gel. Western analysis was performed on serum dilutions of 1 : 200, 1 : 400, 1 : 1000, and 1 : 3000. Positive controls were antirecoverin (23 kDa) 1 : 1000 and antiarrestin (48 kDa) 1 : 000. Negative controls were antibody buffer without serum. Secondary antibody for the canine sera was biotinylated goat antidog, tertiary reagent was streptavidin horseradish peroxidase, and substrate incubation was with diaminobenzidine with nickel enhancement. Results: No pulmonary, pituitary gland, or adrenal gland abnormalities were found on the imaging studies. Positive bands were identified on all sera analyzed by Western blot. Bands were assessed as significant if the density at 1 : 1000 dilution was equivalent to one or both positive controls, and the band was also present at 1 : 3000 dilution. Significant bands were identified in 9/20 normal dogs, 10/20 cancer dogs, and 6/16 SARDS dogs. Location of significant bands varied from greater than 48 kDa to less than 23 kDa. No positive bands were identified at the 23 kDa (recoverin) site in any of the samples. Conclusions: The dogs with SARDS evaluated in this study showed no pulmonary, adrenal gland, or pituitary gland neoplasia as assessed by diagnostic imaging; and did not have an increased number of antiretinal antibodies on Western blot when compared to normal dogs. The dogs with cancer evaluated in this study also did not have an increased number of antiretinal antibodies on Western blot when compared to normal dogs. Supported by a Jules and Doris Stein Professorship from Research to Prevent Blindness (RPB); National Institutes of Health grants EY06085, EY13050, an NEI core grant-EY12190, a P20 RR017703 from the COBRE program of the National Center for Research Resources, general funds from Presbyterian Health Foundation, and by an unrestricted grant from RPB to the Department of Ophthalmology, Dean McGee Eye Institute. Commercial interest: None.

ABSTRACT NO.: 64

Effects of oral meloxicam, deracoxib, tepoxalin and carprofen in reducing intraocular inflammation in the dog

M. A. Gilmour* and G. Kennard†

*Department of Veterinary Clinical Sciences, Oklahoma State University;Eye Care for Animals, Phoenix, Arizona, USA

Purpose: To evaluate the effect of three new oral nonsteroidal anti-inflammatory drugs (NSAIDs), meloxicam (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO USA), deracoxib (Novartis Animal Health US, Inc., Greensboro, NC USA), and tepoxalin (Schering-Plough Animal Health, Union, NJ USA) compared with carprofen (Pfizer Animal Health, Exton, PA USA), on intraocular inflammation in the dog. Methods: Twenty-four dogs undergoing terminal surgery were used in the study. All dogs had an ophthalmic examination including biomicroscopy, indirect ophthalmoscopy, tonometry and fluorescein staining, 2 days prior to the aqueous sampling. The dogs were divided into 5 groups: 4 treatment groups of 5 dogs each, and one control group of 4 dogs. The dogs in the control group received no medication. The dogs in the treatment groups received their respective NSAID on Days 1 and 2 at the following doses: meloxicam 0.2 mg/kg po every 24 h for 2 total doses; deracoxib 3 mg/kg po every 24 h for 2 total doses; tepoxalin 10 mg/kg po every 24 h for 2 total doses; and carprofen 2 mg/kg po every 12 h for 3 total doses. On Day 2, under general anesthesia, aqueocentesis was performed on both eyes of all dogs using a 27-gauge needle inserted through the corneal stroma at the limbus and aspirating 0.2 mL of aqueous into a 1 mL syringe. The aqueocentesis was repeated on all eyes one hour later. The aqueous was frozen at −80 °C for later protein assay. A commercial chemistry analyzer utilizing the biuret reaction and colormetric detection at 550 nm wavelength (total protein) and 600 nm wavelength (micro protein) was used for aqueous protein quantitation. Results: Analysis of variance was used for statistical analysis. Protein levels in the first aqueous samples showed no significant difference between all 5 groups. Average protein values (mg/mL ± SD) were 0.234 ± 0.283 (control), 0.215 ± 0.221 (carprofen), 0.104 ± 0.063 (deracoxib), 0.108 ± 0.033 (meloxicam), and 0.423 ± 0.486 (tepoxalin). There was a significant difference (P-value 0.04) between the protein levels in the first aqueous samples and the second aqueous samples verifying aqueocentesis-induced inflammation. Protein levels in the second aqueous samples showed no significant difference between all 5 groups. Average protein values (mg/mL ± SD) were 16.64 ± 11.69 (control), 19.45 ± 10.39 (carprofen), 14.95 ± 5.93 (deracoxib), 12.21 ± 13.06 (meloxicam), and 10.46 ± 9.83 (tepoxalin). Conclusions: Although the aqueocentesis model created intraocular inflammation as measured by aqueous protein levels, none of the groups receiving an oral NSAID showed significantly less inflammation compared to each other, or compared to the control group receiving no treatment. Supported by Oklahoma State University Office of Veterinary Research funds. Commercial interest: None.

ABSTRACT NO.: 65

The light microscopic appearance of dysplasic zonular ligament protein in breed related lens luxation

R. R. Dubielzig and R. A. Morris

Department of Pathobiological Sciences, University of Wisconsin, Madison WI, USA

Purpose: Lens luxation resulting in secondary glaucoma is common in terrier breeds. This study was done to evaluate whether light microscopy was useful in detecting lesions in the zonular ligament in affected dogs. Methods: Eyes from 63 dogs with lens luxation glaucoma were evaluated for the presence of abnormal zonular ligament protein using H&E. PAS, trichrome and elastin staining was done on selected cases. Results: A distinctive zonular ligament protein dysplasia was found predominantly in Fox Terrier, Jack Russell Terriers and related terrier breeds (18 of 29), and Shar-Pei dogs (4 of 29). Zonular protein dysplasia is characterized by a tightly adherent, acellular protein staining strongly positive with PAS and blue with trichrome stains, and staining negative with elastin stains. Dysplastic protein is regionally adherent to the nonpigmented ciliary body epithelium with a distinct lamellar and cross-hatched morphology. 19 of the remaining 34 dogs had redundant amounts of zonular ligament protein which also had an excessive collagen content. Only 9 of 19 dogs with collogenized zonules were terrier breeds. This type of collagenized excess in zonules is often seen in a variety of abnormalities and we do not consider this to be a specific entity. 15 dogs had normal appearing zonular ligaments. The staining pattern in these dogs matched normal controls and were positive with PAS and elastin and had only minimal positive (blue) staining with trichrome. Only 2 of 15 dogs were terrier breeds. Conclusions: These results suggest that light microscopy is useful in detecting breed related changes in zonular ligament protein which might be important in the pathogenesis of primary lens luxation in terrier (particularly the Fox Terrier family of terrier dogs) and Shar-Pei dogs. Commercial interest: None.

ABSTRACT NO.: 66

Prospective comparison of acell biomaterial to conventional methods for treatment of corneal ulcers

J. A. Hyman, A. R. Hoffman, D. H. Agapito and R. E. Merideth

Eye Care for Animals, Kansas City, KS, USA

Purpose: To prospectively evaluate Acell porcine bladder mucosa (ACell, Inc., West Lafayette, IN) as the sole repair material in an array of corneal ulcers ranging from epithelial erosions to full thickness corneal defects. Methods: Patients that presented with ulcerative corneal disease were categorized into three groups: (1) corneal epithelial erosion (2) corneal stromal ulcer (3) descemetocoele/perforated corneal ulcer. Group 1 was treated using Acell biomaterial or a traditional treatment method such as linear grid keratotomy. Groups 2 and 3 were treated with Acell biomaterial or a conventional method such as conjunctival pedicle graft (CPG) or corneoconjunctival transposition (CCT). This resulted in 6 different treatment groups (1 experimental and 1 conventional for each of the 3 categories). A minimum of 10 patients was randomly assigned to each group. Acell was evaluated against standard repair techniques in several areas: (a) time to complete healing (b) comfort (c) corneal opacification. Results: In all cases of groups 2 and 3 Acell resulted in healing, usually primary, however, 2 of the group 3 cases required a second procedure. One case received debridement and reapplication of Acell; the other case was treated with a CCT since the original ulcer site had contracted enough for this procedure. Thereafter, a double layer of Acell was used for all group 3 cases. Time to complete healing using Acell was longer compared to CPG/CCT. Subjectively, patients were slightly more uncomfortable compared to CPG healing. Patients had the same degree of discomfort during Acell healing compared to our experience with CCT. Corneal opacification in the form of a leukoma occurred in all group 2 and 3 cases. The leukoma was always smaller that the original corneal defect and resulted in less visual obstruction than a CPG, but more obstruction to the visual axis than a CCT. Less corneal distortion occurred during healing with Acell compared to CPG; similar amounts of corneal distortion were noted compared to CCT. Healing and comfort were not optimal for corneal epithelial erosions treated with Acell using the method described for the study. Acell may still be successful for treatment of these erosions but an alternate method of securing the Acell will need to be utilized. Conclusions: Acell biomaterial was successful as a primary repair method for most corneal stromal ulcers/perforations. A few cases needed a second procedure for complete healing. A double layer of Acell is recommended for descemetocoeles/corneal perforations. Healing times were longer compared to CPG/CCT. Corneal opacification was less compared to CPG. Final comfort level was good. Acell needs to be re-evaluated using a different application technique for treatment of corneal epithelial erosion. Commercial interest: None.

ABSTRACT NO.: 67

Congenital cataract and microphthalmia, new spontaneous mutant mouse model for hereditary eye diseases

K. N. Kohale, S. Galande and P. B. Parab

National Center for Cell Science, University Campus, Ganeshkhind, Pune, India

Purpose: Hereditary mouse cataract models have great relevance to humans since it provide an ideal biological system for studying the pathophysiology of human cataract at cellular and molecular level. It was estimated that congenital cataracts comprise approximately 10% of visual loss in humans. We have earlier reported that dense cataract and microphthalmia (dcm), a spontaneous mutant mouse exhibits congenital eye abnormality at 14th postnatal day in BALB/c mice (Kohale et al. 2004, Comparative Medicine 54: 3, 263–267). Methods: We propagated mutant mice colony and established a new inbred strain carrying congenital cataract and microphthalmia by full sib mating. The efforts were being initiated to characterize this mutant mouse model for molecular alterations using SDS-PAGE and High resolution 2D electrophoresis. Results: The colony at present progressing through (F + 25) generations of inbreeding. Present work highlighted the decrease in lens proteins by SDS-PAGE analysis in mutant eye lenses when compared with the wild type mice. High-resolution 2D Electrophoresis indicated appearance of few new species of proteins in mutant lenses. Conclusions: The change in the composition of or synthesis of defective lens proteins could be related with the development of congenital cataract and microphthalmia phenotype in dcm mice. Supported by NCCS funds. Commercial interest: None.

ABSTRACT NO.: 68

Acute corneal hydrops in horses: 16 cases (1996–2004)

U. M. Dietrich,* P. A. Moore,* M. J. Chandler,* C. B. Mosunic,* C. O. Williams,* K. P. Carmichael† and C. L. Martin*

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

Purpose: To retrospectively describe clinical findings, results of corneal cytology, culture and histopathology, as well as treatment and outcome of acute corneal hydrops in horses. Methods: Medical records of 16 horses with acute corneal hydrops, presented to the UGA-VTH, were reviewed. Data collection included signalment, clinical presentation, treatment and outcome. Results of corneal cytology, bacterial and fungal culture, as well as histopathology of corneal biopsies were recorded. Immunohistochemistry (IHC) was performed on 8 tissue samples using a multivalent primary rabbit antileptospira antibody. Results: Mean age was 1.9 years. Study population included 12 females, 2 geldings, and 2 stallions. Breeds represented were 9 Quarter Horses, 3 Thoroughbreds, 2 Arabians, and 2 Tennessee Walker Horses. The clinical presentation was similar in all horses: sudden appearance of a unilateral, well circumscribed, round, edematous, fluorescein positive bullae of the cornea, either located centrally (8/16) or peripherally (8/16). Typically, the surrounding cornea was clear and free of neovascularization. The affected eye appeared comfortable with mild blepharospasm observed in 6/16 horses. Signs of anterior uveitis included mild flare (8/16), traces of fibrin (3/16), mild hypopyon (2/16), and miosis (9/16). The anterior chamber was clear in 8/16 cases. Ophthalmoscopic examination was normal in all cases except in one horse, where chorioretinal scars were detected on a follow-up examination. This horse had a positive titer (1 : 100) for L. grippotyphosa. In all horses corneal cytology revealed abundant neutrophils but no organisms were detected. Bacterial culture was positive in 6/16 cases, revealing very light growth of either Bacillus sp. (3/16), Staphylococcus sp. (2/16), or beta-Streptococcus sp. (1/16). Fungal culture was positive for one horse with light growth of Cladosporium sp. In all cases microorganisms were believed to be contaminants. Eight corneal biopsies were submitted for histopathology and diagnosed as purulonecrotic keratitis. IHC was negative in 7 samples; however, scattered immunopositive areas were observed within macrophages and neutrophils in one sample. Treatment included placement of a subpalpebral lavage system, application of antifungals, antibiotics, nonsteroidal anti-inflammatories, atropine, as well as systemic Banamine (Fort Dodge, Fort Dodge, IA) in 16/16 horses. A temporary tarsorrhaphy was placed in 10/16, in which case the lesion flattened and resolved with only a scar remaining in the cornea. Conjunctival flap surgery was performed in 4/16 cases. Visual outcome was positive in all eyes. Conclusions: Acute corneal hydrops is a corneal disease predominantly observed in young horses, which appears to be similar to acute bullous keratopathy described in cats. The etiology remains unknown. Commercial interest: None.

ABSTRACT NO.: 69

The influence of rainfall on the frequency of equine fungal keratitis

P. A. Moore,* U. M. Dietrich,* M. H. Barton,† C. B. Mosunic,* M. J. Chandler,* C. K. Carmichael‡ and D. E. Stooksbury§

College of Veterinary Medicine, Athens, GA, *Department of Small Animal Medicine and Surgery,Department of Large Animal Medicine and Surgery,Department of Pathology,§Department of Biological and Agricultural Engineering, Athens GA, USA

Purpose: To evaluate the influence of rainfall on the frequency of equine fungal keratitis. Methods: The medical records at The UGA-CVM for a 21-years period were searched for equine fungal keratitis cases confirmed by cytology, culture, and/or histopathology. The historical date for the onset of ophthalmic signs was recorded. The closest NOAA climate station to the horse's location was identified. The rainfall measurements for 90, 60, 30, and 15 days prior to the historical date were compared to the 30 years mean. The number of cases occurring during periods of positive and negative rainfall was recorded. Nonparametic statistically analysis was performed by a Wilcoxon Signed Rank Test. Results: Ninety-six confirmed cases were identified. Ninety medical records had complete historical information, and 79 cases had complete rainfall data. The majority of cases occurred when a negative rainfall measurement was recorded at 90 (58.22%, N = 46), 60 (54.43%, N = 43), 30 (55.69%, N = 44) and 15 days (60.76%, N = 48) prior to the historical date of onset. The majority of case occurred when a negative rainfall measurement was recorded between 90 and 60 (59.5%, N = 47), 90–30 (54.43%, N = 43), 90–15 (56.96%, N = 45), 60–30 (55.69%, N = 44), 60–15 (50.6%, N = 40), and 30–15 days (53.16%, N = 42) prior to the historical date of onset. These results were not statistically significant at any time point. Conclusion: The absence of statistical significance to support the trend for cases to occur during periods of rainfall deficits suggest that other climatological and/or biological factors are involved in the frequency of equine fungal keratitis. Commercial interest: None.

ABSTRACT NO.: 70

Evaluation of tricide™ as an antimicrobial potentiator for antifungal drugs against fungal keratitis isolates

W. L. Weinstein,* P. A. Moore,* U. M. Dietrich,* R. E. Wooley,† B. W. Ritchie*,‡ and S. Sanchez†,§

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

Purpose: To evaluate the in vitro effect of Tricide™ as an antimicrobial potentiator for antifungal drugs against fungal keratitis isolates. Methods: The NCCLS microdilution assay method was performed using reference grade antifungal powders (miconazole, ketoconazole, itraconazole, and natamycin) against equine fungal keratitis isolates (2 Aspergillus sp., 5 Fusarium sp., 1 Penicillium sp., 1 Curvularia sp., and 1 Cladosporium sp.) and ATCC quality control strains. MIC50 and MIC90 were determined for the antifungal drugs alone and in combination with Tricide™. Tricide™ was considered effective as a potentiator when the percent reduction in the MIC50 and MIC90 for the antifungal drugs was reduced by = 50%. Results: For both Aspergillus sp. isolates, the percent reduction in MIC50 and MIC90 was statistically significant for all the antifungal drugs combined with Tricide™ at 200 and 400 µg/mL (P < 0.0033). The percent reduction in MIC50 and MIC90 for the 5 Fusarium sp. isolates was statistically significant for all the antifungal drugs combined with Tricide™ at 540 µg/mL (P < 0.004). For the Penicillium sp., Curvularia sp., and Cladosporium sp. isolates, the percent reduction in MIC50 and MIC90 were statistically significant for all the antifungal drugs combined with Tricide™ at 200 µg/mL (P < 0.005). The correlation between the MIC50 and MIC90 reduction and the Tricide™ concentration was statistically significant for all antifungal drugs tested except for itraconazole MIC90. Conclusion:In vitro, Tricide™ is an effective antimicrobial potentiator for the antifungals drugs (miconazole, ketoconazole, itraconazole, and natamycin) against equine fungal keratitis isolates. Commercial interest: Financial interest (BW Ritchie RE Wooley) Supported by the Veterinary Ophthalmology Research Fund, The University of Georgia. Tricide™ (Molecular Therapeutics, 110 Riverbend Rd, Suite 171, Athens, GA 30602).

ABSTRACT NO.: 71

A novel method for objective vision testing in dogs

P. M. Gearhart, C. C. Gearhart and S. M. Petersen-Jones

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

Purpose: The use of rodent and canine models of retinal disease in the development of therapeutic strategies for inherited retinal disorders is an active area of research today. In order to accurately evaluate the success of these potential vision-enhancing treatments, reliable methods for assessing visual function in these animal models is necessary. Rodent models can be effectively vision tested using variations on the Morris water maze, in which the animals must swim to a submerged platform based on visual cues. The current standard for vision testing in canines is observing the ability to negotiate an obstacle course. For a more objective evaluation, a blinded observer counts the number of times obstacles are bumped in an effort to obtain data suitable for statistical analysis. Problems with these types of tests center on randomization of the obstacle course, dog cooperation, and a potential subjective element even when the observer is blinded to the experimental conditions. Other approaches might involve training dogs to perform a task in response to visual cues. Training dogs to respond to visual cues is time consuming and subject to bias inadvertently introduced by the trainer or nonvisual cues of which the trainer or tester may not be aware. Thus there is a need for an accurate, objective and quantitative method for vision testing in dogs that requires minimal training. Methods: We have developed a simple vision-testing apparatus designed to exploit a dog's natural desire to get out of an enclosed space. The device consists of a 3-foot square junction box with openings to short tunnel sections on each side. The ends of 3 of the 4 tunnels are capped so that there is only one visibly open tunnel, the location of which is randomized for each test. The dog is placed in the junction box and the first tunnel chosen is recorded, as well as the time taken to exit. Normal and affected dogs from our colony of Swedish Briard crosses with the RPE65 mutation were tested with the apparatus set up in a room where the ambient lighting conditions were either bright or dim red light. Next, one affected RPE65 dog was tested through the device under varying white light intensities. Binary logistic regression was used to analyze the proportion of correct choices as a function of room light intensity. Linear regression was used to examine time to exit as a function of room light intensity. Results: Most dogs tested to date performed the task easily with minimal training. A significant difference between the affected and unaffected dogs was found for both exit time and proportion of correct choices at dim and bright light conditions. There was also a positive correlation between room light intensity and performance. Conclusions: Our results indicate that this novel canine vision testing method appears to be an easy, accurate and sensitive method to distinguish between affected and unaffected dogs in bright and dim light conditions using either the correct choice or time to exit the maze. Commercial interest: None.

ABSTRACT NO.: 72

A clinical and manometric evaluation of the Tonovet rebound tonometer in dogs and horses

A. M. Knollinger,* N. C. La Croix,* P. M. Barrett* and P. E. Miller†

*Eye Care for Animals; School of Veterinary Medicine,University of Wisconsin-Madison, Madison WI, USA

Purpose: To determine and compare the accuracy of intraocular pressure (IOP) measurements obtained with the TonoVet® rebound tonometer and the Tono-Pen® VET applanation tonometer in clinically disease-free canine and equine eyes. The TonoVet has been recently marketed to the veterinary community and uses a novel induction-impaction method to determine IOP. There are no previous publications establishing TonoVet IOP measurements of canine or equine eyes. Methods:In vivo: One hundred dogs with normal eyes were divided into 2 groups. The IOP was measured bilaterally in each dog of the first group with the Tono-Pen VET followed by the TonoVet. The IOP of the second canine group was measured with the TonoVet followed by the Tono-Pen VET. Topical anesthesia was instilled in the second group prior to the IOP reading. A 20 minute interval was used between the individual instrument IOP measurements. This interval was determined via esthesiometry measurements (n = 24) of clinically effective topical anesthetic duration. The IOP of normal eyes of 35 horses was measured with the TonoVet followed by the Tono-Pen VET. Topical anesthesia was not instilled prior to either of the equine IOP measurements. In vitro: Ten normal canine eyes and 6 normal equine eyes were freshly exenterated. The anterior chamber of each eye was pressurized from 5 to 80 mmHg (in 5 mm increments) with sterile saline. Absolute IOP was determined by calibrated manometry. Three sequential TonoVet IOP measurements were taken for each eye at calibrated manometric pressure intervals. Results: Manometric and TonoVet IOP measurements were analyzed using a multiple linear regression model, revealing a strong linear trend in the data. The order in which the measurements were taken with either instrument was not statistically significant (P > 0.05). However, there was a statistically significant difference (P < 0.001) between the readings of the tonometers in dogs. In dogs, the Tono-Pen VET on average reads 2.19 units higher than the TonoVet (P < 0.001) for OD measurements, and 2.13 units higher for OS measurements (P < 0.001). Conclusions: The TonoVet rebound tonometer's accuracy is comparable to that of the Tono-Pen VET applanation tonometer when measuring IOP of normal canine and equine eyes. However, preliminary results reveal that the TonoVet is more accurate then the Tono-Pen VET in IOP established by manometry at both low and high pressures. The TonoVet may provide a superior method to the Tono-Pen VET for measuring true IOP, as the latter has been shown to underestimate IOP when compared with direct manometery. The TonoVet is significantly accurate for most of the IOP range routinely encountered by veterinary ophthalmologists, and it allows for tonometry without prior anesthetic instillation. Commercial interest: None.

ABSTRACT NO.: 73

Expression of the chemokine lix is decreased in a mouse model that develops less severe keratitis following infection with HSV-1

L. E. Galle,* E. Chang,† N. S. Taus† and W. J. Mitchell†

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

Purpose: Herpes simplex virus type 1 (HSV-1) induced stromal keratitis (HSK) in the mouse model is an immunopathologic disease which is dependent upon T lymphocytes and results in corneal infiltration of inflammatory cells, vascularization and fibrosis in the cornea. Previous studies have shown that neutrophils are important in the development of HSK. Three chemokines, MIP-2, KC, and lipopolysaccharide-induced chemokine (LIX) have been identified in the mouse as being important in recruitment of neutrophils to sites of inflammation. The perforin-deficient mouse model of HSK develops significantly less severe clinical and histopathological HSK, and has been used to study HSK pathogenesis. Recent studies have shown that expression of neutrophil recruiter MIP-2 is significantly reduced in HSV-1-infected perforin-deficient mice compared to virus-infected wild-type mice. The purpose of this study was to investigate the role of LIX in development of HSK in perforin-deficient mice. Materials and Methods: Perforin-deficient and wild type mice were inoculated with 1 × 107 pfu of HSV-1 strain F per eye via the corneal route of infection. Corneas of infected mice and uninfected controls of both genotypes were harvested at 3, 5, 8, 11, and 23 days postinfection. Expression of mRNA of LIX, MIP-2, and KC were evaluated by RT-PCR and real time RT-PCR. Expression of protein was determined by ELISA. Results: LIX and MIP-2 mRNA were decreased in infected perforin-deficient mice when compared to infected wild type mice. There was a four-fold decrease in expression of LIX mRNA in corneas of infected perforin-deficient mice when compared to infected wild type mice at 11 days post infection. ELISA confirmed that LIX protein expression was decreased in infected perforin-deficient mice when compared to infected wild type mice. Conclusion: LIX expression is decreased in perforin-deficient mice compared to wild type mice following corneal infection with HSV-1. This difference in expression of neutrophil recruiting chemokine, LIX, may be involved in the pathogenesis of corneal inflammation following HSV-1 infection in this model. Supported by a grant from the University of Missouri Research Board. Commercial interest: None.

ABSTRACT NO.: 74

Clinical features and outcomes of phacoemulsification in 39 horses, a retrospective study (1993–2003)

T. M. Fife, A. J. Gemensky-Metzler, I. D. Bras, C. M. H. Colitz, D. A. Wilkie and D. C. Klages

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

Purpose: To identify postoperative complications and outcome following phacoemulsification in horses. Methods: Records of 39 horses (54 eyes) were reviewed. Data collected included age, breed, sex, stage and cause of cataract, unilateral vs. bilateral, ultrasound findings, and postoperative complications. Results: Age ranged from 4 weeks to 15.75 years (mean 2.4 years, median 7 months). Breeds included the Appaloosa (2), Arabian (2), Miniature (1), Paint (5), Pony (1), Quarterhorse (7), Rocky Mountain Horse (1), Saddlebred (3), Standardbred (4), Thoroughbred (11), and Warmblood (2). There were 23 male and 16 female horses. The cataract was congenital in 25/39 horses (bilateral 14/25 and unilateral 11/25), traumatic in 9/39, and uveitis-induced in 5/39. Two horses were diagnosed with anterior segment dysgenesis (ASD) prior to surgery. One horse was microphthalmic OU. Ultrasound findings included detached retina (2/51), vitreal degeneration (9/51), microphakia (1/51) and posterior lenticonus (1/51). Phacoemulsification was performed bilaterally in 8/39 horses and unilaterally in 31/39. Postoperative complications included corneal edema (19/47), vitreal prolapse (10/47), fibrin in the anterior chamber (10/47), postoperative ocular hypertension (9/47), synechia/dyscoria (9/47), hyphema (6/47), corneal ulceration (6/47), decreased menace reflex (4/47), peripapillary depigmentation (4/47), retinal detachment (3/47), partial retinal detachment (1/47), retinal degeneration (1/47), phthisis bulbi (2/47), cyclosporine implant (placed at the time of surgery) dislocation into the anterior chamber (2/2 implants attempted), and fungal keratitis (1/47). Two of the eyes were enucleated due to postoperative complications. At the time of last follow-up 39 eyes (83%) were visual, 2 eyes reportedly had decreased vision, and 6 eyes (of 5 horses) were blind (2 enucleated, 2 phthisical postglaucoma, 1 detached retina postglaucoma, 1 with ASD). Post-operative follow-up times ranged from 1 day to 494 weeks (mean 56 weeks, median 7.5 weeks, with 17 horses lost to follow-up of greater than two weeks). Conclusions: The most common cause for phacoemulsification in the horse is congenital cataract and 88% were visual at last follow-up. Both horses with ASD were blind at the time of last follow-up. All 9 trauma-induced cataracts were visual despite preoperative vitreal degeneration in 5, ruptured lens capsules in 2, and phacoclastic uveitis in 1. Postoperative complications of traumatic cataracts included glaucoma (1/9) and vitreal prolapse (2/9). Peripapillary chorioretinal scarring was noted immediately postoperatively in 3/9 horses. Horses with uveitis-induced cataracts were the oldest population. Of these, 80% (4/5) had corneal ulcers postoperatively and 80% (4/5) were visual. Commercial interest: None.

ABSTRACT NO.: 75

Matrix metalloproteinases in precorneal tear film from cats with ulcerative keratitis

D. H. Agapito,* M. B. Berman,† T. Blocker,‡ G. Kennard,§ R. E. Merideth,§ J. Urbanz,§ C. Warren,§ D. J. Maggs¶ and L. Meade-Tollin†

*Eye Care for Animals, IL;University of Arizona College of Medicine, Department of Surgery;Eye Care for Animals, CA;§Eye Care for Animals, AZ;University of California, Davis School of Veterinary Medicine, Davis CA, USA

Purpose: To identify and semiquantify matrix metalloproteinases (MMP's) in the tears of cats without evidence of ocular disease and cats with ulcerative keratitis or keratoconjunctivitis. Methods: Tear samples were collected from 21 clinically normal cats and from 20 cats with ulcerative keratitis or keratoconjunctivitis. Aerobic bacterial culture and sensitivity were performed OU and a conjunctival cytobrush sample for feline herpesvirus (FHV-1) polymerase chain reaction (PCR) was obtained OU. Protein concentration was determined for each tear sample prior to zymography. An aliquot of each tear sample was equiloaded for protein and volume and activity were assayed on Novex precast gelatin zymogram gels in the presence and absence of EGTA. HT1080 (pro and active MMP-2/MMP-9 standard) and a molecular weight standard were also run on each gel. The gels were analyzed by computer-assisted densitometry and semiquantitative relative values for MMP-2 and MMP-9 were determined. The gelatinolytic activities were further characterized by Western blot analysis using antihuman-MMP-2 monoclonal antibody and rabbit antirat MMP-9 polyclonal antibody. Purified human MMP-9 and MMP-2 were used as MMP standards. Results: Gelatin zymographic data suggested that gelatinase activities exist in tears from normal cats and cats with ulcerative keratitis or keratoconjunctivitis and comigrate with isoforms of human MMP-2 and MMP-9. These activities are inhibited in the presence of EGTA, a typical response of MMPs Comparison of tears from normal cats with tears from cats with ulcerative keratitis demonstrated enhanced gelatinase activity in those cases of ulcerative keratitis/keratoconjunctivitis. Initial Western blot analysis of MMP-9 supports the possibility that this enzyme is feline MMP-9. FHV-1 PCR results are pending. Conclusions: Gelatinolytic activities present in the precorneal tear film of cats, comigrate with human MMP-9 and MMP-2, are inhibited by EGTA and are recognized by MMP antibodies. These results support identity of these two as feline MMP-9 and MMP-2. In addition, MMP-9 and MMP-2 activities are enhanced in tears from ulcerative keratitis/keratoconjunctivitis when compared to tears from clinically normal feline eyes. Supported by the ACVO Vision for Animals Foundation- Resident Grants. Commercial interest: None.

ABSTRACT NO.: 76

Efficacy of prophylactic topical miotic treatment for spontaneous lens luxation in dogs

D. R. Binder, I. P. Herring and D. L. Ward

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

Purpose: To determine the efficacy of topical miotic treatment in delaying contralateral anterior lens luxation and associated complications in dogs following diagnosis of unilateral anterior lens luxation. Methods: Medical records of dogs presenting to the VMRCVM with anterior lens luxation were reviewed. Treatment conditions, visual status, lens position, and intraocular pressures were recorded at initial presentation and subsequent visits. Time to lens luxation, loss of vision, and development of glaucoma in the contralateral eye of dogs with or without miotic treatment was compared using survival analysis. Results: Thirty-three untreated dogs and 16 dogs treated with miotics fit the study inclusion criteria. Mean time (days S.E.) between anterior luxation of the primary lens and luxation of the contralateral lens was numerically greater in dogs treated with miotics (720.7 + 132.5) compared to untreated dogs (631.3 + 90.6). Similar trends were observed for time between anterior lens luxation in the primary eye and development of glaucoma in the contralateral eye (treated: 808.5 + 166.6, untreated: 688.2 + 99.9), anterior lens luxation in the primary eye and vision loss of the contralateral eye (treated: 1015.1 + 136.8, untreated: 921.2 + 111.6), and anterior lens luxation in the contralateral eye and vision loss in that eye (treated: 212 + 80.6, untreated: 89.4 + 19.5). None of these trends were statistically significant. Conclusions: All measures studied portrayed trends toward improved outcomes following prophylactic topical miotic treatment in dogs susceptible to anterior lens luxation. However, no differences achieved statistical significance. Commercial interest: None.

ABSTRACT NO.: 77

Choroidal melanocytoma in a dog: a case report

S. A. Pot,* G. C. M. Grinwis† and M. H. Boevé*

*Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 8, 3584 Cm Utrecht, The Netherlands,Department of Pathology, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 8, 3584 Cm Utrecht, The Netherlands

Purpose: In this report the unique case of a dog with a choroidal melanocytoma in the left eye and an iridal melanocytoma with secondary glaucoma in the right eye is presented. Primary choroidal melanocytomas are relatively uncommon in the dog, with only 27 cases described in the veterinary literature up to this date. Methods: A nine year old female Beagle dog with a history of sudden blindness was referred to the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht. A complete ophthalmic examination was performed, followed by ocular ultrasonography and thoracic radiography. Histopathology was performed on both globes after enucleation. Results: On indirect ophthalmoscopy a black mass was observed protruding from the fundus into the vitreous of the left eye. No further abnormalities were found in this eye. The right eye showed a dorsal temporal iridal mass, enlarged globe, elevated intraocular pressure (54 mmHg), mydriasis with absence of pupillary light reflexes, cupping of the optic disc, hyperreflectivity of the tapetal area and mitigated retinal vasculature. The presumptive diagnosis of a choroidal melanocytoma in the left eye and an iridal melanocytoma with secondary glaucoma in the right eye was made. Since the dog was blind in both eyes, ultrasonography showed no extraocular extension of the masses and thoracic radiography did not reveal any metastases, both eyes were enucleated. On histopathologic examination the diagnosis was confirmed. Both the tumors were classified as benign because of low mitotic index, low to moderate cellular polymorphism and no prominence of nucleoli. Conclusions: The question arises whether these two masses might be interrelated. Metastasis of an iridal melanoma to the contralateral eye has been reported once. Because of the different cellular morphology of the two masses, and the absence of clear cellular indications of malignancy in this case, metastasis seems unlikely. Commercial interest: None.

ABSTRACT NO.: 78

Clinical features and outcomes of equine eyelid squamous cell carcinoma treated with cryotherapy with or without surgical debulking: a retrospective study (1993–2003)

C. Delgado, E. A. Giuliano, P. J. Johnson, J. W. Tyler, G. Klauss, L. E. Galle and C. P. Moore

Veterinary Medicine and Surgery, University of Missouri-Columbia, Columbia, MO, USA

Purpose: Squamous cell carcinoma (SCC) is the most common neoplasm of the equine eye and ocular adnexa. Various treatment modalities have been reported in individual case reports or case series with variable success. This study evaluated risk factors for survival and recurrence of equine eyelid SCC treated with cryotherapy with or without surgical debulking. Methods: Medical records and follow up data from 19 cases from the UMC-VMTH and private veterinary clinics in Missouri (1993–2003) were reviewed. Evaluated data included breed, sex, age at treatment, method of treatment, location of tumor on the eyelid, size of tumor, recurrence rate, and survival time. Follow-up information was obtained from RDVMs and owners to evaluate treatment outcome, complications at the surgical site, time to recurrence, number of recurrences, and survival time. Breed and sex distribution in the case population was compared to a control population comprising all horses presented to the VMTH over a 10-year period. Distributions were compared using a Chi-squared test with 11 degrees of freedom. Additionally, the impact of hypothesized risk factors [whether treatment was performed under general anesthesia or sedation and local anesthesia, whether cryotherapy alone was used, lesion surface area (= 125 mm2 or > 125 mm2), age of the horse (= 15 year or > 15 year), location of SCC (medial, central, temporal, upper or lower lid)] on recurrence and survival time was examined using the Kaplan-Meier Product limit survival Methods: Data are presented as mean ± SD. Results: Breed distribution differed significantly between the total hospital population (n = 13 273) and horses with eyelid SCC (P < 0.001, Chi-squared test with 11 degrees of freedom). Belgian and Appaloosa horses were more likely to develop eyelid SCC. Sex distribution did not differ significantly between the total hospital population and horses with eyelid SCC (P > 0.1 Chi-squared test with 3 degrees of freedom). Age of affected horses was 14.9 ± 6.7 mos (range, 5–24 mos). Seventy-nine percentage (11/14) of horses had one recurrence and 21% (3/14) of horses had more than two recurrences during the study period. Time to recurrence (n = 11) was 9.6 ± 12.0 mos (range, 1–44 mos). Survival time was 36.8 ± 18.0 mos (range, 8–63 mos). None of the hypothesized risk factors was significantly related to either recurrence time or survival time. Conclusions: Reports of recurrence rate and survival time for horses affected with eyelid SCC treated with cryotherapy are limited. Predilection of Belgian and Appaloosa horses for eyelid SCC supports similar conclusions reported previously. Recurrence and survival data reported here should be useful for purposes of comparing other treatment modalities for eyelid SCC under development. Commercial interest: None.

ABSTRACT NO.: 79

Identification of lactoferrin in canine and feline tears

B. J. Holmberg,* D. J. Maggs* and B. L. Lönnerdal†

*School of Veterinary Medicine andDepartment of Nutrition, University of California, Davis CA, USA

Purpose: To identify the iron-binding glycoprotein, lactoferrin, in canine and feline tears. Methods: Forty dogs and 20 cats free of ocular disease as determined by slit lamp biomicroscopy and indirect ophthalmoscopy were included in the study. Animals were placed in right lateral recumbency, and a nonheparinized microhematocrit tube was atraumatically placed at the lateral canthus of the right eye. Approximately 10–50 µL of tears were collected via capillary action from each animal and subsequently pooled by species for an approximate final volume of 0.5 mL (cats) and 1.0 mL (dogs). Tears were placed in a heparin-Sepharose column and agitated for 24 h at 20 °C before a three-step elution was performed. Protein content in eluted fractions was determined by absorption at 280 nm using a spectrophotometer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 10% running gel and 4% stacking gel to separate proteins based on molecular weight (MW). Purified human and bovine lactoferrin were used as standards to screen for bands of appropriate MW. Gels were stained with Coomassie blue and single bands of approximately 70–80 kDa were cut from the gel. Protein was isolated after reduction with DTT, alkylation with iodoacetamide, and finally by digestion with trypsin. Tryptic peptides were desalted, mixed with an equal volume of matrix solution, applied to the target, and analyzed by mass spectrometry (MS) to determine monoisotopic masses. Samples were further analyzed by quadrupole time of flight (qTOF) nanospray MS/MS to determine partial amino acid sequences. These sequences were compared to known amino acid sequences in GenBank. Results: SDS-PAGE analysis revealed a distinct protein band at 69.2 kDa in canine tears and 64.8 kDa in feline tears. The canine tear protein had a MS score of 94 with equine lactoferrin and a gene sequence homology of 88% with human lactoferrin. The protein isolated from feline tears had a significant MS score of 88 with bovine lactoferrin. Partial amino acid analysis revealed 100% homology of the canine peptides to equine and human lactoferrin, 90% homology to bovine, caprine, and camel lactoferrin, and 87% homology to buffalo lactoferrin. The sequenced feline peptides were 100% homologous to bovine, equine, caprine, murine, and camel lactoferrin. Conclusions: Lactoferrin is present in canine and feline tears where it most likely contributes to ocular health through the sequestration of iron from potential pathogens and inhibition of viral adsorption or penetration. Alterations in tear lactoferrin concentration may increase susceptibility to microbial invasion and be used as a marker of ocular surface health. Supported by ACVO Vision for Animals Foundation. Commercial interest: None.

ABSTRACT NO.: 80

Comparison of 6 PCR protocols for detection of feline herpesvirus (FHV-1) DNA in clinical samples and commercial vaccines

D. J. Maggs, H. E. Clarke and S. J. Smith

School of Veterinary Medicine, University of California – Davis, Davis, CA, USA

Purpose: The results of many polymerase chain reaction (PCR) protocols for detection of feline herpesvirus (FHV-1) DNA have been reported for diseased and normal cats. However, to the authors’ knowledge, their relative detection rates and ability to detect vaccine virus have not been assessed. These were the specific aims of this study. Methods: All cats were examined by a veterinary ophthalmologist or resident. Conjunctival biopsies were collected from 15 cats and placed in 1 mL of sterile phosphate buffered saline. Five commercially available vaccines licensed as an aid in preventing feline viral rhinotracheitis (FVR) were purchased from retail outlets or donated by the manufacturers and reconstituted according to each manufacturer's recommendations. DNA was extracted from vaccines and clinical samples using a commercial kit and subjected to PCR using 6 different published protocols for detection of FHV-1 DNA. Three assays were single-phase PCR (Weigler; Reubel; Sykes) and 3 were nested protocols (Hara; Stiles; Burgesser). All protocols targeted regions of the FHV-1 thymidine kinase gene and were performed as previously published with minor exceptions. Bands visible to the naked eye on ethidium bromide-stained agarose gels were considered positive. Results: All 6 protocols detected FHV-1 DNA in all 5 vaccines. However, widely disparate results were obtained for clinical samples. Complete agreement among all 6 protocols occurred for 9/15 samples; 8/9 were negative and 1/9 was positive by all protocols (Table 1). Considering the 7 samples positive by at least one protocol, the FHV-1 DNA detection rates for each protocol were 89% (Stiles), 71% (Hara), 57% (Burgesser; Weigler) 43% (Sykes), and 29% (Reubel). Conclusions: Although all PCR protocols tested detect vaccine virus, they varied greatly in their ability to detect FHV-1 DNA in clinical samples. Commercial interest: None.

Table 1. 
Sample #WeiglerHaraReubelStilesBurgesserSykes
1–8
9++++++
10+++++
11+++++
12++++
13++
14+
15+

ABSTRACT NO.: 81

Effect of shipping conditions on PCR detection of feline herpesvirus (FHV-1) DNA in clinical ocular samples

D. J. Maggs, H. E. Clarke and S. J. Smith

School of Veterinary Medicine, University of California – Davis, Davis, CA, USA

Purpose: Clinical samples are frequently tested by polymerase chain reaction (PCR) for presence of feline herpesvirus (FHV-1) DNA. Shipping methods for such samples vary greatly with respect to temperature, duration, cost, and convenience. This study was designed to assess the effect of shipping conditions on results of a commonly utilized PCR assay. Methods: All cats were examined by a veterinary ophthalmologist or resident and corneal scrapings or swabs, or conjunctival swabs or biopsies were collected based upon disease state and clinician preference. Conjunctival biopsies were divided in half, or two swabs or scraping samples were collected. All samples were placed into 1 mL of sterile phosphate buffered saline. One sample from each pair was stored at ambient temperature and mailed via USPS. The other was stored at −20C and shipped on ice via overnight courier. Transit time, maximum ambient temperature at the shipping city and Davis, CA (National Climatic Data Center), and sample temperature and condition on receipt were recorded. On receipt, samples were stored at −20C until processing to ensure identical handling of all sample pairs. DNA was extracted using a commercial kit and subjected to 40 cycles of a PCR targeting a 322 bp fragment of the FHV-1 thymidine kinase gene. Bands visible to the naked eye on ethidium bromide-stained agarose gels were considered positive. Parameters for each shipping method were compared using the Mann–Whitney rank sum test. Results: Paired samples from 24 cats were submitted. There was no significant difference (P = 0.48) between median (range) ambient temperature at shipping cities on day of dispatch for courier (32; 17–47C) and USPS (31; 17–42C) samples. There was no significant difference (P = 0.42) between median (range) ambient temperature at Davis, CA on day of receipt for courier samples (32; 11–41C) and USPS samples (31; 15–38C). Median shipping time for courier samples (24; 18–43 h) was significantly shorter (P < 0.001) than for USPS samples (144; 72–336 h). Median sample temperature upon receipt for samples sent by courier (6; range 1–23C) was significantly lower (P < 0.001) than for those sent by USPS (23; range: 22–24C). Color or turbidity changes were noted in 1 sample sent by courier and 8 samples sent by USPS. PCR results for paired samples were consistent in 23 cats (6 positive; 17 negative). FHV-1 DNA was detected only in the chilled sample from the remaining cat. For this cat, data for the samples sent by courier and USPS, respectively, were shipping times (24 & 100 h), sample temperatures (6 & 23C), and dispatch (40 & 40C) or receipt (38 & 33C) ambient temperatures. No change in sample quality was visually evident in either of the samples from this cat. Conclusions: Sample handling and shipping temperatures appear to have little effect on FHV-1 DNA detection for the PCR protocol assessed here. Commercial interest: None.

ABSTRACT NO.: 82

Multifocal chorioretinitis in dogs resembling white dot syndromes in humans

S. Pizzirani, F. Maggio and M. G. Davidson

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

Purpose: To describe fundic lesions in dogs represented by idiopathic multifocal chorioretinitis and chorioretinoneuritis similar to posterior segment diseases in humans recognized as White Dot Syndromes (WDS). Methods: Clinical signs, diagnostic procedures and results, therapies and outcomes of dogs presented at Clinica Veterinaria Europa (Florence, Italy) and VTH-NCSU (1999–2004) with multifocal chorioretinitis with or without optic neuritis of the posterior pole were reviewed. Dogs with infectious causes, cerebral masses or other identifiable diseases were excluded. Results: Eleven dogs were included in this study (7 m, 5 F). Ten different breeds were represented. Median age was 5.0 years (range 1–7). Chief complaints at presentation were bilateral (7) or unilateral (1) blindness, and red eyes (3) with no visual deficit. Clinical diagnoses included bilateral optic neuritis/neuropathy (7), unilateral optic neuritis (1), and multifocal peripapillary chorioretinitis/retinopathy (11). Non contrast and contrast brain imaging (4 CT and 1 MRI) was unremarkable in 4 cases while a diffuse, multifocal inflammatory disease of the brain cortex was suspected in 1 patient, who had a mild increase in CSF proteins and cells. CSF analysis was performed in 5 dogs (mean protein content 17.9 ± 12.93 mg/dL and mean cells content 4.8 ± 4.4/µL). Fluorescein angiography was performed in 10 cases and demonstrated early choroidal hypoperfusion and progressive multifocal fluorescein pooling at RPE interface mostly in the nontapetal retina. Therapy was instituted with systemic steroids (prednisone 1–4 mg/kg, tapered) in all patients. Oral azathioprine (2 mg/kg, tapered) was used in 5 cases. Vision was recovered in 6 blind dogs and preserved for a median of 9.5 months (range 2–12) (mean 8.5 ± 4.18). Recurrence and progression of retinal degeneration and optic neuropathy was common. Conclusions: Clinical findings and responses to therapy in these patients were supportive of an immune/autoimmune disease. Close clinical similarities with Acute Placoid Multifocal Posterior Pigmented Epitheliopathy (AMPPPE), Birdshot Choroidopathy, and Multifocal Choroiditis in humans were hypothesized. In case of chorioretinoneuritis in dogs, once infectious and neoplastic causes have been ruled out, immunosuppressive therapy delays the progression of the disease and, in acute cases, may restore and preserve vision for many months. Commercial interest: None.

ABSTRACT NO.: 83

Parasite load in lacrimal glands of dogs affected with leishmaniasis

C. Naranjo,* L. Altet,† M. Leiva,‡ X. Roura‡ and T. Pena*

Departament de Medicina i Cirurgia Animals, *Departament de Ciència Animal i dels Aliments,Hospital Clínic Veterinari,Universitat Autònoma de Barcelona (Spain)

Purpose: Keratoconjuntivitis sicca (KCS) is a potentially blinding disease. In a previous study it was determined that 2.8% of dogs with leishmaniasis have KCS, but no other studies about the pathogenesis of KCS in dogs with leishmaniasis have been performed. The aim of this study was to evaluate the presence of Leishmania infantum in lacrimal glands of dogs affected with leishmaniasis by means of Real-Time PCR. Methods: Twenty-five dogs that died or were euthanized with a diagnosis of leishmaniasis and three control dogs belonging to a research group of the University were included in the study. Eleven of the diseased dogs had ocular signs as reported by the clinicians attending the cases. Control dogs were from an endemic area, had no signs of leishmaniasis and had negative serology and negative delayed hypersensitivity reaction. Meibomian glands, main lacrimal gland and gland of the third eyelid were collected and Real-Time PCR was performed. Standardization of this technique was developed for these tissues and relative quantitation assay and comparative Ct method (2−ΔΔCt) was applied using 18S RNA gene as housekeeping. Results: The standard curves of the three tissues tested had slopes near −3.3, revealing a PCR efficiency of 100% for the two genes (Leishmania and 18S RNA). All samples submitted yielded positive results with varying amounts of parasite. Samples from control dogs were also positive. So, we considered as ‘true positives’ those samples having more parasites than those from the control dogs. According to this criterion, 40 out of 48 Meibomian glands, 35 out of 50 main lacrimal glands and 32 out of 50 glands of the third eyelid had higher amounts of parasite than the corresponding control tissues. There were no differences between parasite presence and location in the right or left eye. Samples from dogs with ophthalmic signs had higher quantities of parasites. Conclusions: Ophthalmic signs compatible with KCS in dogs with leishmaniasis are related to the presence of the parasite in lacrimal glands. Although in an endemic area animals without ocular signs can harbor the parasite in their glands it is possible that a certain amount is necessary in order to produce KCS. Real-Time PCR can help in these areas to determine if ocular signs are really related to leishmaniasis or not. Due to the design of this study, not all the animals underwent a systematic ophthalmic examination, so it is also possible that ocular signs in this study may have been underestimated. Our results show that, as signs of KCS are related to local parasite amount, treatment with cyclosporinee in animals with leishmaniasis and KCS may not be recommended, as this drug inhibits IL-2, a cytokine related to resistance to leishmaniasis. Support: Carolina Naranjo receives doctoral funding from an Autonomous government grant. Commercial interest: None.

ABSTRACT NO.: 84

Aqueous humor and trabecular meshwork myocilin in normal and POAG Beagles

E. O. MacKay,* H. Hart,* K. N. Gelatt,* D. A. Samuelson,* D. W. Esson† and M. B. Sherwood†

Departments of *Small Animal Clinical Sciences, andOphthalmology, University of Florida, Gainesville, FL, USA

Purpose: Spontaneous primary open angle glaucoma (POAG) is inherited as an autosomal recessive trait in the Beagle. Biochemical, histochemical, and ultrastructural studies suggest the increase in aqueous humor (AH) outflow resistance is associated with the accumulation of the extracellular matrix and decline in the number of trabecular meshwork (TM) cells in the outflow pathways. Mutations in the GLC1A gene or myocilin have been associated with juvenile open-angle glaucoma in man. Myocilin was first isolated from human trabecular meshwork cells after induction with dexamethasone (TIGR-trabecular meshwork-induced glucocorticoid response), and has been reported in man, nonhuman primate, cow, pig, rabbit, rat and mouse. The aim of this study was to measure and compare AH and TM myocilin levels in normal Beagles and Beagles with POAG at different levels of ocular hypertension. Methods: AH samples (0.1–0.2 mL) were obtained from the anterior chambers of 10 normal laboratory quality Beagles and 12 Beagles exhibiting early, moderate or severe POAG. Two:L aqueous samples from different dogs were loaded and run on 12% Bis-Tri gels (Invitrogen, Carlsbad CA). The separated proteins were then transferred onto nitrocellulose membranes and the blot incubated with an antimyocilin rabbit polyclonal antibody and stained (Supra Signal West Pico Chemiluminescent, Pierce, Rockford IL) for scanning. The TM and ciliary body tissues were immunolabeled with the same antibody that was secondarily localized by antirabbit goat biotinylated antibody. Results: AH and TM myocilin levels were detected in both normal and glaucomatous beagles. The highest levels of AH myocilin occurred in the glaucomatous dogs and were strongly correlated with the severity of disease. In TM myocilin was localized most intensely within the aqueous outflow pathways. Conclusion: This is the first report of myocilin in the canine eye. AH and TM myocilin levels were present in normal Beagles as well as those affected by POAG. Myocilin levels in glaucomatous dogs were higher than those in the normal animals and increased progressively in the early, moderate and advanced glaucoma stages. Supported by the University of Florida DSR Opportunity Grant # 03090955, Society for the Prevention of Blindness, Merck-Merial Veterinary Scholars Research Grant, Jaqua Foundation, and Alcon Pharmaceuticals. Commercial interest: None.

ABSTRACT NO.: 85

Phenol red thread test and Schirmer tear tests in amazon parrots

E. S. Storey, D. A. Carboni and T. N. Tully

School of Veterinary Medicine, Louisiana State University, Baton Rouge LA, USA

Purpose: (1) To determine the mean Phenol Red Thread Test (PRTT) value in normal Amazon parrots and compare it to the mean Schirmer Tear Test (STT) value before and after topical anesthesia. Methods: Each of 24 Hispanolian parrots (Amazona ventralis) in one colony underwent PRTT and STT before topical anesthesia with topical 1% proparacaine as part of a complete ophthalmic examination. IOP was measured after topical anesthesia with applanation tonometry. Biomicroscopic examination and indirect ophthalmoscopic examination followed. Ten minutes after application of the topical anesthetic, PRTT and STT were repeated. Fluorescein staining of the cornea was then completed. Results: Twenty-three of the 24 parrots were normal on ophthalmic examination. Mean PRTT value prior to topical anesthesia was 15.4 mm/15 s ± 5.3 mm and ranged from 6 to 28 mm/15 s. Following topical anesthesia, mean PRTT value was 16.5 mm/15 s ± 5.2 mm and ranged from 6 to 27 mm/15 s. Mean STT value prior to topical anesthesia was 8.1 mm/minute ± 2.6 mm and ranged from 2 to 12 mm/minute. Following topical anesthesia, mean STT value was 6.0 mm/minute ± 3.7 mm and ranged from 0 to 18 mm/minute. There was no significant difference between the right and left eyes. A Pearson's correlation of only 0.34 (P < 0.04) exists between PRTT and STT values prior to topical anesthesia and 0.46 (P < 0.004) following topical anesthesia. Conclusions: PRTT and STT are both viable methods to measure the tear production in Amazona ventralis. Topical anesthesia did not have a significant effect on the PRTT values (P < 0.15). Topical anesthesia significantly lowered the STT values (P < 0.0001). PRTT and STT values are weakly correlated and should not be used interchangeably or compared directly. Commercial interest: None.

ABSTRACT NO.: 86

Evaluation of digital imaging to document the adnexa and anterior segment in veterinary ophthalmology

N. J. Millichamp,*,† L. Wadsworth,* J. Dziezyc,*,† A. Yu-Speight,† J. Harris* and D. R. Sisley†

*College of Veterinary Medicine, Texas A & M University,Central Texas Veterinary Ophthalmology, Austin TX, USA

Purpose: To evaluate the usefulness of various digital cameras required to image the ocular adnexa and anterior segment in small animals. Methods: Macrophotographs were taken of the eyes of small animals using four different digital cameras ranging from low resolution point and shoot to high resolution professional single lens reflex (SLR) models. The characteristics evaluated included the overall ease of use, the quality of the resulting image for diagnostic interpretation, the simplicity and precision of focus and the influence of the flash arrangement on the image. Results: All of the cameras tested yielded photographs of adequate quality to make an evaluation of the disease process being recorded. The main differences between the quality of images produced by the different cameras were related to the inherent camera resolution, the ease and speed of achieving focus and the effect of flash illumination and artifacts on the image. Higher resolution SLR cameras with ring-flash options provided the best quality images. Conclusions: Digital imaging of the anterior segment of the small animal eye is easily accomplished with currently available digital cameras with attention to a few simple photographic principles. The quality of the images may be adequate for relatively low-cost asynchronous telemedicine applications. Supported by Central Texas Veterinary Ophthalmology. Commercial interest: None.

Ancillary