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Background: Conjunctival pedicle flaps are one of the most frequently employed surgical interventions used to address a variety of sight threatening corneal diseases in veterinary ophthalmic practice. Securing the conjunctiva to the cornea is typically achieved through suturing, which is technically challenging and can result in prolonged surgical times, increased corneal edema, increased scar tissue, foreign body reaction, suture abscess and dehiscence. In human ophthalmology, a number of sutureless techniques to affix ocular tissues are being explored. Specifically, these approaches include synthetic tissue adhesives, bioadhesives, and hydrogel sealants. The proposed advantages of adhesives over suture, include reduced operative times, watertight seals, decreased foreign-body sensation and inflammation, faster healing times and tissue regeneration with original architecture restoration. Objective. To evaluate the maximum tensile force a conjunctival pedicle flap is able to withstand with respect to different fixation methods, i.e., Tisseel® fibrin glue, ethyl cyanoacrylate adhesive, ReSure® hydrogel sealant, or 8-0 VICRYL suture. Animals Studied. Ex-vivo porcine globes Procedures. Following a 500-micron restricted depth lamellar keratectomy, conjunctival pedicle flaps were dissected and secured to corneal defects with either the bioadhesive Tisseel®, or the synthetic adhesives ReSure®, ethyl cyanoacrylate, or 8-0 VICRYL® suture. Harvested corneoconjunctival flap interfaces were clamped to an accelerometer and potentiometer device, and loaded under video surveillance until the point of failure. Peak load at failure was determined for each test and used to compare between sample types. Results. 40 flaps underwent tensile force testing, with 6 being omitted for dehiscence prior to tensile testing. Of the 34 tests included in analysis, 10 conjunctival flaps were secured with suture, 10 with cyanoacrylate, 8 with ReSure® hydrogel sealant, and 6 with Tisseel® fibrin glue. A significant increase in maximum withstood tensile force was recorded between sutured flap fixation when compared with cyanoacrylate glue (p=0.02474), ReSure® hydrogel sealant (p= 0.00000), and Tisseel® fibrin glue (p= 0.00002). Cyanoacrylate fixation was significantly stronger when compared with ReSure® hydrogel sealant and Tisseel fibrin glue (p=0.01194 and 0.01798 respectively). There was no significant difference in adhesion strength between ReSure® hydrogel sealant and Tisseel® fibrin glue (p=0.95675). Conclusions. Conjunctival pedicle flap fixation using 8-0 VICRYL® suture fixation was able to withstand significantly greater maximum tensile force application in comparison with the ReSure®, Tisseel®, or cyanoacrylate adhesives. Master of Science Conjunctival pedicle flaps are one of the most frequently employed surgical interventions to address sight threatening corneal disorders in companion animals. Due to its redundant nature and close proximity to the corneal surface, conjunctival tissue is readily available for grafting to the cornea. It is surgically dissected to appropriate size and repositioned over the corneal defect where it effectively aids in healing through direct provision of structural support and indirectly via its rich blood supply. Securing the conjunctiva to cornea is typically achieved through suturing, which is technically challenging and can result in prolonged surgical times, increased corneal edema, increased scar tissue, foreign body reaction, abscess and dehiscence. In human ophthalmology, a number of sutureless techniques to affix ocular tissues are being explored. Specifically, these approaches include synthetic tissue adhesives, bioadhesives, and hydrogel sealants. The proposed advantages of tissue adhesives over suture, include reduced operation times, watertight closures, decreased foreign-body reaction and inflammatory response, faster healing times and increased ability to induce regeneration of the original tissue architecture. The purpose of this study was to evaluate the maximum tensile force a corneoconjunctial pedicle flap is able to withstand with respect to four different fixation methods, 40 ex-vivo porcine globes underwent conjunctival pedicle flap procedures. Each pedicle flap was secured to cornea with either 8-0 Vicryl® suture, Tisseel®, ethyl cyanoacrylate, or ReSure®. After harvesting from the globe, the corneoconjunctival unions were clamped to an accelerometer and potentiometer device, and loaded under video surveillance until the point of failure. The peak load was determined for each test and used to compare between sample types.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their analgesic, anti-pyretic and anti-inflammatory properties in both human and veterinary patients. Topical ophthalmic NSAIDs are commonly employed in the management of intraocular inflammation (uveitis), corneoconjunctival inflammatory disease and pre-operatively to prevent intraoperative miosis during cataract surgery. Despite their routine application in these clinical scenarios, little is known regarding the corneal penetration and relative anti-inflammatory efficacy of the available topical ophthalmic NSAIDs in the dog. Decisions regarding which of these agents to employ are therefore based upon factors such as cost and ease of acquisition as opposed to established efficacy. Objectives: To investigate the relative intraocular penetration and anti-inflammatory efficacy of two commonly utilized topical ophthalmic NSAIDs in dogs, diclofenac 0.1% and ketorolac 0.5%. Animals: Twenty-two client owned dogs (22 operated eyes) presenting to the VTH ophthalmology service for routine cataract surgery for mature or hypermature cataract. Methods: Subjects were randomized to be treated with either topical ketorolac 0.5% or topical diclofenac 0.1% ophthalmic solutions at specified times in the 24-hour period pre-operatively. Aqueous humor samples were obtained intra-operatively and stored for subsequent evaluation of drug concentrations and prostaglandin E2 (PGE2) concentrations via ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and enzyme-linked immunoassay (ELISA) analysis, respectively. Results: Median aqueous humor drug concentrations were significantly higher in dogs treated with ketorolac 0.5% (1311.6 ng/mL) compared to those treated with diclofenac 0.1% (284.9 ng/mL). There was no significant difference in aqueous humor PGE2 concentrations between the two treatment groups. No significant association was determined between aqueous humor drug concentration and PGE2 concentration. There was no significant association between diabetic status and aqueous humor drug concentration or PGE2 concentration in either group. Conclusions and clinical importance: This study suggests that topical ketorolac 0.5% and diclofenac 0.1% are efficacious in decreasing aqueous humor PGE2 concentrations and are equally suitable for use based on their comparable anti-inflammatory profiles. The results of these assays provide clinically relevant information regarding intraocular penetration and anti-inflammatory efficacy of these medications in dogs with cataract. Master of Science Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their analgesic, anti-pyretic and anti-inflammatory properties in both human and veterinary patients. Topical ophthalmic NSAIDs are commonly employed in the management of intraocular inflammation (uveitis), corneoconjunctival inflammatory disease and pre-operatively to prevent intraoperative miosis during cataract surgery. Despite their routine application in these clinical scenarios, little is known regarding the intraocular penetration and relative anti-inflammatory efficacy of the available topical ophthalmic NSAIDs in the dog. Decisions regarding which of these agents to employ are therefore based upon factors such as cost and ease of acquisition as opposed to established efficacy. Efficacy of topical anti-inflammatory medications in controlling intraocular inflammation is primarily related to the ability of the medication to penetrate the cornea and its efficacy at suppressing inflammatory mediators. The purpose of this study, therefore, is to investigate the relative intraocular penetration and anti-inflammatory efficacy of two commonly utilized topical ophthalmic NSAIDs in dogs, diclofenac 0.1% and ketorolac 0.5%. Twenty-two dogs presenting to the VTH ophthalmology service for routine cataract surgery with the presence of a mature or hypermature cataract were enrolled in a prospective, randomized clinical trial. Subjects were treated with either topical ketorolac 0.5% or topical diclofenac 0.1% ophthalmic solutions at specified times in the 24-hour period pre-operatively. Aqueous humor samples were obtained intra-operatively and stored for subsequent evaluation of drug concentrations (n=22) and prostaglandin E2 (PGE2) concentrations (n=19) via ultra performance liquid chromatography (UPLC) and enzyme-linked immunoassay (ELISA) analysis, respectively. Treatment with topical ketorolac 0.5% resulted in higher median aqueous humor drug concentrations when compared to treatment with diclofenac 0.1% (1311.6 ng/mL vs. 284.9 ng/mL). However, there was no significant difference in anti-inflammatory efficacy when comparing PGE2 concentrations between the two groups. Furthermore, no significant association was determined when drug concentration was directly compared with PGE2 concentration. The results of these assays suggest that topical ketorolac 0.5% and diclofenac 0.1% are equally suitable for use based on their comparable anti-inflammatory profiles, and provides clinically relevant information regarding intraocular penetration and anti-inflammatory efficacy of these medications in dogs with cataract.

Background: Glaucoma is a chronic ocular disease of both dogs and humans that results in blindness and ocular discomfort. Most commonly, end-stage glaucomatous eyes in dogs are enucleated to provide comfort. This intervention requires significant financial investment, general anesthesia, and has a psychological impact on some owners. Retrobulbar neurolytic injections are used in humans to provide immediate and long-acting pain relief, while simultaneously preserving the globe. Objectives: To determine the safety and efficacy of retrobulbar neurolytic ethanol injection in canine eyes with end-stage glaucoma. Animals: 16 client-owned dogs (19 eyes) diagnosed with end-stage glaucoma. Methods: All eyes underwent an ophthalmic examination, including Schirmer Tear Testing (STT), intraocular pressure (IOP) measurement, corneal touch threshold (CTT), anterior and posterior segment examination, and fluorescein staining. Subjects were sedated and administered a retrobulbar block with bupivacaine, followed by retrobulbar injection of ethanol or saline solution. At specified time points after the procedure, clients assessed their pet's comfort and side effects of the injections via survey. Subjects returned for enucleation. Owner perceived comfort after the enucleation was assessed at identical post-procedure time points and compared to that achieved with retrobulbar ethanol injection or control solution. Overall client satisfaction with each procedure, as well as the effects of retrobulbar ethanol injection on STT, IOP, CTT, and histological changes in retrobulbar tissues were investigated. Results: Retrobulbar neurolytic ethanol injections did not signficiantly improve owner perceived comfort compared to control group treatment or provide more comfort than enucleation. Retrobulbar ethanol injections did not signficantly lower IOP, but did significantly elevate CTT. There was a trend towards lower STT in eyes receiving retrobulbar ethanol injections. Retrobulbar ethanol injections were safe, well tolerated, and no differences in client satisfaction with particpation in the study were noted in either injection group. Histologically, globes in the treatment group displayed significantly greater inflammation and fibrosis; retrobublar tissue samples were not significantly different between control and treatment groups with regard to inflammation or fibrosis. Conclusions and Clinical Importance: Retrobulbar neurolytic ethanol injections were not determined to be an effective globe-sparing alternative treatment to provide analgesia for end-stage canine glaucoma. Enucleation remains an effective way to provide comfort to dogs with elevated IOP. Master of Science The glaucomas represent a diverse group of blinding and painful diseases associated with elevated intraocular pressure (IOP). Despite advances in the medical and surgical treatment of glaucoma, the long-term prognosis in dogs remains dismal for IOP control, comfort, and globe retention. Blindness and pain are common long-term outcomes, necessitating surgical salvage procedures aimed at restoring patient comfort, including enucleation (eye removal), intrascleral prosthesis, or intravitreal chemical cyclodestruction. The most commonly performed, effective, and predictable of these options is enucleation, but this requires general anesthesia, a considerable financial investment, risks post-surgical complications, and has a negative psychological impact on some owners. Retrobulbar neurolytic injections with absolute ethanol have been performed in humans with blind, painful eyes since the early 1900s. Immediate and long lasting pain relief can be achieved from 2 weeks to 2 years after a single injection. The purpose of this study is to determine the safety and efficacy of retrobulbar ethanol injections as a globe-sparing therapeutic option for end-stage glaucoma in dogs. Nineteen dogs presenting to the VTH ophthalmology service with end-stage glaucoma were enrolled in a prospective, randomized, double-masked clinical trial. Subjects were sedated and administered a retrobulbar injection of ethanol (n=9) or control saline solution (n=10). At specified time points after the procedure, clients assessed their pet’s comfort and side effects of the injections via survey. Three weeks later, subjects returned for enucleation and the level of comfort after the enucleation was assessed at identical post-procedure time points and compared to that achieved with retrobulbar ethanol injection or control solution. Retrobulbar neurolytic ethanol injections did not significantly improve comfort compared to control group sham treatment or provide more comfort than enucleation. Retrobulbar ethanol injections did not significantly lower IOP, but did significantly elevate corneal touch threshold in treated patients. There was a trend towards lower tear production in eyes receiving retrobulbar ethanol injections. Retrobulbar ethanol injections were safe, well tolerated, and no differences in client satisfaction with participation in the study were noted in either injection group. Further investigation is warranted to determine the optimal volume of retrobulbar ethanol to provide analgesia for patients with end-stage glaucoma as well as to determine the duration of clinical effect of these injections.

Background: Keratoconjunctivitis sicca (KCS) is a chronic ocular disease of both dogs and humans that can result in ocular discomfort, corneal opacification, and vision loss. Lacritin, a protein found in the tears of many species, has been shown to play a role in lacrimation and corneal health. Because of its role as a potential lacrimostimulant, assessment of endogenous lacritin levels could reveal a correlation between lacritin and tear production in the dog. Objectives: To determine if tear lacritin concentrations are decreased in canine eyes affected by KCS. Animals: 58 client-owned dogs (tear samples from 55 eyes with normal tear production and 55 eyes diagnosed with KCS). Methods: All eyes underwent an ophthalmic exam, including Schirmer Tear Testing (STT), anterior segment assessment, and tear sample collection. Tear samples were evaluated for their total protein concentrations via BCA assay and lacritin concentrations via ELISA. Results: Total protein of canine tears is increased in KCS-affected eyes as compared to normal eyes. Tear lacritin as a component of total tear protein is significantly decreased in tears from KCS-affected eyes. When measured as a concentration (mass per volume of aqueous tears), lacritin is not significantly different between KCS-affected eyes and normal eyes, nor were they strongly correlated to STT values. Conclusions and Clinical Importance: Total tear protein levels were significantly increased in canine KCS. When quantified as a proportion of total tear sample protein, tear lacritin levels are decreased in KCS-affected eyes. Relative to tear volume, tear lacritin levels are not significantly different between KCS-affected eyes and normal eyes. Assessment of lacritin supplementation in canine KCS is warranted to evaluate potential effects on lacrimation and ocular surface health. Master of Science

The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. Ph. D.

Combat-related blast injuries are occurring more frequently with the increased use of improvised explosive devices in current military conflicts. Though much research has focused on how the body responds to the relatively low loading rates associated with blunt trauma, little is known regarding the response of the body to the higher loading rates associated with blast trauma. While soldiers are surviving once-lethal blast events due to enhanced protective equipment, injuries such as those to the eye and face that were once considered inconsequential, can now be detrimental to long-term healthcare costs and quality of life. Although it is suggested primary blast overpressure (i.e., the shock wave) can cause severe eye injuries, there remains few empirical data in the literature that confirms this. Adding to this, there are currently no testing standards to assess the effectiveness of personal protective equipment during blast exposure. Expanding upon traditional research techniques within the field of injury biomechanics, the research in this dissertation focuses specifically on developing experimental and physical models of the eye, face, and orbit for blast overpressure exposure. Foremost, a porcine eye model is used to quantify eye injury risk from blast overpressure exposure. Subsequently, these biomechanical data are used to develop a physical model of the eye that can be used in lieu of cadaver specimens for blunt and blast loading. Lastly, military spectacles and goggles are examined for effectiveness at protecting the eye during blast exposure. Combined with detailed computer-aided design geometries, these data can be used to validate computational models of the eye, orbit, and face to blast loading. Results from these tests support one theory that shock waves may enter the skull through the orbit, alluding to future work that is essential to more fully understanding the physiological response of the brain and ocular motor system to blast exposure. Ultimately, the experimental methods and analysis techniques disseminated herein serve as a framework for future experimental work related to blast and other high-rate loading scenarios. Ph. D.

Development of cataracts is a relatively frequent ocular disease of the dog and cataract extraction via phacoemulsification (PE) is commonly performed by veterinary ophthalmologists. Postoperative ocular hypertension (POH) describes the elevation of pressures within the eye during the acute postoperative period and can result in vision loss and poor surgical outcome. Relatively little is known about risk factors or efficacy of prophylactic treatment for POH, and current clinical practice with regard to pressure monitoring and medication administration are highly variable. The literature on POH prophylaxis in humans indicates that improved efficacy may be achieved with a multi-dose approach and that dorzolamide hydrochloride/timolol maleate (DHTM) may be more efficacious than other pressure lowering medications. The canine literature on POH prophylaxis is limited and DHTM has not yet been evaluated despite common use in the clinical setting. Our objectives, therefore, were to investigate risk factors for POH and to test the hypothesis that perioperative topical ophthalmic dorzolamide hydrochloride 2%/timolol maleate 0.5% (DHTM) reduces the prevalence and/or severity of postoperative ocular hypertension (POH) in dogs undergoing cataract extraction by phacoemulsification (PE). We employed a randomized double-masked placebo-controlled study and enrolled 103 dogs (180 eyes) presenting for unilateral or bilateral PE. Select historical, signalment, ophthalmic examination, and surgical data was collected. Dogs were treated with DHTM or Blink Contacts (BC) placebo at 14- and 2-h preoperatively and at conclusion of surgical closure. Intraocular pressures were assessed by rebound tonometry at 2, 4, 6, and 8 hours after surgery and at 8 am the following morning. POH was defined as IOP>25 mmHg and intervention consisted of latanoprost 0.005% if IOP rose to 26 mmHg - 45 mmHg or surgeon treatment of choice if >45 mmHg. Our investigation of risk factors yielded a statistically significant association only with surgeon and surgical time, which were also associated with one another. DHTM significantly reduced the prevalence of POH in comparison with BC (26% versus 49% of eyes, OR=0.36; 34% versus 62% of dogs, OR=0.32). There was also a trend toward reduction of POH severity in DHTM-treated eyes (POH value 37.17±10.47 mmHg with BC, 32.67±6.39 mmHg with DHTM). DHTM-treated eyes that developed POH were significantly more likely to respond favorably (1 hour post-treatment IOP

This body of work encompasses two studies: the collection of canine tears via a novel polyesterrod and the comparison of VEGF-A concentrations in tears from dogs with normal andvascularized corneas. The first study used polyester rods for tear collection in dogs. Fluid volume and VEGF recovery characteristics, as well as potential binding of VEGF to the rod, were determined. Tears were harvested from normal dogs using rods and glass capillary tubes. Tears were assayed for tear film VEGF using a commercial canine VEGF sandwich ELISA kit. Dilutions of VEGF standard were wicked into the rods or drawn into capillary tubes, eluted, and assayed. Percent volume recovery is adequate for polyester rods as is percent VEGF recovery. VEGF is detectable in normal canine tears.The second study harvested tear samples from eyes of dogs with vascularizing corneal disease, as well as the contralateral unaffected eye of unilaterally diseased dogs, and normal dogs. Vascularization scores were assigned to diseased eyes and tear film VEGF concentration was assayed as above. Mean tear film VEGF concentration of diseased eyes did not differ from control eyes, and was not correlated with disease process, extent of vascularization, or other parameters. Tear film VEGF in unaffected eyes was significantly higher than control and vascularized eyes. Canine tear film VEGF exceeds biologically active concentrations, but does not correlate with state of corneal vascularization. VEGF-related control of corneal vascularization may be mediated by other proangiogenic factors. Master of Science

Vascular endothelial growth factor A (VEGF) is a potent mediator of blood vessel formation throughout the body. Intraocular diseases characterized by inflammation, hypoxia or neoplasia induce new blood vessel formation within the eye. The end result of such blood vessel formation may be blinding sequellae such as glaucoma from outflow obstruction or hyphema from intraocular hemorrhage. Elevated VEGF concentrations in the aqueous humor and vitreous are documented in a number of human intraocular disease processes, including tumors, retinal detachment and uveitic glaucoma. Pharmacotherapy inhibiting VEGF expression demonstrates promise for control of some of these ophthalmic conditions. We quantified and compared VEGF concentrations in canine aqueous humor samples from 13 dogs with normal eyes and 226 eyes from 178 dogs with a variety of ophthalmic diseases by ELISA. Dogs with primary cataract, diabetic cataract, primary glaucoma, uveitic glaucoma, aphakic/pseudophakic glaucoma, retinal detachment, lens luxation and neoplasia were evaluated. Elevated VEGF concentrations were found in all disease conditions tested as compared to normal dogs excepting cataracts and diabetic cataracts. Elevated aqueous humor VEGF concentrations were found in dogs with pre-iridal fibrovascular membranes (PIFM) as compared to dogs without PIFM. These results are consistent with the hypothesis that VEGF has a role in the causation or progression of a variety of canine ocular disorders. Master of Science

There are over 1.9 million eye injuries per year in the United States with over 30,000 patients left blind in at least one eye as a result of trauma. Some of the most severe eye injuries can occur in automobile accidents and from sports related impacts. Eye injuries in the military environment are even more prevalent and are generally more severe than eye injuries to civilians. The rate of eye injuries has dramatically increased in warfare in recent years, rising from 2% of all casualties during World War I and World War II to over 13% of all combat injuries in Operation Desert Storm. While many of the conflict-related eye injuries are caused by shrapnel and other debris, nearly 25% of the injuries are also caused by blunt trauma from motor vehicle and helicopter crashes, falling, and direct hits from blunt objects. In order to develop safety countermeasures effective at preventing these eye injuries, as well as evaluate the eye injury potential of different impacts, it is desirable to have the capability for distinguishing between injurious and non-injurious eye impacts. Current anthropometric test device (ATD) headforms lack instrumentation and facial features to allow detailed assessment of eye or discrete facial injuries. Therefore, the purpose of this dissertation is to present the development and validation of the Facial and Ocular CountermeasUre Safety (FOCUS) headform's synthetic eye and orbit and corresponding eye injury risk criteria. Ph. D.

The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, Epilife®, compared to Dulbecco's modification of Eagle's medium (DMEM) with fetal bovine serum (FBS), and to characterize cultured canine corneal cells. Superficial keratectomies were performed on three dogs. Samples were trypsinized to separate cell layers. Post-trypsinization, immunohistochemistry confirmed that epithelial cells had been released from the stroma. Both cell populations (presumed epithelial cells and stromal tissues) were cultured in DMEM with FBS or Epilife®. First passage cells were fixed for immunocytochemistry and prepared for PCR. Immunocytochemical staining for pancytokeratin, vimentin, and E-cadherin was evaluated, and immunofluorescence for zonula occludens-1 was attempted. Amplification of cytokeratin 5 (CK5) mRNA was assessed by PCR. Primary presumed epithelial cells grew faster when cultured in DMEM with FBS compared to Epilife®. Stromal tissue segments in Epilife® medium failed to adhere to culture plates, indicating that this medium may inhibit attachment and growth of non-epithelial tissues. Staining of corneal tissue segments confirmed that epithelial layers were pancytokeratin and E-cadherin positive, while stromal cells were vimentin positive. Immunocytochemistry of cultured cells revealed that epithelial cells stained positively for pancytokeratin, vimentin, and E-cadherin, while stromal cells remained only vimentin positive. Greater amplification of CK5 mRNA occurred from epithelial cells grown in Epilife® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, Epilife® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS. Master of Science

The purpose of this study was to determine the effects of three medical treatments, topical oxytetracycline, topical prednisolone acetate, and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. The study design consisted of twelve ophthalmically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (500mg IM bid), topical 1% prednisolone acetate (0.2ml tid), or topical oxytetracycline (0.2ml tid), followed by a 9-day washout period. All topical medications were applied to the left eye and the right eye was treated with a placebo. Tears were collected before the first treatment on day one and the morning following the last treatment on day 5. Tear film proteinase activity was measured using gelatin zymography and measurements of optical density. Statistical analysis of the difference between the treated and untreated eyes and the eyes before and after treatment was performed using mixed effects model for ANOVA. When eyes were compared after treatment, there was no significant difference between treated and placebo eyes for MMP-2 or MMP-9 for any of the treatments. When post-treated eyes were compared to pre-treated eyes, there was a significant decrease in MMP-2 activity in the left eye of horses treated with flunixin meglumine (P=0.0259). There were no differences in MMP-2 and MMP-9 activity for the other treatments. In conclusion, topical 1% prednisolone acetate and topical oxytetracycline did not significantly change MMP-2 or MMP-9 activity in normal equine tear film. Systemic flunixin meglumine had an inhibitory, but questionable, effect on MMP-2 activity in normal equine tear film. This project was funded by Patricia Bonsall Stuart Award for Equine Research. Master of Science