104 Abstracts � Vol 30, No 2 (2010)

(100 mcg/kg). The plasma drug concentration-time profilewas analyzed using noncompartmental and compartmentalapproaches. Pharmacodynamics were measured using ADP-and collagen-induced platelet aggregation, and a model wasdeveloped to describe the observed drug effects.

RESULTSA two-compartment linear pharmacokinetic model de-scribed ketanserin’s plasma concentration-time profile; ter-minal half-life was 8.25 hour, volume of distribution was11.6 L, AUC was 5.98 mg�min/liter and clearance was0.01 L/min. Two populations of horses were identified,one in which ketanserin reduced agonist-induced plateletaggregation and one in which platelet responses were notaltered. In responding horses, ADP-induced platelet ag-gregation 8 hours after treatment decreased from71.2� 5.3% to 45.7� 9% and collagen-induced aggrega-tion from 72.3� 5.7% to 43.4� 17.2% (p< 0.001). After24 hours, platelet aggregation had returned to baselinevalues. Ketanserin pharmacodynamics exhibits a hysteresisand modeling suggests its dynamics can be described usingan indirect response model.

DISCUSSION, CLINICAL RELEVANCEAND CONCLUSIONA pharmacokinetic model was developed to describe theplasma concentration-time profile of ketanserin in healthyhorses, which suggests ketanserin’s time-course of activitycan be described by an indirect response model. Additionalstudies will be performed to determine whether alterationsin platelet and laminar microvascular function are related orindependent effects of the drug.

Cellular Proliferation Markersin Lamellar Tissue and Skin Measuredby Indirect ImmunofluorescenceRebecca Carter,1 Susan Megee,1 Julie Engiles,1

Makoto Senoo,2 and Hannah Galantino-Homer1,1Laminitis Institute, Department of Clinical Studies/New Bolton Center, 2Department of Animal Biology,School of Veterinary Medicine, University ofPennsylvania, Kennett Square, PA

TAKE HOME MESSAGEThe regional proliferative potential and state of lamellar tis-sue may be characterized by immunolocalization of p63and Ki-67, respectively.

INTRODUCTIONAbnormal epidermal proliferation may contribute to thepathology of chronic laminitis.

MATERIALS & METHODS (INCLUDINGSTATISTICAL ANALYSES)Postmortem (<1h) tissue from skin, coronary, proximal,middle and distal lamellae was collected from 3 horseswith no signs of laminitis (control) and 3 horses withchronic laminitis and P3 rotation. Indirect immunofluores-cence of paraformaldehyde-fixed, OCT-embedded cryo-sections using mouse monoclonal antibody againsthuman p63 (clone 4A4) and rabbit monoclonal antibodyagainst human Ki-67 (clone SP6) was used to determinerelative proportions of epidermal cells positive for p63(proliferative potential) and Ki-67 (cellular proliferation).Triplicate images were counted for each section and dataanalyzed by ANOVA.

RESULTSOverall, Ki-67 expression was higher in coronary comparedto skin and lamellar tissues and both p63 and Ki-67 weregreater in primary epidermal lamellae tips (closest to P3)compared to the base (closest to hoof wall). Consideringall tissues, laminitic horses had a lower percentage of epider-mal cells positive for p63 (40� 28%) and Ki-67(1.4� 3.1%)than controls (72� 19% and 3.0� 4.8%, respectively).

DISCUSSIONActively proliferating epidermal cells are located primarilyin coronary tissue, whereas cells with proliferative potentialare located throughout the hoof. Results indicate a decreasein epidermal proliferation during chronic laminitis; how-ever larger sample sizes should be analyzed to account forhigh inter-horse variability.

CLINICAL RELEVANCECharacterizing the proliferative state of normal and laminiticlamellar tissue will contribute to knowledge of laminitispathogenesis.

CONCLUSIONThese preliminary results indicate a decrease in epidermalcell proliferation during chronic laminitis.

Differences in Lamellar ProteinExpression During Laminitis Inducedby a 48h Prolonged Euglycemic-Hyperinsulinemic ClampRebecca Carter,1 Melody de Laat,2

Christopher Pollitt,1,2 and Hannah Galantino-Homer1,1Laminitis Institute, Department of Clinical Studies/New Bolton Center, School of Veterinary Medicine,University of Pennsylvania, Kennett Square, PA,2Australian Equine Laminitis Research Unit, School of

Abstracts � Vol 30, No 2 (2010) 105

Veterinary Science, The University of Queensland, St.Lucia, Queensland, Australia

TAKE HOME MESSAGEProteomic analysis of lamellar tissue is a useful tool forinvestigating the molecular mechanisms of laminitispathogenesis.

INTRODUCTIONHyperinsulinemia resulting from the prolonged euglyce-mic-hyperinsulinemic clamp (pEHC) induces laminitiswithin 48 h in horses. However, the molecular mechanismlinking hyperinsulinemia to lamellar pathology is unknown.

MATERIALS & METHODS (INCLUDINGSTATISTICAL ANALYSES)Four Standardbred horses were subjected to pEHC and 4matched controls were sham-treated with saline. Horseswere euthanized at 48h and lamellar tissue was extractedand frozen in liquid nitrogen. Protein was extracted fromfrozen tissue, separated by SDS-PAGE, and proteins witha molecular weight between 20 - 50 kDa were trypsin di-gested and peptides identified by HPLC/MS/MS andsearch of the equine protein database. Proteins were identi-fied with probabilities R99%, and counts of peptide spectrafor each protein were used to quantitate differences in pro-tein abundance using Scaffold 2 software and differencesbetween groups were determined by independent t tests.

RESULTS670 proteins were identified and 17 proteins were differen-tially expressed between treatment and control groups(P< 0.05). Differences in protein expression were indica-tive of increased translation, intracellular trafficking, im-mune response, oxidative stress, and cellular proliferation;and decreased post-translational modifications and struc-tural or cell adhesion proteins.

DISCUSSIONFurther investigation into proteomic changes during lam-initis, including expanding the range of molecular weightsanalyzed and evaluating additional time points and modelsof laminitis, will provide invaluable data for determiningthe molecular mechanisms of laminitis pathogenesis.

CLINICAL RELEVANCEUnderstanding the mechanisms of laminitis pathogenesiswill facilitate the prevention, management and treatmentof this devastating disease.

CONCLUSIONDifferences in lamellar protein expression occur during hy-perinsulinemia-induced laminitis.

Genome-Wide TranscriptomeProfiling of Laminar Tissue DuringEarly Stages of CarbohydrateOverload-Induced Equine LaminitisJixin Wang,1 Britta Leise,2 Ashley Gustafson,1

Bindu Nanduri,3 James Belknap,2

and Bhanu Chowdhary1, 1Department of VeterinaryIntegrative Biosciences, College of Veterinary Medicine& Biomedical Sciences, Texas A&M University, CollegeStation, TX 77845, 2College of Veterinary Medicine,Ohio State University, Columbus OH-43210, 3College ofVeterinary Medicine, Mississippi State University,Starkville, Mississippi

TAKE HOME MESSAGEGenomewide expression analysis provides clues for mecha-nisms underlying laminitis pathogenesis.

INTRODUCTIONThe carbohydrate overload (CHO) model of laminitis inhorses closely mimics symptoms and pathology exhibitedby clinical cases. The model provides a basis to follow thepathophysiological events associated with laminitis anddetermine underlying mechanisms that modulate them.

MATERIALS & METHODSAn equine-specific 70-mer oligonucleotide microarray rep-resenting 21,500 genes was used to obtain comparativeprofiles of gene expression patterns in the equine laminartissue at developmental (DEV) and Obel-grade 1 (OG1)stages of laminitis following experimental CHO-induction,in relation to healthy control (CON) horses.

RESULTSThe results showed differential expression (DE) of inflam-matory mediators within the laminar tissues at the DEVstage. A negative regulation of cellular process and an in-creased biological regulation were seen during this stage.In contrast, the OG1 stage was characterized by a strongtranscriptional response with more number of up-regulatedthan down-regulated DE genes. Responses to stimulus anddefense response genes were over-represented among theup-regulated genes in this stage. The microarray results of31 up or down regulated genes were validated by qRT-PCR.

DISCUSSIONS100 gene family such as S100P, S100A8, S100A9, andS100A12 and a novel MMP family member MMP13 arepotentially involved in the early progression of laminitis.Roles of p38 MAPK signal transduction, transcription fac-tors NF-kB and AP-1 pathways were deemed central.