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components can be quantified accurately. Two methodsare mainly used to analyse fructans, an enzymatic/colori-metric method and an HPLC technique. The former relieson quantification of monomers released after fructan hy-drolysis by fructanase. Some values for grass fructan usingthis method seem low. Therefore, the fructan values ob-tained by both techniques were compared.

MATERIALS AND METHODSSamples of Phleum pratense, Dactylis glomerata and Loliumperenne were analysed for fructan by both the colorimetricand HPLC methods. Selected samples were then analysedby HPLC both before and after fructanase treatment.

RESULTSValues obtained by the McCleary method ranged from 10to 94 percent of those obtained by HPLC. Post-fructanasetreatment analysis revealed incomplete (28-50%) hydroly-sis of grass fructan.

DISCUSSIONFructans in these grass samples were differentially sensitiveto the fructanase employed in the McCleary method.

CLINICAL RELEVANCEIf using fructan values for grass forage obtained by the col-orimetric method for IR equid ration formulation, the ef-ficacy of the fructanase against the forage should first bedetermined

CONCLUSIONThe colorimetric method may yield artificially low valuesfor grass fructan.

Intraosseous Infusion of the DistalPhalanx and Lamellar Microdialysis:New Techniques for Lamellar DrugDelivery and AnalysisAli R. Nourian, Paul C. Mills, Don M. Walsh,and Christopher C. Pollitt, Australian Equine LaminitisResearch Unit, School of Veterinary Science, TheUniversity of Queensland, Brisbane, Australia

TAKE HOME MESSAGEIOIDP and LMD are viable techniques for lamellar drugdelivery and analysis.

INTRODUCTIONSeeking a new approach to laminitis drug delivery we devel-oped and validated intraosseous infusion of the distal

phalanx (IOIDP). Lamellar microdialysis (LMD) enabledanalysis of lamellar constituents.

MATERIALS & METHODSUnder regional nerve blockade a microdialysis (MD) probewas implanted into the hoof lamellar tissue of six sedated,healthy, Standardbred horses. An intraosseous screw wasintroduced into the body of the distal phalanx and genta-micin (25 mg/mL at 20 mL/ min) was infused for twohours without a tourniquet. MD and blood samples werecollected and analysed for gentamicin concentrations. Inanother experiment radiopaque contrast media was in-fused, without the application of a distal limb tourniquet,to validate lamellar delivery.

RESULTSGentamicin was present in lamellar tissue at much higherconcentrations than serum. The mean concentration ofgentamicin was 24.4, 20.5 and 4.4 mg/mL in lamellar ex-tracellular fluid (ECF) and 0.28, 0.5 and 0.32 mg/mL inserum samples collected 60, 120 and 150 min after theIO infusion was started, respectively. Contrast mediawas present in the sublamellar dermis immediately afterinfusion.

DISCUSSIONIOIDP delivered gentamicin at high concentration directlyto the lamellar ECF and lamellar delivery was validated bycontrast radiography. A vascular relationship exists be-tween the interior of the distal phalanx and the lamellar cir-culation that can be used for lamellar drug delivery.

CLINICAL RELEVANCESlow IOIDP is a clinically viable method of deliveringdrugs directly to the lamellar circulation in the standing,conscious horse. Changes in the lamellar ECF during lam-initis and the local kinetics of drugs used therapeutically forthis condition can be monitored via LMD.

Gene and Protein Expressionof Polysulfated Proteoglycan ECMComponents in the Equine DigitalLamellaeLe Wang,1 Erica Pawlak,1 Dominique Alfandari,1 PhillipJ. Johnson,2 James K. Belknap,3 and Samuel J. Black1,1Department of Veterinary and Animal Science,University of Massachusetts Amherst, 2Department ofVeterinary Medicine and Surgery, College of VeterinaryMedicine, University of Missouri Columbia, 3School ofVeterinary Medicine, The Ohio State University

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TAKE HOME MESSAGEGenetic and biochemical evidence for large polysulfatedproteoglycans in lamellae, but not liver and lung tissue,show that lamellae are functionally specialized to resistcompressive forces.

INTRODUCTIONOur published and on-going studies show ADAMTS-4(aggrecanase) gene and protein expression is elevated inthe lamellae of laminitic horses. ADAMTS-4 cleaves aggre-can and versican, the core proteins of large polysulfatedproteoglycans that in complex with hyaluronan form hy-drated gels and provide resistance to compressive forces.Here we investigate ADAMTS-4 substrates in lamellae.

MATERIALS & METHODScDNA-specific probes and relative quantitative real timePCR were used to detect the expression levels of genes en-coding polysulfated proteoglycans. Ct values were analyzedusing the DDCt method. Protein and glycosaminoglycanmoieties in lamellar extracts were detected after SDS-PAGE electrophoresis by Western blotting.

RESULTSGenes encoding aggrecan, versican, chondroitin synthaseand hyaluronan synthase were found to be consitutively ex-pressed in normal digital lamellae but not, or to a muchlesser extent in lungs and liver. Aggrecan core proteinwas detected in detergent (0.5%NP-40) lysates of lamellae,whereas keratan- and chondroitin- sulfate aggrecan pro-teoglycans were detected in guanidine hydrochloride ex-tracts of lamellar proteoglycans.

DISCUSSION AND CLINICAL RELEVANCEGene expression and product analyses indicate that the equinedigital lamellae are functionally specialized to resist compres-sive forces. Consequently, degradation of this material by ele-vated ADAMTS-4 may compromise lamellar function.

CONCLUSIONThe presence of large polysulfated proteoglycans and ele-vated ADAMTS-4 in laminitic tissue suggest parallels be-tween the pathophysiology of laminitis and osteoarthritis.

Localization and Function of SulfatedProteoglycans in the Equine DigitalLaminae

Erica Pawlak,1 Le Wang,1 Dominique Alfandari,1 PhillipJ. Johnson,2 James K. Belknap,3 and Samuel J. Black1,

1Department of Veterinary and Animal Science,University of Massachusetts Amherst, 2Department ofVeterinary Medicine and Surgery, College of VeterinaryMedicine, University of Missouri Columbia,3Department of Veterinary Clinical Sciences, College ofVeterinary Medicine, The Ohio State University

TAKE HOME MESSAGEPolysulfatedproteoglycansof the lamellar extracellularmatrix(ECM) are similar to those of articular cartilage and hencemay maintain hydration and resistance to compression.

INTRODUCTIONWe have previously shown that ADAMTS-4 (aggrecanase),which is implicated in the pathophysiology of osteoarthritisthrough cleavage of large polysulfated proteoglycans, is el-evated in experimentally induced and naturally acquiredequine laminitis. To understand the role played byADAMTS-4 in equine laminitis, it is necessary to localizesubstrates within the digital lamellae and to determine theircontribution to lamellar structure and function.

MATERIALS AND METHODSAggrecan, chondroitin-6-sulfate, keratan sulfate, laminin,and hyaluran were detected via immunofluorescence in10mm sections of lamellae. Specificity of antibodieswas confirmed by Western blotting and by peptidecompetition.

RESULTSAggrecan and hyaluran appear to be largely concentrated inthe secondary epidermal lamellae, while keratan sulfate andchondroitin sulfate display varying and unique distribu-tions throughout the tissue.

DISCUSSIONProteoglycan components of the lamellar ECM are similarto those of articular cartilage, consistent with the possibilitythat they protect the lamellae against general and localizedcompression resulting from weight bearing and motion-as-sociated rotation of the third phalanx.

CLINICAL RELEVANCEWe propose that degradation of lamellar ECM large poly-sulfated proteoglycans by ADAMTS-4 reduces lamellar re-sistance to compression, thus exposing tensile elements ofthe ECM to excessive stretch and contributing to failure.

CONCLUSIONLamellar proteoglycans are similar to those of articular car-tilage and hence, likely to play a key role in maintaining la-mellar integrity.