Abstracts / Journal of Equine Veterinary Science 31 (2011) 562-609 589

adhesion molecules (ICAM-1, E-selectin), and COX-2. There wasno change in MCP-1 or IL-10. In the DEV group, mRNA concen-trations of CXCL-1, ICAM-1, IL-1ß, CXCL8, and MCP-2 weredecreased (p < .05), and the anti-inflammatory cytokine IL-10was increased compared with NON-RX limbs (p < .05).Discussion: Cryotherapy is one of the few therapeutic options forthe treatment or prevention of laminitis that has withstoodmultiple scientific investigations. However, there has been a greatdeal of controversy regarding the mechanism(s) by which itexerts a protective effect on the laminae. The current studydemonstrates that cryotherapy blocks multiple inflammatorysignaling pathways, indicating that it most likely inhibits one ofthe central upstream inflammatory signaling mechanisms such asTLR, NFkB, or MAPK signaling. This study provides further supportfor the institution of cryotherapy in any horse with an acute septiccondition which puts it at immediate risk for laminitis. The nextstudy will determine whether cryotherapy is still valuable wheninstituted at the onset of clinical signs of laminitis.Conclusions and Clinical Relevance: Digital hypothermia/cryo-therapy results in a marked decrease in laminar inflammatoryevents during the developmental phase of carbohydrate-inducedlaminitis. It should be instituted in any horse with an acute septiccondition which puts it at immediate risk for laminitis.

An up-close and early look at lamellar pathologycharacteristic of oligofructose-induced laminitis

Julie Engiles 1, Lauren Duffee 2,5, Susan Megee 2,3,Christopher Pollitt 4, Andrew Van Eps 4, Mary Robinson 3,and Hannah Galantino-Homer 2,31 Department of Pathobiology, New Bolton Center, 2 LaminitisInstitute, New Bolton Center, 3 Department of Clinical Studies,New Bolton Center, 5 Veterinary student class of 2013; School ofVeterinary Medicine, University of Pennsylvania, Kennett Squareand Philadelphia, PA, USA, 4 School of Veterinary Science,University of Queensland, Gatton, QLD, Australia

Take-Home Message: Although the developmental phase oflaminitis is clinically silent, pathological changes are occurring atthe microscopic levels, including structural alterations of thesensitive epidermal lamellae as well as inflammation within thedermal lamellae and corium dermis. A comprehensive descriptionof the stepwise progression of microanatomic pathology inexperimental laminitis induction models provides a betterunderstanding of laminitis pathogenesis and potential targets forpreventive or therapeutic intervention.Introduction: The catastrophic outcome of equine laminitis insevere cases is the failure of attachment between the distalphalanx (P3) and the overlying hoof capsule. This biomechanicalfailure follows the onset of acute laminitis, with lameness,bounding digital pulses, and heat in the hoof. The clinical phase ispreceded by a clinically silent developmental phase.Laminitis develops as a result of many different causes, includingtrauma, biomechanical overload (e.g. severe contralateral limblameness), endocrine/metabolic disease (e.g. equine metabolicsyndrome, pituitary pars intermedia dysfunction), orthopedic orvisceral inflammatory disease (e.g. sepsis, systemic inflammatoryresponse syndrome), and hindgut disturbance, particularly due tograzing pasture with a high fructan content. In all cases, theunderlying pathogenesis is not clearly understood.The oligofructose (OF) induction model of laminitis createsa hindgut disturbance, resulting in colitis and laminitis. Thismodel is thought to mimic laminitis due to excess pasture fructancontent. Carbohydrate overload induction model studies havedemonstrated increased inflammatory gene expression andleukocyte accumulation in developmental stages of laminitis;

however, to date the majority of laminitis histopathology hasmainly focused on basement membrane (BM) destruction and thedegree of BM separation from the epidermal basal cells.The goals of this study were to: 1) semi-quantitatively charac-terize the microanatomic alterations of the sensitive lamellae inOF induction model subjects at various time points during thedevelopmental and acute stages of laminitis, and 2) better char-acterize the inflammatory component using both histochemicaland immunohistochemical stains.Materials and Methods: Following either OF administration orsham treatment, 4 treatment groups of horses (n¼6 per group)were euthanized for lamellar tissue retrieval:

� control horses, which underwent sham induction and wereeuthanized at 24 h

� two time points during the developmental phase (12 h and18 h)

� one group that was euthanized at 24 h, which was at theonset of acute laminitis

Both front feet from each horse were harvested and sensitivelamellar tissues were trimmed and processed for routine histo-pathology including hematoxylin and eosin and periodic acid-Schiff stains. Slides of sensitive lamellar tissues were evaluated forquantitative and qualitative parameters. Quantitative parametersincluded the following:

� average primary epidermal lamellar (PEL) length, in mm� average keratinized axis (KA) displacement (mm)� average KA width (mm)� average secondary epidermal lamellar (SEL) length (mm)� average SEL width (mm)� necrotic/apoptotic/dyskeratotic cell index (number per 10high-powered fields [hpf])

� mitotic cell index (number per 10 hpf)

Qualitative parameters were graded on a scale from 1 to 4 (nor-mal¼1, mild¼2, moderate¼3, severe¼4) and included thefollowing:

� degree of PEL branching� degree of SEL distortion� degree of lamellar inflammation� degree of (deep) corium inflammation� degree of BM separation

Additional re-cuts from the original paraffin-embedded blockswere obtained for immunohistochemical stains to characterizethe inflammatory cell infiltrate including CD3 (T-lymphocytemarker), CD79a (B-lymphocyte marker), F4/80+ (macrophagemarker), and caspase-3 (apoptosis marker).Compared parameter means for both front feet harvested fromthe 6 horses in each of the 4 study groups were analyzed byKruskal-Wallis one-way analysis of variance on ranks (due to lackof normality) and pairwise post-hoc tests. The F4/80+ antibodywas validated in the horse by western blotting (protein extractedfrom equine lymph node, cultured macrophages, serum, andsensitive lamellae from a horse with naturally occurring lami-nitis) and indirect immunofluorescence using formalin-fixed,paraffin-embedded sections of equine lymph node.Results: Significant differences in both quantitative and qualita-tive measurements were detected between the sham-treatedcontrols and the OF-treated horses. Among the different treat-ment groups, significant differences in histopathologic parame-ters included SEL length, KA width, degree of SEL distortion, anddegree of inflammation in the corium and lamellar tissues. TheF4/80+ antibody was found to be a valid marker for equinemacrophages.

Abstracts / Journal of Equine Veterinary Science 31 (2011) 562-609590

Discussion: In this study we semi-quantitatively characterizedthe microanatomic alterations in the sensitive lamellae at varioustime points during the developmental and acute stages of lami-nitis induced by OF. We further characterized the inflammatoryevents occurring within these developmental and acute laminiticphases using both histochemical and immunohistochemicalstains. In short, inflammation in the lamellae and corium issignificant in the developmental stage as well as in the clinicallyapparent acute stage of OF-induced laminitis.Our study also validated the F4/80 antibody as a valid marker forequine macrophages, allowing it to be used to identify a specificsubset of inflammatory cells in equine laminitis. Although themajority of histopathologic studies pertaining to the OF inductionmodel have focused on basement membrane pathology, we identi-fied additional parameters that are significantly altered inthedevelopmental and acute phases of OF-induced laminitis. These

quantitative and qualitative pathological features provide importantinsights into laminitis development, and when correlated withtranscriptomic/proteomic data aquired from these same subjects,willprovideamorecompleteunderstandingofdiseasepathogenesis.Conclusions and Clinical Relevance: This study identified bothspecific measurable differences in sensitive lamellar pathologyamong the different groups of horses (control and OF-induced), aswell as inflammatory infiltrates present in developmental stage ofOF-induced laminitis, even before predominant basementpathology occurs. Critical evaluation of lamellar tissues frominduction models can identify certain microanatomic alterationswhich, when correlated with molecular data, can provide a morecomplete understanding of laminitis pathogenesis.This study and related studies were funded by: The Fund forLaminitis Research, Bernice Barbour Foundation, Inc., and NIH andMerial grants (5T35RR007065-14).

Practical – Natural Intervention

Sport horse mechanics II: healing the hoof during therapy foranother injury or lay-up

Aaron Gygax CJF

Take-Home Message: A lay-up in the performance horse'sschedule gives the farrier time to make needed changes to thetrimming or shoeing that may not have been possible while thehorse was in work. Making the most of this time helps speedrecovery and improves the overall health and condition of the feetbefore the horse starts back in work.Introduction: When a performance horse is lame because of footpain, the source of the pain must be localized. Once a diagnosis ismade, the veterinarian and farrier can come upwith a plan for theshoeing mechanics, and hopefully get the horse back to per-forming as soon as possible.Hoof Care During Lay-Up: If a horse is given time off for a hoof-related injury, the shoeing mechanics are selected to support thetreatment of this injury.When a horse is laid up for another sort ofinjury or for any other reason, it can give us enough time to applyshoeing mechanics or even a “barefoot” treatment to improve thehoof balance and condition before the horse starts back in work.When a hoof problem is present, it is important to communicatewith the rider and veterinarian in advance of any lay-up. If thecurrent shoeing is retained because the horse is still in work, thena different shoeing plan can be discussed in case lay-up occurslater. That way, everybody already knows about it, so no time willbe lost.For example, in a horse with severely contracted and shearedheels, I may pull the shoes off as soon as I get a chance. Withrepeated trimming over a short period of time, the problem cannot only be corrected, but the feet can be dramatically improved,which gives the horse a better foundation for the upcomingseason.During a lay-up, I try to remove any kind of glue from the feet, aswell as any shoeing applications that have been used to give thehorse more traction. Bigger changes that would have beenimpractical while the horse was in work can now be applied, andwill allow us to see how the horse is likely to respond when it isback in work.Examples: In my shoeing philosophy, there is always one bigconflict: It is always the goal to align the bony column, butsometimes there is not enough hoof to accomplish that goal withtrimming alone. For example, with low and under-run heels, the

toe is shortened as much as possible. But if I want to keep thecorrect angle through the bony column, the heels cannot betrimmed back to the widest part of the frog. So, the heels are keptfrom functioning normally. If the heels are trimmed back to thewidest part of the frog in one line with the toe, I end up with verylittle “foot” and most likely too low an angle.However, during lay-up I can drastically change my trimmingmethod and trim the foot on two levels. First, I trim the foot fromthe toe to the widest part of the foot, then the heels are trimmedback in a second step to the widest part of the frog. (This strategyis not to be confused with a banana shoe.) Using a rocker shoe, therocker on the shoes will start a little bit behind the centre ofrotation so that the horse cannot tilt back on its shoe.In a horse with sheared heels that must be hand-walked overhard ground for treatment of an injury, going barefoot would notbe a good solution. In such cases, a heartbar shoe with heelflotation may be applied.In horses with little sole depth, I would put in a lot of mechanicsto try to enhance sole growth. For example, a full-rockeraluminium shoe may be used during lay-up. When the horse goesback to work, the mechanics are reduced but kept in place.

The toe crena: gross and microscopic examination of the“normal” white line

Lisa Lancaster PhD, DVMPathobiology and Diagnostic Investigation, Michigan StateUniversity

Take-Home Message: The toe crena may be a marker of toe wallhealth, as evidence suggests that it may be an adaptation tomechanical stress. In feet with pre-existing pathology, monitoringof the toe region may prove clinically useful over the course oflaminitis treatment or other hoof rehabilitation.Introduction: The crena is identified in radiographs of the distalphalanx as a notch at mid-toe on the distal edge. The same term isalso used by farriers to describe a notch on the bearing surface ofthe hoof at mid-toe in the white line region. There is no publisheddata on the correlation between the notch in the bone and that inthe white line, and it is not clear if there is any clinical significanceto either. This paper presents some preliminary histologic data onthe crena in the feet of racehorses.