Journal of Equine Veterinary Science 32 (2012) 641-654

Journal of Equine Veterinary Science

journal homepage: www.j -evs.com

Abstracts

What’s new in laminitis research? I:pathophysiology and prevention

James A. Orsini DVM, DACVS 1, and Nora S. Grenager VMD,DACVIM2

1Associate Professor of Surgery, Director, LaminitisInstitute Penn-Vet, New Bolton Center, School ofVeterinary Medicine, University of Pennsylvania,2 Founder Of Grenager Equine Consulting, LLC,Veterinarian At Harrison Equine In Northern Virginia

Once again, the 2011 Laminitis Conference containeda wealth of new information that helps us move along inour understanding of laminitis. With so many individualsand research groups activelyworking on themany differentfacets of laminitis, it is difficult to summarize the recentadvances succinctly, but we shall try. Recent and on-goingresearch highlights the following themes, with respect tothe pathophysiology, and thus the prevention, of laminitis:

1. Laminitis caused by either the carbohydrate overload(CHO) or black walnut extract (BWE) model representsa local or tissue-specific manifestation of systemicinflammatory response syndrome (SIRS).

2. The mechanisms of structural failure in acute laminitisare multifaceted and somewhat specific to the incitingevent (SIRS-associated laminitis, endocrinopathy-asso-ciated laminitis, supporting limb laminitis, traumaticlaminitis). Treatment and prevention must therefore beboth relatively cause-specific and multimodal.

3. The various mechanisms of structural failure are acti-vated hours or days before the first clinical signs oflaminitis appear, but a narrow window of opportunityto positively affect the outcome after the onset oflameness still exists. Early recognition of imminent riskand initial clinical signs, coupled with an immediateand vigorous treatment response, may greatly improvethe outcome.

4. Although lamellar pathology predominates in acutelaminitis, it is accompanied by significant osteopa-thology of the third phalanx (P3). These osseouschanges may impact response to treatment in acutelaminitis and be key players in the various manifesta-tions of chronic laminitis.

0737-0806/$ – see front matter � 2012 Elsevier Inc. All rights reserved.

5. Evidence of chronic laminitis is common in the feet offeral horses from arid and semi-arid areas, especiallythose traveling over hard ground. This suggests thatphysical/mechanical factors may be more involved inlaminitis development than we have been accustomedto thinking.

Laminitis as a manifestation of SIRS: In recent years,laminitis has been reframed as a manifestation of thesystemic inflammatory response syndrome (SIRS). Thatmay be self-evident in cases of laminitis secondary to aninfectious process, such as pleuropneumonia, enterocolitis,or endometritis, but perhaps less so with carbohydrateoverload (CHO) or bedding on black walnut shavings(BWE). However, the systemic abnormalities that havebeen shown to be characteristic of CHO and exposure toBWE clearly indicate that laminitis must be considereda local manifestation of SIRS in these scenarios as well.In fact, equine laminitis is now described as a clinicalhallmark of multiple organ dysfunction syndrome (MODS),a common sequela of SIRS [1]. During the developmentalphase of experimental CHO-induced laminitis, elevations inthe expression of various inflammatory mediators havebeen documented in the liver, lung, and kidney, as well asin the lamellae. For example, by the time there is anin-crease in body temperature of just 2 degrees Farenheit, theexpression of IL-1ß, IL-10, CXCL-8, and TNF-a in the liverare increased. By the time the horse is showing the firstsigns of lameness there are further indicators of systemicinflammation including elevation of COX-2 in the liver, IL-1ß in the lung and kidney, and an influx of leukocytes in thelung [2]. These other organs may be only subclinicallyeffected, thus the lamellae become the clinical focus due tothe mechanical load of the weight-bearing foot super-imposed on tissue compromise [4].Sepsis vs. SIRS. The term “sepsis” is often used inter-changeably with “SIRS” in laminitis research, followinga convention used in human research. However, sepsis isperhaps not themost clinically useful term for us to proceedwith here. To many of us, sepsis connotes an overwhelmingbacterial infection, as in the case of septic arthritis orsepticemia. However, as we will review a little later, SIRScan occur in the absence of any infectious process. So, SIRSis probably the more appropriate term to describe what

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654642

transpires during the developmental and early clinicalstages of acute laminitis caused by CHO or BWE.It is also useful to think of these disease states as a systemicinflammatory event to focus our clinical assessment andtreatment plans on inflammation and its sequelae, ratherthan on the antimicrobial therapy that would be appro-priate for a classic septic process. (Recent advances in thetreatment of acute laminitis are discussed in part II of thisconference wrap-up.)Leukocyte responses. Leukocyte responses in the lamellardermis during the developmental and early clinical phasesof laminitis provide further evidence that laminitis causedby CHO or BWE is a manifestation of SIRS. They also suggestsome avenues for fruitful research, as human sepsis/SIRSresearch is actively pursuing targeted therapy in this area.The lamellar dermis of healthy feet contain a large residentpool (60–100 cells/mm2) of tissue macrophages of theinactive/anti-inflammatory (M2) phenotype that arediffusely distributed throughout the lamellae, with an evengreater concentration in the deeper dermis (150–200 cells/mm2). In contrast, there are few or no resident neutrophilsor classically activated (M1) macrophages (0–2 cells/mm2)in the healthy lamellar dermis [4].However when the lamellar system is challenged with CHOor BWE to Obel grade 1 laminitis, there is a rapid changefrom anti-inflammatory to pro-inflammatory phenotypewhich triggers the release of potentially destructive prote-ases, reactive oxygen species, and inflammatory mediators.Consequently, or concurrently, there is an influxof activatedleukocytes (both neutrophils andmonocytes/macrophages)into the lamellar region from the local vasculature.Neutrophils are the primary cell type emigrating into thelamellar tissues in the BWE model; monocytes/macro-phages predominate in the CHO model [4].Regardless of these specific profiles, this local leukocyteresponse and influx appear to exacerbate the inflammatoryinjury to the lamellar tissues in both the CHO and BWEmodels, similar to that described in human sepsis/SIRS.There are some interesting temporal changes in cell typesand numbers between the developmental time points andthe onset of Obel grade 1 and Obel grade 3 laminitis thatwill be discussed a little later, as they may have somebearing on specific treatment choices at these various timepoints.Next an equine metabolic syndrome (EMS) model oflaminitis was evaluated, in which lean and obese ponieswere fed a diet that was high in nonstructural carbohy-drates (NSC). Interestingly, there was minimal emigrationof leukocytes of any type, and minimal change in thelamellar expression of pro-inflammatory cytokines andchemokines in these cases, even in the face of moderatelaminar epithelial stress as indicated by calprotectinsignaling in the laminar epithelial cells. Even though onlysome of the ponies showed mild signs of laminitis, itappears that the mechanism of lamellar basal cell injury/failure associated with EMS does not involve inflammatoryevents which trigger leukocyte influx [4]. There will bemore on the mechanisms of endocrinopathic laminitisa little later.Preventing SIRS-associated laminitis. Because the risk forlaminitis is greatly increased in horses with SIRS, it isimperative that we recognize the potential for, and the

clinical signs of, SIRS earlydwell before signs of laminitisdevelop.Mostof us are familiarwith the common triggers forSIRS, infection and endotoxemia, but perhaps less so themany other potential triggers, which include trauma,ischemia, immune-mediated diseases, surgery, hypo- orhyperthermia, and profound hypoxemia (e.g. hemorrhagicshock) [1]. Any of these circumstances or disease states canprecipitate a systemic inflammatory response and thuspredispose the horse to developing laminitis.Proposed diagnostic criteria for SIRS in the adult horseinclude the following [1]:

� rectal temperature above 101.5� F (38.6� C) or below98� F (36.6� C)

� heart rate above 60 beats/min� tachypnea or hyperventilation (PaCO2 less than 32mmHg)

� white cell count above 14,000 cells/ml or below 4,000cells/ml, or more than 10% band neutrophils

Prompt and vigorous treatment at the first signs of SIRSmay prevent or allay the progression to multiple organdysfunction or failure and laminitis.Cryotherapy. It has been repeatedly established that local(digital) hypothermia, or cryotherapy, decreases the severityof the lamellar changes in horses with SIRS-associatedlaminitis by inhibiting inflammatory-signaling mechanisms.The mechanism by which it exerts this effect has recentlybeen investigated further [5]. Compared with the control(uncooled) foot, the foot subjected to digital hypothermiathroughout the developmental (DEV) phase of CHO-inducedlaminitis showed significant reductions in the expression ofthemajority of inflammatorymediatorsmeasured, includingvarious cytokines (IL-1ßand -6), chemokines (CXCL-1, -6, and-8/IL-8, MCP-2), cell adhesion molecules, and COX-2.As will be shown in more detail later, COX-2 is one of theinflammatory mediators that is significantly upregulated inthe laminitic foot. Also of note, digital hypothermia did notsuppress the production of IL-10, an important anti-inflammatory cytokine. In fact, while expressions of theproinflammatory IL-1ß, CXCL-1 and -8, MCP-2, and ICAM-1were decreased during DEV in the cooled foot, theexpression of IL-10 was increased.The fact that digital hypothermia blocks multiple inflam-matory signaling pathways, and may upregulate an anti-inflammatory pathway, makes it an important strategy forpreventing SIRS-associated laminitis in at-risk horses.Cryotherapy’s ability to also halt disease progression ifapplied only once there are early clinical signs of laminitisawaits further study. Anecdotally, it appears to be of somevalue even after the onset of lameness, and theoretically itshould (see Mechanisms below), but we have yet to fullyinvestigate its biochemical and cellular effects at this stageof the disease process.Protease inhibition. As discussed in the next section on themechanisms of structural failure, various proteases areinvolved in the lamellar degradation of SIRS-associatedlaminitis. In an effort to prevent SIRS-associated laminitis,targeted delivery of specific anti-proteases directly to thefoot is currently being investigated [3].Mechanisms of structural failure: The mechanisms ofstructural failure of the lamellar dermal-epidermal cell

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654 643

bond, and thus of the suspensory apparatus of P3, are beingrevealed as still more complex than previously understood,in part because they are somewhat specific to the incitingevent or disease state. In other words, it is rather a case ofmany paths to the same destination, or “all roads lead toRome.” While several different states of disorder or initialinsult result in laminitis, there are some general themes andbroad categorizations of thesepaths that are of practical use.SIRS-associated laminitis. There are some well-defined andintriguing temporal differences in the specific biochemicaland cellular events between the CHO and BWE models oflaminitis. Practically speaking, both of these SIRS-associ-ated causes of laminitis involve a relatively nonspecificinflammatory response and a more targeted enzymaticdestruction of the extracellular matrix (ECM) componentswhich bond the epidermal to the dermal cells across thebasement membrane (BM).Inflammatory response. Most of the specific biochemicaland cellular events involved in the lamellar inflammatoryresponse were discussed in the preceding section on SIRS,and their temporal changes will be discussed in a latersection. One aspect that has not yet been addressed is thatSTAT3 activation. The JAK-STAT cytokine-activated signalingpathway is a feature of SIRS in humans, and occurs inlaminitis caused by CHO or BWE in horses, beginning duringthe developmental phase and persisting into the early clin-ical phase [6]. As the STATs are involved in various cellfunctions, this upregulation may also be instrumental insome of the other mechanisms of structural failure or in thepathology of chronic laminitis, such as apoptosis and cellproliferation. STAT3 inhibitors are currently being used andstudied in human medicine, so STAT3 may be a usefultherapeutic target for SIRS-associated laminitis in horses [6].ECM destruction. During the developmental and early clin-ical phases of acute laminitis, there is upregulation ofa variety of matrix metalloproteinases (MMPs), specificallythose that degrade elastin, collagen, aggrecan, versican, andvarious other adhesion molecules [7]. One of the earliestevents, documented to occur at the onset of feverdi.e. wellbefore the first clinical signs of laminitis appeardis theupregulation of ADAMTS-4 (an aggrecanase) and MMP-12(an elastase). The expression of these enzymes is increasedabout 100-fold at this time point and is sustained thereafter.By the time the horse is showing the first clinical signsof laminitis, there is also a 25-fold increase in MMP-1(a collagenase), whose expression also remains increasedthereafter. TheonsetofObel grade3 laminitis is accompaniedby a 100-fold increase in MMP-13 (another collagenase) [7].ADAMTS-4 cleaves aggrecan and versican, and its upregu-lation alters cell attachment and physiological remodelingof the basal epithelial cells. Thus, its increased expressionresults in the detachment of some epithelial cells from thebasement membranedall of which starts hours before clin-ical signs of laminitis first appear. The concurrent andsubsequent upregulation of elastase and two differentcollagenases further precipitates structural failure underload through loss of the normal resistance to stretch defor-mation and tensile strength in the affected lamellae [7].Oxidative stress. The role of oxidative stress in laminitiswas evaluated due to the current interest in antioxidants,and the fact that oxidative stress/injury is a pathophysio-logical feature of SIRS in humans. Three different markers

of oxidative stress - 3-nitrotyrosine, 4-hydroxynonenal,and protein carbonyl content – were evaluated in bankedlaminar tissues from BWE or CHO-induced laminitis [8].These studies found that oxidative stress/injury is not animportant contributor to lamellar failure in either theCHO or the BWE model of laminitis. It was noted,however, that therapy could be directed at modulation ofintracellular signaling cascades in which reactive oxygenspecies normally play a prominent physiologic signalingrole.Endocrinopathic laminitis. The mechanisms of structuralfailure in horses with endocrinopathic laminitis are quitefascinatingly different from those with SIRS-associatedlaminitis. Whether laminitis is a consequence of equinemetabolic syndrome (EMS) or of equine Cushing’s disease(pituitary pars intermedia dysfunction, or PPID), it iscurrently thought that the precipitating factorappears to bepersistent hyperinsulinemia (HI).The experimental creation of a persistent hyperinsulinemicstate in healthy equids leads to clinical laminitis within 48hrs (horses)[9] to 72 hrs (ponies) [3]. It must be pointed outthat the HI induced in some of these studies was extreme,such as insulin concentrations of >800 mIU/ml, wherecontrol values are in the range of 8.7–11.5 mIU/ml, and thatdegree of HI wasmaintained for at least 48 hrs [9]. Whethersuch a state reflects the typical clinical case of EMS or PPIDis doubtful because naturally occurring cases of EMS havevariable blood insulin concentrations and insulin sensi-tivity over time [10]. Even so, a strong correlation betweenbody condition score (BCS) and laminitis risk remains, suchthat the higher the BCS, the higher the serum insulin, andthe greater the risk for laminitis [11].In the typical prolonged euglycemic, hyperinsulinemicclamp (p-EHC) model of laminitis, HI is induced by an IVinfusion of insulin while maintaining a normal bloodglucose concentration. The goal is to study the effects ofinsulin independent of glucose dynamics. While this modelis useful, it does not precisely replicate the process nor thedynamics of HI in horses with naturally occurring endo-crinopathic laminitis. Alternatively, clinical laminitis doesnot consistently develop when HI is instead induced by anintravenous infusion of glucose, to more closely mimic thehyperglycemic response to a high-carbohydrate meal, .However, the lamellae in this model still show thepathology typical of early endocrinopathic laminitis. Thus,it does seem fair to conclude that insulin has a directpathogenic effect on lamellar integrity [3].Ischemia. Exactly how HI induces laminitis is perhaps themost interesting aspect of the recent research into endo-crinopathic laminitis. Building on earlier research whichshowed that glucose uptake by the cells of the lamellarregion is not insulin-dependent, we now know that insulinreceptors in the lamellar region are confined to the endo-thelium of the dermal vasculature. In ponies overfedcarbohydrates to mimic abrupt exposure to pasture high innonstructural carbohydrates (NSC), the expected rise inserum insulin concentration was accompanied by anincrease in the total number of insulin receptors in thelamellar microvasculature. however no epithelial cellswere positive for the insulin receptor, and neither were anycells in the deeper dermis, including the endothelium. Thedistribution of IGF-1R expressionwasmuchmore extensive

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654644

than that of the insulin receptors, in the vascular elements,on laminar epithelial cells, and on fibroblasts. This mayindicate that insulin signaling through the IGF-1R on thelaminar epithelium is critical for mediating the negativeeffects of HI in the foot [12].In humans with insulin resistance (IR), there is attenuationof activity of endothelial insulin receptor signaling.Endothelial insulin receptor signaling normally causesvasodilation, but in IR individuals it instead causes vaso-constriction [12]. If the same holds true for horses andponies, then HI associated with high-NSC diets could beexpected to cause vasoconstriction and thus ischemia in thelamellar dermis. And if, unlike inhumans, vasodilation is theconsequence of HI in horses, then lamellar ischemia maystill ensue if vasodilation results in opening of the abundantarteriovenous anastomoses in the lamellar dermis.Secondary inflammation. While HI-induced ischemia isassumed as part of the mechanism of endocrinopathiclaminitis, and inflammation and lamellar injury are an ex-pected consequence of that ischemia, this piece of thepuzzle is yet to be studied in full. Most of the work oninsulin receptors and insulin-like growth factor receptors(discussed further below) is focused on the effects ofreceptor stimulation on the lamellar epithelial cells. Asnoted earlier, there is a curious lack of lamellar inflamma-tion and leukocyte influx during the developmental andearly clinical stages of laminitis in the EMS model [4].However, there is some evidence for an innate immune orautoinflammatory response in the developmental andacute phases of experimentally-induced HI.When HI is induced and maintained for at least 24 hrs,there is an increase in the lamellar expression of immu-noglobulins of the G class (IgGs) and various proteinsinvolved in the stress and inflammatory responses [13].These findings are consistent with a response to cell stress,cell damage, and even cell death. A robust cellular stressresponse is to be expected, as there is a concurrent increasein the rate of apoptosis and necrosis, and a loss of celladhesion and cytoskeletal proteins, both of which area forerunner to structural failure.Epithelial proliferation. The most interesting and perhapsunexpected mechanism of structural failure in horses withendocrinopathic laminitis is that of epithelial cell prolifer-ation. Two features characterize the pathology of HI-induced laminitis: lamellar lengthening and narrowing,and a dramatic increase in lamellar cell proliferation [3].As a broad brushstroke, insulin stimulates cell proliferation;it promotes cell survival, growth, and differentiation. Whileinsulin receptors in the lamellar region are restricted to thedermal microvasculature, receptors for the structurallyhomologous insulin-like growth factor type 1 (IGF-1) arewidely distributed throughout the lamellar region: withinvascular elements, on epithelial cells, and on fibroblasts[12]. As their name suggests, IGF-1 receptors respond inawaysimilar to insulin receptors. Practically speaking, it’s asif insulin receptors were plentiful in the lamellar epidermis,so overstimulation of IGF-1 receptors by persistent HIresults in uncontrolled epidermal cell proliferation [9].In a study inwhichHIwas induced andmaintained for up to48 hrs, acute laminitis developed by 48 hrs. Prior to that,after just 24 hrs of HI, there was an increase in the expres-sion of a marker of cell proliferation (TPX2) in the lamellar

epidermal cells and it was maintained in the 48 hr group.Thus, proliferation of epidermal cells was underway by24 hrs of persistent HI. It is likely that this is themechanismby which lengthening of the secondary epidermal lamellae(SEL) that is documented to occur in HI-induced laminitis isstimulated. This may sufficiently weaken the suspensoryapparatus of P3 such that structural failure and laminitisensuesdwithin 48 hrs of HI in this model [9].Blocking the IGF-1 receptor with anti-receptor antibodiesmight be a useful treatment option in horses with, or at riskfor, HI-associated laminitis. This is an approach that iscurrently being studied in human cancer patients [9].The use of quantitative proteomics has revealed severaladditional details of this structural failure and suggestssome serum biomarkers that may be of clinical use inidentifying the horses at imminent risk for endocrinopathiclaminitis. During experimentally induced HI, there is anabnormal keratinization of epidermal cells, an increasein mitotic activity, and an increase in apoptosis andnecrosisdall with minimal BM separation and MMP acti-vation [13]. By 24 hrs of persistent HI, there is an increasedexpression of various proteins involved in protein trans-lation, processing, and cell proliferation; there is alsoa decreased expression of desmosomes, hemidesmosomes,and adherens junctionsdi.e. cell adhesion and cytoskeletalproteins. By 48 hrs of persistent HI, there is a furtherdecrease in the expression of several cytoskeletal proteins(e.g. keratins, tubulin, vimentin) and of proteoglycans [13].These molecules may provide some useful serumbiomarkers for the development of HI-associated laminitis.We could take advantage of this upregulation in acute-phase reactive proteins and IgGs, and the degradation ofvarious lamellar proteins such as cartilage oligomericmatrix protein (COMP) and keratins, because some of themmay be released into the bloodstream [13].For the present, however, the physical appearance of thehorse and its hooves, as well as the measurement of seruminsulin, can serve to identify at-risk individuals and thosealready in the early clinical phase of endocrinopathiclaminitis. Obesity, as evidenced by a high BCS, is a signifi-cant risk factor for HI and thus for laminitis [10,11].Research is revealing that differences for several morpho-metric and biochemical measurements are sufficientlyimportant such that breed-specific criteria are beingdeveloped [11].Clinically apparent abnormalities in the hoof growth ofhorses with naturally occurring EMS reflect and validate themicroscopic changes documented with the HI model. Evenin horses with no history of lameness, there is a pattern ofabnormal hoof growth that is related to HI: abnormal growthrings in the external hoof wall, separation of the wall at thewhite line, and seedy toe, often with small areas of hemor-rhage within the abnormal white line area. This damage iscumulative and at some point culminates in acute laminitis ifnot properly addressed with diet, exercise, and medicationwhere necessary to normalize blood insulin concentrations.The good news is that, with proper management, hoof wallgrowth can normalize over time [14].As for medical treatment of EMS, L-thyroxine may behelpful in weight management when fed at a rate of 48–72mg/day for several weeks. However, in a group of horseswith naturally occurring EMS, the average weight loss on

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654 645

L-thyroxine was only 68 lbs (31 kg) after 24 weeks at 48mg/day and 12 weeks at 72 mg/day, and individual varia-tions were relatively great (68 � 31 lbs) [10]. Thus, on itsown L-thyroxine can be considered only of limited utilitywithout concurrent dietary and management changes.In the same group of horses with EMS, metformin (15–30mg/kg PO q12 h) prevented pasture-induced exacerbationof IR. However, the horses were also moving more when onpasture than when confined to a dry lot, so it was notpossible with the study design to eliminate the confound-ing effect of exercise[10]. Still, metformin may be worthy offurther study in helping to prevent pasture-associatedlaminitis.Bioenergetic failure. A current area of research is lamellarbioenergetics, which usestissue microdialysis to monitorenergy metabolism in the lamellar dermis. This techniqueshould be able to document changes in local perfusion andenergy metabolites associated with ischemia and withnonischemic bioenergetic failure, such as mitochondrialfailure and glycolytic perturbations [15]. Glucose-insulindynamics clearly are abnormal in horses with EMS [16], sobioenergetic failure may be a further mechanism whichcontributes to structural compromise and ultimate failurein endocrinopathic laminitis.Supporting limb laminitis. The pathogenesis of supportinglimb laminitis (SLL) is also unique among the variousforms of laminitis. While a limb is heavily loaded, there isno filling of any artery below the coronary band. When thelimb is persistently loaded in this way, such as occurs inhorses at risk for SLL, lamellar ischemia presumablyensues. Lamellar bioenergetics are currently being inves-tigated in an effort to further elucidate the pathophysi-ology of SLL [3].Traumatic laminitis. As will be discussed in the final sectionof this paper, there was a surprisingly high incidence ofchanges characteristic of chronic laminitis in the feet offeral horses from arid and semi-arid areas in the Australianoutback, particularly in the herds that were traveling longdistances over hard ground [17]. These findings suggesttwo possibilities that may be relevant to the domestichorse.First, there may be a concussive and/or compressiveelement to lamellar pathology that remains to be studied.Not only was the ground hard in these horses’ environ-ments, but the hoof walls of these horses typically wereshort fromexcessivewear, resulting in greater solar loading,and perhaps also concussion and vascular compression,than in the feet of horses living in more forgiving terrains.Second, traumatic laminitis likely represents a separateform of laminitis, complete with its own unique set ofmechanisms (yet to be fully elucidated). It does seempossible that traumatic laminitis may be additive to otherforms of laminitis in domestic horses kept on hard footingand/or trimmed excessively.Timing of structural compromise: As researchers haveinvestigates earlier stages of the developmental (DEV) orpreclinical phase of laminitis, it has become apparent thatthe cascade of lamellar destruction begins within hours ofthe insult, at least with SIRS-associated laminitis. Theprocess leading to structural failure in endocrinopathiclaminitis may be a much slower, yet still at times inexo-rable, decline.

Another important finding is that thewindow for positivelyinfluencing the course of the disease may not becompletely closed at the onset of lameness. There are somedifferences in the profile or intensity of biochemical andcellular events between the two standard observationpoints: onset of Obel grade 1 laminitis (OG-1; lameness atthe trot) and onset of Obel grade 3 laminitis (OG-3; reluc-tance tomove). In short, by OG-3 the lamellar inflammationis subsiding; so by then the initial damage has already beendone. These findings suggest that vigorous anti-inflam-matory therapy initiated at the first signs of lameness mayminimize the deterioration in lamellar structure andfunction.Developmental phase. The developmental phase in theresearch discussed here begins at the onset of fever, prior toany clinical signs of laminitis. As mentioned earlier, acti-vation of the resident tissue macrophage pool and an influxof leukocytes (predominantly neutrophils with BWE andmonocytes/macrophages with CHO) from the vascularpool is a feature of SIRS-associated laminitis. With theBWE model, the leukocyte response is detectable in thelamellar dermis as early as 90 min after the administrationof BWE [4].There is somewhat of a delay, or at least a slower rampingup, in the activation and extravasation of leukocytes in theCHO model. There is only a slight increase in leukocyteresponse and inflammatory mediator expression duringthe developmental phase[4]. There is, however, a signifi-cant increase in the expression of the chemokines CXCL-1,-6, and -8, but not yet upregulation of any proinflammatorycytokines nor of COX-2 [18].Expression of the matrix-destructive ADAMTS-4 andMMP-12 are increased about 100-fold during the developmentalphase in the CHO model, and this upregulation persists [7].It is not surprising, then, that pathological changes to thestructure of the epidermal lamellae, accompanied byinflammation in the dermal lamellae and deeper dermis,first appear during during this stage. These sensitivelamellar pathological changes are evident as early as 12 hrspost-CHOdat least 12 hrs prior to the onset of lame-nessdand even before major basement membranepathology is found [19]. There is alteration in the molecularconformation of the basement membrane by 12 hrs post-CHO, however, at which time one of its major constituents,collagen IV, has begun to disappear [3].In short, the triggering of lamellar inflammation (begin-ning with inflammatory chemokine induction), ECMdestruction, and cellular dysadhesion is well underwayseveral hours before the horse shows the first clinicalsigns of laminitis. While these processes have ramped upby the time the horse first shows signs of lameness or anincrease in digital pulse pressure, there is still a thera-peutic window in which to intervene and halt or mitigatefurther damage prior to progression to Obel grade 3laminitis.Obel grade 1 laminitis. Obel grade 1 laminitis is defined asthe onset of lameness. The whole process of lamellarinflammation is more transient in the BWE model than inthe CHO model. By OG-1 in the BWE model, the earlyincrease in lamellar neutrophils and classically activated(M1) macrophages is still present, but the M2 macrophageactivity has already returned to baseline levels [4].

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654646

By the onset of lameness (OG-1) in the CHO model, whichusually occurs at about 24 hrs post-CHO, there is a vigorousleukocyte response in the lamellar dermis, with activatedneutrophils and macrophages intensely and diffuselydistributed through the lamellar region [4]. By this time,there is a marked increase in the lamellar expression ofmultiple inflammatory mediators, including chemokines(CXCL-1, -6, and -8, CCL-2 and -8), cytokines (IL-1ß, -6, and-12p35), and COX-2 [18]. Furthermore, a 25-fold increase inthe expression of the collagenase MMP-1 joins that ofADAMTS-4 and MMP-12, which began during the devel-opmental phase [7].Thus, Obel grade 1 laminitis, especially in the CHOmodel, ischaracterized bymarked lamellar inflammation, an influxofleukocytes, and more concerted disintegration of thedermal-epidermal cell bond. Or, to turn it around, thesebiochemical and cellular changes evidently cause the footpain which characterizes Obel grade 1 laminitis. The goodnews is that there is yet very little structural damage visiblehistologicallyandno frank lamellar failure at this timepoint.Pathological changes to the dermal-epidermal structurelargely comprise a reduction in the average cross-sectionalareaof individual secondarydermal lamellae [20]. Thereforeif the developmental time periodwasmissed, this is the timeto actively intervenedboth pharmacologically and mechan-icallydto prevent structural failure.Obel grade 3 laminitis. Obel grade 3 laminitis is defined asreluctance to move. Perhaps the most important thing tounderstand about this later clinical phase of acute laminitisis that the lamellar inflammation which characterized theonset of Obel grade 1 laminitis is now subsiding; however,the majority of lamellar injury has already occurred. At thistime point, anti-inflammatory therapy is still useful, butmechanical interventions must predominate if furtherdestruction of the suspensory apparatus of P3 is to be pre-vented (see Part II of this conference wrap-up).In the CHO model of laminitis, the earlier influx ofneutrophils andmacrophages has already receded by OG-3,along with a marked reduction in the expression of most ofthe proinflammatory cytokines noted earlier [4,18]. Theremaining leukocytes have become focally clustered inspecific lamellae, particularly adjacent to areas of greatercellular injury/stress and around specific lamellar vessels,suggestive of vascular involvement. Thus, what began asa generalized inflammatory event affecting all lamellaediffusely has progressed to a more isolated vascular event(i.e. ischemia), with focal leukocyte extravasation andepithelial stress [4,18].By OG-3, all of the inflammatory mediators that wereupregulated during the developmental phase or at OG-1are decreased, with the notable exception of COX-2. Thisinflammatory isoenzyme remains markedly elevated atOG-3 [18]. Note that this waning of the initial inflammationhas occurred on its own, without anti-inflammatorytherapy in this experimental model. Lamellar failurecommonly occurs at OG-3, but surprisingly it does notappear to be accompanied by, nor does it induce, a markedinflammatory cytokine or leukocyte response [18].In other words, diffuse inflammatory injury occurs duringthe developmental phase and at least until OG-1. Itcontributes to, and may even precipitate, lamellar failure,but that failure itself appears to cause little further

inflammatory signaling or response [18]. The fact that thereis a persistent increase in the expression of COX-2 at OG-3,in the absence of a persistent leukocyte response, suggeststhat the lamellar epithelial cells themselves are a majorsource of this protein, perhaps stimulated by the structuralfailure that is occurring by this point [18].At OG-3 there is a 100-fold increase in the lamellarexpression of MMP-13, another collagenase [7]. Dramaticchanges in the lamellar structure are seen, includingmultifocal gaps lacking collagen, fibronectin, and glycos-aminoglycans in the secondary dermal lamellae; significantloss of microvasculature, and detachment of basal epithe-lial cells from the BM and each other [20]. This array ofmicrostructural changes presages the gross failure of thesuspensory apparatus of P3.Even though the majority of the cellular injury associatedwith the inciting event has already occurred by OG-3 andthe inflammatory response has largely abated, COX-2inhibition by nonsteroidal anti-inflammatory drugs(NSAIDs) remains important therapeutically. Of course,vigorous anti-inflammatory therapy is best begun duringthe developmental stage or at the onset of OG-1 laminitis.However when laminitis is not recognized until the horsehas OG-3 laminitis, it is useful to know that NSAID therapyis still of value, not just for pain management, but toameliorate the adverse effects of this persistent over-expression of COX-2 [18].P3 osteopathology: Most of us are familiar with theradiographic changes inP3 that are a characteristic of chroniclaminitis, but recent work has shown that osteopathology ofP3 occurs concurrently with lamellar pathology in acutelaminitis, even before radiographic changes are apparent.Abnormalities may be found in both the trabecular andcompact bone compartments of P3 that correlate with theseverity and progression/stage of lamellar pathology. Earlylaminitis-associated changes found in the medullarycompartments of P3 include marrow “edema” and fibro-myxoid spindle cell proliferation, neovascularization,mononuclear inflammation, reactive osteoproliferation, anda dramatic activation of osteoclasts with osteoclasticresorption [21].Two things are clear from this research: (1) the fate of P3 istied to that of the overlying lamellae, in more ways thanjust their shared anatomy (e.g. collagenous entheses,shared blood supply); and (2) these osseous changes couldadversely affect the overlying lamellae and compound thechallenge of pain management in the laminitic foot.Laminitis in feral horses: Studies of four different pop-ulations of feral horses in New Zealand and Australiaindicate that chronic laminitis is quite common in feralhorses [17]. However, the type of laminitis seen in thesehorses is quite clearly related to environmental factors,especially diet and terrain.Pasture-associated laminitis. Just as in domesticated horses,pasture-associated laminitis apparently occurs in feralhorses grazing high-NSC grasses. Histopathologic andradiographic evidence of chronic laminitis with similarpathology to the HI model of laminitis was found in 45% ofthis population. The laminitis was characterized as mildand the horses were described as gracile and athleticdi.e.they did not show the IR phenotype. These horses werethought to be selectively grazing on high-NSC pastures, so

Abstracts / Journal of Equine Veterinary Science 32 (2012) 641-654 647

perhaps these findings were partially a function of the timeof year at which the horses were sampled. However it isalso possible that there was a protective effect of move-ment (those horses having lived in a mountainous region)and hoof quality on their potential to show clinical signsfrom the lamellar changes that were identified [17].Traumatic laminitis. The more interesting group of feralhorses examinedwere the three populations of horses fromarid and semi-arid regions of the Australian outback,particularly those living in hard, rocky terrain and travelinggreat distances for forage andwater. Depending on the typeof ground surface and distances traveled, the incidence ofchronic laminitis in these populations ranged from 40% to93%, with the highest rates being reported for the horses inthe harshest terrain [17].In all but one of the horses, whose changeswere consideredsevere, the laminitis was categorized as mild or moderate.The most common histopathologic features were attenu-ated secondary epidermal lamellae (SELs), multi-branchedSELs, dystrophic tips of primary epidermal lamellae, and thepresence of cap horn. Radiographic features included anincrease in the “sinker distance” (vertical distance from theproximal margin of the dorsal hoof wall to the top of theextensor process of P3), an increase in the distance betweenthe external hoof wall and P3, remodeling of P3, and calci-fication of the ungual cartilages (sidebone) [17].These changes were more common and more marked inhorses from the regions with hard ground. Together, theselamellar and osseous changes are suggestive of traumaticlaminitis, possibly secondary to overuse or concussiveinjuries to both P3 and to its lamellar suspensory apparatus.The fact that these horses were still apparently soundshould not be interpreted with caution. Feral horses havelittle option but to keep moving and either adapt or makedo the best they can if they are to survive. In a domesticatedhorse, comparable changes typically are accompanied bychronic lameness or stiffness, and are incompatible withoptimal performance.In Part II of this conferencewrap-up, wewill discuss patientassessment and treatment for acute and chronic laminitis.

ReferencesEach of the following papers are to be found in theJournal of Equine Veterinary Science, vol. 31, issue 10(Oct 2011), pages 555–619.

[1] Barton MH. Systemic inflammation, multiple organ dysfunction, andthe hoof.

[2] Leise BS, Watts M, Johnson PJ, et al. Inflammatory gene expression inthe liver, lung, and kidney in the carbohydrate overload model ofequine laminitis.

[3] Pollitt CC, deLaat M, van Eps A, et al. Advances in laminitis researchat the Australian Equine Laminitis Research Unit.

[4] Belknap JK, Faleiros R, Black SJ, et al. The laminar leukocyte: fromsepsis to endocrinopathic models of laminitis.

[5] Belknap JK, van Eps AW, Pollitt CC. Efficacy of cryotherapy inblocking laminar inflammation events in oligofructose-inducedlaminitis.

[6] Leise BS, Watts M, Tanhoff E, et al. STAT1 and STAT3 regulation in thelaminae of horses administered black walnut extract or carbohy-drate overload.

[7] Wang L, Pawlak EA, Johnson PJ, et al. Expression and activity ofmetalloproteinases in the digital laminae of horses with starchgruel-induced laminitis.

[8] Burns TA, Nuovo GJ, Watts MR, Belknap JK. Role of oxidative stress inthe pathophysiology of equine laminitis: a comparison of twoexperimental models.

[9] de Laat M, Kyaw-tanner M, Patterson-Kane J, et al. Insulin and theinsulin-like growth factor system: a novel theory for hyper-insulinemic laminitis pathogenesis.

[10] Chameroy KA, Frank N. Equine metabolic syndrome: clinicalresearch studies.

[11] Schultz N, McCue M, Frank N, Geor R. Phenotypic characterization ofequine metabolic syndrome.

[12] Burns TA, Watts MR, Geor RJ, et al. Insulin targets in the equinedigital laminae: distribution of insulin and insulin-like growthfactor-1 receptors.

[13] Galantino-Homer H, Carter R, Linardi R, et al. The big picture: usingquantitative proteomics to investigate laminitis pathophysiology.

[14] Walsh DM. Metabolic intervention: recognizing and acting on thesubtle early warning signs of insulin resistance.

[15] van Eps AW, Richardson DW, Pollitt CC. Lamellar bioenergeticsstudies using tissue microdialysis.

[16] Lacombe VA. Glucose metabolism in insulin-sensitive tissue: fromhealth to disease.

[17] Hampson B. Laminitis in feral horses: where, when, and why?[18] Belknap JK, Eades S, Johnson PJ, Black SJ. Laminar inflammatory

mediator expression in the CHO model: developmental stage toObel grade 3 laminitis.

[19] Engiles J, Duffee L, Megee S, et al. An up-close and early look atlamellar pathology characteristic of oligofructose-induced laminitis.

[20] Pawlak EA, Wang L, Johnson PJ, et al. Structural changes in thedermal and epidermal laminae of horses with starch gruel-inducedlaminitis.

[21] Engiles JB, Galantino-Homer H, McDonald D, et al. Laminitis-asso-ciated P3 osteopathology.

Abstracts

What’s new in laminitis research? II: advances inlaminitis treatment

Nora S. Grenager VMD, DACVIM 1, andJames A. Orsini DVM, DACVS 2

1 Founder of Grenager Equine Consulting, LLC,Veterinarian At Harrison Equine In Northern Virginia,2 Associate Professor of Surgery, Director, LaminitisInstitute Penn-Vet, New Bolton Center, School ofVeterinary Medicine, University of Pennsylvania

Following on from Part I, which covered recent advances inthe pathophysiology and thus prevention of laminitis, thispaper summarizes recent advances in the treatment ofacute and chronic laminitis. Because the line is somewhatblurred between “acute” and “chronic” laminitis, and someresearchers and clinicians add an intermediate category,“subacute,” it is probably most expedient to discuss each ofthe treatments highlighted at the 2011 Laminitis Confer-ence one by one, and discuss the timing of their optimal usealong the way.Anti-Inflammatory Therapies: As discussed in Part I, anti-inflammatory therapydparticularly cyclo-oxygenase type 2(COX-2) inhibition d remains an essential component ofmanaging acute and subacute laminitis, even days after theinciting event. It is not only important for painmanagement,but suppression of lamellar inflammation may preventstructural failure if initiated before significant damage hasoccurred. In addition, it may limit further lamellar damageeven if the horse is not treated until after the onset of lame-ness. Digital hypothermia (cryotherapy) is perhaps the beststudied anti-inflammatory therapy in this regard.When firstapplied and then maintained throughout the preclinicalphase of laminitis, it can prevent the development of clinicallaminitis. Anecdotally it appears to limit additional damageevenwhen first applied after the onset of lameness.