Equine Laminitis- gaining footholds in a complex disease: a review of pathophysiology,
current experimental models, and future therapeutic interventions.
Julie B. Engiles, VMD, DACVP. University of Pennsylvania, School of Veterinary
Medicine, Department of Pathobiology, New Bolton Center, Kennett Square,
Pennsylvania, USA.
Laminitis is an important cause of lameness, morbidity, and mortality in horses, and is
economically costly to the equine industry. A 1998 USDA survey of 1178 non-racetrack
horse operations, including over 28,000 horses representing 28 states and
approximately 83.9% of non-racehorses in the US, estimated laminitis to account for
7.5-15.7% of lameness problems with death or euthanasia resulting from laminitis in
4.7% of these horses.32 However, the estimated frequency of disease occurrence as
reported in the literature varies from 1.5-34%, depending on underlying etiology or
horse populations.64 A recently published cohort study of equine laminitis in Great
Britain (2009-2011) estimates the prevalence of veterinary-diagnosed active laminitis to
be 0.47% (approximately 1 in 200 horses seen by veterinarians). In comparison, a
retrospective study performed at a tertiary care facility investigating the factors
associated with non-survival in horses treated for laminitis from 1986-2003 reports 247
out of 591 horses hospitalized for laminitis died or were euthanized, and that the
severity of lameness was the most significant prognostic indicator, not the presence of
systemic inflammatory diseases (e.g. phlebitis/ thrombophlebitis, pneumonia, or
treatment with NSAIDs).48
The equine foot is a highly specialized appendage that integrates several different
anatomic elements including a keratinized epidermis, a highly vascularized dermis and
corium having valveless veins, as well as other connective tissue elements such as the
distal phalanx (DP) with its ligamentous entheses, the fibroadipose tissue of the heel
bulbs and digital cushion, the collateral cartilages, and tendons (e.g. the insertion of the
deep digital flexor tendon). [Figure 1] Although the pathogenesis of laminitis is
incompletely understood, the target of the disease is the suspensory apparatus of the
distal phalanx (SADP) that consists of extensively interdigitated epidermal and dermal
lamellae that attach and suspend the DP within the hard keratinized hoof capsule
through this inner portion of the hoof wall (stratum internum).53 The lamellae comprise
500-600 primary lamellae, each having 150-200 secondary lamellae that, via a
basement membrane at the dermo-epidermal junction, provide an large surface area
(~0.8m2) for attachment and suspension.54 [Figure 2] The blood supply to DP that
originates from the palmar/plantar digital arteries continues via vascular canals that
extend through its semi-porous dorsal cortex, to supply the overlying epidermal/dermal
lamellae. The blood vessels supplying the primary and secondary lamellae contain
valveless veins and numerous arteriovenous anastomoses that allow for shunting of
blood within hoof wall for pressure distribution and thermoregulation.54 In fact,
experimental intraosseous infusion of the DP via a cannulated bone screw
demonstrated clinically safe and efficacious delivery of an aminoglycoside (Gentamicin)
to lamellar tissues at much higher concentrations than detected in peripheral serum.45
Given the unique anatomical and functional properties of the SADP, it is not surprising
that unique molecular components of lamellar epithelium and mesenchyme have also
been discovered. Examples include the identification of novel keratin isoforms (K42 and
K124) as the most abundant cytoskelatal proteins of lamellar keratinocytes, the
production of large polysulfated glycosaminoglycans (aggrecan, versican, and
hyaluronic acid: large water-binding molecules predominantly found in cartilage tissues
under extensive compressive mechanical stress) localized within lamellar epidermal
keratinocytes, and ubiquitous expression of proteoglycans that are detectable by
oligosaccharide-binding plant lectins such as wheat germ agglutinin (WGA) throughout
the extracellular matrix of dermal lamellae, as well as delineating intercellular
membranes of epidermal keratinocytes.10, 11, 50 In addition to not containing resident
neutrophils, normal lamellae demonstrate less superoxide dismutase activity, and also
exist in a relative hypoxic state as compared to other equine tissues.19, 51 Histologic
architectural variations within primary and secondary epidermal lamellae (PELs and
SELs, respectively) exist depending on location within the hoof wall as well as age and
athletic history of the horse, likely representing a spectrum of physiologic responses due
to normal growth and remodeling of the hoof wall, as well as pathologic responses to
prior (multi)focal injury. 24, 33
The pathogenesis of equine laminitis is complex, and to date, incompletely understood
with no known cure. The disease predominantly affects the sensitive epidermal and
dermal lamellae of the inner hoof wall that typically results in biomechanical failure of
these tissues with eventual rotation and distal displacement (“sinking”) of the DP, often
necessitating humane euthanasia because of intractable pain and dysfunction.
Inadequate tissue healing and aberrant regenerative responses result in hyperproliferation of epidermal/dermal lamellae, and correspond to the formation of the
lamellar wedge, the most characteristic radiographic and gross lesion of chronic
laminitis.8, 12, 36 [Figure 3] The disease can be divided into stages depending on clinical
presentation, with a developmental, or subclinical “eclipse” phase that precedes clinical
signs manifesting as systemic illness (e.g. fever) or lameness (e.g. Obel Grade
lameness stages 1-4), depending on the inciting cause. Using experimental induction
models of laminitis, early “acute” histololgic changes within the first 48 hours post
induction, coinciding with the initial onset of Obel Grade 1 lameness, include narrowing
and lengthening of the SELs, loss of polarity and rounding of basal keratinocyte nuclei,
and increases in basal keratinocyte necrosis and mitosis.17 Histologic changes
corresponding to the subacute into chronic stages of experimental and natural (e.g.
spontaneous) cases of laminitis include necrosis and fragmentation of SELs with
basement membrane separation at the dermoepidermal junction, displacement of the
keratinized axis with lengthening of PELs, dysplastic epidermal lamellar regeneration,
and dermal inflammation.44, 46, 57 [Figure 4] The initial acute to subacute changes in the
lamellae are typically not detectable by conventional radiology, but can be detected with
more sensitive imaging modalities such as magnetic resonance imaging (MRI). 2
Biomechanical failure of the SADP corresponding to the characteristic distal
displacement, or “sinking” of the distal phalanx often coincides with the proliferation of a
markedly dysplastic, hyperkertotic regenerative reponse of the epidermal lamellae,
corresponding to the lamellar wedge.13, 27 [Figure 5] In addition to structural alterations
in the lamellae, laminitis results in structural alterations to other tissues including the
digital microvasculature, digital nerves and spinal dorsal root ganglia, and the distal
phalanx. More complete characterization of changes in these tissues are ongoing and,
given their potential impact on tissue regeneration, disease progression, and important
co-morbidities such as pain, are considered to have potentially important contributions
to the pathogenesis.20, 31, 65
The development of spontaneous equine laminitis is associated with many different
environmental exposures or clinical risk factors including: deranged endocrine/metabolic
states (e.g. hyperinsulinemia and Equine Metabolic Syndrome-EMS, Pars Pituitary
Intermedia Dysfunction- PPID), high carbohydrate diet (e.g. “pasture founder” or “grain
overload”, systemic inflammatory and infectious diseases (e.g. Salmonellosis, Potomac
Horse Fever, sepsis), vascular disease (e.g. local or generalized ischemic states,
vasculitis/vasculopathy), repetitive local trauma (e.g. “road founder”), and toxic causes
(e.g. Black Walnut Toxicity). 18, 49 Risk factors and clinical outcomes also differ
dramatically among different populations of horses, such as in different breeds of
horses, or hospitalized versus non-hospitalized horses.1, 48, 63 However in spite of these
various and numerous risk factors, very few absolute “causes” of laminitis have been
confirmed by experimental models, making research investigations of laminitis difficult.29
In the past, laminitis research largely depended on anecdotal or observational
descriptive manuscripts; however to better characterize the pathogenesis, more recent
studies have incorporated experimental induction models for investigating the
morphological and molecular differences between normal versus laminitic feet. Current
induction models include: 1) carbohydrate overload (CHO) model, considered to mimic
sepsis-associated laminitis, induced by nasogastric administration of wood starch/gruel
slurry or oligofructose; 2) hyperinsulinemic (HI) model, considered to mimic endocrinemetabolic-associated laminitis, induced by intravenous infusion of insulin and glucose to
achieve a prolonged euglycemic/hyperinsulinemic “clamp”; and 3) Black Walnut Extract
(BWE) model, considered to mimic Black Walnut toxin-associated laminitis, induced by
nasogastric administration of an aqueous extract from Black Walnut heartwood
shavings.6, 26, 37 Since naturally occurring laminitis is most frequently associated with risk
factors and processes similar to the CHO and HI models, the focus of this review will
pertain to these two experimental induction methods.
Experimental introduction of excessive alimentary carbohydrate results in a microflora
shift in the equine hindgut that reliably produces laminitis in most equine subjects.52
Recent studies that include microbiomic analyses of equine hindgut microflora show a
post-induction shift to acid-producing Gram positive bacteria (e.g. Lactobacillus sp. and
Streptococcus sp.) as well as potentially pathogenic Gram negative bacteria (e.g.
Veillonella sp. and Serratia sp.), which are thought to contribute to the intestinal
mucosal damage and the pro-inflammatory state that progresses to laminitis.42, 47
Transcriptomic, proteomic, and immunohistochemical evaluations of laminitis in horses
induced by CHO reveal endotoxemia and a molecular profile consistent with a systemic
as well as lamellar-specific inflammatory response including systemic platelet activation
with release of serotonin and thromboxane, and dysregulation of fatty acid metabolism
with significant depletions in citrulline (a potential biomarker of critically ill animals),
lamellar inflammatory gene expression (IL-1, IL-6, IL12p35, COX2, E-selectin, ICAM-1,
CXC chemokines and CC chemokines), and lamellar infiltration by leukocytes.3, 22, 23, 38,
56 In addition, CHO induced laminitis results in the production of catalytically active
ADAMTS-4 with depletion of versican from basal keratinocytes, accompanied by
regional basement separation of secondary epidermal lamellae.60 Further investigation
showed that versican depletion is associated with suppression of the canonical Wnt
signaling pathway that maintains the epithelial phenotype, as evidenced by concomitant
depletion of -catenin and -integrin, and that an increased expression of MMP-13
collagenase in basal keratinocytes is accompanied by the loss of collagen I, Fibronectin,
chondroitin and keratan sulfate glycosaminoglycans, and eosin-staining material within
adjacent secondary dermal lamellae.61, 62 Evaluation of epidermal lamellar keratinocytes
isolated by laser capture microdissection confirms production of inflammatory mediators
and extracellular matrix regulators by basal keratinocytes, indicating that these cells are
active participants in the molecular and cellular events leading to lamellar structural
failure, and are not merely “innocent bystanders” as once thought.39 However, although
the CHO induction model may resemble sepsis-associated laminitis in horses, a natural
disease model of “pasture associated laminitis” produced by feeding a high nonstructural carbohydrate diet (hay chop supplemented with sweet feed and oligofructose)
to lean and obese mixed breed ponies only resulted in increased COX-2 expression as
quantitatively assessed by immunohistochemistry, not pro-inflammatory mediator
expression or inflammatory infiltration of lamellar tissues, concluding that perhaps the
cellular and molecular events occurring in the model of sepsis-associated laminitis may
not be representative of pasture-associated laminitis.7
A different experimental induction technique thought to model endocrinopathic and
pasture-associated laminitis is the hyperinsulinemia (HI) model that induces a
hyperinsulemic state by prolonged intravenous infusion of insulin (6 mIU/kg bwt/min)
with a variable rate intravenous infusion of 50% glucose to maintain a euglycemic state
until the onset of Obel Grade 2 lameness (~48 hour period).37 Although all treated
horses developed laminitis with degenerative and necrotic changes to the SELs similar
to that described for the CHO model, in contrast to the CHO model, the inflammation
with lamellae induced by HI was much less severe, and there was minimal evidence of
matrix metalloproteinase upregulation.17, 55 Interestingly, a less marked hyperinsulemia
induced in horses by intravenous infusion of glucose for a period of 48 hours did not
result in lameness, and lamellar pathology was much less severe, suggesting that a
glucose overload alone is unlikely to be the underlying mechanism of endocrinopathic
and pasture-associated laminitis.16 In addition, the pro-inflammatory cytokine response
observed in the CHO model, was not observed in either euglycemic or hyperglycemic HI
models.14 Results from another study evaluating different isoforms of glucose
transporters in lamellae, skeletal muscle, and heart from the HI model suggest that
epidermal lamellar tissue may function independently of insulin and that insulin
resistance may not be a necessary component of laminitis pathogenesis in this model,
thus the pathogenesis of endocrinopathic laminitis remains elusive. 5
Although these experimental induction models have provided valuable insight into the
molecular and anatomical events preceding structural failure of the lamellar interface,
their direct translation to events occurring in naturally occurring laminitis is limited, thus
the importance of investigating spontaneous cases of laminitis should not be
overlooked. However, given the nature of clinical laminitis, and that biopsy of the hoof
wall is considered relatively invasive and not routine, thorough pathological evaluation
typically requires euthanasia, which often occurs after the devastating secondary effects
due to complete biomechanical failure of the SADP, precluding of evaluation of
pathogenically important yet clinically silent developmental through acute stages of
laminitis. Perhaps for logistical reasons, investigations of spontaneous laminitis mostly
comprise either case controlled or observational retrospective studies of clinical risk
factors and survival outcomes, with concurrent clinical pathologic parameters.4, 5, 21, 30, 34,
43, 48, 59 Given the limited numbers of studies including anatomic pathologic
investigations in conjunction with detailed historical and clinical pathologic data, a
Laminitis Discovery Database has been developed at New Bolton Center to provide a
comprehensive image, serum and tissue repository for the study of naturally occurring
laminitis.25 This database includes samples from over 125 normal (N=55) and laminitic
(N=70) horses having various associated risk factors (inflammatory-sepsis, support limb
overload, endocrinopathic-metabolic) and in various stages of disease, including
developmental and acute stages as well as subacute and chronic. Two to four front and
hind feet are harvested from each horse, totaling 304 feet. The goal is to correlate
transcriptomic and proteomic data with gross anatomic and histopathologic changes to
more completely evaluate naturally occurring laminitis, and to potentially identify serum
biomarkers for early disease detection and prevention, or novel targets for therapeutic
interventions. In addition to abundant gross and histomorphometrical data, pathologic
evaluation of these tissues has identified reduced expression of the epidermal stem cell
marker p63 in lamellae from horses with chronic laminitis, and medullary activation with
osteoclastic osteolysis of the distal phalanx in early-onset as well as chronic stages of
laminitis.9, 20 The tissue repository has also provided lamellar epidermal cells to develop
an epithelial stem cell-selective and 3-dimensional culture system as an in vitro
organotypic model system for pathophysiologic studies of lamellar epithelial cell-cell
interactions, therapeutic interventions, and epidermal regenerative medicine.40
However, in spite of intense efforts to identify novel therapeutic targets, to date very little
has changed regarding treatment of laminitis. Distal limb cryotherapy (e.g. application of
tall ice boots to the level of carpi or hocks) has been shown to reduce the severity of
lamellar pathology in a CHO experimental induction model, and also reduce the
incidence of laminitis in a clinical population of horses with colitis at risk for sepsisassociated laminitis.35, 58 Regarding dietary and exercise management, one casecontrolled study demonstrated that regular low intensity exercise did significantly
decrease the concentration of two plasma inflammatory markers (serum amyloid AsAA, and haptoglobin- Hp) in both previously laminitic and non-laminitic ponies, and
another in vitro study identified the -acid phytochemical from hops in beer (Humulus
lupulus L.) prevented the growth of fructan-fermenting equine hindgut microflora.28, 41
Experimental investigations for gene therapy delivery to inhibit lamellar matrix
metalloproteinase activation, and mechanically dynamic shoeing to prevent support-limb
laminitis are on-going (personal communications, Dr. Dean Richardson).
Given the complexity of experimental and naturally occurring models of laminitis, the
discovery of a “magic bullet” treatment is unlikely; however continued dedication of
resources to this area of research underscores its importance to the veterinary and
equine community at large. Although good progress has been made since the
application of molecular techniques to laminitis pathophysiologic research, many more
critical investigations will be required before the step-wise molecular and anatomic
progression to lamellar failure is fully elucidated for each associated risk factor, so that
successful targeted therapeutic intervention or reliable disease prevention can occur.
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Figure 1. Normal foot- parasagittal section. DDFT,
Deep Digital Flexor Tendon with insertion (asterisk);
NAV, navicular bone; DC, digital cushion; MP,
middle phalanx; DP, distal phalanx; white arrow and
line, stratum medium; black arrow and line, stratum
internum (lamellae with corium dermis).
Figure 2. Chronically laminitic foot- parasagittal
section. Severe rotation and sinking of the distal
phalanx with subsolar abscess (arrow head); MP,
middle phalanx; DP, distal phalanx; black arrow,
lamellar wedge delineated by dorsal cortex of the
DP (black line).
Figure 3. Normal lamellae, H&E (2X, upper
panel; 20X, lower panel). Interdigitating Primary
Epidermal & Dermal Lamellae (PEL, PDL) merge
abaxially with the stratum medium (SM).
Secondary Epidermal & Dermal lamellae (SEL,
SDL) extend from the keratinized axis (KA).
Figure 4. Acute laminitis. H&E (2X, upper panel;
10X, lower panel). Necrosis with complete
epidermal separation from the Primary Dermal
Lamellae (PDL). Surrounding the keratinized axes
(KA) are necrotic secondary epidermal lamellae
(SEL) with early regeneration (lower right).
Figure 5. Chronic laminitis. Complete abaxial epidermal separation (black arrow), dysplastic lamellar
regeneration (white arrow) with hyperkeratosis (HK), acanthosis and epidermal islands (inset- A, asterisk).