Overview of What We Know About the Pathophysiology of Laminitis

Evidence of Cellular Infiltration 

Systemic sequelae to inflammation (systemic inflammatory response syndrome) commonly plague equine patients undergoing treatment for numerous conditions, including pleuropneumonia, colitis, enteritis, peritonitis, endometritis, and hepatitis. Although end-organ damage from the systemic inflammatory response can include damage to numerous body tissues, there is no complication more common and devastating during equine inflammatory disease than acute laminitis. Although researchers once questioned whether the disease should be called “laminar degeneration” due to the minimal neutrophilic infiltration present histologically, application of more sensitive research tools has produced abundant evidence of inflammatory changes during laminitis. In laminitis, neutrophils become aggregated to platelets in the early prodromal stage and at the onset of lameness.2, 3 Carbohydrate overload laminitis (corn starch) was prevented in eight ponies by pretreatment with an antagonist of platelet aggregation (platelet fibrinogen receptor antagonist peptide).4 Using immunoperoxidase and CD13 monoclonal antibodies, Black et al documented emigration of neutrophils to perivascular tissues of both the skin and laminae during prodromal stages (3–4 hours) and at onset of lameness after administration of black walnut hardwood extract (BWHE).5 Neutrophil emigration from the circulation is accompanied by reduction in numbers of circulating neutrophils and monocytes.6 Neutrophil and/or monocyte activation in skin, plasma, and lamina 3 and 12 hours after BWHE administration confirm the activation of peripheral white blood cells (WBCs) and the initiation of the systemic inflammatory response syndrome.7 These activated neutrophils produce increased quantities of reactive oxygen species.6 Investigators sought to determine whether these emigrated neutrophils could be the source of matrix metalloproteinases (MMPs) that damage the laminar extracellular matrix. It was determined that neutrophil activation and emigration (as measured by CD 13 immunohistochemical staining of tissues obtained from horses euthanized at 3–4 hours after administration of BWHE and at onset of Obel grade I laminitis) occurred concurrent with peaks in MMP-9 activity (measured by gelatin zymography and polymerase chain reaction [PCR]).8 Evidence of the systemic inflammatory response syndrome was confirmed in horses with BWHE-induced laminitis by documentation of neutrophil infiltration (CD13 immunohistochemistry) in liver and lung within 3 hours after BWE administration.9 In addition, laminar immunohistochemical staining of calprotectin, a marker of remote tissue inflammation and damage during the systemic inflammatory response syndrome in people, is increased at 12 hours after onset of black walnut-induced laminitis in horses.10 This immunohistochemical staining of calprotectin was perivascular, near the epithelial basement membrane, and mostly associated with neutrophil emigration. Furthermore, calprotectin signaling of epithelial damage occurred 12 hours after administration of BWHE, which is 9 hours after the initial onset of neutrophil emigration, suggesting that WBCs emigration is a primary event that is not initiated by epithelial damage. In human keratinocyte preparations, inflammatory cytokines induce production of calprotectin, and calprotectin causes further induction of cytokines. Calprotectin can induce cellular apoptosis, a documented event in equine laminitis.11 Taken together, these results confirm that WBC activation is a significant and early event in acute laminitis, and that these WBCs may be a significant source of damage to the extracellular matrix and epithelium.

Enzymatic Dysregulation 

Although these studies document that WBC activation occurs more rapidly, there is substantial evidence that accelerated enzymatic remodeling with degradation of laminin and type IV and type VII collagen is an important event during laminitis with documented increased in MMP-2 and MMP-9.12, 13, 14, 15, 16 Most recently, real-time PCR documented increased tissue expression of MMP-14 concurrent with decreased amounts of tissue inhibitor of metalloproteinases (TIMPs) in horses with laminitis induced by use of fructan administration.17 In contrast MMP-14 content of the basilar epithelial cells near the basement membrane was depleted based on immunohistochemistry.17 Examination of laminar samples (gelatin zymography for MMP-9 and MMP-2 and myeloperoxidase enzyme-linked immunosorbent assay and real-time PCR for proMMP-2 processing genes) from horses with naturally occurring laminitis, and those administered starch gruel revealed that MMP-9 concentrations correlate directly with activation of neutrophils, suggesting production or induction by inflammatory leukocytes. In contrast, MMP-2 regulation occurred independent of myeloperoxidase concentration, suggesting that dysregulation of MMP-2 occurs independent of inflammatory processes.18 A recent study also examined the expression of genes coding for proteins containing a Disintegrin and Metalloproteinase domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (TIMP) in horses with carbohydrate overload, BWHE, and naturally occurring laminitis.19, 20 ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally induced and naturally acquired laminitis. The expression of MMP-9 and ADAMTS-5 was also increased in many of the laminitic horses. Furthermore, TIMP-2 gene expression was decreased in most laminitic horses. It appears that improper regulation of the extracellular matrix is an important event in horses with laminitis, whether as part of or independent of the general inflammatory processes.

Other Inflammatory Mediators 

It was determined fairly early in the history of laminitis research that expression of interleukin (IL)-1 and IL-6 increased in laminar tissue of horses with laminitis induced with BWHE extract.21, 22 More recent studies of BWHE and oligofructan models of laminitis have documented increased lamellar mRNA expression of cytokines important in the innate immune response present at the developmental stage of the BWHE model, and at the onset of acute lameness in both the BWHE model and OF model.23 Cytokines characteristic of the adaptive immune response were present at the onset of lameness in the BWHE model.23

Pivotal to our understanding of the systemic inflammatory response syndrome during equine laminitis is the recent discovery of increased expression of pulmonary and hepatic inflammatory mediators in horses administered BWHE. The pattern of proinflammatory cytokine expression in the lung and liver in the BWE model was similar to that reported in other sepsis models, with increases in expression of tumor necrosis factor-α, IL-6, IL-8, and IL-1β within 1.5 hours after administration of BWHE. However, the increases in IL-1β, IL-6, and IL-8 in the lung and liver were all much smaller in magnitude than those occurring in the laminae at the same time points in the BWHE model.23, 24 The reason for the heightened laminar inflammatory response is not known. Unlike what has been shown in human sepsis models, the expression of anti-inflammatory cytokines IL-10 and IL-4 did not increase after BWHE administration.

In addition to this intense lamellar cytokine response, there is evidence for a diverse laminar inflammatory reaction. Noschka et al used an equine-specific cDNA microarray to screen gene expression in laminar tissues collected at 1.5, 3, and 12 hours after BWHE administration.25 As early as 1.5 hours after BWHE administration, genes associated with leukocyte activation and emigration were upregulated. Other genes involved in inflammatory processes, antioxidant processes, and antimicrobial processes were upregulated from tissues collected at the onset of Obel grade I laminitis. Immunohistochemical analysis has revealed that cyclooxygenase-2 is markedly increased in the basal epithelial cells during the first few hours after BWHE administration coincident with leukopenia.26

Oxidants 

Oxidant injury plays an important role in the end-organ insult resulting from the systemic inflammatory response syndrome. Loftus et al evaluated laminar tissues for presence of xanthine oxidase (XO)–dependent production of superoxide anion after administration of BWHE.24 Tissues from liver, lungs, and skin of control and BWHE-treated horses contained superoxide dismutase (SOD). Laminar samples from both groups of horses were devoid of SOD. Tissues from liver, lung, skin, and laminae of control and BWHE-treated horses all had endogenous XO and catalase. The levels of XO and catalase were similar in extracts of laminae from control and BWHE-treated horses. The absence of increased XO activity suggests against the involvement of this reactive oxygen intermediate-generating system in the development of laminar pathology in BWE-treated horses. However, the absence of SOD suggests that the equine digital laminae are highly susceptible to damage by superoxide anion.24

Yin et al evaluated 4-hydroxy-2-nonenal (4-HNE), a lipid aldehyde that forms due to lipid peroxidation occurring during episodes of oxidant stress that can be used as an index of tissue oxidant stress in laminar, lung, liver, and intestinal tissues of horses undergoing BWHE laminitis.27 The laminar concentrations of 49-HNE increased significantly in horses with laminitis; however, they remained normal in lung, liver, and intestinal tract. It is possible that antioxidant systems prevent lipid peroxidation in these other tissues, while damage occurs in the unprotected intestinal tract.