REVIEW Overview of What We Know About the Pathophysiology of Laminitis Susan C. Eades, DVM, PhD, Diplomate ACVIM-LA Laminitis is frustrating for veterinarians because current knowledge and understanding of the pathophysiology and progression of the disease are incomplete, limiting efforts to prevent and treat this devastating disease successfully. However, scientific investigations have recently occurred at a phenomenal rate shedding light on the pathophysiologic events involved with laminitis. Development of acute laminitis often follows other primary diseases; therefore, the mechanisms involved in the pathogenesis of laminitis are most likely numerous and interrelated. On the basis of the discussion from the 2007 Havemeyer Meeting,1 inflammation, metabolic disease, and endothelial and vascular dysfunction are considered pivotal events in the development of laminitis.
INFLAMMATION 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 Equine Health Studies Program, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA. 0737-0806/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.jevs.2010.01.047
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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 MMP9 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-9.12-16 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
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to that reported in other sepsis models, with increases in expression of tumor necrosis factor-a, IL-6, IL-8, and IL-1b within 1.5 hours after administration of BWHE. However, the increases in IL-1b, 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 antiinflammatory 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 BWEtreated 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
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tissues, while damage occurs in the unprotected intestinal tract.
METABOLIC SYNDROME Endocrinopathic laminitis is a term that has been used to describe laminitis that occurs in horses with obesity, insulin resistance, pituitary dysfunction, and glucocorticoid administration. Insulin resistance is a common factor in the disease in a large number of these horses. A pivotal discovery that has advanced our understanding of the pathogenesis of laminitis in these horses revealed that intravenous infusion of insulin through a euglycemic hyperinsulinemic clamp technique for 72 hours induced Obel grade 2 laminitis with a mean serum insulin concentration at 1036 mU/ mL compared with 14.6 mU/mL in control horses. These results confirm that insulin toxicity is a key factor in triggering laminitis. Serum insulin and leptin concentrations had reproducible accuracy for prediction of laminitis in pastured ponies.28 Furthermore, serum insulin concentrations were significantly higher in ponies that recurrently develop laminitis on pasture than those that do not.29 Mediators of inflammation and oxidant damage may induce lamellar injury increasing the risk for laminitis in obese or insulin-resistant ponies. Treiber et al evaluated markers of inflammation and redox status in pastured ponies with a history of laminitis and determined that there were no differences between markers of antioxidant function and oxidant pressure between ponies with laminitis and those without laminitis.30 However, laminitic ponies had higher serum concentrations of tumor necrosis factor than ponies that were not laminitic.
ALTERATION OF ENDOTHELIAL AND VENOUS FUNCTION Many of the vascular events during equine laminitis were recently summarized by Robertson et al.31 Laminar edema due to venous constriction was among the earliest vascular events identified in the early stages of BWHE and carbohydrate-induced laminitis.32,33 This increased venous resistance is concurrent with increased concentrations of endothelin-1 and can be prevented by administration of an antagonist of endothelin-1.3,34 Endothelin-1 causes intense vasoconstriction of laminar veins, an effect that is enhanced fourfold by L-NG-Nitroargine methyl ester (L-NAME).35 Weiss et al demonstrated platelet activation and platelet-neutrophil activation.4 Localized platelet activation causes vasoconstriction through release of thromboxane and serotonin, which cause laminar vein constriction more than laminar arteriolar constriction. Furthermore, vasoactive amines are formed by bacteria in the gastrointestinal tract and may enter the circulation contributing to the pathogenesis of laminitis.36 Insulin resistance alters endothelial function, which can create
a proinflammatory condition leading to platelet and leukocyte activation, increased endothelin production, and production of mediators of inflammation and oxidant stress.37 The earliest laminar events in BWHE-induced laminitis include activation of endothelial adhesion molecules and leukocyte emigration.24 Altered concentrations of vasoactive substances may not have an important effect on regional blood flow, but likely signal alteration in endothelial function.3 The end result of altered endothelial function during laminitis may be creation of a proinflammatory state with increased oxidant stress rather than oxygen deprivation. The preponderance of these results supports roles for inflammation, endocrinopathic laminitis, and endothelial and venous dysfunction. Inflammation, oxidant stress, and matrix degradation may be factors common to each of these mechanisms that lead to lamellar damage in this devastating disease.
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