Role of oxidative stress in the pathophysiology of equine laminitis: a comparison of two experimental models

s / Journal of Equine Veterinary Science 31 (2011) 562-609 587 Role of oxidative stress in the pathophysiology of equine laminitis: a comparison of two experimental models T.A. Burns , G.J. Nuovo , M.R. Watts , and J.K. Belknap 1 1 College of Veterinary Medicine, The Ohio State University, Columbus, OH, 2 College of Medicine, The Ohio State University, Columbus, OH Take-Home Message: Oxidative stress does not play an important role in laminar failure in experimentally-induced equine laminitis. Introduction: Oxidative stress reportedly plays a role in sepsisinduced organ dysfunction and failure in many species. In septic horses, the laminae are highly vulnerable to injury. Evidence of laminar oxidative stress has been reported experimentally in the black walnut extract (BWE) model of laminitis. However, carbohydrate (CHO)-induced laminitis may be more similar to clinical sepsis-related laminitis than the BWE model, in that animals with CHO-induced disease commonly develop laminar failure. As yet, the role of oxidative stress in the CHOmodel of laminitis is unknown. Oxidative stress, occurring secondary to elaboration of reactive oxygen (ROS) and nitrogen species by activated leukocytes, plays an important role in the initiation and perpetuation of multiple organ dysfunction and failure associated with sepsis in multiple species. In the septic horse, the digital laminae are the tissue most often injured. Laminitis induced with soluble carbohydrate (CHO) may be more similar to clinical disease than the BWE model due to the fact that CHO-induced animals more commonly proceed to laminar failure. However, the role of oxidative stress in the CHO model has not been reported. Materials and Methods: Banked laminar tissues from various time points in animals subjected to BWE (n 1⁄4 15) or CHO (n 1⁄4 20) induced laminitis were used for this study. Immunohistochemistry targeting 3-nitrotyrosine (3-NT; a marker of nitrosative stress) was optimized using the Ventana Ultraview Universal system with Fast Red as the chromogen on paraffin-embedded laminar sections from CHO horses (control, n 1⁄4 3; Obel grade 1 [OG-1], n 1⁄4 5) in order to identify the cell types positive for 3-NT. Laminar sections from BWE subjects were stained as a positive control (control, developmental, and late time points; n 1⁄4 1). Laminar 4-hydroxynonenal (4-HNE; a marker of lipid peroxidation) and protein carbonyl content (a stable marker of tissue oxidation) were evaluated by slot blot analysis. Results: Very few 3-NT-positive cells were visualized in any of the samples from the CHO model compared to the large number of cells observed in the BWE model. Positive cells in the CHO model were predominantly interstitial leukocytes and endothelial cells, and they were strictly limited to the laminar dermis. (In contrast, prominent laminar epithelial signal is present by OG-1 lameness in the BWE model.) The number of laminar 3-NT (+) cells was increased at developmental (DEV) and Obel grade 1 (OG-1) time points in the BWE model (vs. control [CON]; p 1⁄4 .013). It was lower in OG-1 tissues than CON in the CHO model (p 1⁄4 .04). No change in 4-HNE content was observed in the CHO model, and no increase in laminar protein carbonyl content was present in either model (p > 0.05). Discussion: Although the clinical use of several antioxidants (e.g. vitamin E) in horses has been reported, the results of this study do not support their use for modulation of oxidative tissue damage in the pathophysiology of laminar failure in the septic horse. However, modulation of intracellular signaling cascades in which ROS normally play a prominent physiologic signaling role may be important for laminar injury. Therapeutic intervention may be more reasonable in this setting. Additional study is required to evaluate this possibility. Conclusions and Clinical Relevance: These results do not support a prominent role for oxidative stress at examined time points in CHO-induced laminitis and they support only transient oxidative stress in the BWE model. Oxidative stress does not appear to be a central early pathophysiologic event leading to laminar injury and failure in the CHO model of laminitis, so antioxidant therapy for the laminitic patient may not be warranted. Scientific – Alternative Therapies Is there a role for pentoxifylline in laminitis? Michelle Henry Barton DVM, PhD, DACVIM University of Georgia, Athens, GA Take-Home Message: Pentoxifylline (PTX) is sufficiently absorbed from the GI tract of horses at 10 mg/kg PO q12h. Some studies have shown therapeutic promise for the treatment of vascular and inflammatory conditions, though it remains to be determined if PTX has a beneficial role in the treatment of laminitis. Introduction: Pentoxifylline (PTX) is a synthetic derivative of theobromine that was originally marketed as a rheologic agent (Trental ) for people with chronic occlusive peripheral disease or cerebral arterial disease. Since its introduction almost 40 years ago, additional pharmacodynamic effects of PTX have been discovered. Its rheologic, vasodilatory, bronchodilatory, and antiinflammatory properties have sparked interest in its potential for equine patients, including the treatment of laminitis. Mechanisms and Clinical Uses: The hemorheologic properties of PTX appear to be the result of its nonselective inhibition of membrane-bound phosphodiesterase and its activation of protein kinase, thereby increasing intracellular cyclic adenosine monophosphate and decreasing intracellular calcium. As a result, red blood cells are more flexible and blood viscosity, platelet aggregation, fibrinogen concentrations, and thrombus formation are reduced. Its originally marketed uses in people were for the treatment of intermittent claudication resulting from obstructed arteries in the limbs and the treatment of vascular dementia. It has also been used for a variety of other vascular or blood disorders, including the treatment of leg ulcers, stroke, high-altitude sickness, sickle cell disease, ischemic heart disease, ocular and hearing disorders with a vascular component, diabetic neuropathy, vasculitis, and the prevention of reocclusion after vascular surgery. More recently, it was discovered that PTX has anti-inflammatory properties via its ability to inhibit tumor necrosis factor-a (TNF-a), interleukin, and interferon-g synthesis and T cell and natural killer cell cytotoxicity, and its ability to reduce superoxide radical formation and granular release of enzymes by neutrophils. Along with its anti-inflammatory properties, inhibition of phosphodiesterase also induces bronchodilation and thus it may serve a role in the treatment of chronic inflammatory bronchoconstrictive diseases. Lastly, PTX has been shown to be a potent inhibitor of connective tissue and fibroblast growth factor and thus can deter the development of fibrosis in such diseases as alcoholic and nonalcoholic steatohepatitis, tubulointerstitial nephritis, endometriosis, Peyronie's disease, and radiation-induced fibrosis.