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Structural changes in the dermal and epidermal laminae of horses with starch gruel-induced laminitis
Abstracts / Journal of Equine Veterinary Science 31 (2011) 562-609
snap-frozen tissue samples. Using formalin-fixed tissues, MAC387 immunohistochemistry was performed to determine the presence and magnitude of leukocyte migration. Results: Significant increases in pro-inflammatory mediators were present in the liver, lung, and kidney. Hepatic tissue showed the earliest response, with significant increases in mRNA concentrations of IL-1b, CXCL8, IL-10, and TNFa in the DEV horses when compared with controls (p < .05). Increases in these inflammatory mediators, along with COX-2, were also present in the liver at the OG-1 stage. However, only increases in mRNA concentrations of IL-1b were present in all 3 tissues in the OG-1 horses when compared with controls. Increased leukocyte numbers were present in the lung after CHO administration. Discussion: Although no significant differences in leukocyte counts were seen, the liver underwent the greatest inflammatory signaling of the organs evaluated, most likely reflecting its function in processing toxins from the gastrointestinal tract. The inflammatory response seen in all of these organs differed in magnitude and profile when compared with the previously studied main target organ of CHO-induced SIRS, the laminae. Greater increases in cytokine responses are documented in the laminae after CHO administration, without increases in gene expression of TNFa and IL-10. In addition, a large increase in IL-6 mRNA concentration is documented in the laminae at OG-1, but not in the tissues evaluated in this study. Conclusions and Clinical Relevance: The increased expression of inflammatory mediators in the liver, lung, and kidney after carbohydrate overload is much less than what has been reported for the laminae, suggesting that although inflammation is present, it is not sufficient to result in complete organ failure as is commonly seen in the laminae.
Structural changes in the dermal and epidermal laminae of horses with starch gruel-induced laminitis Erica A. Pawlak 1, Le Wang 1, Philip J. Johnson 2, James K. Belknap 3, Dominique Alfandari 1, and Samuel J. Black 1 1 University of Massachusetts, Amherst, MA, 2 University of Missouri, College of Veterinary Medicine, Columbia, MO, 3 The Ohio State University, College of Veterinary Medicine, Columbus, OH Take-Home Message: Few or no signs of structural damage are evident in the digital laminae of horses with starch gruel-induced laminitis at the onset of Obel grade-1 lameness. However, by the onset of Obel grade-3 lameness there are multiple signs of pathology in the secondary dermal and epidermal laminae. Introduction: The microstructure of the laminae in the equine hoof has been described. The digital laminae are composed of epidermal and dermal layers separated by a basement membrane (BM) that is readily detected by a pan-laminin stain. Secondary epidermal laminae abut the BM and are composed of a sheet of basal epithelial cells that contain aggrecan, versican, and
567
hyaluronan, and supra-basal keratinocytes that contain aggrecan and hyaluronan but no versican. Basal epithelial cells and suprabasal keratinocytes are contiguous and have little or no extracellular matrix. The secondary epidermal laminae contain cellular elements (fibroblasts and mononuclear cells) as well as microvasculature defined by a laminin BM and surrounded by a packed extracellular matrix containing collagen I, collagen III, fibronectin, chondroitin sulfate, and keratin sulfate proteoglycans. The purpose of this study was to investigate the development and localization of pathologic changes in these tissues that are associated with structural failure of the laminae in horses with acute laminitis induced by starch gruel. Materials and Methods: Acute laminitis was induced in horses by the nasogastric administration of starch gruel. Extracellular matrix proteins (collagen I, collagen III, fibronectin, laminin), proteoglycans (aggrecan and versican), glycosaminoglycans (chondroitin sulfate, keratin sulfate), and hyaluronan were localized using indirect immunofluorescent microscopy performed on thin sections of frozen OCT-embedded laminae collected from a front foot of each horse. The laminae were examined histologically using thin sections of alcohol-fixed tissue stained with hematoxylin and eosin. The tissues were examined at two time points: at the onset of Obel grade-1 laminitis (OG-1; lameness noticeable only at the trot) and at the onset of Obel grade-3 laminitis (OG-3; stiff, stilted gait at the walk, reluctance to move). Results: When compared with healthy horses, few or no differences were observed in the structural organization of the laminae in horses with OG-1 laminitis, although the average crosssectional area of individual secondary dermal laminae was reduced. In contrast, there were dramatic changes in the laminae from horses with OG-3 laminitis. Multifocal gaps lacking collagen, fibronectin, and glycosaminoglycans appeared in the secondary dermal laminae and there was a significant loss of microvasculature in the region. In addition, basal epithelial cells were detached from the BM and from each other, particularly in the crypt regions of the secondary epidermal laminae. Discussion: Gross changes in laminar structure occur after the onset of OG-1 laminitis. By the onset of OG-3 laminitis, there is extensive loss of microvasculature and multifocal disruption of extracellular matrix in the secondary dermal laminae, along with loss of versican from basal epithelial cells of the secondary epidermal laminae and initial separation of basal epithelial cells from the basement membranedall of which presage structural failure of the laminae. Parallel studies (reported in a companion abstract) implicate ADAMTS-4, MMP-1, MMP-12, MMP-13, and epithelial-tomesenchymal cell transition in these pathologic processes. Conclusions and Clinical Relevance: Pathologic processes that cause structural failure of the laminae develop between OG-1 and OG-3 lameness. There is a need to more finely track the onset of these pathologic changes and to identify associated changes in laminar metalloproteinase expression in order to determine putative cause-and-effect relationships. These are important steps toward the design and implementation of therapeutic interventions that will halt disease progression.
Scientific – Metabolic and Endocrinopathic Part 1 Phenotypic characterization of equine metabolic syndrome
2 3
Nichol Schultz 1, Molly McCue 1, Nicholas Frank 2, and Raymond Geor 3 1 College of Veterinary Medicine, University of Minnesota,
Cummings School of Veterinary Medicine, Tufts University, College of Veterinary Medicine, Michigan State University
Take-Home Message: There is a strong correlation between body condition score (BCS) and serum insulin concentration,
snap-frozen tissue samples. Using formalin-fixed tissues, MAC387 immunohistochemistry was performed to determine the presence and magnitude of leukocyte migration. Results: Significant increases in pro-inflammatory mediators were present in the liver, lung, and kidney. Hepatic tissue showed the earliest response, with significant increases in mRNA concentrations of IL-1b, CXCL8, IL-10, and TNFa in the DEV horses when compared with controls (p < .05). Increases in these inflammatory mediators, along with COX-2, were also present in the liver at the OG-1 stage. However, only increases in mRNA concentrations of IL-1b were present in all 3 tissues in the OG-1 horses when compared with controls. Increased leukocyte numbers were present in the lung after CHO administration. Discussion: Although no significant differences in leukocyte counts were seen, the liver underwent the greatest inflammatory signaling of the organs evaluated, most likely reflecting its function in processing toxins from the gastrointestinal tract. The inflammatory response seen in all of these organs differed in magnitude and profile when compared with the previously studied main target organ of CHO-induced SIRS, the laminae. Greater increases in cytokine responses are documented in the laminae after CHO administration, without increases in gene expression of TNFa and IL-10. In addition, a large increase in IL-6 mRNA concentration is documented in the laminae at OG-1, but not in the tissues evaluated in this study. Conclusions and Clinical Relevance: The increased expression of inflammatory mediators in the liver, lung, and kidney after carbohydrate overload is much less than what has been reported for the laminae, suggesting that although inflammation is present, it is not sufficient to result in complete organ failure as is commonly seen in the laminae.
Structural changes in the dermal and epidermal laminae of horses with starch gruel-induced laminitis Erica A. Pawlak 1, Le Wang 1, Philip J. Johnson 2, James K. Belknap 3, Dominique Alfandari 1, and Samuel J. Black 1 1 University of Massachusetts, Amherst, MA, 2 University of Missouri, College of Veterinary Medicine, Columbia, MO, 3 The Ohio State University, College of Veterinary Medicine, Columbus, OH Take-Home Message: Few or no signs of structural damage are evident in the digital laminae of horses with starch gruel-induced laminitis at the onset of Obel grade-1 lameness. However, by the onset of Obel grade-3 lameness there are multiple signs of pathology in the secondary dermal and epidermal laminae. Introduction: The microstructure of the laminae in the equine hoof has been described. The digital laminae are composed of epidermal and dermal layers separated by a basement membrane (BM) that is readily detected by a pan-laminin stain. Secondary epidermal laminae abut the BM and are composed of a sheet of basal epithelial cells that contain aggrecan, versican, and
567
hyaluronan, and supra-basal keratinocytes that contain aggrecan and hyaluronan but no versican. Basal epithelial cells and suprabasal keratinocytes are contiguous and have little or no extracellular matrix. The secondary epidermal laminae contain cellular elements (fibroblasts and mononuclear cells) as well as microvasculature defined by a laminin BM and surrounded by a packed extracellular matrix containing collagen I, collagen III, fibronectin, chondroitin sulfate, and keratin sulfate proteoglycans. The purpose of this study was to investigate the development and localization of pathologic changes in these tissues that are associated with structural failure of the laminae in horses with acute laminitis induced by starch gruel. Materials and Methods: Acute laminitis was induced in horses by the nasogastric administration of starch gruel. Extracellular matrix proteins (collagen I, collagen III, fibronectin, laminin), proteoglycans (aggrecan and versican), glycosaminoglycans (chondroitin sulfate, keratin sulfate), and hyaluronan were localized using indirect immunofluorescent microscopy performed on thin sections of frozen OCT-embedded laminae collected from a front foot of each horse. The laminae were examined histologically using thin sections of alcohol-fixed tissue stained with hematoxylin and eosin. The tissues were examined at two time points: at the onset of Obel grade-1 laminitis (OG-1; lameness noticeable only at the trot) and at the onset of Obel grade-3 laminitis (OG-3; stiff, stilted gait at the walk, reluctance to move). Results: When compared with healthy horses, few or no differences were observed in the structural organization of the laminae in horses with OG-1 laminitis, although the average crosssectional area of individual secondary dermal laminae was reduced. In contrast, there were dramatic changes in the laminae from horses with OG-3 laminitis. Multifocal gaps lacking collagen, fibronectin, and glycosaminoglycans appeared in the secondary dermal laminae and there was a significant loss of microvasculature in the region. In addition, basal epithelial cells were detached from the BM and from each other, particularly in the crypt regions of the secondary epidermal laminae. Discussion: Gross changes in laminar structure occur after the onset of OG-1 laminitis. By the onset of OG-3 laminitis, there is extensive loss of microvasculature and multifocal disruption of extracellular matrix in the secondary dermal laminae, along with loss of versican from basal epithelial cells of the secondary epidermal laminae and initial separation of basal epithelial cells from the basement membranedall of which presage structural failure of the laminae. Parallel studies (reported in a companion abstract) implicate ADAMTS-4, MMP-1, MMP-12, MMP-13, and epithelial-tomesenchymal cell transition in these pathologic processes. Conclusions and Clinical Relevance: Pathologic processes that cause structural failure of the laminae develop between OG-1 and OG-3 lameness. There is a need to more finely track the onset of these pathologic changes and to identify associated changes in laminar metalloproteinase expression in order to determine putative cause-and-effect relationships. These are important steps toward the design and implementation of therapeutic interventions that will halt disease progression.
Scientific – Metabolic and Endocrinopathic Part 1 Phenotypic characterization of equine metabolic syndrome
2 3
Nichol Schultz 1, Molly McCue 1, Nicholas Frank 2, and Raymond Geor 3 1 College of Veterinary Medicine, University of Minnesota,
Cummings School of Veterinary Medicine, Tufts University, College of Veterinary Medicine, Michigan State University
Take-Home Message: There is a strong correlation between body condition score (BCS) and serum insulin concentration,