Temporal aspects of laminar gene expression during the developmental stages of equine laminitis

The results of recent studies indicate that inflammatory responses occurring in the early stages of equine laminitis lead to downstream events that eventually result in failure of the bond between the hoof wall and the distal phalanx. In order to gain further insights into the molecular mechanisms involved in the development of laminitis, an equine-specific cDNA microarray consisting of transcripts for more that 3000 genes was used to assess temporal changes in gene expression in laminar tissues at 1.5, 3 and 12 h after administration of either a laminitis-inducing agent (black walnut heartwood extract; BWHE) or an equal volume of water (control). As early as 1.5 h after BWHE administration, pro-inflammatory genes associated with leukocyte activation and emigration, including MCP-3/CCL7, MCP-1/CCL2, IP-10/CXCL10 and ICAM-1 were up-regulated. At both 1.5 and 3h after administration of BWHE, expression of B-cell specific transcripts (e.g., Ig-gamma 3, Ig-gamma 1 and lambda-light chain) were decreased in the laminar tissues. At the onset of Obel grade 1 lameness in horses administered BWHE, other genes involved in inflammatory processes (e.g., serum amyloid A, calgranulin C and NFAT-activation molecule 1), regulation of inflammation (e.g., inter-alpha-trypsin inhibitor, BiP/GRP78 [Ig binding protein], L-plastin, serpin and nexin-1), antioxidant responses (e.g., superoxide dismutase), matrix turnover (e.g., MMP-9 and TIMP-1), and anti-microbial responses (e.g., serotransferrin, beta-defensin-1 and elafin) were up-regulated. These results provide convincing evidence that genes associated with inflammation, activation and extravasation of leukocytes, antimicrobial activities, and destruction of the lamellar basement membrane are induced during the early stages of development of laminitis in response to administration of BWHE.Erik Noschka, Michel L. Vandenplas, David J. Hurley, James N. Moor

Evaluation of the possible role of prostaglandin F(2)alpha in laminitis induced in horses by nasogastric administration of black walnut heartwood extract

Obective—To provide insights into the role of prostaglandin F₂α (PGF₂α) in the developmental stages of laminitis induced in horses by ingestion of black walnut heartwood extract (BWHE). Sample Population—10 adult mixed-breed horses. Procedures—Horses were separated into 2 groups and were euthanatized at 12 hours after placebo (water) administration (control horses) or after BWHE administration and development of Obel grade 1 laminitis. Blood samples were obtained to determine plasma PGF2α concentrations hourly for the first 4 hours and subsequently every 2 hours after substance administration. Laminar arteries and veins were isolated, and responses to increasing concentrations of PGF₂α were measured before and after preincubation of blood vessels with prostanoid and thromboxane receptor antagonists SQ 29,548, SC-19220, and AH 6809. Results—Plasma PGF₂α concentrations increased in horses given BWHE; the WBC count decreased concurrently. In control horses, PGF₂α was a potent contractile agonist for laminar veins but not for laminar arteries. In horses given BWHE, PGF₂α was similarly selective for laminar veins; however, the magnitude of PGF2α-induced venoconstriction was less than that in control horses. After preincubation with SQ 29,548, laminar veins from control horses responded to PGF₂α with a small degree of dilation, whereas laminar veins from horses given BWHE did not. Conclusions and Clinical Relevance—PGF₂α may play a role in the inflammatory and vascular dysfunction associated with the prodromal stages of laminitis. Prostanoids such as PGF₂α may be viable targets for the prevention of acute laminitis in horses.Erik Noschka, James N. Moore, John F. Peroni, Tristan H. Lewis, Stephen J. Lewis, Tom P. Robertso

Comparison of characteristics and enzymatic products of leukocytes in the skin and laminar tissues of horses administered black walnut heartwood extract or lipopolysaccharide

Objective—To compare characteristics and enzymatic products of leukocytes detected in the skin and laminar tissues of horses administered black walnut heartwood extract (BWHE) and horses administered purified lipopolysaccharide (LPS). Animals—25 healthy 5- to 15-year-old horses. Procedures—Horses were randomly assigned to receive LPS (20 ng of O55:B5 Escherichia coli endotoxin/kg; n = 5) IV or 6 L of BWHE (10) or water (control group; 10) via nasogastric intubation. Horses were euthanatized 12 hours after treatment or at onset of Obel grade 1 lameness. Laminar tissue samples and skin samples from the middle region of the neck were harvested at the time of euthanasia. Leukocyte emigration (determined via CD13 immunohistochemical analysis) and matrix metalloproteinase (MMP)-2 and MMP-9 gene expressions and activities (determined via reverse transcription PCR assay and gelatin zymography, respectively) were measured in skin and laminar tissue samples. Results—Tissues of horses receiving BWHE contained significantly higher numbers of CD13-positive cells and increased MMP-9 gene expression and activity, compared with findings in the other 2 groups. Values for laminar tissue and skin from LPS-treated horses were not increased, compared with findings in the control group, in any experiment. Conclusions and Clinical Relevance—Results indicated that BWHE administration causes increases in CD13-positive leukocyte numbers and MMP-9 expression and activity in laminar tissue and skin in horses; similar effects were not detected following LPS administration. Leukocyte emigration in horses with experimentally induced endotoxemia and in horses administered BWHE differed markedly, thereby providing additional evidence that the development of laminitis involves more complex mechanisms than endotoxemia-induced leukocyte activation alone.Laura M. Riggs, Thomas M. Krunkosky, Erik Noschka, Lindsay A. Boozer, James N. Moore, Thomas P. Robertso

Thromboxane and isoprostanes as inflammatory and vasoactive mediators in black walnut heartwood extract induced equine laminitis

Inflammation and vascular dysfunction occur concurrently during the prodromal stages of equine laminitis. The aim of this study was to provide insights into the role that thromboxane and isoprostanes may play in the development of black walnut heartwood extract (BWHE)-induced laminitis. Horses were divided into two groups, either control or BWHE-administered horses. Plasma concentrations of thromboxane increased transiently after administration of BWHE and coincided with the nadir in white blood cell counts, whereas plasma concentrations of iso-prostaglandin PGF(2alpha) (iso-PGF(2alpha)) did not change in either group. At 12h (for the control group) or Obel grade 1 laminitis (for the BWHE group) the horses were euthanized and laminar tissue collected. Laminar arteries and veins were used in functional studies with vasoconstrictor substances and tissue samples were used for the determination of laminar iso-PGF(2alpha) concentrations. Laminar tissue concentrations of iso-PGF(2alpha) were significantly greater in BWHE horses when compared to control horses. In parallel studies concentrations of iso-PGF(2alpha) in laminar tissue samples obtained 1.5 and 3h after administration of BWHE were indistinguishable from those for control horses at 3 or 12h after administration of an equal volume of water. Laminar vessel constrictor responses to either a thromboxane mimetic (U46619), iso-prostaglandin PGE(2) (iso-PGE(2)) or iso-PGF(2alpha) were determined using small vessel myographs. In some vessels, the effects of putative prostanoid and thromboxane receptor antagonists, SQ 29,548, SC-19220 and AH 6809, upon contractile responses were determined. In control horses, U46619, iso-PGF(2alpha) and iso-PGE(2) more potently and efficaciously constricted laminar veins when compared to laminar arteries. Responses of laminar veins from BWHE horses to iso-PGE(2) were similar to those of laminar veins from control horses, whereas iso-PGF(2alpha) elicited significantly greater responses in laminar veins from BWHE horses when compared to controls. In contrast, responses to U46619 were smaller in laminar veins isolated from BWHE horses when compared to those in laminar veins from control horses. In the presence of SQ 29,548, iso-PGF(2alpha) elicited a small dilation in laminar veins from control horses, which was not apparent in laminar veins from BWHE horses. These results are consistent with both systemic and local inflammatory events occurring during the prodromal stages of BWHE-induced laminitis. Because laminar veins are sensitive to thromboxane and isoprostanes, these substances may act as conduits between the inflammatory and vascular events occurring in laminitis and may be therapeutic targets for this crippling condition.Erik Noschka, James N. Moore, John F. Peroni, Stephen J. Lewis, Jason D. Morrow and Tom P. Robertsonhttp://www.elsevier.com/wps/find/journaldescription.cws_home/503319/description#descriptio

Evaluation of activation of protein kinase C during agonist-induced constriction of veins isolated from the laminar dermis of horses

Objective—To determine the effects of the protein kinase C (PKC) inhibitor, Ro-31-8220, on agonist-induced constriction of laminar arteries and veins obtained from horses. Sample Population—Laminar arteries and veins obtained from 8 adult mixed-breed horses. Procedures—Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were then obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine, prostaglandin F2α, and endothelin-1. All responses were measured with or without the addition of Ro-31-8220 (3μM). Results—Laminar veins were more sensitive to vasoconstrictor agonists than laminar arteries, and incubation of laminar veins with Ro-31-8220 resulted in significantly smaller agonist-induced contractile responses for all agonists tested. In contrast, Ro-31-8220 had no effect on agonist-induced contractile responses of laminar arteries. Conclusions and Clinical Relevance—Results of the study were consistent with activation of PKC being confined to agonist-induced contraction of laminar veins isolated from the laminar dermis of horses. Consequently, the possible involvement of PKC in the venoconstriction observed during the development of laminitis is worthy of further investigation.Tom P. Robertson, James N. Moore, Erik Noschka, Tristan H. Lewis, Stephen J. Lewis, John F. Peron

Effect of head and neck position on upper airway function in standardbred racehorses

Abstract onlyJohnson K, Noschka E, Allen K, Tilbrook A, Ryan T, Franklin

Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis

Equine laminitis is a crippling condition associated with a variety of systemic diseases. Although it is apparent that the prodromal stages of laminitis involve microvascular dysfunction, little is known regarding the physiology of this vasculature. The aim of the present study was to determine the relative responses of equine laminar arteries and veins to the vasoconstrictor agonists phenylephrine (1 nM–10 µM), 5-HT (1 nM–10 µM), PGF2α (1 nM–100 µM), and endothelin-1 (1 pM–1 µM). We have determined that laminar veins were more sensitive, with respect to the concentration of agonist required to initiate a contractile response and to achieve EC50, for all agonists tested. EC50 values, for veins and arteries, respectively, were 84 ± 7 vs. 688 ± 42 nM for phenylephrine, 35 ± 6 vs. 224 ± 13 nM for 5-HT, 496 ± 43 nM vs. 3.0 ± 0.6 µM for PGF2α, and 467 ± 38 pM vs. 70.6 ± 6.4 nM for endothelin-1. Moreover, when expressed as a percentage of the response to a depolarizing stimulus (80 mM potassium), the maximal contractile response of laminar veins exceeded that for the laminar arteries for each agonist. These results indicate that there may be a predisposition for venoconstriction within the vasculature of the equine digit. While this physiological predisposition for venoconstriction may be important in the regulation of blood flow during exercise, it also may help to explain why laminitis can result from a variety of pathological systemic conditions.John F. Peroni, James N. Moore, Erik Noschka, Megan E. Grafton, Maria Aceves-Avila, Stephen J. Lewis, and Tom P. Robertso