• 1.

    True RG, Lowe JE, Heissen J, et al. Black walnut shavings as a cause of acute laminitis, in Proceedings. 24th Annu Meet Am Assoc Equine Pract 1978;511516.

    • Search Google Scholar
    • Export Citation
  • 2.

    True RG, Lowe JE. Induced juglone toxicosis in ponies and horses. Am J Vet Res 1980; 41:944945.

  • 3.

    Galey FD, Whiteley HE, Goetz TE, et al. Black walnut (Juglans nigra) toxicosis: a model for equine laminitis. J Comp Pathol 1991; 104:313326.

    • Search Google Scholar
    • Export Citation
  • 4.

    Thomsen ME, Davis EG, Rush BR. Black walnut induced laminitis. Vet Hum Toxicol 2000; 42:811.

  • 5.

    Hurley DJ, Parks RJ, Reber AJ, et al. Dynamic changes in circulating leukocytes during the induction of equine laminitis with black walnut extract. Vet Immunol Immunopathol 2006; 110:195206.

    • Search Google Scholar
    • Export Citation
  • 6.

    Fontaine GL, Belknap JK, Allen D, et al. Expression of interleukin-1beta in the digital laminae of horses in the prodromal stage of experimentally induced laminitis. Am J Vet Res 2001; 62:714720.

    • Search Google Scholar
    • Export Citation
  • 7.

    Black SJ, Lunn DP, Yin C, et al. Leukocyte emigration in the early stages of laminitis. Vet Immunol Immunopathol 2006; 109:161166.

  • 8.

    Riggs LM, Franck T, Moore JN, et al. Neutrophil myeloperoxidase measurements in plasma, laminar tissue, and skin of horses given black walnut extract. Am J Vet Res 2007; 68:8186.

    • Search Google Scholar
    • Export Citation
  • 9.

    Inbaraj JJ, Chignell CF. Cytotoxic actin of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 2004; 17:5562.

    • Search Google Scholar
    • Export Citation
  • 10.

    Zhang R, Hirsch O, Mohsen M, et al. Effects of nitroxide stable radicals on juglone cytotoxicity. Arch Biochem Biophys 1994; 312:385391.

  • 11.

    Hurley DJ, Hurley KAE, Galland KL, et al. Evaluation of the ability of aqueous black walnut extracts to induce the production of reactive oxygen species. Am J Vet Res 2011; 72:308317.

    • Search Google Scholar
    • Export Citation
  • 12.

    Parsons CS, Orsini JA, Krafty R, et al. Risk factors for development of acute laminitis in horses during hospitalization: 73 cases (1997–2004). J Am Vet Med Assoc 2007; 230:885889.

    • Search Google Scholar
    • Export Citation
  • 13.

    Tóth F, Frank N, Elliot SB, et al. Effects of an intravenous endotoxin challenge on glucose and insulin dynamics in horses. Am J Vet Res 2008; 69:8288.

    • Search Google Scholar
    • Export Citation
  • 14.

    Peroni JF, Harrison WE, Moore JN, et al. Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature. Equine Vet J 2005; 37:546551.

    • Search Google Scholar
    • Export Citation
  • 15.

    Belknap JK, Giguère S, Pettigrew A, et al. Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response. Equine Vet J 2007; 39:4247.

    • Search Google Scholar
    • Export Citation
  • 16.

    Waguespack RW, Kemppainen RJ, Cochran A, et al. Increased expression of MAIL, a cytokine-associated nuclear protein, in the prodromal stage of black walnut-induced laminitis. Equine Vet J 2004; 36:285291.

    • Search Google Scholar
    • Export Citation
  • 17.

    Loftus JP, Black SJ, Pettigrew A, et al. Early laminar events involving endothelial activation in horses with black walnut–induced laminitis. Am J Vet Res 2007; 68:12051211.

    • Search Google Scholar
    • Export Citation
  • 18.

    Loftus JP, Belknap JK, Stankiewicz KM, et al. Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black-walnut induced equine laminitis. Equine Vet J 2007; 39:4853.

    • Search Google Scholar
    • Export Citation
  • 19.

    Tilton RC, Van Kruiningen HJ, Kwasnik I, et al. Toxigenic Clostridium perfringens from a parvovirus-infected dog. J Clin Microbiol 1981; 14:697698.

    • Search Google Scholar
    • Export Citation
  • 20.

    Moore JN, Norton N, Barton MH, et al. Evaluation of rapid infusion of a phospholipid emulsion in experimental endotoxaemia in horses. Equine Vet J 2007; 39:243248.

    • Search Google Scholar
    • Export Citation
  • 21.

    Okano S, Hurley DJ, Vandenplas ML, et al. Effect of fetal bovine serum and heat-inactivated fetal bovine serum on expression of tissue factor by equine and canine mononuclear cells in vitro. Am J Vet Res 2006; 67:10201024.

    • Search Google Scholar
    • Export Citation
  • 22.

    Frohlich E, Samberger C, Kueznik T, et al. Cytotoxicity of nanoparticles independent from oxidative stress. J Toxicol Sci 2009; 34:363375.

    • Search Google Scholar
    • Export Citation
  • 23.

    Noschka E, Vandenplas ML, Hurley DJ, et al. Assessment of the dynamics of laminar gene expression during the developmental stages of equine laminitis. Vet Immunol Immunopathol 2009; 129:242253.

    • Search Google Scholar
    • Export Citation

Advertisement

Evaluation of the in vitro effects of aqueous black walnut extract on equine mononuclear cells

View More View Less
  • 1 Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 2 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 3 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 4 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

Abstract

Objective—To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects.

Sample—Mononuclear cells separated from blood samples from 8 horses.

Procedures—Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference.

Results—BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively.

Conclusions and Clinical Relevance—Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.

Abstract

Objective—To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects.

Sample—Mononuclear cells separated from blood samples from 8 horses.

Procedures—Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference.

Results—BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively.

Conclusions and Clinical Relevance—Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.

Contributor Notes

Supported by a grant from the American Quarter Horse Foundation and the White Fox Farm Research Fund.

Address correspondence to Dr. David Hurley (djhurley@uga.edu).