• 1. Borena BM, Martens A, Broeckx SY, et al. Regenerative skin wound healing in mammals: state-of-the-art on growth factor and stem cell based treatments. Cell Physiol Biochem 2015;36:123.

    • Search Google Scholar
    • Export Citation
  • 2. Theoret C. Physiology of wound healing. In: Theoret C, Schumacher J, eds. Equine wound management. 3rd ed. Ames, Iowa: John Wiley & Sons Inc, 2017;113.

    • Search Google Scholar
    • Export Citation
  • 3. Grice EA, Segre JA. Interaction of microbiome and the innate immune response in chronic wounds. Adv Exp Med Biol 2012;946:5568.

  • 4. Maher M, Kuebelbeck L. Nonhealing wounds of the equine limb. Vet Clin North Am Equine Pract 2018;34:539555.

  • 5. Lepault E, Celeste C, Dore M, et al. Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse. Wound Repair Regen 2005;13:520529.

    • Search Google Scholar
    • Export Citation
  • 6. Adam EN, Southwood LL. Surgical and traumatic wound infections, cellulitis, and myositis in horses. Vet Clin North Am Equine 2006;22:335361.

    • Search Google Scholar
    • Export Citation
  • 7. Sørensen MA, Pedersen LJ, Bundgaard L, et al. Regional disturbances in metabolism and blood flow in equine limb wounds healing with formation of exuberant granulation tissue. Wound Repair Regen 2014;22:647653.

    • Search Google Scholar
    • Export Citation
  • 8. Celeste CJ, Deschene K, Riley CB, et al. Regional differences in wound oxygenation during normal healing in an equine model of cutaneous fibroproliferative disorder. Wound Repair Regen 2011;19:8997.

    • Search Google Scholar
    • Export Citation
  • 9. Jørgensen E, Bay L, Bjarnsholt T, et al. The occurrence of biofilm in an equine experimental wound model of healing by secondary intention. Vet Microbiol 2017;204:9095.

    • Search Google Scholar
    • Export Citation
  • 10. Bundgaard L, Bendixen E, Sorensen MA, et al. A selected reaction monitoring-based analysis of acute phase proteins in interstitial fluids from experimental equine wounds healing by secondary intention. Wound Repair Regen 2016;24:525532.

    • Search Google Scholar
    • Export Citation
  • 11. Wilmink JM, Van Weeren PR, Stolk PWT, et al. Differences in second-intention wound healing between horses and ponies: histological aspects. Equine Vet J 1999;31:6167.

    • Search Google Scholar
    • Export Citation
  • 12. Wilmink JM, Veenman JN, van den Boom R, et al. Differences in polymorphonucleocyte function and local inflammatory response between horses and ponies. Equine Vet J 2003;35:561569.

    • Search Google Scholar
    • Export Citation
  • 13. Mustoe T. Understanding chronic wounds: a unifying hypothesis on their pathogenesis and implications for therapy. Am J Surg 2004;187:65S70S.

    • Search Google Scholar
    • Export Citation
  • 14. Schultz G, Bjarnsholt T, James GA, et al. Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds. Wound Repair Regen 2017;25:744757.

    • Search Google Scholar
    • Export Citation
  • 15. Freeman K, Woods E, Welsby S, et al. Biofilm evidence and the microbial diversity of horse wounds. Can J Microbiol 2009;55:197202.

  • 16. Jørgensen E, Bay L, Skovgaard LT, et al. An equine wound model to study effects of bacterial aggregates on wound healing. Adv Wound Care (New Rochelle) 2019;8:487498.

    • Search Google Scholar
    • Export Citation
  • 17. Bischofberger AS, Dart CM, Horadagoda N, et al. Effect of Manuka honey gel on the transforming growth factor β and β3 concentrations, bacterial counts and histomorphology of contaminated full-thickness skin wounds in equine distal limbs. Aust Vet J 2016;94:2734.

    • Search Google Scholar
    • Export Citation
  • 18. Harmon CCG, Hawkins JF, Li J, et al. Effects of topical application of silver sulfadiazine cream, triple antimicrobial ointment, or hyperosmolar nanoemulsion on wound healing, bacterial load, and exuberant granulation tissue formation in bandaged full-thickness equine skin wounds. Am J Vet Res 2017;78:638646.

    • Search Google Scholar
    • Export Citation
  • 19. Theoret CL, Barber SM, Moyana TN, et al. Expression of transforming growth factor β1, β3, and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax. Vet Surg 2001;30:269277.

    • Search Google Scholar
    • Export Citation
  • 20. Theoret CL, Olutoye OO, Parnell LKS, et al. Equine exuberant granulation tissue and human keloids: a comparative histopathologic study. Vet Surg 2013;42:783789.

    • Search Google Scholar
    • Export Citation
  • 21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 2001;25:402408.

    • Search Google Scholar
    • Export Citation
  • 22. Wilmink JM, Stolk PWT, Van Weeren PR, et al. Differences in second-intention wound healing between horses and ponies: macroscopic aspects. Equine Vet J 1999;31:5360.

    • Search Google Scholar
    • Export Citation
  • 23. Bischofberger AS, Dart CM, Perkins NR, et al. The effect of short- and long-term treatment with Manuka honey on second intention healing of contaminated and noncontaminated wounds on the distal aspect of the forelimbs in horses. Vet Surg 2013;42:154160.

    • Search Google Scholar
    • Export Citation
  • 24. Fazli M, Bjarnsholt T, Kirketerp-Møller K, et al. Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds. Wound Repair Regen 2011;19:387391.

    • Search Google Scholar
    • Export Citation
  • 25. Trøstrup H, Thomsen K, Christophersen LJ, et al. Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model. Wound Repair Regen 2013;21:292299.

    • Search Google Scholar
    • Export Citation
  • 26. Gurjala AN, Geringer MR, Seth AK, et al. Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing. Wound Repair Regen 2011;19:400410.

    • Search Google Scholar
    • Export Citation
  • 27. Zhao G, Usui ML, Underwood RA, et al. Time course study of delayed wound healing in a biofilm-challenged diabetic mouse model. Wound Repair Regen 2012;20:342352.

    • Search Google Scholar
    • Export Citation
  • 28. Pastar I, Nusbaum AG, Gil J, et al. Interactions of methicillin resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in polymicrobial wound infection. PLoS One 2013;8:e56846.

    • Search Google Scholar
    • Export Citation
  • 29. Seth AK, Geringer MR, Hong SJ, et al. Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model. PLoS One 2012;7:e42897.

    • Search Google Scholar
    • Export Citation
  • 30. Serra R, Grande R, Buffone G, et al. Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines. Int Wound J 2016;13:5358.

    • Search Google Scholar
    • Export Citation
  • 31. Jacobsen SA, Andersen PH. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses. Equine Vet Educ 2007;19:3846.

    • Search Google Scholar
    • Export Citation
  • 32. Wilmink JM, van Herten J, van Weeren PR, et al. Retrospective study of primary intention healing and sequestrum formation in horses compared to ponies under clinical circumstances. Equine Vet J 2002;34:270273.

    • Search Google Scholar
    • Export Citation
  • 33. Jun JI, Kim KH, Lau LF. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing. Nat Commun 2015;6:7386.

    • Search Google Scholar
    • Export Citation
  • 34. Jun JI, Lau LF. Resolution of organ fibrosis. J Clin Invest 2018;128:97107.

  • 35. Babic AM, Kireeva ML, Kolesnikova TV, et al. CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Natl Acad Sci U S A 1998;95:63556360.

    • Search Google Scholar
    • Export Citation
  • 36. Roy S, Patel D, Khanna S, et al. Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue. Proc Natl Acad Sci U S A 2007;104:1447214477.

    • Search Google Scholar
    • Export Citation
  • 37. Jun JI, Lau LF. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 2010;12:676685.

    • Search Google Scholar
    • Export Citation
  • 38. Wetzler C, Kampfer H, Stallmeyer B, et al. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 2000;115:245253.

    • Search Google Scholar
    • Export Citation
  • 39. Karam RA, Rezk NA, Abdel Rahman TM, et al. Effect of negative pressure wound therapy on molecular markers in diabetic foot ulcers. Gene 2018;667:5661.

    • Search Google Scholar
    • Export Citation
  • 40. Jiang L, Dai Y, Cui F, et al. Expression of cytokines, growth factors and apoptosis-related signal molecules in chronic pressure ulcer wounds healing. Spinal Cord 2014;52:145151.

    • Search Google Scholar
    • Export Citation
  • 41. Wang Y, Dai YL, Piao JL, et al. The expressions and functions of inflammatory cytokines, growth factors and apoptosis factors in the late stage of pressure ulcer chronic wounds [in Chinese]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2017;33:181184.

    • Search Google Scholar
    • Export Citation
  • 42. Iqbal J, Bird JL, Hollander AP, et al. Effect of matrix depleting agents on the expression of chondrocyte metabolism by equine chondrocytes. Res Vet Sci 2004;77:249256.

    • Search Google Scholar
    • Export Citation
  • 43. Deschene K, Céleste C, Boerboom D, et al. Constitutive expression of hypoxia-inducible factor-1 α in keratinocytes during the repair of skin wounds in horses. Wound Repair Regen 2011;19:250259.

    • Search Google Scholar
    • Export Citation
  • 44. Dahlgren LA, Mohammed HO, Nixon AJ. Temporal expression of growth factors and matrix molecules in healing tendon lesions. J Orthop Res 2005;23:8492.

    • Search Google Scholar
    • Export Citation

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Histologic changes and gene expression patterns in biopsy specimens from bacteria-inoculated and noninoculated excisional body and limb wounds in horses healing by second intention

Elin J⊘rgensen DVM, PhD1, Freja B. Hjerpe DVM1, Hans P. Hougen MD, DVM, Dr Med Sci2, Thomas Bjarnsholt PhD, Dr Med Sci3,4, Lise C. Berg DVM, PhD1, and Stine Jacobsen DVM, PhD1
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  • 1 1Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark.
  • | 2 2Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark.
  • | 3 3Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark.
  • | 4 4Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.

Abstract

OBJECTIVE

To evaluate histologic changes and gene expression patterns in body and limb wounds in horses in response to bacterial inoculation.

SAMPLE

Wound biopsy specimens from 6 horses collected on days 7, 14, 21, and 27 after excisional wounds (20 wounds/horse) were created over the metacarpal and metatarsal region and lateral thoracic region (body) and then inoculated or not inoculated on day 4 with Staphylococcus aureus and Pseudomonas aeruginosa.

PROCEDURES

Specimens were histologically scored for the amount of inflammation, edema, angiogenesis, fibrosis organization, and epithelialization. Quantitative PCR assays were performed to quantify gene expression of 10 inflammatory, proteolytic, fibrotic, and hypoxia-related markers involved in wound healing.

RESULTS

Except for gene expression of interleukin-6 on day 27 and tumor necrosis factor-α on day 14, bacterial inoculation had no significant effect on histologic scores and gene expression. Gene expression of interleukin-1β and −6, serum amyloid A, and matrix metalloproteinase-9 was higher in limb wounds versus body wounds by day 27. Gene expression of cellular communication network factor 1 was higher in limb wounds versus body wounds throughout the observation period.

CONCLUSIONS AND CLINICAL RELEVANCE

The lack of clear markers of wound infection in this study reflected well-known difficulties in detecting wound infections in horses. Changes consistent with protracted inflammation were evident in limb wounds, and gene expression patterns of limb wounds shared similarities with those of chronic wounds in humans. Cellular communication network factor warrants further investigation and may be useful in elucidating the mechanisms underlying poor limb wound healing in horses.

Abstract

OBJECTIVE

To evaluate histologic changes and gene expression patterns in body and limb wounds in horses in response to bacterial inoculation.

SAMPLE

Wound biopsy specimens from 6 horses collected on days 7, 14, 21, and 27 after excisional wounds (20 wounds/horse) were created over the metacarpal and metatarsal region and lateral thoracic region (body) and then inoculated or not inoculated on day 4 with Staphylococcus aureus and Pseudomonas aeruginosa.

PROCEDURES

Specimens were histologically scored for the amount of inflammation, edema, angiogenesis, fibrosis organization, and epithelialization. Quantitative PCR assays were performed to quantify gene expression of 10 inflammatory, proteolytic, fibrotic, and hypoxia-related markers involved in wound healing.

RESULTS

Except for gene expression of interleukin-6 on day 27 and tumor necrosis factor-α on day 14, bacterial inoculation had no significant effect on histologic scores and gene expression. Gene expression of interleukin-1β and −6, serum amyloid A, and matrix metalloproteinase-9 was higher in limb wounds versus body wounds by day 27. Gene expression of cellular communication network factor 1 was higher in limb wounds versus body wounds throughout the observation period.

CONCLUSIONS AND CLINICAL RELEVANCE

The lack of clear markers of wound infection in this study reflected well-known difficulties in detecting wound infections in horses. Changes consistent with protracted inflammation were evident in limb wounds, and gene expression patterns of limb wounds shared similarities with those of chronic wounds in humans. Cellular communication network factor warrants further investigation and may be useful in elucidating the mechanisms underlying poor limb wound healing in horses.

Supplementary Materials

    • Supplementary Table 1 (PDF 15 kb)
    • Supplementary Table 2 (PDF 15 kb)
    • Supplementary Table 3 (PDF 15 kb)
    • Supplementary Table 4 (PDF 15 kb)
    • Supplementary Table 5 (PDF 23 kb)

Contributor Notes

Address correspondence to Dr. J⊘rgensen (elin.jorgensen@sund.ku.dk).