A porcine model for pathomorphological age assessment of surgically excised skin wounds

A porcine model for pathomorphological age assessment of surgically excised skin wounds A porcine model of second intention wound healing was established and gross and histological changes needed for accurate assessing the age of wounds were determined. Twenty‑ five pigs were anesthetized before incision of four wounds on each. The wounds were left to heal from 1 h to 35 days when the pigs were euthanized. In 14 pigs, biopsies were sampled from two wounds between days 2 and 18. By histological evaluation the following characteris‑ tics were found useful for determining the age of wounds: neutrophil:macrophage ratio, angiogenesis, hyperplasia of fibroblasts, presence of hemosiderophages and granulation tissue. The latter was present from day 4 (n = 8 wounds, 100%), but by gross evaluation it was not recognized until day 5 (n = 4 wounds, 100%). From day 4 to 10, the thick‑ ness of granulation tissue increased by 1.2 ± 2.4 mm/day. The thickness of collagen fibers within granulation tissue increased throughout the study period, and complete epithelization was reached from day 18. Keywords: Age of wounds, Experimental animal model, Pig, Skin, Ulceration, Veterinary forensic pathology Findings individually with bedding of straw and sawdust (Spanvall, Age assessment of skin wounds in pigs is often requested Denmark) and numbered 1–25 in the order they arrived in veterinary forensic pathology [1, 2]. Wounds in pigs to the facility (Table 1). Each pig was anesthetized (Additional file  1), treated are primarily caused by external trauma, and the age of with continuous intravascular infusion of fentanyl (Fen wounds submitted for forensic investigation ranges from - hours  and up to several months [3]. Methods to deter- tanyl 50  µg/mL, Fentanyl-Hameln; Hameln Pharma- mine the age of wounds have been investigated in por- ceuticals gmbh, Hameln, Germany), placed in sternal cine models [4–6]. However, in previous studies and recumbency and prepared for sterile surgery. Four sur- unlike wounds in veterinary forensic cases, these wounds gical areas of 2 × 2  cm (locations 1–4) were drawn on were treated with a epinephrine solution to obtain hemo- the back in the paravertebral area (Fig.  1). By incision, stasis or bandaged during healing [4–6]. The aim of the full thickness wounds (from the epidermis down to and present study was to identify hallmarks for forensic age including the subcutis) were established by removal of assessment of porcine wounds by assessing characteris- 4 cm skin and left to heal by second intention. Pigs 1–5 tics in experimental wounds during healing by second were kept anesthetized for 1–6  h and then euthanized, intention. while pigs 6–25 were transported to their pens to wake Twenty-five specific pathogen free (SPF) female up (Table 1). At 8 h intervals, pigs were given four injec- Yorkshire-Landrace crossbred pigs with body weights tions of buprenorphine (Temgesic, 0.3 mg/mL, Schering- of 23–34  kg were acclimatized for 1  week, housed Plough, NJ, USA). None of the pigs showed sign of pain as no increase  in respiratory rate, depression, reduced feed intake or mobility were observed. At different time *Correspondence: krisb@sund.ku.dk points, pigs 12–25 were anesthetized (Additional file  2) Department of Veterinary and Animal Sciences, Faculty of Health before two full thickness biopsies from each of the wound and Medical Sciences, University of Copenhagen, Ridebanevej 3, margins of locations 2 and 3 were sampled by incision 1870 Frederiksberg C, Denmark © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barington et al. Acta Vet Scand (2018) 60:33 Page 2 of 7 Table 1 Study overview Wound age Pig No. of wounds No. of wounds Histological Average Median Average Median N:M ratio biopsied sampled post- features neutrophil neutrophil score macrophage macrophage mortem relevant for age score + SD (range) score + SD score (range) determination 1 h 1 4 Clot 4.3 ± 1.7 4.5 (2–6) 1 ± 0 1 (1–1) 4:1 3 h 2, 3 8 5.9 ± 0.4 6 (5–6) 1.5 ± 0.5 1.5 (1–2) 4:1 6 h 4, 5 8 4.8 ± 0.9 5 (3–6) 1.8 ± 0.7 2 (3) 3:1 12 h 6, 7 8 Migration of epithe 5.1 ± 0.6 5 (4–6) 1.6 ± 0.5 2 (1–2) 3:1 lial cells 1 day 8, 9 8 5.4 ± 1.2 6 (3–6) 2.9 ± 1.0 2.5 (2–4) 2:1 2 days 10–13 4 8 Angiogenesis 4.8 ± 1.6 6 (2–6) 2.3 ± 0.5 2 (2–3) 2:1 3 days 14, 15 4 Fibroblasts 3.8 ± 0.5 4 (3–4) 3.5 ± 0.6 3.5 (3–4) 1:1 4 days 12, 13, 16, 17 4 4 Granulation tissue 3 ± 1.4 3 (1–5) 2.9 ± 0.6 3 (2–4) 1:1 5 days 18,19 4 2 ± 0.8 2 (1–3) 3.3 ± 0.5 3 (3–4) 1:2 6 days 14, 15 4 2.3 ± 1.0 2.5 (1–3) 3.3 ± 0.5 3 (3–4) 1:1 7 days 20, 21 4 1.8 ± 0.5 2 (1–2) 3.8 ± 0.5 4 (3–4) 1:2 8 days 16, 17 4 Hemosiderophages 2.8 ± 1.0 2.5 (2–4) 3.8 ± 0.5 4 (3–4) 1:1 10 days 18, 19 4 2.8 ± 2.1 2.5 (1–5) 3.8 ± 1.0 3.5 (3–5) 1:1 14 days 22, 23 4 1 ± 0 1 (1–1) 2 ± 0 2 (2–2) 1:2 18 days 20, 21, 24, 25 4 4 Complete epitheliza 1.1 ± 1.0 1 (0–3) 2 ± 0.5 2 (1–3) 1:2 tion 27 days 22, 23 4 0 0 2 ± 0 2 (2–2) – 35 days 24, 25 4 0 0 1.5 ± 0.6 1.5 (1–2) – Study overview: age of wounds; pigs; number of wounds (sampled by biopsy or post-mortem); first appearance of histological features relevant for age determination; average score ± standard deviation (SD) of neutrophils and macrophages; median score and range of neutrophils and macrophages and the neutrophil:macrophage (N:M) ratio Infiltration of neutrophils and macrophages was scored as: (0) absent, (1) < 10, (2) 10–20, (3) 21–50, (4) 51–100, (5) 101–200, (6) > 200 using a 40× objective and 10× ocular with FN 22 mm in one high power field (HPF) of 0.237 mm [9]. The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within areas 1–4 (Fig. 2a, b). From each wound, only the highest score (regardless of area) was registered. At each time point, the average neutrophil and macrophage score, the standard deviation, the median and the neutrophil:macrophage ratio were calculated N neutrophils, M macrophages Barington et al. Acta Vet Scand (2018) 60:33 Page 3 of 7 red and hemorrhagic connective tissue filling the wound bed and recorded as present or absent by gross inspec- tion [7]. In wounds that were biopsied, evaluations were only recorded until the day of biopsy sampling. Histological preparation of the tissue was done as previously described [8]. Sections stained with hematoxylin and eosin were evaluated in four areas (3 × 3  mm) depending on the age of the wounds and therefore not blinded (Fig. 2a, b). All registrations were carried out by a single veterinary pathologist. Infiltra - tion of neutrophils and macrophages was scored as: Fig. 1 Location of wounds 1–4, experimental pig 21. Each wound (0) absent, (1) < 10; (2) 10–20; (3) 21–50; (4) 51–100; was located 4 cm lateral to the spine and 4 cm cranial or caudal to (5) 101–200; (6) > 200 using a 40 × objective and 10× the last rib, respectively. Biopsies were taken from two wound edges ocular with FN 22  mm in one high power field (HPF) at locations 2 and 3 (red boxes) while the pigs were anesthetized. Wounds at location 1 and 4 were sampled post mortem of 0.237  mm [9]. The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within areas 1–4 (Fig.  2a, b). From each (Fig.  1 and Table  1). After 4  h, these pigs received an wound, only the highest score (regardless of area) was intramuscular injection of 0.1  mg/kg buprenorphine. registered, i.e. one score per wound. Similarly, when The pigs were euthanized from 1  h to 35  days after the two biopsies were sampled from one wound both biop- creation of the wounds with an overdose of intravenous sies were evaluated, but only the highest score was pentobarbital (Glostrup Apotek, Glostrup, Denmark) registered. At each time point, the average neutrophil (Table 1). and macrophage score, the standard deviation (SD), Following euthanasia, cross sections of the wounds the median and the neutrophil:macrophage ratio were that had not previously been biopsied were sampled for calculated. Hemorrhage, hyperplasia of fibroblasts and histology. In total, 100 wounds were evaluated, i.e. 28 endothelial cells and angiogenesis were registered as wounds were biopsied and 72 wounds were sampled after present or absent in areas 1–4 (Figs. 2a, b). Granulation euthanasia (Table 1). Granulation tissue was defined as a tissue was defined as fibroblasts and collagen arranged Fig. 2 Areas subjected to histological evaluation of biopsies (a) or cross sections of wounds (b), hematoxylin and eosin stain. a Histological evaluation of wounds aged 1 h to 10 days was carried out in areas 1–3 (3 mm × 3 mm). Area 1: at the wound edge, dermis. Area 2: at the wound edge, subcutis. Area 3: central in the wound bed. b Histological evaluation of wounds aged 14 to 35 days was carried out in area 4 (3 mm × 3 mm), i.e. central in the granulation tissue. The thickness of the granulation tissue was measured at the center of the wound (straight line) Barington et al. Acta Vet Scand (2018) 60:33 Page 4 of 7 Fig. 3 The average score ± standard deviation of neutrophils and Fig. 4 The average thickness ± standard deviation of granulation macrophages in wounds being from 1 h to 35 days tissue in wounds being from 4 to 35 days perpendicular to the new proliferating vessels (angio- where a clot had formed in the wound bed. Infiltration genesis) [7]. When present, the thickness of granulation of neutrophils around vessels and in the dermal and sub- tissue was measured at the center of the wound from cutaneous tissue was seen from 1  h to day 18 (90 of 92 the top of the wound bed (not including epidermis) to wounds, 98%). In comparison, a intravital reaction (i.e. the level of normal tissue components using the soft- active infiltration of leukocytes) is not found in porcine ware Delta Pix 2.3.5 (Smorum, Denmark) (Fig. 2b). Dif- wounds inflicted postmortem or seconds before death [8, ferences in the thickness of granulation tissue, due to 13]. The number of neutrophils peaked after 3 h to 2 days the location of the wound (location 1–4), were evalu- with a score greater than 4.5 (Fig. 3) (Additional file 4). ated by comparing locations 1 and 4 and locations 2 Infiltration of macrophages in the dermis and subcutis and 3 using the paired t test (P < 0.05). Moreover, at was seen in the wounds (n = 100 wounds, 100%) through- each time point the mean thickness of granulation tis- out the experimental period but peaked from day 7 to 10 sue and SD were calculated (Additional file 3 ). with a score greater than 3.5 (Fig.  3)  (Additional file  4). Epithelization was characterized as: (0) absent, (1) In accordance with this, macrophages peaked at day 7 in hyperplasia of epithelial basal cells, (2) migration of epi- wounds in a previous porcine excisional wound model thelial cells, and (3) full epithelization. [14]. From 8 to 35 days, hemosiderophages were present. Randomly selected sections from all time points were In comparison, hemosiderophages, which to the authors stained with Perl’s Prussian blue, Masson’s trichrome, and knowledge have not been noticed in porcine wounds Picrosirius red in order to confirm the presence of hemo - hitherto, have been reported in human wounds already at siderophages and visualization of collagen in the wound day 3 and appeared regularly from day 8 [15]. bed [10, 11]. The change in polarization color from green Based on the neutrophil:macrophage ratio, wounds to yellow to red in the aging wounds stained with Picro- could roughly be assessed as being from 1 to 3  h, 6 to sirius red is due to increasing thickness of collagen [11]. 12 h, 1 to 2 days or more than 3 days (Table 1). Contami- Immunohistochemistry based on polyclonal rabbit anti- nation of the wounds with bedding material was inevita- human von Willebrand Factor antibody (A0082, Agilent, ble and may have affected the inflammatory reaction [16]. USA) was used to confirm the presence of vessels [12]. However, in forensic cases, wounds are also subjected to The first appearance of the most important histologi - infection, foreign bodies and continuous trauma, which cal manifestations for determining the age of wounds is can affect the neutrophil:macrophage ratio [16]. Hyper - presented in Table  1. Hemostasis was present within 1 h plasia of endothelial cells, angiogenesis and hyperplasia Barington et al. Acta Vet Scand (2018) 60:33 Page 5 of 7 of fibroblast were observed in all wounds from 12  h, 2 and 3 days, respectively. Our observations are in accord- ance with previous records on experimental wounds in pigs, in which hyperplasia of fibroblasts was seen within 3  days [17]. Moreover, in forensic porcine wounds, we have previously predicted angiogenesis and hyperplasia of fibroblasts to begin at 16 h to 3 days [3]. Histologically, granulation tissue was present at day 4 (n = 8  wounds, 100%), but at gross evaluation it was not recognized until day 5 (n = 4  wounds, 100%). Granula- tion tissue thickness on days 4 to 7 varied from approxi- mately 2–6  mm and increased to nearly 10  mm on day 10 (growth rate: 1.2 ± 2.4  mm per day). However, from day 10 to 35 the thickness decreased by 0.3 ± 0.3 mm per day (Fig.  4)  (Additional file  3). In forensic cases of por- cine wounds, granulation tissue with thicknesses of 5, 15 and 27  mm have been used to estimate wound age as being 4–7  days, 8–28  days and > 28  days, respectively [3]. The decrease in granulation tissue thickness seen in the experimental wounds but apparently not in foren- sic cases is likely due to the initial depth of the lesion, accompanying infection, sequestration of necrotic tissue and sustained injury [16]. Therefore, the growth rate of granulation tissue up to 10 days should probably be seen as the maximum by which it can be formed. The wide SD of the average growth rate is likely due to inter pig vari- ation as no differences in granulation tissue thickness between locations 1 and 4 (P = 0.26) and locations 2 and 3 (P = 0.07) were found. Masson’s trichrome and Picrosirius red stains both confirmed the presence of newly formed collagen from day 4 (Fig.  5a). Gradually, more collagen was deposited throughout the experimental period (Figs. 5a–c). Hyperplasia and early migration of epithelial basal cells were seen from 12  h  (2/8 wounds, 25%) and complete epithelization was present in wounds being from 18 to 35  days old. This is in agreement with another porcine model based on 2 × 2 cm full thickness wounds, in which complete epithelization was achieved on day 28 [18]. Histological features of significant importance for assessing the age of porcine wounds were identi- fied (Table  1, Fig.  6). Age assessment of wounds with- Fig. 5 Porcine wound beds being 4 days (a), 10 days (b) and 27 days (c) old. a A scarce amount of blue stained collagen is present (arrows), out granulation tissue should be based on the ratio Masson’s trichrome stain. Inset: Green collagen fibers are visible under between neutrophils and macrophages and the pres- polarized light (arrows), Picrosirius red stain. b Massive amounts of ence of angiogenesis and fibroblast hyperplasia (Fig.  6). blue stained collagen are present (arrows), Masson’s trichrome stain. In wounds containing granulation tissue, the thickness Inset: Green, yellow and few red collagen fibers are visible in polarized of it was useful for wound age determination and the light (arrows), Picrosirius red stain. c Massive amounts of blue stained collagen are present (arrows), Masson’s trichrome stain. Inset: Yellow, neutrophil:macrophage ratio, deposition of collagen and red and a few green collagen fibers are visible in polarized light (arrows), Picrosirius red stain Barington et al. Acta Vet Scand (2018) 60:33 Page 6 of 7 Fig. 6 Decision diagram for determining the age of wounds based on gross and histological evaluation histology and immunohistochemistry. Moreover, we wish to thank Frederik presence of hemosiderophages can further support the Andersen and Dennis Brok for skilled technical assistance with photos and assessment (Fig. 6). figures. Additional files Competing interests The authors declare that they have no competing interests. Additional file 1. Sedation and general anesthesia for surgical incision of Availability of data wounds. All data generated or analyzed during this study are included in this published Additional file 2. Anesthesia and reversal of anesthesia for sampling of article and its additional files. biopsies. Consent for publication Additional file 3. Granulation tissue was measured at the center of the Not applicable. wound from the top of the wound bed (not including epidermis) and excluding underlying fat tissue and the fibrous fascia of the muscle. Ethics approval and consent to participate Additional file 4. Infiltration of neutrophils and macrophages was The study and the procedures were approved by the Danish Animal Inspec‑ scored as: (0) absent; (1) < 10; (2) 10–20; (3) 21–50; (4) 51–100; (5) 101–200; torate (2013–15–2934–00849) and were carried out in accordance with all (6) > 200 using a 40× objective and 10× ocular with FN 22 mm in one institutional, local and national guidelines. high power field (HPF) of 0.237 mm . The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within Funding areas 1–4 in the wounds (Fig. 2a, b). From each wound, only the highest The study was funded by University of Copenhagen, Denmark. The funding score was registered, i.e. one neutrophil score and one macrophage score source had no involvement in the experimental design, analysis and interpre‑ per wound. Location of the wound (1–4), wound age, neutrophil score tation of the results. and macrophage score are presented. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Abbreviations SPF: specific pathogen free, i.e. free from infection with Mycoplasma hyo - Received: 16 January 2018 Accepted: 19 May 2018 pneumoniae, Actinobacillus pleuropneumoniae serotype 1–10 and 12, Porcine Reproductive Respiratory Syndrome, Brachyspira hyodysenteriae, Pasteurella multocida, Sarcoptes Scabiei var. Suis. and Haematopinus suis. Authors’ contributions KB and HEJ contributed to the design of the study. All authors contributed to References the experimental work and the gross and histological evaluation of the tissue. 1. de Siqueira A, Cuevas SEC, Salvagni FA, Maiorka PC. Forensic veterinary In addition, KB performed the statistical analyses and drafted the manuscript. pathology. Vet Pathol. 2016;53:979–87. All authors read and approved the final manuscript. 2. Ressel L, Hetzel U, Ricci E. Blunt force trauma in veterinary forensic pathol‑ ogy. Vet Pathol. 2016;53:941–61. 3. Barington K, Dich‑ Jørgensen K, Jensen HE. A retrospective study of Acknowledgements forensic cases of skin ulcerations in Danish pigs from 2000 to 2014. Acta The authors wish to thank Elisabeth Wairimu Petersen and Betina Gjedsted Vet Scand. 2016;58:48. Andersen for skilled technical assistance with preparation of tissue for Barington et al. Acta Vet Scand (2018) 60:33 Page 7 of 7 4. Wang JF, Olson ME, Reno CR, Wright JB, Hart DA. The pig as a model for 11. Montes GS, Junqueira LC. The use of the Picrosirius‑polarization method excisional skin wound healing: characterization of the molecular and for the study of the biopathology of collagen. Mem Inst Oswaldo Cruz. cellular biology, and bacteriology of the healing process. Comp Med. 1991;86:1–11. 2001;51:341–8. 12. Jensen HE, Nielsen OL, Agerholm JS, Iburg T, Johansen LK, Johannesson 5. Rigal C, Pieraggi MT, Vincent C, Prost C, Bouisou H, Serre G. Healing of E, et al. A non‑traumatic Staphylococcus aureus osteomyelitis model in full‑thickness cutaneous wounds in the pig. I. Immunohistochemical pigs. In Vivo. 2010;24:257–64. study of epidermo‑ dermal junction regeneration. J Invest Dermatol. 13. Barington K, Jensen HE. Forensic aspects of agonal wounds in slaughter 1991;96:777–85. pigs. In: 35th ESVP Meet. Eur. Soc. Vet. Pathol. 28th ECVP Meet Eur Coll Vet 6. Wang JF, Olson ME, Reno CR, Kulyk W, Wright JB, Hart DA. Molecular and Pathol. 2017. cell biology of skin wound healing in a pig model. Connect Tissue Res. 14. Elgharably H, Roy S, Khanna S, Abas M, DasGhatak P, Das A, et al. A modi‑ 2000;41:195–211. fied collagen gel enhances healing outcome in a preclinical swine model 7. Ackermann MR. Inflammation and Healing. In: Zachary JF, McGavin MD, of excisional wounds. Wound Repair Regen. 2013;21:473–81. editors. Pathologic basis of veterinary disease. St. Louis: Elsevier Mosby; 15. Betz P. Histological and enzyme histochemical parameters for the age 2012. p. 89–146. estimation of human skin wounds. Int J Legal Med. 1994;107:60–8. 8. Barington K, Jensen HE. Forensic aspects of incised wounds and bruises 16. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular in pigs established post‑mortem. Res Vet Sci. 2017;112:42–5. and cellular mechanisms. J Invest Dermatol. 2007;127:514–25. 9. Meuten DJ, Moore FM, George JW. Mitotic count and the field of view 17. Bouissou H, Pieraggi M, Julian M, Uhart D, Kokolo J. Fibroblasts in dermal area. Vet Pathol. 2016;53:7–9. tissue repair. Electron microscopic and immunohistochemical study. Int J 10. Luna LG. Manual of histological staining methods of the Armed Forces Dermatol. 1988;27:564–70. Institute of Pathology. 3rd ed. New York: McGraw‑Hill Book Company; 18. Gallant‑Behm CL, Hart DA. Genetic analysis of skin wound healing and 1968. scarring in a porcine model. Wound Repair Regen. 2006;14:46–54. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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A porcine model for pathomorphological age assessment of surgically excised skin wounds

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Abstract

A porcine model of second intention wound healing was established and gross and histological changes needed for accurate assessing the age of wounds were determined. Twenty‑ five pigs were anesthetized before incision of four wounds on each. The wounds were left to heal from 1 h to 35 days when the pigs were euthanized. In 14 pigs, biopsies were sampled from two wounds between days 2 and 18. By histological evaluation the following characteris‑ tics were found useful for determining the age of wounds: neutrophil:macrophage ratio, angiogenesis, hyperplasia of fibroblasts, presence of hemosiderophages and granulation tissue. The latter was present from day 4 (n = 8 wounds, 100%), but by gross evaluation it was not recognized until day 5 (n = 4 wounds, 100%). From day 4 to 10, the thick‑ ness of granulation tissue increased by 1.2 ± 2.4 mm/day. The thickness of collagen fibers within granulation tissue increased throughout the study period, and complete epithelization was reached from day 18. Keywords: Age of wounds, Experimental animal model, Pig, Skin, Ulceration, Veterinary forensic pathology Findings individually with bedding of straw and sawdust (Spanvall, Age assessment of skin wounds in pigs is often requested Denmark) and numbered 1–25 in the order they arrived in veterinary forensic pathology [1, 2]. Wounds in pigs to the facility (Table 1). Each pig was anesthetized (Additional file  1), treated are primarily caused by external trauma, and the age of with continuous intravascular infusion of fentanyl (Fen wounds submitted for forensic investigation ranges from - hours  and up to several months [3]. Methods to deter- tanyl 50  µg/mL, Fentanyl-Hameln; Hameln Pharma- mine the age of wounds have been investigated in por- ceuticals gmbh, Hameln, Germany), placed in sternal cine models [4–6]. However, in previous studies and recumbency and prepared for sterile surgery. Four sur- unlike wounds in veterinary forensic cases, these wounds gical areas of 2 × 2  cm (locations 1–4) were drawn on were treated with a epinephrine solution to obtain hemo- the back in the paravertebral area (Fig.  1). By incision, stasis or bandaged during healing [4–6]. The aim of the full thickness wounds (from the epidermis down to and present study was to identify hallmarks for forensic age including the subcutis) were established by removal of assessment of porcine wounds by assessing characteris- 4 cm skin and left to heal by second intention. Pigs 1–5 tics in experimental wounds during healing by second were kept anesthetized for 1–6  h and then euthanized, intention. while pigs 6–25 were transported to their pens to wake Twenty-five specific pathogen free (SPF) female up (Table 1). At 8 h intervals, pigs were given four injec- Yorkshire-Landrace crossbred pigs with body weights tions of buprenorphine (Temgesic, 0.3 mg/mL, Schering- of 23–34  kg were acclimatized for 1  week, housed Plough, NJ, USA). None of the pigs showed sign of pain as no increase  in respiratory rate, depression, reduced feed intake or mobility were observed. At different time *Correspondence: krisb@sund.ku.dk points, pigs 12–25 were anesthetized (Additional file  2) Department of Veterinary and Animal Sciences, Faculty of Health before two full thickness biopsies from each of the wound and Medical Sciences, University of Copenhagen, Ridebanevej 3, margins of locations 2 and 3 were sampled by incision 1870 Frederiksberg C, Denmark © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barington et al. Acta Vet Scand (2018) 60:33 Page 2 of 7 Table 1 Study overview Wound age Pig No. of wounds No. of wounds Histological Average Median Average Median N:M ratio biopsied sampled post- features neutrophil neutrophil score macrophage macrophage mortem relevant for age score + SD (range) score + SD score (range) determination 1 h 1 4 Clot 4.3 ± 1.7 4.5 (2–6) 1 ± 0 1 (1–1) 4:1 3 h 2, 3 8 5.9 ± 0.4 6 (5–6) 1.5 ± 0.5 1.5 (1–2) 4:1 6 h 4, 5 8 4.8 ± 0.9 5 (3–6) 1.8 ± 0.7 2 (3) 3:1 12 h 6, 7 8 Migration of epithe 5.1 ± 0.6 5 (4–6) 1.6 ± 0.5 2 (1–2) 3:1 lial cells 1 day 8, 9 8 5.4 ± 1.2 6 (3–6) 2.9 ± 1.0 2.5 (2–4) 2:1 2 days 10–13 4 8 Angiogenesis 4.8 ± 1.6 6 (2–6) 2.3 ± 0.5 2 (2–3) 2:1 3 days 14, 15 4 Fibroblasts 3.8 ± 0.5 4 (3–4) 3.5 ± 0.6 3.5 (3–4) 1:1 4 days 12, 13, 16, 17 4 4 Granulation tissue 3 ± 1.4 3 (1–5) 2.9 ± 0.6 3 (2–4) 1:1 5 days 18,19 4 2 ± 0.8 2 (1–3) 3.3 ± 0.5 3 (3–4) 1:2 6 days 14, 15 4 2.3 ± 1.0 2.5 (1–3) 3.3 ± 0.5 3 (3–4) 1:1 7 days 20, 21 4 1.8 ± 0.5 2 (1–2) 3.8 ± 0.5 4 (3–4) 1:2 8 days 16, 17 4 Hemosiderophages 2.8 ± 1.0 2.5 (2–4) 3.8 ± 0.5 4 (3–4) 1:1 10 days 18, 19 4 2.8 ± 2.1 2.5 (1–5) 3.8 ± 1.0 3.5 (3–5) 1:1 14 days 22, 23 4 1 ± 0 1 (1–1) 2 ± 0 2 (2–2) 1:2 18 days 20, 21, 24, 25 4 4 Complete epitheliza 1.1 ± 1.0 1 (0–3) 2 ± 0.5 2 (1–3) 1:2 tion 27 days 22, 23 4 0 0 2 ± 0 2 (2–2) – 35 days 24, 25 4 0 0 1.5 ± 0.6 1.5 (1–2) – Study overview: age of wounds; pigs; number of wounds (sampled by biopsy or post-mortem); first appearance of histological features relevant for age determination; average score ± standard deviation (SD) of neutrophils and macrophages; median score and range of neutrophils and macrophages and the neutrophil:macrophage (N:M) ratio Infiltration of neutrophils and macrophages was scored as: (0) absent, (1) < 10, (2) 10–20, (3) 21–50, (4) 51–100, (5) 101–200, (6) > 200 using a 40× objective and 10× ocular with FN 22 mm in one high power field (HPF) of 0.237 mm [9]. The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within areas 1–4 (Fig. 2a, b). From each wound, only the highest score (regardless of area) was registered. At each time point, the average neutrophil and macrophage score, the standard deviation, the median and the neutrophil:macrophage ratio were calculated N neutrophils, M macrophages Barington et al. Acta Vet Scand (2018) 60:33 Page 3 of 7 red and hemorrhagic connective tissue filling the wound bed and recorded as present or absent by gross inspec- tion [7]. In wounds that were biopsied, evaluations were only recorded until the day of biopsy sampling. Histological preparation of the tissue was done as previously described [8]. Sections stained with hematoxylin and eosin were evaluated in four areas (3 × 3  mm) depending on the age of the wounds and therefore not blinded (Fig. 2a, b). All registrations were carried out by a single veterinary pathologist. Infiltra - tion of neutrophils and macrophages was scored as: Fig. 1 Location of wounds 1–4, experimental pig 21. Each wound (0) absent, (1) < 10; (2) 10–20; (3) 21–50; (4) 51–100; was located 4 cm lateral to the spine and 4 cm cranial or caudal to (5) 101–200; (6) > 200 using a 40 × objective and 10× the last rib, respectively. Biopsies were taken from two wound edges ocular with FN 22  mm in one high power field (HPF) at locations 2 and 3 (red boxes) while the pigs were anesthetized. Wounds at location 1 and 4 were sampled post mortem of 0.237  mm [9]. The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within areas 1–4 (Fig.  2a, b). From each (Fig.  1 and Table  1). After 4  h, these pigs received an wound, only the highest score (regardless of area) was intramuscular injection of 0.1  mg/kg buprenorphine. registered, i.e. one score per wound. Similarly, when The pigs were euthanized from 1  h to 35  days after the two biopsies were sampled from one wound both biop- creation of the wounds with an overdose of intravenous sies were evaluated, but only the highest score was pentobarbital (Glostrup Apotek, Glostrup, Denmark) registered. At each time point, the average neutrophil (Table 1). and macrophage score, the standard deviation (SD), Following euthanasia, cross sections of the wounds the median and the neutrophil:macrophage ratio were that had not previously been biopsied were sampled for calculated. Hemorrhage, hyperplasia of fibroblasts and histology. In total, 100 wounds were evaluated, i.e. 28 endothelial cells and angiogenesis were registered as wounds were biopsied and 72 wounds were sampled after present or absent in areas 1–4 (Figs. 2a, b). Granulation euthanasia (Table 1). Granulation tissue was defined as a tissue was defined as fibroblasts and collagen arranged Fig. 2 Areas subjected to histological evaluation of biopsies (a) or cross sections of wounds (b), hematoxylin and eosin stain. a Histological evaluation of wounds aged 1 h to 10 days was carried out in areas 1–3 (3 mm × 3 mm). Area 1: at the wound edge, dermis. Area 2: at the wound edge, subcutis. Area 3: central in the wound bed. b Histological evaluation of wounds aged 14 to 35 days was carried out in area 4 (3 mm × 3 mm), i.e. central in the granulation tissue. The thickness of the granulation tissue was measured at the center of the wound (straight line) Barington et al. Acta Vet Scand (2018) 60:33 Page 4 of 7 Fig. 3 The average score ± standard deviation of neutrophils and Fig. 4 The average thickness ± standard deviation of granulation macrophages in wounds being from 1 h to 35 days tissue in wounds being from 4 to 35 days perpendicular to the new proliferating vessels (angio- where a clot had formed in the wound bed. Infiltration genesis) [7]. When present, the thickness of granulation of neutrophils around vessels and in the dermal and sub- tissue was measured at the center of the wound from cutaneous tissue was seen from 1  h to day 18 (90 of 92 the top of the wound bed (not including epidermis) to wounds, 98%). In comparison, a intravital reaction (i.e. the level of normal tissue components using the soft- active infiltration of leukocytes) is not found in porcine ware Delta Pix 2.3.5 (Smorum, Denmark) (Fig. 2b). Dif- wounds inflicted postmortem or seconds before death [8, ferences in the thickness of granulation tissue, due to 13]. The number of neutrophils peaked after 3 h to 2 days the location of the wound (location 1–4), were evalu- with a score greater than 4.5 (Fig. 3) (Additional file 4). ated by comparing locations 1 and 4 and locations 2 Infiltration of macrophages in the dermis and subcutis and 3 using the paired t test (P < 0.05). Moreover, at was seen in the wounds (n = 100 wounds, 100%) through- each time point the mean thickness of granulation tis- out the experimental period but peaked from day 7 to 10 sue and SD were calculated (Additional file 3 ). with a score greater than 3.5 (Fig.  3)  (Additional file  4). Epithelization was characterized as: (0) absent, (1) In accordance with this, macrophages peaked at day 7 in hyperplasia of epithelial basal cells, (2) migration of epi- wounds in a previous porcine excisional wound model thelial cells, and (3) full epithelization. [14]. From 8 to 35 days, hemosiderophages were present. Randomly selected sections from all time points were In comparison, hemosiderophages, which to the authors stained with Perl’s Prussian blue, Masson’s trichrome, and knowledge have not been noticed in porcine wounds Picrosirius red in order to confirm the presence of hemo - hitherto, have been reported in human wounds already at siderophages and visualization of collagen in the wound day 3 and appeared regularly from day 8 [15]. bed [10, 11]. The change in polarization color from green Based on the neutrophil:macrophage ratio, wounds to yellow to red in the aging wounds stained with Picro- could roughly be assessed as being from 1 to 3  h, 6 to sirius red is due to increasing thickness of collagen [11]. 12 h, 1 to 2 days or more than 3 days (Table 1). Contami- Immunohistochemistry based on polyclonal rabbit anti- nation of the wounds with bedding material was inevita- human von Willebrand Factor antibody (A0082, Agilent, ble and may have affected the inflammatory reaction [16]. USA) was used to confirm the presence of vessels [12]. However, in forensic cases, wounds are also subjected to The first appearance of the most important histologi - infection, foreign bodies and continuous trauma, which cal manifestations for determining the age of wounds is can affect the neutrophil:macrophage ratio [16]. Hyper - presented in Table  1. Hemostasis was present within 1 h plasia of endothelial cells, angiogenesis and hyperplasia Barington et al. Acta Vet Scand (2018) 60:33 Page 5 of 7 of fibroblast were observed in all wounds from 12  h, 2 and 3 days, respectively. Our observations are in accord- ance with previous records on experimental wounds in pigs, in which hyperplasia of fibroblasts was seen within 3  days [17]. Moreover, in forensic porcine wounds, we have previously predicted angiogenesis and hyperplasia of fibroblasts to begin at 16 h to 3 days [3]. Histologically, granulation tissue was present at day 4 (n = 8  wounds, 100%), but at gross evaluation it was not recognized until day 5 (n = 4  wounds, 100%). Granula- tion tissue thickness on days 4 to 7 varied from approxi- mately 2–6  mm and increased to nearly 10  mm on day 10 (growth rate: 1.2 ± 2.4  mm per day). However, from day 10 to 35 the thickness decreased by 0.3 ± 0.3 mm per day (Fig.  4)  (Additional file  3). In forensic cases of por- cine wounds, granulation tissue with thicknesses of 5, 15 and 27  mm have been used to estimate wound age as being 4–7  days, 8–28  days and > 28  days, respectively [3]. The decrease in granulation tissue thickness seen in the experimental wounds but apparently not in foren- sic cases is likely due to the initial depth of the lesion, accompanying infection, sequestration of necrotic tissue and sustained injury [16]. Therefore, the growth rate of granulation tissue up to 10 days should probably be seen as the maximum by which it can be formed. The wide SD of the average growth rate is likely due to inter pig vari- ation as no differences in granulation tissue thickness between locations 1 and 4 (P = 0.26) and locations 2 and 3 (P = 0.07) were found. Masson’s trichrome and Picrosirius red stains both confirmed the presence of newly formed collagen from day 4 (Fig.  5a). Gradually, more collagen was deposited throughout the experimental period (Figs. 5a–c). Hyperplasia and early migration of epithelial basal cells were seen from 12  h  (2/8 wounds, 25%) and complete epithelization was present in wounds being from 18 to 35  days old. This is in agreement with another porcine model based on 2 × 2 cm full thickness wounds, in which complete epithelization was achieved on day 28 [18]. Histological features of significant importance for assessing the age of porcine wounds were identi- fied (Table  1, Fig.  6). Age assessment of wounds with- Fig. 5 Porcine wound beds being 4 days (a), 10 days (b) and 27 days (c) old. a A scarce amount of blue stained collagen is present (arrows), out granulation tissue should be based on the ratio Masson’s trichrome stain. Inset: Green collagen fibers are visible under between neutrophils and macrophages and the pres- polarized light (arrows), Picrosirius red stain. b Massive amounts of ence of angiogenesis and fibroblast hyperplasia (Fig.  6). blue stained collagen are present (arrows), Masson’s trichrome stain. In wounds containing granulation tissue, the thickness Inset: Green, yellow and few red collagen fibers are visible in polarized of it was useful for wound age determination and the light (arrows), Picrosirius red stain. c Massive amounts of blue stained collagen are present (arrows), Masson’s trichrome stain. Inset: Yellow, neutrophil:macrophage ratio, deposition of collagen and red and a few green collagen fibers are visible in polarized light (arrows), Picrosirius red stain Barington et al. Acta Vet Scand (2018) 60:33 Page 6 of 7 Fig. 6 Decision diagram for determining the age of wounds based on gross and histological evaluation histology and immunohistochemistry. Moreover, we wish to thank Frederik presence of hemosiderophages can further support the Andersen and Dennis Brok for skilled technical assistance with photos and assessment (Fig. 6). figures. Additional files Competing interests The authors declare that they have no competing interests. Additional file 1. Sedation and general anesthesia for surgical incision of Availability of data wounds. All data generated or analyzed during this study are included in this published Additional file 2. Anesthesia and reversal of anesthesia for sampling of article and its additional files. biopsies. Consent for publication Additional file 3. Granulation tissue was measured at the center of the Not applicable. wound from the top of the wound bed (not including epidermis) and excluding underlying fat tissue and the fibrous fascia of the muscle. Ethics approval and consent to participate Additional file 4. Infiltration of neutrophils and macrophages was The study and the procedures were approved by the Danish Animal Inspec‑ scored as: (0) absent; (1) < 10; (2) 10–20; (3) 21–50; (4) 51–100; (5) 101–200; torate (2013–15–2934–00849) and were carried out in accordance with all (6) > 200 using a 40× objective and 10× ocular with FN 22 mm in one institutional, local and national guidelines. high power field (HPF) of 0.237 mm . The scoring was carried out in the HPF with the highest number of neutrophils and macrophages within Funding areas 1–4 in the wounds (Fig. 2a, b). From each wound, only the highest The study was funded by University of Copenhagen, Denmark. The funding score was registered, i.e. one neutrophil score and one macrophage score source had no involvement in the experimental design, analysis and interpre‑ per wound. Location of the wound (1–4), wound age, neutrophil score tation of the results. and macrophage score are presented. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Abbreviations SPF: specific pathogen free, i.e. free from infection with Mycoplasma hyo - Received: 16 January 2018 Accepted: 19 May 2018 pneumoniae, Actinobacillus pleuropneumoniae serotype 1–10 and 12, Porcine Reproductive Respiratory Syndrome, Brachyspira hyodysenteriae, Pasteurella multocida, Sarcoptes Scabiei var. Suis. and Haematopinus suis. Authors’ contributions KB and HEJ contributed to the design of the study. All authors contributed to References the experimental work and the gross and histological evaluation of the tissue. 1. de Siqueira A, Cuevas SEC, Salvagni FA, Maiorka PC. Forensic veterinary In addition, KB performed the statistical analyses and drafted the manuscript. pathology. Vet Pathol. 2016;53:979–87. All authors read and approved the final manuscript. 2. Ressel L, Hetzel U, Ricci E. Blunt force trauma in veterinary forensic pathol‑ ogy. Vet Pathol. 2016;53:941–61. 3. Barington K, Dich‑ Jørgensen K, Jensen HE. A retrospective study of Acknowledgements forensic cases of skin ulcerations in Danish pigs from 2000 to 2014. Acta The authors wish to thank Elisabeth Wairimu Petersen and Betina Gjedsted Vet Scand. 2016;58:48. Andersen for skilled technical assistance with preparation of tissue for Barington et al. Acta Vet Scand (2018) 60:33 Page 7 of 7 4. Wang JF, Olson ME, Reno CR, Wright JB, Hart DA. The pig as a model for 11. Montes GS, Junqueira LC. The use of the Picrosirius‑polarization method excisional skin wound healing: characterization of the molecular and for the study of the biopathology of collagen. Mem Inst Oswaldo Cruz. cellular biology, and bacteriology of the healing process. Comp Med. 1991;86:1–11. 2001;51:341–8. 12. Jensen HE, Nielsen OL, Agerholm JS, Iburg T, Johansen LK, Johannesson 5. Rigal C, Pieraggi MT, Vincent C, Prost C, Bouisou H, Serre G. Healing of E, et al. A non‑traumatic Staphylococcus aureus osteomyelitis model in full‑thickness cutaneous wounds in the pig. I. Immunohistochemical pigs. In Vivo. 2010;24:257–64. study of epidermo‑ dermal junction regeneration. J Invest Dermatol. 13. Barington K, Jensen HE. Forensic aspects of agonal wounds in slaughter 1991;96:777–85. pigs. In: 35th ESVP Meet. Eur. Soc. Vet. Pathol. 28th ECVP Meet Eur Coll Vet 6. Wang JF, Olson ME, Reno CR, Kulyk W, Wright JB, Hart DA. Molecular and Pathol. 2017. cell biology of skin wound healing in a pig model. Connect Tissue Res. 14. Elgharably H, Roy S, Khanna S, Abas M, DasGhatak P, Das A, et al. A modi‑ 2000;41:195–211. fied collagen gel enhances healing outcome in a preclinical swine model 7. Ackermann MR. Inflammation and Healing. In: Zachary JF, McGavin MD, of excisional wounds. Wound Repair Regen. 2013;21:473–81. editors. Pathologic basis of veterinary disease. St. Louis: Elsevier Mosby; 15. Betz P. Histological and enzyme histochemical parameters for the age 2012. p. 89–146. estimation of human skin wounds. Int J Legal Med. 1994;107:60–8. 8. Barington K, Jensen HE. Forensic aspects of incised wounds and bruises 16. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular in pigs established post‑mortem. Res Vet Sci. 2017;112:42–5. and cellular mechanisms. J Invest Dermatol. 2007;127:514–25. 9. Meuten DJ, Moore FM, George JW. Mitotic count and the field of view 17. Bouissou H, Pieraggi M, Julian M, Uhart D, Kokolo J. Fibroblasts in dermal area. Vet Pathol. 2016;53:7–9. tissue repair. Electron microscopic and immunohistochemical study. Int J 10. Luna LG. Manual of histological staining methods of the Armed Forces Dermatol. 1988;27:564–70. Institute of Pathology. 3rd ed. New York: McGraw‑Hill Book Company; 18. Gallant‑Behm CL, Hart DA. Genetic analysis of skin wound healing and 1968. scarring in a porcine model. Wound Repair Regen. 2006;14:46–54. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Acta Veterinaria ScandinavicaSpringer Journals

Published: May 30, 2018

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