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Time-course changes in the expression of heme oxygenase-1 in human subcutaneous hemorrhage
Forensic Science International 158 (2006) 157–163 www.elsevier.com/locate/forsciint
Time-course changes in the expression of heme oxygenase-1 in human subcutaneous hemorrhage Toru Nakajima a,b,*, Mutsumi Hayakawa a, Daisuke Yajima a, Hisako Motani-Saitoh a, Yayoi Sato a, Masahiro Kiuchi a, Masaharu Ichinose b, Hirotaro Iwase a a
Department of Legal Medicine, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan b Department of Plastic Surgery, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan Received 26 November 2004; accepted 19 May 2005 Available online 21 July 2005
Abstract To determine the time-course of human subcutaneous hemorrhage, heme oxygenase (HO)-1 expression and macrophage infiltration were observed using an immunohistochemical technique and semiquantitative analysis. The number of immunoreactive cells and the number of all infiltrating cells of each microscopic field were counted, and the ratio of the former to the latter was calculated as the positive cells ratio. An increase in the HO-1-positive cells ratio was observed starting at 3 h after injury, and the maximum ratio was observed 3 days after injury. The pattern of the increase in the macrophage ratio was similar to that of the HO-1-positive cells ratio in the early period after injury. Observation of serial sections revealed that the expression of HO-1 in the cells corresponded to the localization of macrophage. The present results suggest that the determination of HO-1 expression, as derived from macrophages, might be useful for the estimation of the time-course of subcutaneous hemorrhage. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Heme oxygenase; Subcutaneous hemorrhage; Immunohistochemistry; Macropahge; Time-course; Wound age
1. Introduction Subcutaneous hemorrhage is one of the most important findings associated with injuries, and the estimation of the time-course of progression is important in the field of forensic medicine. Especially in abuse cases, both in autopsy cases and in living bodies, the time-course is quite important information in order to make decisions regarding the duration time of the abuse. Most forensic pathologists use * Corresponding author. Tel.: +81 43 226 2078; fax: +81 43 226 2079. E-mail address: [email protected] (T. Nakajima).
practical approaches to estimate the time-dependent changes associated with cases involving subcutaneous hemorrhage; for example, in human cases, it has been reported that particular color changes occur with time [1–3]. However, such approaches are not entirely reliable or reproducible, due to the influence of the pathologists’ subjectivity. There have been many studies on wound age determination using objective methods, including those with a focus on the histological observation of infiltrating cell migration to the wound site, those applying immunohistochemical techniques to determine the expression of chemokines, and quantitative mRNA analyses of inflammatory cytokines [4–28]. However, most of these previous studies have
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.05.028
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focused on open skin wounds, and only a few reports have examined cases involving subcutaneous hemorrhage [29–32]. Heme oxygenase (HO) is the enzyme that catalyzes the degradation of heme into biliverdin, carbon monoxide and iron [33,34]. Recently, three HO isoforms (HO-1, HO-2 and HO-3) were identified, and HO-1 has been reported as an inducible enzyme that is mediated by stress conditions, such as heat shock, hyperoxia, hypoxia, endotoxin, inflammatory cytokines and prostaglandins [35–38]. Moreover, it has been reported that the HO system exists in phagocytic macrophages [34], which infiltrate into hemorrhage sites. Such observations have suggested that there is strong correlation between HO-1 expression and macrophage infiltration. Although there is one previous experimental study on time-dependent HO expression in hematomas of rats using spectrophotometric techniques [29], to the best of our knowledge, there has not yet been a report on time-dependent HO-1 expression in the human body using human subcutaneous hemorrhage specimens taken from dead bodies during forensic autopsy. In this study, we examined subcutaneous hemorrhage not accompanied by an open skin wound. To this end, immunohistochemical study and semiquantitative analysis of HO1 expression and macrophage infiltration were carried out, and we evaluated the usefulness of these approaches in the determination of the age of cases of subcutaneous hemorrhage.
2. Materials and methods 2.1. Materials A total of 65 human subcutaneous hemorrhage specimens were obtained during forensic autopsies. All of the cases had been brought to the hospital immediately after injury (e.g., traffic accident cases). In all cases, medical records were obtained that specifically addressed the wounds in question. The individuals were aged from 7 to 89 years (mean age: 51.9 years). The time from death to autopsy ranged from 11 h to 5 days, and in cases when the time lapse between death until autopsy exceeded 24 h, the cadavers were kept in a refrigerator at 4 8C until autopsy. The time from injury to death ranged from 0 h (i.e., cases of immediate death) to 10 days.
and rabbit anti-HO-1 polyclonal antibody (Alexis Biochemicals, Switzerland) or mouse anti-human macrophage monoclonal antibody (Zymed Laboratories Inc., USA) was used as the primary antibody. The immunostaining was performed according to the protocol recommended by the manufacturer of the detection kit. After the sections were deparaffinized and rehydrated, they were treated with proteinase K (Dako Corporation), and the endogenous peroxidase was inactivated by 3% hydrogen peroxide. Then, primary antibody or tris buffered saline (as a control) was applied, and EnVision + dextran polymer reagent was added to the sections, which were then incubated at room temperature. Positive immunostaining reactions were visualized by diaminobenzidine, and the sections were counterstained with hematoxylin. 2.3. Semiquantitative analysis of immunoreactive cells The existence of subcutaneous hemorrhage in each case was confirmed by microscopic observation of H&E-stained sections. As regards examination of the serially immunostained sections, 10 microscopic fields (400 magnification) were randomly selected; then, the immunoreactive cells (A) and the infiltrating cells (B) were counted, and the ratio of A/ B was calculated. All of the observed fields were located at the margin of the hemorrhage, which contained both the hemorrhage and the adjacent subcutaneous tissue or dermis (Fig. 1). The mean A/B value of 10 fields was regarded as the positive-cells ratio of each specimen, and we examined the relationship between that ratio and the time from injury. Additionally, the same microscopic fields of directly adjacent serial sections that had been immunostained with antiHO-1 and anti-human macrophage antibody were compared, and the distribution of positive cells was examined. 2.4. Statistical analysis Specimens were classified by time from injury as follows: group I = 0–2 h, group II = 3–8 h, group III = 9–24 h,
2.2. Immunohistochemistry The specimens were fixed in 4% formaldehyde solution immediately after autopsy, embedded in paraffin and serial sections were prepared at thickness of 3.5 mm. In each case, one of the serial sections was routinely stained with hematoxylin and eosin (H&E) in order to confirm the existence of subcutaneous hemorrhage. Immunostaining was performed with an EnVision + detection kit (Dako Corporation, USA),
Fig. 1. Selected fields were located at the margin of the hemorrhage; these fields contained both the site of the hemorrhage and the adjacent subcutaneous tissue or dermis.
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Fig. 2. Immunostaining with anti-HO-1 polyclonal antibody. Positive cells were identified as nucleated cells with clearly stained cytoplasm (400).
group IV = 25–48 h, group V = 49–72 h, group VI = 73– 96 h, group VII = 97–144 h, group VIII = 145–240 h. The mean value and standard error of the positive-cells ratio in each group were calculated, and the statistical significance between groups was evaluated by Student’s t-test at a significance level of P < 0.05.
3.3. Comparison of directly adjacent serial sections The HO-1-positive cells were almost all macrophages (Fig. 6). In the serial sections, both the HO-1-positive cells and macrophages appeared to be arranged in a similar pattern, i.e., as immunoreactive cells in groups II–V; moreover, the number of HO-1-negative macrophages increased in groups VI and VIII.
3. Results 3.1. HO-1-positive cells
4. Discussion
HO-1-positive cells were identified as nucleated cells with clearly stained cytoplasm (Fig. 2). They were observed at the site of the adjacent subcutaneous tissue, as well as in the hemorrhage itself. A significant increase in the positive cells ratio was observed in group II, and after 49–72 h (group V), the positive cells ratio reached the maximum level, after which a rapid decrease to the baseline level of group I was observed. Statistical significance was detected between groups I and II, groups I and VII and groups VI and VIII (Fig. 3).
Although HO is known to be the enzyme responsible for the physiological degradation of heme, it is also involved in
3.2. Macrophages As was the case with the HO-1-positive cells, macrophages were identified as cells with clearly stained cytoplasm (Fig. 4). Some macrophages were observed in cases of immediate death, and the ratio of macrophages was increased in group II. After 49–72 h (group V), the ratio of macrophages reached the maximum level, and then this value decreased gradually; however, group VIII still showed a moderate increase in the macrophages ratio (Fig. 5).
Fig. 3. Mean value and standard error of the ratio of HO-1-positive cells in each group.
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Fig. 4. Immunostaining with anti-human macrophage monoclonal antibody. Macrophages were identified as cells in which the cytoplasm was clearly stained (400).
the regulation of activity of second messenger gases, i.e., carbon monoxide and nitric oxide [38,39]. And then, these gases are known to play important role in tissue injury and protection. HO-1, a stress response protein, is among three isoforms of HO identified to date, and it is thought to be induced by local tissue inflammation [35–38]. Laiho and Tenhunen reported the results of a study on the time-dependent HO expression in subcutaneous hematomas using the skin of rats [29]. However, at the time when their report was published, it was not yet known that there are three different isoforms of HO; thus, they reported observing the highest increases in HO expression levels from 2 to 9 days after injury. In the present study, we aimed to observe the time-
Fig. 5. Mean value and standard error of the macrophages ratio in each group.
course of HO-1 expression in particular, and not that of HO expression in general. Using this approach, a significant increase was observed with respect to the ratio of HO-1positive cells from 3 h to 6 days after injury. The serial sections revealed HO-1 expression almost entirely among the infiltrating macrophages. HO-1 has been reported to be widely distributed in a variety of tissues, and is highly inducible in all cells [37]. In our study, HO-1 was not observed in nucleated cells that had infiltrated to the hemorrhage site within 2 h after injury, whereas HO-1 was observed in nearly all infiltrated macrophages starting at 3 h after injury. We therefore suspected that the HO-1 in this study was derived from macrophages. This result is consistent with a report on the skin wound repair process in mice [40], and also with a previous report on HO expression in macrophages [34]. The infiltrative transition of macrophages at wound sites, as well as the role played by macrophages in the wound healing process, have already been reported, not only in the field of forensic medicine, but also in clinical medicine [41– 44]. For example, Betz reported that macrophages were first observed at the wound site 3 h after injury, and that they were commonly seen 15 h after injury; furthermore, macrophage infiltration was found to continue for up to a few months following injury [7]. DiPietro noted that macrophages are the predominant cell type at wound sites from 3 to 5 days after injury [42]. Our observation of macrophages in human subcutaneous hemorrhage specimens revealed a similar infiltrative transition pattern. Macrophages are known to appear at the wound site, and they have been shown to produce various growth factors and cytokines, e.g., TNF-a, IL-1, IL-6, PDGF, TGF-a, TGF-b [42]. In general, macrophages play a major role in the wound healing process. Although subcutaneous hemorrhage is not an open injury, if
Fig. 6. Directly adjacent serial sections are shown. Immunostaining with anti-HO-1 polyclonal antibody (left, 200) and with anti-human macrophage monoclonal antibody (right, 200). The immunoreactive cells are stained, and indicated by arrows.
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we regard it as a wound of the subcutaneous tissue, then it appears that macrophages play a similar role in both subcutaneous hemorrhage and in cases of open injury, and therefore determination of the infiltration of macrophages might be applicable in the subsequent determination of the age of a subcutaneous hemorrhage. The results of this study suggest that the age of a subcutaneous hemorrhage can be estimated as follows; by considering the time-dependent expression of HO-1-positive cells and macrophages: (a) If macrophages have not infiltrated to the subcutaneous hemorrhage site, and HO-1-positive cells have not been observed, then the hemorrhage may have occurred within the past 2 h. (b) If both macrophages and HO-1-positive cells have been observed at a similar ratio, the age of the subcutaneous hemorrhage may range from 3 h to 4 days. (c) If HO-1 is localized in only some of the infiltrated macrophages, the age of the injury may range from 5 to 6 days. (d) If macrophages have infiltrated to a moderate extent, and there is no detectable HO-1 expression, the time lapse since the injury may be 7 days or more. When observing stained sections, it should be noted that the density of infiltrating cells may differ between the margin and at the center of a hemorrhage. As it was expected that the density of infiltrating cells would be higher at the margin, we selected and observed fields located at the margin of each hemorrhage; these fields contained both the hemorrhage and the adjacent subcutaneous tissue or dermis. However, we were unable to determine a precise means of creating equivalent experimental conditions, due to the large variety in the size and shape of each case studied. In cases involving open skin wounds, it would be possible to ensure relatively equivalent experimental conditions by fixing the range from the wound margin. However, this approach would most likely be controversial if applied to cases of subcutaneous hemorrhage. To start this study, there was a problem that it was difficult to specify the time of injury. Here, we examined subcutaneous hemorrhage specimens obtained at forensic autopsy. We selected cases which had been brought to the hospital immediately after injury, and had medical records specific to the wounds evaluated, and therefore we were able to correctly identify the time of injury. Moreover, as Laiho and Tenhunen pointed out in their report, it should be noted that the storage conditions (such as temperature) from death to autopsy might exert an influence on results [29]. Although we calculated the results of this study without regard to the time since death, it might probably be helpful to evaluate the influence of the time lapse between death and autopsy on our results if we could clarify the time lapse. The specimens used in this study were obtained upon forensic autopsy as part of a routine tissue preservation
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protocol. The methods do not require particular experimental instruments, and the techniques involved are not difficult to perform. In this study, we attempted to elucidate the relationship between HO-1 expression and the time since injury using specimens obtained from human forensic autopsy cases, instead of those obtained from animal experiments. The approach used in this study may be helpful for practical forensic estimations. Moreover, the present results suggest that a more reliable estimation of time lapsed since subcutaneous hemorrhagic injury may become possible by determining the time-dependent expression of various growth factors and inflammatory cytokines, i.e., by applying methods already employed in cases investigating the experimental aging of open skin wounds.
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T. Nakajima et al. / Forensic Science International 158 (2006) 157–163 [29] K. Laiho, R. Tenhunen, Hemoglobin-degrading enzymes in experimental subcutaneous hematomas, Z. Rechtsmed. 93 (1984) 193–198. [30] R.N. Thornton, R.D. Jolly, The objective interpretation of histopathological data: an application to the ageing of ovine bruises, Forensic Sci. Int. 31 (1986) 225–239. [31] T. Sawaguchi, B. Jasani, M. Kobayashi, B. Knight, Postmortem analysis of apoptotic changes associated with human skin bruises, Forensic Sci. Int. 108 (2000) 187–203. [32] M. Oehmichen, A. Windisch, C. Meissner, Mononuclear cells in subcutaneous haemorrhage with special consideration of myeloid percursor cells, Med. Sci. Law 40 (2000) 286–292. [33] R. Tenhunen, H.S. Marver, R. Schmid, Microsomal heme oxygenase: characterization of the enzyme, J. Biol. Chem. 244 (1969) 6388–6394. [34] N.R. Pimstone, R. Tenhunen, P.T. Seitz, H.S. Marver, R. Schmid, The enzymatic degradation of hemoglobin to bile pigments by macrophages, J. Exp. Med. 133 (1971) 1264– 1281. [35] D. Willis, A.R. Moore, R. Frederick, D.A. Willoughby, Heme oxygenase: a novel target for the modulation of the inflammatory response, Nat. Med. 2 (1996) 87–90. [36] W.K. McCoubrey Jr., T.J. Huang, M.D. Maines, Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3, Eur. J. Biochem. 247 (1997) 725–732.
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Time-course changes in the expression of heme oxygenase-1 in human subcutaneous hemorrhage Toru Nakajima a,b,*, Mutsumi Hayakawa a, Daisuke Yajima a, Hisako Motani-Saitoh a, Yayoi Sato a, Masahiro Kiuchi a, Masaharu Ichinose b, Hirotaro Iwase a a
Department of Legal Medicine, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan b Department of Plastic Surgery, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan Received 26 November 2004; accepted 19 May 2005 Available online 21 July 2005
Abstract To determine the time-course of human subcutaneous hemorrhage, heme oxygenase (HO)-1 expression and macrophage infiltration were observed using an immunohistochemical technique and semiquantitative analysis. The number of immunoreactive cells and the number of all infiltrating cells of each microscopic field were counted, and the ratio of the former to the latter was calculated as the positive cells ratio. An increase in the HO-1-positive cells ratio was observed starting at 3 h after injury, and the maximum ratio was observed 3 days after injury. The pattern of the increase in the macrophage ratio was similar to that of the HO-1-positive cells ratio in the early period after injury. Observation of serial sections revealed that the expression of HO-1 in the cells corresponded to the localization of macrophage. The present results suggest that the determination of HO-1 expression, as derived from macrophages, might be useful for the estimation of the time-course of subcutaneous hemorrhage. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Heme oxygenase; Subcutaneous hemorrhage; Immunohistochemistry; Macropahge; Time-course; Wound age
1. Introduction Subcutaneous hemorrhage is one of the most important findings associated with injuries, and the estimation of the time-course of progression is important in the field of forensic medicine. Especially in abuse cases, both in autopsy cases and in living bodies, the time-course is quite important information in order to make decisions regarding the duration time of the abuse. Most forensic pathologists use * Corresponding author. Tel.: +81 43 226 2078; fax: +81 43 226 2079. E-mail address: [email protected] (T. Nakajima).
practical approaches to estimate the time-dependent changes associated with cases involving subcutaneous hemorrhage; for example, in human cases, it has been reported that particular color changes occur with time [1–3]. However, such approaches are not entirely reliable or reproducible, due to the influence of the pathologists’ subjectivity. There have been many studies on wound age determination using objective methods, including those with a focus on the histological observation of infiltrating cell migration to the wound site, those applying immunohistochemical techniques to determine the expression of chemokines, and quantitative mRNA analyses of inflammatory cytokines [4–28]. However, most of these previous studies have
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.05.028
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focused on open skin wounds, and only a few reports have examined cases involving subcutaneous hemorrhage [29–32]. Heme oxygenase (HO) is the enzyme that catalyzes the degradation of heme into biliverdin, carbon monoxide and iron [33,34]. Recently, three HO isoforms (HO-1, HO-2 and HO-3) were identified, and HO-1 has been reported as an inducible enzyme that is mediated by stress conditions, such as heat shock, hyperoxia, hypoxia, endotoxin, inflammatory cytokines and prostaglandins [35–38]. Moreover, it has been reported that the HO system exists in phagocytic macrophages [34], which infiltrate into hemorrhage sites. Such observations have suggested that there is strong correlation between HO-1 expression and macrophage infiltration. Although there is one previous experimental study on time-dependent HO expression in hematomas of rats using spectrophotometric techniques [29], to the best of our knowledge, there has not yet been a report on time-dependent HO-1 expression in the human body using human subcutaneous hemorrhage specimens taken from dead bodies during forensic autopsy. In this study, we examined subcutaneous hemorrhage not accompanied by an open skin wound. To this end, immunohistochemical study and semiquantitative analysis of HO1 expression and macrophage infiltration were carried out, and we evaluated the usefulness of these approaches in the determination of the age of cases of subcutaneous hemorrhage.
2. Materials and methods 2.1. Materials A total of 65 human subcutaneous hemorrhage specimens were obtained during forensic autopsies. All of the cases had been brought to the hospital immediately after injury (e.g., traffic accident cases). In all cases, medical records were obtained that specifically addressed the wounds in question. The individuals were aged from 7 to 89 years (mean age: 51.9 years). The time from death to autopsy ranged from 11 h to 5 days, and in cases when the time lapse between death until autopsy exceeded 24 h, the cadavers were kept in a refrigerator at 4 8C until autopsy. The time from injury to death ranged from 0 h (i.e., cases of immediate death) to 10 days.
and rabbit anti-HO-1 polyclonal antibody (Alexis Biochemicals, Switzerland) or mouse anti-human macrophage monoclonal antibody (Zymed Laboratories Inc., USA) was used as the primary antibody. The immunostaining was performed according to the protocol recommended by the manufacturer of the detection kit. After the sections were deparaffinized and rehydrated, they were treated with proteinase K (Dako Corporation), and the endogenous peroxidase was inactivated by 3% hydrogen peroxide. Then, primary antibody or tris buffered saline (as a control) was applied, and EnVision + dextran polymer reagent was added to the sections, which were then incubated at room temperature. Positive immunostaining reactions were visualized by diaminobenzidine, and the sections were counterstained with hematoxylin. 2.3. Semiquantitative analysis of immunoreactive cells The existence of subcutaneous hemorrhage in each case was confirmed by microscopic observation of H&E-stained sections. As regards examination of the serially immunostained sections, 10 microscopic fields (400 magnification) were randomly selected; then, the immunoreactive cells (A) and the infiltrating cells (B) were counted, and the ratio of A/ B was calculated. All of the observed fields were located at the margin of the hemorrhage, which contained both the hemorrhage and the adjacent subcutaneous tissue or dermis (Fig. 1). The mean A/B value of 10 fields was regarded as the positive-cells ratio of each specimen, and we examined the relationship between that ratio and the time from injury. Additionally, the same microscopic fields of directly adjacent serial sections that had been immunostained with antiHO-1 and anti-human macrophage antibody were compared, and the distribution of positive cells was examined. 2.4. Statistical analysis Specimens were classified by time from injury as follows: group I = 0–2 h, group II = 3–8 h, group III = 9–24 h,
2.2. Immunohistochemistry The specimens were fixed in 4% formaldehyde solution immediately after autopsy, embedded in paraffin and serial sections were prepared at thickness of 3.5 mm. In each case, one of the serial sections was routinely stained with hematoxylin and eosin (H&E) in order to confirm the existence of subcutaneous hemorrhage. Immunostaining was performed with an EnVision + detection kit (Dako Corporation, USA),
Fig. 1. Selected fields were located at the margin of the hemorrhage; these fields contained both the site of the hemorrhage and the adjacent subcutaneous tissue or dermis.
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Fig. 2. Immunostaining with anti-HO-1 polyclonal antibody. Positive cells were identified as nucleated cells with clearly stained cytoplasm (400).
group IV = 25–48 h, group V = 49–72 h, group VI = 73– 96 h, group VII = 97–144 h, group VIII = 145–240 h. The mean value and standard error of the positive-cells ratio in each group were calculated, and the statistical significance between groups was evaluated by Student’s t-test at a significance level of P < 0.05.
3.3. Comparison of directly adjacent serial sections The HO-1-positive cells were almost all macrophages (Fig. 6). In the serial sections, both the HO-1-positive cells and macrophages appeared to be arranged in a similar pattern, i.e., as immunoreactive cells in groups II–V; moreover, the number of HO-1-negative macrophages increased in groups VI and VIII.
3. Results 3.1. HO-1-positive cells
4. Discussion
HO-1-positive cells were identified as nucleated cells with clearly stained cytoplasm (Fig. 2). They were observed at the site of the adjacent subcutaneous tissue, as well as in the hemorrhage itself. A significant increase in the positive cells ratio was observed in group II, and after 49–72 h (group V), the positive cells ratio reached the maximum level, after which a rapid decrease to the baseline level of group I was observed. Statistical significance was detected between groups I and II, groups I and VII and groups VI and VIII (Fig. 3).
Although HO is known to be the enzyme responsible for the physiological degradation of heme, it is also involved in
3.2. Macrophages As was the case with the HO-1-positive cells, macrophages were identified as cells with clearly stained cytoplasm (Fig. 4). Some macrophages were observed in cases of immediate death, and the ratio of macrophages was increased in group II. After 49–72 h (group V), the ratio of macrophages reached the maximum level, and then this value decreased gradually; however, group VIII still showed a moderate increase in the macrophages ratio (Fig. 5).
Fig. 3. Mean value and standard error of the ratio of HO-1-positive cells in each group.
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Fig. 4. Immunostaining with anti-human macrophage monoclonal antibody. Macrophages were identified as cells in which the cytoplasm was clearly stained (400).
the regulation of activity of second messenger gases, i.e., carbon monoxide and nitric oxide [38,39]. And then, these gases are known to play important role in tissue injury and protection. HO-1, a stress response protein, is among three isoforms of HO identified to date, and it is thought to be induced by local tissue inflammation [35–38]. Laiho and Tenhunen reported the results of a study on the time-dependent HO expression in subcutaneous hematomas using the skin of rats [29]. However, at the time when their report was published, it was not yet known that there are three different isoforms of HO; thus, they reported observing the highest increases in HO expression levels from 2 to 9 days after injury. In the present study, we aimed to observe the time-
Fig. 5. Mean value and standard error of the macrophages ratio in each group.
course of HO-1 expression in particular, and not that of HO expression in general. Using this approach, a significant increase was observed with respect to the ratio of HO-1positive cells from 3 h to 6 days after injury. The serial sections revealed HO-1 expression almost entirely among the infiltrating macrophages. HO-1 has been reported to be widely distributed in a variety of tissues, and is highly inducible in all cells [37]. In our study, HO-1 was not observed in nucleated cells that had infiltrated to the hemorrhage site within 2 h after injury, whereas HO-1 was observed in nearly all infiltrated macrophages starting at 3 h after injury. We therefore suspected that the HO-1 in this study was derived from macrophages. This result is consistent with a report on the skin wound repair process in mice [40], and also with a previous report on HO expression in macrophages [34]. The infiltrative transition of macrophages at wound sites, as well as the role played by macrophages in the wound healing process, have already been reported, not only in the field of forensic medicine, but also in clinical medicine [41– 44]. For example, Betz reported that macrophages were first observed at the wound site 3 h after injury, and that they were commonly seen 15 h after injury; furthermore, macrophage infiltration was found to continue for up to a few months following injury [7]. DiPietro noted that macrophages are the predominant cell type at wound sites from 3 to 5 days after injury [42]. Our observation of macrophages in human subcutaneous hemorrhage specimens revealed a similar infiltrative transition pattern. Macrophages are known to appear at the wound site, and they have been shown to produce various growth factors and cytokines, e.g., TNF-a, IL-1, IL-6, PDGF, TGF-a, TGF-b [42]. In general, macrophages play a major role in the wound healing process. Although subcutaneous hemorrhage is not an open injury, if
Fig. 6. Directly adjacent serial sections are shown. Immunostaining with anti-HO-1 polyclonal antibody (left, 200) and with anti-human macrophage monoclonal antibody (right, 200). The immunoreactive cells are stained, and indicated by arrows.
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we regard it as a wound of the subcutaneous tissue, then it appears that macrophages play a similar role in both subcutaneous hemorrhage and in cases of open injury, and therefore determination of the infiltration of macrophages might be applicable in the subsequent determination of the age of a subcutaneous hemorrhage. The results of this study suggest that the age of a subcutaneous hemorrhage can be estimated as follows; by considering the time-dependent expression of HO-1-positive cells and macrophages: (a) If macrophages have not infiltrated to the subcutaneous hemorrhage site, and HO-1-positive cells have not been observed, then the hemorrhage may have occurred within the past 2 h. (b) If both macrophages and HO-1-positive cells have been observed at a similar ratio, the age of the subcutaneous hemorrhage may range from 3 h to 4 days. (c) If HO-1 is localized in only some of the infiltrated macrophages, the age of the injury may range from 5 to 6 days. (d) If macrophages have infiltrated to a moderate extent, and there is no detectable HO-1 expression, the time lapse since the injury may be 7 days or more. When observing stained sections, it should be noted that the density of infiltrating cells may differ between the margin and at the center of a hemorrhage. As it was expected that the density of infiltrating cells would be higher at the margin, we selected and observed fields located at the margin of each hemorrhage; these fields contained both the hemorrhage and the adjacent subcutaneous tissue or dermis. However, we were unable to determine a precise means of creating equivalent experimental conditions, due to the large variety in the size and shape of each case studied. In cases involving open skin wounds, it would be possible to ensure relatively equivalent experimental conditions by fixing the range from the wound margin. However, this approach would most likely be controversial if applied to cases of subcutaneous hemorrhage. To start this study, there was a problem that it was difficult to specify the time of injury. Here, we examined subcutaneous hemorrhage specimens obtained at forensic autopsy. We selected cases which had been brought to the hospital immediately after injury, and had medical records specific to the wounds evaluated, and therefore we were able to correctly identify the time of injury. Moreover, as Laiho and Tenhunen pointed out in their report, it should be noted that the storage conditions (such as temperature) from death to autopsy might exert an influence on results [29]. Although we calculated the results of this study without regard to the time since death, it might probably be helpful to evaluate the influence of the time lapse between death and autopsy on our results if we could clarify the time lapse. The specimens used in this study were obtained upon forensic autopsy as part of a routine tissue preservation
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protocol. The methods do not require particular experimental instruments, and the techniques involved are not difficult to perform. In this study, we attempted to elucidate the relationship between HO-1 expression and the time since injury using specimens obtained from human forensic autopsy cases, instead of those obtained from animal experiments. The approach used in this study may be helpful for practical forensic estimations. Moreover, the present results suggest that a more reliable estimation of time lapsed since subcutaneous hemorrhagic injury may become possible by determining the time-dependent expression of various growth factors and inflammatory cytokines, i.e., by applying methods already employed in cases investigating the experimental aging of open skin wounds.
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