Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1beta,IL-6, TNF-alpha) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n = 105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1beta and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30-60 and 60-90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-alpha after 15 and 60-90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1beta and IL-6 appeared after approximately 2 h. CONCLUSION: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

Quantitative analysis of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) in human skin wounds

Proinflammatory cytokines play an important role in the mediation of inflammation and trauma. They could be useful for the determination of vitality and wound age. In the present study, 144 human skin wounds due to sharp force were investigated. The material was collected during operations (N=96) and postmortem examinations (N=48). The wound age varied from several seconds or minutes to 9 days. Control skin was available in each individual. The tissue specimens were homogenized and extracted in a solution of PBS and protease inhibitors. Interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) were measured by quantitative ELISA analysis. Statistical evaluation was performed by the t-test using the quotients of levels (wound sample/control skin). In surgical specimens the cytokine levels revealed a clear tendency to increase with wound age. IL-1beta in early skin wounds (24 h, P<0.05). The quantitative analysis of proinflammatory cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular in the very early post-traumatic interval (classic stab wounds).     

Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n=105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1β and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30–60 and 60–90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-α after 15 and 60–90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1β and IL-6 appeared after approximately 2 h. Conclusion: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

A preliminary study on the changes of expression of PDGF-beta,PDGFR-beta,TGF-beta 1, TGFR,bFGF and its relationship with the wound age in wound healing

OBJECTIVE: To explore the relationship between the expression change of cytokines and the wound age during the healing process of rats skin wound. METHODS: Immunohistochemical and image-analysis methods were performed on vital skin wounds(after incision 0.5-168 h am) and postmortem damage(after incision 0.5-6 h pm). RESULTS: The expression of the cytokines PDGF-beta, PDGFR-beta, TGF-beta 1, and bFGF in the epithelial cells was already enhanced since 0.5 h am after damage and their strongest expression reaction was seen at 24-96 h am. In addition, the expression of PDGF-beta, PDGFR-beta, TGF-beta 1 and bFGF was also found in the macrophages and the fibroblasts of the granulation tissue, and the expression changes in the postmortem damage group showed that the skin tissue within 0.5-3 h after incision showed immunohistochemical changes but weakly expression and 3 h thereafter no any change was found. CONCLUSION: The expression characteristics of the above mentioned cytokines in wound repair should be related to the wound age and it reminds therefore that they may be used as immunohistochemical criteria for accurate determining the wound age.     

Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine

The adhesion molecules identified in recent years can help improve the diagnosis of the wound age, especially of injuries with a short survival time. This is also indicative of the vitality of the wounds. The material investigated in the study originated from 465 skin wounds. The samples were taken from human autopsy material, during the surgical treatment of wounds (excision) of patients and from experimental incised wounds of mice. To judge the age of skin wounds the endothelial adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. Human skin wounds: strong positive staining was observed of ICAM-1 1.5 h at the earliest and 3.5 days at the latest, for the P-selectin 3 min at the earliest and 7 h at the latest, for the E-selectin 1 h at the earliest and 17 days at the latest and for VCAM-1 3 h at the earliest and 3.5 days at the latest after the time of injury. The L-selectin was expressed constitutively. Mice skin wounds: strong positive immunohistochemical reactions were found as a rule earlier than in human skin wounds. The detection of an increased expression of ICAM-1, VCAM-1 and P- and E-selectins can improve the wound age assessment in injuries with short survival times.     

Immunohistochemical detection of fibronectin and tenascin in incised human skin injuries

Immunohistochemical detection of molecules involved in inflammatory reaction can be useful for the diagnosis of vitality in skin wounds. We studied the expression of fibronectin (FN) and tenascin (TN) in 58 human skin wounds (48 vital and 10 postmortem). The age of vital injuries ranged from 3 min to 8 h and postmortem specimens were collected after a postinfliction interval of 15-180 min. One hundred thirty-seven formalin-fixed paraffin-embedded sections (mean: 2.3 sections per case) were stained with each of two monoclonal antibodies against FN and TN using the streptABC technique. A reticular staining for FN in wound edge and dermis was observed in 50% of vital specimens versus 0% in postmortem cases. Immunoreactivity was reduced in 10 autolysed cases. FN positivity exclusively at the injury margin was observed in 39.4% of vital wounds and 10% of postmortem cases. TN was negative in all specimens. Vital and postmortem hemorrhage areas showed positivity for FN and TN. Due to its low sensitivity, immunohistochemical analysis of FN is useful for determining vitality only in a minority of cases. Different factors in everyday practice, including autolysis and technical problems often produce false negative reactions with the result that FN cannot be regarded as a reliable parameter of vitality. Positive reactions (network staining) are more valuable than negativity but are not pathognomonic. Both vital and postmortem hemorrhages show an enhanced positivity for FN and TN, thus impeding the diagnosis.     

Immunohistochemical study of basic fibroblast growth factor and vascular endothelial growth factor expression for age determination of cutaneous wounds

The authors investigated the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) during skin wound healing using immunohistochemical techniques. After a full-thickness incision was made on the dorsal skin, mice were killed 0.5, 1, 3, 8, 24, 72, 144, or 240 hours after incision, and the wound was excised. To evaluate the influences of postmortem degeneration, cutaneous wound excision was performed 1 to 5 days after the mice were killed. The excised wounds were stained by the conventional streptoavidin-biotinylated peroxidase complex method, using specific antibodies, and the ratio of the number of positive cells to total cells was determined. Expression of bFGF was detected in the nuclei of epidermal cells and fibroblasts in the early 0.5- to 1-hour phases and the late 24- to 144-hour phases. Expression of VEGF was detected in the cytoplasm of epidermal cells in the 24- to 144-hour phases. Immunoreactivity of both cytokines was detected 1 day post mortem and was especially well preserved in the fibroblasts. Time-dependent expression of both factors suggested that they would be useful markers for the determination of wound age. However, bFGF should be superior to VEGF because of its earlier expression and because of its more persistent expression in dermal fibroblasts with increasing postmortem interval.     

人体皮肤创伤组织细胞因子表达的时间相关性

目的探讨TGF-β1、b—FGF和VEGF在人体皮肤创伤组织中的表达变化及其与损伤时间的关系。方法收集15例机械性致伤的人体皮肤组织(受伤5—120min内死亡),常规制作组织切片后进行免疫组化染色,并统计分析TGF-β1、b—FGF和VEGF因子的表达情况。结果 TGF-β1在16—50min组表达升高,60～120min组呈阳性至强阳性表达;b—FGF在16—50min组表达升高明显,强阳性表达亦出现在60～120min组;VEGF仅在60—120min组出现阳性表达。统计学分析表明,TGF—β1和b—FGF的表达升高分别在受伤60~120min(P<0.01)和在16—50min(P<0.01)有显著性意义,VEGF表达升高只在60~120min(P<0.05)有显著性意义。结论 TGF-β1、b—FGF和VEGF的表达时间和表达强度与损伤时间有关;TGF-β1、b—FGF和VEGF的表达可为人体短存活期损伤时间的推断提供参考。     

Postmortem stability of some markers of intra-vital wounds

We have studied the diagnostic value of several markers of the intra-vital nature of wounds - cathepsin D (EC 3.4.23.5) and ions (Ca, Mg, Cu, Zn and Fe) - after the influence of putrefaction. For this purpose, we have inflicted vital wounds to six pigs, which were killed 20 min later. Ten minutes after death, wounds were excised with 5-6 cm of skin around the incision and maintained at three different temperatures (4, 18 and 28.5 +/- 13.4 degrees C). After varying periods of postmortem interval from 0 to 48 h, aliquots of each wound were taken and analyzed with atomic absorption spectrophotometry for ions and with UV-spectrophotometry for cathepsin D. Our results demonstrate that ions conserve their diagnostic ability to differentiate vital from postmortem wounds after the influence of putrefaction. Nevertheless, cathepsin D does not show this ability in these experimental conditions.     

人皮肤组织刺、切创后IL-8表达的免疫组织化学研究

为探讨人皮肤刺、切创后白细胞介素 8(interleukin 8,IL 8)在推断皮肤损伤时间中的应用价值 ,本研究应用免疫组化技术对 5 2例不同损伤时间人体皮肤刺、切创组织中IL 8的表达进行了研究。伤后 4h的损伤皮肤组织中可见部分的多核粒细胞表达IL 8。伤后 12～ 2 4h ,大部分浸润的多核粒细胞及部分单核细胞为IL 8阳性。随伤后时间延长 ,IL 8阳性细胞以单核及成纤维细胞为主。伤后 4～ 6h的皮肤中 ,IL 8阳性细胞比率较低 ,为 16 0±10 1%。伤后 1～ 4d达高峰 ,为 5 9 6± 8 7%。其后逐渐减少。本研究结果表明 ,IL 8的表达可用于皮肤刺、切创后损伤时间的推断。     

Localization and quantification of the nonspecific esterase in injured skin for timing of wounds.

Histochemical quantification of the nonspecific esterase (NSE) in injured skin was performed using histochemical demonstration of the enzyme and a microspectrophotometric scanning technique on specimens taken from 32 Hartley guinea-pigs and 8 cases of human skin wounds. In all antemortem incisions and lacerations, including those made at the agonal stage, NSE activity could be observed in the dermal tissue of the wound edge. The enzyme activity increases with the antemortem duration of the injuries. Both total content and mean concentration of NSE in the wound edge between antemortem and postmortem wound groups differ significantly (less than 0.01). Multiple range test shows that significant differences (P less than 0.05 or P less than 0.01) of total content of NSE in the wound edge also exist in 0-5 min, 15-30 min, 1-h, 2-h and 4-h antemortem incised wound groups and in 0-min, 5-min, 15-30 min, 1-h and 4-h antemortem laceration groups. The positive NSE reactions in 8 cases of human skin wounds were similar. The study indicates that histochemical quantification of NSE in injured skin is very useful in timing wounds and is exactly applicable in medicolegal practice. According to the different influences of inhibitors on enzymes, it was inferred that the enzyme activity in wound edges was due to B-esterase.     

皮肤切创组织IL—2、TNF—α时间相关性表达的ELISA分析

目的了解细胞因子在创伤及修复过程中的表达的含量变化与损伤时间的关系。方法用免疫酶联吸附方法(ELISA)检测不同损伤时间(生前伤0.5～168h)实验动物皮肤切创组织中IL-2和TNF-a的表达水平。结果IL-2、TNF-a在造创后0.5h即升高,并分别在造创后3h和1h到达峰值,在造创后48h均有反弹,在72h和168h持续升高。结论上述细胞因子在创口修复中的蛋白表达水平呈时间相关性特点。     

小鼠皮肤创伤愈合过程中IL-10在不同表达部位的组化定量研究

目的探讨小鼠皮肤创伤愈合过程中IL-10在不同表达部位的变化与损伤时间的关系。方法应用免疫组织化学技术和形态计量法,对小鼠不同时程皮肤切创组织中IL-10不同表达部位(表皮细胞及表皮下组织的浸润细胞)的变化进行研究。结果皮肤切创前后表皮层均有阳性表达,切创后1～3h表皮细胞阳性表达水平迅速升高,24h时降至较低水平,伤后48h表达水平再次升高,96h后逐渐回复到正常水平;表皮以下阳性部位主要是浸润的单核/巨噬细胞,损伤6h后阳性细胞在真皮、皮下组织及创口肉芽组织内逐渐增多,于伤后72h达最大值(51.41±3.12)%,96～168h阳性细胞数逐渐减少(29.38±2.64)%～(5.56±4.74)%。结论皮肤创伤修复过程中IL-10在不同表达部位的变化特点与皮肤损伤时间相关,检测其表达变化可望用于损伤时间推断。     

The time-dependent expression of fibronectin,MRP8, MRP14 and defensin in surgically treated human skin wounds

Fibronectin, MRP8, MRP14 and defensin were detected immunohistochemically in 46 surgically treated, fresh (hours old) human wounds and in wounds of 13 individuals who died immediately from fatal trauma (airplane crashes or train rollovers). In immediate fatal trauma, it was not possible to detect fibronectin outside of bleeding areas nor could inflammatory cells be visualized in the interstitium using MRP8, MRP14, or defensin antibodies. Fibronectin staining could be regularly demonstrated in wounds at least 20 min-old. Granulocytic infiltrates limited to the perivasal space could be detected 20-30 min after infliction of the wound expressing MRP8, MRP14 and defensin. It was also possible to detect fibronectin networks and MRP8-, MRP14-, and defensin-positive granulocytes and macrophages in particular wounds up to 30 days-old. No differences between the expression of MRP8 and MRP14 could be demonstrated in the wounds, the majority of which were only several hours old. As wound age increased, the number of defensin-positive granulocytes detected decreased. The immunohistochemical detection of fibronectin is a useful way to demonstrate vitality in fresh wounds, beginning about 20-30 min post-trauma. However, detection of MRP8, MRP14 and defensin provides no advantage over the routine histological detection of granulocytes and macrophages in wounds under 1-2 days old.     

小鼠皮肤切创IL-10和IL-4的表达及其与损伤时间关系

目的　探讨IL 10、IL 4在小鼠皮肤切创愈合过程中的表达变化规律及其与损伤时间的关系。方法　应用免疫组织化学及图像分析技术 ,观察实验小鼠不同时程的生前伤 ( 0 5～ 168h)及死后伤 ( 1～ 6h)皮肤创伤局部IL 10、IL 4的表达情况。结果　生前伤 ,IL 10在伤后 1～ 3h阳性反应逐渐增强 ,3h达峰值 ,2 4h降至较低水平 ,伤后 48h再次升高 ,72h又达新峰值 ,其阳性表达部位主要在表皮细胞及单核 /巨噬细胞 ;IL 4于伤后 2 4h出现明显阳性反应 ,96h表达水平达峰值 ,168h仍维持在较高水平 ,其阳性细胞主要为创伤局部的成纤维细胞。死后伤 ,IL 10仅于死后 1～ 3h呈阳性染色 ,IL 4未见阳性染色。结论　IL 10、IL 4在小鼠皮肤切创愈合过程中的表达呈现一定的时序性变化。     

Forensic Potential of MMPs and CC Chemokines for Wound Age Determination

In this study, we investigated time-dependent expression of matrix metalloproteases (MMP)-2, MMP-9, chemokine CC motif ligand (CCL)-2, CCL-3, CCL-5, IL-1β, IL-6, and TNF-α mRNA at the skin injury site and sought their forensic potentials during the skin wound repair process. The tested wound ages in 42 mouse skin wounds were distributed at 0d, 1d, 3d, 5d, 7d, 10d, and 14d, respectively and then followed by real-time polymerase chain reaction. Ultimately, MMP-2 played an important role in the inflammation phase. On the contrary, MMP-9 became involved at a later phase during wound healing. Meanwhile, CCL-2 and CCL-3 were active throughout almost all of the process. However, CCL-5 mRNA had no significance. Collectively, an MMP-9/MMP-2 ratio of over 0.84 indicated that skin wound healing age was strongly 5 days or less. So elevated gene expressions of cytokines and chemokines in different phases of wound ages implied that combined exploration could make wound age determination more accurate and objective.     

Cathepsin D: a new marker of the vitality of the wound. Comparative study with histamine and serotonin

We have studied the variation of specific activity of the most important lysosomal enzyme: Cathepsin D. This study has been developed in pig skin incision wounds for its application to the differential diagnosis between vital and postmortem wounds. At the same time, we have determined histamine and serotonine levels to make a comparative study with Cathepsin results. Our results show the utility of this new marker to determine the vitality of wounds, even when the time elapsed until death has been very short. Nevertheless, there is no correlation between Cathepsin D results and those of histamine or serotonin, a fact that induces us to think that this new marker is not useful to determine wound age.     

皮肤切创愈合中caspase-3表达的免疫组化研究

目的探讨皮肤切创损伤愈合过程中,caspase-3在损伤区内的表达以及不同损伤时间caspase-3的变化规律。方法应用免疫组织化学技术对33例不同损伤时间小鼠皮肤切创组织中caspase-3的表达进行研究。同时以3例非切创小鼠皮肤组织做对照。结果伤后6h的损伤皮肤组织中可见少量中性粒细胞表达caspase-3,伤后12～24h,大部分浸润的中性粒细胞及部分单核细胞为caspase-3阳性。随伤后时间延长,caspase-3阳性细胞以单核细胞及成纤维细胞为主。伤后0～3h,caspase-3阳性细胞比率较低,为(4.53±6.53)%,12h后逐渐增加,伤后3d达高峰,为(62.66±4.84)%,其后逐渐下降。结论小鼠皮肤损伤愈合过程中,caspase-3可能在诱导损伤区内中性粒细胞、单核巨噬细胞及成纤维细胞发生凋亡过程中发挥重要作用,同时,caspase-3的规律性表达可用于损伤时间的推断。     

The informative aspects of the wound surface for determining vitality and wound age in skin cuts

A histological study was carried out on skin cut wounds of guinea pigs at various time intervals during the first day after infliction of the cut. At the same time intervals, blood cells were obtained from the wound surface prints and analyzed cytologically and cytochemically. Histological examination showed that a central and peripheral zone formed in the wound area, demonstrating that necrobiotic and inflammatory processes were even occurring in the early hours after injury. However, changes in the quantitative relationships between individual kinds of cells were much more differentiated cytologically and reflected much more precisely. Functional and structural macrophage and lymphocyte changes in the wound region during inflammation were also revealed. In correlation, cytochemical analysis confirmed the concept concerning the development of the inflammatory process in the wound area, as enzyme-activity changes clearly detectable, which reflected the rapid, energetic, plastic cellular processes on the wound surface. Our combined results suggest that the wound surface is a zone of vital processes, but that it is not a "dead," "necrobiotic" area. The dynamics of the cellular alterations in the wound surface, reflecting the vital processes developing there, can be successfully used when the problem of vitality, especially the time lapse after the skin injury, is to be resolved.     

纤维联接蛋白的析出与大鼠皮肤早期损伤的时间关系

采用免疫组化(PAP)方法,检测大鼠皮肤损伤区的FN,发现在生前创伤后15min,创壁FN即呈明确阳性;随着伤后经历时间的延长,创壁FN逐渐增多,并滑创壁呈条带状沉积;而在死后5min的创伤,创壁FN则呈阴性。本文为区别生前与死后皮肤创伤提出了一种新的方法。