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).     

细胞因子(IL-6、TNF-α)在损伤时间中原位杂交法研究

采用原位杂交法，研究大鼠切削皮肤中细胞团子IL－6、TNF－αmRNA表达量，旨在探讨IL－6、TNF－α推断法医损伤时间的应用价值以及其在损伤生活反应中的分子机制。研究结果表明，根据大鼠损伤皮肤中TNF－αmRNA表达量能够区别生前伤与死后伤，并可以利用TNF－αmRNA表达量改变准确区别60min内和60min后损伤时间，但IL－6mRNA表达量在研究组内均未见阳性反应，不能推断损伤时间。     

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。     

The time-dependent expressions of IL-1beta, COX-2, MCP-1 mRNA in skin wounds of rabbits

In this study, we investigated the time-dependent expression of IL-1beta, COX-2, MCP-1 mRNA after incised wounds in rabbit skin using real-time fluorescent quantitative PCR. The tested wound ages were distributed as following: <0.5h, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, 24h, 2d, 3d, and 7d. The expressions of three markers in postmortem wounds were determined. Comparison of each wound age with control group, expression of IL-1beta mRNA showed that the significant increase occurred at <0.5h (p<0.01), and the peak level at 2h. The expression was almost normalized at 2d. But for COX-2 and MCP-1 mRNA, the significant increase occurred at 1h for COX-2 mRNA, and 3h for MCP-1 mRNA. The expression peak levels were at 3h and 5h, and were almost normalized at 3d and 7d, respectively. There was no significant increase in all postmortem samples for IL-1beta, COX-2, MCP-1 mRNA compared with control group. Thus, the results of these cytokines and enzyme significant increase at different early wound ages implied that the combined investigation could make wound age determination more objective and accurate. Moreover, the three markers could also be used to distinguish the supravital injuries.     

Transforming growth factors (TGF-alpha and TGF-beta1) in the determination of vitality and wound age: immunohistochemical study on human skin wounds

In continuation of former investigations on proinflammatory cytokines, in the present study the relevance of the transforming growth factors TGF-alpha and TGF-beta1 was evaluated for the diagnosis of vitality and wound age. Paraffin sections from human skin wounds due to sharp force influence, which had been collected in operations and autopsies, were investigated using immunohistochemistry. The wound age varied from a few minutes to a maximum of 6 weeks with focus on the early post-traumatic interval up to 5h. Samples from uninjured skin were available as controls. TGF-alpha (n=74) was weakly expressed in normal skin and showed a marked increase in epidermal reactivity after a wound age of approximately 10 min. The maximum was between 30 and 60 min. TGF-beta1 (n=51) revealed constitutional expression only in connective tissue. An increase of immunohistochemical reaction was partially detected even in classical stab wounds (wound age of several minutes). The immunohistochemically detectable signal concerned--presumably due to an infiltration with TGF-beta-rich thrombocytes--large parts of the traumatized skin and also the epidermal layers (cellular and interstitial marking). TGF-beta1 peaked after a post-traumatic interval of 30-60 min. Both factors, especially TGF-beta1, remained detectable in elevated levels also in older wounds with an age of days to weeks (network in granulation tissue). TGF-alpha and TGF-beta1 can efficiently contribute to the estimation of vitality and wound age based on the evaluation of cytokine patterns. In particular, this applies to TGF-beta1 because of its easier evaluation and rapid up-regulation. Similar to other cytokines, the parallel investigation of control skin from the same individual must be recommended to eliminate variation in the basal expression.     

兔皮肤钝器伤IL-1β、COX-2和MCP-1 mRNA表达与法医学应用

目的应用荧光定量PCR技术检测兔皮肤钝器伤后IL-1β、COX-2和MCP-1mRNA的时序性表达规律,分析其与损伤时间的关系。方法分别在兔耳皮肤钝器伤后<0.5h,0.5h,1h,2h,3h,4h,5h,6h,8h,12h,24h,1d,3d,7d和死后10min,0.5h,1h取材,一步法提取组织总RNA,逆转录合成cDNA第一条链,荧光定量PCR检测IL-1β、COX-2和MCP-1mRNA逆转录合成第一条cDNA链的拷贝数,作统计学分析。结果IL-1βmRNA在钝器伤后<0.5h表达显著增强(P<0.001),0.5h达到峰值,至第2d降至基本水平;COX-2mRNA于创伤后0.5h表达显著增强(P<0.05)并持续强表达至24h开始下降,第2d降至正常;MCP-1 mRNA在钝器伤后于1h表达显著增强(P<0.05),于3h出现表达峰,至第3d降至正常;3个指标在死后各时间组与正常对照无明显表达差异。结论检测皮肤IL-1β、COX-2和MCP-1mRNA可对早期损伤时间的推断提供帮助,并且这三个指标都可以鉴别生前伤和死后伤。     

IL-33在小鼠皮肤创伤后损伤时间推断中的作用

目的通过观察白细胞介素-33(interleukin-33,IL-33)在皮肤创伤后的时序性变化规律,探讨IL-33在法医学实践中用于损伤时间推断的应用价值。方法利用直径为5mm的圆形锉刀在小鼠背部建造皮肤损伤模型,于伤后1h、3h、6h、12h、1d、3d、5d、7d、10d取损伤处组织,对照组在与创伤组小鼠相同部位取同等大小的皮肤样本。采用苏木精-伊红(hematoxylin-eosin,HE)染色法观察皮肤创伤后愈合过程中的形态学变化,通过Western印迹法、免疫组织化学染色和双重免疫荧光染色法检测皮肤创伤样本IL-33的表达变化。结果Western印迹法结果显示,伤后3h,IL-33蛋白表达稍有下降,6h后IL-33蛋白表达逐渐增加,于伤后3d达峰值,随后逐渐减少。免疫组织化学染色结果显示,在对照组皮肤的表皮、毛囊、皮脂腺及真皮中固有细胞有少量IL-33阳性表达,伤后3h,IL-33阳性细胞率开始增加,伤后3d达到峰值,随后逐渐减少。双重免疫荧光染色结果显示,伤后1~3d,IL-33阳性表达细胞主要为巨噬细胞,伤后5~7d,IL-33阳性表达细胞主要为肌成纤维细胞。HE染色结果显示该皮肤损伤模型创口愈合过程符合炎症的病理学发展规律。结论IL-33有望成为法医学推断皮肤损伤时间的参考指标。     

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。     

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。     

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。     

Determination of γ-hydroxybutyrate (GHB) and its precursors in blood and urine samples: A salting-out approach

γ-Hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose–response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 μL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na₂SO₄, incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC–MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC–FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.     

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.     

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.     

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。     

大鼠皮肤切创愈合过程中ICAM-1及P选择素的表达

目的　探讨细胞间粘附分子 1(ICAM 1)及P选择素的表达变化与损伤时间的关系。方法　应用免疫组织化学技术 ,对实验大鼠不同损伤时间的生前伤 ( 5min～ 7d)及死后伤 ( 5～ 3 0min)皮肤组织中ICAM 1及P选择素的表达变化进行观察。结果　在生前损伤组中 ,ICAM 1最早在伤后 1h ,最迟至伤后 3d在表皮层中呈阳性表达 ;P选择素最早在伤后 10min ,最迟至伤后 5h即在血管内皮细胞中呈阳性表达。此外 ,ICAM 1还在炎症细胞及纤维母细胞中表达 ,且随损伤时间变化而呈规律性变化。在时间段分组 ,ICAM 1在第Ⅰ组 ( 5min～ 1h)中阳性细胞率极低( 0 41± 0 73 % ) ,第Ⅱ组 ( 3h～ 7h)及第Ⅲ组 ( 9h～ 12h)均呈显著性增高 ( 9 79± 3 74% ,2 3 3 3± 1 10 % ) ,至第Ⅳ组 ( 1d)达高峰 ( 3 0 5 8± 2 65 % ) ,其后逐渐减少。ICAM 1及P选择素在死后伤中均呈阴性表达。结论　ICAM 1及P选择素可作为法医学损伤时间判定的有效指标。     

用RT-PCR法区别生前与死后伤

12只SD大鼠随机分为生前伤、死后伤、麻醉伤和正常对照四组，损伤时间为15min，取创缘皮肤为检材；IL－6引物为寡聚核着酸引物（27hP），内对照引物为Tx基因（20hP），用TRIZOLTMTotalRNA提取试剂盒抽据总RNA，AMV逆转录酶使mRNA逆转录成cDNA，PCR扩增得出：在生前伤、麻醉伤以及正常皮肤中均能扩增出IL－6cDNA片段（590hp）和内对照Tx基因片段（188b），在死后伤组只能扩增出Tx基因片段；图像分析凝胶灰度扫描；生前损伤组与麻醉组无显著性差异，而二者与正常皮肤组有统计学差异。藉此，运用RT－PCR法可以准确区别大鼠实验性损伤的生前伤与死后伤。     

小鼠皮肤切创愈合过程中caspase-6、-7表达的免疫组织化学研究

目的　观察皮肤切创损伤愈合过程中 ,caspase 6、 7在损伤周边区内的表达及其变化规律。方法　应用免疫组织化学技术观察伤后不同时间小鼠皮肤切创组织中caspase 6、 7的表达 ,以非切创小鼠皮肤组织作对照。 结果　伤后 6h的损伤皮肤组织中可见少量多核粒细胞表达caspase 6、 7,伤后 12～ 2 4h ,大部分浸润的多核粒细胞及部分单核细胞为caspase 6、 7阳性。随伤后时间延长 ,caspase 6、 7阳性细胞以单核细胞及成纤维细胞为主。伤后 0～ 3h ,caspase 6、 7阳性细胞比率较低 ,12h后逐渐增加 ,伤后 3d达高峰 ,其后逐渐下降。 结论　小鼠皮肤损伤愈合过程中 ,caspase 6、 7在损伤周边区内中性粒细胞 ,单核细胞及成纤维细胞中表达 ,其时序性变化规律可望用于皮肤损伤时间的推断。     

Complement component C3a or C3a desArg as a new marker for estimation of local vital reactions in incised skin wounds.

The anaphylatoxin C3a or its desArg form (C3a/desArg) generated during complement activation could be detected in the vicinity of incised skin wounds of guinea pigs using immunoblotting methods. The C3a/desArg peptides were detectable immediately after injury in local sites up to 3 mm from the wound edge. In subsequent determinations of up to at least 3-day-old antemortem wounds, the maximum concentration of these peptides was largely localized up to 6 mm from the wound edge at 2 h after injury. In postmortem wounds, however, these peptides were undetectable. When they were released in antemortem wounded tissues they could be detected up to 1 day at 22 degrees C after death. These results suggest that the detection of C3a/desArg in wounds using immunoblotting methods can be useful for distinguishing ante- from postmortem wounds.     

小鼠皮肤切创愈合过程中caspase-9、-3表达的研究

目的研究caspase-9、-3在小鼠皮肤切创愈合过程中的表达及其变化规律。方法在小鼠背部正中制作皮肤全层切创模型,应用免疫组化染色技术观察切创及切创周边区内caspase-9、-3的表达情况,以无切创的小鼠皮肤作为对照。结果对照组中caspase-9、-3表达于表皮层,毛囊及皮脂腺。伤后3h损伤区及损伤周边区中可见少量多核粒细胞表达caspase-9、-3,6~24h,部分浸润的多核粒细胞和单核细胞caspase-9、-3阳性,此后caspase-9、-3阳性细胞逐渐以单核细胞和成纤维细胞为主。伤后caspase-9、-3阳性细胞率逐渐升高并在3d达到高峰,此后逐渐下降,至14d最低。结论caspase-9、-3可能引发正常小鼠皮肤表皮、毛囊和皮脂腺细胞的凋亡并参与正常皮肤细胞的自我更新;在小鼠皮肤切创愈合过程中,caspase-9、-3在多核粒细胞、单核细胞及成纤维细胞中表达,其时序性变化规律可望用于皮肤切创损伤时间的判定。