人脑挫伤后波形蛋白表达的免疫组化染色观察

目的观察人脑挫伤后波形蛋白（vimentin,Vim）的动态变化,探讨星形胶质细胞Vim表达变化与脑损伤时间的关系。方法42例闭合性脑损伤死亡者,根据伤后死亡时间分为6组（〈2h,〈24h,〈3d,〈7d,〈14d,≤30d）;取大脑挫伤灶进行HE和Vim免疫组织化学染色,对Vim染色阳性细胞面积进行图像分析。结果HE染色显示,脑挫伤后2h挫伤灶内脑组织挫碎、出血,5．5h挫伤灶周出现反应性星形胶质细胞,3d脑水肿明显,7d反应性星形胶质细胞明显增多,14d胶质结节形成,30d出现大量泡沫细胞,胶质瘢痕增多。Vim染色结果显示,脑挫伤后5．5hVim开始表达,少量Vim阳性细胞出现在挫伤灶周围,7d阳性反应性最强,14d逐渐下降,30d胶质瘢痕增多,Vim阳性表达又增强。Vim阳性细胞主要为反应性星形胶质细胞。结论Vim在反应性星形胶质细胞内表达呈现一定波动性,其表达部位在脑挫伤周围,Vim免疫组化染色结合图像分析技术可作为推断脑挫伤时间及部位的参考指标。     

大鼠脑挫伤后组织学及Bax/Bcl-2表达的研究

采用自制大鼠右顶叶局灶性脑挫伤模型,进行组织学和 Bax/Bcl－ 2的免疫组织化学法研究。结果发现 :伤后 12h～ 24h,挫伤灶周围神经细胞和星形胶质细胞形态学发生改变,白细胞附壁和游出;伤后 4d,挫伤灶周围出现大量的泡沫细胞和核大而深染的星形胶质细胞; 8～ 10d,挫伤灶处形成软化灶; 12～ 14d,挫伤灶愈合变成胶质细胞结节,或者变成囊状,可见大量的星型胶质细胞及新生毛细血管,可见含铁血黄素颗粒沉积。 Bax/Bcl－ 2表达水平与伤后经历时间有一定相关性。     

迟发性外伤性脑内血肿脑组织GFAP、S-100和NSE的免疫组织化学研究

应用免疫组织化学ABC法和S－P法，对6例伤后1．5天至2年发生的迟发性外伤性脑内血肿（DTICH）脑组织标本进行神经胶质细胞内胶质纤维酸性蛋白（GFAP）、S－100蛋白（S－100）和神经元细胞内特异性烯醇化酶（NSE）变化和应用价值的研究。尸检和组织学检查发现有脑挫伤等病变。6例DTICH脑组织挫伤及周围区均有明显的GFAP、S－100阳性的细胞变化，细胞数目增多，胞体肥大、增生，免疫组化反应产物增多、深染；神经元细胞NSE免疫组化染色均呈阴性改变。结果表明：6例DTICH脑组织中GFAP、S－100的NSE的免疫组织化学变化是有一定时间间隔的外伤性陈旧性改变，在DTICH的病理诊断上具有重要价值，可作为DTICH法医学鉴定的一个辅助手段。     

Preliminary study on using changes in astrocytes and neurons in the brain-stem of the rat after stab wounds for determining the time of injury

The changes in astrocytes and neurons in the brain-stem of the rat after stab wounds were studied immunohistochemically. Anti-GFAP and anti-albumin antibodies were used to detect the changes of astrocytes and neurons, respectively. The results showed that in normal control group, the numbers of GFAP-positive cells differed in different areas of brain-stem, but remained similar in the same area of different rats. No albumin-positive cells could be found. The number of GFAP-positive cells in antemortem injury groups increased significantly followed by a decrease, and then a further increase. Albumin-positive cells including neurons and glial cells were found after injury in the midbrain, pons and medulla oblongata. In the postmortem injury groups, there were no significant changes in either anti-GFAP or anti-albumin immunoreactivities in any area of brain-stem compared to the control group. These results indicated that the changes in the number of GFAP-positive cells could be used to estimate the time of injury and to diagnose early brain-stem injury. The antemortem and postmortem injuries can also be differentiated by the presence of albumin-positive cells. The authors suggest that the timing and early diagnosis of injury in the brain-stem should be carried out on the basis of comprehensive analyses of changes in both neurons and glial cells.     

大鼠脑挫伤后GFAP和iNOS表达与损伤时间关系的实验研究

目的　观察大鼠实验性脑挫伤后胶质纤维酸性蛋白 (GFAP)和诱导型—氧化氮合成酶 (iNOS)的表达及其时相性的关系 ,试图寻找法医学脑损伤时间的推断方法。方法　建立脑挫伤的动物模型 ,采用免疫组织化学技术 (SABC法 )观察大鼠脑挫伤后GFAP及iNOS在伤后不同时间的表达。染色结果用计算机彩色图像分析技术作定量统计分析处理。结果　 ( 1)伤后 3h时GFAP阳性表达细胞开始增多 ,1d和 5d组单个阳性细胞染色强度达峰值 ,但在 3d组相对邻近组却下降 ,呈现出一双峰波形 ,7d达最大值 ;阳性物质总面积自 3h时表达增多开始后一直增加 ,直到 7d时达高峰。 ( 2 )伤后 12h组可见iNOS活性增高 ,并随伤后存活时间的延长 ,iNOS阳性细胞数目 (或阳性物质总面积 )逐渐增多 ,单个细胞染色强度逐渐加深。伤后 1d至 3d组阳性物质总面积达到高峰 ,5d后开始下降 ,但 7d时仍维持较高水平 ;而单个细胞染色强度随伤后存活时间的延长而加深 ,于 5d时达高峰后显著下降 ,但高于起始水平。结论　脑挫伤后GFAP和iNOS在损伤局部的染色变化有一定的时间规律性 ,可以用于脑挫伤的时间推断。     

脑挫伤大鼠胶质细胞GFAP、S100的表达及其损伤时间的推断

目的观察大鼠实验性脑挫伤后胶质纤维酸性蛋白(GFAP)和S100的表达,观察脑损伤后星形胶质细胞随脑损伤时间的变化规律,研究其在脑损伤及神经修复中的作用。方法建立脑挫伤模型,利用免疫组化染色(SP法)观察大鼠脑挫伤后GFAP、S100在伤后不同时间星形细胞中的表达。并应用图像分析技术对其作定量统计分析处理。结果(1)伤后1h时GFAP阳性表达开始增多,阳性物质表达随时间大致呈线性上升7d达最大值,然后阳性物质染色强度和面积呈下降趋势。(2)伤后12h组可见S100阳性表达活性增高,S100阳性细胞数目逐渐增多,5d时达高峰后显著下降,但仍高于对照组。结论脑挫伤后星形胶质细胞的表达水平的变化有一定时间规律性,在脑挫伤时间推断中及神经组织修复中有一定作用。     

White matter damage following acute head injury

The study of a series of brains from patients who had a severe head injury and died within 72 h without a lucid interval showed that there was a step-wise progression in the development of retraction balls. At 2 h after injury sinusoidal enlargement of the axons was evident. This progressed over 16 h when the lesions appeared as retraction balls which were fully developed at 72 h. There was a similar increase of staining with an immunoperoxidase method for glial fibrillary acid protein (GFAP) initially around blood vessels spreading diffusely into the white matter. The number of reactive astrocytes also increased. In a control case where the corpus callosum was torn at post-mortem there were sinusoidally distended and torn axons in the absence of GFAP staining. It is proposed that there are three components to a head injury. First, mechanical injury as seen in the control case; second, the development of retraction balls which are an active process probably representing damaged axons which cannot undergo repair where the sinusoidal swellings develop into retraction balls and third, an astrocytic reaction. The sinusoidal change, when present on its own, may not be separable from post-mortem trauma. However, when it is associated with an astrocytic response it should be correlated with coma in the same way as retraction balls.     

Effect of toluene inhalation on astrocytes and neurotrophic factor in rat brain

Toluene, an abused substance in Japan, is a neurotoxic chemical that has been shown to have neurobehavioral and electrophysiological effects. In previous work, both acute and chronic effects of toluene on cells have been studied extensively. However, although glial cells are thought to play an important role in the survival of neurons in the brain, the effect of toluene on glial cell function has not yet been characterized. To elucidate this, the effect of toluene inhalation on astrocytes in rat brain was examined. Toluene exposure (1500 ppm for 4 h on 4-10 days) augmented glial fibrillary acidic protein (GFAP) immunoreactivity, particularly in the hippocampus and cerebellum. Quantitative analysis showed that toluene inhalation markedly enhanced GFAP expression in the hippocampus and cerebellum. In both regions, proliferating cell nuclear antigen (PCNA) showed no obvious changes, but glutamine synthetase (GS)-immunoreactive cells were markedly increased by toluene exposure. Thus, the elevation of GFAP expression was induced by astrocyte activation rather than by cell proliferation. If toluene exposure activates astrocytes, astrocytes may play a role in the neurophysiological changes observed in toluene intoxication. A neurotrophic factor, basic fibroblast growth factor (b-FGF) was observed immunohistochemically in the capillary vessel walls in the hippocampus and the cerebellum of toluene-intoxicated rats. Basic-FGF may have induced GFAP expression both in the hippocampus and the cerebellum. So, other neurotrophic factors may affect the difference of GFAP elevation between the hippocampus and the cerebellum. These differences may relate to neurobehavioral function of each brain part after toluene exposure.     

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).     

Quantitative immunohistochemical analysis of human brain basic fibroblast growth factor, glial fibrillary acidic protein and single-stranded DNA expressions following traumatic brain injury

Previous studies demonstrated the healing process after traumatic brain injury (TBI), usually at the site or in the area adjacent to the injury, in connection with wound timing. However, the whole brain condition after TBI has not been elucidated clearly. In the present study, we investigated immunohistochemistry of the basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) in the parietal lobe and hippocampus distant from the primary injury site in forensic autopsy cases of TBI (n=174). Characteristic findings were detected with regard to brain compression signs and survival time (ST). Peracute deaths (n=22) had a lower GFAP positivity in the parietal white matter. Fatalities without a brain compression sign (parahippocampal herniation/Duret hemorrhage; n=53) had a lower brain weight without glial loss; however, GFAP positivity in the parietal white matter was decreased during ST of <12h, and glial bFGF positivity was increased at each site in deaths after 12h to 3 days, followed by a delayed neuronal loss after 3 days. Fatalities with a brain compression sign (n=78) showed a higher brain weight, and gradual glial and neuronal losses with an early increase of glial bFGF positivity in the parietal cerebral cortex (ST <0.5h). This was followed by an increase of glial bFGF positivity in other sites (ST, 6-24h), and final decreases of glial bFGF and GFAP positivities with increased neuronal ssDNA positivity in the parietal lobe and hippocampus (ST >3 days), which were detected in earlier deaths despite decompressive craniectomy (ST, 12-60h; n=21). These observations suggest that the combined use of bFGF, GFAP and ssDNA immunohistochemistry can be used to evaluate the severity of damage and response of brain after TBI.     

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 throttled hemophilic who died due to choking

It is the first reported case, in which death occurred due to asphyxia by choking in a victim of throttling. When an alcoholic hemophilic patient was throttled, he became unconscious. On regaining consciousness he died after approximately 16 h due to sub-mucosal collection of extravasated blood which blocked his upper respiratory tract. This paper stresses this unique danger of trauma to neck in hemophilic patients.     

A Rare Case of an Intact Bone Plug Associated with a Gunshot Exit Wound

This case study presents an unusual manifestation of gunshot trauma in skeletal tissue from a post‐World War II human rights abuse sample uncovered in Vilnius, Lithuania. After briefly reviewing the typical wound appearance of projectile trauma in the cranium, we discuss the presence of an intact bone plug associated with a gunshot exit wound in an individual from the Tuskulenai Case. While this individual demonstrated typical gunshot entry and exit wounds to the cranium consistent with high‐velocity trauma, the bone plug indicates that the projectile likely lost much of its kinetic energy while traveling through the cranium resulting in a low‐velocity impact at the exit site. This study reviews a similar instance of a bone plug recovered from a bioarcheological sample in Peru and emphasizes the importance of thorough archeological excavations of mass graves.     

A Molecular Method to Detect Wound Cells in Bloodstains Resultant of Sharp Force Injuries for Crime Scene Reconstruction

Previous research by the authors on an animal model showed that bloodstains can contain additional information about their somatic origin in the form of wound cells. Bloodstains produced by a gunshot wound to the head were distinguished from bloodstains produced by a gunshot wound to the chest by testing the stains for a brain microRNA marker. In this study, the effectiveness of the technique was examined on blood drops shed externally from a stab wound to the liver of rat carcasses. Specifically, investigations were conducted on the liver microRNA marker, rno‐mir‐122‐3p, with the QIAGEN miScript System, and PCR analysis. Between the two stabbing methods used, 67% of the scalpel blades and 57% of the blood drops tested positive for rno‐mir‐122‐3p; however, other samples tested negative giving inconclusive results as to the wound‐of‐origin. The amount of the liver cells in the bloodstains appeared to be related to the extent of trauma.     

原发性脑干损伤组织学诊断要点

目的 探讨人头面部受击致原发性脑干损伤的组织病理学诊断标准。方法 用171例脑干损伤者的脑干颅神经根部作水平切块,HE染色光镜下观察;另取50例非脑干损伤死亡者作对照。并用统计学方法分析两组所见病变。结果 损伤组见脑干浅表部(32.7％-55.6％)或脑干内部挫伤(57.9％-85.9％)、颅神经根部损伤(动眼神经70.2％,面及位听神经53.8％,三叉神经49.7％等)、星形胶质细胞反应性增生、肥大(34.5％-57.3％)及水肿(19.3％-57.3％)改变,与对照组相比,脑干的挫伤、颅神经根部损伤和星形胶质细胞反应性增生、肥大有显著差异(P<0.001),水肿无明显差异(P>0.05)。结论脑干浅表部和/或脑干内部挫伤、颅神经根部损伤及星形胶质细胞反应性增生肥大可作为原发性脑干损伤的诊断依据。     

A Qualitative Examination of the Self-Medicating Hypothesis Among Female Juvenile Offenders

Substance misuse among criminally delinquent youth has typically been described as a concurrent part of their participation in risky and delinquent behavior. Using Khantzian’s self-medication hypothesis, this article presents an alternative view by presenting qualitative data which suggests that substance misuse for female juvenile offenders may serve as self-medication for mental health problems stemming from early trauma, often at the hands of their families. Based on the narratives of 30 female juvenile offenders, this article examines the lived experiences of girls with childhood trauma and substance misuse, followed by arrest and incarceration. The paper concludes with recommendations for juvenile justice and child welfare practitioners.     

实验性脑挫伤GFAP,PCNA免疫组织化学研究

采用改进的Ｆｅｅｎｅｙ氏落体打击致Ｗｉｓｔａｒ大鼠脑挫伤模型,利用免疫组织化学染色,观察大鼠实验性脑挫伤后ＧＦＡＰ、ＰＣＮＡ的改变,用图像分析仪对免疫组化染色阳性细胞灰度和面积进行测量,ＳＡＳ统计软件分析,得出脑挫伤后ＧＦＡＰ、ＰＣＮＡ变化的时间规律性,为脑挫伤形成时间推断提供新的手段。ＧＦＡＰ阳性细胞面积和灰度伤后呈上升趋势,伤后３ｈ,即有显著增加（Ｐ＜０．０１）,至４天时,阳性细胞灰度,面积达到峰值（Ｐ＜０．０１）,伤后７天仍保持较高水平（Ｐ＜０．０１）。ＰＣＮＡ阳性细胞伤后１２ｈ出现,灰度呈上升趋势,面积呈下降趋势,伤后３天时达到最大值（Ｐ＜０．０１）。因此,ＧＦＡＰ、ＰＣＮＡ免疫组织化学染色阳性细胞的数量,灰度,面积改变有时间性规律;推断２～７天的脑挫伤则以ＧＦＡＰ、ＰＣＮＡ改变为主;脑挫伤后星形胶质细胞数目增多,只有很少一部分为星形胶质细胞增殖而来,且这种增殖在伤后３天时达到高峰     

Validation study of endogenous reference genes for normalization of quantitative real time PCR data in post mortem skin tissue

Gene expression profiling may provide insights into the molecular mechanisms underlying wound skin repair contributing to applications in the field of post mortem forensic wound age estimation. Quantitative real-time PCR (qPCR) is the most sensitive technique for gene expression studies but represents a complex procedure with several requirements for accurate data analysis. One of the major pre-requisites is the selection and validation of appropriate endogenous control genes for normalization in a given set of samples. In this study, the gene expression stabilities of ten endogenous controls ACTß, ß2M, PPIA, GAPDH, HPRT1, PGK1, SDHA, TBP, UBC, and YWHAZ were evaluated in two sets of samples: post mortem human wounded and unwounded skin tissue aimed for subsequent wound age estimation studies. The most stable genes were determined by calculating a gene expression normalization factor for each sample based on the geometric mean of the ten selected genes using the reference gene validation software, geNorm. In uninjured skin, the most stable endogenous controls were YWAZ, with the highest stability, followed by PGK1 and GAPDH. In the wounded samples, GAPDH was the most stable gene followed by PGK1, ACTß and HPRT1. Both groups revealed some differences relative to reference gene expression stabilities, nevertheless, PGK1 and GAPDH could serve as two possible common reference genes for qPCR data normalization as both were identified as stable genes in wounded and unwounded skin tissues. The differences of expression stabilities of the reference genes identified in both groups support the need for accurate normalization of gene-expression levels previous to qPCR studies.     

p38MAPK信号转导通路与组织损伤

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是广泛表达的丝氨酸/酪氨酸激酶,在哺乳动物细胞多种信号转导通路中起重要作用。MAPK有4个主要亚族:ERK、p38MAPK、JNK和ERK5。p38信号通路是MAPK通路的一重要分支,它在炎症、细胞应激、凋亡、细胞周期和生长等多种生理和病理过程中起重要作用。本文就p38MAPK的分型、分布、信号通路的组成及其在法医损伤学领域中的应用现状做一综述,旨在进一步对组织损伤程度和伤后时间推断的法医病理学研究提供参考资料。