溺死大鼠肺组织AQP-1、AQP-4的表达

目的观测生前溺死与死后抛入河水中大鼠肺组织水通道蛋白-1(aquaporin-1,AQP-1)、AQP-4的表达变化。方法将30只健康雄性Wistar大鼠随机分为河水溺死组、抛尸入水组及脱颈致死组。HE染色观察各组大鼠肺组织形态学变化,实时荧光定量PCR法检测各组大鼠肺组织AQP-1、AQP-4 m RNA的表达,免疫组织化学、Western印迹法检测其蛋白表达。结果免疫组织化学法和Western印迹法检测河水溺死组AQP-1蛋白的表达高于抛尸入水组和脱颈致死组(P0.05)。免疫组织化学法显示河水溺死组AQP-4蛋白的表达高于抛尸入水组和脱颈致死组(P0.05),而Western印迹法未检测出三者的差别(P0.05)。河水溺死组大鼠肺组织的AQP-1、AQP-4 m RNA表达高于抛尸入水组(P0.05)。结论河水溺死组的大鼠在急性肺损伤时肺组织AQP-1、AQP-4 mRNA及蛋白表达增高可能对生前溺死与死后抛尸的鉴别有一定意义。     

The usefulness of lung surfactant phospholipids (LSPs) in the diagnosis of drowning

The authors have studied the usefulness of some lung surfactant phospholipids (LSPs) isolated from lung tissues as markers of drowning. Two different groups of rabbits were sacrificed by drowning in fresh and salt water, and their phospholipid compositions were compared with those of a non-drowned control series. For the phospholipids studied in lung lavages (phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl glycerol) the proportions differed between the control group and the drowned group, and between the fresh-water and salt-water drowned animals. According to these results, the lipids we have analyzed can be employed as markers in forensic autopsies, where it is necessary to differentiate between death by drowning and postmortem immersion and between fresh-water and salt-water drowning. In lung tissue, only phosphatidyl choline and phosphatidyl inositol showed significative differences. These results also confirm that LSPs are strongly affected in drowning.     

Microbiota Composition and Pulmonary Surfactant Protein Expression as Markers of Death by Drowning

Pathological diagnosis of drowning remains a challenge for forensic science, because of a lack of pathognomonic findings. We analyzed microbiota and surfactant protein in the lungs for a novel diagnosis of drowning. All rats were divided into drowning, postmortem submersion, and control groups. The water, lungs, closed organs (kidney and liver), and cardiac blood in rats were assayed by targeting 16S ribosomal RNA of Miseq sequencing. Lung samples were analyzed by immunohistochemical staining for surfactant protein A. The closed organs and cardiac blood of drowned group have a lot of aquatic microbes, which have not been detected in postmortem submersion group. Furthermore, intra‐alveolar granular staining of surfactant protein A (SP‐A) was severely observed in the drowned group than the postmortem submersion and control groups. The findings suggested that the presence of aquatic microbiota in the closed organs and increased expression of SP‐A could be markers for a diagnosis of drowning.     

大鼠溺死后肺组织与血清IL-1β、IL-13mRNA表达变化

目的检测大鼠肺组织和血清中IL-1β、1L-13mRNA表达变化,探讨IL-1β、IL-13mRNA的变化情况与溺死的关系。方法 12只SD大鼠随机分为对照组、溺死组(实验组)。分别提取各组大鼠右侧肺下叶及右心室血清,应用TaqMan探针法检测肺组织及右心室血清IL-1β、IL-13mRNA的表达情况。结果 (1)溺死组大鼠肉眼观察:体表征象及解剖所见符合生前溺死特征。(2)肺组织中IL-1β、IL-13mRNA表达检测:与对照组比较,实验组IL-1β、IL-13表达呈下降趋势,差异无统计学意义(p0.05)。(3)血清中IL-1β、IL-13mRNA表达检测:与对照组比较,实验组IL-1β表达显著升高(p0.01)、IL-13表达显著升高(p0.01)。结论 (1)IL-1β、IL-13 mRNA在溺死组肺组织中表达呈下降趋势,可能是大鼠溺死出现代偿性抗炎反应综合症导致免疫无能的表现。(2)IL-1β、IL-13 mRNA在血清中表达明显升高,可能与溺水应激及溺死后急性肺损伤创伤应激有关。     

Potential Forensic Application of Receptor for Advanced Glycation End Products (RAGE) as a Novel Biomarker for Estimating Postmortem Interval

Studies which estimate the postmortem interval (PMI) based on diatoms or bacterial communities have been well‐conducted. However, predicting the PMI based on microorganisms has limited accuracy. The objective of this study was to discover specific biomarkers for estimating the PMI for drowning. The in vivo experimental model consisted of rats classified into nine groups (seven drowned groups from Day 1 to Day 7 and two control groups). Their lung tissues were analyzed for the receptor for advanced glycation end products (RAGE) mRNA expression determined by qRT‐PCR. RAGE protein levels were determined by Western blotting and immunohistochemical (IHC) staining. RAGE mRNA and protein levels gradually degraded over time. Immunostaining analysis revealed that RAGE in the Day 1 sample was significantly higher than that in the Day 6 or Day 7 samples. These results indicate that RAGE in drowned lungs could be a potential biomarker for the determination of the PMI after drowning.     

Morphometric Investigation of Death by Asphyxia

Abstract:  The aim of this study was to investigate the possibility of distinguishing deaths by asphyxia from those due to natural causes by comparing morphometric measurements in lungs. The study population comprised 27 subjects: 14 cases of death by asphyxia (hanging or drowning) and 13 cases of sudden natural death. Eighty parenchyma sections were used for each subject. Data were collected by computerized image analysis. Measurements aimed at quantifying, as percentages, pulmonary parenchyma (mean values of positive-fraction areas [PFA] and their standard deviations). Subjects who died of natural causes and of drowning showed a relative internal homogeneity compared to those who hanged. Results show significant discrimination between drowned subjects and those dying of natural causes (mean of PFA p  = 0.01) and between hanged and drowned subjects (SD of PFA p  = 0.04). Attention must be paid to the possible role played by senile emphysema. The method is proposed as a complementary tool in forensic cases.     

Plasmatic levels of atrial natriuretic peptide (ANP) in drowning. A pilot study

Bodies found in water may cause problems for forensic pathologists who have to differentiate drowning from postmortem immersion or fresh from salt water drowning. The exact physiopathology of drowning is still controversial and complementary tests can not exactly establish the exact cause of death if macroscopic findings at autopsy are not conclusive. We have employed atrial natriuretic peptide (ANP) as a marker in an experimental series of fresh and salt water drowning, comparing their results with a non-drowned control series. There are differences between the plasma basal levels of the control series (79 pg/ml) and the levels in animals drowned in fresh water (358 pg/ml, P less than 0.001) and between control and rabbits drowned in salt water (190 pg/ml, P less than 0.001). According to these values, there are also differences between fresh and salt water drowned animals (P less than 0.001). We propose this peptide as a new marker in cases of drowning, with the ability to differentiate drowning from postmortem immersion and fresh from salt water drowning.     

检测肺锶含量鉴定溺死的进一步研究

探索检测肺锶含量鉴定溺死的法医学意义，及水中浸泡时间因素对肺锶含量的影响和本方法的实际应用价值，用ICP检测溺死及死后入水浸泡时间较久的不同时间组（48～96小时）的家兔肺锶含量。结果表明：溺死组肺锶含量显著高于死后入水组（P＜0．01），死后水中浸泡时间（4天）对肺锶含量无明显影响，3例实际检案检测结果证实，本研究具有良好的应用前景。     

PCR-DGGE法检测浮游生物16S rDNA在溺死鉴定中的应用

目的建立并评估PCR-DGGE法检测浮游生物对溺死鉴定的应用价值。方法大白兔30只随机分为3组:溺死组(n=12),死后抛尸组(n=12)和对照组(n=6);溺死组和死后抛尸组又分为2个亚组:东湖水域组(n=6)和墨水湖水域组(n=6)。死后提取心血和肺、肝、肾、脑等组织,匀浆后,采用Percoll密度梯度离心法分离浮游生物并提取其DNA,PCR扩增浮游生物特异的16S rDNA片段后分别用琼脂糖凝胶电泳及DGGE检测分析。2个溺死案例检材同法检验。结果溺死组各组织器官中浮游生物检测多呈阳性:肺(100%)、肝(83%)、肾(75%)、心血(83%)、脑(42%);死后抛尸组仅2例肺组织(16.7%)检出阳性;对照组全部阴性。溺尸肺组织DGGE分型图谱与相应溺死点水样分型图谱相似,而与非溺死点水样分型结果差异显著。2实际案例均呈阳性。结论本方法不仅有助于定性诊断溺死,而且通过比较产物的多样性可以推断溺死地点,在法医学溺死鉴定中具有较大实用价值。     

Pulmonary surfactant-associated protein A levels in cadaveric sera with reference to the cause of death

Pulmonary surfactant-associated protein A (SP-A) is an exclusively lung specific protein, and is considered to leak into the blood stream in alveolar septal damage. In this study we examined the serum SP-A level in forensic autopsy materials using an enzyme immunoassay with monoclonal antibodies to assess the postmortem level in relation to the cause and mode of death. Although a gradual postmortem degradation should be taken into consideration, topological relationship of serum level seemed to be fairly stable (arterial> or =venous blood in most cases), indicating no evident influence of postmortem diffusion. Significant elevation of serum SP-A (76.7-250 ng/ml in left heart blood) was observed in hyaline membrane diseases from various causes independent of the postmortem intervals (<30 h). However, mean SP-A levels in postmortem heart blood were usually low in asphyxia including hanging, strangulation and choking (left, 25.5 ng/ml; right, 22.3 ng/ml), polytrauma (left, 13.1 ng/ml; right, 9.0 ng/ml) and stab wound to the neck (left, 34.1 ng/ml; right, 29.4 ng/ml). Prominent elevation was noted in a case of fatal strangulation with complication of idiopathic interstitial pneumonia, and also in some deaths due to drowning, burns in fires, blunt and gunshot chest injuries. These findings indicated that postmortem elevation of serum SP-A levels was associated with alveolar septal damage due to inflammation, mechanical and physical stresses, which caused leakage of SP-A into the bloodstream.     

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.     

Organ weight effects of drowning and asphyxiation on the lungs, liver, brain, heart, kidneys, and spleen

An examination of the organ weights associated with victims of drowning, asphyxiation and trauma was undertaken to determine (a) the effects of asphyxiation compared to a trauma group, and in turn, (b) the effects of drowning compared to an asphyxiation group. Included in the study were 217 drowning deaths, 166 pure asphyxiation deaths and 381 trauma deaths. The effects of asphyxiation (compared to trauma) resulted in elevated mean organ weights for the lungs, liver, kidneys and spleen (with mean increases of 17.8, 10.5, 10.3 and 23.4%, respectively). Effects of drowning (compared to asphyxiation) resulted in elevated mean organ weights only with the lungs and kidneys (with mean increases of 30.0 and 4.4%, respectively). Only the mean heart and brain weight remained constant across all experimental groups. A picture of drowning is suggested in which elevated lung and kidney weights are the result of both asphyxiation and the aspiration of water that occurs with drowning, whereas elevated spleen and liver weights in drowning victims are associated with only the effects of asphyxiation. In addition, the common autopsy finding of a small, anemic spleen in drowning, rather than caused by some pathophysiological mechanism of death, is hypothesized to be a postmortem phenomenon.     

Study of the diagnostic value of iron in fresh water drowning

The aim of our study was to test the diagnostic value of iron (Fe) in fresh water drowning by investigating the postmortem levels of hemodilution in drowning cases compared to control cases. Twenty-six typical fresh water drowning cases were selected from 128 immersion cases autopsied in our Department of Forensic Pathology between 1998 and 2004. The exclusion criteria were a long postmortem interval and other causes of death than drowning. For all selected cases, the diagnosis of drowning was based on the presence of autopsy findings and positive diatom test. A control population of 12 cases was also selected. For each drowning and control case, iron blood levels were measured in the left ventricle (LV) and right ventricle (RV) of the heart. The mean difference of iron concentration (RVFe-LVFe) between the drowning group and the control group was statistically compared. Furthermore, iron measurements were performed in 19 drowning cases showing advanced putrefaction. The mean difference of iron concentration was significantly higher in the drowning cases compared with controls (P<0.001). All drowning cases showed hemodilution. No overlap was found in the RVFe-LVFe levels between the two groups. Resuscitation attempts seemed to have no effect on the results. In cases of drowning showing advanced putrefaction, the iron test was not reliable because biochemical iron measurement was often prevented by no sufficient blood in the heart or postmortem clots. In conclusion, according to our results, iron seems to be a good biochemical marker in fresh water drowning with a short postmortem interval.     

What is the incidence and significance of "dry-lungs" in bodies found in water?

BACKGROUND: Drowning without aspiration of liquid, generally attributed to death from asphyxia while submerged and in laryngospasm, has been reported to occur in approximately 10% to 15% of drowning victims. OBJECTIVES: The occurrence of "dry-drowning" recently has been questioned and the hypothesis developed that "dry-lungs" in bodies found dead in the water could conceal more natural deaths than previously recognized. METHODS: Based on 578 selected adult victims who presumably drowned, we analyzed the correlation between the cases with a low combined lung/pleura liquid weight (< 1000 g and < 750 g) and a wide set of individual, circumstantial, and postmortem (PM) variables, using multivariate logistic regression analysis. Victims with lung weight < 1000 g were screened for long-QT syndrome (LQTS) founder mutations in KCNQ1 and KCNH2 genes. RESULTS: Of the 578 victims, 120 (20.7%) had a lung weight of < 1000 g, and 22 of these (3.8%) of < 750 g. Multivariate analysis showed a significant correlation for women (P < 0.001), for women aged 65 years or older (P < 0.001), and for men with prolonged PM submersion time (P < 0.001). "Normal" lungs were found in only 8 (1.4%) victims. Low-weight (< 1000 g), overdistended lungs with no sign of liquid penetration were seen in 11 (1.9%). No LQTS founder mutations were detected. CONCLUSIONS: The actual incidence of death of persons found in water who have normal lungs or do not have penetration of liquid into their airways, based on our study, is much lower (below 2%) than currently assumed.     

Organ weight effects of drowning and asphyxiation on the lungs,liver, brain,heart, kidneys,and spleen

An examination of the organ weights associated with victims of drowning, asphyxiation and trauma was undertaken to determine (a) the effects of asphyxiation compared to a trauma group, and in turn, (b) the effects of drowning compared to an asphyxiation group. Included in the study were 217 drowning deaths, 166 pure asphyxiation deaths and 381 trauma deaths. The effects of asphyxiation (compared to trauma) resulted in elevated mean organ weights for the lungs, liver, kidneys and spleen (with mean increases of 17.8, 10.5, 10.3 and 23.4%, respectively). Effects of drowning (compared to asphyxiation) resulted in elevated mean organ weights only with the lungs and kidneys (with mean increases of 30.0 and 4.4%, respectively). Only the mean heart and brain weight remained constant across all experimental groups. A picture of drowning is suggested in which elevated lung and kidney weights are the result of both asphyxiation and the aspiration of water that occurs with drowning, whereas elevated spleen and liver weights in drowning victims are associated with only the effects of asphyxiation. In addition, the common autopsy finding of a small, anemic spleen in drowning, rather than caused by some pathophysiological mechanism of death, is hypothesized to be a postmortem phenomenon.     

A promising microbiological test for the diagnosis of drowning

A number of biological and chemical tests have been developed over the years to determine whether a person was drowned. This study focuses on the potential of a microbiological test for detecting common bacterial markers of water faecal pollution such as faecal coliforms (FC) and faecal streptococci (FS) as possible indicators of drowning. A promising previous study was carried out on central and peripheral blood samples of 42 drowned victims (20 cases in saltwater and 22 cases in freshwater) and 30 not-drowned bodies. To improve the accuracy of our previous results and also in order to investigate a possible cause of a false positive due to pulmonary passive diffusion and subsequently endogenous or exogenous bacterial invasion of the blood in the post-mortem interval (PMI), the FC and FS test was applied to bodies submerged in water but died from causes other than drowning. In the present study, blood samples collected from the left ventricle (LV), right ventricle (RV), femoral artery (FA) and, femoral vein (FV) of 10 drowned victims (5 cases in freshwater and 5 cases in seawater) and 3 not-drowned individuals with bodies submerged in water for a while after death have been analysed. Preliminary results are in agreement with other reports dealing with diatoms and marine bacteria that suggest to exclude the hypothesis of a passive penetration of sufficient quantities of drowning medium into circulation after death or during the agonal period. Based on our results there is also no evidence of a relevant dissemination of endogenous micro-flora from the gastrointestinal tract affecting the FS and FC test. There are still several other factors that could influence the applicability of post-mortem FS and FC cultures for the diagnosis of drowning and they need further investigations. The present article provides only a glimpse of the potential of the FS and FC test as bacteriological method for the diagnosis of drowning.     

Scanning electron microscope study of the lung in drowning

The authors describe the results obtained using the scanning electron microscope (SEM) to study drowned lungs and control lungs. Results are compared with a previous study carried out using laboratory animals. The results from both studies are analogous, in the absence of chronic pulmonary emphysema. The diagnosis of chronic pulmonary emphysema is simple with the SEM even when dealing with autoptic material obtained 24 to 48 h after death, but the diagnosis of drowning becomes difficult in lungs with chronic emphysema.     

检测浮游生物叶绿素相关基因诊断溺死的实验研究

目的评估浮游生物叶绿素相关基因检测用于溺死诊断的价值。方法将18只大白兔随机分为溺死组（n=10）、死后抛尸组（n=6）和对照组（n=2）,各组分别取心血及肺、肝、肾、脑组织,分离浮游生物并提取其DNA,用PCR技术检测叶绿素相关基因EG（EG1和EG2）及SK（SK1和SK2）。同时用硝酸消化法检验肺和肝组织中的硅藻。结果溺死组心血及肺、肝、肾、脑组织中EG1分别检出9、10、9、7和8例阳性,EG2分别检出8、10、7、5和7例阳性;死后抛尸组仅在心血和肺组织中各检出1例EG1阳性;对照组各组织均未检出EG1和EG2。SK1、SK2除在溺死组心血、肺和肾有少数检出外（≤2例）,在其他组未检出扩增产物。硝酸消化法从溺死组肺、肝组织中分别有9例及3例检出硅藻,死后抛尸组仅在1例肺组织中检出。结论浮游生物叶绿素相关基因EG用于溺死诊断的阳性检出率要高于硝酸消化法,在溺死诊断中具有较高的应用价值。     

溺死尸体器官硅藻检出率季节性差异

目的 研究溺水死亡者肺、肝、肾、骨髓等器官淡水硅藻检出率可能存在的季节性差异,为法医学解决水中尸体死因鉴定提供技术帮助与科学证据.方法 收集2009～2011年1、4、7、10月沈阳市浑河中溺死尸体每月(组)各5例;45只家兔分相应月份生前、死后入水组及对照组,分别于当月发现水中尸体后第2天入水,取人及兔肺、肝、肾、股骨骨髓及现场水样备检,应用酶消化法检测各组器官中硅藻数量、种属并与现场水样比对.结果 各组溺死尸体肺中均检出多量硅藻且种属与现场水样一致,春、秋及冬季溺死尸体肝、肾中仅可检出少量体积较小的小环藻等中心纲硅藻,夏季肝、肾及骨髓中均未检出硅藻;各组溺死家兔肺中均检出少量小环藻等中心纲硅藻,肝、肾及骨髓中未检出硅藻.结论 硅藻种群的季节性变化及不同种群硅藻体积的差异可能造成肝、肾等大循环器官中硅藻检出率的季节性差异;仅肺中检出硅藻,肝、肾中硅藻检验结果阴性并不能排除溺死可能.     

Factors affecting strontium absorption in drownings

This study examines the effects of age, gender, a cold water medium versus warm water medium, and salinity on strontium levels determined in left ventricular blood in drownings. Significant differences in the amount of strontium absorbed into the bloodstream (p<0.001) were detected between individuals who drowned in fresh water versus those drowning in seawater, and between those drowning in cold water versus warm water (p=0.030). However, no significant differences were noted in the strontium concentrations of left ventricular blood according to gender or age.