Shuji Kozawa

Background: Deaths due to heat stroke have surged in recent years in Japan. While the long-term sequelae of heat stroke in its survivors are well understood, our previous studies suggest that heat exposure may cause fatal organ damage before the systemic inflammatory response is triggered. In this study we aimed to provide a comprehensive analysis of cytokines and other signaling factors in sera and myocardial tissues in a rat model of heat stress. Methods: Male Wistar rats were anesthetized and subjected to heat stress (37.0°C, 100% humidity; n = 8) to induce heat stroke (Heat group, n = 4) or normal temperatures (Control group, n = 4) for 90 min, followed by collection of blood sera and heart tissue samples. A multiplex assay system was used to analyze serum markers. Heart tissue was subjected to multiplex RNA sequencing and gene cascade analysis, and combined with iTRAQ (isobaric tags for relative and absolute quantitation) protein pathway analysis. Results: There were significantly higher levels of five interleukins (IL-1α, IL-2, IL-6, IL-17A, IL-18), fractalkine, interferon-γ-inducible protein-10, leptin, tumor necrosis factor α, and vascular endothelial growth factor, and significantly lower levels of LIX and RANTES, in the sera of the Heat group compared with the Control group. From the 2,741 genes that showed a significant difference in myocardial mRNA expression between the groups, we identified 48 transcription factors and 34 binding regions. Pathway analysis identified the involvement of key node proteins including protein kinase C isoforms, calmodulin-dependent protein kinase II, and protein interacting with C-kinase (PICK1). Conclusion: This model of heat stress in rats triggered changes in the mRNA and protein levels of many genes involved in cardiac function, primarily affecting the activity of ion channels and mitogen-activated protein kinase.

Glioblastoma multiforme (GBM) is the most common primary intracranial neoplasm. Although maternal mortality in Japan is among the lowest in the world, the main cause of maternal mortality is pregnancy-related intracranial hemorrhage. This case reports a 30-year-old female pregnant patient who suddenly died at 30 weeks of gestation. She had complained of headache from early stages during pregnancy and reported a headache and stiff neck starting two weeks before her death. Postmortem computed tomography revealed an intracerebral hematoma. The autopsy of the brain revealed severe swelling. The left frontal lobe contained a cavity filled with clear fluid, and a hematoma weighing 24 g. There was also widespread cerebral edema. The histological findings after evaluation of the cavity wall material were indicative of malignancy, and positive immunostaining for alpha thalassemia/mental retardation syndrome X-linked (ATRX), p53 and isocitrate dehydrogenase (IDH-1), was consistent with a diagnosis of GBM. There are studies that indicate pregnancy can promote the clinical and radiological advancement of glioma. The acceleration of tumor growth during pregnancy has been found to depend on several factors (e.g., hormonal factors, growth factors, and hemodynamic changes). Increased cardiac output and blood volume have been shown to promote the size of vascular tumors during pregnancy. Although headaches and neck stiffness are often considered pregnancy-related, this case highlights the importance of its prompt diagnosis, which can also save the fetus’ life. It also emphasizes the need for forensic pathologists to further evaluate intracerebral hematomas identified during investigations of maternal deaths during pregnancy.

This study presents an autopsy case of unexpected sudden death caused by mitochondrial disorders with a subdural hematoma suspected to have manifested from Pearson’s marrow pancreas syndrome in a four-year-old boy. The cause of death was not determined by his history and autopsy, but was later determined via a postmortem examination. The boy was unable to walk unaided four days before death. On the day of death, he developed a fever of 37.4 °C and coughed to the point of vomiting when given water by his mother, after which he became limp with fatigue. He was brought to the hospital by ambulance, but he died without recovering. The boy had many symptoms but no diagnosis. During the autopsy, a subdural hematoma was identified, and severe fatty changes were observed in the liver. Biochemical analysis and molecular testing revealed no particular metabolic disorders. The activity level of mitochondrial respiratory chain complex I was significantly decreased in the liver, whereas that of complex II was slightly decreased. Although the activity levels of complexes I to IV were all decreased in the cardiac muscle, a particularly marked decrease was observed for complex I. It was therefore concluded that a subdural hematoma without cerebral herniation in this case would not normally be a direct cause of death, but it suspected fatal in this case because of the deteriorated systemic condition resulting from the patient’s mitochondrial disorder suspected by Pearson’s marrow pancreas syndrome.

An autopsy case of sudden death due to pulmonary arterial hypertension (PAH) in a 5-year-old boy whose cause of death was not determined during autopsy, but was later determined by postmortem examination, is presented. The boy developed convulsions that subsequently stopped, but remained unconscious. He was transported to hospital by ambulance, but died soon after. The boy had been found to have right ventricular overload on ECG 2 weeks earlier. A plan had been made to consult a doctor for a specialist visit 2 months later. During autopsy, significant abnormalities or injuries were not observed on the body's external surface. Internal examination showed congested organs, and the blood remaining in the body was dark red with fluidity. The heart was significantly enlarged (146 g), with nearly equivalent thickness of the left and right ventricles, showing right ventricular hypertrophy. Obvious macroscopic abnormalities were not observed at the origin and main trunk of the pulmonary artery. The lungs were slightly swollen (right lung 100 g, left lung 95 g), severely congested, and edematous. A postmortem CT scan displayed some patchy shadows in both lungs; however, no significant abnormalities were detected. Histopathological examination suggested a diagnosis of PAH. Three genes (BMPR2, ALK1, and ENG) were tested, revealing a heterozygous insertion of five nucleotides, TTTCC, between nucleotides 2677 and 2678 within exon 12 of the BMPR2 gene. Therefore, the subject was considered to have had heritable PAH due to a BMPR2 gene mutation.

Alcohol intake induces cardiovascular effects. We performed a comprehensive analysis of the gene expression profiles of myocardial tissues from a mouse model of alcohol feeding by microarray to clarify the effect of alcohol on myocardium from the perspective that the acute phase of alcohol use causes changes to myocardial injury related gene expression profiles, while the withdrawal phase is associated with changes in gene expression profiles related to myocardial protection. A model of single alcohol feeding was generated using pathogen-free, 7-week-old, male C57BL/6N mice administered a peroral injection of 7.0 g/kg ethanol (single alcohol-fed group group) or saline (control group). A repeated alcohol feeding group was generated following Lieber’s method. To investigate the effects of alcohol in the alcohol-fed group, the left ventricles of hearts were extirpated 1 h (alcohol group) or 24 h (withdrawal group) after the last alcohol feeding. Myocardial tissues were then subjected to RNA extraction and gene expression analysis by real-time reverse transcription polymerase chain reaction. Sera were also analyzed for various signal transducers, including 22 cytokines. Using RNA extracted from extirpated myocardia, mRNA expressions were comprehensively analyzed by one-color microarray (n=4) using SurePrint G3 Mouse Gene Expression 8x60K (Agilent) containing 38,640 gene probes as the microchip, and gene ontology and gene cascade analyses were performed. Differing gene profiles were seen in the repeated alcohol intake group. In the acute phase of alcohol intake, there were changes in gene profiles related to activation of the STAT pathway and blood cytokine-mediated inflammatory responses to hypercardia. In the withdrawal from alcohol phase, changes in gene profiles related to STAT activity did not persist, although gene profile changes related to apoptosis and the development of sudden death were observed. Further detailed investigations of the effects of alcohol on cardiomyocytes are planned.

Heatstroke damages various organs, such as the brain and heart, either directly by heat exposure or by systemic inflammatory responses because of hypercytokinemia. Electrolyte drink intake before heat exposure might suppress heat exposure induced cardiac stress. This study aimed to determine how electrolyte drink intake before heat exposure modulates heat exposure-induced damage to the diencephalon and brain stem in a rat model. After giving rats an electrolyte drink or water for 1 week, they were exposed to heat in a CO2 incubator for 90 min at 37°C. Following this, they were decapitated and their diencephalon and brain stem immediately harvested. Total RNA was isolated from these tissues and cDNA was synthesized by reverse transcription. In addition, relative mRNA expression of the Finkel–Biskis–Jinkinsosteosarcoma oncogene (c-fos), heat shock 70 kD protein 1A (Hspa1a), heat shock 70 kD protein 1B (Hspa1b), and heat shock protein B1 (Hspb1) were determined using real-time polymerase chain reaction. Results showed that heat exposure significantly pregulates each gene in the diencephalon and brain stem, indicating that heat stress affects these brain areas. Electrolyte drink intake before heat exposure did not modify gene expressions in the brain stem but suppressed the upregulation of c-fos, Hspa1a, and Hspb1 in the diencephalon, the body’s thermoregulatory center. These findings suggested that electrolyte drink intake before heat exposure alleviates heat stress in the diencephalon and might consequently suppress the upregulation of Fos (which regulates biological functions as a transcription factor) and genes related to heat shock proteins, which play a role in cellular protection. Therefore, electrolyte drink intake before heat exposure reduces the intensity of heat stress to the body and improves heat tolerance.

A case of sudden cardiac arrhythmia causing death following volatile substance misuse is presented. A 17-year-old man was found in his dormitory room having suffered a cardiac pulmonary arrest. He was brought to an emergency medical center where he was confirmed dead. The police investigation suggested butane gas aspiration since cans for portable cooking stoves were found in his room, but whether they were involved in his death was unclear. Thus, a judicial autopsy was performed 8 hours after his death. The autopsy found no severe injuries that could have led to death. Inhalation of butane gas was strongly suspected, and a screening test of intratracheal gas and femoral venous blood samples collected at autopsy detected butane. Histopathologically, all organs examined showed congestion. Endocardial hyperplasia on the left ventricular wall was confirmed on cardiac examination, and the lungs showed the transformation of the lumina of the tracheal branches due to constriction, indicating repeated stimuli. Thus, the autopsy and histological findings did not indicate any pre-existing pathological findings as a possible cause of death. Gas chromatography-mass spectrometry showed trace levels of isobutene and n-butane, while gas chromatography analysis showed trace levels of 2-butanone, which is only detected in decedents with a history of chronic butane misuse, in samples of femoral blood, cerebrum, brain stem, heart, lungs, liver, stomach contents, iliopsoas muscle, and fat tissues (from the abdomen). Thus, the autopsy findings led to the conclusion that the decedent habitually inhaled butane gas and died due to butane gas poisoning.

Chronic alcohol consumption remains a global health problem and has been implicated in many chronic and acute diseases. Alcohol consumption is regarded as a major risk factor for sudden cardiac death, with chronic alcohol consumption contributing to collagen accumulation, myocardial fibrosis, and arrhythmias. Although various signal transducers are believed to be involved in the instigation and progression of alcohol-induced cardiac dysfunction, the mechanism remains poorly understood. In the present study, mRNA and protein levels of sarco/endoplasmic reticulum Ca2+ ATPase type 2a (SERCA2a), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-3 (MMP-3) were measured in a repeated-ethanol-exposed mouse model to investigate the effect of chronic alcohol consumption on cardiac tissue. We hypothesized that chronic alcohol consumption brings about an imbalance in the expression of MMPs and TIMPs, and also alters the regulation of proteins like SERCA2a and CTGF, resulting in myocardial remodeling and cardiac dysfunction. Cardiac tissue isolated from ethanol-exposed mice and non-exposed mice, after 6 weeks on diet, were assayed for SERCA2a, CTGF, TIMP-1 and MMP-3 expression by western blotting (for protein expression) and quantitative reverse transcription-polymerase chain reaction (for mRNA expression). Our data revealed that ethanol exposure in C57BL/6N mice yielded a significant accumulation of the transcripts of Ctgf and Mmp3, without significantly affecting the levels of the corresponding proteins. TIMP-1 mRNA and protein expression were not changed by ethanol exposure. Notably, expression of the SERCA2a protein was reduced significantly in the ethanol group compared to the control group, despite the lack of significant changes in accumulation of the corresponding mRNA. Our findings on mRNA expression of the Ctgf, MMP3, and TIMP1 encoding genes are consistent with previous reports on protein expression of these signal transducers, given that the data for the western blots did not reveal significant changes in accumulation of these proteins. Therefore, we conclude that the MMP/TIMP imbalance induced by extended alcohol exposure may contribute significantly to myocardial remodeling and cardiac dysfunction, which is in turn responsible for sudden cardiac death in ethanol abusers.

Acute gastric volvulus resulting in abdominal compartment syndrome was determined to be the cause of death in a 4-year-old girl who presented with abdominal distension. At about 1AM on the day of her death, she was brought to our emergency medical center. Physical examination and plain abdominal X-ray revealed pronounced gastric dilatation. A decompression procedure was performed, followed by observation. She went into cardiopulmonary arrest around 1PM on the same day and died. Postmortem investigation, including an autopsy and computed tomography (CT), was performed to determine the cause of death. The findings included that the stomach was severely distended. Evidence was seen of mucosal hemorrhage in the gastric mucosa on the greater curvature side, which was thinned in places but without perforation. No necrosis of the gastric mucosa was observed; reversible changes were evident on histopathological examination. The postmortem CT images suggested that the pyloric region was positioned cranioventrally to the cardiac region. None of the findings indicated sudden blockage, and the cause of death was determined to be acute gastric volvulus resulting in abdominal compartment syndrome. The abnormal placement of the organs was difficult to determine based on physical examination alone; postmortem CT and careful examination were helpful in conducting the autopsy in this case.

Two newborn infants (one male and one female) were discovered dead and frozen in a home freezer. Although thawing is expected to lead to changes that may hamper postmortem investigations, the victims could not be examined in the frozen state and were thus immersed in saline at 37 degrees C to completely thaw them over about 90 min. Autolysis and putrefaction were not evident, postmortem changes were slight, and the internal organs were soft, allowing a thorough examination, including an autopsy and a histological investigation. Autopsy showed that both infants were full-term at the time of death. Hydrostatic tests of the lung and stomach indicated that the infants had been born alive, and based on histological analyses of pulmonary alveoli and bronchioles, they had started breathing. Malformations, pathological findings, and signs of suspected asphyxia were absent, and we assume that the infants were very likely to have been murdered. However, the cause of death is not yet fully understood. Although a police investigation revealed that the infants were smothered by occlusion of the mouth and nose and then frozen, there were no injuries on the skin around the nose and mouth. Nonetheless, we concluded that none of the postmortem findings were in conflict with this suspected mechanism of death. As these infants had been kept frozen since their death and thawed to body temperature as rapidly as possible in saline at 37 degrees C, their bodies were well preserved, which was helpful for the postmortem investigation.

A 34-year-old man was discovered by his coworkers in a tank filled with 35% (w/w) hydrochloric acid. Despite undergoing intensive treatment, he died one and a half days later. An autopsy revealed generalized high tensity, overall grayish brown skin color, heavy gastric submucosal hemorrhage and heavy pulmonary edema. We concluded that death was caused by burn shock due to wide, generalized chemical burn. Microscopic investigation of the burn in the area with grayish brown skin considered coagulation necrosis of full-thickness of the skin (third-degree or deep burn), revealed that the burn was judged to cover the partial thickness of the skin (second-degree or dermal burn). These findings suggest that chemical burn by hydrochloric acid results in a change of skin color due to chemical reaction so that the appearance of the chemical burn is more severe than the degree assigned by histological examination.

症例は65歳女性で、統合失調症にて長期入院中に胸痛が出現し、呼吸困難となり、酸素投与で一旦は回復したが、その後死亡した。臨床経過からは死因が判然としないため、承諾解剖を実施した。外表検査では前胸上部から顔面のうっ血、眼瞼眼球結膜の溢血点を認めた。解剖の結果、死因は機能性イレウスにより多量の腸管ガスが貯留し、横隔膜が挙上されて腹腔内圧が上昇するで呼吸運動が妨げられ窒息死したものと判断された。本症例では病理組織学的に大腸メラノーシスが認められ、その発生要因としてsennosideの長期服用が示唆された。

We studied the effects of chronic alcohol intake on the disposition of alcohol and its metabolites in the rat. We used male Wistar rats for all of the experiments in this study. Using a pair-feeding process, rats were fed a liquid diet containing alcohol or without alcohol for 6 weeks. Ethanol solutions (0.5, 1.0, 1.5, and 2.0 g/kg body weight [BW]) were administered as a bolus, intravenously. We then measured blood ethanol and acetate concentrations. Simultaneous multiline fitting was performed using mean blood alcohol concentration (BAC)-time curves fitted to the one-compartment open model with parallel first-order and Michaelis-Menten elimination kinetics. At low doses (0.5, 1.0, and 1.5 g/kgBW), no differences were observed between the alcohol group and the control group with respect to ethanol elimination rate, area under the curve of ethanol (AUC(EtOH)), and mean residence time of ethanol (MRT(EtOH)). At higher doses (2.0 g/kgBW), ethanol elimination rate in the alcohol group was significantly higher than in the control group (P<.5%). These findings were also substantiated by corresponding changes in AUC(EtOH) and MRT(EtOH). At low doses, no differences were observed between the alcohol group and the control group with respect to plateau concentration of acetate (AcT) (concentration of steady state=C(ss)AcT), area under the curve of AcT (AUC(AcT)), and mean residence time of AcT (MRT(AcT)). However, at higher doses, although there were no differences in C(ss)AcT, both AUC(AcT) and MRT(AcT) were significantly lower in the alcohol group when compared to the control group (P<.5%). Chronic alcohol consumption increases ethanol oxidation and AcT metabolism in rats, as observed at high blood alcohol concentrations (BACs). These effects were observed at BACs of 3.5-4.5 mg/ml, and were not observed at lower doses. Thus, with general alcohol consumption, interindividual differences and intra-individual changes in alcohol metabolism may not take into account increased or accelerated metabolism due to alcohol tolerance.

低出生体重児で精神障害をもつ3歳男児が、睡眠薬トリクロールシロップを誤飲し死亡した。解剖の結果、顔面及び四肢などに打撲傷が、気管内に吐物が認められた。慢性硬膜下血腫も認められ、その後半部に新しい出血が認められたことから、慢性硬膜下血腫から再出血をきたし、そのため脳浮腫・脳圧亢進が生じて嘔吐し、吐物の吸引により窒息死したものと判断した。本屍は前期破水・切迫早産のため約30週に1740gで出生し、生後は夜間の不眠が続き、母親から「情緒のコントロールがきかない」「言い出したら聞かない」「他の子供に暴力をふるう」「店の商品である食品や、夜中に冷蔵庫の物(生魚など)を手当り次第に食べる」などの訴えがあったが、遺伝子検査を含めた各種検査でも原因は判明しなかった。本件は親による虐待も疑われ、前記の訴えも「代理によるMunchausen症候群」による詐話の疑いがあったが、明らかにはならなかった。

An established approach to forensic bloodstain examinations is to first confirm that the suspected bloodstain is human blood and then to perform tests to determine individual identity. Although the confirmation of human blood step may be skipped in order to secure sufficient sample size for individual identification when the amount of bloodstain available for analysis is very limited, the validity of such partial examinations is likely to be challenged in court trials. Therefore, in order to perform complete examination of minute bloodstains, we developed a one-tube method in which hemoglobin and DNA were simultaneously extracted for use in confirmation of human blood and individual identification, respectively. Blood was diluted in 5 steps to produce 1- to 500-fold dilutions, and 1-μl aliquots were applied to tips of cotton-wool swabs. Prepared swabs were held at room temperature for periods up to 28 days. All swabs emitted light in a darkened room after being sprayed with luminol. Using 1.5-ml microcentrifuge tubes, the sprayed swabs were soaked in 100 μl of blood extraction buffer, one of the reagents in a DNA extraction kit designated for forensic samples. Following incubation, a 3-l aliquot of the resulting blood extraction fluid (bloodstain dissolved in blood extraction buffer) was subjected to immunoblotting against human hemoglobin A in order to confirm the presence of human blood. The remaining blood extraction fluid was used for DNA extraction. After the quantification of DNA, TH01, F13A01 and amelogenin loci, and mtDNA were amplified by PCR. All samples tested positive for human blood by immunoblotting. The amount of DNA extracted using this method was comparable with that of other extraction protocols. TH01 and the amelogenin loci gave similar results in that both loci were amplified in up to 5- or 20-fold dilutions of blood that had been held for 7 days and in 1- or 5-fold dilutions held for 28 days. F13A01 was less successful in that the locus was amplified in 1- or 5-fold dilutions held for 7 days, while amplification was only possible for 40% of samples at 1-fold dilution (no dilution) held for 28 days. In contrast, amplification was possible for mtDNA at concentrations as low as 500-fold dilution held for 28 days. Thus, the one-tube method allowed both confirmation of human blood and individual identification from minute bloodstains on single swabs; however, only mtDNA can be amplified in aged and/or diluted bloodstains.

直腸温(Tr)からの死後経過時間の推定には,Henssgeの式が世界的に用いられている.Henssgeの式は,2つの指数関数からなるため,解析的に解くことは不可能であるため,コンピュータプログラムおよびノモグラムによる解法を開発した.これまでノモグラムを使用してきたが,ノモグラム上で正確に線を引かなければならないという作業に煩わしさを感じていた.そこで,Tr=f(t)における直腸温Trと死後経過時間tとの関係を,Microsoft Excelでテーブルにし,死後経過時間tの概算値を簡便を試みた.各環境温を3度間隔とし,6ページの表に納めた.直腸温の測定値から死後経過時間を読みとるだけでなく,逆に,例えば死後12時間経過しているとすればこの程度の直腸温になるはずであるといった使い方もできた