The application of autophagy to thrombus age estimation in murine deep vein thrombosis model.

We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To perform the immunohistochemical analyses, we used anti-LC3 antibody and anti-p62 antibody for detecting the intrathrombotic autophagic functions. We estimated dynamics of the intrathrombotic autophagy as LC3+ cell number (×1000, five fields) with the thrombus ages (each group n = 5). The number of LC3+ cells was once decreased at 3 days, and then increased until 10 days. On the contrary, the number of p62+ cells progressively increased until 10 days after the inferior vena cava (IVC) ligation, and then gradually decreased. Especially, in all of thrombus samples with the postligation intervals of 5-10 days, both numbers were larger than 10. Subsequently, we compared the number of LC3+ cells to that of p62+ cells. Although, at 1 day after the IVC ligation, LC3+ cell number significantly exceeded p62+ cell number, the former was significantly or relatively less than the latter at 3 days or more after the IVC ligation. Thus, positive cells of > 10 in both LC3 and p62 indicated the thrombus age of 5-10 days. Upon comparison of immunopositive cells in LC3 and p62, the p62/LC3 ratio was > 1.0 in 29 out of 30 thrombus samples aged 3-21 days, and all of 1-day-old thrombus had the p62/LC3 ratio of < 0.5. Thus, the ratio of > 1.0 and that of < 0.5 could indicate thrombus age of 3 days or more and that of 1 day, respectively. Collectively, our study implied that the detection of autophagy-related molecules such as LC3 and p62 would be useful for the determination of thrombus age.

Forensic pathological study on temporal appearance of dendritic cells in skin wounds.

Dendritic cells (DCs) can essentially contribute to innate and adaptive immune system in various organs. A double-color immunofluorescence analysis was carried out with anti-CD11c and -HLA-DRα antibodies to detect DCs in 53 skin wounds (their postinfliction intervals: group I, 0-3 days; group II, 4-7 days; group III, 9-14 days; and group IV, 17-21 days). CD11c+HLA-DRα+ DCs were first observed in skin wounds with postinfliction intervals of 3 days, and the DC numbers were found to be elevated in skin wounds with the subsequent increase in postinfliction intervals. Semi-quantitative morphometric analyses showed that the DC number was the highest in the 12-day-old wound. More than 50 DCs were present in 8 of 10 samples (80%) in group II and 14 of 16 samples (87.5%) in group III, and there was no difference between the two groups. Thus, the presence of DCs in a skin wound was possibly estimated as postinfliction intervals of at least 3 days. Furthermore, when a skin wound contained > 50 DCs, its age would be judged as 4-14 days. Collectively, the appearance of DCs in human skin wounds may provide useful information in determining the age of a wound.

Possible application of CT morphometry of the calcaneus and talus in forensic anthropological identification.

Computed tomography (CT) data provide information for volumetric and radiographic density analysis. The present study investigated the application of virtual CT volumetry of the tarsal bones to estimation of the sex, stature, and body weight using postmortem CT (PMCT) data of forensic autopsy cases. Three-dimensional (3D) images of the bilateral foot bones of intact Japanese subjects after adolescence (age ≥ 15 years, n = 179, 100 males and 79 females) were reconstructed on an automated CT image analyzer system. Measured parameters were mass volume, mean CT value (HU), and total CT value of the talus and calcaneus. Mean CT values of these bones showed age-dependent decreases in elderly subjects over 60 years of age for both sexes, with significant sex-related differences especially in the elderly. The mass volumes and total CT values of the talus and calcaneus showed significant sex-related differences, and also moderate correlations with body height and weight for bilateral bones in all cases (r = 0.58-0.78, p < 0.0001); however, the correlations of these parameters of the female talus with body weight were insufficient (r = 0.41-0.61, p < 0.0001). These observations indicate the applicability of virtual CT morphometry of the talus and calcaneus using an automated analyzer to estimate the sex and stature in forensic identification; however, greater variations should be considered in body weight estimations of females.

Postmortem CT morphometry of great vessels with regard to the cause of death for investigating terminal circulatory status in forensic autopsy.

Postmortem CT (PM-CT) is useful to investigate the viscera in situ before opening the body cavities at autopsy. The present study involved a virtual morphometric analysis of thoracic and abdominal great vessels with regard to the cause of death as a possible index of terminal circulatory status in forensic autopsy cases, using PM-CT data of forensic autopsy cases within 3 days postmortem (n = 93). Perimeters and cross-sectional areas of the aorta and vena cava depended on the age and/or gender of subjects; however, when the vessel flattening index (vFI) was calculated as the ratio of the cross-sectional area (a) to the estimated circle area having the same perimeter (l), using the formula vFI = 4πa/l(2), the vFI showed distinct differences among the causes of death without significant postmortem time dependence. The index was low for each vessel in fatal bleeding, while the vFI of the abdominal aorta and inferior vena cava was low in hyperthermia (heatstroke), but higher in drowning, hypothermia (cold exposure) and sudden cardiac death. These CT findings provide quantitative data as supplementary indicators to reinforce autopsy findings for interpreting terminal circulatory status.

Virtual CT morphometry of lower limb long bones for estimation of the sex and stature using postmortem Japanese adult data in forensic identification.

The application of computed tomography (CT) is useful for the documentation of whole-body anatomical data on routine autopsy, virtual reconstruction of skeletal structure, objective measurements, and reassessment by repetitive analyses. In addition, CT data processing facilitates volumetric and radiographic density analyses. Furthermore, a recently developed automated analysis system markedly improved the performance and accuracy of three-dimensional (3D) reconstruction. The present study investigated virtual CT morphometry of lower limb long bones, including the femur, tibia, fibula, and first metatarsus, to estimate the sex and stature using postmortem CT data of forensic autopsy cases of Japanese over 19 years of age (total n = 259, 150 males and 109 females). Bone mass volumes, lengths, and total CT attenuation values of bilateral femurs, tibias, and fibulas correlated with the stature; however, the mean CT attenuation (HU) values showed age-dependent decreases. Correlations with the stature were similar for the lengths and mass volumes of the femur, tibia, and fibula (r = 0.77-0.85) but were higher for the mass volume of the first metatarsus (r = 0.77 for right and r = 0.58 for left). In addition, the ratio of the bone volume to the length of each bone showed the most significant sex-related differences (males > females with accuracy of 75.8-98.1 %). These findings indicate the usefulness of virtual CT morphometry of individual lower limb long bones, including volumetry, to estimate the sex and stature in identification.

Molecular pathology of brain matrix metalloproteases, claudin5, and aquaporins in forensic autopsy cases with special regard to methamphetamine intoxication.

Methamphetamine (METH) is a highly addictive drug of abuse and toxic to the brain. Recent studies indicated that besides direct damage to dopamine and 5-HT terminals, neurotoxicity of METH may also result from its ability to modify the structure of blood-brain barrier (BBB). The present study investigated the postmortem brain mRNA and immunohistochemical expressions of matrix metalloproteases (MMPs), claudin5 (CLDN5), and aquaporins (AQPs) in forensic autopsy cases of carbon monoxide (n = 14), METH (n = 21), and phenobarbital (n = 17) intoxication, compared with mechanical asphyxia (n = 15), brain injury (n = 11), non-brain injury (n = 21), and sharp instrument injury (n = 15) cases. Relative mRNA quantification using Taqman real-time PCR assay demonstrated higher expression of AQP4 and MMP9, lower expression of CLDN5 in METH intoxication cases and lower expression of MMP2 in phenobarbital intoxication cases. Immunostaining results showed substantial interindividual variations in each group, showing no evident differences in distribution or intensity among all the causes of death. These findings suggest that METH may increase BBB permeability by altering CLDN5 and MMP9, and the self-protective system maybe activated to eliminate accumulating water from the extracellular space of the brain by up-regulating AQP4. Systematic analysis of gene expressions using real-time PCR may be a useful procedure in forensic death investigation.

α-Pyrrolidinononanophenone derivatives induce differentiated SH-SY5Y neuroblastoma cell apoptosis via reduction of antioxidant capacity: Involvement of NO depletion and inactivation of Nrf2/HO1 signaling pathway.

α-Pyrrolidinononanophenone (α-PNP) derivatives are known to be one of the hazardous new psychoactive substances due to the most extended hydrocarbon chains of any pyrrolidinophenones on the illicit drug market. Our previous report showed that 4'-iodo-α-PNP (I-α-PNP) is the most potent cytotoxic compound among α-PNP derivatives and induces apoptosis due to mitochondrial dysfunction and suppression of nitric oxide (NO) production in differentiated human neuronal SH-SY5Y cells. In this study, to clarify the detailed action mechanisms by I-α-PNP, we investigated the mechanism of reactive oxygen species (ROS) -dependent apoptosis by I-α-PNP in differentiated SH-SY5Y with a focus on the antioxidant activities. Treatment with I-α-PNP elicits overproduction of ROS such as H2O2, hydroxyl radical, and 4-hydroxy-2-nonenal, and pretreatment with antioxidant N-acetyl-L-cysteine is attenuated the SH-SY5Y cells apoptosis by I-α-PNP. These results suggested that the overproduction of ROS is related to SH-SY5Y cell apoptosis by I-α-PNP. In addition, I-α-PNP markedly decreased antioxidant capacity in differentiated cells than in undifferentiated cells and inhibited the upregulation of hemeoxygenase 1 (HO1) and glutathione peroxidase 4 (GPX4) expression caused by induction of differentiation. Furthermore, the treatment with I-α-PNP increased the nuclear expression level of BTB Domain And CNC Homolog 1 (Bach1), a transcriptional repressor of Nrf2, only in differentiated cells, suggesting that the marked decrease in antioxidant capacity in differentiated cells was due to suppression of Nrf2/HO1 signaling by Bach1. Additionally, pretreatment with an NO donor suppresses the I-α-PNP-evoked ROS overproduction, HO1 down-regulation, increased nuclear Bach1 expression and reduced antioxidant activity in the differentiated cells. These findings suggest that the ROS-dependent apoptosis by I-α-PNP in differentiated cells is attributed to the inactivation of the Nrf2/HO1 signaling pathway triggered by NO depletion.

Molecular pathology of brain edema after severe burns in forensic autopsy cases with special regard to the importance of reference gene selection.

Brain edema is believed to be linked to high mortality incidence after severe burns. The present study investigated the molecular pathology of brain damage and responses involving brain edema in forensic autopsy cases of fire fatality (n = 55) compared with sudden cardiac death (n = 11), mechanical asphyxia (n = 13), and non-brain injury cases (n = 22). Postmortem mRNA and immunohistochemical expressions of aquaporins (AQPs), claudin5 (CLDN5), and matrix metalloproteinases (MMPs) were examined. Prolonged deaths due to severe burns showed an increase in brain water content, but relative mRNA quantification, using different normalization methods, showed inconsistent results: in prolonged deaths due to severe burns, higher expression levels were detected for all markers when three previously validated reference genes, PES1, POLR2A, and IPO8, were used for normalization, higher for AQP1 and MMP9 when GAPDH alone was used for normalization and higher for MMP9, but lower for MMP2 when B2M alone was used for normalization. Additionally, when B2M alone was used for normalization, higher expression of AQP4 was detected in acute fire deaths. Furthermore, the expression stability values of these five reference genes calculated by geNorm demonstrated that B2M was the least stable one, followed by GAPDH. In immunostaining, only AQP1 and MMP9 showed differences among the causes of death: they were evident in most prolonged deaths due to severe burns. These findings suggest that systematic analysis of gene expressions using real-time PCR might be a useful procedure in forensic death investigation, and validation of reference genes is crucial.

Death following the ingestion of detergent: an autopsy case with special regard to the histochemical findings.

We report an autopsy case of death due to accidental ingestion of a liquid laundry detergent with special regard to the histochemical findings. A female inpatient suffering from schizophrenia in a psychiatric institution, was found unconscious lying on the floor of her room, with a container of detergent nearby, and died despite intensive life-support measures. At autopsy, the stomach and duodenum contained whitish translucent foamy viscous fluid, and the mucous membranes, from the esophagus to the duodenum, had diffuse erosions with congestion and edema. There was otherwise no significant pathology other than signs of acute death and hemolysis. Toxicological investigations detected 1-methyl-4-prop-1-en-2-ylcyclohexene (detergent additive) in the gastric contents using headspace-gas chromatography/mass spectrometry, and a nonionic surfactant by a color identification test. Although these substances could not be detected in the blood, body fluid or viscera, histochemical examination using Oil red O demonstrated droplet-like staining in the pulmonary alveoli, suggesting aspiration of detergent, and similar staining in the vasculature of the lung, Kupffer cells of the liver, Bowman capsules of the kidney, and capillaries of the brain, suggesting the systemic effect of ingested/aspirated detergent. These findings were in keeping with death from ingestion of detergent and demonstrated the importance of preventing accidents such as this in healthcare facilities for elderly people.

Molecular pathology of natriuretic peptides in the myocardium with special regard to fatal intoxication, hypothermia, and hyperthermia.

The present study investigated the molecular pathology of atrial and brain natriuretic peptides (ANP and BNP) in the myocardium to evaluate terminal cardiac function in routine forensic casework with particular regard to fatal drug intoxication (n = 18; sedative-hypnotics, n = 10; methamphetamine, n = 8), hypothermia (cold exposure, n = 13), and hyperthermia (heatstroke, n = 10), compared with that in acute ischemic heart disease (AIHD, n = 35) and congestive heart disease (CHD, n = 11) as controls (total n = 87; within 48 h postmortem). Quantitative analyses of myocardial ANP and BNP messenger RNA demonstrated that their expressions in bilateral atrial and ventricular walls were high in methamphetamine intoxication and hypothermia, comparable to those in AIHD and CHD, but were low in sedative-hypnotic intoxication and hyperthermia. In pericardial fluid, both ANP and BNP levels were increased in hypothermia, while CHD cases had an elevated BNP level, and ANP level showed a tendency to increase in hyperthermia; however, immunohistochemistry showed no evident differences in myocardial ANP and BNP among the causes of death. These findings suggest terminal high cardiac strain in methamphetamine intoxication, decreased cardiac strain in sedative-hypnotic intoxication and hyperthermia (heatstroke), and persistent congestion in hypothermia (cold exposure).

Molecular pathology of pulmonary surfactants and cytokines in drowning compared with other asphyxiation and fatal hypothermia.

Drowning involves complex fatal factors, including asphyxiation and electrolyte/osmotic disturbances, as well as hypothermia in cold water. The present study investigated the molecular pathology of pulmonary injury due to drowning, using lung specimens from forensic autopsy cases of drowning (n = 21), acute mechanical asphyxia due to neck compression and smothering (n = 24), and hypothermia (cold exposure, n = 11), as well as those of injury (n = 23), intoxication (n = 13), fire fatality (n = 18), and acute cardiac death (n = 9) for comparison. TaqMan real-time reverse transcription polymerase chain reaction was used to quantify messenger RNA (mRNA) expressions of pulmonary surfactant-associated proteins A and D (SP-A and SP-D), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10. SP-A and SP-D mRNA levels were lower for drowning, mechanical asphyxiation, fire fatality, and acute cardiac deaths than for hypothermia and injury. TNF-α, IL-1ß, and IL-10 mRNA levels were higher for drowning or for drowning and injury than for other groups; there was no significant difference between fire fatality, involving airway injury due to inhalation of hot/irritant gases, and other control groups. These observations suggest characteristic molecular biological patterns of pulmonary injury involving suppression of pulmonary surfactants and activation of early-phase mediators of inflammation in drowning, with high mRNA expression levels of pulmonary surfactants in fatal hypothermia; however, there was no significant difference among these markers in immunohistochemical detection, except for SP-A. These mRNA expressions can be used as markers of pulmonary injury to assist in investigations of the pathophysiology of drowning and fatal hypothermia in combination with other biochemical and biological markers.

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper.

In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

Molecular pathology of pulmonary edema after injury in forensic autopsy cases.

The lung is vulnerable to trauma; pulmonary edema starts quickly as part of the systemic responses involved in shock. The present study investigated the molecular pathology of posttraumatic alveolar damage and responses involving pulmonary edema in forensic autopsy cases of injury (n = 66) compared with acute cardiac death cases (n = 13). Intrapulmonary mRNA and immunohistochemical expressions of matrix metalloproteinases (MMPs; MMP-2 and MMP-9), intercellular adhesion molecule-1, claudin-5, and aquaporins (AQPs, AQP-1 and AQP-5) were examined. Subacute injury deaths showed an increase in lung weight similar to that in acute cardiac death, but relative mRNA quantification using the Taqman real-time PCR assay demonstrated different findings among the causes of death; higher expressions were detected for all markers, except for AQP-5 in sharp instrument injury, for MMP-2 in blunt brain injury, and for MMP-9 in non-brain blunt injury, but these expression levels were lower in acute cardiac death. In immunostaining, only MMPs showed differences among the causes of death: MMP-2 expression was evident in most subacute deaths due to blunt brain injury and sharp instrument injury, whereas MMP-9 was intensely positive in those of non-brain blunt injury and sharp instrument injury. These findings suggest significant differences in the mechanism of pulmonary edema among fatal injuries and acute cardiac death, especially between blunt and sharp instrument injury. Systematic analysis of gene expressions using real-time PCR in combination with immunohistochemistry may be useful in evaluating pulmonary damage and responses after injury in death investigations, especially in connection with posttraumatic shock.

Postmortem CT investigation of skeletal and dental maturation of the fetuses and newborn infants: a serial case study.

Physical maturation involving bones and teeth is a classic indication of fetal maturity in forensic practice, for which radiological examination is effective. The present study preliminarily investigated the efficacy of postmortem computed tomography (PM-CT) in estimating the gestational age of fetuses (n = 3) and newborn infants (n = 4), compared with plain radiography. Body length and weight estimated in reconstructed CT images approximated the measured values, providing additional data of body mass volume and fat development. The diaphyses of humerus and femur, and the epiphyseal ossification centers of femur and calcaneus were more easily identified and accurately measured in CT images than in plain radiographs. The developmental stage of deciduous teeth was also clearly identified. PM-CT was useful to evaluate the physical maturity of infants.

Postmortem biochemistry and immunohistochemistry of chromogranin A as a stress marker with special regard to fatal hypothermia and hyperthermia.

Chromoganin A (CgA) is widely distributed in the secretory granules of endocrine and neuroendocrine cells and cosecreted with hormones such as catecholamines. The present study investigated postmortem serum and cerebrospinal fluid (CSF) levels of CgA in comparison with those of catecholamines, and also cellular CgA immunopositivity in the hypothalamus, adenohypophysis and adrenal medulla to assess forensic pathological significance. Serial medicolegal autopsy cases (n = 298, within 3 days postmortem) were used. Serum and CSF CgA levels were independent of the gender or age of subjects or postmortem time. The most characteristic findings were seen for fatal hypothermia (cold exposure), hyperthermia (heat stroke) and intoxication. Serum CgA levels were lower for hypothermia and intoxication than for other causes of death (p < 0.05), while CSF CgA levels were higher for hypothermia (p < 0.0001). A negative correlation was detected between serum and CSF CgA levels for hypothermia (R = 0.552, p < 0.05). Correlations between serum levels of CgA and catecholamines (adrenaline, noradrenaline and dopamine) were evident for hyperthermia (R = 0.632-0.757, p < 0.05 to <0.01), but there was no significant correlation between CgA and catecholamine levels in CSF. Cellular CgA immunopositivity in the hypothalamus, adenohypophysis and adrenal medulla varied extensively among cases in each group. However, CgA immunopositivity in hypothalamus neurons was lower for hypothermia than other causes of death including hyperthermia and intoxication. These observations suggest characteristic neuroendocrinal activation in fatal cases of hypo- and hyperthermia and also intoxication. CgA may be a useful biochemical and immunohistochemical marker for investigating these causes of death.

An unusual presentation of thoracic aortic aneurysm rupturing into the esophagus: an autopsy case report.

Ruptured aortic aneurysms often present with sudden death, and have varied signs and symptoms depending on the site of rupture and hemorrhage. We report a case of an aortic aneurysm with an aorto-esophageal fistula, which showed slow gastrointestinal bleeding for days before death. A 79-year-old male was brought to a hospital emergency unit, with a history of melena for about 3 days, and recent hematemesis. He collapsed immediately after endoscopy and died. A forensic autopsy which was performed due to possible medical malpractice demonstrated a large saccular aneurysm of the descending thoracic aorta with a fistula into the esophagus. A significant finding was a lid or valve shaped thrombus covering the aortic orifice of the fistula, which may have partly contributed to slow bleeding, and which may have been dislodged by endoscopy. This case suggests that very careful management of aorto-esophageal fistula is needed in patients with clinical signs of possible thoracic aortic aneurysm with slow hemorrhage.

[Significance of postmortem computed tomography in forensic autopsy and its possible issues].

Postmortem imaging (PMI) including computed tomography (CT) and magnetic resonance imaging (MRI) has become a familiar procedure in forensic casework. We investigated a short term impact of postmortem CT(PM-CT) in routine forensic autopsy cases at our institute during a period of 9 months (n = 121, fetus--92 year-old, 7 h--years postmortem), comparing to autopsy findings. In identification, PM-CT was useful for matching skeletal/dental characteristics, superimposing, and detection of foreign materials. However, conventional X-ray was often more effective for detection of small metallic foreign bodies. In pathomorphology, PM-CT partly demonstrated important findings for determining the immediate cause of death, which were confirmed by autopsy, but interpretation to the underlying/initiating/preceding causes of death or contributory factor(s) was mostly difficult or impossible. However, accumulated PM-CT data were useful for retrospective evaluation and review of autopsy findings. These experiences indicate that PMI is useful for radiographic screening and documentation, to be included in supplementary procedures, employing knowledge and experiences of forensic autopsy.

[Biochemical investigation for clinical diagnosis of drug dependence].

Specific biochemical marker is not available for clinical diagnosis of drug dependence at present. However, drug abuse is accompanied by a decrease in serotonin and the derivative in cerebrospinal fluid, suggesting central serotonergic dysfunction, that is associated with cognitive deficits, alterations in sleep architecture and neuroendocrine function, and increased impulsivity as well as an increase in the risk for aggressive behavior toward the self involving suicide. For central stimulant abusers, elevated plasma catecholamine levels are associated with psychotic episodes and cardiovascular complications involving tachycardia and arrhythmias. Biochemical investigation is useful for predicting drug-induced mental disorders, complications and the prognosis, and also for differentiation from other mental disorders, e.g., secondary to metabolic and infectious diseases, or management of acute intoxication.

Forensic pathological investigation of myocardial hypoxia-inducible factor-1 alpha, erythropoietin and vascular endothelial growth factor in cardiac death.

The present study investigated the immunohistochemical distributions and mRNA expressions of myocardial hypoxia-inducible factor (HIF)-1 alpha and its downstream factors, erythropoietin (Epo) and vascular endothelial growth factor (VEGF), in cardiac deaths. Medico-legal autopsy cases (n=114, within 48-h postmortem) of cardiac deaths (n=58) and control cases (n=56) were examined. Immunohistochemical positivities of HIF-1 alpha, Epo and VEGF were patchily observed in cardiomyocytes in the acute ischemic lesions of myocardial infarction (n=37), showing a relationship to morphological cardiomyocyte damage: the staining was intense in the regions with early ischemic changes and weak in the necrotic regions. Immunopositivities were sporadically detected in cardiomyocytes in some cases of sudden cardiac death without infarction (SCD, n=13). In chronic congestive heart disease (CHD, n=8), weak positivities were diffusely observed in the cardiomyocytes. However, there were no such findings in cases of mechanical asphyxiation (n=16) or drowning (n=18). HIF-1 alpha, Epo and VEGF mRNA expressions, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR), showed localized elevations related to acute myocardial infarction (AMI) lesions, whereas such findings were mild in recurrent myocardial infarction (RMI) and SCD cases. CHD showed significant elevations of these mRNAs irrespective of the sampling site. The mRNA expressions were significantly lower in cases of drowning. These findings suggest that focal immunopositivities and increased mRNAs of these factors are indicative of short and substantial duration of myocardial ischemia, respectively. The combined analyses may not only be useful for investigating the site, phase and severity of acute myocardial ischemia and the severity of chronic ischemic stress, but also contribute to differentiating cardiac deaths from asphyxiation and drowning or interpreting the possible contribution of cardiac disease in traumatic death.

[Immunohistochemical distribution of single-stranded DNA in the brain in medico-legal autopsy cases of carbon monoxide intoxication].

To evaluate apoptotic neuronal damage by carbon monoxide (CO) in medico-legal autopsy cases, we investigated the immunohistochemical distribution of single-stranded DNA (ssDNA) as a marker of apoptosis and programmed cell death in the brain. Formalin-fixed paraffin-embedded brain tissue specimens, including cerebral cortex of frontal lobe, substantia nigra of the midbrain and pallidum, from medico-legal autopsy cases of fire fatality (n=63), including cases with blood carboxyhemoglobin (COHb) of a lower (<60%) and a higher (>60%) level (n = 39 and 24, respectively), and CO intoxication without burns (n = 6) were examined, in comparison with acute ischemic heart disease (IHD, n = 29) and asphyxiation due to strangulation (AS, n= 14). In the pallidum, neuronal immunopositivity for ssDNA was significantly higher in fire fatality with a higher COHb level than in IHD (p<0.0001), and CO intoxication cases showed significantly higher positivity than other groups excluding fire fatality with a higher COHb level (p< 0.05). In cases without cardiopulmonary resuscitation, ssDNA-positivity in the pallidum mildly correlated to COHb concentrations (r = 0.31, p<0.05), and the positivity was significantly higher in higher COHb (>60%) cases than in lower COHb (<30%) cases. In the cerebral cortex and substantia nigra of the midbrain, neuronal ssDNA-positivity showed no significant findings with regard to the cause of death and COHb concentration. These findings suggest that CO causes selective neuronal damage in the pallidum.