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A male in his late 30s was found dead in his home. He was diagnosed with human immunodeficiency virus (HIV) about six years prior. The HIV infection was well controlled before his death. He was 166 cm in height and 75 kg in weight. Aside from discoloration of the skin on the right lower patellar, there were no obvious injuries. His brain weighed 1456 g. A cut surface of the brain revealed left thalamic hemorrhage. Histologically, infiltration of phagocytic cells was observed in the bleeding site. Thalamic hemorrhage was considered to be his cause of death in this case. Due to the effects of anti-HIV therapy, the mortality rate from HIV infection has decreased and the causes of death of HIV-infected persons have changed. HIV-infected persons have been suggested to be associated with cerebrovascular disease, especially juvenile ischemic stroke. Patients with acquired immunodeficiency syndrome (AIDS) have an increased risk of cerebrovascular disease. Possible mechanisms of cerebrovascular disease in HIV-infected individuals include coagulopathy, secondary effects of embolism and central nervous system infection, and direct vascular disease due to HIV. At the time of autopsy, his post-mortem interval was estimated to be approximately two weeks. Therefore, it was difficult to clarify histologically the cerebrovascular disorder that caused his cerebral hemorrhage. In recent years, anti-HIV therapy has reduced the number of AIDS-related deaths, but deaths in HIV-infected people from cardiovascular disease are increasing. This case is considered to be a valuable forensic autopsy case of an HIV-infected patient who actually died due to cerebral hemorrhage in Japan. • Anti-HIV therapy has reduced the number of AIDS-related deaths. • Deaths in HIV-infected people from cardiovascular disease are increasing. • An autopsy case of HIV-infected young patient who died due to cerebral hemorrhage. [ABSTRACT FROM AUTHOR]

Highlights • Catecholamine levels were measured in cerebrospinal fluid (CSF) from autopsy cases. • MAOA-uVNTR polymorphism was associated with CSF adrenaline and dopamine levels in females. • MAOA-uVNTR polymorphism was not associated with CSF noradrenaline levels in females. • MAOA-uVNTR polymorphism was not associated with CSF catecholamine levels in males. Abstract Monoamine oxidase A (MAOA) plays important roles in the metabolism of catecholamines and modulates adrenergic, noradrenergic, and dopaminergic signaling. A polymorphic promoter variable number tandem repeat (VNTR) locus (MAOA-uVNTR) is located approximately 1.2 kb upstream from MAOA exon 1. Functional studies revealed that MAOA-uVNTR affects gene expression. In the present study, we examined the frequencies of MAOA-uVNTR alleles in Japanese autopsy cases, in which amphetamines or psychotropic drugs were not detected. In total, 87 males and 35 females were evaluated and investigated for the possible effect of MAOA-uVNTR polymorphisms on cerebrospinal fluid (CSF) catecholamine concentrations. In males, there was no significant association between MAOA-uVNTR polymorphisms and CSF adrenaline (Adr), noradrenaline (Nad), or dopamine (DA) levels. In contrast, females who were homozygous for the 3-repeat allele (i.e., 3/3 genotype carriers) had higher CSF levels of Adr (p = 0.024) and DA (p = 0.035) than individuals who were heterozygous or homozygous for the 4-repeat allele (3/4 and 4/4, respectively). We found no significant association between MAOA-uVNTR polymorphisms and CSF Nad levels in females. Thus, the results of the present study indicated that MAOA-uVNTR polymorphism influences CSF Adr and DA levels in females. [ABSTRACT FROM AUTHOR]

Graphical abstract A female was found dead in a reverse jack-knife position. The cause of death was considered as positional asphyxia. Highlights • A female was found dead in the reverse jack-knife position. • No fatal trauma or lesions were found. • The effects of tofisopam and the deceased's poor physical condition had restricted her motility. • The cause of death in this case was considered to be positional asphyxia. • We should be aware that chairs with wheels can cause such accidents. [ABSTRACT FROM AUTHOR]

Abstract: We report an autopsy case of a man who died 2days after taking an overdose of vegetamin. The autopsy findings were as follows: the epidermis on the axillary fossa and the inguinal skin had become macerated. Skeletal muscle was discolored. Concentrations of urea nitrogen, creatinine and urine myoglobin were 1.95g/day, 0.66g/day and 1100ng/mL, respectively. Immunohistochemically, myoglobin was strongly stained at the Bowman’s capsule, and tubular lumen and epithelium. 8-OH-dG was strongly stained in renal tubular epithelium in which cell nuclei were strongly stained. ORP-150 was observed in intraglomerular cells and renal tubular epithelium. The concentrations of phenobarbital, promethazine and chlorpromazine ranged from therapeutic to toxic levels, from toxic to lethal levels and toxic level, respectively. His cause of death was considered to be vegetamin-induced rhabdomyolysis. In genetic analysis of this subject, there were two heterozygous silent mutations in the three hot-spot regions in the RYR1 gene. In the CPT II gene, the subject was found to be heterozygous for an amino acid substitution in exon 4, 1203G>A causing a 368Val>Ile amino acid substitution. There was no mutation in the VLCAD gene or CYP2C19 gene. The subject was heterozygous for CYP2D6*1 and CYP2D6*2. [Copyright &y& Elsevier]

Abstract: An 87-year-old female who had been hospitalized due to pneumonia was administered nourishment through a nasogastric tube. She collapsed as a result of dyspnea after the insertion of a new tube and administration of nourishment. Chest X-rays revealed that the tube was inserted into the left pleural cavity passing the trachea and left bronchi and that the nourishment pooled. In spite of immediate treatment including removal of the tube and insertion of a drain, she died 12 days later. Autopsy findings: Both the left pulmonary and parietal pleurae were thickened and covered with a dirty gray-yellowish moss-like paste. The left lower lobe was softened, and this region was suspected as the ruptured site of the pleura. Histological findings: A part of the thick pleura with inflammatory cells, including multinucleated giant cells, was positive-stained for anti α-lactalbumin antibody immunohistochemically. These giant cells are often observed in granulomatous inflammation against a foreign material. It was considered that those in the pleura had been induced by the nourishment, and that those in the pulmonary parenchyma had been affected by the insertion of the tube. The multinucleated giant cells clarified the cause of fatal pleuritis and pneumonia and the misinsertion of the tube. [Copyright &y& Elsevier]

Abstract: In this study, four Y-SNPs (M122, P31, M174 and M216) were analyzed in 207 Japanese males (Gunma, Fukui, Kagoshima and Tokushima Prefecture). Eleven Y-STRs, including DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS389, DYS385 and YCAII were also investigated in each prefecture. As a new technique, we used Cycleave PCR method with real-time PCR, which allows detection of Y-SNPs by an easier and faster method. Regarding Y-SNPs, each region in Japan could be almost classified based on two types, the D and O type. We found that the O type was remarkably higher in Kagoshima Prefecture than in other areas. Moreover, the D type was more prevalent than the O type in Gunma Prefecture, which is geographically located at the center of Japan. These findings of geographical differences may be related to the historical background and population movements of Japanese. Y-STRs were analyzed and data from Tokushima Prefecture that we previously reported was included in the analyses. As a result, DYS385 showed the highest gene diversity (GD) value (GD=0.953), while DYS391 showed the lowest GD value (GD=0.288). Haplotype diversity (HD) value of only Y-STRs (Extended haplotype) was estimated at 0.99981, whereas the HD value of Y-SNPs/Y-STRs was 0.9988. [Copyright &y& Elsevier]

Abstract: FTA® technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA® cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA® cards containing DTT. The FTA® cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5–3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA® card is available for application to routine work. [Copyright &y& Elsevier]

Abstract: In this study, we observed renal damage and peroxidative injury as the acute or sub-acute effect of methamphetamine (MA) to determine whether MA intoxication can be diagnosed from immunohistochemical changes in the kidney. In addition, renal function was investigated in relation to the immunohistochemical changes. A single administration of MA (group I) (50mg/kg/ (i.p.)) and repeated administration (group II) (10mg/kg/day (i.p.) for 5 days) were designed as an acute model and a sub-acute or chronic model. Immunohistochemically, cell damage markers were observed. Then, renal function markers and minerals in blood were measured. Myoglobin and creatinine phosphokinase (CPK) in blood were also analyzed. In group I, ubiquitin immunoreactivity was enhanced only in the renal tubules. Creatinine increased, while K, Ca, and P decreased (P<0.01). CPK increased significantly (P<0.01). Therefore, it was suspected that MA might induce renal dysfunction with renal tubule damage. This damage might be related to leakage of CPK from muscle. In group II, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) increased immunohistochemically and quantitatively (P<0.01). It was considered that oxidative DNA damage might be induced by repeated administration. It was considered that this study offers basic information for the evaluation of pathological changes in the kidney in MA-related autopsy cases. [Copyright &y& Elsevier]

On a cold winter morning, a 35-year-old male was unexpectedly found dead and therefore autopsied. Macro- and microscopically, the lungs were demonstrated bronchopneumonia. On the surface of brain, small blood vessels were slightly congested. Microscopically, brain edema was also observed, and proliferation of lymphocytes was observed around the capillary vessels of the hippocampus. These findings suggested a viral infection of the cerebrum. We conducted an immunohistochemical study with antibody against influenza virus. Influenza A virus antigen was detected in both the lungs and brain. Therefore, findings were compatible with influenza A encephalopathy. Even when serological inspection is not performed, it is useful to inspect localization of the virus antigen immunohistochemically. We considered that it is necessary to perform pathological examination for influenza encephalopathy in sudden death cases when influenza is epidemic. [Copyright &y& Elsevier]

Between 1996 and 2002, we tested a total of 20 unidentified bodies for DNA typing. We describe here the relationships among detection rates achieved by four DNA typing systems (D1S80 typing, TH01 typing, HLA DQA1 typing, and PM typings), the post-mortem interval, types of specimens (bone, nail, and blood), post-mortem changes, and the site at which the corpse was found (indoors, outdoor, or in the sea). Detection rates for PM typings, HLA DQA1 typing, TH01 typing, and D1S80 typing in all cases were 94.7, 90.0, 73.7, and 50.0%, respectively. The success of the typings was highly influenced by the post-mortem interval. Using blood, almost all DNA types were detected, while the nail showed comparatively higher detection rates than bone. The detection rate decreased in order with indoor, outdoor, sea, and soil as the site at which the corpse was found. It is important to consider the specimen, the site at which the corpse was found, and the post-mortem interval to successfully achieve DNA typing. [Copyright &y& Elsevier]

Abstract: Rats were exposed to toluene (1,500 ppm for 4 hr per day) for 7 days. The body weight of the rats was significantly lower and the weight of the adrenal gland was significantly higher in the toluene inhalation group compared to the controls. Microscopically, there was no obvious change in the medulla, but hypertrophy of the cortex was observed in the toluene inhalation group. And, the size of adrenocortical cells in treated-rats was also significantly enlarged than the control. Immunohistochemical staining did not show a clear difference in localization of aldosterone-positive cells between the control and inhalation groups. Expansion of the corticosterone-positive area consistent with the cortical hypertrophy was recognized in the inhalation group. Enhancement of 72 kD-heat-shock protein (HSP70)-expression in the toluene inhalation group was not observed. Neither stress nor damage to cortical cells due directly to toluene exposure was observed in the cortex. Also, there was no obvious difference in the anti-proliferating cell nucleus antigen (PCNA)-immunostaining between control and inhalation groups. Thus, it is suspected that cortical hypertrophy was the result of cell enlargement due to the stimulation of the cortical cells. Corticotropin-releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) was increased in the inhalation group. Concentration of plasma ACTH was elevated significantly by toluene exposure. The amounts of mRNA of adrenocortical steroid metabolism gene, cytochrome side-chain cleavage (P450scc), was also increased by toluene inhalation. Toluene exposure might induce adrenocortical hypertrophy via the hypothalamus-pituitary-adrenal gland (HPA) axis. [Copyright &y& Elsevier]

Abstract: After prolonged toluene-inhalation (for 20 days) at the common abuse density (1500ppm for 4h per day), the effect of toluene on spermatogenesis in rats was investigated. Body weight was significantly decreased in the toluene group (P<0.05). However, the weights of the testis and epididymis were maintained. To confirm whether the toluene-inhalation influences testis and epididymis as a stress factor; anti-70kD heat-shock protein (HSP70) and c-fos gene product (c-Fos) were observed. To observe the change of the cell division and the proliferation in spermatogenesis, proliferating cell nuclear antigen (PCNA) were stained immunohistochemically, and apoptosis was also detected. There was no positive immunoreactivity for HSP70 or c-Fos. There was no significant difference in the PCNA-expression in both groups. It was considered that toluene-inhalation did not have a clear influence in the division of spermatogonium and spermatocytes. On in situ apoptosis detection, slightly enhanced signals were observed in the toluene-inhalation groups. This might have some influence on meiosis from spermatocyte to spermatid. However, in toluene-inhalation rats exposed to a common abuse density, it was considered that spermatogenesis was well maintained and not apparently damaged. [Copyright &y& Elsevier]

Rats were exposed to toluene (1500 ppm for 4 h per day) for 7 days. After toluene inhalation, only granule cells in the dentate gyrus of the hippocampus were slightly shrunken. In the cerebellum, several Purkinje cells were shrunken and lost, and the white matter was thinner than in controls. Microtubule-associated protein 2 (MAP2)-immunopositive filaments of neuronal processes were slightly disarrayed in the radial layer of the hippocampus, and were fragmented in the molecular layer of the cerebellum. It was considered that toluene induced neuronal changes both in the cerebellum and the hippocampus. To elucidate the effect of neurotrophic factors on those neuronal changes, glial cell line-derived neurotrophic factor (GDNF), transforming growth factor (TGF) and tumor necrosis factor (TNF) in rat brain were examined immunohistochemically. In control rats, TNF-α was not stained in either the hippocampus or the cerebellum, while TGF-β1 was scarcely expressed in the cerebellum. GDNF was minimally expressed in the Purkinje cells in the cerebellum. After toluene-treatment, TGF-β1 was over-expressed in the endothelium of the capillary vessel walls in both regions. In the cerebellum, TNF-α was induced only in the granule cells, while GDNF expression was enhanced in the Purkinje cells. These data suggest that toluene induces astrocyte activation through TGF-β1 upregulation, which then induces GDNF in the Purkinje cells and TNF-α in the granule cells of the cerebellum. The differences in the expression of the neurotrophic factors may account for neurobehavioral changes after toluene exposure. [Copyright &y& Elsevier]

Methamphetamine (MA) is an illicit psychostimulant that stimulates the release of catecholamines from sympathetic nerve terminals and is widely abused worldwide. Since catechol-O-methyltransferase (COMT) metabolizes catecholamines and mediates adrenergic, noradrenergic, and dopaminergic signaling responses, we investigated the effects of the COMT polymorphisms rs4633 and rs4680 on cerebrospinal fluid (CSF) catecholamine concentrations in autopsies of subjects who died of drug intoxication. 28 MA abusers and 22 fatal psychotropic drug intoxication cases were evaluated. No correlations were identified between rs4633 or rs4680 polymorphisms and CSF concentrations of adrenaline (Adr), noradrenaline (Nad), or dopamine (DA) in fatal psychotropic cases. However, among MA abusers, DA concentrations in the CSF were significantly higher in those with the T allele (CT and TT) of rs4633 than in CC genotype carriers (p=0.004). Moreover, among MA abusers, DA concentrations were significantly higher in those with the A allele (GA and AA) of rs4680 than in GG genotype carriers (p=0.017). In subsequent haplotype analyses of MA abusers, a strong correlation was identified between two COMT haplotypes and CSF DA concentrations (p=0.002). However, the CSF concentrations of Adr and Nad were not associated with COMT genotypes or haplotypes. The present results indicate that rs4633 and rs4680 polymorphisms influence CSF DA concentrations and MA toxicity in MA abusers. [ABSTRACT FROM AUTHOR]

A female in her thirties fell face down in her room. She was motionless when her sister found her. She was transported to the hospital by ambulance and was in a state of cardiopulmonary arrest on admission. She did not respond to resuscitation. Her abdomen had started to swell 3 years before her death. An autopsy was performed to clarify the decedent’s cause of death. She was 172 cm tall and weighed 146 kg. Her maximum abdominal girth was 172.1 cm. A subcutaneous hemorrhage measuring 4.5 cm in diameter was observed in the epigastric region. The abdominal cavity contained brownish ascites (54.1 L). The left ovary was markedly swollen, and the combined weight of the uterus and right ovary was more than 13.0 kg. A left ovarian tumor consisting of serous and mucinous cysts was detected. There were no metastatic lesions in the peritoneum or other organs. She might have suffered circulatory disturbance caused by the ascites and ovarian tumor. Moreover, being in a prone position would have resulted in an increase in intra-abdominal pressure, further exacerbating her circulatory problems. Therefore, her cause of death was considered to be circulatory failure caused by significant ascites and a large ovarian tumor. [ABSTRACT FROM AUTHOR]

We report the quantification of immunohistochemical findings for a diagnosis of dementia in autopsy cases among older decedents. Autopsy cases were selected with the following requirements: >65 yo; no head injuries, thermal injuries, or heat stroke; no intracranial lesions; and within 48 h of death. Among cases that met all requirements, 10 had a clinical diagnosis of dementia were included in dementia group. Non-dementia group consisted of 38 cases without any record of dementia. To compare these groups, immunohistochemically, beta-amyloid, tau protein, gephyrin, and IL-33 were examined in five regions. Quantitative analysis was performed by collecting with image data analyzed using analysis software. Image data on tau-immunopositive neurofibrillary tangles (NFT) and beta-amyloid-positive senile plaques (SP) were photographed. Criteria for dementia were made by counting and measuring NFT and SP from image data using software. Differences in SP and NFT were effective for discriminating between the two groups. These criteria may reveal the presence and progression of dementia. Total of tau-positive NFT in Ammon’s horn (AH) may be useful for diagnosing dementia. When the total is more than 41 in approximately 6 mm 2 of AH, the possibility of dementia is considered. Total of beta-amyloid-positive SP in the parahippocampal gyrus (PHG) may be useful for diagnosing dementia. When the total in approximately 5 mm 2 of PHG is more than 47, the possibility of dementia is considered. Immunohistochemical staining may be more useful for obtaining image data for quantification than conventional staining techniques, such as Bielschowsky-Hirano’s silver staining. [ABSTRACT FROM AUTHOR]

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC–MS and LC–MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC–MS and LC–MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratory’s daily routine for screening many different compounds from postmortem samples. [ABSTRACT FROM AUTHOR]

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds. [ABSTRACT FROM AUTHOR]

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations. [ABSTRACT FROM AUTHOR]

Abstract: Background/aim: Liver-type fatty acid-binding protein (L-FABP) is a clinical biomarker of the progress of kidney disease. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is known as a biomarker of peroxidative DNA damage. We investigated both urinary L-FABP and 8-OHdG in forensic autopsy cases as biomarkers to elucidate the metabolic changes in survival periods after insults. Methods: In 196 urinary samples from forensic autopsy cases, we measured L-FABP and 8-OHdG by enzyme-linked immunosorbent assay (ELISA) and creatinine by enzymatic assay. Urinary L-FABP/Cr and 8-OHdG/Cr were obtained. Results: No significant correlation was observed between urinary L-FABP/Cr or 8-OHdG/Cr, and gender, age, or postmortem interval. Regarding urinary L-FABP/Cr or 8-OHdG/Cr, there were no significant differences among the causes of death. In the survival/agony period, urinary L-FABP/Cr under the cut-off value 31.3 might show that the survival/agony period was within 1h. Under the cut-off value of urinary 8-OHdG/Cr, 17.8, might indicate that it is within 24h. Conclusion: Urinary L-FABP/Cr may rise within a relatively short survival/agony period, and urinary 8-OHdG/Cr may increase when the damage continues longer. Measuring the urinary L-FABP/Cr and 8-OHdG/Cr might be useful in elucidating the survival/agony period. [ABSTRACT FROM AUTHOR]

Abstract: In fatalities with multiple traumatic injuries, it is important to determine the severity of trauma, the main damaged organ, and the antemortem pathophysiological condition. We examined 63 cases within 48h of the postmortem interval, which included assaults, slips and falls and falls from heights, traffic accidents, and sharp instrumental injuries. Immunohistochemically, each kidney was stained against hemoglobin (Hb), myoglobin (Mb), superoxide dismutase (SOD), 8-hydroxy-2′-deoxyguanosine (8-OHdG), 150kDa oxygen regulated protein (ORP150), pulmonary surfactant A (SP-A), and liver-type fatty acid binding protein (L-FABP). Bleeding or circulatory failure induced ORP150, 8-OHdG, and L-FABP in the kidney. Statistical analysis of the immunoreactivity revealed that in battered and/or abused cases, Hb could be considered a specific marker. Hb and Mb were observed in the cases with general severe trauma, such as slips and falls and falls from heights. In traffic accidents, ORP150 could reflect general circulatory failure with bleeding. SP-A was observed in the cases with severe thoracic injuries, such as lung injuries and multiple thoracic fractures. L-FABP appeared in cases with renal circulatory failure as well as renal injury. These findings suggest that immunohistochemical observation of the kidneys could be a useful tool in determining several key factors, such as the severity of injury, the specific damaged organ, and the pathological condition after injury. [Copyright &y& Elsevier]

Abstract: Methamphetamine (MA) use sometimes causes rhabdomyolysis, which has been associated with mortality. We analyzed potential rhabdomyolysis-susceptibility genes from autopsy samples of 18 methamphetamine abusers. We examined mutations in the ryanodine receptor 1 (RYR 1), carnitine palmitoyltransferase II (CPT II), very long-chain acyl-CoA dehydrogenase (VLCAD), and cytochrome P450 (CYP) 2D6 genes. Different RYR1 mutations that caused amino acid substitutions (612Ala>Thr and 4295Ala>Val) were identified in 2 cases. In the CPT II gene, there was a new mutation (545Glu>Ala) in 1 case and there were mutations that did not change activity in 17 cases. In the VLCAD gene, there were mutations that did not change activity in 6 cases. In the CYP2D6 gene, homozygosity for CYP2D6∗10, which is associated with significantly reduced metabolic activity, was found in 3 cases, while 2 cases carried a different previously unreported missense mutation (344Arg>Gln and 48His>Tyr). RYR1 mutations and the new CPT II mutation identified in this study were not observed in a control group. Eighteen cases that were genetically analyzed were also investigated immunohistochemically to diagnose the possibility of rhabdomyolysis. However, there were no significant mutations that reduced enzyme activity in the suspected cases of rhabdomyolysis. These data suggested no obvious relationship between the genetic mutations observed in this study and rhabdomyolysis. [Copyright &y& Elsevier]

Abstract: Little is known about the role of glial cells in the striatum of chronic methamphetamine (METH) users. In this study, we immunohistochemically examined glial reactions in the striatum of chronic METH users who did not abstain from METH use and died of drug intoxication. Human glucose transporter 5 (hGLUT), a useful marker of microglia, and CR3.43, a major histocompatibility complex class II antigen specific for reactive microglia, were immunostained. Glial fibrillary acidic protein (GFAP) and S100Β were used for astrocyte immunohistochemistry. We analyzed 12 chronic METH users and 13 control subjects, and detected a 200–240% increase in the number of hGLUT5-positive cells in chronic METH users (p< 0.01). However, we did not detect any proliferation of CR3.43-positive cells. The number of GFAP-positive astrocytes increased, but this increase was not significant (p> 0.05). Moreover, S100B-positive cell density between the two groups was not significant (p> 0.05). This study demonstrates the absence of reactive gliosis in the striatum of chronic METH users who did not abstain for prolonged periods from METH use. The results suggest that chronic METH use by itself did not activate glial cells in humans and reactive gliosis may not be involved in the mechanism underlying the loss of control in drug intake, which is a characteristic feature of drug addiction. [Copyright &y& Elsevier]

Abstract: The drowning index (DI) was devised to diagnose drowning deaths, and is the weight ratio of the lungs and pleural effusion to the spleen. Among drowning (94 cases), mechanical asphyxia (47 cases), and acute cardiac (42 cases) deaths, within 2weeks postmortem we compared six markers, the weight of each lung, pleural effusion weight, total weight of the lungs and pleural effusion, spleen weight, heart weight, and the DI. Statistical analysis revealed that the total weight was heavier, while spleen weight was lighter, and the DI was significantly larger in the drowning group (p <0.05). We examined the relation between the postmortem time and these markers. We divided 94 drowning cases into three groups according to the postmortem duration, group A (0–3days; 43 cases), group B (3–7; 29 cases), and group C (7–14; 22 cases). The cut-off point of the DI was analyzed using the receiver operating characteristic (ROC) curve. As a result, the DI cut-off point was 14.1 in cases within two postmortem weeks. Drowning is still a difficult autopsy diagnosis, but in our experience, DI is a valuable indicator. [Copyright &y& Elsevier]

Abstract: Toluene, an abused substance in Japan, is well known as a neurotoxic chemical and has been shown to have neurobehavioral and electrophysiological effects. We used a fluorescence differential display PCR technique to analyze the genes expressed in the brain by toluene inhalation. We found 20 genes that were differentially expressed by toluene exposure. We confirmed by re-amplified PCR, nucleotide sequence and quantitative real-time PCR that of the 20 cDNAs, only 10 showed reproducible expression patterns by toluene inhalation. Of these genes, four had high homology with known genes (MIDA1, PEBP2 β, phosphatidylserine synthase 2 and SKAP55) and six fragments were new sequence tags of unknown genes. This result may contribute to reveal the patho-physiological effects of toluene inhalation on rat brain. [Copyright &y& Elsevier]

Abstract: We investigated the effects of toluene inhalation on neurons and neurotrophic factors in the spinal cord and the relationship between them. Male Wistar rats were exposed to toluene (1500ppm for 4h per day) for 7 days. To observe damage of the neurons in spinal cord with the toluene, expression of microtubule associated protein 2 (MAP2) and 70kDa heat shock protein (HSP70) in spinal cord were performed by immunohistochemistry. MAP2 was degraded and HSP70-immunoreactivity was enhanced in nerve cell bodies of the gray matter in toluene inhalation group. Immunoreactivity of glial fibrillary acidic protein (GFAP), a marker of astrocytes, was enhanced in the toluene-treated group. Furthermore, glial cell line-derived neurotrophic factor (GDNF)- and brain-derived neurotrophic factor (BDNF)-immunoreactivity in spinal cord were slightly decreased in the treated group. In addition, the concentrations of GDNF and BDNF in the spinal cord were determined using enzyme linked immunosorbent assay (ELISA). Concentration of GDNF was reduced significantly by toluene exposure. BDNF also reduced, but not significantly. The toluene inhalation caused the damage of the neuron in the spinal cord, which was accompanied by the decrease in the neurotrophic factors, such as BDNF and GDNF. [Copyright &y& Elsevier]

Abstract: Ischemic neuronal injury induce degradation of microtubule-associated protein 2 (MAP2). In addition to ischemia, postmortem brains show alterations in MAP2 immunoreactivity in the hippocampus, suggesting that the factors inducing cytoskeletal disruption in postmortem brain are similar to those in ischemic brains. Hypothermia reduces the severity of ischemic injury including disruption of MAP2 in the hippocampus. However, whether hypothermia reduces postmortem changes of MAP2 was not clear. In this study, we evaluated the effect of hypothermia on postmortem degradation of MAP2 in the human hippocampus at various postmortem intervals using immunohistochemistry. In postmortem brains without hypothermia (the normothermic group), the locus of MAP2 immunoreactivity moved from the dendrites to the cell bodies prior to becoming undetectable with increasing postmortem interval, particularly in the CA1-subiculum region. On the other hand, the change in MAP2 immunoreactivity was remarkably attenuated in brains of death from cold (the hypothermic group). The present study demonstrated that MAP2 disruption is remarkable in the CA1-subiculum region of autopsied brains and that hypothermia reduces the postmortem change of MAP2, as observed in ischemic brain. Therefore, immunostaining of MAP2 in the hippocampus could be used to diagnose hypothermia. [Copyright &y& Elsevier]

Abstract: In this study, we examined the efficacy and limitations of electron paramagnetic resonance (EPR) for estimating the age of human bloodstains. At 77K, human bloodstains give four striking EPR signals in the g=6.2 (g6), 4.3 (g4), 2.27 (H) and 2.005 (R) regions due to ferric high-spin, ferric non-heme, ferric low-spin and free radical species, respectively. We found that plotting double logarithms of the EPR intensity ratio of H/g4 versus days past bleeding gave a linear correlation up to 432 days with an error range within 25% of the actual number of days under controlled conditions. However, environmental factors such as differences of absorbent, light exposure and fluctuations of storage temperature affected the changes of these EPR-active compounds, which result in misestimation of the time since bleeding occurred. Therefore, one should take such factors into account in estimating the period since bleeding by this method. [Copyright &y& Elsevier]

The effect of toluene inhalation on oxidative damage in rat organs was examined. Male Wistar rats was inhaled toluene (1500 ppm for 4 h a day) for 7 days. Quantitatively and immunohistochemically, oxidative DNA damage, lipid peroxide (LPO) and superoxide dismutase (SOD) were examined. As a marker of the oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) immunoreactivity increased in the lung, liver and kidney. The amount of 8-OH-dG also increased in liver and kidney significantly. In the testis, the amount of 8-H-dG did not increase, however 8-OH-dG immunoreactivity enhanced in the spermatogonia. SOD immunoreactivity increased in the lung, liver and kidney. However, 4-hydroxy-nonenal immunoreactivity and the amount of LPO did not change in each organ. Thus, oxidative damage by toluene is mainly DNA damage, especially, the oxidative DNA damage observed in the lung, liver and kidney for the increase of the immunoreactivity and amount of 8-OH-dG. [Copyright &y& Elsevier]

It is known that methamphetamine (MA) causes rhabdomyolysis, myoglobinuria, and acute renal failure. We conducted an immunohistochemical study on the kidney of 22 forensic autopsy cases in which MA had been detected. Myoglobin was positive in 17 cases. The concentration of the blood MA in the myoglobin-positive cases (8.39±3.43 μmol/dl) was higher than -negative cases (0.198±0.076 μmol/dl). And, the 70 kDa heat shock protein (HSP70), 8-hydroxy-2′-deoxyguanosine (8-OH-dG), 4-hydroxy-2-nonenal (4-HNE), and Cu/Zn superoxide dismutase (SOD) were also stained positively in five, ten, 11, nine cases of examined, respectively. In addition, 80% of HSP70-positive cases were myoglobin-positive. Myoglobin was also observed in 60% of 8-OH-dG-positive, in 82% of 4-HNE-positive, and in 78% of SOD-positive cases, respectively. Therefore, myoglobin rather than MA itself might induce oxidative damage. From these results, it was considered that MA abuse had caused the skeletal muscle damage before death. In forensic autopsy cases of drug abusers, the antemortem situation is not often known. The present research suggested that in addition to the measurement of the concentration of MA, immunohistochemical staining of myoglobin, HSP70, 8-OH-dG, 4-HNE, and SOD offers important information for the diagnosis of MA poisoning. [Copyright &y& Elsevier]

Abstract: SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. SCN5A mutations have been associated with a variety of inherited arrhythmias, including long QT syndrome and Brugada syndrome. We report an autopsy case of sudden unexpected nocturnal death syndrome. A man in his thirties died at night while sleeping. At autopsy, no traumatic injury, disease or drug intake was observed as a possible cause of death. We examined mutations in the SCN5A gene and identified a heterozygous mutation causing an R1193Q amino acid substitution. It was reported that the R1193Q polymorphism in the SCN5A gene destabilizes channel inactivation and may be a risk factor for Brugada and long QT syndrome. It may be considered that the cause of death in this case was sudden cardiac death. [Copyright &y& Elsevier]

Abstract: To diagnose the cause of death in autopsy cases, systematic examinations, such as macroscopic, pathological, biochemical, and toxicological are important. In this case report, drug examinations also gave very useful information to diagnose the cause of death, fatal diabetic ketoacidosis (DKA). A female methamphetamine abuser in her forties was found dead lying on a hotel bed. Diagnosing her cause of death was difficult only from the macroscopic findings because there was no fatal and/or serious injury or disease. On toxicological examination, acetone was detected at a high concentration (682μg/mL in blood, 887μg/mL in urine) using gas chromatography (GC). Using gas chromatography–mass spectrometry (GC–MS), methamphetamine was detected in the blood, urine, hair, and visceral organs; however, these concentrations were low. At the same time, GC–MS examination revealed a high glucose peak. From the results of the biochemical examination of urine, acetoacetic acid was 1940μmol/L, β-hydroxybutyric acid was 14,720μmol/L, and glucose was 4620mg/dL. Histologically, Langerhans’ islets in the pancreas were fibrotic and atrophic, and no insulin-immunoreactive cells were observed. The subsequent police investigation also revealed that she had contracted diabetes mellitus type 1; therefore, we concluded that her cause of death was DKA, due to a lack of insulin injection. [Copyright &y& Elsevier]

Abstract: We report an autopsy case of a man in his forties who died 2days after taking an overdose of vegetamin. The autopsy findings were as follows: externally, the upper epidermis of some parts of the body had become loosened. The epidermis was easily detached from the dermis using the fingers. Viscous fluid adhered around the nose and mouth. The brain was edematous and weighed 1520g. Skeletal muscle was discolored. The urine was a slightly red-tinged yellow. The organs showed congestion. Urine tests: urea nitrogen: 1.95g/day; creatinine: 0.66g/day; urine myoglobin: 1100ng/mL. Blood level of drugs: phenobarbital: 38.2μg/ml; promethazine: 2.22μg/ml; chlorpromazine: 0.96μg/ml. Immunohistochemistry identified myoglobin in the kidney. From these findings, his cause of death was considered to be vegetamin-induced neuroleptic malignant syndrome and rhabdomyolysis. Mutation of the ryanodine receptor 1 gene is associated with malignant hyperthermia. However, there was no mutation which causes amino acid substitution in the three hot-spot regions of the ryanodine receptor 1 gene. Partial deficiency of carnitine palmitoyltransferase II is the commonest cause of recurrent rhabdomyolysis in adults. The subject was found to be heterozygous for an amino acid exchange in exon 4, 1203G→A causing a 368Val→Ile amino acid substitution. It is necessary to examine other candidate gene mutations. [Copyright &y& Elsevier]