Molecular autopsy for sudden death in Japan.

Japan has various death investigation systems; however, external examinations, postmortem computed tomography, macroscopic examinations, and microscopic examinations are performed regardless of the system used. These examinations can reveal morphological abnormalities, whereas the cause of death in cases with non-morphological abnormalities can be detected through additional examinations. Molecular autopsy and postmortem genetic analyses are important additional examinations. They are capable of detecting inherited arrhythmias or inherited metabolic diseases, which are representative non-morphological disorders that cause sudden death, especially in infants and young people. In this review, we introduce molecular autopsy reports from Japan and describe our experience with representative cases. The relationships between drug-related deaths and genetic variants are also reviewed. Based on the presented information, molecular autopsy is expected to be used as routine examinations in death investigations because they can provide information to save new lives.

Autopsy results of a case of ingestion of sodium hydroxide solution.

Sodium hydroxide is a strongly corrosive alkali. We describe herein a case of suicide by ingestion of sodium hydroxide. A man in his 80s was found dead with a mug and a bottle of caustic soda. Macroscopically, liquefaction and/or disappearance of esophagus, trachea and lung tissue and a grayish discoloration of the mucosa of the stomach were seen along with blackish brown coloration of the skin, mouth, and oral cavity. The contents of the gastrointestinal tract showed a pH level of 7-8 on pH indicator strips. Histopathologically, liquefactive necrosis of remnant lung tissue and the stomach were seen. As biological reactions such as vasodilatation and inflammation were not detected in these organs, only a short number of hours must have passed between ingestion and death. This human case provides valuable information concerning the direct irritation induced by systemic exposure to corrosive substances.

Green tea extract attenuates MNU-induced photoreceptor cell apoptosis via suppression of heme oxygenase-1.

The effects of green tea extract (GTE) on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis were examined, and the possible mechanisms of action of GTE were assessed. Alterations in the retinal morphological architecture were determined by hematoxylin-eosin staining, vimentin immunoreactivity, and photoreceptor cell apoptosis (TUNEL labeling). Expression of oxidant marker, heme oxygenase (HO)-1, mRNA levels in outer nuclear cells was assessed by laser capture microdissection (LCM). Sprague-Dawley rats were given 40 mg/kg MNU at 7 weeks of age in the absence and presence of 250 mg/kg GTE treatment (once daily from 3 days prior to MNU for a maximum 10 days). Although photoreceptor cell degeneration began 24 hr after MNU, the morphological effects of GTE at the time point were not definitive. However, GTE lowered TUNEL labeling and HO-1 mRNA expression. At 7 days after MNU, photoreceptor damage was attenuated by GTE treatment. Therefore, the ability of GTE to reduce MNU-induced photoreceptor cell apoptosis may be due to its antioxidant properties.

Susceptibility to N-methyl-N-nitrosourea-induced retinal degeneration in different rat strains.

To evaluate the potential role of genetic background in the susceptibility to retinal degeneration induced by N-methyl-N-nitrosourea (MNU), female rats of the Sprague-Dawley (SD), Long-Evans (LE) and Copenhagen (CH) strains were administered 50 mg/kg MNU or saline at 7 weeks of age. Retina morphology and morphometric analysis of all rats was performed 7 days after MNU administration. Atrophy of both the peripheral and central outer retina occurred in all rat strains exposed to MNU. Decreased photoreceptor cell ratio and increased retinal damage ratio were observed. The severities of the retinal atrophy were similar among all three rat strains. In conclusion, MNU-induced photoreceptor degeneration developed consistently in all three strains regardless of the absence (SD rats) or presence (LE and CH rats) of melanin in the retina, suggesting that genetic and melanin factors did not affect photoreceptor cell death after MNU.

A spontaneously occurring malignant ovarian Sertoli cell tumor in a young Sprague Dawley rat.

Primary ovarian tumors are generally uncommon in rats used in toxicologic studies. A malignant Sertoli cell tumor was present in the ovary of a 19-week-old female Sprague Dawley rat. Macroscopically, the mass was white and firm, 10 × 13 × 17 mm in size, and located in the right ovary. Histopathologically, the mass was composed of nests of pleomorphic cells, which formed seminiferous-like tubules separated by a thin fibrovascular stroma. The tubules were lined by tumor cells, which had basally located nuclei and abundant eosinophilic and vacuolated cytoplasm. In some areas, the tumor cells were arranged in a retiform growth pattern, mimicking a rete testis/ovarii. Disseminated metastases to the surfaces of the mesentery, spleen and liver were also present. Immunohistochemically, many tumor cells were strongly positive for vimentin, estrogen receptor α and Ki 67. Some tumor cells were positive for pancytokeratin and inhibin α. These findings closely resemble those of an ovarian-derived human malignant Sertoli cell tumor. From our review of the literature, we believe this is the first report of a spontaneous malignant Sertoli cell tumor in the ovary of a young laboratory rat. This case might provide useful historical control information for rat toxicity studies.

Mead acid supplementation does not rescue rats from cataract and retinal degeneration induced by N-methyl-N-nitrosourea.

Fatty acids and their derivatives play a role in the response to ocular disease. Our current study investigated the effects of dietary mead acid (MA, 5,8,11-eicosatrienoic acid) supplementation on N-methyl-N-nitrosourea (MNU)-induced cataract and retinal degeneration in Sprague-Dawley rats. Experiment 1 was designed to inhibit cataract formation, with the dams fed a 2.4% MA or basal (<0.01% MA) diet during lactational periods. On postnatal day 7, male pups received a single intraperitoneal (ip) injection of 50 mg/kg MNU or vehicle. Lens opacity and morphology were examined 7 and 14 days after the MNU injection. Experiment 2 was designed to inhibit retinal degeneration and was performed with female postweaning rats. In this experiment, dams were fed the 2.4% MA or basal diet during the lactational periods. Thereafter, the female pups were continuously fed the same diets during their postweaning periods. On postnatal day 21 (at weaning), pups received a single ip injection of 50 mg/kg MNU. Retinal morphology was examined 7 days after the MNU injection. In experiment 3, six-week-old female rats were fed the 2.4% MA or basal diet starting at one week before the MNU injection and were then continuously fed the same diets until sacrifice. Rats at 7 weeks of age were given a single ip injection of 40 mg/kg MNU, and the retina was then examined morphologically one week after the MNU injection. In experiment 1, mature cataract was found in all of the MNU-treated groups, with or without MA supplementation. In experiments 2 and 3, atrophy of both the peripheral and central outer retina occurred in all rats exposed to MNU, with or without MA supplementation, respectively. The severities of the cataracts and retinal atrophy in the rats were similar regardless of MA supplementation. Dietary mead acid, which is used as a substitute in essential fatty acid deficiency in the body, does not modify MNU-induced cataract and retinal degeneration in rat models.

Autopsy report for a caffeine intoxication case and review of the current literature.

Caffeine (1,3,7-trimethylxanthine) is a popular mild central nervous system stimulant found in the leaves, seeds and fruits of various plants and in foodstuffs such as coffee, tea, and chocolate, among others. Caffeine is widely used and is not associated with severe side effects when consumed at relatively low doses. Although rarely observed, overdoses can occur. However, only a few fatal caffeine intoxication cases have been reported in the literature. Herein, we report the pathological examination results and information on caffeine concentrations in the blood, urine and main organs in a fatal caffeine intoxication case. Even though high caffeine concentrations were found in the systemic organs, no caffeine-related pathological changes were detected.

Green tea extract suppresses N-methyl-N-nitrosourea-induced photoreceptor apoptosis in Sprague-Dawley rats.

BACKGROUND: Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and eventual blindness. A single intraperitoneal (ip) injection of N-methyl-N-nitrosourea (MNU) causes photoreceptor cell apoptosis within 7 days in rats. Green tea extract (THEA-FLAN 90S; GTE) is a common herbal supplement with pluripotent properties including antioxidant activity. The purpose of the present study was to evaluate the efficacy of GTE against photoreceptor apoptosis in 7-week-old female Sprague-Dawley rats that received a single ip injection of 40 mg/kg MNU. METHODS: The oral administration of 250 mg/kg/day GTE was initiated 3 days prior to MNU injection and continued once daily throughout the experiment. Rats were sacrificed at 12, 24, and 72 h and 7 days after MNU injection, and the eyes were examined morphologically and morphometrically. The photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio were used to determine the structural and functional alterations. The number of apoptotic photoreceptor cells per mm(2) was determined in situ by TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL). Our results indicated that oral administration of GTE significantly suppressed the loss of photoreceptor cells morphometrically 7 days after MNU injection. The number of TUNEL-positive cells per mm(2) in MNU-exposed rat central retina with or without GTE administration was 981 vs. 2056 at 24 h after MNU injection. CONCLUSIONS: GTE structurally and functionally suppressed MNU-induced photoreceptor cell apoptosis. These findings indicate that GTE may help to ameliorate the onset and progression of human RP.

Green Tea Extract-induced Acute Hepatotoxicity in Rats.

Although green tea is considered to be a healthy beverage, hepatotoxicity associated with the consumption of green tea extract has been reported. In the present study, we characterized the hepatotoxicity of green tea extract in rats and explored the responsible mechanism. Six-week-old IGS rats received a single intraperitoneal (ip) injection of 200 mg/kg green tea extract (THEA-FLAN 90S). At 8, 24, 48 and 72 hrs and 1 and 3 months after exposure, liver damage was assessed by using blood-chemistry, histopathology, and immunohistochemistry to detect cell death (TUNEL and caspase-3) and proliferative activity (PCNA). Analyses of malondialdehyde (MDA) in serum and the liver and of MDA and thymidine glycol (TG) by immunohistochemistry, as oxidative stress markers, were performed. Placental glutathione S-transferase (GST-P), which is a marker of hepatocarcinogenesis, was also immunohistochemically stained. To examine toxicity at older ages, 200 mg/kg green tea extract was administered to 18-wk-old female rats. In 6-wk-old rats, 12% of males and 50% of females died within 72 hrs. In 18-wk-old rats, 88% died within 72 hrs. The serum levels of aspartate aminotransferase, alanine aminotransferase and/or total bilirubin increased in both males and females. Single-cell necrosis with positive signs of TUNEL and caspase-3 was seen in perilobular hepatocytes from 8 hrs onward in all lobular areas. PCNA-positive hepatocytes increased at 48 hrs. MDA levels in the serum and liver tended to increase, and MDA- and TG-positive hepatocytes were seen immunohistochemically. GST-P-positive hepatocellular altered foci were detected in one female rat at the 3-month time point. In conclusion, a single injection of green tea extract induced acute and severe hepatotoxicity, which might be associated with lipid peroxidation and DNA oxidative stress in hepatocytes.

Similarity of GATA-3 Expression between Rat and Human Mammary Glands.

The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. In particular, GATA-3 is necessary for mammary gland maturation and is a useful marker in the characterization of mammary carcinoma in humans. The expression of GATA-3 protein in normal mammary glands, fibroadenomas and carcinomas was immunohistochemically compared in female rats and humans. In normal mammary glands of rats and humans, scattered luminal cells in the acini and whole ductal epithelial cells were positive for GATA-3 in the nuclei. No positive cells were detected in rat or human fibroadenomas. In rat and human mammary carcinomas, the nuclei of proliferating luminal-derived cancer cells expressed GATA-3. Therefore, GATA-3 protein is a candidate marker for mammary carcinoma in rats as well as humans.

Autopsy case of sudden maternal death from thrombotic thrombocytopenic purpura.

A 31-year-old pregnant woman was transferred to the emergency room at 27 weeks of gestation. She had one-day history of fever and upper abdominal pain. Soon after admission, she underwent cardiopulmonary arrest. Autopsy was performed and multiple microthrombi were seen within the small-caliber vessels of many organs, but not in the lungs. Immunohistochemical staining revealed that the thrombi were rich in von Willebrand factor. We also obtained results which showed severely deficient plasma a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS) 13 activity and positive ADAMTS13 inhibitor, confirming a diagnosis of thrombotic thrombocytopenic purpura. As far as we know, in Japan, this is the first autopsy report of sudden maternal death from thrombotic thrombocytopenic purpura. We expect that the routine laboratory application of ADAMTS13 assays for unknown thrombocytopenic patients during pregnancy may help in differential diagnosis at an earlier stage of the disease and facilitate tailor-made therapeutic intervention.

Histopathological and immunohistochemical characterization of spontaneously occurring uterine deciduomas in young adult rats.

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.

N -Methyl- N -nitrosourea-induced Renal Tumors in Rats: Immunohistochemical Comparison to Human Wilms Tumors.

N-Methyl-N-nitrosourea (MNU)-induced renal tumors in rats and Wilms tumors in humans were compared. Renal mesenchymal tumors (RMTs) and nephroblastomas (blastemal and epithelial components) in female Lewis rats treated with a single intraperitoneal injection of 50 mg/kg MNU at birth and Wilms tumors (blastemal, epithelial and mesenchymal components) in humans were analyzed for the expression of pancytokeratin (CK), vimentin, p63, α-smooth muscle actin (SMA), desmin, S-100, CD57, CD117/c-kit, Wilms tumor 1 protein (WT1) and ß-catenin. The mesenchymal components of rat RMTs and human Wilms tumors expressed vimentin, SMA and ß-catenin. The blastemal components of rat nephroblastomas and human Wilms tumors expressed vimentin, CD117/c-kit and ß-catenin. The epithelial components of rat nephroblastomas and human Wilms tumors expressed vimentin and ß-catenin. WT1 was expressed in different cellular components of rat tumors as compared with human Wilms tumors; the expression was seen in mesenchymal tumors and blastemal components of nephroblastomas in rats and epithelial components in human Wilms tumors. CK, p63 and CD57 were not expressed in rat RMTs or nephroblastomas, while CK and WT1 were expressed in epithelial components and CD57 was expressed in blastemal and epithelial components of human Wilms tumors. Rat and human tumors were universally negative for the expression of desmin and S-100. The immunohistochemical characteristics of rat renal tumors and human Wilms tumors may provide valuable information on the differences in renal oncogenesis and biology between the two species.

Metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy: diagnosis of mitochondrial respiratory chain disorders.

Mitochondrial respiratory chain disorders are the most common disorders among inherited metabolic disorders. However, there are few published reports regarding the relationship between mitochondrial respiratory chain disorders and sudden unexpected death in infancy. In the present study, we performed metabolic autopsy in 13 Japanese cases of sudden unexpected death in infancy. We performed fat staining of liver and postmortem acylcarnitine analysis. In addition, we analyzed mitochondrial respiratory chain enzyme activity in frozen organs as well as in postmortem cultured fibroblasts. In heart, 11 cases of complex I activity met the major criteria and one case of complex I activity met the minor criteria. In liver, three cases of complex I activity met the major criteria and four cases of complex I activity met the minor criteria. However, these specimens are susceptible to postmortem changes and, therefore, correct enzyme analysis is hard to be performed. In cultured fibroblasts, only one case of complex I activity met the major criteria and one case of complex I activity met the minor criteria. Cultured fibroblasts are not affected by postmortem changes and, therefore, reflect premortem information more accurately. These cases might not have been identified without postmortem cultured fibroblasts. In conclusion, we detected one probable case and one possible case of mitochondrial respiratory chain disorders among 13 Japanese cases of sudden unexpected death in infancy. Mitochondrial respiratory chain disorders are one of the important inherited metabolic disorders causing sudden unexpected death in infancy. We advocate metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy cases.

Case report of death from falling: Did heart tumor cause syncope?

A healthy man in his 30s was working on the balustrade of stairs on the second floor. He suddenly fell downstairs without saying anything. On emergency hospitalization, chest echogram showed left hemothorax. Cardiac echogram showed a floating mass from the mitral valve in the left ventricle and severe mitral regurgitation. Surgery for hemothorax and pulmonary contusion was immediately undertaken. However, bleeding from pulmonary contusion could not be controlled and he underwent cardiopulmonary arrest. Autopsy showed a white, elastic, pendulous mass in the left atrium and a white mass in the lower lobe of the left lung. Tumor histology showed a reticular pattern, Schiller-Duval bodies, eosinophilic hyaline globules, and positive staining for α-fetoprotein. We diagnosed primary lung yolk sac tumor with metastatic intracardiac yolk sac tumor, a rare and highly malignant germ cell tumor. It usually arises in the ovaries and testes, and intracardiac yolk sac tumor is rare. Intracavitary tumors induce obstruction of inflow into and outflow from the ventricular cavity. The most common clinical presentation is dyspnea and syncope. In the present case, metastatic cardiac yolk sac tumor might have disturbed cardiac outflow and affected hemodynamics, probably causing syncope. Unfortunately, he was in a high place at that time and fell to receive pulmonary contusion that led to death. Autopsy may sometimes reveal latent diseases which might be related to the cause of death. We should perform autopsy thoroughly to diagnose not only the cause of death but also the factors leading to death.

Retrospective review of Japanese sudden unexpected death in infancy: the importance of metabolic autopsy and expanded newborn screening.

Sudden unexpected death in infancy is defined as sudden unexpected death occurring before 12 months of age. The common causes of sudden unexpected death in infancy are infection, cardiovascular anomaly, child abuse, and metabolic disorders. However, the many potential inherited metabolic disorders are difficult to diagnose at autopsy and may therefore be underdiagnosed as a cause of sudden unexpected death in infancy. In the present study we retrospectively reviewed 30 Japanese sudden unexpected death in infancy cases encountered between 2006 and 2009 at our institute. With postmortem blood acylcarnitine analysis and histological examination of the liver, we found two cases of long-chain fatty acid oxidation defects. Molecular analysis revealed that the one patient had a compound heterozygote for a novel mutation (p.L644S) and a disease-causing mutation (p.F383Y) in the carnitine palmitoyltransferase 2 gene. Furthermore, retrospective acylcarnitine analysis of the newborn screening card of this patient was consistent with carnitine palmitoyltransferase II deficiency. Metabolic autopsy and expanded newborn screening would be helpful for forensic scientists and pediatricians to diagnose fatty acid oxidation disorders and prevent sudden unexpected death in infancy.

Prolonged constant eye misalignment leads to failure to develop binocular vision in childhood ocular myasthenia gravis.

PURPOSE: Variable eye misalignment and blepharoptosis in childhood ocular myasthenia gravis can lead to permanent binocular visual loss. However, a standard ophthalmologic intervention for this condition has yet to be fully established. This study investigated the influence of variable eye misalignment and asymmetric blepharoptosis on the development of binocular vision in childhood ocular myasthenia gravis. METHODS: The authors retrospectively reviewed clinical records of consecutive patients with childhood ocular myasthenia gravis whose age of onset was younger than 36 months and who had follow-up periods of more than 1 year. Five patients were enrolled and were treated medically with pyridostigmine or corticosteroids. Eye alignment, eye movements, and blepharoptosis were observed during the follow-up period. Stereoacuity was recorded before and at the end of follow-up using Titmus stereoscopic and major amblyoscopic tests. RESULTS: None of the patients had amblyopia or anisometropia, but all had blepharoptosis that improved within 2 weeks after medication was administered. There was only one case in which stereoacutiy failed to develop, and this patient had a long period of constant exotropia lasting 48 months. In the remaining four cases, there was constant exotropia lasting less than 4 months or intermittent exotropia throughout the follow-up period. All of these patients regained or maintained binocular vision. CONCLUSION: Prolonged constant eye misalignment may be attributable to impaired binocular vision, and prompt eye alignment may be necessary in childhood ocular myasthenia gravis. Therefore, treatment based on precise assessment by a neuro-ophthalmologist or pediatric ophthalmologist that evaluates even slight eye misalignment is essential in ensuring normal development of binocular vision.

Spontaneously occurring lymphohematopoietic tumors in three young Sprague Dawley rats.

To assess the toxicological and pharmacological effects of chemicals, it is important to know what kinds of neoplasms naturally occur in the early life of laboratory animals. In the present study, we identified three spontaneous hematopoietic tumors in three of 52 young female Sprague-Dawley rats used in a pharmacological study. These cases included two rats (Case 1 and 2) from a sesame oil-treated group and one rat (Case 3) from a chemical-treated group in the same single gavage study. Case 1 rapidly lost body weight at 13 weeks of age without any clinical signs and died. Round lymphoid tumor cells were found in the spleen, liver, bone marrow, and pancreas. The tumor cells were immunohistochemically positive for CD3 and PCNA, which is suggestive of malignant T-cell lymphoma. Cases 2 and 3 had rapid body weight loss at 14 and 16 weeks of age, respectively, exhibited severe anemia, hypolocomotion, and decreased body temperature, and were euthanized due to a poor prognosis based on severe clinical signs. Pleomorphic large tumor cells were found in the spleen, liver, bone marrow, lymph nodes, heart, kidneys, lung, pancreas, adrenal glands, pituitary gland, ovaries, Harderian gland, and/or eyes. The tumor cells were immunoreactive for CD34, lysozyme, and PCNA, which is suggestive of myeloid leukemia. These cases might provide useful historical control information for rat toxicity studies.

Autopsy report for chemical burns from cresol solution.

Cresol, which is used as a disinfectant and insecticide, has erosive effects on epidermal and epithelial tissues in the body. Oral exposure causes gastrointestinal corrosive injuries as a direct chemical burn. We report herein a case of suicidal poisoning by ingestion of cresol solution. An octogenarian man with depression was found dead approximately 14 h after exposure to less than 500 mL of saponated cresol solution. Macroscopically, corrosive lesions such as red-to-brown-colored epithelium and edematous thickening of walls were seen in the skin, mouth, oral cavity, esophagus, and stomach. Histopathologically, coagulative necrosis and vascular dilatation were detected from mucosal to muscular layers in the esophagus, stomach, and duodenum. Congestive edema of the lungs, edematous changes in the brain, and proximal tubular necrosis of the kidneys were seen, suggesting acute circulatory disturbance due to shock. This human case offers valuable information on the direct irritation and shock induced by systemic exposure to corrosive substances.