An autopsy case of neuroleptic malignant syndrome (NMS) and its immunohistochemical findings of muscle-associated proteins and mitochondria.

Neuroleptic malignant syndrome (NMS) is a rare but potentially fatal disorder. In forensic cases, post-mortem diagnosis of NMS is sometimes difficult if ante-mortem information, such as neuroleptic ingestion or signs and symptoms, cannot be obtained. A 39-year-old Japanese male on a neuroleptic treatment regimen suddenly became agitated and died. Autopsy revealed muscle rigidity and hyperthermia. Post-mortem examination of blood revealed elevation of creatine phosphokinase-MM (CK-MM) and lactate dehydrogenase-4 and dehydrogenase-5 (LDH-4 and LDH-5). In renal glomeruli and tubules, myoglobin was stained immunohistochemically. From these findings, the cause of death was considered to be NMS. To support the diagnosis of NMS, both skeletal and cardiac muscles were stained with actin, myoglobin, desmin and mitochondria antibodies immunohistochemically. Actin, myoglobin, desmin, and mitochondria had been lost from skeletal, but not from the cardiac muscle, which suggested that only the skeletal muscle was damaged. Moreover, because mitochondria had disappeared only from the skeletal muscle, it was considered that skeletal muscle degeneration was caused by mitochondrial damage. Therefore, it is suggested that immunostaining of skeletal muscle by antibodies for muscle-associated proteins and mitochondria is useful to corroborate a diagnosis of NMS.

Neuronal changes in the arcuate and hypoglossal nuclei of brain stem induced by head injury.

In head injury, assessing the damage not only to the cerebrum and the cerebellum but also to the brain stem is very important. In this paper, we report neuronal changes of the arcuate nucleus (ARC) and the hypoglossal nucleus (HN) in the brain stem. We investigated these changes immunohistochemically with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos), and the 72 kD heat-shock protein (HSP70). We measured the percentage of immunopositive neurons among the total neurons of the ARC and the HN. The investigation of neuronal changes in relation to the type of head injury showed different results. In cases of tonsillar herniation, immunoreactivity to MAP2 and mAChR in the ARC was significantly lower than in the HN (p < 0.01). Moreover, MAP2, HSP70 and c-Fos reactivities in the ARC were significantly lower than in other types of head injuries (p < 0.01). In the HN, diffuse axonal injury produced slightly higher immunoreactivity to mAChR and c-Fos (p < 0.1). Our observations indicate that immunohistochemical examination of brain stem nuclei can provide useful information for estimating damage to the brain stem.

Effect of toluene inhalation on astrocytes and neurotrophic factor in rat brain.

Toluene, an abused substance in Japan, is a neurotoxic chemical that has been shown to have neurobehavioral and electrophysiological effects. In previous work, both acute and chronic effects of toluene on cells have been studied extensively. However, although glial cells are thought to play an important role in the survival of neurons in the brain, the effect of toluene on glial cell function has not yet been characterized. To elucidate this, the effect of toluene inhalation on astrocytes in rat brain was examined. Toluene exposure (1500 ppm for 4 h on 4-10 days) augmented glial fibrillary acidic protein (GFAP) immunoreactivity, particularly in the hippocampus and cerebellum. Quantitative analysis showed that toluene inhalation markedly enhanced GFAP expression in the hippocampus and cerebellum. In both regions, proliferating cell nuclear antigen (PCNA) showed no obvious changes, but glutamine synthetase (GS)-immunoreactive cells were markedly increased by toluene exposure. Thus, the elevation of GFAP expression was induced by astrocyte activation rather than by cell proliferation. If toluene exposure activates astrocytes, astrocytes may play a role in the neurophysiological changes observed in toluene intoxication. A neurotrophic factor, basic fibroblast growth factor (b-FGF) was observed immunohistochemically in the capillary vessel walls in the hippocampus and the cerebellum of toluene-intoxicated rats. Basic-FGF may have induced GFAP expression both in the hippocampus and the cerebellum. So, other neurotrophic factors may affect the difference of GFAP elevation between the hippocampus and the cerebellum. These differences may relate to neurobehavioral function of each brain part after toluene exposure.

HSP70 and c-Fos expression of brain stem hypoglossal nucleus in drowning.

The brain stem hypoglossal nucleus (HN) is the center of nerves innervating the upper respiratory tract and is related to control of mastication, deglutition, speech and respiration. To elucidate the relationship between asphyxia and the HN, we investigated the change of hypoglossal neurons in cases of hanging, strangulation, smothering, choking, drowning and respiratory failure. Using immunohistochemical techniques, we observed the brain stem HN with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). MAP2, a cytoskeletal protein of the neuron, is a marker of neuronal damage. Muscarinic AChR was used as a marker of neuronal membrane and ACh signaling. We employed both HSP70 and c-Fos as markers of stress- or damage-related events. We measured the percentage of immunopositive neurons in total neurons of HN. Drowning produced higher expression of HSP70 and c-Fos than other causes of asphyxia, suggesting that drowning induces more severe damage in HN neurons. Furthermore, it was suspected that neuronal changes in drowning might relate to functions of the HN. These observations indicate that immunohistochemical examination of the brain stem HN could provide useful information for determining the cause of asphyxia.

One autopsy case of an elderly traffic accident victim with tetralogy of Fallot.

The case of a 61-year-old male traffic accident victim with Tetralogy of Fallot (TOF) is reported. The autopsy revealed massive hemorrhages in the subcutaneous tissue, muscle, and subarachnoidal space. Furthermore, multiple fractures of ribs, sternum and thoracic vertebrae were observed. Histopathological examination revealed changes characteristic of trauma, such as acute lung congestion, acute renal cortical necrosis, and embolization in the lungs and kidney. These autopsy and histological observations indicated that traumatic shock was cause of his death. Moreover, histologically, we observed changes due to his congenital heart disease, such as right ventricular hypertrophy, heart failure cells in the lungs, sclerosis of the liver, and hyaline degeneration in the kidney. Furthermore, ischemic changes, shrinkage or loss of neurons, were seen in hippocampus, and swelling of astrocytes in both cortex and hippocampus were also observed. These observations lead us to speculate that a hypoxic episode may have caused his accidental death while driving.

Sudden death of a patient with Crohn's disease.

We describe a case of sudden death caused by a rare complication of Crohn's disease. A 29-year-old man with Crohn's disease who had not taken medications regularly complained of nausea, vomiting, and abdominal pain for 2 days, and then died suddenly. Autopsy revealed protruding intestinal loops filled with gas, internal fistulas between fused loops of the terminal ileum, and complete obstruction of the fistulous tract. The histologic findings of transmural inflammation consisting of lymphocytic infiltration, accumulation of partially hyalinized collagen, and fibrosis in the skip areas from the ileum to the cecum were compatible with Crohn's disease. Furthermore, marked emaciation, atrophic change of the heart muscle, and diffuse fatty change of the liver were found. Although the patient died of obstructive ileus caused by a stricture produced by progression of Crohn's disease, he was severely affected by malnutrition which may have been fatal. This case not only illustrates that Crohn's disease can cause obstructive ileus and sudden death, but also makes the forensic pathologist realize the importance of suspecting the presence of an active inflammatory bowel disease in a patient with internal fistulas or malnutrition.

Autopsy case of duodenal obstruction from impacted mushroom.

We report an autopsy case of duodenal bulb obstruction caused by a shiitake mushroom. A 74-year-old woman with depression was admitted to the hospital after suffering nausea and abdominal fullness for 3 days. Because the physical findings and laboratory data on admission revealed marked dehydration, lactated Ringer's solution was administered. Twelve hours later, the patient suddenly died. Autopsy showed an enlarged stomach filled with 850 ml of partially digested food. In the anal side of the pylorus, a 9-cm-diameter shiitake mushroom had become impacted, causing complete obstruction. We conclude that the patient suffered from duodenal bulb obstruction caused by the impacted mushroom and subsequently died of ileus. This case illustrates a rare cause of duodenal obstruction and emphasizes that ingested food can cause obstructive ileus and death.

Significance of local recurrence of rectal cancer as a local or disseminated disease.

BACKGROUND: The nature of 'local recurrence' of rectal cancer remains unclear. METHODS: Fifty-nine patients with locally recurrent rectal cancer who underwent extended repeat resections including total pelvic exenteration (39) and sacrectomy (43) were reviewed. Twelve patients had distant metastases before or at the time of repeat resection. RESULTS: The 5-year survival rate was 25 per cent. A second recurrence occurred in 45 patients including five of the eight 5-year survivors. Thirty-six of these recurrences had locoregional manifestations and 29 had distant metastases. Of 18 prognostic factors examined, the most significant determinant was the postoperative carcinoembryonic antigen doubling time (CEADT), followed by the preoperative CEADT, carcinoembryonic antigen (CEA) level and occurrence of distant metastases, in decreasing order. Late onset of first recurrence was also a favourable indicator. Thus, the growth rate of the tumour had a more profound impact on survival than the current extent of tumour progression. After operation the CEADT was reduced in patients with second recurrence (P = 0.05). CONCLUSION: Locally recurrent rectal cancer is a manifestation of disseminated disease spreading locoregionally and often to distant organs with a low probability of long-term cure. However, survival varies widely depending on the tumour growth rate, which is biologically predetermined and is also influenced by surgery.

Immunohistochemical diagnosis and significance of forensic neuropathological changes.

Immunohistochemistry is very useful when investigating the cause of death. Ischemic cell changes in the hippocampal neurons were not obvious in the brains damaged by hypoxic injury. However, it is suggested that even a moderate hypoxia, which may affect the neuronal proteins and metabolism, induced astrocytosis in the CA3 and CA4 regions, and that in patients with a history of hypoxic attacks neuronal damage may be severe even several hours after ischemic injury. Furthermore, hsp70 expression was found in the CA2, CA3 and CA4 regions of long-term survivors after severe hypoxic/ischemic injury. In forensic practice, detailed information about the duration and extent of a hypoxic/ischemic injury is often unavailable, so that immunohistochemical detection of hsp70 and glial cell staining can be of great value in diagnosing not only the hypoxic/ischemic injury during the process of death but also the victim's past history of hypoxic attacks. In diffuse axonal injury, degeneration of axon and myelin, such as swelling and waving, were observed in survivors of more than 8 hours. Retraction balls appeared in survivors of more than 1 days. In longer term survivors, such as 3 or 5 months, breakdown of myelin and fat-granule cells were observed. In addition, retraction balls were also found. Immunohistochemical staining of 200 kD neurofilament was a very useful method to examine axonal changes, because antisera is specific for degenerative neurofilaments. In our study, all cases which had pathological findings of diffuse axonal injury (DAI) were associated with focal head injuries. From the immunohistochemical staining of neurons in the hippocampus, it was suggested that neurons in the hippocampus were injured by diffuse brain damage. Furthermore, repairing and protective mechanisms occurred especially from CA2 to CA4. It was considered that neuronal damage in diffuse brain injury was elucidated not only morphologically but also functionally. Therefore, in cases of suspected diffuse brain damage, it is recommended to examine the neuronal changes in addition to observing the findings of diffuse axonal injury. Immunohistochemical staining of the carotid body is potentially very useful for necropsy diagnosis, since it provides a method to detect evidence of mechanical asphyxia in suspected cases of manual and/or ligature strangulation.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. I. Various cases of asphyxia and respiratory disorder].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. To investigate the correlation of brain stem damage with various cause of respiratory disorders, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) were examined using immunohistochemical technique. Based on the cause of death, the forensic autopsy cases were divided into 5 groups as follows. Group I: hanging, ligature strangulation and manual strangulation, Group II: smothering and choking, Group III: drowning, Group IV: respiratory failure, control group: heat stroke and sun stroke. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Three nuclei showed no obvious morphological changes in all examined groups. However, in case of asphyxia (Group I to III), neurons in HN were positively stained with both HSP70 and c-Fos antibodies. This may indicate that the occlusion of upper airway results in the neuronal damage of HN without their morphological changes. Positive staining of HSP70 and c-Fos in IO was more frequently observed in Group III than other 4 groups. Since IO is involved in maintaining body balance which is often disturbed by drowning, it seems possible that neuronal damage in IO observed in drowning may be related to the disturbance of body balance. These observations indicate that immunohistochemical study on the damage to neurons in brain stem nuclei can provide useful information for determining the cause of death.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. II. Sudden infant death syndrome].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. Previously, in sudden infant death syndrome (SIDS) it has been suggested that impaired cardioventilatory control might contribute to cause of death. So, to investigate the brain stem damage in SIDS, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) was examined using immunohistochemical technique. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Morphological changes of neurons in three nuclei were not evident. Moreover, because MAP2 degeneration and expression of HSP70 and c-Fos were not observed, neuronal damage in those nuclei was not suspected. However, although there was no abnormality of mAChR immunostaining in HN and IO, the rate of mAChR-immunopositive neurons in ARC was less than that in control. These observations indicate that immunohistochemical study on the neuronal changes in ARC can provide useful information for diagnosing SIDS.

Isolated adrenocorticotropic hormone deficiency: an autopsy case of adrenal crisis. A case report.

We present a case of fatal adrenal crisis due to isolated adrenocorticotropic hormone (ACTH) deficiency. Autopsy revealed each adrenal gland weighed 0.9 g and the adrenal cortexes were very thin and atrophic. Additionally, cortisol could not be observed in the adrenal cortex by immunohistochemical staining. Furthermore, urine cortisol and 17-OHCS concentration had decreased to a very low level, 20 mg/L and 0.8 mg/L respectively. The anterior pituitary gland was atrophic, and showed fibrosis and lymphocytosis was suspected. Immunohistochemically growth hormone (GH)-stained pituitary gland cells were observed, but there were no cells stained with anti-ACTH antibody. From the history and pathological findings, no other deficiencies of pituitary hormones were evident. Therefore, isolated ACTH deficiency was suspected. Furthermore, as the thyroid gland showed lymphocytic thyroiditis, is was considered that isolated ACTH deficiency was associated with an autoimmune cause. Generally, as patients of chronic adrenocortical insufficiency are exposed to stress and, therefore, have an increased requirement for glucocorticoids, the blood pressure falls, leading to hypovolemic shock called " an adrenal crisis." Without treatment, patients die in crisis within several hours. In our case, the deceased had drunk alcohol without sleep for 2 days. We believe that the stress of drinking and sleeplessness induced adrenal crisis and caused his death.

[Analysis of sudden death caused by intestinal obstruction].

Five autopsy cases of sudden death caused by intestinal obstruction are reported. The causes of death of the cases were duodenal obstruction of impacted food stuff, ileocaecal obstruction caused by Crohn's disease, incarceration of inguinal hernia, intestinal obstruction caused by heterotopic pancreas and paralytic ileus. In three cases, the patient was in cardiopulmonary arrest on arrival at hospital, and in the remaining cases the patient died within 12 hours from the beginning of treatment; therefore, a correct clinical diagnosis was not made before the death in all cases. All the patients had from one to three days history of nausea and abdominal pain, major complications of intestinal obstruction. Among all cases, the duration from the onset to death was the shortest in the case of a patient complicated with schizophrenia. It is characteristic that the patients of all cases died suddenly and resuscitation was not successful. Regarding the laboratory data of a hospitalized patient, marked hemoconcentration and an increased level of BUN/Cr ratio and blood sugar were shown. The patient who died from duodenal obstruction caused by impacted food-stuff had suffered from depression for six years, and the patient who died from paralytic ileus had suffered from schizophrenia for about 23 years. In both cases, it is characteristic that the complaints of the patient were poorer than what would be expected. Furthermore, these patients had been taking medication of psychotic, anti-depressant and anti-parkinsonism drugs; therefore the combination of these drugs was thought to be reflected in the bowel movement.

[A case of dural sinus thrombosis during the medication of medroxyprogesterone acetate].

It is reported that medroxyprogesterone acetate (MPA) causes venous thrombosis as one of the side effects. A 49-year-old woman suffering from metastatic lung carcinoma from breast carcinoma was administered MPA 1200 mg/day for about four months. Thereafter she complained of dizziness about two weeks but the general practitioners could detect no abnormality on physical examinations and on brain CT X-ray findings. Six days later, she died suddenly at her home. Forensic autopsy findings revealed the marked superior sagital sinus thrombosis and the malignant lymphadenomatosis caused by metastatic lung carcinoma. As the patient had not disease or trauma causing dural sinus thrombosis except for the administration of MPA, we concluded that superior sagital sinus thrombosis was due to the medication of MPA. This case illustrates that forensic pathologist should consider the major side effect of some drugs like our case.

[An autopsy case of traumatic subdural hematoma from arterio-venous malformation with diffuse axonal injury].

Diffuse axonal injury (DAI) is defined as widespread damage to axons in the white matter of the brain without focal injury such as contusion and acute subdural hematoma. A case of traumatic subdural hematoma from arterio-venous malformation accompanied by DAI is reported. A 58-year-old man was assaulted, and immediately lost consciousness, and remained unconscious during about 44 hours until his death. The autopsy revealed acute subdural hematoma (about 160 g) on left temporal lobe and left cingular, uncal and cerebellar tonsillar herniation, and tear and hemorrhage of the corpus callosum. Under this subdural hematoma, gray-whitish vascular lesion with subarachnoid hemorrhage was found. Histologically, this lesion was diagnosed as the arterio-venous malformation. Neuropathological examination of the corpus callosum, dorsolateral part of midbrain and superior cerebellar peduncle revealed DAI findings, such as swelling and ballooning of the myelin fibers, swelling and waving of axons, and retraction balls. Axon degenerations were also observed immunohistochemically by anti-200 kD neurofilament antibody. From the results, his unconsciousness from the moment of impact might be occurred from not only subdural hematoma but also DAI.

[An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhage (hematoma)].

An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhages (hematoma) is reported. A 49-year-old woman who had been suffering from Sjögren syndrome had gradually lost her consciousness and was taken to the hospital where she died several hours later. Subsequently a doctor found the subdural hematoma of unknown origin on her Brain CT. At autopsy, her skin was dry and all of her teeth were missing. There were sporadic cutaneous purpura and subcutaneous hemorrhages in her trunk and limbs. The histopathological examination revealed that the submandibular gland had no normal acini, and was replaced by fibrous and adipose tissues with numerous lymphocytes. There were signs of fibrosis with inflammation in her liver, kidneys and lungs. The thyroid gland showed thyroiditis. Serological findings showed a significant high level of antinuclear antibody, positive RA factor and high gamma-globulinemia. The autopsy revealed that her cause of death was acute subdural hematoma and uncal herniation. There were no external injuries on her head or face. It is suggested that her acute subdural hematoma according to the hemorrhagic tendency, affected by her Sjögren syndrome.

Immunohistochemical investigation of hypoxic/ischemic brain damage in forensic autopsy cases.

A neuropathological study of 41 forensic autopsy cases of hypoxic/ischemic brain damage has been undertaken, using immunohistochemical staining to detect the 70-kDa heat shock protein (hsp70) and the status of the glial cells. In cases surviving 2-5 h after hypoxic/ischemic injury, ischemic cell changes were seen whereas glial reactions were not apparent. In cases of longer survival, neuronal necrosis and a loss of neurons were seen, and these changes were accompanied by proliferation of glial fibrillary acidic protein (GFAP), vimentin-positive astrocytes and microglia which transformed into rod cells or lipid-laden macrophages. In cases with a history of hypoxic attacks, GFAP-positive and vimentin-negative astrocytes had proliferated in the CA3 and CA4 regions of hippocampus. The cases of severe hypoxic injury, such as an asthmatic attack and choking, showed no ischemic changes in the hippocampal neurons. On the other hand, the CA1 pyramidal cells showed neuronal necrosis in a patient suffering from tetralogy of Fallot (TOF), who survived for 2 h after a traffic accident. Therefore, it is suggested that even moderate hypoxic injury induces astrocytosis in the CA3 and CA4 regions and may affect the neuronal proteins and the metabolism, and that in cases with a history of hypoxic attacks neuronal damage may be severe even several hours after ischemic injury. The protein hsp70 expression was found in the CA2, CA3 and CA4 regions in cases of long-term survival after severe hypoxic/ischemic injury and in cases of alcoholic intake or toluene abuse just before acute death.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistological investigations of autopsied carotid bodies and their application to diagnosing strangulation.

Using immunohistochemical staining, the histological changes and the presence of neuropeptides (enkephalin and VIP) in the carotid body have been investigated in medico-legal autopsy cases, especially asphyxia cases. Only in cases of manual and/or ligature strangulation cases that sustained a force near the carotid body, were the chief cells mainly lightly stained, indicating that they had been "active" cells. Furthermore, these cells and their nuclei were enlarged in comparison to the chief carotid body cells in other autopsy groups. It was thus felt that these changes had resulted from the force that had directly affected the carotid body. Based on these findings, it was concluded that immunohistochemical investigation of the carotid body offers a useful possibility for diagnosing manual asphyxia, especially in autopsy cases involving strangulation.

[Paternity test by DNA fingerprinting in a sexual assault].

We have applied the method of DNA fingerprinting to resolve a paternity case. DNA samples were extracted from the blood of mother, child (4 months old fetus) and alleged father, digested with the restriction endonuclease Hinf I and Hae III, size separated by agarose gel electrophoresis, and hybridized with the multi-locus minisatellite probe B.E.S.T.-MZ1.3 digoxigenin. DNA fingerprinting patterns in the child and alleged father indicated possibly paternity. Following extraction of DNA from cord blood (infant of 26 to 39 week's gestation), the quantity of DNA isolated was determined on a spectrophotometer at 260 nm and its integrity by electrophoresis in agarose gels. We found that high-molecular-weight DNA could be recovered in large quantities from cord blood as well as from adult blood.

Hereditary recombined three-allele variant of the Gc system.

A three-allele variant with Gc 2, Gc 1F and Gc 1A2 alleles was detected in both a baby and his mother during paternity testing by isoelectric focusing. His father had a normal Gc phenotype, Gc 2-1F. Further examination of his mother's relatives revealed that his grandfather also had the same three-allele variant, while his grandmother and his aunt had normal Gc 2-1F and Gc 2-2. From these results, it was considered that the Gc 1F and Gc 1A2 alleles were on the same single chromosome. It was suggested that recombination had occurred between two chromosomes that had the Gc 1F and Gc 1A2 allele, respectively, forming the variant allele Gc 1F1A2 on a single chromosome.