Chiemi Yoshida

Immunohistochemistry of catecholamines in the hypothalamic–pituitary–adrenal system with special regard to fatal hypothermia and hyperthermia

Catecholamines are involved in various stress responses. Previous studies have suggested applicability of the postmortem blood levels to investigations of physical stress responses or toxic/hyperthermic neuronal dysfunction during death process. The present study investigated cellular immunopositivity for adrenaline (Adr), noradrenaline (Nad) and dopamine (DA) in the hypothalamus, adenohypophysis and adrenal medulla with special regard to fatal hypothermia (cold exposure) and hyperthermia (heat stroke) to examine forensic pathological significance. Medicolegal autopsy cases (n=290, within 3 days postmortem) were examined. The proportions of catecholamine (Adr, Nad and DA)-positive cells (% positivity) in each tissue were quantitatively estimated using immunostaining. Hyperthermia cases (n=12) showed a lower neuronal DA-immunopositivity in the hypothalamus than hypothermia cases (n=20), while Nad- and DA-immunopositivities in the adrenal medulla were higher for hyperthermia than for hypothermia. Rates of Nad-immunopositivity in the adrenal medulla were very low for hypothermia. No such difference between hypothermia and hyperthermia was seen in the adenohypophysis. In hypothermia cases, cellular Nad-immunopositivity in the adrenal medulla correlated with the Nad level in cerebrospinal fluid (r=0.591, p<0.01). These observations suggest a characteristic immunohistochemical pattern of systemic stress response to fatal hypothermia and hyperthermia, involving the hypothalamus and adrenal medulla.

Tissue-specific differences in mRNA quantification of glucose transporter 1 and vascular endothelial growth factor with special regard to death investigations of fatal injuries

Glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) have been established as being responsible for cellular adaptation to oxygen deficiency in tissue ischemia and hypoxia mediated by hypoxia-inducible factor 1. We hypothesized that mRNA quantification of these factors in autopsy tissue specimens could have diagnostic significance for investigating the pathology of death, especially after injury. Various cases (total, n=119; less than 48h postmortem) were examined, including fatal blunt injury (n=71) and sharp instrument injury (n=18), as well as asphyxia (strangulation/hanging, n=12) and acute myocardial infarction/ischemia (n=18) as controls. Quantification of mRNA by TaqMan real-time RT-PCR and immunostaining were performed for GLUT1 and VEGF in lung, kidney, and skeletal muscle specimens. The postmortem interval showed no significant influence on the relative quantification of mRNA during the early postmortem period. Characteristic results were found in blunt injury cases: both GLUT1 and VEGF mRNAs decreased in the lung but increased in the skeletal muscle depending on survival time. In the kidney, subacute deaths showed higher GLUT1 mRNA levels compared with acute deaths from blunt injury, but no significant change was found for VEGF mRNA. Immunohistochemistry showed visually predominant GLUT1 immunoreactivity in the renal cortex for cases with a longer survival time, coincident with the results at the mRNA level. Tissue-specific differences in mRNA quantification of GLUT1 and VEGF shed light on tissue ischemia/hypoxia and subsequent tissue-dependent pathophysiological changes leading to death after injury.

Evaluation of postmortem S100B levels in the cerebrospinal fluid with regard to the cause of death in medicolegal autopsy

Previous studies have suggested the usefulness of the postmortem serum S100B level as a marker of the severity of brain damage. In this study, we investigated the S100B level in the cerebrospinal fluid (CSF) in serial autopsy cases (n=216, within 3 days postmortem), including those of blunt injury (n=34: fatal head injury, n=20; others, n=14), sharp instrument injury (n=9), mechanical asphyxiation (n=19), drowning (n=11), fire fatality (n=26), intoxication (n=20), hypothermia (cold exposure, n=16), hyperthermia (heat stroke, n=9), acute cardiac death (n=52) and pneumonia (n=20). The CSF S100B level showed a moderate postmortem time-dependent increase for acute cardiac death (r=0.58, p<0.0001) and asphyxia (r=0.741, p<0.001). In cases of survival time within 48 h, drowning and hypothermia usually showed a lower CSF S100B level (around 500 ng/ml), and the level was higher for delayed head injury death, asphyxia, intoxication, and hyperthermia (around 1500 ng/ml) (p<0.05). In fatal head injury cases, however, CSF S100B did not correlate with the survival time or postmortem interval. A CSF S100B level of >2000 ng/ml in the early postmortem period might be considered a biochemical sign of fatally severe brain damage.

Evaluation of postmortem calcium and magnesium levels in the pericardial fluid with regard to the cause of death in medicolegal autopsy.

Previous studies have suggested the usefulness of postmortem serum calcium (Ca) and magnesium (Mg) for investigating cause of death. The present study investigated their levels in the pericardial fluid of serial autopsy cases of adults within 48 h postmortem (n=385), including fatalities from blunt injury (n=57), sharp instrument injury (n=9), mechanical asphyxiation (n=28), salt- and freshwater drowning (n=14 and n=61, respectively), fire fatality (n=35), intoxication (n=23), hypothermia (cold exposure, n=12), hyperthermia (heat stroke, n=7), acute cardiac death (ACD, n=86), pneumonia (n=9) and spontaneous cerebral hemorrhage (n=11). The pericardial Ca level was independent of the postmortem interval, showing a value similar to that of the clinical reference range in cases other than saltwater drowning, while the Mg level was higher than the clinical reference range and showed a mild postmortem time-dependent increase. Pericardial Ca was significantly higher for saltwater drowning than other groups, and a lower level was seen for hyperthermia, and some cases of blunt injury and intoxication. The Mg level was also significantly higher for saltwater drowning than the other groups, and showed a higher level for sharp instrument injury, but a lower level for hypothermia. The Mg/Ca ratio was higher for sharp instrument injury and saltwater drowning, but was lower for hypothermia. These findings suggest that postmortem pericardial Ca and Mg can be used to investigate the cause of death, especially for saltwater drowning, hypothermia and hyperthermia.

Evaluation of pulmonary GLUT1 and VEGF mRNA levels in relation to lung weight in medicolegal autopsy cases

The present study focuses attention upon the relationship among postmortem mRNA levels of pulmonary glucose transporter (GLUT1) and vascular endothelial growth factor (VEGF) to lung weight to investigate pulmonary pathophysiology in the death process. Autopsy cases (n=173, within 48 h postmortem) of blunt injury, including head injury (brain contusions and acute subdural hemotoma) and non-head injury, sharp instrument injury, mechanical asphyxiation, drowning, acute myocardial infarction/ischemia (AMI) and idiopathic cerebral hemorrhage (ICH) were examined. GLUT1 and VEGF mRNAs were quantified by TaqMan real-time RT-PCR for the upper lobes of the bilateral lungs. Combined lung weight was normalized against height for statistical analyses. GLUT1 mRNA showed a higher level for ICH. GLUT1 and VEGF mRNA levels were higher for brain contusions than for acute subdural hematoma, which showed a significantly lower VEGF mRNA level. Lung weight showed a larger value for saltwater drowning and ICH, and was larger for acute subdural hematoma than for brain contusions. GLUT1 mRNA level was correlated with lung weight in cases of ICH and brain contusions (survival time <24 h), and VEGF mRNA showed a similar tendency. Such findings were not detected for other groups. These findings indicate parallel increases in hypoxia-induced responses and lung weight in ICH and brain contusions, suggesting different pulmonary hemodynamics with milder alveolar damage compared with other groups, including AMI and acute subdural hematoma. Different mechanisms might be involved in non-cardiogenic or neurogenic pulmonary congestion and edema for ICH/brain contusions and subdural hematoma.

Analyses of cardiac blood cells and serum proteins with regard to cause of death in forensic autopsy cases

To investigate hematological and serum protein profiles of cadaveric heart blood with regard to the cause of death, serial forensic autopsy cases (n=308, >18 years of age, within 48 h postmortem) were examined. Red blood cells (Rbc), hemoglobin (Hb), platelets (Plt), white blood cells (Wbc), total protein (TP) and albumin (Alb) were examined in bilateral cardiac blood. Blood cell counts, collected after turning the bodies at autopsy, approximated to the clinical values. Postmortem changes were not significant for these markers. In non-head blunt injury cases, Rbc counts, Hb, TP and Alb levels in bilateral cardiac blood were lower in subacute deaths (survival time, 1-12 h) than in acute deaths (survival time <1 h). Wbc counts of left cardiac blood were significantly higher for non-head injury than for head injury in subacute deaths. In fire fatality cases, Plt count was markedly higher with an automated hematology analyzer than by using a blood smear test, suggesting Rbc fragmentation caused by deep burns, while increases in Wbc count and decreases in Alb levels were seen for subacute deaths. For asphyxiation, Rbc count, Hb, TP and Alb levels in bilateral cardiac blood were higher than other groups, and TP and Alb levels in the right cardiac blood were higher for hanging than for strangulation. These findings suggest that analyses of blood cells and proteins are useful for investigating the cause of death.

Postmortem serum levels of pulmonary surfactant-associated proteins A and D with regard to the cause of death in medicolegal autopsy

Pulmonary surfactant-associated proteins A and D (SP-A and -D) are tissue-specific components. Previous studies showed an increase in the postmortem serum SP-A level due to acute pulmonary alveolar damage and acute respiratory distress. The present study comparatively investigated serum SP-A and SP-D levels with regard to the cause of death in serial medicolegal autopsy cases (n=679, within 48 h postmortem). SP-A and SP-D levels were usually higher in left cardiac blood than at other sites, independent of postmortem interval. The left-to-right difference was significantly larger for mechanical asphyxiation, drowning, intoxication and spontaneous cerebral hemorrhage. Both SP-A and -D levels in bilateral cardiac blood were significantly higher for drowning and secondary pulmonary damage involving ARDS after traumas, but were lower for hypothermia (cold exposure). SP-A was predominantly elevated in fire fatality and delayed deaths from injury and fires, while pneumonia showed a predominant elevation of SP-D. These findings suggest that comparative analysis of serum SP-D and SP-A is useful for investigating primary or secondary pulmonary alveolar damage in the death process.

Postmortem mRNA quantification for investigation of infantile death: a comparison with adult cases.

Previous studies suggested the diagnostic significance of postmortem mRNA quantification in investigating the death process. The present study investigated infantile cases compared with adult cases. Autopsy cases (n=58, within 48 h postmortem) comprised infants (<1 year of age: mechanical asphyxiation, n=5; pulmonary infection, n=14), elderly children (2-8 years of age; pulmonary infection, n=3), and adults (>20 years of age: mechanical asphyxiation, n=21; pulmonary infection, n=13). Quantitative mRNA analyses of glucose transporter (GLUT1) and vascular endothelial growth factor (VEGF) by TaqMan real-time RT-PCR were performed for the bilateral lungs and skeletal muscle (sartorius). In adult cases, lower levels of VEGF mRNA in the lung and higher GLUT1 mRNA in the skeletal muscle were found for pulmonary infection than for asphyxiation. However, there was no significant difference between these causes of death for infants. Compared with adult cases, infantile cases showed a higher VEGF mRNA level in the lungs for pulmonary infection, and higher GLUT1 and VEGF mRNA levels in the skeletal muscle for asphyxiation. For pulmonary infection, both mRNA levels in the skeletal muscle were similar in infantile and adult cases. In older child cases of pulmonary infection, the pulmonary mRNA level of GLUT1 was similar to that in adult cases, and that of VEGF was intermediate between infant and adult levels. These findings indicate a similar death process due to pulmonary infection and asphyxiation in infants; thus, it may be difficult to distinguish these causes of death.

Immunohistochemical distribution of chromogranin A in medicolegal autopsy materials

Chromogranin A (CgA) was recently reported as a marker of various stress responses. The aim of this study was to investigate the immunohistochemical distribution of CgA in human tissues in medicolegal autopsy cases as a basis for postmortem investigation of stress responses. The autopsy cases (n=30, within 48 h postmortem) comprised cases of mechanical asphyxia (n=15: strangulation, n=8; hanging, n=7) and acute myocardial infarction/ischemia (AMI, n=15). Routinely formalin-fixed paraffin-embedded tissue sections, including those of the hypothalamus, pituitary gland, cardiac muscle, lungs, liver, kidneys, spleen, skeletal muscle, skin, thyroid gland, submandibular gland, pancreas, and adrenal gland, were stained with polyclonal anti-human CgA antibodies and CgA positivity was quantitatively examined. Localization of CgA immunopositivity was clearly demonstrated in specific cell components in all tissue sections. CgA was mainly observed in the anterior lobe of the pituitary, adrenal medulla, neurons and some gliocytes in the hypothalamus, submandibular gland, follicular epithelial cells and connective tissue in the thyroid gland and pancreatic islet cells. CgA immunopositivity showed no significant difference between mechanical asphyxia and AMI cases. Positivity was slightly higher in adenohypophysis, adrenal medullar, and pancreatic islet cells (approximately 50-80%) than in the thyroid and submandibular glands (approximately 30-60%); however, a large case difference was observed in hypothalamic CgA immunopositivity (0-100%). These findings suggest that hypothalamic CgA immunopositivity can be used as a marker for investigating individual differences in stress responses during the death process. Further investigation of other causes of death is needed.

Immunohistochemistry of von Willebrand factor in the lungs with regard to the cause of death in forensic autopsy

Pulmonary microvascular injury is involved in severe trauma or disease. The present study investigated the immunohistochemical distribution of von Willebrand factor (vWF) and platelet CD61 factor in forensic autopsy cases (n=157, >18 years of age, within 48 h postmortem), which comprised fatalities from blunt and sharp instrument injuries, strangulation, fire fatality and acute cardiac death (ACD). vWF immunoreactivity was clearly detected in the endothelia of large vessels (LV, phi>200 microm), small vessels (SV, phi 40-200 microm) and capillaries (Cap, phi<40 microm). Cap-vWF positivity was also detected in microthrombi with CD61 immunopositivity. The vWF positivity was higher in non-edema areas than in the edema area in the lungs. For acute deaths, Cap-vWF positivity of non-edema areas was frequently detected for strangulation (n=8/13, 61.5%), fire fatality (n=11/26, 42.3%) and ACD (n=8/15, 53.3%), but was infrequent for blunt and sharp instrument injuries (n=6/27, 22.5%, and n=2/15, 13.3%, respectively), irrespective of the complication of chest injury. However, for non-acute deaths, Cap-vWF positivity was more frequent for non-chest blunt injury (n=12/27, 44.4%) than for blunt chest injury (n=3/13, 23.1%) and sharp instrument injury (n=0/10, 0%). For fire fatality, Cap-vWF positivity was relatively frequent in cases with a lower blood carboxyhemoglobin (COHb) level of <60% (n=6/14, 42.8%) than in cases with a higher COHb level of >60% (n=3/12, 25.0%). These findings suggest that Cap-vWF positivity is closely related to the death process involving pulmonary microvascular injury.