Takahito Hayashi

Retrospective study on suicidal cases by sharp force injuries.

A total of 65 suicidal cases due to sharp force injuries (cut and/or stab wounds) were investigated. Suicide by sharp force injuries accounted for 2.5% of all suicides in our prefecture during 1995-2005. The 65 victims were composed of 49 males and 16 females, and the age range of 50-70 years was most common. A history of psychiatric disease was found in 11 victims, and depression was the most common disease followed by schizophrenia. Of 65 cases, 8 victims had a history of previously attempting suicide. In 41 cases, a suicide note or will was found. Forty-six cases had pleural sharp force injuries. Interestingly, the remaining 19 deaths were due to a single sharp injury. Cutting injuries were predominantly located at the flexor side of the wrist (11 cases, 38%), followed by the neck (10 cases, 34%). On the other hand, stab wounds were most commonly located in the chest (17 cases, 49%). Tentative wounds, which were superficial cut wounds or stab wounds, were present in 37 cases (57%). In 27 of 37, hesitation marks were observed in close proximity. Tentative wounds more frequently appeared in cut injuries than in stab injuries. Of 28 cases with fatal cut or stab wounds localized in the trunk, 11 cases (39%) had clothing damage. In the discrimination between suicide and homicide, forensic pathologists should obtain information on victims and witnesses as well as investigating the scene and postmortem examination of the victim.

Essential Involvement of CX3CR1-Mediated Signals in the Bactericidal Host Defense during Septic Peritonitis

Cecal ligation and puncture (CLP) caused septic peritonitis in wild-type (WT) mice, with ∼33% mortality within 7 days after the procedure. Concomitantly, the protein level of intraperitoneal CX3CL1/fractalkine was increased, with infiltration by CX3CR1-expressing macrophages into the peritoneum. CLP induced 75% mortality in CX3CR1-deficient (CX3CR1−/−) mice, which, however, exhibited a similar degree of intraperitoneal leukocyte infiltration as WT mice. Despite this, CX3CR1−/− mice exhibited impairment in intraperitoneal bacterial clearance, together with a reduction in the expression of intraperitoneal inducible NO synthase (iNOS) and bactericidal proinflammatory cytokines, including IL-1β, TNF-α, IFN-γ, and IL-12, compared with WT mice. Bactericidal ability of peritoneal phagocytes such as neutrophils and macrophages was consistently attenuated in CX3CR1−/− mice compared with WT mice. Moreover, when WT macrophages were stimulated in vitro with CX3CL1, their bactericidal activity was augmented in a dose-dependent manner, with enhanced iNOS gene expression and subsequent NO generation. Furthermore, CX3CL1 enhanced the gene expression of IL-1β, TNF-α, IFN-γ, and IL-12 by WT macrophages with NF-κB activation. Thus, CX3CL1-CX3CR1 interaction is crucial for optimal host defense against bacterial infection by activating bacterial killing functions of phagocytes, and by augmenting iNOS-mediated NO generation and bactericidal proinflammatory cytokine production mainly through the NF-κB signal pathway, with few effects on macrophage infiltration.

IFN-γ Protects Cerulein-Induced Acute Pancreatitis by Repressing NF-κB Activation

We explored the pathophysiological roles of IFN-γ in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-α, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-γ in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-γ mRNA. Unexpectedly, IFN-γ−/− mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-α, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-γ−/− mice, compared with WT mice. Cerulein activated NF-κB, an indispensable transcription factor for gene transcription of TNF-α, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-κB p65. In comparison with WT mice, IFN-γ−/− mice exhibited exaggerated and prolonged NF-κB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-γ, because acetylated Stat1 can inhibit NF-κB activation. Indeed, IFN-γ acetylated Stat1 and reciprocally reduced NF-κB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-γ remarkably attenuated acute pancreatitis in both WT and IFN-γ−/− mice, with reduced NF-κB activation and COX-2 expression. Thus, IFN-γ can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-κB activation.

Differential diagnosis between freshwater drowning and saltwater drowning based on intrapulmonary aquaporin-5 expression

The intrapulmonary expression of aquaporin-5 (AQP5) was examined in an experimental drowning model and forensic autopsy cases to discuss the possibility for differentiation between freshwater drowning (FWD) and saltwater drowning (SWD). In animal experiments, mice were classified into four groups: (group I: FWD; group II: SWD; group III: postmortem immersion (PI); and group IV: cervical dislocation as controls. In group I, intrapulmonary AQP5 expression was significantly suppressed at both gene and protein levels, compared with the other three groups, and there was no significant difference in AQP5 expression among groups II to IV. In the next series, we examined AQP5 gene expression in human lung samples obtained from forensic autopsies at less than 48 h postmortem (nine FWD cases, five SWD cases, and 14 other cases). Although AQP5 mRNA could be detected in all lung samples under the employed experimental conditions, the intrapulmonary gene expression of AQP5 in FWD was significantly attenuated compared with the other groups. These observations imply that AQP5 expression in type I alveolar epithelial cells was suppressed by hypotonic water to prevent hemodilution from the physiological aspect. Moreover, the analysis of intrapulmonary AQP5 expression would be forensically useful for differentiation between FWD and SWD, or between FWD and PI.

Temporal expression of wound healing-related genes in skin burn injury

Determination of the age of burns, as well as of wounds induced mechanically, is essential in forensic practice, particularly in cases of suspected child abuse. Here, we investigated temporal changes in the expression of 13 genes during wound healing after a burn. The expression of cytokines (IL-1β, IL-6, IL-10, TNF-α, and IFN-γ), chemokines (KC, MCP-1), proliferative factors (TGF-β, VEGF), proteases (MMP-2, 9, 13) and type I collagen in murine skin was examined by real-time PCR at 3, 6, 9, and 12 h and 1, 2, 3, 5, 7, and 14 days after a burn. Based on macroscopic and histological appearance, the healing process of a burn consists of 3 phases: inflammatory (from 3 h to 1 day after the burn), proliferative (from 1 to 7 days), and maturation (from 7 to 14 days). Expression of IL-1β, IL-6, TNF-α, IFN-γ and KC increased significantly in a biphasic pattern from 3 or 6 h to 12 h or 1 day and from 3 or 5 days to 7 days. Expression of MCP-1 increased significantly from 6 h to 5 days. Expression of both IL-10 and TGF-β increased significantly from 12 h to 7 days. Expression of VEGF, MMP-2, MMP-13 and type I collagen increased significantly from 3 days to 7 or 14 days. Expression of MMP-9 increased significantly from 6 h to 14 days. Our results suggest that evaluating the expression of a combination of these genes would enable the exact estimation of the age of a burn.

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist

Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1α and IL-1β were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-κB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1α repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-κB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.

Two patterns of β-amyloid precursor protein (APP) immunoreactivity in cases of blunt head injury

Immunostaining for beta-amyloid precursor protein (APP) is widely recognized as an effective tool for detecting diffuse traumatic axonal injury (TAI). APP selectively labels injured axons, such as axonal bulbs and varicose axons. However, it has been reported that axonal bulbs are detected in cases of cerebral hypoxia without head injury. Therefore, we examined whether there are differences in the morphological pattern of axonal bulbs between trauma and hypoxia. Sections of the corpus callosum from 25 cases of head injury and 23 control cases were immunostained for APP. APP staining detected axonal bulbs in 14 cases of head injury, who survived more than several hours, although it failed to label axons in control cases. In addition, two patterns of immunoreactivity were identified in several cases of head injury. The first pattern showed that labeled axons were oriented along with white matter bundles; the second demonstrated that the axons were scattered irregularly. The first pattern alone was found in 5 of 14 cases, while cases of the second pattern alone were not observed. Both patterns were detected in 5 cases and in the remaining 4 cases, clear patterns were not found. From these findings, we speculated that the first pattern may represent TAI. Further examinations are required for determining whether these two patterns are identical with patterns of trauma and hypoxic brain damage as indicated by [Oehmichen M, Meissner C, Schmidt V, Pedal I, König HG, Saternus KS. Axonal injury--a diagnostic tool in forensic neuropathology? A review. Forensic Sci Int 1998;95:67-83] and [Graham DI, Smith C, Reichard R, Leclercq PD, Gentleman SM. Trials and tribulations of using beta-amyloid precursor protein immunohistochemistry to evaluate traumatic brain injury in adults. Forensic Sci Int 2004;146:89-96].

Aberrant Expression of Histo-blood Group A Type 3 Antigens in Vascular Endothelial Cells in Inflammatory Sites

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1–4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.

Circumstantial and toxicological features of deaths from self-administered intravenous anesthetic/narcotic agents

For a better understanding of circumstantial and toxicological findings of fatalities resulting from self-administration of intravenous anesthetic/narcotic agents, medico-legal autopsy files of the State Institute of Legal and Social Medicine Berlin from 1998 to 2011 were reviewed retrospectively. Of a total of 15,300 autopsies, 9 cases of such deaths were identified, and all were health care professionals. Medical supplies for injection were found still on, or near, the body at the scene. Anesthetic/narcotic agents detected were classified into 3 categories, and administered solely or in combination. Propofol was the most common agent, being detected in 6 cases. In 2 out of 6 cases, propofol was detected substantially above therapeutic levels and was considered the cause of death. In the remaining 4 cases, propofol levels were within the therapeutic range, but propofol intoxication was considered as lethal due to it being administered by rapid continuous injection. In 5 cases, injection of opioid narcotics was fatal. Alongside the 2 propofol-detected cases, there was one case where a higher-than-therapeutic level of piritramide and a therapeutic level of alfentanil was identified. Despite suspected usage, remifentanil was not detected due to its rapid metabolism by elastases in one case, and sufentanil was undetectable due to putrefaction in another, but death was attributed to their potent respiratory depressant effects without respiratory assistance. Benzodiazepines were detected in 4 cases. All of them were used together with propofol or opioids, and contributed to death by inhibiting respiration. It is essential to consider means of administration as well as additive or synergistic effects of combined agents when interpreting toxicological results in such cases.

Positional asphyxia or diabetic ketoacidosis? A case report

We describe an autopsy case in which a patient with diabetic ketoacidosis (DKA) was found in a head-down position. A female in her late 70s was found dead in her home in a supine position on the kitchen floor. The upper part of her body was hanging down over the edge of the kitchen floor to the backyard through the open window. External examination revealed congestion of the head and upper region of the face and neck. There were numerous petechiae on the superior palpebral conjunctivae and upper part of the oral mucosa. On internal examination, extensive hemorrhages in the subcutaneous fat tissues and muscles were observed at the upper part of the neck, although there were no external injuries on the neck. Histopathological examination revealed that hemorrhages were accompanied with infiltration of polymorphonuclear leukocytes both within and around the hemorrhages on the neck skin. Nodular glomerulosclerosis and many fat droplets in the cytoplasm of proximal tubule cells were found in the kidney. Postmortem blood analysis showed acetone (204.2 μg/ml), HbA1c (10.8%), acetoacetate (<2.0 μmol/l), 3-hydroxybutyrate (11,844 μmol/l), blood urea nitrogen (128.9 mg/dl), and creatinine (3.11 mg/dl). The glucose and acetone levels in the urine were 876.7 mg/dl and 201.4 μg/ml, respectively, suggesting that she suffered severe DKA. However, since hemorrhage of the neck could have developed only when she was still alive, asphyxia should have arisen antemortem. Based on these findings, we concluded that the direct cause of her death is positional asphyxia, which was resulted from DKA. It is difficult to diagnose the cause of death when the victim is in an unusual posture. To confirm a suspicion of positional asphyxia, photographs of the undisturbed scene are useful in addition to a precise autopsy and accurate examinations.