Forensic significance of intracardiac expressions of Nrf2 in acute myocardial ischemia.

When exposed to oxidative and electrophilic stress, a protective antioxidant response is initiated by nuclear factor erythroid 2-related factor 2 (Nrf2). However, the extent of its importance in the forensic diagnosis of acute ischemic heart diseases (AIHD), such as myocardial infarction (MI), remains uncertain. On the other hand, immunohistochemical analyses of fibronectin (FN) and the terminal complement complex (C5b-9) prove valuable in identifying myocardial ischemia that precedes necrosis during the postmortem diagnosis of sudden cardiac death (SCD). In this study, we investigated the immunohistochemical levels of Nrf2, FN, and C5b-9 in human cardiac samples to explore their forensic relevance for the identification of acute cardiac ischemia. Heart samples were obtained from 25 AIHD cases and 39 non-AIHD cases as controls. Nrf2 was localized in the nuclei of cardiomyocytes, while FN and C5b-9 were detected in the myocardial cytoplasm. The number of intranuclear Nrf2 positive signals in cardiomyocytes increased in AIHD cases compared to control cases. Additionally, the grading of positive portions of cardiac FN and C5b-9 in the myocardium was also significantly enhanced in AIHD, compared to controls. Collectively, these results indicate that the immunohistochemical investigation of Nrf2 combined with FN, and/or C5b-9 holds the potential for identifying early-stage myocardial ischemic lesions in cases of SCD.

A case of hemoperitoneum after percutaneous radiofrequency ablation in a patient with hepatocellular carcinoma.

We report a case of hemoperitoneum after percutaneous radiofrequency ablation in a patient with hepatocellular carcinoma. A 60-year-old female was hospitalized for the treatment of thrombasthenia and cirrhosis caused by chronic Hepatitis C, and computed tomography revealed hepatocellular carcinoma, which was treated by percutaneous radiofrequency ablation. After the ablation, hemoperitoneum was suspected because of the low hemoglobin level with abdominal pain. Approximately 6 h after the ablation treatment, the patient suddenly fell into a shock state and died. In this case, medical treatment-related death including malpractice was suspected, and forensic autopsy was performed. The abdominal cavity contained 910 mL of dark red fluid blood and 210 g of soft hemocoagula. Moreover, several puncture marks were observed on the liver surface and diaphragm, and there was no clear damage to the main arteries and veins. Considering the macroscopic and microscopic findings, the cause of death was assumed as hemorrhagic shock due to the hemoperitoneum caused by the damage to the liver by radiofrequency ablation. It is important to consider all the indications and adverse effects of radiofrequency ablation.

Relationship between intrathrombotic appearance of HSP27 and HSP70 and thrombus ages in a murine model of deep vein thrombosis.

Heat shock proteins (HSPs) are molecular chaperones whose primary function is cytoprotection, supporting cell survival under (sub) lethal conditions. They have been implicated in various diseases such as inflammatory diseases and cancer due to their cytoprotective and immunomodulatory effects, and their biological mechanisms have been studied. Central family members include, HSP27, which is induced by various stimuli such as heat shock, hypoxia, hyperoxia, ultraviolet exposure, and nutritional deficiency, and HSP70, which is homeostatically expressed in many organs such as the gastrointestinal tract and has anti-cell death and anti-inflammatory effects. In this study, HSP27 and HSP70 were investigated during thrombus formation and dissolution in a deep vein thrombosis model by immunohistochemistry to determine their involvement in this process and whether their expression could be used as a forensic marker. In the process of thrombus formation and lysis, HSP27 and HSP70 were found to be expressed by immunohistochemical analysis. The role of inhibitors of HSP27 and HSP70 in the pathogenesis of thrombosis in mice was also investigated. When HSP27 or HSP70 inhibitors were administered, thrombi were significantly smaller than in the control group on day 5 after inferior vena cava ligation, indicating pro-thrombotic effects HSP27 and HSP70. If HSP27- or HSP70-positive cells were clearly visible and easily identifiable in the thrombus sections, the thrombus was presumed to be more than 10 days old. Thus, the detection of intrathrombotic HSP27 and HSP70 could forensically provide useful information for the estimation of thrombus ages. Collectively, our study implied that both HSP27 and HSP70 might be molecular targets for thrombus therapy and that the detection of HSP-related molecules such as HSP27 and HSP70 could be useful for the determination of thrombus ages.

A case of necrotizing fasciitis following intra-articular injections - Iatrogenic or spontaneous?

Here, we report a case of necrotizing fasciitis following intra-articular injection of hyaluronic acid. A 73-year-old female received intra-articular injections of hyaluronic acid due to arthralgia at the left shoulder and knee, and was found dead in her living room at one day. At the forensic autopsy, injection marks with bullae and erythema were found at the left shoulder and knee and liquefactive necrosis of muscle tissues was observed in the left but not right extremities. Histopathological examinations of the left upper arm and thigh revealed severe rhabdomyolysis with lots of bacterial clusters. Bacteriological examinations detected group A Streptococcus from intracardiac blood and affected muscle tissues. Postmortem biochemical analysis of blood showed escalated blood urea nitrogen (133.8 mg/dL), creatinine (4.57 mg/dL) and C-reactive protein (45.0 mg/dL). The cause of her death was diagnosed as streptococcal toxic shock syndrome (STSS). Moreover, it was suggested that the injection was inappropriately conducted and served as a portal of bacterial entry.

Forensic significance of intracardiac heme oxygenase-1 expression in acute myocardial ischemia.

Heme oxygenase-1 (HO-1), an inducible stress-response protein, exerts anti-oxidant and anti-apoptotic effects. However, its significance in forensic diagnosis of acute ischemic heart diseases (AIHD) such as myocardial infarction (MI) is still unknown. We examined the immunohistochemical expression of HO-1 in the heart samples to discuss their forensic significance to determine acute cardiac ischemia. The heart samples were obtained from 23 AIHD cases and 33 non-AIHD cases as controls. HO-1 positive signals in cardiomyocyte nuclear were detected in 78.2% of AIHD cases, however, that were detected in only 24.2% control cases with statistical difference between AIHD and non-AIHD groups. In contrast to HO-1 protein expression, there was no significant difference in the appearance of myoglobin pallor regions and leukocyte infiltration in the hearts between AIHD and non-AIHD groups. From the viewpoints of forensic pathology, intracardiac HO-1 expression would be considered a valuable marker to diagnose AIHD as the cause of death.

Intrathrombotic appearances of AQP-1 and AQP-3 in relation to thrombus age in murine deep vein thrombosis model.

Aquaporins (AQPs) are membrane-bound proteins for water transportation and are useful for diagnosing drowning and wound vitality in forensic pathology. Here, we examined intrathrombotic expression of AQP-1 and AQP-3 using deep vein thrombosis models in mice. To perform immunohistochemical analyses, we used anti-AQP-1 and anti-AQP-3 antibodies. In thrombus samples with the post-ligation intervals of 1 to 5 days, AQP-1+ areas were over 70%. At 7 days after the IVC ligation, AQP-1+ areas became less than 50%, eventually decreasing to 11% at 21 days. At 3 days after the IVC ligation, AQP-3+ cells started to appear from the peripheral area. Thereafter, the positive cell number progressively increased and reached to a peak at 10 days after the IVC ligation. When the intrathrombotic AQP-1+ area was as large as the intrathrombotic collagen area or smaller, it would indicate a thrombus age of ≥ 10 days. AQP-3+ cell number of > 30 would indicate a thrombus age of 10-14 days. Collectively, our study implied that the detection of AQP-1 and AQP-3 would be useful for the determination of thrombus age.

Complete decapitation by a self-constructed guillotine in a burned body - complex suicide or postmortem burning?

We report a suicide case of complete decapitation using a self-constructed guillotine. A 45-year-old man, whose body was severely burned, was found dead. The head was completely separated from the middle level of the neck, and a sharp blade with a steel frame was placed between the head and neck. The severance plane passed between the C4 and C5 vertebrae. Vital reactions such as hemorrhage could not be confirmed at the decapitated skin edge because the body was severely burned. Both common carotid arteries were sharply transected. Subendocardial hemorrhage was detected in the left ventricle. Only a little blood, but no soot, was detected in the respiratory tract, including the trachea and bilateral bronchi. Subarachnoid hemorrhage was noted at the edge of the cervical spinal cord. The saturation level of CO-Hb was 5.7% in the left cardiac blood, 5.9% in the right cardiac blood, and 5.8% in the peripheral blood from the femoral vein. Cervical transection was diagnosed as the cause of death. We believe that he was unintentionally burned by spread fire from an automobile after decapitation by a self-constructed guillotine.

Forensic pathological study on temporal appearance of dendritic cells in skin wounds.

Dendritic cells (DCs) can essentially contribute to innate and adaptive immune system in various organs. A double-color immunofluorescence analysis was carried out with anti-CD11c and -HLA-DRα antibodies to detect DCs in 53 skin wounds (their postinfliction intervals: group I, 0-3 days; group II, 4-7 days; group III, 9-14 days; and group IV, 17-21 days). CD11c+HLA-DRα+ DCs were first observed in skin wounds with postinfliction intervals of 3 days, and the DC numbers were found to be elevated in skin wounds with the subsequent increase in postinfliction intervals. Semi-quantitative morphometric analyses showed that the DC number was the highest in the 12-day-old wound. More than 50 DCs were present in 8 of 10 samples (80%) in group II and 14 of 16 samples (87.5%) in group III, and there was no difference between the two groups. Thus, the presence of DCs in a skin wound was possibly estimated as postinfliction intervals of at least 3 days. Furthermore, when a skin wound contained > 50 DCs, its age would be judged as 4-14 days. Collectively, the appearance of DCs in human skin wounds may provide useful information in determining the age of a wound.

A case of complex suicide due to acute nicotine intoxication caused by cigarette ingestion.

This paper presents an unusual complex suicide case that died of nicotine addiction. The deceased was a 40-year-old male who was found lying dead on the floor in his room. In external findings, many incision wounds on his forearms and skin discoloration with epidermolysis on his cervical region could be seen. In the room, a blood-stained scissors and electric cord hanged on the exercise bike were found. Moreover, nine cigarette residues which were only the filter part and empty bottle of coffee were found on his side. At autopsy, we found that those injuries were not serious enough to lead him to the death. Toxicologically, caffeine, nicotine, cotinine, mirtazapine, and olanzapine could be detected, and the concentrations of nicotine were 3.740, 2.140, 3.100, and 451.100 µg/ml in cardiac blood, peripheral blood, urine, and stomach contents, respectively. These concentrations were evaluated as the fatal levels, and the cause of his death was diagnosed as acute nicotine intoxication.

Feasibility and Clinical Significance of In Vivo Cholesterol Crystal Detection Using Optical Coherence Tomography.

OBJECTIVE: Cholesterol crystals (CCs) are frequently found at the site of acute myocardial infarctions (AMIs), but the role of CCs in the onset of AMI remains unclear due to the lack of validated in vivo imaging tools. The aim of this study was to validate the ability of optical coherence tomography (OCT) to detect CCs and to compare the prevalence and distribution of CCs in patients with AMIs and stable angina pectoris. Approach and Results: CC assessment using OCT were compared with histopathology results in 45 coronary samples. We investigated 152 consecutive patients with AMIs and 41 patients with single vessel-diseased stable angina pectoris. Based on the presence of plaque ruptures (PR), AMI patients were divided into 2 groups: those with PR (n=112) and those without PR (n=40). CCs invading fibrous caps were defined as superficial-type CCs. A multivariable logistic regression analysis was performed to determine PR predictors. The sensitivity and specificity of OCT for detecting CCs were 68% and 92%, respectively. The prevalence of plaques with CCs was higher in the AMI with PR group (AMI with PR 81%, AMI without PR 48%, stable angina pectoris 39%, P<0.01). A multivariable logistic model showed that superficial-type CCs and thin-cap fibroatheromas were positive predictors for PR. CONCLUSIONS: OCT has a high specificity and modest sensitivity for the detection of CCs. The combination of CCs invading fibrous cap and thin-cap fibroatheromas detected by OCT may better identify rupture-prone plaques.

The application of autophagy to thrombus age estimation in murine deep vein thrombosis model.

We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To perform the immunohistochemical analyses, we used anti-LC3 antibody and anti-p62 antibody for detecting the intrathrombotic autophagic functions. We estimated dynamics of the intrathrombotic autophagy as LC3+ cell number (×1000, five fields) with the thrombus ages (each group n = 5). The number of LC3+ cells was once decreased at 3 days, and then increased until 10 days. On the contrary, the number of p62+ cells progressively increased until 10 days after the inferior vena cava (IVC) ligation, and then gradually decreased. Especially, in all of thrombus samples with the postligation intervals of 5-10 days, both numbers were larger than 10. Subsequently, we compared the number of LC3+ cells to that of p62+ cells. Although, at 1 day after the IVC ligation, LC3+ cell number significantly exceeded p62+ cell number, the former was significantly or relatively less than the latter at 3 days or more after the IVC ligation. Thus, positive cells of > 10 in both LC3 and p62 indicated the thrombus age of 5-10 days. Upon comparison of immunopositive cells in LC3 and p62, the p62/LC3 ratio was > 1.0 in 29 out of 30 thrombus samples aged 3-21 days, and all of 1-day-old thrombus had the p62/LC3 ratio of < 0.5. Thus, the ratio of > 1.0 and that of < 0.5 could indicate thrombus age of 3 days or more and that of 1 day, respectively. Collectively, our study implied that the detection of autophagy-related molecules such as LC3 and p62 would be useful for the determination of thrombus age.

CCL2-Mediated Reversal of Impaired Skin Wound Healing in Diabetic Mice by Normalization of Neovascularization and Collagen Accumulation.

Patients with diabetes frequently present with complications such as impaired skin wound healing. Skin wound sites display a markedly enhanced expression of CCL2, a potent macrophage chemoattractant, together with macrophage infiltration during the early inflammatory phase in skin wound healing of healthy individuals, but the association of CCL2 with delayed skin wound healing in patients with diabetes remains elusive. In this study, we showed that, compared with control mice, mice with streptozotocin-induced diabetes displayed impaired healing after excisional skin injury, with decreased neovascularization, CCL2 expression, and macrophage infiltration. Compromised skin wound healing in mice with diabetes was reversed by the administration of topical CCL2 immediately after the injury, as evidenced by normalization of wound closure rates, neovascularization, collagen accumulation, and infiltration of macrophages expressing vascular endothelial growth factor, a potent angiogenic factor, and transforming growth factor-ß. CCL2 treatment further increased the accumulation of endothelial progenitor cells at the wound sites of mice with diabetes and eventually accelerated neovascularization. Thus, the topical application of CCL2 can be an effective therapeutic option for the treatment of patients with diabetes with defective wound repair, promoting neovascularization and collagen accumulation at skin wound sites.

Postmortem distribution of acephate and its metabolite methamidophos in body fluids and organ tissues of an intoxication case.

We herein report an intoxication case caused by the ingestion of the pesticide Ortoran®, which consists of 50% acephate aqueous solution. A man in his 60 s was found dead in his car with a 100-mL bottle containing approximately 50 mL of Ortoran®. In a gas chromatography - mass spectrometry (GC-MS) screening test, acephate and its metabolite methamidophos were qualitatively detected in his stomach contents. The amounts of acephate and methamidophos (µg/g) in the extract of each body fluid or organ tissue were measured using GC-MS and were as follows: 35.8, 2.84 (heart blood); 44.0, 2.26 (peripheral blood); 2,240, 2.79 (urine); 53.1, 8.91 (brain occipital lobe); 43.7, 2.95 (liver); 102.3, 8.02 (right kidney); and 5450, 22.9 (stomach contents). Based on these results and autopsy findings, the cause of death was concluded to be acute fatal intoxication caused by the pesticide containing acephate and its active metabolite, methamidophos. Concentration ratios between acephate and methamidophos in each body fluid and organ tissue showed higher relative concentrations of brain methamidophos to acephate than those of other organ tissues. A high relative concentration of brain methamidophos may contribute to the intoxication of acephate in humans.

Forensic application of epidermal AQP3 expression to determination of wound vitality in human compressed neck skin.

In forensic practices, it is often difficult to determine wound vitality in compression marks of the neck with naked eyes. AQP1 and AQP3 are the major water channels associated with skin. Thus, we immunohistochemically examined the expression of AQP1 and AQP3 in neck skin samples to discuss their forensic applicability to determination of the wound vitality. Skin samples were obtained from 56 neck compression cases (hanging, 35 cases; strangulation, 21 cases). The intact skin from the same individual was taken as a control. Although AQP1 was immnunostained in dermal capillaries in both the neck compression marks and intact skin samples, there was no significant difference in the magnitude of AQP1 expression between both groups. On the contrary, AQP3-positive signals could be faintly detected in uninjured skin samples, and the positive signals seemed more intense in the keratinocytes in compression regions. Morphometrical analyses revealed that the ratio of AQP3-expressed keratinocytes was significantly enhanced in neck compression regions, compared with control groups. From the viewpoints of forensic pathology, immunohistochemical detection of AQP3 in the neck skin can be considered a valuable marker to diagnose the trace of antemortem compression.

Immunohistochemical analysis on aquaporin-1 and aquaporin-3 in skin wounds from the aspects of wound age determination.

Immunohistochemical investigation of aquaporin (AQP)1 and AQP3 was performed in human skin wounds obtained from forensic autopsy cases. A total of 55 human skin wounds of different postinfliction intervals were collected as follows: group I, 0-3 days (n = 16); II, 4-7 days (n = 11); III, 9-14 days (n = 16); and IV, 17-21 days (n = 12). In uninjured skin samples, AQP1 and AQP3 could be slightly detected in dermal vessels and keratinocytes, respectively. The percentage of AQP1+ vessels and the number of AQP3+ keratinocytes were apparently elevated in accordance with wound ages. The number of AQP3+ keratinocytes was distinctly evident in groups II and III. Morphometrically, both AQP1+ vessel area and AQP3+ cell number were markedly increased in group II, compared with other three groups. With regard to forensic safety, AQP1+ vessel area of over 5% would imply wound ages of 4-12 days. Moreover, the positive area of > 15% would suggest wound age of 7-10 days. Especially, most samples of skin wounds aged 5-10 days except for only one sample (a 10-day-old wound) showed AQP3+ cell number of > 300, and the remaining other samples had that of < 300. Thus, the AQP3+ cell number of > 300 would indicate wound ages of 5-10 days. Collectively, immunohistochemical analyses of AQP1 and AQP3 in human skin wounds would support the objective accuracy of wound age determination.

Detection of intrathrombotic endothelial progenitor cells and its application to thrombus age estimation in a murine deep vein thrombosis model.

Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and Flk-1 (one of the receptors for vascular endothelial growth factor). In this study, double-color immunofluorescence analysis was performed using anti-CD34 and anti-Flk-1 antibodies in order to examine the time-dependent intrathrombotic appearance of EPCs, using the thrombi of DVT model mice with different thrombus ages (1-21 days). In thrombus cross-section specimens with an age of less than 3 days, CD34+/Flk-1+ EPCs were not detected. EPCs were initially observed in wounds aged 5 days, and their number was increased in thrombi with the advance of thrombus ages. The number of EPCs was the largest in the 10-day thrombus. Moreover, all 15 samples aged 7-14 days had an EPC number of more than 10, and, in 9 of them, the number of intrathrombotic EPCs was over 20. In contrast, in all thrombus samples aged 21 days, the number of intrathrombotic EPCs was less than 20. However, in three of them, the intrathrombotic EPC number was ≥ 10. These observations suggested that an intrathrombotic EPC number exceeding 20 would indicate a thrombus age of approximately 7-14 days.

[Preoperative Spirometry Leads Latent COPD Patients to be Discovered and Treated].

BACKGROUND AND METHODS: We have noticed the possibility that preoperative spirometry may discover latent COPD patients. When COPD is discovered before operation, we can start to treat COPD promptly. Consequently, the better perioperative care is given to COPD patients. On discharge, patients are referred to their family doctors, so that they can receive COPD treatment. RESULTS: There activities were started in July 2012. During the initial twelve months, 90 patients of COPD II x III were discovered and treated before admission. FEV1.0 was significantly improved (1.54 l vs. 1.71 l in average) on repeated spirometry on admission. There was no adverse event in COPD treatment All patients have shown good clinical courses. CONCLUSIONS: Latent COPD patients can be discovered by preoperative spirometry.

Detection of endothelial progenitor cells in human skin wounds and its application for wound age determination.

Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and vascular endothelial growth factor (VEGF) receptor (Flk-1). In this study, a double-color immunofluorescence analysis was carried out using anti-CD34 and anti-Flk-1 antibodies to examine the time-dependent appearance of EPCs, using 52 human skin wounds with different wound ages (Group I, 0-1 days; Group II, 2-6 days; Group III, 7-14 days; and Group IV, 17-21 days). In wound specimens with an age of less than one day, CD34(+)/Flk-1(+) EPCs were not detected. EPCs were initially observed in wounds aged two days, and their number was increased in lesions with advances in wound age. In morphometrical analysis, the average number of EPCs was the highest in the wounds of Group III. Especially, 20 out of 21 wounds aged 7-12 days had >20 EPCs, and all wound samples with postinfliction intervals of 14-21 days had <15 EPCs. These observations at least showed that >20 EPCs would indicate a wound age of 7-12 days. Taken together, our observations indicate the detection of EPCs would be useful for wound age determination.

Autophagy in skin wounds: a novel marker for vital reactions.

Detection of vitality of mechanical wounds in human cadavers is one of the important issues in forensic medicine. In order to explore novel markers for vitality of acute mechanical wounds, we investigated autophagy in mouse and human skin wounds. Western blotting analysis of mouse skin wounds showed marked reduction of LC3-II and reciprocal increase of p62 in wound samples with the postinfliction intervals of ≥0.5 h, compared with the uninjured skin tissues. These observations indicated that autophagy level was reduced in the wound sites. In postmortem wound samples, there were no remarkable changes in LC3-II and p62 levels. Furthermore, the postmortem intervals of 1-4 days have no significant effects on the changes of LC3-II and p62 in the antemortem skin wounds. Like murine wound samples, these alterations of LC3-II and p62 could be detected in human skin wound samples. Collectively, our study using animal and human samples implied that the detection of autophagy-related molecules such as LC3-II and p62 might be useful for forensic practice as markers of wound vitality.

Immunohistochemical detection of intrathrombotic IL-6 and its application to thrombus age estimation.

We immunohistochemically examined intrathrombotic IL-6 expression using a murine model of deep vein thrombosis induced by the ligation of the inferior vena cava (IVC) and discussed the availability of intrathrombotic IL-6 for thrombus age estimation. IL-6(+) cells could be first detected at 1 day after IVC ligation in three of five samples, and all samples with postligation intervals of 3 days or more had intrathrombotic IL-6(+) cells. Thereafter, the numbers of IL-6(+) cells were elevated with the increase of postligation intervals. Double-color immunofluorescence analyses demonstrated that IL-6 was mainly expressed by intrathrombotic macrophages. In all samples with postligation intervals of 5 days or less, the IL-6/macrophage ratio (IL-6/Mϕ) was <0.5. In contrast, the IL-6/Mϕ was greater than 0.5 at ≥7 days after the IVC ligation. These observations implied that IL-6/Mϕ ratios of >0.5 would strongly indicate thrombus ages of ≥7 days. Reciprocally, the ratios of less than 0.5 would suggest thrombus ages of ≤5 days. The present study demonstrated that the immunohistochemical detection of IL-6 was suitable to estimate the age of venous thrombi.