Increased fatty acid synthesis and disturbed lipid metabolism in Neuro2a cells after repeated cocaine exposure: A preliminary study.

Chronic use of cocaine prompts neurodegeneration and neuroinflammation. Lipids play pivotal roles in neuronal function and pathology. Although evidence correlates cocaine use with the alteration of lipid metabolism in blood and brain, the precise mechanism remains to be elucidated. In this study, we explore the effect of cocaine on neuronal fatty acid profiles in vitro. Neuro2a cells following seven days of repeated exposure to cocaine (0, 600, 800, 1000 µM) showed apoptosis-irrelevant cell death, dysregulated autophagy, activation of atypical endoplasmic reticulum stress response, increased saturated and unsaturated fatty acid synthesis, and disrupted lipid metabolism. These preliminary findings indicated the association between lipid metabolism and cocaine-induced neurotoxicity, which should be beneficial for understanding the neurotoxicity of cocaine.

Alcohol and toxicological factors influencing fatal falls from height in the Greater Tokyo Area: a retrospective study.

Falls from height pose a significant public health concern in urban regions, including the highly urbanized Greater Tokyo Area. The Japanese population is characterized by high rates of suicide and psychoactive drug usage, underscoring the importance of investigating these attributes in falls from height. This study aimed to retrospectively analyze the alcohol and toxicological aspects influencing falls from height in the Greater Tokyo Area between 2014 and 2022 and compare the findings with existing reports on other populations. In total, 75 cases of falls from height and 159 cases of natural deaths were included. Consistent with previous findings, Fisher's exact test revealed a predominance of males (66.67%, 50/75) and young adults (57.33%, 43/75) in falls from height. Multivariate logistic regression analysis identified antidepressant usage as the most significant risk factor within the target population, while younger individuals under alcohol influence constituted another high-risk group. Notably, contradictory to other populations, female individuals involved in fatal falls in the Greater Tokyo Area exhibited a higher frequency of alcohol consumption than males (48.00%, 12/25 vs. 26.00%, 13/50), and most of them were associated with suicide (83.33%, 10/12). These findings elucidate the population characteristics that pose a high risk for fatal falls from height in Japan and can serve as a reference for other Asian populations residing in similar megacities.

Ectopic myogenesis and fibrosis accompany adipogenesis during thymic involution induced by repeated cocaine administration.

We have shown previously that daily cocaine administration for 7-14 days in rats resulted in the acceleration of thymic involution, which is, alike to age-related thymic involution, accompanied by ectopic adipogenesis. Here we show that the thymic involution caused by repeated cocaine administration is accompanied by not only adipogenesis but also ectopic myogenesis and fibrosis. In accordance with fibrosis, we observed an increase of N-cadherin, a marker of mesenchymal cells, as well as a decrease of E-cadherin, an epithelial cell marker, in the thymus in response to cocaine administration, suggesting the occurrence of epithelial-to-mesenchymal transition (EMT). Levels of fibronectin was also increased in the thymus of cocaine group compared to control group. In addition, increases in the levels of the transcription factors for myogenic differentiation, myogenin, MYF5, and MYF6, were observed in the thymus administered cocaine for 14 days. These results indicate that the thymic involution induced by cocaine administration involves not only adipogenesis and fibrosis but also ectopic myogenesis, which is scarcely observed and rather pathological process during thymic involution.

Case report: Fatal ischemic stroke induced by unruptured traumatic intracranial vertebral artery dissection.

Intracranial vertebral artery dissection (IVAD) is rare and potentially fatal due to the risk of secondary subarachnoid hemorrhage once ruptured. Unruptured traumatic IVAD is even rarer and can result in ischemic stroke, yet mostly benign when timely diagnosed. Herein, we present an uncommon case of a patient who underwent a fatal ischemic stroke induced by unruptured traumatic IVAD. The patient was symptomatic soon after being physically assaulted but left untreated until acute deterioration for multiple brain infarctions occurred, secondary to IVAD-induced cerebellar stroke. Fifteen days later, he died, regardless of an urgently performed thrombectomy. Multiple serial histologic examinations revealed an unruptured dissection of the intracranial vertebral artery with a slit-like tear of the intimal and medial layers, considered to be the culprit lesion. The 15-day prolonged onset of stroke was rare in traumatic IVADs. Furthermore, the slit-like tear of the intimal layer in our case may support the initial intimal laceration hypothesis for VAD pathogenesis. Since limited pathohistological information is available regarding ischemic IVAD, we believe this rare case will be beneficial in understanding the pathophysiology of ischemic IVAD.

Revisiting the canonical definition of heat hematoma: A rare case of postmortem subdural heat hematoma.

Heat hematoma is generally recognized as a postmortem heat-induced artifact in extradural spaces found in burned bodies. Conversely, subdural hematoma in charred bodies is more indicative of antemortem trauma. Here, we present a rare case of a subdural heat hematoma in forensic practice. The subdural hematoma was found in a charred body that was determined to be dead before the fire without findings of antemortem head injury. Furthermore, the detailed determination and formation mechanism of this subdural heat hematoma are discussed. With this rare case, we propose a reconsideration of the canonical definition of heat hematoma. This report envisions benefitting forensic pathologists facing similar cases.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells.

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.

Cocaine induces vascular smooth muscle cells proliferation via DRP1-mediated mitochondrial fission and PI3K/HIF-1α signaling.

Long-term cocaine abuse is associated with cardiovascular and pulmonary vascular complications. The vascular toxicity of cocaine can lead to vascular remodeling characterized by excessive proliferation of vascular smooth muscle cells. Though hypoxia-inducible factor (HIF) signaling and mitochondrial fission have been suggested to play essential roles in the pathogenesis of hypoxia-induced vascular remodeling, pathogenetic mechanism for cocaine-related vascular remodeling remains to be elucidated. In this study, we explore the effect of cocaine on the proliferation of vascular smooth muscle cells by in vitro experiments. The findings indicated that the cocaine-induced vascular smooth muscle cell hyperproliferation is achieved by enhancing DRP1-mediated mitochondrial fission and activating PI3K/HIF-1α signaling. Current findings suggested that mitochondrial fission would play a pivotal role in cocaine-related vascular remodeling and would be helpful in understanding the vascular toxicity of cocaine.

Case Report: Molecular autopsy underlie COVID-19-associated sudden, unexplained child mortality.

Herein, we report a child with COVID-19 and seemingly no underlying disease, who died suddenly. The autopsy revealed severe anemia and thrombocytopenia, splenomegaly, hypercytokinemia, and a rare ectopic congenital coronary origin. Immunohistochemical analysis demonstrated that the patient had acute lymphoblastic leukemia of the B-cell precursor phenotype (BCP-ALL). The complex cardiac and hematological abnormalities suggested the presence of an underlying disease; therefore, we performed whole-exome sequencing (WES). WES revealed a leucine-zipper-like transcription regulator 1 (LZTR1) variant, indicating Noonan syndrome (NS). Therefore, we concluded that the patient had underlying NS along with coronary artery malformation and that COVID-19 infection may have triggered the sudden cardiac death due to increased cardiac load caused by high fever and dehydration. In addition, multiple organ failure due to hypercytokinemia probably contributed to the patient's death. This case would be of interest to pathologists and pediatricians because of the limited number of NS patients with LZTR1 variants; the complex combination of an LZTR1 variant, BCP-ALL, and COVID-19; and a rare pattern of the anomalous origin of the coronary artery. Thus, we highlight the significance of molecular autopsy and the application of WES with conventional diagnostic methods.

The proportion of false-positives in positive Seratec® prostate-specific antigen SemiQuant test results in postmortem screening for seminal fluid.

Prostate-specific antigen (PSA) tests are used in forensics to conduct rapid screening for semen in vaginal swab samples from alleged victims of sexual abuse. Although PSA membrane tests have been applied to autopsy specimens, no study has evaluated predictors of false-positive test results in relation to factors such as age, cause of death, postmortem interval, drugs, and alcohol. This study describes the results obtained with the Seratec® PSA SemiQuant Kit test in 283 deceased women, with or without a history of sexual assault. Overall, 18.4% (52/283) of the vaginal swab samples tested positive for PSA. However, 63.5% (33/52) of the PSA-positive vaginal swab samples had no sperm detected. The proportion of false-positives in positive PSA results was 94.4% in those aged over 60 years. Multivariate logistic regression for PSA-positive samples showed that the proportion of false-positives in positive PSA results increased with the age of the deceased. However, the cause of death, postmortem interval, and presence of drugs or alcohol in the blood or urine of the deceased did not affect the PSA determination. These results show that PSA membrane tests are relatively unreliable and can be misleading, especially when derived from vaginal swab samples of older women, obtained at autopsy. In forensic cases, positive PSA screening test results may have an impact on subsequent legal actions and criminal charges brought against the accused. These findings are important for both forensic pathologists and the police to ensure accurate screening of older women in cases of suspected sex crimes.

Aristolochic acid induces an inflammatory response with prostaglandin E2 production and apoptosis in NRK-52E proximal tubular cells.

Aristolochic acid nephropathy (AAN) is a type of drug-induced nephropathy in which ingestion of aristolochic acid (AA) causes acute kidney injury, with progressive renal fibrosis and upper urothelial carcinoma. Although the pathological features of AAN have been reported to involve significant cell degeneration and loss in the proximal tubules, the details of the toxic mechanism in the acute phase of the disease remain unclear. This study investigates the cell death pathway and intracellular metabolic kinetics of AA exposure in rat NRK-52E proximal tubular cells. AA exposure induces dose- and time-dependent apoptotic cell death in NRK-52E cells. We examined the inflammatory response to further investigate the mechanism of AA-induced toxicity. AA exposure increased the gene expression of inflammatory cytokines IL-6 and TNF-α, suggesting that AA exposure induces inflammation. Furthermore, analysis of lipid mediators by LC-MS revealed increases in intra- and extra-cellular arachidonic acid and prostaglandin E2 (PGE2). To investigate the relationship between the AA-induced increase in PGE2 production and cell death, celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), which is involved in the production of PGE2, was administered, and a marked inhibition of AA-induced cell death was observed. These results suggest that exposure to AA induces concentration- and time-dependent apoptosis in NRK-52E cells, which is attributed to inflammatory responses mediated by COX-2 and PGE2.

Induction of filamin-C and its involvement in the regulation of cellular senescence and apoptosis in Huh-7 hepatoma cells during arsenic trioxide exposure.

Arsenic trioxide (ATO) is one of the most toxic inorganic arsenic compounds. In this study, we examined the effects of long-term (7 days) exposure to low dose (5 µM) ATO on a human hepatocellular carcinoma cell line, Huh-7. Along with apoptosis accompanied by secondary necrosis though GSDME cleavage, we observed enlarged and flattened cells adhering to the culture dish and surviving even after exposure to ATO. An increase in cyclin-dependent kinase inhibitor p21 levels as well as positive staining for senescence-associated ß-galactosidase activity were observed in ATO-treated cells, indicating cellular senescence. Screening for both ATO-inducible proteins by MALDI-TOF-MS analysis and ATO-inducible genes by DNA microarray analysis showed a marked increase in filamin-C (FLNC), an actin cross-linking protein. Interestingly, the increase in FLNC was observed in both dead and surviving cells, suggesting that the upregulation of FLNC by ATO occurs in both apoptotic and senescent cells. Small interference RNA-mediated knock down of FLNC resulted in not only a reduction of senescence-associated enlarged morphology of the cells, but also an exacerbation of cell death. Taken together, these results suggest a regulatory role of FLNC in the execution of senescence as well as apoptosis during ATO exposure.

Thymic involution caused by repeated cocaine administration includes apoptotic cell loss followed by ectopic adipogenesis.

Cocaine abuse has a negative impact on the immune system. To investigate the adverse effects of binge cocaine administration on lymphoid organs such as thymus and spleen, we examined the effects of repeated intravenous (i.v.) administration of cocaine on rats. Sprague Dawley rats (male, 8 weeks old) received 20 mg/kg body weight of cocaine hydrochloride per day for 7 or 14 days. In addition to a significant loss in the weight of the spleen, consistent with our previous intraperitoneal (i.p.) injection model of binge cocaine abuse (50 mg/kg cocaine for 7 days), we also found a significant loss of weight as well as apparent shrinkage of the thymus in the cocaine group. Transcriptome analysis of the thymus revealed increased expressions of genes involved in apoptosis, such as Ifi27 and Traf2, as well as decreased expressions of several genes related to lipid metabolism, such as Cd36, Adipoq, Scd1, and Fabp4, in the thymus of the cocaine group (7 days), suggesting an apoptotic loss of thymic cells as well as alterations in lipid metabolism. Paradoxically, cocaine activates PPARγ, a key transcriptional factor activating lipid metabolism, although ectopic adipogenesis was scarcely observed in the thymus. Further analysis of rats administered 20 mg/kg cocaine for 14 days revealed ectopic adipogenesis, which was accompanied with the activation of PPARγ as well as increased expression of Adipoq and Fabp4, in the thymus. Taken together, these results indicate that repeated cocaine administration induces thymic involution, which is initiated by the loss of thymic cells through apoptosis and subsequent ectopic adipocyte development.

Forensic Characteristics of Physical Elder Abuse and Current Status and Issues of Collaboration between Forensic Medicine Departments and Related Institutions in Japan.

This study sought to clarify the characteristics and trends of physical elder abuse and the status of collaboration between forensic medicine departments and related institutions in Japan. Questionnaires were sent to 82 forensic medicine departments and 2857 institutions randomly selected from hospitals, municipalities and public community general support centers. The survey period was February to June 2021, including an extension period for collection. Responses from 675 facilities were analyzed. The most common finding in cases of physical elder abuse at forensic medicine departments was subcutaneous hemorrhage on the head (85.7%), with mixed old and new injuries most commonly observed in the lower limbs (70%). There were few cases in which there was collaboration between forensic medicine departments and other institutions. Among the issues identified, there is a need to provide related institutions with information obtained in forensic medicine departments. A new collaboration system is needed to achieve this.

Contraction Band Necrosis with Dephosphorylated Connexin 43 in Rat Myocardium after Daily Cocaine Administration.

Contraction band necrosis (CBN) is a common abnormality found in the myocardium of cocaine abusers, but is rarely reported in experimental models of cocaine abuse. Connexin 43 (Cx43) is essential for cardiac intercellular communication and the propagation of CBN. Under stress or injury, cardiac Cx43 is dephosphorylated, which is related to cardiomyocyte dysfunction and pathogenesis, whereas adiponectin exerts beneficial effects in the myocardium. In this study, we explore the effects of cocaine on cardiac Cx43 in vivo. Rats were administered cocaine via the tail vein at 20 mg/kg/day for 14 days, and showed widespread CBN, microfocal myocarditis and myocardial fibrosis, corresponding to a dysfunction of cardiac mitochondria under increased oxidative stress. The increase in dephosphorylated cardiac Cx43 and its negative correlation with the myocardial distribution of CBN after cocaine administration were determined. In addition, apoptosis and necroptosis, as well as increased adiponectin levels, were observed in the myocardium after cocaine exposure. Accordingly, we found altered profiles of cardiac Cx43, CBN and its negative correlation with dephosphorylated cardiac Cx43, and the possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. The latter might play a protective role in the cardiotoxicity of cocaine. The current findings would be beneficial for establishing novel therapeutic strategies in cocaine-induced cardiac consequences.

Accuracy of forensic pathologists in incorporating post-mortem CT (PMCT) in forensic death investigation.

Post-mortem computed tomography (PMCT) is now performed routinely in some medical examiner's offices, and the images are typically interpreted by forensic pathologists. In this study, the question of whether pathologists appropriately identify significant PMCT findings and incorporate them into the death investigation report and the cause and manner of death (COD and MOD) statements was addressed. We retrospectively reviewed 200 cases where PMCT was performed. The cases were divided into four categories: (1) full autopsy without radiology consultation (n = 77), (2) external exam without radiology consultation (n = 79), (3) full autopsy with radiology consultation (n = 26), (4) external exam with radiology consultation (n = 18). A radiologist (not the consult radiologist) read the PMCT images, and a pathologist (not the case pathologist) reviewed the case pathologist's post-mortem examination report in tandem to determine any PMCT findings omitted from the report. Omitted findings were classified into error types according to a modified Goldman classification including Major 1: Unrecognized fatal injury or pathology that would change COD and/or MOD, and Major 2: Unrecognized fatal injury or pathology that would not change COD and/or MOD. A total of 13 Major errors were identified (6.5%), and none definitively changed the MOD. All four Major-1 errors which could change the COD were found in Category 2. Of 9 Major-2 errors, 2 occurred in Category 1, 6 occurred in Category 2, and 1 occurred in Category 4. In conclusion, forensic pathologists who routinely utilize computed tomography (CT) interpret CT images well enough to reliably certify the COD and MOD.

Fatal consequence after MiraDry® treatment: Necrotizing fasciitis complicated with streptococcal toxic shock syndrome.

MiraDry® is a microwave-based cosmetic device commonly used to treat hyperhidrosis and osmidrosis by affecting apocrine and eccrine sweat glands. In most countries, its application is limited to the axillary region. A healthy woman received MiraDry® treatment in the perineal, genital, and perianal regions for body odor in a cosmetic clinic. She experienced severe adverse effects after treatment, including persistent fever, sustained pain, and bleeding in the treated area. The condition deteriorated rapidly with systemic symptoms, and she died on the sixth day. Group A Streptococcus was detected in her skin in the treated areas, and in blood obtained in the hospital and during autopsy. Combined with the clinical diagnosis and autopsy findings, the woman's death was attributed to fatal necrotizing fasciitis (Fournier's gangrene) complicated by streptococcal toxic shock syndrome. Pathogen inoculation was most likely attributable to skin disruption caused by MiraDry® treatment. The MiraDry® application on the genital and perineum is occasionally performed by cosmetic surgeons; however, this case demonstrates the possibility of a rare but fatal complication. Therefore, this case report may be noteworthy and beneficial in forensic practice, and relevant in cosmetic clinical practice.

Two fatal cases due to inadvertent discharge of carbon dioxide fire suppressant: Intoxication or asphyxiation?

Carbon dioxide (CO2) is widely used for fire protection and is lethal when inhaled at the minimum design concentration. Two workers were performing periodic maintenance of isolated stored CO2 cylinders of a fire extinguishing system, and during the maintenance, CO2 was accidently discharged into the storage room. The workers were found unconscious beside the cylinders and were confirmed dead in the hospital soon after. Arterial blood gas analysis revealed severe acute hypercapnia accompanied by respiratory acidosis. Combined with the scene investigation and autopsy findings, the cause of their death was determined to be CO2 intoxication and possible involvement of asphyxiation. Considering the repeated occurrences of unfortunate deaths involving CO2 fire extinguishing systems, this report will be useful for forensic pathologists when dealing with similar cases.

Role of Mitochondrial Dynamics in Cocaine's Neurotoxicity.

The dynamic balance of mitochondrial fission and fusion maintains mitochondrial homeostasis and optimal function. It is indispensable for cells such as neurons, which rely on the finely tuned mitochondria to carry out their normal physiological activities. The potent psychostimulant cocaine impairs mitochondria as one way it exerts its neurotoxicity, wherein the disturbances in mitochondrial dynamics have been suggested to play an essential role. In this review, we summarize the neurotoxicity of cocaine and the role of mitochondrial dynamics in cellular physiology. Subsequently, we introduce current findings that link disturbed neuronal mitochondrial dynamics with cocaine exposure. Finally, the possible role and potential therapeutic value of mitochondrial dynamics in cocaine neurotoxicity are discussed.

Sixty-eight cases of postmortem pink teeth observed in dental autopsies of unidentified cadavers.

The pink teeth phenomenon was first reported in people who died by hanging or drowning in 1829. In recent years, there have been many reports that the occurrence of postmortem pink teeth is closely related to the condition of surroundings and time after death rather than to the cause of death, as had previously been thought. We observed pink teeth in 68 of 324 unidentified cadavers autopsied during 2015-2021, and we investigated the relationship of pink teeth with age, sex, cadaveric condition, time after death, tooth type, and cause of death. The results showed no significant differences in the occurrence of pink teeth according to sex or place of discovery (indoors, outdoors, or in water). Pink teeth tended to be observed below the age of 60 years. In the 68 cases with pink teeth, 29.1% of all vital teeth were affected, with the anterior teeth being most common. No evidence was found that supported an association between pink teeth and cause of death. These results are largely consistent with recent reports on postmortem pink teeth. As it stands, the pink teeth phenomenon may be nonspecific and must not be misinterpreted in medico-legal investigations. This case series provides further evidence supporting recent reports and contributes to clarifying in greater detail the mechanisms of the coloration and fading of pink teeth.

Inverse regulation of GSDMD and GSDME gene expression during LPS-induced pyroptosis in RAW264.7 macrophage cells.

GSDMD and GSDME, members of the gasdermin protein family, are involved in the formation of plasma membrane channels contributing to cell rupture during a certain type of necrosis called pyroptosis. GSDMD is activated in response to immunological stimulation such as lipopolysaccharides (LPS) treatment while GSDME is mainly involved in drug-induced tumor cell death. Here we show that the expression of the GSDMD gene increases significantly during LPS-induced pyroptosis in RAW264.7 murine macrophage cells. In contrast, GSDME expression is decreased in the same cells. The increasing effect of LPS on GSDMD expression was observed only when the cells were cultured in high glucose (4.5 g/l) medium, suggesting that glucose availability is important for this effect. The effect of LPS on GSDMD expression is abolished by 2-deoxyglucose (2DG), confirming that glycolysis plays crucial roles in the increasing effect of LPS. Small interference RNA-mediated knock down of GSDMD or overexpression of GSDME causes LPS-induced pyroptosis to take place through GSDME rather than through GSDMD. Taken together, LPS regulates GSDMD and GSDME expression in opposite directions through, at least in part, its effect on glycolysis. This transcriptional regulation may contribute to the execution of pyroptosis in a GSDMD-dependent manner.