Cardiac SARS-CoV-2 Infection, Involvement of Cytokines in Postmortem Immunohistochemical Study.

In the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, significant attention was given to pulmonary manifestations. However, cardiac involvement is increasingly recognized as a critical factor influencing the prognosis, leading to myocardial damage, heart failure, acute coronary syndromes, potentially lethal arrhythmic events, and sudden cardiac death. Despite these findings, there is a lack of studies detailing the necroscopic, macroscopic, and microscopic cardiac changes associated with SARS-CoV-2. This study aimed to investigate the presence of SARS-CoV-2 viral proteins in cardiac tissue using immunohistochemical techniques to assess viral tropism. The analysis of cardiac tissue samples from deceased subjects, in different stages of conservation, confirmed to be positive for SARS-CoV-2 via reverse transcriptase-polymerase chain reaction (RT-PCR), showed immunopositivity for the SARS-CoV-2-NP viral antigen in 33% of cases. Notably, the presence of leukocyte infiltrates sufficient for diagnosing lymphocytic myocarditis was not observed. The central proinflammatory cytokines involved in the pathogenetic mechanism of coronavirus disease 19 (COVID-19) were researched using the immunohistochemical method. A significant increase in cytokine expression was detected, indicating myocardial involvement and dysfunction during SARS-CoV-2 infection. These findings suggest that the immunohistochemical detection of SARS-CoV-2 viral antigens and inflammatory cytokine expression in cardiac tissue could be crucial for a proper forensic assessment of the cause of death, even in sudden cardiac death.

Macroscopic and Microscopic Cerebral Findings in Drug and Alcohol Abusers: The Point of View of the Forensic Pathologist.

Drug abuse still represents a significant challenge for forensic pathologists; it must always be considered during the autopsy examination when the brain morphological alterations observed are not characteristic of any known disease of the central nervous system (CNS). Nonetheless, no specific brain lesions had been found to characterize the precise drug that caused the poisoning. In fact, a broad spectrum of changes affecting the CNS are seen in drug abusers. Thus, forensic pathology plays a key role in identifying the encephalic morphological alterations underlying the death. The aim of this review is to present an updated overview of the literature regarding the correlation between the main substances of abuse and the morphological alterations of the CNS to help the forensic pathologist to discriminate drug-induced alterations of the brain. The authors used the PRISMA criteriology to perform the bibliographic search for the present review. Among the articles identified according to the selected search criteria, 116 articles were chosen which allow us to define a picture of the main macroscopic and microscopic alterations of the brain in drug abuse.

Application of Aquaporins as Markers in Forensic Pathology: A Systematic Review of the Literature.

The study of aquaporins (AQPs) in various forensic fields has offered a promising horizon in response to the need to have reliable elements for the identification of the manner of death and for the individuation of forensic markers for the timing of lesions and vitality of injury. In the literature, various tissues have been studied; the most investigated are the lungs, brain, kidneys, skin, and blood vessels. A systematic literature review on PubMed following PRISMA 2020 guidelines enabled the identification of 96 articles. In all, 34 of these were enrolled to identify Aquaporin-like (AQP-like) forensic markers. The analysis of the literature demonstrated that the most significant markers among the AQPs are as follows: for the brain, AQP4, which is very important in brain trauma and hypoxic damage; AQP3 in the skin lesions caused by various mechanisms; and AQP5 in the diagnosis of drowning. Other applications are in organ damage due to drug abuse and thrombus dating. The focus of this review is to collect all the data present in the literature about the forensic application of AQPs as forensic markers in the most important fields of application. In the current use, the individuation, validation, and application of markers in forensic investigation are very useful in real forensic applications in cases evaluated in court.

Effect of Repeated Administration of ɣ-Valerolactone (GVL) and GHB in the Mouse: Neuroadaptive Changes of the GHB and GABAergic System.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB's abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. METHODS: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). RESULTS: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. CONCLUSIONS: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile.

Oxidative Stress Markers in Human Brain and Placenta May Reveal the Timing of Hypoxic-Ischemic Injury: Evidence from an Immunohistochemical Study.

During pregnancy, reactive oxygen species (ROS) serve as crucial signaling molecules for fetoplacental circulatory physiology. Oxidative stress is thought to sustain the pathogenesis and progression of hypoxic-ischemic encephalopathy (HIE). A retrospective study was performed on the brains and placentas of fetuses and newborns between 36-42 weeks of gestation (Group_1: Fetal intrauterine deaths, Group_2: Intrapartum deaths, Group_3: Post-partum deaths, Control group sudden neonatal death); all groups were further divided into two subgroups (Subgroup_B [brain] and Subgroup_P [placenta]), and the study was conducted through the immunohistochemical investigations of markers of oxidative stress (NOX2, 8-OHdG, NT, iNOS), IL-6, and only on the brain samples, AQP4. The results for the brain samples suggest that NOX2, 8-OHdG, NT, iNOS, and IL-6 were statistically significantly expressed above the controls. iNOS was more expressed in the fetal intrauterine death (Group_1) and less expressed in post-partum death (Group_3), while in intrapartum death (Group_2), the immunoreactivity was very low. IL-6 showed the highest expression in the brain cortex of the fetal intrauterine death (Group_1), while intrapartum death (Group_2) and post-partum death (Group_3) showed weak immunoreactivity. Post-partum death (Group_3) placentas showed the highest immunoreactivity to NOX2, which was almost double that of the fetal intrauterine death (Group_1) and intrapartum death (Group_2) placentas. Placental tissues of fetal intrauterine death (Group_1) and intrapartum death (Group_2) showed higher expression of iNOS than post-partum death (Group_3), while the IL-6 expression was higher in the fetal intrauterine death (Group_1) than the post-partum death (Group_3). The AQP4 was discarded as a possible marker because the immunohistochemical reaction in the three groups of cases and the control group was negative. The goal of this study, from the point of view of forensic pathology, is to provide scientific evidence in cases of medical liability in the Obstetric field to support the clinical data of the timing of HIE.

Application of Forensic DNA Phenotyping for Prediction of Eye, Hair and Skin Colour in Highly Decomposed Bodies.

In the last few years, predicting externally visible characteristics (EVCs) by adopting informative DNA molecular markers has become a method in forensic genetics that has increased its value, giving rise to an interesting field called "Forensic DNA Phenotyping" (FDP). The most meaningful forensic applications of EVCs prediction are those in which, having only a DNA sample isolated from highly decomposed remains, it is essential to reconstruct the physical appearance of a person. Through this approach, we set out to evaluate 20 skeletal remains of Italian provenance in order to associate them with as many cases of missing persons as possible. To achieve the intended goal, in this work we applied the HIrisPlex-S multiplex system through the conventional short tandem repeats (STR) method to confirm the expected identity of subjects by evaluating phenotypic features. To investigate the reliability and accuracy of the DNA-based EVCs prediction, pictures of the cases were compared as they were available to researchers. Results showed an overall prediction accuracy greater than 90% for all three phenotypic features-iris, hair, and skin colour-at a probability threshold of 0.7. The experimental analysis showed inconclusive results in only two cases; this is probably due to the characteristics of subjects who had an intermediate eye and hair colour, for which the DNA-based system needs to improve the prediction accuracy.

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1ß, IL-6, IL-15 and TNF-α.

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1ß, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1ß in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

Wound Vitality in Decomposed Bodies: New Frontiers Through Immunohistochemistry.

Background: The question about wound vitality and the estimation of wound age of production are two of the classic investigation fields of forensic sciences. To answer this, the techniques most frequently used in research studies are immunohistochemistry (IHC), molecular biology, and biochemistry. Despite the great data on the literature about the usefulness of IHC in forensic pathology, there is always a request for further studies, especially on tissues altered by putrefactive phenomena. In fact, the degradation of the tissues is intended as the main limiting factor to the use of this technique. Scope: The aim of this pilot study was to evaluate the immunohistochemical behavior of samples collected from decomposed bodies (in different putrefaction phases) and to relate these findings to wound vitality and postmortem interval. Materials and Methods: Samples of skin and soft tissues were collected during autopsies, which were executed on decomposed bodies, whose cause of death was concluded to be traumatic. An immunohistochemical study was performed using antibodies against CD15, CD45, IL-15, tryptase, and glycophorin-A MMPs (endopeptidases involved in degrading extracellular matrix proteins: MMP-9 and MMP-2). An immunohistochemistry (IHC) reaction was evaluated according to a qualitative method as the following legend: (0): not expressed, (+): isolated and disseminated expression, (++): expression in groups or widespread foci, and (+++): widespread expression. Results: Most of the tested markers (tryptase, glycophorin, IL15, CD 15, CD 45, and MMP9) showed to be highly expressed in the tissue of putrefied skin for 15 days. Discussion and Conclusion: Although certainly inconclusive, this experimental application demonstrated that a nonexclusive but combined use of multiple antibodies is appropriate to verify wound vitality in decomposed bodies. Among them, GPA exhibited major reliability.

Spine surgery and fat embolism syndrome. Defining the boundaries of medical accountability by hospital autopsy.

BACKGROUND: Fat Embolism Syndrome (FES) is a clinical condition characterized by neurological, respiratory, hematological and cutaneous manifestations. Fatal FES has been described as a rare complication during or after spinal elective surgery. The investigation of the cause of death in fatalities related with spine surgery should be mandatory to exclude or confirm fat embolism; a detailed methodological approach to the body in these cases suggests to provide a cautious dissection of surgical site and collection of samples to detect embolized fat globules in vessels. METHODS: Two fatal cases of fat embolism syndrome after posterior spinal fusion are presented. CONCLUSIONS: A complete post mortem examination by means of histochemical and immunohistochemical analysis explained the cause of death and prevented medical malpractice litigation.