Impairment of the GABAergic system in the anterior insular cortex of heroin-addicted males.

Opioid addiction is a global problem, causing the greatest health burden among drug use disorders, with opioid overdose deaths topping the statistics of fatal overdoses. The multifunctional anterior insular cortex (AIC) is involved in inhibitory control, which is severely impaired in opioid addiction. GABAergic interneurons shape the output of the AIC, where abnormalities have been reported in individuals addicted to opioids. In these neurons, glutamate decarboxylase (GAD) with its isoforms GAD 65 and 67 is a key enzyme in the synthesis of GABA, and research data point to a dysregulation of GABAergic activity in the AIC in opioid addiction. Our study, which was performed on paraffin-embedded brains from the Magdeburg Brain Bank, aimed to investigate abnormalities in the GABAergic function of the AIC in opioid addiction by densitometric evaluation of GAD 65/67-immunostained neuropil. The study showed bilaterally increased neuropil density in layers III and V in 13 male heroin-addicted males compared to 12 healthy controls, with significant U-test P values for layer V bilaterally. Analysis of confounding variables showed that age, brain volume and duration of formalin fixation did not confound the results. Our findings suggest a dysregulation of GABAergic activity in the AIC in opioid addiction, which is consistent with experimental data from animal models and human neuroimaging studies.

Rare case of multiple self-inflicted gunshot wounds to the chest in Poland.

Multiple gunshot suicides are relatively rare and present significant challenges for investigators and forensic pathologists. In such cases, assessing the possibility of more than one shot being fired can be crucial in distinguishing homicide from suicide. We present a rare case of multiple self-inflicted gunshot wounds to the chest with severe injury to the heart and left lung. Both the sudden, unexpected death of the man, the unknown source of the firearm, and the number and nature of the injuries sustained seemed quite unusual. The investigation revealed that the wounds were self-inflicted at close range, and the interval between successive shots (estimated by witnesses at up to 2 min) suggests that even multiple gunshot wounds perforating the heart and lungs may not necessarily cause immediate incapacitation. Forensic investigations in such cases should be multi-faceted and include full autopsy and ballistics expertise, as well as witness testimony and medical history.

Homicide numbers, rates, and victims' characteristics in the Tri-City metropolitan area, Poland, between 2010 and 2019. / Liczby i wskazniki zabójstw oraz charakterystyka ofiar na terenie aglomeracji Trójmiasta w latach 2010-2019.

Despite a clear global downward trend, homicides still account for a relatively high proportion of all violent deaths, making them a serious problem both in Poland and worldwide. The discrepancy in available data prompted the authors of the study to analyse the numbers and rates of homicides and the characteristics of the homicide victims in the Tri-City area of northern Poland. The study was based on data from autopsy reports, supplemented by information from prosecutor's files on all homicides in the Tri-City area between 2010 and 2019. A total of 107 homicides were statistically analysed for age, sex, blood alcohol concentration at the time of death, time and place of death. The annual homicide rate was 1.24 per 100,000 inhabitants, with a clear downward trend over the period analysed. The average age of victims was about 48 years, and the majority of victims were male (70.1%). 92.5% of homicides were committed in the Tri-City, with a clear predominance of Gdansk (49.5%) over other, mostly rural, areas of the analysed agglomeration. The majority of victims (57.8%) whose blood alcohol concentration was measured were intoxicated, with a clear predominance of males in this group (70.9%). Victim characteristics and the homicide rates obtained from the analysed material were similar to other countries in Central and Eastern Europe, which may be related to historical, cultural, and demographic similarities. The study highlights the significant impact of alcohol abuse on the risk of homicide.

Inverse pattern of GABAergic system impairment in the external versus internal globus pallidus in male heroin addicts.

Opioid addiction is a global problem that has been exacerbated in the USA and Europe by the COVID-19 pandemic. The globus pallidus (GP) plays a prominent neurobiological role in the regulation of behaviour as an output station of the striato-pallidal system. GABAergic large projection neurons are the main neuronal type in the external (EGP) and internal (IGP) parts of the GP, where addiction-specific molecular and functional abnormalities occur. In these neurons, glutamate decarboxylase (GAD) with isoforms GAD 65 and 67 is a key enzyme in GABA synthesis, and experimental studies suggest GAD dysregulation in the GP of heroin addicts. Our study, which was performed on paraffin-embedded brains from the Magdeburg Brain Bank, aimed to investigate abnormalities in the GABAergic function of large GP neurons by densitometric evaluation of their GAD 65/67-immunostained thick dendrites. The study revealed a bilaterally decreased fibres density in the EGP paralleled by the increase in the IGP in 11 male heroin addicts versus 11 healthy controls (significant U-test P values). The analysis of confounding variables found no interference of age, brain volume, and duration of formalin fixation with the results. Our findings suggest a dysregulation of GABAergic activity in the GP of heroin addicts, which is consistent with experimental data from animal models and plays potentially a role in the disturbed function of basal ganglia circuit in opioid addiction.

Homicide mechanisms, weapon types and causes of death in the autopsy material of the Department of Forensic Medicine Medical University of Gdansk Poland in the years 2010-2019.

The study objective was the analysis of homicides based on reports of 107 medicolegal autopsies of victims, which were carried out in 2010-2019 at the Department of Forensic Medicine of the Medical University of Gdansk, Poland. The study focused on the analysis of homicide mechanisms, on the weapon type, injuries characteristics, and both pathomechanisms and causes of death. Alcohol and illicit drugs abuse, which is a predisposing factor, was also assessed. In the analysed cohort, 70.1% were males and 29.9% females. The most frequently used weapon was a blunt instrument (accounting for 47.7% of homicides), while the least common was a firearm (accounting for 4.7% of homicides). There were significant differences in homicide mechanisms related to victims' gender. In males, blunt or sharp instruments predominated, while in females violent suffocation and chop injuries were more frequent. Fatal head injuries and their consequences predominated in the pathomechanisms of death (32.7% of cases). It was shown that the highest blood alcohol concentration (BAC) was found in homicides due to violent strangulation and sharp instrument use, while the lowest BAC was found in gunshot victims. Toxicological tests were performed in 18 cases and in 7 cases showed positive results for illicit drugs. The analysis of various elements involved in homicide may contribute to a conceptual framework for preventive measures aiming at the reduction of homicides number.

The left-lateralisation of citrate synthase activity in the anterior cingulate cortex of male violent suicide victims.

The anterior cingulate cortex (AC) as a part of prefrontal cortex plays a crucial role in behavioural regulation, which is profoundly disturbed in suicide. Citrate synthase (CS) is a key enzyme of tricarboxylic acid cycle fundamental for brain energetics and neurotransmitter synthesis, which are deteriorated in suicidal behaviour. However, CS activity has not been yet studied in brain structures of suicide victims. CS activity assay was performed bilaterally on frozen samples of the rostral part of the AC of 24 violent suicide completers (21 males and 3 females) with unknown psychiatric diagnosis and 24 non-suicidal controls (20 males and 4 females). Compared to controls, suicide victims revealed decreased CS activity in the right AC, however, insignificant. Further statistical analysis of laterality index revealed the left-lateralisation of CS activity in the AC in male suicides compared to male controls (U-test P = 0.0003, corrected for multiple comparisons). The results were not confounded by postmortem interval, blood alcohol concentration, age, and brain weight. Our findings suggest that disturbed CS activity in the AC plays a role in suicide pathogenesis and correspond with our previous morphological and molecular studies of prefrontal regions in suicide.

Epidemiology of suicide in the Tri-City metropolitan area in Poland in 2010-2019.

The paper, which is a continuation of our previous epidemiological studies on the phenomenon of suicide in the Tri-City metropolitan area, presents the results of statistical analyses of suicides in the autopsy material of the Department of Forensic Medicine of the Medical University of Gdansk in the years 2010-2019. The purpose of the study was to analyse in detail demographic data of suicides (age, sex, place of death), as well as to assess suicide methods and the impact of alcohol on suicides in the study area. During the 10-year study period, 8495 autopsies were performed, of which 1261 were suicides (14.8%). Statistical analyses were conducted using the statistical data analysis software system STATISTICA, version 13 (StatSoft, Tulsa, Oklahoma, USA). The results of the study indicate a continuing downward trend in the number of suicides since the beginning of the 21th century, with the number of suicides in rural areas increasing over the same period. In the analysed cohort, suicides were committed in particular by middle-aged men and the number of suicides among older people (65 +) increased at the same time. The increase in suicide occurred in late autumn and early spring. The most common method of suicide was hanging. There was a high percentage of inebriated victims (45%), and a comparison of the present studies with previous ones indicates the increasing impact of alcohol on suicide.

Long-Term Stability of Benzodiazepines and Z-Hypnotic Drugs in Blood Samples Stored at Varying Temperatures.

Benzodiazepines (BZDs) and Z-drugs are among the most commonly prescribed pharmaceuticals in the world and are considered standard care for various mental illnesses and for the treatment of sleeping and anxiety disorders, alcohol withdrawal, muscle spasms and epilepsy. Some BZDs are not allowed as pharmaceuticals in many countries, and they are used as designer benzodiazepines (DBZDs). All these compounds are typically screened in routine toxicological analyses for forensic purposes. Knowledge of time-dependent decreases in drug concentrations during storage or transport of samples is of considerable significance and allows forensic toxicologists to achieve reliable data, proper interpretation and high-quality results. The aim of this study was to evaluate changes in the amounts of selected BZDs, DBZDs and Z-drugs in blood samples stored at various temperatures. The study involved BZDs (19), DBZDs (3) and Z-drugs (2) spiked into blank blood. Subsequently, the blood samples were stored at various temperatures (room temperature, 4°C, -20°C and -80°C) for up to 6 months. Analyses were performed at 1- to 2-week intervals using liquid chromatography-tandem mass spectrometry. The stability of compounds was evaluated under four temperature conditions over a 6-month period. Some BZDs were stable at all temperatures tested (e.g., diazepam, oxazepam, nordazepam and prazepam) with a degradation rate of only 0-10%. The highest instability was observed for analyte samples kept at room temperature, and the losses in content for some compounds, e.g., lorazepam and chlordiazepoxide, were almost 100%. For other compounds, the stability was clearly different at each tested temperature. To the best of our knowledge, this is one of the first such comprehensive study of the long-term stability of BZDs covering a wide range of different storage temperatures.

Ribosomal DNA transcription is increased in the left nucleus accumbens of heroin-dependent males.

Opioid addiction is a worldwide problem accentuated in the USA and European countries by the COVID-19 pandemic. The nucleus accumbens (NAc) plays an outstanding neurobiological role in opioid addiction as a part of the striatum and key component of brain reward system. The striatal GABAergic medium spiny projection neurons (MSNs) are the main neuronal type in the NAc where addiction-specific synaptic plasticity occurs. The activity of ribosomal DNA (rDNA) transcription is crucial for neural plasticity and molecular studies suggest its increase in the NAc of heroin addicts. Silver-stained argyrophilic nucleolar organizer region (AgNOR) areas visualised in neuronal nuclei in paraffin-embedded brain sections are reliable morphological estimators of rDNA transcription and thus surrogate markers for the activity of brain regions. Our study revealed increased AgNOR areas in MSNs of the left NAc in 11 heroin addicts versus 11 healthy controls from the Magdeburg Brain Bank (U-test P = 0.007). No differences were observed in another investigated part of the striatum, namely the head of caudate nucleus, which is located closely to the NAc. The results were not confounded by significant differences in the age, brain volume and time of formalin fixation existing between compared groups. Our findings suggest an increased NAc activity in heroin addicts, which is consistent with human and animal experimental data.

Comparative study of the percutaneous permeation and bioaccumulation of a cyclic siloxane using frozen-thawed and nonfrozen ex vivo human skin.

Literature shows contradictory information regarding the effect of freezing the excise skin ex vivo on the diffusion of substances into the skin. Few studies indicate that storing the human or animal skin in a frozen state decreases the barrier properties after thawing. Therefore, to understand the properties of frozen skin, we evaluated the effect of storage of ex vivo human skin (2 weeks at -20 °C) on the penetration of stratum corneum and permeation into deeper skin layers (epidermis, and dermis) as well as to the receptor fluid by octamethylcyclotetrasiloxane (D4) a representative test compound of cyclic siloxanes. The main research were preceded by checking the integrity of nonfrozen ex vivo human skin in comparison to the frozen-thawed one by using the Electrical Resistance technique (ER) and the fluorescence microscopy. Samples collected in the skin absorption experiment were analyzed by gas chromatography equipped with a flame ionization detector (GC-FID). The results of this study demonstrated that freezing of excised ex vivo human skin at -20 °C for up to 14 days does not alter the permeability of D4 in a statistically significant manner. Thus, our results confirmed the validity of using skin storage conditions for testing the penetration and permeation of xenobiotics recommended by the OECD, EMA, and WHO guidelines.

The Application of Metabolomics in Forensic Science with Focus on Forensic Toxicology and Time-of-Death Estimation.

Recently, the diagnostic methods used by scientists in forensic examinations have enormously expanded. Metabolomics provides an important contribution to analytical method development. The main purpose of this review was to investigate and summarize the most recent applications of metabolomics in forensic science. The primary research method was an extensive review of available international literature in PubMed. The keywords "forensic" and "metabolomics" were used as search criteria for the PubMed database scan. Most authors emphasized the analysis of different biological sample types using chromatography methods. The presented review is a summary of recently published implementations of metabolomics in forensic science and types of biological material used and techniques applied. Possible opportunities for valuable metabolomics' applications are discussed to emphasize the essential necessities resulting in numerous nontargeted metabolomics' assays.

Reduced ribosomal DNA transcription in the prefrontal cortex of suicide victims: consistence of new molecular RT-qPCR findings with previous morphometric data from AgNOR-stained pyramidal neurons.

Prefrontal cortical regions play a key role in behavioural regulation, which is profoundly disturbed in suicide. The study was carried out on frozen cortical samples from the anterior cingulate cortex (dorsal and ventral parts, ACd and ACv), the orbitofrontal cortex (OFC), and the dorsolateral cortex (DLC) obtained from 20 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 21 non-suicidal controls. The relative level of ribosomal RNA (rRNA) as a marker of the transcriptional activity of ribosomal DNA (rDNA) was evaluated bilaterally in prefrontal regions mentioned above (i.e. in eight regions of interest, ROIs) by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The overall statistical analysis revealed a decrease in rDNA activity in suicide victims versus controls, particularly in male subjects. Further ROI-specific post hoc analyses revealed a significant decrease in this activity in suicides compared to non-suicides in five ROIs. This effect was accentuated in the ACv, where it was observed bilaterally. Our findings suggest that decreased rDNA transcription in the prefrontal cortex plays an important role in suicide pathogenesis and corresponds with our previous morphometric analyses of AgNOR-stained neurons.

Fatal N-Ethylhexedrone Intoxication.

N-Ethylhexedrone [2-(ethyloamino)-1-phenylhexan-1-one; α-ethylaminohexanophenone (NEH)] is one of the most recent synthetic cathinones that appeared on the illegal market in late 2015. The majority of information concerning the model of consumption of NEH and its impact on the body originates only from self-reports from gray literature websites and drug forums. There are only limited data associated with the concentrations of NEH in blood samples available in the literature. This article presents a case of fatal NEH intoxication and a method for the determination of this substance in whole blood. A 21-year-old man without any diagnosed diseases was admitted to the hospital due to disorientation, aggression and finally loss of consciousness. Hyperthermia (>41°C), tachycardia (>160 beats per minute), tachypnea (20 breaths per minute), blood pressure (110/60 mmHg) and acute kidney failure were diagnosed. After a few hours of hospitalization, the patient died. A plastic bag with a white powder was found in his underwear. Analysis of the powder by another laboratory revealed cocaine hydrochloride; however, no cocaine or its metabolites were found in the biological material upon testing in our laboratory. Therefore, re-analysis of the powder was performed, and NEH was identified. Liquid-liquid extraction followed by liquid chromatography-triple quadrupole-mass spectrometry (LC-MS/MS) analysis were used for the determination of NEH in blood. The validation parameters were as follows: calibration range 1-250 ng/mL, accuracy 106.5-109.9%, precision 3.5-6.3%, recovery 90.1-96.9%, limit of detection 0.07 ng/mL and limit of quantification 1 ng/mL. NEH was quantified in the blood at a concentration of 145 ng/mL. Additionally, amphetamine at low concentrations and 11-nor-9-karboksy-Δ9-tetrahydrokannabinol (THC-COOH) were detected. Our study provided information on the possible lethal concentration and toxidrome that clinicians can observe for NEH-intoxicated patients and can be helpful during the preparation of toxicology analysis reports for a court of law for proper data interpretation.

Fake gunshot wounds in the skull-post-mortem artifact caused by steel probe during police search for a missing body.

The paper presents a case of a forensic autopsy of a young woman who was murdered and her dismembered body was hidden in soil and water. In the skull of the deceased, in the temporal and occipital regions, the autopsy revealed 3 round, almost identical holes, which looked like small caliber gunshot wounds. Doubts about the cause of these injuries were raised by the fact that despite the decomposition of the body, the continuity of the dura at the site of these holes remained undamaged and the absence of any trace of a bullet's wound track in the brain, the absence of a foreign body in the cranial cavity, as well as the absence of wounds on the opposite side of the skull that could be exit wounds. A thorough analysis of the investigation and the activities carried out during the search for the missing body allowed to adopt and finally confirm the hypothesis that the above mentioned skull damage occurred during the search for the cut-off head of the deceased in shallow water by means of special tapered conical steel probes used by the rescue/search teams. Due to the structure of such a spike, i.e., a sharp end and then a wide cone, only a superficial puncture of the steel probe tip three times into the skull had taken place, which caused regular, rounded bone damage without damaging the dura and brain. The presented case indicates that sometimes post-mortem artifacts may suggest a completely different origin of wounds, which emphasizes the need for a comprehensive analysis of all possible causes of their occurrence, particularly data concerning the handling of the corpse before it is delivered to the morgue, so as not to make a diagnostic error during autopsy.

Fatal haemorrhage from an aortoesophageal fistula secondary to button battery ingestion in a 15-month-old child. Case report and literature review.

Small children put various objects into their mouths, which is one of their ways of exploring the surrounding world. Apart from toy parts, e.g. Lego® bricks or magnetic spheres, such objects include coins, small stones, as well as batteries used for different electric devices. Such batteries, especially the flat, round button type, may be ingested and become impacted in one of the physiological narrowing sites of the oesophagus, leading to serious complications. The case of a 15-month-old child is presented, who died due to a massive gastrointestinal haemorrhage from an aortoesophageal fistula that developed at the site of a pressure ulcer caused by an impacted button battery. The presented case shows that ingestion of even a small battery may lead to death. The asymptomatic course of the battery ingestion significantly hinders both the assessment of circumstances and the time of the battery ingestion. Sudden death may result from a massive haemorrhage from a pressure-induced injury to a big blood vessel.

Ribosomal DNA transcription in prefrontal pyramidal neurons is decreased in suicide.

Prefrontal cortical regions, which are crucial for the regulation of emotionally influenced behaviour, play most probably a dominant role in the pathogenesis of suicide. The study was carried out on paraffin-embedded brain tissue blocks containing specimens from the anterior cingulate cortex (dorsal and ventral parts), the orbitofrontal cortex, and the dorsolateral cortex obtained from 23 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 25 non-suicidal controls. The transcriptional activity of ribosomal DNA (rDNA) as a surrogate marker of protein biosynthesis was evaluated separately in layers III and V pyramidal neurons in regions of interest (ROIs) mentioned above by the AgNOR silver staining method bilaterally. The overall statistical analysis revealed a decrease of AgNOR area suggestive of attenuated rDNA activity in suicide victims versus controls, particularly in male subjects. Further ROI-specific post-hoc analyses revealed decreases of the median AgNOR area in suicides compared to non-suicides in all 16 ROIs. However, this effect was only significant in the layer V pyramidal neurons of the right ventral anterior cingulate cortex. Our findings suggest that decreased rDNA transcription in prefrontal pyramidal neurons plays possibly an important role in suicide pathogenesis.

Eye temperature measured after death in human bodies as an alternative method of time of death estimation in the early post mortem period. A successive study on new series of cases with exactly known time of death.

The paper presents a continuation of the studies on time of death (TOD) estimation based on post-mortem temperature measurements in the human eyeball. In the current study, single (in 20 patients) or double (within a 1-hour interval in 10 patients) eyeball and rectal temperature measurements were taken in patients who died in the University Hospital Intensive Therapy Unit. The actual TOD in each patient was exactly known and the body temperature was recorded shortly after (between 50 min and 3 h 30 min). The temperature was measured using pin probes connected to a high precision electronic thermometer. The measured eye temperatures ranged from 27.4 to 33.7°C. The ambient temperature in all cases was stable (22 °C), which corresponded to the usual room temperature. Post mortem interval (PMI) was calculated using a formula based on Newton's law of cooling, previously successfully applied in comprehensive studies on pigs and recent studies on human bodies. Thanks to knowledge of the exact TOD and the stable ambient temperature, the current study on 30 new cases allowed the method to be improved by adjusting the mean value of the cooling constant and the initial eye temperature in comparison to recent studies.

Postmortem Analysis of Electrogram Records from an Implantable Cardioverter-Defibrillator (ICD) in the Reconstruction of a Road Traffic Accident.

The case of a 69-year-old man, equipped with an ICD and suffering from several chronic cardiac diseases, who died in a car accident, was presented. We analyzed electrogram records from the ICD explanted from the body during the autopsy, which showed that the driver had suffered from malignant ventricular arrhythmia-ventricular fibrillation (VF). A thorough analysis of the details of the accident, as well as the timing of VF and the rhythm observed after the discharge of the ICD showed that the direct cause of the accident was the episode of arrhythmia resulting in a loss of consciousness. Therefore, the presented case illustrates the usefulness of postmortem analysis of electrogram records from ICDs in the reconstruction of road traffic accidents. In such cases, if the victims are implanted with ICDs, it should be a routine procedure performed by forensic pathologists.

Determination of tocopherols and tocotrienols in human breast adipose tissue with the use of high performance liquid chromatography-fluorescence detection.

Tocopherols and tocotrienols have been extensively studied owing to their anticancer potential, especially against breast cancer. Therefore, the aim of this study was to quantitatively determine tocochromanols in human breast adipose tissue with the use of HPLC-FLD. The sample preparation procedure included homogenization and solvent extraction with isopropanol-ethanol-0.1% formic acid mixture prior to solid-phase extraction. After implementation of central composite design, satisfactory separation of all eight target compounds was achieved within 10.5 min. Chromatographic runs were carried out with the use of a naphthylethyl chromatographic column with methanol-water mixture (89:11, v/v) as the mobile phase. Fluorescence detection of tocochromanols was performed with excitation and emission wavelengths 298 and 330 nm, respectively. The method was validated in terms of linearity, carryover, recovery, precision, accuracy and stability. Extraction yield was also determined for accurate evaluation of vitamin E content in human breast adipose tissue samples. Finally, concentrations of particular tocochromanols compounds were assessed in human breast adipose tissue samples obtained from 99 patients, including women with breast cancer, healthy volunteers and deceased women who had died as a result of accidents. The raw data was transformed according to the newly developed equation for accurate estimation of the concentrations of tocochromanols in breast adipose tissue samples. Results obtained in the study indicated that the proposed analytical assay could be useful in breast cancer research.