Gene-expression profiling of individuals resilient to Alzheimer's disease reveals higher expression of genes related to metallothionein and mitochondrial processes and no changes in the unfolded protein response.

Some individuals show a discrepancy between cognition and the amount of neuropathological changes characteristic for Alzheimer's disease (AD). This phenomenon has been referred to as 'resilience'. The molecular and cellular underpinnings of resilience remain poorly understood. To obtain an unbiased understanding of the molecular changes underlying resilience, we investigated global changes in gene expression in the superior frontal gyrus of a cohort of cognitively and pathologically well-defined AD patients, resilient individuals and age-matched controls (n = 11-12 per group). 897 genes were significantly altered between AD and control, 1121 between resilient and control and 6 between resilient and AD. Gene set enrichment analysis (GSEA) revealed that the expression of metallothionein (MT) and of genes related to mitochondrial processes was higher in the resilient donors. Weighted gene co-expression network analysis (WGCNA) identified gene modules related to the unfolded protein response, mitochondrial processes and synaptic signaling to be differentially associated with resilience or dementia. As changes in MT, mitochondria, heat shock proteins and the unfolded protein response (UPR) were the most pronounced changes in the GSEA and/or WGCNA, immunohistochemistry was used to further validate these processes. MT was significantly increased in astrocytes in resilient individuals. A higher proportion of the mitochondrial gene MT-CO1 was detected outside the cell body versus inside the cell body in the resilient compared to the control group and there were higher levels of heat shock protein 70 (HSP70) and X-box-binding protein 1 spliced (XBP1s), two proteins related to heat shock proteins and the UPR, in the AD donors. Finally, we show evidence for putative sex-specific alterations in resilience, including gene expression differences related to autophagy in females compared to males. Taken together, these results show possible mechanisms involving MTs, mitochondrial processes and the UPR by which individuals might maintain cognition despite the presence of AD pathology.

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder.

Background: Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5 mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5 hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5 mC and 5 hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Methods: Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5 mC and 5 hmC at the genome-wide level. Differential 5 mC and 5 hmC were evaluated using the methylKit R package and significance was set at false discovery rate < 0.05 and differential methylation > 2. Functional enrichment analyses were performed, and gene-level convergence was evaluated in an independent dataset that assessed 5 mC and 5 hmC of AUD in bulk cortical tissue. Results: We identified 417 5 mC and 363 5hmC significant differential CpG sites associated with AUD, with 59% in gene promoters. Some of the identified genes have been previously implicated in alcohol consumption, including SYK, DNMT3A for 5 mC, GAD1, DLX1, DLX2, for 5 hmC and GATA4 in both. Convergence with a previous AUD 5 mC and 5 hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5 mC and 5 hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5 mC genes. Discussion: This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD, identifying both previously reported and potentially novel gene associations with AUD. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

ADNP dysregulates methylation and mitochondrial gene expression in the cerebellum of a Helsmoortel-Van der Aa syndrome autopsy case.

BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation. RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain. CONCLUSION: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories.

Background: Brain banks provide small tissue samples to researchers, while gross anatomy laboratories could provide larger samples, including complete brains to neuroscientists. However, they are preserved with solutions appropriate for gross-dissection, different from the classic neutral-buffered formalin (NBF) used in brain banks. Our previous work in mice showed that two gross-anatomy laboratory solutions, a saturated-salt-solution (SSS) and an alcohol-formaldehyde-solution (AFS), preserve antigenicity of the main cellular markers (neurons, astrocytes, microglia, and myelin). Our goal is now to compare the quality of histology and antigenicity preservation of human brains fixed with NBF by immersion (practice of brain banks) vs. those fixed with a SSS and an AFS by whole body perfusion, practice of gross-anatomy laboratories. Methods: We used a convenience sample of 42 brains (31 males, 11 females; 25-90 years old) fixed with NBF (N = 12), SSS (N = 13), and AFS (N = 17). One cm3 tissue blocks were cut, cryoprotected, frozen and sliced into 40 µm sections. The four cell populations were labeled using immunohistochemistry (Neurons = neuronal-nuclei = NeuN, astrocytes = glial-fibrillary-acidic-protein = GFAP, microglia = ionized-calcium-binding-adaptor-molecule1 = Iba1 and oligodendrocytes = myelin-proteolipid-protein = PLP). We qualitatively assessed antigenicity and cell distribution, and compared the ease of manipulation of the sections, the microscopic tissue quality, and the quality of common histochemical stains (e.g., Cresyl violet, Luxol fast blue, etc.) across solutions. Results: Sections of SSS-fixed brains were more difficult to manipulate and showed poorer tissue quality than those from brains fixed with the other solutions. The four antigens were preserved, and cell labeling was more often homogeneous in AFS-fixed specimens. NeuN and GFAP were not always present in NBF and SSS samples. Some antigens were heterogeneously distributed in some specimens, independently of the fixative, but an antigen retrieval protocol successfully recovered them. Finally, the histochemical stains were of sufficient quality regardless of the fixative, although neurons were more often paler in SSS-fixed specimens. Conclusion: Antigenicity was preserved in human brains fixed with solutions used in human gross-anatomy (albeit the poorer quality of SSS-fixed specimens). For some specific variables, histology quality was superior in AFS-fixed brains. Furthermore, we show the feasibility of frequently used histochemical stains. These results are promising for neuroscientists interested in using brain specimens from anatomy laboratories.

Antipsychotic-induced epigenomic reorganization in frontal cortex of individuals with schizophrenia.

Genome-wide association studies have revealed >270 loci associated with schizophrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone modifications remain largely plastic during development and adulthood, allowing a dynamic impact of environmental factors, including antipsychotic medications, on access to genes and regulatory elements. However, few studies so far have profiled cell-specific genome-wide histone modifications in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) individuals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT cohorts separately, we identified schizophrenia-associated alterations in specific transcription factors, their regulatees, and epigenomic and transcriptomic features that were reversed by antipsychotic treatment; as well as those that represented a consequence of antipsychotic medication rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia, and remark for the first time on the impact of age and antipsychotic treatment on chromatin organization.

[125I]IPC-Lecanemab: Synthesis and Evaluation of Aß-Plaque-Binding Antibody and Comparison with Small-Molecule [18F]Flotaza and [125I]IBETA in Postmortem Human Alzheimer's Disease.

Therapeutic antibodies for reducing Aß plaque load in Alzheimer's disease (AD) is currently making rapid progress. The diagnostic imaging of Aß plaque load in AD has been underway and is now used in clinical studies. Here, we report our preliminary findings on imaging a therapeutic antibody, Lecanemab, in a postmortem AD brain anterior cingulate. [125I]5-iodo-3-pyridinecarboxamido-Lecanemab ([125I]IPC-Lecanemab) was prepared by coupling N-succinimidyl-5-([125I]iodo)-3-pyridinecarboxylate with Lecanemab in modest yields. The distinct binding of [125I]IPC-Lecanemab to Aß-rich regions in postmortem human AD brains was higher in grey matter (GM) containing Aß plaques compared to white matter (WM) (GM/WM was 1.6). Anti-Aß immunostaining was correlated with [125I]IPC-Lecanemab regional binding in the postmortem AD human brains. [125I]IPC-Lecanemab binding was consistent with the binding of Aß small molecules, [18F]flotaza and [125I]IBETA, in the same subjects. [18F]Flotaza and [125I]IBETA, however, exhibited significantly higher GM/WM ratios (>20) compared to [125I]IPC-Lecanemab. Our results suggest that radiolabeled [125I]IPC-Lecanemab retains the ability to bind to Aß in human AD and may therefore be useful as a PET imaging radiotracer when labeled as [124I]IPC-Lecanemab. The ability to directly visualize in vivo a promising therapeutic antibody for AD may be useful in treatment planning and dosing and could be complimentary to small-molecule diagnostic imaging to assess outcomes of therapeutic interventions.

CT analysis of skull contents in naturally mummified human corpses, a multicentric study.

INTRODUCTION: This study aimed to assess skull contents, brain appearance, and density on postmortem computed tomography in naturally mummified corpses. MATERIAL AND METHODS: For this purpose, a retrospective multicentric study, including mummified corpses from two French centers (Brest and Nantes) and from the New Mexico Decedent Image Database (USA), was performed by analyzing postmortem computed tomography (PMCT) focused on the head and neck of partially or fully mummified corpses discovered between 2011 and 2022. The PMCT analysis provided data on the CT appearance of brains, allowing them to be classified into four different categories (desiccation, liquefaction, dura mater only (DMO), and absence), and to measure densities (HU) of the brain remains. In addition, data on postmortem intervals (PMI) from Nantes and Brest centers were collected and analyzed to test the link between brain densities and PMIs. RESULTS: 54 cases of naturally mummified corpses were included. The brains were classified as liquefied (56%), desiccated (17 %), DMO (20 %), and absent (7 %) based on their CT appearance. Dehydrated brains were significantly (p < 0.004) denser (median 102 HU, interquartile range (IQR) 41) than either liquefied brains (median 39.5 HU, IQR 9) or brains with DMO (median -25 HU, IQR 57). However, the density of brain remains was not significantly affected by where the bodies were found (p = 0,41). Analysis of PMI and brain densities was performed on 22 cases. The results showed that brain remains were significantly (p = 0.039) denser when they were found after a PMI of more than six months. CONCLUSION: Brain desiccation was the aspect with the highest densities on PMCT, and for which we were able to highlight great preservation of anatomical structures observable in living organisms.

Age estimation using postmortem computed tomography-based Hounsfield unit values of the palate and mandibular condyle and the Eichner classification.

Age estimation is important in forensic investigations of unidentified human remains. This study assessed the correlation between age and Hounsfield unit (HU) values of the palate and mandibular condyle based on postmortem computed tomography (CT) and analyzed the influence of occlusal support in developing an age estimation method for Japanese individuals, including older adults. The sample consisted of a training dataset (357 cadavers) and a validation dataset (300 cadavers) that underwent postmortem CT. Three measurements were selected: the respective HU values of the palate and mandibular condyle and the Eichner classification. The correlation coefficients between age and HU values were also evaluated. Multiple stepwise regression analysis was performed to evaluate the significance of four parameters (sex, respective HU values of the palate and mandibular condyle, and the Eichner classification) for age estimation and to determine the best age estimation formula. In the validation tests, inaccuracy and bias were calculated for the groups aged ≥65 or <65 years. Significant correlations between age and HU values of the palate and mandibular condyle were observed, regardless of sex. In multiple stepwise regression analysis, all variables except sex were significantly correlated with age. The age estimation formula from the regression analysis was useful, and the validation test exhibited high accuracy, especially in older adults. The HU values of the palate and mandibular condyle and the Eichner classification are useful for age estimation in Japanese individuals.

Postmortem contrast-enhanced computed tomography via direct large-vessel puncture.

The aim of this study was to assess the usefulness of postmortem contrast-enhanced CT (PMeCT) performed via direct large-vessel puncture when routine postmortem CT suggests a vascular lesion as the cause of death. PMeCT was performed in 9 cases (4 male, 5 female) with a mean age of 76 years (range 52-92) at the time of death. The mean time elapsed since death was 29.1 h (12.0-72.0). The location of the target vessel for puncture was determined based on the CT table position and a grid placed on the body surface. An 18-G spinal needle was advanced to the puncture site, and the needle tip was confirmed to have reached the intended blood vessel. Using negative pressure with a 20-ml syringe, the needle tip was advanced until reverse bleeding was confirmed. Diluted contrast medium was injected slowly to ensure its dispersion within the blood vessels. Following confirmation of no extravasation, additional doses of diluted contrast agent were injected in 3-4 divided doses, with CT scans obtained at each step to track the distribution of contrast agent over time. PMeCT was successful in all cases, revealing cardiac tamponade in 7 (ascending aortic dissection, n = 6; cardiac rupture, n = 1), thoracic aortic aneurysm rupture, n = 1, and iliac artery aneurysm rupture, n = 1. There were no cases of procedure-related extravasation (pseudo-lesions). When postmortem CT reveals pericardial hematoma or bleeding in the thoracic or abdominal cavity, PMeCT can identify the source of bleeding.

Ligand bias and inverse agonism on 5-HT2A receptor-mediated modulation of G protein activity in post-mortem human brain.

BACKGROUND AND PURPOSE: Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. EXPERIMENTAL APPROACH: Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. KEY RESULTS: MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. CONCLUSION AND IMPLICATIONS: The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

Lower levels of TRAF1 in Brodmann's area 24, but not 46, in bipolar disorders are not detectable in major depressive disorders.

INTRODUCTION: Multiple lines of research implicate inflammation-related pathways in the molecular pathology of mood disorders, with our data suggesting a critical role for aberrant cortical tumour necrosis factor α (TNF)-signaling in the molecular pathology of bipolar disorders (BPD) and major depressive disorders (MDD). METHODS: To extend our understanding of changes in TNF-signaling pathways in mood disorders we used Western blotting to measure levels of tumour necrosis factor receptor associated factor 1 (TRAF1) and transmembrane TNF receptor superfamily member 1B (tmTNFRSF1B) in Brodmann's areas (BA) 24 and 46 from people with BPD and MDD. These proteins are key rate-limiting components within TNF-signaling pathways. RESULTS: Compared to controls, there were higher levels of TRAF1 of large effect size (η = 0.19, Cohen's d = 0.97) in BA 24, but not BA 46, from people with BPD. Levels of TRAF1 were not altered in MDD and levels of tmTNFRSF1B were not altered in either disorder. LIMITATIONS: The cases studied had been treated with psychotropic drugs prior to death which is an unresolvable study confound. Cohort sizes are relatively small but not untypical of postmortem CNS studies. CONCLUSIONS: To facilitate post-synaptic signaling, TRAF1 is known to associate with tmTNFRSF1B after that receptor takes its activated conformation which occurs predominantly after it binds to transmembrane TNF (tmTNF). Simultaneously, when tmTNFRSF1B binds to tmTNF reverse signaling through tmTNF is activated. Hence our findings in BA 24 argues that bidirectional TNF-signaling may be an important component of the molecular pathology of BPD.

Development of a protocol for standardized use of a water-soluble contrast agent with polyethylene glycol in post-mortem CT angiography.

Computed tomography angiography (PMCTA) is increasingly used in postmortem cases. Standardized validated protocols permit to compare different PMCTA images and make it more easily to defend a case in court. In addition to the well-known technique by Grabherr et al. (2011) which is using paraffin oil as a carrier substance, water-soluble polyethylene glycol 200 (PEG200) can be used in combination with the contrast agent Accupaque® 300. As to date, there exists no standardized protocol for the use of this contrast agent mixture, the aim of this study was to develop a protocol using it. Between 2012 and 2022, 23 PMCTA with PEG200 and Accupaque®300 were performed at the University Centre of Legal Medicine Lausanne (Switzerland) and the Institute of Forensic Medicine Munich (Germany). The images obtained were evaluated regarding the opacification of the vessels and possible artefacts. The best image quality was obtained with a mixing ratio of 1:15 (Accupaque®300:PEG200) and a perfusion volume of 1000 ml in the arterial, 1400 ml in the venous and 350 ml in the dynamic phase. The infusion rates described by Grabherr et al. were confirmed for the three phases. Overall, the opacification of the vessels was diagnostically sufficient. In 13 cases no opacification of the right coronary artery was observed due to a stratification artefact. By using the PMCTA protocol with PEG200 as a carrier, a good overall image quality can be achieved. This protocol offers the possibility to standardize PMCTA with PEG200.

Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30 mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC-MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine's metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method's applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

Butane-related deaths in post-mortem investigations: A systematic review.

Volatile substance abuse is widespread among adolescents due to its easy availability and methods of consumption. Inhalant abuse represents a current problematic issue, causing significant morbidity and mortality due to direct toxicity on several target organs and displacement of gas which results in a lack of oxygen. This review aims to evaluate post-mortem and toxicological investigations in cases of suspected butane intoxication. We performed comprehensive research using the Preferred Reporting Items for Systematic Review (PRISMA) standards. Forty scientific papers fulfilled the inclusion criteria. A total of 58 cases of butane-related deaths were found. Among these, we found 11 cases of suicide (18%), 1 case of homicide (2%), 44 cases of accidental poisoning (76%), and 2 cases of work-related deaths (4%). Autopsy and post-mortem examinations were performed in 54 cases, whereas toxicological analyses were presented in 56 cases. In autopsy, pulmonary edema (51%) and poli-visceral congestion (59%) were the most common findings. When death by butane inhalation is hypothesized, autopsy and histological findings may be nonspecific, therefore toxicological investigations assume a crucial role along with attention to the methods used to collect biological samples.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h.

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

PURPOSE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. RESULTS: We found that the recombinant ß-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

Availability of individual proteins for quantitative analysis in postmortem brains preserved in two different brain banks.

AIM: Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. METHODS: We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. RESULTS: We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. CONCLUSIONS: When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.

Postmortem concentrations for total blood carbon monoxide (TBCO) as novel biomarker for carbon monoxide (CO) poisonings.

Total Blood Carbon Monoxide (TBCO) showed promising results in improving accuracy of CO determinations in blood and presenting better stability to different storage conditions. Therefore, it was proposed as alternative biomarker to carboxyhemoglobin (COHb) for CO poisoning diagnosis. However, given that current interpretation reference values exist for COHb only, it is difficult to implement TBCO analysis in routine. Therefore, we aimed at determining TBCO reference values for postmortem CO poisoning cases. A previously validated method for TBCO analysis via gas chromatography-mass spectrometry was applied to cardiac, peripheral, cranial and spleen blood samples collected from 92 autopsies. Autopsy cases included 21 non-CO related and 71 CO-related cases with varying postmortem intervals (PMI). Statistical analyses were performed using statistical software R Studio. When comparing lower to higher PMI for non-CO related cases, no significant differences were found, which suggests that CO formation or degradation at low PMIs does not occur. Spleen blood showed potential as alternative matrix for CO determinations in cases with sample availability issues, but needs to be evaluated for CO positive cases. Results for cardiac blood in CO-related autopsies showed a positive correlation between COHb and TBCO values (R = 0.78). This value is lower than what is found in the literature, suggesting that even though COHb and TBCO are correlated, a potential underestimation of the true CO exposure might occur if only COHb values are taken into consideration. Samples were divided into CO exposure groups based on COHb concentrations and with the data obtained, classification into following TBCO concentration groups are proposed: no significant CO exposure case <6 µmol/mL, medium CO exposure case 6-20 µmol/mL, high CO exposure case >20µmol/mL. Even if a higher number of samples in each group would enable to increase the confidence, these results are very promising and highlight the importance of TBCO measurement.

Fatal child abuse detected by systematic post-mortem fundus photograph in sudden death in infancy.

In living children, the use of a wide field fundus camera such as RetCam is the gold standard practice to document retinal haemorrhages in suspected cases of abusive head trauma (AHT). In case of sudden unexpected death in infancy (SUDI), child abuse must be considered as a possible cause of death and an eye examination is required. However, no example of post-mortem fundus photograph (PMFP) of retinal haemorrhages related to AHT is yet available for clinicians.We report a SUDI case, with no external traumatic lesions or limb fractures, for which prompt PMFP showed retinal haemorrhages typical of AHT: child abuse was subsequently confirmed by the forensic investigation. We discuss why PMFP is a relevant screening test to detect retinal haemorrhages in the case of SUDI and why the use of the RetCam should be further investigated.

Owner perceptions and Experiences with Equine Necropsy.

In recent years, the number of horses submitted for necropsy has been in decline. This study aimed to identify the perceptions and barriers of equine necropsy through an online survey of horse owners. The data were analyzed by multivariate models for associations and factors that influenced decision making regarding necropsy. The survey yielded 1,366 usable responses. Respondents had higher familiarity with the term "autopsy" compared to "necropsy" (P<0.001) and showed a greater level of comfort using the terms "body" and "mortality" compared to "carcass" (P<0.001). Most respondents (82%) had experienced euthanizing a horse, but few (27%) were offered a necropsy. Of those offered a necropsy, a majority (81%) opted to perform one. When asked about their reasons for choosing to have a necropsy performed, recommendations from veterinarians (77.87 OR, 95%CI: 49.3, 127.5) and already knowing the cause of death (1.85 OR, 95%CI: 1.2, 30) were positively associated while a benefit to herd health was negatively associated (0.55 OR, 95%CI: 0.3, 1.0). Owning horses for use as competition animals (1.44 OR, 95%CI: 0.9, 2.2), the desire for personal closure (1.76 OR, 95%CI: 1.1, 2.9), and the goals to protect human health (1.29 OR, 95%CI: 1.0, 1.6) and to improve herd health (1.43 OR, 95%CI: 1.1, 1.9) were positively associated with a likelihood of choosing to have a necropsy performed or recommending necropsies in the future. This research highlights the need for education of horse owners about the option and benefits of necropsy, and the use of more societally accepted language.