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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. [ABSTRACT FROM AUTHOR]

Myelin membranes are dominated by lipids while the complexity of their protein composition has long been considered to be low. However, numerous additional myelin proteins have been identified since. Here we revisit the proteome of myelin biochemically purified from the brains of healthy c56Bl/6N-mice utilizing complementary proteomic approaches for deep qualitative and quantitative coverage. By gel-free, label-free mass spectrometry, the most abundant myelin proteins PLP, MBP, CNP, and MOG constitute 38, 30, 5, and 1% of the total myelin protein, respectively. The relative abundance of myelin proteins displays a dynamic range of over four orders of magnitude, implying that PLP and MBP have overshadowed less abundant myelin constituents in initial gel-based approaches. By comparisons with published datasets we evaluate to which degree the CNS myelin proteome correlates with the mRNA and protein abundance profiles of myelin and oligodendrocytes. Notably, the myelin proteome displays only minor changes if assessed after a post-mortem delay of 6 h. These data provide the most comprehensive proteome resource of CNS myelin so far and a basis for addressing proteomic heterogeneity of myelin in mouse models and human patients with white matter disorders.

Abstract Background Epigenetic (including DNA and histone) modifications occur in a variety of neurological disorders. If epigenetic features of brain autopsy material are to be studied, it is critical to understand the post-mortem stability of the modifications. Methods Pig and mouse brain tissue were formalin-fixed and paraffin-embedded, or frozen after post-mortem delays of 0, 24, 48, and 72 h. Epigenetic modifications frequently reported in the literature were studied by DNA agarose gel electrophoresis, DNA methylation enzyme-linked immunosorbent assays, Western blotting, and immunohistochemistry. We constructed a tissue microarray of human neocortex samples with devitalization or death to fixation times ranging from

Myelin membranes are dominated by lipids while the complexity of their protein composition has long been considered to be low. However, numerous additional myelin proteins have been identified since. Here we revisit the proteome of myelin biochemically purified from the brains of healthy c56Bl/6N-mice utilizing complementary proteomic approaches for deep qualitative and quantitative coverage. By gel-free, label-free mass spectrometry, the most abundant myelin proteins PLP, MBP, CNP, and MOG constitute 38, 30, 5, and 1% of the total myelin protein, respectively. The relative abundance of myelin proteins displays a dynamic range of over four orders of magnitude, implying that PLP and MBP have overshadowed less abundant myelin constituents in initial gel-based approaches. By comparisons with published datasets we evaluate to which degree the CNS myelin proteome correlates with the mRNA and protein abundance profiles of myelin and oligodendrocytes. Notably, the myelin proteome displays only minor changes if assessed after a post-mortem delay of 6 h. These data provide the most comprehensive proteome resource of CNS myelin so far and a basis for addressing proteomic heterogeneity of myelin in mouse models and human patients with white matter disorders. [ABSTRACT FROM AUTHOR]

The use of post-mortem human tissue is indispensable in studies investigating alterations in metabolite levels in neurodegenerative conditions such as Alzheimer’s disease (AD). However, variability between samples may have unknown effects on metabolite concentrations. The aim of this study was to characterize the impact of such variables. Cingulate gyrus was obtained from AD cases and controls, from three brain banks. Gas chromatography-mass spectrometry (GC-MS) was used to measure and compare the levels of 66 identifiable metabolites in these tissues to determine effects of tissue-collection variables. The effect of PMD was further investigated by analysis of rat brain cortex and cerebellum collected following post-mortem delays (PMDs) of zero to 72 h. Metabolite levels between cases and controls were not replicable across cohorts with variable age- and gender-matching, PMD, and control Braak staging. Analysis of rat tissues found significant effects of PMD on 31 of 63 identified metabolites over periods up to 72 h. PMD must be kept under 24 h for metabolomics analyses on brain tissues to yield replicable results. Tissues should also be well age- and gender-matched, and Braak stage in controls should be kept to a minimum in order to minimize the impact of these variables in influencing metabolite variability.

Understanding the human brain is the ultimate goal in neuroscience, but this is extremely challenging in part due to the fact that brain tissue obtained from autopsy is practically the only source of normal brain tissue and also since changes at different levels of biological organization (genetic, molecular, biochemical, anatomical) occur after death due to multiple mechanisms. Here we used metabolomic and anatomical techniques to study the possible relationship between post-mortem time (PT)-induced changes that may occur at both the metabolomics and anatomical levels in the same brains. Our experiments have mainly focused on the hippocampus of the mouse. We found significant metabolomic changes at 2 h PT, whereas the integrity of neurons and glia, at the anatomical/ neurochemical level, was not significantly altered during the first 5 h PT for the majority of histological markers. [ABSTRACT FROM AUTHOR]

Human post-mortem brain tissue, mostly provided by brain banks, is precious because most human neurodegenerative diseases are restricted in their complete forms to human beings. However, the agonal state, post-mortem delay between death and tissue processing, variable vulnerability of chromatin, RNAs, proteins and metabolites can compromise the usability of the tissue material. Such factors must be considered and assessed in every experimental approach. The use of controls for comparative purposes to analyze diseased cases is crucial as the basic conditions of controls must be the same as those encountered in problem cases. Age, gender, region, hemisphere, and clear characterisation of objective neuropathological changes (e.g., Alzheimer's disease stages of Braak and Braak) in matching controls and problem cases must be considered in every study, and data must be clearly specified when presenting materials and methods in publications. Additional care must be taken regarding atypical neurodegenerative alterations, concomitant pathologies, and systemic diseases, together with drug consumption and treatments. The absence of neurological and mental symptoms and signs, although a sine qua non condition, is not sufficient to match control and problem cases for research. [ABSTRACT FROM AUTHOR]

Histone acetylation plays a role in the regulation of gene transcription. Yet it is not known whether post-mortem brain tissue is suitable for the analysis of histone acetylation. To examine this question, nucleosomes were isolated from frontal cortex of nine subjects which were obtained at short times after death and immediately frozen at −80°C or maintained at room temperature from 3 h up to 50 h after death and then frozen at −80°C to mimic variable post-mortem delay in tissue processing as currently occurs in normal practice. Chromatin immunoprecipitation assays were performed for two lysine residues, H3K9ac and H3K27ac. Four gene loci were amplified by SyBrGreen PCR: Adenosine A receptor, UCHL1, α-synuclein and β-globin. Results showed variability in the histone acetylation level along the post-mortem times and an increase in the acetylation level at an unpredictable time from one case to another and from one gene to another within the first 24 h of post-mortem delay. Similar results were found with three rat brains used to exclude the effects of agonal state and to normalize the start-point as real time zero. Therefore, the present observations show that human post-mortem brain is probably not suitable for comparative studies of histone acetylation. [ABSTRACT FROM AUTHOR]

Abstract: To microwave or not to microwave, that is the question that has confounded the neurochemist as the quest for reducing changes in neurochemicals associated with post-mortem delay has evolved over the years. Rapid changes in brain constituents during the post-mortem delay have been recognized for years as a problem. What is real and what is artifact? What are true basal levels of molecules found in the brain? In the 1920s, neurochemists recognized this issue and determined freezing of the brain was most advantageous for halting rapid breakdown of some molecules and rapid formation of others. By the early 1970s, a number of laboratories noted that freezing the brain in situ or upon removing it from the cranial vault was not sufficient to reduce alterations in brain chemistry. Groups began experimenting with two different techniques to attack this problem, freeze-blowing and head-focused microwave irradiation. My laboratory and others have found that the utilization of head-focused microwave irradiation to halt enzymic alterations in lipids is an essential tool to limit alterations post-mortem. Recently, we and others have demonstrated that this technique is essential in reliably assessing brain eicosanoid levels, without such fixation true basal levels of eicosanoids are impossible to determine and the high concentrations seen in some paradigms may be merely an artifact produced during handling of the brain. Thus, for eicosanoid analysis and other applications in measuring brain lipid levels, head-focused microwave irradiation is an essential tool for the lipid neurochemist. [Copyright &y& Elsevier]

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies. [ABSTRACT FROM AUTHOR]

Abstract: Gel electrophoresis and Western blotting of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau in Alzheimer disease (AD) have been used to analyze the pattern of phospho-tau by using different antibodies directed to the amino-terminal, core and carboxyl terminus of tau, and by using samples with increased artificial post-mortem delay in order to gain understanding on the characteristics of the band pattern and its vulnerability to post-mortem degradation. In addition to the typical profile of three major bands of 68, 64 and 60kDa, several bands of lower molecular weight have been distinguished in frontal cortex homogenates in four AD cases stage V of Braak and Braak in optimal samples with 2h of post-mortem delay. Lower bands, ranging from 60 to 22kDa, are best seen with antibodies directed to the core of tau protein and, particularly, to the carboxy-terminus, thus suggesting the presence of truncated or cleaved forms of tau containing the C-terminal region. This pattern is not the result of post-mortem degradation, as artificial post-mortem delay of the same sample does not reveal the appearance of new bands with time. On the contrary, tau degradation, manifested as a reduction in the number and intensity of the bands, may occur between 8 and 26h post-mortem and is universal in samples with post-mortem delays of 50h. [Copyright &y& Elsevier]

Assessments of synaptic density in human brain are often based on measurements of synaptic proteins. Little information is available on their post-mortem stability. We have investigated this by ELISAs of the pre-synaptic proteins syntaxin and synaptophysin, and the post-synaptic protein PSD-95, in rat and human cortex. The rat brains were cooled in situ from 37 to 20 or 4 °C over 3 h, and then kept at 20 or 4 °C for a further 24–72 h, to simulate post-mortem storage at room temperature or in a mortuary refrigerator. Synaptophysin and PSD-95 levels in rat cerebral cortex were not significantly decreased after 72 h of incubation at 20 °C. Syntaxin was stable for 24 h but decreased by 39–44% at 48–72 h. Storage at 4 °C resulted in a similar reduction of syntaxin levels over 72 h. In human brain tissue from 160 people aged 24–102 years, post-mortem delay had little effect on synaptic protein levels in superior temporal cortex, but was associated with a decline in PSD-95 and syntaxin in mid-frontal cortex after 24 h. The more robust stability of synaptophysin may be related to its multi-transmembrane structure. [Copyright &y& Elsevier]

The use of post-mortem human tissue is indispensable in studies investigating alterations in metabolite levels in neurodegenerative conditions such as Alzheimer's disease (AD). However, variability between samples may have unknown effects on metabolite concentrations. The aim of this study was to characterize the impact of such variables. Cingulate gyrus was obtained from AD cases and controls, from three brain banks. Gas chromatography-mass spectrometry (GC-MS) was used to measure and compare the levels of 66 identifiable metabolites in these tissues to determine effects of tissue-collection variables. The effect of PMD was further investigated by analysis of rat brain cortex and cerebellum collected following post-mortem delays (PMDs) of zero to 72 h. Metabolite levels between cases and controls were not replicable across cohorts with variable age- and gender-matching, PMD, and control Braak staging. Analysis of rat tissues found significant effects of PMD on 31 of 63 identified metabolites over periods up to 72 h. PMD must be kept under 24 h for metabolomics analyses on brain tissues to yield replicable results. Tissues should also be well age- and gender-matched, and Braak stage in controls should be kept to a minimum in order to minimize the impact of these variables in influencing metabolite variability. [ABSTRACT FROM AUTHOR]

Background: Epigenetic (including DNA and histone) modifications occur in a variety of neurological disorders. If epigenetic features of brain autopsy material are to be studied, it is critical to understand the post-mortem stability of the modifications. Methods: Pig and mouse brain tissue were formalin-fixed and paraffin-embedded, or frozen after post-mortem delays of 0, 24, 48, and 72 h. Epigenetic modifications frequently reported in the literature were studied by DNA agarose gel electrophoresis, DNA methylation enzyme-linked immunosorbent assays, Western blotting, and immunohistochemistry. We constructed a tissue microarray of human neocortex samples with devitalization or death to fixation times ranging from < 60 min to 5 days. Results: In pig and mouse brain tissue, we found that DNA cytosine modifications (5mC, 5hmC, 5fC, and 5caC) were stable for ≥ 72 h post-mortem. Histone methylation was generally stable for ≥ 48 h (H3K9me2/K9me3, H3K27me2, H3K36me3) or ≥ 72 h post-mortem (H3K4me3, H3K27me3). Histone acetylation was generally less stable. The levels of H3K9ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac declined as early as ≤ 24 h post-mortem, while the levels of H3K14ac did not change at ≥ 48 h. Immunohistochemistry showed that histone acetylation loss occurred primarily in the nuclei of large neurons, while immunoreactivity in glial cell nuclei was relatively unchanged. In the human brain tissue array, immunoreactivity for DNA cytosine modifications and histone methylation was stable, while subtle changes were apparent in histone acetylation at 4 to 5 days post-mortem. Conclusion: We conclude that global epigenetic studies on human post-mortem brain tissue are feasible, but great caution is needed for selection of post-mortem delay matched controls if histone acetylation is of interest. [ABSTRACT FROM AUTHOR]