Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

Phosphatidylethanol in post-mortem brain: correlation with blood alcohol concentration and alcohol use disorder.

Phosphatidylethanol (PEth) is an alcohol derivative that has been employed as a blood-based biomarker for regular alcohol use. This study investigates the utility of phosphatidylethanol (PEth) as a biomarker for assessing alcohol consumption in post-mortem brain tissue. Using samples from the New South Wales Brain Tissue Resource Centre, we analysed PEth(16:0/18:1) levels in the cerebellum and meninges of individuals with varying histories of alcohol use, including those diagnosed with alcohol use disorder (AUD) and controls. Our findings demonstrate a significant correlation between PEth levels and blood alcohol content (BAC) at the time of death, supporting the biomarker's sensitivity to recent alcohol intake. Furthermore, this study explores the potential of PEth levels in differentiating AUD cases from controls, taking into consideration the complexities of diagnosing AUD post-mortem. The study also examined the relationship between PEth levels and liver pathology, identifying a link with the severity of liver damage. These results underscore the value of PEth as a reliable indicator of alcohol consumption and its potential contributions to post-mortem diagnostics and consequently, research into alcohol-related brain damage.

Lipidome changes in alcohol-related brain damage.

Alcohol-related brain injury is characterized by cognitive deficits and brain atrophy with the prefrontal cortex particularly susceptible. White matter in the human brain is lipid rich and a major target of damage from chronic alcohol abuse; yet, there is sparse information on how these lipids are affected. Here, we used untargeted lipidomics as a discovery tool to describe these changes in the prefrontal, middle temporal, and visual cortices of human subjects with alcohol use disorder and controls. Significant changes to the lipidome, predominantly in the prefrontal and visual cortices, and differences between the white and grey matter of each brain region were identified. These effects include broad decreases to phospholipids and ceramide, decreased polyunsaturated fatty acids, decreased sphingadiene backbones, and selective decreases in cholesteryl ester fatty acid chains. Our findings show that chronic alcohol abuse results in selective changes to the neurolipidome, which likely reflects both the directs effects on the brain and concurrent effects on the liver.

Matrix phase fractionation: Investigating the compromise between dynamic range of analyte extraction and spatial resolution in mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones. METHODS: A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range. RESULTS: We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a 'fraction' of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique 'matrix phase fractionation'. CONCLUSIONS: We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional 'single-run' workflows.

Severe childhood and adulthood stress associates with neocortical layer-specific reductions of mature spines in psychiatric disorders.

Severe stress exposure causes the loss of dendritic spines on cortical pyramidal neurons and induces psychiatric-like symptoms in rodent models. These effects are strongest following early-life stress and are most persistent on apical dendrites. However, the long-term impacts and temporal effects of stress exposure on the human brain remain poorly understood. Using a novel postmortem cohort of psychiatric cases with severe stress experienced in childhood, adulthood, or no severe stress, and matched controls, we aimed to determine the impact of stress timing on pyramidal neuron structure in the human orbitofrontal cortex (OFC). We performed Golgi Cox staining and manually measured the morphology and density of over 22,000 dendritic spines on layer-specific pyramidal neuron apical dendrites. We also quantified glucocorticoid receptor mRNA and protein as a marker of stress dysregulation. Both childhood and adulthood stress were associated with large reductions in mature mushroom spine density (up to 56% loss) in both the superficial (II/III) and deeper layers (V) of the OFC. However, childhood stress caused more substantial reductions to both total and mature mushroom spines. No difference in glucocorticoid receptor mRNA and protein were seen between groups, although both negatively correlated with total spine density within the whole cohort. These findings indicate that severe stress, especially when experienced during childhood, persistently affects the fine morphological properties of neurons in the human OFC. This may impact on cell connectivity in this brain area, and at least partly explain the social and emotional symptoms that originate in the OFC in psychiatric disorders.

Higher Mass Accuracy MALDI-TOF/TOF Lipid Imaging of Human Brain Tissue in Alzheimer's Disease.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a well-established technique for elucidating the location and relative abundance of a range of biomolecules. More recently, research into this technique has shifted from simple discovery and demonstration of utility to application in biomedical research. Here, we describe a protocol utilizing MALDI-IMS for the spatial mapping of lipids in brain tissue from normal human brains and brains from patients with Alzheimer's disease, in the context of Alzheimer's disease. Improved accuracy calibration of the instrument from the tissue surface is emphasized, as this allows for significantly improved mass determination in time of flight (TOF)-based instruments enabling more confident preliminary lipid identification. This improved initial result allows MALDI-IMS data to be complemented with additional instrumentation, such as liquid chromatography mass spectrometry workflows or specialized non-TOF systems such as Fourier transform cyclotron resonance instruments. This method is not limited to human tissue and can be applied to virtually any lipid-rich formalin-fixed tissue. © 2019 by John Wiley & Sons, Inc.

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

Apoptotic cell death and temporal expression of apoptotic proteins Bcl-2 and Bax in the hippocampus, following binge ethanol in the neonatal rat model.

BACKGROUND: Binge-like ethanol (EtOH) exposure during the early rat neonatal period results in acute cell loss in specific brain regions, but such acute cell death has not been well established in the hippocampus. Binge alcohol exposure can also result in protein expression changes in the cerebellum that could alter cell fate, but this has not been reported for the hippocampal subregions. This study investigates acute apoptotic cell death in hippocampal regions CA1, CA3, and dentate gyrus (DG) following a binge EtOH exposure on postnatal day (PN) 6, PN8, or PN6 + 8 and the alteration in pro- and anti-apoptotic proteins following a single EtOH binge on PN6. METHODS: Apoptotic cell death was quantified 12 hours after EtOH binge exposure using the optical fractionator method. Western blot analysis determined expression of pro-apoptotic Bax and anti-apoptotic Bcl-2, 12, 24, and 48 hours after binge EtOH exposure on PN6. The Bcl-2:Bax ratio was used as a measure of vulnerability to apoptosis. RESULTS: Acute apoptosis increased significantly 12 hours following PN6 or 8 EtOH exposure in CA1, CA3, and DG, but the magnitude of apoptotic cell death was significantly greater in CA1 than in CA3 and DG, which did not differ. Significant cell death was not detected when a PN8 EtOH exposure was preceded by exposure on PN6. Binge EtOH exposure on PN6 resulted in a significant increase in expression of Bcl-2 and the Bcl-2:Bax ratio in the CA1/DG region at 24 hours after EtOH exposure on PN6. The Bcl-2:Bax ratio in the CA3 region was not altered. CONCLUSIONS: This study shows that repeated binge exposure does not have a cumulative effect on the magnitude of acute apoptotic cell death. This finding may be explained in part by changes in the Bcl-2:Bax ratio after a single binge EtOH exposure.