Brain Samples Research Articles

Human Nasal Inferior Turbinate-Derived Neural Stem Cells Improve the Niche of Substantia Nigra Par Compacta in a Parkinson’s Disease Model by Modulating Hippo Signaling

Background:Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer’s disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells.Methods:Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs.Results:In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms.Conclusion:hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions.

Identification of novel endoplasmic reticulum-related genes and their association with immune cell infiltration in major depressive disorder

BackgroundSeveral lines of evidence point to an interaction between genetic predisposition and environmental factors in the onset of major depressive disorder (MDD). This study is aimed to investigate the pathogenesis of MDD by identifying key biomarkers, associated immune infiltration using bioinformatic analysis and human postmortem sample. MethodsThe Gene Expression Omnibus (GEO) database of GSE98793 was adopted to identify hub genes linked to endoplasmic reticulum (ER) stress-related genes (ERGs) in MDD. Another GEO database of GSE76826 was employed to validate the novel target associated with ERGs and immune infiltration in MDD. Moreover, human postmortem sample from MDD patients was utilized to confirm the differential expression analysis of hub genes. ResultsWe discovered 12 ER stress-related differentially expressed genes (ERDEGs). A LASSO Cox regression analysis helped construct a diagnostic model for these ERDEGs, incorporating immune infiltration analysis revealed that three hub genes (ERLIN1, SEC61B, and USP13) show the significant and consistent expression differences between the two groups. Western blot analysis of postmortem brain samples indicated notably higher expression levels of ERLIN1 and SEC61B in the MDD group, with USP13 also tending to increase compared to control group. LimitationsThe utilization of the MDD gene chip in this analysis was sourced from the GEO database, which possesses a restricted number of pertinent gene chip samples. ConclusionsThese findings indicate that ERDEGs especially including ERLIN1, SEC61B, and USP13 associated the infiltration of immune cells may be potential diagnostic indicators for MDD.

Metabolic Profiling in Plasma and Brain Induced by 17β-Estradiol Supplementation in Ovariectomized Mice.

17β-Estradiol is an ovarian hormone that regulates energy circulation and storage by acting on the central nervous system. However, the metabolic differences between the blood and brain when stimulated by 17β-estradiol are poorly understood. Moreover, research using menopause-induced models to investigate primary metabolites in the blood and brain is limited. Thus, this study aimed to identify metabolic changes in the plasma and brain resulting from 17β-estradiol supplementation in an estrogen-deficient mouse model. Three groups of mice were utilized: sham-operated mice (Sham), ovariectomized mice (OVX), and ovariectomized mice that received a weekly supplementation of 17β-estradiol (E2). Plasma and brain samples from these mice were subjected to metabolic analysis using gas chromatography-time-of-flight-mass spectrometry. Compared with the plasma samples from the Sham and OVX groups, the plasma samples from the E2 group contained higher contents of branched-chain amino acids (BCAAs), such as valine, isoleucine, and leucine. Meanwhile, the brain samples from the E2 group contained higher contents of most metabolites, including BCAAs, neurotransmitters, tricarboxylic acid cycle intermediates, and fatty acids, than those from the two other groups. This study is the first to reveal differences in energy metabolism induced by 17β-estradiol supplementation through brain metabolic profiling of ovariectomized mice, emphasizing the importance of brain metabolic profiling in menopausal hormone research.

COMPARATIVE ANALYSIS OF THE EFFECTIVENESS OF USING HISTOLOGICAL AND PHYSICAL-OPTICAL METHODS OF INVESTIGATING OF THE TIME OF HEMORRHAGE FORMATION IN THE SUBSTANCE OF THE HUMAN BRAIN

Important attention in forensic medical practice is given to the solution of issues that arise during the examination in thecase of traumatic brain injury (TBI), as this type of injury leads to high mortality and disability. Conducting an autopsy of thebody of the deceased, whose death occurred from a TBI, there are cases when the injury occurs as a result of a fall of a person who previously developed a cerebral infarction, and sometimes vice versa. In this case, it is important to accurately verify the primary cause of death, therefore, timely and objective determination of the time of formation of a hemorrhage in the human brain is necessary.The purpose and tasks of the research. To evaluate the possibility of detecting time of formation of a hemorrhage of traumatic and non-traumatic genesis by the generally accepted histological method and polarization phase microscopy, and to compare their eff ectiveness.Research materials and methods. For the study, human brain samples were selected from 140 deceased individuals whosedeaths occurred between 1 and 3 days after the onset of bleeding, according to medical records. As a control group, human brain samples were selected from 40 individuals who died of coronary heart disease. Light microscopy was performed by preliminary staining of histologic specimens according to the Perls method. Phase polarization tomography of the samples was performed using a Stokes polarimeter.Research results. Stained human brain preparations of the experimental and control groups were examined and analyzedand it was found that hemosiderin was not present in all experimental samples (only in 31 out of 40 samples were present). It was not possible to establish the corresponding time dependences of the age of the formation of hemorrhages, regardless of the genesis, since the random appearance of the pigment in the experimental samples is noted at diff erent time intervals. Analytical processing of the results of the statistical processing of the topographic structure of tomograms of the anisotropy of linear dichroism of the fi brillar networks of samples of the deceased from the experimental and control groups revealed a wider time range of sensitivity of this method to destructive changes in nervous tissue, in comparison with the polarimetric methods used in previous studies. As a result, there is an accelerated temporal decrease in the absolute values and the range of dispersion of the anisotropy of linear dichroism value with increasing time since the formation of hemorrhage.Conclusions. Polarization- phase microscopy has shown a signifi cant advantage in its use in comparison with the goldstandard – the Perls’ staining method for determining the time of hemorrhage formation in the human brain.

Evaluation of Neuro-Hormonal Dynamics after the Administration of Probiotic Microbial Strains in a Murine Model of Hyperthyroidism.

The microbiota-gut-brain axis has received increasing attention in recent years through its bidirectional communication system, governed by the ability of gut microorganisms to generate and regulate a wide range of neurotransmitters in the host body. In this research, we delve into the intricate area of microbial endocrinology by exploring the dynamic oscillations in neurotransmitter levels within plasma and brain samples. Our experimental model involved inducing hyperthyroidism in mice after a "probiotic load" timeframe using two strains of probiotics (Lactobacillus acidophilus, Saccharomyces boulardii, and their combination). These probiotic interventions continued throughout the experiment and were intended to uncover potential modulatory effects on neurotransmitter levels and discern if certain probiotic strains exhibit any protection from hyperthyroidism. Moreover, we aimed to outline the eventual connections between the gut microbiota and the hypothalamus-pituitary-thyroid axis. As our study reveals, there are significant fluctuations in crucial neurotransmitters within the hyperthyroidism model, related to the specific probiotic strain or combination. These findings could support future therapeutic approaches, help healthcare professionals choose between different probiotic therapies, and also allow us proceed with caution when administering such treatments, depending on the health status of hyperthyroid patients.

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.

Disease and brain region specific immune response profiles in neurodegenerative diseases with pure and mixed protein pathologies

The disease-specific accumulation of pathological proteins has long been the major focus of research in neurodegenerative diseases (ND), including Alzheimer’s disease (AD) and related dementias (RD), but the recent identification of a multitude of genetic risk factors for ND in immune-associated genes highlights the importance of immune processes in disease pathogenesis and progression. Studies in animal models have characterized the local immune response to disease-specific proteins in AD and ADRD, but due to the complexity of disease processes and the co-existence of multiple protein pathologies in human donor brains, the precise role of immune processes in ND is far from understood. To better characterize the interplay between different extracellular and intracellular protein pathologies and the brain’s intrinsic immune system in ND, we set out to comprehensively profile the local immune response in postmortem brain samples of individuals with “pure” beta-Amyloid and tau pathology (AD), “pure” α-Synuclein pathology in Lewy body diseases (LBD), as well as cases with Alzheimer’s disease neuropathological changes (ADNC) and Lewy body pathology (MIX). Combining immunohistochemical profiling of microglia and digital image analysis, along with deep immunophenotyping using gene expression profiling on the NanoString nCounter® platform and digital spatial profiling on the NanoString GeoMx® platform we identified a robust immune activation signature in AD brain samples. This signature is maintained in persons with mixed pathologies, irrespective of co-existence of AD pathology and Lewy body (LB) pathology, while LBD brain samples with “pure” LB pathology exhibit an attenuated and distinct immune signature. Our studies highlight disease- and brain region-specific immune response profiles to intracellular and extracellular protein pathologies and further underscore the complexity of neuroimmune interactions in ND.

Abstract LB432: Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal brain tumor types with a five-year survival rate of 6.9%. In 2023, more than 14,490 Americans were expected to receive a diagnosis. Although great progress has been achieved in protein target identification and treatment over the last few years, current treatment options are limited, and tumor recurrence occurs at a high rate due to treatment resistance. Aptamers, short single-stranded oligonucleotide ligands with high-affinity binding to molecular targets, are well suited tools for exploring novel targets in highly heterogeneous tumors with complex etiologies like GBM, where clinically relevant biomarkers are limited. In this study, aptamer-based cancer-specific drug target identification was performed by Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) Biotargeting System. In short, a GBM-specific library was enriched through a Systematic Evolution of Ligands by EXponential enrichment (SELEX) process using micro dissected Formalin-Fixed Paraffin-Embedded (FFPE) tissues from GBM patients, along with whole body normal tissue for counterselection. Microdissection of tissue allows the partitioning of different types of cells to focus on specific cells of interest. The enriched libraries have been evaluated on clinical GBM and normal brain control samples through aptamer mediated tissue staining, resulting in preferential binding of enriched libraries compared to controls in the vast majority of GBM patients with little binding to normal brain samples. The final library was subsequently applied in an in-house developed library-based affinity pull-down assay using lysates from FFPE tissue of the same group of GBM patients and normal brain samples, followed by unbiased Mass Spectrometry for protein target identification. Combining the results from this proteomics analysis and data from the internal Whole Transcriptome Sequencing (WTS) database, we were able to identify several targets that are highly expressed in GBM and other cancer lineages. Antibody staining provided further validation of the expression levels and the cellular localization. The targets identified in this study include not only potential novel therapeutic targets, but also well-studied biomarkers and targets with available drugs already in clinical trials, demonstrating the platform has great potential for identifying high affinity binding and selectivity compared to traditional proteomics and other methods in drug target discovery. Citation Format: Xixi Wei, Myunggi An, Radhika Santhanam, Barbara Richardson, Lucas Vu, Justin Saul, Anna Walton, Michael Gee, Daniel Martin, Amanda Hubbard, Alexis Rodin, Matthew Rosenow, Tassilo Hornung. Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB432.

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 (5mC), 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 (5hmC) 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 5mC and 5hmC 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 5mC and 5hmC at the genome-wide level. Differential 5mC and 5hmC 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 5mC and 5hmC of AUD in bulk cortical tissue. Results: We identified 417 5mC and 3635hmC 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 5mC, GAD1, DLX1, DLX2, for 5hmC and GATA4 in both. Convergence with a previous AUD 5mC and 5hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5mC and 5hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5mC 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.

Neuroprotective Assessment of Betaine against Copper Oxide Nanoparticle-Induced Neurotoxicity in the Brains of Albino Rats: A Histopathological, Neurochemical, and Molecular Investigation.

Copper oxide nanoparticles (CuO-NPs) are commonly used metal oxides. Betaine possesses antioxidant and neuroprotective activities. The current study aimed to investigate the neurotoxic effect of CuO-NPs on rats and the capability of betaine to mitigate neurotoxicity. Forty rats; 4 groups: group I a control, group II intraperitoneally CuO-NPs (0.5 mg/kg/day), group III orally betaine (250 mg/kg/day) and CuO-NPs, group IV orally betaine for 28 days. Rats were subjected to neurobehavioral assessments. Brain samples were processed for biochemical, molecular, histopathological, and immunohistochemical analyses. Behavioral performance of betaine demonstrated increasing locomotion and cognitive abilities. Group II exhibited significantly elevated malondialdehyde (MDA), overexpression of interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α). Significant decrease in glutathione (GSH), and downregulation of acetylcholine esterase (AChE), nuclear factor erythroid 2-like protein 2 (Nrf-2), and superoxide dismutase (SOD). Histopathological alterations; neuronal degeneration, pericellular spaces, and neuropillar vacuolation. Immunohistochemically, an intense immunoreactivity is observed against IL-1β and glial fibrillary acidic protein (GFAP). Betaine partially neuroprotected against CuO-NPs associated alterations. A significant decrease at MDA, downregulation of IL-1β, and TNF-α, a significant increase at GSH, and upregulation of AChE, Nrf-2, and SOD. Histopathological alterations partially ameliorated. Immunohistochemical intensity of IL-1β and GFAP reduced. It is concluded that betaine neuroprotected against most of CuO-NP neurotoxic effects through antioxidant and cell redox system stimulating efficacy.

Association between hypothalamic Alzheimer's disease pathology and body mass index: The Hisayama study.

The hypothalamus is the region of the brain that integrates the neuroendocrine system and whole-body metabolism. Patients with Alzheimer's disease (AD) have been reported to exhibit pathological changes in the hypothalamus, such as neurofibrillary tangles (NFTs) and amyloid plaques (APs). However, few studies have investigated whether hypothalamic AD pathology is associated with clinical factors. We investigated the association between AD-related pathological changes in the hypothalamus and clinical pictures using autopsied brain samples obtained from deceased residents of a Japanese community. A total of 85 autopsied brain samples were semi-quantitatively analyzed for AD pathology, including NFTs and APs. Our histopathological studies showed that several hypothalamic nuclei, such as the tuberomammillary nucleus (TBM) and lateral hypothalamic area (LHA), are vulnerable to AD pathologies. NFTs are observed in various neuropathological states, including normal cognitive cases, whereas APs are predominantly observed in AD. Regarding the association between hypothalamic AD pathologies and clinical factors, the degree of APs in the TBM and LHA was associated with a lower body mass index while alive, after adjusting for sex and age at death. However, we found no significant association between hypothalamic AD pathology and the prevalence of hypertension, diabetes, or dyslipidemia. Our study showed that a lower BMI, which is a poor prognostic factor of AD, might be associated with hypothalamic AP pathology and highlighted new insights regarding the disruption of the brain-whole body axis in AD.

Histopathological and ultrastructural changes in the rats brain after air shock wave impact

The using of explosive substances and devices can lead to brain injuries, the diagnosis of which requires the determination of primary biomarkers. Therefore, the aim was to establish and study histopathological and ultrastructural changes in the rats brain after exposure to an air blast wave. The study was carried out on 18 male Wistar rats, which were randomly divided into two groups: sham (n=9) and experimental (n=9). The animals of the experimental group were anesthetized with halothane and gently fixed in a horizontal position on the abdomen with the front part of the rat's muzzle at a distance of 5 cm from the device opening and subjected to an excess pressure of 26-36 kPa. Animals of both groups were decapitated, brains were removed, fixed, histopathological and ultrastructural analyzes were conducted using standard methods. Intergroup differences were assessed by Mann-Whitney U-test. Light microscopy revealed primary lesions in the form of small focal, multifocal hemorrhages, cerebral vessel ruptures and microscopic ruptures of the brain substance. In almost all brain samples, there is a significant saturation of the venous vessels with the presence of erythrocyte stasis. Violation of the blood-brain barrier, the presence of edema of the perivascular space, and petechial hemorrhages in the neuropil were registered ultrastructurally. Based on the study results, a morphological algorithm for assessing primary histostructural intracranial brain injuries and their consequences after exposure to an air shock wave was proposed. The injuries were found to be caused by the direct traumatic effect of the air shock wave. In the acute post-traumatic period, histopathological and ultrastructural changes in the brain can manifest as changes in neurons and in the blood-brain barrier and be accompanied by perivascular multifocal small-focal hemorrhages, neuropil ruptures, edema of pericellular and perivascular spaces, which together can be considered as biomarkers of primary traumatic changes after exposure to an air shock wave.

Accelerated Method for Determining Ischemic Brain Damage in Rats and Assessment the Neuroprotective Effect of a Complex Herbal Remedy.

The severity of ischemic injury was evaluated by densitometry of brain samples stained with 2,3,5-triphenyltetrazolium chloride (TTC) on a rat model of cerebral ischemia/reperfusion (common carotid artery occlusion) and the neuroprotective activity of an extract of Astragalus membranaceus, Scutellaria baicalensis, and Phlojodicarpus sibiricus was assessed. Occlusion of the common carotid arteries led to a weakening of TTC staining of the brain tissue: densitometric indicators of the staining intensity for the cortex and striatum were lower than the corresponding indicators of sham-operated rats by 18.3 and 10.4%. The mean intensity of staining of brain samples did not differ in rats treated with the extract and sham-operated animals, which attested to its neuroprotective effect. The applied method is convenient for evaluation of the severity of ischemic brain damage at the early stages and screening potential neuroprotective agents.

Multicausal disruption of complement system activity in schizophrenia: abnormal transcription of C4, complement control proteins and microglia specific genes in brain and blood

IntroductionThe synaptic pruning process is based on the joint action of the complement system and microglia. In schizophrenia, accumulating evidence support that abnormal synaptic pruning during adolescence may be due to an altered Complement system activity. While this hypothesis is supported by C4 overexpression in various brain regions of individuals with schizophrenia, such alterations should be replicated and extended to other brain regions. Moreover, transcriptional studies of genes encoding regulators of the complement system activity (complement control proteins, CCP) and microglia-specific genes are lacking. Furthermore, it remains unknown whether brain and peripheral expression of such genes are related.ObjectivesTo explore expression of C4 as well as 4 CCP encoding genes and 10 microglia-specific genes at the brain and peripheral levels in individuals with schizophrenia as compared to healthy controls.MethodsWe analyzed candidate gene expression from 9 Gene Expression Omnibus datasets obtained from 333 individuals with schizophrenia and 306 healthy controls (HC). We first compared expression of the candidate genes between individuals with schizophrenia and HC in postmortem brain samples from 7 different brain regions. Then, the same comparison was made in 4 different peripheral tissues.ResultsRegarding the complement system, we observed C4 overexpression in the DLPFC, parietal, temporal cortex and associative striatum of individuals with schizophrenia. We report distinct altered expression patterns of CCP genes in the DLPFC, hippocampus and cerebellum of individuals with schizophrenia. Only CD46 expression was altered in the blood of individuals with schizophrenia. Regarding microglia, we report an underexpression of several microglia-specific genes in the cerebellum, associative striatum, hippocampus and parietal cortex of individuals with schizophrenia vs. HC. At the peripheral level, we observed a mixed altered expression pattern in the whole blood of individuals with schizophrenia.ConclusionsFirstly, our results suggest that the CCP-mediated regulatory mechanisms of the Complement system are impaired in the brain of individuals with schizophrenia, potentially contributing to an excessive Complement system activity (CSA). Secondly, our results support the hypothesis of a widespread underexpression of microglia-specific genes in brain tissues of individuals with schizophrenia. Functionally, the observed transcriptional alterations may be related to the synaptic pruning impairment. Alternatively, they may translate a compensatory mechanism for neuroinflammation. In the whole blood, the altered transcriptional pattern may represent a potential peripheral signature of SZ.Disclosure of InterestNone Declared

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

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. 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. 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. 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.

Insights into the Mechanical Characterization of Mouse Brain Tissue Using Microindentation Testing

AbstractIndentation testing is the most common approach to quantify mechanical brain tissue properties. Despite a myriad of studies conducted already, reported stiffness values vary extensively and continue to be subject of study. Moreover, the growing interest in the relationship between the brain's spatially heterogeneous microstructure and local tissue stiffness warrants the development of standardized measurement protocols to enable comparability between studies and assess repeatability of reported data. Here, we present three individual protocols that outline (1) sample preparation of a 1000‐µm thick coronal slice, (2) a comprehensive list of experimental parameters associated with the FemtoTools FT‐MTA03 Micromechanical Testing System for spherical indentation, and (3) two different approaches to derive the elastic modulus from raw force‐displacement data. Lastly, we demonstrate that our protocols deliver a robust experimental framework that enables us to determine the spatially heterogeneous microstructural properties of (mouse) brain tissue. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Mouse brain sample preparationBasic Protocol 2: Indentation testing of mouse brain tissue using the FemtoTools FT‐MTA03 Micromechanical Testing and Assembly SystemBasic Protocol 3: Tissue stiffness identification from force‐displacement data

Long-Term Oral Tamoxifen Administration Decreases Brain-Derived Neurotrophic Factor in the Hippocampus of Female Long-Evans Rats.

Tamoxifen, a selective estrogen receptor modulator (SERM), is commonly used as an adjuvant drug therapy for estrogen-receptor-positive breast cancers. Though effective at reducing the rate of cancer recurrence, patients often report unwanted cognitive and affective side effects. Despite this, the impacts of chronic tamoxifen exposure on the brain are poorly understood, and rodent models of tamoxifen exposure do not replicate the chronic oral administration seen in patients. We, therefore, used long-term ad lib consumption of medicated food pellets to model chronic tamoxifen exposure in a clinically relevant way. Adult female Long-Evans Hooded rats consumed tamoxifen-medicated food pellets for approximately 12 weeks, while control animals received standard chow. At the conclusion of the experiment, blood and brain samples were collected for analyses. Blood tamoxifen levels were measured using a novel ultra-performance liquid chromatography-tandem mass spectrometry assay, which found that this administration paradigm produced serum levels of tamoxifen similar to those in human patients. In the brain, brain-derived neurotrophic factor (BDNF) was visualized in the hippocampus using immunohistochemistry. Chronic oral tamoxifen treatment resulted in a decrease in BDNF expression across several regions of the hippocampus. These findings provide a novel method of modeling and measuring chronic oral tamoxifen exposure and suggest a putative mechanism by which tamoxifen may cause cognitive and behavioral changes reported by patients.

Comprehensive analysis of brain injury parameters in a preclinical porcine model of acute liver failure.

Acute liver failure (ALF) is defined as acute loss of liver function leading to hepatic encephalopathy associated with a high risk of patient death. Brain injury markers in serum and tissue can help detect and monitor ALF-associated brain injury. This study compares different brain injury parameters in plasma and tissue along with the progression of ALF. ALF was induced by performing an 85% liver resection. Following the resection, animals were recovered and monitored for up to 48 h or until reaching the predefined endpoint of receiving standard medical therapy (SMT). Blood and serum samples were taken at Tbaseline, T24, and upon reaching the endpoint (Tend). Control animals were euthanized by exsanguination following plasma sampling. Postmortem brain tissue samples were collected from the frontal cortex (FCTx) and cerebellum (Cb) of all animals. Glial fibrillary acidic protein (GFAP) and tau protein and mRNA levels were quantified using ELISA and qRT-PCR in all plasma and brain samples. Plasma neurofilament light (NFL) was also measured using ELISA. All ALF animals (n = 4) were euthanized upon showing signs of brain herniation. Evaluation of brain injury biomarkers revealed that GFAP was elevated in ALF animals at T24h and Tend, while Tau and NFL concentrations were unchanged. Moreover, plasma glial fibrillary acidic protein (GFAP) levels were negatively correlated with total protein and positively correlated with both aspartate transaminase (AST) and alkaline phosphatase (AP). Additionally, lower GFAP and tau RNA expressions were observed in the FCTx of the ALF group but not in the CB tissue. The current large animal study has identified a strong correlation between GFAP concentration in the blood and markers of ALF. Additionally, the protein and gene expression analyses in the FCTx revealed that this area appears to be susceptible, while the CB is protected from the detrimental impacts of ALF-associated brain swelling. These results warrant further studies to investigate the mechanisms behind this process.

Molecular basis underlying default mode network functional abnormalities in postpartum depression with and without anxiety.

Although Postpartum depression (PPD) and PPD with anxiety (PPD-A) have been well characterized as functional disruptions within or between multiple brain systems, however, how to quantitatively delineate brain functional system irregularity and the molecular basis of functional abnormalities in PPD and PPD-A remains unclear. Here, brain sample entropy (SampEn), resting-state functional connectivity (RSFC), transcriptomic and neurotransmitter density data were used to investigate brain functional system irregularity, functional connectivity abnormalities and associated molecular basis for PPD and PPD-A. PPD-A exhibited higher SampEn in medial prefrontal cortex (MPFC) and posterior cingulate cortex (PPC) than healthy postnatal women (HPW) and PPD while PPD showed lower SampEn in PPC compared to HPW and PPD-A. The functional connectivity analysis with MPFC and PPC as seed areas revealed decreased functional couplings between PCC and paracentral lobule and between MPFC and angular gyrus in PPD compared to both PPD-A and HPW. Moreover, abnormal SampEn and functional connectivity were associated with estrogenic level and clinical symptoms load. Importantly, spatial association analyses between functional changes and transcriptome and neurotransmitter density maps revealed that these functional changes were primarily associated with synaptic signaling, neuron projection, neurotransmitter level regulation, amino acid metabolism, cyclic adenosine monophosphate (cAMP) signaling pathways, and neurotransmitters of 5-hydroxytryptamine (5-HT), norepinephrine, glutamate, dopamine and so on. These results reveal abnormal brain entropy and functional connectivities primarily in default mode network (DMN) and link these changes to transcriptome and neurotransmitters to establish the molecular basis for PPD and PPD-A for the first time. Our findings highlight the important role of DMN in neuropathology of PPD and PPD-A.

Evaluation of neurogranin levels in a rat model of diffuse axonal injury.

Diffuse axonal injury (DAI), one of the most common and devastating type of traumatic brain injury, is the result of the shear force on axons due to severe rotational acceleration and deceleration. Neurogranin (NRGN) is a postsynaptic protein secreted by excitatory neurons, and synaptic dysfunction can alter extracellular NRGN levels. In this study, we examined NRGN levels in serum and cerebrospinal fluid (CSF) after experimental DAI in terms of their diagnostic value. Experimental DAI was induced using the Marmarou technique in male Wistar albino rats. Serum and CSF NRGN levels of the sham group, one‑hour, six‑hour, 24‑hour, and 72‑hour post‑DAI groups were measured by ELISA method. DAI was verified by staining with hematoxylin‑eosin and β‑amyloid precursor protein in the rat brain samples. While no histopathological and immunohistochemical changes were observed in the early hours of the post‑DAI groups, the staining of the β‑APP visibly increased over time, with positivity being most frequent and intense in the 72‑hour group. It was found that serum NRGN levels were significantly lower in the 6‑hour group than in the sham group. The serum NRGN levels in the 24‑hour group were significantly higher than those in the sham group. This study showed a dichotomy of post‑DAI serum NRGN levels in consecutive time periods. NRGN levels in CSF were higher in the one‑hour group than in the sham group and returned to baseline by 72 hours, although not significantly. Our study provides an impression of serum and CSF NRGN levels in a rat DAI model in consecutive time periods. Further studies are needed to understand the diagnostic value of NRGN.