Murine Brain Tissue Research Articles

A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion-critical processes underlying neuronal health and function, and in turn, memory and cognition.

Mass Spectrometry Imaging of Lipids Using MALDI Coupled with Plasma-Based Post-Ionization on a Trapped Ion Mobility Mass Spectrometer.

Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.

Isolation of Exosomes or Extracellular Vesicles from West Nile Virus-Infected N2a Cells, Primary Cortical Neurons, and Brain Tissues.

Several flaviviruses compromise the blood-brain barrier integrity, infect the central nervous system, and elicit neuroinvasion to successfully cause neuropathogenesis in the vertebrate host. Therefore, understanding the pathway(s) and mechanism(s) to block the transmission and/or dissemination of flaviviruses and perhaps other neuroinvasive viruses is considered as an important area of research. Moreover, studies that address mechanism(s) of neuroinvasion by flaviviruses are limited. In this chapter, we discuss detailed methods to isolate exosomes or extracellular vesicles (EVs) from mouse and human N2a cells, primary cultures of murine cortical neurons, and mouse brain tissue. Two different methods including differential ultracentrifugation and density gradient exosome (DG-Exo) isolation are described for the preparation of exosomes/EVs from N2a cells and cortical neurons. In addition, we discuss the detailed DG-Exo method for the isolation of exosomes from murine brain tissue. Studies on neuronal exosomes will perhaps enhance our understanding of the mechanism of neuroinvasion by these deadly viruses.

Spatial Multiomics of Lipids, N-Glycans, and Tryptic Peptides on a Single FFPE Tissue Section.

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

Thymoquinone induces apoptosis in temozolomide-resistant glioblastoma cells via the p38 mitogen-activated protein kinase signaling pathway.

Temozolomide (TMZ) can cross the blood-brain barrier (BBB) and deliver methyl groups to the purine (guanine) bases of DNA, leading to mispairing during DNA replication and subsequent cell death. However, increased expression of the repair enzyme methyl guanine methyltransferase (MGMT), which removes methyl groups from purine bases, counteracts methylation by TMZ. We evaluated the anticancer potential of thymoquinone (TQ), a hydrophobic flavonoid that inhibits resistance and induces apoptosis in various cancer cells, both in vitro and in vivo. In vitro experiments showed that compared with the Hs683 and M059J cell lines, U251 cells were more sensitive to TMZ. Compared to U251 cells, U251R cells, a TMZ drug-resistant strain established in this study, are characterized by increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and MGMT. TQ treatments induced apoptosis in all cell lines. The p38 mitogen-activated protein kinase signal pathway was mainly activated in U251 and U251R cells; however, p-ERK and MGMT upregulation could not suppress TQ effects. Furthermore, si-p38 pretreatment of U251R cells in TQ treatments inhibited cell apoptosis. We speculate that TQ contributed to the phosphorylation and activation of p38, but not of ERK-induced apoptosis (irrespective of TMZ resistance). In vivo, U251R-derived tumors subcutaneously inoculated in nude mice exhibited significant tumor volume reduction after TQ or TQ + TMZ cotreatments. High-performance liquid chromatography assay confirmed the presence of TQ in murine brain tissues. Our findings demonstrate that TQ can effectively cross the BBB and function alone or in combination with TMZ to treat glioblastoma.

Loss of Homeostatic Microglia Signature in Prion Diseases.

Prion diseases are neurodegenerative diseases that affect humans and animals. They are always fatal and, to date, no treatment exists. The hallmark of prion disease pathophysiology is the misfolding of an endogenous protein, the cellular prion protein (PrPC), into its disease-associated isoform PrPSc. Besides the aggregation and deposition of misfolded PrPSc, prion diseases are characterized by spongiform lesions and the activation of astrocytes and microglia. Microglia are the innate immune cells of the brain. Activated microglia and astrocytes represent a common pathological feature in neurodegenerative disorders. The role of activated microglia has already been studied in prion disease mouse models; however, it is still not fully clear how they contribute to disease progression. Moreover, the role of microglia in human prion diseases has not been thoroughly investigated thus far, and specific molecular pathways are still undetermined. Here, we review the current knowledge on the different roles of microglia in prion pathophysiology. We discuss microglia markers that are also dysregulated in other neurodegenerative diseases including microglia homeostasis markers. Data on murine and human brain tissues show that microglia are highly dysregulated in prion diseases. We highlight here that the loss of homeostatic markers may especially stand out.

Characterising murine hippocampal iron homeostasis, in relation to markers of brain inflammation and metabolism, during ageing.

Metal ions (Fe, Cu, and Zn) are essential to a healthy brain function, with the amount, localisation, and chemical form often tightly controlled. Evidence points towards loss of metal ion homeostasis within the ageing brain; in particular brain Fe accumulation appears to be a hallmark of ageing, which may place the brain at a greater risk of neurodegenerative disease. Unfortunately, the cause or consequence of altered brain metal ion homeostasis during ageing remains unknown, and there is a lack of data comparing brain metal ion homeostasis with other events of the ageing process (e.g. brain metabolism, brain inflammation). This study has utilised a multi-modal approach that incorporated: X-ray fluorescence microscopy for elemental mapping of metal ion homeostasis, Perl's Fe histochemistry, FTIR spectroscopic biochemical imaging of lactate and protein aggregates, and immuno-fluorescence analysis of markers of brain inflammation and Fe storage proteins (heavy-chain ferritin, light-chain ferritin, and mitochondrial ferritin). Interestingly, while age-related Fe accumulation was observed in corpus callosum white matter of murine (C56BL/6J) brain tissue (concomitant with elevated levels of markers of brain inflammation and altered metabolism), Fe content was not altered within the hippocampus (a decrease in total Zn within the mossy fibres was observed). Ultimately, the results of this study demonstrate an important association between elevated brain Fe and brain inflammation during natural ageing. This study also highlights that future research is required to image different chemical forms of Fe with respect to changes in brain metabolism and inflammation, as well as localising these changes to specific cell types.

Time-domain feature extraction for target specificity in photoacoustic remote sensing microscopy.

Photoacoustic remote sensing (PARS) microscopy is an emerging label-free optical absorption imaging modality. PARS operates by capturing nanosecond-scale optical fluctuations produced by photoacoustic pressures. These time-domain (TD) variations are usually projected by amplitude to determine optical absorption magnitude. However, valuable details on a target's material properties (e.g., density, speed of sound) are contained within the TD signals. This work uses a novel, to the best of our knowledge, clustering method to learn TD features, based on signal shape, which relate to underlying material traits. A modified K-means method is used to cluster TD data, capturing representative signal features. These features are then used to form virtual colorizations which may highlight tissues based on their underlying material properties. Applied in fresh resected murine brain tissue, colorized visualizations highlight distinct regions of tissue. This may potentially facilitate differentiation of tissue constituents (e.g., myelinated and unmyelinated axons, cell nuclei) in a single acquisition.

Identification of oxytocin expression in human and murine microglia

BackgroundOxytocin is a neuropeptide synthesized in the hypothalamus. In addition to its role in parturition and lactation, oxytocin mediates social behavior and pair bonding. The possibility of using oxytocin to modify behavior in neurodevelopmental disorders, such as autism spectrum disorder, is of clinical interest. Microglia are tissue-resident macrophages with roles in neurogenesis, synapse pruning, and immunological mediation of brain homeostasis. Recently, oxytocin was found to attenuate microglial secretion of proinflammatory cytokines, but the source of this oxytocin was not established. This prompted us to investigate whether microglia themselves were the source. MethodsWe examined oxytocin expression in human and murine brain tissue in both sexes using immunohistochemistry. Oxytocin mRNA expression and secretion were examined in isolated murine microglia from wild type and oxytocin-knockout mice. Also, secretion of oxytocin and cytokines was measured in cultured microglia (MG6) stimulated with lipopolysaccharide (LPS). ResultsWe identified oxytocin expression in microglia of human brain tissue, cultured microglia (MG6), and primary murine microglia. Furthermore, LPS stimulation increased oxytocin mRNA expression in primary murine microglia and MG6 cells, and oxytocin secretion as well. A positive correlation between oxytocin and IL-1β, IL-10 secretion emerged, respectively. ConclusionThis may be the first demonstration of oxytocin expression in microglia. Functionally, oxytocin might regulate inflammatory cytokine release from microglia in a paracrine/autocrine manner.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood–brain barrier in acute ischemic stroke

Blood–brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Opioid-Use, COVID-19 Infection, and Their Neurological Implications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an imminent threat to human health and public safety. ACE2 and transmembrane serine protease 2 proteins on host cells provide the viral entry point to SARS-CoV-2. Although SARS-CoV-2 mainly infects the respiratory system, there have been reports of viral neurotropism and central nervous system injury as indicated by plasma biomarkers, including neurofilament light chain protein and glial fibrillary acidic protein. Even with a small proportion of infections leading to neurological manifestation, the overall number remains high. Common neurological manifestations of SARS-CoV-2 infection include anosmia, ageusia, encephalopathy, and stroke, which are not restricted to only the most severe infection cases. Opioids and opioid antagonists bind to the ACE2 receptor and thereby have been hypothesized to have therapeutic potential in treating COVID-19. However, in the case of other neurotropic viral infections such as human immunodeficiency virus (HIV), opioid use has been established to exacerbate HIV-mediated central nervous system pathogenesis. An analysis of electronic health record data from more than 73 million patients shows that people with Substance Use Disorders are at higher risk of contracting COVID-19 and suffer worse consequences then non-users. Our in-vivo and in-vitro unpublished studies show that morphine treatment causes increased expression of ACE2 in murine lung and brain tissue as early as 24 h post treatment. At the same time, we also observed morphine and lipopolysaccharides treatment lead to a synergistic increase in ACE2 expression in the microglial cell line, SIM-A9. This data suggests that opioid treatment may potentially increase neurotropism of SARS-CoV-2 infection. We have previously shown that opioids induce gut microbial dysbiosis. Similarly, gut microbiome alterations have been reported with SARS-CoV-2 infection and may play a role in predicting COVID-19 disease severity. However, there are no studies thus far linking opioid-mediated dysbiosis with the severity of neuron-specific COVID-19 infection.

The pyrethroids metabolite 3-phenoxybenzoic acid induces dopaminergic degeneration

Exposure to pyrethroids, a significant class of the most widely used agricultural chemicals, has been associated with an increased risk of Parkinson's disease (PD). However, although many different pyrethroids induce roughly the same symptoms of Parkinsonism, the underlying mechanisms remain unknown. To find the shared key features among these mechanisms, we focused on 3-phenoxybenzoic acid (3-PBA), a common and prominent metabolite of most pyrethroids produced via hydrolysis by CEs in mammals. To determine the contribution of 3-PBA to the initiation and progression of PD, we performed in vivo and in vitro experiments, respectively, and found that 3-PBA not only accumulates in murine brain tissues over time but also further induces PD-like pathologies (increased α-syn and phospho-S129, decreased TH) to the same or even greater extent than the precursor pyrethroid. A before-after study of PET-DAT in the same mice revealed that low concentrations of 3-PBA (0.5 mg/kg) could paradoxically cause DAT to increase (22.46% higher than pre-drug test). The intervention of DAT inhibitors and activators respectively alleviated and enhanced the dopaminergic toxicity of 3-PBA, indicating that 3-PBA interacts with DAT. In particular, low concentrations of 3-PBA increase the DAT, which in turn induces 3-PBA to enter the dopaminergic neurons to exert toxic effects. Finally, we described a mechanism underlying this potential role of 3-PBA in the pathological aggregation of α-syn. Specifically, 3-PBA was found to dysregulate C/EBP β levels and further anomalously activate AEP in vivo and in vitro, accompanied by increased accumulation of pathologically cleaved α-syn (N103 fragments) and accelerated α-syn aggregation. All these results suggest that 3-PBA exposure could mimic the pathological and pathogenetic features of PD, showing that this metabolite is a key pathogenic compound in pyrethroid-related pathological effects and a possible dopamine neurotoxin. Additionally, our findings provide a crucial reference for the primary prevention of PD.

Spectrum of Novel Anti–Central Nervous System Autoantibodies in the Cerebrospinal Fluid of 119 Patients With Schizophreniform and Affective Disorders

BackgroundAutoimmune psychosis may be caused by well-characterized anti-neuronal autoantibodies, such as those against the NMDA receptor. However, the presence of additional anti–central nervous system (CNS) autoantibodies in these patients has not been systematically assessed. MethodsSerum and cerebrospinal fluid (CSF) from patients with schizophreniform and affective syndromes were analyzed for immunoglobulin G anti-CNS autoantibodies using tissue-based assays with indirect immunofluorescence on unfixed murine brain tissue as part of an extended routine clinical practice. After an initial assessment of patients with red flags for autoimmune psychosis (n = 30), tissue-based testing was extended to a routine procedure (n = 89). ResultsBased on the findings from all 119 patients, anti-CNS immunoglobulin G autoantibodies against brain tissue were detected in 18% (n = 22) of patients (serum 9%, CSF 18%) following five principal patterns: 1) against vascular structures, most likely endothelial cells (serum 3%, CSF 8%); 2) against granule cells in the cerebellum and/or hippocampus (serum 4%, CSF 6%); 3) against myelinated fibers (serum 2%, CSF 2%); 4) against cerebellar Purkinje cells (serum 0%, CSF 2%); and 5) against astrocytes (serum 1%, CSF 1%). The patients with novel anti-CNS autoantibodies showed increased albumin quotients (p = .026) and white matter changes (p = .020) more frequently than those who tested negative for autoantibodies. ConclusionsThe study demonstrates five novel autoantibody-binding patterns on brain tissue of patients with schizophreniform and affective syndromes. CSF yielded positive findings more frequently than serum analysis. The frequency and spectrum of autoantibodies in these patient groups may be broader than previously thought.

Use of a stable and solvent-soluble variant of Amylo-Glo, Amylo-Glo+, for the high-contrast and resolution fluorescent localization of amyloid plaques in murine and human brain tissue sections

BackgroundAlthough numerous different fluorescent compounds have been used for the fluorescent localization of amyloid plaques, limitations include weak fluorescence, low contrast, low resolution, broad excitation / emission profile and chemical instability. New methodAmylo-Glo+ was reconstituted from a powder into a stable solution for the histological staining of plaques in brain tissue sections from both patients diagnosed with Alzheimer’s disease and APP/PS1 transgenic mice. ResultsAmylo-Glo+ was found to result in high resolution and contrast labeling of amyloid plaques. The tracer was excited exclusively by ultraviolet light illumination. The staining of amyloid plaques corresponded with the labeling pattern seen following staining with HQ-O. Counterstaining with ethidium bromide resulted in no bleed-through of the Amylo-Glo+ under blue or green light excitation. The tracer was stable in both powder and solution form. Comparison with existing methodsAmylo-Glo+ was found to be more stable, in both a powder and solution form, than the original Amylo-Glo, brighter than Congo red, of narrower emission and greater contrast than Thioflavine S and of narrower emission than X-34 and BSB. ConclusionAmylo-Glo+ is a stable and solvent soluble variant of Amylo-Glo that allows for the high contrast and resolution localization of amyloid plaques in tissue sections from the brains of transgenic mice and humans with Alzheimer’s disease. The tracer is also solely excited by ultraviolet light, making it ideal for fluorescent multiple labeling studies in combination with commonly used red or green fluorophores. It is also stable in both solution and powder form, unlike the original Amylo-Glo which is only available as a basic solution that is prone to precipitation.

Development and characterization of a disposable submillimeter fiber optic Raman needle probe for enhancing real-time in vivo deep tissue and biofluids Raman measurements.

We report on the development and characterization of disposable submillimeter fiber optic Raman needle probe for enhancing real-time in vivo tissue and biofluids Raman measurements. The submillimeter Raman probe is designed and fabricated using an aluminum-coated multimode fiber tapered with a semispherical lens, resulting in the coaxial laser excitation/Raman collection configuration for maximizing tissue and biofluid Raman measurements. We demonstrate that, with the use of the Raman needle probe associated with the structured background subtraction algorithms developed, high quality tissue Raman spectra covering both the fingerprint (FP) (800-1800cm-1) and high-wavenumber (HW) (2800-3300cm-1) regions can be acquired within subseconds from different tissue types (e.g., skin, muscle, fat, cartilage, liver, and brain) and biofluids (e.g., blood, urine). By advancing the Raman needle probe into the murine brain tissue model, high quality depth-resolved deep tissue Raman spectra can also be acquired rapidly. This work shows that the submillimeter fiberoptic Raman needle probe is capable of achieving real-time collection of deep tissue and biofluids FP/HW Raman spectra with high signal to noise ratios. This opens a new avenue with dual functioning of Raman optical biopsy and fine needle aspiration biopsy for enhancing in vivo deep tissue and biofluids diagnosis and characterization in different organs.

Role of adipocyte Na,K-ATPase oxidant amplification loop in cognitive decline and neurodegeneration

SummaryRecent studies suggest that a western diet may contribute to clinical neurodegeneration and dementia. Adipocyte-specific expression of the Na,K-ATPase signaling antagonist, NaKtide, ameliorates the pathophysiological consequences of murine experimental obesity and renal failure. In this study, we found that a western diet produced systemic oxidant stress along with evidence of activation of Na,K-ATPase signaling within both murine brain and peripheral tissues. We also noted this diet caused increases in circulating inflammatory cytokines as well as behavioral, and brain biochemical changes consistent with neurodegeneration. Adipocyte specific NaKtide affected by a doxycycline on/off expression system ameliorated all of these diet effects. These data suggest that a western diet produces cognitive decline and neurodegeneration through augmented Na,K-ATPase signaling and that antagonism of this pathway in adipocytes ameliorates the pathophysiology. If this observation is confirmed in humans, the adipocyte Na,K-ATPase may serve as a clinical target in the therapy of neurodegenerative disorders.

Down-Regulation of Inpp5e Associated With Abnormal Ciliogenesis During Embryonic Neurodevelopment Under Inositol Deficiency.

The inositol polyphosphate-5-phosphatase E (Inpp5e) gene is located on chromosome 9q34.3. The enzyme it encodes mainly hydrolyzes the 5-phosphate groups of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) and phosphatidylinositol (4,5)-bisphosphate (PtdIns (4,5)P2), which are closely related to ciliogenesis and embryonic neurodevelopment, through mechanisms that are largely unknown. Here we studied the role of Inpp5e gene in ciliogenesis during embryonic neurodevelopment using inositol-deficiency neural tube defects (NTDs) mouse and cell models. Confocal microscopy and scanning electron microscope were used to examine the number and the length of primary cilia. The dynamic changes of Inpp5e expression in embryonic murine brain tissues were observed during Embryonic Day 10.5–13.5 (E 10.5–13.5). Immunohistochemistry, western blot, polymerase chain reaction (PCR) arrays were applied to detect the expression of Inpp5e and cilia-related genes of the embryonic brain tissues in inositol deficiency NTDs mouse. Real-time quantitative PCR (RT-qPCR) was used to validate the candidate genes in cell models. The levels of inositol and PtdIns(3,4) P2 were measured using gas chromatography-mass spectrometry (GC-MS) and enzyme linked immunosorbent assay (ELISA), respectively. Our results showed that the expression levels of Inpp5e gradually decreased in the forebrain tissues of the control embryos, but no stable trend was observed in the inositol deficiency NTDs embryos. Inpp5e expression in inositol deficiency NTDs embryos was significantly decreased compared with the control tissues. The expression levels of Inpp5e gene and the PtdIns (3,4) P2 levels were also significantly decreased in the inositol deficient cell model. A reduced number and length of primary cilia were observed in NIH3T3 cells when inositol deficient. Three important cilia-related genes (Ift80, Mkks, Smo) were down-regulated significantly in the inositol-deficient NTDs mouse and cell models, and Smo was highly involved in NTDs. In summary, these findings suggested that down-regulation of Inpp5e might be associated with abnormal ciliogenesis during embryonic neurodevelopment, under conditions of inositol deficiency.

A 3D cell culture approach for studying neuroinflammation

BackgroundNeurodegenerative diseases are highly complex making them challenging to model in cell culture. All cell types of the brain have been implicated as exerting an effect on pathogenesis, and disease progression is likely influenced by the cross-talk between the different cell types. Sophisticated investigation of the cellular level consequences of cross-talk between different cells types requires three-dimensional (3D) co-culture systems. New methodMurine neural stem cells were differentiated into mixed-neuronal lineage populations in 3D culture. By seeding these differentiated cultures with microglia from adult brain, we have generated a 3D ex-vivo model of murine brain tissue populated with microglia. ResultsMonitoring the infiltration of GFP-expressing microglia into the 3D neuronal lineage cultures showed population throughout the tissue and assumption of ramified homeostatic morphology by the microglia. The co-cultures showed good longevity and were functionally responsive to external stimuli. Comparison with existing methodsWe have previously used 2-dimensional adhered cultures to model cell-cell interactions between microglia and neuronal lineage cells. While the microglia integrate well into these cultures and demonstrate inter-cellular cross-talk, it is known that adhered culture can change their activation state and therefore a 3D system better represents communication throughout a network of neuronal and support cells. ConclusionsOur system offers a straight-forward and time effective way to model 3D mouse brain tissue that is responsive to external neuroinflammatory stimulus. It not only allows inter-cellular interactions to be studied in live tissue but additionally permits study of changes within any available mouse genotype.

Neuroprotective effects of protocatechuic acid on sodium arsenate induced toxicity in mice: Role of oxidative stress, inflammation, and apoptosis

Arsenic is a toxic metalloid abundantly found in nature and used in many industries. Consumption of contaminated water mainly results in human exposure to arsenic. Toxicity (arsenicosis) resulting from arsenic exposure causes cerebral neurodegeneration. Protocatechuic acid (PCA), a phenol derived from edible plants, has antioxidant properties. The present study investigated the neuroprotective potential of PCA against arsenic-induced neurotoxicity in mice. Male Swiss albino mice were divided into four groups: (i) orally administered physiological saline, (ii) orally administered 100 mg/kg PCA, (iii) orally administered 5 mg/kg NaAsO2, and (iv) orally administered 100 mg/kg PCA 120 min prior to oral administration of 5 mg/kg NaAsO2. Each group received its respective treatment for 1 week, after which cortical tissues from each group were analyzed for various parameters of oxidative stress, proinflammatory cytokines, apoptosis-related proteins, and changes in histopathology. NaAsO2-treatment resulted in a significant increase in lipid peroxidation (LPO), inducible nitric oxide synthetase (iNOs), and NO levels, with a decrease in the levels of both enzymatic (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and non-enzymatic (glutathione) antioxidant markers. Arsenic increased proinflammatory cytokine (tumor necrosis factor-α and interleukin-1β) levels, enhanced caspase-3 and Bax expression, and reduced Bcl-2 expression. Furthermore, arsenic-exposure in mice decreased significantly acetylcholinesterase activity and brain-derived neurotrophic factor level in the cerebral cortex. Histopathological examination revealed changes in nerve cell cyto-architecture and distribution in arsenic-exposed brain tissue sections. PCA treatment before arsenic administration resulted in a positive shift in the oxidative stress and cytokine levels with decreased levels of LPO, iNOS, and NO. PCA pre-treatment considerably attenuated arsenic-associated histopathological changes in murine brain tissue. This study suggested that the presence of PCA may be responsible for the prevention of arsenic-induced neurotoxicity.

Correlation of Aβ‐pE(3) and ptau in human and mouse brain

AbstractBackgroundSenile plaques frequently contain pyroglutamate amyloid β (Aβ‐pE(3)), a N‐terminally truncated Aβ species that is more closely linked to Alzheimer’s Disease (AD) compared to other Aβ species. Tau protein is highly phosphorylated at several residues in AD, specifically tau protein phosphorylated (ptau) at Ser202/Thr205 is known to be increased in AD. First studies suggest that the two pathologies of plaques and tau phosphorylation might be linked to each other.MethodTo evaluate if Aβ‐pE(3) and ptau Ser202/Thr205 levels correlate in human and transgenic AD mouse models we analyzed human cortical and hippocampal brain tissue of different Braak stages as well as murine brain tissue of two transgenic mouse models for levels of Aβ‐pE(3) and ptau Ser202/Thr205 and correlated the data.ResultOur results show that Aβ‐pE(3) formation increases already at early Braak stages while ptau Ser202/Thr205 mostly increases at later stages. Further analyses revealed strong correlations between the two pathologies in the temporal, frontal, cingulate and occipital cortex. However, correlation in the hippocampus was weaker. Evaluation of murine transgenic brain tissue demonstrated a slow but steady increase of Aβ‐pE(3) from 6 to 12 months of age in the cortex and hippocampus of APPSL mice and a very early and strong Aβ‐pE(3) increase in 5xFAD mice. ptau Ser202/Thr205 levels increased at the age of 9 months in APPSL mice and already at 6 months in 5xFAD mice.ConclusionOur results show that Aβ‐pE(3) and ptau Ser202/Thr205 levels strongly correlate in human as well as murine brain tissues, suggesting that tau phosphorylation might be amplified by Aβ‐pE(3).