scholarly journals Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation

2020 ◽  
Vol 14 ◽  
Author(s):  
Alex R. D. Delbridge ◽  
Dann Huh ◽  
Margot Brickelmaier ◽  
Jeremy C. Burns ◽  
Chris Roberts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Brain Slice ◽  
Initial Period ◽  
Slice Culture ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures ◽  
The Impact ◽  
Over Time

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

scholarly journals Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 592
Author(s):  
Cara L. Croft ◽  
Wendy Noble
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Brain Slice ◽  
Synaptic Dysfunction ◽  
Slice Culture ◽  
Therapeutic Effects ◽  
Culture Model ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures

Alzheimer's disease, the most common cause of dementia, is a progressive neurodegenerative disorder characterised by amyloid-beta deposits in extracellular plaques, intracellular neurofibrillary tangles of aggregated tau, synaptic dysfunction and neuronal death. There are no cures for AD and current medications only alleviate some disease symptoms. Transgenic rodent models to study Alzheimer’s mimic features of human disease such as age-dependent accumulation of abnormal beta-amyloid and tau, synaptic dysfunction, cognitive deficits and neurodegeneration. These models have proven vital for improving our understanding of the molecular mechanisms underlying AD and for identifying promising therapeutic approaches. However, modelling neurodegenerative disease in animals commonly involves aging animals until they develop harmful phenotypes, often coupled with invasive procedures. In vivo studies are also resource, labour, time and cost intensive. We have developed a novel organotypic brain slice culture model to study Alzheimer’ disease which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. We obtain 36 brain slices from each mouse pup, considerably reducing the numbers of animals required to investigate multiple stages of disease. This tractable model also allows the opportunity to modulate multiple pathways in tissues from a single animal. We believe that this model will most benefit dementia researchers in the academic and drug discovery sectors. We validated the slice culture model against aged mice, showing that the molecular phenotype closely mimics that displayed in vivo, albeit in an accelerated timescale. We showed beneficial outcomes following treatment of slices with agents previously shown to have therapeutic effects in vivo, and we also identified new mechanisms of action of other compounds. Thus, organotypic brain slice cultures from transgenic mouse models expressing Alzheimer’s disease-related genes may provide a valid and sensitive replacement for in vivo studies that do not involve behavioural analysis.

scholarly journals Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 592 ◽  
Author(s):  
Cara L. Croft ◽  
Wendy Noble
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Brain Slice ◽  
Synaptic Dysfunction ◽  
Slice Culture ◽  
Therapeutic Effects ◽  
Culture Model ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures

Alzheimer's disease, the most common cause of dementia, is a progressive neurodegenerative disorder characterised by amyloid-beta deposits in extracellular plaques, intracellular neurofibrillary tangles of aggregated tau, synaptic dysfunction and neuronal death. Transgenic rodent models to study Alzheimer’s mimic features of human disease such as age-dependent accumulation of abnormal beta-amyloid and tau, synaptic dysfunction, cognitive deficits and neurodegeneration. These models have proven vital for improving our understanding of the molecular mechanisms underlying AD and for identifying promising therapeutic approaches. However, modelling neurodegenerative disease in animals commonly involves aging animals until they develop harmful phenotypes, often coupled with invasive procedures. We have developed a novel organotypic brain slice culture model to study Alzheimer’s disease using 3xTg-AD mice which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. Using a McIllwain tissue chopper, we obtain 36 x 350 micron slices from each P8-P9 mouse pup for culture between 2 weeks and 6 months on semi-permeable 0.4 micron pore membranes, considerably reducing the numbers of animals required to investigate multiple stages of disease. This tractable model also allows the opportunity to modulate multiple pathways in tissues from a single animal. We believe that this model will most benefit dementia researchers in the academic and drug discovery sectors. We validated the slice culture model against aged mice, showing that the molecular phenotype closely mimics that displayed in vivo, albeit in an accelerated timescale. We showed beneficial outcomes following treatment of slices with agents previously shown to have therapeutic effects in vivo, and we also identified new mechanisms of action of other compounds. Thus, organotypic brain slice cultures from transgenic mouse models expressing Alzheimer’s disease-related genes may provide a valid and sensitive replacement for in vivo studies that do not involve behavioural analysis.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

Neuroprotective Effects of the FGF21 Analogue LY2405319

2021 ◽  
pp. 1-13
Author(s):  
Claire Rühlmann ◽  
David Dannehl ◽  
Marcus Brodtrück ◽  
Andrew C. Adams ◽  
Jan Stenzel ◽  
...  
Keyword(s):  
Glial Cells ◽  
Neuroprotective Effect ◽  
Amyloid Β ◽  
Fibroblast Growth Factor 21 ◽  
Neuroprotective Effects ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures ◽  
Abca1 Mrna

Background: To date, there are no effective treatments for Alzheimer’s disease (AD). Thus, a significant need for research of therapies remains. Objective: One promising pharmacological target is the hormone fibroblast growth factor 21 (FGF21), which is thought to be neuroprotective. A clinical candidate for medical use could be the FGF21 analogue LY2405319 (LY), which has a specificity and potency comparable to FGF21. Methods: The present study investigated the potential neuroprotective effect of LY via PPARγ/apoE/abca1 pathway which is known to degrade amyloid-β (Aβ) plaques by using primary glial cells and hippocampal organotypic brain slice cultures (OBSCs) from 30- and 50-week-old transgenic APPswe/PS1dE9 (tg) mice. By LY treatment of 52-week-old tg mice with advanced Aβ deposition, we further aimed to elaborate the effect of LY on AD pathology in vivo. Results: LY application to primary glial cells caused an upregulation of pparγ, apoE, and abca1 mRNA expression and significantly decreased number and area of Aβ plaques in OBSCs. LY treatment in tg mice increased cerebral [18F] FDG uptake and N-acetylaspartate/creatine ratio indicating enhanced neuronal activity and integrity. Although LY did not reduce the number of Aβ plaques in tg mice, the number of iba1-positive cells was significantly decreased indicating reduced microgliosis. Conclusion: These data identified LY in vitro as an activator of Aβ degrading genes leading to cerebral Aβ load amelioration in early and late AD pathology. Although Aβ plaque reduction by LY failed in vivo, LY may be used as therapeutic agent to treat AD-related neuroinflammation and impaired neuronal integrity.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 739-739
Author(s):  
Vijay P. S. Rawat ◽  
Natalia Arseni ◽  
Farid Ahmed ◽  
Medhanie A. Mulaw ◽  
Silvia Thoene ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
The Impact

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Time Course of Microbiologic Outcome and Gene Expression in Candida albicans during and following In Vitro and In Vivo Exposure to Fluconazole

2006 ◽  
Vol 50 (4) ◽  
pp. 1311-1319 ◽  
Author(s):  
A. Lepak ◽  
J. Nett ◽  
L. Lincoln ◽  
K. Marchillo ◽  
D. Andes
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Time Course ◽  
Dependent Manner ◽  
Time Points ◽  
In Vivo Expression ◽  
The Impact ◽  
The Relationship

ABSTRACT Pharmacodynamics (PD) considers the relationship between drug exposure and effect. The two factors that have been used to distinguish the PD behaviors of antimicrobials are the impact of concentration on the extent of organism killing and the duration of persistent microbiologic suppression (postantibiotic effect). The goals of these studies were (i) to examine the relationship between antimicrobial PD and gene expression and (ii) to gain insight into the mechanism of fluconazole effects persisting following exposure. Microarrays were used to estimate the transcriptional response of Candida albicans to a supra-MIC F exposure over time in vitro. Fluconazole at four times the MIC was added to a log-phase C. albicans culture, and cells were collected to determine viable growth and for microarray analyses. We identified differential expression of 18% of all genes for at least one of the time points. More genes were upregulated (n = 1,053 [16%]) than downregulated (174 [3%]). Of genes with known function that were upregulated during exposure, most were related to plasma membrane/cell wall synthesis (18%), stress responses (7%), and metabolism (6%). The categories of downregulated genes during exposure included protein synthesis (15%), DNA synthesis/repair (7%), and transport (7%) genes. The majority of genes identified at the postexposure time points were from the protein (17%) and DNA (7%) synthesis categories. In subsequent studies, three genes (CDR1, CDR2, and ERG11) were examined in greater detail (more concentration and time points) following fluconazole exposure in vitro and in vivo. Expression levels from the in vitro and in vivo studies were congruent. CDR1 and CDR2 transcripts were reduced during in vitro fluconazole exposure and during supra-MIC exposure in vivo. However, in the postexposure period, the mRNA abundance of both pumps increased. ERG11 expression increased during exposure and fell in the postexposure period. The expression of the three genes responded in a dose-dependent manner. In sum, the microarray data obtained during and following fluconazole exposure identified genes both known and unknown to be affected by this drug class. The expanded in vitro and in vivo expression data set underscores the importance of considering the time course of exposure in pharmacogenomic investigations.

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