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Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.

ABSTRACT: Among the top ten causes of death in the United States, Alzheimer's disease (AD) is the only one that cannot be cured, prevented, or even slowed down at present. Significant efforts have been exerted in generating model systems to delineate the mechanism as well as establishing platforms for drug screening. In this study, a promising candidate model utilizing primary mouse brain organotypic (MBO) cultures is reported. For the first time, we have demonstrated that the MBO cultures exhibit increased blood brain barrier (BBB) permeability as shown by IgG leakage into the brain parenchyma, astrocyte activation as evidenced by increased expression of glial fibrillary acidic protein (GFAP), and neuronal damage-response as suggested by increased vimentin-positive neurons occur upon histamine treatment. Identical responses-a breakdown of the BBB, astrocyte activation, and neuronal expression of vimentin-were then demonstrated in brains from AD patients compared to age-matched controls, consistent with other reports. Thus, the histamine-treated MBO culture system may provide a valuable tool in combating AD.

SUBMITTER: Sedeyn JC

PROVIDER: S-EPMC4677161 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.

Sedeyn Jonathan C JC Wu Hao H Hobbs Reilly D RD Levin Eli C EC Nagele Robert G RG Venkataraman Venkat V

BioMed research international 20151130

Among the top ten causes of death in the United States, Alzheimer's disease (AD) is the only one that cannot be cured, prevented, or even slowed down at present. Significant efforts have been exerted in generating model systems to delineate the mechanism as well as establishing platforms for drug screening. In this study, a promising candidate model utilizing primary mouse brain organotypic (MBO) cultures is reported. For the first time, we have demonstrated that the MBO cultures exhibit increas ...[more]

PMID: 26697497

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Project description: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.

Project description:BackgroundMethylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage.MethodsTo explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3.ResultsAmong the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA.ConclusionSurprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

Project description:BackgroundHerpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro.MethodsCell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections.ResultsSlicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE.ConclusionsWe developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Project description:BackgroundOrganotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer's disease (AD). AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs), surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S.Methodology/principal findingsIn this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1) and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH)- and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO(2) concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue.Conclusion/significanceUntil now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study provides evidence that adult organotypic brain slice cultures from 7- to 10-month-old mice independently of the transgenic modification undergo slow programmed cell death, caused by a dysfunction of the neuronal repair systems.

Dataset Information

Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.

Publications

Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.

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