Abstract
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 CO2 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.
Highlights
The ex vivo culture system of organotypic brain slices was first introduced by Gähwiler establishing the roller-tube method [1].After embedding in a plasma clot, the slices are cultivated on glass coverslips in a slowly rotating tube
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
We present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue
Summary
The ex vivo culture system of organotypic brain slices was first introduced by Gähwiler establishing the roller-tube method [1].After embedding in a plasma clot, the slices are cultivated on glass coverslips in a slowly rotating tube. A second method for brain slice cultivation using semiporous membranes was established by Stoppini and coworkers [2] In this model the three-dimensional tissue structure is well preserved, enabling morphological, biochemical and electrophysiological studies. Both methods have been applied to prepare organotypic brain slice cultures from neonatal rodents and the technique has become a sophisticated tool in neuroscience. AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid b 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
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