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A versatile prion replication assay in organotypic brain slices.

ABSTRACT: Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrP(Sc), >10(5)-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrP(Sc) accumulated predominantly in the molecular layer, as in infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines, with variable detection efficiency. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrP(Sc) concentrations over those in microglia-containing slices, as well as an increase in susceptibility to infection. This suggests that the extensive microglial activation accompanying prion diseases represents an efficacious defensive reaction.

SUBMITTER: Falsig J

PROVIDER: S-EPMC2754795 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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A versatile prion replication assay in organotypic brain slices.

Falsig Jeppe J Julius Christian C Margalith Ilan I Schwarz Petra P Heppner Frank L FL Aguzzi Adriano A

Nature neuroscience 20071209 1

Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrP(Sc), >10(5)-fold. T ...[more]

PMID: 18066056

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Project description:BackgroundOrganotypic brain slices (OTBS) are an excellent experimental compromise between the facility of working with cell cultures and the biological relevance of using animal models where anatomical, morphological, and cellular function of specific brain regions can be maintained. The biological characteristics of OTBS can subsequently be examined under well-defined conditions. They do, however, have a number of limitations; most brain slices are derived from neonatal animals, as it is difficult to properly prepare and maintain adult OTBS. There are ample problems with tissue integrity as OTBS are delicate and frequently become damaged during the preparative stages. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells, and cells imbedded within tissues, have been consistently difficult to achieve.ResultsFollowing the ex vivo extraction of adult mouse brains, mounted inside a medium-agarose matrix, we have exploited a precise slicing procedure using a custom built vibroslicer. To transfect these slices we used an improved biolistic transfection method using a custom made low-pressure barrel and novel DNA-coated nanoparticles (40 nm), which are drastically smaller than traditional microparticles. These nanoparticles also minimize tissue damage as seen by a significant reduction in lactate dehydrogenase activity as well as propidium iodide (PI) and dUTP labelling compared to larger traditional gold particles used on these OTBS. Furthermore, following EYFP exogene delivery by gene gun, the 40 nm treated OTBS displayed a significantly larger number of viable NeuN and EYFP positive cells. These OTBS expressed the exogenous proteins for many weeks.ConclusionsOur described methodology of producing OTBS, which results in better reproducibility with less tissue damage, permits the exploitation of mature fully formed adult brains for advanced neurobiological studies. The novel 40 nm particles are ideal for the viable biolistic transfection of OTBS by reducing tissue stress while maintaining long term exogene expression.

Dataset Information

A versatile prion replication assay in organotypic brain slices.

Publications

A versatile prion replication assay in organotypic brain slices.

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