title: Modelling of amyloid beta-peptide induced lesions using roller-drum incubation of hippocampal slice cultures from neonatal rats.
abstract: Pronounced neurodegeneration of hippocampal pyramidal neurons has been shown in Alzheimer's disease. The aim of this study was to establish an organotypic in vitro model for investigating effects of the amyloid beta (Abeta)-peptide on pyramidal neuron degeneration, glial cell activation and tau phosphorylation. Tissue cultures in a quasi-monolayer were obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Neuronal populations identified included N-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons, and neurons immunopositive for glutamic acid decarboxylase-65 (GAD65) or gamma amino butyric acid (GABA). Many neurons expressed phosphorylated tau as shown by pS(396), AD2 and PHF-tau immunostaining. Astrocytes, microglial cells and macrophages were also identified. The Abeta(25-35) peptide formed fibrillar networks within 2 days as demonstrated by electron microscopy. In the presence of the neurotoxic Abeta(25-35) peptide, but not Abeta(35-25), deposits developed in the tissue that were stainable with Thioflavine T and Congo red and showed the characteristic birefringence of Abeta plaques. Following Abeta(25-35) exposure, neurodegenerative cells were observed with Fluoro-Jade B staining. Further characterization of pyramidal neurons immunopositive for NMDA-R1 showed a decrease of cell number in the immediate surrounding of Abeta(25-35) deposits in a time- and concentration-dependent fashion. Similar effects on pyramidal neurons were obtained following exposure to the full-length, Abeta(1-40) peptide. Also, a loss of neuronal processes was seen with GAD65, but not GABA, immunohistochemistry after exposure to Abeta(25-35). Abeta(25-35)-exposed neurons immunopositive for phospho-tau showed degenerating, bent and often fragmented processes. Astrocytes showed increased GFAP-positive reactivity after Abeta(25-35) exposure and formation of large networks of processes. No obvious effect on microglial cells and macrophages could be seen after the Abeta(25-35) exposure. The developed in vitro system may constitute a useful tool for screening novel drugs against Abeta-induced alterations of tau and degeneration of hippocampal neurons.
authors.affiliation: Local Discovery Research Area CNS& Pain Control, AstraZeneca, S-151 85, Södertälje, Sweden. Sara.Johansson@astrazeneca.com
authors.forename: Sara
authors.initials: S
authors.lastname: Johansson
authors.forename: Ann-Cathrin
authors.initials: AC
authors.lastname: Radesäter
authors.forename: Richard F
authors.initials: RF
authors.lastname: Cowburn
authors.forename: Johan
authors.initials: J
authors.lastname: Thyberg
authors.forename: Johan
authors.initials: J
authors.lastname: Luthman
mesh_terms: D016229:Amyloid beta-Peptides / Q000633:toxicity*; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D039481:CD11b Antigen / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D004305:Dose-Response Relationship, Drug; D053331:Ectodysplasins; D005452:Fluoresceins; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D006624:Hippocampus / Q000473:pathology*; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009924:Organ Culture Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D010446:Peptide Fragments / Q000633:toxicity; D058225:Plaque, Amyloid / Q000473:pathology / Q000648:ultrastructure; D011108:Polymers / Q000378:metabolism; D017966:Pyramidal Cells / Q000187:drug effects* / Q000473:pathology / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013997:Time Factors; D048069:Tumor Necrosis Factors / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D039481:CD11b Antigen; D053331:Ectodysplasins; D005452:Fluoresceins; D005904:Glial Fibrillary Acidic Protein; D007527:Isoenzymes; D008565:Membrane Proteins; D009930:Organic Chemicals; D010446:Peptide Fragments; D011108:Polymers; D016194:Receptors, N-Methyl-D-Aspartate; D048069:Tumor Necrosis Factors; C435731:fluoro jade; C506479:poly(1-hydroxymethylethylene hydroxymethylformal); D016875:tau Proteins; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2