Fig. 2

Detection of A-beta 42 peptides within brain after intranasal acute application. a Mice (wt: FVB/N; ko; Pgp knock-out) were treated with NEM and mannitol as indicated. Subsequently, A-beta was intranasally administered and animals were sacrificed after 1 h. Brains were dissected and proteins extracted by formic acid treatment. 20 µg of proteins were subjected to 4–12 % SDS–polyacrylamide gel and transferred to nitrocellulose membrane. Signals were obtained as described in Fig. 1. As a control, brain extracts from a 5xFAD Alzheimer model mouse (AD) and the corresponding wild type littermates (wt, C57B6/J) were used (aged 9 months). Additionally, pure A-beta (in vitro, iv) and wild type brain spiked with A-beta during the extraction process (s) were analyzed. A band potentially representing A-beta oligomers from the 5xFAD or A-beta/mannitol treated animal is depicted by an arrow. Unspecific signals that also occur in a “secondary antibody only” incubation (Additional file 2) are indicated by curly brackets). b For assessment of A-beta distribution upon intranasal delivery, 10 µg of TAMRA-labeled A-beta 42 peptide or water in diluent solution (control) were administered to C57Bl6/J wt mice pre-treated with mannitol (one exemplary animal for each treatment). 1 h after the application, slices from brains (cut positions indicated in the scheme) were used for detection of light emission at 580 nm in technical replicates of tissue homogenates. RFU (relative fluorescence units) were calculated by substracting values obtained from TAMRA-A-beta 42 treated animal by values measured for the control animal (mean and SD). c To analyze distribution of intranasally administered A-beta 42 peptides, animals were treated as described in B with unlabeled A-beta 42 peptide (i.n.) or solvent (control) and brains dissected 1 h after treatment. Staining of sagittal sections was performed with primary antibody 6E10. For comparison, a “secondary antibody only” staining (2°Ab) and samples from a wild type as well as a 5xFAD transgenic mouse (4 months of age) are shown. An overview of the midbrain region of intranasally treated mice is given (4 × 10 magnification) as well as hippocampal (H) and cerebellar (C) region in higher magnification (10 × 10)