Mice
All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and following local ethical advice. Experimental procedures were approved by the UK Home Office and ethical approval was granted through consultation with veterinary staff at University College London (UCL). For all experiments, mice were obtained from the following sources and subsequently bred side by side in cages: C57BL/6J (WT) mice were obtained from Charles River UK, C57BL/6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber/J (3KL) were obtained from The Jackson Laboratory. VHD (B6.tg(HD2)) mice were obtained from V. Cerovic (Institute of Molecular Medicine, RWTH Aachen). Sncatm1.1Kluk/J (SncaWT/GFP) were obtained from K. Luk (Perelman School of Medicine, University of Philadelphia). Cx3cr1CreERT2.Tgfb1LoxP animals were obtained from M. Greter (University of Zurich). Different strains were bred in the same location to harmonize environmental factors. All animals were housed under temperature-controlled (temperature, 23.1 °C; humidity, 30–60%) and pathogen-free conditions with 12 h light–12 h dark cycle with an ad libitum supply of food and water. Both male and female age-matched mice were used in this study: WT, VHD, SncaWT/GFP. Both male and female 3KL mice were used. Animals of 3–4 months were used for ME injection experiments.
Human tissue samples
All tissue samples were donated with the full, informed consent. Accompanying clinical and demographic data of all cases used in this study were stored electronically in compliance with the 1998 Data Protection Act. Ethical approval for the study was obtained from the NHS Research Ethics Committee and in accordance with the Human Tissue Authority’s code of practice and standards under licence number 12198, with an approved material transfer agreement. Consent has been obtained for sharing of individual-level data. Use of Translational Pathology Core Laboratory-derived human tissues was approved by the University of California Los Angeles Institutional Review Board, which waived the informed consent requirement for specimens acquired from the Translational Pathology and Core Laboratory (TPCL) (IRB 11-002504). Specimens were deidentified and age was provided as a 5-year age range.
Tamoxifen treatment
Cx3cr1CreERT2.Tgfb1LoxP and littermate control mice were orally gavaged 3 times (every other day) with 5 mg of tamoxifen (Sigma-Aldrich T5648) dissolved in corn oil at around 6 weeks of age.
Tissue isolation for histology
Mice were deeply anaesthetized using pentobarbital and were transcardially perfused with 15 ml of ice-cold filtered PBS (Gibco) followed by 15 ml of ice-cold 4% paraformaldehyde (PFA) (ThermoFisher). To obtain tissue from the ME, the small intestine was isolated, stored in ice-cold PBS and cut open longitudinally. Ice-cold PBS was used to clean the tissue from luminal debris. The tissue was stretched on a Sylgard plate, and the ME was carefully peeled off using surgical tools. Isolated ME was further fixed in ice-cold 4% PFA for 20 min and preserved at −20 °C in cryoprotectant solution (10% sucrose, ethylene glycol) until stained. From the lamina propria (cross-sections), 2-cm long pieces of small intestine were isolated and luminal contents were gently flushed with ice-cold PBS using a syringe. The tissue was further fixed in 4% PFA at 4 °C overnight and excess PFA was washed with 3 × 10 min in ice-cold PBS. The tissue was dehydrated using 30% sucrose solution in PBS and frozen vertically in embedding moulds (Epredia) with optimal cutting temperature medium (Sakura) before cryo-sectioning. Sections of 20–25 μm were mounted on microscopy slides (Epredia) until staining. The brain was isolated from the skull and was further fixed, frozen and sectioned as described for cross-sections above. Sections were preserved at −20 °C in cryoprotectant solution until stained. To obtain whole mounts of dura mater, the skull cap containing the dura mater was removed from the mouse skull and stored fixed in ice-cold 4% PFA at 4 °C for 2 h. After fixation, the dura mater was carefully removed.
Tissue isolation for FACS and cell sorting
Mice were anaesthetized using pentobarbital and were transcardially perfused with 15 ml of ice-cold filtered PBS. Collected tissues were stored in ice-cold Roswell Park Memorial Institute medium 1640 (RPMI-1640) (Gibco) completed with 5% FBS (Gibco) and 20 mM HEPES (Gibco). To obtain single-cell suspensions from the ME, peeled ME was cut in 1–2-mm pieces and digested with 2 mg ml−1 Collagenase type IV and 0.8 mg ml−1 dispase in RPMI (Gibco) supplemented with 2% HEPES (Gibco), 2% FBS (Gibco) and 50 μg ml−1 DNase for 1 h at 37 °C with continuous agitation. The single-cell suspensions were then homogenized with a potter grinder and strained through 70-μm cell strainers (BD Falcon). From the lamina propria, the remaining tissue following isolation of the ME, containing the lamina propria and submucosa layers, was washed with ice-cold Hank’s buffered saline solution (Gibco) supplemented with 1 mM dithiothreitol (Sigma-Aldrich), 1 mM EDTA (Invitrogen) and 20 mM HEPES for 8 min. Single-cell suspensions of lamina propria were prepared by digestion with 0.85 mg ml−1 Collagenase Type V (Sigma-Aldrich) in MEMα (Lonza) supplemented with 2% HEPES, β-mercaptoethanol (Gibco) and DNase for 30 min at 37 °C with continuous agitation. For the brain and dura, brains were quickly isolated from the skull, and brainstem was dissected from the rest of the brain tissue on ice using chilled instruments. The dorsal part of the skull was carefully removed, and meningeal dura mater was peeled off of the skull cap. The tissues were finely chopped using chilled razorblades and transferred to tubes containing ice-cold RPMI-1640 supplemented with 10% FBS and 20 mM HEPES medium. Single-cell suspensions from brainstem were prepared using the Dissociation kit (Miltenyi Biotech) according to the manufacturer’s instructions. Tissue chunks were pelleted by centrifugation at 300g for 2 min at 4 °C followed by medium removal and resuspension in a mix of buffer Z with enzymes A, P and Y. For digestion of meningeal dura mater, chopped tissue was resuspended in 0.5 mg ml−1 Collagenase P, 0.8 mg ml−1 Dispase II and 250 U ml−1 DNase1 and digested for 30 min. Single-cell suspensions from all tissues were then blocked with rat anti-mouse CD16/CD32 antibodies (BD Biosciences) used at the recommended dilution for 12 min before incubation with primary antibodies diluted at recommended dilutions (Supplementary Table 3) in FACS buffer (PBS, 2% FBS, 0.78 mM EDTA) containing Fc block for 20 min at 4 °C. Dead cells were excluded using Live/Dead near-infrared dye staining (Invitrogen) diluted in PBS or 4,6-diamidino-2-phenylindole (DAPI). Precision Count Beads (BioLegend) were used for absolute cell counting and analysed using the following formula: absolute cell count (cells per μm) = cell count/beads count × bead concentration. Flow cytometry data were analysed using FACSDiva software (v.4.0) and FlowJo software (Treestar).
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