296 volumes of 10% potassium ferrocyanide (K4Fe(CN)6 tri- hydrate) in d.d. H2O and 20% hydrochloric acid (HCl) in d.d. H2O for 30 min. Brain sections were subsequently washed with d.d. H2O, counterstained with nuclear fast red solution for 10 min and washed with d.d. H2O. To co-stain for CAA/ﬁbrillar Ab, sections were subsequently incubated in a 0.25% aqueous solution of potassium permanganate for 20 min, washed, incubated in an aqueous solution of 2% potassium disulphite and 1% oxalic acid for 2 min; washed, incubated in 0.25% acetic acid for 5 s; washed, incubated in a 50% ethanol solution of 0.0125% Thioﬂavin S; washed and mounted as described above. All chemicals were purchased from Sigma-Aldrich, Switzerland. Prussian Blue was detected using bright ﬁeld microscopy; ThioS using ﬂuorescence microscopy with excitation and emis- sion wavelengths at 490 and 514 nm, respectively. Ex vivo astrocyte cultures Astrocytes were cultured from early and mid-stage pathol- ogy TG and arcAb NTG mice. For the isolation of adult mouse brain astrocytes, mice were deeply anaesthetised using a cocktail of 1.25% ketamine and 0.25% xylazine. Working on ice, meninges and cerebellum ? brain stem were carefully removed, the remaining brain tissue minced into small pieces and homogenised in culture medium (DMEM/F12 containing L-glutamine 2 mM, FCS 10%, PenStrep 1.0% (all from Invitrogen, Switzerland)) using a glass homogeniser with Teﬂon pestle (Wheaton, USA). Brain homogenates were cultured in poly-L-lysine-coated 75 cm2 cell culture ﬂasks (TPP, Switzerland) at 37 C, 5.0% CO2 and 100% humidity. After the cells had reached 80–90% conﬂuence (about 4–6 weeks), the oligodendro- cytes layer was detached and removed by shaking of the culture ﬂasks and the medium aspirated. Trypsin/EDTA buffer (Invitrogen, Switzerland) was added to the remaining astrocytes and left for 2 min at 37 C. Culture medium was added and the astrocytes in the medium centrifuged at 1,250 rpm for 10 min. Pellets were homogenised in a fresh culture medium and astrocytes were seeded in poly-L- lysine-coated 6-well culture plates (TPP, Switzerland), 1.0 9 106 cells in 2 ml culture medium per well or 10 cm culture Petri dishes (2.0 9 106 cells in 10 ml culture med- ium per dish). Cells were grown to 80–90% conﬂuence and split or frozen and stored in liquid nitrogen at -196 C. Microdialysis surgery Surgery for microdialysis studies was performed in mid-stage pathology arcAb mice which were deeply anaesthetised by an intraperitoneal injection (100 ll/10 g body weight) of a cocktail consisting of fentanyl 50 ll/ml, midazolam 5.0 mg/ ml and medetomidin 1 mg/ml in 0.9% sterile saline. To 123 Acta Neuropathol (2011) 122:293–311 prevent drying of the cornea, an eye cream (Viscotears , carbomerum 980, 2.0 mg) was applied onto the eyes. Body temperature was controlled throughout all the surgical pro- cedures by means of a rectal probe connected to a whole body heating pad set at 37 C. Mice were placed in a stereotactic frame and the skull was exposed. A small hole was drilled to allow the implantation of a microdialysis guide cannula (CMA/7, Aurora Borealis Control B.V. Schoonebeek, The Netherlands) in the hippocampus [relative to bregma (in mm): AP: -3.1; L: ?3.0; V: -2.0]. Two support screws were placed to hold the guide, which was glued to the skull with dental acrylic cement (Pattern Resin, GC Europe, Switzer- land). A subcutaneous injection of the analgesic Metacam (1 lg/g body weight) was administered directly after surgery followed by an antidote (intraperitoneal injection of 100 ll/ 10 g body weight of a mixture consisting of naloxon 0.4 mg/ ml, ﬂumazenil 0.1 mg/ml and atipamezol 5.0 mg/ml in 0.9% sterile saline) to antagonise the anaesthetic. Microdialysis experiments Eighteen hours prior to the experiment, the microdialysis probe (CMA/7, cut-off 6 kDa, membrane length 1.0 mm, Aurora Borealis Control B.V., Schoonebeek, the Nether- lands) was inserted into the guide cannula. The inlet of the microdialysis probe was connected by ﬂuorinated ethylene propylene tubing (FEP tubing, CMA, Sweden) to a syringe pump (Beehive, Bas Technicol, Congleton, UK). The probe was perfused with artiﬁcial extracellular ﬂuid (aCSF, com- position in mM: NaCl 145; KCl 2.7; CaCl2 1.2; MgCl2 1.0 in a 2 mM phosphate buffer, pH 7.4) at a ﬂow rate of 1 ll/min. The outlet was connected to another piece of FEP tubing with sample vial attached. Eighteen hours after insertion and start of perfusion of the probe, the actual experiment was started (between 08:00 and 09:00 a.m.). To assess the baseline glucose differences between TG and NTG arcAb mice, 20 samples with 30-min intervals were collected. Each sample vial was put on dry ice directly after the sampling time was over and exchanged for a new, empty one. For glucose uptake measurements, mice were fasted for 12 h prior to the experiment. After collection of three 30-min interval microdialysis samples (for baseline glucose measurements), mice were given an intraperitoneal injection of 250 mg glucose in PBS/kg body weight during continuous microdi- alysis sampling. Twelve 5-min interval microdialysis samples were collected, followed by four 30-min samples. Fifteen min before the sampling time of the last 30-min interval sample had ended, aCSF perfusate was changed to aCSF containing potassium chloride (KCl) 120 mM using a liquid switch (CMA, Sweden). These 15 min were calcu- lated based on the dead volume of the FEP tubing and microdialysis probe in- and outlet. Two 5-min samples were collected after which an intraperitoneal injection of 250 mg