Acta Neuropathol (2011) 122:293–311 glucose in PBS/kg body weight was given during continuous microdialysis sampling. Twelve more 5-min interval sam- ples were taken followed by two 30-min collections. Each sample vial was put on dry ice immediately after its sampling time was over and exchanged for a new, empty one. Glucose and lactate assays Glucose was measured using the Amplex Red Glucose/ Glucose Oxidase Assay Kit from Invitrogen, Switzerland. Microdialysis samples were diluted 1:5 and blood samples 1:20, cell culture media were used undiluted; processing of samples and assay was performed according to the manu- facturer’s directions. Lactate assays were performed with an in-house made lactate assay: cell culture media samples were used undiluted whereas microdialysis samples were diluted 1:10 in reaction buffer (KH2PO4 10 mM, pH 6.6, with ﬂavin adenine dinucleotide 0.01 mM) and 50 ll of these samples and 50 ll of lactate calibration standards (L-lactate, Sigma-Aldrich, Switzerland) were mixed with 50 ll lactate assay reaction mix [5 ml lactate reaction mix contained 30 ll Amplex Red 10 mM (Invitrogen, Switzerland), 80 ll horseradish peroxidase 10 U/ml (Invitrogen, Switzerland), 30 ll lactate oxidase 100 U/ml (Sigma-Aldrich, Switzerland) in 4.86 ml reaction buffer] followed by a 20 min incubation at 37 C. Fluorescence was read using a ﬂuorescence reader (SpectraMax Gemini XS, Bucher Biotec AG, Switzerland). Excitation and emission were set at 540 and 590 nm, respectively. Primary antibodies Primary antibodies used for immunohistochemistry and western blotting: glucose transporter 1 (GLUT1, Alpha Diagnostic, GT11-A; GLUT1, Abcam, ab40084), glucose transporter 3 (GLUT3, Alpha Diagnostic, GT31-A), endo- thelial cells (CD31/PECAM1, BD Pharmingen, 553370), APP/Ab (6E10, Covance/Signet, SIG-39300), Ab (in-house produced antibody recognising ﬁbrillar Ab), laminin (Sigma- Aldrich, L9393), b actin (Abcam, ab6276), lactate transporter 1/monocarboxylate transporter 1 (MCT1, Alpha Diagnostic, MCT-13A), glial ﬁbrillary acidic protein (GFAP, Advanced Immunochemical Inc., 031223), alpha smooth muscle actin (Sigma-Aldrich, A2547), b dystroglycan (Abcam, ab49515). Results Decreased endothelial and astrocytic GLUT1 expression in mid-stage arcAb mice Western blot analysis of cortex and brain homogenates from mid-stage TG arcAb mice and their NTG littermates 297 revealed that the expression of the endothelial GLUT1 protein (55 kDa isoform) was reduced in the TG animals (Fig. 1d). This phenomenon was observed starting in TG mice around mid-stage disease pathology, an age group at which diffuse and vascular Ab plaque pathology becomes wide-spread in our TG arcAb mice. The decrease in GLUT1 protein expression was more pronounced in the hippo- campus than in the cortex. Moreover, ex vivo astrocytic cultures from these mid-stage TG arcAb mice were found to express less GLUT1 protein (45 kDa isoform) as compared to astrocytes from their NTG littermates (Fig. 3a). Immu- nohistochemical staining of free-ﬂoating brain sections revealed that the expression of GLUT1 protein but not that of the endothelial marker CD31 was signiﬁcantly reduced mainly on blood vessels affected by CAA (Fig. 1a, c). Yet, in brain regions with a dense population of diffuse plaques GLUT1 staining pattern on blood vessels was affected as well, being more intervallic in contrast to the continuous expression of CD31 (Fig. 1b). No differences in expression level of the neuronal GLUT3 protein between TG and NTG brain homogenates were observed (Fig. 1d). The apparent difference in GLUT1 reduction between biochemical anal- ysis (Fig. 1d) and histochemical quantiﬁcation (Fig. 1c) is due to the fact that biochemical analysis was performed on whole brain extracts (excluding cerebellum) and histo- chemical quantiﬁcation took account of GLUT1 reduction on CAA-affected vessels only. As can be seen from Fig. 1b also in areas with high abundance of diffuse Ab plaques endothelial GLUT1 was decreased, an additional decrease that has to be added to the GLUT1 reduction as found on CAA-laden vessels. Reduced brain baseline glucose concentrations and impaired blood-to-brain glucose uptake in mid-stage arcAb mice To investigate whether reduced GLUT1 protein expression would cause decreased glucose concentration in hippocampal extracellular ﬂuid (ECF), in vivo hippocampal microdialysis was performed. Baseline hippocampal glucose concentra- tions measured continuously over 10 h were found to be lower in TG animals as compared to their NTG littermates (Fig. 2a). No such differences were found in plasma glucose levels (Fig. 2b) and a systemic cause for the hippocampal glucose deﬁciencies as present in the TG animals could hence be ruled out. To investigate if these chronically decreased ECF glucose baseline concentrations in the TG arcAb mice were indeed caused by insufﬁcient blood-to-brain glucose uptake due to decreased GLUT1 expression, glucose uptake was assessed (Fig. 2c). After a 12 h period of fasting and collection of four 30-min interval microdialysis samples (for baseline glucose measurements), mice were given an intra- peritoneal injection of 250 mg glucose/kg body weight 123