294 possibility of an early involvement of vascular dysfunction in the pathophysiology of the disease with disturbances in angiogenesis [11, 54]. Cerebral hypoperfusion and hypo- metabolism occur in patients with mild cognitive impairment (MCI), and were also observed in transgenic mouse models of AD in which they precede the onset of b-amyloid plaque pathology [5, 10]. While vascular and metabolic disorders including atherosclerosis and diabetes can strongly impair cerebral circulation [25], neurotoxic soluble Ab species can also diminish performance of the cerebral vasculature [16, 18], presumably already at early and pre-pathological stages of b-amyloid pathology. As a consequence, Ab-related disruption of brain blood ﬂow could lead to neuronal impairments by compromising a steady vascular supply of essential metabolic precursors such as oxygen, amino acids and glucose [4, 24, 65], as well as by decreasing cerebral lymphatic drainage of metabolic products [58, 59]. Astrocytes play a pivotal role in regulating these functions by mediating signalling and nutrient exchange between neurons and vascular endothe- lium [1, 23] via astrocyte endfeet that contain numerous nutrient transporters including the glucose transporter GLUT1 and the monocarboxylate transporter 1 (MCT1), in addition to many receptors essential for signal transduction [1, 41]. Interactions of astrocyte endfeet with the VBM are mediated via the dystroglycan protein complex that binds to laminin and neuronal-expressed neurexin [2, 56, 63]. Astrocyte endfeet can retract and swell during pathological conditions including neuroinﬂammation in multiple scle- rosis and in experimental autoimmune encephalomyelitis [63]. Because both astrogliosis surrounding b-amyloid plaques and neuroinﬂammation occur in AD, alterations in astrocyte endfeet structure and function are likely to be present [13, 63, 64]; such changes consistent with loss of contact between astrocyte endfeet and the cerebrovascu- lature were indeed observed in a transgenic mouse model of AD [61]. To further understand the role of the cere- brovasculature and astrocytes as well as their inﬂuence on cerebral metabolism in b-amyloid pathology, we charac- terised cerebral blood vessels and astrocytes in transgenic arcAb mice expressing both the Swedish and Arctic APP mutations. This mouse model exhibits severe vascular AD pathology with signiﬁcant electrophysiological and behavioural deﬁcits preceding b-amyloid deposition [28, 35]. The Swedish double mutation (K670N and M671L) together with the arctic mutation (E693G) leads to an over-production of aggregation-prone Ab42 and accumu- lation of Ab at brain blood vessels [28, 35]. Similar as was described for other mouse models of AD [22, 42, 55, 62], our studies identiﬁed severe morphological and bio- chemical abnormalities of CAA-affected vessels including stenoses, leakages, loss of vascular smooth muscle cells, and decreased GLUT1 expression. We also observed a 123 Acta Neuropathol (2011) 122:293–311 unique astrocytic pathology commencing at the early dis- ease stages. These included reduced GLUT1 and MCT1 expression, decreased astrocytic release of lactate in vivo and ex vivo, increased laminin secretion and retraction and swelling of astrocyte endfeet surrounding diffuse and vas- cular Ab depositions, signs of neurovascular uncoupling and BBB disruption. In vivo microdialysis revealed severe reductions in brain glucose concentrations at baseline during neuronal stimulation and impaired blood-to-brain glucose uptake. These ﬁndings establish an astrocytic involvement in the early stages of the cerebrovascular and metabolic pathology in this transgenic mouse model of AD. Materials and methods Animals ArcAb mice were produced as described previously by Knobloch and colleagues [28]. Non-transgenic littermates were used as controls when speciﬁed. All animals were housed 3–5 per cage and had access to food and water ad libitum under a 12-h light/dark cycle. All in vivo experiments conducted complied with Swiss federal and cantonal law and were performed with the approval of the Swiss Veterinary Council, approval number 47/2008, 3781. Classiﬁcation of disease pathology Transgenic arcAb mice were grouped according to the dis- ease pathology in the following groups: early-stage pathology: 6 months old, mid-stage pathology: 9–13 months old and late-stage disease pathology: 16–22 months old. Disease stage pathology was based on overall cerebral Ab plaque burden including CAA and cognitive performance according to Knobloch et al. [28]. Perfusion of mice and collection of brain material Mice were deeply anaesthetised using a cocktail of 1.25% ketamine and 0.25% xylazine. Mice used for histochem- istry purposes were transcardially perfused with ice-cold PBS (10 mM Na2HPO4, 2.5 mM NaH2PO4, 150 mM NaCl, 3 mM KCl) followed by ice-cold paraformaldehyde (PFA) 4.0% in phosphate buffer 0.15 M (Na2HPO4 ? NaH2PO4 in double-distilled water), pH 7.4. Brains were incubated in PFA 4.0% in PBS for 4 h at 4 C followed by incubation in sucrose 30% in PBS for 24–36 h. Mice serving biochemistry purposes were transcardially perfused with ice-cold PBS only. Hippocampi were dissected out and cortices, hippocampi and cerebella were separately snap-frozen in liquid nitrogen and stored at -80 C.