*2.5. Systolic Blood Pressure*

Measurement of systolic blood pressure was performed in conscious NTg and 3xTg-AD mice using the tail-cuff method (NIPREM 645; Cibertec, Madrid, Spain). The average systolic blood pressure of each mouse was determined from six consecutive measurements after habituation, as described [32].

#### *2.6. MRI-ASL—Relative Cerebral Blood Flow*

MRI was carried out at the joint nuclear magnetic resonance facility of the Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red—Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Cerdanyola del Vallès, Spain) in a 7-Tesla horizontal magnet (BioSpec 70/30, Bruker BioSpin, Ettlingen Germany), equipped with actively shielded gradients (B-GA12 gradient coil inserted into a B-GA20S gradient system). For signal reception, a mouse brain surface coil was used actively decoupled from a 72 mm

inner diameter volume resonator. Animals were anaesthetized using an average 2.5% isoflurane in O2, and both animal respiration and temperature were constantly monitored with a preclinical monitoring and gating system (SA Instruments, New York, USA). Highresolution T2-weighted images (T2w) were acquired for anatomical references using rapid acquisition with relaxation enhancement sequence with double echoes. The acquisition parameters were the following: orientation = axial plane, echo train length or rare factor = 8, field of view (FOV) = 1.92 × 1.92 cm2, matrix size (MTX) = 256 × 256 (75 × <sup>75</sup> <sup>μ</sup>m/pixel), number of slices = 11, slice thickness = 1 mm, interslice distance = 1 mm, repetition time (TR)/ effective echo time (TEeff) = 5000/36 ms, number of averages = 1, and total acquisition time (TAT) = 2 min. Perfusion-weighted imaging was obtained using the MRI arterial spin labelling (MRI-ASL) technique without contrast. Two consecutive axial slices were placed in two different sections (Bregma −1.5 mm, −2.5 mm) according to the mouse brain atlas by Paxinos and Franklin [33] using a T2w sagital plane image as anatomic reference. The vendor provided ASL protocol using a flow-sensitive alternating inversion-recovery rapid acquisition with relaxation enhancement (FAIR-RARE) sequence. The parameters were the following: echo train length = 72, TR/TEeff = 16,000/ 50 ms, slice thickness = 1 mm, thickness of the selective inversion slice = 4 mm, FOV = 1.92 × 1.92 cm<sup>2</sup> (150 × 150 μm/ pixel), MTX = 128 × 128, inversion recovery time (TIR) = 30 ms, increment of TIR = 100 ms, number of TIR = 22, and TAT = 13 min. The obtained ASL images were analyzed using the workstation software Paravision 5.1 (Bruker Española S.A., Madrid, Spain) to generate rCBF images. rCBF values were measured using a region of interest (ROI) created corresponding to cortex (Bregma −1.5 mm, −2.5 mm), striatum (Bregma −1.5 mm, −2.5 mm), caudate putamen (Bregma −1.5 mm), basolateral amygdala (−1.5 mm), and hippocampus (−2.5 mm) in both hemispheres (Figure 5). Bilateral ROIs from the same mouse were analyzed together as a mean value and separately to evaluated left-right rCBF asymmetries between hemispheres. For correlations, asymmetry index (AI) defined as (right-left)/(right + left) \* 100 was used.

## *2.7. Angiogenesis*

Segments of MCA and the descending thoracic aorta were dissected in ice-cold physiological salt solution (PSS; composition in mM: NaCl 112.0; KCl 4.7; CaCl2 2.5; KH2PO4 1.1; MgSO4 1.2; NaHCO3 25.0 and glucose 11.1) supplemented with amphotericin B (15 mg/l) (Biowhittaker ®, Lonza, Basel, Switzer- land) and gentamicine (30mg/l) (Genta-gobens ®, Laboratorios Normon SA, Tres Cantos, Madrid, Spain) and gassed with 95% O2 and 5% CO2. Afterwards, vessels were immersed into Matrigel® (50 μL; BD Bioscience, San Jose, CA, USA) following the protocol previously described [34,35]. Angiogenic growth was measured using an inverted microscope equipped with a camera (10× objective; TE2000 Nikon Eclipse-S, Nikon España, Madrid, Spain) at day 4, 5, 6, and 6 after seeding. The longest vessel sprouting from the artery's outer surfacy (starting point) determined the angiogenic growth [34,35], and at least three fields per arterial segment were measured.
