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, Physiopathology and Imaging of Neurological Disorders (PhIND)
, CNRS UMR 7587, INSERM U979
, * Corresponding Author Information
Bd Becquerel, BP5229, 14074 Caen, France. Phone, fax and e-mail: +33 2 31 47 01 66, +33 2 31 47 02 22, vivien@cyceron.fr 1467, Santa-Cruz) primary antibodies, FITC-conjugated mouse anti-?SMA (1:500, ab8211, Abcam) primary antibodies and rabbit anti-laminin (1:2000, ab11575, Abcam) primary antibodies. Primary antibodies were revealed using Fab'2 fragment anti rabbit and goat IgG linked to CY3 and Alexa Fluor 647 (1:600, Jackson ImmunoResearch) co-incubated 90 min at room temperature. Vessels were then coverslipped using mounting medium containing DAPI, GIP Cyceron ,
, Fluorescent intensity of the GluN1 staining in vessels were assessed using ImageJ software (NIH)
, Arginine content in Actilyse was removed by dialysis at 4°C overnight (see rtPA preparation section). Then, purified rtPA was mixed with the N-succinimidyl ester of Alexa 555 for 4 h at 4°C with continuous stirring. The resulting solution was dialyzed in bicarbonate buffer overnight at 4°C to remove unbound dyes
, Cryomicrotome-cut sections (10 ?m) were collected on polylysine slides and stored at -80°C before processing. Sections were incubated overnight with FITC-conjugated mouse anti-?SMA (1:500, ab8211, Abcam) primary antibodies. Sections were then coverslipped using mounting medium containing DAPI
Analyzing data: A.A and V.H. Writing the manuscript ,
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, whisker stimulation in tPA -/-mice before (?) and after (?) IV injection of rtPA (10mg/kg) and saline, HEPES (0.3M) and MK-801 (0.4mg/kg), rtPA (10mg/kg) and MK-801
, Circles represent values for each mouse (Mean ? SEM, Wilcoxon test, n = 9 for the tPA + saline group, n = 10 for the HEPES + MK-801 group, n = 8 for the rtPA + MK-801 group, n = 9 for the HEPES + AP5 group and n = 8 for the rtPA + AP5 group). (c) Mean CBF signal trace from the S1bf during whisker stimulation of tPA -/-mice before (?) and after (?) the injection of rtPA (10mg/kg) and saline, HEPES (0.3M) and AP5 (0.4mg/kg), or rtPA (10mg/kg) and AP5 (0.4mg/kg)
, HEPES (0.3M) and AP5 (0.4mg/kg), or rtPA (10mg/kg) and AP5 (0.4mg/kg
, = 10 for the HEPES + MK-801 group, n = 8 for the rtPA + MK-801 group, n = 9 for the HEPES + AP5 group and n = 8 for the rtPA + AP5 group). (d) Diagram showing the evolution of the hemodynamic response of each mice between the control condition and after the IV injection of rtPA (10mg/kg) and saline, HEPES (0.3M) and MK-801 (0.4mg/kg), rtPA (10mg/kg) and MK-801 (0.4mg/kg), Doppler speckle images (curves in transparency define the SEM, n = 9 for the rtPA + saline group
, Lines are colored according to whether the response is more than 5% higher (?), between +5% and -5% (?), or decreased for more than 5% (?) after the injection
, Expression of NMDARs is restricted to arteries and arterioles, vol.8
, Only vessels over 10µm in diameter express ?-SMA. In b (scale bar = 100µm), immunostainings reveal GluN1 subunit of the NMDAR (red), ?-SMA (green), laminin (magenta) and cells nuclei (blue). (c) Quantification ?-SMA positive vessels (left) and in ?-SMA negative vessels (right) separately. (d) Quantification of the GluN1 staining fluorescence intensity in ?-SMA positive vessels and in ?-SMA negative vessels (Mean ? SEM, ANOVA test, n = 34 for the ?-SMA + group and n = 29 for the ?-SMA -group, from 4 different mice). (e) Epifluorescence images of isolated vessels from C57BL/6 mice brain revealing Glunomab® staining (red), Col IV (green) and cells nuclei (blue), and corresponding schematic representation. Supplementary figure 1: Doppler Laser speckle imaging allows measurement of the CBF directly through the intact skull. (a) Representative images of a mouse skull, intact (bottom right) or thinned (top right) and corresponding laser Doppler speckle images. (b) Mean CBF 2: Physiological parameters. (a) Schematic representation of the set of physiological parameters, Epifluorescence images of isolated vessels from C57BL/6 mice brain revealing in a (scale bar = 50µ m): PDGF-R? (red), ?-SMA (green) and cells nuclei (blue)