Experimental procedure.

(A) Workflow for brain imaging of awake head-fixed rats including, from left to right: animal preparation (habituation to the bench, implantation of cranial windows, training), functional ultrasound (fUS) imaging of stroke induction and brain functions, data processing, and histopathology. (B) Overview of the headpost implantation and cranial windows developed for combined MCAo (left) and brain imaging (right) under awake conditions. (C) Computer-aided design of the experimental apparatus where the animal is placed and secured in a suspended sling suit and the head fixed by the means of clamps holding the headpost implanted to the rat skull. A, Anterior; D, Dorsal; L, Left.

FeCl3-stroke induction under awake conditions.

(A) Front view representation of functional ultrasound (fUS) imaging during live chemo-thrombosis of the left MCA with FeCl3 in awake head-fixed rats. (B) Set of typical coronal μDoppler images of the brain microvasculature (top row) and morphological Bmode images (bottom row) before stroke (left), 3hrs (middle), and 5d after stroke onset (right) from the same animal. μDoppler images (top left) were registered and segmented based on a digital version of the rat brain atlas (white outlines). Colored outlines (cyan, purple, and black) delineate regions of interest plotted in (C) and (D). The white dotted region of interest highlights the ischemia in μDoppler images (Top row) and tissue hyper-echogenicity in Bmode (Bottom row). (C) Temporal plot of the average signal (ΔrCBV (%), mean±95%CI, n=5) in the barrel-field primary somatosensory cortex (S1BF, cyan) from the left hemisphere, affected by the MCAo. (D) Temporal plots of the average signal (ΔrCBV (%)) in the retrosplenial granular cortex (RSGc) from the affected (purple) and non-affected hemisphere (black) from the same animal. (E) Occurrence of spreading depolarizations after MCAo. Each horizontal line represents one rat; each triangle marker depicts the occurrence of one spreading depolarization. (F) Temporal plots of the average signal change (ΔrCBV (%), mean±95%CI) of the spreading depolarizations (centered on the peak) per rat. (G) Typical rat brain cross-sections stained by cresyl violet to evaluate the tissue infarction at 24hrs after FeCl3-induction occlusion of MCA. The infarcted territory is delineated in red. Scale bars: 1mm. D: Dorsal; L: left; R: right.

Early post-stroke alteration of whisker-to-barrel thalamo-cortical circuit.

(A) Front view representation of functional ultrasound (fUS) imaging during repetitive stimulation of the left (orange) or right whisker pad (green) with a mechanical comb in awake head-fixed rats. Whisker stimulations were delivered alternately between left and right whisker pads before and early after MCAo. Each rat receives 45 stimuli per whisker pad each hour of imaging. (B) Average activity maps (z-score) from one rat depicting evoked functional responses to either left (orange) or right whisker pads stimulation (green) registered with a digital version of the rat Paxinos atlas (white outlines) and overlaid with the corresponding coronal μDoppler image, before (left; Pre-stroke, average of 45 trials) and after stroke induction in the left hemisphere (right; Post-stroke, average of 125 trials). (C) Time-course traces of the average hemodynamic changes (ΔrCBV(%)) in response to right (green) or left whisker stimulation (orange) extracted from the contralateral hemisphere (left and right, respectively) before (left; Pre-stroke, n=5, 45 trials/rat) and after stroke induction in the left hemisphere (right; Post-stroke, n=5, 135 trials/rat). Brain regions are ordered by major anatomical structures (see Supplementary Table 2). The vertical line represents the stimulus start. S1BF, S2, AuD, VPM, VPL and Po regions are brain regions significatively activated (all pvalue<0.01). (D) Left, Average response curves from the S1BF, the VPM, and Po regions before (Pre-stroke, black, n=5, 45 trials/rat), and from first to third hour after stroke induction (0-1hr, 1-2hr, 2-3hr Post-stroke, orange and green, n=5, 45 trials/hr/rat). Data are mean±95%CI. The vertical bar represents the whisker stimulus. Right, Statistical comparison between pre-stroke and post-stroke response curves for S1BF, VPM, and Po regions (Two-way ANOVA with Fisher’s LSD test for multiple comparisons; ns: non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Scale bars: 1mm. D: Dorsal; L: left; R: right; Ctx: Cortex; Hpc: Hippocampus; Th: Thalamus; CPu: Caudate Putamen; HTh: Hypothalamus; S1BF: barrel-field primary somatosensory cortex; S2: Secondary somatosensory cortex; AuD: Dorsal auditory cortex; VPM: Ventral postero-medial nucleus of the thalamus; ; VPL: Ventral postero-lateral nucleus of the thalamus; Po: Posterior nucleus of the thalamus.

Late post-stroke alteration of whisker-to-barrel thalamo-cortical circuit.

(A) Activity maps (z-score; average of 45 trials) depicting evoked functional responses to left (orange) or right whisker pads stimulation (green) 5d after stroke induction. Z-score maps are registered with the Paxinos atlas (white outlines; Left) and overlaid with the corresponding coronal μDoppler image (Right). (B) Left; Average response curves to left and right whisker stimulation (orange and green; respectively) extracted from S1BF, VPM, and Po before (Pre-stroke, black, n=2, 45 trials/rat), 0-3hr (0-3hr Post-Stroke; light orange/green, n=2, 45 trials/hr/rat) and 5d after stroke induction (5d Post-stroke, dark orange/green, n=2, 45 trials/rat). Data are mean±95%CI. The vertical bar represents the whisker stimulus. Right; Statistical comparison between response curves for S1BF, VPM, and Po pre-stroke, 0-3hr and 5d post-stroke (Two-way ANOVA with Fisher’s LSD test for multiple comparisons; ns: non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Scale bars: 1mm. D: Dorsal; R: right; S1BF: barrel-field primary somatosensory cortex; VPM: Ventral postero-medial nucleus of the thalamus; Po: Posterior nucleus of the thalamus.

Example of hemodynamic changes (rCBV in %) induced by MCAo in 69 regions located in the ischemic hemisphere of the imaged coronal cross-section. Regions are organized by main anatomical structures (see Supplementary Table 2). SDs stands for spreading depolarizations.

Averaged hemodynamic response curves (ΔrCBV in %) of 45 consecutive right (green) or left whisker stimulation (orange; 1hr recording) extracted in the contralateral S1BF, VPM and Po regions (top to bottom). The corresponding individual trials presented below confirmed the stability across the recording. Vertical grey bar, the period of whisker stimulation.

Reporting on animal use, experimentation, inclusion/exclusion criteria, and figure association.

List of the 69 brain regions/hemisphere from the coronal cross-section μDoppler imaged in each rat organized by main anatomical structures. Adapted from the Paxinos rat brain atlas35.