(a) 3D view of the rat head showing the coronal craniotomy
(brown-shaded band) drilled between bregma −2.0 and −4.0 mm and
extending 6.5 mm on each side from the sagittal suture for fUSi
acquisition. The ultrasonic probe was positioned above the coronal
craniotomy. This coronal plane was selected based on the Paxinos and
Watson stereotaxic atlas32 in order to include the S1 barrel field, i.e. the stimulation
target. A smaller round-shaped 1 mm2 craniotomy was
performed at bregma +2.0 mm, lateral +7.0 mm to access the left MCA
to place the microclip used for occlusion (black dot). Two pairs of
stimulation electrodes (blue and red) were inserted in the whisker
pads for stimulation of the unaffected and affected side,
respectively. The stimulated area is shown in light gray shade. (b)
Schematic representation of the experiment timelines. After baseline
fUSi data acquisition (duration: 30 min which included the three
runs of left and right whisker pad stimulation in the last 5 min),
the left MCA was occluded (black arrowhead) for 90 min and the clip
was then released (black arrowhead). Whisker pad stimulations (red
and blue bars for left and right whisker pad stimulation,
respectively) are depicted across the baseline, occlusion and
reperfusion conditions. Each side was alternatively stimulated three
times during the baseline period, and every 2 min throughout the
occlusion and reperfusion periods. Interruptions in the stimulation
bars represent the short time periods where the rat was removed from
the experimental set-up to allow proceeding with MCAo and
recanalization. Throughout the baseline, occlusion and reperfusion
periods fUS images were acquired continuously every 0.7 s, except
during the MCAo and reperfusion clip manipulations. After fUS
imaging, rats were returned to their home cage for 48 h until
perfusion-fixation for and immuno-histochemistry (see Methods) to
assess infarction and selective neuronal loss (SNL); (c) Coronal
fUSi image with superimposed “activated” pixels in response to
whisker pad stimulation of the unaffected-side (left) whisker pad
during baseline (image shown on the left), generated according to
the voxel-based image processing procedures described in Methods.
Activations are shown in % CBV increase compared to baseline
(pseudo-colour scale on right of image). For illustration, similar
images obtained during MCA occlusion and after reperfusion are also
presented (middle and right, respectively), showing markedly
increased contralesional rCBV responses to whisker stimulation in
both conditions. Scale bar = 2.5 mm.