Brain-wide continuous functional ultrasound imaging for real-time monitoring of hemodynamics during ischemic stroke

Abstract

Ischemic stroke occurs abruptly causing sudden neurologic deficits, and therefore, very little is known about hemodynamic perturbations in the brain immediately after stroke onset. Here, functional ultrasound imaging was used to monitor variations in relative cerebral blood volume (rCBV) compared to baseline. rCBV levels were analyzed brain-wide and continuously at high spatiotemporal resolution (100 μm, 2 Hz) until 70mins after stroke onset in rats. We compared two stroke models, with either a permanent occlusion of the middle cerebral artery (MCAo) or a tandem occlusion of both the common carotid and middle cerebral arteries (CCAo + MCAo). We observed a typical hemodynamic pattern, including a quick drop of the rCBV after MCAo, followed by spontaneous reperfusion of several brain regions located in the vicinity of the ischemic core. The severity and location of the ischemia were variable within groups. On average, the severity of the ischemia was in good agreement with the lesion volume (24 hrs after stroke) for MCAo group, while larger for the CCAo + MCAo model. For both groups, we observed that infarcts extended to initially non-ischemic regions located rostrally to the ischemic core. These regions strongly colocalize with the origin of transient hemodynamic events associated with spreading depolarizations.

Keywords: Brain-wide imaging; functional ultrasound imaging; ischemic lesion prediction; ischemic stroke; spreading depolarization.

Figure 1.

Experimental workflow and timeline. From left to right. An 11 × 13-mm cranial window was performed to access the whole brain by functional ultrasound imaging before occluding the middle cerebral artery occlusion (MCAo) or MCAo combined with the common carotid artery (CCAo + MCAo). Image acquisition has been performed continuously for 90 mins by scanning the brain repeatedly in the anteroposterior direction (23 secs/scan) before, during, and after stroke onset. After the experiments, rats returned to their home cage and were euthanized 24 hrs after occlusion to quantify the infarct size using cresyl violet staining. We developed a digital version of the rat Paxinos atlas for the registration, segmentation, and temporal analysis of ischemia using a dedicated software solution.

Figure 2.

Brain-wide continuous and real-time monitoring of hemodynamics during ischemic stroke. (a) A typical image of the brain microvasculature for one coronal cross-section from the 2 D scan before the stroke. Only the left hemisphere is imaged entirely here because of the too-small size of the ultrasound transducer used for this study. All images were registered and segmented based on a digital version of the rat Paxinos atlas (white outlines). Time series plots of the average signal in the primary somatosensory barrel-field cortex (S1BF; green and orange region) in the affected hemisphere and the control ROI located in the opposite hemisphere (black region) for each stroke model (MCAo in green; CCAo + MCAo in orange). (b) Global hemodynamic changes (rCBV) in 115 regions located in the ischemic hemisphere for both the MCAo (left and green panel) and CCAo + MCAo groups (right and orange panel). Regions were clustered according to the extent of rCBV signal loss (from more to less pronounced; find red-to-blue color-coded labels in Supplementary Table 1). (c) Left to right. Plot showing the total volume of brain region corresponding to a given rCBV decrease (%; mean ± sd) and associated statistical difference (P-value, black square Unpaired t-test, *p < 0.05; left panel) between ischemic territory volumes in the MCAo (green, n = 7) and CCAo + MCAo groups (orange, n = 10). (d) Typical 2D brain scan of hemodynamics showing the loss in rCBV induced by either MCAo (left panel) or CCAo + MCAo (right panel). 22 cross-sections out of 23 are represented here. (e) Unrolled-cortex projection (see Supplementary Figure 1) showing the loss in rCBV induced by either MCAo (left panel) or CCAo + MCAo (right panel). D: dorsal, R: right, A: anterior, P: posterior, ß: Bregma reference point. Scale bars: 100 µm.

Figure 3.

Size and location of the region with a low rCBV signal at 70mins do partially overlap with the infarct at 24 hrs. (a) Typical rat brain cross-section stained by cresyl violet to evaluate the infarct size at 24 hrs after MCAo (left) or CCAo + MCAo (right). The infarcted territory is highlighted for each stroke model (green and orange, respectively). (b) Comparison of the infarct volume (mm³) between the two models, showing the CCAo + MCAo (orange) display a statistically more extensive infarct than the MCAo group (green; Unpaired t-test, ***p = 0.0003). (c) The infarct (colored shadings) and the region with low rCBV levels (colored outlines) are overlaid on the unrolled-cortex projection for one typical rat. (d) Same as in (c) but for all rats used in the study. D: dorsal, R: right, A: anterior, P: posterior, ß: Bregma reference point.

Figure 4.

Real-time monitoring of spreading depolarizations using functional ultrasound imaging. (a) A coronal cross-section extracted from the 2D functional ultrasound imaging scan after stroke onset shows the substantial reduction of the rCBV signal in the cortex (red area). The time series plot of the average hemodynamic signal (rCBV) in the retrosplenial granular cortex (green and orange dotted line) from the ischemic hemisphere for each stroke model (MCAo in green; CCAo + MCAo in orange). (b) Monitoring of SDs. Each horizontal line represents one animal; each dot corresponds to the transient hemodynamic increase associated with an SD. The average SDs are not statistically different between the two groups (Unpaired t-test, ns = 0.7557). (c) Location of the epicenter of SDs for the MCAo (green dots) and CCAo + MCAo (orange dots) models with respective centroids (mean ± sd). Red star locates the approximate site of clip placement on the MCA. (d) A plot of the distribution of the average velocity of SDs for MCAo (green) and CCAo + MCAo models (orange). Each point represents one rat. The average SD velocity is not statistically different between the two groups (Unpaired t-test, ns = 0.5683) and (e) Color-coded map showing the propagation of an SD in a concentric manner around the ischemic core from its epicenter located in the anterior part of the brain to the posterior part. D: dorsal, R: right, ß: Bregma reference point. Scale bar: 100 µm.