Introduction

Stroke is a multifaceted and multiphasic pathology, complex to mimic under experimental conditions. Indeed, when compared to clinic, preclinical stroke models suffer from several limitations that narrow the experimental focus to few conditions^1–3. Among these limitations, one can highlight the complexity to combine (i) imaging stroke in conscious animal models, (ii) addressing post-stroke brain functions and (iii) recording of hyperacute stroke hemodynamics, all crucial to design timely effective therapeutic strategies.

As first limitation, the use of anesthesia in preclinical studies seems to hamper the transition from animal to patient as stroke rarely occurs in sedated patients. Moreover, anesthetics disrupt the brain functionality, alterates neurovascular coupling^4,5 and affects the stroke by acting as neurotoxic or neuroprotective agents^6,7. To date, only a few groups succeeded in inducing a stroke in awake rodents^8–11. Moreover, post-stroke network and functional alterations have been addressed by few preclinical studies, providing evidence of functional network reorganization from minutes^12,13 to days^14–17 following stroke onset. However, these studies mostly focused on the cortical readouts and were unable to capture how deeper brain regions, like thalamic relays, were functionally and/or temporally affected remotely from the stroke insult (e.g., diaschisis)^18–20. Furthermore, these studies were always conducted using various anesthetics (e.g., ventilated with halothane or isoflurane; medetomidine, urethane) known to differentially impact brain functions, as mentioned above.

Until recently, live monitoring of the hyperacute stroke-induced hemodynamics was restricted to few methods but often focused to the brain surface^10,21,22. On the other hand, functional ultrasound (fUS), a recent neuroimaging modality measuring cerebral blood volume changes (CBV)^23,24, was successfully employed to measure brain functions of awake rodents^25–28, to address early post-stroke functional reorganization¹³, and to track stroke-induced hemodynamics at the brain-wide scale (i.e., ischemia and spreading depolarization)²⁹. However, no study has further exploited such strategies to combine together stroke hemodynamics and brain-wide functional alteration in awake rodents.

In this study, we report on the stroke induction and the alteration of somatosensory brain functions in awake rats. Using the latest improvements toward imaging of awake rodents^25–27 combined with chemo-thrombotic agent directly applied to the middle cerebral artery (MCA)^30,31, we were able to induce MCA occlusion (MCAo) in awake rats while capturing continuous hemodynamic changes, including ischemia and spreading depolarization, in +65 brain regions for up to 3hrs after stroke onset. Finally, we investigated on how somatosensory thalamo-cortical functional reponses were progressively altered from early (0-3hrs) to late post-stroke (5d) timepoints.

Materials and methods

Animals

The experimental procedures were approved by the Committee on Animal Care of the Katholieke Universiteit Leuven, following the national guidelines on the use of laboratory animals and the European Union Directive for animal experiments (2010/63/EU). The manuscript was written according to the ARRIVE Essential 10 checklist for reporting animal experiments³². Adult male Sprague-Dawley rats weighed between 250–400g (n=9; Janvier Labs, France) were used. During habituation rats were housed two per cage kept in a 12-hr dark/light cycle at 23°C with ad libitum access to water and controlled access to food (15g/rat/day). After the initial surgical procedure, rats were housed alone. See Supplementary Table 1 reporting on animal use, experimentation, inclusion/exclusion criteria.

Body restraint and head fixation

Rats were habituated to the workbench and to be restrained in a sling suit (Lomir Biomedical inc, Canada), progressively increasing the restraining period from minutes to hours^33,34. After the headpost implantation (see below), rats were habituated to be head-fixed while restrained in the sling. The period of fixation was progressively increased from minutes to hours. Water and food gel (DietGel, ClearH₂O, USA) were provided along the habituation session. Once habituated, the cranial window for imaging was performed as described below (Figure 1A-C).

Figure 1.

Surgical procedures

Cranial window over the MCA: Rats were anesthetized with isoflurane (5% for induction, 2% for maintenance; Iso-Vet, 1000 mg/g, Dechra, Belgium) and fixed in a stereotaxic frame. The depth of anesthesia was confirmed by the absence of reflex during paw pinching. After scalp removal and tissue cleaning, a 1-mm² cranial window was performed at coordinates bregma +2mm and lateral 7mm, over the left distal branch of the MCA as reported in Brunner, Korostelev et al.¹³. A silicone plug (Body Double-Fast Set, Smooth-on, Inc., USA) was used to protect the window and ease the access to the MCA before the occlusion procedure. Then, a stainless-steel custom designed headpost was fixed with bone screws (19010-00, FST, Germany) and dental cement (Super-Bond C&B, Sun Medical Co., Japan) to the animal skull (Figure 1B, left) as previously described by Brunner, Grillet et al.,²⁷.

Cranial window for imaging: After recovery and habituation to head-fixation, a second cranial window was performed between bregma -2 to -4mm and 6mm apart from the sagittal suture (same anesthesia settings as the first cranial window; see above) following the procedure described in Brunner, Grillet et al.,²⁸ (Figure 1B, right). A silicone plug was also used to protect the window and a headshield was added to secure it²⁵.

For both cranial windows, the dura mater was kept intact. After each surgery, rats were placed in their home cage and monitored until they woke up. Rats were medicated with analgesic (Buprenorphine, 0.1mg/kg, Ceva, France), anti-inflammatory (Dexamethasone, 0.5mg/kg, Dechra, Belgium) drugs injected directly after the surgery, at 24hrs and 48hrs after the surgery. An antibiotic (Emdotrim, 5%, Ecuphar, The Netherland) was added to the water bottle.

Chemo-thrombotic stroke induction with ferric chloride solution

Once body restrained and head-fixed the silicone plug covering the MCA window was removed allowing the application of a drop of 20% ferric chloride solution (FeCl₃; Sigma Aldrich, USA) to the MCA^30,31 (Figure 2). Once the ischemia was visually detected using the real-time display of μDoppler images, the solution was washed out with saline to stop the reaction.

Figure 2.

Whisker stimulation paradigm

Two stimulation combs individually controlled by a stepper motor (RS Components, UK) were used to deliver mechanical 5-Hz sinusoidal deflection of ∼20° of amplitude for 5s, alternatively to left and right whisker pads. For each whisker pad, trials were spaced by a 1.3-min period without stimulation. The blocks of stimulation were continuously delivered throughout the imaging sessions, time-locked with the fUS acquisition (Figure 3) to allow the subsequent analysis of hemodynamic responses within the fUS time-series.

Figure 3.

Functional ultrasoung imaging acquisition

Coronal μDoppler images were acquired using a 15-MHz linear probe composed of 128 piezo-elements spaced by 100μm (L22-14Vx, Vermon, France) connected to a dedicated ultrasound scanner (Vantage 128, Verasonics, USA) and controlled by a high-performance computing workstation (fUSI-2, AUTC, Estonia). This configuration allowed to image the brain vasculature with a resolution of 100μm laterally, 110μm in depth, and 300μm in elevation²⁸. The ultrasound sequence generated by the software is the same as in Macé et al.,²⁶ and Brunner, Grillet et al.,²⁸. Briefly, the ultrafast scanner images the brain with 5 tilted plane-waves (−6°, -3°, +0.5°, +3°, +6°) at a 10-kHz frame rate. The 5 plane-wave images are added to create compound images at a frame rate of 500Hz. Each set of 250 compound images is filtered to extract the blood signal. Finally, the intensity of the filtered images is averaged to obtain a vascular image of the rat brain at a frame rate of 1.25Hz. Then, the acquired images are processed with a dedicated GPU architecture, displayed in real-time for data visualization, and stored for subsequent off-line analysis²⁸.

Control sessions lasted between 2.5hrs (Rat#1) and 4hrs (Rat#5,8,9). Continuous imaging session combining pre-stroke, MCA occlusion and early post-stroke recording consisted in 4hrs recording (Rat#2-4, 6-9) and late post-stroke recordings were 2 sessions of 1hr (Rat#6,8) performed 5d after the stroke onset. See Supplementary Table 1 reporting on animal use, experimentation, inclusion/exclusion criteria.

fUS data processing and analysis

The data processing was performed following the procedure described by Brunner, Grillet et al.,²⁸.

Registration to Paxinos rat brain atlas and data segmentation

We registered the fUS dataset to a custom digital rat brain atlas used in Brunner et al.²⁹ and with one coronal plane (bregma -3.4mm) from the stereotaxic atlas of Paxinos³⁵. Briefly, a spatial transformation matrix was computed on the imaged coronal cross-section that was manually aligned on the atlas by affine transformation (i.e., translation, rotation, and scaling). The dataset was segmented into 69 anatomical regions/hemisphere listed following the major anatomical structures (see Supplementary Table 2). The software for data registration and segmentation is open-access³⁶.

Relative cerebral blood volume (rCBV)

We used the relative cerebral blood volume (rCBV, expressed in % as compared to baseline) to analyze ischemia, spreading depolarizations (SDs) and functional changes. rCBV is defined as the signal in each voxel compared to its average level during the baseline period. After registration and segmentation, the rCBV signal was averaged in each individual region.

Analysis of stroke hemodynamics

The extraction of the temporal traces from the ischemic area and the SDs was performed based on the temporal analysis of the rCBV signal as described in Brunner et al.²⁹. Briefly the temporal traces were extracted by averaging the rCBV signal in the primary somatosensory barrel-field cortex (S1BF) for the ischemia and the retrosplenial granular cortex (RSGc) for detecting the SDs, both located in the left hemisphere. This procedure allowed to measure the occurrence of SDs over the recording period. Live recording of ischemia and spreading depolarizations can be visualized in Movie 1.

Activity maps

Pre- and post-stroke recordings are reshaped in shorter 40-s sessions, i.e., 50 frames, and averaged based on the whisker stimulation paradigm (left or right). In each voxel, we compared signals along the recording in a time window before the stimulus onset and a time window after stimulus onset using two-tailed Wilcoxon rank sum test. We obtained the z-statistics of the test for each voxel, and consequently a z-score for the coronal cross-section. Mean activity maps for left or right whisker stimulation (Figure 3B and 4A) show z-score value calculated using a Fisher’s transform for all voxels across the coronal cross-section. Only voxels with a z-score>1.6 were considered significantly activated (p<0.05 for a one-tailed test).

Hemodynamic response time-courses

The relative hemodynamic time course ΔrCBV was computed for each brain regions (after registration and segmentation; Figure 3C-D and 4B), as the rCBV change compared to baseline at each time point. No additional filtering was used, and no trial was removed from the analysis.

Statistical analysis

Average rCBV changes were compared with a two-way ANOVA and a Fisher’s LSD test for multiple comparisons using GraphPad Prism 9.2.0.

Results

Real-time imaging of stroke induction in awake rats

We first developed a dedicated procedure for real-time imaging of stroke induction and associated evoked functional deficits in awake head-fixed rats (Figure 1A). Each rat was subjected to two cranial windows accessing independently the distal branch of the left MCA (Figure 1B, Left) and the selected brain regions to image (Figure 1B, Right). The latter was performed between bregma -2 and -4mm allowing for jointly monitoring the bilateral thalamo-cortical circuits of the somatosensory whisker-to-barrel pathway, including the ventroposterior medial nucleus of the thalamus (VPM) and the primary somatosensory barrel-field cortex (S1BF). Moreover, the selected coronal cross-section includes the posterior nucleus of the thalamus (Po), the reticular nucleus of the thalamus, and the ventral part of the zona incerta known for relaying information related to whiskers^37,38, and also direct efferent projections from the S1BF to other cortical and subcortical regions³⁹. Prior to imaging sessions, rats were extensively trained to accept comfortable restraints in the experimental apparatus (Figure 1C), suitable for fUS recording of brain functions and stroke induction under awake conditions. After data acquisition, the coronal cross-section was registered and segmented on a custom-developed digital rat atlas²⁹ to provide a dynamic view of the changes in perfusion induced either by the stroke or evoked activity.

To overcome the limitations of conventional stroke models, we occluded the distal branch of the MCA by the mean of a chemo-thrombotic ferric chloride solution (FeCl₃)^30,31 while performing fUS imaging in awake rats (Figure 2A). It should be mentioned that the rats did not show any obvious signs of pain or discomfort (e.g., vocalization, aggressiveness) during the restrain period and occlusion procedure. The MCA occlusion (MCAo) was captured live with fUS and confirmed by the large drop of signal, i.e., ischemia, localized in the cortex of the left hemisphere (Figure 2B, C and Movie 1) as shown with μDoppler image taken 3hrs and 5d after the stroke onset (dashed outline, Figure 2B, Top row). Bmode images accounting for the brain tissue echogenicity remain unchanged early after stroke onset (3hrs) while showing focal hyper-echogenicity (dashed outline, Figure 2B, Bottom row) lately after stroke onset (5d) as a marker of focal lesion⁴⁰. The stroke-induced hemodynamic changes have been continuously recorded for up to 3hrs after stroke onset, registered and segmented into 69 regions (Supplementary Figure 1). We first extracted the average change in rCBV (ΔrCBV in %) in the S1BF cortex of the left hemisphere (blue region, Figure 2B) and detected an abrupt drop of rCBV down to ∼40% of the baseline level after the occlusion of the MCA, followed by a progressive decrease of the rCBV to 30% of baseline level 3hrs after the stroke onset (Figure 2C and Movie 1). Second, we extracted the average rCBV change from a cortical region supplied by the anterior cerebral artery directly after the MCAo. The signal extracted from the retrosplenial granular cortex (RSGc; purple and black regions in Figure 2B) shows successive and transient increases of signal. It characterizes spreading depolarizations (SDs) in the left hemisphere (in purple; Figure 2D and Movie 1) while resulting in a slight and stable oligemia in the right hemisphere (in black; Figure 2D). SDs were observed in the peri-ischemic territory of all rats subjected to MCAo and occurred in an ostensibly random fashion (Figure 2E); however, SDs showed a similar bell shape and time-course across animals (Figure 2F). On average, we detected 5 SDs per hour per rat. Finally, we stained brain slices 24hrs after MCAo and confirmed that FeCl₃-induced ischemia turned into tissue infarction (red delineation; Figure 2G).

Stroke-induced alterations of the thalamo-cortical functions

One hour before and during 3hrs after the occlusion of the MCA, rats received mechanical stimulation of the whisker alternately delivered to the left and right pad using motorized combs (5-Hz sinusoidal deflection, 20° amplitude, 5-s duration; Figure 3A) to capture the spatiotemporal dynamics of the functional circuit. Before stroke, the sensory-evoked stimulations elicited a robust and statistically significant functional response (z-score>1.6, see Material and Methods) for both left and right stimulation (orange and green respectively; z-score maps; Pre-stroke panel, Figure 3B and Movie 2) with the activity spatially confined in the contralateral dorsal part of the VPM and S1BF. The temporal analysis of the somatosensory evoked responses in the contralateral hemisphere confirmed that VPM, Po, and S1BF regions were significantly activated and for both left and right stimuli (z-score, ****p<0.0001, ***p<0.001 and ****p<0.0001 respectively; Left panel, Figure 3C). We also detected significant increase of activity in S2, AuD, Ect (****p<0.0001) and PRh (***p<0.001) cortices and VPL nucleus (**p<0.01; the list of acronyms is provided in Supplementary Table 2), brain regions receiving direct efferent projections from the S1BF^39,41,42, VPM or Po nuclei^43–45. It is worth noted that no habituation or sensitzation due to the repetitiveness of whiskers stimulation was observed in cortical and subcortical regions over the pre-stroke sessions (Supplementary Figure 2).

After the stroke, the activity map from the left pad stimulation elicited a similar response pattern as pre-stroke; however, the right pad stimulation showed a total absence of functional response in the S1BF cortex and a significant reduction of the response in the VPM (z-score maps; Post-stroke panel, Figure 3B and Movie 2). Over the 3hrs following stroke onset, functional responses to left whisker stimulation were still detected in the cortical and thalamic regions of the contralateral (right) hemisphere; however, functional responses to right whisker stimulation were only detected in subcortical nuclei (i.e., VPM, Po, VPL), while attenuated when compared with the responses from the pre-stroke period and from the other hemisphere (Figure 3B, C). Furthermore, no responses were detected at the cortical level (S1BF, S2, and AuD; right panel, Figure 3B, C).

To better evaluate how the functional responses were affected by the stroke, we have divided the post-stroke recording period into 3 sections of 1hr each and compared them with the 1-hr pre-stroke period (Figure 3D). Temporal plots from the pre-stroke period showed robust increases in signal during the stimulus in S1BF, VPM, and Po regions and high consistency between left and right stimuli (black plots, Figure 3D and Supplementary Figure 2); fitting well the hemodynamic response functions as previously observed^13,46. Indeed, the hemodynamic responses were characterized by a quick increase in signal during whisker stimulation reaching a peak after 4.0s at 18.2±1.3% (4.0s, 18.6±1.2%) of baseline level for S1BF, 4.0s at 4.6±0.5% (3.2s, 5.8±0.7%) for VPM, and 2.4s at 2.9±0.7% (3.2s, 4.0±0.8%) for Po from the left stimulation (right, respectively; mean±95%CI) before slowly returning to baseline level (black plots, Figure 3D).

During the first hour following the stroke onset, functional responses in the left hemisphere (i.e., ipsilesional) were abolished in the S1BF, S2, and AuD (0-1hr Post-stroke, ****pvalue<0.0001), significantly decreased in the VPM (0-1hr Post-stroke, ***pvalue<0.001), and unchanged in Po and VPL (0-1hr Post-stroke, ns; Figure 3D) when compared with the pre-stroke responses (Pre-stroke, black plots, Figure 3D). Over the two following hours (i.e., 1-2hr and 2-3hr Post-stroke), the hemodynamic responses captured in these regions remained similar as those detected during the first post-stroke hour (green plots, Figure 3D).

Regarding the right hemisphere (i.e., contralesional), the functional responses of S1BF and VPM were conserved during the first hour after the stroke onset (ns, 0-1hr Post-stroke; orange plots, Figure 3D). During the two following hours, signal changes in S1BF show a significant and progressive decrease of activity (1-2hr Post-stroke **pvalue<0.01, 2-3hr Post-stroke ****pvalue<0.0001; orange plots, Figure 3D; Similar observation were made for S2 and AuD) whereas responses in VPM remained stable during the second hour post-stroke (1-2hr, ns) before significantly decreasing during the third hour (2-3hr Post-stroke *pvalue<0.05; orange plots, Figure 3D). Finally, the functional responses in VPM and Po remained unchanged over the 3hrs following the stroke onset (bottom panel, Figure 3D).

Delayed alteration of the somatosensory thalamo-cortical pathway

A secondary objective of this work was to evaluate the fUS ability to identify potential delayed functional alteration within a few days after the initial injury. Two animals were imaged five days after the MCAo following the same experimental, stimulation, imaging, and processing conditions as for the early post-stroke session.

First, we used the vascular markers and the shape of the hippocampus²⁸ to find back the coronal cross-section imaged during the pre-stroke session. Five days after the MCA occlusion, the functional responses to left whisker stimulation were still detected in the right hemisphere (i.e., contralesional), at the cortical and subcortical levels (orange; Figure 4A). As for the early post-stroke imaging period, the functional responses to right whisker stimulation were only detected in the subcortical nuclei and not at the cortical level (green; Figure 4A).

Figure 4.

Second, we extracted and compared the temporal plots of the functional responses gathered 5d after the stroke with the one obtained from the same two animals at the pre-stroke and 3hrs post-stroke timepoints (Figure 4B). At this later time point, the functional responses in the left S1BF (dark green plot, left panel, Figure 4B. Similar observation were made for the S2 and AuD) remained abolished when compared with the pre-stroke period (black plot; ****pvalue<0.0001), while slightly increased when compared with the 3hrs post-stroke timepoint (green plot; **pvalue<0.01). The responses detected in the VPM 5d after the stroke onset (dark green plot, left panel, Figure 4B) were largely decreased not only when compared with the pre-stroke signal (black plot; ****pvalue<0.0001) but also with the 3hrs post-stroke trace (green plot; ****pvalue<0.0001). Interestingly, both the amplitude and time-to-peak of the hemodynamic response function were very similar to those from the early post-stroke signal (i.e., 3hrs post-stroke); however, the post-peak period was largely dampened in the 5d post-stroke signal. A similar alteration of the hemodynamic response function was also observed for the 5d post-stroke signal extracted from the Po nucleus when compared to the pre-stroke and 3hrs post-stroke signals (**pvalue<0.01 and ***pvalue<0.001, respectively; left panel, Figure 4B. Similar observation were made for the VPL).

Regarding the right hemisphere (i.e., non-ischemic; right panel, Figure 4B), the S1BF functional responses to left whisker stimulation were still significantly reduced when compared with pre-stroke responses (black plot, ****pvalue<0.0001) but remained similar to the traces detected at 3-hr post-stroke (orange plot, non-significant). As for the left VPM, both the amplitude and time-to-peak of the hemodynamic responses from the right VPM responses were consistent with pre-stroke and 3hr post-stroke values but the post-peak signal was decreased (brown plot, *pvalue<0.05 and **pvalue<0.01 respectively). The functional responses extracted from the Po and VPL did not show significant changes when compared to pre-stroke and 3hrs post-stroke responses.

Discussion

With this proof-of-concept study, we document on the feasibility of the continuous brain hemodynamics recording of a focal cerebral ischemia after MCAo in conscious rats. Using functional ultrasound imaging, we were able to extract multiple parameters (i.e., ischemia, location and spreading depolarization), characteristic of such cortical stroke. Then, we report on how the functional sensorimotor thalamo-cortical circuit was altered at early and late post-stroke stages.

Compared to highly-invasive conventional strategies such as clipping or suturing^1,2, the FeCl₃ model used here, is well suited to study stroke under awake conditions. Indeed, the use of FeCl₃ requires less manipulation, allows to maintain the dura intact and strongly reduces the risk of hemorrhage^30,31 and animal loss. Furthermore, the FeCl₃ model closely mimics key human stroke features including focal ischemia, creation of blood clot, possibility of vessel recanalization, and penumbral tissue^30,31.

The FeCl₃-induced MCAo showed an abrupt and massive drop of blood perfusion remaining constant during the entire recording period. The ischemia was confined within the cortical territory perfused by the MCA (Figure 2B), and the infarct (location and size; Figure 2G) is in agreement with previous observations^29,31. We also detected transient hyperemic events associated with spreading depolarizations (SDs) within the peri-ischemic territory, with occurence, frequency and amplitude of the hemodynamic waves (Figure 2D-F) consistent with prior observation^29,47–52. Moreover, the spatiotemporal dynamic of the FeCl₃-induced MCAo is consistent with previous fUS imaging reports on cortical ischemia with various stroke models^13,29,53.

On top of tracking large hemodynamic variation (i.e. ischemia, SDs), one asset of the fUS imaging technology relies on its ability to track subtle hemodynamic changes in sparse brain^13,23–28. Therefore, we have evaluated how evoked functional responses reorganize at early and late timepoints after stroke induction. Functional responses to mechanical whisker stimulation were detected in several regions relaying the information from the whisker to the cortex, including the VPM and Po nuclei of the thalamus, and S1BF, the somatosensory barrel-field cortex. Responses were also observed in the S2 cortex involved in the multisensory integration of the information^37,38,54, the auditory cortex as it receives direct efferent projection from S1BF^39,54, and the VPL nuclei of the thalamus connected via corticothalamic projections³⁹.

Functional responses extracted in the left hemisphere affected by the focal ischemia (i.e., ipsilesionel) show a primary alteration of the whisker-to-barrel pathway within the first hour after the stroke onset. While the abrupt loss in S1BF responses was mainly driven by the focal ischemia, the immediate but partial drop in VPM responses (Figure 3D) might result from the direct the loss of the excitatory corticothalamic feedback to the VPM^45,55,56. The absence of such cortical feedback suggests that the dampened functional responses might be driven by the intrinsic activity of the VPM in response to whisker stimulation. Five days after the initial injury, nuclei of the thalamus (VPM and Po) were subjected to a delayed and robust functional alteration (Figure 4B), probably associated with diaschisis, as previously characterised by tissue staining, reduction of metabolism, and perfusion^18–20,43. Functional responses of the S1BF extracted from the right hemisphere (i.e., contralesionel) show a significant decrease shortly after the stroke onset (Figure 3D), and still detected at day 5, could be provoked by a loss of transcortical excitability^57,58. The late drop in VPM responses might be explained by corticothalamic modulation of the projections toward VPM^37,58.

Further work will need to dissect the complex and long-lasting post-stroke alterations of the functional whisker-to-barrel pathway, including at the neuronal level, as fUS only reports on hemodynamics as a proxy of local neuronal activity^{24,26,59–61}. Another limitation relies on the experimental condition as our brain imaging paradigm was constrained to a single cross-section, thus missing out-of-plane brain regions also affected by the stroke (e.g., ischemic size, infract extension, origin, and propagation pattern of SDs34) or involved in the whisker network (e.g., superior colliculus, striatum, amygdala and cerebellum)38. To overcome such limitation, one can extend the size of the cranial window to allow for larger scale imaging either by sequentially scanning the brain^{26,28,29,53,59,60,62}, or by using the recently developed volumetric fUS which provides whole-brain imaging capabilities in anesthetized⁶³ and awake rats²⁷.

Beyond studying the whisker-to-barrel somatosensory circuit, the brain-wide capability of fUS opens the door to investigate on stroke-affected brain circuits and functions using transgenic lines combined with opto-/chemo-genetic strategies as the technology is fully mature for mice studies^26–28,60.

Supplementary Materials

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Table 1.

Supplementary Table 2.

Movie 1. Movie of hemodynamic changes induced by MCA occlusion using FeCl₃ in awake head-fixed rats. Raw images.

Movie 2. Movie of thalamo-cortical functional responses to left and right whisker stimulation before and 3hrs after stroke onset.

Author contribution

Funding

This work is supported by grants from the Fondation Leducq (15CVD02) and KU Leuven (C14/18/099-STYMULATE-STROKE). The functional ultrasound imaging platform is supported by grants from FWO (MEDI-RESCU2-AKUL/17/049, G091719N, and 1197818N), VIB Tech-Watch (fUSI-MICE), Neuro-

Electronics Research Flanders TechDev fund (3D-fUSI project).

Acknowledgements

The authors thank the members of the Fondation Leducq network #15CVD02, Dr. M. Grillet, T. Lambert and lab members for their insightful comments and discussions. We thank NERF animal caretakers, including I. Eyckmans, F. Ooms, and S. Luijten, for their help with the management of the animals. Figures 1-3 use BioRender.com icons.

Competing interests

A.U. is the founder and a shareholder of AUTC company commercializing functional ultrasound imaging solutions for preclinical and clinical research.