Functional ultrasound imaging of stroke in awake rats

Reviewer #1 (Public Review):

The authors apply a new approach to monitor brain-wide changes in sensory-evoked hemodynamic activity after focal stroke in fully conscious rats. Using functional ultrasound (fUS), they report immediate and lasting (up to 5 days) depression of sensory-evoked responses in somatosensory thalamic and cortical regions.

Strengths: This a technically challenging and proof-of-concept study that employs new methods to study brain-wide changes in sensory-evoked neural activity, inferred from changes in cerebral blood flow. Despite the minor typos/grammatical errors and small sample size, the authors provide compelling images and rigorous analysis to support their conclusions. Overall, this was a very technically difficult study that was well executed. I believe that it will pave the way for more extensive studies using this methodological approach. Therefore I support this study and my recommendations to improve it are relatively minor in nature and should be simple for the authors to address.

Weaknesses: The primary weakness of this paper is the small sample sizes. Drawing conclusions based on the small sham control group (n=2) or 5-day stroke recovery group (n=2), is rather tenuous. One way to alleviate some uncertainty with regard to the conclusions would be to state in the discussion that the findings (ie. loss of thalamocortical function after stroke) are perfectly consistent with previous studies that examined thalamocortical function after stroke. The authors missed some of these supporting studies in their reference list (see PMID: 28643802, 1400649). A second issue that can easily be resolved is their analysis of the 69 brain regions. This seems like a very important part of the study and one of the primary advantages of employing efUS. As presented, I had difficulty seeing the data. I think it would be worthwhile to expand Fig 3 (especially 3C) into a full-page figure with an accompanying table in the Supplementary info section describing the % change in CBF for each brain region.

Other Recommendations for the authors:.
- Since there is variability in spreading depolarizations, was there any trend in the relationship between # SD's and ischemic volume? I know there are few data points but a scatterplot might be of interest.
- For statistical comparisons of 'response curves' in Fig 3 and 4, what exactly was the primary dependent measure: changes in peak amplitude (%) or area under the curve?
- There are several typos and minor grammatical errors in the manuscript. Some editing is recommended.

Reviewer #2 (Public Review):

Brunner et al. present a new and promising application of functional ultrasound (fUS) imaging to follow the evolution of perfusion and haemodynamics upon thrombotic stroke in awake rats. The authors leveraged a chemically induced occlusion of the rat Medial Cerebral Artery (MCA) with ferric chloride in awake rats, while imaging with fUS cerebral perfusion with high spatio and temporal resolution (100µm x 110µm x 300µm x 0.8s). The authors also measured evoked haemodynamic response at different timepoints following whisker stimulation.

As the fUS setup of the authors is limited to 2D imaging, Brunner and colleagues focused on a single coronal slice where they identified the primary Somatosensory Barrel Field of the Cortex (S1BF), directly perfused by the MCA and relay nuclei of the Thalamus: the Posterior (Po) and the Ventroposterior Medial (VPM) nuclei of the Thalamus. All these regions are involved in the sensory processing of whisker stimulation. By investigating these regions the authors present the hyper-acute effect of the stroke with these main results:

- MCA occlusion results in a fast and important loss of perfusion in the ipsilesional cortex.
- Thrombolysis is followed by Spreading Depolarisation measured in the Retrosplenial cortex.
- Stroke-induced hypo-perfusion is associated with a significant drop in ipsilesional cortical response to whisker stimulation, and a milder one in ipsilesional subcortical relays.
- Contralesional hemisphere is almost not affected by stroke with the exception of the cortex which presents a mildly reduced response to the stimulation.

In addition, the authors demonstrate that their protocol allows to follow up stroke evolution up to five days post-induction. They further show that fUS can estimate the size of the infarcted volume with brilliance mode (B-mode), confirming the presence of the identified lesional tissue with post-mortem cresyl violet staining.

Upon measuring functional response to whisker stimulation 5 days after stroke induction, the authors report that:
- The ipsilesional cortex presents no response to the stimulation
- The ipsilesional thalamic relays are less activated than hyper acutely
- The contralesional cortex and subcortical regions are also less activated 5d after the stroke.

These observations mainly validate the new method as a way to chronically image the longitudinal sequelae of stroke in awake animals. However, the potentially more intriguing results the authors describe in terms of functional reorganization of functional activity following stroke appear to be preliminary, and underpowered ( N = 5 animals were imaged to describe hyper-acute session, and N = 2 in a five day follow-up). While highly preliminary, the research model proposed by the author (where the loss of the infarcted cortex induces reduces activity in connected regions, whether by cortico-thalamic or cortico-cortical loss of excitatory drive), is interesting. This hypothesis would require a greatly expanded, sufficiently powered study to be validated (or disproven).

Reviewer #3 (Public Review):

The authors set out to demonstrate the utility of functional ultrasound for evaluating changes in brain hemodynamics elicited acutely and subacutely by the middle cerebral artery occlusion model of ischemic stroke in awake rats.

Functional ultrasound affords a distinct set of tradeoffs relative to competing imaging modalities. Acclimatization of rats for awake imaging has proven difficult with most, and the high quality of presented data in awake rats is a major achievement. The major weakness of the approach is in its being restricted to single-slice acquisitions, which also complicates the registration of acquisition across multiple imaging sessions within the same animal. Establishing that awake imaging represents an advancement in relation to studies under anesthesia hinges upon the establishment of the level of stress experienced by the animals in the course of imaging, i.e., requires providing data on the assessment of stress over the course of these long imaging sessions. This is particularly significant given how significant a stressor physical restraint has been established to be in rodent models of stress. Furthermore, assessment of the robustness of these measurements is of particular significance for supporting the wide applicability of this approach to preclinical studies of brain injury: the individual animal data (effect sizes, activation areas, kinetics) should thus be displayed and the statistical analysis expanded. Both within-subject, within/across sessions, and across-subjects variability should be evaluated. Thoughtful comments on the relationship between power doppler signal and cerebral blood volume are important to include and facilitate comparisons to studies recording other blood volume-weighted signals. Finally, the contextualization of the observations with respect to other studies examining acute and subacute changes in brain hemodynamics post focal ischemic stroke in rats is needed. It is also quite helpful, for establishing the robustness of the approach, when the statistical parametric maps are shown in full (i.e. unmasked).