Gabriel Montaldo

Linking circuit level activity to large scale functional organization requires imaging methods combining high spatial resolution, broad coverage, and single trial sensitivity. We present volumetric functional ultrasound imaging (3D-fUS) in behaving macaques, enabling imaging of ~1 cm³ cortical volumes at high spatiotemporal resolution (100 × 150 × 150 μm³ voxels, 1.67 Hz). Visually evoked responses were reliably detected at the level of single trials and single voxels, substantially reducing experimental time. To enable model-based analyses analogous to functional magnetic resonance imaging (fMRI), we estimated a fUS hemodynamic response function (fUS-HRF) that was consistent across subjects, cortical areas, and visual stimuli and was well approximated by a gamma function. Compared with fMRI HRFs, the fUS-HRF exhibited faster dynamics, enabling shorter and more closely spaced stimulus presentations. Together, these results establish 3D-fUS as a fast, volumetric, and circuit relevant imaging modality for efficient investigation of distributed cortical dynamics in primates.

Linking circuit level activity to large scale functional organization requires imaging methods combining high spatial resolution, broad coverage, and single trial sensitivity. We present volumetric functional ultrasound imaging (3D-fUS) in behaving macaques, enabling imaging of ~1 cm ³ cortical volumes at high spatiotemporal resolution (100 × 150 × 150 μm ³ voxels, 1.67 Hz). Visually evoked responses were reliably detected at the level of single trials and single voxels, substantially reducing experimental time. To enable model-based analyses analogous to functional magnetic resonance imaging (fMRI), we estimated a canonical fUS hemodynamic response function (fUS-HRF) that was consistent across subjects, cortical areas, and visual stimuli and was well approximated by a gamma function. Compared with canonical fMRI HRFs, the fUS-HRF exhibited faster dynamics, enabling shorter and more closely spaced stimulus presentations. Together, these results establish 3D-fUS as a fast, volumetric, and circuit relevant imaging modality for efficient investigation of distributed cortical dynamics in primates.

Functional ultrasound (fUS) imaging is rapidly gaining interest for its unprecedented ability to study large-scale brain dynamics, yet its adoption and broader dissemination have been hindered by the lack of standardized tools and methodologies to analyze and interpret its rich datasets. We present OpenfUS Analyzer (OfUSA), a companion software suite designed to help researchers quickly engage with fUS data and perform the full range of analyses needed to generate publication-ready results and figures without relying on additional software. OfUSA offers a versatile and modular architecture including preprocessing, recording quality assessment, signal dynamics exploration, statistical analysis and visualization. These functions are separated yet easily combined into analytic pipelines through a programming-free graphical interface. The framework can be applied across species and experimental contexts, either by registering data to anatomical atlases, as shown here for the mouse brain, or by analyzing data without atlas constraints, as illustrated in a primate dataset. This flexibility, together with its comprehensive functionality, makes OfUSA a practical solution for standardized and reproducible analysis of fUS data in both preclinical and translational research. Using OfUSA, we demonstrate the capacity to detect stimulus-evoked responses with high sensitivity, identify their spatial localization within brain networks, and quantify both their extent and temporal dynamics. These results highlight the software’s ability to capture robust activation patterns and provide detailed insights into brain function, thereby accelerating the use of fUS as a powerful tool for systems neuroscience. Highlights: We present OpenfUS Analyzer (OfUSA), a novel software platform for the complete analysis of functional ultrasound (fUS) datasets. OfUSA combines a user-friendly graphical interface with a standardized, flexible workflow and powerful visualization tools, making it an ideal solution for fUS researchers at all experience levels. The software’s utility is first demonstrated through the analysis of rodent fUS data using a standardized atlas, while its versatility is further emphasized by the successful analysis of a primate fUS dataset without a template, thereby illustrating its adaptability to non-standard experimental conditions.

Continuous real-time assessment of cerebral blood flow (CBF) and cerebrovascular autoregulation (CA) remains a major unmet clinical need in acute brain injury. Methods such as laser Doppler flowmetry (LDF), transcranial Doppler, or indirect indices lack accuracy and robustness. Functional ultrasound (fUS) is an emerging modality combining high spatiotemporal resolution, large field-of-view, and sensitivity to blood velocity and volume, making it a promising neuromonitoring tool. Piglets were equipped with arterial blood pressure (ABP), intracranial pressure (ICP), and LDF probes, plus cranial windows for fUS and red blood cell (RBC) flux imaging. CA was challenged by non-pharmacological ABP manipulation via intraaortic or intracaval balloon inflation. fUS hemodynamic parameters were compared with other modaliters across a CPP range of 10–150 mmHg. fUS provided continuous, stable intensity- and velocity-derived parameters across vessels types. CBF estimates correlated strongly with RBC flux and showed reproducibility comparable to LDF, with lower inter-animal variability. Autoregulation breakpoints were reliably identified by fUS, particularly the lower limit, while the upper limit was more variable. Parcellation confirmed robustness of fUS across brain regions. fUS images CBF and CA with higher stability and reproducibility than standard approaches, supporting its applicability for bedside neuromonitoring and clinical translation.

Anesthesia is a major confounding factor in preclinical stroke research as stroke rarely occurs in sedated patients. Moreover, anesthesia affects both brain functions and the stroke outcome acting as neurotoxic or protective agent. So far, no approaches were well suited to induce stroke while imaging hemodynamics along with simultaneous large-scale recording of brain functions in awake animals. For this reason, the first critical hours following the stroke insult and associated functional alteration remain poorly understood. Here, we present a strategy to investigate both stroke hemodynamics and stroke-induced functional alterations without the confounding effect of anesthesia, i.e., under awake condition. Functional ultrasound (fUS) imaging was used to continuously monitor variations in cerebral blood volume (CBV) in +65 brain regions/hemisphere for up to 3hrs after stroke onset. The focal cortical ischemia was induced using a chemo-thrombotic agent suited for permanent middle cerebral artery occlusion in awake rats, and followed by ipsi- and contralesional whiskers stimulation to investigate on the dynamic of the thalamo-cortical functions. Early (0-3hrs) and delayed (day 5) fUS recording enabled to characterize the features of the ischemia (location, CBV loss), spreading depolarizations (occurrence, amplitude) and functional alteration of the somatosensory thalamo-cortical circuits. Post-stroke thalamo-cortical functions were affected not only early after the stroke onset but were also altered secondarly and remotely from the initial insult. Overall, our procedure enables early, continuous, and chronic evaluations of hemodynamics and brain functions which, combined to stroke or other pathologies, aims to better understand physiopathologies toward the development of clinically relevant therapeutic strategies.

Anesthesia is a major confounding factor in preclinical stroke research as stroke rarely occurs in sedated patients. Moreover, anesthesia affects both brain functions and the stroke outcome acting as neurotoxic or protective agent. So far, no approaches were well suited to induce stroke while imaging hemodynamics along with simultaneous large-scale recording of brain functions in awake animals. For this reason, the first critical hours following the stroke insult and associated functional alteration remain poorly understood. Here, we present a strategy to investigate both stroke hemodynamics and stroke-induced functional alterations without the confounding effect of anesthesia, i.e., under awake condition. Functional ultrasound (fUS) imaging was used to continuously monitor variations in cerebral blood volume (CBV) in +65 brain regions/hemisphere for up to 3hrs after stroke onset. The focal cortical ischemia was induced using a chemo-thrombotic agent suited for permanent middle cerebral artery occlusion in awake rats, and followed by ipsi- and contralesional whiskers stimulation to investigate on the dynamic of the thalamo-cortical functions. Early (0-3hrs) and delayed (day 5) fUS recording enabled to characterize the features of the ischemia (location, CBV loss), spreading depolarizations (occurrence, amplitude) and functional alteration of the somatosensory thalamo-cortical circuits. Post-stroke thalamo-cortical functions were affected not only early after the stroke onset but were also altered secondarly and remotely from the initial insult. Overall, our procedure enables early, continuous, and chronic evaluations of hemodynamics and brain functions which, combined to stroke or other pathologies, aims to better understand physiopathologies toward the development of clinically relevant therapeutic strategies.

The brain is composed of a dense and ramified vascular network comprising various sizes of arteries, veins, and capillaries. One way to assess the risk of cerebrovascular pathologies is to use computational models to predict the physiological effects of a reduction of blood supply and correlate these responses with observations of brain damage. Therefore, it is crucial to establish a detailed 3D organization of the brain vasculature, which could be used to develop more accurate in silico models. For this purpose, we have adapted our open-access functional ultrasound imaging platform previously designed for recording brain-wide activity that is now capable of fast and reproducible acquisition, segmentation, and reconstruction of the cortical vasculature. For the first time, it allows us to digitize the cortical vasculature in awake rodents with a ∼100 µm ³ spatial resolution. Contrary to most available strategies, our approach can be performed in vivo within minutes. Moreover, it is easy to implement since it neither requires exogenous contrast agents nor long post-processing time. Hence, we performed a cortex-wide reconstruction of the vasculature and its quantitative analysis, including i) classification of descending arteries versus ascending veins in more than 1500 vessels/animal, ii) quick estimation of their length. Importantly, we confirmed the relevance of our approach in a model of cortical stroke, which enables quick visualization of the ischemic lesion. This development contributes to extending the capabilities of ultrasound neuroimaging to understand better cerebrovascular pathologies such as stroke, vascular cognitive impairment, and brain tumors and is highly scalable for the clinic.

Positioning and navigation are essential components of neuroimaging as they improve the quality and reliability of data acquisition, leading to advances in diagnosis, treatment outcomes, and fundamental understanding of the brain. Functional ultrasound (fUS) imaging is an emerging technology providing high-resolution images of the brain vasculature, allowing for the monitoring of brain activity. However, as the technology is relatively new, there is no standardized tool for inferring the position in the brain from the vascular images. This study presents a deep learning-based framework designed to address this challenge. Our approach uses an image classification task coupled with a regression on the resulting probabilities to determine the position of a single image. We conducted experiments using a dataset of 51 rat brain scans to evaluate its performance. The training positions were extracted at intervals of 375 µm, resulting in a positioning error of 176 µm. Further GradCAM analysis revealed that the predictions were primarily driven by subcortical vascular structures. Finally, we assessed the robustness of our method in a cortical stroke where the brain vasculature is severely impaired. Remarkably, no specific increase in the number of misclassifications was observed, confirming the method’s reliability in challenging conditions. Overall, our framework provides accurate and flexible positioning, not relying on a pre-registered reference but on conserved vascular patterns.

Ischemic stroke occurs with no warning, 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 (100μm, 2Hz) 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 highly variable between animals. Still, both parameters were, on average, in good agreement with the final ischemic lesion volume measured 24hrs after stroke onset for the MCAo but not the CCAo+MCAo model. For the latter, we observed that the infarct was extended to regions that were initially not ischemic and located rostrally of the ischemic core. These regions strongly colocalize with the origin of transient hemodynamic events associated with spreading depolarizations.