Transcranial functional ultrasound imaging of the brain using microbubble-enhanced ultrasensitive Doppler

Abstract

Functional ultrasound (fUS) is a novel neuroimaging technique, based on high-sensitivity ultrafast Doppler imaging of cerebral blood volume, capable of measuring brain activation and connectivity in rodents with high spatiotemporal resolution (100μm, 1ms). However, the skull attenuates acoustic waves, so fUS in rats currently requires craniotomy or a thinned-skull window. Here we propose a non-invasive approach by enhancing the fUS signal with a contrast agent, inert gas microbubbles. Plane-wave illumination of the brain at high frame rate (500Hz compounded sequence with three tilted plane waves, PRF=1500Hz with a 128 element 15MHz linear transducer), yields highly-resolved neurovascular maps. We compared fUS imaging performance through the intact skull bone (transcranial fUS) versus a thinned-skull window in the same animal. First, we show that the vascular network of the adult rat brain can be imaged transcranially only after a bolus intravenous injection of microbubbles, which leads to a 9dB gain in the contrast-to-tissue ratio. Next, we demonstrate that functional increase in the blood volume of the primary sensory cortex after targeted electrical-evoked stimulations of the sciatic nerve is observable transcranially in presence of contrast agents, with high reproducibility (Pearson's coefficient ρ=0.7±0.1, p=0.85). Our work demonstrates that the combination of ultrafast Doppler imaging and injection of contrast agent allows non-invasive functional brain imaging through the intact skull bone in rats. These results should ease non-invasive longitudinal studies in rodents and open a promising perspective for the adoption of highly resolved fUS approaches for the adult human brain.

Keywords: Blood volume; Functional ultrasound imaging; Microbubbles; Primary sensory cortex; Somatosensory activation; Transcranial.

Fig. 1

Schematic view of the fUS imaging setup and fUS imaging protocol. A: The 15 MHz probe was placed on the top of the animals' heads and coupled via ultrasound gel. A stereotaxic frame immobilized the skull. An isolated constant current stimulator delivered five electrical stimuli alternatively on the left and right sciatic nerves (LSN and RSN, respectively). The panel presents the stimulation parameters. B: The continuous-fUS insonification sequence consisted in three tilted plane waves (− 3°, 0°and 3°). The frame rate was set to 500 Hz to sample the blood flow without aliasing. The resulting three low quality images were coherently summed to obtain a single high quality B-mode image of the brain. The sequence was repeated 374 times leading to 150 s acquisition time to stack a set of 200 high quality images per each sequence. The power Doppler images were acquired over 150 s in order to fully recover the electrical-evoked activation of the brain.

Fig. 2

High-resolution ultrafast Doppler imaging of the rat brain vasculature through the intact skull (IS) after intravenous injection of microbubble contrast agents. A–C: Schematic of the three imaging configurations used: bilateral thinned skull window (TSW, A), intact skull (IS, B) or unilateral thinned skull window (C). D–F: Examples of vascular maps obtained with the different imaging modalities in absence (1) or presence of microbubbles (2). D: High quality imaging of both superficial and deep cerebral structures was obtained in the control configuration, i.e. without microbubbles through the bilateral thinned-skull window. E–F: In absence of contrast agent, the microvascular structures of the brain are no longer distinguishable through the intact skull (IS). However, the loss of signal due to the cranial bone is fully compensated by the injection of 150 μl of microbubble contrast agents. The yellow boxes in D–F show the fUS signal in the choroid plexus of the lateral ventricle, allowing the correct US probe placement above the S1HL at Bregma − 1 mm. Scale bar: 2 mm.

Fig. 3

Antero-posterior fUS scans allow to map the vasculature of the rat brain through the intact skull (IS) after an intravenous injection of microbubbles. Injection of 150 μl of contrast agent led to a clear ultrasound signal at all different coronal imaging planes, at full depth. The stereotaxic coordinates (antero-posterior distances to Bregma) are reported on the top of each Doppler acquisition. Scale bar: 2 mm.

Fig. 4

Microbubble contrast agents allow fUS imaging of cortical hyperemia evoked by electrical stimulation of the sciatic nerve, through the intact skull (IS). A–E, The left column shows the vascularization maps obtained by power Doppler imaging of the investigated coronal plane at Bregma − 1 mm in representative animals for each imaging condition. The middle and the right columns show the hemodynamic responses evoked by stimulation of either the right (RSN) or the left sciatic nerves (LSN). The correlation coefficient shown in the color bar represents the temporal correlation between the evoked task and the hemodynamic response recorded in the S1HL. Scale bar: 2 mm.

Fig. 5

Summary of successful trials on each animal for the different fUS imaging configurations. Functional activations of the S1HL were performed on n = 7 animals. A distinction between transcranial hemispheres and thinned skull hemispheres led to a total of n = 12 transcranial S1HL activation through the intact skull when boluses of microbubbles were delivered in the bloodstream and n = 12 activations on the thinned skull windows (n = 6 without microbubbles and n = 6 with injection of contrast agent). fUS imaging through the intact skull in absence of microbubbles was not applicable (NA).

Fig. 6

Sensitivity and robustness of contrast-agent enhanced transcranial Doppler imaging. A: Averaged hemodynamic response function (n = 6, black curve) evaluated on the thinned-skull hemispheres without injection of microbubbles (control imaging protocol). The stimulation pattern is shown in red. Due to physiological adaptation to the electrical stimulations, the peak amplitude displays a decrease of ~ 2% from the first to the last activation. B: Time evolution of the hemodynamic responses of a representative animal with the transcranial imaging protocol (blue curve). The red curve shows the fUS signal profile corrected for the signal diminution, due to the dissolution of the injected boluses of contrast agent. C: Mean evoked hemodynamic response function of the three imaging protocols: thinned-skull window (TSW) without (black curve, n = 6) and with (blue curve, n = 6) microbubbles and intact skull (IS) transcranial hemispheres with microbubbles (red curve, n = 12). The fUS signal in response to the neuronal stimulation is represented as the mean ± standard deviation across trials and across subjects. D: Peak enhancement of the three imaging protocols: TSW hemispheres with microbubbles (blue, n = 6); 2, transcranial (IS) hemispheres with microbubbles (red, n = 12); 3, TSW hemispheres without microbubbles (black, n = 6). Statistics: paired Student's t-test, compared to the control condition (TSW no bubbles). P values: ****P < 0.0001.