Introduction

Focused ultrasound (FUS) with the intravascular presence of circulating microbubbles (MBs) can noninvasively and locally disrupt the blood-brain barrier (BBB) as a result of ultrasonic cavitation effect.1-3 However, clinical implementation of FUS requires careful evaluation of its safety and efficacy, including the identifications of brain tissue injury and neuromodulation effect. Ultra-high field magnetic resonance imaging (MRI) has been demonstrated as a precise tool to provide BLOD change in such a local BBB disruption.4,5 The aim of this study was identifying the duration of BBB opening after FUS sonication via contrast-enhanced T1-weighted images (T1-WIs) and confirming the accompanied with neuromodulation effect by measuring the blood-oxygen-level-dependent (BOLD) responses using the visual evoked fMRI in 7T MRI.

Methods

Two adult male crab-eating macaques (weight 3.8 kg and 3 kg) were studied. Animals were placed in a MR-compatible stereotaxic frame after initially anesthesia with 10 mg/kg ketamine and then were maintained a stable state of anesthesia with isoflurane (0.2-0.3%), ketamine (0.1 mg/kg*h) and vecuronium (0.1 mg/kg*h) during functional MRI data acquisition. EtCO2 of 23-27 mHg and SPO2 above 96%, and a circulated warm water pad was used to maintain rectal temperature at 37±0.5°C. Immediately after microbubble administration, the 305 kHz lab-designed MRI-compatible FUS transducer (Figure 1) applied transcranial ultrasound exposure under the following parameters: mechanical index (MI) = 0.55, sonic duration = 90 s, pulse-repetition frequency = 1 Hz, targeting at right primary visual cortex. The visual binocularly block stimulus were performed pre- and post- FUS by a Visual Stimulation System (SINORAD, China) with a block-design with 36 s initial rest, 36 s stimulation on followed by 36 s OFF. All MRI images were acquired using 7T research system (Siemens, Erlangen, Germany) for up to 48 h. The contrast-enhanced T1-WIs by a turbo spin echo (TSE) sequence (TR=2300 ms, TE=18 ms, BW=100 Hz, Voxel size: 1.5×1.5×1.5 mm3)g. The MRI contrast-agents, Gd-DTPA (Gd-DTPA, 0.3 mmol/kg), were IV injected at 0.1-h, 3-h, 6-h and 48-h post ultrasound to evaluate the BBB permeability. BOLD-fMRI data were acquired by a prototype multiband echo planar imaging (EPI) sequence (TE = 24.2 ms, TR = 2000 ms, BW =1710 Hz, voxel size = 1.5×1.5×1.5 mm3). Diffusion Weighted Imaging (DWI) sequence (TE = 63 ms, TR = 5000 ms, BW =908 Hz, voxel size = 1.5×1.5×1.5 mm3). BOLD signals were analyzed by the seed-based correlation analysis with a 2 x 2 pixel region of interest (ROI) in the visual cortex using AFNI.

Results and Discussion

Contrast-enhanced T1-WIs at different time points revealed the local BBB opening in the right primary visual cortex (Figure 2A). Gd-DTPA leakage at the FUS exposure site was evaluated via SI changes obtained from the subtracted T1-WIs between pre- and post-FUS exposure to identify BBB-opening. The SI change significantly decreased at 5 h post-FUS exposure and show a more obvious decrease trend at 6 h after Gd injection, representing recovery of the BBB (Figure 2B). Figure 3 shows no edema in the FUS targeted area compared with the DWI images pre FUS exposure, proving that the process of BBB opening didn’t result in brain tissue injury. Figure 3 is the comparison of visual stimulus-evoked fMRI BOLD responses pre- and post- FUS-induced BBB opening in the macaque brain. According to the results of visual stimulus at different time points, the group-averaged BOLD signal change values significantly increased in both FUS and non-FUS side (Figure 4A). The light Yellow-shaded area indicates the visual stimulus duration. Figure 4B showed that the average BOLD responses peaked at 1h at FUS side while the BOLD changes continually increased at 1 h and 2 h and the maximum at 6 h at non-FUS side. The BOLD change may caused by animal physiological status even EtCO2 and SPO2 were maintained stably. The results indicated the FUS-induced BBB opening didn't cause any tissue damage and functional influence in monkey brain.

Conclusion

We confirmed that MB-presented FUS exposure could successfully induce temporal and noninvasive BBB opening in monkeys monitoring by 7T MRI. It is also inferred that the BBB-opened process didn't induce additional neuromodulation. This research, together with 7T MRI, might potentially provide the basis for clinical application.

References

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