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Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation
Journal Pre-proofs Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation Zhiwei Cui, Dapeng Li, ShanShan Xu, Tianqi Xu, Shan Wu, Ayache Bouakaz, Mingxi Wan, Siyuan Zhang PII: DOI: Reference:
S1350-4177(19)31418-X https://doi.org/10.1016/j.ultsonch.2019.104935 ULTSON 104935
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
8 September 2019 12 December 2019 17 December 2019
Please cite this article as: Z. Cui, D. Li, S. Xu, T. Xu, S. Wu, A. Bouakaz, M. Wan, S. Zhang, Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.104935
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Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation Zhiwei Cui1, #, Dapeng Li1, #, ShanShan Xu1, Tianqi Xu1, Shan Wu1, Ayache Bouakaz2, Mingxi Wan1, * and Siyuan Zhang1, *
1. The Key Laboratory of Biomedical Information Engineering of the Ministry of Education, Department of Biomedical Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China 2. UMR 1253, iBrain, Université de Tours, Inserm, France
*Corresponding
author:
Mingxi Wan, Prof. Email: http://invalid.uri/[email protected] Siyuan Zhang, Associate Prof. Email: [email protected] # These
authors contributed equally to this work.
1
Abstract Previous studies have indicated that the presence of microbubbles (MBs) during sonication has an impact on neuronal activity, while the underlying mechanisms remain to be revealed. In this study, a model for the scattered pressures produced by the pulsating lipid-encapsulated MBs in mouse brain was developed to numerically investigate the effect of MBs on neuronal activity during transcranial focused ultrasound stimulation. The additional summed scattered pressure (𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡) from the oscillating MBs was calculated from the model. The level of neuronal activity was experimentally verified using an immunofluorescence assay with antibodies against cfos. The pressure difference (∆P) between acoustic pressures at which the same level of neuronal activity is excited by ultrasound stimulation with and without MBs was obtained from the experiments. The results showed that 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 accounts for about half of the ∆P when the MBs experience a “compression-only” response. The 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 suddenly increased at a critical acoustic pressure, around which a rapid enhancement of ∆P obtained from experiment also occurred. This work suggested that the additional scattered pressures from pulsating MBs is probably a mechanism that affects neuronal activity under transcranial focused ultrasound stimulation. Keywords: Bubble dynamics; Neuronal activity; Ultrasound stimulation; Microbubble oscillation
2
1. Introduction Transcranial ultrasound combined with microbubbles (MBs) has been extensively investigated to address the treatment of some neurological diseases [1-3], and the technology have begun clinical testing [4, 5]. The safety of this technology has been evaluated using various histological evaluations in different animal models, and no overt tissue and/or cell-level damage has been observed under a wide range of ultrasound parameters [2, 6, 7]. Meanwhile, considering that MBs are administrated in central nervous system (CNS), increasing attention has been paid to the effect of ultrasound combined with MBs on neurological function. Ultrasound has been proven to be an effective neuromodulation technique, and this technique attracts increasing attention due to its high spatial selectivity and high penetrating power into the brain [8, 9]. Previous studies have shown that ultrasonic neuromodulation can alter the central and peripheral nervous systems neuronal activity including the motor and sensory cortex, thalamus, retina, and other brain regions of several animal models [10-13]. Furthermore, Deffieux et al. [14] demonstrated for the first time that US neuromodulation can affect brain cognitive function in primates. Tyler et al. [15] reported that US stimulation of the sensory cortex in humans can enhance their sensory resolution recently. Some recent studies have indicated that the oscillation of MBs might also have an impact on neuronal activity [16-19]. Ibsen et. al. [19] demonstrated that the mechanical deformations induced by MB-ultrasound interaction can amplify the response of Caenorhabditis elegans to ultrasound stimulation. Several experimental 3
evidences indicated that blood-brain barrier (BBB) opening induced by ultrasound stimulation coupled with MBs can be also capable of altering the neuronal response and neurological function in mammals [16-18]. Moreover, the alterations in neural activity can occur only when microbubbles are present [16-18], which indicates that the presence of MBs has a direct or indirect impact on neuronal activity. Existing studies have suggested that the mechanical effect of ultrasound plays an important role in the mechanism for ultrasonic neuromodulation [19-23]. However, few studies have investigated the mechanism of sonicated MBs impacting neuronal activity. Previous studies have indicated that the BBB opening is always accompanied with ultrasound sonication with MBs, however, BBB opening can result in enriching the parenchymal concentration of oxygen, glucose, ions, nutrients, and hormones [1, 16, 24]. These blood constituents diffused into the brain tissue are able to affect neuronal function [1, 16, 18, 24], thus, it is unclear that whether the blood constituents such as ions and nutrients or the oscillation of MBs influence the neuronal activity. Our previous work [25] showed that the presence of MBs can enhance the neuromodulatory effect evoked by ultrasound stimulation, and it appears that the enhancement is not dependent on BBB opening. Additionally, the neural circuit in C. elegans response to ultrasound stimuli can be amplified by the oscillation of MBs, while C. elegans have no circulatory system [19]. Therefore, it is more likely that the oscillation of MBs has a direct effect on neuronal activity. Considering the suggestion that the mechanical effects of ultrasound might be the main potential mechanism for ultrasonic 4
neuromodulation, the additional mechanical effects induced by the oscillating MBs are probably the main reason for the alteration of neuronal activity. In the acoustic field, oscillating MBs produce mechanical effects, such as shear forces, microstreaming, and microjetting, affecting the cells extremely close to or in contact with the MBs [2, 22, 23]. In general, MBs are injected intravenously, but the distance from the neuron to the nearest vessel is 13-20 μm [24, 25]. In other word, intravenous MBs and neurons are at least 13-20 μm apart, therefore, it is too long for the aforementioned mechanical effects produced by MBs to affect the neurons. Previous studies have indicated that oscillating MBs can radiate acoustic pressure into the surrounding medium [26-28]. Considering a volume of MBs were intravenously injected, the scattered pressures from MBs, the extra pressure additional to the transmitted acoustic pressure from the ultrasound transducer, should be noticeable and is supposed to be a potential mechanism of sonicated MBs affecting neuronal activity. In this paper, a model of the pulsating encapsulated bubble driven by a focused acoustic field in mouse brain was developed to investigate the effects of microbubble oscillation on neuronal activity, and the summed scattered pressures from all the MBs was calculated. The neuronal activity induced by ultrasound stimulation with and without MBs was measured by an immunofluorescence assay (IFA) with antibodies against cfos. The oscillating MBs are able to radiate pressures into the surrounding medium, generating additional pressure to the neurons, which might affect neuronal activity. The pressure difference (𝛥𝑃) between acoustic driven pressures at which the same level of 5
neuronal activity is excited by ultrasound stimulation with and without MBs was obtained. The pressure difference is likely to be produced by the pressure radiated from oscillating MBs. Therefore, in order to relate the experimental results to the numerical studies, the pressure difference was compared with the summed scattered pressures from all the MBs.
2. Theory The scattered pressures from the encapsulated MBs pulsating near a neuron in mouse cortex was estimated in order to investigate the effects of oscillating MBs on neuronal activity (Fig. 1). SonoVue® MBs were used in the experimental section of this work, which are coated by lipid. Compared to unshelled MBs, the coating alters the effective surface tension of MBs and strongly influence the bubble dynamics. The acoustic pressure used in this work might produce high-amplitude oscillations of MBs. Marmottant model [29] proposed the effective surface tension, and takes into account the physical properties of MBs’ coating. Three parameters, a buckling radius, the compressibility of the shell, and a break-up shell tension, are used to describe the physical properties of MBs’ coating. This proposition enables this model to describe large amplitude oscillations of lipid coated MBs. Therefore, Marmottant model was used in this work to describe the bubble dynamics of SonoVue® MBs. The oscillation of the MBs is defined by the following equation [29], 2𝜎(𝑅0) 𝑅 3 𝜌𝑙 𝑅𝑅 + 𝑅2 = 𝑃0 + 2 𝑅0 𝑅0
) [
(
― 𝑃0 ―
2𝜎(𝑅) 𝑅
―
4𝜇𝑙𝑅 𝑅
―
4𝜅𝑠𝑅 𝑅2
―3𝜅
]( ) (
― 𝑃𝑎𝑐(𝑡),
1―
3𝜅 𝑅 𝑐𝑙
) (1) 6
where ρl is the liquid density, P0 is the ambient pressure, R0 is the equilibrium bubble radius, R is the instantaneous bubble radius, κ is the polytropic gas exponent, μ is the surrounding liquid viscosity, κs is the shell surface dilatational viscosity, 𝑐𝑙 is the speed of sound in the liquid, and Pac is the acoustic pressure. The surface tension of the encapsulated bubble σ is defined by three states, which are described in terms of the bubble radius, 0, if R Rbuckling R2 R 2 -1 , if Rbuckling R Rbreak -up , Rbuckling liquid , if ruptured and R Rruptured
(2)
where χ is the elastic modulus of the shell, Rbuckling and Rbreak - up are the lower and upper limit radius for the elastic state of the bubble, respectively, and Rruptured is the lower limit radius for the raptured state of the bubble. The pressure scattered by an encapsulated bubble can be expressed as [28] 𝑃𝑠𝑐𝑎𝑡(𝑡, 𝑑) =
𝜌𝑙(𝑅2𝑅 + 2𝑅𝑅2) 𝑑
,
(3)
where 𝑑 is the distance from the bubble center. For the MBs at different locations, the phases of transmitted ultrasound are different. The phases of scattered pressure are also different due to the different distances between MBs and neurons. Therefore, for a neuron at the origin of coordinates, the scattered pressure (𝑃𝑠𝑐𝑎𝑡) from a MB located at (𝑥, 𝑦, 𝑧) can be described as
(
𝑧
𝑃𝑠𝑐𝑎𝑡 𝑡 ― 𝑐𝑙 +
𝑥2 + 𝑦2 + 𝑧2 𝑐𝑙
),
(4)
It is assumed that intravenously injected MBs are uniformly distributed in the 7
circulating blood. The number of all the sonicated MBs can be expressed as 𝑁𝑀𝐵 = 𝑉𝑟𝑒𝑔𝑖𝑜𝑛 × 𝐶𝐵𝑉 × 𝑐𝑀𝐵,
(5)
where 𝑉𝑟𝑒𝑔𝑖𝑜𝑛 is the volume of sonicated brain tissue, 𝐶𝐵𝑉 is the cerebral blood volume, and 𝑐𝑀𝐵 is the concentration of MBs in the circulating blood. Thus, for the neuron at the origin of coordinates, the summed scattered pressures from the uniformly distributed MBs can be estimated by the following equation,
(
𝑧
𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 = ∑𝑁 𝑃𝑠𝑐𝑎𝑡 𝑡 ― 𝑐𝑙 + 𝑀𝐵
𝑥2 + 𝑦2 + 𝑧2 𝑐𝑙
).
(6)
In order to be consistent with the experimental work described below, the corresponding physical constants were chosen in the numerical simulations. For SonoVue® MBs, the average shell elastic modulus was 𝜒 = 0.55 𝑁/𝑚 and the shell dilatational viscosity was κs = 7.2 × 10 ―9 𝑁 [30]. The break-up radius (𝑅𝑏𝑟𝑒𝑎𝑘 ― 𝑢𝑝) is associated with the limit surface tension (σbreak - up). Here, the value of σbreak - up was set to be 0.13 N/m based on BR14® MBs [29], due to the similarity in the lipid shell composition between BR14® and SonoVue® MBs [31]. The value of the other parameters used in the Marmottant model were 𝜌𝑙 = 1.06 × 103 𝑘𝑔/𝑚3, 𝑃0 = 1.013 × 105 𝑃𝑎, 𝑅0 = 1.25 × 10 ―6 𝑚, 𝜅 = 1.095, 𝜇 = 0.0035 𝑃𝑎/𝑠, 𝑐 = 1580 𝑚/ 𝑠, and σliquid = 0.056 𝑁/𝑚, which were obtained from published studies [28, 29]. Assuming that the circulating blood volume in the mouse was 1 mL [32], the concentration of MBs in the circulating blood was 𝑐𝑀𝐵 = 1.6 × 107 /𝑚𝐿 after intravenously injecting 80 μL SonoVue® MBs. The cerebral blood volume (CBV) in the mouse brain has shown significant differences between brain regions, which are 7.9% 8
and 4% in the cerebral cortex and other brain regions, respectively [33]. CBV was set as 7.9% to calculate the number of MBs in the cerebral cortex, and 4% was used when calculating the number of MBs in other brain regions. Thus, bubble separation is 20 times higher than bubble radius in this condition, which makes the interaction of bubbles is weaker [34]. This is the reason that the interaction of bubbles was not taken into account when calculating the summed scattered pressures from all MBs. Taking the FWHM of the FUS transducer and the dorsoventral distance between the upper and lower mouse brain surface of 5.5 mm into consideration, the scattered pressures from the MBs in a cylindrical region with a diameter of 4 mm and a height of 5.5 mm were used to calculate the summed scattered pressures.
3. Material and methods 3.1. Animal preparation All procedures described in this study were approved by the Institutional Animal Care and Use Committee of School of Life Science and Technology of Xi’an Jiaotong University. Adult BALB/c mice were anesthetized in an induction chamber with isoflurane (2%, 0.5 L/min O2), and then were housed in a mouse stereotaxic apparatus, where the anesthesia was continued through a custom anesthesia mask. The hair on the mice head was shaved by scissors. A heating pad was used to keep mouse body temperature at 37℃. 3.2. Ultrasound stimulation
9
A custom 620 kHz focused ultrasound (FUS) transducer (100 mm in aperture, 80 mm in focal depth) was used to sonicate the motor cortex of mice. The focus of the transducer was positioned at bregma, 1.5 mm lateral to the midline and 0.75 mm under the surface of the motor cortex. The acoustic pressure field of the FUS transducer was measured separately using a needle hydrophone (HNR-1000) in degassed water, and the full width at half maximum (FWHM) in lateral and axial dimensions was 2 mm and 10.5 mm. The acoustic pressures used in this study were peak rarefactional pressures at the focus of the transducer after mouse skull attenuation. Referring to our previous study [25], pulsed ultrasound with 2 ms pulse width, 250 Hz pulse repetition frequency, and 400 ms total sonication duration were applied in 8 s intervals. The pulses were generated by a two-channel function generator (DG5072, Rigol Inc., Beijing, China) and amplified by an RF power amplifier (25A250A, AR Europe, Bothell, USA) before being sent to the FUS transducer. 3.3. Motor response evaluation Motor responses evoked by ultrasound stimulation with and without MBs were compared to investigate the enhanced neuromodulatory effect induced by the oscillation of MBs. The motor cortex of the mouse was selected as the simulation target. Electromyogram (EMG) signals evoked by ultrasound stimulation were recorded by the tungsten electrodes inserted into the triceps muscles of mouse forelimbs. EMG signals were amplified with a gain of 1000 and bandpass filtered between 300 Hz and 5 kHz using an amplifier (model 1700, A-M Systems, Inc., Sequim, WA, USA), and 10
acquired at a 10 kHz sampling frequency using a data acquisition card (1550A, Axon Instruments, San Jose, CA, USA). Twenty ultrasound stimulations as described above in 8 s intervals were performed for each condition. For the group of ultrasound stimulation with MBs, 80 μl SonoVue® MBs (Bracco SpA, Milan, Italy) were administered intravenously, and then ultrasound stimulation was performed immediately. 3.4. Histological evaluation C-fos expression is always regarded as a marker of neuronal activity, which is a more direct evaluation revealing the neuronal activity. Therefore, in order to experimentally measure the neuronal activities under ultrasound stimulation with and without MBs, IFA with antibodies against c-fos was performed. The same ultrasound stimulation parameters as described above were applied and the motor cortex of mice was stimulated for 30 min in 8 s intervals. For the stimulation with MBs group, 80 μl SonoVue® MBs were administered by intravenous injection every 10 minutes to ensure the presence of MBs during total stimulation. The mice were sacrificed after a 30-min recovery period. Following perfusion with 4% paraformaldehyde, mouse brains were removed and fixed in 4% paraformaldehyde at 4℃ for 24 hours. The staining brain sections were cut at 40 μm in thickness using a cryotome. Then, the brain sections were incubated with the primary antibody against cfos (1:500, ab214672, Abcam) at 4℃ for 12 hours, and were washed and incubated with Invitrogen Alexa Fluor Plus 555 donkey anti-rabbit IgG secondary antibody (1:1000, 11
A32794, Invitrogen) for 1 hour at room temperature. Histological images were acquired using a fluorescence microscope, and analyzed with the ImageJ software (NIH).
4. Results and Discussion The bubble responses to 620 kHz ultrasound are shown in Fig. 2(a) and 2(b). As it can be seen, a much stronger positive radius excursion and higher scattered pressure was observed at an acoustic pressure of 0.22 MPa than under a 0.12 MPa ultrasound stimulation. The maximum and minimum radii of the bubble sonicated by the 0.12 MPa ultrasound were 1.327 μm and 0.986 μm, respectively, while, under a 0.22 MPa ultrasound stimulation, they expanded to 2.315 μm and 0.827 μm, respectively. The simulation results demonstrated that in the ultrasound field with pressure of 0.12 MPa, the corresponding peak positive and negative scattered pressures, 𝜆 4 away from the pulsating bubble, were 21.294 Pa and 64.810 Pa, respectively, which increased to 799.511 Pa and 340.597 Pa, respectively, under the 0.22 MPa acoustic driven pressure. At lower acoustic pressures, MBs experience a “compression-only” response as mentioned by Marmottant et al. [29], in which the negative MB radius excursion is much higher than the positive radius excursion. With increasing acoustic pressure, a fast expansion is likely to result in the break-up of the MBs at a critical tension σbreak - up. In this state, the surface tension of the MBs will be decreased to the surface tension of the surrounding liquid σliquid, and the MBs will oscillate as free bubbles [29]. Therefore, a sudden maximum bubble radius increase appears when the critical acoustic pressure is reached (Fig. 2(c)). Meanwhile, large oscillation of MBs above a critical pressure can 12
produce much higher velocity and acceleration of MB oscillation, which can generate higher scattered pressure (Fig. 2 (d)). In addition, for higher acoustic pressures, a much larger maximum bubble radius gives rise to a faster contraction as compression pressure arrives, which does not appear when MBs experience the “compression-only” response. This is the reason why the scattered pressure exhibits a higher peak positive pressure above the critical acoustic driven pressure. Undoubtedly, MBs cannot expand without limit. Flynn et al. indicated that the collapse threshold of gas bubbles for inertial cavitation is 2𝑅0 [35, 36], thereby, the results in this letter are presented by a dotted line when the bubble radius is greater than twice the initial radius. The FUS transducer forms a cigar-shaped focus and the acoustic driven pressure depends on the MB locations. Moreover, the phase difference between the scattered pressures produced by MBs at different locations should be taken into consideration. For a neuron at the origin of coordinates, scattered pressures from MBs at five different locations are shown in Fig. 3. The amplitude and phase of acoustic driven pressure varied with the locations in acoustic field, resulting in the various amplitude and phase of scattered pressure radiated from the MBs at different locations. Figure 4(a) shows the summed scattered pressures from all the uniformly distributed MBs under sonication, which contains strong signals at multiples of the fundamental frequency (Fig. 4(b)). In order to demonstrate the effect of MB oscillation on neuronal activity experimentally, motor responses evoked by ultrasound stimulation with and without MBs were 13
measured. Fig. 5 shows the representative EMG signals evoked by the 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The results indicated the success rates of motor response to ultrasound stimulation were significantly elevated for the group of ultrasound stimulation with MBs. The c-fos expression is considered to be a marker of neural activation [37]. Fig. 6 demonstrates the representative results of the c-fos expression in response to 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The presence of MBs increased the c-fos+ cells in the region of ultrasound stimulation, suggesting a higher level of neuronal activity. The mean densities of c-fos+ cells with and without the presence of MBs under different acoustic pressures are shown in Fig. 7(a), where the data points were fitted to a sigmoidal curve. The neuronal activity in the group of ultrasound stimulation with MBs was higher than that in the group without MBs under all acoustic pressures. In addition, for both groups, the increasing acoustic pressure induced higher level of neuronal activity, while a plateau occurred when the acoustic pressure reached near 0.36 MPa. If a level of neuronal activity is excited by the ultrasound stimulation with MBs at a certain acoustic pressure 𝑃, then ultrasound with the higher acoustic pressure 𝑃 +∆𝑃 is required in order to achieve the same level of neuronal activity when ultrasound stimulation is performed without MBs, which defines the 𝛥𝑃 at the acoustic pressure 𝑃 in Fig. 7. In other words, if acoustic pressure radiated from pulsating MBs is the main potential mechanism of enhancing neuronal activity, the 𝛥𝑃 means the experimentally obtained summed scattered pressures from all the MBs under sonication. 14
As shown in Fig. 7(b), the 𝛥𝑃 was increased from 52 kPa to 63 kPa as acoustic pressure increased from 0.12 MPa to 0.23 MPa. The peak positive and negative summed scattered pressures were 24.2 kPa and 19.9 kPa, respectively, under the acoustic pressure of 0.12 MPa, and increased to 41.9 kPa and 30.7 kPa, respectively, when the acoustic pressure was 0.23 MPa. In general, the amplitude of summed scattered pressures accounts for about half of the 𝛥𝑃, suggesting that the scattered pressures are likely to contribute to the effect of MBs on neuronal activity. Moreover, since increased acoustic pressure caused the break-up of the MBs at the critical tension σbreak - up, the summed scattered pressures exhibited a sudden enhancement at the acoustic pressure of 0.24 MPa. Around this acoustic pressure magnitude, 𝛥𝑃 also rapidly increased. The numerical calculations results demonstrated that, above the critical pressure, the summed scattered pressures were much higher than 𝛥𝑃. In this condition, the maximum bubble radius was larger than 2𝑅0, which was likely to result in the collapse of MBs. Thus, the period during which the MBs radiate acoustic pressure is much shorter than that when MBs experience the “compression-only” response, and fewer MBs might be considered when calculating the sum of scattered pressures from MBs. The simulated and experimental results demonstrated that the scattered pressures from pulsating MBs were likely to affect MBs on the neuronal activity. However, the additional acoustic pressure induced by MB oscillation might not be the only reason, since the amplitude of the summed scattered pressures resulted from simulations, was 15
not completely consistent with the experimental results. More importantly, if the additional acoustic pressure induced by MB oscillation is the only reason that enhanced neuronal activity, the same level of neuronal activity evoked by ultrasound stimulation with MBs can be achieved by improving the acoustic pressure from the ultrasound transducer when neurons stimulated without MBs. However, when a plateau occurred at the higher acoustic pressure, the neuronal activity induced by ultrasound stimulation with MBs was higher than that without MBs, suggesting that the effect of MBs on neuronal activity cannot be achieved by just improving the acoustic pressure from the ultrasound transducer when neurons stimulated without MBs. Therefore, there must be other potential effects produced by the oscillation of MBs impacting the neuronal activity. In a previous study [19], it was demonstrated that neurons in C. elegans could be activated by low-intensity ultrasound with the presence of microbubbles, while in the ultrasound-alone group insensitive neuromodulation was observed. Mechanical deformation of C. elegans cuticle is supposed to be the mechanism of amplifying neuromodulatory effect [19]. Vessel deformation can be produced by MB oscillation and acoustic radiation [38-40]. Since the mechanical deformation have capable to excite the neurons 25 μm from C. elegans cuticle [19], the distance between neurons and capillaries in mouse cortex might not be an obstacle to the propagation of vessel deformations to neurons. Multi-frequency stimulation might be other potential mechanisms for pulsating MBs affecting neuronal activity. The nonlinear volumetric 16
and nonspherical oscillation of bubbles produce emissions of harmonics, subharmonics, and ultraharmonics [41, 42]. Our simulation results showed that the summed scattered pressure also contains strong signals at harmonics. Most studies on ultrasonic neuromodulation use low frequency ultrasound (< 0.7 MHz) as they offer optimal compromise between transcranial transmission and skull absorption [43, 44]. However, recent investigations have focused on the neuromodulation achieved by high frequency ultrasound for higher spatial resolutions [45, 46]. Ye et al. [47] indicated that in order to achieve equivalent efficacy with increased ultrasound frequency, significantly higher intensities are required, thus it seems that low frequency ultrasound results in neuromodulatory efficacy. But so far, very little research has addressed the neuromodulatory efficacy of multi-frequency ultrasound stimulation. However, it has been indicated that multi-frequency sonication can provide enhanced efficiency in other therapeutic applications of ultrasound, such as thrombolysis and ablation [48, 49], and some of these are based on the mechanical effect of ultrasound. Thus it would be reasonable to believe that the harmonics, subharmonics, and ultraharmonics from scattered pressures might be a more effective stimulation than single frequency ultrasound to neuronal activity.
5. Conclusions In conclusion, the effects of oscillating MBs on neuronal activity during transcranial focused ultrasound stimulation were numerically and experimentally investigated. When the MBs experienced a “compression-only” response, the summed scattered 17
pressures estimated numerically accounted for a relatively large proportion of 𝛥𝑃, which was obtained experimentally. With increasing acoustic pressure and more specifically, near the critical acoustic pressure, both 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 and 𝛥𝑃 exhibited a sudden enhancement. These results indicated that scattered pressures from pulsating MBs are likely to be a potential mechanism of oscillating MBs that affects neuronal activity, providing a further understanding of the effects of MB oscillation on neuronal activity.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 11874297 and 81827801) and the Welfare Technology Research Plan of Zhejiang Province (LGF20A040001).
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Hilgenfeldt, D. Lohse, A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture, J. Acoust. Soc. Am., 118 (2005) 3499-3505. [30] J.-M. Gorce, M. Arditi, M. Schneider, Influence of Bubble Size Distribution on the Echogenicity of Ultrasound Contrast Agents: A Study of SonoVue™, 35 (2000) 661-671. [31] N. Lamanauskas, A. Novell, J.M. Escoffre, M. Venslauskas, S. Satkauskas, A. Bouakaz, Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue(R) or BR14(R) microbubbles, J Drug Target, 21 (2013) 407-414. [32] K.A. Sheikh, G. Zhang, Y.P. Gong, R.L. Schnaar, J.W. Griffin, An antiganglioside antibody-secreting hybridoma induces neuropathy in mice, Annals of Neurology, 56 (2004) 228-239. [33] B.P. Chugh, J.P. Lerch, L.X. Yu, M. Pienkowski, R.V. Harrison, R.M. Henkelman, J.G. Sled, Measurement of cerebral blood volume in mouse brain regions using microcomputed tomography, Neuroimage, 47 (2009) 1312-1318. [34] E.M.B. Payne, S.J. Illesinghe, A. Ooi, R. Manasseh, Symmetric mode resonance of bubbles attached to a rigid boundary, J. Acoust. Soc. Am., 118 (2005) 2841-2849. [35] H.G. Flynn, C.C. Church, Erratum: Transient pulsations of small gas bubbles in water, The Journal of the Acoustical Society of America, 84 (1988) 1863-1876. [36] H.G. Flynn, Cavitation dynamics. I. A mathematical formulation, The Journal of the Acoustical Society of America, 57 (1975) 1379-1396. [37] M.K. Lobo, H.E. Covington, D. Chaudhury, A.K. Friedman, H. Sun, D. DamezWerno, D.M. Dietz, S. Zaman, J.W. Koo, P.J. Kennedy, Cell type–specific loss of BDNF signaling mimics optogenetic control of cocaine reward, Science, 330 (2010) 385-390. [38] H. Chen, A.A. Brayman, T.J. Matula, Characteristic microvessel relaxation timescales associated with ultrasound-activated microbubbles, Appl. Phys. Lett. , 101 (2012) 163704. [39] H. Chen, W. Kreider, A.A. Brayman, M.R. Bailey, T.J. Matula, Blood vessel deformations on microsecond time scales by ultrasonic cavitation, Phys. Rev. Lett. , 106 (2011) 034301. [40] H. Koruk, A. El Ghamrawy, A.N. Pouliopoulos, J.J. Choi, Acoustic particle palpation for measuring tissue elasticity, Appl. Phys. Lett. , 107 (2015). [41] S.Y. Wu, Y.S. Tung, F. Marquet, M.E. Downs, C.S. Sanchez, C.C. Chen, V. Ferrera, E. Konofagou, Transcranial cavitation detection in primates during blood-brain barrier opening-a performance assessment study, IEEE Trans. Ultrason. Ferroelectr. Freq. Control 61 (2014) 966-978. [42] B. Dollet, S.M. van der Meer, V. Garbin, N. de Jong, D. Lohse, M. Versluis, Nonspherical Oscillations of Ultrasound Contrast Agent Microbubbles, Ultrasound in Medicine & Biology, 34 (2008) 1465-1473. [43] M. Hayner, K. Hynynen, Numerical analysis of ultrasonic transmission and absorption of oblique plane waves through the human skull, J. Acoust. Soc. Am., 110 (2001) 3319-3330. 21
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22
Figure captions Figure 1. Schematic of the model. Figure 2. Encapsulated bubble dynamics under ultrasound stimulation. Bubble radius and scattered pressures (λ 4 away from the bubble) under an acoustic pressure of (a) 0.12 MPa and (b) 0.22 MPa. (c) Maximum bubble radius, (d) peak positive scattered pressure (𝑃𝑃𝑃𝑠𝑐𝑎𝑡), and peak negative scattered pressure (𝑃𝑁𝑃𝑠𝑐𝑎𝑡) as a function of acoustic pressure. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius. Figure 3. Scattered pressure from MBs at five different locations. Figure 4. (a) Sum of the scattered pressures radiated from all the uniformly distributed MBs under sonication for a neuron at the origin of coordinates and (b) its spectrogram. Figure 5. Representative EMG signals evoked by ultrasound stimulation with and without MBs. The synchronous signals of ultrasound stimulation indicating the onset time of stimuli shown at the bottom. Figure 6. Representative fluorescent images of c-fos+ cells (red) obtained from the region of 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The rectangle indicates the region of the obtained fluorescent images of c-fos+ cells. Cell nuclei were stained by DAPI (blue). Figure 7. (a) Mean density of c-fos+ cells in response to ultrasound stimulation with and without MBs (n = 3 mice per group). The data were fitted using a sigmoidal curve, and 23
the fitting degree R2 for the group of control and MBs are 0.995 and 0.993, respectively. (b) Pressure difference (𝛥𝑃), peak positive summed scattered pressures (𝑃𝑃 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡), and peak negative summed scattered pressures (𝑃𝑁𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡) as a function of acoustic pressure. Pressure difference (𝛥𝑃) is defined as the pressure difference between acoustic pressures at which the same level of neuronal activity is excited by ultrasound stimulation with and without MBs. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius.
6. Highlights
Psummed_scat accounts for about half of ∆P while acoustic pressure below 0.23 MPa.
Psummed_scat and ∆P show the same trend with the increasing acoustic pressure.
Psummed_scat is supposed to be a potential mechanism of enhanced neuronal activity.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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S1350-4177(19)31418-X https://doi.org/10.1016/j.ultsonch.2019.104935 ULTSON 104935
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
8 September 2019 12 December 2019 17 December 2019
Please cite this article as: Z. Cui, D. Li, S. Xu, T. Xu, S. Wu, A. Bouakaz, M. Wan, S. Zhang, Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.104935
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© 2019 Published by Elsevier B.V.
Effect of scattered pressures from oscillating microbubbles on neuronal activity in mouse brain under transcranial focused ultrasound stimulation Zhiwei Cui1, #, Dapeng Li1, #, ShanShan Xu1, Tianqi Xu1, Shan Wu1, Ayache Bouakaz2, Mingxi Wan1, * and Siyuan Zhang1, *
1. The Key Laboratory of Biomedical Information Engineering of the Ministry of Education, Department of Biomedical Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China 2. UMR 1253, iBrain, Université de Tours, Inserm, France
*Corresponding
author:
Mingxi Wan, Prof. Email: http://invalid.uri/[email protected] Siyuan Zhang, Associate Prof. Email: [email protected] # These
authors contributed equally to this work.
1
Abstract Previous studies have indicated that the presence of microbubbles (MBs) during sonication has an impact on neuronal activity, while the underlying mechanisms remain to be revealed. In this study, a model for the scattered pressures produced by the pulsating lipid-encapsulated MBs in mouse brain was developed to numerically investigate the effect of MBs on neuronal activity during transcranial focused ultrasound stimulation. The additional summed scattered pressure (𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡) from the oscillating MBs was calculated from the model. The level of neuronal activity was experimentally verified using an immunofluorescence assay with antibodies against cfos. The pressure difference (∆P) between acoustic pressures at which the same level of neuronal activity is excited by ultrasound stimulation with and without MBs was obtained from the experiments. The results showed that 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 accounts for about half of the ∆P when the MBs experience a “compression-only” response. The 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 suddenly increased at a critical acoustic pressure, around which a rapid enhancement of ∆P obtained from experiment also occurred. This work suggested that the additional scattered pressures from pulsating MBs is probably a mechanism that affects neuronal activity under transcranial focused ultrasound stimulation. Keywords: Bubble dynamics; Neuronal activity; Ultrasound stimulation; Microbubble oscillation
2
1. Introduction Transcranial ultrasound combined with microbubbles (MBs) has been extensively investigated to address the treatment of some neurological diseases [1-3], and the technology have begun clinical testing [4, 5]. The safety of this technology has been evaluated using various histological evaluations in different animal models, and no overt tissue and/or cell-level damage has been observed under a wide range of ultrasound parameters [2, 6, 7]. Meanwhile, considering that MBs are administrated in central nervous system (CNS), increasing attention has been paid to the effect of ultrasound combined with MBs on neurological function. Ultrasound has been proven to be an effective neuromodulation technique, and this technique attracts increasing attention due to its high spatial selectivity and high penetrating power into the brain [8, 9]. Previous studies have shown that ultrasonic neuromodulation can alter the central and peripheral nervous systems neuronal activity including the motor and sensory cortex, thalamus, retina, and other brain regions of several animal models [10-13]. Furthermore, Deffieux et al. [14] demonstrated for the first time that US neuromodulation can affect brain cognitive function in primates. Tyler et al. [15] reported that US stimulation of the sensory cortex in humans can enhance their sensory resolution recently. Some recent studies have indicated that the oscillation of MBs might also have an impact on neuronal activity [16-19]. Ibsen et. al. [19] demonstrated that the mechanical deformations induced by MB-ultrasound interaction can amplify the response of Caenorhabditis elegans to ultrasound stimulation. Several experimental 3
evidences indicated that blood-brain barrier (BBB) opening induced by ultrasound stimulation coupled with MBs can be also capable of altering the neuronal response and neurological function in mammals [16-18]. Moreover, the alterations in neural activity can occur only when microbubbles are present [16-18], which indicates that the presence of MBs has a direct or indirect impact on neuronal activity. Existing studies have suggested that the mechanical effect of ultrasound plays an important role in the mechanism for ultrasonic neuromodulation [19-23]. However, few studies have investigated the mechanism of sonicated MBs impacting neuronal activity. Previous studies have indicated that the BBB opening is always accompanied with ultrasound sonication with MBs, however, BBB opening can result in enriching the parenchymal concentration of oxygen, glucose, ions, nutrients, and hormones [1, 16, 24]. These blood constituents diffused into the brain tissue are able to affect neuronal function [1, 16, 18, 24], thus, it is unclear that whether the blood constituents such as ions and nutrients or the oscillation of MBs influence the neuronal activity. Our previous work [25] showed that the presence of MBs can enhance the neuromodulatory effect evoked by ultrasound stimulation, and it appears that the enhancement is not dependent on BBB opening. Additionally, the neural circuit in C. elegans response to ultrasound stimuli can be amplified by the oscillation of MBs, while C. elegans have no circulatory system [19]. Therefore, it is more likely that the oscillation of MBs has a direct effect on neuronal activity. Considering the suggestion that the mechanical effects of ultrasound might be the main potential mechanism for ultrasonic 4
neuromodulation, the additional mechanical effects induced by the oscillating MBs are probably the main reason for the alteration of neuronal activity. In the acoustic field, oscillating MBs produce mechanical effects, such as shear forces, microstreaming, and microjetting, affecting the cells extremely close to or in contact with the MBs [2, 22, 23]. In general, MBs are injected intravenously, but the distance from the neuron to the nearest vessel is 13-20 μm [24, 25]. In other word, intravenous MBs and neurons are at least 13-20 μm apart, therefore, it is too long for the aforementioned mechanical effects produced by MBs to affect the neurons. Previous studies have indicated that oscillating MBs can radiate acoustic pressure into the surrounding medium [26-28]. Considering a volume of MBs were intravenously injected, the scattered pressures from MBs, the extra pressure additional to the transmitted acoustic pressure from the ultrasound transducer, should be noticeable and is supposed to be a potential mechanism of sonicated MBs affecting neuronal activity. In this paper, a model of the pulsating encapsulated bubble driven by a focused acoustic field in mouse brain was developed to investigate the effects of microbubble oscillation on neuronal activity, and the summed scattered pressures from all the MBs was calculated. The neuronal activity induced by ultrasound stimulation with and without MBs was measured by an immunofluorescence assay (IFA) with antibodies against cfos. The oscillating MBs are able to radiate pressures into the surrounding medium, generating additional pressure to the neurons, which might affect neuronal activity. The pressure difference (𝛥𝑃) between acoustic driven pressures at which the same level of 5
neuronal activity is excited by ultrasound stimulation with and without MBs was obtained. The pressure difference is likely to be produced by the pressure radiated from oscillating MBs. Therefore, in order to relate the experimental results to the numerical studies, the pressure difference was compared with the summed scattered pressures from all the MBs.
2. Theory The scattered pressures from the encapsulated MBs pulsating near a neuron in mouse cortex was estimated in order to investigate the effects of oscillating MBs on neuronal activity (Fig. 1). SonoVue® MBs were used in the experimental section of this work, which are coated by lipid. Compared to unshelled MBs, the coating alters the effective surface tension of MBs and strongly influence the bubble dynamics. The acoustic pressure used in this work might produce high-amplitude oscillations of MBs. Marmottant model [29] proposed the effective surface tension, and takes into account the physical properties of MBs’ coating. Three parameters, a buckling radius, the compressibility of the shell, and a break-up shell tension, are used to describe the physical properties of MBs’ coating. This proposition enables this model to describe large amplitude oscillations of lipid coated MBs. Therefore, Marmottant model was used in this work to describe the bubble dynamics of SonoVue® MBs. The oscillation of the MBs is defined by the following equation [29], 2𝜎(𝑅0) 𝑅 3 𝜌𝑙 𝑅𝑅 + 𝑅2 = 𝑃0 + 2 𝑅0 𝑅0
) [
(
― 𝑃0 ―
2𝜎(𝑅) 𝑅
―
4𝜇𝑙𝑅 𝑅
―
4𝜅𝑠𝑅 𝑅2
―3𝜅
]( ) (
― 𝑃𝑎𝑐(𝑡),
1―
3𝜅 𝑅 𝑐𝑙
) (1) 6
where ρl is the liquid density, P0 is the ambient pressure, R0 is the equilibrium bubble radius, R is the instantaneous bubble radius, κ is the polytropic gas exponent, μ is the surrounding liquid viscosity, κs is the shell surface dilatational viscosity, 𝑐𝑙 is the speed of sound in the liquid, and Pac is the acoustic pressure. The surface tension of the encapsulated bubble σ is defined by three states, which are described in terms of the bubble radius, 0, if R Rbuckling R2 R 2 -1 , if Rbuckling R Rbreak -up , Rbuckling liquid , if ruptured and R Rruptured
(2)
where χ is the elastic modulus of the shell, Rbuckling and Rbreak - up are the lower and upper limit radius for the elastic state of the bubble, respectively, and Rruptured is the lower limit radius for the raptured state of the bubble. The pressure scattered by an encapsulated bubble can be expressed as [28] 𝑃𝑠𝑐𝑎𝑡(𝑡, 𝑑) =
𝜌𝑙(𝑅2𝑅 + 2𝑅𝑅2) 𝑑
,
(3)
where 𝑑 is the distance from the bubble center. For the MBs at different locations, the phases of transmitted ultrasound are different. The phases of scattered pressure are also different due to the different distances between MBs and neurons. Therefore, for a neuron at the origin of coordinates, the scattered pressure (𝑃𝑠𝑐𝑎𝑡) from a MB located at (𝑥, 𝑦, 𝑧) can be described as
(
𝑧
𝑃𝑠𝑐𝑎𝑡 𝑡 ― 𝑐𝑙 +
𝑥2 + 𝑦2 + 𝑧2 𝑐𝑙
),
(4)
It is assumed that intravenously injected MBs are uniformly distributed in the 7
circulating blood. The number of all the sonicated MBs can be expressed as 𝑁𝑀𝐵 = 𝑉𝑟𝑒𝑔𝑖𝑜𝑛 × 𝐶𝐵𝑉 × 𝑐𝑀𝐵,
(5)
where 𝑉𝑟𝑒𝑔𝑖𝑜𝑛 is the volume of sonicated brain tissue, 𝐶𝐵𝑉 is the cerebral blood volume, and 𝑐𝑀𝐵 is the concentration of MBs in the circulating blood. Thus, for the neuron at the origin of coordinates, the summed scattered pressures from the uniformly distributed MBs can be estimated by the following equation,
(
𝑧
𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 = ∑𝑁 𝑃𝑠𝑐𝑎𝑡 𝑡 ― 𝑐𝑙 + 𝑀𝐵
𝑥2 + 𝑦2 + 𝑧2 𝑐𝑙
).
(6)
In order to be consistent with the experimental work described below, the corresponding physical constants were chosen in the numerical simulations. For SonoVue® MBs, the average shell elastic modulus was 𝜒 = 0.55 𝑁/𝑚 and the shell dilatational viscosity was κs = 7.2 × 10 ―9 𝑁 [30]. The break-up radius (𝑅𝑏𝑟𝑒𝑎𝑘 ― 𝑢𝑝) is associated with the limit surface tension (σbreak - up). Here, the value of σbreak - up was set to be 0.13 N/m based on BR14® MBs [29], due to the similarity in the lipid shell composition between BR14® and SonoVue® MBs [31]. The value of the other parameters used in the Marmottant model were 𝜌𝑙 = 1.06 × 103 𝑘𝑔/𝑚3, 𝑃0 = 1.013 × 105 𝑃𝑎, 𝑅0 = 1.25 × 10 ―6 𝑚, 𝜅 = 1.095, 𝜇 = 0.0035 𝑃𝑎/𝑠, 𝑐 = 1580 𝑚/ 𝑠, and σliquid = 0.056 𝑁/𝑚, which were obtained from published studies [28, 29]. Assuming that the circulating blood volume in the mouse was 1 mL [32], the concentration of MBs in the circulating blood was 𝑐𝑀𝐵 = 1.6 × 107 /𝑚𝐿 after intravenously injecting 80 μL SonoVue® MBs. The cerebral blood volume (CBV) in the mouse brain has shown significant differences between brain regions, which are 7.9% 8
and 4% in the cerebral cortex and other brain regions, respectively [33]. CBV was set as 7.9% to calculate the number of MBs in the cerebral cortex, and 4% was used when calculating the number of MBs in other brain regions. Thus, bubble separation is 20 times higher than bubble radius in this condition, which makes the interaction of bubbles is weaker [34]. This is the reason that the interaction of bubbles was not taken into account when calculating the summed scattered pressures from all MBs. Taking the FWHM of the FUS transducer and the dorsoventral distance between the upper and lower mouse brain surface of 5.5 mm into consideration, the scattered pressures from the MBs in a cylindrical region with a diameter of 4 mm and a height of 5.5 mm were used to calculate the summed scattered pressures.
3. Material and methods 3.1. Animal preparation All procedures described in this study were approved by the Institutional Animal Care and Use Committee of School of Life Science and Technology of Xi’an Jiaotong University. Adult BALB/c mice were anesthetized in an induction chamber with isoflurane (2%, 0.5 L/min O2), and then were housed in a mouse stereotaxic apparatus, where the anesthesia was continued through a custom anesthesia mask. The hair on the mice head was shaved by scissors. A heating pad was used to keep mouse body temperature at 37℃. 3.2. Ultrasound stimulation
9
A custom 620 kHz focused ultrasound (FUS) transducer (100 mm in aperture, 80 mm in focal depth) was used to sonicate the motor cortex of mice. The focus of the transducer was positioned at bregma, 1.5 mm lateral to the midline and 0.75 mm under the surface of the motor cortex. The acoustic pressure field of the FUS transducer was measured separately using a needle hydrophone (HNR-1000) in degassed water, and the full width at half maximum (FWHM) in lateral and axial dimensions was 2 mm and 10.5 mm. The acoustic pressures used in this study were peak rarefactional pressures at the focus of the transducer after mouse skull attenuation. Referring to our previous study [25], pulsed ultrasound with 2 ms pulse width, 250 Hz pulse repetition frequency, and 400 ms total sonication duration were applied in 8 s intervals. The pulses were generated by a two-channel function generator (DG5072, Rigol Inc., Beijing, China) and amplified by an RF power amplifier (25A250A, AR Europe, Bothell, USA) before being sent to the FUS transducer. 3.3. Motor response evaluation Motor responses evoked by ultrasound stimulation with and without MBs were compared to investigate the enhanced neuromodulatory effect induced by the oscillation of MBs. The motor cortex of the mouse was selected as the simulation target. Electromyogram (EMG) signals evoked by ultrasound stimulation were recorded by the tungsten electrodes inserted into the triceps muscles of mouse forelimbs. EMG signals were amplified with a gain of 1000 and bandpass filtered between 300 Hz and 5 kHz using an amplifier (model 1700, A-M Systems, Inc., Sequim, WA, USA), and 10
acquired at a 10 kHz sampling frequency using a data acquisition card (1550A, Axon Instruments, San Jose, CA, USA). Twenty ultrasound stimulations as described above in 8 s intervals were performed for each condition. For the group of ultrasound stimulation with MBs, 80 μl SonoVue® MBs (Bracco SpA, Milan, Italy) were administered intravenously, and then ultrasound stimulation was performed immediately. 3.4. Histological evaluation C-fos expression is always regarded as a marker of neuronal activity, which is a more direct evaluation revealing the neuronal activity. Therefore, in order to experimentally measure the neuronal activities under ultrasound stimulation with and without MBs, IFA with antibodies against c-fos was performed. The same ultrasound stimulation parameters as described above were applied and the motor cortex of mice was stimulated for 30 min in 8 s intervals. For the stimulation with MBs group, 80 μl SonoVue® MBs were administered by intravenous injection every 10 minutes to ensure the presence of MBs during total stimulation. The mice were sacrificed after a 30-min recovery period. Following perfusion with 4% paraformaldehyde, mouse brains were removed and fixed in 4% paraformaldehyde at 4℃ for 24 hours. The staining brain sections were cut at 40 μm in thickness using a cryotome. Then, the brain sections were incubated with the primary antibody against cfos (1:500, ab214672, Abcam) at 4℃ for 12 hours, and were washed and incubated with Invitrogen Alexa Fluor Plus 555 donkey anti-rabbit IgG secondary antibody (1:1000, 11
A32794, Invitrogen) for 1 hour at room temperature. Histological images were acquired using a fluorescence microscope, and analyzed with the ImageJ software (NIH).
4. Results and Discussion The bubble responses to 620 kHz ultrasound are shown in Fig. 2(a) and 2(b). As it can be seen, a much stronger positive radius excursion and higher scattered pressure was observed at an acoustic pressure of 0.22 MPa than under a 0.12 MPa ultrasound stimulation. The maximum and minimum radii of the bubble sonicated by the 0.12 MPa ultrasound were 1.327 μm and 0.986 μm, respectively, while, under a 0.22 MPa ultrasound stimulation, they expanded to 2.315 μm and 0.827 μm, respectively. The simulation results demonstrated that in the ultrasound field with pressure of 0.12 MPa, the corresponding peak positive and negative scattered pressures, 𝜆 4 away from the pulsating bubble, were 21.294 Pa and 64.810 Pa, respectively, which increased to 799.511 Pa and 340.597 Pa, respectively, under the 0.22 MPa acoustic driven pressure. At lower acoustic pressures, MBs experience a “compression-only” response as mentioned by Marmottant et al. [29], in which the negative MB radius excursion is much higher than the positive radius excursion. With increasing acoustic pressure, a fast expansion is likely to result in the break-up of the MBs at a critical tension σbreak - up. In this state, the surface tension of the MBs will be decreased to the surface tension of the surrounding liquid σliquid, and the MBs will oscillate as free bubbles [29]. Therefore, a sudden maximum bubble radius increase appears when the critical acoustic pressure is reached (Fig. 2(c)). Meanwhile, large oscillation of MBs above a critical pressure can 12
produce much higher velocity and acceleration of MB oscillation, which can generate higher scattered pressure (Fig. 2 (d)). In addition, for higher acoustic pressures, a much larger maximum bubble radius gives rise to a faster contraction as compression pressure arrives, which does not appear when MBs experience the “compression-only” response. This is the reason why the scattered pressure exhibits a higher peak positive pressure above the critical acoustic driven pressure. Undoubtedly, MBs cannot expand without limit. Flynn et al. indicated that the collapse threshold of gas bubbles for inertial cavitation is 2𝑅0 [35, 36], thereby, the results in this letter are presented by a dotted line when the bubble radius is greater than twice the initial radius. The FUS transducer forms a cigar-shaped focus and the acoustic driven pressure depends on the MB locations. Moreover, the phase difference between the scattered pressures produced by MBs at different locations should be taken into consideration. For a neuron at the origin of coordinates, scattered pressures from MBs at five different locations are shown in Fig. 3. The amplitude and phase of acoustic driven pressure varied with the locations in acoustic field, resulting in the various amplitude and phase of scattered pressure radiated from the MBs at different locations. Figure 4(a) shows the summed scattered pressures from all the uniformly distributed MBs under sonication, which contains strong signals at multiples of the fundamental frequency (Fig. 4(b)). In order to demonstrate the effect of MB oscillation on neuronal activity experimentally, motor responses evoked by ultrasound stimulation with and without MBs were 13
measured. Fig. 5 shows the representative EMG signals evoked by the 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The results indicated the success rates of motor response to ultrasound stimulation were significantly elevated for the group of ultrasound stimulation with MBs. The c-fos expression is considered to be a marker of neural activation [37]. Fig. 6 demonstrates the representative results of the c-fos expression in response to 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The presence of MBs increased the c-fos+ cells in the region of ultrasound stimulation, suggesting a higher level of neuronal activity. The mean densities of c-fos+ cells with and without the presence of MBs under different acoustic pressures are shown in Fig. 7(a), where the data points were fitted to a sigmoidal curve. The neuronal activity in the group of ultrasound stimulation with MBs was higher than that in the group without MBs under all acoustic pressures. In addition, for both groups, the increasing acoustic pressure induced higher level of neuronal activity, while a plateau occurred when the acoustic pressure reached near 0.36 MPa. If a level of neuronal activity is excited by the ultrasound stimulation with MBs at a certain acoustic pressure 𝑃, then ultrasound with the higher acoustic pressure 𝑃 +∆𝑃 is required in order to achieve the same level of neuronal activity when ultrasound stimulation is performed without MBs, which defines the 𝛥𝑃 at the acoustic pressure 𝑃 in Fig. 7. In other words, if acoustic pressure radiated from pulsating MBs is the main potential mechanism of enhancing neuronal activity, the 𝛥𝑃 means the experimentally obtained summed scattered pressures from all the MBs under sonication. 14
As shown in Fig. 7(b), the 𝛥𝑃 was increased from 52 kPa to 63 kPa as acoustic pressure increased from 0.12 MPa to 0.23 MPa. The peak positive and negative summed scattered pressures were 24.2 kPa and 19.9 kPa, respectively, under the acoustic pressure of 0.12 MPa, and increased to 41.9 kPa and 30.7 kPa, respectively, when the acoustic pressure was 0.23 MPa. In general, the amplitude of summed scattered pressures accounts for about half of the 𝛥𝑃, suggesting that the scattered pressures are likely to contribute to the effect of MBs on neuronal activity. Moreover, since increased acoustic pressure caused the break-up of the MBs at the critical tension σbreak - up, the summed scattered pressures exhibited a sudden enhancement at the acoustic pressure of 0.24 MPa. Around this acoustic pressure magnitude, 𝛥𝑃 also rapidly increased. The numerical calculations results demonstrated that, above the critical pressure, the summed scattered pressures were much higher than 𝛥𝑃. In this condition, the maximum bubble radius was larger than 2𝑅0, which was likely to result in the collapse of MBs. Thus, the period during which the MBs radiate acoustic pressure is much shorter than that when MBs experience the “compression-only” response, and fewer MBs might be considered when calculating the sum of scattered pressures from MBs. The simulated and experimental results demonstrated that the scattered pressures from pulsating MBs were likely to affect MBs on the neuronal activity. However, the additional acoustic pressure induced by MB oscillation might not be the only reason, since the amplitude of the summed scattered pressures resulted from simulations, was 15
not completely consistent with the experimental results. More importantly, if the additional acoustic pressure induced by MB oscillation is the only reason that enhanced neuronal activity, the same level of neuronal activity evoked by ultrasound stimulation with MBs can be achieved by improving the acoustic pressure from the ultrasound transducer when neurons stimulated without MBs. However, when a plateau occurred at the higher acoustic pressure, the neuronal activity induced by ultrasound stimulation with MBs was higher than that without MBs, suggesting that the effect of MBs on neuronal activity cannot be achieved by just improving the acoustic pressure from the ultrasound transducer when neurons stimulated without MBs. Therefore, there must be other potential effects produced by the oscillation of MBs impacting the neuronal activity. In a previous study [19], it was demonstrated that neurons in C. elegans could be activated by low-intensity ultrasound with the presence of microbubbles, while in the ultrasound-alone group insensitive neuromodulation was observed. Mechanical deformation of C. elegans cuticle is supposed to be the mechanism of amplifying neuromodulatory effect [19]. Vessel deformation can be produced by MB oscillation and acoustic radiation [38-40]. Since the mechanical deformation have capable to excite the neurons 25 μm from C. elegans cuticle [19], the distance between neurons and capillaries in mouse cortex might not be an obstacle to the propagation of vessel deformations to neurons. Multi-frequency stimulation might be other potential mechanisms for pulsating MBs affecting neuronal activity. The nonlinear volumetric 16
and nonspherical oscillation of bubbles produce emissions of harmonics, subharmonics, and ultraharmonics [41, 42]. Our simulation results showed that the summed scattered pressure also contains strong signals at harmonics. Most studies on ultrasonic neuromodulation use low frequency ultrasound (< 0.7 MHz) as they offer optimal compromise between transcranial transmission and skull absorption [43, 44]. However, recent investigations have focused on the neuromodulation achieved by high frequency ultrasound for higher spatial resolutions [45, 46]. Ye et al. [47] indicated that in order to achieve equivalent efficacy with increased ultrasound frequency, significantly higher intensities are required, thus it seems that low frequency ultrasound results in neuromodulatory efficacy. But so far, very little research has addressed the neuromodulatory efficacy of multi-frequency ultrasound stimulation. However, it has been indicated that multi-frequency sonication can provide enhanced efficiency in other therapeutic applications of ultrasound, such as thrombolysis and ablation [48, 49], and some of these are based on the mechanical effect of ultrasound. Thus it would be reasonable to believe that the harmonics, subharmonics, and ultraharmonics from scattered pressures might be a more effective stimulation than single frequency ultrasound to neuronal activity.
5. Conclusions In conclusion, the effects of oscillating MBs on neuronal activity during transcranial focused ultrasound stimulation were numerically and experimentally investigated. When the MBs experienced a “compression-only” response, the summed scattered 17
pressures estimated numerically accounted for a relatively large proportion of 𝛥𝑃, which was obtained experimentally. With increasing acoustic pressure and more specifically, near the critical acoustic pressure, both 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡 and 𝛥𝑃 exhibited a sudden enhancement. These results indicated that scattered pressures from pulsating MBs are likely to be a potential mechanism of oscillating MBs that affects neuronal activity, providing a further understanding of the effects of MB oscillation on neuronal activity.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 11874297 and 81827801) and the Welfare Technology Research Plan of Zhejiang Province (LGF20A040001).
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Figure captions Figure 1. Schematic of the model. Figure 2. Encapsulated bubble dynamics under ultrasound stimulation. Bubble radius and scattered pressures (λ 4 away from the bubble) under an acoustic pressure of (a) 0.12 MPa and (b) 0.22 MPa. (c) Maximum bubble radius, (d) peak positive scattered pressure (𝑃𝑃𝑃𝑠𝑐𝑎𝑡), and peak negative scattered pressure (𝑃𝑁𝑃𝑠𝑐𝑎𝑡) as a function of acoustic pressure. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius. Figure 3. Scattered pressure from MBs at five different locations. Figure 4. (a) Sum of the scattered pressures radiated from all the uniformly distributed MBs under sonication for a neuron at the origin of coordinates and (b) its spectrogram. Figure 5. Representative EMG signals evoked by ultrasound stimulation with and without MBs. The synchronous signals of ultrasound stimulation indicating the onset time of stimuli shown at the bottom. Figure 6. Representative fluorescent images of c-fos+ cells (red) obtained from the region of 0.12 MPa and 0.25 MPa ultrasound stimulation with and without MBs. The rectangle indicates the region of the obtained fluorescent images of c-fos+ cells. Cell nuclei were stained by DAPI (blue). Figure 7. (a) Mean density of c-fos+ cells in response to ultrasound stimulation with and without MBs (n = 3 mice per group). The data were fitted using a sigmoidal curve, and 23
the fitting degree R2 for the group of control and MBs are 0.995 and 0.993, respectively. (b) Pressure difference (𝛥𝑃), peak positive summed scattered pressures (𝑃𝑃 𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡), and peak negative summed scattered pressures (𝑃𝑁𝑃𝑠𝑢𝑚𝑚𝑒𝑑_𝑠𝑐𝑎𝑡) as a function of acoustic pressure. Pressure difference (𝛥𝑃) is defined as the pressure difference between acoustic pressures at which the same level of neuronal activity is excited by ultrasound stimulation with and without MBs. Results are presented by a dotted line when the bubble radius is greater than twice the initial radius.
6. Highlights
Psummed_scat accounts for about half of ∆P while acoustic pressure below 0.23 MPa.
Psummed_scat and ∆P show the same trend with the increasing acoustic pressure.
Psummed_scat is supposed to be a potential mechanism of enhanced neuronal activity.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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