Theranostics

2020; 10(12):5514-5526. doi:ten.7150/thno.40520 This result

Research Paper

Non-invasive ultrasonic neuromodulation of neuronal excitability for treatment of epilepsy

Zhengrong Lin1,5*, Long Mengone,two,v*, Junjie Zoui,3*, Wei Zhou1,v, Xiaowei Huang1,v, Shan Xue3, Tianyuan Bianone, Tifei Yuaniv, Lili Niu1,2,v

, Yanwu Guoiii

, Hairong Zhengi,ii

1. Institute of Biomedical and Health Engineering science, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China, 518055.
ii. CAS Key Laboratory of Health Informatics, Shenzhen Institutes of Advanced Applied science, Shenzhen, Prc, 518055.
3. The National Fundamental Dispensary Specialty; The Engineering Applied science Research Center of Teaching Ministry building of China; Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration; Department of Neurosurgery, Zhujiang Infirmary, Southern Medical University, Guangzhou, China, 510282.
4. Shanghai Mental Health Middle, Shanghai Jiaotong University School of Medicine, Shanghai, China, 200030.
five. Guangdong-Hong Kong-Macao Greater Bay Surface area Middle for Brain Scientific discipline and Brain-Inspired Intelligence, Guangzhou, Prc, 510515
*These authors contributed equally to this work.

This is an open access article distributed under the terms of the Creative Eatables Attribution License (https://creativecommons.org/licenses/by/iv.0/). See http://ivyspring.com/terms for full terms and conditions.

Citation:

Lin Z, Meng L, Zou J, Zhou W, Huang X, Xue Southward, Bian T, Yuan T, Niu L, Guo Y, Zheng H. Non-invasive ultrasonic neuromodulation of neuronal excitability for treatment of epilepsy.
Theranostics
2020; 10(12):5514-5526. doi:10.7150/thno.40520. Available from https://www.thno.org/v10p5514.htm

Abstract

Graphic abstract

Not-invasive low-intensity pulsed ultrasound has been employed for straight neuro-modulation. However, its range and effectiveness for unlike neurological disorders take not been fully elucidated.

Methods:
We used multiple approaches of electrophysiology, immunohistochemistry, and behavioral tests as potential epilepsy treatments in not-man primate model of epilepsy and human epileptic tissues. Low-intensity pulsed ultrasound with a frequency of 750 kHz and acoustic pressure of 0.35 MPa (the spatial peak pulse average intensity, ISPPA
= 2.02 W/cm2) were delivered to the epileptogenic foci in v penicillin-induced epileptic monkey models. An ultrasound neuro-modulation system with a frequency of 28 MHz and acoustic pressure of 0.13 MPa (ISPPA
= 465 mW/cm2) uniform with patch-clench systems was used to stimulate the brain slices prepared from fifteen patients with epilepsy.

Results:
After 30 min of depression-intensity pulsed ultrasound treatment, full seizure count for xvi hours (sham group: 107.7 ± one.2, ultrasound grouping: 66.0 ± 7.9, P < 0.01) and seizure frequency per hour (sham group: fifteen.6 ± i.two, ultrasound group: nine.vi ± one.v, P < 0.05) were significantly reduced. The therapeutic efficacy and underlying potential mechanism of low-intensity pulsed ultrasound handling were studied in biopsy specimens from epileptic patients
in vitro. Ultrasound stimulation could inhibit epileptiform activities with an efficiency exceeding 65%, potentially due to adjusting the balance of excitatory-inhibitory (E/I) synaptic inputs by the increased activity of local inhibitory neurons.

Determination:
Herein, nosotros demonstrated for the outset time that low-intensity pulsed ultrasound improves electrophysiological activities and behavioral outcomes in a non-man primate model of epilepsy and suppresses epileptiform activities of neurons from human epileptic slices. The study provides testify for the potential clinical use of non-invasive low-intensity pulsed ultrasound stimulation for epilepsy treatment.

Keywords: pulsed ultrasound treatment, epilepsy, electrophysiological activities

Introduction

Epilepsy is one of the most prevalent neurological disorders characterized past recurrent seizures resulting from excessive excitation or inadequate inhibition of neurons [i-5]. During the seizures, abnormal synchronized activities in the epileptic focus may spread to other encephalon regions, causing behavioral disorders [6-8]. Neuro-modulation techniques have recently been employed to modulate abnormal neuronal activity and decrease the frequency and/or elapsing of seizures [9]. For case, vagus nerve stimulation (VNS) is an established and condom process to suppress seizure activities past delivering electric impulses to the encephalon [10-12]. Besides, deep encephalon stimulation (DBS) reduces seizure frequency and severity in both animal and human studies with implantation of one or more electrodes in specific encephalon regions [13, 14]. Optogenetics, the use of lite to modulate neural circuits via viral transduction of protein channels, has emerged as a potential method for treating epilepsy. Combined with a closed-loop seizure detection arrangement, optogenetics has been shown to command spontaneous seizures in animal models of epilepsy [7, xv]. Besides, transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS) are non-invasive methods that modulate regional cortical excitability through electric current, and accept been shown to finer decrease epileptic seizures and suppress epileptiform activities [sixteen-xviii]. However, each technique has certain limitations, such as lacking spatial specificity/depth (TMS and tDCS), or containing invasive procedures (DBS and VNS).

Not-invasive depression-intensity pulsed ultrasound with high spatial specificity and penetration depth has emerged as a novel neuromodulation technique [xix]. The ultrasound waves tin penetrate the intact skull to specific brain regions, causing modulatory effects of neuronal activity or behavioral result [20-29]. Pioneering studies in monkeys have provided evidence that low-intensity pulsed ultrasound tin exist used to modify perception and behavior [22, 26]. Also, human studies accept demonstrated that low-intensity pulsed ultrasound tin can not-invasively attune the function of the chief somatosensory cortex and cause significant changes in electroencephalograph responses by enhancing the discrimination abilities [23, 30]. Although recent studies take shown that ultrasound stimulation can suppress epileptic seizures in animal models [28, 31], the therapeutic potential of low-intensity pulsed ultrasound for non-human primate epileptic model or human epileptic tissues remains to exist elucidated.

Nosotros aimed to investigate whether low-intensity pulsed ultrasound was capable of modulating epilepsy. First, the effectiveness of ultrasound stimulation was identified by a penicillin-induced epilepsy model in non-human primates
in vivo. Subsequently, brain tissues from patients with temporal lobe epilepsy (TLE) undergoing surgery were tested by miniaturized ultrasound stimulation systems uniform with patch-clench technique
in vitro. Our results showed that ultrasound stimulation exerted an inhibitory influence on epileptiform discharges and improved behavioral seizures in a non-human primate epileptic model. Furthermore, ultrasound stimulation could potentially modulate neuronal excitability to inhibit epileptiform activities in human epileptic tissues. The results from epileptic monkey models
in vivo
and human epileptic tissues
in vitro
suggest that depression-intensity pulsed ultrasound could suppress epileptiform activities and may provide a potential clinical treatment for epilepsy.

Materials and Methods

Ultrasound stimulation in monkeys

The study was approved by the Ethics Committee of the Center of Guangdong Landao Biotechnology in Guangzhou, China (LDACU20170306-01) and was performed in accordance with recommendations from the Guidelines for the Use and Care of Experimental Animals. Every endeavor was fabricated to minimize suffering. Five monkeys (Macaca fascicularis, labeled from 1 to v, four-7 years old, 5.4-5.9 kg) were provided by the Center of Guangdong Landao Biotechnology in Guangzhou, Prc. They were individually housed in a temperature (24 ± i°C) and humidity (fifty ± v%) controlled facility with a 12 h lite nighttime bike (lights on viii:00 a.m.). Each monkey had free admission to standard primate chow and water.

The furnishings of ultrasound stimulation on epileptic monkey models were assessed past electrophysiological recording and behavioral outcome analysis. A unmarried-chemical element focused ultrasound transducer (H116, Sonic Concept) was placed on the site of penicillin injection to evangelize ultrasound energy to the epileptogenic foci (Figure 1A; see also in Supplementary Figure S1). We performed a full of 12 electrophysiological trials in three monkeys, six sham trials without ultrasound stimulation, six with 30 min ultrasound stimulation, and six behavioral monitoring in two monkeys. Anesthesia was induced by injection of ketamine (ten mg/kg, i.m.) and atropine (0.05 mg/kg, i.m.) for ane.5 hours. The heads of the monkeys were fixed, and surgery was performed using a stereotaxic apparatus (68901, RWD) for nonhuman primates. All hair of the monkeys was shaved, and the pare was fully disinfected and separated to betrayal the skull. The right frontal lobe was targeted according to the Macaca fascicularis Brain in Stereotaxic Coordinates [32, 33], with the stereotactic coordinates 30 mm posterior to the bregma, 15 mm lateral to the midline, and 3 mm from the dura (Figure 1A). A department of the skull, called a bone flap about 30 mm * 15 mm, was removed to evangelize drugs locally. The monkeys were given sufficient nutrition after surgery and antibiotics were practical for seven days.

Experiments on the monkeys were started after a 14-twenty-four hour period postoperative recovery period. Penicillin was diluted to 250 IU/uL, and 2000 to 3000 IU was practical to the brain surface using an injection cannula with a microsyringe (l μl, 1705RN, Hamilton) at 1μl/min. Focal seizures were induced by penicillin injection in the right frontal lobe (3mm depth) lasting less than 48 hours. Subsequently penicillin injection, the monkeys were monitored for epileptiform activities with video-EEG for 8 hours paying attention to containment and comfort of the animal in a restrained position [34, 35]. Subsequently, we brought back the monkeys in the cage to monitor behavioral seizures using video recording for sixteen hours. Behavioral seizure evaluation index included seizure count in 16 hours, frequency per hour, and duration and seizure interval time which was calculated by the average time between 2 seizures. A single-element ultrasound transducer (H116, Sonic Concept) with a cardinal frequency of 750 kHz, TBD of 300 μs, PRF of 1000 Hz, sonication elapsing (SD) of 200 ms, and inter-stimulation interval (ISI) of 5 seconds was used for stimulation of the epileptic focus. The monkeys were treated for xxx min with ultrasound treatment (Figure 1B). In the sham stimulation group, the ultrasound transducer was fixed to the monkeys like to the stimulated group for 30 minutes without ultrasound signal output. The ultrasound transducer was stock-still on the operating arm of the stereotactic device, the probe and the dura mater were kept perpendicular, and the coupling agent was filled between the ultrasonic probe outlet and the membrane meninges. One experiment in monkey five was carried out to evaluate the effect of any audible sound produced by ultrasound transducer on epileptiform activities. The ultrasound transducer was placed outside away from the monkey brain and the monkey was monitored for epileptiform activity for 8 h continuously. The transducer was actuated by an electrical signal generated by a role generator (AFG3101, Tektronix) which was meantime amplified using a power amplifier (2100L, E&I, NY, USA).

Subsequently the end of the ultrasound stimulation, we removed the monkeys from the stereo positioner and moved them to the monkey chair, fixing the limbs to the monkey chair with a neckband on the cervix and the neckband continued to the chair. The audio-visual intensity (ISPPA) was evaluated to be approximately ii.02 Due west/cmii
(the acoustic pressure = 0.35 MPa), equally measured with a needle hydrophone (Precision Acoustics, Dorchester, Dorset, UK). Ultrasound force per unit area mapping was acquired past a commercial finite element method (FEM) software COMSOL Multiphysics and the acoustic field was measured in XY plane by the OptiSon® Ultrasound Beam Analyzer (Onda, USA). The electrophysiological data from monkeys 1, two, 4, and five and behavioral seizure information from monkeys 1, and 3 were farther calculated.

Evaluation of temperature elevation and prophylactic in the monkeys

To evaluate the thermal consequence of the ultrasound, we placed a 5mm polyvinyl alcohol on the probe exit plane and recorded the temperature of the ultrasonic probe exit using a thermal infrared imager (R300, NEC Avio, Tokyo, Japan).

One epileptic monkey model after thirty min ultrasound stimulation was sacrificed and histological examinations were performed to evaluate the safe of ultrasound stimulation. Following deep anesthesia, 0.9% NaCl and 4% paraformaldehyde (ph 7.four) solution were immediately perfused through the heart. After fixation, the brain was removed and immersed in iv% paraformaldehyde. The coronal section was taken in the prefrontal cortex area by ultrasound stimulation. Brain sections were then subjected to histological staining and were analyzed using a conventional microscope.

Evaluation of epilepsy patients

This study was reviewed and approved by the Ethics Committee of the Zhujiang Infirmary of Southern Medical Academy, Guangzhou, People’s republic of china (2016-SJWK-005). Written informed consent and a statement confirming consent to publish were obtained from 19 participants. A total of 15 TLE patients and 4 glioma patients without epilepsy were included in the preliminary study. The details of these patients are summarized in Supplementary Table S1. Based on the systematic assessment, including magnetic resonance imaging, the scalp electroencephalograms (EEGs), semiology, and pathology, the patients were diagnosed with medically intractable focal epilepsy in the temporal lobe (Supplementary Figure S4). Consequent with previous studies, the histopathological findings showed the key features of TLE, including neuronal loss, the layer dispersion, cellular morphology changes, reactive gliosis, and focal cortical dysplasia [36-38]. Moreover, epileptiform discharges in the TLE patient were observed in inter-ictal scalp EEGs. Hematoxylin and Eosin (HE) staining and Bielschowsky argent staining demonstrated that neuronophagia and microvacuoles occurred in the neurons of epileptic tissues (Supplementary Figure S4D-E).

Preparation of encephalon slices

Biopsy specimens removed from fifteen TLE patients or 4 glioma patients without epilepsy were rapidly immersed in ice-cold, oxygenated high-sucrose solution (0 – 2°C) containing (in mM): sixty NaCl, 3 KCl, seven MgClii, ane.25 NaH2PO4, 25 NaHCO3, 10 D-glucose, 115sucrose, and 0.five CaCl2. Coronal slices (300 μm thick) were prepared with a Vibratome (VT-1200 Series, Leica) instrument. The encephalon slices were equilibrated and incubated in the ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCOthree, ten D-glucose, ii sodium pyruvate, 0.5 50-ascorbic acid, and two CaClii
and saturated continuously with 95% O2
– five% CO2, pH 7.3-7.4. The osmolality was 290 – 300 mOsm/L and the temperature was kept at 35°C before the slices reached the recording chamber. Ultrasound stimulation was shown to exist effective in heady or reversibly suppressing neuronal activity. For the choice of ultrasonic parameters, four C57BL/six mice were sacrificed under deep anesthesia with 20% urethane (x mL kg-1) and slices were prepared as described above. All procedures were performed according to the guidelines of The Institutional Review Board at Zhujiang Hospital of Southern Medical University, Guangzhou, China and the Commission for Animate being Experimentation at Shenzhen Institutes of Advanced Applied science, Chinese Academy of Sciences.

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Application of ultrasound stimulation in epileptic slices

An ultrasound neuro-modulation chip with 28 MHz resonant frequency uniform with the patch-clamp systems due to the small size and transparent character was used for stimulation of slices
in vitro
(Figure S5)[39-41]. The scrap consisted of interdigital transducers (IDTs) and a recording chamber (a polydimethylsiloxane ring-shaped bedchamber). The finger-electrodes of IDTs were deposited using micro-electromechanical systems (MEMS) techniques on a piezoelectric 128o
Y-rotated, X-propagating lithium niobate (LN, LiNbO3) sub-strate (1 mm thick, transparent) with an aluminium layer of 200 nm [42]. Pulsed ultrasound waveforms were generated by an capricious waveform generator (AFG 3102, Tektronix, Beaverton, Oregon) and amplified by a power amplifier (ZHL-1-2W+, Mini-Circuits, Brooklyn, NY, USA). The displacement of the piezoelectric substrate perpendicular to the surface plane was twenty pm measured by a Laser Doppler Velocimetry (UHF-120 Ultra High-Frequency Vibrometer, Polytec, Germany) and the acoustic force per unit area equal to 0.13 MPa (the spatial-peak pulse-average intensity (ISPPA) was evaluated to be approximately 465 mW/cm2. The spatial top time average intensity (ISPTA) was equal to 233 mW/cm2
and was calculated by multiplying duty cycle to the ISPPA
[43].

Electrophysiological recording and histology

The brain slices were transferred to the recording chamber in the ultrasound neuro-modulation chip after 1-hr incubation and were perfused with ACSF flowing at a rate of ii – three ml/min and maintained at xxx°C by an automated temperature controller (TC-324C, WARNER) throughout the experiment. Traditional cell-attached and whole-jail cell recordings were performed to tape the spontaneous action using a patch-clamp system (AXON 1550A and 700B, United states). Records were filtered at 5 kHz and digitized at a sampling rate of three kHz. The series resistance was compensated, and leakage and capacitive currents were subtracted on-line. Patch glass microelectrodes were pulled past a micropipette puller (P-97, Sutter Instrument Co., Novato, CA, USA) and the resistance ranged from v to 10 MΩ later on filling with the internal solution.

Upon the traditional jail cell-fastened recordings, the spontaneous activity was recorded before and later ultrasound stimulation in 60 seconds duration. The internal solution contained the post-obit (in mM): 126 NaCl, two.five KCl, one MgCl2, ane.25 NaHtwoPO4, 26 NaHCO3, 10 D-glucose, 2 sodium pyruvate, 0.5 50-ascorbic acid, and ii CaCltwo. Whole-cell electric current-clamps were used to record evoked action potentials in response to dissimilar injection currents (ranging betwixt -100 and 400pA). The voltage-clamp internal solution contained the following (in mM): 140 K-gluconate, iv.5 MgClii, 5 EGTA, 4 Mg-ATP, 0.three GTP, 4.iv phosphocreatine disodium common salt hydrate, and 9 HEPES. Further identification of neuronal morphology was carried out by intracellular injection of 0.25% biocytin (Sigma, USA). Whole-jail cell recordings in the voltage-clamp fashion were used to record isolated excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) at holding potentials of -70, and 0 mV. The current and voltage (I-V) curves showed a linear correlation of postsynaptic currents with the holding potentials. In the -70-mV property membrane potential, which was close to the reversal potentials for GABAA
receptor-mediated Cl
currents, only the EPSCs could be recorded. In the 0-mV property membrane potential, which was close to the reversal potentials for glutamatergic currents, only the IPSCs could exist recorded. To further verify the recording currents, nosotros found that the EPSCs could exist fully blocked past applying DNQX (6.vii-dinitroquinoxaline-2.3-dione) and AP-5 (DL-two-amino-5-phosphonopentanoic acid). The recording of IPSCs could besides exist blocked by strychnine and bicuculline. The voltage-clench internal solution contained the following (in mM): 125 cesium gluconate, five TEA-Cl, 4 MgATP, 0.3 GTP, 10 phosphocreatine, ten HEPES, 10 EGTA, 2 CsCl, 1.5 QX-314, and pH 7.3. Using the same method, the EPSCs and IPSCs were separated in the ‘epileptic neurons ‘ of TLE slices by holding at unlike membrane potentials.

Histological experiments were carried out to place the neuronal morphology. Human encephalon slices of patients were immersed with 4 % paraformaldehyde (for neuronal morphology, 15 min subsequently patch-clamp recording). Brain sections were stained for neuronal morphology (biocytin, Sigma) and pathological assessments (HE staining and Bielschowsky argent staining). The full biopsy specimens for slice preparations were harvested from 15 patients with temporal lobe epilepsy and 4 glioma patients without epilepsy. Biopsy specimens removed from six TLE patients and 2 gliomas patients were used for slice preparations for the traditional cell-attached recordings. Biopsy specimens removed from 4 TLE patients and other two gliomas were used to ready slices for whole-cell recordings in the voltage-clamp mode to isolate excitatory and inhibitory postsynaptic currents. Biopsy specimens removed from ii TLE patients were used for slices preparations for whole-cell recordings in the current-clench way to investigate the neuronal excitability in dissimilar blazon of neurons. Some leftover tissues were used for histological experiments. Biopsy specimens removed from 3 TLE patients were used to prepare slices for patch-clamp recording to evaluate temperature top produced by ultrasound transducer on epileptiform activities.

Statistical analysis

All statistical procedures were performed using SPSS (13.0) statistical software parcel. Values were expressed as means ± standard mistake of the hateful (SEM). The effects of the ultrasound stimulation on spike frequency and aamplitude were statistically evaluated by the Pearson correlation coefficient, Student’s paired
t-examination, and contained samples
t-examination. One-mode ANOVA followed past LSD was used for differences between groups. Statistical significance was defined equally a value of
P
< 0.05.

Results and Discussion

Ultrasound stimulation reduces epileptiform activities and behavioral seizures in epileptic monkeys

Although non-invasive low-intensity pulsed ultrasound is often used for modulation of different neurological disorders also as for straight neuromodulation, its range and effectiveness for epilepsy in non-human primate models have not been elucidated. Therefore, nosotros beginning tested whether ultrasound stimulation could influence epileptiform activities in epileptic monkeys (Figure 1A-B, Supplementary Effigy S1). Ultrasound stimulation for xxx minutes finer suppressed electrographic frequency of ictal spikes per infinitesimal in all monkeys tested (Sham: 29.3 ± three.eight; The states: 12.62 ± three.9. n = 6 experiments,
P
< 0.01, contained samples
t-test, Effigy 1C). For behavioral testing, a full of half-dozen experiments were performed in two monkeys including iii sham experiments without ultrasound stimulation and 3 with ultrasound stimulation. The mean values of the total seizure counts in 16 hours were significantly reduced (107.seven ± 1.2 in the sham group and 66.0 ± seven.9 in the ultrasound grouping, northward = two animals,
P
< 0.01, independent samples
t-test, Effigy 1D) upon ultrasound stimulation. Independent samples
t-test revealed that the monkeys in the ultrasound stimulation grouping had lower seizure frequency per hour, lower seizure elapsing, and longer seizure interval (all
P
< 0.05, Figure 1E-G). These results showed that ultrasound stimulation could effectively suppress epileptiform activities and amend the behavioral effect in epileptic monkey models. To evaluate the safety of thirty minutes ultrasound stimulation in epileptic monkeys, temperature monitoring and Hematoxylin and Eosin (HE) staining were performed for bear witness of tissue damage. Relatively small-scale temperature top (0.69 ± 0.044°C) and intact cortex structure and neurons indicated that 30 min ultrasound stimulation in this study was safe for the treatment in epileptic monkey models (Supplementary Figure S2).

Previous studies have suggested two possible explanations for ultrasound neuro-modulation: ultrasound straight activates a localized expanse or indirectly impacts auditory pathways which further propagate to other cortical networks [44-46]. The outcome of ultrasound-induced auditory indirect activation is mainly related to acoustic parameters, specially pulse repetition frequencies (PRF). Although our experiment in epileptic monkey models employed ultrasound PRF of m Hz within the hearing range of primates, another experiment was carried out to evaluate the effect of any audible sound produced by ultrasound transducer on epileptiform activities. The ultrasound transducer was placed outside away from the monkey encephalon and the monkey was monitored for epileptiform activity for 8 h continuously (Supplementary Figure S3A). The upshot showed that ultrasound had no meaning consequence on the epileptiform activities of epileptic monkey (Supplementary Figure S3B). Further investigation is needed to clarify whether or non the inhibition of epileptiform activities or behavioral seizures in monkey models is induced by the possible effect of ultrasound-induced auditory indirect activation. Besides, ultrasound exposure promoted drug accumulation in cells through downregulation of P-glycoprotein. Thus, modulation of p-glycoprotein expression might be role of the ultrasound-induced furnishings [47].

 Figure 1

Ultrasound stimulation improves electrophysiological activities and behavioral outcomes in penicillin-induced epileptic monkey models. (A) Schematic illustration depicting the system used for stimulating the monkey. A single-element focused ultrasound transducer with fundamental frequency of 750 kHz, audio-visual pressure of 0.35 MPa (ISPPA= 2.02 Due west/cm2), TBD of 300 μs, PRF of 1000 Hz, SD of 200 ms, and ISI of 5 due south was placed on the site of penicillin injection to deliver ultrasound free energy to the epileptogenic foci (The correct frontal lobe). A depth-microelectrode was placed to record the electrophysiological activities. (B) Flowchart of the experimental procedure. (C) Representative EEG traces in the baseline, Before United states of america, US, Sham and After The states indicate that a thirty-min US stimulation decreased ictal fasten activities in penicillin-induced epileptic monkey models. (D-Yard) Bar charts of the data measuring different behavioral seizure parameters in the U.s. and Sham groups. D, total seizure count (sixteen hours); E, seizure frequency per hour; F, seizure duration; Yard, seizure interval time. The results showed that ultrasound stimulation enabled to improve the behavioral outcomes in penicillin-induced epileptic monkey models. *
P
< 0.05, **
P
< 0.01.

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 Figure 2

Inhibitory effect of ultrasound stimulation on epileptiform discharges of TLE patients. (A) Cell-fastened recording of neurons from epileptic patients has epileptiform discharges consisting of few to dozens of spikes while neurons from glioma patients without epilepsy have few discharges. (B) PSTH (pre-stimulation spikes time histogram) of xiii ‘epileptic neurons’ and xv normal neurons showing the epileptiform discharges in the ‘epileptic neurons’ of TLE patients compared with the few discharges in glioma patients. (C) Comparing of the frequency of spikes showing that the firing frequency of neurons from epileptic patients was significantly college than that of neurons from glioma patients. (D) Representative traces of epileptiform discharge recorded (center) in unlike recording phases (upper, 60 seconds before ultrasound stimulation, 60 seconds ultrasound stimulation and after ultrasound stimulation). Spike frequency of epileptiform discharges was significantly decreased during ultrasound stimulation (lower). The inhibitory effect lasted 75 seconds and rapidly resumed after ultrasound stimulation. (E) Thirteen neurons recorded from TLE patient’s slices showing an effective subtract in the frequency of epileptiform discharges during ultrasound stimulation. The frequency of epileptiform discharges resumed after ultrasound stimulation. (F) Ultrasound stimulation significantly decreased the normalized spikes frequency of epileptiform discharges. (G) No meaning change was observed earlier United states of america and during United states in the amplitude of the spikes. (H) No significant change was observed before US and U.s. in the one-half-width of spikes. Sixty seconds pulsed ultrasound waveforms with a fundamental frequency of 28 MHz, audio-visual pressure level of 0.xiii MPa (ISPPA= 465 mW/cm2), TBD of 5 ms and PRF of 100 Hz were used to stimulate the human being epileptic slices.

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Ultrasound stimulation straight inhibits the epileptiform discharges in neurons from TLE patients

To investigate whether ultrasound stimulation could suppress epileptiform activities in human being epileptic slices and uncover the underlying mechanisms, an experimental system consisting of an ultrasound neuro-modulation chip and patch-clamp systems was used for stimulation of brain slices
in vitro
(Supplementary Figure S5). Using cell-fastened recording, we start detected the epileptiform discharges in neurons from TLE patients while there were few spontaneous discharges in neurons from glioma patients (Figure 2A-C). The neurons from TLE patients produced a 0.3 – 0.5 Hz epileptiform discharge with few to dozens of spikes (n = 13), while the neurons in glioma patients did not prove such epileptiform discharges (n = 15). The blueprint of epileptiform discharges in neurons from human epileptic tissue
in vitro
and those seen in neurons recorded in both acute and chronic epileptogenic foci
in vivo
was similar[48, 49].

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Previous studies take indicated that acoustic parameters are associated with the bimodal modulatory effects of ultrasound on neuronal activeness [l-52]. We used mice brain slices to test the excitatory or inhibitory outcome of ultrasound stimulation with different acoustic parameters (Supplementary Figure S6). 60 second depression-intensity pulsed ultrasound waveforms with frequency of 28 MHz, ISPPA
of 465 mW/cm2
tone flare-up duration (TBD) of five ms and pulse repetition frequency (PRF) of 100 Hz indicated inhibitory outcome on neuronal excitability in mice cortical slices and were, therefore used to attune the human epileptic slices. Effigy 2d shows that the epileptiform discharges of neurons could be effectively inhibited during ultrasound stimulation. The inhibitory effect lasted about 75 seconds, and so speedily resumed after ultrasound stimulation. The histogram of spikes frequency displays the inhibitory procedure of ultrasound stimulation. Recording from a population of 13 neurons from TLE patients showed that the relative frequency of epileptiform discharges rapidly decreased several-fold (Effigy 2E) and the inhibitory issue lasted 63 ± 4 seconds afterwards ultrasound stimulation. As displayed in Figure 2F, ultrasound stimulation significantly decreased the relative frequency of spikes compared to before ultrasound stimulation (during U.s.a.: 0.303 ± 0.064 Hz; subsequently US: 0.936 ± 0.041 Hz.
P
< 0.01, one-way ANOVA followed past LSD). Furthermore, analysis of individual spike waveform revealed that both the amplitude and half-width were unchanged before and during ultrasound treatment (150.16 ± 12.77 pA to 152.45 ± 12.27 pA,
P
= 0.18 for mean amplitude of spikes; 0.41 ± 0.015 ms to 0.36 ± 0.011 ms,
P
= 0.13 for mean one-half-width of spikes, Figure 2G-H). Previous results indicated that disruptions of the synaptic inputs could rapidly produce epileptiform activities and seizures within minutes [53, 54]. To chop-chop re-stabilize neural circuits before the onset of pathological activities, an constructive method is required for compensatory adjustments of synaptic inputs. In cultured hippocampal and cortical neurons, restoration of the synaptic inputs occurred over hours to days through homeostatic adjustments [55-58]. Ultrasound stimulation used in this report could quickly conform the synaptic inputs inside seconds. Therefore, noninvasive ultrasound stimulation might take significant translational therapeutic potential equally an antiepileptic treatment modality.

The ultrasound wave used in this study was generated past an ultrasound neuro-modulation flake (cardinal frequency, 28 MHz). The obvious question and so was whether the bit had the same issue on neurons as the traditional ultrasound transducer used in the not-man primate model of epilepsy (fundamental frequency, 750 kHz). Our previous studies have demonstrated that the ultrasound neuro-modulation fleck transmitting surface acoustic waves (SAW) could directly modulate the neurons of hippocampal slices or from
C. elegans
[39, 41]. When the SAW propagates along the substrate inbound the bars fluid in the microcavity, the refraction of SAW occurs at the interface of the fluid and substrate. Part of the SAW free energy converts to bulk moving ridge propagating in the fluid, and the rest of audio-visual free energy lies in the leaky SAW propagating in the substrate [59-61]. Furthermore, SAW every bit a form of ultrasound has similar bio-furnishings on the cells compared to the longitudinal moving ridge generated by the traditional ultrasound transducer [59, 62]. Therefore, due to its compatibility with patch clamp and standard calcium imaging, the ultrasound neuro-modulation fleck was starting time used to written report the inhibitory furnishings of ultrasound stimulation and the underlying mechanism on inhibiting epileptiform discharges.

Ultrasound stimulation re-adjusts the imbalance of synaptic inputs to inhibit the epileptiform discharges

The interplay of cortical excitation and inhibition is a fundamental feature of cortical function processing [63, 64]. The cortical neuronal activity is dynamically adjusted past synaptic inputs, including the excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs), which is often disrupted in the neurons of TLE patients [65-68]. To exam whether disturbed synaptic inputs in the neurons from TLE patients can be adjusted by ultrasound stimulation, the EPSCs and IPSCs of the neurons from TLE patients (n = 9) and glioma patients (northward = 11) were separated by holding the neurons at different membrane potentials. A synaptic input was observed in xi neurons from 4 glioma patients (Effigy 3A left) while both the frequency and amplitude of the EPSCs were higher than those of IPSCs in the neurons from TLE patients (Figure 3A right). Ultrasound stimulation progressively restored the imbalance of E/I frequency from 2.10 ± 0.10 Hz to 0.99 ± 0.05 Hz (P
< 0.01, paired t test, Figure 3B). However, ultrasound stimulation did not change the Due east/I of amplitude (Before U.s.: 1.64 ± 0.06, after US: 1.65 ± 0.06,
P
= 0.xix, paired t test, Effigy 3C).

 Figure 3

Ultrasound stimulation re-adjusts the imbalance of excitatory and inhibitory inputs in the neurons from TLE epileptic slices. (A) Representative electric current traces of EPSCs and IPSCs were recorded in the neurons from TLE patients and neurons from glioma patients separated by property in different membrane potentials. (B-C) 9 neurons from TLE epileptic slices showed an imbalance in Eastward/I of frequency or amplitude compared with the neurons from glioma patients (unpaired
t
test,
P
< 0.01). Ultrasound stimulation re-adapted the balance of Due east/I in frequency, but non aamplitude (x neurons from TLE epileptic slicesultrasound stimulation in the E/I of frequency, paired
t
exam,
P
< 0.01; 1.64 ± 0.06 to 1.65 ± 0.06 after 60 seconds ultrasound stimulation in the E/I of amplitude, paired
t
exam,
P
= 0.37 ).

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The observed inhibitory effect of epileptiform discharges induced past ultrasound stimulation might be result from reduced excitatory inputs, increased inhibitory inputs or modulating both excitatory and inhibitory inputs. To exam this hypothesis, the belongings membrane potential was set at -70 mV for recording excitatory post-synaptic currents during three phases (Figure 4A). Ultrasound stimulation had no significant issue on the events and relative frequency of EPSCs compared to those before ultrasound (US: i.024 ± 0.035 Hz; after US: 1.029 ± 0.016 Hz;
P
= 0.49, ane-way ANOVA followed by LSD, n = 12, Figure 4B-C). Furthermore, analysis of the EPSCs waveform revealed that the mean of amplitude, x% – 90% rise time, and one-half-width were unchanged before ultrasound and in ultrasound stimulated groups (Supplementary Figure S7A).

We further tested the effects of ultrasound stimulation on inhibitory synaptic activities. The holding membrane potential was prepare at 0 mV for recording inhibitory mail-synaptic currents during three phases (Effigy 4D). A population of 13 neurons from TLE patients showed a rapid and significant increment in the relative frequency of IPSC events (Figure 4E) compared to before ultrasound (Usa: 2.467 ± 0.075 Hz; afterward United states: 3.241 ± 0.143 Hz;
P
< 0.01, one-way ANOVA followed by LSD, Figure 4F). As was the example with EPSCs, there was no alter in the hateful of aamplitude, 10%-90% of ascent time, and half-width in IPSC waveform before and afterward the ultrasound stimulation (Supplementary Effigy S7B). Pretreatment with SR-95531, a specific GABA receptor antagonist, prevented the effect of ultrasound stimulation on inhibition of epileptiform activities (Supplementary Figure S8). Handling with the GABA receptor antagonist hardly changed the relative frequency of ultrasound stimulation (during Usa: ane.02 ± 0.027 Hz; Afterwards Us: 1.04 ± 0.026 Hz;
P
= 0.29, one-way ANOVA followed by LSD, Supplementary Figure S8A-C) and individual spike waveform (326.18 ± 46.43 pA to 328.54 ± 41.62 pA,
P
= 0.31, Student’s paired t-test for mean spikes amplitude; 0.525 ± 0.03 ms to 0.515 ± 0.05ms,
P
= 0.39, Educatee’s paired t-examination for mean spikes half-width, Supplementary Figure S8D-E). The results demonstrated that ultrasound stimulation could induce an increase in inhibitory inputs, thus adjusting the synaptic inputs to inhibit the epileptiform discharges.

 Effigy 4

Ultrasound stimulation directly increases the inhibitory synaptic inputs. (A) Representative current traces of spontaneous EPSCs recorded in the neurons from TLE epileptic slices in unlike recording phases. (B-C) Ultrasound stimulation did not change the events and normalized frequency of EPSCs in neurons. (D) Representative current traces of spontaneous IPSCs recorded from TLE epileptic slices in different recording phases. (East-F) Ultrasound stimulation significantly increased the events and normalized frequency of IPSCs.

Theranostics Image

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Ultrasound stimulation activates the interneurons to increase the inhibitory synaptic inputs

We tested the possibility that ultrasound stimulation directly activated the interneurons of epileptic tissues to increase inhibitory inputs. Whole-cell patch-clench recording of typical interneurons was identified by morphology and firing pattern in response to injected currents and indicated that ultrasound stimulation increased the inter-neuronal excitability of epileptic tissues (Pupil’south paired
t-exam, **
P
< 0.01, Figure 5A-C). Consistent with these findings, ultrasound stimulation decreased the activity of pyramidal neurons in epileptic slices (Pupil’s paired
t-test, *
P
< 0.05, **
P
< 0.01, Figure 5D-F).

Three potential physical mechanisms could contribute to the modulation of neuronal excitability induced by ultrasound stimulation including thermal, mechanical, and cavitation effects [69]. Pulsed ultrasound stimulation with 60 second elapsing could accrue heat in the recording chamber and the temperature meridian might exert an important influence on the activity of neurons [20, 70, 71]. The results showed that the temperature superlative during the perfusion of artificial cerebrospinal fluid (ACSF) during ultrasound stimulation was relatively small (less than 0.64 ± 0.036°C; Supplementary Figure S9). To evaluate the effect of temperature induced past ultrasound, we also stimulated the neurons obtained from human epileptic slices using heated ACSF (ii°C temperature height) in a water bath. The results showed that 2°C temperature pinnacle of ACSF perfusion during the recording had no significant effect on the spontaneous activity and evoked firing of recording neurons in TLE slices (Supplementary Effigy S10), indicating that temperature superlative of 0.64 ±0.036 °C might not inhibit the epileptiform discharge in human TLE slices. The frequency used in the brain slices (28 MHz) was relatively loftier. Therefore, we also made a neuro-modulation scrap with
a
frequency of six.57 MHz to investigate the effect of the frequency on the suppression of neuronal activities in pyramidal neurons from homo epileptic slices. The results demonstrated that the ultrasound with a frequency of 6.57 MHz could likewise decrease the firing frequency of pyramidal neurons in homo epileptic slices. For the animal study or realistic man therapy, a lower frequency is needed to reduce skull attenuation (Supplementary Figure S11).

 Effigy v

Ultrasound stimulation modulates the neuronal excitability in epileptic slices. (A) Representative voltage traces recorded from interneurons of epileptic slices in response to a sequence of sustained currents injection (50, 150, and 250 pA). During ultrasound stimulation, interneurons could activate more action potentials. (B) Double-staining of biocytin-injected neurons with GAD 67 antibody showing a typical morphology of interneuron. Scale bar = 10 μm. (C) Ultrasound stimulation (red) significantly increased the firing frequency of interneurons compared with Before US (black, Student’s paired t-test, **
P
< 0.01). (D) Representative voltage traces recorded from pyramidal neurons of epileptic slices in response to a sequence of sustained currents injection (150, 250, and 350 pA). Ultrasound stimulation suppressed the firing frequency of pyramidal neurons. (Eastward) Biocytin-injected staining showing a typical morphology of pyramidal neurons. Scale bar = 20 μm. (F) Ultrasound stimulation (red) significantly decreased the firing frequency of pyramidal neurons compared with Before United states of america (black, Student’s paired t-test, *
P
< 0.05; **
P
< 0.01).

Theranostics Image

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Due to the high frequency of 28 MHz, the pulsed ultrasound used in the nowadays study could non generate acoustic cavitation without the microbubbles [72, 73]. It is, therefore, likely that the mechanical effect mediated the inhibitory effects of ultrasound on neuronal activities. These results were consistent with previous reports that the electrical activity in the epileptic network was associated with disruption of synaptic inputs, which promoted neuronal hyperexcitability and hyper-synchronization through an increase in excitatory neurotransmission besides equally a decrease of inhibitory neurotransmission or GABA, leading to neuronal hyper-excitability [56, 74]. Because of differences in the audio-visual modal, frequency, audio-visual intensity etc. used in the primate experiments equally well as homo epileptic slice, the inhibitory machinery of ultrasound might be entirely different. Farther studies are needed to understand the different furnishings.

Conclusions

Herein, nosotros demonstrated, for the first fourth dimension, that low-intensity pulsed ultrasound could improve behavioral outcomes in the non-human primate models of epilepsy and suppress abnormal epileptiform activities on slices harvested from epileptic patients. In the non-human primate model of epilepsy, 30 minutes of ultrasound treatment significantly reduced total seizure counts for 8 hours and seizure frequency per hour. In human epileptic slices, ultrasound stimulation could inhibit epileptiform activities with an efficiency exceeding 65%, probably due to the aligning of synaptic inputs by the increased neuronal excitability of local inhibitory neurons. Our study suggests that low-intensity pulsed ultrasound could suppress epileptiform activities and might provide a potential clinical treatment for epilepsy.

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Supplementary Material

Supplementary figures and tables.

Attachment

Acknowledgements

The work was supported by the National Natural Science Foundation of China (Grant No. 81527901; 81671193; 81501046; 11904380); Guangdong-Hong Kong-Macao Greater Bay Area Heart for Brain Science and Brain-Inspired Intelligence Fund (NO. 2019024); Guangdong grant ‘Key technologies for handling of encephalon disorders’ (No.2018B030332001); and CAS Key Laboratory of Health Computer science Fund (2011DP173015). We thank Dr. Xiaoming Li (Zhe Jiang University), Dr. Jie Tang (Southern Medical University) and Dr. Jiandong Yu (Jinan University) for helpful discussions. Z. L., L. N., L. K., and H. Z. designed experiments. Z. L., Due west. Z., and J. Z. conducted the experiments. Due south. Ten. and Y. G. designed and performed epilepsy surgery. Z. L., L. Northward., L. M., T. Y. and H. Z. wrote the manuscript.

Competing Interests

The authors have declared that no competing involvement exists.

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Author contact


Corresponding author: Electronic mail: ll.niuac.cn; eguoyanwucom; hr.zhengac.cn


Received 2019-nine-21

Accepted 2020-1-10

Published 2020-4-12

Source: https://www.thno.org/v10p5514.htm