Original Contribution
Image-Guided Focused Ultrasound-Mediated Regional Brain Stimulation in Sheep

https://doi.org/10.1016/j.ultrasmedbio.2015.10.001Get rights and content

Abstract

Non-invasive brain stimulation using focused ultrasound has largely been carried out in small animals. In the present study, we applied stimulatory focused ultrasound transcranially to the primary sensorimotor (SM1) and visual (V1) brain areas in sheep (Dorset, all female, n = 8), under the guidance of magnetic resonance imaging, and examined the electrophysiologic responses. By use of a 250-kHz focused ultrasound transducer, the area was sonicated in pulsed mode (tone-burst duration of 1 ms, duty cycle of 50%) for 300 ms. The acoustic intensity at the focal target was varied up to a spatial peak pulse-average intensity (Isppa) of 14.3 W/cm2. Sonication of SM1 elicited electromyographic responses from the contralateral hind leg, whereas stimulation of V1 generated electroencephalographic potentials. These responses were detected only above a certain acoustic intensity, and the threshold intensity, as well as the degree of responses, varied among sheep. Post-sonication animal behavior was normal, but minor microhemorrhages were observed from the V1 areas exposed to highly repetitive sonication (every second for ≥500 times for electroencephalographic measurements, Isppa = 6.6–10.5 W/cm2, mechanical index = 0.9–1.2). Our results suggest the potential translational utility of focused ultrasound as a new brain stimulation modality, yet also call for caution in the use of an excessive number of sonications.

Introduction

The development of a method that enables modulation of regional brain activity is sought after as a potential neurotherapeutic modality for neurologic and psychiatric disorders (George and Aston-Jones, 2010, Hoy and Fitzgerald, 2010), as well as a tool for functional brain mapping (Hallett, 2000, Min et al., 2011b). Deep brain stimulation (DBS) and epidural cortical stimulation (EpCS) can modulate the region-specific function of the brain, but the range of utilization is limited because of the invasive surgeries required (Hoy and Fitzgerald 2010). Non-invasive techniques, such as transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS), lack spatial specificity and penetration depth (Fregni and Pascual-Leone, 2007, Loo and Mitchell, 2005). Optogenetic techniques are capable of controlling the neural activity in the brain on a cellular level (Deisseroth, 2011, Miesenböck, 2009), yet the genetic modification of neurons needed to introduce the stimulatory response to an external light stimulus, along with the limited transcranial penetration of the stimulatory light, may impede its prompt utilization in humans.

Focused ultrasound (FUS) techniques deliver acoustic pressure waves to a small, localized area (on the order of a few millimeters in diameter) of biological tissue (Fry et al., 1955, Fry and Fry, 1960, Hynynen et al., 1996, Jolesz et al., 2005, Lele, 1962, Lynn et al., 1942, Vallancien et al., 1992, Yang et al., 1992). Advancement in FUS technology has enabled the transcranial application of highly focused ultrasound to region-specific brain areas in a non-invasive manner (Elias et al., 2013, Hynynen et al., 2004, Martin et al., 2009). With advantages of spatial specificity and depth penetration over existing methods, FUS has been investigated as a new mode of brain stimulation (Bystritsky et al., 2011, Tufail et al., 2011, Yoo et al., 2011). After early seminal work by Fry et al. (1958), who reported that the sonication of the lateral geniculate nucleus of cats can temporarily modify visual evoked potentials (VEPs), the neuromodulatory effects of ultrasound were illustrated by sonicating excised ex vivo rodent brain tissue (Bachtold et al., 1998, Rinaldi et al., 1991, Tyler et al., 2008). Subsequent in vivo studies have revealed that FUS applied to region-specific brain areas reversibly modulates the excitability of the motor and visual areas in rabbits (Yoo et al. 2011), stimulates the various motor areas (Mehić et al. 2014), suppresses epileptic electroencephalogram (EEG) activity (Min et al. 2011a) and alters the extracellular levels of neurotransmitters in rats (Min et al., 2011b, Yang et al., 2012). The effects of sonication parameters on the effectiveness of neuromodulation have also been investigated using small animals (Kim et al., 2014, Kim et al., 2015, King et al., 2013). Although the stimulatory effects of FUS have been reported in humans (Lee et al., 2015, Legon et al., 2014) and non-human primates (Deffieux et al. 2013), studies on large animals are warranted to validate the stimulatory findings from small animals, as well as to establish important translational tolerability information for human studies.

As the size of the acoustic focus and concomitant stimulatory area is small (Kim et al. 2013), the use of large animal species (with large brain volumes) is helpful in validating the stimulatory effects of FUS on a discrete region-specific area of the brain. Furthermore, the effect of acoustic reverberations, which may result in less accurate spatial localization of the acoustic energy in a small cranium (Younan et al. 2013), is of less concern in larger cranial structures. In the study described here, we explored the administration of transcranial FUS to region-specific (i.e., primary sensorimotor [SM1] and visual [V1]) cortical areas of sheep. Sheep were chosen as a study model because of their large brain volume with distinct neuroanatomic structures. Unlike pigs (having a flat and thick skull), sheep have a relatively round skull with a thickness (on the order of 4–5 mm) similar to that of humans. Also, its availability in various brain disease/injury models, such as stroke (Boltze et al. 2008), epilepsy (Stypulkowski et al. 2014) and brain injury (Van den Heuvel et al. 1999), makes sheep an attractive species for translational research of FUS.

The hypothesis tested in the present study is that pulsed application of the FUS transcranially delivered to the SM1 and V1 of the sheep brain would stimulate the regional brain tissue. Our aim was to illustrate that the stimulation elicits corresponding electromyogram (EMG)-based motor evoked potentials (MEPs) and EEG-based VEPs. To distinguish the VEPs elicited by the FUS from the traditional nomenclature describing the EEG potentials evoked by external visual stimulation, a term, sonication-triggered VEPs (sVEPs), was employed throughout the text. Placement of the FUS focus at the desired SM1 and V1 areas was achieved using anatomic and functional magnetic resonance imaging (MRI) data obtained from each sheep brain to promote spatial accuracy of the sonication. Different acoustic intensities (AIs) were applied to probe their effect on the magnitude of the evoked potentials. We also assessed the behavior of each animal at different time points for up to 2 mo after sonication and conducted histologic analysis on the sonicated brain tissue.

Section snippets

Animal preparation

All animal procedures were performed under the approval of and according to the ethical standards set forth by the Harvard Medical Area Standing Committee. Each sheep (Dorset, all female, weight = 32.6 ± 4.4 kg, mean ± SD, 25–38 kg, n = 8, numbered S1 through S8 herein) underwent two separate procedures: (i) identification of the anatomic and functional locations of the SM1 and V1 areas for sonication using MRI, and (ii) FUS stimulation sessions. For all procedures, the animals were sedated and

Sensorimotor area (SM1) and visual area (V1) stimulation

Sonication-related EMG signals were detected for all sheep, except S6. MEPs were not detected when there was no sonication. Elicited MEPs were detected only from the right hind limb muscle contralateral to the side of sonication; no MEPs were detected from the left (ipsilateral) hind limb (Fig. 2a). MEPs were not accompanied by actual muscle or limb movement (data not shown). Sonication elicited signals over a certain AI threshold, which varied among sheep (Fig. 2b). For example, the threshold

Discussion

Image-guided transcranial administration of FUS to specific areas of the sheep brain induced active physiologic responses, which suggests successful stimulation of the corresponding brain areas. We observed that the responses were detected only over a certain AI threshold level. The results are in good agreement with those for acoustic stimulation in small animal models, in which the success rate for eliciting motor movement in rodents exhibited similar threshold effects (King et al., 2013,

Acknowledgments

This study was supported by National Institutes of Health Grant R21 NS074124 (partial salary support) and KIST Grant to S.S.Y. and by the Focused Ultrasound Surgery Foundation (to S.S.Y.).

We thank Dr. Yongzhi Zhang and Dr. Mimi Lam for helpful technical advice on sheep procedures. The initial help and support by Mr. Jeffrey Pettit and Ms. Rita G. Laurence are also acknowledged. We also thank Mr. Matthew J. Marzelli for editorial assistance.

References (70)

  • W. Muellbacher et al.

    Effects of low-frequency transcranial magnetic stimulation on motor excitability and basic motor behavior

    Clin Neurophysiol

    (2000)
  • W.D. O'Brien

    Ultrasound—Biophysics mechanisms

    Prog Biophys Mol Biol

    (2007)
  • L.W. Ostrow et al.

    Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    Biochem Biophys Res Commun

    (2011)
  • P.C. Rinaldi et al.

    Modification by focused ultrasound pulses of electrically evoked responses from an in vitro hippocampal preparation

    Brain Res

    (1991)
  • P.H. Stypulkowski et al.

    Brain stimulation for epilepsy—Local and remote modulation of network excitability

    Brain Stimul

    (2014)
  • T. Tsurugizawa et al.

    Effects of isoflurane and alpha-chloralose anesthesia on BOLD fMRI responses to ingested L-glutamate in rats

    Neuroscience

    (2010)
  • Y. Tufail et al.

    Transcranial pulsed ultrasound stimulates intact brain circuits

    Neuron

    (2010)
  • C. Van den Heuvel et al.

    Upregulation of amyloid precursor protein messenger RNA in response to traumatic brain injury: An ovine head impact model

    Exp Neurol

    (1999)
  • S.S. Yoo et al.

    Focused ultrasound modulates region-specific brain activity

    Neuroimage

    (2011)
  • J. Boltze et al.

    Permanent middle cerebral artery occlusion in sheep: A novel large animal model of focal cerebral ischemia

    J Cereb Blood Flow Metab

    (2008)
  • P.G. Clarke et al.

    The cortical visual areas of the sheep

    J Physiol

    (1976)
  • C.M. Collins et al.

    Model of local temperature changes in brain upon functional activation

    J Appl Physiol

    (2004)
  • C. Deblieck et al.

    Correlation between motor and phosphene thresholds: A transcranial magnetic stimulation study

    Hum Brain Mapp

    (2008)
  • K. Deisseroth

    Optogenetics

    Nat Methods

    (2011)
  • T. Eken

    Spontaneous electromyographic activity in adult rat soleus muscle

    J Neurophysiol

    (1998)
  • W.J. Elias et al.

    A pilot study of focused ultrasound thalamotomy for essential tremor

    N Engl J Med

    (2013)
  • J.M. Fitzpatrick et al.

    The distribution of target registration error in rigid-body point-based registration

    IEEE Trans Med Imaging

    (2001)
  • F. Fregni et al.

    Technology insight: Noninvasive brain stimulation in neurology—Perspectives on the therapeutic potential of rTMS and tDCS

    Nat Clin Pract Neurol

    (2007)
  • W.J. Fry et al.

    Ultrasonically produced localized selective lesions in the central nervous system

    Am J Phys Med

    (1955)
  • F.J. Fry et al.

    Production of reversible changes in the central nervous system by ultrasound

    Science

    (1958)
  • W.J. Fry et al.

    Fundamental neurological research and human neurosurgery using intense ultrasound

    IRE Trans Med Electron

    (1960)
  • R.H. Garman

    Artifacts in routinely immersion fixed nervous tissue

    Toxicol Pathol

    (1990)
  • M.S. George et al.

    Noninvasive techniques for probing neurocircuitry and treating illness: Vagus nerve stimulation (VNS), transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS)

    Neuropsychopharmacology

    (2010)
  • M. Hallett

    Transcranial magnetic stimulation and the human brain

    Nature

    (2000)
  • A.M. Harper et al.

    Effect of alterations in the arterial carbon dioxide tension on the blood flow through the cerebral cortex at normal and low arterial blood pressures

    J Neurol Neurosurg Psychiatry

    (1965)
  • Cited by (145)

    View all citing articles on Scopus
    View full text