Original ContributionImage-Guided Focused Ultrasound-Mediated Regional Brain Stimulation in Sheep
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.
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