Original ContributionNoninvasive Transcranial Stimulation of Rat Abducens Nerve by Focused Ultrasound
Introduction
The cranial nerves, as part of the peripheral nervous system, dictate important sensory motor functions of the head ranging from vision, audition and olfaction, to the movement of ocular as well as facial/vocal muscles. In addition, cranial nerves subserve autonomic nervous functions, such as innervations of abdominal viscera in the case of the vagus nerve. Aberrant or disrupted cranial nerve function ramifies into pathologic sensory conditions (e.g., trigeminal neuralgia), impaired facial expression or abnormal eye movement (e.g., eye movement disorders associated with cerebral nerve palsy) (Wilson-Pauwels 2010).
Progress has been made in an attempt to gain control over aberrant cranial neural transmissions, mainly through surgical or pharmacologic interventions. Electrical neuromodulation of cranial nerves, such as vagus nerve stimulation (VNS) and trigeminal nerve stimulation (TNS), have been applied using surgically implanted electrodes to treat epilepsy and trigeminal neuralgia (DeGiorgio et al. 2003; Uthman et al. 2004). Direct electrical stimulation of optic and auditory nerves has also been proposed to augment corresponding impaired sensory functions (Simmons et al. 1965; Killian and Fromm 1968; Humayun et al. 1999). Pharmacologic treatments aiming for the modulation of neural excitability, such as administration of anticonvulsants for trigeminal neuralgia, have been deployed (Blom 1962); however, they lack selectivity toward the specific neural tissue-of-interest and have potential adverse effects (e.g., aplastic anemia) (Donaldson and Graham 1965; Dyer et al. 1966). Therefore, noninvasive and nonpharmacologic means to harness cranial nerve function, with the spatial specificity to target the anatomy-of-interest, have been warranted.
As a noninvasive alternative to the surgical interventions, transcranial magnetic stimulation (TMS) of cranial nerves has been proposed (Wessel and Kompf 1991; Roth et al. 1994). TMS relies on inducing electrical current on biological tissue by applying strong time-variant magnetic fields over the skin surface, and has been used for noninvasive stimulation of peripheral nerves as well as brain cortices (Roth et al. 1994). However, the inductive nature of TMS lacks sufficient penetration depth to reach the deep brain area (because of the rapid reduction of the magnetic field away from the coil) and also lacks spatial specificity during the stimulation (i.e., the modulatory area is wide, on the order of a few centimeters in diameter) (Kobayashi and Pascual-Leone 2003).
With recent advancements in the focused ultrasound (FUS) technique, highly focused acoustic energy can be delivered to specific areas of biological tissues, as small as a few millimeters in diameter. The acoustic energy is deposited to the tissues at the focus as thermal or mechanical energy, which can be used to ablate tumorous tissue via hyperthermia or to break down crystalline structures such as kidney stones. Ultrasound, typically operating at a frequency <1 MHz, can also be delivered through the skull to specific areas of the brain, such as the thalamus, in a focused manner using helmetlike multi-array ultrasound transducers (Clement et al. 2005) or through thin temporal bone (Gavrilov et al. 1996). Most of the acoustic energy given >1 MHz is attenuated during the transmission through the skull. The lower acoustic frequency (compared with the one used in diagnostic imaging, i.e., on the order of 1 to 15 MHz) (O’Brien 2007) favors the transcranial application of the FUS because of longer wavelength, e.g., only mild attenuation (22.5%) of the acoustic intensity has been observed at 120 kHz through a human skull (Coussios et al. 2002). For these reasons, commercially available transcranial FUS equipment (for human use) uses the frequencies in the range of 200–700 kHz and have been used in hyperthermic ablative treatments for brain tumors (McDannold and Jolesz 2000) and functional neurosurgery (Martin et al. 2009). Regarding its extended potential in modulating peripheral nerve function, Colluci and colleagues have shown that pulsed high-intensity (high enough to raise the temperature of the nerve bundle) FUS, applied to excised frog sciatic nerves, supressed the magnitude and latencies in action potential propagation (Colucci et al. 2009).
Pulsed application of the ultrasound at low acoustic intensity, under the threshold for heat generation or mechanical damage in biological tissue, has been shown to modulate the excitability of the brain tissue both ex vivo (Gavrilov et al. 1996; Bachtold et al. 1998) as well as in vivo (Tufail et al. 2010). We have also recently demonstrated that FUS, when applied in short bursts of pulses, modulates neural tissue excitability in the motor and visual cortices of rabbits without changing tissue temperature (Yoo et al. 2011). This exciting feature has also been applied to decrease the electrographic seizure activites from chemically-induced epileptic rats (Min et al. 2011a), and to modify the extracelluar level of neurotransmitters (Min et al. 2011b; Yang et al. 2012). Converging evidence from these studies indicates that application of FUS at low acoustic intensity, typically under the FDA limit for diagnostic imaging (720 mW/cm2 Ispta; spatial-peak temporal-average intensity) (AIUM Clinical Standards Committee 2004), temporarily and reversibly modified neural function in vivo.
In the present study, we were motivated to extend the applicability of FUS in stimulating the peripheral nervous system, specifically the cranial nerves. The ability to stimulate intracranial nerves using focused ultrasound was explored at two different frequencies (350 kHz and 650 kHz), falling within the ranges of frequencies adopted in human transcranial FUS systems. We hypothesized that the successful stimulation of the abducens nerve by pulsed FUS sonication would induce corresponding abductive eyeball movements.
Section snippets
Materials and Methods
All experiments were conducted under institutional review and approval by the Harvard Medical Area Standing Committee on Animals. Male Sprague-Dawley rats (weight 290 to 405 g; n = 22) were used in this study. All animals were anesthetized with an intraperitoneal injection of a ketamine/xylazine mixture of 80:10 mg/kg before sonication.
Effect of sonication on abductive eye movement
The application of 650 kHz FUS to the same area, across the ranges of acoustic intensities, from 0.5 up to 20 W/cm2 Isppa, did not elicit eye movement in any of the tested animals (n = 3). On the other hand, abductive eyeball movement, ipsilateral to the site of sonication, was observed during the sonication at 350 kHz, as shown in Figure 2a and b (and in Supplemental Movie S1). The eyeball movement was discrete and occurred upon each sonication, which was given at 1-s intervals. Figure 2c
Discussion
The application of low-intensity FUS, transcranially delivered to the abducens nerve, elicited the corresponding abductive ipsilateral eyeball movement in rats. The acoustic intensity used in the present study for successful stimulation (4.6 W/cm2 Ispta) was comparable with the intensity used in a previous study on excitation of the motor area of the rabbit brain (6.3 W/cm2 Ispta) (Yoo et al. 2011), suggesting that acoustic intensity, on the order of 4.6–6.3 W/cm2 Ispta, successfully stimulates
Acknowledgments
This work was supported by grants from the National Institute of Health (R21 NS074124 to Yoo) and the National Research Foundation of Korea (Korean Ministry of Education, Science and Technology, 2010-0027294 to Park). The authors gratefully acknowledge the assistance of Drs. Yong-Zhi Zhang and Byoung-Kyong Min in data acquisition and animal preparation. The authors also thank Alan Chiu for editorial support.
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