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Ultrasound-Induced Heart Rate Decrease: Role of the Vagus Nerve

olivia c. coiado, Elaine b. buiochi, and William d. o’brien Jr., Life Fellow, IEEE

Abstract—The goal of this study is to investigate the role of the vagus nerve (VN) in the ultrasound (US)-induced negative chronotropic effect (deceased heart rate). One of the functions of the VN is to mediate lowering of the heart rate. A previous study showed a decrease of ~20% in the heart rate but the mechanism of the effect was not investigated. Sprague Dawley rats (n = 20) were exposed transthoracically to ultrasonic puls-es at an approximate duty factor of 1% with sequentially 2.0, 2.5, and 3.0 MPa peak rarefactional pressure amplitudes (PRPAs). The ultrasonic exposure parameters herein were chosen to match those of the previous study to have confidence that an ultrasound-induced negative chronotropic effect would occur. For each of the three PRPA sequences, the pulse repeti-tion frequency (PRF) started slightly greater than the rat’s heart rate and then was decreased sequentially in 1-Hz steps every 10 s (i.e., 6, 5, and 4 Hz for a total duration of 30 s). The experiments were organized in a standard (2 × 2) factorial design with VN (cut versus intact) as one factor and US (on versus off) as another factor. VN (intact/cut) and US (on/off) groups were divided into four groups each consisting of 5 ani-mals: 1) VN intact-US off, 2) VN intact-US on, 3) VN cut-US off, and 4) VN cut-US on. Two-way analysis of variance for repeated measures was used to compare heart rate, cardiac output, systolic volume, ejection fraction, end-diastolic vol-ume, end-systolic volume, respiratory rate, and arterial pres-sure before and after ultrasound stimulation. In this study, the heart rate decreased ~4% for the non-vagotomy and vagotomy groups. The ultrasound effect was significant for heart rate (p = 0.02) and cardiac output (p = 0.005) at 3 min post US expo-sure; the vagotomy effect was not significant. For heart rate, the Bonferroni test showed no differences between the four groups. The vagotomy group showed similar ultrasound-in-duced cardiac effects compared with the non-vagotomy group, suggesting that the vagus nerve is not influenced by the ultra-sound exposure procedures. The US application caused a nega-tive chronotropic effect of the rat heart without affecting the hemodynamic conditions. The results at this point are sugges-tive for an alternative cardiac pacing capability.

I. Introduction

Therapeutic ultrasound applications continue to in-crease and include, for example, physiotherapy, litho-

tripsy, hyperthermia, focused-ultrasound surgery, pain relief, and bone healing [1]. Therapeutic effects of cardiac ultrasound have been examined through in vitro and in vivo studies [2]–[9]. In vitro cardiac tissue observations

in frogs, guinea pigs, rats, dogs, and pigs included defi-brillation [3], premature contraction of myocardium [7], [10]–[12], negative chronotropic effect [9], and positive ino-tropic/lusitropic effect [13]. In vivo observations of pulsed ultrasound (Us) delivered to the animal heart caused a variety of effects such as intravascular bubbles [4], ar-rhythmias [5], changes in cardiac rhythm and aortic pres-sure [6], and cardiac pacing [8]. such effects depended on variations in the Us parameters, such as pulse repetition frequency (PrF), magnitude (peak rarefactional pressure amplitude, PrPa), frequency, and exposure duration.

Ultrasound is mechanical energy that propagates longi-tudinally through elastic media, such as tissues [14]. The heart rhythm can be affected by mechanical disturbances and influence the cardiac electrical excitation by intra- or extra-cardiac mechanisms [9].

Mechanical pertubation of the vagus nerve during neck dissection may stimulate actions that cause changes in the cardiac rhythm and rate. also traction of the vagus nerve has often resulted in bradycardia (slow heart rate) and decreased blood pressure [15]. The general role of the vagus nerve acts to lower the heart rate. The question to be addressed herein is whether these ultrasound-induced cardiac observations were induced directly by the activa-tion of efferent vagal fibers (carries nerve impulses toward muscles/glands—such as the heart—from the central ner-vous system), or by a reflex response resulting from exci-tation of afferent nerve fibers (carries nerve impulses in the opposite direction, that is, toward the central nervous system) [15], [16]. The vagus nerve consists of efferent parasympathetic fibers and afferent sensory fibers.

In this study, a bilateral vagotomy was performed to evaluate whether the vagus nerve was involved in the ultrasound-induced negative chronotropic effect. If the heart rate was depressed in a vagotomized animal, then the mechanism is likely a reflex response. If so, then one of the causes is likely that the direct ultrasonic stimulation of aortic baroreceptors triggers the baroreceptor reflex, increasing the parasympathetic tone and decreasing the sympathetic tone, with resultant bradycardia. another possible cause is the bezold–Jarisch reflex, an eponym for a triad of responses (apneia, bradycardia, and hypoten-sion) [17]. This cardio-inhibitory reflex results in brady-cardia, vasodilation, and hypotension. The bezold–Jarisch reflex originates in cardiac receptors that are mainly lo-cated in the posterior-inferior wall of the left ventricle, and is activated by mechanical or chemical stimulation, leading to increased parasympathetic activity and inhibi-tion of sympathetic activity [18].

Manuscript received september 25, 2014; accepted november 12, 2014. This work was supported by sao Paulo research Foundation (FaPEsP 2008/54165-2) and by the national Institutes of Health (r37 Eb002641).

The authors are with the bioacoustics research laboratory, depart-ment of Electrical and computer Engineering, University of Illinois at Urbana-champaign, Urbana, Il (e-mail: coiado@illinois.edu).

doI http://dx.doi.org/10.1109/TUFFc.2014.006755

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The potential of cardiac pacing using pulsed ultrasound is clinically important as an alternative and leadless source of energy. all pacing leads are associated with complica-tions such as infection, fracture, and dislodgment, so there is a clinical need to develop a pacing system that reduces the problem inherent in the pacing leads [19], [20]. The extraction of a failed lead that has been implanted for a long time could be a high-risk procedure, including serious arterial injuries or even death [21]–[23].

In a previous study [9], a negative chronotropic effect was shown to be reproducible, but the origin of the mech-anism was not investigated. The previous study showed a decrease in the heart rate by ~20% in 3-mo-old female sprague dawley rats. The objective of this study is to investigate the role of the vagus nerve (Vn) on the ultra-sound-induced depression of the heart rate in 3-mo-old female sprague-dawley rats using the same transthoracic Us exposure conditions as those of the previous study. The same exposure conditions were used because they produced a significant negative chronotropic effect. com-paring with other studies [1]–[10], this is the first set of animal experiments to investigate the mechanism that induces a decreased heart rate. The Us protocol in this study involves a specific combination of PrF sequences and amplitudes [9]; this unique sequence has never been shown before in other studies [1]–[8], [10].

II. Methodology

A. Animals

Experimental conditions were approved by the Univer-sity of Illinois Institutional animal care and Use commit-tee (protocol #10104). rats of the same age and strain as those from previous study [9] were used. The experiments were organized in a standard (2 × 2) factorial design with Vn (cut versus intact) as one factor and Us (on versus off) as another factor. Twenty 3-mo-old 250- to 300-g fe-male sprague dawley rats (Harlan laboratories Inc., Indi-anapolis, In) were divided into four groups (n = 5 ea): 1) Vn intact-Us off, 2) Vn intact-Us on, 3) Vn cut-Us off, and 4) Vn cut-Us on; see Fig. 1. animals received 5% iso-flurane for inhalation anesthesia induction, and then 1.5% to 2% isoflurane for anesthesia maintenance. The level of anesthesia was monitored by pedal reflex. The thoracic re-gion was shaved and depilated to maximize transthoracic acoustic transmission. Gel was used for acoustic coupling. rats were placed on a temperature-controlled platform in dorsal recumbency for transthoracic Us exposure of the

heart. The animals’ limbs were secured to the electrocar-diogram (EKG) pads (4 leads) on the animal platform, a capability connected to the small animal Vevo 2100 high-frequency (13 to 24 MHz) ultrasound imaging system (Vi-sualsonics, Toronto, on, canada) so that physiological data could be monitored in real time and also recorded. The Vevo 2100 was used to monitor the heart via b-mode and M-mode displays. Ejection fraction and other cardiac parameters were calculated by the ventricular trace tool from the Vevo 2100 workstation. This tool is used to trace the position of the inner and outer ventricular walls over one or more heart cycles on a long-axis M-mode tracing of the left ventricle [9].

In physiologic and cardiologic studies, it is often the practice to record arterial pressure from an easily acces-sible artery such as the femoral, tail, or carotid artery, and to use that pressure to represent the systemic arte-rial pressure or mean pressure [10]. In our previous study, arterial pressure was not monitored [9]. In this study, ar-terial pressure was monitored to assess possible changes that might elicit reflex responses that affect the heart. The right carotid artery of all rats was cannulated for arterial pressure measures. The cannula was connected to a pressure transducer (samba Preclin 420lP, samba sensors, Gothenburg, sweden) and a control unit (samba 201, samba sensors). also, the intercostal space (away from the ultrasound path) and rectal temperatures were monitored in all rats.

The objective of the vagotomy group was to clarify if the increase in vagal tone induced by reflex pathways was involved in the cardiac response to ultrasound. For the vagotomy groups (Vn cut-Us off and Vn cut-Us on), a bilateral vagotomy (the vagus nerve is paired) was performed before the ultrasound exposure session (Fig. 2). For the vagotomy, a small vertical midline incision 1 cm superior to the sternum was performed to expose the sternomastoid muscle up to the trachea. The muscle was reflected back to expose and ligate with silk suture the vagus nerve of both sides. The vagus nerve was then cut with small scissors. The animal’s neck region remained open for the balance of the experimental procedures.

For the non-vagotomy groups (Vn intact-Us off and Vn intact-Us on), the same surgical procedure was per-formed but following the ligation with silk suture, the va-gus nerve was not cut; the Vn remained intact.

For all rats, arterial pressure, intercostal temperature, and EKG were recorded continuously from before the ini-tiation of the Us application until about 18 min after the Us exposure ceased (see Fig. 2). Then (≈18 min), rats were euthanized (co2 inhalation for 5 min), and the lung

Fig. 1. 2 × 2 factorial design with vagus nerve (cut versus intact) as one factor and ultrasound (on versus off) as another factor.

coiado et al.: ultrasound-induced heart rate decrease: role of the vagus nerve 331

and heart were removed, fixed, and processed for evalua-tion by a board-certified pathologist.

B. Ultrasound

a commercially available transducer was used, with a 25-mm-diameter circular geometry aperture and a center frequency of 1 MHz (Pro seven 997, quark Medical, Pi-racicaba, brazil). For either reflex or direct mechanism, the size of the transducers may result in different Us stim-ulations on the heart and neighboring structures; however, the previous study [9] and this study used the same diam-eter and frequency of unfocused Us transducers.

The transducer was calibrated at 1 MHz in a tank containing distilled, degassed 22°c water. The calibrated hydrophone was a polyvinylidene fluoride (PVdF) mem-brane hydrophone (y-34-3598 EW295, GEc Marconi, chelmsford, UK) with a 0.5-mm-diameter active element. The transducer was held in a fixed position while the hy-drophone was moved perpendicular to the beam axis at a distance of 1 cm from the transducer surface (in the near field) by a micropositioning system (2-µm translational accuracy) [12].

The transducer was driven by a function generator (33250a, agilent Technologies Inc., santa clara, ca) and an rF power amplifier (a150, Electronic navigation In-dustries, rochester, ny; 0.3 to 35 MHz; 55 db). The Vevo 2100 ultrasound imaging system, operated by a registered diagnostic medical sonographer (rdMs), was used to monitor the heart via b-mode and M-mode displays. The Vevo 2100 workstation has a tool to calculate the ejec-tion fraction and other cardiac parameters by ventricular trace. To have an increased likelihood that the ultrasound-induced negative chronotropic effect would result in this experiment, the same exposure conditions of a previous experiment [9] were applied (Fig. 2): 1) bursts of 2.0 MPa

PrPa were delivered consecutively in three 10-s intervals; that is, 10 s for each PrF (6, 5, and 4 Hz) for a 30-s expo-sure duration. 2) bursts of 2.5 MPa PrPa were delivered consecutively in three 10-s intervals; that is, 10 s for each PrF (6, 5, and 4 Hz) for a 30-s exposure duration. 3) bursts of 3.0 MPa PrPa were delivered consecutively in three 10-s intervals; that is, 10 s for each PrF (6, 5, and 4 Hz) for a 30-s exposure duration. The duty factor was approximately 1%, which means that for a PrF sequence of 6, 5, and 4 Hz (i.e., 167, 200, and 250-ms pulse repeti-tion periods, correspondingly), a 2-ms pulse duration was used. The interval between the three-pulse sequences was 10 s (when ultrasound was turned off), and the total ultra-sound exposure duration was 90 s (plus the two 10-s no-ultrasound intervals). The same protocol as the previous study [9] was used, except that in this study the animals were also cannulated for arterial pressure measurement and a bilateral vagotomy was performed.

The consideration for the PrF sequence is that start-ing at a PrF slightly above the heart rate and then decreasing the PrF will likely be able to capture and pace the heart. The 2.0, 2.5, and 3.0 MPa PrPas are equivalent to spatial peak intensities of 133, 208, and 300 W/cm2, respectively. The ultrasonic parameters such as frequency, PrF, duty factor, and PrPa were studied pre-viously [9], [24]. a major issue is to avoid thermal dam-age, a temporal average concept. a theoretical evaluation of temperature increase was published wherein, for a 3.0-MPa PrPa, ΔTmax ≈ 0.014°c for a 2-ms pulse duration [9]. also, considering the peak pressure from which the mechanical índex (MI) is estimated, at 3.0 MPa and 1 MHz, the MI is around 3. because the Vevo 2100 is not an Fda-approved system, the MI is not reported, but it is likely quite low given the ~20 MHz frequency (a Vevo 2100 MI of 1.9 would be consistent with an 8-MPa PrPa, and is thus highly unlikely). Thus, it is prudent to

Fig. 2. Timeline for the 1-MHz transthoracic high-amplitude ultrasound stimulation. baseline data were acquired before the initial ultrasound exposure. Post-exposure data were acquired at 3 and 15 min after ultrasound ceased. Three ultrasonic exposure sequences of 30 s each (each with a different peak rarefactional pressure amplitude, 2.0, 2.5, and 3.0 MPa) were used to stimulate the heart, during which time three separate and decreasing PrFs were used, as noted (6, 5, and 4 Hz). The total duration of the stimulation protocol was 110 s. duty factor (dF) is approximate because the pulse duration was 2 ms.

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have the exposed cardiac tissue pathologically evaluated, which was done in this study.

C. Statistical Analysis

The data were organized in a standard (2 × 2) factorial design with Vn (cut versus intact) as one factor and Us (on versus off) as another factor (n = 5 per group), Fig. 1.

an unpaired student’s t-test was used to compare the absolute baseline values between the vagotomy and non-vagotomy groups. note that at baseline, there were only two groups, the vagotomy group (Vn cut-Us off and Vn cut-Us on) and the non-vagotomy group (Vn intact-Us off and Vn intact-Us on), n = 10 per group. The variables evaluated at baseline were heart rate, cardiac output, sys-tolic volume, ejection fraction, end-diastolic volume, end-systolic volume, respiratory rate, and arterial pressure.

Two-way analysis of variance (r version 3.0.2, “Fris-bee sailing,” The r Foundation for statistical comput-ing, auckland, new Zealand) for repeated measures was performed at 3 and 15 min post Us exposure and com-pared between 1) Vn intact-Us off, 2) Vn intact-Us on, 3) Vn cut-Us off, and 4) Vn cut-Us on, n = 5 per group. The variable values used for the statistical comparisons were normalized to the baseline values for each respective animal. The variables evaluated at 3 and 15 min post Us exposure were heart rate, cardiac output, systolic volume,

ejection fraction, end-diastolic volume, end-systolic vol-ume, respiratory rate, and arterial pressure. bonferroni post hoc tests were applied to compare the results.

all results were expressed as mean and standard error of the mean (sEM). The significance level was set at 0.05.

III. results

Tables I and II list the absolute values of physiologi-cal parameters at baseline and at 3 and 15 min post Us exposure for each of the four groups (n = 5 ea): 1) Vn intact-Us off, 2) Vn intact-Us on, 3) Vn cut-Us off, and 4) Vn cut-Us on, see Fig. 1. Fig. 3 graphically shows the heart rate, cardiac output, end-diastolic volume, and arte-rial pressure at 3 and 15 min post Us exposure normalized to their baseline values.

baseline: There were no significant differences between the vagotomy and non-vagotomy groups, n = 10 per group, that is, heart rate, cardiac output, systolic volume, ejection fraction, end-diastolic volume, end-systolic vol-ume, respiratory rate, and arterial pressure.

Heart rate: at 3 min post Us exposure, the ultrasound effect was significant (p = 0.02), a decrease of ~2% in the heart rate; the vagotomy effect was not significant. The interactions were not significant. at 15 min post Us exposure, there were no significant effects or interactions.

TablE I. absolute Values of Physiological Parameters at baseline and at 3 and 15 min Post Us Exposure (Fig. 1) for the non-Vagotomy Groups.

Parameters

Vn intact-Us off (n = 5) Vn intact-Us on (n = 5)

baseline 3 min post Us 15 min post Us baseline 3 min post Us 15 min post Us

Heart rate (bPM) 288.8 ± 10.54 276.0 ± 9.50* 270.4 ± 9.20 308.8 ± 13.24 302.8 ± 11.90* 297.0 ± 9.13cardiac output (ml/min) 52.74 ± 3.14 47.64 ± 2.70** 49.06 ± 2.54 53.52 ± 3.00 51.88 ± 1.25** 53.82 ± 1.18systolic volume (µl) 182.6 ± 9.40 173.4 ± 11.17 182.4 ± 9.14 174.4 ± 12.08 172.4 ± 5.78 181.8 ± 6.00Ejection fraction (%) 78.46 ± 3.95 80.48 ± 4.08 82.18 ± 4.00 85.92 ± 1.00 85.78 ± 2.50 89.44 ± 2.00End-diastolic volume (µl) 233.1 ± 6.90 217.0 ± 14.51 223.4 ± 11.70 203.8 ± 16.40 201.6 ± 7.72 203.6 ± 6.87End-systolic volume (µl) 50.43 ± 8.92 43.56 ± 10.75 41.04 ± 10.56*** 29.47 ± 4.40 29.22 ± 5.65 21.70 ± 4.47***respiratory rate (min−1) 29.20 ± 0.33 25.60 ± 0.22 21.40 ± 2.00 28.80 ± 1.75 27.20 ± 1.66 24.60 ± 1.22arterial pressure (mmHg) 88.60 ± 3.15 84.80 ± 4.74 85.20 ± 5.15 96.60 ± 2.73 96.00 ± 2.72 94.60 ± 3.00

all values are expressed as mean and standard error of the mean (sEM).*, **, and *** denote, respectively, p < 0.05, 0.01, and 0.001 significant changes relative to baseline.

TablE II. absolute Values of Physiological Parameters at baseline and at 3 and 15 min Post Us Exposure (Fig. 1) for the Vagotomy Groups.

Parameters

Vn cut-Us off (n = 5) Vn cut-Us on (n = 5)

baseline 3 min post Us 15 min post Us baseline 3 min post Us 15 min post Us

Heart rate (bPM) 317.6 ± 8.10 304.6 ± 8.09 299.2 ± 8.95 287.8 ± 9.22 282.8 ± 7.22 276.6 ± 9.05cardiac output (ml/min) 53.66 ± 6.60 40.46 ± 1.74 47.28 ± 3.06 52.28 ± 1.58 56.48 ± 1.58 54.28 ± 1.89systolic volume (µl) 154.1 ± 10.65 146.3 ± 6.01 158.6 ± 11.34 182.1 ± 7.87 200.7 ± 10.1 196.9 ± 11.35Ejection fraction (%) 82.12 ± 3.33 82.32 ± 3.68 81.84 ± 2.86 84.78 ± 2.00 87.64 ± 1.10 89.68 ± 1.63End-diastolic volume (µl)* 190.0 ± 16.84 179.5 ± 10.34 195.8 ± 16.77 216.0 ± 13.27 229.8 ± 13.47 220.9 ± 16.43End-systolic volume (µl) 35.86 ± 8.56 33.22 ± 8.00 37.20 ± 7.04 33.90 ± 6.40*** 28.96 ± 3.94 24.00 ± 5.39***respiratory rate (min−1) 29.40 ± 1.18 26.60 ± 1.84 25.80 ± 1.96 26.80 ± 1.24 25.80 ± 0.95 22.20 ± 2.2arterial pressure (mmHg) 101.0 ± 7.2 106.2 ± 7.32 105.4 ± 7.21 93.20 ± 3.29 89.80 ± 2.83 84.80 ± 4.00

all values are expressed as mean and standard error of the mean (sEM).* and *** denote, respectively, p < 0.05 and 0.001 significant changes relative to baseline.

coiado et al.: ultrasound-induced heart rate decrease: role of the vagus nerve 333

The bonferroni test showed no differences between the four groups.

cardiac output: at 3 min post Us exposure, the ul-trasound effect was significant (p = 0.005); the vagotomy effect was not significant. The interactions were not sig-nificant. at 15 min post Us exposure, the ultrasound ef-fect was significant (p = 0.03); the vagotomy effect was not significant. The interactions were not significant. The bonferroni test showed a significant effect after 3 and 15 min post Us exposure between Vn intact-Us off and Vn intact-Us on groups.

End-diastolic volume: at 3 min post Us exposure, there were no significant effects or interactions. at 15 min post Us exposure, the ultrasound effect was significant (p < 0.001); the vagotomy effect was significant (p < 0.001). The interactions were significant (p < 0.001). bonferroni test showed a significant effect at 3 min post Us exposure between Vn intact-Us off and Vn intact-Us on groups.

arterial pressure: at 3 and 15 min post Us exposure, there were no significant effects or interactions. The bon-ferroni test showed no differences between the four groups.

systolic volume, ejection fraction, end-systolic volume, and respiratory rate: at 3 and 15 min post Us exposure, there were no significant effects or interactions.

Intrathoracic and rectal temperatures did not show any significant change after the ultrasound exposure. Histo-logical examination of the heart and lungs of all animals exposed to ultrasound did not show any gross abnormali-ties following the ultrasonic exposure procedures.

IV. discussion

The goal of this study was to investigate the role of the vagus nerve on the ultrasound-mediated depression of the heart rate. The Us exposure parameters caused a negative chronotropic effect and did not exhibit any adverse outcomes on cardiac function or structure. The baseline results showed that there were no differences be-tween whether or not the vagus nerve was cut, suggesting possibly that the vagus nerve might not have a signifi-cant ultrasound-induced cardiac effect on this rat model. Further, the results showed that the interaction of ultra-sound and vagotomy was not significant for heart rate and cardiac output at 3 and 15 min post Us exposure. The end-diastolic volume showed a significant interaction at 15 min post Us exposure but not at 3 min post Us exposure, and overall, it is likely not to be an important observation considering that the other cardiac outcome variables did not show significance.

as the heart rate decreased, the end-diastolic volume (ventricular filling) is expected to increase. In the previ-ous study [9], the end-diastolic volume increased, but in this study, the end-diastolic volume did not increase. It thus might be that ultrasonic exposure somehow inhibited full ventricular relaxation or affected to some extent the venous return. Thus, there is a similar qualitative (nega-tive) chronotropic effect in both experiments, but in each experiment the hemodynamic behaviors are different, the causes of which are not yet known.

Fig. 3. normalized to the respective baseline values of heart rate,* cardiac output,* end-diastolic volume,* and arterial pressure at 3 and 15 min post Us exposure for all 4 groups, n = 5 per group. * denotes physiological parameters with statistically significant changes.

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a negative chronotropic effect (~18.7% of the basal heart rate) was observed at 15 min post Us exposure in the previous study [9]. In this study, a decrease in the heart rate (~4%) was observed for both groups in which Us was on (Vn intact-Us on and Vn cut-Us on) at 15 min post Us exposure. There were significant animal pro-tocol differences between the two studies: previously [9], the rats were not cannulated for arterial pressure mea-sures, whereas in this study all the rats were cannulated and had open chests. Thus, in the two distinctly different groups for which Us was turned on, a relative weak, but significant, negative chronotropic effect was observed. ca-rotid cannulation in rats appears to produce effects such as hemorrhagic shock [25] or increase in the arterial pressure [26]. In this study, a significant change in the arterial pres-sure was not observed but ~17% of the rats died before the end of the experiment. In the previous study [9], there were no animal deaths. The cannulation procedure re-quired ~30 min and the baseline heart rate (before versus after cannulation) decreased ~4% to 16%; this observation could be an explanation for the lesser decrease in the heart rate after Us exposure compared with previous study [9]. also note that there were no significant baseline heart rate changes between the two groups for which Us was turned off (Vn intact-Us off and Vn cut-Us off). Thus, whether or not the vagus nerve was cut, the vagus nerve did not appear to affect change in the negative chronotropic effect; also, there were no significant changes in arterial pressure, heart rate, or cardiac output (Fig. 3). The blood pressure is affected by baroreflex-mediated changes in efferent au-tonomic activity to the heart [15], [16]. The monitoring of arterial pressure did not show any significant change, thus suggesting that the baroreceptors are not directly involved in the negative chronotropic effect.

animal models are widely used in cardiovascular dis-ease studies. rat models are generally inexpensive, rela-tively easy to hande, and their large size facilitates surgi-cal and postsurgical procedures [27]–[29]. However, there are limitations regarding differences in myocardial func-tion compared with the human heart [27]. In this study, it was not determined if the origin of the negative chrono-tropic effect was evoked by direct or indirect ultrasound stimulation. It is logical, therefore, to speculate that the rat model or its age, or both, might not be appropriate for this study. age-related peculiarities of cardiac function are related to the development of sympathethic and parasym-pathetic innervation of the heart [30]. a study investigat-ed the age-dependent heart rate after bilateral vagotomy: in 2-week-old rats, bilateral vagotomy reduced the heart rate; the same effect was observed in 3-mo-old rats. How-ever, in 6-week-old rats, the heart rate was significantly increased within 5 min of vagotomy and then decreased after 60 min [31]. In adult (4-mo-old) rats, an increase in the heart rate and stroke volume was observed 1 min after bilateral vagotomy, returned to the initial value in 5 min and then gradually decreased [31]. In this study, 3-mo-old vagotomized rats showed a negative chronotropic effect. after the bilateral vagotomy it was not possible to identify

the role of the vagus nerve in the negative chronotropic effect after ultrasound application because there were no significant changes. The formation of the cardiac sym-pathic innervation at this age [32] or enhanced endocrine function during puberty [33] might explain the decrease of heart rate after bilateral vagotomy. For this study (3-mo-old rats), even after the bilateral vagotomy it is possible that the innervation of the parasympathic system could be responsible for the negative chronotropic effect; the ef-fect might not be due to the ultrasound exposure. This might explain why the vagotomized and non-vagotomized rats showed the same results. It is possible that the inner-vation of the parasympathic system played a role in the effect as if the vagus nerve were intact. an experimental approach could be to use rats older than 5 mo to eliminate the puberty interference or the immature parasympathic system might answer such questions.

consistent with the previous study [9], the ultrason-ic exposure studies have not identified any gross cardiac thermal damage. However, it is important to continue to study the risk-based exposure limits. These results raise the possibility of circulatory depression resulting from pulsed ultrasonic stimulation.

V. conclusion

a cause that was hypothesized to affect the ultrasound-induced negative chronotropic effect in rats, namely the vagus nerve, was investigated. Unfortunately, there were no negative chronotropic effect changes between whether or not the vagus nerve was cut. although the cause of the ultrasound-induced negative chronotropic effect remains yet to be determined, the negative chronotropic effect still represents a promising new therapy to pace the heart in cardiac diseases that involve abnormal cardiac rhythms. Future research might usefully be directed at further ex-amining the cellular and molecular physiology of the ac-tions of the cardiac vagal stimulation in animal models and at methods of increasing cardiac parasympathetic activity in patients with heart disease. additional stud-ies are required to elucidate the physiological mechanisms involved in the production of these effects, such as sex and age differences.

acknowledgments

This work was supported by sao Paulo research Foun-dation (FaPEsP, 2008/54165-2) and by the national In-stitutes of Health (r37 Eb002641). The authors acknowl-edge the technical assistance by r. J. Miller, dVM, E. Hartman, bs, rdMs, and s. sarwate, Md.

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Olivia C. Coiado received her b.s. degree in medical physics in 2005 from the University of são Paulo, ribeirão Preto, brazil; her M.sc. degree in electrical engineering from the University of campinas, brazil, in 2008; and her doctorate de-gree in electrical engineering from the University of campinas, brazil, in 2012. she developed part of her doctoral research at the bioacoustics re-search laboratory (brl), University of Illinois at Urbana-champaign (UIUc). currently she is a postdoctoral research associate at the brl, de-

partment of Electrical and computer Engineering, UIUc. Her research interests include bioacoustics, therapeutic ultrasound, animal models, and cardiology studies.

Elaine B. Buiochi received a degree in dental surgery from the Federal University of rio de Ja-neiro, brazil, in 2001; an M.sc. degree in biomedi-cal engineering from the alberto luiz coimbra Institute–Graduate school and research in Engi-neering, Federal University of rio de Janeiro, bra-zil, in 2004; and a doctorate degree in electrical engineering (biomedical engineering) from the University of campinas, brazil, in 2011. she de-veloped part of her doctorate research at the acoustics Institute, spanish national research

council (csIc), Madrid, spain, and at the bioacoustics research labo-ratory, University of Illinois at Urbana-champaign. Her research inter-ests include the development of ultrasonic transducers and medical ap-plications of ultrasound.

IEEE TransacTIons on UlTrasonIcs, FErroElEcTrIcs, and FrEqUEncy conTrol, vol. 62, no. 2, FEbrUary 2015336

William D. O’Brien Jr. (s’64–M’70–sM’79–F’89–lF’08) received the b.s., M.s., and Ph.d. degrees from the University of Illinois at Urbana-champaign. From 1971 to 1975, he worked with the bureau of radiological Health (currently the center for devices and radiological Health) of the U.s. Food and drug administration. In 1975, he joined the faculty at the University of Illinois. He is currently research Professor of Electrical and computer Engineering and director of the bio-acoustics research laboratory. His research inter-

ests involve the many areas of ultrasound–tissue interaction, including biological effects and quantitative ultrasound imaging, for which he has published 393 papers. dr. o’brien is a life Fellow of the Institute of Electrical and Electronics Engineers, a Fellow of the acoustical society of america, and a Fellow of the american Institute of Ultrasound in Medicine, and is a Founding Fellow of the american Institute of Medical and biological Engineering. He was recipient of the IEEE centennial

Medal in 1984, the aIUM Presidential recognition awards in 1985 and 1992, the aIUM/WFUMb Pioneer award in 1988, the IEEE outstand-ing student branch counselor award for region 4 in 1989, the aIUM Joseph H. Holmes basic science Pioneer award in 1993, the IEEE Ultra-sonics, Ferroelectrics, and Frequency control society distinguished lec-turer for 1997–1998, the IEEE Ultrasonics, Ferroelectrics, and Frequency control society’s achievement award in 1998, the IEEE Millennium Medal in 2000, the IEEE Ultrasonics, Ferroelectrics, and Frequency con-trol society’s distinguished service award in 2003, the aIUM William J. Fry Memorial lecture award in 2007, and the IEEE Ultrasonics, Fer-roelectrics, and Frequency control society’s rayleigh award in 2008. He has served as President of the IEEE sonics and Ultrasonics Group (cur-rently the IEEE Ultrasonics, Ferroelectrics, and Frequency control soci-ety) from 1982 to 1983, Editor-in-chief of the IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control from 1984 to 2001, and President of the american Institute of Ultrasound in Medicine from 1988 to 1991.