Ann Thorac Surg 2004;77:126-132
© 2004 The Society of Thoracic Surgeons
Original article: cardiovascular
Nitric oxide and prostacyclin in ultrasonic vasodilatation of the canine internal mammary artery
Ayako Maruo, MDa,
Chad E. Hamner, MDa,
Alfredo J. Rodrigues, MD, PhDa,
Tetsuya Higami, MD, PhDb,
James F. Greenleaf, PhDb,
Hartzell V. Schaff, MDa*
a division of Cardiovascular Surgery, Mayo Clinic, Rochester, Minnesota, USA
b Division of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota, USA
Accepted for publication July 8, 2003.
* Address reprint requests to Dr Schaff, Division of Cardiovascular Surgery, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
e-mail: schaff{at}mayo.edu
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Abstract
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BACKGROUND: Investigators recently demonstrated increased free blood flow from radial artery free grafts harvested using ultrasonic technology. We investigated the mechanism underlying this phenomenon.
METHODS: Canine internal mammary artery segments (with and without intact endothelium) were precontracted with norepinephrine and sonicated 3 seconds in organ chambers with ultrasonic coagulating shears (Harmonic Scalpel; Ethicon Endo-Surgery, Cincinnati, OH) functioning at level 2. Vessel tension was continuously measured to examine vasoactivity in response to sonication alone (control) or with Nù-Nitro-l-arginine (l-NNA) and indomethacin added to the chamber medium individually or in combination. Tissue heating, acoustic pressure, and endothelial damage as detected by scanning electron micrography were also assessed.
RESULTS: In vitro sonication with the Harmonic Scalpel induced predominately endothelium-dependent internal mammary artery vasorelaxation but a small endothelium-independent contribution was also observed. Early vasorelaxation (1 minute after stimulus) was maximally inhibited by l-NNA alone and in combination with indomethacin. Relaxation during this period was insignificantly affected by indomethacin alone. Only the combination of l-NNA and indomethacin maximally inhibited late vasorelaxation (5 minutes after stimulus), whereas inhibitory effects of l-NNA diminished during this time period. Indomethacin inhibited relaxation substantially during this phase, although significantly less than did l-NNA alone. The Harmonic Scalpel minimally heated the tissue surface (0.3 ± 0.03°C) and did not disrupt endothelial cell integrity while operating at 50 mW/cm2 intensity (acoustic pressure 40 kPa).
CONCLUSIONS: Sonication induces vasorelaxation almost completely by time-dependent endothelial nitric oxide and prostacyclin release, which appears unrelated to tissue heating or endothelial architectural disruption.
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Introduction
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The use of ultrasound technology for excising and coagulating tissue during surgery has gained popularity in cardiac surgery as a safe and beneficial means of dissecting arterial conduits for coronary artery bypass grafting such as the internal mammary artery (IMA) [1–3], radial artery [4, 5], and gastroepiploic artery [6]. An ultrasonically activated scalpel can provide simultaneous incision and coagulation by transfer of mechanical and thermal energy to tissues [7] with less endothelial injury [2] and vasospasm [8] than that produced by conventional dissection techniques.
Recent reports [9] demonstrating excellent short-term patency rates in skeletonized IMA grafts harvested with ultrasonic technology have rekindled interest among investigators regarding the interactions of ultrasonic energy and vascular cellular function. Prompted by observations of reduced vasospasm in radial artery grafts dissected with ultrasonic instruments, Ronan and colleagues [8] compared harvest of radial artery grafts with traditional techniques or with an ultrasonic scalpel and found significantly greater free blood flow in vessels dissected with the ultrasonic device. However their study was not designed to analyze the underlying mechanism by which ultrasonic energy promotes arterial vasodilatation, and to date this process remains unknown. Therefore to gain insight into this phenomenon we investigated the biologic effects of in vitro sonication produced by an ultrasonic scalpel on canine IMA preparations.
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Material and methods
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Drugs
Acetylcholine chloride, norepinephrine, indomethacin, and sodium nitroprusside were obtained from Sigma-Aldrich, St. Louis, Missouri. The Nù-nitro-l-arginine (l-NNA) was obtained from Calbiochem-Novabiochem Corporation, San Diego, California. All drugs were prepared with distilled water except for indomethacin, which was dissolved in Na2CO3 (10-5 M).
Tissue harvest and preparation
All animals were treated in accordance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985), the Animal Welfare Act of 1-66 (PL 89-544), and the Animal Welfare Act amendment of 1985 (PL 99-198). The Animal Care and Use Committee of Mayo Foundation approved all procedures and protocols.
Nine heartworm-free, mongrel canine subjects (25 to 30 kg) of either sex were euthanized with sodium thiopental injection (30 mg/kg given intravenously; Abbott Laboratories, Chicago, IL) followed by exsanguination. A segment of anterior chest wall including sternum and IMAs was excised en bloc and immediately immersed in cool, oxygenated, buffered physiologic salt solution (BPS) of the following composition: 118.3 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.22 mmol/L KH2PO4, 2.5 mmol/L CaCl2, 25.0 mmol/L NaHCO3, and 11.1 mmol/L glucose; pH, 7.4.
Both IMAs were skeletonized from the chest wall segment under magnification, with care taken not to manipulate the intimal surface. Then IMA segments 5 mm in length were prepared and placed in BPS. Half of the segments were denuded of endothelium by gently rubbing the intimal surface with watchmakers' forceps; these segments allowed assessment of vascular smooth muscle relaxation independent of endothelial influences [10].
Organ chamber preparation
Four pairs of IMA segments with intact or denuded endothelium from each animal were studied in parallel. All IMA segments were suspended in 25-mL organ chambers filled with BPS (temperature regulated at 37°C) and continuously oxygenated by a bubbled 95% O2/5% CO2 gas admixture. Two stainless steel clips, one anchored and the other connected to a strain gauge for isometric tension measurement, were passed through the vessel lumen to suspend each segment. The segments were brought to the optimal point in their length-tension relation by progressive stretching until the point of maximum contraction with potassium ions (20 mmol/L) was achieved. The presence or absence of endothelium in each segment was verified by determining the response to acetylcholine (10-6 M) after precontraction. (Vessels without endothelium fail to relax after acetylcholine application.) The segments were then allowed to equilibrate for 30 to 45 minutes in oxygenated BPS before further stimuli were applied.
To investigate the mechanism by which an ultrasonic energy stimulus (sonication) produces vascular relaxation, each segment pair was allocated to 1 of 3 treatment groups or a control group. Treatments were indomethacin (10-5 M), an inhibitor of prostacyclin production, l-NNA (10-4 M), an inhibitor of nitric oxide (NO) production, and a combination of indomethacin and l-NNA at similar concentrations. All segments were precontracted with norepinephrine (3 x 10-6 M) and then stimulated with an ultrasonic device in the presence of the allotted treatment. Vessel tension was continuously monitored, and the percent of relaxation of each segment was determined at the point of maximal relaxation and at 1 and 5 minutes after ultrasonic scalpel application. All studies were performed in duplicate.
Harmonic scalpel application
The ultrasonic device used in this study was the Harmonic Scalpel (Ethicon Endo-Surgery, Cincinnati, OH). After precontraction with norepinephrine, all segments were stimulated for 3 seconds with ultrasonic energy applied by the Harmonic Scalpel operating at level 2 in the presence of the allotted treatment. Level 2 was selected because it is the level most often used for clinical harvesting of skeletonized IMA. Preliminary studies performed in our laboratory indicated no difference in vascular reactivity among various levels. To apply the ultrasonic stimulus we placed the 5-mm dissecting hook (DH105) in the organ chamber so that the blade face was oriented parallel and adjacent to the arterial wall without directly contacting the tissue surface (Fig 1).
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Fig 1. Schematic diagram of organ chamber preparation and Harmonic Scalpel application. The face of the dissecting hook was placed parallel to the wall of the internal mammary artery (IMA) at a distance of 1 cm.
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Thermal effects
To investigate the thermal effects that ultrasonic energy produces on vascular endothelium, vessel segments with intact endothelium were suspended in organ chambers and stimulated with the Harmonic Scalpel for 3 seconds at level 2 in the absence of precontraction. The temperature of the segments and BPS was measured with a digital temperature probe before and after stimulation.
Scanning electron micrography
After the thermal studies the segments were prepared for scanning electron micrography to evaluate endothelial disruption. Two IMA segments were fixed in Trump solution for scanning electron micrography. Longitudinal sections were dehydrated in graded alcohol and transferred to a critical-point freezing-drying apparatus. The sections were mounted on an aluminum stub and sputter coated with gold/palladium. These preparations were examined in a Hitachi 4700 scanning electron microscope and assessed for endothelial cellular damage.
Measurement of acoustic intensity
Acoustic intensity produced by the Harmonic Scalpel was measured continuously across a broad spectrum of ultrasonic frequencies while the device was operated at level 2. The 5-mm dissecting hook was immersed in a 200-L volume anechoic-lined water tank containing a 9-cm diameter thin plastic membrane stretched tightly across a Plexiglas frame. The blade face was oriented parallel to the center of the membrane surface at a distance of 10 cm. Membrane deflection in response to the acoustic pressure field was measured using a laser vibrometer (Polytec PI, Tustin, CA) capable of detecting displacements below 1 nm. The laser vibrometer output is bandpass filtered to a range of 5 to 70 kHz and digitized by a 12-bit analog-to-digital converter. Acoustic pressure versus frequency was computed by using the Fast Fourier transformation. It should be noted that cavitation produced by the Harmonic Scalpel contributes to acoustic pressure over a broad frequency range.
Data analysis
Results were expressed as mean ± SEM. In all experiments, "n" referred to the number of animals from which vessel segments were obtained. Analysis of variance (ANOVA) was used to analyze continuous variables for comparisons among multiple groups. The two-tailed Student t test was used to analyze continuous variables for between-group comparisons. Values were considered significant at an alpha level less than 0.05.
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Results
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Ultrasonic energy produced by the Harmonic Scalpel induced IMA smooth muscle relaxation in vitro in an endothelium-dependent fashion. The maximal relaxation of untreated endothelium-intact segments was significantly greater than that observed for endothelium-denuded segments (50.4% ± 2.8% versus 10.5% ± 1.6%; p < 0.0001). Relaxation in segments having intact endothelium typically began within seconds of Harmonic Scalpel stimulation, lasted approximately 5 minutes, and was followed by a gradual return to a state of maximum contraction (Fig 2).
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Fig 2. Example of original tracings from canine internal mammary artery segments with intact and denuded endothelium. After precontraction with norepinephrine (3 x 10-6 M), segments were exposed for 3 seconds to stimulation with the Harmonic Scalpel operating at level 2. Maximum relaxation occurred rapidly in segments with intact endothelium, lasted approximately 5 minutes, and gradually returned to the point of maximal contraction.
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Addition of prostacyclin and NO production inhibitors affected endothelium-dependent IMA relaxation in a time-dependent fashion (Fig 3).
At 1 minute after Harmonic Scalpel stimulation IMA relaxation was insignificantly inhibited by indomethacin (40.7% ± 5.1%; p > 0.05) but was maximally inhibited by l-NNA (7.2% ± 1.1%; p < 0.0001) and by the combination of indomethacin and l-NNA (6.6% ± 1.0%; p < 0.0001; Fig 4). At 5 minutes indomethacin partially inhibited relaxation to a significant degree (30.5% ± 3.2%; p < 0.0001), and l-NNA inhibition remained (18.3% ± 1.2%; p < 0.0001) but no longer at the maximum level (p < 0.001 versus combination indomethacin and l-NNA; Fig 5).
Maximum inhibition by a combination of indomethacin and l-NNA remained at 5 minutes (6.15% ± 1.0%; p < 0.0001).
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Fig 3. Time-dependent inhibition of endothelium-dependent internal mammary artery relaxation. At 1 minute relaxation was insignificantly inhibited by indomethacin (Indo [circles]) and maximally inhibited by Nù-nitro-l-arginine (l-NNA [triangles]) and a combination of Indo and l-NNA ([squares] p < 0.0001 versus control [diamonds]). At 5 minutes Indo partially inhibited relaxation (p < 0.001 versus control) whereas l-NNA inhibition remained (p < 0.0001 versus control) but was no longer maximal (p < 0.0001 versus Indo + l-NNA). The combination of Indo and l-NNA continued to maximally inhibit relaxation at 5 minutes (p < 0.0001 versus control). Data are presented as average ± SEM and represent percent relaxation after norepinephrine (3 x 10-6 M) precontraction.
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Fig 4. Endothelium-dependent internal mammary artery relaxation 1 minute after 3-second stimulation with the Harmonic Scalpel at level 2. Relaxation was insignificantly inhibited by indomethacin (Indo) but was maximally inhibited by Nù-nitro-l-arginine (L-NNA) and a combination of Indo and L-NNA. Data are presented as average ± SEM and represent percent relaxation after norepinephrine (3 x 10-6 M) precontraction. *p less than 0.0001 versus control and Indo.
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An increase in temperature that was similar in both the organ chamber solution and IMA segments (0.3 ± 0.03°C and 0.4 ± 0.03°C, respectively) was observed after Harmonic Scalpel stimulation at level 2 for 3 seconds. Scanning electron micrography studies of these segments demonstrated an intact endothelial monolayer without evidence of cellular damage (Fig 6).
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Fig 6. Representative scanning electron micrographs of internal mammary artery segments. (A) Without sonication. (B) After 3-second stimulation with the Harmonic Scalpel at level 2. Both micrographs display an intact endothelium without features of endothelial cell injury. (Gold/palladium sputter coated: original magnification x500.)
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The acoustic pressure produced by the Harmonic Scalpel operating at level 2 was 4 kPa (peak at about 55 kHz) at a distance of 10 cm from the detector (Fig 7).
Extrapolated to a distance of 1 cm, which was the distance of the instrument from the IMA segments in the organ chambers, the acoustic pressure experienced by the segments would be approximately 10 times higher, or 40 kPa, equivalent to an intensity of 50 mW/cm2.
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Fig 7. Quantification of acoustic pressure produced by the Harmonic Scalpel. Acoustic pressure was highest (4 kPa) at a frequency of 5.5 kHz.
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Comment
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The results of this in vitro study demonstrated that IMA relaxation in response to short periods (3 seconds) of sonication with the Harmonic Scalpel occurs mostly by an endothelium-dependent process from combined release of NO and prostacyclin. The early phase of vasodilatation (within 1 minute after stimulation) appears to be almost completely dependent on NO activity. Adding l-NNA to the organ chamber bath nearly abolished IMA relaxation whereas indomethacin failed to significantly inhibit relaxation alone and provided no additional inhibition when used in combination with l-NNA. In contrast the late vasodilatory phase (at 5 minutes after stimulation) demonstrated a relative increase in prostacyclin activity and reduction in NO activity, with maximal relaxation occurring when both compounds acted in concert. Indomethacin alone blunted IMA relaxation to 33% and in combination with l-NNA almost abolished relaxation at 5 minutes after stimulation whereas l-NNA alone continued to profoundly inhibit relaxation at 5 minutes but to a significantly lesser degree than that observed at 1 minute.
Regarding the role of endothelium in mediating the effects of sonication on arterial systems, other in vitro models have generated conflicting results. Chokshi and colleagues [11] noted endothelium-dependent relaxation in rabbit aorta preparations after exposure to ultrasonic energy at various intensities produced by a thin intraluminal wire. In distinct contrast Fischell and coworkers [12] demonstrated endothelium-independent relaxation in a similar rabbit aorta model. The discrepancy between these reports may arise from notable differences in study design including the frequency, intensity, and duration of the ultrasonic stimulus and the method for measuring changes in vessel reactivity. Likewise the results of these studies are difficult to compare with our findings because of potential interspecies variation in vasoactive mechanisms and differences between aorta and IMA cellular function.
The nature of interaction between ultrasonic energy and endothelial cells promoting release of vasoactive substances remains ill defined. However investigators have observed that cellular changes occurring after a period of sonication are quite similar to those promoted by mechanical sheer stress [13] and cavitation. As the ultrasonic pressure wave propagates through the fluid media and contacts the endothelial surface it likely induces changes in cellular membrane permeability with an increase in ionic and molecular flux across this barrier, a process previously demonstrated both in vitro and in vivo [14]. Intracellular second messenger systems are activated, most notably the calcium-calmodulin pathway [14–16], creating a chain of cellular alterations including activation of nitric oxide synthase (NOS) and cyclooxygenase. As long as cellular integrity is not lost permeability alterations to calcium appear to be reversible, with a decay time of approximately 10 minutes [15].
In addition some investigators have recently identified specific tyrosine kinase systems that react to sheer stress independently of calcium signaling pathways and result in activation of constitutive NOS in rabbit carotid arteries [16]. Others have demonstrated increased NOS phosphorylation [17] and NOS mRNA expression [18] induced by sheer stress. Although the time course of IMA relaxation and the integrity of the endothelial monolayer observed in this study fit the altered membrane permeability mechanism we cannot rule out other processes on the basis of our results. Whatever the process, previous studies have similarly identified either NO or prostacyclin release from other in vitro cellular preparations stimulated by sonication including human osteoblasts [19] and human umbilical vein endothelium.
Although the endothelium plays a substantial role in sonication-induced IMA relaxation our results suggest that endothelium-independent mechanisms provide a small but consequential influence as well. The IMA segments denuded of endothelium did relax by 5% to 10% at all time points evaluated and endothelium-intact segments similarly demonstrated this degree of relaxation despite addition of NO and prostacyclin inhibitors. Direct effects of ultrasonic energy upon vascular smooth muscle cells may possibly have contributed to this endothelium-independent component. Evidence exists from published in vitro models that high-intensity ultrasonic energy (> 5.5 W) exposure for 5 minutes produces smooth muscle cell lysis and impaired vasoconstrictor responses in rabbit aortas [12]. From our acoustic pressure studies we were able to determine that Harmonic Scalpel vibration at 55.5 kHz generated a pressure of approximately 40 kPa, equivalent to an intensity of 50 mW/cm2 experienced by the tissue. Although our stimulus was applied for only 3 seconds this intensity certainly does not fall within the range capable of producing irreversible smooth muscle cell damage. However the return of maximum contraction after relaxation that occurred in our organ chamber preparations and the absence of endothelial or smooth muscle cell damage seen on scanning electron micrography argue against this as an explanation for our findings.
Smooth muscle cells within certain vascular beds have been shown to dilate in vitro in response to warming above physiologic temperature (from 37° to 41°C) by a direct effect on 
2-adrenergic pathways [20]. As such, tissue warming after sonication offers a plausible explanation for the endothelium-independent contribution to IMA vasodilatation. However only a miniscule average increase in tissue temperature (0.3 ± 0.03°C) was observed during the short period of Harmonic Scalpel stimulation. Further in a limited number of separate studies measuring IMA vasoactivity in response to a thermal probe energy source, a tissue temperature increase of 1°C failed to induce any significant IMA segment relaxation (data not shown). Although these additional studies were limited they are consistent with other published studies evaluating ultrasonic energy stimuli that also failed to produce substantial relaxation in arterial preparations after an identical increase in tissue temperature in vitro [12]. Therefore we conclude that thermal effects of sonication do not adequately account for these results.
A direct effect of ultrasonic energy upon smooth muscle contractile protein complexes may provide an alternative explanation for the endothelium-independent component observed in this study. Ultrasonic frequencies less than that used in our model are known to reversibly disrupt actin-actin bonds [12], leading to actin filament fragmentation [21]. After fragmentation by sonication actin polymers spontaneously reanneal into functional filaments that can generate effective although diminished contractile force after a brief recovery period [22–24]. Annealing occurs rapidly in vitro after sonication with peak activity observed within 5 minutes [24], which closely approximates the point in our model at which IMA segments began to return to their maximal state of contraction after the Harmonic Scalpel stimulus. Although this is an attractive hypothesis we cannot exclude other possible explanations for this phenomenon.
These results although compelling are limited by the in vitro design of the study. The ultrasonic stimulus was propagated to the tissue through a fluid medium. This differs from the usual technique of surgical dissection, in which the instrument directly contacts the arterial wall. The biologic effects of ultrasonic energy including mechanical sheer stress and cavitation may have become amplified as the pressure wave was propagated through the medium. Furthermore our results reflect the application of only one intensity level to the tissue for a short, constant duration. Other reports have shown dose-dependent variation in cellular changes [12] so mechanistic processes other than those we observed may become apparent when the ultrasonic stimulus is applied over increasing intensity levels or for a longer duration. Finally although interspecies variation may exist in the physiologic responses of vascular segments to sonication previous studies in our laboratory have established the canine model as an appropriate model for study of human IMA vascular reactivity [25, 26].
In conclusion IMA dilatation in vitro in response to ultrasonic stimulation appears to occur for the most part from endothelium-dependent release of NO and prostacyclin in a time-dependent manner. A small but consequential endothelium-independent component also exists. Although neither process appears to be related to thermal effects we can only speculate about the true interaction of ultrasonic energy with vascular endothelium and smooth muscle cells from our results and other published studies. However these findings provide insight into the augmentation of free flow in radial arteries and the excellent clinical short-term patency rates observed after coronary artery bypass with skeletonized IMA grafts harvested with ultrasonic technology.
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Acknowledgments
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We sincerely thank Randall R. Kinnick and Linda L. Cornelius, RMT, for providing technical assistance with acoustic pressure measurement and scanning electron micrography.
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Top
Abstract
Introduction
p less than 0.0001 versus control, p less than 0.001 versus Indo; 
p less than 0.0001 versus control.