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Nitric Oxide Generation Directly Responds to Ultrasound Exposure
Ultrasound in Med. & Biol., Vol. 34, No. 3, pp. 487– 493, 2008 Copyright © 2008 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/08/$–see front matter
doi:10.1016/j.ultrasmedbio.2007.08.008
● Original Contribution NITRIC OXIDE GENERATION DIRECTLY RESPONDS TO ULTRASOUND EXPOSURE YOICHI SUGITA,* SATOKO MIZUNO,* NAOTO NAKAYAMA,* TAKAMASA IWAKI,† EIICHI MURAKAMI,‡ ZUOJUN WANG,* REIKO ENDOH,* and HIROSHI FURUHATA* *Medical Engineering Laboratory and †Laboratory Animal Facilities, Jikei University School of Medicine, Tokyo, Japan; and ‡Eikoukagaku Co. Ltd., Tokyo, Japan (Received 18 February 2007; revised 6 August 2007; in final form 22 August 2007)
Abstract—Recently, several reports have been published on ultrasonic vascular dilation produced with relatively low-frequency ultrasound. It has been speculated that nitric oxide (NO) is an important factor for this ultrasonic vascular dilation. However, a quantitative relationship between the ultrasound intensity and NO generation was not clarified in these reports. We investigated the quantity of NO generated by various ultrasonic intensities by means of real-time measurement of NO concentration in the adductor muscles of the thigh of New Zealand white rabbits exposed to a continuous-wave ultrasound (490 kHz). In the quantitative relationship between NO generation and ultrasonic intensity, the percent increase in NO concentration was 1.25% ⴞ 1.25%, 10.6% ⴞ 2.9% and 20.1% ⴞ 3.5%, with the maximum muscle temperature increase 0.5 ⴞ 0.2°C, 0.7 ⴞ 0.2°C, and 0.8 ⴞ 0.3°C at the ultrasonic intensity (SPTA) of 0.21, 0.35 and 0.48 W/cm2, respectively. The effect of ultrasound on NO generation was intensity-dependent with a progressive increase from 0.21 W/cm2 to 0.48 W/cm2 without significant thermal effect. Ultrasonic NO generation was partially reduced by NOS inhibitor of L-NMMA, clarifying that ultrasound can activate both NOS-dependent and NOS-independent NO generation. These new findings provided scientific basis for ultrasonic vasodilatation and support the potentiality of a new ultrasonic technology for the treatment and prevention of the ischemic tissue based on the new concept of NO generated angiogenesis. (E-mail: [email protected]) © 2008 World Federation for Ultrasound in Medicine & Biology. Key Words: Nitric Oxide (NO), Nitric Oxide Synthase (NOS), Ultrasound, Muscle, Biochemical effect.
the release of Ca2⫹ from intracellular stores, with consequent elevation of intracellular free Ca2⫹ concentration, which increases the enzymatic activity of endothelial NO synthase (eNOS) to generate NO. Recently, however, several investigations (Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998) have reported Ca2⫹ independent activation of the eNOS in response to fluid shear stress. They have suggested that tyrosine phosphorylation and intracellular pH (Ayajiki et al. 1996), phosphorylation of the eNOS (Corson et al. 1996) and tyrosine kinase inhibitor-sensitive pathway (Fleming et al. 1998) play an important role in NO generation. In addition to this physiological stimulation, recent reports have pointed out that external ultrasonication of living tissue induced vasodilatation, blood flow increases and pH changes because of NO generation from the blood vessels, muscular cells and other tissue externally stimulated by the ultrasonic kinetic effect (Chokshi et al. 1989; Fischell et al. 1991; Steffen et al. 1994; Suchkova et al. 2002; Maruo et al. 2004). However, a quantitative rela-
INTRODUCTION Nitric oxide (NO) is now known to control vascular function, biological function, neural signaling and immunologic function (Furchgott and Zawadzki 1980; Palmer et al. 1987). In the field of vascular function, it has been reported that NO generation is stimulated physiologically when the endothelium is exposed to shear stress induced by blood flow and changes in the blood flow (Taso et al. 1995; Uematsu et al. 1995; Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998). There have been two possible mechanisms of endothelial NO generation in response to fluid shear stress. Previous investigations (Ando et al. 1988; Geiger et al. 1992; James et al. 1995) have established that the exposure of endothelium to increased fluid shear stress may stimulate
Address correspondence to: Hiroshi Furuhata, Professor, Medical Engineering Laboratory, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461 Japan. E-mail: [email protected] 487
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tionship between the ultrasound intensity and NO generation was not clarified in these reports as NO was not directly measured. Accordingly, we investigated the quantity of NO generated by various ultrasonic intensities by means of real-time measurement of NO concentration in tissue exposed to ultrasound. MATERIALS AND METHODS All animal procedures were performed under the guidance of the Animal Research Committee of Jikei University School of Medicine, Tokyo, Japan. Twentyone New Zealand white rabbits weighing 3400 g to 4080 g were used in the study. The rabbits were anesthetized with 5% isoflurane and the anesthesia was maintained with 2% to 2.5% isoflurane by a gas mask. The abdominal of the thigh skin was shaved and prepared. The animals were kept at the normothermic core body temperature (38 ⫾ 0.3°C) by means of a heating blanket and the core body temperature was measured with a rectal digital thermometer (Model PTW-100A, Unique Medical Co. Ltd., Tokyo, Japan). NO measuring system NO was measured with a commercially available NO measuring system (Eikoukagaku Co., Ltd. Tokyo, Japan), which is composed of an NO meter (NO-502), a counter electrode (NOR-20) and a working electrode (NOE-47). The principles behind the measurements and the method of measuring the NO concentration with this system have been described elsewhere (Ichimori et al. 1994; Mitsuhata et al. 1994; Miyoshi and Nakaya 1994; Saitoh et al. 1996; Tamaoki et al. 1995; Wang et al. 1995; Zeng and Quon 1996). The NO measuring system is based on the measurement of pA-order currents between the two electrodes. The counter electrode (diameter 500 m) is made of carbon. The working electrode (diameter 200 m) is made of a platinum-iridium (Pt-IR) alloy with a three-layer coating at its detection tip. The electrode was calibrated to NO using S-nitroso-N-acetyl-dlpenicillamine, which serves as a stable standard NO generator, according to the method described by Ichimori et al. (1994). Using this measuring instrument, a current of 1000 pA is approximately equivalent to an NO concentration of 1 Mol/L (Saitoh et al. 1996). The response time of the electrode was 1.14 ⫾ 0.09 s (Ichimori et al. 1994). Ultrasound system The ultrasound system is composed of a ceramic transducer (diameter: 10 mm) consisting of PZT and an ultrasound generator designed by the authors (manufactured by Honda Denshi Co. Ltd. Toyohashi, Japan). A continuous waveform (490 kHz) was applied and three different intensities (0.75, 1.25 and 1.7 W/cm2) were
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selected and applied. Acoustic powers were measured before and after experiments with an ultrasonic power meter (Model UPM-DT-1, Ohmic Instruments Co., Easton, MD, USA) to confirm the exposure conditions. The UPM-DT-1 power meter was calibrated at a frequency of 490 KHz with the accuracy of ⫾3% and resolution of 2 mW by the manufacturer. The actual ultrasound intensity through the spacer was measured by the acoustic intensity measurement system (NTR Systems, Inc. Seattle, WA, USA). The axial intensity (SPTA) at 2.5 cm distance from the probe surface was decreased to 0.21, 0.35 and 0.48 W/cm2 corresponding to the applied power of 0.75, 1.25 and 1.7 W/cm2, respectively. Experimental protocol The NO working electrode was inserted into the adductor muscles of the thigh percutaneously through the 22 G intravenous catheter. The counter electrode was placed under the skin approximately 5 mm away from the working electrode. The needle type electrode of a digital thermometer (DT-300 Eikoukagaku Co. Ltd., Tokyo, Japan) was inserted into a position close to the NO electrode percutaneously through the 22 G intravenous catheter. A low-frequency continuous ultrasound waveform (490 kHz) was applied for 10 min percutaneously to the adductor muscles of the thigh in 11 rabbits through ultrasound gel and a spacer (Acoustic Standoffs, ATS Laboratories, Inc., Bridgeport, CT, USA). The spacer is composed of flexible low attenuation sonolucent gel, covered with a pliable film. The spacer (2 cm thickness) was used to avoid a surface heat affect of the transducer and the near field effect of the ultrasound. The schematic of experimental model is shown in Fig. 1. In three different emitted intensities (0.75, 1.25 and 1.7 W/cm2), the spatial acoustic intensity of the US probe
Ultrasound generator
Digital Thermometer DT-300 Muscle
NO Meter NO-502
Fig. 1. Scheme of experimental set-up. (1) working electrode (NOE-47); (2) counter electrode (NOR-20); (3) needle type electrode of thermometer; (4) spacer (Acoustic Standoffs); (5) ceramic type transducer.
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Fig. 2. The acoustic power profile of the ultrasound transducer (10 ) in the water measured by the hydrophone (Onda Co., Sunnyvale, CA, USA) and the AIMS (1dB per contour). (a) The acoustic power profile without the spacer. (b) The acoustic power profile through the spacer (2 cm thickness).
was calculated from the beam profile of the transducer measured by Acoustic Intensity Measurement System (AIMS) (Fig. 2a). The US intensity in the body at the 5 mm depth from the skin was 0.21 W/cm2 in the emission intensity 0.75 W/cm2 through the spacer 0.35 W/cm2 in 1.25 W/cm2, 0.48 W/cm2 in 1.7 W/cm2 (Fig. 2b). The NO concentration (pA) was measured by a NO meter during the ultrasonication for 10 min. Percent increase of NO concentration was calculated. In another five rabbits, the Thermal Stimulator (BTL-201, Unique Medical Co. Ltd., Tokyo, Japan) was applied directly to the adductor muscles of the thigh for approximately 10 min to examine the relationship between temperature rise and NO generation. The temperature of the adductor muscles of the thigh was increased
gradually to the maximum of 3°C to avoid burning of the muscle. To examine the inherent effect of NO synthase (NOS) on ultrasonic NO generation, an NOS inhibitor of NG-monomethyl-L-arginine (L-NMMA) was used in another five rabbits. Before administration of L-NMMA, the same ultrasonic procedure (490 kHz, 1.7 W/cm2) was performed for 10 min and the percent increase of NO concentration was measured. In this experiment, because the temperature rise of the ultrasonic probe did not affect NO generation as shown by the results, we applied the probe directly to the adductor muscles of the thigh without a tissue spacer to maximize the accuracy of test results. Subsequently, L-NMMA was administrated IV (5 mg/Kg) 5 min before ultrasonication and the same
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Fig. 3. Real-time record of NO concentration in the muscular tissue produced by ultrasonication (490 kHz, 1.7 W/cm2, 10 min). NO generation was increased gradually with the application of ultrasound and immediately decreased after the cessation of ultrasonication.
ultrasonic procedure (490 kHz, 1.7 W/cm2) was performed for 10 min. On this case, the US intensity in the tissue at 5 mm depth from the skin was calculated to 0.83 W/cm2 in the emitted intensity of 1.7 W/cm2 from the beam profile (Fig. 2a). The percent increase of NO concentration was compared between L-NMMA(⫹) and LNMMA(⫺). Results were expressed as mean ⫾ SEM. The Student’s t-test and paired t-test were used to analyze intergroup comparison. Values were considered significant at an alpha level of less than 0.05.
creased at 0.35 and 0.48 W/cm2 compared with 0.21 W/cm2 (p ⫽ 0.019 and p ⫽ 0.0004, Student’s t-test). A regression curve was calculated as y ⫽ 69.776x ⫺ 13.539 (Fig. 4). Maximum muscle temperature increases at the ultrasonic intensity (SPTA) of 0.21, 0.35 and 0.48 W/cm2 were 0.5 ⫾ 0.2°C, 0.7 ⫾ 0.2°C, and 0.8 ⫾ 0.3°C, respectively. Increase in temperature was less than 1°C.
RESULTS One example for the typical time course of NO generation at the insonation intensity of 1.7 W/cm2 is shown in Fig. 3. NO generation was gradually increased with the application of ultrasound of 490 kHz at the ultrasonic intensity of 0.48 W/cm2 in the tissue and gradually decreased after the cessation of ultrasonication as shown in Fig. 3. The quantitative relationship between NO generation and ultrasonic intensity (SPTA) in the tissue was that the percent increase in NO concentration was 1.25% ⫾ 1.25%, 10.6% ⫾ 2.9% and 20.1% ⫾ 3.5% at the ultrasonic intensity (SPTA) in the tissue of 0.21, 0.35 and 0.48 W/cm2, respectively ( n ⫽ 11, mean ⫾ SE). The effect of ultrasound on NO generation was intensitydependent with a progressive increase from 0.21 to 0.48 W/cm2 (Fig. 4). NO concentration was significantly in-
Fig. 4. Regression curve of percent increase in NO concentration and ultrasonic intensity (SPTA).
Nitric oxide generation by ultrasound exposure ● Y. SUGITA et al.
Fig. 5. The effect of L-NMMA on ultrasonic NO generation. NO generation was significantly reduced from 32.2 ⫾ 6.8% of L-NMMA(⫺) to 18.3 ⫾ 4.3% of L-NMMA(⫹) at the emission intensity of 1.7 W/cm2 (p ⫽ 0.029).
A temperature increase of 3°C in the adductor muscles of the thigh by the thermal stimulator produced 0% increase in NO concentration. In the NO synthase (NOS) inhibition condition using L-NMMA, ultrasonic NO generation was significantly reduced from 32.2 ⫾ 6.8% of L-NMMA(⫺) to 18.3 ⫾ 4.3% of L-NMMA(⫹) at the intensity of 0.83 W/cm2 in the tissue (p ⫽ 0.029, paired t-test, n ⫽ 5) (Fig. 5). It was observed that ultrasonic NO generation can not be completely inhibited by L-NMMA. Maximum muscle temperature increases in L-NMMA(⫹) and L-NMMA(⫺) were 2.2 ⫾ 0.6°C and 2.1 ⫾ 0.5°C, respectively. There was no significant difference in muscle temperature increase between L-NMMA(⫹) and L-NMMA(⫺) (p ⫽ 0.96, Student’s t-test). DISCUSSION Results of the present study demonstrated that 490 kHz ultrasound gradually induced NO generation in accordance with an intensity increase ranging from 0.21 to 0.48 W/cm2 without any thermal effect and gradually decreased after cessation of ultrasonography as shown in Fig. 3 and Fig. 4. Our data calculation showed that the minimum intensity (SPTA) required to generate NO was 0.19 W/cm2 at 490 kHz in the rabbit thigh adductor muscles as can be seen in the regression curve (Fig. 4). It is noticed that since the rabbit was placed on a bench surface in air (i.e., not in a water bath), the estimates of in vivo intensity might be affected by the reflection from the posterior surface. Ultrasonic NO generation was significantly reduced by an NOS inhibitor (L-NMMA) (p ⫽ 0.029, paired t-test). The results clarified that ultrasound stimulated NOS mechanism to generate NO. The mechanism of ultrasonic NO generation in the muscle is considered to be related to the endothelium stimulated by the shear stress caused from ultrasonication. The mechanism of NO generation in which endo-
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thelial cells release NO in response to increased fluid shear stress is reported in Ayajiki et al. (1996), Corson et al. (1996), and Fleming et al. (1998) with fluid shear stress of 15 dyne/cm2, 25 dyne/cm2 and 45 dyne/cm2, respectively. In our experimental condition, the peripheral blood flow direction was randomly settled for making various biochemical balances in the thigh adductor muscle. The acoustic pressure might stress the endothelium in various directions, including the perpendicular or the parallel to the cell surface, and the main pressure was calculated to be 0.16 MPa corresponding to 0.83 W/cm2 and the shear stress of the beam was estimated 434 Pa in water, which value was negligibly small. At present, the relationship between the NO generation and the force direction to the endothelial surface has not been clarified well from the basic investigation. It is natural to speculate from these facts that ultrasound induced sufficient stress in the endothelial cell to activate NOS function for the generation of NO. Moreover, there are two possible mechanisms for endothelial NO generation in response to fluid shear stress. Previous investigations (Ando et al. 1988; Geiger et al. 1992; James et al. 1995) have established that the exposure of endothelium to increased fluid shear stress may stimulate the release of Ca2⫹ from intracellular stores, with consequent elevation of intracellular free Ca2⫹ concentration, which increases the enzymatic activity of endothelial NOS (eNOS) for generation of NO. However, several investigations (Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998) have reported Ca2⫹ independent activation of the eNOS in response to fluid shear stress. They have suggested that tyrosine phosphorylation and intracellular pH (Ayajiki et al. 1996), phosphorylation of the eNOS (Corson et al. 1996) and the tyrosine kinase inhibitor-sensitive pathway (Fleming et al. 1998) play an important role in NO generation. Regarding the mechanism of NO generation in the tissue in response to ultrasound exposure, Mortimer et al. (1988) demonstrated that exposure to ultrasound led to an increase in the amount of intracellular Ca2⫹ and that the cell was able to pump out the Ca2⫹ from the cell once the ultrasound was no longer present. Therefore, NO generation in response to ultrasound exposure is that ultrasonically increased intracellular Ca2⫹ concentration may increase the enzymatic activity of eNOS in generating NO. On the other hand, another mechanism of inducible NOS (iNOS) stimulated by a rapid increase in temperature in the tissue to generate NO through the heat shock protein response may be considered (Yoshida and Xia 2003). However, our ultrasound did not increase the tissue temperature rapidly so that the heat shock protein did not affect NO generation as was shown by the
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thermal examination results. Therefore, the thermal effect of ultrasound does not account for our results. It has been generally accepted that NO is exclusively generated in biological tissues by specific NOS. Initially, this ultrasonic NO generation was thought to be NOS-dependent NO generation due to ultrasonic stress to the endothelium. However, L-NMMA, one of the NOS inhibitors, could not completely inhibit the NO generation by ultrasound. This finding led us to speculate NOS-independent NO generation in the muscle due to ultrasound. Recently, there have been several reports about NOS-independent generation of NO in biological tissues. Zweier et al. (1995) reported that NO can be generated in ischemic tissue by direct chemical mechanism formation of nitrate to NO under acidotic conditions. Furthermore, Jia et al. (1996) discovered that a dynamic cycle exists in which hemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated and the NO group is released during arterial-venous transit in peripheral tissues. Ishida et al. (1999) reported NO production through catalase, myoglobin, nitrite and hemeproteins in vascular smooth muscle. In their study, they mentioned that myoglobin plays an important roll in generation of NO from nitrite. Myoglobin was identified in the vessel tissue by the immunohistological method (Ishida et al. 1999) and the tissue nitrite concentration was measured using a Sievers 270B nitric oxide chemiluminescence analyzer (Sievers Instruments) (Zweier et al. 1995). Therefore, the mechanism of NOS-independent ultrasonic NO generation is thought to be one in which ultrasound might stimulate the chemical mechanism underlying the conversion of nitrate to NO in the muscles (Zweier et al. 1995). Ultrasound might accelerate red blood cells to release NO in the capillary of the muscles (Jia et al. 1996) or ultrasound might activate the catalytic function of myoglobin to generate NO from nitrite in the muscles (Ishida et al. 1999). From a therapeutic ultrasound point of view, this new finding supports scientific evidence for conventional ultrasonic therapy for tissue repair and wound healing with soft tissue injuries. Endothelium-derived NO is known to be a mediator of angiogenesis (Cook and Losordo 2002; Matsunaga et al. 2002). Vascular endothelial growth factor (VEGF) stimulates the release of NO from cultured human umbilical venous endothelial cells and upregulates the expression of NOS. (Hood et al. 1998 and van der Zee et al. 1997) The release of NO by VEGF, growth factor  and basic fibroblast growth factors play a critical role in their angiogenic action (Cook and Losordo 2002). In a three-dimensional fibrin gel, human umbilical venous endothelial cells elaborate NO and form capillary-like structures when stimulated by basic fibroblast growth factor or VEGF, effect that were
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blocked by the NOS antagonist (Babaei et al. 1998; Papapetropoulos et al. 1997). Our research findings support the new concept of angiogenesis based on ultrasonic generated NO and the possibility of a new ultrasonic technology for the treatment and prevention of the ischemic tissue. This is the first report on the direct and real-time measurement of NO generated in the muscle by ultrasound alone. Further studies are necessary, especially to clarify the mechanism of NO generation by ultrasound in the muscle. Acknowledgments—The authors thank Shinichirou Umemura, PhD (Faculty of Engineering, Tohoku University), for his advice in calculating the shear stress by ultrasonic beam. This study was supported by Health and Labor Sciences research grants from the Ministry of Health, Labor and Welfare of Japan. The authors also thank Dr. Janet S. Denny for her assistance in editing this article.
REFERENCES Ando J, Komatsuda T, Kamiyama A. Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells. In Vitro Cell Dev Biol 1988;24:871– 877. Ayajiki K, Kindemann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide Production in native endothelial cells. Circ Res 1996;78:750 –758. Babaei S, Teichert-Kuliszewska K, Monge JC, Mohamed F, Bendeck MP, Stewart DJ. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res 1998;82:1007– 1015. Chokshi SK, Rongione AJ, Freeman I, Gal D, Grunwald AM, Alliger H. Ultrasonic energy produces endothelium dependent vasomotor relaxation in vitro. Circulation 1989;80(Suppl 2):565. Cook JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;105:2133–2135. Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG. Phosphorylation of endothelial nitric oxide synthase in fluid shear stress. Circ Res 1996;79:984 –991. Fischell TA, Abbas MA, Grant GW, Siegel RJ. Ultrasonic energy: Effects on vascular function and integrity. Circulation 1991;84: 1783–1795. Fleming I, Bauersachs J, Fisslthaler B, Busse R. Ca2⫹-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res 1998;82:686 – 695. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373–376. Geiger RV, Berk BC, Alexander RW, Nerem RM. Flow-induced calcium transients in single endothelial cells: Spatial and temporal analysis. Am J Physiol 1992;262:C1411–C1417. Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol 1998;274:H1054 –H1058. Ichimori K, Ishida H, Fukahori M, Nakazawa H, Murakami E. Practical nitric oxide measurement employing a nitric oxide-selective electrode. Rev Sci Instrum 1994;64:2714 –2718. Ishida Y, Tomoda A, Momose K. NO production through catalase and myoglobin, hemoproteins, in vascular smooth muscle. Folia Pharmacol Jpn 1999;114(Suppl 1):27–32. James N, Harrison D, Nerem R. Effects of shear on endothelial cell calcium in the presence and absence of ATP. FASEB J 1995;9: 968 –973.
Nitric oxide generation by ultrasound exposure ● Y. SUGITA et al. Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: A dynamic activity of blood involved in vascular control. Nature 1996;380:221–226. Maruo A, Hammer CE, Rodrigues AJ, Higami T, Greenleaf JF, Schaff HV. Nitric oxide and prostacyclin in ultrasonic vasodilation of the canine internal mammary artery. Ann Thorac Surg 2004;77:126 – 132. Matsunaga T, Weihrauch DW, Moniz MC, Tessmer J, Warltier DC, Chilian WM. Angiostatin inhibits coronary angiogenesis during impaired production of nitric oxide. Circulation 2002;105:2185– 2191. Mitsuhata H, Saitoh J, Takeuchi H, Hasom N, Horiguchi Y, Shimizu R. Production of nitric oxide in anaphylaxis in rabbits. Shock 1994; 2:381–384. Miyoshi H, Nakaya Y. Endotoxin-induced nonendothelial nitric oxide activated the Ca2⫹-activated K⫹ channel in cultured vascular smooth muscle cells. J Mol Cell Cardiol 1994;26:1487–1495. Mortimer AJ, Dyson M. The effect of therapeutic ultrasound on calcium uptake in fibroblasts. Ultrasound Med Biol 1988;14:499 –506. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524 –526. Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997;100:3131–3139. Saitoh J, Mitsuhata H, Takeuchi H, Hasome N, Shimizu R. In vivo production of nitric oxide in the canine heart in IgE-mediated anaphylaxis. Shock 1996;6:66 –70. Steffen W, Cumberland D, Gaines P, Luo H, Nita H, Maurer G, Fishbein MC, Siegel RJ. Catheter-delivered high intensity, low
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frequency ultrasound induces vasodilation in vivo. Eur Heart J 1994;15:369 –376. Suchkova VN, Baggs RB, Sahni SK, Francis SC. Ultrasound improves tissue perfusion in ischemic tissue through a nitric oxide dependent mechanism. Thromb Haemost 2002;88:865– 870. Tamaoki J, Chiyotani A, Konndo K, Konno K. Role of NO generation in adrenoreceptor-mediated stimulation of rabbit airway ciliary motility. Am J Physiol 1995;268:C1342–C1347. Taso PS, Lewis NP, Alpert S, Cook JP. Exposure to shear stress alters endothelial adhesiveness: Role of nitric oxide. Circulation 1995;92: 3513–3519. Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, Nerem RM, Herrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol 1995;269:C1371–C1378. Wang D, Hsu K, Hwang C-P, Chen HI. Measurement of nitric oxide release in the isolated perfused rat lung. Biochem Biophys Res Commun 1995;208:1016 –1020. Yoshida M, Xia Y. Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase. J Biol Chem 2003;278: 36953–36958. van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 1997;95:1030 –1037. Zeng G, Quon MJ. Insulin-stimulated production of nitric oxide is inhibited by wortmannin. J Clin Invest 1996;98:894 – 898. Zweier JL, Wang P, Samouilov A, Kuppusamy P. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med 1995;1: 804 – 809.
doi:10.1016/j.ultrasmedbio.2007.08.008
● Original Contribution NITRIC OXIDE GENERATION DIRECTLY RESPONDS TO ULTRASOUND EXPOSURE YOICHI SUGITA,* SATOKO MIZUNO,* NAOTO NAKAYAMA,* TAKAMASA IWAKI,† EIICHI MURAKAMI,‡ ZUOJUN WANG,* REIKO ENDOH,* and HIROSHI FURUHATA* *Medical Engineering Laboratory and †Laboratory Animal Facilities, Jikei University School of Medicine, Tokyo, Japan; and ‡Eikoukagaku Co. Ltd., Tokyo, Japan (Received 18 February 2007; revised 6 August 2007; in final form 22 August 2007)
Abstract—Recently, several reports have been published on ultrasonic vascular dilation produced with relatively low-frequency ultrasound. It has been speculated that nitric oxide (NO) is an important factor for this ultrasonic vascular dilation. However, a quantitative relationship between the ultrasound intensity and NO generation was not clarified in these reports. We investigated the quantity of NO generated by various ultrasonic intensities by means of real-time measurement of NO concentration in the adductor muscles of the thigh of New Zealand white rabbits exposed to a continuous-wave ultrasound (490 kHz). In the quantitative relationship between NO generation and ultrasonic intensity, the percent increase in NO concentration was 1.25% ⴞ 1.25%, 10.6% ⴞ 2.9% and 20.1% ⴞ 3.5%, with the maximum muscle temperature increase 0.5 ⴞ 0.2°C, 0.7 ⴞ 0.2°C, and 0.8 ⴞ 0.3°C at the ultrasonic intensity (SPTA) of 0.21, 0.35 and 0.48 W/cm2, respectively. The effect of ultrasound on NO generation was intensity-dependent with a progressive increase from 0.21 W/cm2 to 0.48 W/cm2 without significant thermal effect. Ultrasonic NO generation was partially reduced by NOS inhibitor of L-NMMA, clarifying that ultrasound can activate both NOS-dependent and NOS-independent NO generation. These new findings provided scientific basis for ultrasonic vasodilatation and support the potentiality of a new ultrasonic technology for the treatment and prevention of the ischemic tissue based on the new concept of NO generated angiogenesis. (E-mail: [email protected]) © 2008 World Federation for Ultrasound in Medicine & Biology. Key Words: Nitric Oxide (NO), Nitric Oxide Synthase (NOS), Ultrasound, Muscle, Biochemical effect.
the release of Ca2⫹ from intracellular stores, with consequent elevation of intracellular free Ca2⫹ concentration, which increases the enzymatic activity of endothelial NO synthase (eNOS) to generate NO. Recently, however, several investigations (Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998) have reported Ca2⫹ independent activation of the eNOS in response to fluid shear stress. They have suggested that tyrosine phosphorylation and intracellular pH (Ayajiki et al. 1996), phosphorylation of the eNOS (Corson et al. 1996) and tyrosine kinase inhibitor-sensitive pathway (Fleming et al. 1998) play an important role in NO generation. In addition to this physiological stimulation, recent reports have pointed out that external ultrasonication of living tissue induced vasodilatation, blood flow increases and pH changes because of NO generation from the blood vessels, muscular cells and other tissue externally stimulated by the ultrasonic kinetic effect (Chokshi et al. 1989; Fischell et al. 1991; Steffen et al. 1994; Suchkova et al. 2002; Maruo et al. 2004). However, a quantitative rela-
INTRODUCTION Nitric oxide (NO) is now known to control vascular function, biological function, neural signaling and immunologic function (Furchgott and Zawadzki 1980; Palmer et al. 1987). In the field of vascular function, it has been reported that NO generation is stimulated physiologically when the endothelium is exposed to shear stress induced by blood flow and changes in the blood flow (Taso et al. 1995; Uematsu et al. 1995; Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998). There have been two possible mechanisms of endothelial NO generation in response to fluid shear stress. Previous investigations (Ando et al. 1988; Geiger et al. 1992; James et al. 1995) have established that the exposure of endothelium to increased fluid shear stress may stimulate
Address correspondence to: Hiroshi Furuhata, Professor, Medical Engineering Laboratory, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461 Japan. E-mail: [email protected] 487
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tionship between the ultrasound intensity and NO generation was not clarified in these reports as NO was not directly measured. Accordingly, we investigated the quantity of NO generated by various ultrasonic intensities by means of real-time measurement of NO concentration in tissue exposed to ultrasound. MATERIALS AND METHODS All animal procedures were performed under the guidance of the Animal Research Committee of Jikei University School of Medicine, Tokyo, Japan. Twentyone New Zealand white rabbits weighing 3400 g to 4080 g were used in the study. The rabbits were anesthetized with 5% isoflurane and the anesthesia was maintained with 2% to 2.5% isoflurane by a gas mask. The abdominal of the thigh skin was shaved and prepared. The animals were kept at the normothermic core body temperature (38 ⫾ 0.3°C) by means of a heating blanket and the core body temperature was measured with a rectal digital thermometer (Model PTW-100A, Unique Medical Co. Ltd., Tokyo, Japan). NO measuring system NO was measured with a commercially available NO measuring system (Eikoukagaku Co., Ltd. Tokyo, Japan), which is composed of an NO meter (NO-502), a counter electrode (NOR-20) and a working electrode (NOE-47). The principles behind the measurements and the method of measuring the NO concentration with this system have been described elsewhere (Ichimori et al. 1994; Mitsuhata et al. 1994; Miyoshi and Nakaya 1994; Saitoh et al. 1996; Tamaoki et al. 1995; Wang et al. 1995; Zeng and Quon 1996). The NO measuring system is based on the measurement of pA-order currents between the two electrodes. The counter electrode (diameter 500 m) is made of carbon. The working electrode (diameter 200 m) is made of a platinum-iridium (Pt-IR) alloy with a three-layer coating at its detection tip. The electrode was calibrated to NO using S-nitroso-N-acetyl-dlpenicillamine, which serves as a stable standard NO generator, according to the method described by Ichimori et al. (1994). Using this measuring instrument, a current of 1000 pA is approximately equivalent to an NO concentration of 1 Mol/L (Saitoh et al. 1996). The response time of the electrode was 1.14 ⫾ 0.09 s (Ichimori et al. 1994). Ultrasound system The ultrasound system is composed of a ceramic transducer (diameter: 10 mm) consisting of PZT and an ultrasound generator designed by the authors (manufactured by Honda Denshi Co. Ltd. Toyohashi, Japan). A continuous waveform (490 kHz) was applied and three different intensities (0.75, 1.25 and 1.7 W/cm2) were
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selected and applied. Acoustic powers were measured before and after experiments with an ultrasonic power meter (Model UPM-DT-1, Ohmic Instruments Co., Easton, MD, USA) to confirm the exposure conditions. The UPM-DT-1 power meter was calibrated at a frequency of 490 KHz with the accuracy of ⫾3% and resolution of 2 mW by the manufacturer. The actual ultrasound intensity through the spacer was measured by the acoustic intensity measurement system (NTR Systems, Inc. Seattle, WA, USA). The axial intensity (SPTA) at 2.5 cm distance from the probe surface was decreased to 0.21, 0.35 and 0.48 W/cm2 corresponding to the applied power of 0.75, 1.25 and 1.7 W/cm2, respectively. Experimental protocol The NO working electrode was inserted into the adductor muscles of the thigh percutaneously through the 22 G intravenous catheter. The counter electrode was placed under the skin approximately 5 mm away from the working electrode. The needle type electrode of a digital thermometer (DT-300 Eikoukagaku Co. Ltd., Tokyo, Japan) was inserted into a position close to the NO electrode percutaneously through the 22 G intravenous catheter. A low-frequency continuous ultrasound waveform (490 kHz) was applied for 10 min percutaneously to the adductor muscles of the thigh in 11 rabbits through ultrasound gel and a spacer (Acoustic Standoffs, ATS Laboratories, Inc., Bridgeport, CT, USA). The spacer is composed of flexible low attenuation sonolucent gel, covered with a pliable film. The spacer (2 cm thickness) was used to avoid a surface heat affect of the transducer and the near field effect of the ultrasound. The schematic of experimental model is shown in Fig. 1. In three different emitted intensities (0.75, 1.25 and 1.7 W/cm2), the spatial acoustic intensity of the US probe
Ultrasound generator
Digital Thermometer DT-300 Muscle
NO Meter NO-502
Fig. 1. Scheme of experimental set-up. (1) working electrode (NOE-47); (2) counter electrode (NOR-20); (3) needle type electrode of thermometer; (4) spacer (Acoustic Standoffs); (5) ceramic type transducer.
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Fig. 2. The acoustic power profile of the ultrasound transducer (10 ) in the water measured by the hydrophone (Onda Co., Sunnyvale, CA, USA) and the AIMS (1dB per contour). (a) The acoustic power profile without the spacer. (b) The acoustic power profile through the spacer (2 cm thickness).
was calculated from the beam profile of the transducer measured by Acoustic Intensity Measurement System (AIMS) (Fig. 2a). The US intensity in the body at the 5 mm depth from the skin was 0.21 W/cm2 in the emission intensity 0.75 W/cm2 through the spacer 0.35 W/cm2 in 1.25 W/cm2, 0.48 W/cm2 in 1.7 W/cm2 (Fig. 2b). The NO concentration (pA) was measured by a NO meter during the ultrasonication for 10 min. Percent increase of NO concentration was calculated. In another five rabbits, the Thermal Stimulator (BTL-201, Unique Medical Co. Ltd., Tokyo, Japan) was applied directly to the adductor muscles of the thigh for approximately 10 min to examine the relationship between temperature rise and NO generation. The temperature of the adductor muscles of the thigh was increased
gradually to the maximum of 3°C to avoid burning of the muscle. To examine the inherent effect of NO synthase (NOS) on ultrasonic NO generation, an NOS inhibitor of NG-monomethyl-L-arginine (L-NMMA) was used in another five rabbits. Before administration of L-NMMA, the same ultrasonic procedure (490 kHz, 1.7 W/cm2) was performed for 10 min and the percent increase of NO concentration was measured. In this experiment, because the temperature rise of the ultrasonic probe did not affect NO generation as shown by the results, we applied the probe directly to the adductor muscles of the thigh without a tissue spacer to maximize the accuracy of test results. Subsequently, L-NMMA was administrated IV (5 mg/Kg) 5 min before ultrasonication and the same
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Fig. 3. Real-time record of NO concentration in the muscular tissue produced by ultrasonication (490 kHz, 1.7 W/cm2, 10 min). NO generation was increased gradually with the application of ultrasound and immediately decreased after the cessation of ultrasonication.
ultrasonic procedure (490 kHz, 1.7 W/cm2) was performed for 10 min. On this case, the US intensity in the tissue at 5 mm depth from the skin was calculated to 0.83 W/cm2 in the emitted intensity of 1.7 W/cm2 from the beam profile (Fig. 2a). The percent increase of NO concentration was compared between L-NMMA(⫹) and LNMMA(⫺). Results were expressed as mean ⫾ SEM. The Student’s t-test and paired t-test were used to analyze intergroup comparison. Values were considered significant at an alpha level of less than 0.05.
creased at 0.35 and 0.48 W/cm2 compared with 0.21 W/cm2 (p ⫽ 0.019 and p ⫽ 0.0004, Student’s t-test). A regression curve was calculated as y ⫽ 69.776x ⫺ 13.539 (Fig. 4). Maximum muscle temperature increases at the ultrasonic intensity (SPTA) of 0.21, 0.35 and 0.48 W/cm2 were 0.5 ⫾ 0.2°C, 0.7 ⫾ 0.2°C, and 0.8 ⫾ 0.3°C, respectively. Increase in temperature was less than 1°C.
RESULTS One example for the typical time course of NO generation at the insonation intensity of 1.7 W/cm2 is shown in Fig. 3. NO generation was gradually increased with the application of ultrasound of 490 kHz at the ultrasonic intensity of 0.48 W/cm2 in the tissue and gradually decreased after the cessation of ultrasonication as shown in Fig. 3. The quantitative relationship between NO generation and ultrasonic intensity (SPTA) in the tissue was that the percent increase in NO concentration was 1.25% ⫾ 1.25%, 10.6% ⫾ 2.9% and 20.1% ⫾ 3.5% at the ultrasonic intensity (SPTA) in the tissue of 0.21, 0.35 and 0.48 W/cm2, respectively ( n ⫽ 11, mean ⫾ SE). The effect of ultrasound on NO generation was intensitydependent with a progressive increase from 0.21 to 0.48 W/cm2 (Fig. 4). NO concentration was significantly in-
Fig. 4. Regression curve of percent increase in NO concentration and ultrasonic intensity (SPTA).
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Fig. 5. The effect of L-NMMA on ultrasonic NO generation. NO generation was significantly reduced from 32.2 ⫾ 6.8% of L-NMMA(⫺) to 18.3 ⫾ 4.3% of L-NMMA(⫹) at the emission intensity of 1.7 W/cm2 (p ⫽ 0.029).
A temperature increase of 3°C in the adductor muscles of the thigh by the thermal stimulator produced 0% increase in NO concentration. In the NO synthase (NOS) inhibition condition using L-NMMA, ultrasonic NO generation was significantly reduced from 32.2 ⫾ 6.8% of L-NMMA(⫺) to 18.3 ⫾ 4.3% of L-NMMA(⫹) at the intensity of 0.83 W/cm2 in the tissue (p ⫽ 0.029, paired t-test, n ⫽ 5) (Fig. 5). It was observed that ultrasonic NO generation can not be completely inhibited by L-NMMA. Maximum muscle temperature increases in L-NMMA(⫹) and L-NMMA(⫺) were 2.2 ⫾ 0.6°C and 2.1 ⫾ 0.5°C, respectively. There was no significant difference in muscle temperature increase between L-NMMA(⫹) and L-NMMA(⫺) (p ⫽ 0.96, Student’s t-test). DISCUSSION Results of the present study demonstrated that 490 kHz ultrasound gradually induced NO generation in accordance with an intensity increase ranging from 0.21 to 0.48 W/cm2 without any thermal effect and gradually decreased after cessation of ultrasonography as shown in Fig. 3 and Fig. 4. Our data calculation showed that the minimum intensity (SPTA) required to generate NO was 0.19 W/cm2 at 490 kHz in the rabbit thigh adductor muscles as can be seen in the regression curve (Fig. 4). It is noticed that since the rabbit was placed on a bench surface in air (i.e., not in a water bath), the estimates of in vivo intensity might be affected by the reflection from the posterior surface. Ultrasonic NO generation was significantly reduced by an NOS inhibitor (L-NMMA) (p ⫽ 0.029, paired t-test). The results clarified that ultrasound stimulated NOS mechanism to generate NO. The mechanism of ultrasonic NO generation in the muscle is considered to be related to the endothelium stimulated by the shear stress caused from ultrasonication. The mechanism of NO generation in which endo-
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thelial cells release NO in response to increased fluid shear stress is reported in Ayajiki et al. (1996), Corson et al. (1996), and Fleming et al. (1998) with fluid shear stress of 15 dyne/cm2, 25 dyne/cm2 and 45 dyne/cm2, respectively. In our experimental condition, the peripheral blood flow direction was randomly settled for making various biochemical balances in the thigh adductor muscle. The acoustic pressure might stress the endothelium in various directions, including the perpendicular or the parallel to the cell surface, and the main pressure was calculated to be 0.16 MPa corresponding to 0.83 W/cm2 and the shear stress of the beam was estimated 434 Pa in water, which value was negligibly small. At present, the relationship between the NO generation and the force direction to the endothelial surface has not been clarified well from the basic investigation. It is natural to speculate from these facts that ultrasound induced sufficient stress in the endothelial cell to activate NOS function for the generation of NO. Moreover, there are two possible mechanisms for endothelial NO generation in response to fluid shear stress. Previous investigations (Ando et al. 1988; Geiger et al. 1992; James et al. 1995) have established that the exposure of endothelium to increased fluid shear stress may stimulate the release of Ca2⫹ from intracellular stores, with consequent elevation of intracellular free Ca2⫹ concentration, which increases the enzymatic activity of endothelial NOS (eNOS) for generation of NO. However, several investigations (Ayajiki et al. 1996; Corson et al. 1996; Fleming et al. 1998) have reported Ca2⫹ independent activation of the eNOS in response to fluid shear stress. They have suggested that tyrosine phosphorylation and intracellular pH (Ayajiki et al. 1996), phosphorylation of the eNOS (Corson et al. 1996) and the tyrosine kinase inhibitor-sensitive pathway (Fleming et al. 1998) play an important role in NO generation. Regarding the mechanism of NO generation in the tissue in response to ultrasound exposure, Mortimer et al. (1988) demonstrated that exposure to ultrasound led to an increase in the amount of intracellular Ca2⫹ and that the cell was able to pump out the Ca2⫹ from the cell once the ultrasound was no longer present. Therefore, NO generation in response to ultrasound exposure is that ultrasonically increased intracellular Ca2⫹ concentration may increase the enzymatic activity of eNOS in generating NO. On the other hand, another mechanism of inducible NOS (iNOS) stimulated by a rapid increase in temperature in the tissue to generate NO through the heat shock protein response may be considered (Yoshida and Xia 2003). However, our ultrasound did not increase the tissue temperature rapidly so that the heat shock protein did not affect NO generation as was shown by the
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thermal examination results. Therefore, the thermal effect of ultrasound does not account for our results. It has been generally accepted that NO is exclusively generated in biological tissues by specific NOS. Initially, this ultrasonic NO generation was thought to be NOS-dependent NO generation due to ultrasonic stress to the endothelium. However, L-NMMA, one of the NOS inhibitors, could not completely inhibit the NO generation by ultrasound. This finding led us to speculate NOS-independent NO generation in the muscle due to ultrasound. Recently, there have been several reports about NOS-independent generation of NO in biological tissues. Zweier et al. (1995) reported that NO can be generated in ischemic tissue by direct chemical mechanism formation of nitrate to NO under acidotic conditions. Furthermore, Jia et al. (1996) discovered that a dynamic cycle exists in which hemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated and the NO group is released during arterial-venous transit in peripheral tissues. Ishida et al. (1999) reported NO production through catalase, myoglobin, nitrite and hemeproteins in vascular smooth muscle. In their study, they mentioned that myoglobin plays an important roll in generation of NO from nitrite. Myoglobin was identified in the vessel tissue by the immunohistological method (Ishida et al. 1999) and the tissue nitrite concentration was measured using a Sievers 270B nitric oxide chemiluminescence analyzer (Sievers Instruments) (Zweier et al. 1995). Therefore, the mechanism of NOS-independent ultrasonic NO generation is thought to be one in which ultrasound might stimulate the chemical mechanism underlying the conversion of nitrate to NO in the muscles (Zweier et al. 1995). Ultrasound might accelerate red blood cells to release NO in the capillary of the muscles (Jia et al. 1996) or ultrasound might activate the catalytic function of myoglobin to generate NO from nitrite in the muscles (Ishida et al. 1999). From a therapeutic ultrasound point of view, this new finding supports scientific evidence for conventional ultrasonic therapy for tissue repair and wound healing with soft tissue injuries. Endothelium-derived NO is known to be a mediator of angiogenesis (Cook and Losordo 2002; Matsunaga et al. 2002). Vascular endothelial growth factor (VEGF) stimulates the release of NO from cultured human umbilical venous endothelial cells and upregulates the expression of NOS. (Hood et al. 1998 and van der Zee et al. 1997) The release of NO by VEGF, growth factor  and basic fibroblast growth factors play a critical role in their angiogenic action (Cook and Losordo 2002). In a three-dimensional fibrin gel, human umbilical venous endothelial cells elaborate NO and form capillary-like structures when stimulated by basic fibroblast growth factor or VEGF, effect that were
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blocked by the NOS antagonist (Babaei et al. 1998; Papapetropoulos et al. 1997). Our research findings support the new concept of angiogenesis based on ultrasonic generated NO and the possibility of a new ultrasonic technology for the treatment and prevention of the ischemic tissue. This is the first report on the direct and real-time measurement of NO generated in the muscle by ultrasound alone. Further studies are necessary, especially to clarify the mechanism of NO generation by ultrasound in the muscle. Acknowledgments—The authors thank Shinichirou Umemura, PhD (Faculty of Engineering, Tohoku University), for his advice in calculating the shear stress by ultrasonic beam. This study was supported by Health and Labor Sciences research grants from the Ministry of Health, Labor and Welfare of Japan. The authors also thank Dr. Janet S. Denny for her assistance in editing this article.
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