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Frequency dependence of excitation–contraction of multicellular smooth muscle preparations: the relevance to bipolar electrosurgery
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 1 9 e1 2 5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Frequency dependence of excitationecontraction of multicellular smooth muscle preparations: the relevance to bipolar electrosurgery Irina A. Vladimirova, PhD,a,c Yuri N. Lankin, PhD,b Igor B. Philyppov, PhD,a,c Lyudmyla F. Sushiy, MS,b and Yaroslav M. Shuba, PhDa,c,* a
Bogomoletz Institute of Physiology NASU, Kyiv, Ukraine Paton Electric Welding Institute NASU, Kyiv, Ukraine c State Key Laboratory of Molecular and Cellular Physiology, Kyiv, Ukraine b
article info
abstract
Article history:
Background: Bipolar electrosurgical tissue welding uses forceps-like electrodes for grasping
Received 1 March 2013
the tissues and delivering high-frequency electric current (HFEC) to produce local heat,
Received in revised form
desiccation, and protein denaturation, resulting in the fusion of the contacting tissues.
22 July 2013
Although in this technique no electric current is flowing through the whole body to cause
Accepted 12 August 2013
electric injury, depending on the frequency of applied energy, it may produce local exci-
Available online 3 September 2013
tation of intramural nerves, which can propagate beyond the surgical site potentially causing harmful effects.
Keywords:
Materials and methods: The effects of varying frequency of HFEC on tissue excitability in
Electrosurgery
bipolar electrosurgical modality were studied in vitro using electric field stimulation (EFS)
Electrosurgical tissue welding
method on multicellular smooth muscle strips of rat vas deferens. Contractile response to
Electric field stimulation
5-s-long sine wave EFS train was taken as the measure of excitation of intramural nerves.
Vas deferens
Results: EFS-induced contraction consisted of phasic and tonic components. The amplitude
Excitation
of both components decreased with increasing frequency, with tonic component dis-
Contraction
appearing at about 10 kHz and phasic component at about 50 kHz. Because components of
Frequency
EFS-induced contraction depend on different neurotransmitters, this indicates that various neurotransmitter systems are characterized by distinct frequency dependence, but above 50 kHz they all become inactivated. Bipolar electrosurgical sealing of porcine gut showed no difference in the structure of seal area at HFEC of 67 and 533 kHz. Conclusions: EFS frequency of 50 kHz represents the upper limit for excitation. HFEC above 50 kHz is safe to use for bipolar electrosurgical tissue welding without concerns of excitation propagating beyond the surgical site. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Electrosurgery uses high-frequency electric current (HFEC) passed through the tissue to create the desired clinical effect
via locally induced diathermy [1]. Among its common applications, such as cutting, coagulation, desiccation, and fulguration, electrosurgical tissue welding is increasingly viewed as a viable alternative to the traditional mechanical means of
* Corresponding author. Bogomoletz Institute of Physiology NASU, Bogomoletz Street 4, Kyiv 01024, Ukraine. Tel.: þ380 44 2562048; fax: þ380 44 2562435. E-mail address: [email protected] (Y.M. Shuba). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.08.012
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reconnecting the tissues, which are based on using the suture, metal staples, or clips [2]. In its bipolar mode, this technique uses HFEC to produce diathermy between tightly pressed tissues against each other [1e3]. The HFEC is usually delivered using bipolar forceps-like electrodes for grasping the tissues and applying pressure to them [4,5]. The two tines of the forceps perform the active and return electrode functions, and only the tissue grasped is included in the electric circuit, making separate patient “return” electrode characteristic of monopolar electrosurgery mode unnecessary. Localized HFEC passing through the grasped tissues induces thermal tissue damage, which depends on the size and shape of the bipolar electrodes, exerted pressure, and the parameters (power, frequency, waveform, and duration) of the high-frequency (HF) electric energy. If all parameters are optimal, the heat generation, desiccation, and protein denaturation results in fusion or “welding” of the contacting tissues confined to the area of the electrodes, which can withstand distraction force comparable to the force achieved for traditional sutured joints [6]. Power and duration of the applied electric energy have to be such that to prevent overheating and tissue burning to minimize necrotic damage and at the same time providing the strongest fusion possible. As these parameters change, so will the corresponding tissue effects [7]. Animal studies and clinical surgical experiences indicate that optimal frequency of the applied electric energy must range between 60 and 75 kHz [6]. However, despite bipolar welding technique does not require separate patient return electrode, which prevents net current flow through the patient’s body thereby negating many of the safety precautions related to the clinical use of electric current, the International Electrotechnical Commission (IEC) and the Association for the Advancement of Medical Instrumentation only approve the use of the devices with the frequencies above 300 kHz [8]. Technically, the bipolar electrosurgical mode is similar to the electric field stimulation (EFS) method widely used in science to stimulate contractions of multicellular muscle preparation in vitro via excitation of intramural nerve fibers [9]. Thus, confined application of HF electric energy with the purpose of welding the tissues can also produce local excitation of intramural nerves, which can propagate to the central nervous system structure potentially causing undesirable effects. Because of the phenomenon of refractoriness, a short-term decrease in the excitability of nerve and muscle tissue occurs immediately after the manifestation of action potential [10]. The possibility of excitation decreases with increasing frequency of EFS. However, to our knowledge, systematic study of this phenomenon in the context of bipolar electrosurgical techniques, with the purpose of establishing the lowest frequency limit at which no potentially harmful excitation can occur, was not performed. Here, we have used rat vas deferens smooth muscle preparations with and without epithelial cell layers to establish frequency dependence of the contractions in response to the sine wave EFS in vitro. Our results show that at frequencies above 50 kHz no contraction can be induced, indicating that this frequency represents the lowest safe limit, which can be used for bipolar HFECemediated electrosurgical procedures.
2.
Materials and methods
2.1. Vas deferens preparation and recording of contraction Experiments were conducted on smooth muscle strips from the vas deferens of male Wistar rats weighing 200e250 g. Animals were killed by decapitation, their vas deferens was removed, and placed in the warm (37 C), oxygenated (95% O2 and 5% CO2) Krebs solution (in mM): 120.4 NaCl, 5.9 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.8 CaCl2, 15.5 NaHCO3, and 11.5 glucose (pH 7.4). Vas deferens was cleaned from connective tissue, its wall was cut along the axis, and longitudinal strips of 0.2e0.3 cm in diameter and 0.7e1.0 cm in length were excised from the parts adjacent to the prostate (prostatic portion). Smooth muscle strips with both epithelial layers removed and retained were used in the experiments. Schematic diagram of the experimental arrangement is presented in Figure 1. For the recording of contractions, the strip was placed in the acrylic glass chamber continuously superfused with Krebs solution at 37 C with one end of the strip fixed still and another one attached to the capacitative force sensor with the baseline load of 3 mN applied to the strip. EFS was delivered via two Ag/AgCl wires positioned at one end of the strip from its top and bottom with direct contact with the tissue. The EFS consisted of sine wave trains of various frequencies (0.02e200 kHz), amplitudes (10e60 V), and durations (2-10 s) applied every 3 min, which was sufficient for complete restoration of basal tone. Contractile
Fig. 1 e Schematic diagram of experimental arrangement. Acrylic glass experimental chamber (1) is continuously superfused via inlet and outlet tubes with Krebs solution (2). One end of the smooth muscle strip (3) is fixed still to the chamber wall via retainer (4) and another end via retainer (5) is attached to the lever (6) of the capacitative force sensor (7). EFS is delivered via Ag/AgCl electrodes (8) connected to the generator of HFEC (9). The drawing is not to scale.
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responses were recorded on chart recorder (model H3030-4, Russia) and computer via analog-digital converter (DigiData 1200, Axon Instruments Inc, Union City, CA).
2.2.
washed in water. They were stained with hematoxylin and eosin, sandwiched between microscope glass and coverslip, and used for microscopic examination.
Electrosurgical tissue welding and histology
Frequency dependence of electrosurgical tissue welding was assessed in gut sealing experiments on an isolated portion of porcine gut using the custom designed and manufactured electrosurgical apparatus with variable output frequency. The gut was placed transversely between the two electrodes, and the force was applied to electrodes sufficient to bring opposite gut’s walls into the tight contact. HFEC with root mean square (RMS) value of 2 A was passed through electrodes and the gut tissue resulted in tissue heating and desiccation. HFEC was turned off shortly after desiccation, before any signs of tissue burning were evident. After HFEC termination, the tissue was let to cool down, electrodes were removed, and the tissue was used for the preparation of histologic slides. Briefly, gut tissue was fixed in 10% formalin for 48 h, dehydrated by processing through a series of ethanol baths of increasing concentration (50e96%), infiltrated with paraffinechloroform mixture, and embedded in blocks of paraffin and bee wax. Embedded gut specimens were cooled on ice and sectioned in longitudinal direction onto 10-mmthick slices on a microtome. Slices were deparaffinised in xilol, passed through decreasing alcohol concentrations, and
3.
Results
3.1. Frequency dependence of the contractions at weak stimulation In the first series of experiments, we have applied sine wave EFS of effective amplitude 15 V, which according to our pilot studies was insufficient to produce tissue damage and was optimal for ensuring long-lasting viability of the strip (up to 3 h) and reproducibility of contractile responses. In this series, the frequency of the sine wave within EFS train varied from 20 Hz to 100 kHz. With the 5-s-long EFS, independently of the frequency within EFS, typical contractile response consisted of three components: the first (I) component of transient (phasic) contraction lasting for about 2 s, the second (II) component of tonic contraction lasting till the end of EFS, and the third (III) component of relaxation to the baseline after EFS termination (Fig. 2A). In visceral smooth muscle, the phasic and tonic components of EFS-evoked contractions are usually attributed to the release from intramural efferent nerve fibers excited by EFS of the main efferent transmitters, adenosine-triphosphate
A I
B
II
III
Normalized amplitude, %
140 120 100 Overall
80 60 40
Sustained
20 0 10
100
1000
10000
100000
Frequency, Hz Fig. 2 e Frequency dependence of EFS-induced contractions of rat vas deferens. (A) Representative original recordings of the contractions induced by 5-s-long sine wave EFS of 15 V and variable frequencies (time of EFS application is shown by horizontal bars above recordings); transient (I), tonic (II), and relaxation (III) phases of the contractile response, its overall amplitude (filled symbol), and amplitude of tonic component (open symbol) are indicated on 2 kHz recording. (B) Frequency dependence of overall amplitude (filled symbols) and amplitude of tonic component (open symbols) of EFS-induced contraction (mean ± standard error of the mean, n [ 8); all amplitudes for each preparation were normalized to the overall amplitude at 500 Hz and then averaged. (Color version of figure is available online.)
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(ATP), acetylcholine, and noradrenalin (NA), which are characterized by different kinetics of contractile action on smooth muscle cells (SMCs) [11e14]. The transient component typically consisted 70%e80% and the tonic component 20%e30% of the overall amplitude of contractile response. To be able to compare frequency dependence of the amplitudes of the contractions of various preparations, all amplitudes for a given preparation were normalized to the overall amplitude of the contraction at 500 Hz (Fig. 2B). Increasing the frequency of EFS from 20e100 Hz resulted in about 10% enhancement of the overall amplitude of the contractions (Fig. 2). Furthermore increase of the frequency produced almost no change in overall amplitude up to 500 Hz with the progressive decrease observed at higher frequencies. The reduction in the overall amplitude of EFS-evoked contractions at frequencies above 500 Hz progressed at a rate of about 60% per decade and eventually brought the amplitude to zero at 50 kHz (Fig. 2B). The amplitude of the tonic component of the contractions showed more monotonic but notably accelerated decline, gradually decreasing to zero in the range of frequencies from 20e10 kHz with the rate of about 10% per decade (Fig. 2B). Higher “resistance” of the phasic component of EFS-evoked contractions to the increased frequencies compared with the tonic component may likely reflect the differences in the release and contractile action of the “fast” transmitter ATP compared with the “slower” neurotransmitters, such as acetylcholine and NA [13]. Overall, these results indicate that at the frequencies of EFS above 50 kHz intramural nerves do not excite most likely
because of the property of refractoriness, and as a result do not release transmitters that can cause muscle contraction. Consequently, EFS at the frequencies above 50 kHz cannot evoke harmful excitation that can propagate in the ascending direction from the site of its application.
3.2.
Contractions at HF and strong stimulation
Tissue fusion or welding during localized passing of HFEC requires induction of tissue heating accompanied by desiccation, cell damage and protein denaturation. This can be achieved only by applying sufficiently high power of electric energy, which in turn can theoretically induce harmful excitation propagating from the site of application even at HFs that are unable to do so at low power. To check for such possibility, we have conducted experiments with application of 50-kHz sine wave EFS of increasing effective amplitude from 15e60 V. Figure 3 presents the original recordings of a typical experiment of this kind. As it can be seen, weak 15-V EFS of 50 kHz produced only miniscule transient contraction compared with 500 Hz with no tonic component present (Fig. 3). Increasing EFS to 25 V with 5-V increment first produced gradually higher transient contraction, which decreased again at EFS above 30 V (Fig. 3B, upper row). However, furthermore increase of EFS to 40 V and above produced drastically different contractile responses. These responses were characterized by much slower onset amplitude that kept creeping up during whole duration of EFS, and strongly impaired relaxation leading to the development of ever accumulating poststimulatory tonic contraction (Fig. 3).
Fig. 3 e Dependence of EFS-induced contractions of rat vas deferens on EFS power. (A) Representative original recording of the reference contraction induced by 5-s-long sine wave EFS of 15 V at 500 Hz (shown by horizontal bars). (B) Representative original recording of EFS-induced (5-s-long sine wave) contractions from the same preparations at 50 kHz and increasing power from 15e60 V showing initial gradual increase in the amplitude of transient contractile response and its irreversible transformation into constant nonrelaxing tone at EFS power of 40 V.
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Induction of such response was irreversible in terms of returning the muscle preparation to normal contractility even after prolonged rest. Such behavior is most likely explained by the ability of HF EFS of up to 35 V still to produce small graded contractile responses via excitation of intramural nerves, however, with delivering even more power intramural nerves may likely get damaged, and EFS induces tonic contraction by acting directly on SMCs, which eventually results in SMC damage as well. Thus, although increasing the power of HF EFS may enhance excitation of intramural nerves, damage of the nerves at powers that are required for tissue fusion prevent negative impact on the ascending pathways.
3.3. Frequency dependence of electrosurgical tissue fusion To test whether the frequency of HFEC influences the quality of tissue fusion, we have performed experiments on electrosurgical sealing of porcine gut at two frequencies, the low frequency of 67 kHz and the HF of 533 kHz. These experiments were conducted on the adjacent portions of the gut, using the same electrosurgical apparatus, with equal mechanical force applied to the tissue-grasping bipolar electrodes, identical RMS value of HFEC passing through the tissues, and the same time of exposure to HFEC. Tissue fusion occurred in the area of contact of two mucous layers of the gut walls. Figure 4 shows representative longitudinal histologic sections of the seal area obtained under such conditions at HFEC of 67 kHz and 533 kHz. Inspection of these sections reveals no essential difference in the structure of the seal area between the two frequencies, suggesting that at both frequencies HFEC produces desirable tissue fusion effects.
4.
Discussion
One of the useful clinical applications of HFEC is related to blood coagulation, electrosurgical tissue cutting, and welding
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[7]. Localized delivery of HFEC for such purposes is usually achieved with “monopolar” electrosurgical modality in which one “active” electrode with the small surface of the tip is used to apply HFEC directly to the surgical site, while the second much bigger return electrode is placed at the remote body area, most commonly on the patient’s back or limb. Because of the small contact area of the active electrode with the tissue, even miniscule net electric current flowing from the active electrode through the patient’s body to the return electrode may have local density at the tip of the active electrode high enough to produce the thermal effect sufficient for blood coagulation or even tissue cutting and fusion. However, along with the possibility of achieving desired local effects, the flow of the electric current through the whole body in monopolar configuration inevitably raises the basic safety issues. Because the harm to the organism induced by electric current flowing through the body is known to increase with decreased frequency, whereas at very HFs “skin effect,” a phenomenon that tends to displace alternating current to the surface of conducting media, plays protective role, IEC standards limit the lower frequency of electrosurgical equipment by 300 kHz [8]. At the same time, according to some studies and empirical clinical experience [6], application of HFEC with the frequency around or slightly higher than 50 kHz produces stronger and more stable connections of intestinal, stomach, skeletal muscle, and skin tissues compared with the frequencies of several hundreds of kilohertz, prompting to search for the solutions satisfying both safety requirements and surgical practicality. In the event of “bipolar” electrosurgical modality, HFEC almost completely passes through the tissue grasped between two electrodes and “classic” remote return electrode is absent, making safety concerns related to the flow of electric current through the whole body largely insignificant. However, the possibility remains that the locally applied HFEC will cause undesirable excitation of intramural nerves producing harm to the ascending centers. Here, we show that such excitation may occur only at HFEC frequencies below 50 kHz and completely refute such concerns for higher frequencies. In our
Fig. 4 e Frequency of HFEC does not influence the quality of electrosurgical tissue fusion. Representative longitudinal histologic sections of the electrosurgical seal area (marked by braces) of porcine gut obtained at 67 kHz (left) and 533 kHz (right) HFEC showing no principal difference in the seal structure for the two frequencies. The inset shows schematic drawing (not to scale) of the electrosurgical procedure: (1) gut portion, (2) bipolar electrodes, (3) generator of HFEC, and (4) switch; dashed line box denotes the area shown in histologic slices. (Color version of figure is available online.)
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studies, we mimicked bipolar electrosurgical modality by using EFS on multicellular preparations (strips) of rat vas deferens, and recorded EFS-evoked contractions as the measure of functional response. Rat vas deferens can be viewed as the most representative model tissues, as it consists of SMCs that are directly activated by neurotransmitter released from nerve varicosities and SMCs which activation is dependent on gap junctionemediated cell-to-cell communication [14]. Moreover, specifically in rat and mouse vas deference passive propagation of excitation is basically absent [15], indicating predominance of direct activation of SMCs by neurotransmitter and giving this tissue resemblance to both individually innervated skeletal muscle and classical smooth muscle syncytium. Our results demonstrate that contractile response of the prostatic portion of the vas deferens completely disappears at EFS frequency above 50 kHz, whereas tonic component of this response diminishes at even lower frequencies, already above 10 kHz. Pharmacologic characterization of the biphasic contraction of rat vas deferens induced by EFS of intramural sympathetic nerve fibers supplying vas deferens established that the first, transient component has purinergic nature, whereas the second, sustained one is adrenergic [12,14,16]. The adrenergic component predominates in the epididymal segment of the duct and purinergic in the prostatic segment [12]. The fact that transient and sustained components of EFSevoked contractions revealed divergent sensitivity to the frequency of EFS indicates that synaptic releases of ATP and NA are characterized by different coupling efficiency with fiber excitation. As frequency-dependent decrease of contractions is explained by refractoriness of nerve excitability, this suggests that the purinergic system is more resistant to the deterioration of action potential parameters with increasing EFS frequency. In addition, the disparity in SMC activation via direct innervation and gap junctionemediated mechanisms may contribute to the observed frequency dependence of various components of neurogenic contractile response [17]. Thus, although various types of nerve fibers and neurotransmitter systems may exhibit differential frequency sensitivity of excitation during local application of HFEC by means of bipolar electrosurgical technique, 50 kHz seems to represent the upper limit for the frequency when excitation may propagate beyond the surgical site. Increasing the power of EFS to the level that is required for electrosurgical tissue fusion induces damage to intramural nerves and irreversible tonic contraction in smooth muscle strip via direct stimulation of SMCs with subsequent damage to SMCs as well. Thus, induction of nerves excitation and its propagation beyond the surgical site at such powers is prevented by local tissue damage. It should be noted that bipolar electrosurgical devices generating HFEC of 50e100 kHz are already successfully used in clinics in Ukraine since the second half of 1990s with tens of thousands of operations on humans already performed. The results of these practical experiences for various types of tissues are reported in Refs. [2,6]. Nevertheless, at the beginning of the adoption of bipolar electrosurgical tissue welding
in clinics in early 1990s, because of safety concerns, only the frequencies of 0.8e1.1 MHz were used in Ukraine as well. However, with time the frequency was gradually brought down to 66 6 kHz, which was found empirically to be optimal for providing quality tissue fusion with no evident side effects. In addition, from engineering standpoint electrosurgical devices with such frequencies are simpler to design and manufacture. Unfortunately, the results of clinical trials and countless animal experiments carried out in Ukraine were not published in international scientific literature and so far are best described in Refs. [2,6]. Our own experiments on electrosurgical sealing of porcine gut revealed no essential differences in the structure of the seal area produced at HFEC of 67 kHz and 533 kHz and otherwise identical conditions. This is not surprising given that tissue heating is determined by the RMS value of HFEC, which was kept constant in our trials, and not by its frequency or the waveform. Besides, at the used frequencies, the so-called skin effect, which tends to displace HFEC density toward the surface of a conductor with increasing frequencies, is not prominent enough to cause nonuniformity of tissue heating in bipolar electrosurgical configuration. However, we have found that the losses in electrosurgical apparatus circuitry, their heating, and consequently the power required to produce output HFEC with the same RMS value are much reduced at lower frequency compared with higher frequency: at RMS of 2 A the output voltage consisted 8 V at 67 kHz increasing to 18.5 V (i.e., 2.3-fold) at 533 kHz. The purpose of our study was to demonstrate that HFEC at frequencies just above 50 kHz is not harmful in bipolar electrosurgical configuration, because local excitation cannot propagate beyond the surgical site. At the same time, these frequencies are well suited for producing quality electrosurgical HFECemediated tissue fusion. Our study makes recommendations of the IEC and Advancement of Medical Instrumentation, regarding the HF surgical equipment, at least in the context of bipolar electrosurgery, obsolete and calls for the revision.
Acknowledgment This study was supported by the National Academy of Sciences of Ukraine and F46.2/001 grant from State Fund for Fundamental Research, Ukraine (YMS). The authors declare no conflicts of interest.
references
[1] Massarweh NN, Cosgriff N, Slakey DP. Electrosurgery: history, principles, and current and future uses. J Am Coll Surg 2006;202:520. [2] Paton BE. Electric welding of soft tissues in surgery. Paton Weld J; 2004:6 [in Russian].
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[3] Linchevskyy O, Makarov A, Getman V. Lung sealing using the tissue-welding technology in spontaneous pneumothorax. Eur J Cardiothorac Surg 2010;37:1126. [4] Kennedy JS, Buysse SP, Lawes KR, Ryan TR. Recent innovations in bipolar electrosurgery. Minim Invasive Ther Allied Technol 1999;8:95. [5] Stenquist BC, Holt PJ, Motley RJ. Computerized bipolar diathermy with scissors and forceps in cutaneous surgery. Dermatol Surg 2002;28:601. [6] Paton BE, Ivanova ON. Tissue-preserving high-frequency electro-welding surgery. Kiev: Naukova Dumka; 2009 [in Russian]. [7] Principles of electrosurgery, http://www.valleylab.com/ education/poes/index.html. [8] IEC 60601-2-2 ED. 5.0 B:2009, Medical electrical equipmentdPart 2-2: Particular requirements for the basic safety and essential performance of high frequency surgical equipment and high frequency surgical accessories. [9] Durand DM. Electric stimulation of excitable tissue. In: Bronzino JD, editor. The biomedical engineering handbook. 2nd ed. Boca Raton, FL: CRC Press LLC; 2000. [10] Smith MC. Theories of the psychological refractory period. Psychol Bull 1967;67:202.
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[11] Burnstock G. Cholinergic, adrenergic, and purinergic neuromuscular transmission. Fed Proc 1977;36:2434. [12] Rohde GC, Venezian E, Huidobro-Toro JP. Asymmetric distribution of purinergic and adrenergic neurotransmission cooperates in the motor activity along the rat vas deferens. Neurosci Lett 1986 11;71:197. [13] Sneddon P. Electrophysiology of autonomic neuromuscular transmission involving ATP. J Auton Nerv Syst 2000;81:218. [14] Burnstock G, Verkhratsky A. Vas deferensda model used to establish sympathetic cotransmission. Trends Pharmacol Sci 2010;31:131. [15] Kuriyama H, Kitamura K, Itoh T, Inoue R. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev 1998; 78:811. [16] Ventura S. Autoinhibition, sympathetic cotransmission and biphasic contractile responses to trains of nerve stimulation in the rodent vas deferens. Clin Exp Pharmacol Physiol 1998; 25:965. [17] Palani D, Manchanda R. Effects of heptanol on neurogenic contractions of vas deferens: a comparative study of stimulation frequency in guinea pig and rat. J Physiol Sci 2006;56:21.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Frequency dependence of excitationecontraction of multicellular smooth muscle preparations: the relevance to bipolar electrosurgery Irina A. Vladimirova, PhD,a,c Yuri N. Lankin, PhD,b Igor B. Philyppov, PhD,a,c Lyudmyla F. Sushiy, MS,b and Yaroslav M. Shuba, PhDa,c,* a
Bogomoletz Institute of Physiology NASU, Kyiv, Ukraine Paton Electric Welding Institute NASU, Kyiv, Ukraine c State Key Laboratory of Molecular and Cellular Physiology, Kyiv, Ukraine b
article info
abstract
Article history:
Background: Bipolar electrosurgical tissue welding uses forceps-like electrodes for grasping
Received 1 March 2013
the tissues and delivering high-frequency electric current (HFEC) to produce local heat,
Received in revised form
desiccation, and protein denaturation, resulting in the fusion of the contacting tissues.
22 July 2013
Although in this technique no electric current is flowing through the whole body to cause
Accepted 12 August 2013
electric injury, depending on the frequency of applied energy, it may produce local exci-
Available online 3 September 2013
tation of intramural nerves, which can propagate beyond the surgical site potentially causing harmful effects.
Keywords:
Materials and methods: The effects of varying frequency of HFEC on tissue excitability in
Electrosurgery
bipolar electrosurgical modality were studied in vitro using electric field stimulation (EFS)
Electrosurgical tissue welding
method on multicellular smooth muscle strips of rat vas deferens. Contractile response to
Electric field stimulation
5-s-long sine wave EFS train was taken as the measure of excitation of intramural nerves.
Vas deferens
Results: EFS-induced contraction consisted of phasic and tonic components. The amplitude
Excitation
of both components decreased with increasing frequency, with tonic component dis-
Contraction
appearing at about 10 kHz and phasic component at about 50 kHz. Because components of
Frequency
EFS-induced contraction depend on different neurotransmitters, this indicates that various neurotransmitter systems are characterized by distinct frequency dependence, but above 50 kHz they all become inactivated. Bipolar electrosurgical sealing of porcine gut showed no difference in the structure of seal area at HFEC of 67 and 533 kHz. Conclusions: EFS frequency of 50 kHz represents the upper limit for excitation. HFEC above 50 kHz is safe to use for bipolar electrosurgical tissue welding without concerns of excitation propagating beyond the surgical site. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Electrosurgery uses high-frequency electric current (HFEC) passed through the tissue to create the desired clinical effect
via locally induced diathermy [1]. Among its common applications, such as cutting, coagulation, desiccation, and fulguration, electrosurgical tissue welding is increasingly viewed as a viable alternative to the traditional mechanical means of
* Corresponding author. Bogomoletz Institute of Physiology NASU, Bogomoletz Street 4, Kyiv 01024, Ukraine. Tel.: þ380 44 2562048; fax: þ380 44 2562435. E-mail address: [email protected] (Y.M. Shuba). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.08.012
120
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 6 ( 2 0 1 4 ) 1 1 9 e1 2 5
reconnecting the tissues, which are based on using the suture, metal staples, or clips [2]. In its bipolar mode, this technique uses HFEC to produce diathermy between tightly pressed tissues against each other [1e3]. The HFEC is usually delivered using bipolar forceps-like electrodes for grasping the tissues and applying pressure to them [4,5]. The two tines of the forceps perform the active and return electrode functions, and only the tissue grasped is included in the electric circuit, making separate patient “return” electrode characteristic of monopolar electrosurgery mode unnecessary. Localized HFEC passing through the grasped tissues induces thermal tissue damage, which depends on the size and shape of the bipolar electrodes, exerted pressure, and the parameters (power, frequency, waveform, and duration) of the high-frequency (HF) electric energy. If all parameters are optimal, the heat generation, desiccation, and protein denaturation results in fusion or “welding” of the contacting tissues confined to the area of the electrodes, which can withstand distraction force comparable to the force achieved for traditional sutured joints [6]. Power and duration of the applied electric energy have to be such that to prevent overheating and tissue burning to minimize necrotic damage and at the same time providing the strongest fusion possible. As these parameters change, so will the corresponding tissue effects [7]. Animal studies and clinical surgical experiences indicate that optimal frequency of the applied electric energy must range between 60 and 75 kHz [6]. However, despite bipolar welding technique does not require separate patient return electrode, which prevents net current flow through the patient’s body thereby negating many of the safety precautions related to the clinical use of electric current, the International Electrotechnical Commission (IEC) and the Association for the Advancement of Medical Instrumentation only approve the use of the devices with the frequencies above 300 kHz [8]. Technically, the bipolar electrosurgical mode is similar to the electric field stimulation (EFS) method widely used in science to stimulate contractions of multicellular muscle preparation in vitro via excitation of intramural nerve fibers [9]. Thus, confined application of HF electric energy with the purpose of welding the tissues can also produce local excitation of intramural nerves, which can propagate to the central nervous system structure potentially causing undesirable effects. Because of the phenomenon of refractoriness, a short-term decrease in the excitability of nerve and muscle tissue occurs immediately after the manifestation of action potential [10]. The possibility of excitation decreases with increasing frequency of EFS. However, to our knowledge, systematic study of this phenomenon in the context of bipolar electrosurgical techniques, with the purpose of establishing the lowest frequency limit at which no potentially harmful excitation can occur, was not performed. Here, we have used rat vas deferens smooth muscle preparations with and without epithelial cell layers to establish frequency dependence of the contractions in response to the sine wave EFS in vitro. Our results show that at frequencies above 50 kHz no contraction can be induced, indicating that this frequency represents the lowest safe limit, which can be used for bipolar HFECemediated electrosurgical procedures.
2.
Materials and methods
2.1. Vas deferens preparation and recording of contraction Experiments were conducted on smooth muscle strips from the vas deferens of male Wistar rats weighing 200e250 g. Animals were killed by decapitation, their vas deferens was removed, and placed in the warm (37 C), oxygenated (95% O2 and 5% CO2) Krebs solution (in mM): 120.4 NaCl, 5.9 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.8 CaCl2, 15.5 NaHCO3, and 11.5 glucose (pH 7.4). Vas deferens was cleaned from connective tissue, its wall was cut along the axis, and longitudinal strips of 0.2e0.3 cm in diameter and 0.7e1.0 cm in length were excised from the parts adjacent to the prostate (prostatic portion). Smooth muscle strips with both epithelial layers removed and retained were used in the experiments. Schematic diagram of the experimental arrangement is presented in Figure 1. For the recording of contractions, the strip was placed in the acrylic glass chamber continuously superfused with Krebs solution at 37 C with one end of the strip fixed still and another one attached to the capacitative force sensor with the baseline load of 3 mN applied to the strip. EFS was delivered via two Ag/AgCl wires positioned at one end of the strip from its top and bottom with direct contact with the tissue. The EFS consisted of sine wave trains of various frequencies (0.02e200 kHz), amplitudes (10e60 V), and durations (2-10 s) applied every 3 min, which was sufficient for complete restoration of basal tone. Contractile
Fig. 1 e Schematic diagram of experimental arrangement. Acrylic glass experimental chamber (1) is continuously superfused via inlet and outlet tubes with Krebs solution (2). One end of the smooth muscle strip (3) is fixed still to the chamber wall via retainer (4) and another end via retainer (5) is attached to the lever (6) of the capacitative force sensor (7). EFS is delivered via Ag/AgCl electrodes (8) connected to the generator of HFEC (9). The drawing is not to scale.
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responses were recorded on chart recorder (model H3030-4, Russia) and computer via analog-digital converter (DigiData 1200, Axon Instruments Inc, Union City, CA).
2.2.
washed in water. They were stained with hematoxylin and eosin, sandwiched between microscope glass and coverslip, and used for microscopic examination.
Electrosurgical tissue welding and histology
Frequency dependence of electrosurgical tissue welding was assessed in gut sealing experiments on an isolated portion of porcine gut using the custom designed and manufactured electrosurgical apparatus with variable output frequency. The gut was placed transversely between the two electrodes, and the force was applied to electrodes sufficient to bring opposite gut’s walls into the tight contact. HFEC with root mean square (RMS) value of 2 A was passed through electrodes and the gut tissue resulted in tissue heating and desiccation. HFEC was turned off shortly after desiccation, before any signs of tissue burning were evident. After HFEC termination, the tissue was let to cool down, electrodes were removed, and the tissue was used for the preparation of histologic slides. Briefly, gut tissue was fixed in 10% formalin for 48 h, dehydrated by processing through a series of ethanol baths of increasing concentration (50e96%), infiltrated with paraffinechloroform mixture, and embedded in blocks of paraffin and bee wax. Embedded gut specimens were cooled on ice and sectioned in longitudinal direction onto 10-mmthick slices on a microtome. Slices were deparaffinised in xilol, passed through decreasing alcohol concentrations, and
3.
Results
3.1. Frequency dependence of the contractions at weak stimulation In the first series of experiments, we have applied sine wave EFS of effective amplitude 15 V, which according to our pilot studies was insufficient to produce tissue damage and was optimal for ensuring long-lasting viability of the strip (up to 3 h) and reproducibility of contractile responses. In this series, the frequency of the sine wave within EFS train varied from 20 Hz to 100 kHz. With the 5-s-long EFS, independently of the frequency within EFS, typical contractile response consisted of three components: the first (I) component of transient (phasic) contraction lasting for about 2 s, the second (II) component of tonic contraction lasting till the end of EFS, and the third (III) component of relaxation to the baseline after EFS termination (Fig. 2A). In visceral smooth muscle, the phasic and tonic components of EFS-evoked contractions are usually attributed to the release from intramural efferent nerve fibers excited by EFS of the main efferent transmitters, adenosine-triphosphate
A I
B
II
III
Normalized amplitude, %
140 120 100 Overall
80 60 40
Sustained
20 0 10
100
1000
10000
100000
Frequency, Hz Fig. 2 e Frequency dependence of EFS-induced contractions of rat vas deferens. (A) Representative original recordings of the contractions induced by 5-s-long sine wave EFS of 15 V and variable frequencies (time of EFS application is shown by horizontal bars above recordings); transient (I), tonic (II), and relaxation (III) phases of the contractile response, its overall amplitude (filled symbol), and amplitude of tonic component (open symbol) are indicated on 2 kHz recording. (B) Frequency dependence of overall amplitude (filled symbols) and amplitude of tonic component (open symbols) of EFS-induced contraction (mean ± standard error of the mean, n [ 8); all amplitudes for each preparation were normalized to the overall amplitude at 500 Hz and then averaged. (Color version of figure is available online.)
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(ATP), acetylcholine, and noradrenalin (NA), which are characterized by different kinetics of contractile action on smooth muscle cells (SMCs) [11e14]. The transient component typically consisted 70%e80% and the tonic component 20%e30% of the overall amplitude of contractile response. To be able to compare frequency dependence of the amplitudes of the contractions of various preparations, all amplitudes for a given preparation were normalized to the overall amplitude of the contraction at 500 Hz (Fig. 2B). Increasing the frequency of EFS from 20e100 Hz resulted in about 10% enhancement of the overall amplitude of the contractions (Fig. 2). Furthermore increase of the frequency produced almost no change in overall amplitude up to 500 Hz with the progressive decrease observed at higher frequencies. The reduction in the overall amplitude of EFS-evoked contractions at frequencies above 500 Hz progressed at a rate of about 60% per decade and eventually brought the amplitude to zero at 50 kHz (Fig. 2B). The amplitude of the tonic component of the contractions showed more monotonic but notably accelerated decline, gradually decreasing to zero in the range of frequencies from 20e10 kHz with the rate of about 10% per decade (Fig. 2B). Higher “resistance” of the phasic component of EFS-evoked contractions to the increased frequencies compared with the tonic component may likely reflect the differences in the release and contractile action of the “fast” transmitter ATP compared with the “slower” neurotransmitters, such as acetylcholine and NA [13]. Overall, these results indicate that at the frequencies of EFS above 50 kHz intramural nerves do not excite most likely
because of the property of refractoriness, and as a result do not release transmitters that can cause muscle contraction. Consequently, EFS at the frequencies above 50 kHz cannot evoke harmful excitation that can propagate in the ascending direction from the site of its application.
3.2.
Contractions at HF and strong stimulation
Tissue fusion or welding during localized passing of HFEC requires induction of tissue heating accompanied by desiccation, cell damage and protein denaturation. This can be achieved only by applying sufficiently high power of electric energy, which in turn can theoretically induce harmful excitation propagating from the site of application even at HFs that are unable to do so at low power. To check for such possibility, we have conducted experiments with application of 50-kHz sine wave EFS of increasing effective amplitude from 15e60 V. Figure 3 presents the original recordings of a typical experiment of this kind. As it can be seen, weak 15-V EFS of 50 kHz produced only miniscule transient contraction compared with 500 Hz with no tonic component present (Fig. 3). Increasing EFS to 25 V with 5-V increment first produced gradually higher transient contraction, which decreased again at EFS above 30 V (Fig. 3B, upper row). However, furthermore increase of EFS to 40 V and above produced drastically different contractile responses. These responses were characterized by much slower onset amplitude that kept creeping up during whole duration of EFS, and strongly impaired relaxation leading to the development of ever accumulating poststimulatory tonic contraction (Fig. 3).
Fig. 3 e Dependence of EFS-induced contractions of rat vas deferens on EFS power. (A) Representative original recording of the reference contraction induced by 5-s-long sine wave EFS of 15 V at 500 Hz (shown by horizontal bars). (B) Representative original recording of EFS-induced (5-s-long sine wave) contractions from the same preparations at 50 kHz and increasing power from 15e60 V showing initial gradual increase in the amplitude of transient contractile response and its irreversible transformation into constant nonrelaxing tone at EFS power of 40 V.
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Induction of such response was irreversible in terms of returning the muscle preparation to normal contractility even after prolonged rest. Such behavior is most likely explained by the ability of HF EFS of up to 35 V still to produce small graded contractile responses via excitation of intramural nerves, however, with delivering even more power intramural nerves may likely get damaged, and EFS induces tonic contraction by acting directly on SMCs, which eventually results in SMC damage as well. Thus, although increasing the power of HF EFS may enhance excitation of intramural nerves, damage of the nerves at powers that are required for tissue fusion prevent negative impact on the ascending pathways.
3.3. Frequency dependence of electrosurgical tissue fusion To test whether the frequency of HFEC influences the quality of tissue fusion, we have performed experiments on electrosurgical sealing of porcine gut at two frequencies, the low frequency of 67 kHz and the HF of 533 kHz. These experiments were conducted on the adjacent portions of the gut, using the same electrosurgical apparatus, with equal mechanical force applied to the tissue-grasping bipolar electrodes, identical RMS value of HFEC passing through the tissues, and the same time of exposure to HFEC. Tissue fusion occurred in the area of contact of two mucous layers of the gut walls. Figure 4 shows representative longitudinal histologic sections of the seal area obtained under such conditions at HFEC of 67 kHz and 533 kHz. Inspection of these sections reveals no essential difference in the structure of the seal area between the two frequencies, suggesting that at both frequencies HFEC produces desirable tissue fusion effects.
4.
Discussion
One of the useful clinical applications of HFEC is related to blood coagulation, electrosurgical tissue cutting, and welding
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[7]. Localized delivery of HFEC for such purposes is usually achieved with “monopolar” electrosurgical modality in which one “active” electrode with the small surface of the tip is used to apply HFEC directly to the surgical site, while the second much bigger return electrode is placed at the remote body area, most commonly on the patient’s back or limb. Because of the small contact area of the active electrode with the tissue, even miniscule net electric current flowing from the active electrode through the patient’s body to the return electrode may have local density at the tip of the active electrode high enough to produce the thermal effect sufficient for blood coagulation or even tissue cutting and fusion. However, along with the possibility of achieving desired local effects, the flow of the electric current through the whole body in monopolar configuration inevitably raises the basic safety issues. Because the harm to the organism induced by electric current flowing through the body is known to increase with decreased frequency, whereas at very HFs “skin effect,” a phenomenon that tends to displace alternating current to the surface of conducting media, plays protective role, IEC standards limit the lower frequency of electrosurgical equipment by 300 kHz [8]. At the same time, according to some studies and empirical clinical experience [6], application of HFEC with the frequency around or slightly higher than 50 kHz produces stronger and more stable connections of intestinal, stomach, skeletal muscle, and skin tissues compared with the frequencies of several hundreds of kilohertz, prompting to search for the solutions satisfying both safety requirements and surgical practicality. In the event of “bipolar” electrosurgical modality, HFEC almost completely passes through the tissue grasped between two electrodes and “classic” remote return electrode is absent, making safety concerns related to the flow of electric current through the whole body largely insignificant. However, the possibility remains that the locally applied HFEC will cause undesirable excitation of intramural nerves producing harm to the ascending centers. Here, we show that such excitation may occur only at HFEC frequencies below 50 kHz and completely refute such concerns for higher frequencies. In our
Fig. 4 e Frequency of HFEC does not influence the quality of electrosurgical tissue fusion. Representative longitudinal histologic sections of the electrosurgical seal area (marked by braces) of porcine gut obtained at 67 kHz (left) and 533 kHz (right) HFEC showing no principal difference in the seal structure for the two frequencies. The inset shows schematic drawing (not to scale) of the electrosurgical procedure: (1) gut portion, (2) bipolar electrodes, (3) generator of HFEC, and (4) switch; dashed line box denotes the area shown in histologic slices. (Color version of figure is available online.)
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studies, we mimicked bipolar electrosurgical modality by using EFS on multicellular preparations (strips) of rat vas deferens, and recorded EFS-evoked contractions as the measure of functional response. Rat vas deferens can be viewed as the most representative model tissues, as it consists of SMCs that are directly activated by neurotransmitter released from nerve varicosities and SMCs which activation is dependent on gap junctionemediated cell-to-cell communication [14]. Moreover, specifically in rat and mouse vas deference passive propagation of excitation is basically absent [15], indicating predominance of direct activation of SMCs by neurotransmitter and giving this tissue resemblance to both individually innervated skeletal muscle and classical smooth muscle syncytium. Our results demonstrate that contractile response of the prostatic portion of the vas deferens completely disappears at EFS frequency above 50 kHz, whereas tonic component of this response diminishes at even lower frequencies, already above 10 kHz. Pharmacologic characterization of the biphasic contraction of rat vas deferens induced by EFS of intramural sympathetic nerve fibers supplying vas deferens established that the first, transient component has purinergic nature, whereas the second, sustained one is adrenergic [12,14,16]. The adrenergic component predominates in the epididymal segment of the duct and purinergic in the prostatic segment [12]. The fact that transient and sustained components of EFSevoked contractions revealed divergent sensitivity to the frequency of EFS indicates that synaptic releases of ATP and NA are characterized by different coupling efficiency with fiber excitation. As frequency-dependent decrease of contractions is explained by refractoriness of nerve excitability, this suggests that the purinergic system is more resistant to the deterioration of action potential parameters with increasing EFS frequency. In addition, the disparity in SMC activation via direct innervation and gap junctionemediated mechanisms may contribute to the observed frequency dependence of various components of neurogenic contractile response [17]. Thus, although various types of nerve fibers and neurotransmitter systems may exhibit differential frequency sensitivity of excitation during local application of HFEC by means of bipolar electrosurgical technique, 50 kHz seems to represent the upper limit for the frequency when excitation may propagate beyond the surgical site. Increasing the power of EFS to the level that is required for electrosurgical tissue fusion induces damage to intramural nerves and irreversible tonic contraction in smooth muscle strip via direct stimulation of SMCs with subsequent damage to SMCs as well. Thus, induction of nerves excitation and its propagation beyond the surgical site at such powers is prevented by local tissue damage. It should be noted that bipolar electrosurgical devices generating HFEC of 50e100 kHz are already successfully used in clinics in Ukraine since the second half of 1990s with tens of thousands of operations on humans already performed. The results of these practical experiences for various types of tissues are reported in Refs. [2,6]. Nevertheless, at the beginning of the adoption of bipolar electrosurgical tissue welding
in clinics in early 1990s, because of safety concerns, only the frequencies of 0.8e1.1 MHz were used in Ukraine as well. However, with time the frequency was gradually brought down to 66 6 kHz, which was found empirically to be optimal for providing quality tissue fusion with no evident side effects. In addition, from engineering standpoint electrosurgical devices with such frequencies are simpler to design and manufacture. Unfortunately, the results of clinical trials and countless animal experiments carried out in Ukraine were not published in international scientific literature and so far are best described in Refs. [2,6]. Our own experiments on electrosurgical sealing of porcine gut revealed no essential differences in the structure of the seal area produced at HFEC of 67 kHz and 533 kHz and otherwise identical conditions. This is not surprising given that tissue heating is determined by the RMS value of HFEC, which was kept constant in our trials, and not by its frequency or the waveform. Besides, at the used frequencies, the so-called skin effect, which tends to displace HFEC density toward the surface of a conductor with increasing frequencies, is not prominent enough to cause nonuniformity of tissue heating in bipolar electrosurgical configuration. However, we have found that the losses in electrosurgical apparatus circuitry, their heating, and consequently the power required to produce output HFEC with the same RMS value are much reduced at lower frequency compared with higher frequency: at RMS of 2 A the output voltage consisted 8 V at 67 kHz increasing to 18.5 V (i.e., 2.3-fold) at 533 kHz. The purpose of our study was to demonstrate that HFEC at frequencies just above 50 kHz is not harmful in bipolar electrosurgical configuration, because local excitation cannot propagate beyond the surgical site. At the same time, these frequencies are well suited for producing quality electrosurgical HFECemediated tissue fusion. Our study makes recommendations of the IEC and Advancement of Medical Instrumentation, regarding the HF surgical equipment, at least in the context of bipolar electrosurgery, obsolete and calls for the revision.
Acknowledgment This study was supported by the National Academy of Sciences of Ukraine and F46.2/001 grant from State Fund for Fundamental Research, Ukraine (YMS). The authors declare no conflicts of interest.
references
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