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Cardiac nociceptors innervated by vagal afferents in rats
Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 174 – 178 www.elsevier.com/locate/autneu
Cardiac nociceptors innervated by vagal afferents in rats Yoichi Hisata a,b, Jorge L. Zeredo b, Kiyoyuki Eishi a, Kazuo Toda b,* b
a Cardiovascular Surgery, Graduate School of, Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan Division of Integrative Sensory Physiology, Graduate School of Biomedical, Sciences, Nagasaki University, Sakamoto 1-7-1, Nagasaki 852-8588, Japan
Received 1 November 2005; received in revised form 17 February 2006; accepted 20 February 2006
Abstract It is reported that cardiac afferent information is transmitted through at least three different pathways to the CNS: sympathetic, parasympathetic, and somatic; however, there are few studies concerning the role of afferent fibers of vagus nerves for eliciting cardiac sensation including pain. Especially, receptive field properties innervated by single vagal nerve fiber and mechanical threshold of nociceptors on the cardiac surface are not yet quantitatively studied. Therefore, in this study, we systematically investigated characteristics of vagal units innervating cardiac nociceptors in rats. Using anesthetized and artificially ventilated rats, 37 single unit recordings were made from fine nerve filaments of the left vagal nerve. For quantitative mechanical stimulation, the cardiac surface was stimulated by a von Frey type device. In addition, bradykinin was used for checking the chemical sensitivity of the nociceptor. Electrical stimulation was used to estimate the conduction velocity of the recorded nerve fiber. All units recorded from the vagal nerve were either Ay- or C-polymodal nociceptors. About 70% of the afferents had conduction velocities in the C-fiber range. In 60% of the units, the peripheral receptive field covered spot-like areas, but we also found larger and continuous receptive fields. Our results show that a majority of nociceptors innervated by vagal afferents are the C-polymodal type with spot-like receptive fields. We consider it to relate to the ambiguous and dull pain of angina pectoris. D 2006 Elsevier B.V. All rights reserved. Keywords: Cardiac pain; Vagal nerve; Receptive field; Polymodal nociceptor; Angina pectoris; Rat
1. Introduction The cardiac pain known as angina pectoris is a common symptom of ischemic heart disease, and has been described as a retrosternal, characteristically crushing, burning, or squeezing (Foreman, 1999). Pain may radiate to the throat, neck, or ulnar aspect of the left arm, sometimes reaching to the little finger. Less often, it radiates to the neck and lower jaw or either the right or both arms. It is also characteristic of angina pectoris to be variable in intensity and location from person to person and from time to time (Foreman, 1999; Mody and Faxon, 2000). Surgical interventions aiming at the sympathetic afferent pathway are reported to produce complete relief of angina pectoris in about half of the cases (Meller and Gebhart, 1992).
* Corresponding author. Tel.: +81 95 849 7636; fax: +81 95 849 7639. E-mail address: [email protected] (K. Toda). 1566-0702/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.02.008
However, symptoms remain in 40 –50% of the patients, including 10– 20% who report no pain relief at all, thus suggesting participation of vagal afferents for eliciting cardiac pain (Meller and Gebhart, 1992). In fact, a recent study in the rat by Qin et al. (2001) estimated 55% of the pericardial chemonociceptors to be innervated by vagal afferents. Despite numerous experimental and clinical investigations, the exact mechanisms involved in the development of cardiac pain, and particularly the involvement of vagal afferents, are not completely understood (Barber et al., 1984; Bassenge and Munzel, 1988). Veelken et al. (2003), and Ditting et al. (2004) reported a role for vagal C fibers in neurogenic cardiovascular regulation, however, characteristics of receptive field innervated by single vagal nerve fiber and mechanical threshold of nociceptors on the cardiac surface are still unknown. Therefore, this study was done to determine more clearly the characteristics of cardiac nociceptors innervated by vagal afferents in rats.
Y. Hisata et al. / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 174 – 178
2. Material and methods 2.1. Animal preparation Twenty-five Wistar albino rats (weighing 260– 650 g) were anesthetized with thiamylal sodium (initial dose of 80 mg/kg, i.p.; Isozol\, Yoshitomi Pharmaceutical, Tokyo, Japan). Artificial ventilation was established through a tracheostomy (3.0 ml/stroke, 60 strokes/min) using artificial ventilator for small animals (SN-480-7, Shinano, Tokyo, Japan). The thorax was opened by a full sternotomy, and the beating heart was exposed. Then, the left vagal nerve was identified next to left common carotid artery, and isolated from the surrounding tissues. The thoracic cavity pool made by surrounding skin was filled with pre-warmed (37 -C) physiological saline to prevent tissue dehydration. The proximal end of the vagal nerve was cut and ligated with a cotton thread to interrupt efferent nerve information. The fine filament was isolated from the vagal nerve bundle and was covered with liquid paraffin. The methods described here follow the ethical guidelines proposed by the International Association for the Study of Pain and received approval by the Animal Welfare Committee of Nagasaki University. 2.2. Study design We selected the right ventricle, which is innervated by a branch of the vagal nerve (Benson et al., 1999), as the site of stimulation. For quantitative mechanical stimulation, a von Frey type device with metal needle tip (diameter: 0.2mm) was used and stimulus intensities were monitored by a strain gauge amplifier (TBM-4, World Instruments Inc, FL, USA). This signal was displayed on the DOS-V computer through a CED 1401 interface (Cambridge Electronic Design Limited, Cambridge, UK). We found that stimuli over 20 mN applied to buccal skin surface always evoked pain sensation. Therefore, cardiac units having von Frey mechanical threshold over 20 mN were considered to be nociceptive. Bradykinin (BK, 10 4 M in Krebs– Henseleit solution, Sigma, USA), was applied topically with a volume of 1.0 ml using a syringe attached to a metal canula (tip diameter: 2.0 mm) closely attached to the mechanical receptive field. With the use of a bipolar concentric electrode (interpolar distance: 1.0 mm), electrical stimulation was applied to the receptive field on the cardiac surface. Conduction velocity was estimated using the distance and conduction delay between the stimulating and recording electrode. In the rat, the estimation of conduction velocity adopted classification reported by Koltzenburg et al. (1992). A cutoff of 2.0m/s was used to distinguish between myelinated (A fiber) and unmyelinated fiber (C fiber). Units conducting faster than 10.0 m/s were considered to be large myelinated (Ah) fibers, whereas units with conduction velocity below this value
175
were considered thin myelinated (Ay) fibers. In the present study, we could not find Ah innervated units as vagal nociceptors. Functional single unit responses from fine filaments of the vagal nerve were recorded with a tungsten hook electrode. The spike signal was fed into a high-impedance, low-noise amplifier (DAM-80E, World Precision Instruments, Sarasota, USA) and displayed on a DOS-V computer through the CED 1401. When two or three spikes were recorded simultaneously, units were separated using the Spike 2 software (Version 2.01) for Windows. 2.3. Data analysis The paired t-test was used to determine the statistical significance of the data from before and after chemical stimulation. Statistical significance was set at the 5% level ( P < 0.05). The software Statview version 5.0 (SAS Institute, Cary, NC) aided in statistical analysis. All values are displayed as mean T S.E.M.
3. Results We recorded activities of 37 single units from cardiac nociceptors on the right ventricle, all of which were of the polymodal type, being 11 Ay- and 26 C-fiber innervated. The conduction velocities of innervated Ay and C units were 3.8 T 0.4 and 1.4 T 0.1 m/s, respectively. 3.1. Threshold to mechanical stimulation Fig. 1 shows a typical example of Ay fiber-innervated single unit responses of a cardiac nociceptor after mechanical stimulation. Mechanical threshold was determined during the ramp phase of the von Frey stimulation, at the point where the first spike was fired (Fig. 1A, oblique arrow). The threshold level is also shown by horizontal continuous dotted line. This unit had von Frey threshold of 23.1 mN. The throbbing on the mechanical stimulation trace (upper line) was caused by the cardiac contractions against
Fig. 1. Typical response of a single Ay fiber innervated cardiac nociceptor. (A) von Frey pressure; an oblique arrow indicates threshold for this nociceptor (threshold: 23.1mN). Dotted line indicates the threshold level of this unit. (B) Phasic spike response of the cardiac nociceptors.
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Table 1 Characteristics of cardiac nociceptors innervated by vagal nerve
Ay C a
n
Conduction velocity (m/s)
Threshold (mN)
Spontaneous discharge (Hz)
Receptive fields (C/S)a
11 26
3.8 T 0.4 1.4 T 0.1
38.0 T 3.1 43.6 T 3.3
0.37 T 0.1 0.46 T 0.1
5/6 11/15
C: continuous type, S: separate type.
the von Frey stimulation. The spike activity was typically phasic during mechanical stimulation, and not synchronized with the beating of the heart, showing that these responses were evoked by mechanical stimulation, not by cardiac contraction. In the present study, as shown in Table 1, the threshold value of Ay units were 38.0 T 3.1 mN (n = 11), that of C units were 43.6 T 3.3 mN (n = 26). 3.2. Receptive field properties Receptive field properties were not quantitatively shown in the previous studies concerning cardiac nociceptors. We investigated receptive field properties of cardiac nociceptors innervated by the vagal nerve by using threshold intensity of von Frey stimulation. Typically, we could distinguish two types of receptive fields: continuous and spot-like separate as shown in Fig. 2. 45% of the Ay-innervated and 42% of the C-innervated nociceptive units responded to stimuli over one wide, continuous receptive field; however, about 60% of the units showed non-responding spaces and a separate receptive field (Table 1).
Fig. 3. Discharge frequencies before and after bradykinin application. Data are means T S.E.M. Asterisks indicate P < 0.001 in the paired t-test.
increased the on-going discharge frequency of both Ay- and C-fiber innervated nociceptive units (Fig. 3). As shown in Fig. 4, mechanical threshold was significantly lowered by BK application in both Ay- and C-fiber innervated units. The average decrease was about 25% compared with the pre-BK value (38.0 T 3.1 mN to 28.0 T 3.3 mN in Ay units, and 43.6 T 3.3 mN to 32.8 T 3.1mN in C units). Table 1 summarizes the characteristics of nociceptors on the cardiac surface, indicating the following results: (1) Vagal nociceptors were innervated by Ay with slower conduction velocity or C fibers. (2) About 70% of the afferents were C fibers. (3) The receptive fields of Ay- and C-innervated units covered more often separate areas. (4) There was no significant difference in mechanical threshold between Ay and C nociceptors.
3.3. Chemical stimulation 4. Discussion Topical application of BK evoked tonic spike responses with an about 30-s delayed onset. The evoked responses gradually increased within 180 s and disappeared after washing out with physiological saline. BK significantly
The present study first revealed the receptive-field characteristics of cardiac nociceptors innervated by vagal afferents. This seemed to be important to recognize basic
Fig. 2. Types of receptive fields. (A) In the continuous type, units responded to stimulus from a wider continuous area. (B) In the separate type, units responded to stimulus from a spotted area. (a) Superior vena cava, (b) right atrium, (c) inferior vena cava, (d) aorta, (e) pulmonary artery, (f) right ventricle, (g) left ventricle, (h) left atrium, (i) pulmonary vein.
Y. Hisata et al. / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 174 – 178
Fig. 4. Mechanical thresholds before and after bradykinin application. Data are means T S.E.M. Asterisks indicate P < 0.001 in the paired t-test.
mechanisms of evoking cardiac pain. Larger receptive field of single cardiac unit is associated with an ambiguous and dull pain sensation. Two hypotheses have been formulated to explain ischemic cardiac pain: one hypothesis proposes that pain sensation would be brought up by a distention of the ventricular wall (‘‘mechanical hypothesis’’) (Foreman, 1999), and the other, by the intramyocardial release of pain-producing substances induced by ischemia (‘‘chemical hypothesis’’) (Benson et al., 1999; Crea and Gaspardone, 1997). However, it is not established which mechanism is important for producing ischemic cardiac pain. As previously reported, nociceptors in the skin, deep tissue or viscera are subdivided into several subtypes, the frequency distribution of which is different between body sites and animal species (Kumazawa and Mizumura, 1977, 1980; Perl, 1980). The relative distribution of each subtype is supposed to determine characteristics of pain sensation (Perl, 1980; Zimmermann, 1981). Some reports showed that the ventricular endings of afferent sympathetic fibers act as polymodal type nociceptors (Lombardi et al., 1981; Pan and Chen, 2002). In our investigation of the vagal nerve, likewise, all nociceptors were of the polymodal type in accordance with previous studies (Ditting et al., 2004; Veelken et al., 2003). This finding, therefore, favors the ‘‘chemical hypothesis’’ of ischemic cardiac pain partly evoked by inflammatory mediators. This point of view is also supported by several reports that correlate cardiac pain from angina pectoris with ischemic chemical mediators BK, PGE2, adenosine, histamine, 5-HT, or K+ (Baker et al., 1980; Bassenge and Munzel, 1988; Lombardi et al., 1981; Meller et al., 1990; Minisi and Thames, 1993; Nerdrum et al., 1986). In addition, the mechanical threshold of cardiac vagal nociceptors was lower than that reported for other body sites in the rat: for example, joint (Takeuchi et al., 2001), oral mucosa (Toda et al., 1997). This suggests that vagalinnervated nociceptors may be very sensitive to changes in mechanical stimuli such as, shape or volume of the cardiac wall. In this study, the mean conduction velocity of Ay-fiber innervated polymodal nociceptors was slower than that
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reported for other tissues and organs. The conduction velocities of Ay afferents from polymodal nociceptors in testis, oral mucosa were 11.2 T 0.3 and 9.1 T 2.0, respectively (Kumazawa et al., 1987; Toda et al., 1997), while our results showed 3.8 T 0.4 m/s (see Table 1). In a clinical situation, this finding may reflect the dullness of cardiac pain when compared to pain sensations from other deep tissues or internal organs (Russek, 1970). We observed that chemical stimulation with BK lowered the mechanical threshold and increased the discharge frequency of both Ay- and C-innervated nociceptors. This is in agreement with previous reports, which indicate BK to be a mediator of angina pectoris (Baker et al., 1980; Lombardi et al., 1981; Minisi and Thames, 1993). On the other hand, Koltzenburg et al. (1992) reported that BK did not contribute appreciably to the sensitization of sympathetic nociceptors in the skin. Further studies shall determine whether this discrepancy would contribute to the functional distinction between parasympathetic and sympathetic neurons, or between deep and superficial tissues. In summary, our results indicate that cardiac nociceptors innervated by the vagal nerve would be associated with an ambiguous and dull pain sensation, because the receptive field of single units was somewhat larger. This finding partly reflects the characteristics of cardiac pain, especially that related to angina pectoris and myocardial infarction.
References Baker, D.G., Coleridge, H.M., Coleridge, J.C., Nerdrum, T., 1980. Search for a cardiac nociceptor: stimulation by bradykinin of sympathetic afferent nerve endings in the heart of the cat. J. Physiol. (Lond.) 306, 519 – 536. Barber, M.J., Mueller, T.M., Davies, B.G., Zipes, D.P., 1984. Phenol topically applied to canine left ventricular epicardium interrupts sympathetic but not vagal afferents. Circ. Res. 55, 532 – 544. Bassenge, E., Munzel, T., 1988. Cardiac mechanisms for the development of angina pectoris pain. Z. Kardiol. 77 (Suppl. 5), 5 – 14. Benson, C.J., Eckert, S.P., McCleskey, E.W., 1999. Acid-evoked currents in cardiac sensory neurons: a possible mediator of myocardial ischemic sensation. Circ. Res. 84, 921 – 928. Crea, F., Gaspardone, A., 1997. New look to an old symptom: angina pectoris. Circulation 96, 3766 – 3773. Ditting, T., Hilgers, K.F., Scrogin, K.E., Stetter, A., Linz, P., Veelken, R., 2004. Mecanosensitive cardiac C-fiber response to change in ventricular filling, coronary perfusion pressure, hemorrhage, and volume expansion in rats. Am. J. Physiol., Heart Circ. Physiol. 288, H541 – H552. Foreman, R.D., 1999. Mechanisms of cardiac pain. Annu. Rev. Physiol. 61, 143 – 167. Koltzenburg, M., Kress, M., Reeh, P.W., 1992. The nociceptor sensitization by bradykinin does not depend on sympathetic neurons. Neuroscience 46, 465 – 473. Kumazawa, T., Mizumura, K., 1977. Thin-fibre receptors responding to mechanical, chemical, and thermal stimulation in the skeletal muscle of the dog. J. Physiol. (Lond.) 273, 179 – 194. Kumazawa, T., Mizumura, K., 1980. Chemical responses of polymodal receptors of the scrotal contents in dogs. J. Physiol. 299, 219 – 231.
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Kumazawa, T., Mizumura, K., Sato, J., 1987. Response properties of polymodal receptors studied using in vitro testis superior spermatic nerve preparations of dogs. J. Neurophysiol. 57, 702 – 711. Lombardi, F., Della Bella, P., Casati, R., Malliani, A., 1981. Effects of intracoronary administration of bradykinin on the impulse activity of afferent sympathetic unmyelinated fibers with left ventricular endings in the cat. Circ. Res. 48, 69 – 75. Meller, S.T., Gebhart, G.F., 1992. A critical review of the afferent pathways and the potential chemical mediators involved in cardiac pain. Neuroscience 48, 501 – 524. Meller, S.T., Lewis, S.J., Ness, T.J., Brody, M.J., Gebhart, G.F., 1990. Vagal afferent-mediated inhibition of a nociceptive reflex by intravenous serotonin in the rat: I. Characterization. Brain Res. 524, 90 – 100. Minisi, A.J., Thames, M.D., 1993. Distribution of left ventricular sympathetic afferents demonstrated by reflex responses to transmural myocardial ischemia and to intracoronary and epicardial bradykinin. Circulation 87, 240 – 246. Mody, V.H., Faxon, D.P., 2000. Management of unstable angina: integrating the new approaches. Rev. Cardiovasc. Med. 1, 104 – 119. Nerdrum, T., Baker, D.G., Coleridge, H.M., Coleridge, J.C., 1986. Interaction of bradykinin and prostaglandin E1 on cardiac pressor reflex and sympathetic afferents. Am. J. Physiol. 250, R815 – R822.
Pan, H.L., Chen, S.R., 2002. Myocardial ischemia recruits mechanically insensitive cardiac sympathetic afferents in cats. J. Neurophysiol. 87, 660 – 668. Perl, E.R., 1980. Afferent basis of nociception and pain: evidence from the characteristics of sensory receptors and their projections to the spinal dorsal horn. Res. Publ. - Assoc. Res. Nerv. Ment. Dis. 58, 19 – 45. Qin, C., Chandler, M.J., Miller, K.E., Foreman, R.D., 2001. Responses and afferent pathways of superficial and deeper c(1) – c(2) spinal cells to intrapericardial algogenic chemicals in rats. J. Neurophysiol. 85, 1522 – 1532. Russek, H.I., 1970. Intractable angina pectoris. Med. Clin. North Am. 54, 333 – 348. Takeuchi, Y., Ishii, N., Toda, K., 2001. An in vitro temporomandibular joint-nerve preparation for pain study in rats. J. Neurosci. Methods 109, 123 – 128. Toda, K., Ishii, N., Nakamura, Y., 1997. Characteristics of mucosal nociceptors in the rat oral cavity: an in vitro study. Neurosci. Lett. 228, 95 – 98. Veelken, R., Stetter, A., Dickel, T., Hilgers, K.F., 2003. Bimodality of cardiac vagal afferent C-fibres in the rat. Pflugers Arch. 446, 516 – 522. Zimmermann, M., 1981. Physiological mechanisms of pain and pain therapy. Triangle 20, 7 – 18.
Cardiac nociceptors innervated by vagal afferents in rats Yoichi Hisata a,b, Jorge L. Zeredo b, Kiyoyuki Eishi a, Kazuo Toda b,* b
a Cardiovascular Surgery, Graduate School of, Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan Division of Integrative Sensory Physiology, Graduate School of Biomedical, Sciences, Nagasaki University, Sakamoto 1-7-1, Nagasaki 852-8588, Japan
Received 1 November 2005; received in revised form 17 February 2006; accepted 20 February 2006
Abstract It is reported that cardiac afferent information is transmitted through at least three different pathways to the CNS: sympathetic, parasympathetic, and somatic; however, there are few studies concerning the role of afferent fibers of vagus nerves for eliciting cardiac sensation including pain. Especially, receptive field properties innervated by single vagal nerve fiber and mechanical threshold of nociceptors on the cardiac surface are not yet quantitatively studied. Therefore, in this study, we systematically investigated characteristics of vagal units innervating cardiac nociceptors in rats. Using anesthetized and artificially ventilated rats, 37 single unit recordings were made from fine nerve filaments of the left vagal nerve. For quantitative mechanical stimulation, the cardiac surface was stimulated by a von Frey type device. In addition, bradykinin was used for checking the chemical sensitivity of the nociceptor. Electrical stimulation was used to estimate the conduction velocity of the recorded nerve fiber. All units recorded from the vagal nerve were either Ay- or C-polymodal nociceptors. About 70% of the afferents had conduction velocities in the C-fiber range. In 60% of the units, the peripheral receptive field covered spot-like areas, but we also found larger and continuous receptive fields. Our results show that a majority of nociceptors innervated by vagal afferents are the C-polymodal type with spot-like receptive fields. We consider it to relate to the ambiguous and dull pain of angina pectoris. D 2006 Elsevier B.V. All rights reserved. Keywords: Cardiac pain; Vagal nerve; Receptive field; Polymodal nociceptor; Angina pectoris; Rat
1. Introduction The cardiac pain known as angina pectoris is a common symptom of ischemic heart disease, and has been described as a retrosternal, characteristically crushing, burning, or squeezing (Foreman, 1999). Pain may radiate to the throat, neck, or ulnar aspect of the left arm, sometimes reaching to the little finger. Less often, it radiates to the neck and lower jaw or either the right or both arms. It is also characteristic of angina pectoris to be variable in intensity and location from person to person and from time to time (Foreman, 1999; Mody and Faxon, 2000). Surgical interventions aiming at the sympathetic afferent pathway are reported to produce complete relief of angina pectoris in about half of the cases (Meller and Gebhart, 1992).
* Corresponding author. Tel.: +81 95 849 7636; fax: +81 95 849 7639. E-mail address: [email protected] (K. Toda). 1566-0702/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.02.008
However, symptoms remain in 40 –50% of the patients, including 10– 20% who report no pain relief at all, thus suggesting participation of vagal afferents for eliciting cardiac pain (Meller and Gebhart, 1992). In fact, a recent study in the rat by Qin et al. (2001) estimated 55% of the pericardial chemonociceptors to be innervated by vagal afferents. Despite numerous experimental and clinical investigations, the exact mechanisms involved in the development of cardiac pain, and particularly the involvement of vagal afferents, are not completely understood (Barber et al., 1984; Bassenge and Munzel, 1988). Veelken et al. (2003), and Ditting et al. (2004) reported a role for vagal C fibers in neurogenic cardiovascular regulation, however, characteristics of receptive field innervated by single vagal nerve fiber and mechanical threshold of nociceptors on the cardiac surface are still unknown. Therefore, this study was done to determine more clearly the characteristics of cardiac nociceptors innervated by vagal afferents in rats.
Y. Hisata et al. / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 174 – 178
2. Material and methods 2.1. Animal preparation Twenty-five Wistar albino rats (weighing 260– 650 g) were anesthetized with thiamylal sodium (initial dose of 80 mg/kg, i.p.; Isozol\, Yoshitomi Pharmaceutical, Tokyo, Japan). Artificial ventilation was established through a tracheostomy (3.0 ml/stroke, 60 strokes/min) using artificial ventilator for small animals (SN-480-7, Shinano, Tokyo, Japan). The thorax was opened by a full sternotomy, and the beating heart was exposed. Then, the left vagal nerve was identified next to left common carotid artery, and isolated from the surrounding tissues. The thoracic cavity pool made by surrounding skin was filled with pre-warmed (37 -C) physiological saline to prevent tissue dehydration. The proximal end of the vagal nerve was cut and ligated with a cotton thread to interrupt efferent nerve information. The fine filament was isolated from the vagal nerve bundle and was covered with liquid paraffin. The methods described here follow the ethical guidelines proposed by the International Association for the Study of Pain and received approval by the Animal Welfare Committee of Nagasaki University. 2.2. Study design We selected the right ventricle, which is innervated by a branch of the vagal nerve (Benson et al., 1999), as the site of stimulation. For quantitative mechanical stimulation, a von Frey type device with metal needle tip (diameter: 0.2mm) was used and stimulus intensities were monitored by a strain gauge amplifier (TBM-4, World Instruments Inc, FL, USA). This signal was displayed on the DOS-V computer through a CED 1401 interface (Cambridge Electronic Design Limited, Cambridge, UK). We found that stimuli over 20 mN applied to buccal skin surface always evoked pain sensation. Therefore, cardiac units having von Frey mechanical threshold over 20 mN were considered to be nociceptive. Bradykinin (BK, 10 4 M in Krebs– Henseleit solution, Sigma, USA), was applied topically with a volume of 1.0 ml using a syringe attached to a metal canula (tip diameter: 2.0 mm) closely attached to the mechanical receptive field. With the use of a bipolar concentric electrode (interpolar distance: 1.0 mm), electrical stimulation was applied to the receptive field on the cardiac surface. Conduction velocity was estimated using the distance and conduction delay between the stimulating and recording electrode. In the rat, the estimation of conduction velocity adopted classification reported by Koltzenburg et al. (1992). A cutoff of 2.0m/s was used to distinguish between myelinated (A fiber) and unmyelinated fiber (C fiber). Units conducting faster than 10.0 m/s were considered to be large myelinated (Ah) fibers, whereas units with conduction velocity below this value
175
were considered thin myelinated (Ay) fibers. In the present study, we could not find Ah innervated units as vagal nociceptors. Functional single unit responses from fine filaments of the vagal nerve were recorded with a tungsten hook electrode. The spike signal was fed into a high-impedance, low-noise amplifier (DAM-80E, World Precision Instruments, Sarasota, USA) and displayed on a DOS-V computer through the CED 1401. When two or three spikes were recorded simultaneously, units were separated using the Spike 2 software (Version 2.01) for Windows. 2.3. Data analysis The paired t-test was used to determine the statistical significance of the data from before and after chemical stimulation. Statistical significance was set at the 5% level ( P < 0.05). The software Statview version 5.0 (SAS Institute, Cary, NC) aided in statistical analysis. All values are displayed as mean T S.E.M.
3. Results We recorded activities of 37 single units from cardiac nociceptors on the right ventricle, all of which were of the polymodal type, being 11 Ay- and 26 C-fiber innervated. The conduction velocities of innervated Ay and C units were 3.8 T 0.4 and 1.4 T 0.1 m/s, respectively. 3.1. Threshold to mechanical stimulation Fig. 1 shows a typical example of Ay fiber-innervated single unit responses of a cardiac nociceptor after mechanical stimulation. Mechanical threshold was determined during the ramp phase of the von Frey stimulation, at the point where the first spike was fired (Fig. 1A, oblique arrow). The threshold level is also shown by horizontal continuous dotted line. This unit had von Frey threshold of 23.1 mN. The throbbing on the mechanical stimulation trace (upper line) was caused by the cardiac contractions against
Fig. 1. Typical response of a single Ay fiber innervated cardiac nociceptor. (A) von Frey pressure; an oblique arrow indicates threshold for this nociceptor (threshold: 23.1mN). Dotted line indicates the threshold level of this unit. (B) Phasic spike response of the cardiac nociceptors.
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Y. Hisata et al. / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 174 – 178
Table 1 Characteristics of cardiac nociceptors innervated by vagal nerve
Ay C a
n
Conduction velocity (m/s)
Threshold (mN)
Spontaneous discharge (Hz)
Receptive fields (C/S)a
11 26
3.8 T 0.4 1.4 T 0.1
38.0 T 3.1 43.6 T 3.3
0.37 T 0.1 0.46 T 0.1
5/6 11/15
C: continuous type, S: separate type.
the von Frey stimulation. The spike activity was typically phasic during mechanical stimulation, and not synchronized with the beating of the heart, showing that these responses were evoked by mechanical stimulation, not by cardiac contraction. In the present study, as shown in Table 1, the threshold value of Ay units were 38.0 T 3.1 mN (n = 11), that of C units were 43.6 T 3.3 mN (n = 26). 3.2. Receptive field properties Receptive field properties were not quantitatively shown in the previous studies concerning cardiac nociceptors. We investigated receptive field properties of cardiac nociceptors innervated by the vagal nerve by using threshold intensity of von Frey stimulation. Typically, we could distinguish two types of receptive fields: continuous and spot-like separate as shown in Fig. 2. 45% of the Ay-innervated and 42% of the C-innervated nociceptive units responded to stimuli over one wide, continuous receptive field; however, about 60% of the units showed non-responding spaces and a separate receptive field (Table 1).
Fig. 3. Discharge frequencies before and after bradykinin application. Data are means T S.E.M. Asterisks indicate P < 0.001 in the paired t-test.
increased the on-going discharge frequency of both Ay- and C-fiber innervated nociceptive units (Fig. 3). As shown in Fig. 4, mechanical threshold was significantly lowered by BK application in both Ay- and C-fiber innervated units. The average decrease was about 25% compared with the pre-BK value (38.0 T 3.1 mN to 28.0 T 3.3 mN in Ay units, and 43.6 T 3.3 mN to 32.8 T 3.1mN in C units). Table 1 summarizes the characteristics of nociceptors on the cardiac surface, indicating the following results: (1) Vagal nociceptors were innervated by Ay with slower conduction velocity or C fibers. (2) About 70% of the afferents were C fibers. (3) The receptive fields of Ay- and C-innervated units covered more often separate areas. (4) There was no significant difference in mechanical threshold between Ay and C nociceptors.
3.3. Chemical stimulation 4. Discussion Topical application of BK evoked tonic spike responses with an about 30-s delayed onset. The evoked responses gradually increased within 180 s and disappeared after washing out with physiological saline. BK significantly
The present study first revealed the receptive-field characteristics of cardiac nociceptors innervated by vagal afferents. This seemed to be important to recognize basic
Fig. 2. Types of receptive fields. (A) In the continuous type, units responded to stimulus from a wider continuous area. (B) In the separate type, units responded to stimulus from a spotted area. (a) Superior vena cava, (b) right atrium, (c) inferior vena cava, (d) aorta, (e) pulmonary artery, (f) right ventricle, (g) left ventricle, (h) left atrium, (i) pulmonary vein.
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Fig. 4. Mechanical thresholds before and after bradykinin application. Data are means T S.E.M. Asterisks indicate P < 0.001 in the paired t-test.
mechanisms of evoking cardiac pain. Larger receptive field of single cardiac unit is associated with an ambiguous and dull pain sensation. Two hypotheses have been formulated to explain ischemic cardiac pain: one hypothesis proposes that pain sensation would be brought up by a distention of the ventricular wall (‘‘mechanical hypothesis’’) (Foreman, 1999), and the other, by the intramyocardial release of pain-producing substances induced by ischemia (‘‘chemical hypothesis’’) (Benson et al., 1999; Crea and Gaspardone, 1997). However, it is not established which mechanism is important for producing ischemic cardiac pain. As previously reported, nociceptors in the skin, deep tissue or viscera are subdivided into several subtypes, the frequency distribution of which is different between body sites and animal species (Kumazawa and Mizumura, 1977, 1980; Perl, 1980). The relative distribution of each subtype is supposed to determine characteristics of pain sensation (Perl, 1980; Zimmermann, 1981). Some reports showed that the ventricular endings of afferent sympathetic fibers act as polymodal type nociceptors (Lombardi et al., 1981; Pan and Chen, 2002). In our investigation of the vagal nerve, likewise, all nociceptors were of the polymodal type in accordance with previous studies (Ditting et al., 2004; Veelken et al., 2003). This finding, therefore, favors the ‘‘chemical hypothesis’’ of ischemic cardiac pain partly evoked by inflammatory mediators. This point of view is also supported by several reports that correlate cardiac pain from angina pectoris with ischemic chemical mediators BK, PGE2, adenosine, histamine, 5-HT, or K+ (Baker et al., 1980; Bassenge and Munzel, 1988; Lombardi et al., 1981; Meller et al., 1990; Minisi and Thames, 1993; Nerdrum et al., 1986). In addition, the mechanical threshold of cardiac vagal nociceptors was lower than that reported for other body sites in the rat: for example, joint (Takeuchi et al., 2001), oral mucosa (Toda et al., 1997). This suggests that vagalinnervated nociceptors may be very sensitive to changes in mechanical stimuli such as, shape or volume of the cardiac wall. In this study, the mean conduction velocity of Ay-fiber innervated polymodal nociceptors was slower than that
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reported for other tissues and organs. The conduction velocities of Ay afferents from polymodal nociceptors in testis, oral mucosa were 11.2 T 0.3 and 9.1 T 2.0, respectively (Kumazawa et al., 1987; Toda et al., 1997), while our results showed 3.8 T 0.4 m/s (see Table 1). In a clinical situation, this finding may reflect the dullness of cardiac pain when compared to pain sensations from other deep tissues or internal organs (Russek, 1970). We observed that chemical stimulation with BK lowered the mechanical threshold and increased the discharge frequency of both Ay- and C-innervated nociceptors. This is in agreement with previous reports, which indicate BK to be a mediator of angina pectoris (Baker et al., 1980; Lombardi et al., 1981; Minisi and Thames, 1993). On the other hand, Koltzenburg et al. (1992) reported that BK did not contribute appreciably to the sensitization of sympathetic nociceptors in the skin. Further studies shall determine whether this discrepancy would contribute to the functional distinction between parasympathetic and sympathetic neurons, or between deep and superficial tissues. In summary, our results indicate that cardiac nociceptors innervated by the vagal nerve would be associated with an ambiguous and dull pain sensation, because the receptive field of single units was somewhat larger. This finding partly reflects the characteristics of cardiac pain, especially that related to angina pectoris and myocardial infarction.
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