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Involvement of two different mechanisms in trigeminal ganglion-evoked vasodilatation in the cat lower lip: role of experimental conditions
Journal of the Autonomic Nervous System 79 (2000) 84–92 www.elsevier.com / locate / jans
Involvement of two different mechanisms in trigeminal ganglion-evoked vasodilatation in the cat lower lip: role of experimental conditions a b c, Hisashi Date , Masato Kato , Hiroshi Izumi * a
Department of Pain Control, Tohoku University School of Medicine, Sendai 980 -8574, Japan Department of Anesthesiology, Tohoku University School of Medicine, Sendai 980 -8574, Japan c Department of Orofacial Functions, Tohoku University School of Dentistry, Sendai 980 -8575, Japan b
Received 18 September 1999; received in revised form 29 October 1999; accepted 29 October 1999
Abstract The present study was designed to examine the vasodilator mechanisms elicited by electrical stimulation of trigeminal ganglion (TG) in cat lower lip of the cats. When vago-sympathectomized cats were fixed into a stereotaxic frame by means of ear-bars, etc., the lip blood flow (LBF) increase evoked by lingual nerve (LN) stimulation (parasympathetic reflex response) was almost abolished in 15 out of 34 animals, but unaffected in the other 19. With the animal in the stereotaxic frame, electrical stimulation at sites within the TG evoked an LBF increase whether or not the LN stimulation-induced reflex response was intact. However, hexamethonium abolished the TG stimulation-induced LBF increase in animals whose brainstem parasympathetic reflex was intact, but reduced it by only 50% in animals whose reflex was impaired. This difference was seen in all experiments in which the electrode site was within the TG proper, regardless of its exact position. Although the underlying mechanism is unclear, these data suggest that when the TG is stimulated the LBF increase is entirely mediated via the parasympathetic reflex mechanism in animals whose brainstem reflex is intact, and that an antidromic vasodilatation occurs only in animals whose brainstem parasympathetic reflex is impaired. 2000 Elsevier Science B.V. All rights reserved. Keywords: Trigeminal ganglion; Parasympathetic; Vasodilatation
1. Introduction Flushing occurs on one side of the face following an attack of trigeminal neuralgia, injection of alcohol into the ipsilateral trigeminal ganglion (TG) (Jefferson, 1931; Rowbotham, 1939), or thermocoagulation of the ipsilateral TG (Drummond et al., 1983). Thus, the mechanisms underlying flushing and the effects of electrical stimulation of TG on the orofacial region are of clinical importance. However, the precise mechanisms remain unclear at the present time because of the presence of many complicating factors (such as experimental conditions). Several possible mechanisms have been suggested to explain this blood flow increase: (i) a centrally mediated parasympathetic reflex (Lambert et al., 1984); (ii) a release of tonic sympathetic vasoconstriction (Fox et al., 1962; Izumi and *Corresponding author. Tel.: 181-22-717-8321; fax: 181-22-7178322. E-mail address: [email protected] (H. Izumi)
Karita, 1991); (iii) the antidromic release of a vasodilator substance from the trigeminal nerve (Lambert et al., 1984); (iv) direct stimulation of parasympathetic or sympathetic vasodilator fibers (Gonzalez et al., 1975); or (v) an effect secondary to the arterial blood pressure increase elicited by TG stimulation [since laser-Doppler flowmeter (LDF) is very sensitive to such changes]. Each mechanism has its advocates, but none is yet widely accepted. During the course of studies regarding the vasodilator mechanisms in response to TG stimulation in the cat lower lip, we found that the effect of the autonomic ganglion blocker, hexamethonium (C 6 ), on this response was not consistent among the individual animals. Indeed, it had one of two effects: it either caused a complete abolition of the response or it had a moderate effect. Although there could be a number of reasons for this discrepancy, we noticed in the present experiments that the effect of C 6 seemed to differ depending on whether the brainstem parasympathetic reflex response elicited by lingual nerve (LN) stimulation was or was not intact. The abolition of the parasympathetic
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reflex vasodilatation seems to occur in cats after placing the stereotaxic frame with use of mouth-piece, eye-pieces and ear-bars. Having made this observation, we designed the present study to examine in more detail the ways in which experimental conditions might influence the lip blood flow (LBF) increase elicited by electrical stimulation within the TG in vago-sympathectomized cats.
2. Methods
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experiment, by an overdose (about 150 mg) of sodium pentobarbital. The experimental protocols were reviewed by the Committee on the Ethics of Animal Experiments in Tohoku University School of Medicine, and they were carried out in accordance with both the Guidelines for Animal Experiments issued by Tohoku University School of Medicine and The Law (No. 105) and Notification (No. 6) issued by the Japanese Government. All animals were cared for and used in accordance with the recommendations in the current National Research Council guide.
2.1. Preparation of animals Thirty-four adult cats, unselected as to sex and of 2.5 to 4.8 kg body weight, were initially sedated with ketamine hydrochloride (30 mg / kg, i.m.) and then anesthetized with a mixture of a-chloralose (50 mg / kg, i.v.) and urethane (100 mg / kg, i.v.). These anesthetics were supplemented if and when necessary throughout the experiment. The anesthetized animals were intubated, paralyzed by intravenous injection of pancuronium bromide (Mioblock; Organon, Teknika, Netherlands; 0.4 mg / kg initially, supplemented with 0.2 mg / kg every hour or so after testing the level of anesthesia; see below) and artificially ventilated via the tracheal cannula with a mixture of 50% air–50% O 2 . The ventilator (Model SN-480-6; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 10–12 cm 3 / kg at a rate of 20 breaths / min, and the end-tidal concentration of CO 2 was determined by means of an infrared analyzer (Capnomac Ultima; Datex, Helsinki, Finland) as reported previously (Izumi and Ito, 1998). Blood pH, PaO 2 , and PaCO 2 data were obtained at intervals of 90 min using a blood-gas analyzer (Model 148; Ciba-Corning, Medfield, MA, USA), and ventilation was adjusted to keep these parameters within normal limits. Ringers solution was continuously infused at a rate of approximately 8 ml / h, and 8.4% NaHCO 3 solution was added if necessary (both solutions from Otsuka Pharmaceutical, Tokyo, Japan). Rectal temperature was maintained at 37–388C using a heating pad. Depth of anesthesia was assessed and adjusted as in our previously published studies (Izumi, 1999a; Izumi and Ito, 1998; Izumi et al., 1997). The criteria for maintenance of an adequate depth of anesthesia were the persistence of miotic pupils and the absence of a reflex elevation of heart rate and arterial blood pressure during stimulation of the central end of the LN. If the depth of anesthesia was considered inadequate, additional a-chloralose and urethane (i.e., intermittent doses of 5 and 10 mg / kg i.v., respectively) was administered. Once an adequate depth of anesthesia had been attained, supplementary doses of pancuronium were given approximately every 60 min to maintain immobilization during periods of stimulation. In all experiments, the vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation. All cats were killed, at the end of the
2.2. Electrical stimulation of the LN ( Fig. 1 A) To elicit a parasympathetic reflex vasodilatation in the lower lip, the central cut end of the LN was electrically stimulated. The routine stimulus parameters were: a 20-s train of 2-ms rectangular pulses at a frequency of 10 Hz and at supramaximal intensity (usually 30 V), as described elsewhere (Izumi, 1999a,b; Izumi and Ito, 1998; Izumi and Karita, 1994a,b; Izumi et al., 1995, 1997). A bipolar silver electrode attached to a Nihon Kohden Model SEN-7103 Stimulator (Tokyo, Japan) was used for stimulation.
Fig. 1. Schematic representation of the sites of electrical stimulation. Stimulation sites: the central cut end of the lingual nerve (A), and the trigeminal ganglion (B). The diagram illustrates pathways in a schematic way; it is not intended to be a detailed representation of synaptic connections within the brainstem. The broken lines indicate the parasympathetic vasodilator fibres from the inferior salivatory nucleus. The solid lines indicate trigeminal and facial sensory inputs to the brain stem. Abbreviations: ISN, inferior salivatory nucleus; LDF, laser-Doppler flowmeter; NST, nucleus of solitary tract; OG, otic ganglion; SSN, superior salivatory nucleus; TG, trigeminal ganglion; TN, trigenimal nucleus; V, trigeminal nerve root; V1 –V3 , branches of the trigeminal nerve; VII, facial nerve root; IX, glossopharyngeal nerve root.
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2.3. Stimulation of the TG ( Fig. 1 B)
2.5. Statistical analysis
The animals were mounted in a stereotaxic frame (Narishige, Tokyo, Japan), and a concentric bipolar electrode (Inter Medical, Tokyo, Japan), insulated with enamel except at the tip, was positioned within the TG via a small burr hole in the skull. The electrode was advanced by means of a micromanipulator to the intended location, which was at stereotaxic coordinates 8-mm rostral to the interaural line, 9-mm lateral to the midline (atlas of Schneider et al., 1981, unless otherwise stated). In each penetration, the electrode was advanced carefully until it made contact with that portion of the skull surface (the alisphenoid) that overlies the TG, after which it was withdrawn 0.8 mm, to place it within the TG [as the TG is approximately 1.2 mm in depth (Schneider et al., 1981)]. We routinely used a 20-s train of rectangular pulses generated by a Nihon Kohden Model SEN-7103 stimulator through an isolation unit (Nihon Kohden Model SS-202J), usually with an amplitude of 10 V and a duration of 2 ms, at a frequency of 20 Hz, unless otherwise stated. Reference sites were marked by passing a 1 mA current for 60 s after the i.v. injection of an overdose of pentobarbital (see above). The location of the stimulation site was ascertained by the naked eye after an extensive craniectomy and excision of the brain rostral to the colliculi.
All numerical data are given as the mean6S.E. The significance of changes in the test responses was assessed using an analysis of variance (ANOVA) followed by a contrast test. Differences between groups were assessed using a contrast test. Differences were considered significant at the level P,0.05. Data were analyzed using a Macintosh Computer with StatView 5.0 and Super ANOVA (Abacus Concepts, CA, USA).
2.4. Measurement of lower LBF and of systemic arterial blood pressure Changes in blood flow in the lower lip adjacent to the canine tooth were monitored on one side using a LDF (model ALF21R; Advance, Tokyo, Japan) as described before (Izumi, 1999b; Izumi and Ito, 1998; Izumi and Karita, 1991, 1992, 1993; Izumi et al., 1997). The probe was placed against the lower lip without exerting any pressure on the tissue. The LDF values obtained in this way represent the blood flow in superficial vessels. Previous studies have indicated a significant correlation between blood flow recordings obtained from oral tissues by laserDoppler flowmetry and by other well-established methods (Edwall et al., 1987; Kim et al., 1990). The analog output of the equipment does not give absolute values, but shows relative changes in blood flow (for technical details and evaluation of the LDF method, see Stern et al., 1977). The output from the various devices was continuously displayed on an eight-channel chart recorder (Model W5000; Graphtec, Tokyo, Japan) at a speed of 10 mm / min. The magnitude of the blood flow changes elicited by nerve stimulation, and the amplitude of the basal LBF level, were assessed by making measurements in millimeters on the chart record. Systemic arterial blood pressure was recorded from the femoral catheter via a Statham pressure transducer. A tachograph (Model AT-610G; Nihon Kohden) triggered by the arterial pulse was used to monitor heart rate.
3. Results Animals were mounted in the stereotaxic frame by means of ear-bars, eye-pieces, and a mouth-piece in the conventional way. However, we shall refer simply to ‘‘ear-bars’’ hereafter both for simplicity and because we suspect (see Section 4) that their insertion into the earcanal was the important factor in producing the effects described below.
3.1. Effects produced by changes of posture and insertion of ear-bars Fig. 2 shows typical examples of the effects produced by changes of posture and insertion of ear-bars on the LBF increase evoked by LN stimulation in our vago-sympathectomized cats. Mean data are shown in Fig. 3. Changing the posture from the supine to the prone position, or vice versa, did not induce any significant change in the LBF increase. On the other hand, when the animals were mounted in the stereotaxic frame by the use of ear-bars (in the prone position in this case), the magnitude of the LBF increase was significantly reduced compared to the responses previously recorded in the supine and prone positions (on average, reduced by 35.3610.0% compared to supine control, n534, P,0.05). However, the size of the reduction varied widely from animal to animal, and the effect could be classified into two types: no effect [Fig. 2A and ‘‘ear-bars (A)’’ in Fig. 3; 19 out of 34 animals] (NS, n519) or marked inhibition [Fig. 2B and ‘‘ear-bars (B)’’ in Fig. 3; 15 out of 34 animals] (reduced by 86.063.9%, n515, P,0.001). Only a very small LN stimulationinduced LBF increase [reduced by 92.965.3%, n58, P, 0.001 when compared with control (supine)] was evoked after decerebration via an extensive craniectomy even in those animals in which the LN stimulation-induced LBF increase was not reduced by insertion of ear-bars. No statistically significant effect on systemic arterial blood pressure were observed upon changing the posture from the supine to the prone position without stereotaxic earbars inserted [82.44610.48 mmHg in the supine position (n512) vs. 82.06610.67 mmHg in the prone without stereotaxic ear-bars (n512), NS by ANOVA]. On the other hand, statistically significant increases in systemic arterial
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Fig. 2. Typical examples of effects of change from the supine to the prone position, and of the insertion of the ear-bars on lip blood flow (LBF) response to lingual nerve (LN) stimulation in vago-sympathectomized cats, and on resting systemic arterial blood pressure (BP). The change of posture or insertion of ear-bars is indicated by an arrow above the blood pressure record. A, B: traces from animals in which the LBF response was (A) or was not (B) intact after ear-bar insertion. The LN was stimulated where indicated (filled circles) for 20 s at a supramaximal voltage (30 V) at 10 Hz with pulses of 2-ms duration. a.u., arbitrary units.
blood pressure were observed upon firm insertion of the ear-bars into the external auditory canals of each cat regardless of whether it was classified as being of type A or B on the basis of the response-types shown in Fig. 2. The blood pressure increase was by 40.44610.95 mmHg (n512) on average when calculated for types A and B together (P,0.01 vs. control). However, marked differences in the magnitude of the arterial blood pressure increase evoked by ear-bar insertion were observed between type A and B animals (15.7861.91 mmHg for type A animals, n56 vs. 65.11616.74 mmHg for type B, n56; P,0.001, ANOVA).
3.2. Effects of autonomic ganglion blockade
Fig. 3. Mean data (6S.E.M) for the effects of lingual nerve (LN) stimulation on lip blood flow (LBF) with the animal in the supine or prone position, and with the animal fixed in the stereotaxic frame by means of ear-bars (all vago-sympathectomized cats). Data obtained after ear-bar insertion are shown for the entire group (n534), and separately for animals in which the LBF response was (A) (n519) or was not (B) (n515) intact after ear-bar insertion. The LN was stimulated for 20 s at a supramaximal voltage (30 V) at 10 Hz with pulses of 2-ms duration. The ordinate shows the LN-stimulated increases in LBF hexpressed as a percentage of the response to LN stimulation in the supine position (control)j, each value being given as mean6S.E. Statistical significance was assessed by means of ANOVA followed by a contrast test. *P,0.05, **P,0.001 vs. the response in the supine position (control). Bracket indicates significant difference between groups (ANOVA followed by contrast test). Number of animals is shown in parentheses.
Fig. 4 shows the effects of the autonomic ganglion blocker, C 6 , on the LBF increase elicited by electrical stimulation within the TG in animals in which the LN stimulation-induced LBF increase was [see Fig. 3, ear-bars (A) and Fig. 4A] or was not [see Fig. 3, ear-bars (B) and Fig. 4B] largely intact after ear-bar insertion. The dose of C 6 chosen for the present study was 10 mg / kg since this dose has been shown to produce a complete inhibition of the LBF increases evoked reflexly via parasympathetic vasodilator fibers by LN stimulation (Izumi and Karita, 1992; Karita et al., 1995) or directly by stimulation of the parasympathetic presynaptic fibers originating from the glossopharyngeal nerve (Izumi and Karita, 1991, 1992). Electrical stimulation at sites within the TG evoked an
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Fig. 4. Typical examples of the effects of the autonomic ganglion blocker, hexamethonium (C 6 , 10 mg / kg, i.v.), on the lip blood flow (LBF) increase evoked by either lingual nerve (LN) or trigeminal ganglion (TG) stimulation in vago-sympathectomized cats in which the LN stimulation-induced LBF increase was not (A) or was (B) affected by insertion of ear-bars. Electrical stimulation was at 30 V, 10 Hz, 2-ms pulse duration for 20 s (LN, filled circles) and at 10 V with pulses of 2-ms duration at 20 Hz for 20 s (TG, open circles). a.u., arbitrary units.
LBF increase regardless of whether a significant LN stimulation-induced LBF increase did (Fig. 4A) or did not occur (Fig. 4B) when the ear-bars were in position. However, the magnitude of the reduction in the TG stimulation-induced LBF increase that was induced by C 6 varied: in some animals it was completely inhibited and in others it was reduced by only 50% or so. In fact, in each of the 19 animals in which the LN stimulation-induced LBF increase was unaffected by the insertion of ear-bars, the LBF increase elicited by TG stimulation was almost completely inhibited by prior administration of C 6 (19 out
of 19 animals; type A; Fig. 4A). On the other hand, when we tested the animals in which the LN stimulation-induced LBF increase was impaired by insertion of ear-bars (Fig. 4B), the TG stimulation-induced LBF increase was found to be reduced by approximately 50% by C 6 in each and every animal (15 out of 15; type B, Fig. 4B). Fig. 5 shows the time course of the effects of C 6 on the TG stimulationinduced LBF increase in type A and type B animals. In type A, the reduction lasted for 100 min after C 6 administration [F(12,36)551.4, n57, P,0.001 by ANOVA for repeated measurements]. In type B in which the LN-
Fig. 5. Time course of the effects of hexamethonium (10 mg / kg, i.v.) on the lip blood flow (LBF) increase elicited by stimulation of the trigeminal ganglion (TG) in animals in which the lingual nerve stimulation-induced LBF increase was not (Fig. 4A) (filled circles) or was (Fig. 4B) (open circles) affected by insertion of ear-bars. The TG was stimulated for 20 s at 10 V with pulses of 2-ms duration at 20 Hz. Each value is expressed as a percentage of the pre-C 6 control response (at time 0), and is given as mean6S.E. Statistical significance from control (0 min) was assessed by means of ANOVA followed by a contrast test (*P,0.05, **P,0.01, ***P,0.001). 1 P,0.01, 11 P,0.001 indicate significant differences between effects of C 6 in animals of A and B types. Number of animals used is shown in parentheses.
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induced vasodilatation was reduced by only 50%, the reduction again lasted for 100 min [F(12,12)58.09, n54, P,0.001 by ANOVA for repeated measurements].
3.3. Sites of TG stimulation The sites of electrical stimulation within the TG are shown in Fig. 6 (produced by superimposing the individual maps for 21 animals). The animals in which the electrode sites were confined to the peripheral end of V3 rather than the TG proper were excluded from this part of the study. No clear difference between the sites at which trigeminal stimulation elicited type A or type B responses was observed in terms of their proximity to V3 (the branch carrying the fibers that run in the LN). It is apparent that the LBF increases elicited by TG stimulation could be abolished by prior treatment with C 6 regardless of whether the electrode site was close to V1 , V2 , or V3 of the TG. Fig. 6. Map of the stimulation sites. The location of the stimulation site in the TG was marked by making a lesion, and its location was ascertained by the naked eye after an extensive craniectomy and excision of the brain rostral to the colliculi (see Section 2). The maps for each of 21 animals are shown superimposed. The symbols (filled and open circles) indicate the electrode sites at which the trigeminal ganglion was electrically stimulated in type A and B animals, respectively (see Fig. 4). V1 2V3 , branches of the trigeminal nerve.
3.4. Effects of stimulus intensity and frequency Fig. 7 shows stimulus intensity (A, C)–response and frequency (B, D)–response relationships for changes in LBF in those animals in which the brainstem parasympathetic reflex function was intact after insertion of ear-bars (Fig. 4A). Data is shown for electrical stimulation of TG
Fig. 7. Stimulus intensity (A, C)–response and frequency (B, D)–response relationships for changes in lip blood flow evoked by electrical stimulation either (A, B) of the trigeminal ganglion (TG) (filled and open squares) or (C, D) of the central cut end of the lingual nerve (LN) (filled and open circles) in animals in which the brainstem parasympathetic reflex function was intact after ear-bar insertion (Fig. 4A). Stimulation of either structure was at various intensities (0 to 40 V) and at various frequencies (0.5 to 100 Hz) with 2-ms pulses for 20s. Intensity–response curves were generated using stimulus trains at 20 Hz (TG, panel A) or 10 Hz (LN, panel C). Frequency response curves were generated using stimulus trains at 10 V (TG, panel B) or 30 V (LN, panel D). Each value on the ordinate is expressed as a percentage of the maximum increase in blood flow at 20 V (for TG stimulation) or 40 V (for LN stimulation) (A and C, respectively) and at 20 Hz (for TG stimulation) or 10 Hz (for LN stimulation) (B and D, respectively). Each value is given as mean6S.E. The number of animals used is shown in parentheses.
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(Fig. 7A, B) and of the central cut end of the LN (Fig. 7C, D). Electrical stimulation at 1–2 V had negligible effects in both cases, whereas increasing the stimulus voltage over the range from 5 to 20 V produced successively bigger blood flow increases; both intensity–response graphs show saturation at 10 V (Fig. 7A, C) [for TG, F(6,30)522.2, n59, P,0.001; for LN, F(5,25)5481.3, n56, P,0.001; ANOVA for repeated measurements]. The optimal frequency for an LBF increase in response to TG or LN stimulation was 20 or 10 Hz, respectively (Fig. 7B, D) [for TG, F(7,49)522.7, n57, P,0.001; for LN, F(6,42)5 27.7, n58, P,0.01; ANOVA for repeated measurements].
3.5. Effects of drugs The blood flow responses evoked by electrical stimulation within the TG were studied after administration of phentolamine (an a-adrenoceptor blocking agent, 1 mg / kg), propranolol (a b-adrenoceptor blocking agent, 0.1 mg / kg), or atropine (a muscarinic cholinoceptor blocker, 0.2 mg / kg) in animals in which the LN stimulationinduced LBF increase was not affected by insertion of ear-bars (Fig. 4A). The values obtained under these three drugs were 106.8611.0 (n54, NS), 103.165.2 (n54, NS), and 91.466.4% (n54, NS), respectively (expressed as a percentage of the control response elicited by TG stimulation). In animals in which the LN stimulation-induced LBF increase was affected by insertion of ear-bars (Fig. 4B), the corresponding values were 119.5614.1 (n54, NS), 94.266.0 (n54, NS), and 96.061.3% (n54, NS). These results indicate that these blocking agents were all ineffective against the response to TG stimulation in type A and type B preparations.
4. Discussion Our previous and present experiments have shown that, in the cat lower lip: (i) the vasodilatation elicited by electrical stimulation of the trigeminal nerve root or TG (when these are separated from the brainstem) is not affected by superior cervical sympathectomy or use of an autonomic ganglion blocker, an a-blocker (phentolamine), or a b-adrenergic blocker (propranolol) (Izumi et al., 1990; Izumi and Karita, 1991), and (ii) that the vasodilatation evoked by afferent trigeminal nerves such as LN and the infraorbital nerve is completely abolished by C 6 in sympathectomized cats (Izumi, 1995; Izumi, 1999b). These findings exclude the involvement of the superior cervical sympathetic nerve in the TG stimulation-induced LBF increase (Izumi and Karita, 1992). The TG is reported to be approximately 1.2 mm in depth (Schneider et al., 1981), and the LBF increase evoked by electrical stimulation of TG disappeared when electrode was moved upwards by 1–2 mm. For these reasons, it seems unlikely that the LBF increase evoked by TG stimulation is due to stimulus
spread to the otic ganglion or the parasympathetic vasodilator fibers which lie in proximity to the TG in man and cat (Williams and Warwick, 1980; Kuchiiwa et al., 1992). Under the conditions of the present study, LN and TG stimulation each evoked a fall in arterial blood pressure, showing that the LBF increase elicited by LN or TG stimulation was not secondary to a rise in arterial blood pressure. From these results, the most plausible mechanism for the vasodilatation in the lower lip elicited by TG stimulation appears to be either a centrally mediated parasympathetic mechanism or an antidromic mechanism. Our original reason for performing the present study was to determine whether the LN-induced reflex vasodilatation might be influenced by the posture of the cat (viz. the supine or prone position). We also studied the effect produced by the use of a stereotaxic frame on this vasodilator response. In practice, the reflex parasympathetic LBF increase was not affected by either a change of posture or by positioning the cat in the stereotaxic frame without ear-bars (but with mouth-piece and eye-pieces in place). However, mounting the animal in the stereotaxic frame by means of ear-bars (as well as mouth-piece and eye-pieces) reduced the reflex response evoked by LN stimulation by approximately 85% in 15 out of 34 animals (Fig. 4B; type B), but had no effect in 19 out of 34 animals (Fig. 4A; type A). This inhibition lasted for several hours and recovery had not occurred by the end of the experiment regardless of whether the ear-bars etc. were or were not removed. As mentioned in Section 3, we suspect that the insertion of ear-bars was the important factor in inducing the effects we observed since the reflex response evoked by LN stimulation was not affected by mounting the animal in the stereotaxic frame with only the eyepieces and mouth-piece in place. For these reasons, we shall assume that ear-bar insertion was the important maneuver in this respect. With this proviso, the above result seems to indicate that firm insertion of ear-bars into the external auditory canals sometimes impairs a brainstem reflex whose afferent limb is the LN, a V3 branch of the trigeminal nerve, and whose efferent limb is the cranial parasympathetic outflow running via the glossopharyngeal nerve. As far as we are aware, there have been no previous reports of a depressive effect on a brainstem reflex caused by the use of ear-bars. It has been reported that the chorda tympani is inevitably injured by the use of ear-bars in the rat (Hosoya et al., 1983). However, to judge from our previous reports showing that dual afferent pathways are involved in the vasodilator reflex induced by LN stimulation in the cat (Karita and Izumi, 1993), a complete inhibition of this brainstem reflex by ear-bar insertion could not be attributed to a loss of the gustatory information that passes via the chorda tympani to the brainstem. At present, the mechanism and functional significance (if any) of this depression are not clear. As mentioned above, the virtual abolition of the LN-induced
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vasodilatation in type B animals showed no recovery within the time scale of the experiments. This may suggest that damage to, rather than stimulation of, some structure(s) within the ear canal was the important factor. A stereotaxic frame and a craniectomy are commonly used in animal experiments designed to study the response to stimulation of the TG and the effects of drugs on this response (Gonzalez et al., 1975; Lambert and Michalicek, 1996; Lambert et al., 1984; Goadsby et al., 1986; Lee et al., 1995). However, as shown in Figs. 2B, 3 and 4B, mounting the cat by use of ear-bars in the stereotaxic frame was itself sufficient to abolish the LN-induced reflex LBF increase in 15 out of 34 animals. Furthermore, the effects of C 6 on the response to TG stimulation were markedly different depending on whether the brainstem parasympathetic reflex was or was not intact (Figs. 4 and 5). This indicates that the mechanism by which TG stimulation elicited a vasodilatation differed between the two types of animals illustrated by Fig. 5; in the former type, it was apparently entirely mediated through the parasympathetic reflex mechanism, while in the latter it was mediated partly via antidromic and partly via parasympathetic reflex mechanisms. These data suggest that antidromic vasodilatation could occur only in those animals whose brainstem reflex activity was depressed. The fact that antidromic vasodilatation can be elicited by TG stimulation in preparations such as ours is supported by our previous observation that the magnitude of the vasodilatations in the lip and gingiva on TG (or mandibular nerve) stimulation was almost unchanged following section of the trigeminal root (Izumi and Karita, 1992). In the present study, antidromic vasodilatation was elicited by TG stimulation only in those animals in which the brainstem parasympathetic reflex was impaired by the use of ear-bars, whereas in animals in which the reflex was intact, TG stimulation evoked a vasodilatation that was entirely mediated via the parasympathetic reflex pathway. We have no way of knowing what neural effects occur in the brainstem upon insertion of ear-bars. However, it seems unlikely that the marked increase in arterial blood pressure itself (Fig. 2) caused the long-lasting inhibition of the parasympathetic reflex vasodilatation seen here since the arterial blood pressure returned to control within a few minutes (7.6760.33 min and 9.0061.52 min after insertion of the ear-bars in types A and B, respectively). At the present time, we can offer no convincing explanation for these findings. However, it may be relevant that recent reports have suggested that the trigemino-vascular system is not involved in setting the baseline flow level or in the cerebral vasodilator response to hypercapnia, but rather acts as an emergency system serving to counterbalance, for example, severe vasoconstriction (McCulloch et al., 1986; Juul et al., 1995. These reports and the present ones suggest to us the hypothesis that in some preparations, a strong and / or noxious afferent input, arising following ear-bar insertion: (i) suppresses parasympathetic reflex
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vasodilatation (presumably via an action within the brainstem), and simultaneously (ii) somehow permits antidromic vasodilatation to occur (presumably by an action in the periphery). Why this happens in some preparations and not in others is unclear, although it may depend on the degree of afferent stimulation or tissue damage produced by ear-bar insertion. Whatever the explanation for our findings, they should serve as a warning to other investigators (and to us) that the very presence of a response to stimulation of a peripheral nerve or ganglion, and the mechanism(s) by which it is mediated, may depend on whether an on-going (or even intermittent) strong and / or noxious afferent input is or is not being evoked by such maneuvers as the use of ear-bars.
Acknowledgements This study was supported by Grants-in Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan [Nos. 08672115, 09557161 and 09671886 (H. Izumi)].
References Drummond, P.D., Gonski, A., Lance, J.W., 1983. Facial flushing after thermocoagulation of the Gasserian ganglion. J. Neurol. Neurosurg. Psych. 46, 611–616. Edwall, B., Gazelius, B., Berg, J.O., Edwall, L., Hellander, K., Olgart, L., 1987. Blood flow changes in the dental pulp of the cat and rat measured simultaneously by laser-Doppler flowmetry and local 125I clearance. Acta Physiol. Scand. 131, 81–91. Fox, R.H., Goldsmith, R., Kidd, D.J., 1962. Cutaneous vasomotor control in the human head, neck and upper chest. J. Physiol. (London) 161, 298–312. Goadsby, P.J., Lambert, G.A., Lance, J.W., 1986. Stimulation of the trigeminal ganglion increases flow in the extracerebral but not the cerebral circulation of the monkey. Brain Res. 381, 63–67. Gonzalez, G., Onofrio, B.M., Kerr, F.W.L., 1975. Vasodilator system for the face. J. Neurosurg. 42, 693–703. Hosoya, Y., Matsushita, M., Sugiura, Y., 1983. A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods., Brain Res. 266, 329–333. Izumi, H., 1995. [Review] Reflex parasympathetic vasodilatation in facial skin. Gen. Pharmac. 26, 237–244. Izumi, H., 1999a. Functional roles played by the sympathetic supply to lip blood vessels in the cat. Am. J. Physiol. 277, R682–R689, Regulatory Integrative Comp. Physiol. 46. Izumi, H., 1999b. [Review] Nervous control of blood flow in the orofacial region. Pharmacol. Ther. 81, 141–161. Izumi, H., Ito, Y., 1998. Sympathetic attenuation of parasympathetic vasodilatation in oro-facial areas in the cat. J. Physiol. (London) 510, 915–921. Izumi, H., Karita, K., 1991. Vasodilator responses following intracranial stimulation of the trigeminal, facial and glossopharyngeal nerves in the cat gingiva. Brain Res. 560, 71–75. Izumi, H., Karita, K., 1992. Somatosensory stimulation causes autonomic vasodilatation in cat lip. J. Physiol. (London) 450, 191–202. Izumi, H., Karita, K., 1993. Innervation of the cat lip by two groups of
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parasympathetic vasodilator fibres. J. Physiol. (London) 465, 501– 512. Izumi, H., Karita, K., 1994a. The parasympathetic vasodilator fibers in the trigeminal portion of the distal lingual nerve in the cat tongue. Am. J. Physiol. 266, R1517–R1522, Regulatory Integrative Comp. Physiol. 35. Izumi, H., Karita, K., 1994b. Parasympathetic-mediated reflex salivation and vasodilatation in the cat submandibular gland. Am. J. Physiol. 267, R747–R753, Regulatory Integrative Comp. Physiol. 36. Izumi, H., Kuriwada, S., Karita, K., Sasano, T., Sanjo, D., 1990. The nervous control of gingival blood flow in cats. Microvasc. Res. 39, 94–104. Izumi, H., Nakamura, I., Karita, K., 1995. Effects of clonidine and yohimbine on parasympathetic reflex salivation and vasodilatation in cat SMG. Am. J. Physiol. 268, R1196–R1202, Regulatory Integrative Comp. Physiol. 37. Izumi, H., Ito, Y., Sato, M., Karita, K., Iwatsuki, N., 1997. Effects of inhalation anesthetics on the parasympathetic reflex vasodilatation in the lower lip and palate of the cat. Am. Physiol. 273, R168–R174, Regulatory Integrative Comp. Physiol. 42. Jefferson, G., 1931. Observations on trigeminal neuralgia. Br. Med. J. 2, 879–883. Juul, R., Hara, H., Gisvold, S.E., Brubakk, A.O., Fredriksen, T.A., Waldemar, G., Schmidt, J.F., Ekman, R., Edvinsson, L., 1995. Alterations in perivascular dilatory neuropeptides (CGRP. SP, VIP) in the external jugular vein and in the cerebrospinal fluid following subarachnoid haemorrhage in man. Acta Neurochir. 132, 32–41. Karita, K., Izumi, H., 1993. Dual afferent pathways of vasodilator reflex induced by lingual stimulation in the cat. J. Auton. Nerv. Sys. 45, 235–240. Karita, K., Takahashi, H., Yasui, T., Izumi, H., 1995. Effects of the autonomic ganglion blocking agent hexamethonium on vasodilator responses mediated by the parasympathetic ganglion on the chorda tympani pathway of the cat. J. Auton. Nerv. Sys. 52, 65–70.
Kim, S., Liu, M., Markowitz, K., Bilotto, G., Dorscher-Kim, J., 1990. Comparison of pulpal blood flow in dog canine teeth determined by the laser-Doppler and the 133xenon washout methods. Arch. Oral Biol. 35, 411–413. Kuchiiwa, S., Izumi, H., Karita, K., Nakagawa, S., 1992. Origins of parasympathetic postganglionic vasodilator fibers supplying the lips and gingivae; an WGA-HRP study in the cat. Neurosci. Lett. 142, 237–240. Lambert, G., Michalicek, J., 1996. Effect of antimigraine drugs on dural blood flows and resistances and the responses to trigeminal stimulation. Eur. J. Pharmac. 311, 141–151. Lambert, G.A., Bogduk, N., Goadsby, P.J., Duckworth, J.W., Lance, J.W., 1984. Decreased carotid arterial resistance in cats in response to trigeminal stimulation. J Neurosurg. 61, 307–315. Lee, W.S., Limmroth, V., Ayata, C., Cutrer, F.M., Waeber, C., Yu, X., Moskowitz, M.A., 1995. Peripheral GABAA receptor-mediated effects of sodium valproate on dural plasma protein extravasation to substance P and trigeminal stimulation. Br. J. Pharmacol. 116, 1661– 1667. McCulloch, J., Uddman, R., Kingman, T.A., Edvinsson, L., 1986. Calcitonin gene-related peptide: functional role in cerebrovascular regulation. Proc. Natl. Acad. Sci. U.S.A. 83, 5731–5735. Rowbotham, G.F., 1939. Observations on the effects of trigeminal denervation. Brain 62, 364–380. Schneider, J.S., Denaro, F.J., Olazabal, U.E., Leard, H.O., 1981. Stereotaxic atlas of the trigeminal ganglion in rat, cat, and monkey. Brain Res. Bull. 7, 93–95. Stern, M.D., Lappe, D.L., Bowen, P.D., Chimosky, J.E., Holloway, G.A.J., Keiser, H.R., Bowman, R.L., 1977. Continuous measurement of tissue blood flow by laser-Doppler spectroscopy. Am. J. Physiol. 232, H441–H448. Williams, P.L., Warwick, R., Gray’s Anatomy, 36th edn. ChurchillLivingstone, Melbourne, Victoria 3004, Australia, 1980.
Involvement of two different mechanisms in trigeminal ganglion-evoked vasodilatation in the cat lower lip: role of experimental conditions a b c, Hisashi Date , Masato Kato , Hiroshi Izumi * a
Department of Pain Control, Tohoku University School of Medicine, Sendai 980 -8574, Japan Department of Anesthesiology, Tohoku University School of Medicine, Sendai 980 -8574, Japan c Department of Orofacial Functions, Tohoku University School of Dentistry, Sendai 980 -8575, Japan b
Received 18 September 1999; received in revised form 29 October 1999; accepted 29 October 1999
Abstract The present study was designed to examine the vasodilator mechanisms elicited by electrical stimulation of trigeminal ganglion (TG) in cat lower lip of the cats. When vago-sympathectomized cats were fixed into a stereotaxic frame by means of ear-bars, etc., the lip blood flow (LBF) increase evoked by lingual nerve (LN) stimulation (parasympathetic reflex response) was almost abolished in 15 out of 34 animals, but unaffected in the other 19. With the animal in the stereotaxic frame, electrical stimulation at sites within the TG evoked an LBF increase whether or not the LN stimulation-induced reflex response was intact. However, hexamethonium abolished the TG stimulation-induced LBF increase in animals whose brainstem parasympathetic reflex was intact, but reduced it by only 50% in animals whose reflex was impaired. This difference was seen in all experiments in which the electrode site was within the TG proper, regardless of its exact position. Although the underlying mechanism is unclear, these data suggest that when the TG is stimulated the LBF increase is entirely mediated via the parasympathetic reflex mechanism in animals whose brainstem reflex is intact, and that an antidromic vasodilatation occurs only in animals whose brainstem parasympathetic reflex is impaired. 2000 Elsevier Science B.V. All rights reserved. Keywords: Trigeminal ganglion; Parasympathetic; Vasodilatation
1. Introduction Flushing occurs on one side of the face following an attack of trigeminal neuralgia, injection of alcohol into the ipsilateral trigeminal ganglion (TG) (Jefferson, 1931; Rowbotham, 1939), or thermocoagulation of the ipsilateral TG (Drummond et al., 1983). Thus, the mechanisms underlying flushing and the effects of electrical stimulation of TG on the orofacial region are of clinical importance. However, the precise mechanisms remain unclear at the present time because of the presence of many complicating factors (such as experimental conditions). Several possible mechanisms have been suggested to explain this blood flow increase: (i) a centrally mediated parasympathetic reflex (Lambert et al., 1984); (ii) a release of tonic sympathetic vasoconstriction (Fox et al., 1962; Izumi and *Corresponding author. Tel.: 181-22-717-8321; fax: 181-22-7178322. E-mail address: [email protected] (H. Izumi)
Karita, 1991); (iii) the antidromic release of a vasodilator substance from the trigeminal nerve (Lambert et al., 1984); (iv) direct stimulation of parasympathetic or sympathetic vasodilator fibers (Gonzalez et al., 1975); or (v) an effect secondary to the arterial blood pressure increase elicited by TG stimulation [since laser-Doppler flowmeter (LDF) is very sensitive to such changes]. Each mechanism has its advocates, but none is yet widely accepted. During the course of studies regarding the vasodilator mechanisms in response to TG stimulation in the cat lower lip, we found that the effect of the autonomic ganglion blocker, hexamethonium (C 6 ), on this response was not consistent among the individual animals. Indeed, it had one of two effects: it either caused a complete abolition of the response or it had a moderate effect. Although there could be a number of reasons for this discrepancy, we noticed in the present experiments that the effect of C 6 seemed to differ depending on whether the brainstem parasympathetic reflex response elicited by lingual nerve (LN) stimulation was or was not intact. The abolition of the parasympathetic
0165-1838 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-1838( 99 )00084-3
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reflex vasodilatation seems to occur in cats after placing the stereotaxic frame with use of mouth-piece, eye-pieces and ear-bars. Having made this observation, we designed the present study to examine in more detail the ways in which experimental conditions might influence the lip blood flow (LBF) increase elicited by electrical stimulation within the TG in vago-sympathectomized cats.
2. Methods
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experiment, by an overdose (about 150 mg) of sodium pentobarbital. The experimental protocols were reviewed by the Committee on the Ethics of Animal Experiments in Tohoku University School of Medicine, and they were carried out in accordance with both the Guidelines for Animal Experiments issued by Tohoku University School of Medicine and The Law (No. 105) and Notification (No. 6) issued by the Japanese Government. All animals were cared for and used in accordance with the recommendations in the current National Research Council guide.
2.1. Preparation of animals Thirty-four adult cats, unselected as to sex and of 2.5 to 4.8 kg body weight, were initially sedated with ketamine hydrochloride (30 mg / kg, i.m.) and then anesthetized with a mixture of a-chloralose (50 mg / kg, i.v.) and urethane (100 mg / kg, i.v.). These anesthetics were supplemented if and when necessary throughout the experiment. The anesthetized animals were intubated, paralyzed by intravenous injection of pancuronium bromide (Mioblock; Organon, Teknika, Netherlands; 0.4 mg / kg initially, supplemented with 0.2 mg / kg every hour or so after testing the level of anesthesia; see below) and artificially ventilated via the tracheal cannula with a mixture of 50% air–50% O 2 . The ventilator (Model SN-480-6; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 10–12 cm 3 / kg at a rate of 20 breaths / min, and the end-tidal concentration of CO 2 was determined by means of an infrared analyzer (Capnomac Ultima; Datex, Helsinki, Finland) as reported previously (Izumi and Ito, 1998). Blood pH, PaO 2 , and PaCO 2 data were obtained at intervals of 90 min using a blood-gas analyzer (Model 148; Ciba-Corning, Medfield, MA, USA), and ventilation was adjusted to keep these parameters within normal limits. Ringers solution was continuously infused at a rate of approximately 8 ml / h, and 8.4% NaHCO 3 solution was added if necessary (both solutions from Otsuka Pharmaceutical, Tokyo, Japan). Rectal temperature was maintained at 37–388C using a heating pad. Depth of anesthesia was assessed and adjusted as in our previously published studies (Izumi, 1999a; Izumi and Ito, 1998; Izumi et al., 1997). The criteria for maintenance of an adequate depth of anesthesia were the persistence of miotic pupils and the absence of a reflex elevation of heart rate and arterial blood pressure during stimulation of the central end of the LN. If the depth of anesthesia was considered inadequate, additional a-chloralose and urethane (i.e., intermittent doses of 5 and 10 mg / kg i.v., respectively) was administered. Once an adequate depth of anesthesia had been attained, supplementary doses of pancuronium were given approximately every 60 min to maintain immobilization during periods of stimulation. In all experiments, the vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation. All cats were killed, at the end of the
2.2. Electrical stimulation of the LN ( Fig. 1 A) To elicit a parasympathetic reflex vasodilatation in the lower lip, the central cut end of the LN was electrically stimulated. The routine stimulus parameters were: a 20-s train of 2-ms rectangular pulses at a frequency of 10 Hz and at supramaximal intensity (usually 30 V), as described elsewhere (Izumi, 1999a,b; Izumi and Ito, 1998; Izumi and Karita, 1994a,b; Izumi et al., 1995, 1997). A bipolar silver electrode attached to a Nihon Kohden Model SEN-7103 Stimulator (Tokyo, Japan) was used for stimulation.
Fig. 1. Schematic representation of the sites of electrical stimulation. Stimulation sites: the central cut end of the lingual nerve (A), and the trigeminal ganglion (B). The diagram illustrates pathways in a schematic way; it is not intended to be a detailed representation of synaptic connections within the brainstem. The broken lines indicate the parasympathetic vasodilator fibres from the inferior salivatory nucleus. The solid lines indicate trigeminal and facial sensory inputs to the brain stem. Abbreviations: ISN, inferior salivatory nucleus; LDF, laser-Doppler flowmeter; NST, nucleus of solitary tract; OG, otic ganglion; SSN, superior salivatory nucleus; TG, trigeminal ganglion; TN, trigenimal nucleus; V, trigeminal nerve root; V1 –V3 , branches of the trigeminal nerve; VII, facial nerve root; IX, glossopharyngeal nerve root.
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2.3. Stimulation of the TG ( Fig. 1 B)
2.5. Statistical analysis
The animals were mounted in a stereotaxic frame (Narishige, Tokyo, Japan), and a concentric bipolar electrode (Inter Medical, Tokyo, Japan), insulated with enamel except at the tip, was positioned within the TG via a small burr hole in the skull. The electrode was advanced by means of a micromanipulator to the intended location, which was at stereotaxic coordinates 8-mm rostral to the interaural line, 9-mm lateral to the midline (atlas of Schneider et al., 1981, unless otherwise stated). In each penetration, the electrode was advanced carefully until it made contact with that portion of the skull surface (the alisphenoid) that overlies the TG, after which it was withdrawn 0.8 mm, to place it within the TG [as the TG is approximately 1.2 mm in depth (Schneider et al., 1981)]. We routinely used a 20-s train of rectangular pulses generated by a Nihon Kohden Model SEN-7103 stimulator through an isolation unit (Nihon Kohden Model SS-202J), usually with an amplitude of 10 V and a duration of 2 ms, at a frequency of 20 Hz, unless otherwise stated. Reference sites were marked by passing a 1 mA current for 60 s after the i.v. injection of an overdose of pentobarbital (see above). The location of the stimulation site was ascertained by the naked eye after an extensive craniectomy and excision of the brain rostral to the colliculi.
All numerical data are given as the mean6S.E. The significance of changes in the test responses was assessed using an analysis of variance (ANOVA) followed by a contrast test. Differences between groups were assessed using a contrast test. Differences were considered significant at the level P,0.05. Data were analyzed using a Macintosh Computer with StatView 5.0 and Super ANOVA (Abacus Concepts, CA, USA).
2.4. Measurement of lower LBF and of systemic arterial blood pressure Changes in blood flow in the lower lip adjacent to the canine tooth were monitored on one side using a LDF (model ALF21R; Advance, Tokyo, Japan) as described before (Izumi, 1999b; Izumi and Ito, 1998; Izumi and Karita, 1991, 1992, 1993; Izumi et al., 1997). The probe was placed against the lower lip without exerting any pressure on the tissue. The LDF values obtained in this way represent the blood flow in superficial vessels. Previous studies have indicated a significant correlation between blood flow recordings obtained from oral tissues by laserDoppler flowmetry and by other well-established methods (Edwall et al., 1987; Kim et al., 1990). The analog output of the equipment does not give absolute values, but shows relative changes in blood flow (for technical details and evaluation of the LDF method, see Stern et al., 1977). The output from the various devices was continuously displayed on an eight-channel chart recorder (Model W5000; Graphtec, Tokyo, Japan) at a speed of 10 mm / min. The magnitude of the blood flow changes elicited by nerve stimulation, and the amplitude of the basal LBF level, were assessed by making measurements in millimeters on the chart record. Systemic arterial blood pressure was recorded from the femoral catheter via a Statham pressure transducer. A tachograph (Model AT-610G; Nihon Kohden) triggered by the arterial pulse was used to monitor heart rate.
3. Results Animals were mounted in the stereotaxic frame by means of ear-bars, eye-pieces, and a mouth-piece in the conventional way. However, we shall refer simply to ‘‘ear-bars’’ hereafter both for simplicity and because we suspect (see Section 4) that their insertion into the earcanal was the important factor in producing the effects described below.
3.1. Effects produced by changes of posture and insertion of ear-bars Fig. 2 shows typical examples of the effects produced by changes of posture and insertion of ear-bars on the LBF increase evoked by LN stimulation in our vago-sympathectomized cats. Mean data are shown in Fig. 3. Changing the posture from the supine to the prone position, or vice versa, did not induce any significant change in the LBF increase. On the other hand, when the animals were mounted in the stereotaxic frame by the use of ear-bars (in the prone position in this case), the magnitude of the LBF increase was significantly reduced compared to the responses previously recorded in the supine and prone positions (on average, reduced by 35.3610.0% compared to supine control, n534, P,0.05). However, the size of the reduction varied widely from animal to animal, and the effect could be classified into two types: no effect [Fig. 2A and ‘‘ear-bars (A)’’ in Fig. 3; 19 out of 34 animals] (NS, n519) or marked inhibition [Fig. 2B and ‘‘ear-bars (B)’’ in Fig. 3; 15 out of 34 animals] (reduced by 86.063.9%, n515, P,0.001). Only a very small LN stimulationinduced LBF increase [reduced by 92.965.3%, n58, P, 0.001 when compared with control (supine)] was evoked after decerebration via an extensive craniectomy even in those animals in which the LN stimulation-induced LBF increase was not reduced by insertion of ear-bars. No statistically significant effect on systemic arterial blood pressure were observed upon changing the posture from the supine to the prone position without stereotaxic earbars inserted [82.44610.48 mmHg in the supine position (n512) vs. 82.06610.67 mmHg in the prone without stereotaxic ear-bars (n512), NS by ANOVA]. On the other hand, statistically significant increases in systemic arterial
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Fig. 2. Typical examples of effects of change from the supine to the prone position, and of the insertion of the ear-bars on lip blood flow (LBF) response to lingual nerve (LN) stimulation in vago-sympathectomized cats, and on resting systemic arterial blood pressure (BP). The change of posture or insertion of ear-bars is indicated by an arrow above the blood pressure record. A, B: traces from animals in which the LBF response was (A) or was not (B) intact after ear-bar insertion. The LN was stimulated where indicated (filled circles) for 20 s at a supramaximal voltage (30 V) at 10 Hz with pulses of 2-ms duration. a.u., arbitrary units.
blood pressure were observed upon firm insertion of the ear-bars into the external auditory canals of each cat regardless of whether it was classified as being of type A or B on the basis of the response-types shown in Fig. 2. The blood pressure increase was by 40.44610.95 mmHg (n512) on average when calculated for types A and B together (P,0.01 vs. control). However, marked differences in the magnitude of the arterial blood pressure increase evoked by ear-bar insertion were observed between type A and B animals (15.7861.91 mmHg for type A animals, n56 vs. 65.11616.74 mmHg for type B, n56; P,0.001, ANOVA).
3.2. Effects of autonomic ganglion blockade
Fig. 3. Mean data (6S.E.M) for the effects of lingual nerve (LN) stimulation on lip blood flow (LBF) with the animal in the supine or prone position, and with the animal fixed in the stereotaxic frame by means of ear-bars (all vago-sympathectomized cats). Data obtained after ear-bar insertion are shown for the entire group (n534), and separately for animals in which the LBF response was (A) (n519) or was not (B) (n515) intact after ear-bar insertion. The LN was stimulated for 20 s at a supramaximal voltage (30 V) at 10 Hz with pulses of 2-ms duration. The ordinate shows the LN-stimulated increases in LBF hexpressed as a percentage of the response to LN stimulation in the supine position (control)j, each value being given as mean6S.E. Statistical significance was assessed by means of ANOVA followed by a contrast test. *P,0.05, **P,0.001 vs. the response in the supine position (control). Bracket indicates significant difference between groups (ANOVA followed by contrast test). Number of animals is shown in parentheses.
Fig. 4 shows the effects of the autonomic ganglion blocker, C 6 , on the LBF increase elicited by electrical stimulation within the TG in animals in which the LN stimulation-induced LBF increase was [see Fig. 3, ear-bars (A) and Fig. 4A] or was not [see Fig. 3, ear-bars (B) and Fig. 4B] largely intact after ear-bar insertion. The dose of C 6 chosen for the present study was 10 mg / kg since this dose has been shown to produce a complete inhibition of the LBF increases evoked reflexly via parasympathetic vasodilator fibers by LN stimulation (Izumi and Karita, 1992; Karita et al., 1995) or directly by stimulation of the parasympathetic presynaptic fibers originating from the glossopharyngeal nerve (Izumi and Karita, 1991, 1992). Electrical stimulation at sites within the TG evoked an
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Fig. 4. Typical examples of the effects of the autonomic ganglion blocker, hexamethonium (C 6 , 10 mg / kg, i.v.), on the lip blood flow (LBF) increase evoked by either lingual nerve (LN) or trigeminal ganglion (TG) stimulation in vago-sympathectomized cats in which the LN stimulation-induced LBF increase was not (A) or was (B) affected by insertion of ear-bars. Electrical stimulation was at 30 V, 10 Hz, 2-ms pulse duration for 20 s (LN, filled circles) and at 10 V with pulses of 2-ms duration at 20 Hz for 20 s (TG, open circles). a.u., arbitrary units.
LBF increase regardless of whether a significant LN stimulation-induced LBF increase did (Fig. 4A) or did not occur (Fig. 4B) when the ear-bars were in position. However, the magnitude of the reduction in the TG stimulation-induced LBF increase that was induced by C 6 varied: in some animals it was completely inhibited and in others it was reduced by only 50% or so. In fact, in each of the 19 animals in which the LN stimulation-induced LBF increase was unaffected by the insertion of ear-bars, the LBF increase elicited by TG stimulation was almost completely inhibited by prior administration of C 6 (19 out
of 19 animals; type A; Fig. 4A). On the other hand, when we tested the animals in which the LN stimulation-induced LBF increase was impaired by insertion of ear-bars (Fig. 4B), the TG stimulation-induced LBF increase was found to be reduced by approximately 50% by C 6 in each and every animal (15 out of 15; type B, Fig. 4B). Fig. 5 shows the time course of the effects of C 6 on the TG stimulationinduced LBF increase in type A and type B animals. In type A, the reduction lasted for 100 min after C 6 administration [F(12,36)551.4, n57, P,0.001 by ANOVA for repeated measurements]. In type B in which the LN-
Fig. 5. Time course of the effects of hexamethonium (10 mg / kg, i.v.) on the lip blood flow (LBF) increase elicited by stimulation of the trigeminal ganglion (TG) in animals in which the lingual nerve stimulation-induced LBF increase was not (Fig. 4A) (filled circles) or was (Fig. 4B) (open circles) affected by insertion of ear-bars. The TG was stimulated for 20 s at 10 V with pulses of 2-ms duration at 20 Hz. Each value is expressed as a percentage of the pre-C 6 control response (at time 0), and is given as mean6S.E. Statistical significance from control (0 min) was assessed by means of ANOVA followed by a contrast test (*P,0.05, **P,0.01, ***P,0.001). 1 P,0.01, 11 P,0.001 indicate significant differences between effects of C 6 in animals of A and B types. Number of animals used is shown in parentheses.
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induced vasodilatation was reduced by only 50%, the reduction again lasted for 100 min [F(12,12)58.09, n54, P,0.001 by ANOVA for repeated measurements].
3.3. Sites of TG stimulation The sites of electrical stimulation within the TG are shown in Fig. 6 (produced by superimposing the individual maps for 21 animals). The animals in which the electrode sites were confined to the peripheral end of V3 rather than the TG proper were excluded from this part of the study. No clear difference between the sites at which trigeminal stimulation elicited type A or type B responses was observed in terms of their proximity to V3 (the branch carrying the fibers that run in the LN). It is apparent that the LBF increases elicited by TG stimulation could be abolished by prior treatment with C 6 regardless of whether the electrode site was close to V1 , V2 , or V3 of the TG. Fig. 6. Map of the stimulation sites. The location of the stimulation site in the TG was marked by making a lesion, and its location was ascertained by the naked eye after an extensive craniectomy and excision of the brain rostral to the colliculi (see Section 2). The maps for each of 21 animals are shown superimposed. The symbols (filled and open circles) indicate the electrode sites at which the trigeminal ganglion was electrically stimulated in type A and B animals, respectively (see Fig. 4). V1 2V3 , branches of the trigeminal nerve.
3.4. Effects of stimulus intensity and frequency Fig. 7 shows stimulus intensity (A, C)–response and frequency (B, D)–response relationships for changes in LBF in those animals in which the brainstem parasympathetic reflex function was intact after insertion of ear-bars (Fig. 4A). Data is shown for electrical stimulation of TG
Fig. 7. Stimulus intensity (A, C)–response and frequency (B, D)–response relationships for changes in lip blood flow evoked by electrical stimulation either (A, B) of the trigeminal ganglion (TG) (filled and open squares) or (C, D) of the central cut end of the lingual nerve (LN) (filled and open circles) in animals in which the brainstem parasympathetic reflex function was intact after ear-bar insertion (Fig. 4A). Stimulation of either structure was at various intensities (0 to 40 V) and at various frequencies (0.5 to 100 Hz) with 2-ms pulses for 20s. Intensity–response curves were generated using stimulus trains at 20 Hz (TG, panel A) or 10 Hz (LN, panel C). Frequency response curves were generated using stimulus trains at 10 V (TG, panel B) or 30 V (LN, panel D). Each value on the ordinate is expressed as a percentage of the maximum increase in blood flow at 20 V (for TG stimulation) or 40 V (for LN stimulation) (A and C, respectively) and at 20 Hz (for TG stimulation) or 10 Hz (for LN stimulation) (B and D, respectively). Each value is given as mean6S.E. The number of animals used is shown in parentheses.
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(Fig. 7A, B) and of the central cut end of the LN (Fig. 7C, D). Electrical stimulation at 1–2 V had negligible effects in both cases, whereas increasing the stimulus voltage over the range from 5 to 20 V produced successively bigger blood flow increases; both intensity–response graphs show saturation at 10 V (Fig. 7A, C) [for TG, F(6,30)522.2, n59, P,0.001; for LN, F(5,25)5481.3, n56, P,0.001; ANOVA for repeated measurements]. The optimal frequency for an LBF increase in response to TG or LN stimulation was 20 or 10 Hz, respectively (Fig. 7B, D) [for TG, F(7,49)522.7, n57, P,0.001; for LN, F(6,42)5 27.7, n58, P,0.01; ANOVA for repeated measurements].
3.5. Effects of drugs The blood flow responses evoked by electrical stimulation within the TG were studied after administration of phentolamine (an a-adrenoceptor blocking agent, 1 mg / kg), propranolol (a b-adrenoceptor blocking agent, 0.1 mg / kg), or atropine (a muscarinic cholinoceptor blocker, 0.2 mg / kg) in animals in which the LN stimulationinduced LBF increase was not affected by insertion of ear-bars (Fig. 4A). The values obtained under these three drugs were 106.8611.0 (n54, NS), 103.165.2 (n54, NS), and 91.466.4% (n54, NS), respectively (expressed as a percentage of the control response elicited by TG stimulation). In animals in which the LN stimulation-induced LBF increase was affected by insertion of ear-bars (Fig. 4B), the corresponding values were 119.5614.1 (n54, NS), 94.266.0 (n54, NS), and 96.061.3% (n54, NS). These results indicate that these blocking agents were all ineffective against the response to TG stimulation in type A and type B preparations.
4. Discussion Our previous and present experiments have shown that, in the cat lower lip: (i) the vasodilatation elicited by electrical stimulation of the trigeminal nerve root or TG (when these are separated from the brainstem) is not affected by superior cervical sympathectomy or use of an autonomic ganglion blocker, an a-blocker (phentolamine), or a b-adrenergic blocker (propranolol) (Izumi et al., 1990; Izumi and Karita, 1991), and (ii) that the vasodilatation evoked by afferent trigeminal nerves such as LN and the infraorbital nerve is completely abolished by C 6 in sympathectomized cats (Izumi, 1995; Izumi, 1999b). These findings exclude the involvement of the superior cervical sympathetic nerve in the TG stimulation-induced LBF increase (Izumi and Karita, 1992). The TG is reported to be approximately 1.2 mm in depth (Schneider et al., 1981), and the LBF increase evoked by electrical stimulation of TG disappeared when electrode was moved upwards by 1–2 mm. For these reasons, it seems unlikely that the LBF increase evoked by TG stimulation is due to stimulus
spread to the otic ganglion or the parasympathetic vasodilator fibers which lie in proximity to the TG in man and cat (Williams and Warwick, 1980; Kuchiiwa et al., 1992). Under the conditions of the present study, LN and TG stimulation each evoked a fall in arterial blood pressure, showing that the LBF increase elicited by LN or TG stimulation was not secondary to a rise in arterial blood pressure. From these results, the most plausible mechanism for the vasodilatation in the lower lip elicited by TG stimulation appears to be either a centrally mediated parasympathetic mechanism or an antidromic mechanism. Our original reason for performing the present study was to determine whether the LN-induced reflex vasodilatation might be influenced by the posture of the cat (viz. the supine or prone position). We also studied the effect produced by the use of a stereotaxic frame on this vasodilator response. In practice, the reflex parasympathetic LBF increase was not affected by either a change of posture or by positioning the cat in the stereotaxic frame without ear-bars (but with mouth-piece and eye-pieces in place). However, mounting the animal in the stereotaxic frame by means of ear-bars (as well as mouth-piece and eye-pieces) reduced the reflex response evoked by LN stimulation by approximately 85% in 15 out of 34 animals (Fig. 4B; type B), but had no effect in 19 out of 34 animals (Fig. 4A; type A). This inhibition lasted for several hours and recovery had not occurred by the end of the experiment regardless of whether the ear-bars etc. were or were not removed. As mentioned in Section 3, we suspect that the insertion of ear-bars was the important factor in inducing the effects we observed since the reflex response evoked by LN stimulation was not affected by mounting the animal in the stereotaxic frame with only the eyepieces and mouth-piece in place. For these reasons, we shall assume that ear-bar insertion was the important maneuver in this respect. With this proviso, the above result seems to indicate that firm insertion of ear-bars into the external auditory canals sometimes impairs a brainstem reflex whose afferent limb is the LN, a V3 branch of the trigeminal nerve, and whose efferent limb is the cranial parasympathetic outflow running via the glossopharyngeal nerve. As far as we are aware, there have been no previous reports of a depressive effect on a brainstem reflex caused by the use of ear-bars. It has been reported that the chorda tympani is inevitably injured by the use of ear-bars in the rat (Hosoya et al., 1983). However, to judge from our previous reports showing that dual afferent pathways are involved in the vasodilator reflex induced by LN stimulation in the cat (Karita and Izumi, 1993), a complete inhibition of this brainstem reflex by ear-bar insertion could not be attributed to a loss of the gustatory information that passes via the chorda tympani to the brainstem. At present, the mechanism and functional significance (if any) of this depression are not clear. As mentioned above, the virtual abolition of the LN-induced
H. Date et al. / Journal of the Autonomic Nervous System 79 (2000) 84 – 92
vasodilatation in type B animals showed no recovery within the time scale of the experiments. This may suggest that damage to, rather than stimulation of, some structure(s) within the ear canal was the important factor. A stereotaxic frame and a craniectomy are commonly used in animal experiments designed to study the response to stimulation of the TG and the effects of drugs on this response (Gonzalez et al., 1975; Lambert and Michalicek, 1996; Lambert et al., 1984; Goadsby et al., 1986; Lee et al., 1995). However, as shown in Figs. 2B, 3 and 4B, mounting the cat by use of ear-bars in the stereotaxic frame was itself sufficient to abolish the LN-induced reflex LBF increase in 15 out of 34 animals. Furthermore, the effects of C 6 on the response to TG stimulation were markedly different depending on whether the brainstem parasympathetic reflex was or was not intact (Figs. 4 and 5). This indicates that the mechanism by which TG stimulation elicited a vasodilatation differed between the two types of animals illustrated by Fig. 5; in the former type, it was apparently entirely mediated through the parasympathetic reflex mechanism, while in the latter it was mediated partly via antidromic and partly via parasympathetic reflex mechanisms. These data suggest that antidromic vasodilatation could occur only in those animals whose brainstem reflex activity was depressed. The fact that antidromic vasodilatation can be elicited by TG stimulation in preparations such as ours is supported by our previous observation that the magnitude of the vasodilatations in the lip and gingiva on TG (or mandibular nerve) stimulation was almost unchanged following section of the trigeminal root (Izumi and Karita, 1992). In the present study, antidromic vasodilatation was elicited by TG stimulation only in those animals in which the brainstem parasympathetic reflex was impaired by the use of ear-bars, whereas in animals in which the reflex was intact, TG stimulation evoked a vasodilatation that was entirely mediated via the parasympathetic reflex pathway. We have no way of knowing what neural effects occur in the brainstem upon insertion of ear-bars. However, it seems unlikely that the marked increase in arterial blood pressure itself (Fig. 2) caused the long-lasting inhibition of the parasympathetic reflex vasodilatation seen here since the arterial blood pressure returned to control within a few minutes (7.6760.33 min and 9.0061.52 min after insertion of the ear-bars in types A and B, respectively). At the present time, we can offer no convincing explanation for these findings. However, it may be relevant that recent reports have suggested that the trigemino-vascular system is not involved in setting the baseline flow level or in the cerebral vasodilator response to hypercapnia, but rather acts as an emergency system serving to counterbalance, for example, severe vasoconstriction (McCulloch et al., 1986; Juul et al., 1995. These reports and the present ones suggest to us the hypothesis that in some preparations, a strong and / or noxious afferent input, arising following ear-bar insertion: (i) suppresses parasympathetic reflex
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vasodilatation (presumably via an action within the brainstem), and simultaneously (ii) somehow permits antidromic vasodilatation to occur (presumably by an action in the periphery). Why this happens in some preparations and not in others is unclear, although it may depend on the degree of afferent stimulation or tissue damage produced by ear-bar insertion. Whatever the explanation for our findings, they should serve as a warning to other investigators (and to us) that the very presence of a response to stimulation of a peripheral nerve or ganglion, and the mechanism(s) by which it is mediated, may depend on whether an on-going (or even intermittent) strong and / or noxious afferent input is or is not being evoked by such maneuvers as the use of ear-bars.
Acknowledgements This study was supported by Grants-in Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan [Nos. 08672115, 09557161 and 09671886 (H. Izumi)].
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