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RESEARCH REPORT |
1 Division of Dento-Oral Anesthesiology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba, Sendai, 980-8575, Japan;
2 Research Fellow of the Japan Society for the Promotion of Science; and
3 Department of Oral Physiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, Hokkaido, 061-0293, Japan
* corresponding author, izumih{at}hoku-iryo-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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1-adrenoceptor agonist methoxamine were markedly different: The reflex vasodilatation was not affected by methoxamine, while the direct vasodilatation was significantly reduced. Picrotoxin (GABAA receptor antagonist) attenuated the inhibitory effect of isoflurane on direct vasodilatation and the systemic arterial blood pressure. These findings suggest that the isoflurane-induced inhibitory effects on direct vasodilatation are produced by a decrease of peripheral vascular tone by GABAergic mechanisms, whereas those on the reflex vasodilatation are produced exclusively by the inhibition of the reflex center.
KEY WORDS: parasympathetic reflex • vasodilatation • isoflurane • submandibular gland • vascular tone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Deep anesthesia has been considered to reduce reflex responses involving salivation and blood flow changes in the submandibular gland (Al-Gailani et al., 1981). Similarly, volatile anesthetics—such as isoflurane, sevoflurane, and halothane—have been shown to suppress the lingual-nerve-evoked parasympathetic reflex vasodilatation in the lower lip and palate of cats (Izumi et al., 1997; Ito et al., 1998; Izumi and Ito, 1999), and their inhibitory effects have been deduced to act on the reflex center in the medulla (Izumi et al., 1997; Ito et al., 1998). However, in our preliminary experiments in the rat submandibular gland, not only the lingual-nerve-evoked parasympathetic reflex vasodilatation but also the chorda-lingual-nerve-evoked non-reflex parasympathetic vasodilatation were inhibited by the inhalation of isoflurane at higher concentrations. These findings suggested that there is a difference in the site of action of isoflurane for these two parasympathetic vasodilatations.
Isoflurane has been reported to decrease systemic arterial blood pressure by decreasing vascular tone (Bernard et al., 1990, 1992; Conzen et al., 1992; Malan et al., 1995), and the vascular tone affects the magnitude of the vasodilatation (Karita and Izumi, 1995). These findings led us to hypothesize that the peripheral vascular tone plays a role in the suppressive effects of isoflurane on the parasympathetic vasodilatations. To test this hypothesis, we have examined whether alterations of the vascular tone induced by the inhalation of isoflurane would affect the parasympathetic vasodilator response in the rat submandibular gland. In addition, we compared the effects of isoflurane on lingual-nerve- and chorda-lingual-nerve-evoked parasympathetic vasodilatations with and without the continuous administration of methoxamine to maintain peripheral vascular tone.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments were performed on 21 male Wistar rats weighing from 320 to 400 g each. After induction of anesthesia by diethyl ether, urethane (1.0 g/kg) was injected subcutaneously, and both femoral veins were cannulated for drug injection. Urethane was supplemented as necessary throughout the experiments (see below) to produce background anesthesia. Urethane was selected because it can induce deep anesthesia with minimal effects on the circulatory dynamics (Farber et al., 1995; Saito et al., 1995).
A femoral artery was also cannulated for measurement of the systemic arterial blood pressure. The anesthetized animals were intubated, paralyzed by an intravenous injection of pancuronium bromide (Mioblock; Organon, Teknica, Netherlands; 0.6 mg/kg initially, supplemented with 0.4 mg/kg/hr continuously), and artificially ventilated via a tracheal cannula with a mixture of 50% air-50% O2. The ventilator (Model SN-480-7; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 1 mL/100 g body weight at a rate of 50–60 breaths/min, and the end-tidal concentration of CO2 was measured with an infrared analyzer (Capnomac Ultima; Datex Co., Helsinki, Finland), as reported previously (Izumi, 1999; Izumi and Nakamura, 2000). The end-tidal CO2 was kept at 35–40 mm Hg. The rectal temperature was maintained at 37–38°C by means of a heating pad.
The criterion we used to determine whether the depth of anesthesia was adequate was whether a reflex elevation of systemic arterial blood pressure occurred in response to a noxious stimulus (such as pinching the upper lip for approximately 2 sec). If the depth of anesthesia was considered inadequate, additional urethane (100 mg/kg i.v.) was administered. Once an adequate depth of anesthesia had been attained, pancuronium was continuously administered to maintain immobilization during the periods of stimulation.
In all experiments, the cervical vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation, to eliminate the reflex actions of the vagus nerve on the cardiovascular system and the effects of sympathetic vasoconstrictor fibers on the orofacial area, respectively. This ensured that only non-vagal parasympathetic effects were being studied. All rats were killed at the end of the experiment by an overdose of pentobarbital sodium.
Electrical Stimulation of the Lingual Nerve and the Chorda-lingual Nerve
For lingual nerve stimulation, the central cut end of the lingual nerve was placed on a bipolar electrode (site A in Fig. 1A
) to elicit a parasympathetic reflex vasodilatation in the submandibular gland, as has been described (Mizuta et al., 2000). For chorda-lingual nerve stimulation, the peripheral cut end of the chorda-lingual nerve was reflected on the submandibular gland duct, and then both the submandibular gland duct and the chorda-lingual nerve were placed on a bipolar electrode (site B in Fig. 1A) to elicit a non-reflex vasodilatation in the submandibular gland (Mizuta et al., 2000).
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Measurement of Blood Flow in the Submandibular Gland and of Systemic Arterial Blood Pressure
The blood flow in the submandibular gland was monitored (Fig. 1A
) with a laser-Doppler flowmeter (model ALF21D; Advance, Tokyo, Japan), and recorded on a chart recorder as described (Izumi, 1999; Izumi and Nakamura, 2000). The probe was placed against the submandibular gland without exerting any pressure on the tissue. We assessed the blood flow changes by measuring the height of the response on the chart and expressed them in arbitrary units. We regarded increases in the submandibular gland blood flow as significant when the ratio between the magnitude of the blood flow increase and the amplitude of the baseline fluctuations ("signal-to-noise ratio") was more than 3 when either the lingual nerve or the chorda-lingual nerve was stimulated with supramaximal intensity.
The systemic arterial blood pressure was recorded from the femoral catheter via a pressure transducer (model TD-400T, Nihon Kohden, Tokyo, Japan).
Pharmacological Agents
Isoflurane, methoxamine, and picrotoxin were purchased from Abbott Japan (Tokyo, Japan), Nippon Shinyaku (Tokyo, Japan), and Wako Pure Chemical (Osaka, Japan), respectively.
Statistical Analyses
All numerical data are given as the mean ± SE. The significance of changes in the test responses was assessed by means of an unpaired Student’s t test or an analysis of variance (ANOVA), followed by a contrast test. Differences were considered significant at the P < 0.05 level. Data were analyzed in a Macintosh Computer equipped with StatView 5.0 and Super ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 2A, 2B). Inhalation of a low concentration of 1.0% isoflurane for 30 min significantly reduced the lingual-nerve-evoked reflex vasodilatation in the submandibular gland [F (6, 18) = 6.949, n = 4, P < 0.001] (Fig. 2A), but this level of isoflurane did not alter the chorda-lingual-nerve-evoked non-reflex vasodilatation [F (6, 30) = 1.854, n = 6, P = 0.122] (Fig. 2B). However, when isoflurane was inhaled at higher concentrations, 1.5 or 2.0%, not only the lingual-nerve-evoked but also the chorda-lingual-nerve-evoked vasodilatation in the submandibular gland were significantly reduced [lingual nerve; 1.5%, F (6, 24) = 14.586, n = 5, P < 0.001: 2.0%, F (6, 30) = 15.748, n = 6, P < 0.001: chorda-lingual nerve; 1.5%, F (6, 24) = 11.848, n = 5, P < 0.001; 2.0%; F (6, 24) = 27.769, n = 5, P < 0.001] (Figs. 2A, 2B). The inhibitory effect of isoflurane reached its maximum approximately 20 min after the start of isoflurane inhalation, and it had disappeared by 30 min after isoflurane inhalation stopped.
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1-adrenoceptor agonist, intravenously (4–10 µg/kg/min) to determine whether the isoflurane-induced reduction in the peripheral vascular tone contributed to the isoflurane-induced inhibition of vasodilatation in the submandibular gland.
Under these conditions, the lingual-nerve-evoked reflex vasodilatation in the submandibular gland was markedly reduced by the inhalation of isoflurane at any concentration [1.0%, F (6, 18) = 9.793, n = 4, P < 0.001: 1.5%, F (6, 18) = 7.664, n = 4, P < 0.001: 2.0%, F (6, 18) = 8.621, n = 4, P < 0.001] (Fig. 2C), whereas those evoked by the chorda-lingual nerve were not significantly reduced [1.0%, F (6, 18) = 1.486, n = 4, P = 0.239: 1.5%, F (6, 18) = 1.698, n = 4, P = 0.179: 2.0%, F (6, 18) = 2.521, n = 4, P = 0.060] (Fig. 2D).
Inhibitory Effect of Isoflurane on Systemic Arterial Blood Pressure with or without Continuous Administration of Methoxamine
Systemic arterial blood pressure was significantly decreased after the inhalation of isoflurane for 30 min in a concentration-dependent manner [1.0%, n = 8, P < 0.05: 1.5%, n = 7, P < 0.05: 2.0%, n = 8, P < 0.001; ANOVA followed by a contrast test] (Fig. 3). The maximum inhibitory effects of isoflurane on systemic arterial blood pressure had a time-course similar to those of lingual-nerve- or chorda-lingual-nerve-evoked blood flow increases in the submandibular gland (Fig. 1B). In contrast, when methoxamine was continuously infused, the change in systemic arterial blood pressure by the inhalation of isoflurane was not significant at any dose [1.0%, n = 8, P = 0.680: 1.5%, n = 5, P = 0.621: 2.0%, n = 5, P = 0.704; ANOVA followed by a contrast test].
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-aminobutyric acid type A (GABAA) receptor antagonist (2 mg/kg iv), prior to the inhalation of isoflurane. Picrotoxin substantially reversed the inhibitory effects of 30 min of isoflurane (2%) on the chorda-lingual-nerve-evoked non-reflex vasodilator response in the submandibular gland [from 69.17 ± 3.03%, n = 5, to 20.81 ± 7.03%, n = 5, of the pre-isoflurane response; P < 0.001; unpaired t test], as well as on mean arterial blood pressure (MAP) [from –45.00 ± 5.17 mm Hg, n = 8, to –25.00 ± 4.76 mm Hg, n = 4, changes from the pre-isoflurane level; P < 0.05; unpaired t test] (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 2A, 2B). These findings suggest that not only the reflex center within the brain stem but also the efferent parts of the reflex arc, such as the parasympathetic vasodilator fibers and/or effector tissues (blood vessels), are involved in the isoflurane-induced inhibition of parasympathetic vasodilatation.
We have examined whether peripheral vascular tone contributed to the isoflurane-induced inhibition of parasympathetic vasodilatation in the submandibular gland elicited by the lingual nerve or the chorda-lingual nerve stimulations. Our results showed that isoflurane inhibited the lingual-nerve-evoked parasympathetic reflex vasodilatation in the submandibular gland in a concentration- and time-dependent manner, and that these inhibitory effects were not affected by alteration of the peripheral vascular tone by the continuous administration of methoxamine (Figs. 2A, 2C). These results suggest that attenuation of peripheral vascular tone plays no role in the isoflurane-induced inhibition of lingual-nerve-evoked reflex vasodilatation. In other words, isoflurane has a direct effect on the reflex center for LN-evoked vasodilatation.
In contrast, the isoflurane-induced suppression of chorda-lingual-nerve-evoked non-reflex vasodilatation in the submandibular gland and systemic arterial blood pressure was significantly reversed to basal levels by the continuous administration of methoxamine (Figs. 2B, 2D, 3, 4). We have previously reported that when baseline blood flow was low (the blood vessels constricted), the amplitudes of the vasodilator responses evoked by parasympathetic nerve fibers were greater, and when the baseline blood flow was higher (the blood vessels are relaxed), the amplitudes of the vasodilator responses evoked via parasympathetic nerve fibers were weaker (Karita and Izumi, 1995). Our present results indicate that the peripheral vascular tone is important for the degree of parasympathetic vasodilator response.
Isoflurane has been reported to decrease the systemic vascular tone (Bernard et al., 1990, 1992; Conzen et al., 1992; Malan et al., 1995). The results of our study suggest that the isoflurane-induced suppression of chorda-lingual-nerve-evoked vasodilatation is due to a decrease of peripheral vascular tone induced by the inhalation of isoflurane. Prior to the administration of picrotoxin, a non-competitive GABAA receptor antagonist significantly attenuated the inhibitory effect of isoflurane on the chorda-lingual-nerve-evoked non-reflex vasodilator response in the submandibular gland, as well as on systemic arterial blood pressure (Fig. 4A). GABA has been shown to decrease vascular tone by suppressing the release of noradrenaline in the isolated rabbit ear artery and rat kidney (Manzini et al., 1985; Monasterolo et al., 1996; Fujimura et al., 1999). Isoflurane was also reported to decrease vascular tone through an alteration of vascular smooth-muscle cells’ vasomotor response to noradrenaline (Brendel and Johns, 1992; Flynn et al., 1992; Ozhan et al., 1994). These findings suggest that the decrease of peripheral vascular tone induced by the inhalation of isoflurane is largely due to GABAergic mechanisms.
The drop in systemic arterial blood pressure was not completely diminished after the administration of picrotoxin (Fig. 4B). From these data, it seems that the suppressive effects on systemic arterial blood pressure are induced by GABAergic mechanisms in addition to as-yet-unknown mechanisms.
In conclusion, there are at least two different mechanisms inducing the inhibitory effects of isoflurane on parasympathetic vasodilatation in the submandibular gland: One is due to a decrease of peripheral vascular tone by the GABAergic mechanism, and the other is due to the inhibition of the reflex center. Thus, we must be cautious in identifying the mechanisms by which inhalation anesthetics as well as general anesthetics evoke inhibitory effects on parasympathetic-mediated vasodilatation in the submandibular gland.
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ACKNOWLEDGMENTS |
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Received June 8, 2005; Last revision October 24, 2005; Accepted November 9, 2005
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REFERENCES |
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