The inhalation of high concentrations of isoflurane has been reported to increase the heart rate and the concentration of serum catecholamines. Although the precise mechanisms for the sympathetic activation of isoflurane have yet to be clearly elucidated, they are considered to possibly originate from the stimulation of airway sensory afferents, the baroreceptor reflex, or the direct stimulation of the central nervous system. To determine how these three mechanisms contribute to sympathetic augmentation, the effects of lower airway deafferentation and baroreceptor deafferentation on the isoflurane-induced changes in the renal sympathetic nerve activity (RSNA) in tracheally intubated rabbits were examined.
Twenty rabbits were given basal anesthesia. After tracheotomy and during mechanical ventilation, the changes in the heart rate, mean arterial pressure, and RSNA in response to random exposures to 1%, 2%, 3%, and 4% isoflurane were examined. The animals were assigned to one of three groups; 1, the intact group (n = 6); 2, the baroreceptor-deafferented group (n = 9), in which the sinoaortic plus vagal nerves were cut; and 3, the lower airway-deafferented group (n = 5), which underwent a bilateral vagotomy. The exposure to isoflurane was for 10 min in group 1 and 5 min in groups 2 and 3. At least 1 h was allowed for the recovery interval between exposures to isoflurane.
The inhalation of isoflurane caused dose-dependent increases in RSNA in all three groups. RSNA during high concentrations of isoflurane began to increase at 1 min, reaching the maximum at 4 or 5 min in group 1 (2.8- and 3.8-fold at 3% and 4% isoflurane, respectively) and group 3 (2.8- and 4.5-fold at 3% and 4% isoflurane, respectively), but it reached the peak at 2 or 3 min in group 2 (1.7- and 2.4-fold at 3% and 4% isoflurane, respectively) after the initiation of inhalation, in association with early slight increases followed by decreases of mean arterial pressure in groups 1 and 2 but only gradual decreases of mean arterial pressure in group 3. The increases in RSNA in group 3 were similar to group 1, however, those in group 2 were significantly attenuated compared with group 1.
The inhalation of isoflurane caused an increase of RSNA in intact, baroreceptor-deafferented, and lower airway-deafferented rabbits. The extent of the increases in RSNA was greater in intact and lower airway-deafferented rabbits than in baroreceptor-deafferented rabbits. Therefore, it is suggested that isoflurane may increase the efferent sympathetic nerve activity via the direct stimulation of the central nervous system and via the arterial baroreceptor reflex reflecting the reduction in arterial blood pressure. The stimulation of the vagally innervated airway may not contribute to the increase in the sympathetic nerve activity by isoflurane.
Key words: Airway stimulation. Anesthetics, volatile: isoflurane. Central nervous system: excitation. Nerve: activity; recording. Reflexes: baroreceptor. Sympathetic nervous system: reflexes.
ISOFLURANE has been reported to increase the heart rate (HR) in animals [1,2]and humans. [3-7]The concomitant increases in plasma norepinephrine and epinephrine concentrations also have been reported, especially when the inhaled concentration of isoflurane is rapidly increased. [3,5,8-10]These findings suggest that isoflurane inhalation during a rapid change of concentration may stimulate the sympathetic nervous system. The origin of this potential stimulation due to the administration of isoflurane could be related to the stimulation of airway sensory afferents, [3-5]the baroreceptor reflex, [11,12]and/or the direct stimulation of the central nervous system. This study was designed to document the presence and the time course of sympathetic activation of isoflurane by direct recordings of nerve outflow to the renal circulation and to dissect potential mechanisms by doing lower airway deafferentation and baroreceptor deafferentation in rabbits.
Methods and Materials
This study was approved by our institutional animal care committee. The experiments were performed on 20 Japanese white rabbits weighing from 2.7 to 3.8 kg. The animals were anesthetized initially with pentobarbital sodium (30 mg *symbol* kg sup -1). alpha-Chloralose (10 mg *symbol* kg sup -1 bolus and 10 mg *symbol* kg sup -1 *symbol* h sup -1) was given intravenously as basal anesthesia. After tracheostomy, the lungs were mechanically ventilated with a Harvard respirator (Mills, MA). A ligation of the trachea above the tracheostomy was performed to rule out the effect of isoflurane on the nasal mucosa, pharynx, and larynx. The arterial blood gases and pH were measured periodically to confirm appropriate ventilation. Body temperature was maintained above the 37 degrees C by a heating pad and a heating lamp. A 16-G double-lumen catheter was placed into the right femoral vein as a route for drug administration. An 18-G catheter was placed into the right femoral artery and connected to a pressure transducer (Uniflow, Baxter, CA) and a carrier amplifier (AP 601G, Nihon Kohden, Tokyo, Japan). HR was determined from the arterial pressure wave with a HR counter (AT 601G, Nihon Kohden).
The method for nerve recording has been reported. [13,14]Briefly, the left renal sympathetic nerves were exposed under a microscope, cut distally, and covered with warm mineral oil. The efferent renal sympathetic nerve activity (RSNA) was recorded with a bipolar stainless steel electrode. Impulses were amplified and fed into a oscilloscope (VC-10, Nihon Kohden), which was connected to a nerve spike counter (MET-1100, Nihon Kohden) with a window discriminator set just above the noise level. The spikes were counted and integrated at 2-s intervals. The raw electroneurogram, the output from the spike counter, HR, and arterial pressure, were displayed on a Macintosh-IISi computer (Apple, Cupertino, CA) through an analog-to-digital converter (Mac-Lab, Analog Digital Instruments, Castle Hill, Australia). Changes in RSNA were expressed as %RSNA, which was determined based on changes from the baseline level, designated as 100%.
The vagi were exposed and cut through a mid cervical incision. Sinoaortic denervation was performed by making a bilateral section of the aortic nerves, interrupting all nerve fibers between the internal and external carotid artery, and stripping the adjacent adventitia in the region of the carotid sinus. The effectiveness of sinoaortic denervation was confirmed by the absence of a decrease in HR and RSNA after a phenylephrine-induced increase in arterial pressure.
After the completion of surgical procedures, at least 1 h was allowed for the stabilization of hemodynamic variables and RSNA under basal anesthesia of alpha-chloralose. The study was divided into the following three groups.
Group 1. Isoflurane in Intact Rabbits (n = 6). Four concentrations of isoflurane--1%, 2%, 3%, and 4%--were randomly inhaled for 10 min in each animal, and changes in RSNA, HR, and mean arterial pressure (MAP) were examined. At least 1 h was allowed for the washout of isoflurane to be achieved after each change in the inspired concentration.
Group 2. Isoflurane in Baroreceptor-deafferented Rabbits (n = 9). After sinoaortic denervation and vagotomy, 1%, 2%, 3%, and 4% isoflurane were randomly inhaled for 5 min, and changes in RSNA, HR, and MAP were examined. One hour was allowed between each exposure.
Group 3. Isoflurane in Lower Airway-deafferented Rabbits (n = 5). After a bilateral vagotomy, 1%, 2%, 3%, and 4% isoflurane were randomly inhaled for 5 min, and the changes in RSNA, HR, and MAP were examined.
In a preliminary study, we demonstrated that MAP severely declined in the baroreceptor-deafferented rabbits if 4% isoflurane was administered for 10 min, whereas RSNA demonstrated a maximal change within 5 min. Therefore, the exposures to isoflurane in groups 2 and 3 were set for 5 min, rather than 10 min as in group 1. The vaporizer was calibrated over the appropriate range of concentrations using a gas analyzer (5250 RGM Ohmeda, Liberty Corner, NJ), and the ability of the vaporizer to maintain these concentrations over the course of the study was established. The end-tidal concentrations of isoflurane were measured in five rabbits of group 1.
All data were expressed as the mean+/-SEM. Statistical comparisons of the baseline values, those of the maximum changes in %RSNA between different groups of rabbits, and those of the hemodynamic and %RSNA changes in repeated measures were performed with an analysis of variance, and post hoc analyses were performed with unpaired or paired t test followed by Bonferroni's correction. A value of P < 0.05 was considered statistically significant.
(Figure 1) shows the time courses of end-tidal concentrations of isoflurane during 10 min inhalation of 1%, 2%, 3%, and 4% isoflurane. The end-tidal concentrations steeply increased for the initial 1-2 min and relatively reached a plateau at 3-5 min after the start of inhalation.
(Table 1) shows the baseline values of HR, MAP, and RSNA in each group. HR in group 1 was significantly lower than that in groups 2 and 3, whereas MAP and RSNA in group 2 were significantly higher than those in groups 1 and 3.
(Figure 2) shows the effects of isoflurane on MAP in the three groups. In groups 1 and 3, MAP initially decreased at 1 min, recovered or reached a plateau from 2 to 3 min, then decreased. In group 2, isoflurane decreased MAP, although the decrease during 3% and 4% isoflurane reached a plateau from 2 to 3 min.
(Figure 3) shows the effects of isoflurane on HR in the three groups. HR increased during the inhalation of isoflurane in groups 1 and 3 but decreased in group 2.
(Figure 4) shows the effects of isoflurane on %RSNA in the three groups. Isoflurane increased %RSNA in a dose-related manner in the three groups within 5 min.
(Table 2) shows the maximum changes in %RSNA from the baseline values in response to the inhalation of 1%, 2%, 3%, and 4% isoflurane. Isoflurane dose-dependently increased %RSNA in the three groups. The maximum increases in %RSNA in group 3 were similar to those in group 1, whereas those in group 2 were significantly less than in groups 1 and 3.
(Figure 5) shows the comparisons among maximum changes of MAP, HR, and %RSNA during inhalation of 2% and 4% in three groups. It appeared that the maximum decreases in MAP in group 2 were significantly greater than group 3. HR increased in groups 2 and 3 but decreased in group 2. The maximum increases in %RSNA in group 3 were similar to those in group 1, whereas in group 2 were less than those of groups 1 and 3.
(Figure 6), Figure 7, Figure 8show the representative tracings of responses in MAP and RSNA to 4% isoflurane in each group. In group 1 (Figure 6), RSNA increased gradually, reached a maximum, and gradually declined thereafter, whereas MAP initially decreased, recovered or even exceeded the baseline level, and then gradually but substantially decreased. In group 2 (Figure 7), RSNA increased and MAP began to decrease after the start of inhalation with a small notch. In group 3 (Figure 8), RSNA increased, and MAP slightly decreased, recovered, and substantially decreased thereafter.
This study provided three principal findings. First, inhaled isoflurane caused a dose-dependent increase in RSNA in tracheally intubated rabbits. Second, the increase in RSNA was not attenuated in the lower airway-deafferented rabbits. Third, the increase in RSNA was attenuated but remained present in the baroreceptor-deafferented rabbits.
An increase in the sympathetic nerve activity during the inhalation of a high concentration of isoflurane has been postulated by many investigators. [3-5,8-10]An increase in HR often is seen during isoflurane anesthesia. [1-7]The plasma concentrations of norepinephrine and epinephrine have been reported to increase during acute exposure to 5% isoflurane. On the other hand, Ebert and Muzi were unable to demonstrate changes in sympathetic nerve activity directed to the vasculature of human skeletal muscles when isoflurane was increased from 1% to 1.8%, contrasted to a 2.5-fold increase in muscle sympathetic nerve activity during equipotent increases in the inspired desflurane inhalation. However, the same authors reported that a rapid exposure to a 5% inspired concentration of isoflurane caused a brief but substantial increase in muscle sympathetic nerve activity in humans. In this study, we demonstrated using a direct recording of the renal sympathetic nerve that isoflurane-induced sympathetic activation occurred in a dose-dependent manner and reached a maximum of 1.5-, 2.1-, 2.8-, and 3.8-fold during the inhalation of 1%, 2%, 3%, and 4% isoflurane, respectively. These peak effects occurred 4 or 5 min after initiating the isoflurane at a time coincident with a significant decrease in blood pressure and a significant increase in HR. Our results about RSNA changes during 4% isoflurane inhalation in rabbits were similar to those of Ebert and Muzi in terms of the time course and magnitude in which the skeletal sympathetic nerve activity increased with a maximum of 3.5-fold at 3-4 min after advancing the isoflurane vaporizer from 1.2% to 5%.
Regarding the mechanism of sympathoexcitation produced by inhalation of isoflurane, at least three possible causes are considered, including irritation of the airway, the baroreceptor reflex, and direct stimulation of the central nervous system. First, irritation of the airway by isoflurane inhalation may initiate reflex increases in sympathetic nerve activity. Isoflurane is a pungent anesthetic associated with laryngospasm and breath-holding during the induction of anesthesia. We used rabbits with a tracheostomy and ligation of the trachea above the tracheostomy to rule out the effect of isoflurane on the nasal mucosa, pharynx, and larynx. Thus, the receptors of the upper airways were bypassed in this study, however, the receptors of the lower airways would be the site of irritation if the observed sympathoaugmentation is attributable to airway irritation. The vagus nerves supply the respiratory tract from the larynx down to the smallest airways and alveoli. Irritant receptors connected with vagal myelinated nerve fibers and other receptors attached to vagal C-fibers are thought to be activated by mechanical stimuli, the inhalation of various irritant gases, such as cigarette smoke, sulfur dioxide, and ammonia, and other chemical mediators. [19-21]Coleridge et al. showed that high concentrations of halothane, ether, chloroform, or trichloroethylene initially produce an excitation of pulmonary stretch receptors innervated by vagal fibers. Accordingly, a bilateral vagotomy appears to eliminate the conduction of sensory and/or afferent information from nerve receptors in the walls of the lower airways and lungs. Therefore, the finding that the increases in %RSNA after exposure to isoflurane was not attenuated in the lower airway-deafferented rabbits (group 3) compared with the intact rabbits (group 1) suggests that airway stimulation is not the major source of the sympathoexcitation. However, our study could not exclude the possibility of different efferent pathways from the lungs other than those traveling in the vagus nerve as a possible source contributing to responses via lower airway stimulation.
Second, the unloading of baroreceptors in association with a reduction of blood pressure during isoflurane inhalation may result in a baroreflex-induced increase in sympathetic efferent nerve activity. Isoflurane decreases blood pressure by its direct depressant effect on the myocardium and vascular smooth muscle. Isoflurane has been reported to cause a lesser attenuation of baroreflex function than halothane or enflurane. [11,12,25]Thus, the isoflurane-induced increase in RSNA might be attributable to unloading of baroreceptors. To exclude the contribution of the baroreflex, we performed sinoaortic plus vagal deafferentation. The carotid sinus, aortic, and vagal nerves carry the afferent information from the carotid sinus baroreceptors, the aortic baroreceptors, and the cardiopulmonary baroreceptors, respectively. The elimination of the baroreflex was confirmed by a lack of decrease in HR and RSNA in response to an increase in the blood pressure by phenylephrine. Therefore, the finding that the increases in %RSNA regarding exposure to isoflurane were significantly attenuated in the baroreceptor-deafferented rabbits (group 2) compared to the intact rabbits (group 1) thus suggests that the baroreflex plays an important role in sympathoexcitation.
Third, isoflurane may directly stimulate the central nervous system. The finding that the increase in RSNA was observed even in the baroreceptor-deafferented rabbits (group 2) suggests a direct stimulating effect of isoflurane on the central nervous system. Volatile anesthetics, such as cyclopropane, diethyl ether, fluroxene, halothane, have been reported to cause direct central sympathetic excitation, in which inhalation of high concentrations of these anesthetics revealed an increase of cervical sympathetic activity in baroreceptor-denervated and decerebrated cats. The exact site of the sympathoexcitation in the central nervous system remains unclear, but the ventral medulla that regulates the tone of the sympathetic nervous system activity may be a candidate.
The baseline RSNA was significantly higher in group 2 than group 1 (Table 1), suggesting that the afferent input from baroreceptors exerts a tonic inhibition of RSNA. The maximum increases in %RSNA in groups 1 (intact) and 2 (baroreceptor-deafferented) during 4% isoflurane were 285% and 140% (Figure 4and Figure 5and Table 2), whereas the baseline values of RSNA were 72 versus 134 counts/s (Table 1). Accordingly, the maximum counts/second of RSNA in both groups were observed to be at almost equivalent levels. It is suggested that the sympathoexcitation of 4% isoflurane in intact rabbits may arise from the full unloading effects on the baroreceptor reflex plus the direct stimulation of the central nervous system. Figure 4shows that RSNA increases were equivalent in all three groups at 2 min, but baroreceptor-deafferented rabbits (group 2) did not increase further; in contrast, groups 1 and 3 continued to increase their RSNA thereafter. Thus, we can speculate that the early increase in RSNA at either 2 or 3 min after inhalation may be primarily due to central nervous system stimulation, whereas the later augmentation in RSNA may be largely due to the baroreflex.
We measured RSNA as a representative of regional sympathetic outflows. However, there is spatial and temporal differing control of regional sympathetic outflow. It has been reported, for example, that an initial phase of sympathoexcitation in response to hemorrhage was unidirectional in renal, cardiac, liver, splenic, and adrenal sympathetic nerve activity, but a late phase of the changes was different among them. Therefore, the increase of RSNA observed in this study could be considered to reflect qualitatively the overall sympathetic activation, but it should be taken into account that the extent and time course of sympathetic activation might be different among different organs.
In conclusion, this study indicates that inhalation of isoflurane is associated with increases in RSNA in rabbits. This response can be attributed to a baroreceptor reflex mechanism responding to the simultaneous hypotension or direct stimulation of the central nervous system. The response does not appear to be mediated by stimulation of airway receptors that mediate their effects via vagal mechanisms.