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Anesthesia affects respiratory and sympathetic nerve activities differentially
Journal of the Autonomic Nervous System, 36 (1991) 225-236
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© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50 JANS 01222
Anesthesia affects respiratory and sympathetic nerve activities differentially Paul G. Wagner, F r e d e r i c L. Eldridge and Russell T. Dowell Departments of Physiology and Medicine, Universityof North Carolina, Chapel Hill, North Carolina, U.S.A. (Received 4 September 1990) (Revision received 13 August 1991) (Accepted 20 August 1991)
Key words: Control of breathing; Phrenic activity; Sympathetic activity; Anesthetic; Pentobarbital; Chloralose/urethane Abstract Phrenic and cervical sympathetic nerve responses to hypercapnia were examined before and after anesthesia in twelve midcollicularly decerebrated, vagotomized, glomectomized, paralyzed and ventilated cats. We measured responses of integrated phrenic and cervical sympathetic nerve activities to increases in end-tidal Pco 2 (PETCO2) from apneic threshold to approximately 30 torr above threshold. All cats were studied first in the unanesthetized state. Six cats were then restudied after a quarter of a usual dose of chloralose/urethane (10 mg/kg and 62.5 mg/kg, respectively) and then after half the usual dose of chloralose/ urethane (20 mg/kg and 125 mg/kg). The other six animals were restudied after quarter of a standard dose of pentobarbital (9 mg/kg), after half the standard dose (18 mg/kg) and then after the full (35 mg/kg) dose. Both anesthetic agents led to significant increases in apneic thresholds for both phrenic and sympathetic nerve activities. These agents also caused dose-dependent decreases in peak, tonic and respiratory-related sympathetic nerk,e activities. Peak (tidal) phrenic nerve activities, in comparison, were much less affected by the anesthetic agents. CO 2 response curves showed that both of these anesthetic agents depressed, at any given level of PETCO2, respiratory-related sympathetic nerve responses more than the responses found in the phrenic nerve. We conclude that the relations between peak, tonic (i.e. between phasic bursts) and respiratory-related sympathetic nerve activities and phrenic nerve activity can be altered by anesthesia.
Introduction Many questions being asked in the areas of respiratory and cardiovascular control cannot be readily studied in awake, conscious animals. This has necessitated the use of anesthetic agents which may have a depressant effect upon neurons
Correspondence." F.L. Eldridge, Department of Physiology CB#7545, University of North Carolina, Chapel Hill, NC 27599, U.S.A.
of the central nervous system that are involved in the control of breathing and circulation. Some of the effects of anesthesia on the responses of the respiratory and cardiovascular systems have been well-documented [7,4,17,21,28]. However, these studies only examined the effects of anesthesia on either respiratory or cardiovascular variables but not both. Caille et al. [4] have shown that anesthetic agents can have different effects on cells of different brain regions. It is known also that relations between phrenic and hypoglossal nerve activities are dramatically al-
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tered by anesthesia, the phasic inspiratory-related component of hypoglossal activity showing a much greater depression than the phrenic [4,14,21]. We also know that sympathetic nerves, like the hypoglossal, show respiratory-related phasic activity associated with each inspiration [1,19,22]. We therefore hypothesized that the relations between respiratory-related phrenic and sympathetic nerve activities would also be affected by anesthesia. If this were the case, then conclusions concerning the origin of the respiratory-related sympathetic activity, drawn from studies in which anesthesia was used, might need to be re-evaluated. We therefore studied phrenic and cervical sympathetic nerve responses to increasing respiratory drive produced by hypercapnia in decerebrate cats, first before they were anesthetized and then after anesthesia with increasing doses of either c h l o r a l o s e / u r e t h a n e ( C / U ) or pentobarbital. We found that the responses of these two systems are indeed differentially affected by anesthesia.
Materials and Methods Studies were performed in 12 healthy adult cats weighing between 2.6 and 3.4 kg. Each was anesthetized with ether and both carotid arteries ligated. A midcollicular decerebration was then performed under direct vision; the entire brain rostral to the section was removed by suction. To control bleeding, Gelfoam ® was placed over the exposed end of the brainstem. Once decerebrated, no more ether was given and the animals were allowed to recover for at least 2 h before the beginning of any experimental procedure. A femoral artery was cannulated for measurement of arterial pressure by means of a strain gauge. A femoral vein was also cannulated for later administration of anesthetic agents. Body temperature was monitored with a rectal thermistor and kept between 37 ° and 38°C by means of a servo-controlled DC heating pad. The trachea was cannulated through a neck incision and continuous sampling of airway P c o 2 was accomplished by means of a catheter placed in the airway. Analysis was made by an infrared CO 2
analyzer (Beckman LB-2). The animals were placed supine on a table equipped with a rigid head mount. The carotid sinus and vagus nerves were visually identified and sectioned in the neck region. Both cervical sympathetic nerve trunks were also visually identified, separated from the vagi and cut. A central end of one of these sympathetic nerves was desheathed and placed on a bipolar recording electrode and covered with mineral oil. One phrenic nerve root (C 5) was also exposed in the neck, cut, desheathed and placed on a bipolar recording electrode in a pool of warm mineral oil. These recording electrodes were built into small acrylic platforms that moved with the cat and allowed stable recording conditions throughout the experiments. Animals were ventilated with 100% oxygen using a volume-cycled ventilator. They were paralyzed with gallamine triethiode, 3 m g / k g initially, followed by a continuous infusion at a rate of 3 m g / k g / h to maintain paralysis. To prevent significant changes of end-tidal Pc'o: (PETCO2) and arterial P c o 2, a servo-controlled ventilator was used that allowed maintenance of PETCO2 within a narrow range ( + 0 . 5 torr) around any desired level [24]. When hypercapnia was to be induced, the set point of the servo-controller was changed to obtain the desired level. To determine responses to hypercapnia, the apneic threshold (the PETCO 2 at which rhythmic phrenic activity first appeared) was first determined. We then raised P c o 2 1 to 2 torr. After at least 5 rain, when the level of phrenic activity had become stable, we recorded all variables over a period of 1 rain. This was followed by progressive step increases in Pco 2 to more than 30 tort above threshold. At each level of Pco 2, all variables were recorded for I min after being allowed to stabilize for at least 5 rain.
Experimental protocol After all preparations had been completed, cardiovascular and respiratory variables were allowed to stabilize. A CO 2 response test was then performed. Following completion of this test, the first dose of one of the anesthetic agents was given intravenously and all variables were allowed
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to stabilize for at least 20 min before another CO 2 response curve, started again at apneic threshold, was generated. In six animals, responses to hypercapnia were determined under the following three conditions: (1) unanesthetized (control); (2) anesthetized (10 m g / k g chloralose/ 62.5 m g / k g urethane); and (3) after an additional dose (cumulative 20 m g / k g chloralose/125 m g / k g urethane). These doses of C / U anesthesia correspond to 1/4 and 1/2 the standard full dose. In the other six animals, the effects of the anesthetic pentobarbital (Nembutal, Abbott Laboratories) on the responses to progressive changes in PETCO: were tested in a similar manner. The following doses of pentobarbital were used: 1/4 (9 mg/kg) of a full dose, 1/2 dose (18 m g / k g cumulative) and a full dose (35 m g / k g cumulative). The final cumulative doses (i.e. C / U of 20 m g / k g and 125 m g / k g respectively, and pentobarbital of 35 mg/kg) used in the present study were chosen on the basis of their reported ability to induce similar levels of surgical anesthesia.
Data analysis Arterial pressure, airway Pco2, phrenic nerve impulses, integrated phrenic activity, integrated sympathetic nerve activity and ventilator rate were recorded on a Gould chart recorder (Model TA2000) and stored on magnetic tape. Breathby-breath analysis of phrenic activity was performed by computer. Both phrenic and sympathetic nerve activities were filtered (30-3 000 Hz), amplified, half-wave rectified and then integrated for each 0.1 s. Integration was performed by means of a voltage-to-frequency converter, whose output frequency is proportional to input voltage, and a digital counter (Hewlett-Packard Model 3525A). This combination of instruments is the equivalent of an integrating digital voltmeter [8]; the advantage of this type of integration is that averaged activity of each 0.1 s is discrete and not subject to temporal distortion. It has been shown that the peak value for each breath is the neural equivalent of tidal volume of breathing. Minute phrenic activity (frequency × tidal) was also calculated. We chose not to use minute activity for presenting our findings for two reasons: (1) we were more interested in the effects of anesthesia
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Fig. 1. Recording of integrated cervical sympathetic nerve activity showing its division into the components (peak, tonic and respiratory-related) used in this study. Baseline refers to the amount of activity recorded after section of the cervical sympathetic trunk central to the electrode.
on the magnitudes of phrenic nerve activity and those of the respiratory-related component of sympathetic nerve activity; and (2) since the rhythmic activities of the phrenic and sympathetic nerves were so tightly coupled, either tidal or minute activities could have been used without materially affecting the results. Peak sympathetic nerve activity was divided into two components: tonic and respiratory-related activities (see Fig. 1). Since sympathetic activity has a rhythm similar to respiration, we measured the level of activity of each of the components from the integrated analog recordings using-an IBM P C / X T , a digitizing tablet (Jandel, Inc.) and appropriate software (Sigmascan, Jandel, Inc.) for each respiratory cycle. Peak activity was defined as the highest level of activity recorded during a given cycle and usually corresponded to the peak of inspiration. Tonic activity was that which occurred between phasic bursts; its average integrated value was visually estimated from the analog recording and was defined as the level of activity above zero. The value for zero activity (or 'baseline') was obtained at the end of each experiment by sectioning the sympathetic nerve central to the recording electrode; the remaining level of activity was used as the zero level. Respiratory-related activity was calculated as the difference between peak and tonic activities.
228 F o r each 1-min r e c o r d i n g t a k e n at each step i n c r e a s e in P c o 2 , an average value for each variable was calculated. T h e s e d a t a w e r e t h e n norm a l i z e d by assigning 100 units to the largest tidal phrenic, p e a k s y m p a t h e t i c a n d r e s p i r a t o r y - r e l a t e d s y m p a t h e t i c nerve activities o b t a i n e d d u r i n g hy-
p e r c a p n i a in the u n a n e s t h e t i z e d state, i.e. control, as previously d e s c r i b e d by E l d r i d g e et al. [9]. A l l s u b s e q u e n t activities w e r e scaled accordingly. T o n i c s y m p a t h e t i c nerve activities w e r e scaled to the largest p e a k value o f s y m p a t h e t i c nerve activity o b t a i n e d d u r i n g h y p e r c a p n i a in the u n a n e s -
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END-TIDAL Pco2 (torr) CHANGE FROM THRESHOLD Fig. 2. Integrated phrenic and cervical sympathetic nerve responses to changes in PETCO2of l, 4, 13 and 35 torr above the apneic threshold (obtained in the unanesthetized state) in one animal under the following three conditions: (A) unanesthetized (decerebrate); (B) anesthetized with 1/4 of a standard dose of chloralose/urethane; (C) anesthetized with a 1/2 dose of chloralose/urethane. Note that with anesthesia the magnitude of sympathetic nerve activity is disproportionately more depressed than the phrenic.
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thetized state. Respiratory-related activities were normalized separately in order that comparisons between the respiratory and sympathetic responses could be made. In addition to normalization, respiratory and sympathetic nerve activities and mean arterial pressures from all animals were grouped into 10 bins based on the changes of P c o 2 from threshold. It should be noted that the 'change', from threshold in the present study refers to the changes from 'control' apneic threshold for phrenic nerve activity determined prior to the administration of any anesthetic agent. The bins used were (0), (0.1-1), (1.1-2), (3.1-4), (4.1-6), (6.1-10), (10.2-18), (18.1-29) and ( > 2 9 ) torr
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Fig. 3. Responses of tidal phrenic and sympathetic nerve activities to changes in PETCO2 plotted for the same animal represented in Fig. 2: unanesthetized (A and A'), anesthetized with 1/4 standard dose of chloralose/urethane (B and B'), anesthetized with 1/2 dose of C / U , (C and C'). (A-C) tidal phrenic (circles) and respiratory-related sympathetic (triangles) activities; (A'-C') peak (circles) and tonic (triangles) sympathetic activities. Note the dose-dependent decreases of sympathetic activities while, in comparison, phrenic activity is minimally affected.
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Results
Chloralose / urethane Fig. 2 shows an example in one cat of the effects of chloralose/urethane ( C / U ) on the responses of integrated phrenic and sympathetic nerve activities to hypercapnia, which is quantified by the increments (1, 4, 13, 35 torr) of Pco 2 above phrenic apneic threshold in the control unanesthetized state; in this cat control apneic threshold was 28 tort. The hypercapnic responses and relations between the two activities before anesthesia are shown in Fig. 2A. It can be seen that phrenic activity increases more relative to sympathetic at low increments of Pco~ and less relative to sympathetic at high increments of Pco 2.
C / U anesthesia led to increases of apneic threshold over control for both phrenic and respiratory-related sympathetic activities, +4.5 torr Pco 2 after the ~/4 dose (fig. 2B) and + 9 torr after the ~/2 dose (Fig. 2C). Threshold changes for phrenic and respiratory-related sympathetic activities were not different. However, it is obvious from the recordings shown in Fig. 2 that the effects on the activities of increasing C / U are different: the sympathetic decreasing more than the phrenic at the same level of hypercapnia. These differences in this cat are more clearly seen in Fig. 3, where tidal phrenic and respiratory-related sympathetic responses to hypercapnia are plotted in panels 3A (control), 3B (J/4 dose C / U ) and 3C (1/2 dose C / U ) . Responses of peak and tonic sympathetic activities are plot-
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Fig. 4. Average data from six animals showing effects of chloralose/urethane anesthesia on hypercapnic responses in the phrenic nerve (A-C, circles), respiratory-related (A-C, triangles), peak (A'-C', circles) and tonic (A'-C', triangles) sympathetic nerve activities. (A-A') unanesthetized; (B-B') anesthetized n/4 dose; (C-C') I/2 dose. Error bars represent SEM.
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ted in panels 3A' (control), 3B' (1/4 dose) and 3C' (1/2 dose). We have already noted that C / U caused rightward shifts in the thresholds for phrenic and sympathetic nerve activities. The whole CO 2 response curves are similarly shifted to the right. In addition to affecting the thresholds, C / U affected the magnitudes of responses of these nerves. Tidal phrenic activities observed with the highest levels of PETCO2 were: control = 100 (panel 3A), 1/4 dose = 95.7 (panel 3B) and 1/2 dose = 67.4 units (panel 3C), only a modest decrease with increasing C / U . Respiratory-related sympathetic activities for the same changes in PETCO 2, on the other hand, were disproportionately more depressed: control = 98.2 units (panel 3A); I/4 dose = 57.7 (panel 3B) and 1/2 dose = 19.7 (panel 3C), largely because of marked decreases in peak sympathetic activity: control = 99.9 units (panel 3A'), 1/4 dose = 60.4 (panel 3B') and 1/2 dose = 18.2 (panel 3C'). Tonic sympathetic activities also decreased with increasing A
B
C / U : (control = 26.6 units (panel 3A'), 1/4 dose = 17.6 (panel 3B') and 1/2 dose =3.6 (panel 3C'). Averaged data from the six cats for tidal phrenic and respiratory-related sympathetic activities (Fig. 4, panels A, B, C) yielded similar findings. Thresholds for phrenic and sympathetic activities after the 1/4 dose of C / U were not different from control but were higher (P < 0.05) than the control with the 1/2 dose. The findings were similar to the cat as represented in Fig. 3 in showing similar disproportionate decreases, relative to phrenic, of respiratory-related sympathetic activity (panels A, B, C), again associated with decreases of peak (panels 4A', 4B' and 4C') and tonic (panels 4A', 4B' and 4C'). Phrenic nerve responses to hypercapnia showed a change in the shape of the curve after the 1/4 dose of C / U (panel 4B) but no decrease of maximal response. There was a small and significant decrease (26.3%) in maximal activity following the 1/2 dose (panel 4C). In contrast, C
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Fig. 5. Average data from six animals showing effects of pentobarbital anesthesia on hypercapnic responses in phrenic nerve (A-D, circles), respiratory-related (A-D, triangles), peak (A'-I)', circles) and tonic (A'-I)', triangles) sympathetic activities. (A-A') unanesthetized; (B-B') anesthetized 1/4 dose; (C-C') 1/2 dose. (D-I)') full dose. Error bars represent SEM.
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the respiratory-related component of sympathetic activity exhibited significant decreases in maximal activity after 1/4 dose (39.6%, panel 4B) and after the 1/2 dose of C / U (67.3%, panel 4C). Peak and tonic sympathetic activities (Figs. 4A'C') also showed decreases after each increment of anesthetic. Maximal peak sympathetic activity decreased by 34.4% after the 1/4 dose and by 62.3% after the 1/2 dose. The tonic level of activity dropped by 26.9% after the 1/4 dose and an additional 27.9% following the 1/2 dose.
increased by only 4.9 torr but the threshold for sympathetic nerve activity increased by 15.2 torr. After a full dose of pentobarbital, both thresholds were significantly greater than their respective controls. Maximal phrenic activity showed no significant changes until after the full dose of anesthetic which caused a 36.3% decrease. However, respiratory-related sympathetic nerve activity decreased by 38.7% after only a 1/2 dose and by 69.9% following a full dose. Levels of peak and tonic sympathetic activity also fell progressively with increasing doses of pentobarbital (Fig. 5, panels B', C' and D').
Pentobarbital Data from the six cats in which pentobarbital was given are plotted in Fig. 5. Panels 5A-5D show the responses to hypercapnia for tidal phrenic and respiratory-related sympathetic activities and panels 5A'-5D' the responses of peak and tonic sympathetic nerve activities. The curves in panels 5A/5A' show the hypercapnic responses of the unanesthetized animals; they are followed by plots of the 1/4 dose (panels 5B/5B'), 1/z dose (panels 5C/5C') and full dose (panels 5D/5D') of pentobarbital. As with C / U , thresholds for rhythmic phrenic and sympathetic nerve activities were not different, except after the full dose of pentobarbital, when the threshold for phrenic nerve activity
Phrenic-sympathetic relations The relationships between respiratory (phrenic) activity and respiratory-related sympathetic activity in the unanesthetized animals and after the various doses of pentobarbital or chloralose/ urethane are shown in Fig. 6. In the unanesthetized decerebrate animals there was a curvilinear relationship, with phrenic activity increasing more than sympathetic activity at low Pco 2 levels but sympathetic activity increasing more rapidly than phrenic when Pco 2 was at high levels. This relationship was unaltered after the ~/4 and ~/2 doses of pentobarbital but shifted to the right after the full dose, which is consistent with
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Fig. 6. Effects of various doses of pentobarbital and c h l o r a l o s e / u r e t h a n e anesthesia on relations between normalized tidal phrenic and respiratory-related sympathetic nerve activities. Dotted lines represent lines of identity.
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Fig. 7. Effects of changes in PETCO2 and different doses of the two anesthetic agents studied (six cats each) on mean arterial pressure.
the observed changes in thresholds and with an interpretation that sympathetic activity is depressed more than phrenic. The relatively greater decrease of respiratory-related sympathetic activity than phrenic with increasing doses of C / U is also seen in the figure. In Fig. 7, average mean arterial pressures are plotted against the changes of CO 2 that occurred during hypercapnic response tests in the animals anesthetized with pentobarbital (left) and those with C / U (right). Mean arterial pressures tended to increase in response to hypercapnia and to decrease in response to anesthesia, but the decreases from control with anesthesia were not significant ( P > 0.05) despite the significant changes in cervical sympathetic nerve activity.
Discussion
In this study we have shown that the anesthetic agents c h l o r a l o s e / u r e t h a n e and pentobarbital have differential effects on the CO 2 reponses of phrenic and cervical sympathetic nerves. Cervical sympathetic activities (peak, tonic and respiratory-related) were disproportionately more depressed by increasing large doses of anesthesia to changes in central chemoreceptor input than were the activities of the phrenic nerve. This difference in sensitivity to anesthesia altered the relations between the phrenic and sympathetic
nerves (Fig. 6) and suggests that autonomic neurons (involved in control of cardiovascular and other organs) are more sensitive to anesthetics than those controlling respiration. Qualitatively, these anesthetic agents (chlor a l o s e / u r e t h a n e and pentobarbital) produced similar results. Unfortunately, we had no method for quantitating the depth of anesthesia produced by a particular dose of anesthetic in these decerebrate animals. We therefore are unable to compare the effects of these anesthetics. However, it appears (Figs. 4 and 5) that a 1/2 dose of chlor a l o s e / u r e t h a n e is able to depress phrenic and sympathetic activities as much as a full dose of pentobarbital. We reported that anesthesia caused significant decreases of sympathetic nerve activity but not of mean arterial pressures. However, the mean data in Fig. 7 show that mean arterial pressures tended to decrease with increasing doses of anesthesia. There are several possible explanations for the lack of a significant effect. One is that mean arterial pressure is not a very satisfactory measure of sympathetic nerve output. A second is that the cervical sympathetic nerve does not efficiently reflect the sympathetic activity responsible for control of arterial pressure. Finally, since CO 2 can cause peripheral vasodilation [25], it is possible that the dilation may oppose the neurogenic vasoconstriction caused by sympathetic activity and thus offset changes.
234 Many studies have looked at the effects of anesthesia on either the cardiovascular [7,17,28] or respiratory [4,14,20] systems but few have looked at the effects of anesthesia on the relations between the two systems. Many studies examining the relations between the respiratory and cardiovascular systems have done so in anesthetized animals [2,11,19,26]. H a n n a et al. [11], using cats anesthetized with a full dose of pentobarbital and under conditions similar to the ones in our study (buffer nerves cut and hyperoxic), showed that the P c o 2 threshold for sympathetic nerve activity was at least 5 torr higher than the threshold for phrenic activity. These authors also plotted phrenic nerve activity against sympathetic nerve activity for various levels of P c o 2 and the resulting relationship was similar to that shown in Fig. 6. Koepchen et al. [15] found, in cats anesthetized with a full dose of c h l o r a l o s e / u r e t h a n e , that cervical sympathetic nerve activity correlated well with changes in the fractional concentration of Pco 2 in alveolar air and that the percent increase in sympathetic activity with increasing C O : was distinctly less than percent increase of phrenic activity. These data are consistent with the findings of the present study and highlight the fact that the relationship between the respiratory and cardiovascular systems is altered by anesthesia. On the other hand, Bachoo and Polosa [2] reported that increasing levels of pentobarbitone anesthesia in cats equally depressed phrenic and cervical sympathetic nerve activities. However, since the amount of data given in support of this interpretation is very limited, it is difficult to compare their results with ours. Our observations that anesthetics can have different effects depending on the cell populations being studied are not unique. Collins et al. [6], reported that chloralose depressed spontaneous and evoked activities in the dorsal horn of the spinal cord and, at similar doses, produced even greater depression of neurons in the nucleus reticularis gigantoeellaris. Caille et al. [4] examined the effects that pentobabitone had on various parts of the brain involved in the control of respiration. They found that cells in different regions of the brain, the mesencephalon, the
brainstem reticular formation and medullary respiratory groups, exhibited different sensitivities to pentobarbital; it had much less effect on the cells of the medullary respiratory groups than on cells of other regions. A similar effect of anesthesia on hypoglossal and phrenic nerve activities has been described [3,14,21]. We have found that hypoglossal activity is much more sensitive to anesthetic depression than either phrenic nerve or sympathetic nerve activities (unpublished data). It is becoming clear that anesthesia should not be used when one is studying the interactions between different neural populations because these agents modify the relationships of the unanesthetized state. Furthermore, conclusions drawn from data in which anesthetics have been used must be evaluated with caution. For example, differences in thresholds for sympathetic and phrenic nerve activities have been used as evidence that respiratory-related sympathetic nerve activity originates from an 'overflow' of activity from the respiratory system [19,22]. However, we found that the thresholds for sympathetic and phrenic nerve activities in the unanesthetized decerebrate animals were not different. Our findings therefore do not give support to the 'overflow' hypothesis and are consistent with the observed changes in threshold being due to the effects of anesthesia. Koepchen et al. [15] reported that even though the two systems are tightly linked they can be separated. He found that graded increases in Pco 2 from apneic threshold resulted in increases of sympathetic nerve activity which sometimes could be detected before phrenic bursts appeared. Trzebski and Kubin [261 reported that tonic levels of sympathetic nerve activity increased in response to changes in Pco 2 even though phrenic activity was below threshold. In designing this study, we were most interested in the changes in amplitudes of the rhythmic bursts of the phrenic and cervical sympathetic nerves. Though we did not specifically examine the questions of phase shifts or coupling between the two systems, we did not observe any obvious differences like those seen by the above mentioned authors [15,26]. At this time there exists no good explanation
235
as to why sympathetic nerve or hypoglossal nerve activities are more sensitive to anesthesia than the activities of the phrenic nerve. However, data from a number of laboratories suggest that the anesthetics pentobarbital and alpha-chloralose may mimic or enhance the actions of G A B A [13,16]. G A B A and its agonists like muscimol have been shown to depress both respiratory and cardiovascular activities [10,12,27]. Unfortunately, studies in which the effects of these substances on phrenic nerve and sympathetic nerve activities were quantitated and compared have not been done. However, it is interesting to speculate that the differences in sensitivity observed in our study are due to differences in either the number of receptors or subtypes of receptors that a particular cell population has. Though both systems may be affected by substances known to be selective for GABA-B receptors [5,23], it is known that GABA-B receptors are insensitive to barbiturate [10]. Further studies will be needed to ascertain what role G A B A receptors play, if any, in determining a system's sensitivity to a particular anesthetic and how this change in sensitivity is accomplished. The present study shows that anesthetic agents such as c h l o r a l o s e / u r e t h a n e and pentobarbital depress cervical sympathetic nerve responses to changes in P¢o 2 more than those of the phrenic nerve and that this difference in sensitivity to anesthesia alters the relations between the two systems. We therefore conclude that the unanesthetized decerebrate preparation must be considered when designing studies in which the interactions between the cardiovascular and respiratory control systems are involved and that data from studies in which anesthetics have been used must be interpreted with caution.
Acknowledgements The authors express their appreciation to Mrs. Lynn M. Houser for her excellent technical assistance. This work was supported by USPHS M E R I T Award Grant HL-17689. Paul Wagner also received doctoral scholarship support from
Glaxo Pharmaceutical, Research Triangle Park, NC.
References 1 Adrian, E.D., Bronk, D.W. and Phillips, G., Discharges in mammalian sympathetic nerves, J. Physiol. (Lond.), 74 (1932) 115-133. 2 Bachoo, M. and Polosa, C., Properties of the inspirationrelated activity of sympathetic preganglionic neurones of the cervical trunk in the cat, J. Physiol.(Lond.), 385 (1987) 545-564. 3 Bennett, F.M and St. John, W.M., Anesthesia selectively reduces hypoglossal nerve activity by actions upon the brain stem, Pfliigers Arch., 401 (1984) 421-423. 4 Caille, D., Vibert, J.F., Bertrand, F., Gromysz, H. and Hugelin, A., Pentobarbitone effects on respiration related units; selective depression of bulbopontine reticular neurones, Respir. Physiol., 36 (1979) 201-216. 5 Chahl, L.A. and Walker, S.B., The effect of baclofen on the cardiovascular system of the rat, Br. J. Pharmacol., 69 (1980) 631-637. 6 Collins, J.G., Kawahara, M., Homma, E. and Kitahata, L.M., Alpha-chloralose suppression of neuronal activity, Life Sci., 32 (1983) 2995-2999. 7 Covert, R.F., Drummond, W.H. and Gimotty, P.A., Chloralose alters circulatory responses to alpha-receptor stimulation and blockade, Am. J. Physiol., 255 (1988) H419H425. 8 Eldridge, F.L., Relationship between respiratory nerve and muscle activity and muscle force output, J. Appl. Physiol., 39 (1975) 567-574. 9 Eldridge, F.L., Gill-Kumar, P. and Millhorn, D.E., Inputoutput relationships of central neural circuits involved in respiration in cats, J. Physiol.(Lond.), 39 (1981) 81-95. 10 Gillis, R.A., Yamada, K.A., Dimicco, J.A., Williford, D.J., Segal, S.A., Hamosh, P. and Norman, W.P., Central gamma-aminobutyric acid involvement in blood pressure control, Fed. Proc., 43 (1984) 32-38. 11 Hanna, B.D., Lioy, F. and Polosa, C., Role of carotid and central chemoreceptors in the CO 2 response of preganglionic neurons, J. Auton. Nerv. Syst., 3 (1981) 421-435. 12 Holtman, J.R. Jr. and King, K.A., Regulation of respiratory motor outflow to the larynx and diaphragm by GABA receptors, Eur. J. Pharmacol., 156 (1988) 181-187. 13 Hurle, M.A., Dierssen, M.N. and Florez, J., Mechanism of the respiratory action of pentobarbital at the medullary and pontine levels, Eur. J. Pharm., 125 (1986) 225-232. 14 Hwang, J.C., St. John, W.M. and Bartlett, D. Jr., Respiratory-related hypoglossal nerve activity: influence of anesthetics, J. Appl. Physiol., 55 (1983) 785-792. 15 Koepchen, H.P., Klussendorf, D. and Sommer, D., Neurophysiological background of central neural cardiovascular-respiratory coordination: Basic remarks and experimental approaches, J. Auton. Nerv. Syst., 3 (1981) 355-368.
236 16 Lalley, P.M., Inhibition of depressor cardiovascular reflexes by a derivative of gamma-aminobutyric acid (GABA) and by general anesthetics with suspected GABA-mimetic effects, J. Pharmacol. Exp. Ther., 215 (1980) 418-425. 17 Matsukawa, K. and Ninomiya, I., Anesthetic effects on tonic and reflex renal sympathetic nerve activity in awake cats, Am. J. Physiol., 256 (1989) R371-R378. 18 Matsumoto, R.R., GABA receptors: are cellular differences reflected in function? Brain Res. Rev., 14 (1989) 203-225. 19 Millhorn, D.E., Neural respiratory and circulatory interaction during chemoreceptor stimulation and cooling of ventral medulla in cats, J. Physiol. (Lond.), 370 (1986) 217-231. 20 Mitra, J., Prabhakar, N.R., Haxhiu, M. and Cherniack, N.8., Comparison of the effects of hypercapnia on phrenic and hypoglossal activity in anesthetized decerebrate and decorticate animals, Brain Res. Bull., 17 (1986) 181-187. 21 Nishino, T., Shirahaata, M., Yonezawa, T. and Honda, Y., Comparison of changes in the hypoglossal and phrenic nerve activity in response to increasing depth of anesthesia in cats, Anesthesiology, 60 (1984) 19-24. 22 Preiss, G., Kirchner, F. and Polosa, C., Patterning of
23
24
25
26
27
28
sympathetic preganglionic neuron firing by the central respiratory drive, Brain Res., 87 (1975) 363-374. Schmid, K., Bohmer, G. and Gebauer, K., GABA-B receptor mediated effects on central respiratory system and their antagonism by phaclofen, Neurosci. Lett. 99 (1989) 305-310. Smith, D.M., Mercer, R.R. and Eldridge, F.L., Servo-control of end-tidal CO 2 in paralyzed animals, J. Appl. Physiol., 45 (1978) 133-136. Somlyo, A.P. and Somlyo, A.V., Vascular smooth muscle. II. Pharmacology of normal and hypertensive vessels. Pharmacological Rev., 22 (1970) 249-353. Trzebski, A. and Kubin, L., Is the central inspiratory activity responsible for Pco2-dependent drive of the sympathetic discharge? J. Auton. Nerv. Syst., 3 (1981) 401-420. Yamada, K.A., Norman, W.P., Hamosh, P. and Gillis, R,A., Medullary ventral surface GABA receptors affect respiratory and cardiovascular function, Brain Res., 248 (1982) 71-78. Zimpfer, M., Sit, S.P. and Vatner, S.F., Effects of anesthesia on the canine carotid chemoreceptor reflex, Circ. Res., 48 (1981) 400-406.
225
© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50 JANS 01222
Anesthesia affects respiratory and sympathetic nerve activities differentially Paul G. Wagner, F r e d e r i c L. Eldridge and Russell T. Dowell Departments of Physiology and Medicine, Universityof North Carolina, Chapel Hill, North Carolina, U.S.A. (Received 4 September 1990) (Revision received 13 August 1991) (Accepted 20 August 1991)
Key words: Control of breathing; Phrenic activity; Sympathetic activity; Anesthetic; Pentobarbital; Chloralose/urethane Abstract Phrenic and cervical sympathetic nerve responses to hypercapnia were examined before and after anesthesia in twelve midcollicularly decerebrated, vagotomized, glomectomized, paralyzed and ventilated cats. We measured responses of integrated phrenic and cervical sympathetic nerve activities to increases in end-tidal Pco 2 (PETCO2) from apneic threshold to approximately 30 torr above threshold. All cats were studied first in the unanesthetized state. Six cats were then restudied after a quarter of a usual dose of chloralose/urethane (10 mg/kg and 62.5 mg/kg, respectively) and then after half the usual dose of chloralose/ urethane (20 mg/kg and 125 mg/kg). The other six animals were restudied after quarter of a standard dose of pentobarbital (9 mg/kg), after half the standard dose (18 mg/kg) and then after the full (35 mg/kg) dose. Both anesthetic agents led to significant increases in apneic thresholds for both phrenic and sympathetic nerve activities. These agents also caused dose-dependent decreases in peak, tonic and respiratory-related sympathetic nerk,e activities. Peak (tidal) phrenic nerve activities, in comparison, were much less affected by the anesthetic agents. CO 2 response curves showed that both of these anesthetic agents depressed, at any given level of PETCO2, respiratory-related sympathetic nerve responses more than the responses found in the phrenic nerve. We conclude that the relations between peak, tonic (i.e. between phasic bursts) and respiratory-related sympathetic nerve activities and phrenic nerve activity can be altered by anesthesia.
Introduction Many questions being asked in the areas of respiratory and cardiovascular control cannot be readily studied in awake, conscious animals. This has necessitated the use of anesthetic agents which may have a depressant effect upon neurons
Correspondence." F.L. Eldridge, Department of Physiology CB#7545, University of North Carolina, Chapel Hill, NC 27599, U.S.A.
of the central nervous system that are involved in the control of breathing and circulation. Some of the effects of anesthesia on the responses of the respiratory and cardiovascular systems have been well-documented [7,4,17,21,28]. However, these studies only examined the effects of anesthesia on either respiratory or cardiovascular variables but not both. Caille et al. [4] have shown that anesthetic agents can have different effects on cells of different brain regions. It is known also that relations between phrenic and hypoglossal nerve activities are dramatically al-
226
tered by anesthesia, the phasic inspiratory-related component of hypoglossal activity showing a much greater depression than the phrenic [4,14,21]. We also know that sympathetic nerves, like the hypoglossal, show respiratory-related phasic activity associated with each inspiration [1,19,22]. We therefore hypothesized that the relations between respiratory-related phrenic and sympathetic nerve activities would also be affected by anesthesia. If this were the case, then conclusions concerning the origin of the respiratory-related sympathetic activity, drawn from studies in which anesthesia was used, might need to be re-evaluated. We therefore studied phrenic and cervical sympathetic nerve responses to increasing respiratory drive produced by hypercapnia in decerebrate cats, first before they were anesthetized and then after anesthesia with increasing doses of either c h l o r a l o s e / u r e t h a n e ( C / U ) or pentobarbital. We found that the responses of these two systems are indeed differentially affected by anesthesia.
Materials and Methods Studies were performed in 12 healthy adult cats weighing between 2.6 and 3.4 kg. Each was anesthetized with ether and both carotid arteries ligated. A midcollicular decerebration was then performed under direct vision; the entire brain rostral to the section was removed by suction. To control bleeding, Gelfoam ® was placed over the exposed end of the brainstem. Once decerebrated, no more ether was given and the animals were allowed to recover for at least 2 h before the beginning of any experimental procedure. A femoral artery was cannulated for measurement of arterial pressure by means of a strain gauge. A femoral vein was also cannulated for later administration of anesthetic agents. Body temperature was monitored with a rectal thermistor and kept between 37 ° and 38°C by means of a servo-controlled DC heating pad. The trachea was cannulated through a neck incision and continuous sampling of airway P c o 2 was accomplished by means of a catheter placed in the airway. Analysis was made by an infrared CO 2
analyzer (Beckman LB-2). The animals were placed supine on a table equipped with a rigid head mount. The carotid sinus and vagus nerves were visually identified and sectioned in the neck region. Both cervical sympathetic nerve trunks were also visually identified, separated from the vagi and cut. A central end of one of these sympathetic nerves was desheathed and placed on a bipolar recording electrode and covered with mineral oil. One phrenic nerve root (C 5) was also exposed in the neck, cut, desheathed and placed on a bipolar recording electrode in a pool of warm mineral oil. These recording electrodes were built into small acrylic platforms that moved with the cat and allowed stable recording conditions throughout the experiments. Animals were ventilated with 100% oxygen using a volume-cycled ventilator. They were paralyzed with gallamine triethiode, 3 m g / k g initially, followed by a continuous infusion at a rate of 3 m g / k g / h to maintain paralysis. To prevent significant changes of end-tidal Pc'o: (PETCO2) and arterial P c o 2, a servo-controlled ventilator was used that allowed maintenance of PETCO2 within a narrow range ( + 0 . 5 torr) around any desired level [24]. When hypercapnia was to be induced, the set point of the servo-controller was changed to obtain the desired level. To determine responses to hypercapnia, the apneic threshold (the PETCO 2 at which rhythmic phrenic activity first appeared) was first determined. We then raised P c o 2 1 to 2 torr. After at least 5 rain, when the level of phrenic activity had become stable, we recorded all variables over a period of 1 rain. This was followed by progressive step increases in Pco 2 to more than 30 tort above threshold. At each level of Pco 2, all variables were recorded for I min after being allowed to stabilize for at least 5 rain.
Experimental protocol After all preparations had been completed, cardiovascular and respiratory variables were allowed to stabilize. A CO 2 response test was then performed. Following completion of this test, the first dose of one of the anesthetic agents was given intravenously and all variables were allowed
227
to stabilize for at least 20 min before another CO 2 response curve, started again at apneic threshold, was generated. In six animals, responses to hypercapnia were determined under the following three conditions: (1) unanesthetized (control); (2) anesthetized (10 m g / k g chloralose/ 62.5 m g / k g urethane); and (3) after an additional dose (cumulative 20 m g / k g chloralose/125 m g / k g urethane). These doses of C / U anesthesia correspond to 1/4 and 1/2 the standard full dose. In the other six animals, the effects of the anesthetic pentobarbital (Nembutal, Abbott Laboratories) on the responses to progressive changes in PETCO: were tested in a similar manner. The following doses of pentobarbital were used: 1/4 (9 mg/kg) of a full dose, 1/2 dose (18 m g / k g cumulative) and a full dose (35 m g / k g cumulative). The final cumulative doses (i.e. C / U of 20 m g / k g and 125 m g / k g respectively, and pentobarbital of 35 mg/kg) used in the present study were chosen on the basis of their reported ability to induce similar levels of surgical anesthesia.
Data analysis Arterial pressure, airway Pco2, phrenic nerve impulses, integrated phrenic activity, integrated sympathetic nerve activity and ventilator rate were recorded on a Gould chart recorder (Model TA2000) and stored on magnetic tape. Breathby-breath analysis of phrenic activity was performed by computer. Both phrenic and sympathetic nerve activities were filtered (30-3 000 Hz), amplified, half-wave rectified and then integrated for each 0.1 s. Integration was performed by means of a voltage-to-frequency converter, whose output frequency is proportional to input voltage, and a digital counter (Hewlett-Packard Model 3525A). This combination of instruments is the equivalent of an integrating digital voltmeter [8]; the advantage of this type of integration is that averaged activity of each 0.1 s is discrete and not subject to temporal distortion. It has been shown that the peak value for each breath is the neural equivalent of tidal volume of breathing. Minute phrenic activity (frequency × tidal) was also calculated. We chose not to use minute activity for presenting our findings for two reasons: (1) we were more interested in the effects of anesthesia
RESP.RELATED ACTIVITY
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Fig. 1. Recording of integrated cervical sympathetic nerve activity showing its division into the components (peak, tonic and respiratory-related) used in this study. Baseline refers to the amount of activity recorded after section of the cervical sympathetic trunk central to the electrode.
on the magnitudes of phrenic nerve activity and those of the respiratory-related component of sympathetic nerve activity; and (2) since the rhythmic activities of the phrenic and sympathetic nerves were so tightly coupled, either tidal or minute activities could have been used without materially affecting the results. Peak sympathetic nerve activity was divided into two components: tonic and respiratory-related activities (see Fig. 1). Since sympathetic activity has a rhythm similar to respiration, we measured the level of activity of each of the components from the integrated analog recordings using-an IBM P C / X T , a digitizing tablet (Jandel, Inc.) and appropriate software (Sigmascan, Jandel, Inc.) for each respiratory cycle. Peak activity was defined as the highest level of activity recorded during a given cycle and usually corresponded to the peak of inspiration. Tonic activity was that which occurred between phasic bursts; its average integrated value was visually estimated from the analog recording and was defined as the level of activity above zero. The value for zero activity (or 'baseline') was obtained at the end of each experiment by sectioning the sympathetic nerve central to the recording electrode; the remaining level of activity was used as the zero level. Respiratory-related activity was calculated as the difference between peak and tonic activities.
228 F o r each 1-min r e c o r d i n g t a k e n at each step i n c r e a s e in P c o 2 , an average value for each variable was calculated. T h e s e d a t a w e r e t h e n norm a l i z e d by assigning 100 units to the largest tidal phrenic, p e a k s y m p a t h e t i c a n d r e s p i r a t o r y - r e l a t e d s y m p a t h e t i c nerve activities o b t a i n e d d u r i n g hy-
p e r c a p n i a in the u n a n e s t h e t i z e d state, i.e. control, as previously d e s c r i b e d by E l d r i d g e et al. [9]. A l l s u b s e q u e n t activities w e r e scaled accordingly. T o n i c s y m p a t h e t i c nerve activities w e r e scaled to the largest p e a k value o f s y m p a t h e t i c nerve activity o b t a i n e d d u r i n g h y p e r c a p n i a in the u n a n e s -
A
INTEGRATED PHRENIC NERVE ACTIVITY
INTEGRATED SYMPATHETIC NERVE ACTIVITY
INTEGRATED PHRENIC NERVE ACTIVnY
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END-TIDAL Pco2 (torr) CHANGE FROM THRESHOLD Fig. 2. Integrated phrenic and cervical sympathetic nerve responses to changes in PETCO2of l, 4, 13 and 35 torr above the apneic threshold (obtained in the unanesthetized state) in one animal under the following three conditions: (A) unanesthetized (decerebrate); (B) anesthetized with 1/4 of a standard dose of chloralose/urethane; (C) anesthetized with a 1/2 dose of chloralose/urethane. Note that with anesthesia the magnitude of sympathetic nerve activity is disproportionately more depressed than the phrenic.
229
thetized state. Respiratory-related activities were normalized separately in order that comparisons between the respiratory and sympathetic responses could be made. In addition to normalization, respiratory and sympathetic nerve activities and mean arterial pressures from all animals were grouped into 10 bins based on the changes of P c o 2 from threshold. It should be noted that the 'change', from threshold in the present study refers to the changes from 'control' apneic threshold for phrenic nerve activity determined prior to the administration of any anesthetic agent. The bins used were (0), (0.1-1), (1.1-2), (3.1-4), (4.1-6), (6.1-10), (10.2-18), (18.1-29) and ( > 2 9 ) torr
A
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above control phrenic threshold. In the figures these bins are represented by the mean values of 0, 0.5, 1.5, 2.5, 3.5, 4.5, 5.0, 8.0, 23, and 30 torr. Analysis of variance was performed on the mean data to determine if values obtained after anesthesia were significantly different from the control (unanesthetized) state and multiple comparisons were made using a Dunnett's test. Student's t-tests for paired data were used in determining if there were differences between the threshold for sympathetic and phrenic nerve activities at each level of anesthesia. Differences were considered significant with P values < 0.05. Error bars represent standard error of the mean (SEM).
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Fig. 3. Responses of tidal phrenic and sympathetic nerve activities to changes in PETCO2 plotted for the same animal represented in Fig. 2: unanesthetized (A and A'), anesthetized with 1/4 standard dose of chloralose/urethane (B and B'), anesthetized with 1/2 dose of C / U , (C and C'). (A-C) tidal phrenic (circles) and respiratory-related sympathetic (triangles) activities; (A'-C') peak (circles) and tonic (triangles) sympathetic activities. Note the dose-dependent decreases of sympathetic activities while, in comparison, phrenic activity is minimally affected.
230
Results
Chloralose / urethane Fig. 2 shows an example in one cat of the effects of chloralose/urethane ( C / U ) on the responses of integrated phrenic and sympathetic nerve activities to hypercapnia, which is quantified by the increments (1, 4, 13, 35 torr) of Pco 2 above phrenic apneic threshold in the control unanesthetized state; in this cat control apneic threshold was 28 tort. The hypercapnic responses and relations between the two activities before anesthesia are shown in Fig. 2A. It can be seen that phrenic activity increases more relative to sympathetic at low increments of Pco~ and less relative to sympathetic at high increments of Pco 2.
C / U anesthesia led to increases of apneic threshold over control for both phrenic and respiratory-related sympathetic activities, +4.5 torr Pco 2 after the ~/4 dose (fig. 2B) and + 9 torr after the ~/2 dose (Fig. 2C). Threshold changes for phrenic and respiratory-related sympathetic activities were not different. However, it is obvious from the recordings shown in Fig. 2 that the effects on the activities of increasing C / U are different: the sympathetic decreasing more than the phrenic at the same level of hypercapnia. These differences in this cat are more clearly seen in Fig. 3, where tidal phrenic and respiratory-related sympathetic responses to hypercapnia are plotted in panels 3A (control), 3B (J/4 dose C / U ) and 3C (1/2 dose C / U ) . Responses of peak and tonic sympathetic activities are plot-
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END-TIDAL Pco 2 (torr) (change from t h r e l h o l d )
10
20
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END-TIDAL Pco 2 ( t o r t )
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Fig. 4. Average data from six animals showing effects of chloralose/urethane anesthesia on hypercapnic responses in the phrenic nerve (A-C, circles), respiratory-related (A-C, triangles), peak (A'-C', circles) and tonic (A'-C', triangles) sympathetic nerve activities. (A-A') unanesthetized; (B-B') anesthetized n/4 dose; (C-C') I/2 dose. Error bars represent SEM.
231
ted in panels 3A' (control), 3B' (1/4 dose) and 3C' (1/2 dose). We have already noted that C / U caused rightward shifts in the thresholds for phrenic and sympathetic nerve activities. The whole CO 2 response curves are similarly shifted to the right. In addition to affecting the thresholds, C / U affected the magnitudes of responses of these nerves. Tidal phrenic activities observed with the highest levels of PETCO2 were: control = 100 (panel 3A), 1/4 dose = 95.7 (panel 3B) and 1/2 dose = 67.4 units (panel 3C), only a modest decrease with increasing C / U . Respiratory-related sympathetic activities for the same changes in PETCO 2, on the other hand, were disproportionately more depressed: control = 98.2 units (panel 3A); I/4 dose = 57.7 (panel 3B) and 1/2 dose = 19.7 (panel 3C), largely because of marked decreases in peak sympathetic activity: control = 99.9 units (panel 3A'), 1/4 dose = 60.4 (panel 3B') and 1/2 dose = 18.2 (panel 3C'). Tonic sympathetic activities also decreased with increasing A
B
C / U : (control = 26.6 units (panel 3A'), 1/4 dose = 17.6 (panel 3B') and 1/2 dose =3.6 (panel 3C'). Averaged data from the six cats for tidal phrenic and respiratory-related sympathetic activities (Fig. 4, panels A, B, C) yielded similar findings. Thresholds for phrenic and sympathetic activities after the 1/4 dose of C / U were not different from control but were higher (P < 0.05) than the control with the 1/2 dose. The findings were similar to the cat as represented in Fig. 3 in showing similar disproportionate decreases, relative to phrenic, of respiratory-related sympathetic activity (panels A, B, C), again associated with decreases of peak (panels 4A', 4B' and 4C') and tonic (panels 4A', 4B' and 4C'). Phrenic nerve responses to hypercapnia showed a change in the shape of the curve after the 1/4 dose of C / U (panel 4B) but no decrease of maximal response. There was a small and significant decrease (26.3%) in maximal activity following the 1/2 dose (panel 4C). In contrast, C
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Fig. 5. Average data from six animals showing effects of pentobarbital anesthesia on hypercapnic responses in phrenic nerve (A-D, circles), respiratory-related (A-D, triangles), peak (A'-I)', circles) and tonic (A'-I)', triangles) sympathetic activities. (A-A') unanesthetized; (B-B') anesthetized 1/4 dose; (C-C') 1/2 dose. (D-I)') full dose. Error bars represent SEM.
232
the respiratory-related component of sympathetic activity exhibited significant decreases in maximal activity after 1/4 dose (39.6%, panel 4B) and after the 1/2 dose of C / U (67.3%, panel 4C). Peak and tonic sympathetic activities (Figs. 4A'C') also showed decreases after each increment of anesthetic. Maximal peak sympathetic activity decreased by 34.4% after the 1/4 dose and by 62.3% after the 1/2 dose. The tonic level of activity dropped by 26.9% after the 1/4 dose and an additional 27.9% following the 1/2 dose.
increased by only 4.9 torr but the threshold for sympathetic nerve activity increased by 15.2 torr. After a full dose of pentobarbital, both thresholds were significantly greater than their respective controls. Maximal phrenic activity showed no significant changes until after the full dose of anesthetic which caused a 36.3% decrease. However, respiratory-related sympathetic nerve activity decreased by 38.7% after only a 1/2 dose and by 69.9% following a full dose. Levels of peak and tonic sympathetic activity also fell progressively with increasing doses of pentobarbital (Fig. 5, panels B', C' and D').
Pentobarbital Data from the six cats in which pentobarbital was given are plotted in Fig. 5. Panels 5A-5D show the responses to hypercapnia for tidal phrenic and respiratory-related sympathetic activities and panels 5A'-5D' the responses of peak and tonic sympathetic nerve activities. The curves in panels 5A/5A' show the hypercapnic responses of the unanesthetized animals; they are followed by plots of the 1/4 dose (panels 5B/5B'), 1/z dose (panels 5C/5C') and full dose (panels 5D/5D') of pentobarbital. As with C / U , thresholds for rhythmic phrenic and sympathetic nerve activities were not different, except after the full dose of pentobarbital, when the threshold for phrenic nerve activity
Phrenic-sympathetic relations The relationships between respiratory (phrenic) activity and respiratory-related sympathetic activity in the unanesthetized animals and after the various doses of pentobarbital or chloralose/ urethane are shown in Fig. 6. In the unanesthetized decerebrate animals there was a curvilinear relationship, with phrenic activity increasing more than sympathetic activity at low Pco 2 levels but sympathetic activity increasing more rapidly than phrenic when Pco 2 was at high levels. This relationship was unaltered after the ~/4 and ~/2 doses of pentobarbital but shifted to the right after the full dose, which is consistent with
CHLORALOSE/URETHANE.
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,
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,
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TIDAL PHRENIC ACTIVITY (unlfs)
Fig. 6. Effects of various doses of pentobarbital and c h l o r a l o s e / u r e t h a n e anesthesia on relations between normalized tidal phrenic and respiratory-related sympathetic nerve activities. Dotted lines represent lines of identity.
233
CHLORALOS'E/URETHANE
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Fig. 7. Effects of changes in PETCO2 and different doses of the two anesthetic agents studied (six cats each) on mean arterial pressure.
the observed changes in thresholds and with an interpretation that sympathetic activity is depressed more than phrenic. The relatively greater decrease of respiratory-related sympathetic activity than phrenic with increasing doses of C / U is also seen in the figure. In Fig. 7, average mean arterial pressures are plotted against the changes of CO 2 that occurred during hypercapnic response tests in the animals anesthetized with pentobarbital (left) and those with C / U (right). Mean arterial pressures tended to increase in response to hypercapnia and to decrease in response to anesthesia, but the decreases from control with anesthesia were not significant ( P > 0.05) despite the significant changes in cervical sympathetic nerve activity.
Discussion
In this study we have shown that the anesthetic agents c h l o r a l o s e / u r e t h a n e and pentobarbital have differential effects on the CO 2 reponses of phrenic and cervical sympathetic nerves. Cervical sympathetic activities (peak, tonic and respiratory-related) were disproportionately more depressed by increasing large doses of anesthesia to changes in central chemoreceptor input than were the activities of the phrenic nerve. This difference in sensitivity to anesthesia altered the relations between the phrenic and sympathetic
nerves (Fig. 6) and suggests that autonomic neurons (involved in control of cardiovascular and other organs) are more sensitive to anesthetics than those controlling respiration. Qualitatively, these anesthetic agents (chlor a l o s e / u r e t h a n e and pentobarbital) produced similar results. Unfortunately, we had no method for quantitating the depth of anesthesia produced by a particular dose of anesthetic in these decerebrate animals. We therefore are unable to compare the effects of these anesthetics. However, it appears (Figs. 4 and 5) that a 1/2 dose of chlor a l o s e / u r e t h a n e is able to depress phrenic and sympathetic activities as much as a full dose of pentobarbital. We reported that anesthesia caused significant decreases of sympathetic nerve activity but not of mean arterial pressures. However, the mean data in Fig. 7 show that mean arterial pressures tended to decrease with increasing doses of anesthesia. There are several possible explanations for the lack of a significant effect. One is that mean arterial pressure is not a very satisfactory measure of sympathetic nerve output. A second is that the cervical sympathetic nerve does not efficiently reflect the sympathetic activity responsible for control of arterial pressure. Finally, since CO 2 can cause peripheral vasodilation [25], it is possible that the dilation may oppose the neurogenic vasoconstriction caused by sympathetic activity and thus offset changes.
234 Many studies have looked at the effects of anesthesia on either the cardiovascular [7,17,28] or respiratory [4,14,20] systems but few have looked at the effects of anesthesia on the relations between the two systems. Many studies examining the relations between the respiratory and cardiovascular systems have done so in anesthetized animals [2,11,19,26]. H a n n a et al. [11], using cats anesthetized with a full dose of pentobarbital and under conditions similar to the ones in our study (buffer nerves cut and hyperoxic), showed that the P c o 2 threshold for sympathetic nerve activity was at least 5 torr higher than the threshold for phrenic activity. These authors also plotted phrenic nerve activity against sympathetic nerve activity for various levels of P c o 2 and the resulting relationship was similar to that shown in Fig. 6. Koepchen et al. [15] found, in cats anesthetized with a full dose of c h l o r a l o s e / u r e t h a n e , that cervical sympathetic nerve activity correlated well with changes in the fractional concentration of Pco 2 in alveolar air and that the percent increase in sympathetic activity with increasing C O : was distinctly less than percent increase of phrenic activity. These data are consistent with the findings of the present study and highlight the fact that the relationship between the respiratory and cardiovascular systems is altered by anesthesia. On the other hand, Bachoo and Polosa [2] reported that increasing levels of pentobarbitone anesthesia in cats equally depressed phrenic and cervical sympathetic nerve activities. However, since the amount of data given in support of this interpretation is very limited, it is difficult to compare their results with ours. Our observations that anesthetics can have different effects depending on the cell populations being studied are not unique. Collins et al. [6], reported that chloralose depressed spontaneous and evoked activities in the dorsal horn of the spinal cord and, at similar doses, produced even greater depression of neurons in the nucleus reticularis gigantoeellaris. Caille et al. [4] examined the effects that pentobabitone had on various parts of the brain involved in the control of respiration. They found that cells in different regions of the brain, the mesencephalon, the
brainstem reticular formation and medullary respiratory groups, exhibited different sensitivities to pentobarbital; it had much less effect on the cells of the medullary respiratory groups than on cells of other regions. A similar effect of anesthesia on hypoglossal and phrenic nerve activities has been described [3,14,21]. We have found that hypoglossal activity is much more sensitive to anesthetic depression than either phrenic nerve or sympathetic nerve activities (unpublished data). It is becoming clear that anesthesia should not be used when one is studying the interactions between different neural populations because these agents modify the relationships of the unanesthetized state. Furthermore, conclusions drawn from data in which anesthetics have been used must be evaluated with caution. For example, differences in thresholds for sympathetic and phrenic nerve activities have been used as evidence that respiratory-related sympathetic nerve activity originates from an 'overflow' of activity from the respiratory system [19,22]. However, we found that the thresholds for sympathetic and phrenic nerve activities in the unanesthetized decerebrate animals were not different. Our findings therefore do not give support to the 'overflow' hypothesis and are consistent with the observed changes in threshold being due to the effects of anesthesia. Koepchen et al. [15] reported that even though the two systems are tightly linked they can be separated. He found that graded increases in Pco 2 from apneic threshold resulted in increases of sympathetic nerve activity which sometimes could be detected before phrenic bursts appeared. Trzebski and Kubin [261 reported that tonic levels of sympathetic nerve activity increased in response to changes in Pco 2 even though phrenic activity was below threshold. In designing this study, we were most interested in the changes in amplitudes of the rhythmic bursts of the phrenic and cervical sympathetic nerves. Though we did not specifically examine the questions of phase shifts or coupling between the two systems, we did not observe any obvious differences like those seen by the above mentioned authors [15,26]. At this time there exists no good explanation
235
as to why sympathetic nerve or hypoglossal nerve activities are more sensitive to anesthesia than the activities of the phrenic nerve. However, data from a number of laboratories suggest that the anesthetics pentobarbital and alpha-chloralose may mimic or enhance the actions of G A B A [13,16]. G A B A and its agonists like muscimol have been shown to depress both respiratory and cardiovascular activities [10,12,27]. Unfortunately, studies in which the effects of these substances on phrenic nerve and sympathetic nerve activities were quantitated and compared have not been done. However, it is interesting to speculate that the differences in sensitivity observed in our study are due to differences in either the number of receptors or subtypes of receptors that a particular cell population has. Though both systems may be affected by substances known to be selective for GABA-B receptors [5,23], it is known that GABA-B receptors are insensitive to barbiturate [10]. Further studies will be needed to ascertain what role G A B A receptors play, if any, in determining a system's sensitivity to a particular anesthetic and how this change in sensitivity is accomplished. The present study shows that anesthetic agents such as c h l o r a l o s e / u r e t h a n e and pentobarbital depress cervical sympathetic nerve responses to changes in P¢o 2 more than those of the phrenic nerve and that this difference in sensitivity to anesthesia alters the relations between the two systems. We therefore conclude that the unanesthetized decerebrate preparation must be considered when designing studies in which the interactions between the cardiovascular and respiratory control systems are involved and that data from studies in which anesthetics have been used must be interpreted with caution.
Acknowledgements The authors express their appreciation to Mrs. Lynn M. Houser for her excellent technical assistance. This work was supported by USPHS M E R I T Award Grant HL-17689. Paul Wagner also received doctoral scholarship support from
Glaxo Pharmaceutical, Research Triangle Park, NC.
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