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Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat
European Journal of Pharmacology, 134 (1987) 15-24
15
Elsevier EJP 00641
Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat Michele O. Carruba *, GianPietro Bondiolotti, Giovanni B. Picotti Nicoletta Catteruccia 2 and Mos6 Da Prada 2
1,
Department of Pharmacology, Chemotherapy and Medical Toxicology, School of Medicine, Unioersity of Milan, via Vanvitelli 32, 20129 Milan, Italy," I Institute of Pharmacology, School of Medicine, University of Genooa, viale Benedetto XV 2, 16132 Genooa, Italy," and 2 Pharmacology Research Department, F. Hoffmann-La Roche & Co., CH 4002 Basel Switzerland
Received 21 July 1986, revised MS received 27 October 1986, accepted 4 November 1986
The present paper describes the effects of different general anaesthetics on plasma catecholamine (CA) concentrations taken as biochemical index of peripheral sympathetic activity. In chronically catheterized rats, diethyl ether, ketamine and urethane increased plasma adrenaline (A) and noradrenaline (NA) concentrations, indicating that these drugs stimulate both neurosympathetic and adrenomedullary functions. These effects appear to be centrally mediated, since ganglionic blockade or spinal transection completely counteracted the diethyl ether- and ketamine-induced increases in plasma CA levels. Halothane induced a transient decrease in circulating A and an increase in NA. These results support the concept that general anaesthetics may have different effects on sympathetic function. Arterial blood pressure and heart rate were also measured to look for possible correlations with peripheral sympathetic activity. The enhanced release of peripheral CAs seemed to be the determining factor for increasing blood pressure and heart rate with ketamine only. In the other instances the activation of the peripheral sympathetic system appeared to maintain homeostasis by counterbalancing the various depressive effects of anaesthetics on the cardiovascular system. Diethyl ether; Halothane; Ketamine; Urethane; Plasma catecholamines; Sympathetic activity
1. Introduction The sympathetic system is known to play a primary role in the maintainance of pressor homeostasis and in cardiovascular side-effects appearing during general anaesthesia (Price, 1960). Plasma catecholamines (CA) in both humans and experimental animals have frequently been measured in an attempt to clarify the effects of different general anaesthetics on CA release from the peripheral sympathetic system. Contradictory re* To whom all correspondence should be addressed: Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita' degli Studi di Milano, via Vanvitelli, 32, 20129 Milano, Italy.
sults have been reported in some instances, perhaps because of the heterogeneity of the experimental approaches. In humans, peripheral sympathetic activity may be altered by differences in pathological conditions, premedication and surgical manoeuvers, hemorrhage, metabolic or respiratory acidosis, hypothermia, hypoxia and hypercapnia, by the methodology used or the condition of the patient (Millar and Morris, 1961). In experimental animals, species differences a n d / o r improper, stressful conditions of blood collection may in some instances have led to inconsistent results (Biihler et al., 1978; Depocas and Behrens, 1977; Perry et al., 1974; Picotti et al., 1979; Popper et al., 1977; Roizen et al., 1974;
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
16 Roizen et al., 1978). Moreover, there has been no systematic analysis of the effects of several general anaesthetic agents under standard experimental conditions. The present investigation was aimed at evaluating the changes in peripheral release of CA induced in rats by different general anaesthetics. For this purpose, in order to avoid stress, sequential blood samples were collected from chronic indwelling jugular catheters (Carruba et al., 1981) in rats before and during general anaesthesia induced by diethyl ether, halothane, ketamine and urethane.
2. Materials and methods
2.1. Animals Male albino rats (270-330 g body weight) of Wistar (Fullinsdorf, SPF) and Sprague-Dawley (Nossan) strains were used. The animals were housed individually in cages 22 × 17 X 13 cm under controlled conditions: 20-1°C room temperature, 65% humidity and 14 h per day of artificial light, i.e. light on at 6 a.m. and light off at 8 p.m. Food and water were available ad libitum. 2.2. Experimental procedure Chronic Silastic jugular or carotid catheters were implanted about 24 h before the experiments under methitural (Thiogenal, Merck) anaesthesia, as previously described (Carruba et al., 1981). Before their recovery from the anaesthesia some of the animals were bilaterally adrenalectomized (24 h before) and others underwent spinal section at the C7 level (5 h before). Experiments were performed in the morning hours in order to avoid possible variations due to diurnal rhythms. On the day of the experiment, some to the animals were placed in a plastic box connected to the appropriate vaporizer for volatile anaesthetic administration while the others, to be treated with ketamine or urethane, were left in their home cages. A sampling cannula was then connected to the jugular- or carotid-implanted catheter according to Carruba et al. (1981). At least 30 rain were
allowed for the rats to recover from the stress associated with such manipulations and a first blood sample (0 time, i.e. basal sample) was then collected. Drugs were administered immediately thereafter and blood samples were collected at various time intervals, leaving the animals untouched and undisturbed until the end of the experiment. The blood samples (0.4-0.5 ml) were collected with a plastic syringe containing 50 NIH units of heparin (Liquemin, Roche). Some of the blood (0.25 ml) was used immediately for haemo-gas analysis and the rest was transferred to ice-cold Eppendorf plastic tubes. The volume of blood withdrawn was replaced each time with saline. After centrifugation (Eppendorf 5412, 12000 r.p.m, for 2 min), the plasma was separated and stored at - 7 0 ° C until assayed for CA levels. Rectal temperature was measured continuously in some of the animals by using an electronic thermometer (Laboratorio Ricerche Elettroniche, Milano) with a rectal probe. 2.3. Plasma catecholamine assay Adrenaline (A) and noradrenaline (NA) were measured simultaneously in 50 #1 of plasma by the sensitive catechol-O-methyl-transferase radioenzymatic method of Da Prada and Ziircher (1976) with slight modifications (Da Prada and Ztircher, 1979). 2. 4. Blood pressure recording Some of the animals, previously cannulated into a carotid artery (Carruba et al., 1981), had their blood pressure (MAP) and heart rate (HR) measured by Grass Polygraph recording immediately before blood sampling for CA assay. 2.5. Haemo-gas analysis Blood haemoglobin, pH, pCO2, pO 2 and bicarbonates were determined by means of a blood gas analyzer radiometer (ABL-2) operating from 0.25 ml samples.
17
2.6. Drugs and administrations ~-
The following drugs were used: diethyl ether (Siegfried), administered with a saturation-type vaporizer (Aether Narkose Topfe 154, A. Riegger, Basel, Switzerland) at 58% for 5 min for induction and at 24% for 25 min for maintenance using 0 2 / N 2 at a flow rate of 5 l / r a i n as carrier; halothane (Hoechst), administered with a calibrated vaporizer (Fluotec, Cyprane Ltd., Keighley, England) at 1.5% for 5 min for induction and at 1% for 55 min for maintenance using O 2 / N 2 20 : 80 at a flow rate of 5 l / r a i n as carrier; ketamine (Ketalar, Parke Davis; 60 mg/kg, i.v.); urethane (Fluka; 1.2 g / k g i.p.); methitural (Thiogenal Merck; 100 m g / k g i.p.). In some experiments chlorisondamine hydrochloride (Giba-Geigy) was given i.p. 5 h before the experiment at the dose of 15 mg/kg. All drug doses refer to the free bases.
2.7. Analysis of data Data are presented as means + S.E.M. Differences between means were analyzed statistically by means of the two-tailed Dunnett's t-test (Dunnett, 1964).
3. Results
As shown in fig. 1, diethyl ether caused a marked increase in both plasma A and NA concentrations. The increase in circulating NA was already evident 5 rnin after drug administration and lasted more than 4 h. Plasma A concentrations were increased at 15 min and remained elevated (although not significantly) up to 4 h. Halothane caused a two-fold increase in plasma NA concentrations lasting for 2 h whereas it decreased plasma A concentrations only at 15 min (fig. 2). A prompt and marked increase in plasma N A concentrations was observed 5 min after ketamine (60 m g / k g i.v.) followed by a progressive decline with a return to basal values after 4 h (fig. 3). A delayed and less marked increase in plasma A concentrations was also observed after ketamine (fig. 3). Urethane (1.2 g / k g i.p.) caused a dramatic and persistent increase in both plasma A and NA concentrations (fig. 4).
800
6OO
400
iI
300] lOO 1
0.
awlzkening
~,
~
12 " Ilr
16
Fig. 1. Effects of diethyl ether anaesthesia on plasma catecholamine concentrations in catheterized rats. Induction 58% for 5 min and maintenance 24% for 25 min, cartier O 2 / N 2 20:80, flow rate 5 l / m i n . * P < 0.05 vs. basal values by Dunnett t-test. Means_+ S.E.M. for 4-10 animals per group.
Table 1 shows the values for body temperature, blood pH, pCO 2, pO 2, bicarbonates and haemoglobin in rats at different times before, during and after anaesthesia with the various drugs. As expected, some of the parameters changed during
E 600
400
100. 500
1 ,,,
awakening
2
'
§
h'r
Fig. 2. Effects of halothane anaesthesia on plasma catecholamine concentrations in catheterized rats. Induction 1.5% for 5 m i n and maintenance 1% for 55 min, carrier O 2 / N 2 20:80, flow rate 5 l/rain. * P < 0.05 vs. basal by Dunnett t-test. M e a n s + S.E.M. for 4-9 animals per group.
18
1200. E . z
150
* -_”
50 IL--I\? 0
Fig. 3. plasma < 0.05 animals
500-
,
,
2
-1 awakening
3
Effects of ketamine anaesthesia (60 mg/kg catecholamine concentrations in catheterized vs. bsal by Dunnett t-test. Means+S.E.M. per group. MET,, ETH
A
,
:
hr
4 i.v.) on rats. * P for 4-10
HALOTHANE
10
Fig. 4. plasma < 0.05 animals
Effects of urethane anaesthesia (1.2 g/kg catecholamine concentrations in catheterized vs. basal by Dunnett t-test. Means5S.E.M. per group.
i.p.) on rats. * P for 4-10
URETHANE
KETAMINE
130.
k-r---+
s 0'
30
0”
30
0"
80
0
min
120
Fig. 5. Effects of different anaesthetic agents on plasma adrenaline (A) and noradrenaline (NA) concentrations, mean arterial pressure (MAP) and heart rate (HR) in catheterized rats. Diethyl ether: induction, 58% for 5 min and maintenance, 24% for 25 a flow rate of 5 l/min with 0,/N, 20: 80 as carrier; halothane: induction, 1.5% for 5 min and maintenance, 1% for 25 min, 0,/N, 20: 80, flow rate 5 l/mm; ketamine: 60 mg/kg i.v.; urethane: 1.2 g/kg i.p. Rats had catheters chronically implanted carotid artery for MAP and HR recording and blood collection. Means + S.E.M. for 5 animals per group.
blood min at carrier in the
19 TABLE 1 Values for blood biochemical parameters and rectal temperature in rats anaesthetized with diethyl ether, halothane, ketamine or urethane. Haem. = haemoglobin; R.T. = rectal temperature. Diethyl ether anaesthesia: induction, 58% for 5 min and maintenance, 24% for 25 min, carrier 0 2 / N 2 20: 80, flow rate 5 1/min; halothane anaesthesia: induction, 1.5% for 5 min and maintenance, 1% for 55 rain, carrier O 2 / N 2 20: 80, flow rate 5 ;/min; ketamine; 60 mg/kg i.v.; urethane: 1.2 g/kg i.p. Basal
Haem. pH pCO 2 pO 2 HCO 3 R.T.
Diethyl ether 15 min
30 min
60 min
120 rain
13.4 +-0.3 7.42 4- 0.03 37.6 +-3.6 85.0 +-6.0 24.3 +-3.8 36.8 +-0.4
13.2 +1.0 7.45 -+0.02 33.9 -+0.2 98.9 -+6.7 23.5 _+1.0 36.1 -+0.9
13.6 +-1.2 7.46 +-0.01 31.6 _+1.2 102.0 +-6.3 22.2 _+0.3 35.8 _+0.7
14.6 _+1.3 7.44 +-0.04 36.3 +-2.9 99.3 _+4.0 24.6 +-4.7 36.4 +-0.3
13.8 +-0.9 7.46 +-0.03 37.2 _+2.6 89.2 +-5.2 26.0 +-3.9 36.9 +-0.5
Basal
Halothane 30 min
60 min
120 min
14.2 +-1.0 7.45 +-0.03 47.2 +-1.6 * 84.5 +-1.0 28.4 +-0.5 35.5 _+0.1 *
14.8 +-1.0 7.43 -4-0.02 40.7 +-2.0 95.6 +-6.0 30.9 +-2.5 35.3 +-0.2 *
30 min
60 rain.
15.7 +-0.8 7.44 +-0.02 40.9 +-0.5 80.8-+1.7 26.8 -+ 1.4 36.3 + 0 . 1 "
14.3 +-0.6 7.42 +-0.03 37.7 +-1.7 85.8 +-1.6 24.6 _4-2.1 35.7 +-0.1 *
120 min
240 min
480 min
14.2 _+0.9 7.35 + 0.04 43.2 + 1.5 94.9 _+5.8 22.9 -+4.4 35.6 -+0.2*
14.0 _+1.5 7.40 + 0.03 48.9 ± 3.0 * 88.6 +_3.7 29.7 +1.1 34.8 +-0.3*
15.1 +1.3 7.42 _+0.02 48.6 + 1.8 * 104.5 +-6.6 * 33.3 +-2.4 * 34.9 +-0.1 *
15 min Haem. pH pCO 2 pO 2 HCO 3 R.T.
15.7 +-0.4 7.46 + 0.03 35.8 +-1.8 86.8 -+3.4 25.3 +-1.4 36.9 +-0.1 Basal
15.6 ±0.5 7.43 ± 0.02 42.4 _+2.4 79.0 _+7.5 27.4 _+1.3 36.5 _+_0.3 Ketamine 5 min
Haem. pH pCO 2 pO 2 HCO 3 R.T.
15.3 +-0.6 7.46 +-0.01 34.1 _+3.5 86.0 +-1.8 27.9 _+0.2 37.4 +0.1 Basal
15.5 +0.7 7.41 ± 0.02 36.9 +1.6 75.5 +-3.5 24.4 +2.5 37.4 +-0.2
14.8 +1.0 7.48 + 0.03 40.1 _+3.4 79.9 +5.2 29.8 +-2.2 37.4 -+0.3
15 min 15.4 +-0.8 7.42 +-0.03 35.2 -+3.4 81.9 _+2.0 26.3 +- 1.6 36.7 +-0.1
14.1 +-1.0 7.48 _+0.03 31.4 +-1.4 97.1 +-5.8 23.4 +-2.2 36.7 +-0.6
240 min 15.3 +-1.2 7.48 +-0.03 38.3 +-1.8 91.6 +-6.9 28.3 +-0.7 36.0 _+0.4 120 min 14.8 +-0.5 7.43 +-0.03 39.5 +-2.2 84.8 +-1.9 26.2 +- 1.8 35.6 +-0.2*
Urethane 30 min
Haem. pH pCO 2 pO~ HCO 3 R.T.
14.6 +-1.5 7.40 ± 0.01 42.8 -+1.7 * 85.5 _+4.5 28.4 +-2.1 36.1 +-0.1
240 min
15.0 _+0.8 7.42 -+0.03 46.8 + 2.4 90.3 +7.2 27.8 -+1.8 36.6 +-0.3
60 min 14.6 _+1.2 7.40 -+ 0.04 45.1 -+ 2.9 84.8 _+4.3 24.6 +-1.3 36.4 +-0.4
* p < 0.05 vs. basal values by Dunnett t-test. Means_+ S.E.M. for 4-10 animals pgr group.
anaesthesia, however the various modifications had time courses completely different from those of the changes in plasma CA. Bilateral adrena l e c t o m y c a u s e d a fall i n c i r c u l a t i n g A t o v e r y l o w l e v e l s a n d a s i g n i f i c a n t rise i n p l a s m a N A c o n c e n t r a t i o n s as c o m p a r e d t o s h a m - o p e r a t e d r a t s ( t a b l e s 2 a n d 3). I n a n i m a l s a d r e n a l e c t o m i z e d 2 4 h before, diethyl ether and ketamine did not alter t h e p l a s m a A c o n c e n t r a t i o n s b u t still c a u s e d a less
rapid but pronounced elevation of circulating NA ( t a b l e s 2 a n d 3). G a n g l i o n i c b l o c k a d e b y c h l o r i s o n d a m i n e (15 m g / k g i.p. 5 h b e f o r e ) w h i c h p e r se d e c r e a s e d p l a s m a C A levels, o r s p i n a l t r a n s e c t i o n (5 h b e f o r e ) w h i c h d i d n o t a f f e c t t h e p l a s m a CA, completely counteracted the increase in plasma A and NA concentrations induced by either d i e t h y l e t h e r ( t a b l e 2), k e t a m i n e ( t a b l e 3) o r u r e t h a n e ( t a b l e 4).
20 TABLE2 Effects of diethyl ether anaesthesia on plasma catecholamine concentrations in normal rats and in rats either adrenalectomized or spinalized or pretreated with a ganglionic blocker. Thirty minutes diethyl ether exposure (5 min at 58% for induction and 25 min at 24% for maintenance), carrier O 2 / N 2 20 : 80, flow rate 5 1/min. Adrenalectomy 24 h before, spinal section at the C7 level 5 h before and chlorisondamine (15 m g / k g i.p.) was given 5 h before diethyl ether. Basal
Diethyl ether 15 rain
30 rain
60 rain
Normal A(pg/ml) NA (pg/ml)
102.-+ 32 299_+ 34
309_+ 86 a 764--+ 10() a
367-+109 a 884 -+ 190 a
255--+ 82 a 899.-+ 102 a
A drenalectomy A(pg/ml) NA (pg/ml)
20+ 3b 1160 + 279 b
23-+ 3 1 248 -+ 245
28± 3 1 894 _+382
31+ 5 2 198 ± 514 a
115__+ 49 235-+ 58
130-+ 38 284_+ 65
132-+ 46 207_+ 52
101+ 47 191_+ 55
56.-+ 14 111.-+ 27
32.-+ 6 138-+ 36
48-+ 16 120.-+ 34
Spinal section A(pg/ml) NA(pg/ml)
Chlorisondamine A(pg/ml) NA(pg/ml)
30_+ 7 b 89:[: 24 b
a p < 0.05 vs. basal and b p < 0.05 vs. basal values of normal rats by Dunnett t-test. Means.-+ S.E.M. for 4-10 animals per group. TABLE 3 Effects of ketamine anaesthesia on plasma catecholamine concentrations in normal rats and in rats either adrenalectomized or pretreated with a ganglionic blocker. Ketamine was given i.v. at the dose of 60 mg/kg. Adrenalectomy 24 h before and chlorisondamine (15 m g / k g i.p.) 5 h before ketamine. Basal
Ketamine 15 min
30 min
60 min
Normal A(pg/ml) NA(pg/ml)
93+ 36 402.-+ 55
120_+ 26 938.-+190 a
179-+ 33 ~ 873-+137 a
177_+ 34 700_+ 60 a
12+ 3 2 394 + 435 ~
15--+ 3 1595 -+ 340 a
33-+ 10 119-+ 45
29+ 8 167-+ 70
A drenalectomy A (pg/ml) NA (pg/ml)
10__+ 2 b 1045 -+ 281 b
9--+ 2 1282 +- 280
27--+ 6 t' 95_+ 25 b
31--+ 9 132-+ 37
Chlorisondamine A(pg/ml) NA(pg/ml)
a p < 0.05 vs. basal and b p < 0.05 vs. basal values from normal rats by Dunnett t-test. Means+- S.E.M. for 4-10 animals per group. TABLE 4 Effects of urethane anaesthesia on plasma catecholamine concentrations in normal rats and in rats either spinalized or pretreated with a ganglionic blocker. Urethane was given at the dose of 1.2 g / k g i.p. Spinal section at the C7 level and ganglionic blockade by chlorisondamine (15 m g / k g i.p.) 5 h before urethane. Basal
Urethane 30 min
60 min
120 rain
70+26 275 + 32
292+46 a 534 4- 80 a
348__+ 71 a 824 _+126 a
355_+ 72 a 1019 + 172 a
90+41 260 + 43
110+ 38 255 + 76
112+- 46 238 ___ 73
100+ 43 241 + 65
28+15 128 + 33
31___ 16 116___ 41
36+ 23 130 + 44
Normal A (pg/ml) NA (pg/ml)
Spinal section A (pg/ml) N A (pg/ml)
Chlorisondamine A(pg/ml) N A (pg/ml)
22+ 8 b 99 + 21 b
a p < 0.05 vs. basal and b p < 0.05 vs. basal values from normal rats by Dunnett t-test. Means + S.E.M. for 4-10 animals per group.
21 Figure 5 shows the effect of the various anaesthetics on MAP, HR and plasma CA concentrations measured concomitantly in the same animal. Diethyl ether increased both plasma A and NA levels with a concomitant decrease in MAP and no change in HR. Halothane caused reductions in MAP and HR at times when plasma NA concentrations were increased and plasma A was decreased. Ketamine caused a parallel increase in plasma CA concentrations and in both MAP and HR. Urethane increased the plasma CA concentrations with no concomitant modifications of MAP and HR.
servation that diethyl ether did not increase plasma A concentrations in adrenalectomized rats but still caused a less rapid but pronounced elevation of circulating NA, indicates that activation of adrenomedullary function by the drug accounts only for the increase in plasma A concentrations but does not participate to a major extent in enhancing NA levels. Since ganglionic blockade and spinal transection completely counteracted the increases in plasma A and NA induced by diethyl ether, this anaesthetic agent appears to enhance adrenomedullary and neurosympathetic activities by supraspinal mechanisms. 4.2. Halothane
4. Discussion
All the general anaesthetic agents studied induced significant modifications of peripheral sympathetic function in the rats, as judged from changes in circulating CA levels. The nature, magnitude and duration of these changes varied with the agent used. Simultaneous determination of plasma NA and A concentrations allows the effects on the neuronal and humoral components of the peripheral sympathetic system to be differentiated. Circulating NA was increased by all the anaesthetics examined. Circulating A was reduced after halothane, whereas it was increased by all the other agents. Measurements of body temperature, blood pH, pCO 2, pO 2 and bicarbonates ruled out the possibility that the increases in plasma CA induced by anaesthetics could have been the result of conditions such as acidosis, hypothermia, hypoxia or hypercapnia. Indeed, even though slight changes occurred in some instances these modifications had time courses that ruled out a clear cause-effect relationship to the changes in plasma CA. 4.1. Diethyl ether As judged by its effects on plasma A and NA concentrations, diethyl ether seemed to markedly enhance both adrenomedullary and neurosympathetic function. This activation persisted for some hours even when anaesthesia was discontinued and the animals had woken up. The ob-
Contradictory results on the effect of halothane on plasma CA have been reported. For instance Price et al. (1959) observed no changes in plasma A and NA levels in man during halothane anaesthesia and Millar and Morris (1961) reported that halothane did not cause significant increases in plasma A and NA concentration in either premedicated surgical patients or in dogs. Hamelberg et al. (1960) reported slightly increased concentrations of plasma A and NA in man during surgery under light halothane anaesthesia but no statistically significant increases during surgery with deep halothane anaesthesia, both with premedication. Anton et al. (1964) found increased CA in blood and urine of patients during heart surgery with halothane anaesthesia. The above studies, however, were done with relatively insensitive fluorometric assays for CA. More recently, Perry et al. (1974) reported that halothane did not change the levels of plasma CA in dogs, while Depocas and Behrens (1977), using a radioenzymatic assay, found elevated plasma NA concentrations in rats under halothane anaesthesia. Our results agree with those of Depocas and Behrens (1977) for the effect of halothane on plasma NA concentrations in rats. Furthermore, we found decreased plasma A levels during the first phase of halothane anaesthesia, evidence of an inhibitory effect of the anaesthetics on adrenomedullary secretion. This observation is consistent with and supports the in vivo and in vitro findings of G/Sthert and associates (G~Sthert
22 and Dreyer, 1973; G~thert, 1974; Gt~thert et al., 1976), which show that inhalation of halothane decreases both the spontaneous release of CA from the adrenal medulla and the release evoked by splanchnic nerve stimulation, but does not affect the stimulated NA discharge from the sympathetic nerve terminals of the isolated rabbit heart.
4.3. Ketamine Both increases (Takki et al., 1972; Zsigmond et al., 1980) and decreases (Craft et al., 1980) in plasma CA levels have been reported to occur in humans during ketamine anaesthesia. Under our experimental conditions, ketamine increased both plasma A and NA concentrations in rats. This agent, as opposed to other anaesthetics such as diethyl ether and halothane which do not affect the metabolism and the reuptake of NA (Brown et al., 1972; Naito and Gillis, 1968; Ngai et al., 1969a; Ngai et al., 1969b), is known to inhibit NA reuptake (Nedergaard, 1973). Thus, this effect of ketamine may be at least in part responsible for the observed rise in plasma CA levels. The drug, however, only blocked CA uptake at high concentrations in vitro or during continuous infusion in vivo (Montel et al., 1973; Nedergaard, 1973). This, together with the observation that the ketamine-induced increase in plasma CA concentrations was abolished by ganglionic blockade, suggests that the increase of plasma CA was mainly the result of a centrally mediated activation of sympathetic neuronal and adrenal medullary functions. This is consistent with studies from other laboratories indicating that ketamine exerts its sympathomimetic effects via direct stimulation of neurons located in the central nervous system (Ivankovich et al., 1974; Roizen et al., 1981; Traber et al., 1970).
4.4. Urethane Although urethane has been widely used as anaesthetic in animal experiments for physiological and pharmacological studies of the cardiovascular system, its effects on sympathetic activity have been little studied. However, the possibility that an effect of the drug on peripheral
CAs may modify the experimental results has been pointed out since 1962 (Bowman et al., 1962). Spriggs (1965), who measured A and NA levels in various peripheral tissues in rats under urethane anaesthesia, first hypothesized that the drug may release CA, at least from the adrenal medulla. The results of the present study indicate clearly that urethane activates both the neuronal and adrenomedullary components of the sympathetic system, as judged by the dramatic and long-lasting increase in both plasma A and NA concentrations, confirming our previous data (Picotti et al., 1979). The observation that urethane failed to increase plasma CA levels in rats with the spinal cord transected at the low cervical level indicates that the mechanism responsible for the enhanced secretion of A and NA into the blood is of central origin and reflects an increased efferent sympathetic drive. Armstrong et al. (1982) reported an increase in plasma A without changes in plasma NA concentrations in Sprague-Dawley rats under urethane anaesthesia. In contrast to our results with Wistar SPF rats, where MAP was unchanged, Armstrong et al. (1982) also reported reduced values of MAP in their urethane-anaesthetized animals. In this respect it is worth noting that urethane induced a huge fall of MAP and no increase in circulating N A in spontaneously hypertensive rats (SHR) (Memo et al., 1985; our unpublished observations). Since Armstrong et al. (1982) have shown that urethane blocks cardiovascular responses mediated by stimulation of a2-adrenoceptors and since hyperreactivity of a-adrenoceptors-mediated responses has been reported to occur in SHR (Yamaguchi and Kopin, 1980), strain differences in t h e response of the neurosympathetic system and/or in adrenoceptor sensitivity may account for the different response of MAP to urethane in the various rat strains.
4. 5. Relationship between sympatho-adrenal and hemodynamic effects of anaesthetics Although the main interest of the present study was not the delineation of the exact mechanisms by which the anaesthetic-induced changes in peripheral CA release alter circulatory homeostasis,
23 the s i m u l t a n e o u s m e a s u r e m e n t s of p l a s m a C A levels, M A P a n d H R in the s a m e a n i m a l s p e r m i t s o m e speculation. S y m p a t h e t i c activation, resulting f r o m direct effects on central nervous system centers, seems to o c c u r after k e t a m i n e when the increase in p l a s m a C A c o n c e n t r a t i o n s is c o n c o m i t a n t with p a r a l l e l changes in M A P a n d H R . This w o u l d i n d i c a t e t h a t k e t a m i n e has n o i m p o r t a n t d e p r e s s a n t effects o n p e r i p h e r a l c a r d i o v a s c u l a r effectors in rats a n d therefore the changes in p e r i p h e r a l C A release i n d u c e d b y the d r u g a p p e a r to b e the p r o m i n e n t factor in m o d i f y i n g b l o o d pressure a n d heart rate. It has a l r e a d y b e e n h y p o t h e s i z e d that a direct or reflex centrally m e d i a t e d increase in symp a t h o - a d r e n a l activity w o u l d c o u n t e r b a l a n c e the p e r i p h e r a l d e p r e s s a n t effects of diethyl ether (Brewster et al., 1953; H a m e l b e r g et al., 1960; MiUar a n d Morris, 1961; Price, 1960; Price et al., 1959). It should b e e m p h a s i z e d that n o t only d i e t h y l e t h e r b u t also h a l o t h a n e a n d u r e t h a n e can d e p r e s s m y o c a r d i a l c o n t r a c t i l i t y (Brown a n d Crout, 1971; Price, 1960; W h i t e et al., 1982). Effects on a d r e n o c e p t o r s a n d p e r i p h e r a l vasculature have also b e e n r e p o r t e d ( A l t u r a a n d W e i n berg, 1979; A r m s t r o n g et al., 1982; Pasch a n d Renkl, 1979; Price, 1960; W h i t e et al., 1982). Therefore, it a p p e a r s that activation of the p e r i p h eral s y m p a t h e t i c system b y these anaesthetics serves in p a r t to c o u n t e r b a l a n c e the various depressive effects of the drugs on the c a r d i o v a s c u l a r system. I n conclusion, general anaesthetic agents do h a v e effects on p e r i p h e r a l s y m p a t h e t i c activity t h a t m u s t b e t a k e n into a c c o u n t in a n y i n t e r p r e t a tion of the relative c o n t r i b u t i o n s of the different m e c h a n i s m s b y w h i c h these drugs can affect circul a t o r y homeostasis. A s it n o w seems that anaesthetics m a r k e d l y affect p e r i p h e r a l a d r e n o m e d u l l a r y a n d n e u r o s y m p a t h e t i c activity, it is felt that the anaesthetic a g e n t to b e e m p l o y e d in c a r d i o v a s c u l a r studies s h o u l d be c h o s e n with great care. I n d e e d , even w h e n the a n a e s t h e t i c p e r se a p p e a r s to b e d e v o i d of h e m o d y n a m i c effects, as seems to b e the case with u r e t h a n e in W i s t a r S P F rats, it can well b e t h a t it modifies e x p e r i m e n t a l p a r a m e t e r s b y interfeting with s y m p a t h e t i c activity.
Acknowledgements The authors thank Professors W.E. Haefely and P. Mantegazza for helpful criticism of the manuscript and Mr. F. D'Agostini for skilful technical assistance. Partial support from the Italian Ministry of Education (MPI) and the Italian Research Council (CNR) is gratefully acknowledged,
References Altura, B.M. and J. Weinberg, 1979, Urethane and contraction of vascular smooth muscle, Br. J. Pharmacol. 67, 255. Anton, A.H., J.S. Gravenstein and M.W. Wheat, 1964, Extracorporeal circulation and endogenous epinephrine and norepinephrine in plasma, atrium and urine in man, Anesthesiology 25, 262. Armstrong, J.M., F. Lefevre-Borg, B. Scatton and I. Cavero, 1982, Urethane inhibits cardiovascular responses mediated by the stimulation of alpha-2 adrenoceptors in the rat, J. Pharmacol. Exp. Ther. 223, 524. Bowman, W.C., A.A.J. Goldberg and C. Raper, 1962, A comparison between the effects of a tetanus and the effects of sympathomimetic amines on fast- and slow-contracting mammalian muscles, Br. J. Pharmacol. 19, 464. Brewster, W.R., J.P. Isaac and T. Waino-Anderson, 1953, Depressant effect of ether on the myocardium of the dog and its modification by reflex release of epinephrine and norepinephrine, Am. J. Physiol. 175, 399. Brown, B.R., Jr. and J.R. Crout, 1971, A comparative study of the effects of five general anesthetics on myocardial contractility: isometric conditions, Anesthesiology 34, 236. Brown, B.R., Jr., E.N. Tatum and G.R. Crout, 1972, The effects of inhalation anesthetics on the uptake and metabolism of 1-3H-norepinephrine in guinea pig atria, Anesthesiology 36, 263. Bi~hler, H.U., M. Da Prada, W. Haefely and G.B. Picotti, 1978, Plasma adrenaline, noradrenaline and dopamine in man and different animal species, J. Physiol. 276, 311. Carruba, M.O., G.B. Picotti, P. Miodini, W. Lotz and M.. Da Prada, 1981, Blood sampling by chronic cannulation technique for reliable measurements of catecholamines and other hormones in plasma of conscious rats, J. Pharmacol. Meth. 5, 293. Craft, J.B., Jr., S.D. Dao, M.L. Yonekura, D.A. Mackinnon, E.G. Co, M. Roizen, P. Mazel, R. Gilman and L. Shokes, 1980, Maternal and fetal catecholamine responses to ketamine, Anesthesiology 53, $319. Da Prada, M. and G. Ziarcher, 1976, Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range, Life Sci. 19, 1161. Da Prada, M. and G. Ziarcher, 1979, Radioenzymatic assay of plasma and urinary catecholamines in man and various animal species: physiological and pharmacological applications, in: Radioimmunoassay of Drugs and Hormones in
24 Cardiovascular Medicine, eds. A. Albertini, M. Da Prada and B.A. Preskar (Elsevier, Amsterdam) p. 175. Depocas, F. and W.A. Behrens, 1977, Effects of handling, decapitation, anesthesia and surgery on plasma noradrenaline levels in the white rat, Can. J. Physiol. Pharmacol. 55, 212. Dunnett, C.W., 1964, New tables for multiple comparisons with a control, Biometrics 20, 482. G~thert, M., 1974, Effects of halothane on the sympathetic nerve terminals of the rabbit heart. Differences in membrane actions of halothane and tetracaine, NaunynSchmiedeb. Arch. Pharmacol. 286, 125. G~Sthert, M., W. Dorn and I. Loewenstein, 1976, Inhibition of catecholamine release from the adrenal medulla by halothane, Naunyn-Schmiedeb. Arch. Pharmacol. 294, 239. G~thert, M. and C. Dreyer, 1973, Inhibitory effect of halothane anaesthesia on catecholamine release from the adrenal medulla, Naunyn-Schmiedeb. Arch. Pharmacol. 277, 253. Hamelberg, W., J.E. Sprouse, J.E. Mahaffey and J.A. Richardson, 1960, Catecholamine levels during light and deep anesthesia, Anesthesiology 21,297. Ivankovich, A.D., D.J. Miletich, C. Reimann, R.F. Albrecht and B. Zahed, 1974, Cardiovascular effects of centrally administered ketamine in goats, Anesth. Analg. 53, 924. Memo, M., G. Sagheddu, M.O. Carruba and P.F. Spano, 1985, Dihydroergotoxine decreases blood pressure in spontaneously hypertensive rats by interacting with peripheral dopamine receptors, Life Sci. 36, 1515. Millar, R.A. and M.E. Morris, 1961, Sympatho-adrenal responses during general anaesthesia in the dog and man, Can. Anaesth. Soc. J. 8, 356. Montel, H., K. Starke, B.-D. Garlitz and H.L. Schiimann, 1973, Tierexperimentelle Untersuchungen zur Wirkung des Ketamins auf periphere sympathische Nerven, Anaesthetist 22, 111. Naito, H. and C.M. GilEs, 1968, Anesthetics and response of atria to sympathetic nerve stimulation, Anesthesiology 29, 259. Nedergaard, O.A., Cocaine-like effect of ketamine on vascular adrenergic neurons, European J. Pharmacol. 23, 153. Ngai, S.H., P.M. Diaz and S. Ozer, 1969a, The uptake and release of norepinephrine: effects of cyclopropane and halothane, Anesthesiology 31, 45. Ngal, S.H., N.H. Neff and E. Costa, 1969b, The effects of cyclopropane and halothane on the biosynthesis of norepinephrine in vivo, Anesthesiology 31, 53. Pasch, T. and F. Renkl, 1979, Wirkung verschiedener Narkotika und Relaxantien auf den glatten Gefabmuskel, Anaesthetist 28, 390.
Perry, L.B., R.A. Van Dyke and R.A. Theye, 1974, Sympathoadrenal and hemodynamic effects of isoflurane, halothane and cyclopropane in dogs, Anesthesiology 40, 465. Picotti, G.B., M.O. Carruba, M.D. Galva, C. Ravazzani, G.P. Bondiolotti and M. Da Prada, 1979, Drug-induced changes of plasma catecholamine concentrations, in: Radioimmunoassay of Drugs and Hormones in Cardiovascular Medicine, eds. A. Albertini, M. Da Prada and B.A. Peskar (Elsevier, Amsterdam) p. 133. Popper, C.W., C.C. Chiueh and I.J. Kopin, 1977, Plasma catecholamine concentrations in unanesthetized rats during sleep, wakefulness, immobilization and after decapitation, J. Pharmacol. Exp. Ther. 202, 144. Price, H.L., 1960, General anesthesia and circulatory homeostasis, Physiol. Rev. 40, 187. Price, H.L., H.W. Linde, R.E. Jones, G.W. Black and M.L. Price, 1959, Sympatho-adrenal responses to general anesthesia in man and their relation to hemodynamics, Anesthesiology 20, 563. Roizen, M.F., C. Helke, E.I. Eger, B. Frazer and B. Johnson, 1981, Dissimilar effects of cyclopropane, halotane and ketamine on brainstem nuclei associated with cardiovascular and respiratory control, Anesthesiology 55, A288. Roizen, M.F., J. Moss, D.P. Henry and I.J. Kopin, 1974, Effects of halothane on plasma catecholamines, Anesthesiology 41,432. Roizen, M.F., J. Moss, D.P. Henry, V. Weise and I.J. Kopin, 1978, Effects of general anesthetics on handling- and decapitation-induced increases in sympathoadrenal discharge, J. Pharmacol. Exp. Ther. 204, 11. Spriggs, T.L.B., 1965, The effects of anaesthesia induced by urethane or phenobarbitone upon the distribution of peripheral catecholamines in the rat, Br. J. Pharmacol. 24, 752. Takki, S., P. Nikki, A. J~i~itteliiand T. Tammisto, 1972, Ketamine and plasma catecholamines, Br. J. Anaesth. 44, 1318. Traber, D.L., R.D. Wilson and L.L. Priano, 1970, Blockade of the hypertensive response to ketamine, Anesth. Analg. 49, 430. White, P.F., W.L. Way and A.J. Trevor, 1982, Ketamine: its pharmacology and therapeutic uses, Anesthesiology 56, 119. Yamaguchi, I. and I.J. Kopin, 1980, Blood pressure, plasma catecholamines and sympathetic outflow in pithed SHR and WKY rats, Am. J. Physiol. 238, H365. Zsigmond, E.K., S.P. Kothary, S.M. Kumar and R.C. Kelsch, 1980, Counteraction of circulatory side effects of ketamine by pretreatment with diazepam, Clin. Ther. 3, 28.
15
Elsevier EJP 00641
Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat Michele O. Carruba *, GianPietro Bondiolotti, Giovanni B. Picotti Nicoletta Catteruccia 2 and Mos6 Da Prada 2
1,
Department of Pharmacology, Chemotherapy and Medical Toxicology, School of Medicine, Unioersity of Milan, via Vanvitelli 32, 20129 Milan, Italy," I Institute of Pharmacology, School of Medicine, University of Genooa, viale Benedetto XV 2, 16132 Genooa, Italy," and 2 Pharmacology Research Department, F. Hoffmann-La Roche & Co., CH 4002 Basel Switzerland
Received 21 July 1986, revised MS received 27 October 1986, accepted 4 November 1986
The present paper describes the effects of different general anaesthetics on plasma catecholamine (CA) concentrations taken as biochemical index of peripheral sympathetic activity. In chronically catheterized rats, diethyl ether, ketamine and urethane increased plasma adrenaline (A) and noradrenaline (NA) concentrations, indicating that these drugs stimulate both neurosympathetic and adrenomedullary functions. These effects appear to be centrally mediated, since ganglionic blockade or spinal transection completely counteracted the diethyl ether- and ketamine-induced increases in plasma CA levels. Halothane induced a transient decrease in circulating A and an increase in NA. These results support the concept that general anaesthetics may have different effects on sympathetic function. Arterial blood pressure and heart rate were also measured to look for possible correlations with peripheral sympathetic activity. The enhanced release of peripheral CAs seemed to be the determining factor for increasing blood pressure and heart rate with ketamine only. In the other instances the activation of the peripheral sympathetic system appeared to maintain homeostasis by counterbalancing the various depressive effects of anaesthetics on the cardiovascular system. Diethyl ether; Halothane; Ketamine; Urethane; Plasma catecholamines; Sympathetic activity
1. Introduction The sympathetic system is known to play a primary role in the maintainance of pressor homeostasis and in cardiovascular side-effects appearing during general anaesthesia (Price, 1960). Plasma catecholamines (CA) in both humans and experimental animals have frequently been measured in an attempt to clarify the effects of different general anaesthetics on CA release from the peripheral sympathetic system. Contradictory re* To whom all correspondence should be addressed: Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita' degli Studi di Milano, via Vanvitelli, 32, 20129 Milano, Italy.
sults have been reported in some instances, perhaps because of the heterogeneity of the experimental approaches. In humans, peripheral sympathetic activity may be altered by differences in pathological conditions, premedication and surgical manoeuvers, hemorrhage, metabolic or respiratory acidosis, hypothermia, hypoxia and hypercapnia, by the methodology used or the condition of the patient (Millar and Morris, 1961). In experimental animals, species differences a n d / o r improper, stressful conditions of blood collection may in some instances have led to inconsistent results (Biihler et al., 1978; Depocas and Behrens, 1977; Perry et al., 1974; Picotti et al., 1979; Popper et al., 1977; Roizen et al., 1974;
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
16 Roizen et al., 1978). Moreover, there has been no systematic analysis of the effects of several general anaesthetic agents under standard experimental conditions. The present investigation was aimed at evaluating the changes in peripheral release of CA induced in rats by different general anaesthetics. For this purpose, in order to avoid stress, sequential blood samples were collected from chronic indwelling jugular catheters (Carruba et al., 1981) in rats before and during general anaesthesia induced by diethyl ether, halothane, ketamine and urethane.
2. Materials and methods
2.1. Animals Male albino rats (270-330 g body weight) of Wistar (Fullinsdorf, SPF) and Sprague-Dawley (Nossan) strains were used. The animals were housed individually in cages 22 × 17 X 13 cm under controlled conditions: 20-1°C room temperature, 65% humidity and 14 h per day of artificial light, i.e. light on at 6 a.m. and light off at 8 p.m. Food and water were available ad libitum. 2.2. Experimental procedure Chronic Silastic jugular or carotid catheters were implanted about 24 h before the experiments under methitural (Thiogenal, Merck) anaesthesia, as previously described (Carruba et al., 1981). Before their recovery from the anaesthesia some of the animals were bilaterally adrenalectomized (24 h before) and others underwent spinal section at the C7 level (5 h before). Experiments were performed in the morning hours in order to avoid possible variations due to diurnal rhythms. On the day of the experiment, some to the animals were placed in a plastic box connected to the appropriate vaporizer for volatile anaesthetic administration while the others, to be treated with ketamine or urethane, were left in their home cages. A sampling cannula was then connected to the jugular- or carotid-implanted catheter according to Carruba et al. (1981). At least 30 rain were
allowed for the rats to recover from the stress associated with such manipulations and a first blood sample (0 time, i.e. basal sample) was then collected. Drugs were administered immediately thereafter and blood samples were collected at various time intervals, leaving the animals untouched and undisturbed until the end of the experiment. The blood samples (0.4-0.5 ml) were collected with a plastic syringe containing 50 NIH units of heparin (Liquemin, Roche). Some of the blood (0.25 ml) was used immediately for haemo-gas analysis and the rest was transferred to ice-cold Eppendorf plastic tubes. The volume of blood withdrawn was replaced each time with saline. After centrifugation (Eppendorf 5412, 12000 r.p.m, for 2 min), the plasma was separated and stored at - 7 0 ° C until assayed for CA levels. Rectal temperature was measured continuously in some of the animals by using an electronic thermometer (Laboratorio Ricerche Elettroniche, Milano) with a rectal probe. 2.3. Plasma catecholamine assay Adrenaline (A) and noradrenaline (NA) were measured simultaneously in 50 #1 of plasma by the sensitive catechol-O-methyl-transferase radioenzymatic method of Da Prada and Ziircher (1976) with slight modifications (Da Prada and Ztircher, 1979). 2. 4. Blood pressure recording Some of the animals, previously cannulated into a carotid artery (Carruba et al., 1981), had their blood pressure (MAP) and heart rate (HR) measured by Grass Polygraph recording immediately before blood sampling for CA assay. 2.5. Haemo-gas analysis Blood haemoglobin, pH, pCO2, pO 2 and bicarbonates were determined by means of a blood gas analyzer radiometer (ABL-2) operating from 0.25 ml samples.
17
2.6. Drugs and administrations ~-
The following drugs were used: diethyl ether (Siegfried), administered with a saturation-type vaporizer (Aether Narkose Topfe 154, A. Riegger, Basel, Switzerland) at 58% for 5 min for induction and at 24% for 25 min for maintenance using 0 2 / N 2 at a flow rate of 5 l / r a i n as carrier; halothane (Hoechst), administered with a calibrated vaporizer (Fluotec, Cyprane Ltd., Keighley, England) at 1.5% for 5 min for induction and at 1% for 55 min for maintenance using O 2 / N 2 20 : 80 at a flow rate of 5 l / r a i n as carrier; ketamine (Ketalar, Parke Davis; 60 mg/kg, i.v.); urethane (Fluka; 1.2 g / k g i.p.); methitural (Thiogenal Merck; 100 m g / k g i.p.). In some experiments chlorisondamine hydrochloride (Giba-Geigy) was given i.p. 5 h before the experiment at the dose of 15 mg/kg. All drug doses refer to the free bases.
2.7. Analysis of data Data are presented as means + S.E.M. Differences between means were analyzed statistically by means of the two-tailed Dunnett's t-test (Dunnett, 1964).
3. Results
As shown in fig. 1, diethyl ether caused a marked increase in both plasma A and NA concentrations. The increase in circulating NA was already evident 5 rnin after drug administration and lasted more than 4 h. Plasma A concentrations were increased at 15 min and remained elevated (although not significantly) up to 4 h. Halothane caused a two-fold increase in plasma NA concentrations lasting for 2 h whereas it decreased plasma A concentrations only at 15 min (fig. 2). A prompt and marked increase in plasma N A concentrations was observed 5 min after ketamine (60 m g / k g i.v.) followed by a progressive decline with a return to basal values after 4 h (fig. 3). A delayed and less marked increase in plasma A concentrations was also observed after ketamine (fig. 3). Urethane (1.2 g / k g i.p.) caused a dramatic and persistent increase in both plasma A and NA concentrations (fig. 4).
800
6OO
400
iI
300] lOO 1
0.
awlzkening
~,
~
12 " Ilr
16
Fig. 1. Effects of diethyl ether anaesthesia on plasma catecholamine concentrations in catheterized rats. Induction 58% for 5 min and maintenance 24% for 25 min, cartier O 2 / N 2 20:80, flow rate 5 l / m i n . * P < 0.05 vs. basal values by Dunnett t-test. Means_+ S.E.M. for 4-10 animals per group.
Table 1 shows the values for body temperature, blood pH, pCO 2, pO 2, bicarbonates and haemoglobin in rats at different times before, during and after anaesthesia with the various drugs. As expected, some of the parameters changed during
E 600
400
100. 500
1 ,,,
awakening
2
'
§
h'r
Fig. 2. Effects of halothane anaesthesia on plasma catecholamine concentrations in catheterized rats. Induction 1.5% for 5 m i n and maintenance 1% for 55 min, carrier O 2 / N 2 20:80, flow rate 5 l/rain. * P < 0.05 vs. basal by Dunnett t-test. M e a n s + S.E.M. for 4-9 animals per group.
18
1200. E . z
150
* -_”
50 IL--I\? 0
Fig. 3. plasma < 0.05 animals
500-
,
,
2
-1 awakening
3
Effects of ketamine anaesthesia (60 mg/kg catecholamine concentrations in catheterized vs. bsal by Dunnett t-test. Means+S.E.M. per group. MET,, ETH
A
,
:
hr
4 i.v.) on rats. * P for 4-10
HALOTHANE
10
Fig. 4. plasma < 0.05 animals
Effects of urethane anaesthesia (1.2 g/kg catecholamine concentrations in catheterized vs. basal by Dunnett t-test. Means5S.E.M. per group.
i.p.) on rats. * P for 4-10
URETHANE
KETAMINE
130.
k-r---+
s 0'
30
0”
30
0"
80
0
min
120
Fig. 5. Effects of different anaesthetic agents on plasma adrenaline (A) and noradrenaline (NA) concentrations, mean arterial pressure (MAP) and heart rate (HR) in catheterized rats. Diethyl ether: induction, 58% for 5 min and maintenance, 24% for 25 a flow rate of 5 l/min with 0,/N, 20: 80 as carrier; halothane: induction, 1.5% for 5 min and maintenance, 1% for 25 min, 0,/N, 20: 80, flow rate 5 l/mm; ketamine: 60 mg/kg i.v.; urethane: 1.2 g/kg i.p. Rats had catheters chronically implanted carotid artery for MAP and HR recording and blood collection. Means + S.E.M. for 5 animals per group.
blood min at carrier in the
19 TABLE 1 Values for blood biochemical parameters and rectal temperature in rats anaesthetized with diethyl ether, halothane, ketamine or urethane. Haem. = haemoglobin; R.T. = rectal temperature. Diethyl ether anaesthesia: induction, 58% for 5 min and maintenance, 24% for 25 min, carrier 0 2 / N 2 20: 80, flow rate 5 1/min; halothane anaesthesia: induction, 1.5% for 5 min and maintenance, 1% for 55 rain, carrier O 2 / N 2 20: 80, flow rate 5 ;/min; ketamine; 60 mg/kg i.v.; urethane: 1.2 g/kg i.p. Basal
Haem. pH pCO 2 pO 2 HCO 3 R.T.
Diethyl ether 15 min
30 min
60 min
120 rain
13.4 +-0.3 7.42 4- 0.03 37.6 +-3.6 85.0 +-6.0 24.3 +-3.8 36.8 +-0.4
13.2 +1.0 7.45 -+0.02 33.9 -+0.2 98.9 -+6.7 23.5 _+1.0 36.1 -+0.9
13.6 +-1.2 7.46 +-0.01 31.6 _+1.2 102.0 +-6.3 22.2 _+0.3 35.8 _+0.7
14.6 _+1.3 7.44 +-0.04 36.3 +-2.9 99.3 _+4.0 24.6 +-4.7 36.4 +-0.3
13.8 +-0.9 7.46 +-0.03 37.2 _+2.6 89.2 +-5.2 26.0 +-3.9 36.9 +-0.5
Basal
Halothane 30 min
60 min
120 min
14.2 +-1.0 7.45 +-0.03 47.2 +-1.6 * 84.5 +-1.0 28.4 +-0.5 35.5 _+0.1 *
14.8 +-1.0 7.43 -4-0.02 40.7 +-2.0 95.6 +-6.0 30.9 +-2.5 35.3 +-0.2 *
30 min
60 rain.
15.7 +-0.8 7.44 +-0.02 40.9 +-0.5 80.8-+1.7 26.8 -+ 1.4 36.3 + 0 . 1 "
14.3 +-0.6 7.42 +-0.03 37.7 +-1.7 85.8 +-1.6 24.6 _4-2.1 35.7 +-0.1 *
120 min
240 min
480 min
14.2 _+0.9 7.35 + 0.04 43.2 + 1.5 94.9 _+5.8 22.9 -+4.4 35.6 -+0.2*
14.0 _+1.5 7.40 + 0.03 48.9 ± 3.0 * 88.6 +_3.7 29.7 +1.1 34.8 +-0.3*
15.1 +1.3 7.42 _+0.02 48.6 + 1.8 * 104.5 +-6.6 * 33.3 +-2.4 * 34.9 +-0.1 *
15 min Haem. pH pCO 2 pO 2 HCO 3 R.T.
15.7 +-0.4 7.46 + 0.03 35.8 +-1.8 86.8 -+3.4 25.3 +-1.4 36.9 +-0.1 Basal
15.6 ±0.5 7.43 ± 0.02 42.4 _+2.4 79.0 _+7.5 27.4 _+1.3 36.5 _+_0.3 Ketamine 5 min
Haem. pH pCO 2 pO 2 HCO 3 R.T.
15.3 +-0.6 7.46 +-0.01 34.1 _+3.5 86.0 +-1.8 27.9 _+0.2 37.4 +0.1 Basal
15.5 +0.7 7.41 ± 0.02 36.9 +1.6 75.5 +-3.5 24.4 +2.5 37.4 +-0.2
14.8 +1.0 7.48 + 0.03 40.1 _+3.4 79.9 +5.2 29.8 +-2.2 37.4 -+0.3
15 min 15.4 +-0.8 7.42 +-0.03 35.2 -+3.4 81.9 _+2.0 26.3 +- 1.6 36.7 +-0.1
14.1 +-1.0 7.48 _+0.03 31.4 +-1.4 97.1 +-5.8 23.4 +-2.2 36.7 +-0.6
240 min 15.3 +-1.2 7.48 +-0.03 38.3 +-1.8 91.6 +-6.9 28.3 +-0.7 36.0 _+0.4 120 min 14.8 +-0.5 7.43 +-0.03 39.5 +-2.2 84.8 +-1.9 26.2 +- 1.8 35.6 +-0.2*
Urethane 30 min
Haem. pH pCO 2 pO~ HCO 3 R.T.
14.6 +-1.5 7.40 ± 0.01 42.8 -+1.7 * 85.5 _+4.5 28.4 +-2.1 36.1 +-0.1
240 min
15.0 _+0.8 7.42 -+0.03 46.8 + 2.4 90.3 +7.2 27.8 -+1.8 36.6 +-0.3
60 min 14.6 _+1.2 7.40 -+ 0.04 45.1 -+ 2.9 84.8 _+4.3 24.6 +-1.3 36.4 +-0.4
* p < 0.05 vs. basal values by Dunnett t-test. Means_+ S.E.M. for 4-10 animals pgr group.
anaesthesia, however the various modifications had time courses completely different from those of the changes in plasma CA. Bilateral adrena l e c t o m y c a u s e d a fall i n c i r c u l a t i n g A t o v e r y l o w l e v e l s a n d a s i g n i f i c a n t rise i n p l a s m a N A c o n c e n t r a t i o n s as c o m p a r e d t o s h a m - o p e r a t e d r a t s ( t a b l e s 2 a n d 3). I n a n i m a l s a d r e n a l e c t o m i z e d 2 4 h before, diethyl ether and ketamine did not alter t h e p l a s m a A c o n c e n t r a t i o n s b u t still c a u s e d a less
rapid but pronounced elevation of circulating NA ( t a b l e s 2 a n d 3). G a n g l i o n i c b l o c k a d e b y c h l o r i s o n d a m i n e (15 m g / k g i.p. 5 h b e f o r e ) w h i c h p e r se d e c r e a s e d p l a s m a C A levels, o r s p i n a l t r a n s e c t i o n (5 h b e f o r e ) w h i c h d i d n o t a f f e c t t h e p l a s m a CA, completely counteracted the increase in plasma A and NA concentrations induced by either d i e t h y l e t h e r ( t a b l e 2), k e t a m i n e ( t a b l e 3) o r u r e t h a n e ( t a b l e 4).
20 TABLE2 Effects of diethyl ether anaesthesia on plasma catecholamine concentrations in normal rats and in rats either adrenalectomized or spinalized or pretreated with a ganglionic blocker. Thirty minutes diethyl ether exposure (5 min at 58% for induction and 25 min at 24% for maintenance), carrier O 2 / N 2 20 : 80, flow rate 5 1/min. Adrenalectomy 24 h before, spinal section at the C7 level 5 h before and chlorisondamine (15 m g / k g i.p.) was given 5 h before diethyl ether. Basal
Diethyl ether 15 rain
30 rain
60 rain
Normal A(pg/ml) NA (pg/ml)
102.-+ 32 299_+ 34
309_+ 86 a 764--+ 10() a
367-+109 a 884 -+ 190 a
255--+ 82 a 899.-+ 102 a
A drenalectomy A(pg/ml) NA (pg/ml)
20+ 3b 1160 + 279 b
23-+ 3 1 248 -+ 245
28± 3 1 894 _+382
31+ 5 2 198 ± 514 a
115__+ 49 235-+ 58
130-+ 38 284_+ 65
132-+ 46 207_+ 52
101+ 47 191_+ 55
56.-+ 14 111.-+ 27
32.-+ 6 138-+ 36
48-+ 16 120.-+ 34
Spinal section A(pg/ml) NA(pg/ml)
Chlorisondamine A(pg/ml) NA(pg/ml)
30_+ 7 b 89:[: 24 b
a p < 0.05 vs. basal and b p < 0.05 vs. basal values of normal rats by Dunnett t-test. Means.-+ S.E.M. for 4-10 animals per group. TABLE 3 Effects of ketamine anaesthesia on plasma catecholamine concentrations in normal rats and in rats either adrenalectomized or pretreated with a ganglionic blocker. Ketamine was given i.v. at the dose of 60 mg/kg. Adrenalectomy 24 h before and chlorisondamine (15 m g / k g i.p.) 5 h before ketamine. Basal
Ketamine 15 min
30 min
60 min
Normal A(pg/ml) NA(pg/ml)
93+ 36 402.-+ 55
120_+ 26 938.-+190 a
179-+ 33 ~ 873-+137 a
177_+ 34 700_+ 60 a
12+ 3 2 394 + 435 ~
15--+ 3 1595 -+ 340 a
33-+ 10 119-+ 45
29+ 8 167-+ 70
A drenalectomy A (pg/ml) NA (pg/ml)
10__+ 2 b 1045 -+ 281 b
9--+ 2 1282 +- 280
27--+ 6 t' 95_+ 25 b
31--+ 9 132-+ 37
Chlorisondamine A(pg/ml) NA(pg/ml)
a p < 0.05 vs. basal and b p < 0.05 vs. basal values from normal rats by Dunnett t-test. Means+- S.E.M. for 4-10 animals per group. TABLE 4 Effects of urethane anaesthesia on plasma catecholamine concentrations in normal rats and in rats either spinalized or pretreated with a ganglionic blocker. Urethane was given at the dose of 1.2 g / k g i.p. Spinal section at the C7 level and ganglionic blockade by chlorisondamine (15 m g / k g i.p.) 5 h before urethane. Basal
Urethane 30 min
60 min
120 rain
70+26 275 + 32
292+46 a 534 4- 80 a
348__+ 71 a 824 _+126 a
355_+ 72 a 1019 + 172 a
90+41 260 + 43
110+ 38 255 + 76
112+- 46 238 ___ 73
100+ 43 241 + 65
28+15 128 + 33
31___ 16 116___ 41
36+ 23 130 + 44
Normal A (pg/ml) NA (pg/ml)
Spinal section A (pg/ml) N A (pg/ml)
Chlorisondamine A(pg/ml) N A (pg/ml)
22+ 8 b 99 + 21 b
a p < 0.05 vs. basal and b p < 0.05 vs. basal values from normal rats by Dunnett t-test. Means + S.E.M. for 4-10 animals per group.
21 Figure 5 shows the effect of the various anaesthetics on MAP, HR and plasma CA concentrations measured concomitantly in the same animal. Diethyl ether increased both plasma A and NA levels with a concomitant decrease in MAP and no change in HR. Halothane caused reductions in MAP and HR at times when plasma NA concentrations were increased and plasma A was decreased. Ketamine caused a parallel increase in plasma CA concentrations and in both MAP and HR. Urethane increased the plasma CA concentrations with no concomitant modifications of MAP and HR.
servation that diethyl ether did not increase plasma A concentrations in adrenalectomized rats but still caused a less rapid but pronounced elevation of circulating NA, indicates that activation of adrenomedullary function by the drug accounts only for the increase in plasma A concentrations but does not participate to a major extent in enhancing NA levels. Since ganglionic blockade and spinal transection completely counteracted the increases in plasma A and NA induced by diethyl ether, this anaesthetic agent appears to enhance adrenomedullary and neurosympathetic activities by supraspinal mechanisms. 4.2. Halothane
4. Discussion
All the general anaesthetic agents studied induced significant modifications of peripheral sympathetic function in the rats, as judged from changes in circulating CA levels. The nature, magnitude and duration of these changes varied with the agent used. Simultaneous determination of plasma NA and A concentrations allows the effects on the neuronal and humoral components of the peripheral sympathetic system to be differentiated. Circulating NA was increased by all the anaesthetics examined. Circulating A was reduced after halothane, whereas it was increased by all the other agents. Measurements of body temperature, blood pH, pCO 2, pO 2 and bicarbonates ruled out the possibility that the increases in plasma CA induced by anaesthetics could have been the result of conditions such as acidosis, hypothermia, hypoxia or hypercapnia. Indeed, even though slight changes occurred in some instances these modifications had time courses that ruled out a clear cause-effect relationship to the changes in plasma CA. 4.1. Diethyl ether As judged by its effects on plasma A and NA concentrations, diethyl ether seemed to markedly enhance both adrenomedullary and neurosympathetic function. This activation persisted for some hours even when anaesthesia was discontinued and the animals had woken up. The ob-
Contradictory results on the effect of halothane on plasma CA have been reported. For instance Price et al. (1959) observed no changes in plasma A and NA levels in man during halothane anaesthesia and Millar and Morris (1961) reported that halothane did not cause significant increases in plasma A and NA concentration in either premedicated surgical patients or in dogs. Hamelberg et al. (1960) reported slightly increased concentrations of plasma A and NA in man during surgery under light halothane anaesthesia but no statistically significant increases during surgery with deep halothane anaesthesia, both with premedication. Anton et al. (1964) found increased CA in blood and urine of patients during heart surgery with halothane anaesthesia. The above studies, however, were done with relatively insensitive fluorometric assays for CA. More recently, Perry et al. (1974) reported that halothane did not change the levels of plasma CA in dogs, while Depocas and Behrens (1977), using a radioenzymatic assay, found elevated plasma NA concentrations in rats under halothane anaesthesia. Our results agree with those of Depocas and Behrens (1977) for the effect of halothane on plasma NA concentrations in rats. Furthermore, we found decreased plasma A levels during the first phase of halothane anaesthesia, evidence of an inhibitory effect of the anaesthetics on adrenomedullary secretion. This observation is consistent with and supports the in vivo and in vitro findings of G/Sthert and associates (G~Sthert
22 and Dreyer, 1973; G~thert, 1974; Gt~thert et al., 1976), which show that inhalation of halothane decreases both the spontaneous release of CA from the adrenal medulla and the release evoked by splanchnic nerve stimulation, but does not affect the stimulated NA discharge from the sympathetic nerve terminals of the isolated rabbit heart.
4.3. Ketamine Both increases (Takki et al., 1972; Zsigmond et al., 1980) and decreases (Craft et al., 1980) in plasma CA levels have been reported to occur in humans during ketamine anaesthesia. Under our experimental conditions, ketamine increased both plasma A and NA concentrations in rats. This agent, as opposed to other anaesthetics such as diethyl ether and halothane which do not affect the metabolism and the reuptake of NA (Brown et al., 1972; Naito and Gillis, 1968; Ngai et al., 1969a; Ngai et al., 1969b), is known to inhibit NA reuptake (Nedergaard, 1973). Thus, this effect of ketamine may be at least in part responsible for the observed rise in plasma CA levels. The drug, however, only blocked CA uptake at high concentrations in vitro or during continuous infusion in vivo (Montel et al., 1973; Nedergaard, 1973). This, together with the observation that the ketamine-induced increase in plasma CA concentrations was abolished by ganglionic blockade, suggests that the increase of plasma CA was mainly the result of a centrally mediated activation of sympathetic neuronal and adrenal medullary functions. This is consistent with studies from other laboratories indicating that ketamine exerts its sympathomimetic effects via direct stimulation of neurons located in the central nervous system (Ivankovich et al., 1974; Roizen et al., 1981; Traber et al., 1970).
4.4. Urethane Although urethane has been widely used as anaesthetic in animal experiments for physiological and pharmacological studies of the cardiovascular system, its effects on sympathetic activity have been little studied. However, the possibility that an effect of the drug on peripheral
CAs may modify the experimental results has been pointed out since 1962 (Bowman et al., 1962). Spriggs (1965), who measured A and NA levels in various peripheral tissues in rats under urethane anaesthesia, first hypothesized that the drug may release CA, at least from the adrenal medulla. The results of the present study indicate clearly that urethane activates both the neuronal and adrenomedullary components of the sympathetic system, as judged by the dramatic and long-lasting increase in both plasma A and NA concentrations, confirming our previous data (Picotti et al., 1979). The observation that urethane failed to increase plasma CA levels in rats with the spinal cord transected at the low cervical level indicates that the mechanism responsible for the enhanced secretion of A and NA into the blood is of central origin and reflects an increased efferent sympathetic drive. Armstrong et al. (1982) reported an increase in plasma A without changes in plasma NA concentrations in Sprague-Dawley rats under urethane anaesthesia. In contrast to our results with Wistar SPF rats, where MAP was unchanged, Armstrong et al. (1982) also reported reduced values of MAP in their urethane-anaesthetized animals. In this respect it is worth noting that urethane induced a huge fall of MAP and no increase in circulating N A in spontaneously hypertensive rats (SHR) (Memo et al., 1985; our unpublished observations). Since Armstrong et al. (1982) have shown that urethane blocks cardiovascular responses mediated by stimulation of a2-adrenoceptors and since hyperreactivity of a-adrenoceptors-mediated responses has been reported to occur in SHR (Yamaguchi and Kopin, 1980), strain differences in t h e response of the neurosympathetic system and/or in adrenoceptor sensitivity may account for the different response of MAP to urethane in the various rat strains.
4. 5. Relationship between sympatho-adrenal and hemodynamic effects of anaesthetics Although the main interest of the present study was not the delineation of the exact mechanisms by which the anaesthetic-induced changes in peripheral CA release alter circulatory homeostasis,
23 the s i m u l t a n e o u s m e a s u r e m e n t s of p l a s m a C A levels, M A P a n d H R in the s a m e a n i m a l s p e r m i t s o m e speculation. S y m p a t h e t i c activation, resulting f r o m direct effects on central nervous system centers, seems to o c c u r after k e t a m i n e when the increase in p l a s m a C A c o n c e n t r a t i o n s is c o n c o m i t a n t with p a r a l l e l changes in M A P a n d H R . This w o u l d i n d i c a t e t h a t k e t a m i n e has n o i m p o r t a n t d e p r e s s a n t effects o n p e r i p h e r a l c a r d i o v a s c u l a r effectors in rats a n d therefore the changes in p e r i p h e r a l C A release i n d u c e d b y the d r u g a p p e a r to b e the p r o m i n e n t factor in m o d i f y i n g b l o o d pressure a n d heart rate. It has a l r e a d y b e e n h y p o t h e s i z e d that a direct or reflex centrally m e d i a t e d increase in symp a t h o - a d r e n a l activity w o u l d c o u n t e r b a l a n c e the p e r i p h e r a l d e p r e s s a n t effects of diethyl ether (Brewster et al., 1953; H a m e l b e r g et al., 1960; MiUar a n d Morris, 1961; Price, 1960; Price et al., 1959). It should b e e m p h a s i z e d that n o t only d i e t h y l e t h e r b u t also h a l o t h a n e a n d u r e t h a n e can d e p r e s s m y o c a r d i a l c o n t r a c t i l i t y (Brown a n d Crout, 1971; Price, 1960; W h i t e et al., 1982). Effects on a d r e n o c e p t o r s a n d p e r i p h e r a l vasculature have also b e e n r e p o r t e d ( A l t u r a a n d W e i n berg, 1979; A r m s t r o n g et al., 1982; Pasch a n d Renkl, 1979; Price, 1960; W h i t e et al., 1982). Therefore, it a p p e a r s that activation of the p e r i p h eral s y m p a t h e t i c system b y these anaesthetics serves in p a r t to c o u n t e r b a l a n c e the various depressive effects of the drugs on the c a r d i o v a s c u l a r system. I n conclusion, general anaesthetic agents do h a v e effects on p e r i p h e r a l s y m p a t h e t i c activity t h a t m u s t b e t a k e n into a c c o u n t in a n y i n t e r p r e t a tion of the relative c o n t r i b u t i o n s of the different m e c h a n i s m s b y w h i c h these drugs can affect circul a t o r y homeostasis. A s it n o w seems that anaesthetics m a r k e d l y affect p e r i p h e r a l a d r e n o m e d u l l a r y a n d n e u r o s y m p a t h e t i c activity, it is felt that the anaesthetic a g e n t to b e e m p l o y e d in c a r d i o v a s c u l a r studies s h o u l d be c h o s e n with great care. I n d e e d , even w h e n the a n a e s t h e t i c p e r se a p p e a r s to b e d e v o i d of h e m o d y n a m i c effects, as seems to b e the case with u r e t h a n e in W i s t a r S P F rats, it can well b e t h a t it modifies e x p e r i m e n t a l p a r a m e t e r s b y interfeting with s y m p a t h e t i c activity.
Acknowledgements The authors thank Professors W.E. Haefely and P. Mantegazza for helpful criticism of the manuscript and Mr. F. D'Agostini for skilful technical assistance. Partial support from the Italian Ministry of Education (MPI) and the Italian Research Council (CNR) is gratefully acknowledged,
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