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Effects of isoflurane, enflurane, and halothane on skeletal muscle microcirculation in the endotoxemic rat
Journal of Critical Care III
VOL 16, NO 1
M A R C H 2001
ORIGINAL INVESTIGATIONS
Effects of Isoflurane, Enflurane, and Halothane on Skeletal Muscle Microcirculation in the Endotoxemic Rat Jan Schumacher, Matthies P6rksen, and KarI-E Klotz Purpose: The cardiovascular effects of volatile anesthetics during sepsis sets patients at high risk for hemodynamic deterioration. We compared the microcirculatory alterations in skeletal muscle under anesthesia w i t h isoflurane, enflurane, and halothane in an endotoxemic rat preparation. Materials and Methods: Twenty-one Sprague-Dawley rats under continuous hemodynamic monitoring and intravital microscopy of the spinotrapezius muscle were studied during t w o level lipopolysaccharide (0.2 mg/kg and 2 mg/kg) induced sepsis. The effects of equianesthetic concentrations (1.5 minimum alveolar concentration [MAC]) of either isoflurane [n:7], enflurane [n:7], or halothane [n:7] on microcirculatory vasoregulation were measured and histopathologic changes were evaluated.
Results: During low-dose endotoxemia, arteriolar vasodilation under isofiurane was nearly abolished (P < .05). At high-dose endotoxemia, this lack of vasodilatory effect was similar (P < .05). Animals receiving 1.5 MAC of enflurane during lowdose endotoxin presented a significant decrease in arteriolar diameter by - 11.3 (+-2.9%), this response was less during high-dose endotoxemia (-7.0, _+2.9%). Halothane caused pronounced vasoconstriction by - 2 0 (_+3.7%) during low-dose endotoxemia and moderate but significant constriction during high-dose endotoxemia (-7.9, _+2.6%). Conclusions: Isoflurane, enflurane, and halothane exert significantly different effects on vasoregulation of skeletal muscle arterioles in the endotoxemic rat. Copyright 9 2001 by W.B, Saunders Company
EPSIS IS THE leading cause of death in nonk.~ coronary intensive care units.'12 Septic shock arises often as a result of a surgical condition that requires an operative procedure. Volatile anesthetics are known to affect the cardiovascular system) Halothane alters the EDRF/NO pathway by decreasing synthesis, transport, release, or action of EDRF. 4-7 In contrast to this, Greenblatt et al 8 showed that isoflurane compared with halothane is associated with greater EDRF/NO action in the coronary, renal, splanchnic, hepatic, and cutaneous vasculature. In addition, enflurane 9 and halothane block calcium channels in vascular smooth muscle, whereas isoflurane exhibits this effect only in clinically irrelevant concentrations, l~ NO appears also to be an important mediator of septic vascular dysfunction. H The pathophysiologic interactions between the cardiovascular effects of volatile anesthetics during septic shock are incompletely
understood. This may place patients undergoing general anesthesia at high risk for hemodynamic deterioration. Recently, Imai et all2 demonstrated that different anesthetic methods affected outcome of severe sepsis after cecal ligation and puncture in endotoxin-resistant and endotoxin-susceptible strains of mice. Prior animal studies used endotoxin-exposed
Journal of Critical Care,Vol 16, No 1 (March), 2001: pp 1-7
From the Department of Anesthesiology, Medical University of Luebeck, Luebeck, Germany. Supported by the Medical University of Luebeck, Luebeck, Germany. Received August 28, 2000. Accepted November 1, 2000. Address reprint requests to Jan Sehumacher, MD, Department of Anesthesiology, Medical University of Luebeck, Ratzebarger Allee 160, D-23538 Luebeck, Germany. Copyright 9 2001 by W.B. Saunders Company 0883-9441/01/1601-0001 $35.00/0 doi: lO.1053/jcrc.2001.21790 1
2
SCHUMACHER, PORKSEN,AND KLOTZ
r a t a o r t i c r i n g s 13 o r i n v e s t i g a t e d c h a n g e s i n c e n t r a l hemodyfiamics
a n d o x y g e n b a l a n c e . 14'15 H o w e v e r ,
no reports are available that compare the effects of v o l a t i \l e a n e s t h e t i c s o n t h e r e g u l a t i o n o f m i c r o c i r -
earcorder Mark 7; Graphtec Co, Yokohama, Japan). Systemic vascular resistance (SVR) was calculated from the quotient of MAP and CO. Body core temperature was measured via a rectal probe and maintained at 37~ with an overhead heating lamp and an underlaying heating element.
c u l a t i o n d u r i n g s e p s i s . T h e a i m o f t h e s t u d y w a s to compare the microcirculatory
alterations in skele-
tal m u s c l e a n d t h e h i s t o p a t h o l o g i c anesthesia
with isoflurane,
thane in an endotoxemic
changes under
enflurane,
and halo-
rat preparation.
MATERIALS A N D METHODS
Animals and Anesthesia The care of the animals was in full accordance with German animal protection laws, and the experiments were officially approved by the governmental animal care and use committee. Female Sprague-Dawley rats (70 to 110 g, aged 4-6 weeks) were allowed to acclimate for a minimum of 7 days after arrival to our laboratory. They were maintained on alternating 12 h/12 h light/dark cycles, housed at 22 ~ (45% relative humidity) with free access to water and rat chow. Food, but not water was withheld at least 12 hours before experimentation. Premedication was achieved by intramuscular (IM) injection of pentobarbital (80 mg/kg) (Sanofi, Ceva, Hannover, Germany). Ten minutes later, anesthesia was induced by ketamine (100 mg/kg) IM (Parke Davis, Freiburg, Germany). Tracheostomy was performed while the animals breathed room air spontaneously. Afterwards the animals were ventilated with a small animal respirator (HSE Schuler, Typ 811; H. Sachs Electronics, Freiburg, Germany) with 100% oxygen. Arterial blood gases were analyzed by an ABL 500 (Radiometer Copenhagen, Denmark). The animals were ventilated at a rate of 85 to 90 breaths/rain, and tidal volume was set to keep the arterial partial pressure of C02 at 35 m m Hg. The volatile anesthetics were administered by means of previously calibrated standard vaporizers (Draeger Medizintechnik, Luebeck, Germany), whereas the anesthetic concentrations were additionally analyzed continuously (Irina, Draeger Medizintechnik, Luebeck, Germany). During the whole experiment the basic level of anesthesia was achieved by isofhirane 0.3 vol% (Abbott, Wiesbaden, Germany), enflurane 0.4 vol% (Abbott, Wiesbaden, Germany) or halothane 0.2 vol% (ICI-Pharma, Planckstadt, Germany), respectively. Fluid therapy consisted of Ringer's solution (5 mL/kg/h) continuously intravenously.
Animal Preparation and Hemodynamic Measurement After preparation of the external jugular vein, a portex catheter (O.D. 0.61 ram) was inserted in the brachiocephalic 9ein under sterile conditions. The right carotid artery was cannulated, and direct arterial pressure and heart rate were monitored via a pressure transducer (Isotec-healthdyne cardiovascular, Marietta, GA). After instrumentation of the two vessels, the skin of the cervical region was sutured. Following thoracotomy a perivascular Doppler flow probe (T106, Transonic Instruments, Utica, NY) was wrapped around the proximal ascending aorta. Mean arterial pressure (MAP), hear{ rate, and ultrasonographically determined CO were continuously recorded (Lin-
Intravital Microscopy Microvascular blood flow in the left spinotrapezius muscle was studied using intravital microscopy. The detailed preparation has been previously described. 16 After the preparation, which was performed in prone position on the microscope stage, the spinotrapezius muscle was placed on a cover glas (6 • 6 mm). This glas was clamped into a holder, which in turn was mounted to the support stage. Therefore, the position of the cover slip was fixed relatively to the muscle tissue even during movement of the stage. This position resulted in a deflection of the muscle from the thoracic wall by approximately 35 ~ to 45 ~. During the preparation and the following experiment, the muscle was superfused with a Tyrode's solution at 37~ temperature, pH-equilibrated (CO2 5% with N2), and flow controlled. Two Tyrode solutions were used. The Tyrode 1 solution contained NaC1 0.133 mol/L, KC1 0:004 mol/L, NaH2PO4 0.013 mol/L, NaHCO3 0.016 mol/L, the Tyrode 2 solution contained CaC12 0.1673 mol/L and MgC12 0,0733 mol/L. The prepared muscle was observed using an intravital microscope (Wild M650; Leitz, Wetzlar, Germany) with a 40• objective lens (L25/0.22P) modified for telescopic viewing and an additional upper platform supporting a low light level video and a photographic camera. Illumination through the muscle was achieved by a prism arrangement using a 150 W xenon lamp and a L25/0.22P-UT40/0.34 Leitz lens combination (Leitz, Wetzlar, Germany). The chosen field of view was visualized and recorded on videotape with a CCD-camera (77CE-Field; PCO Computer, Kehlheim, Germany). The tapes were analyzed by a single observer in a blinded fashion. We analyzed transversal arterioles with a diameter of 10 to 20 ixmat rest. The determination of arteriolar diameter changes during the experimental steps was achieved by computerized video image analysis (Summasketch II; Summagraphics, WDV, Garching, Muenchen, Germany) in 10-second intervals. Representative mean values during each experimental step were collected during every third minute after volatile anesthetic exposition and during every fourth minute after the application of the substances acetylcholine, nitroglycerin, and phenylephrine used for monitoring of vasoregulatory capacity.
Experimental Protocol We used seven Female Sprague-Dawley rats in every volatile anesthetic group. The protocol (Fig 1) was divided in three periods, during which every animal received only one of the tested volatile anesthetics. After a control period, a low dose of endotoxin was injected (low endotoxin period). We used lipopolysaccharities from Escherichia coli (Lipopolysaccharlde E. coli, [LPS], serotype 055-B5, Fhika-Feinchemikalien, Neu-Ulm, Germany) in a dosage of 0.2 mg/kg of body weight. A third period (high endotoxiu period) was established by an additional injection of LPS 2 mg/kg. After the LPS administration, a period of 60 minutes was allowed for developing circulatory changes. During each of the three periods, a set of substances was applied. Two concentrations of the three volatile anesthetics were
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
control period
==.==.l=.=========m
v
3
low endotoxin period highendotoxin period
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Jt
/ /
t
P='''''''''''''''"
~lr
~36 min~
12 rag-k0' , p s
"*%*********
"..,~
~1
0 2
wash
6
10
wash
14
18
wash
22
26
wash
30
34
min
Fig 1. Experimental protocol, Anesthetized rats received either 1.5 MAC of isoflurane, enflurane, or halothane during control, a low-dose endotoxin period, and a high-dose endotoxin period. In every study period acetylcholine (ACh), nitroglycerin (NTG), and phenyl-ephrin (PE) were applied to monitor endothelium-dependent and endothelium-independent mechanisms of vasoregulation.
used. A basal minimum alveolar concentration (MAC) of 0.25 was applied continuously throughout the study and was interrupted by periods of 4 minutes of high concentrations using 1.5 MAC. The respective concentrations are presented in Table 1. After the volatile anesthetic peak exposure, a washout period of 6 minutes was allowed. To test the vasoactive potency of the arteriole under observation, three test substanc~es were superfused during each period. Two vasodilators were used: (1) Acetylcholine (Sigma Chemie, Deisenhofen, Germany) (endothelium
dependent) reaching a concentration of 10 - s mol/L at the muscle preparation and (2) Nitroglycerin (Nitro-Pohl, Pohl, Hohenlockstedt, Germany) (endothelium independent) reaching a concentration of 10 5 mol/L at the muscle. To show vasoc0nstrictory function, phenylephrine (Neosynephrine; SanofiWinthrop, Muenchen, Germany) was superfused gaining a concentration of 10 .5 mol/L at the preparation. Each drug was superfused for 4 minutes, with intermittent washout periods of 4 minutes.
Histopathology Table 1. Dosages of the Studied Volatile Anesthetic Agents
Basal concentration [vol/%] 1.5 MAC [vol-%]
Isoflurane
Enflurane
Halothane
0.3 1.8
0.4 2,6
0.2 1.2
NOTE. Data are expressed in vol%.
After the study, the anesthetized animals were killed by bolus application of KC1, in accordance to the guidelines of the governmental animal care and use committee. Autopsies were performed immediately afterwards, and specimens of hearts, lungs, kidneys, spleens, and livers were fixed in formalin and later embedded in paraffin. Histologic study was performed on 10-t~m-thick sections stained with hematoxylin-eosin.
4
SCHUMACHER, PORKSEN, A N D KLOTZ
Statistical Analysis Data are expressed as mean _+ SEM. To analyze the change in central hemodynamic and skeletal muscle microcirculation during the experiment, we compared the mean values of the three ~olatile anesthetic groups. To determine the effects of LPS on the central hemodynamic and skeletal muscle microcirculation, we compared the mean values of all 21 animals horizontally before and after the LPS administrations. The matched pair ranked test (Friedman and subsequent Wilcoxon tests) Was applied to compare values of different observation time points with initial values. Comparisons between the groups were performed using the Mann-Whitney U test. Probability values <.05 were considered to show significance.
RESULTS
Table 2 presents the absolute values of the central hemodynamic parameters due to LPS and volatile anesthetics during the observation time.
1. Systemic Hemodynamic Response to Volatile Anesthetics Before Endotoxemia During 1.5 MAC of enflurane and halothane anesthesia, HR was significantly reduced, MAP declined with all tested volatile anesthetics (P < .05). Isoflurane induced the smallest CO reduction, which was significantly different to the halothane group. SVR alterations were similar, isoflurane induced the strongest decrease, also significantly different to halothane.
2. Systemic Hemodynamic Response to Endotoxemia During basic level of inhalational anesthesia (0.25 MAC), we found a significant increase in HR after the high dose LPS application (P < .05). MAP and SVR showed a moderate decrease, whereas CO remained constant.
3. Microcirculatory Responses to Endotoxemia and to Monitor Substances During the control period we found a mean arteriolar diameter of 12.7 (_+ 2.8 Ixm). After the application of LPS 0.2 mg/kg mean diameter increased significantly to 14.0 ( + 2.8 ~m). Following the later injection of the high dose of LPS 2.0 mg/kg arteriolar diameter decreased significantly to 11.8 (-+ 3.8 p~m) compared with the diameter during the low dose endotoxin period. Table 3 presents the changes resulting from superfusion of the monitor substances after the induction of endotoxemia. All induced changes were significant compared with baseline. The dilatatory potency of acetylcholine (10 -5 tool/L) and nitroglycerine (10 -5 mol/L) was found to be significantly diminished during the two different endotoxin periods; however, the vasoconstrictive potency of phenylephrine (10 -5 mol/L) appeared unchanged.
Table 2. Change of HemodynamicVariables 0.25 MAC Basic Anesthesia
1.5 MAC Enflurane [n: 7]
Mean, -+ SEM HR [1 beat/rain] M A P [ m m Hg] CO [ m L / m i n ] SVR [ m m Hg 9 m L 9 m i n ]
1.5 MAC Halothane [n: 7]
1.5 MAC Isoflurane [n: 7]
Control period 294 _+ 32
275 • 39*
255 • 18"
64 § 14
48 + 10"
37 • 10"
291 • 33
13.2 + 3.4
8.9 § 3.7*
9.4 • 4,7"
12,2 _+ 3.1"#
5.2 _+ 1,7
5.8 • 1.4
4,7 _+ 2.0
3.7 • 1.1"#
43 • 7*
Low endotoxin period HR [1 beat/min]
309 • 47
287 -- 31"
272 -+ 29*
62 • 16
48 • 10"
37 + 13"
48 • 10"
13.4 • 4.5
9.0 • 2,9*
9.2 • 5.8*
13.8 -+ 3 . 7 " # t
5.1 _+ 1.8
5.6 -+ 0.9
5.2 • 2.6
3.7 • 1.3"#t
HR [1 beat/min]
330 • 64w
290 • 33*
293 • 56*
M A P [ram Hg]
60 • 17
45 • 12"
40 • 14"
42 • 7*
13.2 • 4.6
8.1 • 3.3*
9.9 • 5.9*
13.3 • 3 . 5 t
5.0 • 1.7
6.0 • 1.6
5.0 + 2.1
M A P [ m m Hg] CO [mL/min] SVR [ram Hg . m L 9 rain]
320 • 62
High endotoxin period
CO [ m L / m i n ] SVR [ram Hg 9 m L 9 min]
332 • 66
3.5 • 1.4"#
NOTE. Mean values _+ SEM, * P < .05 c o m p a r e d w i t h basic volatile anesthesia, # P < .05 c o m p a r e d w i t h halothane, t P < .05 c o m p a r e d w i t h enflurane, w < ,05 c o m p a r e d w i t h the control period.
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
Table 3. Change of Arteriolar Diameter (l,m) After Monitor-Substances Acetylcholine
Mean • SEM Control period Low endotoxin period High endotoxin period
Nitroglycerine
Phenylephrine
(10 Stool/L) [changein t~m]
(10-Smol/L) [changein p,m]
(10 Smol/L) [changein l~rn]
+6,1 -+ 1.3"
+3.0 _+ 0.9*
- 4 . 2 -+ 0.5*
+3.4 -+ 0,8*#
+1.4 +_ 0.3*#
- 6 . 5 + 0.6*#
+2.3 -+ 0.8*#
+0,8 _+ 0.4*#
- 4 , 3 + 0.7"1"
NOTE. Mean values are given in ~m _+ SEM. * P < .05 compared with pre-superfusion with monitor-substance. #P < .05 compared with controt period change, t P < .05 compared with low endotoxin period change.
4. Systemic Hemodynamic Response to Volatile Anesthetics During Endotoxemia Table 2 presents the absolute values of the systemic hemodynamic parameters during the observation time. HR was reduced by 5% to 10% during exposure to 1.5 MAC of enflurane and halothane. No significant differences between anesthetics were found. MAP declined in a similar extent of about 30% during the application of the different volatile anesthetics throughout all study periods. CO did not decrease in the isoflurane exposed animals. Enflurane and halothane produced a decline of 30% in CO in all study periods. SVR decreased in the isoflurane group by 15% to 20%, but not in the enflurane and halothane groups.
4O
5
5. MicrocircuIatory Responses to Volatile Anesthetics During Endotoxemia Figure 2 presents the changes in arteriolar diameter due to exposure to isoflurane, enflurane, and halothane during the observation period. In the control period, application of 1.5 MAC of isoflurane dilated the mean arteriolar diameter by 26 (+ 6.2%). After establishment of a low-dose endotoxemia, vasodilation during exposure to 1.5 MAC isofturane was nearly abolished compared with control (P < .05). In the state of high-dose endotoxemia, this lack of vasodilatory effect was similar (P < 0.5). Animals who received 1.5 MAC of enflurane showed only a small increase in arteriolar diameter of 4.5 (+_ 3.6%) during control. In contrast to this, during the low-dose endotoxin period exposure to enflurane resulted in a significant decrease in diameter by -11.3 (_+ 2.9%), this response was less during high-dose endotoxemia (-7.0, +_ 2.9%). Halothane induced virtually no change in microvascular diameter during the control period. We found a pronounced vasoconstriction by - 2 0 (-+ 3.7%) during the low-dose endotoxin period, and a moderate but significant constriction during high-dose endotoxemia ( - 7 . 9 , + 2,6%).
Pathology and Histopathology The macroscopic evaluation presented no abnormalities without signs of hyperyolemia, such as liver congestion or pulmonary edema. The histo-
~B
Isoflurane, n: 7 E n f l u r a n e , n: 7
L
E
H a l o t h a n e , n: 7
30 20
c 0 e-
Fig 2. Skeletal muscle arterial diameter in response to exposure to 1.5 MAC of isoflurane,
-10
L)
-20 -30
i
Control
i
Low endotoxin
i
High endotoxin
enflurane, and halothane during endotoxemia. Change of diameter is given in percent -+ SEM. * P < .05 compared w i t h the control period. #P < .05 compared with the low endotoxin period. +P < .05 compared w i t h the high endotoxin period.
6
SCHUMACHER, PORKSEN,AND KLOTZ
logic investigation of the organ specimens revealed no pattiology. The liver biopsies of the halothane exposed animals showed centrilobular fatty degeneration. DISCUSSION
The aim of this study was to compare the effects of isoflurane, enflurane, and halothane on skeletal muscle microcirculafion in an endotoxininduced rat sepsis preparation. Various models of experimental sepsis have been described in animals: Cecal ligation and puncture, !2 injection of bacteria, iv or the administration of isolated endotoxin intraperitoneally is or intravenously. 14 The latter septic challenge may have advantages in comparing different hemodynamic or microcirculatory effects that may be observed. Additionally, the time course of the Septic vasoregulatory changes is faster and more Consistent. Due to this rationale, we selected the intravenous LPS injection as the sepsis trigger. In our study application of the low dose of LPS (0.2 mg/kg) induced only minor hemodynamic changes, the injection of the higher LPS dose (2 mg/kg) resulted in significant tachycardia. Similar findings have been reported by Stewart et a117 who induced tachycardia without MAP alteration by infusion of E. coli isolates in conscious rats. In contrast to our findings, hemodynamic measurements from late onset septicemic experiments showed a reduction of M A P . t9 The effects of the tested volatile anesthetics on the systemic-hemodynamics are in accordance to the literature. 2~ During endotoXemia, isoflurane induced SVR decline was only 50% from control values. Neither enflurane nor halothane did alter SVR after LPS application. In contrast, Van der Linden et al a4 observed a decrease of SVR following the application of 0.5 MAC of halothane in an endotoxin septic shock dog model. The postmortem evaluation showed none of the abnormalities seen in chronic and fatal sepsis. The presented model focused on the early changes of septic vascular dysfunction, and therefore signs of chronic organ malperfusion and failure were missing. In this study, microcirculatory responses to endotoxemia exhibited the typical pattern of septic vascular dysfunction. After the application of lowdose LPS, mean arteriolar diameter increased significantly. Following the later injection of the high dose of endotoxin, arterioiar diameter decreased
significantly compared with the diameter during the low-dose endotoxin period. This finding of impaired vasoconstriction and later attenuation of vasorelaxafion during sepsis is a persisting observation of microcirculatory studies. This septic vascular dysfunction has recently been reviewed by Murray et al. 11 Additionally, our results confirm that the dilatatory potency of acetylcholine and nitroglycerine were significantly diminished during the two different endotoxin periods. This loss of dilatory potency may be explained by endotoxin enhanced synthesis of nitric oxide (NO) during sepsis.11'21 This might impair further vasodilafion because both superfused monitor substanCes result in the same mediator, either endothelial-dependent, like acetylcholine, or endothelial-independent, like nitroglycerin. From this observation, we conclude that septic vascular dysfunction was achieved by the LPS dosages used, the maintained vasoconstrietory potency of phenylephrine shows that vasoregulation was not completely abolished in this sepsis model. Furthermore, the discrepancy between the muscular microcirculatory vasoregulation and the systemic vascular resistance during sepsis indicates a heterogenity of perfusion in different organ systems. The dosages of the applied volatile anesthetic agents in our study were chosen to induce comparable depressions in MAP during the control period. This was achieved by similar MAC values known from humans. Exposure to 1.5 MAC of either isoflurane, enflurane, or halothane reduced MAP by 30% in all animals during control. This depression of MAP was also found in the low and the high endotoxin periods when the animals were exposed to 1.5 MAC of either isoflurane and enflurane. The halothane group showed significant less reduction of MAP during high-dose endotoxemia. Microcirculatory data, acquired in nonsepfic organisms, 5'9'1~ may explain our observation. Isoflurane dilates skeletal muscle arterioles by enhancing the release of the endothelial mediator NO. s During sepsis, with increased stimulation of the inducible-NO-synthase levels, this mechanism of action is limited, resulting in a lack of vasodilatory potency. Hence, the vasodilatory effect of isoflurane is nearly abolished during endotoxinderived septicemic syndromes. In contradiction to this, halothane is known to hinder the release of the endothelial mediator NO. 5'22 This results in a constrictory influence on
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
v a s o r e g u l a t i o n , w h e r e a s i n n o n s e p t i c states o n e finds a c o m p e t i t i o n w i t h t h e v a s o d i l a t i n g d i r e c t C a 2 + - c h a n n e l b l o c k i n g p r o p e r t i e s t~ o f h a l o t h a n e . I n c a s e s Of a septic s y n d r o m e w i t h a u g m e n t e d N O d e l i v e r a n c e , t h e i n h i b i t i n g effect o n N O s y n t h e s i s outweighs the Ca2+-channel derived vasorelaxation. T h i s m i g h t e x p l a i n o u r f i n d i n g o f a r t e r i o l a r vasoconstriction under halothane exposition during endotoxemia. T h e i n f l u e n c e o f e n f l u r a n e o n skeletal m u s c l e m i c r o c i r c u l a t i o n a p p e a r s a n a l o g o u s to the h a l o t h a n e g e n e r a t e d effects. S u e t al 9 r e p o r t e d a b l o c k a d e o f C a 2+ i n f l u x in v a s c u l a r s m o o t h m u s c l e u n d e r the i n f l u e n c e o f e n f l u r a n e . W e s u g g e s t t h a t a s i m i l a r i n h i b i t i n g effect o n N O s y n t h e s i s prevails, e q u a l l y r e s u l t i n g in arteriolar v a s o c o n s t r i c t i o n .
7
I n s u m m a r y , w e f o u n d t h a t the v a s o a c t i v e effect o f i s o f l u r a n e k n o w n f r o m n o n s e p t i c o b s e r v a t i o n s is a b o l i s h e d d u r i n g e n d o t o x i n - i n d u c e d sepsis. W e also f o u n d t h a t e n f l u r a n e a n d h a l o t h a n e i n d u c e a s i g n i f i c a n t v a s o c o n s t r i c t i o n o f skeletal m u s c l e arterioles in the e n d o t o x e m i c rat. T h e s e f i n d i n g s m a y shed light on the problems of choosing the approp r i a t e volatile a n e s t h e t i c a g e n t i n states o f septic vascular dysfunction.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the support of the Institute of Pathology, Medical University of Luebeck, Germany for the histopathological investigations and the technical support of Dunja Leffler from the Department of Anesthesiology, Medical University of Luebeck.
REFERENCES
1. Bone RC, Grodzin CJ, Balk RA: Sepsis: A new hypothesis for pathogenesis of the disease process. Chest 112:235-243, 1997 2. Friedman G, Silva E, Vincent J-L: Has the mortality of septic shock changed within time? Crit Care Med 26:2078-2086, 1998 3. Hoffman WD, Banks SM, Alling DW, et al: Factors that determine the hemodynamic response to inhalational anesthetics. J Appl Physiol 70:2155-2163, 1991 4. Stone DJ, Johns RA: Endothelium-dependent effects of halothane, enflurane, and isoflurane on isolated rat aortic vascular rings. Anesthesiology 71:126-132, 1989 5. Muldoon SM, Hart JL, Bowen KA, et al: Attenuation of endothelium-mediated vasodilation by halothane. Anesthesiology 68:31-37, 1988 6. Uggeri MJ, Proctor GJ, Johns RA: Halothane, enflurane, and isoflurane attenuate both receptor and non-receptor-mediated EDRF production in rat thoracic aorta. Anesthesiology 76:1012-1017, 1992 7. Toda H, Nakamura K, Hatano Y, et al: Halothane and isofiurane inhibit endothelium-dependent relaxation elicited by acetylcholine. Anesth Analg 75:198-203, 1992 8. Greenblatt EP, Loeb AL, Longnecker DE: Endotheliumdependent circulatory control--A mechanism for the differing peripheral vascular effects of isoflurane versus halothane. Anesthesiology 77:1178-1185, 1992 9. Su JY, Chang YI, Tang LJ: Mechanisms of action of enflurane on vascular smooth muscle. Anesthesiology 81:700-709, 1994 10.. Buljubasic N, Rusch NJ, Mafijic J, et al: Effects of halothane and isofluraue on calcium and potassium currents in canine coronary arterial cells. Anesthesiology 76:990-998, 1992 I 1. Murray PT, Wylam ME, Umans JG: Nitric oxide and septic vascular dysfunction. Anesth Analg 90:89-i01, 2000 12. Imai T, Takahashi K, Masuo F, et al: Anaesthesia affects outcome of sepsis in mice. Can J Anaesth 45:360-366, 1998
13. Grissom TE, Bina S, Hart J, et al: Effects of halothane on phenylephrine-induced vascular smoothe muscle contractions in endotoxin-exposed rat aortic rings. Crit Care Med 24:287-293, 1996 14. Van der Linden R Gilbart E, Engelman E, et al: Comparison of halothane, isoflurane, alfentanyl, and ketamine in experimental septic shock. Anesth Analg 70:608-617, 1990 15. Gill R, Martin C, McKinnon T, et al: Sepsis reduces isoflurane MAC in a normotensive animal model of sepsis. Can J Anaesth 42:631-635, 1995 16. Klotz KF, Pries AR, Jepsen H, et al: A new approach to intravital videomicroscopy of rat spinotrapezius muscle. Int J Microcirc Clin Exp 10:205-218, 1991 17. Stewart KD, Brackett DJ, Lemer MR, et al: Comparison of Staphylococcus aureus and Escherichia coli infusion in conscious rats. J Surg Res 56:60-66, 1994 ' 18. Fischer LG, Horstmann DJ, Hahnenkamp K, et al: Selective iNOS inhibition attenuates acetylcholine- and bradykinin-induced vasoconstriction in lipopolysaccharideexposed rat lungs. Anesthesiology 91:1724-1732, 1999 19. Pastor C, Teisseire B, Vicaut E, et al: Effects of L-arginine and L-nitro-arginine treatment on blood pressure and cardiac output in a rabbit endotoxin shock model. Crit Care Med 22:465-469, 1994 20. Pagel PS, Farber NE, Warltier DC: Cardiovascular pharmacology, in Miller RD (ed): Anesthesia. New York, NY, Churchill Livingstone, 2000, pp 96-124 21. Evans T, Carpenter A, Kinderman H, et al: Evidence of increased nitric oxide production in patients with sepsis syndrome. Circ Shock 41:77-81, 1993 22. Meyer J, Lentz CW, Herndon DN, et al: Effects of halothane anesthesia on vasoconstrictor response to NG-nitroL-arginine methyl ester, an inhibitor of nitric oxide synthesis, in sheep. Anesth Analg 77:1215-1221, 1993
VOL 16, NO 1
M A R C H 2001
ORIGINAL INVESTIGATIONS
Effects of Isoflurane, Enflurane, and Halothane on Skeletal Muscle Microcirculation in the Endotoxemic Rat Jan Schumacher, Matthies P6rksen, and KarI-E Klotz Purpose: The cardiovascular effects of volatile anesthetics during sepsis sets patients at high risk for hemodynamic deterioration. We compared the microcirculatory alterations in skeletal muscle under anesthesia w i t h isoflurane, enflurane, and halothane in an endotoxemic rat preparation. Materials and Methods: Twenty-one Sprague-Dawley rats under continuous hemodynamic monitoring and intravital microscopy of the spinotrapezius muscle were studied during t w o level lipopolysaccharide (0.2 mg/kg and 2 mg/kg) induced sepsis. The effects of equianesthetic concentrations (1.5 minimum alveolar concentration [MAC]) of either isoflurane [n:7], enflurane [n:7], or halothane [n:7] on microcirculatory vasoregulation were measured and histopathologic changes were evaluated.
Results: During low-dose endotoxemia, arteriolar vasodilation under isofiurane was nearly abolished (P < .05). At high-dose endotoxemia, this lack of vasodilatory effect was similar (P < .05). Animals receiving 1.5 MAC of enflurane during lowdose endotoxin presented a significant decrease in arteriolar diameter by - 11.3 (+-2.9%), this response was less during high-dose endotoxemia (-7.0, _+2.9%). Halothane caused pronounced vasoconstriction by - 2 0 (_+3.7%) during low-dose endotoxemia and moderate but significant constriction during high-dose endotoxemia (-7.9, _+2.6%). Conclusions: Isoflurane, enflurane, and halothane exert significantly different effects on vasoregulation of skeletal muscle arterioles in the endotoxemic rat. Copyright 9 2001 by W.B, Saunders Company
EPSIS IS THE leading cause of death in nonk.~ coronary intensive care units.'12 Septic shock arises often as a result of a surgical condition that requires an operative procedure. Volatile anesthetics are known to affect the cardiovascular system) Halothane alters the EDRF/NO pathway by decreasing synthesis, transport, release, or action of EDRF. 4-7 In contrast to this, Greenblatt et al 8 showed that isoflurane compared with halothane is associated with greater EDRF/NO action in the coronary, renal, splanchnic, hepatic, and cutaneous vasculature. In addition, enflurane 9 and halothane block calcium channels in vascular smooth muscle, whereas isoflurane exhibits this effect only in clinically irrelevant concentrations, l~ NO appears also to be an important mediator of septic vascular dysfunction. H The pathophysiologic interactions between the cardiovascular effects of volatile anesthetics during septic shock are incompletely
understood. This may place patients undergoing general anesthesia at high risk for hemodynamic deterioration. Recently, Imai et all2 demonstrated that different anesthetic methods affected outcome of severe sepsis after cecal ligation and puncture in endotoxin-resistant and endotoxin-susceptible strains of mice. Prior animal studies used endotoxin-exposed
Journal of Critical Care,Vol 16, No 1 (March), 2001: pp 1-7
From the Department of Anesthesiology, Medical University of Luebeck, Luebeck, Germany. Supported by the Medical University of Luebeck, Luebeck, Germany. Received August 28, 2000. Accepted November 1, 2000. Address reprint requests to Jan Sehumacher, MD, Department of Anesthesiology, Medical University of Luebeck, Ratzebarger Allee 160, D-23538 Luebeck, Germany. Copyright 9 2001 by W.B. Saunders Company 0883-9441/01/1601-0001 $35.00/0 doi: lO.1053/jcrc.2001.21790 1
2
SCHUMACHER, PORKSEN,AND KLOTZ
r a t a o r t i c r i n g s 13 o r i n v e s t i g a t e d c h a n g e s i n c e n t r a l hemodyfiamics
a n d o x y g e n b a l a n c e . 14'15 H o w e v e r ,
no reports are available that compare the effects of v o l a t i \l e a n e s t h e t i c s o n t h e r e g u l a t i o n o f m i c r o c i r -
earcorder Mark 7; Graphtec Co, Yokohama, Japan). Systemic vascular resistance (SVR) was calculated from the quotient of MAP and CO. Body core temperature was measured via a rectal probe and maintained at 37~ with an overhead heating lamp and an underlaying heating element.
c u l a t i o n d u r i n g s e p s i s . T h e a i m o f t h e s t u d y w a s to compare the microcirculatory
alterations in skele-
tal m u s c l e a n d t h e h i s t o p a t h o l o g i c anesthesia
with isoflurane,
thane in an endotoxemic
changes under
enflurane,
and halo-
rat preparation.
MATERIALS A N D METHODS
Animals and Anesthesia The care of the animals was in full accordance with German animal protection laws, and the experiments were officially approved by the governmental animal care and use committee. Female Sprague-Dawley rats (70 to 110 g, aged 4-6 weeks) were allowed to acclimate for a minimum of 7 days after arrival to our laboratory. They were maintained on alternating 12 h/12 h light/dark cycles, housed at 22 ~ (45% relative humidity) with free access to water and rat chow. Food, but not water was withheld at least 12 hours before experimentation. Premedication was achieved by intramuscular (IM) injection of pentobarbital (80 mg/kg) (Sanofi, Ceva, Hannover, Germany). Ten minutes later, anesthesia was induced by ketamine (100 mg/kg) IM (Parke Davis, Freiburg, Germany). Tracheostomy was performed while the animals breathed room air spontaneously. Afterwards the animals were ventilated with a small animal respirator (HSE Schuler, Typ 811; H. Sachs Electronics, Freiburg, Germany) with 100% oxygen. Arterial blood gases were analyzed by an ABL 500 (Radiometer Copenhagen, Denmark). The animals were ventilated at a rate of 85 to 90 breaths/rain, and tidal volume was set to keep the arterial partial pressure of C02 at 35 m m Hg. The volatile anesthetics were administered by means of previously calibrated standard vaporizers (Draeger Medizintechnik, Luebeck, Germany), whereas the anesthetic concentrations were additionally analyzed continuously (Irina, Draeger Medizintechnik, Luebeck, Germany). During the whole experiment the basic level of anesthesia was achieved by isofhirane 0.3 vol% (Abbott, Wiesbaden, Germany), enflurane 0.4 vol% (Abbott, Wiesbaden, Germany) or halothane 0.2 vol% (ICI-Pharma, Planckstadt, Germany), respectively. Fluid therapy consisted of Ringer's solution (5 mL/kg/h) continuously intravenously.
Animal Preparation and Hemodynamic Measurement After preparation of the external jugular vein, a portex catheter (O.D. 0.61 ram) was inserted in the brachiocephalic 9ein under sterile conditions. The right carotid artery was cannulated, and direct arterial pressure and heart rate were monitored via a pressure transducer (Isotec-healthdyne cardiovascular, Marietta, GA). After instrumentation of the two vessels, the skin of the cervical region was sutured. Following thoracotomy a perivascular Doppler flow probe (T106, Transonic Instruments, Utica, NY) was wrapped around the proximal ascending aorta. Mean arterial pressure (MAP), hear{ rate, and ultrasonographically determined CO were continuously recorded (Lin-
Intravital Microscopy Microvascular blood flow in the left spinotrapezius muscle was studied using intravital microscopy. The detailed preparation has been previously described. 16 After the preparation, which was performed in prone position on the microscope stage, the spinotrapezius muscle was placed on a cover glas (6 • 6 mm). This glas was clamped into a holder, which in turn was mounted to the support stage. Therefore, the position of the cover slip was fixed relatively to the muscle tissue even during movement of the stage. This position resulted in a deflection of the muscle from the thoracic wall by approximately 35 ~ to 45 ~. During the preparation and the following experiment, the muscle was superfused with a Tyrode's solution at 37~ temperature, pH-equilibrated (CO2 5% with N2), and flow controlled. Two Tyrode solutions were used. The Tyrode 1 solution contained NaC1 0.133 mol/L, KC1 0:004 mol/L, NaH2PO4 0.013 mol/L, NaHCO3 0.016 mol/L, the Tyrode 2 solution contained CaC12 0.1673 mol/L and MgC12 0,0733 mol/L. The prepared muscle was observed using an intravital microscope (Wild M650; Leitz, Wetzlar, Germany) with a 40• objective lens (L25/0.22P) modified for telescopic viewing and an additional upper platform supporting a low light level video and a photographic camera. Illumination through the muscle was achieved by a prism arrangement using a 150 W xenon lamp and a L25/0.22P-UT40/0.34 Leitz lens combination (Leitz, Wetzlar, Germany). The chosen field of view was visualized and recorded on videotape with a CCD-camera (77CE-Field; PCO Computer, Kehlheim, Germany). The tapes were analyzed by a single observer in a blinded fashion. We analyzed transversal arterioles with a diameter of 10 to 20 ixmat rest. The determination of arteriolar diameter changes during the experimental steps was achieved by computerized video image analysis (Summasketch II; Summagraphics, WDV, Garching, Muenchen, Germany) in 10-second intervals. Representative mean values during each experimental step were collected during every third minute after volatile anesthetic exposition and during every fourth minute after the application of the substances acetylcholine, nitroglycerin, and phenylephrine used for monitoring of vasoregulatory capacity.
Experimental Protocol We used seven Female Sprague-Dawley rats in every volatile anesthetic group. The protocol (Fig 1) was divided in three periods, during which every animal received only one of the tested volatile anesthetics. After a control period, a low dose of endotoxin was injected (low endotoxin period). We used lipopolysaccharities from Escherichia coli (Lipopolysaccharlde E. coli, [LPS], serotype 055-B5, Fhika-Feinchemikalien, Neu-Ulm, Germany) in a dosage of 0.2 mg/kg of body weight. A third period (high endotoxiu period) was established by an additional injection of LPS 2 mg/kg. After the LPS administration, a period of 60 minutes was allowed for developing circulatory changes. During each of the three periods, a set of substances was applied. Two concentrations of the three volatile anesthetics were
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
control period
==.==.l=.=========m
v
3
low endotoxin period highendotoxin period
/ /
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Jt
/ /
t
P='''''''''''''''"
~lr
~36 min~
12 rag-k0' , p s
"*%*********
"..,~
~1
0 2
wash
6
10
wash
14
18
wash
22
26
wash
30
34
min
Fig 1. Experimental protocol, Anesthetized rats received either 1.5 MAC of isoflurane, enflurane, or halothane during control, a low-dose endotoxin period, and a high-dose endotoxin period. In every study period acetylcholine (ACh), nitroglycerin (NTG), and phenyl-ephrin (PE) were applied to monitor endothelium-dependent and endothelium-independent mechanisms of vasoregulation.
used. A basal minimum alveolar concentration (MAC) of 0.25 was applied continuously throughout the study and was interrupted by periods of 4 minutes of high concentrations using 1.5 MAC. The respective concentrations are presented in Table 1. After the volatile anesthetic peak exposure, a washout period of 6 minutes was allowed. To test the vasoactive potency of the arteriole under observation, three test substanc~es were superfused during each period. Two vasodilators were used: (1) Acetylcholine (Sigma Chemie, Deisenhofen, Germany) (endothelium
dependent) reaching a concentration of 10 - s mol/L at the muscle preparation and (2) Nitroglycerin (Nitro-Pohl, Pohl, Hohenlockstedt, Germany) (endothelium independent) reaching a concentration of 10 5 mol/L at the muscle. To show vasoc0nstrictory function, phenylephrine (Neosynephrine; SanofiWinthrop, Muenchen, Germany) was superfused gaining a concentration of 10 .5 mol/L at the preparation. Each drug was superfused for 4 minutes, with intermittent washout periods of 4 minutes.
Histopathology Table 1. Dosages of the Studied Volatile Anesthetic Agents
Basal concentration [vol/%] 1.5 MAC [vol-%]
Isoflurane
Enflurane
Halothane
0.3 1.8
0.4 2,6
0.2 1.2
NOTE. Data are expressed in vol%.
After the study, the anesthetized animals were killed by bolus application of KC1, in accordance to the guidelines of the governmental animal care and use committee. Autopsies were performed immediately afterwards, and specimens of hearts, lungs, kidneys, spleens, and livers were fixed in formalin and later embedded in paraffin. Histologic study was performed on 10-t~m-thick sections stained with hematoxylin-eosin.
4
SCHUMACHER, PORKSEN, A N D KLOTZ
Statistical Analysis Data are expressed as mean _+ SEM. To analyze the change in central hemodynamic and skeletal muscle microcirculation during the experiment, we compared the mean values of the three ~olatile anesthetic groups. To determine the effects of LPS on the central hemodynamic and skeletal muscle microcirculation, we compared the mean values of all 21 animals horizontally before and after the LPS administrations. The matched pair ranked test (Friedman and subsequent Wilcoxon tests) Was applied to compare values of different observation time points with initial values. Comparisons between the groups were performed using the Mann-Whitney U test. Probability values <.05 were considered to show significance.
RESULTS
Table 2 presents the absolute values of the central hemodynamic parameters due to LPS and volatile anesthetics during the observation time.
1. Systemic Hemodynamic Response to Volatile Anesthetics Before Endotoxemia During 1.5 MAC of enflurane and halothane anesthesia, HR was significantly reduced, MAP declined with all tested volatile anesthetics (P < .05). Isoflurane induced the smallest CO reduction, which was significantly different to the halothane group. SVR alterations were similar, isoflurane induced the strongest decrease, also significantly different to halothane.
2. Systemic Hemodynamic Response to Endotoxemia During basic level of inhalational anesthesia (0.25 MAC), we found a significant increase in HR after the high dose LPS application (P < .05). MAP and SVR showed a moderate decrease, whereas CO remained constant.
3. Microcirculatory Responses to Endotoxemia and to Monitor Substances During the control period we found a mean arteriolar diameter of 12.7 (_+ 2.8 Ixm). After the application of LPS 0.2 mg/kg mean diameter increased significantly to 14.0 ( + 2.8 ~m). Following the later injection of the high dose of LPS 2.0 mg/kg arteriolar diameter decreased significantly to 11.8 (-+ 3.8 p~m) compared with the diameter during the low dose endotoxin period. Table 3 presents the changes resulting from superfusion of the monitor substances after the induction of endotoxemia. All induced changes were significant compared with baseline. The dilatatory potency of acetylcholine (10 -5 tool/L) and nitroglycerine (10 -5 mol/L) was found to be significantly diminished during the two different endotoxin periods; however, the vasoconstrictive potency of phenylephrine (10 -5 mol/L) appeared unchanged.
Table 2. Change of HemodynamicVariables 0.25 MAC Basic Anesthesia
1.5 MAC Enflurane [n: 7]
Mean, -+ SEM HR [1 beat/rain] M A P [ m m Hg] CO [ m L / m i n ] SVR [ m m Hg 9 m L 9 m i n ]
1.5 MAC Halothane [n: 7]
1.5 MAC Isoflurane [n: 7]
Control period 294 _+ 32
275 • 39*
255 • 18"
64 § 14
48 + 10"
37 • 10"
291 • 33
13.2 + 3.4
8.9 § 3.7*
9.4 • 4,7"
12,2 _+ 3.1"#
5.2 _+ 1,7
5.8 • 1.4
4,7 _+ 2.0
3.7 • 1.1"#
43 • 7*
Low endotoxin period HR [1 beat/min]
309 • 47
287 -- 31"
272 -+ 29*
62 • 16
48 • 10"
37 + 13"
48 • 10"
13.4 • 4.5
9.0 • 2,9*
9.2 • 5.8*
13.8 -+ 3 . 7 " # t
5.1 _+ 1.8
5.6 -+ 0.9
5.2 • 2.6
3.7 • 1.3"#t
HR [1 beat/min]
330 • 64w
290 • 33*
293 • 56*
M A P [ram Hg]
60 • 17
45 • 12"
40 • 14"
42 • 7*
13.2 • 4.6
8.1 • 3.3*
9.9 • 5.9*
13.3 • 3 . 5 t
5.0 • 1.7
6.0 • 1.6
5.0 + 2.1
M A P [ m m Hg] CO [mL/min] SVR [ram Hg . m L 9 rain]
320 • 62
High endotoxin period
CO [ m L / m i n ] SVR [ram Hg 9 m L 9 min]
332 • 66
3.5 • 1.4"#
NOTE. Mean values _+ SEM, * P < .05 c o m p a r e d w i t h basic volatile anesthesia, # P < .05 c o m p a r e d w i t h halothane, t P < .05 c o m p a r e d w i t h enflurane, w < ,05 c o m p a r e d w i t h the control period.
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
Table 3. Change of Arteriolar Diameter (l,m) After Monitor-Substances Acetylcholine
Mean • SEM Control period Low endotoxin period High endotoxin period
Nitroglycerine
Phenylephrine
(10 Stool/L) [changein t~m]
(10-Smol/L) [changein p,m]
(10 Smol/L) [changein l~rn]
+6,1 -+ 1.3"
+3.0 _+ 0.9*
- 4 . 2 -+ 0.5*
+3.4 -+ 0,8*#
+1.4 +_ 0.3*#
- 6 . 5 + 0.6*#
+2.3 -+ 0.8*#
+0,8 _+ 0.4*#
- 4 , 3 + 0.7"1"
NOTE. Mean values are given in ~m _+ SEM. * P < .05 compared with pre-superfusion with monitor-substance. #P < .05 compared with controt period change, t P < .05 compared with low endotoxin period change.
4. Systemic Hemodynamic Response to Volatile Anesthetics During Endotoxemia Table 2 presents the absolute values of the systemic hemodynamic parameters during the observation time. HR was reduced by 5% to 10% during exposure to 1.5 MAC of enflurane and halothane. No significant differences between anesthetics were found. MAP declined in a similar extent of about 30% during the application of the different volatile anesthetics throughout all study periods. CO did not decrease in the isoflurane exposed animals. Enflurane and halothane produced a decline of 30% in CO in all study periods. SVR decreased in the isoflurane group by 15% to 20%, but not in the enflurane and halothane groups.
4O
5
5. MicrocircuIatory Responses to Volatile Anesthetics During Endotoxemia Figure 2 presents the changes in arteriolar diameter due to exposure to isoflurane, enflurane, and halothane during the observation period. In the control period, application of 1.5 MAC of isoflurane dilated the mean arteriolar diameter by 26 (+ 6.2%). After establishment of a low-dose endotoxemia, vasodilation during exposure to 1.5 MAC isofturane was nearly abolished compared with control (P < .05). In the state of high-dose endotoxemia, this lack of vasodilatory effect was similar (P < 0.5). Animals who received 1.5 MAC of enflurane showed only a small increase in arteriolar diameter of 4.5 (+_ 3.6%) during control. In contrast to this, during the low-dose endotoxin period exposure to enflurane resulted in a significant decrease in diameter by -11.3 (_+ 2.9%), this response was less during high-dose endotoxemia (-7.0, +_ 2.9%). Halothane induced virtually no change in microvascular diameter during the control period. We found a pronounced vasoconstriction by - 2 0 (-+ 3.7%) during the low-dose endotoxin period, and a moderate but significant constriction during high-dose endotoxemia ( - 7 . 9 , + 2,6%).
Pathology and Histopathology The macroscopic evaluation presented no abnormalities without signs of hyperyolemia, such as liver congestion or pulmonary edema. The histo-
~B
Isoflurane, n: 7 E n f l u r a n e , n: 7
L
E
H a l o t h a n e , n: 7
30 20
c 0 e-
Fig 2. Skeletal muscle arterial diameter in response to exposure to 1.5 MAC of isoflurane,
-10
L)
-20 -30
i
Control
i
Low endotoxin
i
High endotoxin
enflurane, and halothane during endotoxemia. Change of diameter is given in percent -+ SEM. * P < .05 compared w i t h the control period. #P < .05 compared with the low endotoxin period. +P < .05 compared w i t h the high endotoxin period.
6
SCHUMACHER, PORKSEN,AND KLOTZ
logic investigation of the organ specimens revealed no pattiology. The liver biopsies of the halothane exposed animals showed centrilobular fatty degeneration. DISCUSSION
The aim of this study was to compare the effects of isoflurane, enflurane, and halothane on skeletal muscle microcirculafion in an endotoxininduced rat sepsis preparation. Various models of experimental sepsis have been described in animals: Cecal ligation and puncture, !2 injection of bacteria, iv or the administration of isolated endotoxin intraperitoneally is or intravenously. 14 The latter septic challenge may have advantages in comparing different hemodynamic or microcirculatory effects that may be observed. Additionally, the time course of the Septic vasoregulatory changes is faster and more Consistent. Due to this rationale, we selected the intravenous LPS injection as the sepsis trigger. In our study application of the low dose of LPS (0.2 mg/kg) induced only minor hemodynamic changes, the injection of the higher LPS dose (2 mg/kg) resulted in significant tachycardia. Similar findings have been reported by Stewart et a117 who induced tachycardia without MAP alteration by infusion of E. coli isolates in conscious rats. In contrast to our findings, hemodynamic measurements from late onset septicemic experiments showed a reduction of M A P . t9 The effects of the tested volatile anesthetics on the systemic-hemodynamics are in accordance to the literature. 2~ During endotoXemia, isoflurane induced SVR decline was only 50% from control values. Neither enflurane nor halothane did alter SVR after LPS application. In contrast, Van der Linden et al a4 observed a decrease of SVR following the application of 0.5 MAC of halothane in an endotoxin septic shock dog model. The postmortem evaluation showed none of the abnormalities seen in chronic and fatal sepsis. The presented model focused on the early changes of septic vascular dysfunction, and therefore signs of chronic organ malperfusion and failure were missing. In this study, microcirculatory responses to endotoxemia exhibited the typical pattern of septic vascular dysfunction. After the application of lowdose LPS, mean arteriolar diameter increased significantly. Following the later injection of the high dose of endotoxin, arterioiar diameter decreased
significantly compared with the diameter during the low-dose endotoxin period. This finding of impaired vasoconstriction and later attenuation of vasorelaxafion during sepsis is a persisting observation of microcirculatory studies. This septic vascular dysfunction has recently been reviewed by Murray et al. 11 Additionally, our results confirm that the dilatatory potency of acetylcholine and nitroglycerine were significantly diminished during the two different endotoxin periods. This loss of dilatory potency may be explained by endotoxin enhanced synthesis of nitric oxide (NO) during sepsis.11'21 This might impair further vasodilafion because both superfused monitor substanCes result in the same mediator, either endothelial-dependent, like acetylcholine, or endothelial-independent, like nitroglycerin. From this observation, we conclude that septic vascular dysfunction was achieved by the LPS dosages used, the maintained vasoconstrietory potency of phenylephrine shows that vasoregulation was not completely abolished in this sepsis model. Furthermore, the discrepancy between the muscular microcirculatory vasoregulation and the systemic vascular resistance during sepsis indicates a heterogenity of perfusion in different organ systems. The dosages of the applied volatile anesthetic agents in our study were chosen to induce comparable depressions in MAP during the control period. This was achieved by similar MAC values known from humans. Exposure to 1.5 MAC of either isoflurane, enflurane, or halothane reduced MAP by 30% in all animals during control. This depression of MAP was also found in the low and the high endotoxin periods when the animals were exposed to 1.5 MAC of either isoflurane and enflurane. The halothane group showed significant less reduction of MAP during high-dose endotoxemia. Microcirculatory data, acquired in nonsepfic organisms, 5'9'1~ may explain our observation. Isoflurane dilates skeletal muscle arterioles by enhancing the release of the endothelial mediator NO. s During sepsis, with increased stimulation of the inducible-NO-synthase levels, this mechanism of action is limited, resulting in a lack of vasodilatory potency. Hence, the vasodilatory effect of isoflurane is nearly abolished during endotoxinderived septicemic syndromes. In contradiction to this, halothane is known to hinder the release of the endothelial mediator NO. 5'22 This results in a constrictory influence on
EFFECTS OF ISOFLURANE, ENFLURANE, AND HALOTHANE DURING ENDOTOXEMIA
v a s o r e g u l a t i o n , w h e r e a s i n n o n s e p t i c states o n e finds a c o m p e t i t i o n w i t h t h e v a s o d i l a t i n g d i r e c t C a 2 + - c h a n n e l b l o c k i n g p r o p e r t i e s t~ o f h a l o t h a n e . I n c a s e s Of a septic s y n d r o m e w i t h a u g m e n t e d N O d e l i v e r a n c e , t h e i n h i b i t i n g effect o n N O s y n t h e s i s outweighs the Ca2+-channel derived vasorelaxation. T h i s m i g h t e x p l a i n o u r f i n d i n g o f a r t e r i o l a r vasoconstriction under halothane exposition during endotoxemia. T h e i n f l u e n c e o f e n f l u r a n e o n skeletal m u s c l e m i c r o c i r c u l a t i o n a p p e a r s a n a l o g o u s to the h a l o t h a n e g e n e r a t e d effects. S u e t al 9 r e p o r t e d a b l o c k a d e o f C a 2+ i n f l u x in v a s c u l a r s m o o t h m u s c l e u n d e r the i n f l u e n c e o f e n f l u r a n e . W e s u g g e s t t h a t a s i m i l a r i n h i b i t i n g effect o n N O s y n t h e s i s prevails, e q u a l l y r e s u l t i n g in arteriolar v a s o c o n s t r i c t i o n .
7
I n s u m m a r y , w e f o u n d t h a t the v a s o a c t i v e effect o f i s o f l u r a n e k n o w n f r o m n o n s e p t i c o b s e r v a t i o n s is a b o l i s h e d d u r i n g e n d o t o x i n - i n d u c e d sepsis. W e also f o u n d t h a t e n f l u r a n e a n d h a l o t h a n e i n d u c e a s i g n i f i c a n t v a s o c o n s t r i c t i o n o f skeletal m u s c l e arterioles in the e n d o t o x e m i c rat. T h e s e f i n d i n g s m a y shed light on the problems of choosing the approp r i a t e volatile a n e s t h e t i c a g e n t i n states o f septic vascular dysfunction.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the support of the Institute of Pathology, Medical University of Luebeck, Germany for the histopathological investigations and the technical support of Dunja Leffler from the Department of Anesthesiology, Medical University of Luebeck.
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