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Anesth Analg 2000;90:1331-1335
© 2000 International Anesthesia Research Society

PEDIATRIC ANESTHESIA

The Hemodynamic Effects of Halothane and Isoflurane in Chick Embryo

Jacek A. Wojtczak, MD, PhD

Departments of Anesthesiology and Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York

Address correspondence and reprint requests to Jacek A. Wojtczak, MD, PhD, Department of Anesthesiology, Box 604, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The cardiovascular effects of volatile anesthetics in prenatal hearts are not well investigated. The purpose of this study was to determine whether the embryonic cardiovascular system is sensitive to an exposure to clinically relevant, equipotent concentrations of halothane and isoflurane. Stage 24 (4-day-old) chick embryos were exposed to 0.09 and 0.16 mM of halothane and 0.17 and 0.29 mM of isoflurane. Dorsal aortic blood velocity was measured with a pulsed-Doppler velocity meter. Halothane, but not isoflurane, caused a significant decrease in cardiac stroke volume and maximum acceleration of blood (dV/dtmax), an index of cardiac performance. This effect was reversible, and during washout, stroke volume and dV/dtmax increased above control levels. Embryonic heart rate was not affected by either drug. Chick and human embryos are similar during early stages of development; therefore, chick embryo may be a useful model to study the cardiovascular effects of anesthetics.

Implications: In equipotent, clinically relevant concentrations, halothane, but not isoflurane, markedly decreased aortic blood flow and cardiac performance measured with ultrasound techniques in chick embryos. Chick and human embryos are similar during early stages of development; therefore, chick embryo may be a useful model to study the cardiovascular effects of anesthetics.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
An estimated 0.75%–2% of pregnant women require nonobstetric surgery and general anesthesia for incidental illness or trauma (1). Volatile anesthetics—often used during general anesthesia—are known to produce very pronounced cardiovascular depression, especially in newborns and infants (2–4). Their cardiovascular effects in the fetal period have been studied (2–4), but their effects in the embryonic period are unknown. The embryonic period refers to the period between two and eight weeks of human prenatal development, during which morphogenesis (development of shape) and organogenesis (formation of organs) occur (5). Consequently, it is the time of maximum susceptibility to factors, called teratogens, that may interfere with development (6). Establishment of circulation is one of the most crucial physiological requirements of the developing embryo, because it delivers nutrients and allows communication between various tissues (7). Cardiovascular depression induced by volatile anesthetics can cause hypoperfusion and transient global ischemia. Short episodes of ischemia activate immediate early genes (8) that may be harmful to the embryo by initiating a pathway of apoptosis (programmed cell death), causing unplanned deletion of cells. Thus, it is important to identify anesthetics that can cause excessive cardiovascular depression in the embryo. Hemodynamic investigations in mammalian embryos are technically difficult. Therefore, in this study, chick embryos were used as a model to compare the cardiovascular effects of halothane and isoflurane. During early developmental stages, avian and human embryos are similar morphologically and at the cellular and organ levels (5,7,9–12).


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Fertilized white Leghorn chicken eggs were incubated, blunt end up, in a forced draft incubator to Hamburger-Hamilton (9) Stage 24 (4 days) of a 46-stage (21 days) incubation period. Developmental landmarks included somite number, limb size, and cardiac morphology. Because the fertilized chicken egg is considered to be a nonliving vertebrate, this study was not reviewed by the Institutional Animal Investigation Review Committee.

For physiologic measurements, an egg was removed from the incubator and positioned on the stage of a dissecting microscope (Fig. 1). When incubated, blunt end up, the blastodisc floats to the top of the egg and the embryo develops beneath the air cell. Immediately before recordings, a window was made in the shell, and the inner and outer shell membranes that overlay the embryo were removed (11,12). Eggs were placed in a sealed chamber, and their temperature was maintained at 37°C with an air curtain incubator. Halothane (Halocarbon Laboratories, River Edge, NJ) and isoflurane (Abbott Laboratories, North Chicago, IL) were vaporized in Drager vaporizers using ambient air as a carrier. Their concentration in the air flowing out of the chamber was constantly monitored with a Raman spectroscopy gas analyzer (Rascal, Albion Electronics, Salt Lake City, UT). The desired concentration of anesthetics in the chamber was achieved in about 30 s after turning on the vaporizer.



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  		Figure 1. A, Diagram of the workstation for blood flow data acquisition and analysis. B, Analog waveforms of dorsal aortic velocity in control conditions—no air flow (a), flowing ambient air (b), after 15 min exposure to large-dose halothane (c), and during recovery (d).

 
Experiments were performed in 24 embryos. Each embryo was exposed for 15 min to only one concentration (six in each group) of halothane or isoflurane and discarded after 15 min washout. The recordings were performed in the following sequence: 1) baseline in ambient air (no air flow) at -5 min; 2) baseline in flowing ambient air at -2 min (used as baseline control for statistical analysis); 3) every 5 min for 15 min during exposure to 1.5% or 3% halothane and 2.5% or 5% isoflurane in flowing ambient air; and 4) every 5 min for 15 min during washout with ambient air. Seven additional embryos were studied for 30 min in flowing ambient air without anesthetics.

Mean dorsal aortic blood flow was measured with a 20-MHz directional pulsed Doppler velocity meter. A 0.75-mm piezoelectric crystal was positioned at a 45° angle toward the dorsal aorta at the level of sinus venosus. The Doppler velocity meter was validated to be accurate in the range of 0–16 mm/s (11,12). The measurement of dorsal aortic blood velocity includes all blood ejected from the heart except blood flow to the head. From plastic casts of the cardiovascular system, it was previously estimated that less than 10% of chick embryo cardiac output flows to the head (11,12). Dorsal aortic diameter was measured by using a micrometer filar eyepiece. The aortic area was calculated from the equation, area = pd2/4, where d is the aortic diameter. The flow was calculated as mean dorsal aortic velocity x aortic area.

Cycle length was calculated by measuring the interval between pulse waves (11,12). Stroke volume per beat was determined from the quotient of mean dorsal aortic blood flow divided by the heart rate (11,12).

To assess ventricular performance, aortic maximum blood acceleration (dV/dtmax) was digitally derived from the digitized velocity waveforms (11,12). Aortic blood acceleration was demonstrated to be a sensitive index of ventricular performance during induced alterations in the inotropic state (13).

The diameter of vitelline arteries during exposure to halothane and isoflurane was measured using digital microscopy in 10 embryos, 5 in each group. Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) was used to capture and process the digitized images (magnification 45x).

Halothane and isoflurane concentrations in the egg white were determined by gas chromatography-mass spectrometry in 20 eggs, 5 eggs in each group. A 4-mL sample of egg white was aspirated into the glass syringe after 15-min exposure to halothane or isoflurane. It was injected into 10-mL crimped glass headspace vials. Each vial contained 4 mL of hexane. The vial content was mixed in the shaker water bath for 4 h and then allowed to settle for 2 h. Just before sampling, the vials were centrifuged for 30 min to allow clear separation of hexane and water phases. A 10-µL sample of hexane phase was aspirated with a gas-tight syringe (Dynatech GC, Baton Rouge, LA) and injected into the column of a Hewlett-Packard (Palo Alto, CA) Model 5880 gas chromatograph connected to a HP Model 5970 mass spectrometer. A fused-silica capillary column coated with cross-linked methyl silicone was used. The carrier gas (helium) flow rate was 1 mL/min. The injector, column, and detector temperatures were held at 150°C, 60°C, and 50°C, respectively. A single-ion detection technique was used. A gas chromatograph-mass spectrometer was calibrated with the samples of standard egg white solution. This solution was prepared by adding different amounts of halothane and isoflurane to large, crimped headspace vials totally filled with egg white. After overnight mixing, a 4-mL sample of egg white solution was withdrawn and processed in the same way as the biological samples. The completeness of extraction was determined by analyzing identical amounts of halothane and isoflurane added directly to hexane.

Data were presented as the means ± SEM. Statistical analysis was performed by using one-way and two-way analysis of variance. A P value of less than 5% (P < 0.05) was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Exposure of control embryos to the flowing ambient air without anesthetics for 30 min produced no significant changes in stroke volume (from 0.32 ± 0.02 µL in control versus 0.3 ± 0.03 µL) and dV/dtmax (from 821 ± 55 mm/s2 in control versus 789 ± 72 mm/s2), which suggests that the current model is appropriately stable for hemodynamic studies.

Chick embryos were exposed to concentrations of halothane and isoflurane that are equipotent as anesthetics in adult chickens (15,16). Peak concentrations of halothane and isoflurane in egg white, after 15-min exposure to a steady concentration in ambient air, are presented in Table 1.


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  		Table 1. Halothane and Isoflurane Concentrations
 
Exposure to small-dose halothane produced no change in stroke volume (Fig. 2A). Large-dose halothane produced a large decrease in stroke volume (Fig. 2A) and a rebound increase during washout. Changes in dV/dtmax were similar to changes in stroke volume (Fig. 2B), but a rebound during washout after large-dose halothane exposure was more pronounced. Both small-dose and large-dose isoflurane concentrations produced no significant changes in stroke volume or dV/dtmax (Fig. 3).



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  		Figure 2. Effects of small-dose ({blacksquare}) and large-dose ({blacklozenge}) halothane concentrations on stroke volume (A) and maximum acceleration of blood (B). Data are expressed as the means ± SEM (*P < 0.05, compared with baseline measurements at -2 min, n = 6).

 


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  		Figure 3. Effects of small-dose ({blacksquare}) and large-dose ({blacklozenge}) isoflurane concentrations on stroke volume (A) and maximum acceleration of blood (B). Data are expressed as the means ± SEM. There were no differences compared with baseline measurements at -2 min (P > 0.05, n = 6).

 
Halothane and isoflurane had minimal effects on heart rate even in higher concentrations. Heart rate was also stable in the embryos exposed to ambient air flow without anesthetics.

To assess whether halothane or isoflurane exert any peripheral vascular effects and therefore affect the afterload, measurements of the vitelline artery diameter were performed during exposure and washout of halothane and isoflurane. Large-dose halothane concentration induced a small change in the arterial diameter, from 83 ± 4 µm in control to 92 ± 5 µm (n = 5) after 15-min exposure and to 84 ± 4 after 15-min washout. Large-dose isoflurane concentration had no effect on the vitelline artery diameter.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The development of the chick heart parallels that of the human heart. At Stage 24, as used in this study, the embryonic chick heart is a looped tubular structure about 0.8 mm in diameter, with the muscular ventricle tapering off into the truncus arteriosus (7,9). Coordinated contraction of the heart and valve-like function of endocardial and bulbar cushions results in unidirectional flow (11). The human embryo achieves a similar developmental stage about 35–40 days after conception (5,7).

This study shows that in equipotent, clinically relevant concentrations, halothane but not isoflurane can depress the avian embryonic cardiovascular system. Halothane caused a significant decrease in cardiac stroke volume and maximum acceleration of blood (dV/dtmax), a sensitive index of cardiac performance during induced alterations in the inotropic state (13).

Changes in ventricular afterload could not be accurately assessed because the aortic pressure was not simultaneously measured. However, measurements of the diameter of vitelline arteries showed that halothane produced minimal vasodilation that was reversible during washout. Isoflurane had no effect.

Aortic diameter used for calculation of flow was measured at the end of each experiment after washout of volatile anesthetics. Although aortic diameter could be changed by halothane exposure, calcium-entry blockers that have similar pharmacologic effects to volatile anesthetics were shown to induce only a very small decrease in aortic diameter (14).

Halothane and isoflurane were used in concentrations that are equipotent and clinically relevant. The minimum alveolar anesthetic concentration (MAC) of halothane and isoflurane in adult chickens was 0.85% (15) and 1.24% (16), respectively, which is similar to human MAC (17). In this study, the embryonic membranes were removed and only egg white separated the embryonic cardiovascular system from the gas phase. The solubility coefficients of volatile anesthetics in plasma with the addition of albumin (total 8.45% albumin) were shown to be much higher than in blood (18). Egg white of the four-day-old embryo contains approximately 11% albumin (10); therefore, solubility coefficients must be even higher. Because the heart and embryonic vessels are in direct contact with egg white, it is very likely that the egg white was the main route of delivery of volatile anesthetics to the heart.

In mammals, blood concentrations reach about 0.3 mM during exposure to 1 MAC halothane and 0.5 mM during exposure to 1 MAC of isoflurane (2,3,17,19). Concentrations achieved in this study in egg white were considerably lower, which indicates that the halothane-induced cardiovascular depression was achieved with the concentrations that are equivalent to a fraction of MAC in mammals and birds. As shown by Gregory et al. (20) and Bachman et al. (3), mammalian fetal anesthetic requirements for halothane and isoflurane are reduced; however, the anesthetic requirements of the embryo are unknown. Chick embryos may have different requirements due to the absence of the mother–embryo humoral interactions and placental circulation.

Biehl et al. (2) and Bachman et al. (3) showed that, in the fetal lamb, both halothane and isoflurane decreased fetal arterial pressure without decreasing cardiac output, which differs from our results. However, increased sensitivity of the embryonic cardiovascular system to halothane, compared with isoflurane, is consistent with the results obtained in mammalian immature hearts by other groups. Palmisano et al. (21) found a similar difference of potency in isolated infant rabbit hearts exposed to equipotent concentrations of halothane and isoflurane. This difference was less pronounced in isolated papillary muscles from infant rabbits (22). The difference in cardiovascular potency has been also demonstrated clinically. Gallagher et al. (23) studied hemodynamic effects of 1 MAC halothane and isoflurane in healthy children using pulsed Doppler echocardiography. As in our study, aortic dV/dtmax was used to assess cardiac performance in children and was found to be decreased by halothane and unchanged by isoflurane (23). These authors proposed that the difference might be caused by increased ß-sympathetic activity caused by isoflurane. However, the sympathetic system is not developed in the early chick embryo (7,9). Therefore, the difference in potency is likely to be the result of the direct effect of anesthetics on the embryonic heart (e.g., differential inhibition of the reversed mode sodium/calcium exchanger, intracellular Ca2+ release mechanisms, or contractile proteins).

In conclusion, this study shows that halothane, but not isoflurane, markedly decreased aortic blood flow and cardiac performance in chick embryos, but more studies will be required to determine if halothane (or any other anesthetic) has adverse effects on the human cardiovascular system during embryogenesis. Chick and human embryos are similar during early stages of development; therefore, chick embryo may be a useful model for studying the cardiovascular effects of anesthetics.


   Acknowledgments
 
I thank M. Norman Hu (Pediatric Cardiology) for help in performing blood flow experiments, Dr. Edward B. Clark (Pediatric Cardiology) for guidance, Dr. Mazzaz Hashmi (Pharmacology) for help in performing the analysis of volatile anesthetics, and Dr. Michael G. Richardson (Obstetric Anesthesia) for his gracious reviews of the manuscripts.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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  8. Tsuchiya T, Abe T, Saito T, et al. Induction of immediate early genes and apoptosis after ischemia/reperfusion in fatty liver rats. Transplant Proc 1998;30:2919–22.[Web of Science][Medline]
  9. Hamburger V, Hamilton HL. A series of normal stages in the development of chick embryo. J Morphol 1951;88:49–92.[Web of Science]
  10. Romanoff AL. Biochemistry of the avian embryo. New York:Interscience Publishers, 1967:179–94.
  11. Clark EB, Hu N, Dummett JL, et al. Ventricular function and morphology in chick embryo from stages 18 to 29. Am J Physiol 1986;250:H407–13.[Abstract/Free Full Text]
  12. Hu N, Clark EB. Hemodynamics of the stage 12 to stage 29 chick embryo. Circ Res 1989;65:1665–70.[Abstract/Free Full Text]
  13. Nutter DO, Noble RJ, Hurst VW. Peak aortic flow and acceleration as indices of ventricular performance in the dog. J Lab Clin Med 1971;77:307–17.[Web of Science][Medline]
  14. Yano M, Kumada M, Matsuzaki M, et al. Effects of diltiazem on aortic pressure-diameter relationship in dogs. Am J Physiol 1989;256:H1580–7.[Abstract/Free Full Text]
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Accepted for publication February 25, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press