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Isoflurane as an anesthetic for experimental animal surgery.

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THE ANATOMICAL RECORD 218:116-122 (1987)
Isoflurane as an Anesthetic for Experimental Animal
Cell Biology (S.E. R., M. E.B., S.J.B., A . L.J.) and Surgery (S.E. R.), Veterans Administration
Medical Center, S u n Francisco, C A 94121; Department of Surgery, Medicine and Anatomy
and Liver Center (M.E.B., S.J. B., A. L.J.), University of California, S u n Francisco, CA 94143
Isoflurane is an inhalational anesthetic agent associated with no
known hepatic toxicity. Despite this fact, isoflurane has not been widely utilized as
an anesthetic agent in studies of liver structure and function in experimental
animals. For this reason, livers from rats treated with pentobarbital or diethylether
were compared to those from rats treated with isoflurane to determine differences
in biochemical and morphologic parameters.
Liver from pentobarbital-treated rats showed a significant decline in glutathioneS-transferase activity compared to liver from isoflurane/Oz or ether-treated rats.
Liver microsomes from isoflurane/Oz-treatedrats retained more cytochrome-C(P450)reductase activity than did those from pentobarbital-treated, ether-treated, or decapitated rats. Despite these biochemical alterations, morphometric analysis of liver
from isoflurane/Oz and pentobarbital-treated rats showed no quantitative or qualitative differences in liver structure or organelle volume densities. Neither were
differences detected in uptake and distribution of 1251-epidermalgrowth factor when
analyzed by electron microscopic autoradiography. These data show that isoflurane
with supplemental 0 2 has no effects on hepatic structure and fewer effects on hepatic
function than other anesthetics and may be a better experimental anesthetic than
any currently in use.
The liver, as the primary organ for drug metabolism,
is particularly sensitive to pharmacological agents, including anesthetics. Therefore, investigators using laboratory animals in the experimental setting to study
liver physiology must be particularly aware of the influence an anesthetic may have on the outcome of a study.
Experiments with laboratory animals to study liver
structure and function traditionally have been carried
out with barbiturates or diethylether anesthesia. Certain effects of these anesthetics on the liver have been
well documented (Bolender and Weibel, 1973; Eriksson
and Strath, 1981; Kuriyama et al., 1969; Vermeulen et
al., 1983; Watkins et al., 1984).
Isoflurane (1-chloro-2, 2, 2-trifluoroethyl difluoromethy1 ether), a relatively new anesthetic, has gained wide
acceptance as the general anesthetic of choice in clinical
surgery (Eger, 1981). It was quickly recognized to have
several advantages over other commonly used
It is not broken down by sunlight and does not oxidize
spontaneously (Eger, 1981). Less than 0.2% of inhaled
isoflurane is recovered in metabolized form, and the
most common metabolites are triflouroacetic acid and
inorganic fluoride (Holaday et al., 1975). This level of
metabolism is 1/100 that of other ether anesthetics. Isoflurane does not need to be stabilized with preservatives
and is nonflammable over all ranges of application. Prolonged and/or repetitive isoflurane anesthesia does not
cause hepatorenal damage even when administered to
animals for up to 35 consecutive days (Byles et al., 1971).
No hepatic necrosis is seen with isoflurane administra0 1987 ALAN R. LISS, INC
tion even under conditions where other anesthetics do
cause hepatic necrosis (Harper et al., 1981). However,
isoflurane has not yet become widely used for anesthesia
of laboratory animals in the experimental setting.
The purpose of the experiments reported here was to
evaluate the suitability of isoflurane as an anesthetic
for studies of liver structure and function in the laboratory rat. Isoflurane was compared to pentobarbital and
diethylether with respect to the effects these anesthetics
have on blood gas parameters, hepatocyte ultrastructure, drug-metabolizing enzyme activities, and ability of
the liver to carry out receptor-mediated endocytosis.
Male 3-4 month old Sprague-Dawley rats (275-325
gm) (Bantin & Kingman, Fremont, CA) were used. The
animals were not fasted prior to sacrifice, and all procedures were performed between 8 A.M. and 12 noon. All
enzyme assays utilized a mean of six animals; five Forane-treated and four pentobarbital-treated animals were
used for the electron microscopic autoradiography.
Anesthetic Techniques
A simple anesthetic apparatus for the delivery of isoflurane (Forane, Ohio Medical Corp.) was designed (Fig.
1).Approximately 1.25% isoflurane with supplemental
oxygen (95%) was administered. Pentobarbital-treated
rats received a standard intraperitoneal dose of 65 mg/
Received October 6, 1986; accepted December 30, 1986
kg. Diethylether was administered via a vapor-filled
chamber for narcosis, followed by a nose cone containing
ether-saturated gauze.
Light and Electron Microscopy and Autoradiography
After 60 minutes of continuous anesthesia, the peritoneal cavity was opened with a midline incision, and the
liver was perfused via the portal vein with ice-cold 0.9%
saline. Thereafter, all steps were carried out at 4°C.
Livers were removed, and homogenized in 2 vol. of 0.25
M sucrose. Homogenates were centrifuged a t 10,OOOgfor
20 minutes. The 10,OOOg supernatant was centrifuged
at 100,OOOgfor 60 minutes. The 100,OOOgsupernatants
were saved for cytosol enzyme determinations; the pellets were washed, and then resuspended in a homogenization buffer (0.25 M sucrose, 0.05 M KHP04, pH 7.38).
Tissue selected for microscopy was postfixed in 1%
Os04 with 1.5% K4Fe(CN)~,dehydrated into 100%
ethanol, and embedded in Epon. One-micrometer-thick
sections were used to select tissue for light and electron
microscopic autoradiography on the basis of fixation and
presence of portal zones. For light microscopic autoradiography 0.5-pm-thick sections were coated with Kodak
NTB-3 emulsion, exposed for 2-6 weeks, and developed
with Kodak D-19. For electron microscopy thin sections
of 900 A were collected on parlodiodcarbon-coated grids,
overlaid with Ilford L4 emulsion, exposed for 6 weeks,
and developed with Kodak D-19. Grids were then stained
with lead citrate and examined in a Philips 300 or 201
transmission electron microscope operated at 60kV.
Enzyme and Cytochrome P450 Determinations
Stereological Procedures
Microsome Isolation
Two blocks from each of three animals treated with
The 100,OOOg supernatant was assayed for aspartate
amino transferase with a commerical kit (Sigma Tech- isoflurane or pentobarbital were selected. Ten micro0 20 micrographs
nical Bulletin No. 505). Glutathione-S-transferase [E.C. graphs at a magnification of ~ 4 , 6 0 and activity was measured spectrophotometrically at x 15,000 were taken of sections from each tissue block,
a t 340 nm and 25°C: with dinitrochlorobenzene as the and analysis was done according to Weibel's method
substrate (Vessey and Boyer, 1984). Alkaline phospha- (Bolender and Weibel, 1973).An unbiased sampling protase, a plasma membrane marker, was measured with a cedure was employed. Volume densities of hepatocyte
Sigma commercial kit and found to be undetectable in cytoplasm, nuclei, mitochondria, lysosomes, multivesicular bodies, peroxisomes, and cytoplasmic lipid were
all 100,OOOgsupernatants.
Liver microsomes were assayed for NADPH cyto- determined with a coherent double lattice test system
chrome-c (P450)-reductase [E.C. by mea- superimposed over the low power micrographs. A cohersuring the rate of cytochrome C reduction, as described ent multipurpose test system was used to estimate surby Masters et al. (1976). Cytochrome P450 content in face areas of the rough (RER) and smooth (SER)
microsomes was measured by the reduced carbon mon- endoplasmic reticulum. Estimates of intracellular memoxide difference spectrum (Dignam and Strobel, 1977). brane surface density are expressed as a ratio of surface
Protein content in 100,OOOg supernatant and micro- to volume density (M2/cm3hepatocyte cytoplasm).
somes was measured according to Lowry (1951).
Autoradiographic Grain Analysis
Blood Gas Analysis
One-half milliliter of whole blood was collected in a
heparinized syringe and kept on ice until analysis. Aortic, portal venous, and vena caval whole blood were
analyzed on a n ABL 1or 2 Acid Base Laboratory (Radiometer America, Burbank, CAI.
Two blocks were sampled from each of two animals
sacrificed at 3 and 15 minutes after injection of 1251EGF. Enough micrographs were taken so that approximately 400 grains were counted for each block of tissue.
Grain distribution was determined by concentric circle
analysis (Renston et al., 1980).
Radioiodination of Epidermal Growth Factor
Statistical Analyses
Epidermal growth factor (EGF), a gift of Dr. Denis
Gospodarowicz, Universit of California, San Francisco,
was radiolabelled with B51-Na (Amersham, Arlington
Heights, IL) by the chloramine-T method (Vlodavsky et
al., 1978). The unreacted lZ5I-Na was adsorbed onto a
Sephadex G-25 column. Labelled EGF, eluted in the void
volume, was 90% immunoprecipitable and had a specific
activity of 0.95 mCihimole.
Volume and surface density meaurements and the percent of total grains per organelle were compared by
Student's t-test for unpaired data. Where three groups
were compared, analysis of variance was performed; a
difference was considered significant at the P < 0.05
In Srtu Perfusion Fixation of Rat Liver
After 30 minutes of continuous anesthesia, 35 pCi of
1251-EGFwere injected into the portal vein of each rat.
At 3 or 15 minutes postinjection, the vena cava was cut
and PE 205 tubing was inserted into the portal vein.
Perfusion-fixation with 2.5'370 glutaraldehyde and 0.7%
paraformaldehyde in freshly gassed (95%02:5%c02)0.13
M NaHC03 buffer was carried out at 5 cc/midlOO gm
body weight. After the liver was weighed, portions with
the highest specific activity were selected, cut into 1mm3 pieces, and placed in fixative for a n additional 2
Blood Gas Analysis
Oxygen and carbon dioxide tension in blood were compared in pentobarbital- and isoflurane /Oz-treated rats
(Table 1). p02 was significantly higher in isoflurane/O~treated than in pentobarbital-treated rats for all three
major compartments of the circulation; aortic blood
showed the largest increase in pOz. There was no significant difference in pC02 or acid-base balance (as measured by pH) between the two groups. Resting, awake
blood gases were not determined, but published data
suggest that the level of 0 2 tension seen here in the
pentobarbital-treated animals was consistent with hypoxia (Low and Tuck, 1984).The difference in p02 levels
between the isoflurane/Oz- and pentobarbital-treated
animals is most probably due to the administration of
supplemental oxygen along with the isoflurane. Since
isoflurane anesthesia is routinely administered with
supplementary oxygen and pentobarbital anesthesia is
not, the comparison of blood gases is valid in terms of
the conditions that would normally prevail with the use
of either anesthetic.
Cytosolic and Microsomal Enzyme Activities
Glutathione-S-transferase (GSH-transferase) activity
found in rat liver cytosol was inhibited significantly by
pentobarbital treatment when compared to diethylether
or isoflurane treatment (Table 2). Inhibition of GSHtransferase activity in vitro could also be obtained by
introducing serial dilutions of pentobarbital to liver cytosol derived from ether-treated rats (Table 3). The nonlinearity of inhibition of enzyme activity may reflect the
fact that the four or more forms of GSH-transferase
present in rat liver cytosol may show different sensitivities to pentobarbital (Vessey and Boyer, 1984). In contrast to the differences in GSH-transferase seen with
anesthetic treatment, another cytosolic enzyme (unrelated to drug metabolism), aspartate amino transferase,
showed no differences in any of the treatment groups
(Table 2).
The activity of NADPH cytochrome-C(P450)-reductase
(cytochrome-C-reductase)was increased in liver microsomes from isoflurane/Oa-treated rats as compared to
liver microsomes from ether- or pentobarbital-treated
rats (Table 2). Cytochrome-C-reductase activity in microsomes from decapitated rats was similar to that of the
pentobarbital- and ether-treated rats (Table 2). This isoflurane/Oz-induced increase in cytochrome-C-reductase
activity was not accompanied by any differences in cytochrome P450 content of the microsomes from all four
treatment groups (Table 2).
TABLE 1. Blood gas analysis'
Fig. 1. a: Experimental subject placed in restraining chamber for
induction of anesthesia. b: Once asleep, subject is placed in a mask to
allow surgery.
pCOz (torr)
Portal vein
p 0 2 (torr)
pCOa (torr)
Vena cava
D C O (torr)
361.7 f 67.2*
53.9 f 5.2
7.29 f 0.05
77.7 f 8.7
44.3 + 6.0
7.32 i 0.02
59.9 f 3.6*
60.3 k 8.2
7.25 f 0.03
47.8 rt 7.2
53.2 f 5.6
7.29 f 0.04
68.7 f 5.2*
50.1 f 6.2
7.33 0.04
52.8 & 5.0
44.7 k 2.7
7.38 $ 0.03
'Values expressed as mean
S.D. for five or more rats.
TABLE 2. Cvtosolic and membrane-bound enzvme activities'
Glutathione S transferase
(pmolesimidmg prot3)
Aspartate amino transferase
(pmolesimidmg prot)
(nmolesimidmg prot)
Cytochrome P450 content
(nmolesimg prot)
0.92 k 0.06
0.99 f 0.08
0.60 k 0.13*
0.48 k 0.05
0.41 f 0.10
0.50 k 0.06
109.62 f 10.38*
73.21 f 10.42
80.73 f 14.91
0.75 & 0.27
1.02 f 0.37
lValues expressed as mean + S.D. for five or more rats.
2No anesthetic was used. Liver microsomes were prepared from freshly decapitated rats.
"Microsomal protein.
* P < .05.
+ 22.50
0.89 f 0.34
Qualitative and Quantitative Morphometric Analysis
Livers from isoflurane/Oz- and pentobarbital-treated
rats were compared by quantitative morphometric analysis (Table 4). No significant differences in volume or
surface density were demonstrated in any of the analyzed compartments. Organelle distributions in liver obtained from rats treated with isofluranel02 or
pentobarbital were similar to data obtained by other
investigators (Bolender and Weibel, 1973).A qualitative
morphologic comparison of the two groups revealed no
fragmentation or dilatation of SER or RER, and no dispersal of ordered aggregates of ribosomes nor detachment of ribosomes from RER. Mitochondria1 structure,
including intramitochondrial cristae and granules, appeared normal, and the Golgi apparatus was most prominent in the pericanalicular cytoplasm with both
treatments. Lysosomes, peroxisomes, autophagic vacuoles, and multivesicular bodies were similar in size,
structure, and number in both groups, as was nuclear
size, shape, and chromatin distribution. There was no
alteration of microvillus shape or dilatation of the biliary canaliculi.
The data presented here establish that isoflurane is
a n appropriate anesthetic for use in studies on rat liver
structure and function. The parameters assessed were
blood gases, liver drug-metabolizing enzymes, hepatocyte ultrastructure, and receptor-mediated endocytic
Blood Gases
Blood gas analysis was performed because of the profound effects of hypoxia on a variety of physiological
functions. The routine administration of supplemental
oxygen with isoflurane is most likely the key factor in
preventing the hypoxia that may often accompany other
kinds of anesthesia. Hypoxia with or without anesthesia
causes several well-defined alterations in liver cells, including surface bleb formation, vacuolization, cell
shrinkage, enzyme release, and ultimately, cell death
(Lemasters et al., 1981). Hypoxia may also potentiate
the hepatotoxic effects of other agents (Degroot and Noll,
The administration of supplementary oxygen with
pentobarbital would be expected to elevate blood pOz
Autoradiographic Grain Analysis
levels and eliminate the possible complications of hyLight microscope autoradiography showed 1251-EGF poxia. However, our intention was to compare blood
distributed in a steep portal to central venous gradient gases and other parameters under the conditions of usual
in livers from rats treated with either pentobarbital or administration of the anesthetics being studied. Since
isoflurane (Fig. 2). At 3 or 15 minutes after injection of supplementary oxygen is not routinely administered
lZ5I-EGF, no significant differences in electron micro- with pentobarbital or diethylether anesthesia, we chose
scopic autoradiographic grain distribution were noted to omit supplementary oxygen in our experiments with
these anesthetics.
(Table 5).
Drug-metabolizing Enzymes
The microsomal cytochrome P450 system, the most
important drug-metabolizing system in the liver, cataTABLE 3. Inhibition of glutathione-S-transferase by
lyzes the monooxygenation of a wide variety ofzenobiotpentobarbital in vitro'
ics via the reduction of NADPH to NADP. Cytochrome
.P450 content and NADPH cytochrome-C(P450)-reducPentobarbital
(u moles/min/me/urot)
%Inhibition tase are commonly used to detect changes in microsomal
P450 function in response to a variety of chemical and/
or environmental agents (Vessey and Boyer, 1984). The
glutathione-S-transferases comprise a predominantly
cytosolic drug-metabolizing enzyme system which cata0.62
lyzes the conjugation of glutathione (GSH) with a number of potentially toxic electrophiles (Kaplowitz, 1980).
'Pentobarbital was added to liver cytosol derived from ether-treated
Glutathione transferase activity can also be altered by
a variety of chemical agents (Vessey, 1982).
TABLE 4. Morphometric comparison of liver from isoflurane- and
Dentobarbital-treated rats'
Cytoplasm (Vv)'
Smooth endoplasmic reticulum ( S V ) ~
Rough endoplasmic reticulum (Sv)
Golgi apparatus (Sv)
Mitochondria (Vv)
Nucleus (Vv)
Autophagic vacuoles (Vv)
Secondary lysosomes (Vv)
Multivesicular bodies (Vv)
Peroxisomes (Vv)
Lipid (Vv)
Extrahepatocyte space (Vv)
0.62 k 0.02
5.97 f 0.26
3.50 k 0.33
0.29 k 0.07
0.18 k 0.01
0.05 f 0.01
0.003 f 0.001
0.003 + 0.001
0.002 i 0.001
0.013 + 0.001
0.005 f 0.001
0.11 0.02
0.64 f 0.01
6.27 0.26
2.86 f 0.25
0.23 k 0.05
0.20 k 0.01
0.04 k 0.01
0.003 f 0.001
0.006 k 0.001
0.004 i 0.001
0.012 f 0.001
0.011 f 0.001
0.08 k 0.01
'All values expressed as mean i SEM.
'VV = volume density (volume of cell organelle or inclusion per volume of total liver
tissue), a relative mode of data expression (cm3/cm3).
3Sv = surface density (surface area of membrane per volume of hepatocyte ground
substance), a relative mode of data expression (m2/cm3).
Fig. 2. Light microscopic autoradiography of the lobular gradient of EGF uptake in liver from isofluranetreated rats. The liver was perfusion-fixed 3 minutes after intraportal injection of 12’II-EGF. ~ 3 6 0PV
. =
portal vein; CV = central vein.
TABLE 5. IIz5-EGF Autoradiographic grain distribution in liver from isoflurane- and
pentobarbital-treated rats’
3 minutes
Isoflurane, Pentobarbital,
%) grains
15 minutes
Isoflurane, Pentobarbital,
% grains
% grains
Smooth endoplasmic reticulum
Rough endoplasmic reticulum
Multivesicular bodies
Autophagic vacuoles
Secondary lysosomes
Plasma membrane
Endocytic vesicles
Mean grains scored per animal
3.0 k 1.0
19.8 k 2.3
6.8 f 1.9
29.6 k 0.6
0.3 f 0.2
0.5 f 0.1
0.6 f 0.2
9.4 f 0.2
0.4 f 0.2
0.6 f 0.5
8.6 i 0.6
12.9 k 0.4
4.8 k 1.4
21.6 k 0.8
8.8 f 0.2
25.7 k 2.5
2.7 k 0.9
2.4 f 0.5
0.8 f 0.3
9.4 i 2.8
1.9 k 0.5
0.3 i 0.2
5.6 f 1.0
10.4 f 0.8
4.0 f 0.6
26.4 f 0.8
5.2 0.1
30.5 f 2.0
0.8 k 0.5
0.6 & 0.5
0.7 f 0.2
7.2 1.1
1.7 5 0.7
0.02 f 0.02
11.1i 0.6
8.5 f 2.8
‘Values are mean i SEM (isoflurane n = 5 , pentobarbital n=4).
1.6 k 0.6
24.9 k 3.1
6.4 f 0.1
31.6 f 1.4
1.6 5 0.7
2.1 f 0.5
1.5 f 0.4
9.9 + 0.7
3.9 k 1.3
0.1 k 0.1
7.5 f 1.3
6.2 k 1.3
Liver microsomes from isoflurane/Oz-treated rats
demonstrated more cytochrome-C(P45O)-reductaseactivity than those from pentobarbital- or diethylethertreated or decapitated rats. GSH transferase activity
was inhibited by pentobarbital anesthesia but not by
isoflurane/Oz or diethylether anesthesia.
Certain fluorinated ether anesthetics have been shown
to cause the destruction of' purified cytochromes P450
(Murphy et al., 1981). The fact that livers from isoflurane-treated rats do not have decreased cytochrome P450
levels probably reflects a lack of active isoflurane metabolites capable of reacting with nucleophilic moieties on
the cytochrome P450 (Holaday et al., 1975). The unexpected finding that isoflurane/02 administration actually enhanced cytochrome-C-reductase activity may
be due to stabilization of the enzyme by some as yet
unknown mechanism.
Pentobarbital appears to be a potent inhibitor of GSHtransferase activity when compared to the inhalational
anesthetics isoflurane or diethylether. The in vitro inhibition of GSH-transferase activity by pentobarbital suggests that pentobarbital inhibits GSH-transferase
activity by nonsubstrate ligand binding. Other hydrophobic nonsubstrate ligands have been shown to bind
GSH transferase a t the catalytic site and inhibit activity
(Vanderjagt et al., 1982; Vessey, 1982).
The hepatocyte continuously adapts the appearance,
distribution, and number of its subcellular organelles or
inclusions to changes in the external milieu (Jones and
Schmucker, 1977). This adaptive ability allows correlation of structure with certain functions, such as metabolism of a variety of compounds (Jones and Spring-Mills,
1973) and enhanced bile secretion (Jones et al., 1978) as
well as the pathophysiological conditions of aging
(Schmucker et al., 19771, cholestasis (Jones et al., 1976),
and regeneration (Murray et al., 1981).
Isoflurane/02 anesthesia did not cause any alterations
of liver cell ultrastructure, as revealed by both quantitative and qualitative morphometric analysis.
Receptor-Mediated Endocytosis
When 1251-EGFwas administered to the liver via the
portal vein, it was taken up and formed a steep lobular
concentration gradient from portal to central areas, as
previously described (St. Hilaire et al., 1983). At the
electron microscopic level, no statistically significant
difference in organelle volumes or autoradiographic
grain distribution were noted when either pentobarbital
or isoflurane/Oz had been used for anesthesia (Burwen
et al., 1984).
In summary, we have shown that isoflurane/Oz anesthesia is appropriate for studies on rat liver structure
and function, and is more suitable than the more commonly used anesthetics, pentobarbital and diethylether.
These latter two anesthetics caused changes in drugmetabolizing enzyme activities, the magnitude of which
may have been increased by the additional insult of
hypoxia. Isoflurane/02 had no detectable effects on morphological parameters and did not appear to alter the
process of receptor-mediated endocytosis.
This study was supported in part by the Veterans
Administration and NIH g a n t s AM26743, AM33004,
and by a grant from the American Liver Foundation.
We thank Drs. E.I. Eger and R.E. Hickey, Department
of Anesthesia, University of California, San Francisco,
for help in designing the isoflurane apparatus; R. Wang
and D.L. Schmucker, PhD, for assistance with the cytochrome-C-reductase assay; T.D. Boyer, M.D., for invaluable aid with the GSH-transferase assays; and D.A.
Vessey, PhD, for helpful discussion. We are also grateful
to G.T. Hradek for additional micrographs, and to Josephine Ansari and Brigitta Burr for preparation of the
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experimentov, anesthetic, animals, isoflurane, surgery
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