THE ANATOMICAL RECORD 218:116-122 (1987) Isoflurane as an Anesthetic for Experimental Animal Surgery STEVEN E. RAPER, MARY E. BARKER, SUSAN J. BURWEN, AND ALBERT L. JONES 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 ABSTRACT 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 anesthetics. 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. MATERIALS AND METHODS Animals 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 ANESTHETICS FOR EXPERIMENTAL ANIMAL SURGERY kg. Diethylether was administered via a vapor-filled chamber for narcosis, followed by a nose cone containing ether-saturated gauze. 117 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 18.104.22.168 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. 22.214.171.124activity 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 level. ' 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 hours. RESULTS 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 118 S.E. RAPER, M.E. BARKER, S.J. BURWEN, AND A.L. JONES 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' Source 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. Isoflurane Aorta POZ(torr) pCOz (torr) PH Portal vein p 0 2 (torr) pCOa (torr) VH Vena cava POZ(torr) D C O (torr) ~ PH - Pentobarbital 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 7 'Values expressed as mean *Pi.01. S.D. for five or more rats. TABLE 2. Cvtosolic and membrane-bound enzvme activities' Isoflurane Glutathione S transferase (pmolesimidmg prot3) Aspartate amino transferase (pmolesimidmg prot) Cvtochrome-C-reductase (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 0.87 0.27 Ether PentobarbitaI Enzyme 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. Decapitated' 81.78 + 22.50 0.89 f 0.34 119 ANESTHETICS FOR EXPERIMENTAL ANIMAL SURGERY Qualitative and Quantitative Morphometric Analysis DISCUSSION 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 function. 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, 1983). 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 .V (umoles) (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/ 0 1.18 0 or environmental agents (Vessey and Boyer, 1984). The 1 0.83 30 glutathione-S-transferases comprise a predominantly 3 0.80 33 cytosolic drug-metabolizing enzyme system which cata0.62 5 47 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 rats. 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' ComDonent 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) Isoflurane Pentobarbital 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). 120 S.E. RAPER, M.E. BARKER, S.J. BURWEN, AND A.L. JONES 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’ Component 3 minutes Isoflurane, Pentobarbital, %) grains 70grains 15 minutes Isoflurane, Pentobarbital, % grains % grains Cytoplasm Smooth endoplasmic reticulum Rough endoplasmic reticulum Multivesicular bodies Autophagic vacuoles Secondary lysosomes Peroxisomes Mitochondria Golgi Nucleus Plasma membrane Endocytic vesicles Other 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 7.5 800 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 5.6 800 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 3.3 1,000 ‘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 2.7 1,000 ANESTHETICS FOR EXPERIMENTAL ANIMAL SURGERY 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). Ultrastructure 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. ACKNOWLEDGMENTS This study was supported in part by the Veterans Administration and NIH g a n t s AM26743, AM33004, 121 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 manuscript. LITERATURE CITED Bolender, R.P., and E.R. Weibel (1973) A morphometric study of the removal of phenobarbital induced membranes from hepatocytes after cessation of treatment. J. Cell. 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