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GENERAL ARTICLES

The Effects of Pentobarbital, Isoflurane, and Propofol on Immediate-Early Gene Expression in the Vital Organs of the Rat

Yoshihiro Hamaya, MD^*, Tomoo Takeda, MD^*, Shuji Dohi, MD^*, Shigeru Nakashima, MD, and Yoshinori Nozawa, MD

Departments of *Anesthesiology and Critical Care Medicine and Biochemistry, Gifu University School of Medicine, Gifu, Japan

Address correspondence and reprint requests to Dr. S. Dohi, Department of Anesthesiology and Critical Care Medicine, Gifu University School of Medicine, 40 Tsukasamachi, Gifu City, Gifu 500, Japan. Address e-mail to shu-dohi{at}cc.gifu-u.ac.jp

	Abstract

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General anesthetics are known to transiently increase the expression of messenger ribonucleic acids (mRNAs) of immediate-early genes in the brain. We investigated whether the expression of two immediate-early genes in vital organs were modulated by various anesthetics. Inhaled isoflurane (n = 20), intraperitoneal pentobarbital (n = 20), and IV propofol (n = 20) were administered to male Sprague-Dawley rats, and five from each group were decapitated at 5, 30, 60, or 120 min after the induction of anesthesia. Control, nonanesthetized rats (n = 5) were handled gently and then decapitated. Reverse transcriptase-polymerase chain reactions were performed on total RNA from samples of the brain, heart, liver, and kidney to detect the expressions of c-fos and c-jun mRNAs. As internal control, cyclophilin mRNA was amplified simultaneously. The products were separated by electrophoresis, and the optical density of the bands was quantified. The expression of c-fos mRNA was transiently increased in the brain, and more strikingly and for longer times, in the kidney with all three anesthetics; the expression of c-fos mRNA was decreased in the heart with isoflurane and pentobarbital and increased in the liver with isoflurane and propofol. The expression of c-jun mRNA was increased in the heart, liver, and kidney with isoflurane, increased in the heart and kidney with pentobarbital, increased in the heart, liver, and kidney with propofol, and decreased in the brain with pentobarbital. Our results suggest that the appropriate anesthetics to be used to anesthetize animals differ in accord with the target organs in which the expressions of immediate-early genes in response to stimuli were studied.

Implications: In this study, there were quantitative and qualitative interanesthetic and interorgan differences in the expression of immediate-early genes, showing that general anesthetics can stimulate, rather than suppress, some intracellular events. Our results suggest that the appropriate anesthetics to be used to anesthetize animals differ in accord with the target organs in which the expressions of immediate-early genes in response to stimuli were studied.

	Introduction

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General anesthetics are administered to patients and animals with the aim of minimizing the effect of external stimuli, such as surgical incisions. Cells respond to external stimuli with a cascade of intracellular events that can involve the cell membrane, cytoplasm, and nucleus. Although anesthetics can affect various steps in this series of molecular events, our knowledge of the mechanism of action of general anesthetics at the cellular level is, at best, fragmentary (1). Within the nucleus, there is a family of proto-oncogenes whose transcription (which can be activated quickly, within minutes) precedes changes in the expression of other stress-induced genes, which are therefore called "immediate-early genes" (2). There is increasing evidence that immediate-early genes are expressed in neurons in response to many types of stimuli (3), and some studies (4,5) have demonstrated in rats that the expression of the immediate-early genes c-fos and jun-B in the brain is affected by a variety of general anesthetics in vivo. Increased expression of the immediate-early genes in response to the administration of general anesthetics provides further evidence that anesthetics can stimulate, rather than suppress, the central nervous system. This enhanced excitability by general anesthetics has been shown in an electrophysiological approach (6,7). These facts indicate that, as far as neural cells are concerned, general anesthetics themselves can induce a type of stress, or a stimulus at least, that evokes some specific intracellular events, rather than suppressing them.

If anesthetics do induce immediate-early gene expression and consequent intracellular events, putting animals under anesthesia could have confusing effects in studies of the components of the cascade of intracellular messengers. Takayama et al. (5) indeed concluded their report that neuroleptanalgesia with fentanyl and midazolam could be appropriate for preparing rats for examining the brain, because these drugs have a minimal effect on the expression of the immediate-early genes. We thought it would be useful to know if certain anesthetics have an inductive effect on the immediate-early genes in organs other than the brain. By knowing the effect of anesthetics on the expression of immediate-early genes, we can avoid the use of certain anesthetics to negate their influence in the investigation of the pure effects of stimuli on immediate-early gene expression.

There have been no reports on whether the effects of general anesthetics per se affect immediate-early gene expression other than in the central nervous system. We were interested, therefore, to find out if such changes occur in other vital organs, and if there are interanesthetic and interorgan variations in the induced changes in the expression of these genes. We designed our study using the rat to examine whether immediate-early gene expression varies among the vital organs (in this case, the brain, heart, liver, and kidney) when the animals have received various general anesthetics. We chose pentobarbital, isoflurane, and propofol as the general anesthetics; pentobarbital as one of the classical anesthetics for animal experiments, and the others as anesthetics commonly used in modern clinical practice.

	Methods

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With the approval of our institutional animal care committee, 65 male Sprague-Dawley rats (280–350 g) were used. They were kept for more than 2 days in a 12-h light-dark cycle with a 25°C ambient temperature and free access to food and water until the experiment.

In vivo Procedures
Isoflurane-anesthetized rats (n = 20) were kept in a plexiglas box flushed continuously at 5 L/min with warmed (37°C) 98% oxygen containing 2% (=1.5 minimum alveolar concentration) isoflurane. Pentobarbital-anesthetized rats (n = 20) received a bolus intraperitoneal injection of pentobarbital 50 mg/kg and were then placed in a plexiglas box flushed with 100% oxygen at 5 L/min. Propofol-anesthetized rats (n = 20) received an IV infusion of propofol at a rate of 10 mg · kg^-1 · min^-1 for 1 min, followed by a consecutive infusion at a rate of 1 mg · kg^-1 · min^-1 via the tail vein, and they were supplied with oxygen as described above. Rats were decapitated at 5, 30, 60, or 120 min after the induction of anesthesia; five rats under each anesthetic were killed at each time-point. All rats were breathing steadily throughout the experimental period and showed no signs of awakening before the time of decapitation. As a control, nonanesthetized rats (n = 5) were handled gently by avoiding painful stimulation and were placed in a plexiglas box flushed with 100% oxygen at 5 L/min before decapitation. The brain, heart, liver, and kidney were rapidly resected and frozen in liquid nitrogen, then stored at –50°C until used in the in vitro assay.

In vitro Procedures
Total ribonucleic acid (RNA) was extracted from the cerebral hemisphere, the left ventricle of the heart, the liver, and the kidney, according to the instructions of the manufacturer of ISOGEN^® (Nippon Gene, Toyama, Japan), which is a conditioned solution for extracting RNA from tissues. Briefly, each frozen tissue was weighed, then homogenized to a slurry by using a homogenizer (overhead stirrer and tissue grinder with Teflon^®-pestle; Wheaton Science Products, Millville, NJ) in the presence of ISOGEN^® (1 mL/50 mg tissue). After chloroform (0.2 mL/1 mL suspension) had been added and thoroughly mixed, each sample was centrifuged at 12,000g for 15 min at 4°C. The supernatant was transferred to a clean microtube, and 0.5 mL isopropanol was added for 15 min to precipitate RNA, followed by centrifugation at 12,000g for 10 min at 4°C. The precipitate was washed in 1 mL of 75% ethanol, centrifuged at 7,500g for 5 min at 4°C, dried briefly, and resuspended in 50 µL of 0.1% diethylpyrocarbonate in water. Total RNA content was quantified by absorbance at 260 nm (1 optical density = 40 µg RNA/mL) by using a spectrophotometer (GeneQuant^®; Pharmacia, Uppsala, Sweden).

A quantitative assessment of the relative expressions of the mRNAs of c-fos, c-jun, and cyclophilin was made by using the reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA (2 µg) was reverse transcribed by Molony Murine Leukemia Virus reverse transcriptase (Gibco/BRL, Grand Island, NY) by using random hexamer primers (Takara Shuzou, Ohtsu, Japan). Each complementary deoxyribonucleic acid (cDNA) obtained from the RNA samples was prepared for PCR by an assembly of reagents including deoxynucleotide triphosphate, Taq polymerase (Takara Shuzou), and oligonucleotide primers. PCR was performed by means of an automated thermal cycler (Quick Thermo Personal^®, Nippon Genetics, Japan); it involved 30 cycles of heating to 95°C for 30 s (denaturing), cooling to 58°C for 60 s (annealing), and heating to 72°C for 60 s (extending). The rat c-fos oligonucleotide primers used were: 5'-AGCCGACTCCTTCTCCAGCAT-3' (sense) and 5'-CAGATAGCTGCTCTACTTTGC-3' (antisense), corresponding to bases 235–533 (8). The rat c-jun oligonucleotide primers used were: 5'-GCGCCGCCGG-AGAACCTCTGTC-3' (sense) and 5'-CAGCTCCGGCG-ACGCCAGCTTG-3' (antisense), corresponding to bases 577-1227 (9). The oligonucleotide primers for rat cyclophilin, used as aninternal standard for normalization purposes, were: 5'-GCTGATGGCGAGCCCTTGG-GTC-3' (sense) and 5'-ACCAGTGCCATTATGGCG-TGTG-3' (antisense), cor-responding to bases 76–264 (10). Those primers were designed to span at least one splice junction to differentiate between amplification from cDNA and genomic DNA, of which unspliced PCR products must be longer to eliminate the colinearity with the products from cDNA.

The PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The optical density of the various bands was quantified using an image-analysis system (Densitograph AE-6900M; Atto, Tokyo, Japan). During electrophoresis, a single PCR product of the cyclophilin from one sample (the brain of Rat 1) was always applied to one lane of each agarose gel plate and run with the other PCR products. This yielded ratios between the optical density of each of the samples on a given plate and that of cyclophilin, and thus allowed us to normalize interplate variance. The optical density values obtained for c-fos and c-jun in each organ from each rat were divided by the value obtained for the cyclophilin from the same organ in the same rat, thereby normalizing intersample variance. The results were expressed (mean ± SEM) relative to the corresponding value obtained for samples from control, nonanesthetized rats (which were given the value 1.0). Analysis of variance was used for statistical comparisons, followed by Fisher’s protected least significant difference test. P values < 0.05 were considered statistically significant.

	Results

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Each of the primer sets amplified a single sharp band of the expected size for each cDNA amplification product. For each of the primer sets, no amplification of nonspecific or genomic DNA contaminants was visualized after electrophoresis and ethidium bromide staining of agarose gels.

Expression of Cyclophilin mRNA
The expression of cyclophilin mRNA, as quantified by RT-PCR, was stable with respect to time, and the coefficient of variation for the five samples from the same group (for example, the brains of the 5 rats decapitated after 5 min of isoflurane anesthesia) was 6.1%–21.7%. Consequently, we considered cyclophilin mRNA an appropriate internal standard for the normalization of the values obtained for c-fos and c-jun.

Expression of c-fos mRNA
The c-fos mRNA expression in the organs removed from anesthetized rats differed depending on the organ and the anesthetic used (Fig. 1).

View larger version (34K): [in this window] [in a new window]   Figure 1. Time-related c-fos mRNA expression in the brain, heart, liver, and kidney of rats anesthetized with isoflurane, pentobarbital, or propofol, as detected by reverse transcriptase-polymerase chain reaction. Control indicates nonanesthetized rats. Data are expressed relative to the control value (which is given the value 1.0). Each column represents mean ± SEM of data for five rats. *P < 0.05 versus Control.

In pentobarbital-anesthetized rats, the expression of c-fos mRNA was increased in the brain at 30 min (1.4 ± 0.3; P < 0.05) and at 60 min (1.4 ± 0.2; P < 0.05). The c-fos mRNA expression was decreased under pentobarbital in the heart from 30 min (0.6 ± 0.1; P < 0.03) to 120 min (0.5 ± 0.1; P < 0.01). In the kidney, c-fos mRNA expression increased and stayed high throughout the experiment (P < 0.01). The expression of c-fos mRNA in the liver did not show any significant change with pentobarbital anesthesia.

In propofol-anesthetized rats, c-fos mRNA expression was increased at 5 min in the brain (1.6 ± 0.5; P < 0.05) and liver (2.5 ± 1.0; P < 0.04). In the kidney, c-fos mRNA expression was raised from 5 min (5.2 ± 2.1; P < 0.005) to 120 min (5.7 ± 2.2; P < 0.005). Propofol anesthesia did not cause any significant change in the expression of c-fos mRNA in the heart.

Expression of c-jun mRNA
The effects of anesthetics on expression of c-jun mRNA differed in several ways from those obtained for c-fos mRNA (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Figure 2. Time-related c-jun mRNA expression in the brain, heart, liver, and kidney of rats anesthetized with isoflurane, pentobarbital, or propofol, as detected by reverse transcriptase-polymerase chain reaction. Control indicates nonanesthetized rats. Data are expressed relative to the control value (which is given the value 1.0). Each column represents mean ± SEM of data for five rats. *P < 0.05 versus Control.

In rats anesthetized with pentobarbital, the expressions of c-jun mRNA was decreased in the brain at 5 min (0.6 ± 0.1; P < 0.05) and 30 min (0.5 ± 0.2; P < 0.05), but increased in the heart at 5 min (1.6 ± 0.4; P < 0.05) and in the kidney at 60 min (1.7 ± 0.5; P < 0.05). In the liver, no significant changes in the expression of c-jun mRNA were detected under pentobarbital.

Under propofol, c-jun mRNA expression was increased in the heart at 30 min (1.5 ± 0.3; P < 0.05), in the liver at 60 min and 120 min (2.0 ± 0.6 and 2.6 ± 1.2, respectively; P < 0.05), and in the kidney also at 60 min and 120 min (2.0 ± 0.4 and 1.6 ± 0.5, respectively; P < 0.05). In propofol-anesthetized rats, the brain showed no significant change in the expression of c-jun mRNA.

	Discussion

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The present study demonstrated that, in rats, the effects of general anesthetics on the expression of the mRNAs of the immediate-early genes c-fos and c-jun differed both in magnitude and time course, depending on which of four vital organs was under study. The effects on the expression of c-fos and c-jun mRNAs also differed among the anesthetics used, namely isoflurane, pentobarbital, and propofol. The expression of c-fos in the brain increased within 30 min with all three drugs, but brain c-jun mRNA, which did not change under isoflurane or propofol, decreased under pentobarbital. The heart was the only organ in which c-fos expression decreased on exposure to isoflurane and pentobarbital, whereas c-jun expression in the heart increased at some point with all three drugs. The greatest increases in immediate-early gene expression were those seen for c-fos in the kidney, the most marked changes being seen in rats anesthetized with propofol.

This is the first report to show that the effects of general anesthetics on the expression of the immediate-early genes c-fos and c-jun differ among the vital organs (brain, heart, liver, and kidney) in the rat. Previous reports referred only to the effect of general anesthesia on immediate-early gene expression in the brain (4,5). The results obtained in those earlier studies are in general agreement with our results in the brain.

It is well known that stimulation of cells with growth factors, for instance, provokes within a few minutes an increased expression of the mRNAs and proteins related to c-fos (11) and c-jun (12). c-fos protein and c-jun protein constitute heterodimers, and each of them makes homodimers as well, as in other members of the Fos and Jun families (e.g., Fos B and Fos-related antigens, and Jun B and Jun D), and they bind to specific DNA sequences called activator protain-1 sites (13) to act as transcription factors (14). Their binding affinities to the activator protain-1 sites differ with the heterodimeric or homodimeric constituents of the complex (13). An increased expression of an immediate-early gene is not in itself a specific response to a stimulus to the cell, but rather a way of regulating and conducting signals from upstream, such as mitogen-activated protein kinases (15) and protein kinase C (PKC) (16), to downstream in the intracellular signal cascade (leading, for example, to an increased expression of the later response genes that control specific cellular events). The Fos/Jun complex also governs the activity of cyclic adenosine monophosphate response element binding protein and so regulates gluconeogenesis, neuronal excitation, the establishment of circadian rhythms, pituitary proliferation, and opiate tolerance (14).

The present data showed a transient increase in c-fos mRNA expression in the brain at 5–30 minutes with all three anesthetics. This may be related to the fact that there is a pathway through which stimulation of the cell activates PKC to induce an increased expression of c-fos mRNA (16), and that both halothane and propofol activate purified rat brain PKC (17). In the present study, the expression of c-fos mRNA in the brain under both isoflurane and propofol had returned to about the control level in one hour or less (Fig. 3), even though these anesthetics were administered continuously for two hours. The explanation for this remains speculative: possibly, the neurons may have adjusted to stimulation by isoflurane and propofol under steady state conditions of general anesthesia, or c-fos mRNA may exist only briefly in the brain. The present results with pentobarbital, which was given as a single intraperitoneal injection of 50 mg/kg, also showed a time-related increase in c-fos mRNA expression in the brain, the timing of which differed from that described by Marota et al. (4), who observed a significant increase in c-fos mRNA expression in the brain at 120 minutes in rats anesthetized with 65 mg/kg of intraperitoneal pentobarbital. The discrepancy between their results and ours may be related to the dose of pentobarbital. The larger dose might have had a more prolonged effect on c-fos mRNA expression in the brain, or it might have depressed spontaneous respiration more than our dose, with a consequent hypercarbia, as indicated in their report (4), an effect that might have altered the results.

View larger version (43K): [in this window] [in a new window]   Figure 3. Representative characterization of time course of the immediate-early gene expression in the brain of rats. Total RNA was extracted from the brain of rats anesthetized with isoflurane, pentobarbital, or propofol for 5, 30, 60, or 120 min, and the reverse transcribed cDNA were polymerase chain reaction (PCR)-amplified with oligonucleotide primers for cyclophilin, c-fos, and c-jun mRNAs. The reverse transcriptase (RT)-PCR products ran on 1.5% agarose gels and were visualized with ethidium bromide to be analyzed with optical densitometry. Lane 1 is a DNA molecular weight marker (M); Lane 2 is from a nonanesthetized rat (N); Lanes 3–6 are from rats killed at 5, 30, 60, or 120 min of isoflurane anesthesia; Lanes 7–10 are from rats with pentobarbital; Lanes 11–14 are from rats with propofol; Lane 15 is a RT-PCR product of cyclophilin from Rat 1, loaded as a standard for inter-gel normalization.

Although we determined the doses of the anesthetics used by modifying the suggested doses (19) of these drugs according to our preliminary observations of the rat’s vital signs, variations in anesthetic depth between animals and between anesthetics may have biased our results. Variable effects on immediate-early gene expression may have been induced by hypoxia, ischemia, oxidative stress, and hyperthermia (2), none of which we tried to prevent by monitoring or controlling the physiological status of the rats. This may limit the interpretation of our data and prevent us from comparing our results with previous reports. However, this does not negate our general finding, namely that there were induced changes in the expression of the mRNAs of the two immediate-early genes, and that these changes showed interorgan and interanesthetic variability. Because we used PCR for the quantification of mRNA expression, there might have been quantitative variability between samples and even duplicate determinations using the same sample (20). However, we considered the coefficient of variation of <22% for the cyclophilin mRNA expression determined by RT-PCR to be rather small, and the fact that stable values were obtained for cyclophilin mRNA expression could be an indirect demonstration of the quantitative reliability of RT-PCR for our purpose. Different anesthetics have different effects on Fos protein in given regions in the rat brain (5), and this raises the possibility of regional differences in the susceptibility to anesthetics within the brain, and conceivably within organs other than the brain.

Another shortcoming of our study is that we have not proven the translation of the immediate-early gene mRNA into protein in any way. Because transcription of genomic DNA to mRNA does not necessarily lead to translation to protein (21), increased translation of c-fos or c-jun mRNA shown in our data may not mean accumulation of the Fos or Jun protein. It would be important to demonstrate via in situ hybridization, immunohistochemistry, and/or any other suitable methods that increased transcription leads to increased translation of the immediate-early genes. One may also argue that mRNA from the whole organ does not discriminate mRNA from the interstitial tissue or endothelial cells in the blood vessels rather than the specifically differentiated cells in that organ (e.g., neurons in the brain or hepatocytes in the liver). Using an in situ technique would also enable us to examine specific cell types and organ regions for the immediate-early gene expression. A report by Nakao et al. (22) on the inhibitory effect of halothane and diazepam on ketamine-induced c-fos expression in the rat cingulate cortex, or a report by Nagata et al. (23) on the inhibitory effect of propofol on ketamine-induced c-fos expression in the rat posterior cingulate cortex, are good examples of this methodological issue. Interestingly, propofol itself was reported not to induce c-fos expression in the cingulated cortex of the rat brain (23), contradicting our data on the whole brain of the rat.

In conclusion, we found interanesthetic and interorgan differences in the effect of general anesthetics on the expression of two immediate-early genes. Our results would seem to be another indication that general anesthetics can themselves stimulate some intracellular events rather than suppress them. We must choose the appropriate anesthetics that have minimal effects on the target organs when we give them to animals to prepare for a study of the immediate-early genes, because anesthetic preparation per se can influence the results rather than the drugs or stimuli as the study objects. Thus the anesthetics we used seem inappropriate, in particular when the study subject is the kidney. Our results suggest that the appropriate anesthetics to be used to anesthetize animals differ in accord with the target organs in which the expressions of immediate-early genes in response to stimuli are studied.

	Acknowledgments

 
This investigation was supported in part by Ministry of Education Grant 11307027 to SD.

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