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Anesth Analg 2003;97:1046-1052
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Flumazenil Attenuates Development of Tolerance to Diazepam After Chronic Treatment of Mice with Either Isoflurane or Diazepam

Ron Flaishon, MD*, Avi A. Weinbroum, MD*, Leo Veenman, PhD{dagger}, Svetlana Leschiner, PhD{dagger}, Valerie Rudick, MD*, and Moshe Gavish, PhD{dagger},{ddagger}

*Department of Anesthesiology and Critical Care Medicine, Tel Aviv Sourasky Medical Center, and The Sackler Faculty of Medicine, Tel Aviv, Israel; {dagger}Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; and {ddagger}Rappaport Family Institute for Research in the Medical Sciences, Haifa, Israel

Address correspondence and reprint requests to Moshe Gavish, PhD, Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, 31096 Haifa, Israel. Address e-mail to mgavish{at}tx.technion.ac.il


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In an effort to clarify the mechanism of action of isoflurane, we studied the effect of flumazenil on mice chronically treated with isoflurane or diazepam. Mice were pretreated with diazepam, isoflurane, or saline, with and without flumazenil. After 2 wk, responses to isoflurane and diazepam were assessed, and central benzodiazepine receptor (CBR) binding characteristics were assayed. Mice pretreated with isoflurane failed the horizontal wire test at a larger isoflurane concentration (0.5%) compared with saline-pretreated mice (0.4%) (P < 0.05). These differences did not occur when flumazenil was added to the pretreatment. After the administration of diazepam, 20% of diazepam- and 11% of isoflurane-pretreated mice failed the horizontal wire test, versus 50% and 44% when flumazenil was added to either drug (P < 0.002) and 80% and 100% in the saline and saline plus flumazenil-treated mice. The increased CBR density due to flumazenil was attenuated by the coadministration of isoflurane or diazepam. Flumazenil attenuated the development of tolerance to diazepam after chronic treatment with diazepam or isoflurane and attenuated the development of tolerance to isoflurane. Isoflurane, like diazepam, attenuated the effect of flumazenil on CBR ligand binding. These findings suggest that isoflurane shares a mechanism of action with diazepam, probably via the gamma-aminobutyric acid system, most probably the CBR.

IMPLICATIONS: Flumazenil attenuates the development of tolerance to isoflurane and diazepam after chronic isoflurane pretreatment. Isoflurane, like diazepam, attenuates the increase in central benzodiazepine receptor (CBR) density caused by flumazenil. These findings suggest that isoflurane and diazepam share a mechanism of action, most probably via the gamma-aminobutyric acid system and the CBR.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Several theories have been proposed to explain the mechanism of action of inhaled anesthetics (IA) (1–5). The gamma-aminobutyric acid (GABA) system has been suggested as a major substrate for the anesthetic effects of IA (1).

Central benzodiazepine receptors (CBR), on the GABAA receptor complex (6), are located on neurons in the central nervous system (7). Peripheral benzodiazepine receptors are located in peripheral tissues and on glial cells in the central nervous system (8), forming a distinct receptor population (9). Chronic benzodiazepine treatment results in tolerance and dependence (10–12). We previously demonstrated that mice that were tolerant to diazepam were less sensitive to isoflurane than control mice (12). Chronic isoflurane exposure resulted in subsensitivity to isoflurane and tolerance to diazepam (12). On the basis of these findings of "cross-tolerance," we suggested a shared mechanism of action of both drugs via the GABA system and probably the CBR (12). In this study, we go one step further in strengthening this conclusion.

The benzodiazepine antagonist flumazenil was found to attenuate the development of tolerance to benzodiazepines after chronic administration (10,11). A case report indicated that tolerance to isoflurane might also occur, although this was not considered to be a significant clinical problem (13). We have speculated that flumazenil would attenuate the development of tolerance to both isoflurane and diazepam after chronic exposure. The horizontal wire test (HWT) has been described as an assessment tool of coordination and hind paw strength (12,14). We used this test, because tolerance interferes with all aspects of benzodiazepine effects, including muscle strength and coordination (11,12,15). Righting reflex (RR) and recovery time (RT) were used as well, because RT was found to be a good indicator of sensitivity to isoflurane (12). We assumed that chronic benzodiazepine treatment would affect the CBR density. Therefore, we assessed whether chronic isoflurane treatment would affect the CBR ligand-binding density the same way that diazepam did and whether flumazenil would attenuate these changes. We expected that, taken together, the behavioral study and CBR assay would indicate whether CBR might indeed form a common binding site for both isoflurane and diazepam and thereby form a common substrate for the development of behavioral cross-tolerance for these drugs.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The Tel Aviv Sourasky Medical Center Animal Research Committee approved the study. CD1 mice (bred at Tel Aviv University) weighing 28 ± 2 g were studied. The mice were housed 10 animals per cage, in air-conditioned rooms, on a 12:12-h light/dark cycle, with free access to food and water.

Diazepam was donated by Teva Pharmaceuticals (Petah Tiqva, Israel). Flumazenil was donated by Hoffmann-La Roche (Nutley, NJ). [3H]Ro 15-1788 (75.2 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Clonazepam was kindly supplied by Drs. H. Gutmann and E. Kyburz, Hoffmann-La Roche (Basel, Switzerland). The isoflurane concentration was monitored with a Capnomac gas monitor (Datex, Helsinki, Finland).

The mice were divided into 6 groups and were pretreated for 14 days.

  1. Group 1: twice-daily intraperitoneal (IP) injections of diazepam 10 mg/kg in 0.2 mL of normal saline containing 0.1% Tween 80, to facilitate dissolution (referred to hereafter as "saline"), as described previously (12).
  2. Group 2: mice were pretreated as in Group 1, but flumazenil 5 mg/kg in 0.2 mL of saline was injected IP once every other day (referred to hereafter as "DZ/Flu"). The dose of flumazenil was based on previous studies (11).
  3. Group 3: mice were anesthetized in a Plexiglas chamber with 3% isoflurane in air for 25 min (after the desired concentration was reached) twice daily, as described previously (12); 0.2 mL of saline was injected once every other day.
  4. Group 4: mice were pretreated with isoflurane as in Group 3, but flumazenil was added as in Group 2 (Iso/Flu).
  5. Group 5: mice were injected IP twice daily with 0.2 mL of saline.
  6. Group 6: mice received saline as in Group 5, plus flumazenil as in Groups 2 and 4 (saline/Flu).

After 14 days of pretreatment, the responses to isoflurane and diazepam were assessed. To assess the response of mice to isoflurane (Groups 3–6), we used the HWT, RR, and RT, as detailed below. The response of all groups to diazepam after 2 wk of pretreatment according to groups was assessed with the HWT. At the end of the study, mice were killed, and their brains were assayed for CBR ligand-binding densities. Each mouse was assessed only once. Table 1 demonstrates the timetable for the assessment of mice of all groups.


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  		Table 1. Timetable of the Study Procedures
 
Assessment of the response to isoflurane took place 24 h (on the 15th day) after the last exposure to 3% isoflurane or to saline injection (Groups 3–6). Half of the pretreated mice in Groups 3–6 were assessed in this phase of the study, and the rest of the mice were pretreated for an additional day (a total of 15 days). Eight mice (two of each group) were placed in a Plexiglas chamber into which 0.3% isoflurane in air was administered. The mice were marked on their tails and backs by a technician of the animal laboratory who was not a member of the study group. The investigator was blinded to the group to which each mouse belonged. After 10 min of exposure to the desired concentration, the HWT and RR were assessed. In the HWT, a mouse is lifted by its tail, allowed to grasp a horizontal wire by its forepaws, and then released. A naïve mouse heaves itself to grasp the wire with at least one hind paw. This reflex is lost after the administration of an anesthetic or sedative drug (12). The RR is the reflex of a mouse as it scrambles to its feet when placed on its back. After the reflexes were assessed, the isoflurane concentration was increased by 0.1%, and mice were reexamined. When all the mice had lost the RR, the isoflurane was turned off, the chamber was opened to air, and the RT was recorded (the time at which a mouse spontaneously regained the RR). The number of mice that lost each reflex at every concentration was recorded. A mouse that lost a reflex was not reexamined at the next concentration for the same reflex. All the mice were exposed to all concentration points and were exposed to isoflurane for the same duration. Thus, the maximal isoflurane concentration was the same for all mice. At the end of this phase, the assessed mice were killed.

The investigator was blinded as to which group a mouse belonged in (Groups 1–6). The assessment took place on the 17th day, 24 h after the last pretreatment exposure to isoflurane and 48 h after the last pretreatment with diazepam. In Groups 3–6, we assessed the mice that were not examined for their response to isoflurane in the previous phase. The difference in the delay between the isoflurane- and diazepam-pretreated groups was due to the difference in the pharmacokinetics of both drugs and was based on our previous study (12). In the process of the assessment, diazepam 5 mg/kg in 0.2 mL of saline was injected IP. Ten minutes later, mice were examined on the HWT. The lag time of 10 min between injection and assessment was based on our previous study (12), because this was the time to peak diazepam effect.

After decapitation, the brain was removed and frozen at -80°C until it was assayed. Before the binding assay, brains were defrosted and homogenized in 50 vol of 50 mM potassium phosphate buffer, pH 7.4, at 4°C and centrifuged for 20 min at 49,000g. The pellets were washed twice as described above. The final pellet was homogenized (4 mg/mL) in potassium phosphate buffer and used for the [3H]Ro 15-1788 binding assay, as previously described (16). Binding was determined in a final volume of 500 µL containing [3H]Ro 15-1788 (0.1–3.0 nM final concentration) in the absence (total binding) or presence (nonspecific binding) of unlabeled 10 µM clonazepam. After incubation at 4°C for 1 h, samples were filtered under a vacuum over Whatman GF/C filters. The filters were placed in vials containing 4 mL of Opti-Fluor (Packard, Groningen, The Netherlands) for 12 h, and the amount of radioactivity per vial was counted.

Data are expressed as mean ± SD. The responses (HWT and RR) of mice from the different groups to increasing concentrations of isoflurane were compared by using one-way of variance and the Kaplan-Meier estimate for survival analysis. The number of mice in the different groups that failed the HWT in response to diazepam after 2 wk of treatment was compared by using the {chi}2 test. RT among groups was compared by using one-way analysis of variance. The benzodiazepine receptor density between groups was compared with one-way analysis of variance, by using the Tukey-Kramer multiple comparison test for post hoc analysis. Differences were considered significant when P values were <0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Mice chronically treated with isoflurane failed the HWT at a larger isoflurane concentration compared with saline-treated mice. This difference was lost when flumazenil was added to the therapeutic regimen (Fig. 1). Specifically, the mean isoflurane concentration for failure of the HWT for isoflurane-treated mice was 0.53% ± 0.1% (n = 18), compared with 0.41% ± 0.07% for the saline group (n = 10) (P < 0.02). Mice in the Iso/Flu group lost the reflex at an average isoflurane concentration of 0.47% ± 0.1% (n = 18), which was not different from the saline group and the saline/Flu group (n = 10; 0.44% ± 0.08%).



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  		Figure 1. Percentages of mice per experimental group that failed the horizontal wire test (HWT) at each isoflurane (ISO) concentration after 2 wk of chronic ISO or saline treatment with or without the coadministration of flumazenil (FL). The mean ISO concentration for failure of the HWT of the ISO group was significantly larger than that of saline-treated mice (P < 0.02). This difference was lost when FL was added to the therapeutic regimen.

 
Mice in the isoflurane group lost the RR at a significantly higher mean isoflurane concentration (0.75% ± 0.08%) compared with the saline-treated mice (0.67% ± 0.01%) ( P < 0.01) (Fig. 2). This difference was lost when the Iso/Flu group (0.71% ± 0.07%) was compared with the saline group. Moreover, with the Kaplan-Meier estimate it was found that the curve of the isoflurane group was different from that of all the other groups (P < 0.05). Furthermore, at 0.7% isoflurane, all the control mice lost the RR, whereas only 55% of the isoflurane-treated mice did (P < 0.05). Again, such a significant difference did not occur when mice pretreated with both isoflurane and flumazenil were compared.



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  		Figure 2. Percentages of mice per experimental group that lost the righting reflex (RR) at each isoflurane (ISO) concentration after 2 wk of chronic ISO or saline treatment with or without the coadministration of flumazenil (FL). ISO-treated mice lost the RR at a significantly larger ISO concentration compared with saline-treated mice. No such difference occurred in the ISO+FL group. The ISO curve differed significantly from that of the other three groups (P < 0.05). At an ISO concentration of 0.7%, significantly fewer ISO-treated mice lost the RR compared with saline-treated mice (P < 0.05). This significance was lost when FL was added to the treatment.

 
Isoflurane-treated mice recovered after 130.2 ± 83 s (n = 18), significantly faster than the saline group (n = 10; 211.8 ± 87.5 s; P =0.02) (Fig. 3). There was no such difference between the Iso/Flu and saline groups. The Iso/Flu group recovered after a longer average time compared with the isoflurane group (222.8 ± 201 s; n = 18), although without statistical significance (P < 0.08). The Iso/Flu-treated mice did not show any differences in RT compared with either saline- or saline/Flu-treated mice.



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  		Figure 3. Time to recovery from isoflurane (ISO) anesthesia after 2 wk of chronic ISO or saline treatment with or without the coadministration of flumazenil (FL) of mice examined for the horizontal wire test, righting reflex, and seconds of recovery time (mean ± SD). The ISO-treated mice recovered significantly faster than the mice from each of the other three groups (P < 0.05).

 
Figure 4 demonstrates the response to 5 mg/kg of diazepam, as assessed by the HWT, in all 6 groups after 2 weeks of pretreatment. Mice treated with diazepam or isoflurane for 2 weeks were tolerant to diazepam compared with saline-treated or saline/Flu-treated mice. Thus, 20% (4 of 20) of the diazepam-treated mice and 11% (2 of 18) of the isoflurane-treated mice failed the HWT, compared with 80% (8 of 10) of the saline-treated mice (P < 0.02) or 100% (10 of 10) of the saline/Flu-treated mice (P < 0.001). The addition of flumazenil to the chronic treatment significantly attenuated the development of tolerance to diazepam. Thus, 50% (10 of 20) of the DZ/flu-treated mice lost the reflex compared with 20% of the diazepam-treated mice (P < 0.05). Flumazenil also attenuated the development of tolerance to diazepam after chronic exposure to isoflurane; thus, 44% (8 of 18) of the Iso/Flu-treated mice failed the HWT, compared with 11% of the isoflurane-treated mice (P < 0.05). There was no difference between the saline- and saline/Flu-treated mice, indicating that flumazenil did not have a net effect on the response of mice to diazepam.



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  		Figure 4. Percentages of mice per group that failed the horizontal wire test (HWT) 10 min after the injection of diazepam (DZ) 5 mg/kg. Before this assessment, mice were treated for 2 wk with DZ, isoflurane (ISO), or saline, either with or without the coadministration of flumazenil (FL). Four of 20 DZ-treated mice, 10 of 20 DZ and FL-treated mice, 2 of 18 ISO-treated mice, 8 of 18 ISO and FL-treated mice, 8 of 10 saline-treated mice, and all 10 saline and FL-treated mice failed the HWT. *P < 0.05; **P < 0.002; #P < 0.001.

 
The results of the CBR ligand-binding study are presented in Figure 5. No difference was observed in CBR density among the isoflurane-, diazepam-, diazepam/flumazenil-, and saline-treated groups. Flumazenil, however, caused a significant increase in CBR density in the saline-treated mice (P < 0.001). This density was also higher compared with that of diazepam- and isoflurane-treated mice (P < 0.001). The coadministration of isoflurane with flumazenil also resulted in a significant increase with regard to saline-treated mice (P < 0.01), although this was not as pronounced as with saline plus flumazenil. Such an increase, however, was not observed with the coadministration of diazepam and flumazenil. It is interesting to note that the coadministration of isoflurane or diazepam with flumazenil significantly reduced the effect of flumazenil; i.e., CBR density was not increased as it was after treatment with saline plus flumazenil (P < 0.001 in both instances). In addition, isoflurane/flumazenil- and diazepam/flumazenil-treated mice did not display enhanced levels of CBR density compared with isoflurane- and diazepam-treated mice, respectively. These data suggest that both diazepam and isoflurane attenuate the effect of flumazenil.



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  		Figure 5. Central benzodiazepine receptor (CBR) density in brains of mice in the six study groups. The CBR density in the isoflurane/flumazenil (ISO+FL)-treated group was significantly higher than that of the saline-treated mice. The receptor density in the saline/flumazenil (saline+FL)-treated group was significantly higher than in the rest of the groups. *Significantly higher compared with Group 5 (saline; P < 0.01); **significantly higher compared with the following experimental groups: Group 1 (diazepam [DZ]; P < 0.001), Group 2 (DZ+FL; P < 0.001), Group 3 (ISO; P < 0.001), Group 4 (ISO+FL; P < 0.01), and Group 5 (saline; P < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our group previously demonstrated that mice chronically treated with isoflurane or diazepam became less sensitive or even tolerant to both drugs (12). We have now demonstrated that coadministration of the CBR antagonist flumazenil to mice chronically treated with diazepam attenuated the development of tolerance (Fig. 4). The period of treatment and the doses of isoflurane and diazepam were based on our experience (12). While developing the model, we observed that a longer period of treatment and a larger dose did not produce more tolerance and were not tolerated by the animals. The 5-mg dose of diazepam was used for the assessment of HWT because at a larger dose, most of the mice of all groups lost the HWT and the RR.

In agreement with our previous study (12), we found that mice exposed to isoflurane for 2 weeks failed the HWT and lost the RR (Figs. 1 and 2) at larger isoflurane concentrations compared with untreated mice. In addition, the coadministration of flumazenil with isoflurane pretreatment attenuated the development of the subsensitivity to isoflurane (Figs. 1, 2, and 3) and the development of tolerance to diazepam (Fig. 4). Thus, the results from the HWT, RR, and RT suggest that flumazenil indeed interferes with the development of tolerance to isoflurane and probably with the mechanisms of its action.

Development of tolerance to benzodiazepines has been demonstrated in humans (10) and animals (11,12,15) and was attenuated by the coadministration of flumazenil in animals (17) and humans (10). Savic et al. (10) concluded that there might be a rationale for the use of flumazenil to avoid tolerance to both the sedative and antiepileptic effects of benzodiazepines. Other studies, however, suggested that flumazenil might not always prevent the development of tolerance (11). This may be due to methodological differences.

Anecdotal evidence suggests that prolonged exposure to isoflurane in humans may lead to the development of tolerance (13). We now sought to clarify whether the coadministration of flumazenil would affect the development of tolerance to isoflurane the way it affects that of diazepam, an issue that has not been studied before. Several animal studies that dealt with the issue of the effect of flumazenil on acute exposure to IA yielded conflicting results (18,19). This study demonstrates that flumazenil attenuates the development of tolerance to isoflurane as well as to diazepam after chronic exposure to isoflurane in mice.

Flumazenil did not completely prevent the development of tolerance to either isoflurane or diazepam. These findings may be explained by the possibility that, although the GABAA receptor is the prime target for IA (1), these anesthetics may also act via several other receptors (2–5). Thus, because flumazenil is a specific CBR antagonist, it may not be capable of blocking all the actions of isoflurane.

We also investigated the effect of flumazenil on the response of mice to diazepam after chronic administration of the benzodiazepine. We used the HWT to assess several aspects of tolerance to benzodiazepines, including sedation, muscle strength, and coordination. The incomplete blockade by flumazenil of the development of tolerance to diazepam may be because diazepam also acts at the peripheral structures (20), which would not be subject to the antagonistic action of flumazenil on CBR. Thus, as with isoflurane, more than one mechanism may be involved in the development of tolerance to benzodiazepines, and flumazenil may act on only one of them.

In accordance with other reports (21,22), treatment of saline-injected mice with flumazenil caused an increase in CBR density. Also in agreement with previous studies (12,19,22), the results from our CBR-binding assay indicated a lack of difference between the CBR-binding characteristics of mice treated with isoflurane, diazepam, or saline. Other studies, however, reported an increase (23) or decrease (24) in CBR density in response to chronic benzodiazepine treatment. One study (25) has shown that isoflurane exposure enhanced CBR-specific 11C-flumazenil binding. Fujita et al. (24) provided an explanation for the lack of such an effect in our study. They showed that an increase in CBR density, observed after 10 days of benzodiazepine treatment in humans, normalized on Day 17. Our mice were treated for 14 days and were killed for the receptor assay 2–3 days later. This period may have been long enough for normalization of the CBR density, i.e., a reduction in the effects of isoflurane and diazepam on CBR ligand-binding density. Despite the lack of an observable effect on CBR ligand binding, co-treatment with isoflurane or diazepam nonetheless significantly reduced the increase of CBR ligand-binding density by flumazenil. A 20% difference in CBR density seems small, but it is significant both statistically and biologically. Gyulai et al. (25) demonstrated that isoflurane exposure enhanced receptor-specific 11C-flumazenil binding. The difference found in that study was at the same order as in our study. Matheja et al. (26) detected foci of decreased benzodiazepine receptor binding at the onset of temporal lobe epilepsy. A maximal reduction of 22% was found in only one patient. On the basis of these data, we suggest that the difference in CBR found in our study is biologically significant. A larger difference may even result in seizures.

Our CBR ligand-binding study suggests that isoflurane, like diazepam, indeed acts via the CBR. Several other studies also demonstrated functional changes in the CBR-GABAA receptor complex after chronic benzodiazepine treatment, without accompanying changes in CBR abundance (27). Thus, although the administration of diazepam may not necessarily cause a change in CBR abundance in the brain, diazepam may cause functional changes in CBR that prevent flumazenil from increasing CBR density, and the same may be true for isoflurane.

In summary, first, this study has affirmed previous findings that flumazenil attenuates the development of tolerance to diazepam. Second, we observed that flumazenil attenuates the development of subsensitivity to isoflurane after chronic exposure to this drug. The third and most interesting finding was that flumazenil also attenuates the development of tolerance to diazepam after chronic exposure to isoflurane. Our fourth finding was that both isoflurane and diazepam attenuate the increase in CBR ligand-binding density caused by flumazenil administration. Taken together, these data suggest that the GABA system and the CBR indeed form a common site of action for isoflurane and diazepam. Isoflurane probably acts also via other sites apart from CBR, but the fact that flumazenil, being a specific CBR antagonist, affects isoflurane and diazepam in a similar way strengthens our hypothesis that CBR is one of the sites involved in the mechanism of action of isoflurane.


   Acknowledgments
 
SL and LV were supported by the Center for Absorption in Science, Ministry of Immigrant Absorption, State of Israel.

The authors thank Ruth Singer for editing the manuscript.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication May 15, 2003.




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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