This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chenel, M.
Right arrow Articles by Couet, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chenel, M.
Right arrow Articles by Couet, W.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, November 2003, p. 3660-3662, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3660-3662.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Norfloxacin-Induced Electroencephalogram Alteration and Seizures in Rats Are Not Triggered by Enhanced Levels of Intracerebral Glutamate{dagger}

Marylore Chenel,1,2 Anne Limosin,1,2 Sandrine Marchand,1 Joël Paquereau,3 Olivier Mimoz,1,4 and William Couet1*

Equipe Médicaments anti-infectieux et barrière hémato-encéphalique, PBS, Faculté de Médecine & Pharmacie, 86022 Poitiers Cedex,1 Laboratoire de Pharmacocinétique, PBS, CHU La Milétrie, 86022 Poitiers Cedex,2 Equipe Sommeil: Attention et Respiration, PBS, Faculté de Médecine & Pharmacie, 86022 Poitiers Cedex,3 Département d'Anesthésie et Réanimation Chirurgicale, CHU La Milétrie, 86000 Poitiers Cedex, France4

Received 10 March 2003/ Returned for modification 9 July 2003/ Accepted 20 August 2003


arrow
ABSTRACT
 
Pharmacokinetic-pharmacodynamic modeling of the electroencephalogram (EEG) effect was combined with intracerebral glutamate determinations using microdialysis for rats receiving norfloxacin intravenously (150 mg/kg of body weight). The EEG effect (accompanied by tremors and seizures) was consistently observed without glutamate level modifications. Therefore, norfloxacin-inducted seizures are not triggered by intracerebral glutamate level enhancement.


arrow
TEXT
 
Fluoroquinolone antibiotics may induce central nervous system excitatory side effects, including anxiety, nervousness, hallucination, and even seizures on rare occasions (3, 13). It is usually asserted that these excitatory side effects result from inhibition of GABA ({gamma}-aminobutyric acid) binding to its receptors (1, 11). Using intracerebral microdialysis, however, Smolders et al. have previously observed in conscious rats norfloxacin-induced convulsions accompanied not by any apparent effect on the extracellular hippocampal GABA levels but rather by enhanced intracerebral glutamate concentrations (10). Studies of the relationship between glutamate concentrations and epileptic seizures have yielded conflicting observations, as recently reviewed (12). Smolders et al. have hypothesized that enhanced glutamate levels might be essential for the induction of norfloxacin seizures (10), but it could also be argued that the generation, spreading, and maintenance of seizures eventually leads to glutamate release and elevation (9). Quantitative electroencephalogram (EEG) recording combined with intracerebral microdialysis offers a unique opportunity to address this question. In rats treated with imipenem (5) and norfloxacin (2), increases in the total power of the EEG signal are related to behavioral modifications and the occurrence of tremor and partial seizures and can therefore be considered an appropriate surrogate pharmacodynamic (PD) end point for the investigation of the convulsant activity of these antibiotics in rats. Furthermore, the kinetics of this effect can be very precisely evaluated using pharmacokinetic (PK)-PD modeling techniques (2, 5).

The objective of this study was therefore to combine quantitative EEG recording (for the characterization of seizure induction and maintenance) with intracerebral microdialysis using the hippocampus (for the measurement of glutamate levels) of rats (after the administration of norfloxacin) to look for a causality relationship between the two factors.

The experiment was conducted in accordance with the Principles of Laboratory Animal Care (National Institutes of Health), and ethical approval was obtained from the Animal Ethics Committee of the Faculty of Pharmacy (BHE 2001/12/AE). Norfloxacin was obtained from Sigma (Saint-Quentin Fallavier, France), and a salt was prepared as previously described (4). L-glutamate standards were supplied by Sigma. All chemicals used were of analytical grade, and the analytical solvents used were of high-performance liquid chromatography grade. Six male Sprague-Dawley rats (weighing 299 ± 15 g) from Depres Breeding Laboratories (St. Doulchard, France) were used and handled as previously described (2). At 5 days before drug administration, a microdialysis CMA/12 guide cannula was implanted into the left dorsal hippocampus for glutamate determinations (7) and cortical electrodes were implanted for EEG recording (2). The animals were placed in a thermostatted chamber (27°C) and were allowed to recover for 4 days before implantation of femoral catheters for drug administration and blood sampling was performed (2). On the day of the experiment, rats were maintained in a plastic bowl, the guide canula obturator was replaced by a CMA/12 microdialysis probe (Phymep, Paris, France) (0.5 mm in diameter by 3 mm long), and immediately thereafter the miniature plug was connected to a moving connector to record and analyze the EEG signal as previously described (2). The microdialysis probe was infused with a Ringer solution (6) at a flow rate of 2 µl · min-1 for 1 h to stabilize the system. Then, two blank microdialysate samples were taken in preweighed vials at 15-min intervals. Norfloxacin was then injected as an intravenous bolus at a dose of 150 mg · kg-1 (422 µM · kg-1) between 10:30 and 11:00 a.m. Blood samples were drawn for norfloxacin high-performance liquid chromatography determinations in plasma (2). Dialysates were collected every 15 min for glutamate determinations (8). PK-PD modeling was conducted as previously described (2). A two-compartment open model was used to characterize the plasma drug concentration versus time profiles. PK parameters were then fixed, and a nonlinear least-squares program (WinNonlin version 1.1; SCI Software, Carry, N.C.) was used to regress the PD model to the EEG data for each individual rat. An effect compartment model was applied for analysis of the PK-PD relationship, leading to an estimate of the rate constant for the elimination of the drug from the effect compartment ke0. The profile of the EEG effect was described using a spline function derived from the Hill equation:

(1)
In this equation, P represents the total power (EEG effect) corresponding to the drug concentration in the effect compartment (Ce), P0 represents the baseline effect value, Bn represents the combined Emax-(50% effective concentration)n parameters, and n represents a factor determining the steepness of the curve. The goodness of fit of each model was assessed by visual inspection and analysis of the residuals and of the percentage of the coefficient of variation associated to parameter estimates (2, 5). Two control animals underwent the same experimental protocol except that norfloxacin administration was replaced by isotonic saline administration.

All rats exhibited mild-to-severe nonlethal seizures accompanied by a marked effect on the EEG signal. A PK-PD model (with an effect compartment linked to a two-open compartment model and a spline function to characterize the relationship between effect and concentration at the effect site) was successfully fitted to the data. Corresponding parameter estimates (Table 1) were consistent with previously reported values. Extracellular glutamate concentrations in the hippocampus had a tendency to fluctuate with time but no general trend could be observed, and similar fluctuations were observed in the two control rats. This observation may seem to be inconsistent with the previous report of enhancement of glutamate concentrations occurring with seizures (10). However, the present study was conducted with Sprague-Dawley rats receiving norfloxacin at 150 mg/kg of body weight compared to Wistar rats treated with 300 mg/kg of norfloxacin in the previous experiment (10), which could partially explain this apparent discrepancy. The difficulty of finding doses appropriate for the inducement of reversible and therefore nonlethal seizures but sufficiently severe to have an effect on the EEG signal was previously mentioned (2, 5). Preliminary tests suggested that a 300 mg/kg dose of norfloxacin was too high for the present study, and the 150 mg/kg dose was considered a better compromise. In such circumstances, significant alteration of the EEG signal and seizure induction characteristics were consistently observed; these effects did not require any previous observable alteration of hippocampal glutamate levels, as illustrated in Fig. 1. It can therefore be concluded that as previously suggested (10), enhancement of glutamate levels is not essential for the induction of seizures by norfloxacin treatment. The alternative hypothesis, stating that glutamate release and elevation are, rather, a consequence of norfloxacin-induced seizures, should now be preferred. Yet no glutamate alteration was observed in the present study, even after the clinical symptoms appeared. A possible explanation is that the intensity of the seizures that followed the administration of the 150 mg/kg dose might not have been sufficient to induce an enhancement of intracerebral glutamate levels.


View this table:
[in this window]
[in a new window]
  		TABLE 1. PK and PK-PD parameters (estimated using an effect compartment model with a spline function) after intravenous bolus administration of norfloxacin to six rats at a dose equal to 150 mg · kg-1



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1. Concentrations of norfloxacin in plasma and EEG effect versus time for a representative rat. In the upper panel, the broken line represents the best PK fit to the measured norfloxacin plasma concentrations, with the following values for PK parameters: clearance = 18.4 ml · min-1 · kg-1, volume of distribution at steady state = 4,648 ml · kg-1, and half-life at ß phase = 191 min. The solid line represents the best fit to the measured total power of the EEG signal effect according to the effect compartment model, with the following values for the PD parameters: P0 = 0.583 mV2, Bn = 0.015 mV2 · µM-1, and n = 2.6. The rate constant for elimination of the drug from the effect compartment ke0 was equal to 0.023 min-1 for this particular rat. In the lower panel, corresponding glutamate concentrations determined in brain microdialysates and expressed as percentages of fluctuation around basal values are presented versus time and connected for identification purposes.

In conclusion, this study has demonstrated (for the first time to our knowledge) the benefit of combining intracerebral microdialysis for neuromediator determinations and quantitative EEG recording with PK-PD modeling to assess causality relationships between these various parameters.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Equipe Médicament Anti-infectieux et Barrière Hémato Encéphalique, PBS, Faculté de Médecine & Pharmacie, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex, France. Phone: 33 5 49 45 43 79. Fax: 33 5 49 45 43 78. E-mail: william.couet{at}univ-poitiers.fr. Back

{dagger} This article is dedicated to the memory of our colleague Serge Bouquet, deceased on 14 January 2003. Back


arrow
REFERENCES
 
    1
  1. Akahane, K., M. Sekiguchi, T. Une, and Y. Osada. 1989. Structure-epileptogenicity relationship of quinolones with special reference to their interaction with {gamma}-aminobutyric acid receptor sites. Antimicrob. Agents Chemother. 38:2323-2329.
    2. 2
  2. Chenel, M., A. Barbot, A. Dupuis, O. Mimoz, J. Paquereau, S. Bouquet, and W. Couet. 2003. Pharmacokinetic-pharmacodynamic modeling of the electroencephalogram effect of norfloxacin in rats. Antimicrob. Agents Chemother. 47:1952-1957.[Abstract/Free Full Text]
    3. 3
  3. Christ, W. 1990. Central nervous system toxicity of quinolones: human and animal findings. J. Antimicrob. Chemother. 26(Suppl. B):219-225.
    4. 4
  4. Delon, A., F. Huguet, P. Courtois, J. M. Vierfond, S. Bouquet, and W. Couet. 1997. Pharmacokinetic-pharmacodynamic contributions to the convulsant activity of pefloxacin and norfloxacin in rats. J. Pharmacol. Exp. Ther. 280:983-987.[Abstract/Free Full Text]
    5. 5
  5. Dupuis, A., W. Couet, J. Paquereau, S. Debarre, A. Portron, C. Jamois, and S. Bouquet. 2001. Pharmacokinetic-pharmacodynamic modeling of the electroencephalogram effect of imipenem in healthy rats. Antimicrob. Agents Chemother. 45:1682-1687.[Abstract/Free Full Text]
    6. 6
  6. Moghaddam, B., and B. S. Bunney. 1989. Ionic composition of microdialysis perfusing solution alters the pharmacological responsiveness and basolateral outflow of striatal dopamine. J. Neurochem. 53:652-654.[CrossRef][Medline]
    7. 7
  7. Paxinos, G., and C. Watson. 1986. The rat brain in stereotaxic coordinates, 2nd ed. Academic Press, Inc., San Diego, Calif.
    8. 8
  8. Rabouan, S., J. C. Olivier, H. Guillemin, and D. Barthes. Validation of HPLC analysis of aspartate and glutamate neurotransmitters after o-phthaldialdehyde-mercaptoethanol derivatization. J. Liq. Chromatogr. Relat. Technol., in press.
    9. 9
  9. Rowley, H. L., K. F. Martin, and C. A. Marsden. 1995. Decreased GABA release following tonic-clonic seizures is associated with an increase in extracellular glutamate in rat hippocampus in vivo. Neuroscience 68:415-422.[CrossRef][Medline]
    10. 10
  10. Smolders, I., C. Gousseau, S. Marchand, W. Couet, G. Ebinger, and Y. Michotte. 2002. Convulsant and subconvulsant doses of norfloxacin in the presence and absence of biphenylacetic acid alter extracellular hippocampal glutamate but not gamma-aminobutyric acid levels in conscious rats. Antimicrob. Agents Chemother. 46:471-477.[Abstract/Free Full Text]
    11. 11
  11. Tsuji, A., H. Sato, Y. Kume, I. Tamai, and E. Okezaki. 1988. Inhibitory effects of quinolones antibacterial agents on {gamma}-aminobutyric acid binding to receptor sites in rat brain membranes. Antimicrob. Agents Chemother. 32:190-194.[Abstract/Free Full Text]
    12. 12
  12. Ueda, Y., H. Yokoyama, A. Nakajima, J. Tokumaru, T. Doi, and Y. Mitsuyama. 2002. Glutamate excess and free radical formation during and following kainic acid-induced status epilepticus. Exp. Brain Res. 147:219-226.[CrossRef][Medline]
    13. 13
  13. Wallace, K. L. 1997. Antibiotic-induced convulsions. Crit. Care Clin. 13:741-762.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, November 2003, p. 3660-3662, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3660-3662.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chenel, M.
Right arrow Articles by Couet, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chenel, M.
Right arrow Articles by Couet, W.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»