Address correspondence and reprint requests to Dr. M. Holtkamp at Department of Neurology, University Hospital Charité, Schumannstr. 20/21, 10117 Berlin, Germany. E-mail: firstname.lastname@example.org
Summary: Purpose: To evaluate the anticonvulsant properties of furosemide and to determine sedative side effects compared with pentobarbital and diuretic side effects compared with saline-treated controls in an experimental model of limbic status epilepticus.
Methods: Self-sustaining status epilepticus was induced in rats by continuous electrical stimulation of the perforant path. Five minutes after the end of the stimulation, animals were given 100 mg/kg furosemide, 30 mg/kg pentobarbital, or an equal amount of saline, intraperitoneally. After administration of the substance, animals were monitored clinically and electrographically for 3 h regarding status epilepticus, level of sedation, and diuresis.
Results: In seven of 10 animals, furosemide terminated status epilepticus after 68 ± 26 min, whereas pentobarbital was successful in all animals after 5 ± 0.8 min. In contrast to pentobarbital, sedation did not occur with furosemide. Weight loss after furosemide was 10.2 ± 1.7% compared with 6.5 ± 1.1% in animals given saline (p < 0.001).
Conclusions: The results suggest that furosemide may serve as an alternative or additional agent for refractory complex partial status epilepticus in patients in whom common anesthetics are not justifiable.
Despite the advent of novel substances, the management of status epilepticus (SE) still represents an important challenge to neurology. Drug treatment is hampered by a number of problems, including the fact that first-line anticonvulsants such as benzodiazepines (BZDs) and phenytoin (PHT) fail to terminate SE in 30–50% of cases (1,2). In addition, treatment escalation and application of general anesthesia may be associated with harmful side effects such as deep sedation and severe hypotension (3,4). Therefore, drugs are needed that possess strong anticonvulsant properties and have no or few sedative side effects.
In a clinical case–control study, it was recently demonstrated that furosemide and thiazides are protective against the occurrence of unprovoked epileptic seizures (5). Potent anticonvulsive properties of furosemide have been shown in experimental studies employing in vitro models of epileptiform activity (6,7). However, little is known about the efficiency of the substance in vivo and possible associated side effects such as sedation and fluid loss.
It has repeatedly been demonstrated by us and other groups that anticonvulsants used in clinical practice may well be evaluated in a model system of limbic self-sustaining status epilepticus (SSSE) in freely moving animals resembling complex partial SE (8–10). Unfortunately, in spite of powerful anticonvulsant properties of recently tested substances, pronounced sedative side effects were seen with animals lacking spontaneous activity and having lost the righting reflex (8).
Because of these and other side effects of common anesthetics such as pentobarbital (PTB), propofol (PRO), and midazolam (MDL) (4) in refractory SE, the use of these agents in ongoing complex partial SE has recently been discouraged (11,12). In an attempt to identify a substance with strong anticonvulsant effects and few sedative or other side effects, we evaluated the diuretic furosemide in a rat model of SSSE, comparing it with PTB and saline.
Male and female Wistar rats aged 10–12 weeks, weighing 300–400 g, were implanted with intracerebral electrodes under deep anesthesia with PTB [52 mg/kg intraperitoneally (i.p.); Synopharm, Barsbüttel, Germany] following stereotactic coordinates. In brief, a stimulation electrode was implanted in the right perforant path 6.9 mm posterior and 4.1 mm lateral of bregma; a recording electrode was placed in the granule cell layer of the ipsilateral dentate gyrus 3.1 mm posterior and 1.9 mm lateral of bregma. Potentials were amplified via a NeuroLog amplifier (Digitimer, Welwyn Garden City, U.K.) onto an oscilloscope and then via an analog-to-digital interface onto a computer by using WinTIDA (HEKA Electronic, Lambrecht, Germany; sampling rate, 1 kHz). The depth of electrodes was adjusted following the maximal population spike after single electrical test stimuli with 50- to 150-μs monopolar pulses and 3 to 5 mA. Rats were allowed to recover, and 8 to 10 days after the operation, the perforant path of the freely moving animals was stimulated electrically for 2 h at 20 Hz with 3 to 5 mA and 50- to 150-μs monopolar pulses. The presence of SSSE was defined electrographically if discharges occurred at a frequency of ≥1 Hz (13). Because this model resembles nonconvulsive complex partial SE in patients, those animals were excluded from the experiments that developed motor signs such as head nodding, forelimb clonus, or more severe forms of motor seizures as defined by Racine (14) during SSSE. These strict criteria were necessary to obtain as pure a sample of nonconvulsive SE as possible. Five minutes after the end of stimulation, animals were treated with i.p. furosemide. In preliminary experiments, furosemide in dosages of 10 and 20 mg/kg, i.p. (10 mg/ml; Ratiopharm, Ulm, Germany) did not have any influence on epileptic discharges in four animals of each group; in two of four animals, 50 mg/kg, i.p., resulted in a mild to moderate reduction of the amplitude of the discharges, but SSSE was not terminated (data not shown). This prompted us to treat one group of SSSE animals (n = 10) with 100 mg/kg furosemide, i.p. A second group of animals (n = 5) with SSSE was given an equal amount of saline, i.p. (10 ml/kg). In a third group, rats (n = 4) were treated with 30 mg/kg PTB, i.p. The animals were monitored clinically and electrographically for 3 h after the treatments. Termination of SE was defined electrographically as follows: the frequent regular spontaneous discharges had to cease without recurrence within the monitored period of 3 h. The animals were observed clinically concerning their level of sedation by using a well-established scale (score range, 0–5; 0, spontaneous movements; 5, loss of tail-pinch response) (15). Finally, we estimated the urinary fluid loss induced by application of furosemide by weighing each rat before stimulation and at the end of the experiment. The weight loss in the group of furosemide-treated animals as compared with rats given saline was assumed to represent the furosemide-induced diuresis. Data were tested for significance by using analysis of variance (ANOVA)/Fishers PLSD. The animal experiments were conducted in accordance with the German Animal Protection Act and were approved by the regional authority.
A total of 30 animals had been stimulated electrically. In 27 animals, ∼30–60 min after the onset of stimulation, first large-amplitude spontaneous discharges occurred, and all these animals developed SSSE. Eight rats were excluded as they exhibited motor signs. Thus 19 animals were included in this study. Five rats with limbic SSSE were injected with i.p. saline, and as expected, all displayed unchanged epileptic activity for the following 3 h (Fig. 1). In seven of 10 animals, limbic SE was terminated by furosemide (Fig. 1) and did not reoccur in the period monitored. In three other animals at variance to controls, amplitudes of the spontaneous discharges as monitored by the intracerebral EEG showed fluctuations and decreased intermittently over the 3-h observation period. However, seizure activity was not terminated by the substance. SSSE was terminated in all four animals given PTB. Although termination of SSSE was achieved with either substance, the time course of action was markedly different in each. After injection of furosemide, we saw the following pattern: within ∼20–50 min, there was a significant reduction of 20–30% in amplitude of the spontaneous discharges. The amplitude reduction progressed, and seizure activity was further suppressed until termination was achieved. In each individual animal, the time until SSSE was terminated was roughly twice the time that took to achieve the described 20–30% reduction in amplitude of the spontaneous discharges (Fig. 2). Lasting suppression of SSSE after furosemide was seen after 68 ± 26 min (range, 30–100 min). In contrast, PTB terminated SSSE more rapidly within 5 ± 0.8 min after application. All rats given furosemide or saline did not show any signs of sedation (score, 0), whereas all animals treated with PTB did not respond to auditory stimuli (score, 3).
At the end of the experiments, all controls and furosemide-treated rats were weighed. Furosemide-treated animals lost 35.2 ± 6.8 g, and controls lost 23.2 ± 6.2 g. We found significant differences in relative weight loss in the furosemide-treated animals compared with controls, respectively (10.2 ± 1.7% vs. 6.5 ± 1.1%; p < 0.001). We did not find any influence of the animals' sex on response rate, time to cessation of seizure activity, and weight loss.
Nonconvulsive complex partial SE continues to pose therapeutic uncertainties. A major question is how to balance possible severe side effects resulting from aggressive treatment approaches such as anesthesia against deleterious consequences of the condition itself (11,12). Therefore, substances with strong antiepileptic properties and few or minimal side effects are needed. The current experiments were carried out in a model system of limbic nonconvulsive SE. We found a substantial anticonvulsive effect of the diuretic furosemide. Interestingly, other diuretics too have been shown to exhibit strong anticonvulsant actions. The carboanhydrase inhibitor acetazolamide (ACZ) is well known to be effective in focal and generalized epilepsies. Indeed, the substance has been suggested as an additional treatment option in nonconvulsive status epilepticus (16).
The current experiments demonstrate potent anticonvulsant properties of furosemide in freely moving rats, with termination of SE in seven of 10 cases. Furosemide has been evaluated before in two studies using a chemoconvulsant in vivo model of SE induced by kainic acid, in which similar anticonvulsive effects were seen (6,17). In these two studies, furosemide was administered intravenously, and blockade of seizure activity was achieved at dosages of 10 mg/kg (17) and 40–60 mg/kg (6). In the current study, a higher dosage of 100 mg/kg was required, which is most likely because of the different pharmacokinetics of the intraperitoneal route of administration that we used. This also may explain the observed variability, in that 100 mg/kg furosemide did not terminate SSSE in all animals. However, those experiments were performed under urethane anesthesia, which itself may provide some anticonvulsant properties (18), and thus confound the effects of furosemide. In addition, such anesthesia precludes proper clinical evaluation of the animals, in particular, regarding their level of sedation (6,17). In the current study, all animals remained awake after furosemide injection. The lack of sedative side effects is a striking advantage of furosemide as compared with all other anticonvulsant agents currently used in refractory SE. Because of harmful side effects, the use of anesthetics in complex partial SE is controversially discussed (11,12), and the results of the current study represent an experimental basis for a possible future clinical use of furosemide in SE. Thus furosemide may serve as an option after failure of first-line substances. Alternatively, the substance may serve as a supplementary drug given together with first-line agents to reduce the rate of refractory cases in SE. Our results may be regarded as incentives for additional experimental combination studies that should be done to determine if this substance may serve as an add-on anticonvulsant in the initial treatment of SE. We have shown that 5 mg/kg diazepam (DZP) given 5 min after the end of stimulation terminates SSSE in 40% of cases (8). Future studies should investigate whether additional furosemide augments the DZP success rate.
Furosemide in this study successfully terminated SSSE in animals of either sex. Although there is some evidence that female rats need lower dosages of the anticonvulsant anesthetic PTB for sedation (19), influence of sex on the anticonvulsant effect of furosemide remains to be determined.
Previous in vitro studies have demonstrated that the substance before inhibition causes pronounced hyperexcitability, seen with spontaneous bursting (6) and stimulation-induced potentials (7). This effect has not been described in previously performed in vivo experiments with kainate-induced seizure activity (6) and was not seen in the current study. In contrast to PTB, the anticonvulsant effects of furosemide unfold gradually (Fig. 2), in keeping with our previous in vitro studies (7). This retarded onset of action is unlikely to be explained by lipid solubility, as furosemide is more lipophilic than PTB (20). However, the marked difference in plasma protein binding (∼5% PTB vs. >90% furosemide) may serve as an explanation for such different latencies. Such retarded effect may well be tolerated in patients in view of the fact that an aggressive treatment of nonconvulsive status has recently been challenged (11). It is important to note that it is still unclear whether the harmful consequences of general anesthesia outweigh those of the condition itself (21).
In a further step, we estimated furosemide-induced urinary fluid loss. Rats that were treated with furosemide on average lost 10.2% of their body weight compared with the 6.5% in rats that received saline. To our knowledge, weight loss in the model system used in this study has not been reported previously. We assume that it reflects a combination of perspiratioinsensibilis and salivation in the absence of compensational spontaneous water intake. The significant difference in weight loss of 3.7% between the two groups most likely indicates furosemide-induced diuresis.
The anticonvulsive mechanisms of action of furosemide are still elusive. Fluid loss with consequent changes in extracellular volume and cerebrospinal fluid (CSF) osmolality has been suggested to contribute to the anticonvulsant action (6). However, because both furosemide and mannitol increase CSF osmolality, whereas only furosemide exhibits anticonvulsant properties, this assumption is unlikely (17). At variance to ACZ, furosemide does not appear to act through an acidification of the extracellular fluid (22). The anticonvulsant properties are more likely related to direct CNS effects such as reduction in neuronal excitability and consequent reduction of activity-induced potassium increases (7).
In summary, we demonstrated that furosemide exhibits strong anticonvulsive properties in a model of limbic SE. Application of the substance is not accompanied by sedation but induced significant diuresis. Because under physiologic conditions and more so during SE, furosemide crosses the blood–brain barrier (23,24), the results of the current study may be regarded as an experimental basis for incorporating furosemide into the clinical management of refractory complex partial SE when anesthetics are not justifiable.
Acknowledgment: We thank Uwe Heinemann for helpful comments. This study was supported by the DFG grant Bu 1331-1 (K.B. und H.M.).