Neurol Sci (2014) 35:1903–1908 DOI 10.1007/s10072-014-1858-6
ORIGINAL ARTICLE
Electroencephalographic and behavioral effects of intracerebroventricular or intraperitoneal injections of toxic honey extract in adult Wistar rats and GAERS Pinar Kuru • Merve Torun • Hande Melike Halac • Gozde Temiz • Ece Iskender • Tugba Karamahmutoglu Medine Gulcebi Idrizoglu • Filiz Yilmaz Onat
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Received: 3 March 2014 / Accepted: 17 June 2014 / Published online: 14 August 2014 Ó Springer-Verlag Italia 2014
Abstract Toxic honey, containing grayanotoxin, is obtained from nectar and polen of rhododendron. Consumed in excess it produces seizures and convulsions. In order to investigate whether the toxic honey extract can be used as a seizure model, we examined the electroencephalographic (EEG) and motor effects of intracerebroventricular (icv) or intraperitoneal (ip) injection of toxic honey extract in Wistar rats or in genetic absence epilepsy rats from Strasbourg (GAERS). Male Wistar rats or GAERS were stereotaxically implanted with bilateral cortical recording electrodes in all ip groups and cannula in the icv groups. Based on the previous study, an extract was obtained from the non-toxic and toxic honey. After the injection of the non-toxic or toxic honey extract, seizure stages and changes in EEG were evaluated from 9 am to noon. The icv administration of toxic honey extract produced stage 4 seizures and bilateral cortical spikes within 30–60 min and these effects disappeared after 120 min in Wistar rats or GAERS. The mean of bilateral cortical spike acitivity in EEG of Wistar rats was 804.2 ± 261.0 s in the 3-h period. After the icv administration of toxic honey extract to GAERS, the mean duration of spike-and-wave discharges (SWDs) in GAERS significantly decreased during the first 60 min and then returned to baseline level. Ip injection of toxic honey extract caused no seizure and no change in EEG in either GAERS or Wistars. These results
P. Kuru M. Torun H. M. Halac G. Temiz School of Medicine, Marmara University, Istanbul, Turkey E. Iskender T. Karamahmutoglu M. G. Idrizoglu F. Y. Onat (&) School of Medicine, Department of Pharmacology, Marmara University, Istanbul, Turkey e-mail:
[email protected];
[email protected]
suggest that the icv administration of toxic honey extract can be used as a seizure model. Keywords Grayanotoxin Absence epilepsy Absence seizure Mad honey
Introduction Excessive consumption of a particular type of honey obtained from nectar and pollen of rhododendron and rhododendron-like plants containing grayanotoxin (GTX) isoforms causes acute intoxication [1]. It induces hypersalivation, vomiting, hypotension, bradycardia, atrioventricular block, paresthesia, seizures, and convulsions in human [2, 3]. Most rhododendron species, over 900 are found in Southwest Asia ranging from the Himalayas to Tibet, China, the Philippines and Turkey [1, 4] and acute intoxication due to its excessive consumption is known as mad honey poisoning [1, 5]. It has been reported that GTX binds to voltage-dependent Na? channels resulting in a prolonged depolarization of the cell membrane [4]. Since the discovery of phenytoin in the late 1930s, the development of animal models for convulsive and nonconvulsive seizures has become a major focus of epilepsy research. In this regard, neurotoxins including tetanus toxin or scorpion toxin have been used for induction of seizures and ictogenesis [6, 7]. As GTX can be considered to be a neurotoxin, the observation of patients having seizures [8, 9] after the ingestion of the honey extracts containing GTX raised a question whether this toxic honey extract can be used as neurotoxin to develop a seizure model in rat. Therefore, we explored this question in the present study. We first investigated the changes in EEG activities and accompanying motor movements when the extracts
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Fig. 1 Experimental protocol of the honey extraction and the icv or ip administration
containing GTX were administered icv or ip to adult Wistar rats. When we observed that the icv injection of the toxic honey extract to Wistar rats caused convulsive seizure stages in accordance with the Racine scale [10], we decided to examine the effect of this extract in a genetic rat model with absence seizures in the second group of experiments. Therefore, in order to examine the effect of icv or ip administration of toxic honey extract on absence seizures, the toxic honey extract was administered icv or ip to genetic absence epilepsy rats from Strasbourg (GAERS), which are a well-validated genetic model of typical absence epilepsy and show spontaneously occurring spikeand-wave discharges (SWDs) in their EEG [11, 12].
Materials and methods Animals Adult male Wistar albino rats and GAERS (4–5 months old, 250–300 g, total number = 24) were obtained from the Marmara University Experimental Animal Research Center (Ethics approval; 09.12.2010- 90.2010.mar). The animals were housed in a temperature-controlled room (20 ± 3 8C) with a 12-h light/dark cycle with unlimited access to food and water. Preparation of the extract As reported in our previous study, 50 ml of methanol– water (1:3, v/v) solution was added to 25 g of honey. The solution was mixed until it became homogenized and the pH was adjusted to 6.5 with sodium hydroxide solution. The solution was filtered with Whatman filter paper (No: 46). Chloroform (75 ml) was added to the solution and mixed. Then, time was allowed for the phase separation in
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a separation funnel. This solution was extracted three times with same amount of chloroform. The residual organic phases were collected into another glass container. This final solution was evaporated in a 50 8C water bath and was dissolved in 2.5 ml of distilled water (Fig. 1) [5]. In our previous study, microscopic examination of the honey showed the presence of rhododendron pollens. Verification of contents of the extract In order to test and verify the effect of the toxic honey extract on blood pressure as previously reported by our group, an iliac arterial catheter was inserted to the right iliac artery of a Wistar rat under light ether anesthesia [5]. The catheter was routed subcutaneously to exit at the back of the neck and fixed to the skin. Then the animal was placed in a plexiglass cage. After a 2-h recovery period in freely moving rats, the toxic honey extract was applied (5 g/ml, 0.1 ml/100 g, ip). The toxic honey extract lowered blood pressure and caused hypotension reaching its maximum after 60 min as previously reported [5]. The heart rate dropped from 320 to 180 and the blood pressure fell from 135/85 mmHg to 90/45 mmHg. Experimental protocol The Wistar rats in the first group of experiments and GAERS in the second group were randomly grouped as icv (n = 6) or ip (n = 6) with each group first receiving nontoxic (flower) honey and then after an interval of 2 days receiving toxic honey containing GTX extract (Fig. 1). Seizures were evaluated according to Racine’s scale as follows: stage 1, facial twitches; stage 2, chewing and head nodding; stage 3, unilateral forelimb clonus; stage 4, bilateral forelimb clonus, body jerks and rearing; stage 5, imbalance [10].
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Stereotaxic surgery The animals were anesthetized with ketamine (100 mg/kg, ip) and xylazine (10 mg/kg, ip). In the icv groups, a stainless-steel guide icv cannula (C313; Plastics One) was stereotaxically (Stoelting Model 51600, Illinois, USA) placed to 1 mm above the right lateral ventricle to prevent any mechanical damage. Icv coordinates (0.8 mm posterior, and -1.5 mm lateral from the bregma, and -3.5 mm ventral from the surface of the skull) were calculated according to the rat brain atlas of Paxinos and Watson [13]. In both the ip and icv groups, four stainless-steel screws with soldered insulated wires were bilaterally implanted to the frontal and parietal cortex to record the cortical EEG. They were fixed to the skull with dental acrylic. EEG recording The EEG activity of the cortex was amplified (through BioAmp ML 136) and recorded with a PowerLab 8S System running Chart v.5 (ADI Instruments, UK) continuously for 30 min before and 180 min after the injection between 9:00 and 12:30 am. The EEG signals were measured using LabChart Pro v7.1 (PowerLab soft ware, AD Instruments) and filtered between 1 and 100 Hz, digitized at 200 samples per second and stored on a hard disk for offline analyses. The number and total duration of generalized bilateral cortical spike activities were evaluated in the Wistar and GAERS groups. An amplitude at least twofold higher than the basal EEG was accepted as the criteria to measure the duration of cortical spike activity. Additionally the mean duration of cumulative SWDs was analyzed in the GAERS group. Icv injection An internal cannula (C313; Plastics One) with a polyethylene tube (PE 50) extension was put into the guide cannula and fixed by a captive collar of the external tube to allow the icv injections to be made in freely moving rats. The polyethylene tube extension was connected to a Hamilton syringe. The infusion cannula assembly was removed 5 min after the completion of the injection to prevent damage of the tubes. Microinjection of 5 ll of nontoxic (standard flower honey as a control) and toxic honey into the lateral ventricle was made slowly during 1 min. Histological verification for the icv placement The rats injected properly analysis.
were anesthetized and 5 ll methylene blue was for the verification. Only the rats that had a implanted cannula were included in the data After the verification of the icv placement, the
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number of animals in the icv groups were Wistar (n = 3) and GAERS (n = 5). Statistical analysis The results are given as mean ± standard error of the mean (SEM). Statistical comparisons were performed using GraphPad Prism. Data analysis was performed using ANOVA and Tukey post hoc tests for comparisons. Statistical significance was set at p \ 0.05.
Results The first group of experiments: Wistar rats The icv or ip administration of non-toxic honey extract did not cause any seizure or any changes in EEG of the Wistar rats. However, the icv administration of the toxic honey extract in Wistar rats induced convulsive seizure stages and bilateral cortical spike activity in the EEG during the 30–60 min after the injection (Fig. 2a–c). The icv injection of the toxic honey extract produced a maximum of stage 4 (time to reach the first stage 4 seizure, 13.8 ± 3.6 min). After the icv injection of the toxic honey extract, the latency to the first bilateral cortical spikes in the EEG, accompanied by stage 1, was 5.3 ± 0.9 min in Wistar rats. The mean of bilateral cortical spike acitivity in EEG of Wistar rats was 804.2 ± 261.0 s in the 3-h period (minimum seizure duration, 9.2 s; mean duration, amplitude and frequency of cortical spike activity were 77.4 ± 7.6 s, 270 ± 0.2 microV and 5.9 ± 0.3 Hz, respectively). These effects were most frequently seen at 30–60 min and disappeared by 120 min after the administration. The ip injection of the toxic honey extract did not cause any change in EEG, but local muscle twitchings of the extremities were observed. The second group of experiments: GAERS The icv or ip administration of non-toxic honey extract did not cause any seizure activity and produced no changes in EEG in GAERS. However, the icv administration of the toxic honey extract in GAERS caused convulsive seizure stages in accordance with Racine’s scale. The latency to the first seizure stage was 3.9 ± 1.6 min after the administration. Seizure stages reached a maximum of stage 4 in the 30–60 min period (time to reach the first stage 4, 16.9 ± 7.3 min) and disappeared by 90 min after the administration (Fig. 3a). Before the toxic honey extract administration, the mean duration of baseline SWDs was 713.0 ± 76.6 s per 30 min of EEG recording in GAERS (Fig. 3b). After the icv administration of extract, the mean
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Fig. 2 Seizure stages (a), mean duration of the bilateral cortical spikes (b), and mean of the number of bilateral cortical spikes (c) of Wistar rats after the icv administration of the toxic honey extract and representative EEG activity before and after the icv administration
(d). Data are expressed as mean ±SEM. A one-way ANOVA followed by the post hoc Tukey test revealed significant differences between groups, *p \ 0.05
duration of SWDs significantly decreased during the 60-min period (p \ 0.05) and thereafter returned to baseline level during the 60–90 min period while convulsive seizures disappeared in GAERS. The ip injection of the toxic honey extract to GAERS did not cause any change in behaviour and EEG activity.
the present study show that this toxic honey extract, administered by the icv route, can be a seizure model. New seizure models can contribute towards an understanding of the mechanism of seizures, epilepsy and clinically related important issues as reported in case reports in patients experiencing epileptic seizures after ingestion of toxic honey [8, 9]. In urethane anesthetized rats, a dose-dependent decrease in the penicilin-induced epileptiform activity was observed by the injection of GTX III [14]. The icv injection of GTX III (2 lg/2 ll) resulted in a significant decrease in the mean spike frequency and amplitude of the epileptiform activity. The results seem to be in contrast to our findings. This difference could possibly be methodological. First, the toxic honey extract was given to the freely moving rats in our study, whereas urethane anesthetized rats were used in the study of Gunduz and co-workers. Next, while the toxic honey extract was given in our study, GTX was used in the other study. In the second part of the present study, GAERS received the toxic honey extract by icv or ip route. Only icv administration produced convulsive seizures ranging from
Discussion We aimed to translate the development of seizure by the administration of the toxic honey (known as mad honey in Turkey) into a basic science platform. The present study shows that whereas the ip administration causes no effect in animals, the icv injection of the toxic honey extract produces seizures in Wistar rats and GAERS. Bilateral cortical spikes in the EEG and seizure stages, reaching a maximum of stage 4, were observed in the 30–60 min period and disappeared by 120 min after the icv administration of the toxic honey extract. Similar to some toxins such as tetanus or scorpion toxin that have been used for induction of seizures and ictogenesis [6, 7], the results of
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Fig. 3 Seizure stages of GAERS (a) and the mean duration of SWDs of GAERS after the icv administration of the toxic honey extract (b). Representative EEG activity before and 49 min after the icv
administration (c). Data are expressed as mean ± SEM. A one-way ANOVA followed by the post hoc Tukey test revealed significant differences between groups, *p \ 0.05
stage 1 to stage 4 and an effect on SWDs. The icv administration of the toxic honey extract suppressed the duration of SWDs in GAERS. The disappearence of convulsive seizure stages occurred simultaneously with the return of SWDs to the baseline levels in GAERS. Several studies carried out by our group showed a convulsive state induced by kindling or intra-amygdaloid kainic acid injection supresses SWDs in GAERS [15–17]. It seems that the convulsive state and absence epilepsy interact with each other. However, a mechanism underlying this interaction and its site of action remains to be clarified. Previously, our group [5] reported that the icv or ip administration of toxic honey extract causes bradicardia and respiratory depression. In the present study, these effects were also obvious as clinically reported [1]. Indeed, there are some limitations in the present study. There is no laboratory in our university for the analysis GTX content and the subtype of the toxic honey. Additionally, the different doses of the toxic honey extract need to be administered in order to determine a precise seizure model. In conclusion, the icv administration of the toxic honey extract causes generalized seizures characterized by bilateral cortical spikes in EEG and accompanying convulsive seizure stages in adult Wistar rats and GAERS. These results suggest that the icv administration of the toxic honey extract can serve as a new seizure model. Additionally, a decrease in the duration of SWDs in EEG in the
GAERS group after the icv injection of the toxic honey extract can contribute towards an understanding of the mechanism of the interaction between the convulsive states and absence seizures. Acknowledgments This study was supported by the Marmara University Scientific Research Committee.
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