Central effect of crocin on penicillin-inducedepileptiform activity in rats

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Central effect of crocin on penicillin-induced epileptiform activity in rats

Esmaeal Tamaddonfard, Nasrin Hamzeh Gooshchi, Sona Seiednejad-Yamchi

Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia 57153-1177, Iran

Correspondence: Esmaeal Tamaddonfard, e-mail: e_tamaddonfard@yahoo.com and e.tamaddonfard@urmia.ac.ir

Abstract:

In the present study, the effects of separate and combined intracerebroventricular (icv) injections of crocin and diazepam were inves- tigated on penicillin-induced epileptiform activity. In urethane-anesthetized rats, epileptiform activity was induced by intracortical (ic) administration of penicillin (200 IU, 1 µl) and was analyzed using electrocorticographic (ECoG) recordings. The icv injections of crocin at doses of 25, 50 and 100 µg and diazepam at a dose of 10 µg increased the latency time to onset of first spike wave, and decreased the frequency and amplitude of spike waves. Co-administration of an effective dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg), increased the latency time to onset of first spike wave and decreased frequency and amplitude of spike waves as compared with crocin (50 µg). These results indicated that crocin and diazepam produced antiepileptic activities at the levels of the brain. Crocin potentiated the antiepileptic effect of diazepam. A GABA_A-benzodiazepine receptor-mediated mechanism may be involved in the antiepileptic activity of crocin.

Key words:

crocin, diazepam, penicillin-induced epileptiform activity, rats

Abbreviations: ic – intracortical, icv – intracerebroventricular, PE – penicillin-induced epilepsy, PTZ – pentylenetetrazole

Introduction

Epilepsy is a complex neurological disorder characterized by recurrent seizures of cerebral origin, pre- senting with episodes of sensory, motor and auto- nomic disturbances with or without loss of conscious- ness [31]. Epileptic seizures result from excessive discharge in a population of hyperexcitable neurons in cortical and hippocampal structures [4, 34]. Conven-

tional treatment of epilepsy consists primarily of anticonvulsant medications [10]. Although these drugs often control or reduce the frequency of seizures, but possess many side effects, and some patients show lit- tle or no improvement [15, 20]. Hence, there is a need to address a potent alternative as antiepileptic agent with minimal side effects.

Crocin is one of the active substances of gardenia yellow and saffron, the extracts of Gardenia jasminoi- des fruits and Crocus sativus stigmas, respectively [3, 22]. The involvement of crocin in some biological phenomena such as learning and memory, anxiety and pain have been reported [1, 28, 32, 33]. Saffron extracts and safranal (an active constituent of saffron)

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exhibited anticonvulsant effects in both pentylenetetrazole (PTZ) and maximal electroshock (MES) models of seizure in mice [18, 19].

Penicillin-induced epileptiform (PE) activity has been established as a model of epilepsy in rats for studying the effects of antiepileptic drugs [6, 11, 13, 16]. The present study was designed to investigate the effects of icv injection of crocin on penicillin-induced epilepsy. In addition, to identify the mechanism that possibly mediates the effect of crocin on epilepsy, the contribution of GABA_A-benzodiazepine receptor sys- tem was assessed using icv injection of diazepam (a GABA_A-benzodiazepine receptor agonist) with and without crocin.

Materials and Methods

Animals

Healthy adult male Wistar rats, weighing 220–250 g were used in this study. Rats were maintained in poly- ethylene cages with food and water available ad libi- tum in a laboratory with controlled ambient tempera- ture (22 ± 0.5°C) and under a 12 h light-dark cycle (lights on 7:00 a.m.). Experiments were carried out between 11:00 a.m. and 5:00 p.m. Six rats were used in each experiment. The experimental protocol was approved by the Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University and was performed in accordance with the National Institutes of Health Guide for Care and Use of Labo- ratory Animals.

Drugs

Drugs used in the present study included urethane, crocin, diazepam and penicillin G potassium. The drugs were purchased from Sigma-Aldrich Co., St Louis, MO, USA. The drugs were dissolved in normal saline. A drop of Tween 80 was added to diazepam plus normal saline solution.

Treatment groups

The rats were divided into 8 groups with 6 rats in each group. Group A received icv normal saline prior to ic injection of penicillin. In groups B, C, D and E icv in- jections of crocin at doses of 12.5, 25, 50 and 100 µg

were performed before ic injection of penicillin, re- spectively. Groups F and G treated with icv injection of 2.5 and 10 µg of diazepam prior to ic injection of peni- cillin. Group H received icv co-administartion of an ef- fective dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg) before ic injection of penicillin.

In all groups, the icv injection of drug solutions was performed 10 min before ic injection of penicillin.

Study protocol

The animals were anesthetized with intraperitoneal (ip) injection of urethane (1.2 g/kg), and placed in a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA). Rectal temperature was measured by a digital thermometer and was maintained between 36 and 37°C using a controlled heating pad system. Thereaf- ter, the scalp was incised, and the skull was leveled off around the bregma.

For icv injections of normal saline, crocin and di- azepam, a hole with 0.8 mm in diameter was drilled in the left parietal bone according to the following coordinates: 0.8 mm posterior to the bergma and 2 mm lateral to the midline [27]. The tip of the needle of a 5 µl Hamilton’s syringe was introduced through the hole into the brain and was placed at 4 mm below the surface of the skull in the left lateral ventricle of the brain. The volume of solutions to be injected into lateral ventricle was 1 µl and injection was made over a period of 30 s. The injection needle was left in place for a further 30 s after completion of injection to fa- cilitate diffusion of the drug. After injection, the hole was closed using acrylic cement (Acropars, Tehran, Iran). In the present study, we used icv injection route of administration of crocin and diazepam, because it may achieve a greater drug concentration at the epi- leptogenic area.

The epileptic focus was produced by ic injection of penicillin. For this purpose, an additional hole with 0.8 mm in diameter was made in the right parietal bone overlying the right sensory-motor cortex (2 mm posterior to the bregma and 3 mm lateral to the midline) [27]. Penicillin G potassium (200 IU, 1 µl) was injected 1.2 mm beneath the surface of the skull using a 5 µl Hamilton’s syringe in a period of 90 s. [5–7, 12].

For ECoG recordings, two 5-mm height pin electrodes (0.5 mm in diameter) were inserted into the right frontal and parietal bones according to the following coordinates: first electrode, 1 mm anterior to the bregma and 2 mm lateral to the midline (frontal

Crocin and penicillin epilepsy

Esmaeal Tamaddonfard et al.

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on the left pinna. The electrodes were connected to a 4-channel physiograph (Physiograph 4-channels, MK-III-P, NARCO Boi-systems, USA) via a univer- sal coupler (Universal coupler, type 7189, NARCO Boi-systems, USA) for ECoG activity recordings. The ECoG recordings were performed at 20^th, 10^thmin before and at 10^th, 20^th, 30^th, 40^th, 50^thand 60^thmin after ic injection of penicillin. In each of above mentioned times, ECoG activity was recorded for a period of 1 min with two speeds (0.5 and 2 mm/s). To obtain the latency time to onset of first spike wave, the recording of ECoG activity was continuously performed for a period of 5 min after ic injection of penicillin. The latency time to onset of first spike wave, the frequency and amplitude of spike waves were calculated from the recorded ECoGs.

Injection sites verification

At the end of experiments, the rats were icv and ic in- jected with 10 and 1 µl methylene blue, respectively, and they were then deeply anesthetized with intracar- diac high dose injection of thiopental sodium (Bio- chemie GmbH, Vienna, Austria) and decapitated. The brains were removed and placed in a formaldehyde (10%) solution. After 24 h, the brains were sliced into 1 mm slices and the distribution of the dye in the injection sites were controlled under a loup.

Statistical analyses

Data obtained from latency time to onset of first spike wave were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s test. Data obtained from frequency and amplitude of spike waves were analyzed by factorial analysis of variance (ANOVA) and Duncan’s test All the values are expressed as the mean ± SEM. Statistical significance was set at p < 0.05.

Results

Figure 1 shows the ECoG recordings obtained from the present study. Baseline activities of each animal were recorded before the administration of sub-

tivity characterized by spike waves with high frequency and high amplitude (Fig. 1A). Epileptiform activity began 3–5 min after penicillin application and continued with constant levels of frequency and ampli- tude to the end of experiment (Fig. 1A). The icv injec- tions of crocin at doses of 25, 50 and 100 µg, diazepam at a dose of 10 µg and crocin (50 µg) plus diazepam (2.5 µg) decreased the frequency and amplitude of spike waves (Fig. 1C, D, E, G and H). Crocin at a dose of 12.5 µg and diazepam at a dose of 2.5 µg had no effects (Fig. 1B, F). The latency time to the onset of the first spike was obtained 146.7 ± 17.9 s after ic injection of penicillin. The icv injection of crocin at doses of 50 and 100 µg, but not at doses of 12.5 and 25 µg, significantly increased the latency time to onset of first spike induced by penicillin [F (4, 25) = 23.449, p < 0.05] (Fig. 2). The icv injection of diaze- pam at a dose of 10 µg, but not at a dose of 2.5 µg, significantly increased the latency time to onset of first spike [F (2, 15) = 40.424, p < 0.05] (Fig. 2). The icv co-administration of an effective dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg) significantly increased the latency time to onset of first spike as compared with normal saline and diazepam (2.5 µg) and crocin (50 µg) [F (3, 20) = 22.240, p < 0.05] (Fig. 2).

The frequency of spike waves was 42.3 ± 5.6 at 10^th min after ic application of penicillin and with a constant level reached to 41.3 ± 7 at the end of experiment (60^thmin) (Fig. 3A, B and C). The icv injections of crocin at doses of 25, 50 and 100 µg decreased 10^th, 10^th– 30^th, and 10^th– 60^thmin of the frequency of spike waves, respectively [F (4, 150) = 21.739, p < 0.05] (Fig. 1B, C, D, E and Fig. 3A). The icv injection of diazepam at a dose of 2.5 µg was without effect, whereas at a dose of 10 µg, diazepam significantly reduced 10^th– 60^thmin of the frequency of spike waves [F (2, 90) = 37.840, p < 0.05] (Fig. 1F, G and Fig. 3B). The icv co-administration of an effec- tive dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg) significantly decreased 10^th– 40^thmin of the frequency of spike waves as compared with normal saline, diazepam (2.5 µg) and crocin (50 µg) [F (3, 20) = 22.240, p < 0.05] (Fig. 1H and Fig. 3C).

The amplitude of spike waves was 1542 ± 105 µV at 10^thmin after ic application of penicillin and with a constant level reached to 1,400 ± 121 µV at the end

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Fig. 1. Electrocorticogram (ECoG) re- cording samples obtained from right sensory-motor cortex of anesthetized rats treated with crocin and diazepam before penicillin. a) Showing the baseline activity recoded at 20^thmin before penicillin. (A) Showing the first spike and 30^thmin of spike waves induced by penicillin. (C, D, E, G, H) Showing the suppressive effects of crocin at doses of 25, 50 and 100 µg, diazepam at a dose of 10 µg and crocin (50 µg) plus diazepam (2.5 µg) on 10^th, 30^th and 60^th min of epileptiform activity induced by penicillin, respectively.

(B, F) Showing no significant effects of crocin (12.5 µg) and diazepam (2.5 µg) on penicillin-induced epilepsy. Normal saline, crocin, diazepam and crocin plus diazepam were intracerebroven- tricularly (icv) injected 10 min before intracortical (ic) injection of penicillin.

Icvinjections of normal saline, crocin and diazepam did not change the baseline activity (data not shown).

ECoG was recorded with two speeds (0.5 and 2 mm/s) under calibration of 100 µV/1 mm (i.e., 500 µV/5 mm)

Fig. 2. The effects of separate and combined intracerebroventricular (icv) injections of crocin and diazepam on the latency time to onset of first spike wave induced by intracortical (ic) in- jection of penicillin in rats. Normal saline, crocin, diazepam and crocin plus diazepam were injected 10 min before injection of penicillin. Values are expressed as the mean ± SEM (n = 6);

* p < 0.05 compared with normal saline,

p < 0.05 compared with diazepam (2.5 µg) and crocin (50 µg)

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of experiment (60^thmin) (Fig. 1A and Fig. 4A, B, C).

The icv injection of crocin at doses of 25, 50 and 100 µg, but not at a dose of 12.5 µg, significantly decreased 10^th, 10^th–30^th, and 10^th–40^thmin of the amplitude of spike waves, respectively [F (4, 150) = 8.373, p < 0.05] (Fig. 1B, C, D, E and Fig. 4A). The icv in- jection of diazepam at a dose of 2.5 µg was without

effect, whereas at a dose of 10 µg, diazepam significantly reduced 10^th – 50^th min of the amplitude of spike waves [F (2, 90) = 26.923, p < 0.05] (Fig. 1F, G and Fig. 4B). The icv co-administration of an effec- tive dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg) significantly decreased the 20^thand 30^thmin of the amplitude of spike waves as compared

penicillin in rats. Normal saline, crocin, diazepam and crocin plus diazepam were injected 10 min before injection of penicillin. Values are expressed as the mean ± SEM (n = 6); * p < 0.05 compared with normal saline,p < 0.05 compared with diazepam (2.5 µg) and crocin (50 µg)

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with normal saline, diazepam (2.5 µg) and crocin (50 µg) [F (3, 20) = 10.286, p < 0.05] (Fig. 1H and Fig. 4C).

Discussion

In the present study, ic injection of penicillin (200 IU, 1 µl) produced an epileptiform ECoG activity charac-

terized by high frequency and amplitude spike waves.

In addition, appearance of first spike started at 146.7

± 17.9 s after penicillin injection. Our results are approximately consistent with other findings in which the doses of ic injected penicillin were 200–500 IU [5–7, 12]. Clinical experience has indicated that high systemic doses of penicillin in humans can produce myoclonus, generalized tonic-clonic seizures and en- cephalopathy [14]. In addition, it is well known that ic or systemically administered penicillin results in

Fig. 4. The effects of intracerebroven- tricular (icv) injections of crocin (A), di- azepam (B) and crocin plus diazepam (C) on the amplitude of spike waves in- duced by intracortical (ic) injection of penicillin in rats. Normal saline, crocin, diazepam and crocin plus diazepam were injected 10 min before injection of penicillin. Values are expressed as the mean ± SEM (n = 6); * p < 0.05 compared with normal saline,p < 0.05 compared with diazepam (2.5 µg) and crocin (50 µg)

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in cortical tissues by inhibiting GABA receptors, ow- ing to its structural resemblance to a specific GABA_A receptor antagonist, bicuculline, and thus leads to rhythmic epileptiform discharges [13].

In this study, icv injections of crocin and diazepam increased the latent period to appearance of the first spike wave and decreased the frequency and amplitude of spike waves induced by ic injection of penicillin.

The effect of crocin (100 µg) on PE was approximately similar to that of diazepam (10 µg). On the other hand, icv injections of normal saline, crocin and diazepam did not change the basal electrical activity (data not shown). In other words, at the level of the brain, crocin and diazepam showed a similar antiepileptic effect in PE model of epilepsy. To our knowledge, this is the first report that provided an evidence of antiepileptic effect of crocin in a rat model of acute epilepsy. The water-soluble carotenoid crocin, is one of constituents of saffron (Crocus sativus L.) stigma and has neuropro- tective activities [2, 25]. It was reported that Crocus sa- tivus L. extract (saffron) was used in allaying fear, cur- ing trances and some other disorders of central nervous system such as convulsion caused by fear and depres- sion [35]. The antiepileptic activity of saffron extracts was attributed to its active substance, safranal [18, 19].

Diazepam and other benzodiazepines mostly exert their pharmacological actions by allosterically modu- lating the GABA_A-benzodiazepine receptor complex to produce a facilitation effect on the GABA-mediated in- hibitory neurotransmission in the central nervous sys- tem [24]. Intravenous (iv) injection of diazepam re- duced frequency and amplitude of spike waves induced by ic injection of penicillin in rats [16]. Moreover, icv injection of diazepam produced an antiepileptic effect in PTZ-induced epilepsy and icv pretreatment with flu- mazenil (a GABA_A-benzodiazepine receptor antagonist) prevented diazepam-induced antiepileptic effect [26]. Clonazepam, the other benzodiazepine, exhibited a definite anticonvulsant activity in PTZ-induced seizures in mice [23]. However, the results of the present study suggest that crocin and diazepam at the level of the brain can exert antiepileptic activities in PE in rats.

In the present study, icv co-administration of an ef- fective dose of crocin (50 µg) with an ineffective dose of diazepam (2.5 µg) produced a more potent antiepileptic effect as compared with crocin (50 µg) used alone. This result indicates that a potentiation effect

the antiepileptic effect of crocin. The involvement of the GABA_A-benzodiazepine receptor system has been reported in the antiepileptic effects of the ethanolic leaf extracts of Rhus tridentate, Rhus rehmanniana and Hoslundia opposite and the ethanolic corm ex- tract of Hypoxis clochicifolia [29]. Apigenin isolated from the extract of Tanacetum parthenium, the plant used in traditional Danish folk medicine for the treatment of epilepsy, showed high affinity for the benzodiazepine site on the GABA_Areceptor [21]. Moreover, icv pretreatment with flumazenil reversed the anticon- vulsant effects induced by icv injections of diazepam and Pasipay in PTZ-induced convulsion model in rats [26]. In addition, it has been reported that the GABA- ergic system involves in the suppressive effect of Annona diversifolia Saff. on behavioral and electroen- cephalographical (EEG) seizures induced by microin- jection of penicillin into the central amygdala nucleus of brain in rats [17]. The involvement of GABA_A- benzodiazepine receptor has been reported in the antiepileptic effect of safranal, an active substance of saffron, in experimental absence seizures [30].

In conclusion, the results of the present study indi- cate that at the level of the brain, both crocin and diazepam produced antiepileptic effect in PE model of epilepsy. Moreover, a potentiation effect was ob- served between crocin and diazepam in producing antiepileptic effect. The GABA-benzodiazepine receptor-mediated mechanism may be involved in the antiepileptic activity of crocin.

Acknowledgment:

This study was supported by the Office of Vice Chancellor for Research of the Urmia University Research Project No.

002/D/88.

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Received: April 17, 2011; in the revised form: September 7, 2011;

accepted: October 17, 2011.