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The effect of vitamin D3 and paricalcitol on penicillin-induced epileptiform activity in rats
Epilepsy Research 159 (2020) 106262
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The effect of vitamin D3 and paricalcitol on penicillin-induced epileptiform activity in rats
T
Orhan Sumbula, Hatice Aygunb,* a b
Department of Neurology, Faculty of Medicine, Tokat Gaziosmanpasa University, Tokat, Turkey Department of Physiology, Faculty of Medicine, Tokat Gaziosmanpasa University, Tokat, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Electroencephalogram Paricalcitol Penicillin epilepsy model Rat Vitamin D3
Aim: Epilepsy is a disease characterized by seizures which impair human life considerably. Vitamin D is of different systemic effects on metabolism and its deficiency is known to have a high prevalence among epilepsy patients. Paricalcitol, a vitamin D receptor agonist, has relatively fewer side effects. This study aimed to investigate the anticonvulsant effect of vitamin D3 (cholecalciferol) and paricalcitol on penicillin-induced epileptiform activity. Method: 21 male Wistar rat weighing 180−240 g were used. After anesthetized by 1.25 g/kg urethane intraperitoneally (i.p.), rats were placed in the stereotaxic frame and tripolar electrodes were placed on the skull. The single microinjection of penicillin (2.5 μl, 500 IU, i.c.) into left sensorimotor cortex induced epileptiform activity. A single dose of 60.000 IU/kg (i.p.) vitamin D3 was administered 14 days before intracortical penicillin (500 IU) injection. Paricalcitol (10 μg/kg, i.p.) was administered 30 min before intracortical penicillin (500 IU) administration and recorded for the following 180 min. Results: Vitamin D3 pretreatment and paricalcitol diminished the frequency of epileptiform activity (p < 0.001) without changing the amplitude (p > 0.05) compared to the penicillin-injected group. Vitamin D3 pretreatment and paricalcitol led to an important delay in the onset of penicillin-induced epileptiform activity (p < 0.001 and p < 0.05, respectively). Vitamin D3 increased the latency of penicillin-induced epileptic activity compared to paricalcitol group (p < 0.001). Conclusion: Results indicate that vitamin D3 and paricalcitol decreased the frequency and increased the latency of the penicillin-induced epileptic activity. Vitamin D3 was more effective than paricalcitol.
1. Introduction Epilepsy is a common chronic disease characterized by unprovoked, recurrent spontaneous seizures that disrupt the nervous system and can lead to physical and mental dysfunction (Dichter, 1994; Andrade and Minassian, 2007). Although drug therapy provides optimal seizure control nearly 80 % of patients, it may not control seizure activity in some patients (Dichter, 2007). Thus, further investigation is highly needed for the alternative therapies for epilepsy. Vitamin D is a fundamental nutrient because it maintains phosphorous levels and the homeostasis of calcium in the body. At this point, many human tissues have nuclear vitamin D receptor (VDR). VDR is also highly expressed in kidney, skin, bone, small intestine, immune system, colon, brain, endocrine organs and muscle (Lips, 2006; Wrzosek et al., 2013). Several studies have demonstrated a significant association between
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vitamin D deficiency and many neurological disorders including Parkinson, epilepsy and Alzheimer (Peterson, 2014; Al-Temaimi et al., 2015; Keeney and Butterfield, 2015). For example, Sheth (2002) and Pack (2008) have reported that adult epilepsy patients can exhibit vitamin D deficiency. Similarly, Kalueff et al. (2005) and Borowicz et al. (2015) disclosed anticonvulsant properties of 1,25-dihydroxy vitamin D. A study investigating the possible association between epilepsy and VDR genetic variations in 82 patients with the temporal lobe epilepsy (TLE) found an important association between VDR genetic variation and TLE risk (Jiang et al., 2014). Vitamin D was shown to have an anticonvulsant effect in various experimental animal models. AbdelWahab et al. (2017) demonstrated that 1.5 mg/kg/day vitamin D administration significantly reduced seizure frequency of epileptic episodes in a PTZ-induced experimental epilepsy model. It was shown that long term use of antiepileptic drugs caused anomalies in bone metabolism. Serum 1,25- (OH) 2D3 levels were
Corresponding author. E-mail address: [email protected] (H. Aygun).
https://doi.org/10.1016/j.eplepsyres.2019.106262 Received 16 August 2019; Received in revised form 11 December 2019; Accepted 21 December 2019 Available online 23 December 2019 0920-1211/ © 2019 Elsevier B.V. All rights reserved.
Epilepsy Research 159 (2020) 106262
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with frontal bone) was identified. With reference to bregma with the stereotaxic device, three holes of 0.2 mm diameter were opened using microdrill. Bleeding which might occur in bone tissue during the procedure was controlled using bone wax (W810, ETHICON). Electric blankets (Harvard 7087) were used to keep the body temperature of rats stable. Tripolar electrodes were mounted over the left somatomotor cortex. The first electrode was placed in a position 3 mm lateral to sagittal suture and 4 mm anterior to bregma, while the second one was mounted in a position 3 mm lateral to sagittal suture and 4 mm posterior to bregma. A third electrode was used as a reference and implanted in the skin. Continuous monitoring of ECoG activity was performed using a PowerLab 16/35 data acquisition system. Recordings were transferred to a computer, and then offline analysis of frequency and amplitude of epileptiform activity was carried out.
reported to decrease in patients using carbamazepine, an antiepileptic drug (Välimäki et al., 1994). Verrotti et al. (2002) reported that bone turnover increased in therapeutic levels of carbamazepine. Similarly, another study found that serum vitamin D3 level was very low in patients receiving carbamazepine or oxcarbazepine, and that these patients developed secondary hyperparathyroidism (Mintzer et al., 2006). Considering its role in bone health and seizures, vitamin D could produce promising outcomes in epilepsy patients. However, vitamin D may have side effects. For instance, vitamin D intake might cause vomiting, stomach cramps, abdominal pain, bone loss, electrolyte imbalances and ultrafiltration loss. Therefore, paricalcitol, which is a vitamin D receptor agonist, is preferred in longterm treatment (Teng et al., 2003). Administration of paricalcitol has been shown to increase serum vitamin D levels in a more subtle and controlled manner. Studies have shown that paricalcitol has significantly fewer side effects than vitamin D (Balint et al., 2000; Andress, 2007). Studies have disclosed that vitamin D has anticonvulsant effects in epilepsy. However, it is not known which effect is more pronounced on epileptiform activity. For that reason, the efficacy of vitamin D and paricalcitol, a vitamin D receptor, was studied electrophysiologically in the same epilepsy model in this study.
2.3. Drug administration and induction of epileptiform activity Vitamin D3 and paricalcitol were used in the experiments. On the first day of the experiment, some rats were pretreated with a single dose of vitamin D3 (60.000 IU/kg, i.p.), and 14 days later the penicillin injection (500 IU, 2.5 μl, i.c.) were administered. Other rats were pretreated with paricalcitol (10 μg/kg, i.p.) 30 min before penicillin injection (500 IU, 2.5 μl, i.c.) was administered. Penicillin solution in sterile distilled water was intracortical injected (i.c.) in neocortex (by a Hamilton microsyringe type 701 N with an infusion rate of 0.5 μl/min) using a stereotaxic apparatus. Injections were made at a position about 2 mm posterior to the bregma, 2 mm lateral to the midline and 3.2 mm ventral to the surface of the skull. The epileptic focus was induced by 500 international units (IU) of penicillin G potassium injection. Within 1–3 min, epileptiform activity was observed in ECoG. ECoG recordings continued for three hours after penicillin injection. The data recorded by PowerLab system was used to calculate frequencies and amplitudes of epileptic spikes using Labchart v7.3.7 software (ADInstruments, Australia). Average spike and frequency were calculated for each 10 min intervals of the 180 min after penicillin injection, and statistical analyses were performed on these data (Fig. 1).
2. Materials and methods 2.1. Animals Adult male Wistar rats (200−250 g) obtained from Experimental Research Centre of Tokat Gaziosmanpaşa University were used in the experiments. Regulations of Experimental Animal Administration were observed in all animal procedures, and the study was approved by the Institutional Animal Ethics Committee with the approval number of 2018/HAYDEK-25. The rats were kept individually in a 12:12 h light/ dark cycle (lights on at 07.00 h). Constant temperature (23 °C ± 2 °C) and relative humidity (60 ± 15 %) regimes were used throughout the study, and rats had ad libitum access to water and food. Three study groups were established with seven rats in each group. (1) 500 IU penicillin (2.5 μl, i.c.) (2) 60.000 IU/kg vitamin D3 (i.p.) + 500 IU penicillin (2.5 μl, i.c.) (3) 10 μg/kg paricalcitol (i.p.) + 500 IU penicillin (2.5 μl, i.c.)
2.4. Statistical analyses Data were expressed as means ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) first and then using Tukey test for multiple comparisons of treatments as a post hoc test. SPSS statistical software package (15.0, SPSS Inc., USA) was used in all statistical procedures. p < 0.05 was considered statistically significant.
2.2. Surgical procedure and ECoG recordings The rats were fasted 24 h before the operation. The animals were anesthetized with urethane (25 % solution) at a dose of 1.25 g/kg administered intraperitoneally (i.p.). Anesthetized rats were placed in a stereotaxic frame (Harvard Stereotaxic Instrument). The hair on the scalp was shaved. The head skin was cut off with a middle line scraper and opened to the side. The periosteum of the skull was removed by scraping and small bleeding foci were cauterized by electrocautery to prevent bleeding. Thereafter (bregma point: junction of parietal bones
3. Results 3.1. Effects of Vitamin D3 and paricalcitol on epileptiform ECoG activity Intracortical injection of penicillin (500 IU) induced epileptiform ECoG activity characterized by bilateral spikes and spike-wave
Fig. 1. Experimental timeline diagram. Single dose vitamin D3 (60.000 IU/kg, i.p.) injection was made. On the 14th day, paricalcitol was administered first. Thirty minutes after the paricalcitol, penicillin was injected, and recording was made for three hours. Spike frequency and amplitude were measured with 10-minute intervals. 2
Epilepsy Research 159 (2020) 106262
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Fig. 2. Representative illustration of ECoG recordings for all groups in the 90th minute. A: Intracortical injection of the penicillin (500 IU) induced epileptiform activity on ECoG. B). A single dose of vitamin D3 (60.000 IU/kg) decreased the frequency of penicillin-induced epileptiform activity without changing amplitude. C) 10 μg/kg paricalcitol decreased frequency of penicillin-induced epileptiform activity without changing the amplitude.
Fig. 3. The effects of intraperitoneal injection of vitamin D3 and paricalcitol on the mean spike frequency of the penicillin-induced epileptiform activity. Pretreatment with vitamin D3 (60.000 IU/kg, i.p.) significantly decreased mean spike frequency of epileptiform activity compared to penicillin-injected groups (***p < 0.001). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly decreased mean spike frequency of the epileptiform activity compared to penicillin-injected groups (***p < 0.001).
min; 62 ± 2 spike/min in the 10th, 30th, 90th and 180th minutes after penicillin injection (i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes were 1072 ± 9 μV; 1007 ± 29 μV; 1006 ± 6 μV and 933 ± 42 μV 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 4, Table 2). The 60.000 IU/kg vitamin D3 and 10 μg/kg paricalcitol importantly decreased the frequency of the epileptiform activity starting from 10th minute, and this decrease continued for 180 min (Figs. 3, 4, Tables 1 and 2). However, vitamin D3 and paricalcitol did not significantly alter the amplitude of epileptiform activity in any of the groups during the experiments. The mean frequency of epileptiform activity of 60.000 IU/kg vitamin D3 (i.p.) injection groups were 24 ± 4, 17 ± 1, 13 ± 2 and 12 ± 2 spike/min 10, 30, 90 and 180 min after penicillin injection
Table 1 Penicillin-induced epileptiform activity’s mean spike frequency (spike/min). Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used: a: p < 0.001 vitamin D3 and paricalcitol groups compared to control b: p < 0.05 vitamin D3 group compared to paricalcitol groups. Groups
10th
30th
90th
180th
Control Vitamin D3 Paricalcitol
80 ± 2 24 ± 4;a 38 ± 3;a; b
81 ± 3 17 ± 1;a 30 ± 3;a;b
75 ± 2 13 ± 2;a 21 ± 4;a
62 ± 2 12 ± 2;a 14 ± 2;a
complexes (Fig. 2A–C). This ECoG activity began within 1–3 min after penicillin application and lasted for 180 min. The mean spike frequencies were 80 ± 2 spike/min; 81 ± 3 spike/min; 75 ± 2 spike/ 3
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Fig. 4. The effects of vitamin D3 and paricalcitol on the mean spike amplitude of penicillin-induced the epileptiform activity. Vitamin D3 at 60.000 IU/kg rate did not alter the epileptiform activity’s mean amplitude. Paricalcitol at 10 μg/kg rate did not alter the mean amplitude of epileptiform activity (p > 0.05). Table 2 Penicillin-induced epileptiform activity’s mean spike amplitude. Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used. There was no statistically significant difference in mean spike amplitude values among the groups (p > 0.05). Groups
10th
30th
90th
180th
Control Vitamin D3 Paricalcitol
1072 ± 9 990 ± 45 1091 ± 21
1007 ± 29 985 ± 35 1044 ± 64
1006 ± 34 944 ± 38 967 ± 30
933 ± 42 896 ± 29 851 ± 38
(i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes of epileptiform activity of 60.000 IU/kg vitamin D3 (i.p.) injection groups were 990 ± 45, 985 ± 35, 944 ± 38 and 896 ± 29 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively. Vitamin D3 at a rate of 60.000 IU/kg (i.p.) significantly decreased the mean frequency of epileptiform activity without changing the amplitude of epileptiform activity compared to penicillin-injected group (p < 0.001) (Figs. 2A and B, , Tables 1 and 2). The mean frequencies of epileptiform activity of 10 μg/kg paricalcitol (i.p.) injection groups were 38 ± 3; 30 ± 3; 21 ± 4 and 14 ± 2 spikes/minute 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes of epileptiform activity of 10 μg/kg paricalcitol (i.p.) injection groups were 1091 ± 21, 1044 ± 64, 967 ± 30 and 851 ± 3810 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 4, Table 2). Paricalcitol at a rate of 10 μg/kg (i.p.) significantly decreased the mean frequency of epileptiform activity without changing the amplitude of epileptiform activity compared to penicillin-injected group (p < 0.001) (Figs. 2A, C, , Tables 1 and 2). Vitamin D3 at a 60.000 IU/kg rate (i.p.) significantly decreased the mean frequency of epileptiform activity between 10th and 70th minutes without changing the amplitude of epileptiform activity compared to a paricalcitol injected group (p < 0.05) (Figs. 2B, C, , Tables 1 and 2). Pretreatment with vitamin D3 (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity from 90.61 ± 14.86–347.3 ± 32.14 s compared to penicillin-injected
Fig. 5. Vitamin D3 pretreatment (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity compared to penicillin-injected groups, (***p < 0.001). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly increased the latency of the epileptiform activity compared to penicillin-injected groups (*p < 0.05). The latency of the epileptiform activity was significantly lower in paricalcitol treatment group (10 μg/kg, i.p.) compared to vitamin D3 (60.000 IU/kg, i.p.) treatment groups (••• = p < 0.001). Data are given as mean ± SEM. Table 3 The latency of penicillin-induced epileptiform activity. Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used: a: p < 0.05, b: p < 0.001 vitamin D3 and paricalcitol groups compared to the control c: p < 0.001 vitamin D3 group compared to paricalcitol groups. Groups
The latency of epileptiform activity (seconds)
Control Vitamin D3 Paricalcitol
90.61 ± 14.86 347.3 ± 32.14; b 156.3 ± 25.35; a; c
groups (p < 0.001) (Fig. 5, Table 3). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly increased the latency of the epileptiform activity from 90.61 ± 14.86–156.3 ± 25.35 s compared to penicillin4
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glial cells and peripheral nerves of the brain. Genomic and non-genomic pathways of VDR activation were shown to play a role in vitamin Dinduced neuroprotection. Vitamin D can trigger genotypic antiepileptic properties by inducing genes that encode enzymes and cytokines in the neurotransmitter metabolism via the VDR (Garcion et al., 2002). These genomic actions include the binding of 1,25 (OH) D to the nuclear vitamin D receptor and the release of neurotrophin-3, neurotrophin-4, glial cell-derived neurotrophic factor, nerve growth factor, parvalbumin and nitric oxide synthase from the nervous system (Schwaller et al., 2004). These genomic actions occur with a time delay of hours to days (Garcion et al., 2002). Nonetheless, faster vitamin D motions suggest the presence of non-genomic pathways. An anticonvulsant effect was reported in an animal study, which was realized five minutes prior to vitamin D3 on electroconvulsive seizure. It was stated that this effect was caused by non-genomic mechanisms (Borowicz et al., 2007). The GABAergic system has a significant role in the pathogenesis of epilepsy (Holló et al., 2012). GABA-A receptors are a significant target for the non-genomic effect of various neurosteroids and neuroactive hormones, including vitamin D. A number of clinical and experimental epilepsy studies suggested that seizure susceptibility could be caused by the attenuated GABAergic and increased glutamatergic neurotransmission (Schwartzkroin, 1994; Hoogland et al., 2004; Mahfoz et al., 2017). Penicillin is an antagonists of GABA neurotransmitter. The penicillin injection via microsyringe into the neocortex produced epileptiform activity with decreased GABA level and increased glutamate in brain (Fisher, 1989). Sikoglu et al. (2015) demonstrated that vitamin D treatment increased GABA level in patients with bipolar disorders. A study by Kalueff et al. (2006) suggested that Vitamin D receptors are allosteric modulators of GABA receptor and they reduce neuronal excitability. Another study (Jiang et al., 2014) demonstrated that pretreatment with a higher dose of calcitriol (1,25-dihydroxyvitamin D), another vitamin D agonist, elevated GABA level in the hippocampus. Mahfoz et al. (2017) reported that pretreatment with vitamin D significantly increased GABA levels in the brain and decreased the brain glutamate level in lithium-pilocarpine induced status epilepticus in rats. Pretreatment with vitamin D and paricalcitol may reduce penicillin-induced epileptiform activity by increasing GABA levels. In the present study, vitamin D appeared to be more effective on seizure activity than paricalcitol. This can be explained by the fact that vitamin D3 was administered much earlier than paricalcitol. DNA repair, differentiation, apoptosis and cell membrane integrity, and the suppression of the release of proinflammatory cytokines are the other important effects of vitamin D (Bikle, 2009). A number of studies pointed to the essential roles of oxidative stress in the pathophysiology of epilepsy (Royes et al., 2007; Aguiar et al., 2012). A clinical study showed that oxidative stress markers increased in the hippocampus of surgically resected drug-resistant epilepsy patients (Pecorelli et al., 2015). In many experimental studies, increasing oxidative stress was shown in pentylenetetrazole-induced kindling seizures (Rauca et al., 2004), pentylenetetrazole‐induced seizures (Uma Devi et al., 2006) pilocarpine-induced epilepsy model (Freitas et al., 2005; 2009), kainic acid-induced seizures (Shin et al., 2008), spontaneously epileptic rat (Shin et al., 2011), audiogenic seizures in rats (Parreira et al., 2018) and penicillin-induced epileptiform activity in rats (Tubaş et al., 2017). In addition, Ayyildiz et al. (2007) demonstrated that penicillin induced seizures triggered oxidative stress along with an increase in oxidative stress marker malondialdehyde (MDA) and a decrease in antioxidant glutathione (GSH) in brain tissue of epileptic rats. Paricalcitol is an active vitamin D analog that exhibits biological activities similar to what vitamin D does (Drüeke, 2005). Recent studies have shown that vitamin D and paricalcitol have a protective effect on oxidative stress in several organs such as the brain, liver, kidney, pancreas and lung (Garcion et al., 2002; Östman et al., 2009; Ali et al., 2018). Similarly, Mahfoz et al. (2017) reported that pretreatment with vitamin D significantly decreased lithium-pilocarpine induced epileptiform activity by reduced oxidative stress and
injected groups (p < 0.05) (Fig. 5, Table 3). Pretreatment with Vitamin D3 (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity from 347.3 ± 32.14–156.3 ± 25.35 s compared to paricalcitol groups (10 μg/kg, i.p.) (p < 0.001) (Fig. 5, Table 3). 4. Discussion In the present study, 10 μg/kg intraperitoneal (i.p.) paricalcitol and 60.000 IU/kg (i.p.) vitamin D3 significantly decreased seizure frequency compared to the saline administration and delayed spike activity and seizure onset. When vitamin D3 and paricalcitol applications were compared, delay in seizure onset was more pronounced in vitamin D3 administration. There is no study in the literature comparing vitamin D analogues in a penicillin epilepsy model. In topical administration of penicillin, a GABA receptor antagonist, seizures start focally but continue in a generalized fashion. Wallenstein (1987) found that after the topical administration of 250 IU penicillin in right hemisphere, spike discharges were observed in the 119th second, which started with 10 s delays in the opposite (left) hemisphere. Seizure activity that arose with penicillin administration to the cortex of experimental animals was classified as simple partial epilepsy model by Fisher (1989). In the last epilepsy classification by ILAE, focal term was used instead of partial. Delayed discharges in the opposite hemisphere after topical penicillin administration showed that penicillin could be used as an experimental model for focal epilepsy (Sagratella et al., 1985; Wallenstein, 1987; Fisher, 1989; Aygun et al., 2019). Cholecalciferol (vitamin D3) is produced from 7- dehydrocholesterol on the skin under the influence of ultraviolet radiation at 290−315 nm wavelength, and this endogenous production is the main source of the vitamin D (Glerup et al., 2000; Holick, 2005). Cholecalciferol is a natural vitamin D. Many clinical and experimental studies reported anticonvulsant effects of vitamin D. In a pilot study, vitamin D combined with antiepileptic drugs reduced seizures in patients with pharmaco-resistant epilepsy (Holló et al., 2012). Anticonvulsant activity of vitamin D was shown in lithium-pilocarpine induced status epilepticus rats (Mahfoz et al., 2017), PTZ-induced seizures (Kalueff et al. (2005) and generalized seizures in PTZ-kindled rat model of epilepsy (AbdelWahab et al., 2017). Siegel et al. (1984) found that intrahippocampal stereotaxic injection of vitamin D3 increased the hippocampal seizure threshold in rats. In addition, Tetich et al. (2005) demonstrated the modulating effects of vitamin D3 treatment for the seizure-related changes in pilocarpine-induced epileptic rat brain. While many studies found positive effects of vitamin D on epileptic convulsions, we have come across only two studies (Uyanıkgil et al., 2016) reporting a positive effect of paricalcitol on epileptic convulsions. In their study using an experimental epilepsy model induced by PTZ (pentilentetrazol), a generalized epilepsy model, Uyanıkgil et al. (2016) evaluated EEG recordings, oxidative stress markers and behavioral conditions. In that study, Racine's Convulsion Scale (RCS) score was importantly lower in groups treated with 5 and 10 μg/kg paricalcitol. In addition, the duration of the first myoclonic jerk (FMJ) latencies were prolonged and a significant reduction was observed in spike percentages in EEG recordings. They also found that this effect was more pronounced in the 10 μg/kg paricalcitol group and concluded that paricalcitol was effective in PTZ-induced seizures (Uyanıkgil et al., 2016). Recently Aygun et al. (2019) demonstrated that acute and chronic treatment of vitamin D and paricalcitol decreased absence-like seizures in genetic epileptic WAG/Rij rat. In the present study, treatment with vitamin D and paricalcitol reduced seizure epileptiform activity and delayed seizure formation in the epileptic rats with penicillin induced seizures. These findings are consistent with the previous studies. Different mechanisms were proposed to explain the anticonvulsant effects of vitamin D. This vitamin exerts its effect through vitamin D receptors, and vitamin D receptors are present in neurons, spinal cord, 5
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increased antioxidants activity in the epileptic rat brain. In addition, various previous studies showed antioxidant characteristic of paricalcitol. It was demonstrated that paricalcitol treatment decreased the levels of oxidative stress markers including MDA, oxidative DNA damage, NADPH oxidase, iNOS levels and increased endogenous antioxidant levels including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and glutathione (GSH) (Husain et al., 2015; Bulut et al., 2016; Ali et al., 2018). Uyanıkgil et al. (2016) reported that acute paricalcitol treatment decreased MDA and increased SOD activity in PTZ-induced epileptic rat brain. In the present study, the antioxidant properties of paricalcitol and vitamin D may have been effective in decreasing the penicillin-induced epileptiform activity. Paricalcitol is an agent with clinical and paraclinical advantages over other vitamin D preparations (Finch et al., 1999; Sprague et al., 2003). Additionally, we have also revealed that paricalcitol is as effective as vitamin D3, whose effect on epileptic seizures has previously been proven. Although many new antiepileptic drugs (AEDs) have been introduced in recent years, the search for new therapies with better tolerability and efficacy remains a significant target. The development and innovation of the new AED are based on preclinical use of the animal models. In the present study, vitamin D and paricalcitol has been shown to be effective on focal epileptic seizures. We conclude that vitamin D and paricalcitol could be effective antiepileptic agents in focal epilepsy. Because of their strong antiepileptogenic effects as well as their costeffectiveness, easy access, high efficiency and good safety considerations, vitamin D and paricalcitol merit further clinical studies. Therefore, a more comprehensive examination of animal models is important in terms of evaluating the mechanisms underlying epileptogenesis and ictogenesis.
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Funding None. Declaration of Competing Interest No potential conflict of interest was reported by the authors. References Abdel-Wahab, A.F., Afify, M.A., Mahfouz, A.M., Shahzad, N., Bamagous, G.A., Al Ghamdi, S.S., 2017. Vitamin D enhances antiepileptic and cognitive effects of lamotrigine in pentylenetetrazole-kindled rats. Brain Res. 1673, 78–85. Aguiar, C.C.T., Almeida, A.B., Araújo, P.V.P., Abreu, R.N.D.C.D., Chaves, E.M.C., Vale, O.C.D., et al., 2012. Oxidative stress and epilepsy: literature review. Oxid. Med. Cell. Longev. 2012, 795259. Ali, T.M., El Esawy, B., Elaskary, A., 2018. Effect of paricalcitol on pancreatic oxidative stress, inflammatory markers, and glycemic status in diabetic rats. Irish J. Med. Sci. 187 (1), 75–84 (1971-). Al-Temaimi, R.A., Al-Enezi, A., Al-Serri, A., Alroughani, R., Al-Mulla, F., 2015. The association of vitamin D receptor polymorphisms with multiple sclerosis in a casecontrol study from Kuwait. PLoS One 10 (11), e0142265. Andrade, D.M., Minassian, B.A., 2007. Genetics of epilepsies. Expert Rev. Neurother. 7 (6), 727–734. Andress, D., 2007. Nonclassical aspects of differential vitamin D receptor activation. Drugs 6 (14), 1999–2012. Aygun, H., Ayyildiz, M., Agar, E., 2019. Effects of vitamin D and paricalcitol on epileptogenesis and behavioral properties of WAG/Rij rats with absence epilepsy. Epilepsy Res. 157, 106208. Ayyildiz, M., Coskun, S., Yildirim, M., Agar, E., 2007. The effects of ascorbic acid on penicillin‐induced epileptiform activity in rats. Epilepsia 48 (7), 1388–1395. Balint, E., Marshall, C.F., Sprague, S.M., 2000. Effect of the vitamin D analogues paricalcitol and calcitriol on bone mineral in vitro. Am. J. Kidney Dis. 36 (4), 789–796. Bikle, D.D., 2009. Vitamin D and immune function: understanding common pathways. Current Osteoporos Rep. 7 (2), 58–63. Borowicz, K.K., Morawska, D., Morawska, M., 2015. Effect of cholecalciferol on the anticonvulsant action of some second-generation antiepileptic drugs in the mouse model of maximal electroshock. Pharmacol. Rep. 67 (5), 875–880. Borowicz, K.K., Morawska, M., Furmanek-Karwowska, K., Luszczki, J.J., Czuczwar, S.J., 2007. Cholecalciferol enhances the anticonvulsant effect of conventional antiepileptic drugs in the mouse model of maximal electroshock. Eur. J. Pharmacol. 573 (1-3), 111–115.
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of Cornus mas L. and Morus rubra L. extracts on penicillin-induced epileptiform activity: an electrophysiological and biochemical study. Acta Neurobiol. Exp. (Wars) 77, 45–56. Uma Devi, P., Kolappa Pillai, K., Vohora, D., 2006. Modulation of pentylenetetrazole‐induced seizures and oxidative stress parameters by sodium valproate in the absence and presence of N‐acetylcysteine. Fundamental Clin. Pharmacol. 20 (3), 247–253. Uyanıkgil, Y., Solmaz, V., Çavuşoğlu, T., Çınar, B.P., Çetin, E.Ö., Sur, H.Y., et al., 2016. Inhibitor effect of paricalcitol in rat model of pentylenetetrazol-induced seizures. Naunyn Schmiedebergs Arch. Pharmacol. 389 (10), 1117–1122. Välimäki, M.J., Tiihonen, M., Laitinen, K., Tähtelä, R., Kärkkäinen, M., Lamberg-Allardt, C., et al., 1994. Bone mineral density measured by dual-energy x-ray absorptiometry and novel markers of bone formation and resorption in patients on antiepileptic drugs. J. Bone Miner. Res. 9, 631–637. Verrotti, A., Greco, R., Latini, G., Morgese, G., Chiarelli, F., 2002. Increased bone turnover in prepubertal, pubertal, and postpubertal patients receiving carbamazepine. Epilepsia 43 (12), 1488–1492. Wallenstein, M.C., 1987. Attenuation of penicillin models of epilepsy by nonsteroidal anti-inflammatory drugs. Exp. Neurol. 98 (1), 152–160. Wrzosek, M., Łukaszkiewicz, J., Wrzosek, M., Jakubczyk, A., Matsumoto, H., Piątkiewicz, P., et al., 2013. Vitamin D and the central nervous system. Pharmacol. Rep. 65 (2), 271–278.
Shin, E.J., Jeong, J.H., Chung, Y.H., Kim, W.K., Ko, K.H., Bach, J.H., et al., 2011. Role of oxidative stress in epileptic seizures. Neurochem. Int. 59 (2), 122–137. Shin, E.J., Jeong, J.H., Bing, G., Park, E.S., Chae, J.S., Yen, T.P., et al., 2008. Kainateinduced mitochondrial oxidative stress contributes to hippocampal degeneration in senescenceaccelerated mice. Cell. Signal. 20, 645–658. Siegel, A., Malkowitz, L., Moskovits, M.J., Christakos, S., 1984. Administration of 1, 25dihydroxyvitamin D3 results in the elevation of hippocampal seizure threshold levels in rats. Brain Res. 298 (1), 125–129. Sikoglu, E.M., Navarro, A.A.L., Starr, D., Dvir, Y., Nwosu, B.U., Czerniak, S.M., et al., 2015. Vitamin D3 supplemental treatment for mania in youth with bipolar spectrum disorders. J. Child and Adolescent Psychopharmacol. 25 (5), 415–424. Sprague, S.M., Llach, F., Amdahl, M., Taccetta, C., Batlle, D., 2003. Paricalcitol versus calcitriol in the treatment of secondary hyperparathyroidism. Kidney Int. 63 (4), 1483–1490. Teng, M., Wolf, M., Lowrie, E., Ofsthun, N., Lazarus, J.M., Thadhani, R., 2003. Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N. Engl. J. Med. 349 (5), 446–456. Tetich, M., Dziedzicka Wasylewska, M., Kuśmider, M., Kutner, A., Leśkiewicz, M., Jaworska-Feil, L., et al., 2005. Effects of PRI-2191—a low-calcemic analog of 1,25dihydroxyvitamin D3 on the seizureinduced changes in brain gene expression and immune system activity in the rat. Brain Res. 1039 (1–2), 1–13. Tubaş, F., Per, S., Taşdemir, A., Bayram, A.K., Yıldırım, M., Uzun, A., et al., 2017. Effects
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Epilepsy Research journal homepage: www.elsevier.com/locate/epilepsyres
The effect of vitamin D3 and paricalcitol on penicillin-induced epileptiform activity in rats
T
Orhan Sumbula, Hatice Aygunb,* a b
Department of Neurology, Faculty of Medicine, Tokat Gaziosmanpasa University, Tokat, Turkey Department of Physiology, Faculty of Medicine, Tokat Gaziosmanpasa University, Tokat, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Electroencephalogram Paricalcitol Penicillin epilepsy model Rat Vitamin D3
Aim: Epilepsy is a disease characterized by seizures which impair human life considerably. Vitamin D is of different systemic effects on metabolism and its deficiency is known to have a high prevalence among epilepsy patients. Paricalcitol, a vitamin D receptor agonist, has relatively fewer side effects. This study aimed to investigate the anticonvulsant effect of vitamin D3 (cholecalciferol) and paricalcitol on penicillin-induced epileptiform activity. Method: 21 male Wistar rat weighing 180−240 g were used. After anesthetized by 1.25 g/kg urethane intraperitoneally (i.p.), rats were placed in the stereotaxic frame and tripolar electrodes were placed on the skull. The single microinjection of penicillin (2.5 μl, 500 IU, i.c.) into left sensorimotor cortex induced epileptiform activity. A single dose of 60.000 IU/kg (i.p.) vitamin D3 was administered 14 days before intracortical penicillin (500 IU) injection. Paricalcitol (10 μg/kg, i.p.) was administered 30 min before intracortical penicillin (500 IU) administration and recorded for the following 180 min. Results: Vitamin D3 pretreatment and paricalcitol diminished the frequency of epileptiform activity (p < 0.001) without changing the amplitude (p > 0.05) compared to the penicillin-injected group. Vitamin D3 pretreatment and paricalcitol led to an important delay in the onset of penicillin-induced epileptiform activity (p < 0.001 and p < 0.05, respectively). Vitamin D3 increased the latency of penicillin-induced epileptic activity compared to paricalcitol group (p < 0.001). Conclusion: Results indicate that vitamin D3 and paricalcitol decreased the frequency and increased the latency of the penicillin-induced epileptic activity. Vitamin D3 was more effective than paricalcitol.
1. Introduction Epilepsy is a common chronic disease characterized by unprovoked, recurrent spontaneous seizures that disrupt the nervous system and can lead to physical and mental dysfunction (Dichter, 1994; Andrade and Minassian, 2007). Although drug therapy provides optimal seizure control nearly 80 % of patients, it may not control seizure activity in some patients (Dichter, 2007). Thus, further investigation is highly needed for the alternative therapies for epilepsy. Vitamin D is a fundamental nutrient because it maintains phosphorous levels and the homeostasis of calcium in the body. At this point, many human tissues have nuclear vitamin D receptor (VDR). VDR is also highly expressed in kidney, skin, bone, small intestine, immune system, colon, brain, endocrine organs and muscle (Lips, 2006; Wrzosek et al., 2013). Several studies have demonstrated a significant association between
⁎
vitamin D deficiency and many neurological disorders including Parkinson, epilepsy and Alzheimer (Peterson, 2014; Al-Temaimi et al., 2015; Keeney and Butterfield, 2015). For example, Sheth (2002) and Pack (2008) have reported that adult epilepsy patients can exhibit vitamin D deficiency. Similarly, Kalueff et al. (2005) and Borowicz et al. (2015) disclosed anticonvulsant properties of 1,25-dihydroxy vitamin D. A study investigating the possible association between epilepsy and VDR genetic variations in 82 patients with the temporal lobe epilepsy (TLE) found an important association between VDR genetic variation and TLE risk (Jiang et al., 2014). Vitamin D was shown to have an anticonvulsant effect in various experimental animal models. AbdelWahab et al. (2017) demonstrated that 1.5 mg/kg/day vitamin D administration significantly reduced seizure frequency of epileptic episodes in a PTZ-induced experimental epilepsy model. It was shown that long term use of antiepileptic drugs caused anomalies in bone metabolism. Serum 1,25- (OH) 2D3 levels were
Corresponding author. E-mail address: [email protected] (H. Aygun).
https://doi.org/10.1016/j.eplepsyres.2019.106262 Received 16 August 2019; Received in revised form 11 December 2019; Accepted 21 December 2019 Available online 23 December 2019 0920-1211/ © 2019 Elsevier B.V. All rights reserved.
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with frontal bone) was identified. With reference to bregma with the stereotaxic device, three holes of 0.2 mm diameter were opened using microdrill. Bleeding which might occur in bone tissue during the procedure was controlled using bone wax (W810, ETHICON). Electric blankets (Harvard 7087) were used to keep the body temperature of rats stable. Tripolar electrodes were mounted over the left somatomotor cortex. The first electrode was placed in a position 3 mm lateral to sagittal suture and 4 mm anterior to bregma, while the second one was mounted in a position 3 mm lateral to sagittal suture and 4 mm posterior to bregma. A third electrode was used as a reference and implanted in the skin. Continuous monitoring of ECoG activity was performed using a PowerLab 16/35 data acquisition system. Recordings were transferred to a computer, and then offline analysis of frequency and amplitude of epileptiform activity was carried out.
reported to decrease in patients using carbamazepine, an antiepileptic drug (Välimäki et al., 1994). Verrotti et al. (2002) reported that bone turnover increased in therapeutic levels of carbamazepine. Similarly, another study found that serum vitamin D3 level was very low in patients receiving carbamazepine or oxcarbazepine, and that these patients developed secondary hyperparathyroidism (Mintzer et al., 2006). Considering its role in bone health and seizures, vitamin D could produce promising outcomes in epilepsy patients. However, vitamin D may have side effects. For instance, vitamin D intake might cause vomiting, stomach cramps, abdominal pain, bone loss, electrolyte imbalances and ultrafiltration loss. Therefore, paricalcitol, which is a vitamin D receptor agonist, is preferred in longterm treatment (Teng et al., 2003). Administration of paricalcitol has been shown to increase serum vitamin D levels in a more subtle and controlled manner. Studies have shown that paricalcitol has significantly fewer side effects than vitamin D (Balint et al., 2000; Andress, 2007). Studies have disclosed that vitamin D has anticonvulsant effects in epilepsy. However, it is not known which effect is more pronounced on epileptiform activity. For that reason, the efficacy of vitamin D and paricalcitol, a vitamin D receptor, was studied electrophysiologically in the same epilepsy model in this study.
2.3. Drug administration and induction of epileptiform activity Vitamin D3 and paricalcitol were used in the experiments. On the first day of the experiment, some rats were pretreated with a single dose of vitamin D3 (60.000 IU/kg, i.p.), and 14 days later the penicillin injection (500 IU, 2.5 μl, i.c.) were administered. Other rats were pretreated with paricalcitol (10 μg/kg, i.p.) 30 min before penicillin injection (500 IU, 2.5 μl, i.c.) was administered. Penicillin solution in sterile distilled water was intracortical injected (i.c.) in neocortex (by a Hamilton microsyringe type 701 N with an infusion rate of 0.5 μl/min) using a stereotaxic apparatus. Injections were made at a position about 2 mm posterior to the bregma, 2 mm lateral to the midline and 3.2 mm ventral to the surface of the skull. The epileptic focus was induced by 500 international units (IU) of penicillin G potassium injection. Within 1–3 min, epileptiform activity was observed in ECoG. ECoG recordings continued for three hours after penicillin injection. The data recorded by PowerLab system was used to calculate frequencies and amplitudes of epileptic spikes using Labchart v7.3.7 software (ADInstruments, Australia). Average spike and frequency were calculated for each 10 min intervals of the 180 min after penicillin injection, and statistical analyses were performed on these data (Fig. 1).
2. Materials and methods 2.1. Animals Adult male Wistar rats (200−250 g) obtained from Experimental Research Centre of Tokat Gaziosmanpaşa University were used in the experiments. Regulations of Experimental Animal Administration were observed in all animal procedures, and the study was approved by the Institutional Animal Ethics Committee with the approval number of 2018/HAYDEK-25. The rats were kept individually in a 12:12 h light/ dark cycle (lights on at 07.00 h). Constant temperature (23 °C ± 2 °C) and relative humidity (60 ± 15 %) regimes were used throughout the study, and rats had ad libitum access to water and food. Three study groups were established with seven rats in each group. (1) 500 IU penicillin (2.5 μl, i.c.) (2) 60.000 IU/kg vitamin D3 (i.p.) + 500 IU penicillin (2.5 μl, i.c.) (3) 10 μg/kg paricalcitol (i.p.) + 500 IU penicillin (2.5 μl, i.c.)
2.4. Statistical analyses Data were expressed as means ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) first and then using Tukey test for multiple comparisons of treatments as a post hoc test. SPSS statistical software package (15.0, SPSS Inc., USA) was used in all statistical procedures. p < 0.05 was considered statistically significant.
2.2. Surgical procedure and ECoG recordings The rats were fasted 24 h before the operation. The animals were anesthetized with urethane (25 % solution) at a dose of 1.25 g/kg administered intraperitoneally (i.p.). Anesthetized rats were placed in a stereotaxic frame (Harvard Stereotaxic Instrument). The hair on the scalp was shaved. The head skin was cut off with a middle line scraper and opened to the side. The periosteum of the skull was removed by scraping and small bleeding foci were cauterized by electrocautery to prevent bleeding. Thereafter (bregma point: junction of parietal bones
3. Results 3.1. Effects of Vitamin D3 and paricalcitol on epileptiform ECoG activity Intracortical injection of penicillin (500 IU) induced epileptiform ECoG activity characterized by bilateral spikes and spike-wave
Fig. 1. Experimental timeline diagram. Single dose vitamin D3 (60.000 IU/kg, i.p.) injection was made. On the 14th day, paricalcitol was administered first. Thirty minutes after the paricalcitol, penicillin was injected, and recording was made for three hours. Spike frequency and amplitude were measured with 10-minute intervals. 2
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Fig. 2. Representative illustration of ECoG recordings for all groups in the 90th minute. A: Intracortical injection of the penicillin (500 IU) induced epileptiform activity on ECoG. B). A single dose of vitamin D3 (60.000 IU/kg) decreased the frequency of penicillin-induced epileptiform activity without changing amplitude. C) 10 μg/kg paricalcitol decreased frequency of penicillin-induced epileptiform activity without changing the amplitude.
Fig. 3. The effects of intraperitoneal injection of vitamin D3 and paricalcitol on the mean spike frequency of the penicillin-induced epileptiform activity. Pretreatment with vitamin D3 (60.000 IU/kg, i.p.) significantly decreased mean spike frequency of epileptiform activity compared to penicillin-injected groups (***p < 0.001). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly decreased mean spike frequency of the epileptiform activity compared to penicillin-injected groups (***p < 0.001).
min; 62 ± 2 spike/min in the 10th, 30th, 90th and 180th minutes after penicillin injection (i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes were 1072 ± 9 μV; 1007 ± 29 μV; 1006 ± 6 μV and 933 ± 42 μV 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 4, Table 2). The 60.000 IU/kg vitamin D3 and 10 μg/kg paricalcitol importantly decreased the frequency of the epileptiform activity starting from 10th minute, and this decrease continued for 180 min (Figs. 3, 4, Tables 1 and 2). However, vitamin D3 and paricalcitol did not significantly alter the amplitude of epileptiform activity in any of the groups during the experiments. The mean frequency of epileptiform activity of 60.000 IU/kg vitamin D3 (i.p.) injection groups were 24 ± 4, 17 ± 1, 13 ± 2 and 12 ± 2 spike/min 10, 30, 90 and 180 min after penicillin injection
Table 1 Penicillin-induced epileptiform activity’s mean spike frequency (spike/min). Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used: a: p < 0.001 vitamin D3 and paricalcitol groups compared to control b: p < 0.05 vitamin D3 group compared to paricalcitol groups. Groups
10th
30th
90th
180th
Control Vitamin D3 Paricalcitol
80 ± 2 24 ± 4;a 38 ± 3;a; b
81 ± 3 17 ± 1;a 30 ± 3;a;b
75 ± 2 13 ± 2;a 21 ± 4;a
62 ± 2 12 ± 2;a 14 ± 2;a
complexes (Fig. 2A–C). This ECoG activity began within 1–3 min after penicillin application and lasted for 180 min. The mean spike frequencies were 80 ± 2 spike/min; 81 ± 3 spike/min; 75 ± 2 spike/ 3
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Fig. 4. The effects of vitamin D3 and paricalcitol on the mean spike amplitude of penicillin-induced the epileptiform activity. Vitamin D3 at 60.000 IU/kg rate did not alter the epileptiform activity’s mean amplitude. Paricalcitol at 10 μg/kg rate did not alter the mean amplitude of epileptiform activity (p > 0.05). Table 2 Penicillin-induced epileptiform activity’s mean spike amplitude. Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used. There was no statistically significant difference in mean spike amplitude values among the groups (p > 0.05). Groups
10th
30th
90th
180th
Control Vitamin D3 Paricalcitol
1072 ± 9 990 ± 45 1091 ± 21
1007 ± 29 985 ± 35 1044 ± 64
1006 ± 34 944 ± 38 967 ± 30
933 ± 42 896 ± 29 851 ± 38
(i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes of epileptiform activity of 60.000 IU/kg vitamin D3 (i.p.) injection groups were 990 ± 45, 985 ± 35, 944 ± 38 and 896 ± 29 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively. Vitamin D3 at a rate of 60.000 IU/kg (i.p.) significantly decreased the mean frequency of epileptiform activity without changing the amplitude of epileptiform activity compared to penicillin-injected group (p < 0.001) (Figs. 2A and B, , Tables 1 and 2). The mean frequencies of epileptiform activity of 10 μg/kg paricalcitol (i.p.) injection groups were 38 ± 3; 30 ± 3; 21 ± 4 and 14 ± 2 spikes/minute 10, 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 3, Table 1). The mean spike amplitudes of epileptiform activity of 10 μg/kg paricalcitol (i.p.) injection groups were 1091 ± 21, 1044 ± 64, 967 ± 30 and 851 ± 3810 30, 90 and 180 min after penicillin injection (i.c.), respectively (Fig. 4, Table 2). Paricalcitol at a rate of 10 μg/kg (i.p.) significantly decreased the mean frequency of epileptiform activity without changing the amplitude of epileptiform activity compared to penicillin-injected group (p < 0.001) (Figs. 2A, C, , Tables 1 and 2). Vitamin D3 at a 60.000 IU/kg rate (i.p.) significantly decreased the mean frequency of epileptiform activity between 10th and 70th minutes without changing the amplitude of epileptiform activity compared to a paricalcitol injected group (p < 0.05) (Figs. 2B, C, , Tables 1 and 2). Pretreatment with vitamin D3 (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity from 90.61 ± 14.86–347.3 ± 32.14 s compared to penicillin-injected
Fig. 5. Vitamin D3 pretreatment (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity compared to penicillin-injected groups, (***p < 0.001). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly increased the latency of the epileptiform activity compared to penicillin-injected groups (*p < 0.05). The latency of the epileptiform activity was significantly lower in paricalcitol treatment group (10 μg/kg, i.p.) compared to vitamin D3 (60.000 IU/kg, i.p.) treatment groups (••• = p < 0.001). Data are given as mean ± SEM. Table 3 The latency of penicillin-induced epileptiform activity. Data are presented as mean ± SEM. One-way ANOVA and multiple comparison tests were used: a: p < 0.05, b: p < 0.001 vitamin D3 and paricalcitol groups compared to the control c: p < 0.001 vitamin D3 group compared to paricalcitol groups. Groups
The latency of epileptiform activity (seconds)
Control Vitamin D3 Paricalcitol
90.61 ± 14.86 347.3 ± 32.14; b 156.3 ± 25.35; a; c
groups (p < 0.001) (Fig. 5, Table 3). Pretreatment with paricalcitol (10 μg/kg, i.p.) significantly increased the latency of the epileptiform activity from 90.61 ± 14.86–156.3 ± 25.35 s compared to penicillin4
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glial cells and peripheral nerves of the brain. Genomic and non-genomic pathways of VDR activation were shown to play a role in vitamin Dinduced neuroprotection. Vitamin D can trigger genotypic antiepileptic properties by inducing genes that encode enzymes and cytokines in the neurotransmitter metabolism via the VDR (Garcion et al., 2002). These genomic actions include the binding of 1,25 (OH) D to the nuclear vitamin D receptor and the release of neurotrophin-3, neurotrophin-4, glial cell-derived neurotrophic factor, nerve growth factor, parvalbumin and nitric oxide synthase from the nervous system (Schwaller et al., 2004). These genomic actions occur with a time delay of hours to days (Garcion et al., 2002). Nonetheless, faster vitamin D motions suggest the presence of non-genomic pathways. An anticonvulsant effect was reported in an animal study, which was realized five minutes prior to vitamin D3 on electroconvulsive seizure. It was stated that this effect was caused by non-genomic mechanisms (Borowicz et al., 2007). The GABAergic system has a significant role in the pathogenesis of epilepsy (Holló et al., 2012). GABA-A receptors are a significant target for the non-genomic effect of various neurosteroids and neuroactive hormones, including vitamin D. A number of clinical and experimental epilepsy studies suggested that seizure susceptibility could be caused by the attenuated GABAergic and increased glutamatergic neurotransmission (Schwartzkroin, 1994; Hoogland et al., 2004; Mahfoz et al., 2017). Penicillin is an antagonists of GABA neurotransmitter. The penicillin injection via microsyringe into the neocortex produced epileptiform activity with decreased GABA level and increased glutamate in brain (Fisher, 1989). Sikoglu et al. (2015) demonstrated that vitamin D treatment increased GABA level in patients with bipolar disorders. A study by Kalueff et al. (2006) suggested that Vitamin D receptors are allosteric modulators of GABA receptor and they reduce neuronal excitability. Another study (Jiang et al., 2014) demonstrated that pretreatment with a higher dose of calcitriol (1,25-dihydroxyvitamin D), another vitamin D agonist, elevated GABA level in the hippocampus. Mahfoz et al. (2017) reported that pretreatment with vitamin D significantly increased GABA levels in the brain and decreased the brain glutamate level in lithium-pilocarpine induced status epilepticus in rats. Pretreatment with vitamin D and paricalcitol may reduce penicillin-induced epileptiform activity by increasing GABA levels. In the present study, vitamin D appeared to be more effective on seizure activity than paricalcitol. This can be explained by the fact that vitamin D3 was administered much earlier than paricalcitol. DNA repair, differentiation, apoptosis and cell membrane integrity, and the suppression of the release of proinflammatory cytokines are the other important effects of vitamin D (Bikle, 2009). A number of studies pointed to the essential roles of oxidative stress in the pathophysiology of epilepsy (Royes et al., 2007; Aguiar et al., 2012). A clinical study showed that oxidative stress markers increased in the hippocampus of surgically resected drug-resistant epilepsy patients (Pecorelli et al., 2015). In many experimental studies, increasing oxidative stress was shown in pentylenetetrazole-induced kindling seizures (Rauca et al., 2004), pentylenetetrazole‐induced seizures (Uma Devi et al., 2006) pilocarpine-induced epilepsy model (Freitas et al., 2005; 2009), kainic acid-induced seizures (Shin et al., 2008), spontaneously epileptic rat (Shin et al., 2011), audiogenic seizures in rats (Parreira et al., 2018) and penicillin-induced epileptiform activity in rats (Tubaş et al., 2017). In addition, Ayyildiz et al. (2007) demonstrated that penicillin induced seizures triggered oxidative stress along with an increase in oxidative stress marker malondialdehyde (MDA) and a decrease in antioxidant glutathione (GSH) in brain tissue of epileptic rats. Paricalcitol is an active vitamin D analog that exhibits biological activities similar to what vitamin D does (Drüeke, 2005). Recent studies have shown that vitamin D and paricalcitol have a protective effect on oxidative stress in several organs such as the brain, liver, kidney, pancreas and lung (Garcion et al., 2002; Östman et al., 2009; Ali et al., 2018). Similarly, Mahfoz et al. (2017) reported that pretreatment with vitamin D significantly decreased lithium-pilocarpine induced epileptiform activity by reduced oxidative stress and
injected groups (p < 0.05) (Fig. 5, Table 3). Pretreatment with Vitamin D3 (60.000 IU/kg, i.p.) significantly increased the latency of epileptiform activity from 347.3 ± 32.14–156.3 ± 25.35 s compared to paricalcitol groups (10 μg/kg, i.p.) (p < 0.001) (Fig. 5, Table 3). 4. Discussion In the present study, 10 μg/kg intraperitoneal (i.p.) paricalcitol and 60.000 IU/kg (i.p.) vitamin D3 significantly decreased seizure frequency compared to the saline administration and delayed spike activity and seizure onset. When vitamin D3 and paricalcitol applications were compared, delay in seizure onset was more pronounced in vitamin D3 administration. There is no study in the literature comparing vitamin D analogues in a penicillin epilepsy model. In topical administration of penicillin, a GABA receptor antagonist, seizures start focally but continue in a generalized fashion. Wallenstein (1987) found that after the topical administration of 250 IU penicillin in right hemisphere, spike discharges were observed in the 119th second, which started with 10 s delays in the opposite (left) hemisphere. Seizure activity that arose with penicillin administration to the cortex of experimental animals was classified as simple partial epilepsy model by Fisher (1989). In the last epilepsy classification by ILAE, focal term was used instead of partial. Delayed discharges in the opposite hemisphere after topical penicillin administration showed that penicillin could be used as an experimental model for focal epilepsy (Sagratella et al., 1985; Wallenstein, 1987; Fisher, 1989; Aygun et al., 2019). Cholecalciferol (vitamin D3) is produced from 7- dehydrocholesterol on the skin under the influence of ultraviolet radiation at 290−315 nm wavelength, and this endogenous production is the main source of the vitamin D (Glerup et al., 2000; Holick, 2005). Cholecalciferol is a natural vitamin D. Many clinical and experimental studies reported anticonvulsant effects of vitamin D. In a pilot study, vitamin D combined with antiepileptic drugs reduced seizures in patients with pharmaco-resistant epilepsy (Holló et al., 2012). Anticonvulsant activity of vitamin D was shown in lithium-pilocarpine induced status epilepticus rats (Mahfoz et al., 2017), PTZ-induced seizures (Kalueff et al. (2005) and generalized seizures in PTZ-kindled rat model of epilepsy (AbdelWahab et al., 2017). Siegel et al. (1984) found that intrahippocampal stereotaxic injection of vitamin D3 increased the hippocampal seizure threshold in rats. In addition, Tetich et al. (2005) demonstrated the modulating effects of vitamin D3 treatment for the seizure-related changes in pilocarpine-induced epileptic rat brain. While many studies found positive effects of vitamin D on epileptic convulsions, we have come across only two studies (Uyanıkgil et al., 2016) reporting a positive effect of paricalcitol on epileptic convulsions. In their study using an experimental epilepsy model induced by PTZ (pentilentetrazol), a generalized epilepsy model, Uyanıkgil et al. (2016) evaluated EEG recordings, oxidative stress markers and behavioral conditions. In that study, Racine's Convulsion Scale (RCS) score was importantly lower in groups treated with 5 and 10 μg/kg paricalcitol. In addition, the duration of the first myoclonic jerk (FMJ) latencies were prolonged and a significant reduction was observed in spike percentages in EEG recordings. They also found that this effect was more pronounced in the 10 μg/kg paricalcitol group and concluded that paricalcitol was effective in PTZ-induced seizures (Uyanıkgil et al., 2016). Recently Aygun et al. (2019) demonstrated that acute and chronic treatment of vitamin D and paricalcitol decreased absence-like seizures in genetic epileptic WAG/Rij rat. In the present study, treatment with vitamin D and paricalcitol reduced seizure epileptiform activity and delayed seizure formation in the epileptic rats with penicillin induced seizures. These findings are consistent with the previous studies. Different mechanisms were proposed to explain the anticonvulsant effects of vitamin D. This vitamin exerts its effect through vitamin D receptors, and vitamin D receptors are present in neurons, spinal cord, 5
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increased antioxidants activity in the epileptic rat brain. In addition, various previous studies showed antioxidant characteristic of paricalcitol. It was demonstrated that paricalcitol treatment decreased the levels of oxidative stress markers including MDA, oxidative DNA damage, NADPH oxidase, iNOS levels and increased endogenous antioxidant levels including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and glutathione (GSH) (Husain et al., 2015; Bulut et al., 2016; Ali et al., 2018). Uyanıkgil et al. (2016) reported that acute paricalcitol treatment decreased MDA and increased SOD activity in PTZ-induced epileptic rat brain. In the present study, the antioxidant properties of paricalcitol and vitamin D may have been effective in decreasing the penicillin-induced epileptiform activity. Paricalcitol is an agent with clinical and paraclinical advantages over other vitamin D preparations (Finch et al., 1999; Sprague et al., 2003). Additionally, we have also revealed that paricalcitol is as effective as vitamin D3, whose effect on epileptic seizures has previously been proven. Although many new antiepileptic drugs (AEDs) have been introduced in recent years, the search for new therapies with better tolerability and efficacy remains a significant target. The development and innovation of the new AED are based on preclinical use of the animal models. In the present study, vitamin D and paricalcitol has been shown to be effective on focal epileptic seizures. We conclude that vitamin D and paricalcitol could be effective antiepileptic agents in focal epilepsy. Because of their strong antiepileptogenic effects as well as their costeffectiveness, easy access, high efficiency and good safety considerations, vitamin D and paricalcitol merit further clinical studies. Therefore, a more comprehensive examination of animal models is important in terms of evaluating the mechanisms underlying epileptogenesis and ictogenesis.
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