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Toxicology journal homepage: www.elsevier.com/locate/toxicol
Prevention of organophosphate-induced chronic epilepsy by early benzodiazepine treatment
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Shai Shrot a,b,c,∗,1 , Erez Ramaty a,b,c,1 , Yoav Biala d , Guy Bar-Klein e , Moshe Daninos d , Lyn Kamintsky e , Igor Makarovsky a , Liran Stadlander a , Yossi Rosman a,b,c , Amir Krivoy a,f , Ophir Lavon a , Michael Kassirer a , Alon Friedman e , Yoel Yaari d a
Medical Corps HQ, IDF, Israel, PO Box 02149, Tel-Hashomer Base, Ramat-Gan, Israel Sheba Medical Center, Tel Hashomer, affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel c 2 Sheba Road, Ramat-Gan 52621, Israel 8 9 Q3 d Department of Medical Neurobiology, Institute for Medical Research Israel – Canada, Hebrew University-Hadassah Faculty of Medicine, PO Box 12272, Jerusalem 91121, Israel 10 e Departments of Physiology and Biomedical Engineering, Faculty of Health Sciences, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 8410501, 11 Israel 12 f Sackler Faculty of Medicine, Tel-Aviv University, PO Box 39040, Tel-Aviv 69978, Israel 13 6 7
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Article history: Received 8 April 2014 Received in revised form 10 May 2014 Accepted 28 May 2014 Available online xxx
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Keywords: Paraoxon Pesticide Poisoning Neurotoxicity Midazolam Epileptogenesis
Poisoning with organophosphates (OPs) may induce status epilepticus (SE), leading to severe brain damage. Our objectives were to investigate whether OP-induced SE leads to the emergence of spontaneous recurrent seizures (SRSs), the hallmark of chronic epilepsy, and if so, to assess the efficacy of benzodiazepine therapy following SE onset in preventing the epileptogenesis. We also explored early changes in hippocampal pyramidal cells excitability in this model. Adult rats were poisoned with the paraoxon (450 g/kg) and immediately treated with atropine (3 mg/kg) and obidoxime (20 mg/kg) to reduce acute mortality due to peripheral acetylcholinesterase inhibition. Electrical brain activity was assessed for two weeks during weeks 4–6 after poisoning using telemetric electrocorticographic intracranial recordings. All OP-poisoned animals developed SE, which could be suppressed by midazolam. Most (88%) rats which were not treated with midazolam developed SRSs, indicating that they have become chronically epileptic. Application of midazolam 1 min following SE onset had a significant antiepileptogenic effect (only 11% of the rats became epileptic; p = 0.001 compared to non-midazolam-treated rats). Applying midazolam 30 min after SE onset did not significantly prevent chronic epilepsy. The electrophysiological properties of CA1 pyramidal cells, assessed electrophysiologically in hippocampal slices, were not altered by OP-induced SE. Thus we show for the first time that a single episode of OP-induced SE in rats leads to the acquisition of chronic epilepsy, and that this epileptogenic outcome can be largely prevented by immediate, but not delayed, administration of midazolam. Extrapolating these results to humans would suggest that midazolam should be provided together with atropine and an oxime in the immediate pharmacological treatment of OP poisoning. © 2014 Published by Elsevier Ireland Ltd. 31 32
Abbreviations: OP, organophosphate; SE, status epilepticus; SRS, spontaneous relapsing seizures; ACh, acetylcholine; BZD, benzodiazepines; ECoG, electrocorticographic. ∗ Corresponding author at: 2 Sheba Road, Ramat Gan 52621, Israel. Tel.: +972 3 5302530; fax: +972 3 5357315. E-mail addresses: [email protected] (S. Shrot), [email protected] (E. Ramaty), [email protected] (Y. Biala), [email protected] (G. Bar-Klein), [email protected] (M. Daninos), [email protected] (L. Kamintsky), [email protected] (I. Makarovsky), [email protected] (L. Stadlander), [email protected] (Y. Rosman), [email protected] (A. Krivoy), [email protected] (O. Lavon), [email protected] (M. Kassirer), [email protected] (A. Friedman), [email protected] (Y. Yaari). 1 Denotes equal contribution.
1. Introduction Organophosphates are toxic compounds commonly used as pesticides in agriculture, but can also be used in chemical warfare. In some parts of the developing world, poisoning by OPs causes more deaths than infectious diseases (Eddleston et al., 2002). The warfare related OPs, such as sarine, soman and VX, also known as nerve agents, are extremely toxic and are considered to be among the deadliest agents. The main mechanism of action of OPs is irreversible inhibition of the ACh degrading enzyme,
http://dx.doi.org/10.1016/j.tox.2014.05.010 0300-483X/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Shrot, S., et al., Prevention of organophosphate-induced chronic epilepsy by early benzodiazepine treatment. Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.05.010
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acetylcholinesterase, leading to ACh accumulation in muscarinic and nicotinic cholinergic synapses in both the peripheral and central nervous system. Clinically, respiratory failure is the main cause of death in severe OP poisoning and is attributed to a combination of bronchoconstriction, respiratory muscle paralysis and damage to the medullary respiratory centers (Eddleston et al., 2006). Central nervous system effects of OPs include nonspecific symptoms, such as irritability, restlessness, disorientation and confusion, which can evolve into generalized seizures and SE (Marrs et al., 2007). If the poisoned animal is rescued by relieving peripheral symptoms with atropine and an oxime, SE may continue for several hours causing severe brain damage (Gilat et al., 2005; White et al., 2012). The level of neuronal damage was found to be in correlation with duration and intensity of the SE (McDonough and Shih, 1997). Thus, it is assumed that most of the neuronal damage can be avoided if seizures are controlled promptly. BZDs, potent GABAA receptor enhancers, are highly effective in arresting OP-induced SE when administered early after SE initiation (McDonough and Shih, 1997). However, delayed BZD treatment only temporarily impedes SE and only partially prevents brain damage (de Araujo et al., 2012; Gilat et al., 2005). There are several animal models for studying the consequences of SE. In rodents, SE is commonly induced by chemoconvulsants, e.g. kainic acid and pilocarpine, or by electrical stimulation of the amygdala or hippocampus (Rubio et al., 2010). SE per se can cause a significant excitotoxicity associated with neuronal cell death, regardless of the initial insult. This neuropathology is due to the excessive release of excitatory amino acids from neurons and astrocytes leading to the prolonged depolarization of neurons, increased intracellular calcium and activation of a cascade of metabolic changes that cause neuronal cell death (Holmes, 2002). A high proportion of animals that survive SE develop spontaneous recurrent seizures (SRSs), i.e. chronic epilepsy after a latent period of days to weeks, i.e. a process referred to as epileptogenesis. Previous studies have demonstrated that intrinsic changes in firing characteristics of CA1 hippocampal neurons, in conjunction with changes in network synaptic function, might contribute to the development of chronic epilepsy following pilocarpine-induced SE (Su et al., 2002). Chronic epilepsy induced by acute OP poisoning has not been fully studied nor characterized. Recently, de Araujo et al. (de Araujo et al., 2010) demonstrated that rats poisoned with the OP nerve agent soman, who had experienced SE, showed electrographic SRSs 15 days after poisoning. Here we used paraoxon, a commonly used agricultural OP, to investigate whether paraoxon-induced SE also leads to long-term SRSs and to chronic epilepsy, and if so, to characterize these SRSs and assess whether post-poisoning BZD treatment has an effect on their development. We also attempted to compare this model with the widely used pilocarpine model of chronic epilepsy (Rubio et al., 2010) with respect to changes in hippocampal pyramidal cells excitability that may contribute to the emergence of SRSs.
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2.1. Study design and animals
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The study had two separate parts; (1) prolonged ECoG recordings in awake rats several weeks after poisoning, and (2) intracellular electrophysiological recordings and analysis of intrinsic properties of CA1 pyramidal neurons in control versus OP-poisoned rats. All experiments were performed according to the institute’s guidelines for animal care and use. Adult SpragueDawley rats (300 ± 20 g) were randomly divided according to treatment into four groups (Table 1). (1) Non-poisoned rats, treated
Table 1 Study groups in the two stages of the study. Group Treatment
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ATOX PXN + ATOX PXN + ATOX + MID 1 a PXN + ATOX + MID 30 a
Intracellular recordings
In vivo (ECoG) recording
Number of animals
(Total number of neurons)
Number of animals
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(18) (29) (30) (33)
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ATOX – atropine + obidoxime, PXN – paraoxon, MID – midazolam, ECoG – electrocorticography. a Time in minutes post seizure onset until MID was injected.
solely with antidotes (ATOX: atropine 3 mg/kg, i.m. and obidoxime 20 mg/kg, i.m.), served as controls; (2) paraoxon (450 g/kg; ∼1.4 LD50 i.m.) poisoned rats treated with ATOX 1 min later; (3) paraoxon (450 g/kg), ATOX (1 min) and midazolam (MID, 1 mg/kg, i.m.) 1 min after the onset of convulsions; and (4) paraoxon (450 g/kg), ATOX (1 min) and midazolam (MID, 1 mg/kg, i.m.) 30 min after the onset of convulsions. ATOX treatment was given to all poisoned rats in order to reduce acute mortality due to peripheral acetylcholine-esterase inhibition. 2.2. Clinical evaluation Animals were observed continuously for at least an hour after intoxication and scored for motor manifestations of seizure activity on 10, 20 and 60 min. Scores were assigned as follows: (0) no convulsions, (1) chewing and facial clonus, (2) tremor and focal convulsions, and (3) tonic-clonic generalized convulsions. Midazolam was injected in experimental groups 3 and 4, 1 and 30 min after appearance of chewing and facial clonus (score 1 according to our scale), respectively. 2.3. In vivo recordings and analysis ECoG was recorded 4–6 weeks after poisoning using established methods (Bastlund et al., 2004; Levi et al., 2012; Timofeeva and Gordon, 2001). In short, 21 days following drug treatment, rats were deeply anesthetized with ketamine (75 mg/kg, IP) and xylazine (5 mg/kg, IP) and placed into a stereotactic frame. The skin was disinfected and a sagittal incision was made. Chronically implanted electrodes were placed in the epidural space; 3 mm caudal and ± 2.5 mm lateral to bregma. Following a recovery period of 7 days, electrical activity was acquired (1 KHz) for 2 weeks using a telemetric ECoG system (CTA-F40 transmitter and RPC1 receiver, Data Science International, United States). Recordings were analyzed off-line using a custom seizure detection algorithm developed in house. Briefly, ECoG signals were band-pass filtered (2–100 Hz), and five features were extracted from 2 s long epochs (with 1 s overlap), representing signal properties both in the time and frequency domains. The features were then fed to an artificial neural network (ANN)-based classifier, pre-trained to distinguish between epochs corresponding to seizure and non-seizure activity. We defined “seizure like events” (SLEs) as a minimum of 6 consecutive epochs with ‘positive’ ANN detections. An animal was classified as “epileptic” when the algorithm detected at least 2 unprovoked SLEs over 1 h apart. Algorithm performance has been previously validated in 3 different models of epilepsy (namely, genetic (Ketzef et al., 2011), SE-induced (Becker et al., 2008) and albumin-induced epilepsy (Weissberg et al., 2011)), revealing overall sensitivity and positive predictive value over 98% (in a total of >2800 h of ECoG, n = 15).
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2.4. Electrophysiological recordings from hippocampal slices Electrophysiological recordings were performed in transverse hippocampal slices (400 m thick) resected from rats one to two weeks after drug treatments as previously reported (Becker et al., 2008). Briefly, rats were decapitated under deep isoflurane anesthesia and their brains were quickly removed and immersed in ice-cold, carbogenated (95% O2 , 5% CO2 ) modified artificial cerebrospinal fluid (aCSF), comprising (in mM): NaCl 130, NaHCO3 24, KCl 3.5, NaH2 PO4 1.25, d-Glucose 10, MgSO4 5, and CaCl2 0.2 for several minutes. Transverse hippocampal slices were prepared with a vibrating microslicer (Leica VT 1200 S, Solms, Germany), and the hippocampal region was isolated. Slices were maintained in room temperature incubated in carbogenated aCSF composed of (in mM): NaCl 130, NaHCO3 24, KCl 3.5, d-Glucose 10, MgSO4 2, and CaCl2 1.6. Slices were then placed one at a time in an interface slice chamber (33 ◦ C) and perfused with carbogenated aCSF. After a recovery period of 30 min to 1 h in the recording chamber, intracellular electrophysiological recordings were obtained from CA1 pyramidal cells using borosilicate glass capillaries (80–100 M), filled with intracellular solution composed of 4 M K-Acetate and 0.1 M KCl. Only neurons with resting potential more negative than −60 mV and overshooting action potentials were included in this study. 2.5. Measurement of intrinsic properties of CA1 pyramidal cells
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In order to elicit action potentials, positive current pulses were injected into CA1 pyramidal cells in increasing magnitude. Short (4 ms long) pulses were used to elicit single action potentials. Longer (180 ms) pulses were used to elicit repetitive firing. The active and passive properties of single neurons were measured as previously described (Chen et al., 2011). Briefly, we measured resting potential (Vrest), input resistance (RN ), spike threshold, amplitude (measured from Vrest to spike peak) and width (measured at half spike amplitude), fast after-hyperpolarization (fAHP; the membrane voltage at the end of fast spike repolarization), spike afterdepolarization (ADP; measured as the integral or “area under the curve” from the fAHP until the ADP attains Vrest). The firing mode of CA1 pyramidal cells was assessed by injecting 180-ms long excitatory current pulses incrementing in steps of 50 pA. The firing patterns of CA1 pyramidal cells varied along a spectrum of “burstiness”(Azouz et al., 1997; Jensen et al., 1994; Su et al., 2001): (1) Regular firing neurons, or nonbursters, which fired only one spike in response to threshold stimuli. Stronger stimuli evoked trains of spikes whose frequency increased with stimulus strength and accommodated with time. (2) Low-threshold bursters (LTBs) that fired a high-frequency cluster of 2–7 spikes overriding a distinct depolarizing potential already in response to threshold stimuli. Stronger stimuli evoked additional solitary spikes or short spike bursts, but these spikes were clearly separated from the primary burst. (3) Neurons with intermediate bursting capabilities, referred to collectively as high-threshold bursters (HTBs). These neurons fired solitary or unclustered spikes in response to 1–2× threshold stimuli, but stronger stimuli evoked a primary burst followed by several separated spikes. A bursting index (BI) developed by Chen et al. (2011) for distinguishing between nonbursters HTBs was used. The threshold BI value was 2, so that neurons with BI ≥ 2 were classified as HTBs.
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2.6. Statistical analyses
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Fisher Exact Probability Test was used to assess the significance of differences between incidences of epilepsy in various treatment groups in the ECoG studies. Student’s t-test and ANOVA were used for analysis of changes in the electrophysiological parameters of
Fig. 1. Clinical score of animals in different experimental groups in the first hour after paraoxon poisoning. No convulsions were seen in animals treated only with peripheral antidotal treatment (ATOX, blue bars). Data is shown as mean score and standard deviation. Survival rate is shown in brackets. *p < 0.05, **p < 0.005 (Mann–Withney U test), when the comparison is relative to the bar whose color is identical to the color of the asterisk. (For interpretation of the references to color Q6 in this figure legend, the reader is referred to the web version of the article.)
hippocampal slices (MS-Excel, Microsoft Inc., and SPSS, IBM Inc.). Mann–Whitney U test was used for analysis of the clinical score and the mean SRS frequencies in various treatment groups (SPSS). The significance level was set to p < 0.05.
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All OP-poisoned animals developed generalized tonic-clonic convulsions, starting within 1–2 min following the paraoxon injection. Mean clinical scores and survival rates are presented in Fig. 1. 3.2. In vivo ECoG recordings None of the non-poisoned rats (group 1) was found epileptic 4–6 weeks after treatment (Fig. 2B). Seven out of eight (88%) of the OP-poisoned rats who did not receive midazolam treatment (group 2) were classified as epileptic based on recordings of at least two spontaneous, unprovoked seizures within the observation time window (p = 0.001, Fisher Exact Probability Test, when compared with non-poisoned animals). Average number of seizures per day was 0.49 ± 0.13 (Fig. 2C). Although video monitoring was not performed for all rats in this study, behavioral tonic-clonic seizures were observed occasionally in most poisoned rats (see supplementary materials). Importantly, treatment with midazolam 1 min after appearance of convulsions (group 3, PXN + ATOX + MID 1 ) resulted in a significant reduction in the number of epileptic animals, as only one out of nine (11%) rats showed spontaneous seizures 4–6 weeks after poisoning (p = 0.015 when compared with group 2, PXN + ATOX). The average daily number of seizures was also lower in the animals treated with midazolam 1 min after seizures’ onset (0.1 ± 0.09 seizure/day, p = 0.02 compared with group 2, i.e. PXN + ATOX). Midazolam injection 30 min following appearance of convulsions (group 4) resulted in 50% (4/8) of the rats being epileptic, with a mean of 0.24 ± 0.1 seizures/day; however, not statistically significant compared to group 2, i.e. PXN + ATOX (p = 0.57 and p = 0.15, respectively). The individual number of seizures in animals in different experimental groups is presented in Fig. 2D. Together, these data show that midazolam is effective in suppression of epileptogenesis induced by paraoxon when given within 1 min after poisoning.
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Fig. 2. Incidence of epilepsy in the four groups of rats as revealed by in vivo ECoG recordings. (A) Representative ECoG recordings showing a seizure 4–6 weeks after paraoxon poisoning. (B) Incidence of epileptic animals in the four groups. (C) Average number of daily SRSs in four groups. (D) The number of seizures in individual animals within the four groups. *p < 0.05, **p < 0.005, when the comparison is relative to the bar whose color is identical to the color of the asterisk. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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3.3. Electrophysiological properties of CA1 pyramidal cells in control and OP-poisoned rats Intracellular recordings were performed in a total of 110 CA1 pyramidal cells from the four groups of rats (Table 1) between 1 to 2 weeks after paraoxon poisoning. Passive and active neuronal properties in the four groups were not significantly different, except for two minor differences in action potential amplitudes (Table 2). We next compared the four groups of rats with respect to the firing mode of CA1 pyramidal cells. The firing patterns of CA1 pyramidal cells in the four groups, varied along a spectrum of “burstiness” (see Section 2) (Azouz et al., 1997; Jensen et al., 1994; Su et al., 2001). Fig. 3 A shows different firing patterns in representative CA1 neurons in the current study. The mean BI values in the four groups were 1.9 ± 0.3 (ATOX), 2.0 ± 0.05 (paraoxon + ATOX), 1.9 ± 0.04
(paraoxon + ATOX + MID 1 ) and 2.7 ± 0.4 (paraoxon + ATOX + MID 30 ). No significant differences were found between the groups (Fig. 3B). A BI value of 2 was used as a threshold to discriminate between nonbursters (Fig. 3A(I)) and bursters. The latter group of neurons was further subdivided into HTBs (Fig. 3A(II)) and LTBs (Fig. 3A(III)) according to their minimal firing response (see Section 2). The percent of bursters in the four groups was as follows: 50% (9/18 cells; group 1), 38% (11/29 cells; group 2), 41% (12/29 cells; group 3) and 48% (16/33 cells; group 4). These values were not significantly different from each other. Likewise, the percentages of HTBs (38, 24, 30 and 33%, respectively) and LTBs (11, 14, 11 and 15%, respectively) were not significantly different amongst the four groups (Fig. 3C). Thus, we could not detect significant changes in the intrinsic excitability of CA1 pyramidal cells early in epileptogenesis.
Table 2 Active and passive properties of neuronal pyramidal cells in hippocampal CA1 region tested in four groups. The results described as average ± standard error.
Vrest (mV) RN (M) Spike threshold (mV) Spike amplitude (mV) Spike width (ms) fAHP (mV) ADP
Group 1 (ATOX)
Group 2 (PXN + ATOX)
Group 3 (PXN + ATOX + MID 1 )
Group 4 (PXN + ATOX + MID 30 )
−67.3 50.5 −57.7 95.0 1.3 −62.7 187.4
−68.2 45.3 −58.3 98.3 1.4 −64.2 183.4
−68.1 42.3 −57.3 96.9 1.4 −60.8 192.0
−66.4 46.6 −52.2 91.0 1.3 −61.5 170.2
± ± ± ± ± ± ±
0.7 4.4 1.1 1.6 0.0 0.7 17.8
± ± ± ± ± ± ±
0.2 0.7 0.2 0.2* 0.0 0.2 3.3
± ± ± ± ± ± ±
0.1 0.8 0.1 0.2* 0.0* 0.1 3.0
± ± ± ± ± ± ±
0.6 2.6 4.1 1.2 0.0 1.0 18.5
PXN – paraoxon. * Significantly change from group 4, P < 0.05.
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Fig. 3. (A) Firing patterns of three CA1 pyramidal cells in hippocampal slices resected from animals belonging to group 3 (paraoxon + ATOX + MID 1 ). In each panel, the responses of the neuron to threshold (lower voltage trace) and suprathreshold (upper voltage trace) depolarizing current stimuli are shown. The resting membrane potential of each neuron is depicted to the left of the upper trace. (I) This neuron manifested a regular firing pattern. The BI value was 1.0 and the neuron was classified as a regular firing (nonbursting) neuron. (II) This neuron tended to fire an early burst upon strong stimulation, whereas threshold stimuli evoked only one action potential. The BI value of this neuron was 2.8 classifying it as a HTB. (III) This neuron fired bursts of at least 3 spikes even at threshold stimulation and was classified as an LTB. Its BI value was 5.8. (B) Bursting indices of the hippocampal CA1 neurons. The bar diagrams depict the mean BI values for each group of neurons. The differences between the mean values were not statistically significant. (C) The incidence of bursting neurons. The bar diagram depicts the percentage of bursters in each group, comprised of the percentages of LTBs (dark gray) and HTBs (light gray). The differences between the mean values were not statistically significant.
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We show here that rapid antidotal treatment (ATOX) of rats severely poisoned with the OP agent paraoxon results in a high survival rate, but does not prevent the onset of a prolonged SE. Furthermore, most surviving animals undergo epileptogenesis leading to the emergence of SRSs, the hallmark of epilepsy, within several weeks. Both SE and the ensuing epileptogenesis can be dramatically halted by injection of the BZD midazolam immediately upon SE onset. Delaying midazolam treatment by 30 min appears to have only a marginal effect in preventing OP-induced epilepsy.
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4.1. OP-induced epilepsy
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We detected chronic epilepsy in 88% of non-BZD treated rats during the 2 weeks ECoG observation period performed 4–6 weeks following OP poisoning. The presence of SRSs in our study is in accordance with a recently published study by de Araujo et al. (2012) in which the OP nerve agent soman (s.c. 1 LD50 ) was used to induce SE and diazepam was administered 30 min thereafter to arrest the SE. They reported that 15 days following OP poisoning only 28.9% of rats experiencing SE acquired chronic epilepsy. The relatively low incidence of epileptic animals following soman poisoning compared to our results may reflect their early time window of ECoG recording, as epileptogenesis is a progressive process which may develop over several weeks (Becker et al., 2008). Notwithstanding, the fact that two different OPs can lead to chronic epilepsy in a substantial fraction of poisoned animals suggests that chronic
epilepsy may be a pathological consequence of OP poisoning in general. Although not classified as seizures, long-lasting ECoG changes following OP exposure were demonstrated in humans and animals exposed to OPs (Carpentier et al., 2001; Sekijima et al., 1995). Although the initial trigger for SE in OP poisoning is predominately cholinergic, the associated excess release of glutamate is considered the main contributor to OP-induced SE perpetuation and excitotoxicity (McDonough and Shih, 1997). High levels of extracellular glutamate lead to severe neuronal damage due to increased influx of calcium into the cells, activating intracellular catalytic enzymes that damage cell membranes, cytoskeleton and organelles structure. Swollen astrocytes may release their load of glutamate thus amplifying the excitotoxic neuronal damage (Kimelberg, 1995). The mechanisms underlying the development of chronic epilepsy following OP poisoning are not understood, and could be related to glutamate excitotoxicity causing neuronal damage (McDonough and Shih, 1997), blood–brain barrier penetration (Bolwig, 1988; Grauer et al., 2001), inflammatory responses (Collombet, 2011) and reactive gliosis (Britt et al., 2000). 4.2. Prevention of OP-induced SRSs We show here for the first time the dramatic suppression of OP-induced chronic epilepsy by administrating midazolam early after poisoning. The early injection of midazolam was critical for its antiepileptogenic action: midazolam injection 30 min after SE onset, while suppressing the SEs, only partially (and not significantly) prevented the emergence of SRSs. We thus conclude that
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the Henri and Erna Leir Chair for Research in Neurodegenerative Q5 Diseases.
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a 30 min episode of OP-induced SE is sufficient to trigger epileptogenesis, which is not reversible even if the acute seizures are suppressed. This finding establishes a relationship between SE duration and subsequent chronic epilepsy in OP-poisoned rats. Congruently, McDonough and colleagues showed that at least 20 min of seizure activity is necessary for neuropathological damage to occur in rats following OP exposure (McDonough et al., 1995). Klitgaard et al. using the pilocarpine model did not find a difference in SRSs frequencies in animals experiencing 30 versus 120 min of SE, even though SRSs emerged only after a minimal SE duration of 30 min (Klitgaard et al., 2002). The time window for effectively preventing OP-induced chronic epilepsy with midazolam, as demonstrated in our study, was less than 30 min. This is much shorter than the 1–2 h time-window reported for the electrical stimulation-induced SE model using phenobarbital, phenytoin (Prasad et al., 2002) or diazepam (Pitkanen et al., 2005) for SE suppression. Possibly the systemic OP poisoning causes more extensive changes in the brain than those produced by focal electrical stimulation.
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4.3. Pilocarpine and OP-induced changes in intrinsic excitability
Azouz, R., Alroy, G., Yaari, Y., 1997. Modulation of endogenous firing patterns by osmolarity in rat hippocampal neurones. J. Physiol. 502 (Pt 1), 175– 187. Bastlund, J.F., Jennum, P., Mohapel, P., Vogel, V., Watson, W.P., 2004. Measurement of cortical and hippocampal epileptiform activity in freely moving rats by means of implantable radiotelemetry. J. Neurosci. Methods 138 (1–2), 65–72. Becker, A.J., Pitsch, J., Sochivko, D., Opitz, T., Staniek, M., Chen, C.C., Campbell, K.P., Schoch, S., Yaari, Y., Beck, H., 2008. Transcriptional upregulation of Cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy. J. Neurosci. 28 (49), 13341–13353. Bolwig, T.G., 1988. Blood–brain barrier studies with special reference to epileptic seizures. Acta Psychiatr. Scand. Suppl. 345 (15–20), 15–20. Britt Jr., J.O., Martin, J.L., Okerberg, C.V., Dick Jr., E.J., 2000. Histopathologic changes in the brain, heart, and skeletal muscle of rhesus macaques, ten days after exposure to soman (an organophosphorus nerve agent). Comp. Med. 50 (2), 133– 139. Carpentier, P., Foquin, A., Dorandeu, F., Lallement, G., 2001. Delta activity as an early indicator for soman-induced brain damage: a review. Neurotoxicology 22 (3), 299–315. Chen, S., Su, H., Yue, C., Remy, S., Royeck, M., Sochivko, D., Opitz, T., Beck, H., Yaari, Y., 2011. An increase in persistent sodium current contributes to intrinsic neuronal bursting after status epilepticus. J. Neurophysiol. 105 (1), 117–129. Collombet, J.M., 2011. Nerve agent intoxication: recent neuropathophysiological findings and subsequent impact on medical management prospects. Toxicol. Appl. Pharmacol. 255 (3), 229–241. de Araujo, F.M., Lumley, L.A., Robison, C., Tong, L.C., Lichtenstein, S., Yourick, D.L., 2010. Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats. Epilepsia 51 (8), 1503–1510. de Araujo, F.M., Rossetti, F., Chanda, S., Yourick, D., 2012. Exposure to nerve agents: from status epilepticus to neuroinflammation, brain damage, neurogenesis and epilepsy. Neurotoxicology 33 (6), 1476–1490. Eddleston, M., Karalliedde, L., Buckley, N., Fernando, R., Hutchinson, G., Isbister, G., Konradsen, F., Murray, D., Piola, J.C., Senanayake, N., Sheriff, R., Singh, S., Siwach, S.B., Smit, L., 2002. Pesticide poisoning in the developing world – a minimum pesticides list. Lancet 360 (9340), 1163–1167. Eddleston, M., Mohamed, F., Davies, J.O., Eyer, P., Worek, F., Sheriff, M.H., Buckley, N.A., 2006. Respiratory failure in acute organophosphorus pesticide self-poisoning. QJM 99 (8), 513–522. Gilat, E., Kadar, T., Levy, A., Rabinovitz, I., Cohen, G., Kapon, Y., Sahar, R., Brandeis, R., 2005. Anticonvulsant treatment of sarin-induced seizures with nasal midazolam: an electrographic, behavioral, and histological study in freely moving rats. Toxicol. Appl. Pharmacol. 209 (1), 74–85. Grauer, E., Ben, N.D., Lustig, S., Kobiler, D., Kapon, J., Danenberg, H.D., 2001. Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors. Life Sci. 68 (9), 985–990. Holmes, G.L., 2002. Seizure-induced neuronal injury: animal data. Neurology 59 (9 Suppl. 5), S3–S6. Jensen, M.S., Azouz, R., Yaari, Y., 1994. Variant firing patterns in rat hippocampal pyramidal cells modulated by extracellular potassium. J. Neurophysiol. 71 (3), 831–839. Ketzef, M., Kahn, J., Weissberg, I., Becker, A.J., Friedman, A., Gitler, D., 2011. Compensatory network alterations upon onset of epilepsy in synapsin triple knock-out mice. Neuroscience 189, 108–122, http://dx.doi.org/ 10.1016/j.neuroscience.2011.05.030. Kimelberg, H.K., 1995. Current concepts of brain edema. Review of laboratory investigations. J. Neurosurg. 83 (6), 1051–1059. Klitgaard, H., Matagne, A., Vanneste-Goemaere, J., Margineanu, D.G., 2002. Pilocarpine-induced epileptogenesis in the rat: impact of initial duration of status epilepticus on electrophysiological and neuropathological alterations. Epilepsy Res. 51 (1–2), 93–107.
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The pilocarpine model of SE is widely used in the study of epileptogenesis leading to chronic epilepsy (Rubio et al., 2010). Pilocarpine is a muscarinic agonist which triggers SE by initially activating muscarinic receptors, leading to the emergence of SRSs within a week or two. Not surprisingly, the pilocarpine model replicates also the neuropathological changes that are caused by OPs (Tang et al., 2011). It has been shown that pilocarpine-induced SE alters the intrinsic excitability of principal hippocampal neurons. Most dramatically, a single episode of pilocarpine induced SE, causes a large fraction of CA1 pyramidal cells to convert from regular firing to burst-firing mode, a change that appears within 3 days and persists for many weeks after its induction (Sanabria et al., 2001). Intriguingly, we show here that OP-induced SE, even though caused by cholinergic stimulation, was not followed by a change in the intrinsic electrophysiological properties of CA1 pyramidal cells. The proportion of bursters did not increase even in the group of rats that did not receive midazolam and thus experienced a prolonged SE (group 2). This dissimilarity between the two models is intriguing, as both involve cholinergic stimulation and epileptogenesis and suggest that the pilocarpine model may not be a surrogate for OP-induced chronic epilepsy and for the screening of effective neuroprotecting agents against OP poisoning (Tang et al., 2011; Tetz et al., 2006).
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We show that severe paraoxon poisoning associated with SE induces chronic epilepsy, leading to the emergence of SRSs in most poisoned rats. Midazolam is effective in preventing paraoxoninduced chronic epilepsy if administered 1 min after seizures onset, but not 30 min thereafter. Extrapolating these results to humans would indicate that people at risk to agricultural or warfare OP poisoning, may benefit from the availability of an injection device containing not only the classical OP antidotes, atropine and an oxime, but also midazolam. Further research is necessary to fully characterize the OP-induced epileptogenesis leading to chronic epilepsy and its pathogenesis to allow for more efficient preventive treatments at later stages. Funding This work was supported by a grant from the Medical Corps of the Israeli Defense Force. YB, MD and YY were supported by
Conflicts of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2014.05.010. References
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Levi, H., Schoknecht, K., Prager, O., Chassidim, Y., Weissberg, I., Serlin, Y., Friedman, A., 2012. Stimulation of the sphenopalatine ganglion induces reperfusion and blood–brain barrier protection in the photothrombotic stroke model. PLoS. ONE 7 (6), e39636. Marrs, T., Maynard, C., Sidell Frederick, R.L.R., 2007. Chemical Warefare Agents Toxicology and Treatment. John Wiley & Sons, Chichester. McDonough Jr., J.H., Dochterman, L.W., Smith, C.D., Shih, T.M., 1995. Protection against nerve agent-induced neuropathology, but not cardiac pathology, is associated with the anticonvulsant action of drug treatment. Neurotoxicology 16 (1), 123–132. McDonough Jr., J.H., Shih, T.M., 1997. Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology. Neurosci. Biobehav. Rev. 21 (5), 559–579. Pitkanen, A., Kharatishvili, I., Narkilahti, S., Lukasiuk, K., Nissinen, J., 2005. Administration of diazepam during status epilepticus reduces development and severity of epilepsy in rat. Epilepsy Res. 63 (1), 27–42. Prasad, A., Williamson, J.M., Bertram, E.H., 2002. Phenobarbital and MK-801, but not phenytoin, improve the long-term outcome of status epilepticus. Ann. Neurol. 51 (2), 175–181. Rubio, C., Rubio-Osornio, M., Retana-Marquez, S., Veronica Custodio, M.L., Paz, C., 2010. In vivo experimental models of epilepsy. Cent. Nerv. Syst. Agents Med. Chem. 10 (4), 298–309. Sanabria, E.R., Su, H., Yaari, Y., 2001. Initiation of network bursts by Ca2+ -dependent intrinsic bursting in the rat pilocarpine model of temporal lobe epilepsy. J. Physiol. 532 (Pt 1), 205–216. Sekijima, Y., Morita, H., Shindo, M., Okudera, H., Shibata, T., 1995. A case of severe sarin poisoning in the sarin attack in Matsumoto – one-year follow-up
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Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Prevention of organophosphate-induced chronic epilepsy by early benzodiazepine treatment
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Shai Shrot a,b,c,∗,1 , Erez Ramaty a,b,c,1 , Yoav Biala d , Guy Bar-Klein e , Moshe Daninos d , Lyn Kamintsky e , Igor Makarovsky a , Liran Stadlander a , Yossi Rosman a,b,c , Amir Krivoy a,f , Ophir Lavon a , Michael Kassirer a , Alon Friedman e , Yoel Yaari d a
Medical Corps HQ, IDF, Israel, PO Box 02149, Tel-Hashomer Base, Ramat-Gan, Israel Sheba Medical Center, Tel Hashomer, affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel c 2 Sheba Road, Ramat-Gan 52621, Israel 8 9 Q3 d Department of Medical Neurobiology, Institute for Medical Research Israel – Canada, Hebrew University-Hadassah Faculty of Medicine, PO Box 12272, Jerusalem 91121, Israel 10 e Departments of Physiology and Biomedical Engineering, Faculty of Health Sciences, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 8410501, 11 Israel 12 f Sackler Faculty of Medicine, Tel-Aviv University, PO Box 39040, Tel-Aviv 69978, Israel 13 6 7
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Article history: Received 8 April 2014 Received in revised form 10 May 2014 Accepted 28 May 2014 Available online xxx
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Keywords: Paraoxon Pesticide Poisoning Neurotoxicity Midazolam Epileptogenesis
Poisoning with organophosphates (OPs) may induce status epilepticus (SE), leading to severe brain damage. Our objectives were to investigate whether OP-induced SE leads to the emergence of spontaneous recurrent seizures (SRSs), the hallmark of chronic epilepsy, and if so, to assess the efficacy of benzodiazepine therapy following SE onset in preventing the epileptogenesis. We also explored early changes in hippocampal pyramidal cells excitability in this model. Adult rats were poisoned with the paraoxon (450 g/kg) and immediately treated with atropine (3 mg/kg) and obidoxime (20 mg/kg) to reduce acute mortality due to peripheral acetylcholinesterase inhibition. Electrical brain activity was assessed for two weeks during weeks 4–6 after poisoning using telemetric electrocorticographic intracranial recordings. All OP-poisoned animals developed SE, which could be suppressed by midazolam. Most (88%) rats which were not treated with midazolam developed SRSs, indicating that they have become chronically epileptic. Application of midazolam 1 min following SE onset had a significant antiepileptogenic effect (only 11% of the rats became epileptic; p = 0.001 compared to non-midazolam-treated rats). Applying midazolam 30 min after SE onset did not significantly prevent chronic epilepsy. The electrophysiological properties of CA1 pyramidal cells, assessed electrophysiologically in hippocampal slices, were not altered by OP-induced SE. Thus we show for the first time that a single episode of OP-induced SE in rats leads to the acquisition of chronic epilepsy, and that this epileptogenic outcome can be largely prevented by immediate, but not delayed, administration of midazolam. Extrapolating these results to humans would suggest that midazolam should be provided together with atropine and an oxime in the immediate pharmacological treatment of OP poisoning. © 2014 Published by Elsevier Ireland Ltd. 31 32
Abbreviations: OP, organophosphate; SE, status epilepticus; SRS, spontaneous relapsing seizures; ACh, acetylcholine; BZD, benzodiazepines; ECoG, electrocorticographic. ∗ Corresponding author at: 2 Sheba Road, Ramat Gan 52621, Israel. Tel.: +972 3 5302530; fax: +972 3 5357315. E-mail addresses: [email protected] (S. Shrot), [email protected] (E. Ramaty), [email protected] (Y. Biala), [email protected] (G. Bar-Klein), [email protected] (M. Daninos), [email protected] (L. Kamintsky), [email protected] (I. Makarovsky), [email protected] (L. Stadlander), [email protected] (Y. Rosman), [email protected] (A. Krivoy), [email protected] (O. Lavon), [email protected] (M. Kassirer), [email protected] (A. Friedman), [email protected] (Y. Yaari). 1 Denotes equal contribution.
1. Introduction Organophosphates are toxic compounds commonly used as pesticides in agriculture, but can also be used in chemical warfare. In some parts of the developing world, poisoning by OPs causes more deaths than infectious diseases (Eddleston et al., 2002). The warfare related OPs, such as sarine, soman and VX, also known as nerve agents, are extremely toxic and are considered to be among the deadliest agents. The main mechanism of action of OPs is irreversible inhibition of the ACh degrading enzyme,
http://dx.doi.org/10.1016/j.tox.2014.05.010 0300-483X/© 2014 Published by Elsevier Ireland Ltd.
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acetylcholinesterase, leading to ACh accumulation in muscarinic and nicotinic cholinergic synapses in both the peripheral and central nervous system. Clinically, respiratory failure is the main cause of death in severe OP poisoning and is attributed to a combination of bronchoconstriction, respiratory muscle paralysis and damage to the medullary respiratory centers (Eddleston et al., 2006). Central nervous system effects of OPs include nonspecific symptoms, such as irritability, restlessness, disorientation and confusion, which can evolve into generalized seizures and SE (Marrs et al., 2007). If the poisoned animal is rescued by relieving peripheral symptoms with atropine and an oxime, SE may continue for several hours causing severe brain damage (Gilat et al., 2005; White et al., 2012). The level of neuronal damage was found to be in correlation with duration and intensity of the SE (McDonough and Shih, 1997). Thus, it is assumed that most of the neuronal damage can be avoided if seizures are controlled promptly. BZDs, potent GABAA receptor enhancers, are highly effective in arresting OP-induced SE when administered early after SE initiation (McDonough and Shih, 1997). However, delayed BZD treatment only temporarily impedes SE and only partially prevents brain damage (de Araujo et al., 2012; Gilat et al., 2005). There are several animal models for studying the consequences of SE. In rodents, SE is commonly induced by chemoconvulsants, e.g. kainic acid and pilocarpine, or by electrical stimulation of the amygdala or hippocampus (Rubio et al., 2010). SE per se can cause a significant excitotoxicity associated with neuronal cell death, regardless of the initial insult. This neuropathology is due to the excessive release of excitatory amino acids from neurons and astrocytes leading to the prolonged depolarization of neurons, increased intracellular calcium and activation of a cascade of metabolic changes that cause neuronal cell death (Holmes, 2002). A high proportion of animals that survive SE develop spontaneous recurrent seizures (SRSs), i.e. chronic epilepsy after a latent period of days to weeks, i.e. a process referred to as epileptogenesis. Previous studies have demonstrated that intrinsic changes in firing characteristics of CA1 hippocampal neurons, in conjunction with changes in network synaptic function, might contribute to the development of chronic epilepsy following pilocarpine-induced SE (Su et al., 2002). Chronic epilepsy induced by acute OP poisoning has not been fully studied nor characterized. Recently, de Araujo et al. (de Araujo et al., 2010) demonstrated that rats poisoned with the OP nerve agent soman, who had experienced SE, showed electrographic SRSs 15 days after poisoning. Here we used paraoxon, a commonly used agricultural OP, to investigate whether paraoxon-induced SE also leads to long-term SRSs and to chronic epilepsy, and if so, to characterize these SRSs and assess whether post-poisoning BZD treatment has an effect on their development. We also attempted to compare this model with the widely used pilocarpine model of chronic epilepsy (Rubio et al., 2010) with respect to changes in hippocampal pyramidal cells excitability that may contribute to the emergence of SRSs.
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The study had two separate parts; (1) prolonged ECoG recordings in awake rats several weeks after poisoning, and (2) intracellular electrophysiological recordings and analysis of intrinsic properties of CA1 pyramidal neurons in control versus OP-poisoned rats. All experiments were performed according to the institute’s guidelines for animal care and use. Adult SpragueDawley rats (300 ± 20 g) were randomly divided according to treatment into four groups (Table 1). (1) Non-poisoned rats, treated
Table 1 Study groups in the two stages of the study. Group Treatment
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ATOX PXN + ATOX PXN + ATOX + MID 1 a PXN + ATOX + MID 30 a
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ATOX – atropine + obidoxime, PXN – paraoxon, MID – midazolam, ECoG – electrocorticography. a Time in minutes post seizure onset until MID was injected.
solely with antidotes (ATOX: atropine 3 mg/kg, i.m. and obidoxime 20 mg/kg, i.m.), served as controls; (2) paraoxon (450 g/kg; ∼1.4 LD50 i.m.) poisoned rats treated with ATOX 1 min later; (3) paraoxon (450 g/kg), ATOX (1 min) and midazolam (MID, 1 mg/kg, i.m.) 1 min after the onset of convulsions; and (4) paraoxon (450 g/kg), ATOX (1 min) and midazolam (MID, 1 mg/kg, i.m.) 30 min after the onset of convulsions. ATOX treatment was given to all poisoned rats in order to reduce acute mortality due to peripheral acetylcholine-esterase inhibition. 2.2. Clinical evaluation Animals were observed continuously for at least an hour after intoxication and scored for motor manifestations of seizure activity on 10, 20 and 60 min. Scores were assigned as follows: (0) no convulsions, (1) chewing and facial clonus, (2) tremor and focal convulsions, and (3) tonic-clonic generalized convulsions. Midazolam was injected in experimental groups 3 and 4, 1 and 30 min after appearance of chewing and facial clonus (score 1 according to our scale), respectively. 2.3. In vivo recordings and analysis ECoG was recorded 4–6 weeks after poisoning using established methods (Bastlund et al., 2004; Levi et al., 2012; Timofeeva and Gordon, 2001). In short, 21 days following drug treatment, rats were deeply anesthetized with ketamine (75 mg/kg, IP) and xylazine (5 mg/kg, IP) and placed into a stereotactic frame. The skin was disinfected and a sagittal incision was made. Chronically implanted electrodes were placed in the epidural space; 3 mm caudal and ± 2.5 mm lateral to bregma. Following a recovery period of 7 days, electrical activity was acquired (1 KHz) for 2 weeks using a telemetric ECoG system (CTA-F40 transmitter and RPC1 receiver, Data Science International, United States). Recordings were analyzed off-line using a custom seizure detection algorithm developed in house. Briefly, ECoG signals were band-pass filtered (2–100 Hz), and five features were extracted from 2 s long epochs (with 1 s overlap), representing signal properties both in the time and frequency domains. The features were then fed to an artificial neural network (ANN)-based classifier, pre-trained to distinguish between epochs corresponding to seizure and non-seizure activity. We defined “seizure like events” (SLEs) as a minimum of 6 consecutive epochs with ‘positive’ ANN detections. An animal was classified as “epileptic” when the algorithm detected at least 2 unprovoked SLEs over 1 h apart. Algorithm performance has been previously validated in 3 different models of epilepsy (namely, genetic (Ketzef et al., 2011), SE-induced (Becker et al., 2008) and albumin-induced epilepsy (Weissberg et al., 2011)), revealing overall sensitivity and positive predictive value over 98% (in a total of >2800 h of ECoG, n = 15).
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2.4. Electrophysiological recordings from hippocampal slices Electrophysiological recordings were performed in transverse hippocampal slices (400 m thick) resected from rats one to two weeks after drug treatments as previously reported (Becker et al., 2008). Briefly, rats were decapitated under deep isoflurane anesthesia and their brains were quickly removed and immersed in ice-cold, carbogenated (95% O2 , 5% CO2 ) modified artificial cerebrospinal fluid (aCSF), comprising (in mM): NaCl 130, NaHCO3 24, KCl 3.5, NaH2 PO4 1.25, d-Glucose 10, MgSO4 5, and CaCl2 0.2 for several minutes. Transverse hippocampal slices were prepared with a vibrating microslicer (Leica VT 1200 S, Solms, Germany), and the hippocampal region was isolated. Slices were maintained in room temperature incubated in carbogenated aCSF composed of (in mM): NaCl 130, NaHCO3 24, KCl 3.5, d-Glucose 10, MgSO4 2, and CaCl2 1.6. Slices were then placed one at a time in an interface slice chamber (33 ◦ C) and perfused with carbogenated aCSF. After a recovery period of 30 min to 1 h in the recording chamber, intracellular electrophysiological recordings were obtained from CA1 pyramidal cells using borosilicate glass capillaries (80–100 M), filled with intracellular solution composed of 4 M K-Acetate and 0.1 M KCl. Only neurons with resting potential more negative than −60 mV and overshooting action potentials were included in this study. 2.5. Measurement of intrinsic properties of CA1 pyramidal cells
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In order to elicit action potentials, positive current pulses were injected into CA1 pyramidal cells in increasing magnitude. Short (4 ms long) pulses were used to elicit single action potentials. Longer (180 ms) pulses were used to elicit repetitive firing. The active and passive properties of single neurons were measured as previously described (Chen et al., 2011). Briefly, we measured resting potential (Vrest), input resistance (RN ), spike threshold, amplitude (measured from Vrest to spike peak) and width (measured at half spike amplitude), fast after-hyperpolarization (fAHP; the membrane voltage at the end of fast spike repolarization), spike afterdepolarization (ADP; measured as the integral or “area under the curve” from the fAHP until the ADP attains Vrest). The firing mode of CA1 pyramidal cells was assessed by injecting 180-ms long excitatory current pulses incrementing in steps of 50 pA. The firing patterns of CA1 pyramidal cells varied along a spectrum of “burstiness”(Azouz et al., 1997; Jensen et al., 1994; Su et al., 2001): (1) Regular firing neurons, or nonbursters, which fired only one spike in response to threshold stimuli. Stronger stimuli evoked trains of spikes whose frequency increased with stimulus strength and accommodated with time. (2) Low-threshold bursters (LTBs) that fired a high-frequency cluster of 2–7 spikes overriding a distinct depolarizing potential already in response to threshold stimuli. Stronger stimuli evoked additional solitary spikes or short spike bursts, but these spikes were clearly separated from the primary burst. (3) Neurons with intermediate bursting capabilities, referred to collectively as high-threshold bursters (HTBs). These neurons fired solitary or unclustered spikes in response to 1–2× threshold stimuli, but stronger stimuli evoked a primary burst followed by several separated spikes. A bursting index (BI) developed by Chen et al. (2011) for distinguishing between nonbursters HTBs was used. The threshold BI value was 2, so that neurons with BI ≥ 2 were classified as HTBs.
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Fisher Exact Probability Test was used to assess the significance of differences between incidences of epilepsy in various treatment groups in the ECoG studies. Student’s t-test and ANOVA were used for analysis of changes in the electrophysiological parameters of
Fig. 1. Clinical score of animals in different experimental groups in the first hour after paraoxon poisoning. No convulsions were seen in animals treated only with peripheral antidotal treatment (ATOX, blue bars). Data is shown as mean score and standard deviation. Survival rate is shown in brackets. *p < 0.05, **p < 0.005 (Mann–Withney U test), when the comparison is relative to the bar whose color is identical to the color of the asterisk. (For interpretation of the references to color Q6 in this figure legend, the reader is referred to the web version of the article.)
hippocampal slices (MS-Excel, Microsoft Inc., and SPSS, IBM Inc.). Mann–Whitney U test was used for analysis of the clinical score and the mean SRS frequencies in various treatment groups (SPSS). The significance level was set to p < 0.05.
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All OP-poisoned animals developed generalized tonic-clonic convulsions, starting within 1–2 min following the paraoxon injection. Mean clinical scores and survival rates are presented in Fig. 1. 3.2. In vivo ECoG recordings None of the non-poisoned rats (group 1) was found epileptic 4–6 weeks after treatment (Fig. 2B). Seven out of eight (88%) of the OP-poisoned rats who did not receive midazolam treatment (group 2) were classified as epileptic based on recordings of at least two spontaneous, unprovoked seizures within the observation time window (p = 0.001, Fisher Exact Probability Test, when compared with non-poisoned animals). Average number of seizures per day was 0.49 ± 0.13 (Fig. 2C). Although video monitoring was not performed for all rats in this study, behavioral tonic-clonic seizures were observed occasionally in most poisoned rats (see supplementary materials). Importantly, treatment with midazolam 1 min after appearance of convulsions (group 3, PXN + ATOX + MID 1 ) resulted in a significant reduction in the number of epileptic animals, as only one out of nine (11%) rats showed spontaneous seizures 4–6 weeks after poisoning (p = 0.015 when compared with group 2, PXN + ATOX). The average daily number of seizures was also lower in the animals treated with midazolam 1 min after seizures’ onset (0.1 ± 0.09 seizure/day, p = 0.02 compared with group 2, i.e. PXN + ATOX). Midazolam injection 30 min following appearance of convulsions (group 4) resulted in 50% (4/8) of the rats being epileptic, with a mean of 0.24 ± 0.1 seizures/day; however, not statistically significant compared to group 2, i.e. PXN + ATOX (p = 0.57 and p = 0.15, respectively). The individual number of seizures in animals in different experimental groups is presented in Fig. 2D. Together, these data show that midazolam is effective in suppression of epileptogenesis induced by paraoxon when given within 1 min after poisoning.
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Fig. 2. Incidence of epilepsy in the four groups of rats as revealed by in vivo ECoG recordings. (A) Representative ECoG recordings showing a seizure 4–6 weeks after paraoxon poisoning. (B) Incidence of epileptic animals in the four groups. (C) Average number of daily SRSs in four groups. (D) The number of seizures in individual animals within the four groups. *p < 0.05, **p < 0.005, when the comparison is relative to the bar whose color is identical to the color of the asterisk. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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3.3. Electrophysiological properties of CA1 pyramidal cells in control and OP-poisoned rats Intracellular recordings were performed in a total of 110 CA1 pyramidal cells from the four groups of rats (Table 1) between 1 to 2 weeks after paraoxon poisoning. Passive and active neuronal properties in the four groups were not significantly different, except for two minor differences in action potential amplitudes (Table 2). We next compared the four groups of rats with respect to the firing mode of CA1 pyramidal cells. The firing patterns of CA1 pyramidal cells in the four groups, varied along a spectrum of “burstiness” (see Section 2) (Azouz et al., 1997; Jensen et al., 1994; Su et al., 2001). Fig. 3 A shows different firing patterns in representative CA1 neurons in the current study. The mean BI values in the four groups were 1.9 ± 0.3 (ATOX), 2.0 ± 0.05 (paraoxon + ATOX), 1.9 ± 0.04
(paraoxon + ATOX + MID 1 ) and 2.7 ± 0.4 (paraoxon + ATOX + MID 30 ). No significant differences were found between the groups (Fig. 3B). A BI value of 2 was used as a threshold to discriminate between nonbursters (Fig. 3A(I)) and bursters. The latter group of neurons was further subdivided into HTBs (Fig. 3A(II)) and LTBs (Fig. 3A(III)) according to their minimal firing response (see Section 2). The percent of bursters in the four groups was as follows: 50% (9/18 cells; group 1), 38% (11/29 cells; group 2), 41% (12/29 cells; group 3) and 48% (16/33 cells; group 4). These values were not significantly different from each other. Likewise, the percentages of HTBs (38, 24, 30 and 33%, respectively) and LTBs (11, 14, 11 and 15%, respectively) were not significantly different amongst the four groups (Fig. 3C). Thus, we could not detect significant changes in the intrinsic excitability of CA1 pyramidal cells early in epileptogenesis.
Table 2 Active and passive properties of neuronal pyramidal cells in hippocampal CA1 region tested in four groups. The results described as average ± standard error.
Vrest (mV) RN (M) Spike threshold (mV) Spike amplitude (mV) Spike width (ms) fAHP (mV) ADP
Group 1 (ATOX)
Group 2 (PXN + ATOX)
Group 3 (PXN + ATOX + MID 1 )
Group 4 (PXN + ATOX + MID 30 )
−67.3 50.5 −57.7 95.0 1.3 −62.7 187.4
−68.2 45.3 −58.3 98.3 1.4 −64.2 183.4
−68.1 42.3 −57.3 96.9 1.4 −60.8 192.0
−66.4 46.6 −52.2 91.0 1.3 −61.5 170.2
± ± ± ± ± ± ±
0.7 4.4 1.1 1.6 0.0 0.7 17.8
± ± ± ± ± ± ±
0.2 0.7 0.2 0.2* 0.0 0.2 3.3
± ± ± ± ± ± ±
0.1 0.8 0.1 0.2* 0.0* 0.1 3.0
± ± ± ± ± ± ±
0.6 2.6 4.1 1.2 0.0 1.0 18.5
PXN – paraoxon. * Significantly change from group 4, P < 0.05.
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Fig. 3. (A) Firing patterns of three CA1 pyramidal cells in hippocampal slices resected from animals belonging to group 3 (paraoxon + ATOX + MID 1 ). In each panel, the responses of the neuron to threshold (lower voltage trace) and suprathreshold (upper voltage trace) depolarizing current stimuli are shown. The resting membrane potential of each neuron is depicted to the left of the upper trace. (I) This neuron manifested a regular firing pattern. The BI value was 1.0 and the neuron was classified as a regular firing (nonbursting) neuron. (II) This neuron tended to fire an early burst upon strong stimulation, whereas threshold stimuli evoked only one action potential. The BI value of this neuron was 2.8 classifying it as a HTB. (III) This neuron fired bursts of at least 3 spikes even at threshold stimulation and was classified as an LTB. Its BI value was 5.8. (B) Bursting indices of the hippocampal CA1 neurons. The bar diagrams depict the mean BI values for each group of neurons. The differences between the mean values were not statistically significant. (C) The incidence of bursting neurons. The bar diagram depicts the percentage of bursters in each group, comprised of the percentages of LTBs (dark gray) and HTBs (light gray). The differences between the mean values were not statistically significant.
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4. Discussion
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We show here that rapid antidotal treatment (ATOX) of rats severely poisoned with the OP agent paraoxon results in a high survival rate, but does not prevent the onset of a prolonged SE. Furthermore, most surviving animals undergo epileptogenesis leading to the emergence of SRSs, the hallmark of epilepsy, within several weeks. Both SE and the ensuing epileptogenesis can be dramatically halted by injection of the BZD midazolam immediately upon SE onset. Delaying midazolam treatment by 30 min appears to have only a marginal effect in preventing OP-induced epilepsy.
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4.1. OP-induced epilepsy
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We detected chronic epilepsy in 88% of non-BZD treated rats during the 2 weeks ECoG observation period performed 4–6 weeks following OP poisoning. The presence of SRSs in our study is in accordance with a recently published study by de Araujo et al. (2012) in which the OP nerve agent soman (s.c. 1 LD50 ) was used to induce SE and diazepam was administered 30 min thereafter to arrest the SE. They reported that 15 days following OP poisoning only 28.9% of rats experiencing SE acquired chronic epilepsy. The relatively low incidence of epileptic animals following soman poisoning compared to our results may reflect their early time window of ECoG recording, as epileptogenesis is a progressive process which may develop over several weeks (Becker et al., 2008). Notwithstanding, the fact that two different OPs can lead to chronic epilepsy in a substantial fraction of poisoned animals suggests that chronic
epilepsy may be a pathological consequence of OP poisoning in general. Although not classified as seizures, long-lasting ECoG changes following OP exposure were demonstrated in humans and animals exposed to OPs (Carpentier et al., 2001; Sekijima et al., 1995). Although the initial trigger for SE in OP poisoning is predominately cholinergic, the associated excess release of glutamate is considered the main contributor to OP-induced SE perpetuation and excitotoxicity (McDonough and Shih, 1997). High levels of extracellular glutamate lead to severe neuronal damage due to increased influx of calcium into the cells, activating intracellular catalytic enzymes that damage cell membranes, cytoskeleton and organelles structure. Swollen astrocytes may release their load of glutamate thus amplifying the excitotoxic neuronal damage (Kimelberg, 1995). The mechanisms underlying the development of chronic epilepsy following OP poisoning are not understood, and could be related to glutamate excitotoxicity causing neuronal damage (McDonough and Shih, 1997), blood–brain barrier penetration (Bolwig, 1988; Grauer et al., 2001), inflammatory responses (Collombet, 2011) and reactive gliosis (Britt et al., 2000). 4.2. Prevention of OP-induced SRSs We show here for the first time the dramatic suppression of OP-induced chronic epilepsy by administrating midazolam early after poisoning. The early injection of midazolam was critical for its antiepileptogenic action: midazolam injection 30 min after SE onset, while suppressing the SEs, only partially (and not significantly) prevented the emergence of SRSs. We thus conclude that
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the Henri and Erna Leir Chair for Research in Neurodegenerative Q5 Diseases.
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a 30 min episode of OP-induced SE is sufficient to trigger epileptogenesis, which is not reversible even if the acute seizures are suppressed. This finding establishes a relationship between SE duration and subsequent chronic epilepsy in OP-poisoned rats. Congruently, McDonough and colleagues showed that at least 20 min of seizure activity is necessary for neuropathological damage to occur in rats following OP exposure (McDonough et al., 1995). Klitgaard et al. using the pilocarpine model did not find a difference in SRSs frequencies in animals experiencing 30 versus 120 min of SE, even though SRSs emerged only after a minimal SE duration of 30 min (Klitgaard et al., 2002). The time window for effectively preventing OP-induced chronic epilepsy with midazolam, as demonstrated in our study, was less than 30 min. This is much shorter than the 1–2 h time-window reported for the electrical stimulation-induced SE model using phenobarbital, phenytoin (Prasad et al., 2002) or diazepam (Pitkanen et al., 2005) for SE suppression. Possibly the systemic OP poisoning causes more extensive changes in the brain than those produced by focal electrical stimulation.
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4.3. Pilocarpine and OP-induced changes in intrinsic excitability
Azouz, R., Alroy, G., Yaari, Y., 1997. Modulation of endogenous firing patterns by osmolarity in rat hippocampal neurones. J. Physiol. 502 (Pt 1), 175– 187. Bastlund, J.F., Jennum, P., Mohapel, P., Vogel, V., Watson, W.P., 2004. Measurement of cortical and hippocampal epileptiform activity in freely moving rats by means of implantable radiotelemetry. J. Neurosci. Methods 138 (1–2), 65–72. Becker, A.J., Pitsch, J., Sochivko, D., Opitz, T., Staniek, M., Chen, C.C., Campbell, K.P., Schoch, S., Yaari, Y., Beck, H., 2008. Transcriptional upregulation of Cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy. J. Neurosci. 28 (49), 13341–13353. Bolwig, T.G., 1988. Blood–brain barrier studies with special reference to epileptic seizures. Acta Psychiatr. Scand. Suppl. 345 (15–20), 15–20. Britt Jr., J.O., Martin, J.L., Okerberg, C.V., Dick Jr., E.J., 2000. Histopathologic changes in the brain, heart, and skeletal muscle of rhesus macaques, ten days after exposure to soman (an organophosphorus nerve agent). Comp. Med. 50 (2), 133– 139. Carpentier, P., Foquin, A., Dorandeu, F., Lallement, G., 2001. Delta activity as an early indicator for soman-induced brain damage: a review. Neurotoxicology 22 (3), 299–315. Chen, S., Su, H., Yue, C., Remy, S., Royeck, M., Sochivko, D., Opitz, T., Beck, H., Yaari, Y., 2011. An increase in persistent sodium current contributes to intrinsic neuronal bursting after status epilepticus. J. Neurophysiol. 105 (1), 117–129. Collombet, J.M., 2011. Nerve agent intoxication: recent neuropathophysiological findings and subsequent impact on medical management prospects. Toxicol. Appl. Pharmacol. 255 (3), 229–241. de Araujo, F.M., Lumley, L.A., Robison, C., Tong, L.C., Lichtenstein, S., Yourick, D.L., 2010. Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats. Epilepsia 51 (8), 1503–1510. de Araujo, F.M., Rossetti, F., Chanda, S., Yourick, D., 2012. Exposure to nerve agents: from status epilepticus to neuroinflammation, brain damage, neurogenesis and epilepsy. Neurotoxicology 33 (6), 1476–1490. Eddleston, M., Karalliedde, L., Buckley, N., Fernando, R., Hutchinson, G., Isbister, G., Konradsen, F., Murray, D., Piola, J.C., Senanayake, N., Sheriff, R., Singh, S., Siwach, S.B., Smit, L., 2002. Pesticide poisoning in the developing world – a minimum pesticides list. Lancet 360 (9340), 1163–1167. Eddleston, M., Mohamed, F., Davies, J.O., Eyer, P., Worek, F., Sheriff, M.H., Buckley, N.A., 2006. Respiratory failure in acute organophosphorus pesticide self-poisoning. QJM 99 (8), 513–522. Gilat, E., Kadar, T., Levy, A., Rabinovitz, I., Cohen, G., Kapon, Y., Sahar, R., Brandeis, R., 2005. Anticonvulsant treatment of sarin-induced seizures with nasal midazolam: an electrographic, behavioral, and histological study in freely moving rats. Toxicol. Appl. Pharmacol. 209 (1), 74–85. Grauer, E., Ben, N.D., Lustig, S., Kobiler, D., Kapon, J., Danenberg, H.D., 2001. Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors. Life Sci. 68 (9), 985–990. Holmes, G.L., 2002. Seizure-induced neuronal injury: animal data. Neurology 59 (9 Suppl. 5), S3–S6. Jensen, M.S., Azouz, R., Yaari, Y., 1994. Variant firing patterns in rat hippocampal pyramidal cells modulated by extracellular potassium. J. Neurophysiol. 71 (3), 831–839. Ketzef, M., Kahn, J., Weissberg, I., Becker, A.J., Friedman, A., Gitler, D., 2011. Compensatory network alterations upon onset of epilepsy in synapsin triple knock-out mice. Neuroscience 189, 108–122, http://dx.doi.org/ 10.1016/j.neuroscience.2011.05.030. Kimelberg, H.K., 1995. Current concepts of brain edema. Review of laboratory investigations. J. Neurosurg. 83 (6), 1051–1059. Klitgaard, H., Matagne, A., Vanneste-Goemaere, J., Margineanu, D.G., 2002. Pilocarpine-induced epileptogenesis in the rat: impact of initial duration of status epilepticus on electrophysiological and neuropathological alterations. Epilepsy Res. 51 (1–2), 93–107.
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The pilocarpine model of SE is widely used in the study of epileptogenesis leading to chronic epilepsy (Rubio et al., 2010). Pilocarpine is a muscarinic agonist which triggers SE by initially activating muscarinic receptors, leading to the emergence of SRSs within a week or two. Not surprisingly, the pilocarpine model replicates also the neuropathological changes that are caused by OPs (Tang et al., 2011). It has been shown that pilocarpine-induced SE alters the intrinsic excitability of principal hippocampal neurons. Most dramatically, a single episode of pilocarpine induced SE, causes a large fraction of CA1 pyramidal cells to convert from regular firing to burst-firing mode, a change that appears within 3 days and persists for many weeks after its induction (Sanabria et al., 2001). Intriguingly, we show here that OP-induced SE, even though caused by cholinergic stimulation, was not followed by a change in the intrinsic electrophysiological properties of CA1 pyramidal cells. The proportion of bursters did not increase even in the group of rats that did not receive midazolam and thus experienced a prolonged SE (group 2). This dissimilarity between the two models is intriguing, as both involve cholinergic stimulation and epileptogenesis and suggest that the pilocarpine model may not be a surrogate for OP-induced chronic epilepsy and for the screening of effective neuroprotecting agents against OP poisoning (Tang et al., 2011; Tetz et al., 2006).
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5. Conclusions
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We show that severe paraoxon poisoning associated with SE induces chronic epilepsy, leading to the emergence of SRSs in most poisoned rats. Midazolam is effective in preventing paraoxoninduced chronic epilepsy if administered 1 min after seizures onset, but not 30 min thereafter. Extrapolating these results to humans would indicate that people at risk to agricultural or warfare OP poisoning, may benefit from the availability of an injection device containing not only the classical OP antidotes, atropine and an oxime, but also midazolam. Further research is necessary to fully characterize the OP-induced epileptogenesis leading to chronic epilepsy and its pathogenesis to allow for more efficient preventive treatments at later stages. Funding This work was supported by a grant from the Medical Corps of the Israeli Defense Force. YB, MD and YY were supported by
Conflicts of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2014.05.010. References
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