Epilepsia, 50(4):801–810, 2009 doi: 10.1111/j.1528-1167.2008.01815.x
FULL-LENGTH ORIGINAL RESEARCH
Single injection of a low dose of pentylenetetrazole leads to epileptogenesis in an animal model of cortical dysplasia *1Roger O. Oghlakian, *1Cristiane Q. Tilelli, yGirish K. Hiremath, *Andreas V. Alexopoulos, and *Imad M. Najm *Cleveland Clinic, Neurological Institute, Epilepsy Center, Cleveland, Ohio, U.S.A.; and yCleveland Clinic, Department of Neurosurgery, Cleveland, Ohio, U.S.A.
SUMMARY Purpose: Cortical dysplasia (CD) is one of the most frequent causes of pharmacoresistent focal epilepsy. Despite significant advances in various diagnostic and therapeutic methods, the basic mechanisms of higher susceptibility for seizures in patients with CD are unknown. Animal models of CD present with a lower threshold for seizure induction. The purpose of this study is to further characterize the animal model of in utero radiation-induced CD and to illustrate the effect of a late postnatal second hit (low dose of pentylenetetrazole, PTZ) on the development of spontaneous seizures. Methods: Pregnant Sprague–Dawley rats were irradiated on E17 (145 cGy; control group was left untreated). Litters were implanted with bifrontal epidural and hippocampal depth electrodes. After baseline electroencephalography (EEG) recording, animals received 30 mg/kg PTZ and were
Cortical dysplasia (CD) is a confirmed histopathologic finding in many cases of medically intractable epilepsy (Taylor et al., 1971; Corsellis et al., 1973). Many reports from epilepsy surgery centers describe a high correlation between different types of cortical dysplasia and epileptogenicity in patients with intractable epilepsy. Retrospectively, a large percentage of patients with epilepsy have been diagnosed with malformations of cortical development (MCD). As witnessed in clinical practice, these patient populations with congenital or at times possiAccepted August 4, 2008; Early View publication October 14, 2008. Address correspondence to Imad M. Najm, M.D., Director, Cleveland Clinic Epilepsy Center, 9500 Euclid Avenue, S51, Cleveland, OH 44195, U.S.A. E-mail:
[email protected] 1 These authors contributed equally to this work. Wiley Periodicals, Inc. ª 2008 International League Against Epilepsy
chronically monitored. Histopathology of the brains was verified. Results: No seizures were detected in animals that did not receive PTZ. PTZ-injected irradiated (XRT) rats showed severe prolonged, repetitive seizures during the acute period. During the chronic period, XRT rats had recurrent seizures and epileptiform spikes. PTZ-injected control animals exhibited milder and fewer acute seizures and did not show seizures during the chronic period. Histology was consistent with cortical and hippocampal dysplasia. Conclusions: This study shows that a single treatment with a low dose of PTZ renders XRT rats (but not age-matched controls) epileptic, exhibiting spontaneous epileptiform spikes and seizures on EEG. These results might mirror the natural history of patients with CD thought to be caused by prenatal/congenital or perinatal insults. KEY WORDS: Cortical dysplasia, In utero radiation, Animal model, Second hit, Epilepsy.
bly perinatal lesions develop seizures at different ages, starting during the neonatal period all the way to adulthood (Widdess-Walsh et al., 2005). Several mechanisms may play a role in triggering the onset of seizures in patients who are at particularly high risk, such as those with preexisting cortical dysplasia. In fact, patients with an underlying MCD substrate may have a higher risk of developing epilepsy (Porter et al., 2003). Triggers for the development of epilepsy in susceptible patients include febrile seizures in children, trauma, infection involving the brain or meninges, stroke, hypoglycemia or other metabolic disturbances, and a variety of other insults causing damage to the brain or surrounding structures (WiddessWalsh et al., 2005). The study of such conditions is limited by the availability of surgically resected tissue from epileptic patients, as well as the technical and ethical issues involved in dealing with human subjects. Another concern
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802 R. O. Oghlakian et al. is the controversy surrounding the procurement of valid controls from patients in order to investigate the differences between control and dysplastic/epileptic tissues. These difficulties warrant the development and characterization of animal models of cortical dysplasia and spontaneous epilepsy that closely resemble the human condition, both histologically and electroencephalographically, and correlate well with the progression and natural evolution of the disease, as observed in clinical practice. To date, a number of animal models of CD have been described. Models with some of the histopathologic changes that are observed in human CD tissue include the perinatal cortical freeze lesion model (Rosen et al., 1992; Jacobs et al., 1996; Luhmann & Raabe, 1996) the alkylating agent—methylazoxymethanol acetate—model (Dambska et al., 1982; Baraban & Schwartzkroin, 1995), the in utero ethanol exposure model (Miller, 1986), and the in utero radiation-induced model (Riggs et al., 1956; Hicks et al., 1959; Roper et al., 1995, 1997b). So far, spontaneous long-term seizures have been described in only a small number of the genetic models (Lee et al., 1997; Fleck et al., 2000; Wenzel et al., 2001), and in the irradiation model of CD, both in slices (Roper, 1998) and in intact animals (Kondo et al., 2001; Kellinghaus et al., 2004). In addition, in vitro electrophysiology studies performed on brain slices from the radiation-induced CD model showed increased neuronal excitability and a high susceptibility for seizure development (Roper et al., 1997b). Our report focuses on the acute as well as chronic electroencephalography (EEG) and clinical manifestations of the radiation-induced CD rat model after intraperitoneal injection of a single, low dose of pentylenetetrazole (PTZ), a well-described proconvulsant agent used in rat kindling models and in the generation of acute short-lived seizures in animals. Although PTZ has been widely used in several animal models, the exact mechanisms of PTZ-induced convulsions are not clear. The most accepted theory is attributed to PTZ’s action as an antagonist to the picrotoxinin-sensitive site of the c-aminobutyric acid (GABA) receptor complex (Chweh et al., 1983; Ramanjaneyulu & Ticku, 1984). The threshold for the development of acute seizures in normal adult rats is between 40 and 50 mg/kg (Ramzan & Levy, 1985). In this study, we first evaluated chronic EEG characteristics of a group of rats that exhibit cortical dysplasia. We then studied the effect of a single, low dose (30 mg/kg) injection of PTZ on the later occurrence of spontaneous seizures in the same model.
Materials and Methods Animals Six time-pregnant Sprague–Dawley rats (Charles River, Wilmington, MA, U.S.A.) and their litters were included Epilepsia, 50(4):801–810, 2009 doi: 10.1111/j.1528-1167.2008.01815.x
in this study. The day at which a vaginal plug was detected was designated embryonic day 0 (E0). Date of birth, which usually occurred around E22, was designated postnatal day 0 (PND0). Rat pups were weaned at PND21. All animals were maintained on a 12-h light–dark cycle with food and water available ad libitum. This study was approved by the Cleveland Clinic Animal Research Committee. In utero exposure to irradiation Animals were treated using the protocol that was previously described by Roper et al. (1995), with some modifications (Kondo et al., 2001; Kellinghaus et al., 2004). In brief, on E17, four pregnant rats were exposed to c-irradiation using a Shepherd Irradiator containing a 4,000 Ci cesium-137 source (Shepherd and Associates, San Fernando, CA, U.S.A.). The time-pregnant rats were restrained in a perforated plastic box and placed in the irradiator device, and then exposed to a single dose of 145 cGy. The total duration of the single irradiation session was about 35 s. Two other time-pregnant rats were not exposed to irradiation, and their pups were used as age-matched controls. Stereotactic implantation of recording electrodes All rat pups were born on E22, considered as PND0. At PND45, rats were divided into two experimental groups: nonirradiated rats (control group; n = 17) and irradiated rats (XRT group; n = 29), delivered from the two nonirradiated pregnant rats and the four irradiated pregnant rats, respectively. Stereotactic electrode implantation was performed under pentobarbital anesthesia (50 mg/kg, i.p.). For hippocampal recordings, bipolar twisted stainless-steel electrodes (0.2 mm in diameter; Medwire, New York, NY, U.S.A.) were placed bilaterally in the hippocampi (LH, left hippocampus; RH, right hippocampus; A: )4.3 mm, L: €2.5 mm, D: )2.5 mm from Bregma) as previously described using the photographic atlas of rat brain (Paxinos & Watson, 1995; Kondo et al., 2001; Kellinghaus et al., 2004). Stainless-steel screws (MX-0090-2; Small Parts Inc., Miami, FL, U.S.A.) were placed epidurally and bilaterally over the frontal cortices (LF, left frontal; RF, right frontal, A: 1 mm, L: €2.5 mm from Bregma). An additional screw was placed just to the right of the frontal sinus and served as a referential electrode (REF). The electrodes were then connected to a plastic plug (SMB-06V-BC, Japan; Solderless Terminal MFG., Tokyo, Japan), which was fixed to the skull with dental cement (Hygenic repair resin; Hygenic, Akron, OH, U.S.A.). Rats were left unrestrained for at least 72 h to allow for recovery from surgery before further manipulation and prolonged EEG monitoring were started.
803 PTZ-Induced Epileptogenesis in the Dysplastic Brain Chronic EEG recording prior to PTZ injection (Group 1) For the first set of rats (control n = 8 and XRT n = 16), EEG monitoring started 72–96 h after surgery. Monitoring consisted of 48 h of recording alternating with 48 h of no recording for each rat. All rats were sequentially exchanged in four custom-made electrically shielded boxes with simultaneous video recordings. Implanted recording electrodes were connected to pendulous electroslip rings (MRS 35-06P; MÆT GIKEN, Tokyo, Japan), which permitted acquisition of real-time noise and artifact-free EEGs, when the rats were moving and during the seizures. During the recording, rats were unrestrained in the monitoring boxes with freedom of movement and access to food and water ad libitum. Total monitored time ranged from 96–264 h (mean 176 h) during a range of 8–76 days (mean 34.3 days) after surgery. Digital EEG recordings (five channels per rat) were performed using a Vangard system (Lamont, Madison, WI, U.S.A.), which consists of a recording country. Hewlett-Packard (Palo Alto, CA, U.S.A.) workstation, and a similar review system connected to four 21-inch monitors. A total of two rats were monitored at any given time: five channels each, 10-channel acquisition at a sampling rate of 100 Hz for a total of 2 h. The recording filter settings were: low-frequency filter of 1 and high-frequency filter of 70 Hz. PTZ injection and EEG recording (group 2) A second set of implanted control (n = 9) and XRT (n = 13) rats was monitored for a period of 2 and 3 weeks, respectively. PTZ injection and EEG monitoring started 72–96 h after surgery. The same EEG acquisition settings were applied to these animals during the 48-h on and off cycle, as described previously. After this initial monitoring period, all 22 rats were subjected to a single dose of 30 mg/kg PTZ given intraperitoneally. Continuous digital EEG recording of each rat started 5 min before the PTZ injection and continued for up to 2 h for acute monitoring. After the initial acute EEG recording, 18 rats (7 from the control group, and 11 from the XRT group) were monitored for prolonged periods using the same digital EEG/ video system and the same settings and cycles as with the first set of animals. Four rats were monitored at any given time for a total monitoring time of 72–216 h (mean of 96 h). Recording periods ranged from 48 h to 50 days after PTZ injection (mean of 28.6 days). The entire set of EEG data was stored on digital audiotape (DAT) or magneto-optical disks (MODs), and was later blindly analyzed by three different trained neurophysiologists (R.O., I.N., and C.L.D.), who were unaware of the treatment groups. EEG data analysis The digitized EEG signals were reformatted to referential and bipolar montages. Whole EEG samples were ana-
lyzed by visual inspection for the presence of epileptiform activity and were correlated with samples of simultaneous video. Epileptiform spikes were defined as paroxysmal electrical sharp activity lasting 20–150 ms, with an amplitude that was five times the background EEG activity as previously defined (Kondo et al., 2001; Kellinghaus et al., 2004). These spikes were mapped to the area of maximal amplitude (on referential montage) and to the electrode of maximal phase reversal (on bipolar montage), and were divided into four regions (LF, left frontal; RF, right frontal; LH, left hippocampal; RH, right hippocampal). EEG seizures were defined as repetitive spikes with interruption of the background predominant activity for more than 10 s. The seizure-onset zone was mapped to the electrode of maximal amplitude and phase reversal of the first spike. Analysis of the early post-PTZ injection data was based on several EEG and behavioral features that were compared between control and experimental groups. These features included the following: latency to the first unequivocal epileptic discharge and/or first clinical manifestation; latency to the onset of the first EEG seizure; duration of the first seizure; clinical semiology of the first seizure; and number and duration of subsequent EEG seizures and corresponding semiology. Analysis of the prolonged chronic EEG monitoring data was based primarily on the presence or absence of epileptiform seizure patterns or spikes. The frequency of the EEG spikes (spikes/h) and seizures (seizures/24 h) and their behavioral characteristics and corresponding duration were also examined and analyzed. Histologic studies At the end of the EEG monitoring period, rats were deeply anesthetized with pentobarbital (60 mg/kg i.p.) and perfused intracardially with 4% paraformaldehyde in phosphate buffered saline solution (PBS, pH 7.4). The brains were saved in 4% paraformaldehyde in PBS for 2 days and then transferred to 20% sucrose in PBS for 48 h before processing. Thereafter, brains were frozen and serially sectioned in the coronal plane into 30-lm slices using a cryostat (Leica CM-1850; Heidelberg, Germany). Sections were then saved in cryostorage solution and stored at )20C until further processing. One set of sections representative of various anterior–posterior brain regions was thaw-mounted onto silane-coated slides and stained with cresyl violet (CV) for histologic examination. Stained sections from animals of both groups were blindly analyzed according to the same criteria as previously described (Kondo et al., 2001; Kellinghaus et al., 2004). In order to evaluate for the presence of dysplasia in various brain areas, the following criteria were used: (1) presence of cortical neuronal clustering, (2) persistence of neurons in the molecular layer, (3) lack of columnar and layering organization, and/or (4) presence of neuronal dispersion in any of the hippocampal subfields. Epilepsia, 50(4):801–810, 2009 doi: 10.1111/j.1528-1167.2008.01815.x
804 R. O. Oghlakian et al. Statistical analysis Statistical analysis was performed using JMP 7.0 Software (SAS Institute Inc., Cary, NC, U.S.A.) using the Pearson chi-square test for ordinal and the t-test for numeric data, with p-values
