Posttraumatic seizures and epilepsy in adult rats after controlled cortical impact

Epilepsy Research 117 (2015) 104–116

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Posttraumatic seizures and epilepsy in adult rats after controlled cortical impact Kevin M. Kelly a,b,c,d,∗ , Eric R. Miller a , Eka Lepsveridze e , Elena A. Kharlamov a,b,c , Zakaria Mchedlishvili a,b,c a

Center for Neuroscience Research, Allegheny Health Network Research Institute, Pittsburgh, PA, United States Department of Neurology, Allegheny General Hospital, Pittsburgh, PA, United States c Department of Neurology, Philadelphia, PA, United States d Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, United States e Ilia State University, Faculty of Natural Sciences and Engineering, Tbilisi, Georgia b

a r t i c l e

i n f o

Article history: Received 25 February 2015 Received in revised form 26 August 2015 Accepted 10 September 2015 Available online 11 September 2015 Keywords: Traumatic brain injury Video-EEG monitoring Mossy fiber sprouting

a b s t r a c t Posttraumatic epilepsy (PTE) has been modeled with different techniques of experimental traumatic brain injury (TBI) using mice and rats at various ages. We hypothesized that the technique of controlled cortical impact (CCI) could be used to establish a model of PTE in young adult rats. A total of 156 male Sprague-Dawley rats of 2–3 months of age (128 CCI-injured and 28 controls) was used for monitoring and/or anatomical studies. Provoked class 3–5 seizures were recorded by video monitoring in 7/57 (12.3%) animals in the week immediately following CCI of the right parietal cortex; none of the 7 animals demonstrated subsequent spontaneous convulsive seizures. Monitoring with video and/or video-EEG was performed on 128 animals at various time points 8–619 days beyond one week following CCI during which 26 (20.3%) demonstrated nonconvulsive or convulsive epileptic seizures. Nonconvulsive epileptic seizures of >10 s were demonstrated in 7/40 (17.5%) animals implanted with 2 or 3 depth electrodes and usually characterized by an initial change in behavior (head raising or animal alerting) followed by motor arrest during an ictal discharge that consisted of high-amplitude spikes or spike-waves with frequencies ranging between 1 and 2 Hz class 3–5 epileptic seizures were recorded by video monitoring in 17/88 (19%) and by video-EEG in 2/40 (5%) CCI-injured animals. Ninety of 156 (58%) animals (79 CCI-injured, 13 controls) underwent transcardial perfusion for gross and microscopic studies. CCI caused severe brain tissue loss and cavitation of the ipsilateral cerebral hemisphere associated with cell loss in the hippocampal CA1 and CA3 regions, hilus, and dentate granule cells, and thalamus. All Timm-stained CCI-injured brains demonstrated ipsilateral hippocampal mossy fiber sprouting in the inner molecular layer. These results indicate that the CCI model of TBI in adult rats can be used to study the structure–function relationships that underlie epileptogenesis and PTE. © 2015 Elsevier B.V. All rights reserved.

1. Introduction According to the Centers for Disease Control and Prevention, 1.7 million Americans sustain traumatic brain injury (TBI) each

Abbreviations: TBI, traumatic brain injury; PTE, posttraumatic epilepsy; CCI, controlled cortical impact; FPI, fluid percussion injury; POD, postoperative day; SWD, spike-wave discharges. ∗ Corresponding author at: Allegheny General Hospital, 940 South Tower, 320 E. North Avenue, Pittsburgh, PA 15212-4772, United States. Tel.: +1 412 359 3467; fax: +1 412 359 6127. E-mail addresses: [email protected] (K.M. Kelly), [email protected] (E.R. Miller), eka [email protected] (E. Lepsveridze), [email protected] (Z. Mchedlishvili). http://dx.doi.org/10.1016/j.eplepsyres.2015.09.009 0920-1211/© 2015 Elsevier B.V. All rights reserved.

year. Of these individuals, 52,000 die, 275,000 are hospitalized, and 1365 million are treated and released from an emergency department (Faul et al., 2010). An estimated 1.1% of the U.S. civilian population lives with a long-term disability from TBI (Zaloshnja et al., 2008). One of the sequelae of TBI is the development of posttraumatic epilepsy (PTE). In a population-based study, the cumulative incidence of PTE in the first three years after hospitalization per 100 persons was 4.4 for mild TBI, 7.6 for moderate TBI, and 13.6 for severe TBI (Ferguson et al., 2010). The risk of PTE is especially high in the military, where the incidence can be up to 53% after penetrating head trauma (Raymont et al., 2010). Although the link between head trauma and seizures has been recognized for millennia, successful animal modeling of TBI and the subsequent development of PTE has occurred only within the recent past

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(D’Ambrosio et al., 2004a,b; Pitkänen and McIntosh, 2006; Statler et al., 2009; Hunt et al., 2009; Pitkänen et al., 2009, 2011; Curla et al., 2011; Bolkvadze and Pitkänen, 2012). Building on the substantial database of clinical, experimental, and pathological findings associated with TBI, biological mechanisms underlying PTE are actively being explored (D’Ambrosio et al., 1999; Norris and Scheff, 2009; Prince et al., 2009; Willmore and Ueda, 2009; Hunt et al., 2010, 2011; Kharatishvili and Pitkänen, 2010a; Mtchedlishvili et al., 2010; Kharlamov et al., 2011; Belousov et al., 2012; D’Ambrosio et al., 2013a,b; Hunt et al., 2013). Experimental models of TBI simulate human TBI with varying degrees of accuracy (Morales et al., 2005). Two of the most commonly used models of TBI are fluid percussion injury (FPI) and controlled cortical impact (CCI). The advantage of the FPI model is its relative simplicity and its ability to produce significant injury in the brain, including axonal injury and intraparenchymal hemorrhages (Povlishock et al., 1983). The FPI model has been used successfully to demonstrate the development of PTE (D’Ambrosio et al., 2004a,b; Kharatishvili et al., 2006; Kharatishvili and Pitkänen, 2010b; Bolkvadze and Pitkänen, 2012). Alternatively, CCI produces a relatively precise and reproducible injury and can simulate various degrees of TBI severity by titration of the velocity, force, and depth of impact (Dixon et al., 1991). The CCI model has been well characterized histopathologically (Lighthall, 1988; Hall et al., 2005; Saatman et al., 2006) and has been used successfully in mice (Hunt et al., 2009, 2010) and in postnatal day 7 rats to demonstrate the development of PTE (Statler et al., 2009). We hypothesized that the technique of CCI could be used to develop a model of PTE in young adult rats. The main objectives of this study were to characterize: (1) the frequency and severity of both acute symptomatic seizures (i.e., provoked, occurring within the first week following acute brain injury) and epileptic seizures (i.e., unprovoked, occurring after the first week following acute brain injury); (2) the electrographic signatures of epileptogenesis and the epileptic state; and (3) the associated neuropathological changes that occurred following extended periods of animal survival after CCI.

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majority of animals (n = 116) the impactor velocity was adjusted to 4 m/s, the impact duration was 100 ms, and the depth of tissue depression was 2.8 mm (Mtchedlishvili et al., 2010; Kharlamov et al., 2011). Following CCI and the cessation of cortical bleeding, the scalp was sutured, carprofen 5 mg/kg was administered subcutaneously, and the animal was returned to the vivarium for recovery or to satellite vivaria of the Neurophysiology Laboratory for video monitoring for up to 7 days post-lesioning. Carprofen use was continued each day for 48–72 h as judged necessary for recovery. Sham-operated animals underwent identical anesthetic and surgical procedures without CCI. 2.2. Electrode implantation

2. Materials and methods

Cortical and hippocampal recording depth electrodes and anchoring screws were placed about one week after CCI or sham operation, or at various time points during extended videoonly monitoring, as previously described for the photothrombosis method (Kelly et al., 2001), with modifications. Animals were anesthetized with intraperitoneal injections of a 9:1 mixture of ketamine and xylazine, and after the loss of the tail pinch reflex, positioned in a stereotaxic frame. A sterile artificial tear ointment was applied over the eyes and a midline incision was made along the scalp, which was reflected bilaterally. Burr holes were drilled for 3 depth electrodes (0.25 mm; Plastics One Inc., Roanoke, VA), 1 reference electrode, and 4–5 anchoring screws using stereotactic coordinates. Cortical depth electrodes were placed rostral to the lesion and homotopically (AP 3.2 mm, lateral 1.5 mm, vertical −3.5 mm). A hippocampal depth electrode was placed in the ventral hippocampus contralateral to the injury (AP −5.3 mm, lateral 4.9 mm, vertical −6 mm to dura). A hippocampal electrode was not placed on the side of the lesion due to the craniectomy defect. A single skull screw electrode overlying the cerebellum was used as a reference electrode. Anchoring screws were placed posterolaterally; one anchoring screw was used as a ground electrode. All electrodes were crimped into a six-conductor modular plug and secured to the skull surface with dental acrylic (Lang Dental Mfg., Wheeling, IL). The skin around the headset was sutured and bipolar EEG recordings were initiated following full recovery of the animal.

2.1. Controlled cortical impact (CCI)

2.3. Video and video-EEG monitoring

All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Allegheny Health Network Research Institute and were conducted according to NIH guidelines and regulations. Animals were housed individually in the ASRI vivarium, maintained in a 12 h light/12 h dark cycle environment with controlled temperature (23 ± 2 ◦ C), and food and water were given ad libitum. The CCI procedure was performed according to Dixon et al. (1991) with some modifications (Mtchedlishvili et al., 2010) using aseptic technique. Briefly, male Sprague-Dawley rats (2–3-mo old) were anesthetized with an initial dose of 4% isoflurane mixed with oxygen and positioned in a stereotaxic frame (David Kopf Instruments, Tujunga, CA); surgical depth of anesthesia was maintained with 2–3% of isoflurane. Body temperature was monitored throughout the procedure using a rectal probe and maintained at 37 ± 2 ◦ C with a heating pad (Harvard Apparatus). A craniectomy was performed over the right parietal cortex within the boundaries of the bregma and lambda while leaving the dura intact. CCI was performed immediately after removal of the bone flap and exposure of the dura. A 1.975cm-diameter pneumatic impactor, attached to a double-acting, stroke-constrained, pneumatic cylinder with a 5.0 cm stroke (Pittsburgh Precision Instruments, Pittsburgh, PA) was used to deliver CCI. The impactor tip was positioned 4–5 mm laterally to the midline. Following initial titration of the impact procedure, for the

Animals were housed individually in 12 monitoring chambers in satellite vivaria of the Neurophysiology Laboratory and maintained on the 12 h light/12 h dark cycle. Animal behavior was variably monitored around-the-clock by closed-circuit television cameras that were connected to video splitter units (Advanced Technology Video, Inc., Redmond, Washington). Digital video files (Diva, Stellate Systems) were recorded directly to high capacity hard disk drives using removable hard drive bays. Daily recordings were reviewed visually offline to detect any behavioral seizure activity according to a modified classification scale (Racine, 1972), including forelimb clonus (class 3); running and rearing (class 4); and jumping and falling (class 5). Seizures that occurred during the first week following CCI were considered provoked seizures; these animals underwent video-only monitoring. Seizures that occurred after the first week following CCI were considered epileptic (spontaneous) whether they were isolated or recurrent events, consistent with the proposed conceptual definition of epilepsy by the International League Against Epilepsy as “an enduring alteration in the brain and at least one seizure” (Fisher et al., 2005). Specific criteria for video and video-EEG was not adopted for the first 30 animals, for which monitoring was sporadic, i.e., varying numbers of days monitored per month for 3 months, and was skewed to specific CCI-injured animals that displayed potentially abnormal behavior (12/30; 40%). We commenced acute, post-CCI

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injury monitoring with animal 75 and continued this monitoring for all animals throughout the remainder of the study. We began routine, monthly, week-long recording sessions starting with animal 106. Following this, all CCI-injured animals underwent a week of video or video-EEG recording each month until perfusion; animals that subsequently lost their electrode headsets underwent video-only monitoring. Electrode cables were connected via custom-designed swivel commutators (Dragonfly Inc., Ridgeley, WV or Plastics One, Roanoke, VA) to the input boxes of three Stellate 16- or 32-channel EEG acquisition setups. EEG signals were obtained without filtering but for display purposes were standardized with filtering at 1 and 70 Hz and 60 Hz (notch filter). Animals demonstrating electrographic and/or behavioral ictal activity were often monitored more frequently than those that did not in order to assess the potential evolution of the epileptic state. 2.4. Perfusion and preparation of tissue sections At the end of the monitoring period, animals were deeply anesthetized intraperitoneally with ketamine and xylazine and transcardially perfused with 0.9% saline in 0.15 M phosphate buffer (PB, pH 7.4) followed by 4% ice-cold buffered paraformaldehyde solution. The brains were rapidly removed, post-fixed in the same solution at 4 ◦ C overnight, and cryoprotected in 30% sucrose solution in PB until they sank. Gross examination of the brains was performed to confirm the presence or absence of TBI and possible pathology associated with the skull drilling for the placement of the screws and implantation of the cortical and hippocampal depth electrodes. Coronal 40 ␮m-thick sections were cut from frozen brains with a sliding microtome, collected, and stored in cryoprotectant solution (phosphate buffer/ethylene glycol/glycerol) at −20 ◦ C until used. One series of sections was Nissl-stained (0.25% thionin) to determine cytoarchitectural alterations caused by CCI and the location of depth electrodes, whereas adjacent or other sections were immunostained with selected biomarkers. 2.5. Immunohistochemistry Sections were removed from the cryoprotectant solution and rinsed in PB followed by incubation in 0.3% hydrogen peroxide in PB. After several rinses in 0.1 M Tris-buffered saline (TBS, pH 7.4), free-floating sections were incubated in TBS containing 3% normal goat serum and 0.25% triton X-100 (TX-100). Tissue sections were incubated overnight at 4 ◦ C with antineuron-specific nuclear protein (anti-NeuN, 1:1000, Chemicon International, Temecula, CA; neuronal marker) or anti-glial fibrillary acidic protein (anti-GFAP, 1:500, Chemicon; glial marker) diluted with TBS containing 1% normal goat serum and 0.25% TX-100. After several rinses, brain sections were incubated for 2 h at room temperature with biotinylated goat anti-mouse or goat anti-rabbit IgGs (Jackson ImmunoResearch), respectively, diluted 1:500 in TBS containing 1% goat serum followed by a 1h incubation in avidin–biotin-peroxidase complex (ABC Elite Kit, Vector Laboratories, Burlingame, CA). The bound peroxidase was treated with imidazole acetate buffer (pH 9.6) containing 0.05% diaminobenzidine, 2.5% nickel ammonium sulfate, and 0.005% hydrogen peroxide. Immunostaining specificity was assessed by either the omission of primary or secondary antibodies. No positive immunoreactivity or recognizable background staining was observed under these conditions. Sections were mounted on gelatin-coated slides, air-dried, dehydrated, and coverslipped. 2.6. Timm staining For Timm staining (sulphide/silver technique for visualization of heavy metals; Sloviter, 1982), a small set of CCI-injured and

control animals were anesthetized and perfused through the ascending aorta for 2 min at 30 ml/min with 0.9% NaCl, for 30 min at 20 ml/min with 0.37% sodium sulfide solution, for 1 min with 0.9% NaCl, and for 30 min with 4% paraformaldehyde in 0.15 M PB (Buckmaster et al., 2009). Perfused brains were removed and post-fixed in 4% paraformaldehyde at 4 ◦ C overnight, rinsed three times with PB, and cryoprotected in 30% sucrose solution. Coronal 40 ␮m-thick sections from control and CCI-injured with and without PTE animals were processed together. The presence of aberrant mossy fiber sprouting was compared at different levels of the septotemporal axis of the hippocampal formation ipsilaterally and contralaterally to the injury site. Brain sections were washed in PB, mounted on charged slides (Superfrost Plus; Fisher Scientific), and air dried at room temperature overnight. Mounted sections were placed in a solution consisting of 180 ml of 50% gum arabic, 30 ml of 2.25 M citrate buffer, 50 ml of 0.04% silver lactate, and 45 ml of 0.5 M hydroquinone (Babb et al., 1991) and were incubated for ∼90 min in the dark at room temperature. After thorough washing in water, the series of Timm-stained sections were counterstained with Nissl staining, dehydrated, and coverslipped. The adjacent sections were processed for Nissl only. Timm staining was graded according to a modified rating scale of Tauck and Nadler (1985) by an investigator blinded to the surgical and video- and/or video-EEG monitoring data. Sections were analyzed using a light microscope equipped with 4, 10, and 20x objectives. The scores for mossy fiber sprouting were assigned based on the following 0–3 rating scale: 0 – little to no Timm granules in the granule cell layer; 1 – mild staining in the granule cell layer but not the inner third of the molecular layer (IML); 2 – moderate, continuous staining throughout the granule cell layer with a discontinuous band of dense staining in the IML; and 3 – a continuous band of dense staining throughout the IML layer extending along the inner (suprapyramidal) to the outer (infrapyramidal) blade. If Timm-positive staining was variable between the inner and outer blades of the dentate gyrus, then an average score was used (e.g., if the lower blade had a score of 2 while the upper blade had a score of 3, the slice would be given an overall grade of 2.5). Timm scores >1 were considered to have an abnormal degree of mossy fiber sprouting (Patrylo and Dudek, 1998; Shibley and Smith, 2002; Hunt et al., 2009).

2.7. Digital images Light microscopy and anatomic digital images were produced using the Bioquant computer-based imaging system (Bioquant Image Analysis Corporation, Nashville, TN), a microscope (Nikon Eclipse E600, Japan) equipped with a motorized stage (Applied Scientific Instrument, ASI), and a “Qimaging” color camera (“Qimaging”, Canada). The images were captured using a Nikon microscope, and figures were generated by using Adobe Photoshop. Laser scan (LS-2000) and VueScan scanning software (version 8.4.79) was used for obtaining high-quality images of whole coronal brain sections.

2.8. Data analysis Qualitative EEG analysis was performed offline via a complete visual scanning of the records (2–8× speed with up to 4 animals simultaneously). EEG non-ictal activities were distinguished from ictal activities based on waveform morphology and frequency and the associated absence or presence of behavioral change, respectively. Following completion of analysis, records were removed from computer hard drives and transferred to external hard drives for storage and re-review as needed. Descriptive statistics related to animal behavior and/or ictal activities are presented as mean, median, and range values.

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3. Results 3.1. General considerations Out of a total of 199 animals, 156 animals (128 CCI-injured and 28 controls) were used for monitoring and/or anatomical studies; 43 animals were not used, the majority of which had undergone lesioning during the initial titration of the CCI procedure. Total video and video-EEG monitoring time was 28,912 h:27,789 h for CCI-injured animals and 1123 h for controls. Video monitoring was performed during the first week after CCI in 57 (45%) animals to assess for provoked seizure activity; a total of 3212 h of recordings was obtained with a mean recording time of 56.36 ± 4.24 h. Video and/or video-EEG monitoring was performed beyond the first week in 128/128 (100%) animals after CCI to assess for unprovoked (spontaneous) seizure activity, totaling 24,577 h of recordings. Premature mortality was very low, occurring in 5/128 (4%) animals within 2–3 months of lesioning, typically due to anesthesia-related complications during electrode implantation in the initial set of animals studied. No animal died during the first week following CCI. Table 1 is included summarizing the electrical, behavioral, and anatomical findings of the study.

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day 3 of the week following CCI. Provoked seizures were recorded in 4 animals on postoperative day (POD) 1, in 2 animals on PODs 1and 2, and 1 animal on POD 3. Mean seizure occurrence was 4.57 ± 1.45 (median 3, range 1–11) and mean seizure duration was 24.84 ± 5.17 s (range 3–113 s). One of the 7 animals had 11 provoked seizures, ranging in duration from 3 to 7 s (mean 4.5 s). These seizures were different from the provoked events recorded in the other 6 animals in that for each of the events, the animal rolled onto its side, had forelimb or hindlimb clonus, and then propelled itself in a circular fashion while still on its side. Gross and microscopic posttraumatic morphological alterations of the animal’s brain are shown in Supplementary Material #1. A different animal with 2 provoked seizures was monitored for only 3 days during the week following CCI and died 1 day after electrode implantation (∼3 months after CCI) prior to any further monitoring. All of the surviving animals (6/7) with provoked seizures had additional video or video-EEG monitoring beyond the first week post-CCI with a mean monitoring time of 270.05 ± 79.67 h (range 69–566 h) obtained over an average of 216.86 ± 78.92 days post-CCI (range 3–619 days); none of these animals demonstrated spontaneous convulsive or nonconvulsive seizures.

3.2. Provoked seizures

3.3. Epileptic seizures

Video monitoring of 57 animals was performed immediately after CCI for up to 7 days to assess for the occurrence of provoked seizures (seizures occurring within the first week following acute brain injury); no animal experienced convulsive status epilepticus during this time. Provoked seizures (class 3–5) were recorded in 7/57 (12.3%) animals; no provoked seizure was recorded beyond

Monitoring with video and/or video-EEG was performed on 128 animals at time points beyond one week following CCI: video alone (n = 88); video-EEG (n = 40). Epileptic seizures were demonstrated in 26 (20.3%) animals: video (n = 17) and video-EEG (n = 9). No seizure was recorded in any sham-operated animal (n = 16) other than absence seizures (see below).

Table 1 Summary of findings. Seizure subcategories

Types of events recorded

Monitoring data

Anatomical findings

Additional information

Provoked

- Class 3–5 convulsive seizures in 7/57 (12.3%) animals monitored acutely following CCI

Mean monitoring time: 56.36 ± 4.24 h Mean seizure duration: 24.84 ± 5.17 s Mean seizure rate: 4.57 ± 1.45

Epileptic video-only

- Class 3–5 in 17/88 (19%) CCI-injured injured animals

Mean monitoring time: 362.78 ± 40.01 h Mean seizure duration: 87.24 ± 7.53 s

- 6/7 animals demonstrating provoked seizures were monitored beyond one week post-CCI (none demonstrated spontaneous seizures) - 15/57 animals monitored for provoked seizures also underwent subsequent video-EEG monitoring - 1/17 animals demonstrated 3 video-only class 5 seizures over a 5 day period after removal of electrode headset (#175)

Epileptic video-EEG

- Nonconvulsive seizures in 7/40 (17.5%), characterized by initial behavior change followed by motor arrest during ictal discharge (1–2 Hz)

Mean monitoring time: 356.44 ± 80.03 h Mean seizure duration: 31.89 ± 3.21 s

- Class 5 convulsive seizures in 2/40 animals w/left-sided emphasis/onset, generalized ictal/convulsive period and post-ictal slowing - Generalized 8–11 Hz SWDs (absence seizures) and generalized pseudoperiodic spike discharges

Mean monitoring time: 404.38 ± 47.99 h Mean seizure duration: 91.00 ± 12.34 s

- Necrotic cavitation in ipsilateral hemisphere - Decreased Nissl staining/NeuN IR in ipsilateral hemisphere (dentate gyrus and thalamus) - Enhanced GFAP expression in ipsilateral/contralateral hippocampus - Necrotic cavitation in ipsilateral hemisphere - Robust mossy fiber sprouting in inner third of molecular layer of the dentate gyrus (both contra- and ipsilateral hemispheres) (#175) - Necrotic cavitation in ipsilateral hemisphere - Timm staining present in hilus of dentate gyrus and stratum lucidum of CA3 and less apparent in contralateral hippocampus - One animal demonstrated aberrant Timm staining in the IML of dentate gyrus - Cell loss in ipsilateral hippocampus (CA1, CA3) and dentate gyrus - Necrotic cavitation in ipsilateral hemisphere - Timm staining present in hilus of dentate gyrus and stratum lucidum of CA3

- Serial ictal discharges were observed in 4/7 animals demonstrating nonconvulsive seizures and were separated by interictal periods ranging from several seconds to minutes - 4/7 animals had only 2 depth electrodes implanted (contralateral to lesion), while the remaining 3 had 3 (2 contralateral and 1 ipsilateral)

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Fig. 1. (A) Absence seizure (8 Hz) recorded in a CCI-injured animal 135 days after injury. (B) Generalized pseudoperiodic spike discharges occurring in the same animal during wakefulness 143 days after CCI injury. Inset shows magnified, stereotypic SWDs. The animal demonstrated mild body twitches that were synchronous with the spike discharges.

3.4. Video-only epileptic seizures Convulsive epileptic seizures (class 3–5) were observed in 17/88 (19%) CCI-injured animals with a mean duration of 87.24 ± 7.53 s (range 11–349 s). The animals had a mean monitoring time of 362.78 ± 40.01 h (range 100.95–648.40 h). The first day of monitoring (excluding the 1-week period following CCI) ranged from 19 to 503 days with a mean first day monitoring time of 119.59 ± 30.12 days post-CCI. The first recorded seizure ranged from 72 to 507 days with a mean time of 196.41 ± 33.99 days to first seizure. The last day of monitoring for the group ranged from 119 to 521 days after CCI with a mean last day monitoring time of 251.18 ± 33.20 days. 3.5. Video-EEG epileptic seizures 3.5.1. Absence seizures and pseudoperiodic spike discharges Both sham-operated and CCI-injured animals demonstrated numerous generalized 8–11 Hz spike-wave discharges (SWDs;

genetic absence seizures) associated with motor arrest of the animal (Fig. 1A). In addition, generalized pseudoperiodic spike discharges were recorded associated with mild body twitches of the animal that were synchronous with the spike discharges (Fig. 1B). Similar absence seizures and pseudoperiodic spike discharges were recorded in control and lesioned animals in another study (Kharlamov et al., 2003) and were not quantified or further analyzed in the present study. 3.5.2. Lesion-induced nonconvulsive seizures Nonconvulsive epileptic seizures were demonstrated in 7/40 (17.5%) implanted animals usually characterized by an initial change in behavior (head raising or animal alerting) followed by motor arrest during an ictal discharge that consisted of high-amplitude spikes or spike-waves with frequencies ranging from 1 to 2 Hz (Figs. 2A, B and 3A); importantly, the recurrent discharges shown in 4A were associated with uninterrupted eating, drinking, and ambulation of the animal. Representative

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Fig. 2. Nonconvulsive seizures (A and B) characterized by rhythmic 1–1.5 Hz ictal discharges associated with motor arrest of the animal observed 125 days after CCI. Note the relatively abrupt initiation and termination of the discharges. Perfused brain (B) demonstrates a necrotic cavity in the ipsilateral injured hemisphere that is very close to the brain middle line. Comparison of GFAP IR in the contra- (D) and ipsi- (E) lateral hippocampus, and GFAP expression in the area close to the impact site (F; GS = glial scar). Timm-stained (G–N) coronal sections from sham control (G–J) and CCI-injured animal demonstrated epileptic nonconvulsive seizures (K–N). In both group of animals, Timm staining was present in the hilus of the dentate gyrus and in the stratum lucidum of CA3. In the CCI-injured animal (K–N), aberrant Timm staining was observed additionally in the inner molecular layer of the dentate gyrus (arrows). Timm staining was much less apparent in the contralateral hippocampus (CH; K and M) compared with the ipsilateral injured hippocampus (IH; L and N). The asterisk (*) marks the necrotic cavity at the cortical impact site.

histopathological findings are shown in Figs. 2C–N and 3B–D. Serial ictal discharges were recorded in 4 of 7 animals, separated by interictal periods ranging from several seconds to minutes. Each of these 4 animals had at least 10 nonconvulsive events during monitoring. Immediately after the ictal discharge, the animal frequently demonstrated head movement or a mild wet dog shake before returning to normal behavior. Seizure durations ranged from 10 to 155 s, with a mean duration of 31.89 ± 3.21 s. There was significant intra- and inter-animal variability with regard to the time and laterality of ictal onsets. These findings were constrained by the fact that 4 of the 7 animals had only 2 electrodes implanted (contralateral to the lesion), whereas the other 3 animals had 2 contralateral and 1 ipsilateral (cortical) electrodes. For example, one animal with 2 electrodes demonstrated ictal onsets in the contralateral cortical electrode early in the monitoring period but eventually showed contralateral hippocampal-dominant ictal discharges (Fig. 3A). Animals with 3 electrodes could demonstrate

ictal discharges primarily on the contralateral side, in a generalized fashion (Fig. 2A and B), or confined to the ipsilateral cortical electrode. Four of the 7 (23.5%) animals exhibited epileptiform discharges with durations of less than 10 s (duration range 3.00–9.00 s, mean duration 6.66 ± 0.43 s); epileptiform waveforms during these discharges resembled those that exceeded 10 s in duration. Discernible changes in the animals’ behavior during the onset and offset of these discharges were not typically apparent. Of the 81 nonconvulsive events recorded, 18 (22%) had durations of less than 10 s. Mean monitoring time of these animals was 356.44 ± 80.03 h (range 46.83–688.56 h). The first day of monitoring following CCI ranged from 22 to 71 days with a mean time point of 44.14 ± 7.93 days post-CCI. The first recorded seizure ranged from 39 to 146 days with a mean time of 80.57 ± 15.95 days to first seizure. The last day of monitoring for the group ranged from 53 to 352 days with a mean time point of 183 ± 43.60 days after CCI.

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Fig. 3. Nonconvulsive seizure (A) demonstrating left-sided low-voltage fast activity evolving into a rhythmic higher amplitude lower frequency discharge that ends abruptly before the pattern is reinitiated. Note prominence of the discharge in the hippocampal electrode; no electrode was placed ipsilateral to the lesion. Normal animal behavior was not interrupted during these discharges. Nissl-stained coronal brain section (B) demonstrates evidence of cell loss in the ipsilateral hippocampus (D) in CA1, CA3, and dentate gyrus compared to the contralateral side (C). An asterisk (*) marks the necrotic cavity at the cortical impact site (D). Hippocampal depth electrode tract in the contralateral dorsal hippocampus (B – arrow 1) and glial scar formation in the overlying cortex (B – arrow 2). Abbreviations: H: hilus; DG: dentate granule cell layer.

3.5.3. Lesion-induced convulsive seizures One animal lost its headset 23 days after implantation (without discernible injury to the skull or subsequent evidence of intracranial hemorrhage) and demonstrated three video-only convulsive seizures (duration range 52–147 s, mean duration 102.33 ± 27.57 s) over a 5-day period approximately one month later. The animal brain and posttraumatic morphological changes are shown in Supplementary Material #2. Two of 40 (5%) of CCI-injured animals demonstrated ictal discharges with convulsive seizure activity (class 5; Fig. 4) with a mean duration of 91.00 ± 12.34 s (range 74–115 s). Ictal discharges consisted of high-amplitude spikes or spike-waves with clear electrographic onsets and terminations and postictal slowing. No interictal epileptiform activity was recorded in either of these animals. Mean monitoring time was 404.38 ± 47.99 h (range 356.39–452.37 h). The first day of monitoring following CCI ranged from 24 to 240 days with a mean time to first monitoring of 132 ± 108.00 days post-CCI. The first recorded seizures occurred on days 162 and 349 post-CCI; the last days of monitoring were 387 and 462 days post-CCI, respectively. 3.6. Morphological and cytoarchitectural changes following CCI and in PTE Ninety-two of 156 (59%) animals (79 CCI-injured, 11 shamoperated, 2 naïve) underwent transcardial perfusion for gross and microscopic studies. All 79 CCI-injured animals demonstrated morphological alterations in the ipsilateral hemisphere; in some cases, presumably due to the impact site being closer to the midline than was usual, there was minimal tissue loss in the contralateral hemisphere. Otherwise, there was little variation in the severity of the alterations in the ipsilateral hemisphere. Because there was little variation in the size of the lesions, there was no apparent correlation between the severity of the morphological alterations and the expression of acute seizures or PTE. Many animals with CCI-induced lesion sizes comparable to those of animals showing provoked seizures or PTE demonstrated neither provoked seizures nor evidence of the development of PTE.

Gross examination of the brains of sham-operated animals revealed no abnormality other than the dorsal sites of depth electrode penetration. CCI-injured animals had necrotic cavities and brain tissue loss in the ipsilateral (right) hemisphere (Figs. 2C, L, N, 3B, D and 5A–C); in almost all CCI-injured animals there was no sign of gross tissue loss in the contralateral hemisphere. Histological evaluation revealed some variability in the location of the necrotic cavity as well as the extent of the tissue injury. The development of tissue cavities close to the brain midline and cerebellum (Fig. 2C) may have caused or contributed to morphological changes in adjacent areas of the contralateral hemisphere and cerebellum, respectively. In addition, the differences in the appearance of the cavities and brain tissue loss suggest that some animals may have experienced a more severe progression of secondary injury mechanisms compared to others following CCI. However, in most animals, CCI resulted in a consistently large and reproducible focal injury in the ipsilateral hemisphere. Nissl (Figs. 3B–D; 5B, D–G) and NeuN immunostaining (Fig. 5C, H–K) demonstrated tissue loss in the ipsilateral neocortex (Figs. 3B, D and 5B, C) and cell loss in the hippocampal CA1 and CA3 regions, hilus, and dentate granule cells (Figs. 3B, D and 5E, I), and thalamus (Figs. 3B and 5G, K). Nissl staining (Fig. 3B) shows the tract of a hippocampal depth electrode in the dorsal hippocampus contralateral to the injury site and a glial scar in the overlying cortex. GFAP staining of CCI-injured brains resulted in reactive gliosis that developed during long-term survival (Figs. 2D–F and 6D–F). GFAP-positive cells were found throughout the neocortex, corpus callosum, hippocampus, and thalamus, and their expression was slightly enhanced in the ipsilateral hemisphere close to the impact site where reactive astrocytes formed dense scar tissue (Fig. 2F). In addition to the assessment of neuronal loss and gliosis, the possibility of aberrant sprouting of granule cell axons (mossy fibers) was investigated with Timm staining in 12 animals: 7 CCI-injured and 5 naïve or sham controls. All animals demonstrated dark Timm staining in the hilus and stratum lucidum of CA3. Control animals revealed no or minimal Timm-positive staining in the granule cell

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Fig. 4. Discontinuous video-EEG epochs taken from a class 5 seizure in a CCI-injured animal that show (A) brief low voltage fast activity preceding a 4 Hz rhythmic discharge in the cortical electrodes with left-sided emphasis, (B) generalized ictal/convulsive period, and (C) termination of the ictal discharge/post-ictal period.

layer (Fig. 2G–J; grade 0–1 on a 0–3 rating scale). In contrast, all CCI-injured animals displayed dark Timm staining (grade 2–3) in the IML of the ipsilateral hippocampus (Fig. 2L). In the contralateral hippocampus, the presence of mossy fiber sprouting varied from no staining (grade 0; n = 3), minimal staining (grade 0–1; n = 3; Fig. 2K, M) to dense staining (grade 3; n = 1; Supplementary Material #2). Fig. 2K–N illustrates an example of a Timm-stained section of dentate gyrus of a CCI-injured animal following longterm survival. The extent of mossy fiber sprouting and its presence in the inner and/or outer blades of the dentate gyrus was variable. Robust mossy fiber sprouting was found in both septal and temporal areas of the ipsilateral hippocampus of most CCI-injured animals. Brains of CCI-injured animals that did not have identified seizure activity demonstrated greater variability in the distribution and progression of mossy fiber sprouting between ipsi- and

contralateral hippocampus and within the inner and outer blades of the dentate gyrus (Fig. 2K–N). 4. Discussion 4.1. Main findings This study was undertaken to determine whether CCI in young adult rats could result in the development of PTE. The main findings of this study were: (1) provoked seizures occurred in 12.3% of animals studied during the week following CCI, none of which subsequently exhibited convulsive epileptic seizures; (2) epileptic seizures were recorded in 20.3% of the animals, including both nonconvulsive and convulsive events recorded at different times following injury; (3) CCI caused structural alterations and severe

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Fig. 5. Brain of a rat sacrificed 5 months post-CCI (A). The pair of adjacent sections (B and C) demonstrates structural and morphological changes with Nissl (B; D–G) and NeuN (C; H–K) immunostaining. Alterations of Nissl staining and the decrease of NeuN immunoreactivity were found in the ipsilateral hemisphere (IH) in the dentate gyrus (DG; E and I), and the thalamus (G and K) when compared to the contralateral hemisphere (CH) DG (D, H) and thalamus (F, J), respectively. An asterisk (*) marks brain tissue loss at the impact site. Abbreviations: H: hilus; DG: dentate gyrus.

brain tissue loss that resulted in a large cavity in the ipsilateral hemisphere; (4) all CCI animals that underwent Timm staining demonstrated ipsilateral mossy fiber sprouting, including those that demonstrated nonconvulsive or convulsive epileptic seizures, and some also demonstrated staining of the contralateral hippocampus. 4.2. Provoked seizures A subset (57/128, 45%) of CCI-injured animals were videomonitored variably for up to 7 days following lesioning to determine whether provoked convulsive seizure activity occurred and, if so, whether these seizures would predispose to the subsequent development of epileptic seizures. Provoked seizure activity has been reported in only a few previous studies using CCI, including rats and mice. Nilsson et al. (1994), using a weight-drop variation of CCI, recorded electrographic generalized ictal discharges in anesthetized Sprague-Dawley rats within 2 h following injury, whereas Yang et al. (2010) observed spontaneous generalized convulsive seizures immediately after CCI in only one Sprague-Dawley rat from a small group of animals lesioned at day P24. In a study using A1AR knock out mice, Kochanek et al. (2006) observed only 1–2-sec episodes of twitching in wild type litter mates within the immediate 2-h period following CCI. In the present study, provoked convulsive seizures (class 3–5) were

captured in only 7/57 (12.3%) of the animals, similar to the relatively low frequency of occurrence as reported in the studies noted above. No provoked seizure was recorded beyond the third day post-lesioning, likely associated with attenuation of the immediate effects of the acute trauma and inflammatory response. Beyond the first week of monitoring, none of these animals demonstrated spontaneous convulsive seizures; however, the subsequent monitoring of these animals was far from comprehensive in that it was variably intermittent, not adequately extensive, and did not include video-EEG monitoring, the latter’s omission precluding the possibility of providing electrographic evidence of nonconvulsive epileptic seizures. These considerations would suggest that nonconvulsive seizures may have occurred in both the early and late periods following CCI and without EEG monitoring would have gone unrecognized and resulted in an underreporting of actual seizure activity. 4.3. Epileptic seizures Video and/or video-EEG monitoring was performed in all (128/128, 100%) of the CCI-injured animals after the first week post-lesioning. Although the recordings were not performed in a uniformly standardized manner, variably intermittent monitoring provided clear evidence of both nonconvulsive and convulsive epileptic seizures.

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Fig. 6. Low (A, D, G) and higher (B–I) power photomicrographs of glial fibrillary acidic protein (GFAP) expression in the contralateral hippocampus (CH; A, B), and thalamus (CH; C) vs. ipsilateral hippocampus (IH; D, E; G, H) and thalamus (IH; F; I) at 5 months post-CCI (A–F; same animal as in Fig. 5) or sham operation (G–I). GFAP-positive astrocytes were found in the hippocampus and thalamus, but showed more hyperplasia and hypertrophy in the injured tissue (D–F).

4.4. Absence seizures and pseudoperiodic spike discharges Inherent 8–11 Hz SWDs (genetic absence seizures) were recorded in both sham-operated and lesioned (Fig. 1A) animals, virtually identical to those recorded in control and lesioned animals in the photothrombosis model of poststroke epilepsy (Kharlamov et al., 2003). These SWDs are typically episodes of abrupt onset, variable duration (seconds to minutes), and abrupt termination, usually occurring during passive wakefulness and light sleep (Coenen et al., 1992; Willoughby and Mackenzie, 1992). The discharges are associated with decreased responsiveness (behavioral arrest) with or without rhythmic whisker twitching (Willoughby and Mackenzie, 1992; Buzsaki et al., 1990). These SWDs are commonly recorded in both inbred and outbred rat strains. Wistarderived inbred strains include GAERS (genetic absence epilepsy rats from Strasbourg) and WAG/Rij (Wistar Albino Glaxo strain, bred in Rijswijk, Netherlands); Wistar-unrelated inbred strains include Fischer 344, Brown Norway, and dark agouti (Coenen et al., 1992; Willoughby and Mackenzie, 1992; van Luijtelaar and Coenen, 1986). Common outbred strains include Sprague-Dawley, Wistar,

and Long–Evans (Semba et al., 1980; Willoughby and Mackenzie, 1992; Buzsaki et al., 1990; Vergnes et al., 1982). Importantly, these SWDs are distinctive, stereotypic generalized discharges and are easily distinguished from lesion-induced ictal discharges (Kelly, 2004). In addition to the SWDs recorded in this study, generalized pseudoperiodic spike discharges – hypothesized to represent incompletely propagated absence seizures – were recorded in both sham-operated and lesioned animals (Fig. 1B), as previously reported (Kharlamov et al., 2003). Given that the focus of this study was to characterize posttraumatic seizures, neither SWDs nor the pseudoperiodic spike discharges were further analyzed. 4.5. Lesion-induced nonconvulsive and convulsive seizures Lesion-induced nonconvulsive seizures were recorded in 7 animals, typically involving 1–2 Hz high-amplitude generalized spike or spike-wave discharge patterns lasting variably from 10 s to 2.5 min associated with behavioral arrest of the animal (Fig. 2A and B); some of these discharge patterns were associated with uninterrupted animal behavior and therefore are considered as

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electrographic seizures (Fig. 3A). As stated above, these ictal discharges were easily distinguished from the generalized 8–11 Hz SWDs of absence seizures (Fig. 1A) based on waveform morphology, topography, and frequency. Lesion-induced convulsive seizures (Fig. 4A–C) were recorded by video-EEG in only 2 animals; surprisingly, no interictal focal or generalized epileptiform activity was seen in either animal. In general, interictal spiking was most apparent in those animals that demonstrated nonconvulsive seizures. These results compare favorably to those obtained in Sprague-Dawley rats lesioned by CCI at P17 and recorded with serial epochs of continuous video-EEG over a 3-month period initiated 4–8 months after lesioning during which 1/8 (12.5%) animals demonstrated class 4–5 seizures (Statler et al., 2009). In a study using young adult CD-1 mice lesioned by CCI and monitored beginning 42 days after lesioning, Hunt et al. (2009) reported class 2 seizures in 2/10 (20%) animals that sustained mild injury, whereas up to class 3 seizures were observed in 4/11 (36%) animals with severe injury. There were several shortcomings to the video and video-EEG monitoring performed in this study. Although the study provided direct evidence of both provoked and unprovoked seizure activity following CCI, it is likely that numerous ictal events were missed given the intermittency of the recordings. We recognize that the variable schedules of recordings ran the risk of missing substantial numbers of ictal events, which could skew interpretation of potential time-dependent effects on epileptogenesis following CCI. However, at the current stage of the model’s development, and consistent with monitoring practices in exploratory animal studies, the study allowed representative sampling of seizure occurrence at monthly time points and provided a first approximation of the potential time dependency of posttraumatic epileptogenesis. One specific limitation of the study was that we were unable to obtain reliable EEG recordings beyond 6 months following placement of the depth electrodes. Another problem encountered during long-term monitoring was the interruption or premature termination of longitudinal video-EEG data caused by the unexpected loss of an animal’s electrode headset. Although headset loss was relatively infrequent, it occurred secondary to scalp infection, or to mechanical dislodgement by the animal’s scratching or overall body movements. In addition, we recognize that the limited depth electrode recordings were not able to reliably localize ictal onsets. Recorded electrographic seizures may have been generalized (bilateral), focal with rapid secondary generalization, or originated in areas not detected by the 2 or 3 recording depth electrodes used in the study. However, some, if not most, of the recordings provided evidence of lateralized, if not localized, seizure onset. 4.6. Brain pathology The use of neuronal and glial markers and Timm staining was an important component in the interpretation of the behavioral and EEG monitoring data and the establishment of PTE following CCI. CCI-injured brains revealed well-demarcated lesion-induced cavities and associated injury of the adjacent hippocampus and thalamus. Brain structural alterations, including substantial neuronal loss and reactive gliosis, were observed in the injured cerebral cortex, hippocampal CA1–CA3 regions, dentate gyrus, and thalamus. The anatomical extent of brain injury was dependent on the consistency of the craniectomy and the reproducibility of the position, direction, duration, and magnitude of the cortical impact. In addition, it is possible that PTE may have resulted in additional cell loss. Because post-CCI survival time was longer than 12 months for most of the animals studied, a small increase of GFAP immunoreactivity may have been due to the implantation of depth electrodes and normal aging (Jucker et al., 1994; Yu et al., 2002). Brain tissue

injury was not assessed quantitatively in this study largely due to significant differences in post-injury survival times, and some variability in CCI parameters and differences in the size and location of tissue cavities. The Timm stain is a specific marker for granule cell axons (mossy fibers) in which zinc is concentrated in synaptic vesicles (Wenzel et al., 1997; Buckmaster et al., 2009). Although the specific role of aberrant mossy fiber sprouting remains somewhat unclear in epileptogenesis and PTE, the degree of mossy fiber sprouting may correlate with the extent of hilar neuron loss (Buckmaster and Dudek, 1997; Nissinen et al., 2001; Jiao and Nadler, 2007); mossy fiber sprouting becomes permanent once established. Different cellular mechanisms can be involved in the initiation and generation of robust mossy fiber sprouting in the IML of the hippocampus following TBI. Thus, CCI-induced loss of hilar neurons in the ipsilateral hippocampus may lead to deafferentation of the contralateral granule cell layer and subsequent mossy fiber sprouting. Our results support previously published observations that demonstrated robust mossy fiber sprouting in the IML of the hippocampus following FPI and/or CCI (Golarai et al., 2001; Santhakumar et al., 2001; Hunt et al., 2009, 2010). Several important factors might affect mossy fiber sprouting and seizure incidence after brain injury, such as impact parameters, injury location, animal age, and rodent species (Hunt et al., 2012). In addition, excitatory drive to surviving hilar GABAergic interneurons after CCI may be enhanced by convergent input from both pyramidal and granule cells resulting in network destabilization (Hunt et al., 2011). The presence of aberrant mossy fiber sprouting in the IML of the hippocampus is typically considered as a marker for temporal lobe epilepsy and as one of several abnormal excitatory networks that may underlie seizure generation (Gorter et al., 2001; Williams and Dudek, 2007).

5. Conclusions The behavioral, electrographic, and anatomical abnormalities observed in this study reflect dynamic functional reorganization of the brain following traumatic injury that can result in epileptogenesis and PTE. The CCI model of TBI can be a useful tool in promoting an improved understanding of the mechanisms underlying PTE in adult rats and in the testing of antiepileptogenic compounds as well as novel approaches to the treatment of PTE.

Disclosure We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. None of the authors has any conflict of interest to disclose.

Acknowledgements The authors would like to thank Bo Lu, M.D., who provided technical support for the animal studies, and to Teresa Hentosz for clerical support in the preparation of the manuscript. The study was supported by Health Research Formula Fund RFA 01-07-26, Pennsylvania Department of Health, and a Research Grant from the Epilepsy Foundation to ZM.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.eplepsyres.2015. 09.009.

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