Effect of lacosamide on structural damage and functional recovery after traumatic brain injury in rats

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EPIRES-5096; No. of Pages 13 Epilepsy Research (2014) xxx, xxx—xxx

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Effect of lacosamide on structural damage and functional recovery after traumatic brain injury in rats A. Pitkänen a,b,∗, R. Immonen a, X. Ndode-Ekane a, O. Gröhn a, T. Stöhr c,1, J. Nissinen a a

Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland b Department of Neurology, Kuopio University Hospital, PO Box 1777, FI-70211 Kuopio, Finland c Schwarz BioSciences GmbH (a member of the UCB group), Germany Received 21 March 2013; received in revised form 31 January 2014; accepted 1 February 2014

KEYWORDS Antiepileptic drug; Lateral fluid-percussion; Magnetic resonance imaging; Neurodegeneration

Summary In a subgroup of patients, traumatic brain injury (TBI) results in the occurrence of acute epileptic seizures or even status epilepticus, which are treated with antiepileptic drugs (AEDs). Recent experimental data, however, suggest that administration of AEDs at the early post-injury phase can compromise the recovery process. The present study was designed to assess the profile of a novel anticonvulsant, lacosamide (Vimpat® ) on post-TBI structural, motor and cognitive outcomes. Moderate TBI was induced by lateral fluid-percussion injury in adult rats. Treatment with 0.9% saline or lacosamide (30 mg/kg, i.p.) was started at 30 min post-injury and continued at 8 h intervals for 3 d (total daily dose 90 mg/kg/d). Rats were randomly assigned to 4 treatment groups: sham-operated controls treated with vehicle (Sham-Veh) or lacosamide (Sham-LCM) and injured animals treated with vehicle (TBI-Veh) or lacosamide (TBI-LCM). As functional outcomes we tested motor recovery with composite neuroscore and beam-walking at 2, 7, and 15 d post-injury. Cognitive recovery was tested with the Morris water-maze at 12—14 d post-TBI. To assess the structural outcome, animals underwent magnetic resonance imaging (MRI) at 2 d post-TBI. At 16 d post-TBI, rats were perfused for histology to analyze cortical and hippocampal neurodegeneration and axonal damage. Our data show that at 2 d post-TBI, both the TBI-Veh and TBI-LCM groups were equally impaired in neuroscore. Thereafter, motor recovery occurred similarly during the first week. At 2 wk post-TBI, recovery of the TBI-LCM group lagged behind that in the TBI-VEH group (p < 0.05). Performance in beam-walking did not differ between the TBI-Veh and TBI-LCM groups. Both TBI groups were similarly impaired in the Morris water-maze at 2 wk post-TBI. MRI and histology did not reveal any differences in the

∗ Corresponding author at: Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland. Tel.: +358 50 517 2091; fax: +358 17 16 3025. E-mail address: asla.pitkanen@uef.fi (A. Pitkänen). 1 Present address: A2M Pharma GmbH, Alfred-Nobel-Strasse 10, 40789 Monheim, Germany.

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A. Pitkänen et al. cortical or hippocampal damage between the TBI-Veh and TBI-LCM groups. Taken together, acute treatment with LCM had no protective effects on post-TBI structural or functional impairment. Composite neuroscore in the TBI-LCM group lagged behind that in the TBI-Veh group at 15 d postinjury, but no compromise was found in other indices of post-TBI recovery in the LCM treated animals. © 2014 Elsevier B.V. All rights reserved.

Introduction Antiepileptic drugs (AEDs) are used to treat several types of co-morbidities caused by traumatic brain injury (TBI). For example, phenytoin, fos-phenytoin, valproate, carbamazepine, phenobarbital, and levetiracetam have been used to treat early post-traumatic seizures both in children and adults (Brain Injury Special Interest Group, 1998; Jones et al., 2008; Wang et al., 2008; Temkin, 2009; Chan et al., 2010; Ma et al., 2010; Pangilinan et al., 2010; Debenham et al., 2011; Liesemer et al., 2011; Steinbaugh et al., 2012) and various AEDs have been used in the treatment of post-traumatic epilepsy (Han et al., 2008; Yasseen et al., 2008). Gabapentin, topiramate, and carbamazepine have been used to treat different types of pain (Childers and Holland, 1997; Ivanhoe and Hartman, 2004; Sherman et al., 2006; Patil et al., 2011) and lamotrigine can be used to treat behavioral disorders like agitation and aggression in patients with TBI (Pachet et al., 2003). As many of these co-morbidities develop and occur in parallel with recovery processes, a question arises; does the use of AEDs to control early post-traumatic seizures compromise structural or functional outcomes after TBI? Also, are there differences between the AEDs? This concern was already raised more than 20 years ago when Hernandez and colleagues demonstrated that diazepam and phenobarbital delayed the somatosensory recovery after anteromedial cortical injury in rats (Schallert et al., 1986; Hernandez, 1997; Monta˜ nez et al., 2001). Lacosamide (LCM, SPM927, Vimpat® ) is a novel AED with anticonvulsant efficacy in several preclinical rodent models of focal and generalized epilepsy, including audiogenic seizures, maximal electroshock seizures, and limbic seizures in the 6 Hz model (for review, see Stöhr et al., 2007). It has now been approved for use as an add-on treatment for partial onset seizures in Europe and the United States (Cross and Curran, 2009). In addition to epilepsy, it has been tested to treat migraine, neuropathic pain, fibromyalgia, and osteoarthritic pain (Zaccara et al., 2013). The antiepileptic effect of LCM has been linked to its ability to enhance slow inactivation of voltage-gated fast transient sodium currents, rendering sodium channels less available during the high-frequency firing and depolarization shifts encountered during epileptiform activity (Errington et al., 2008; Sheets et al., 2008). Recently, LCM was also shown to maintain its activity under conditions with a lack of accessory sodium channel subunit ␤1, a mutation that renders these channels insensitive to carbamazepine (Uebachs et al., 2012). LCM was also reported to have affinity for collapsing response mediator protein-2 (CRMP-2), an axonal growth and guidance protein (Beyreuther et al., 2007; Park

et al., 2009; Wang et al., 2010). However, these data were recently challenged by Wolff et al. (2012). The present study was designed to investigate the effect of LCM treatment on post-TBI structural damage and functional impairment, and also, whether the treatment would affect functional recovery. TBI was induced by lateral FPI in rats. Monotherapy with a clinically relevant anticonvulsant dose of LCM (30 mg/kg, t.i.d.; see Beyreuther et al., 2007) was started at 30 min post-injury and continued at 8 h intervals for 3 d. Animals were followed-up for 2 wk after TBI. The effect on neuronal and axonal damage was assessed using magnetic resonance imaging (MRI) and histology. The preservation and recovery of motor and memory functions was assessed using neuroscore, beam-walking tests, and the Morris water-maze.

Materials and methods The study design is summarized in Fig. 1.

Animals Adult male Sprague-Dawley rats (332 ± 38 g, Harlan, The Netherlands) were used in the study. Rats were housed in individual cages at a temperature of 19—21 ◦ C, with humidity maintained at 50—60% and lights on 7.00—19.00. Standard food pellets and water were freely available. All animal experiments were approved by the Committee for the Welfare of Laboratory Animals at the University of Kuopio and Neuroscore

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Figure 1 Study design. After sham-operation or traumatic brain injury (TBI) induced by lateral fluid-percussion injury, rats were treated with vehicle [Sham-Veh (n = 7), TBI-Veh (n = 12)] or lacosamide [Sham-LCM (n = 8), TBI-LCM (n = 12)] for 3 d (30 mg/kg, i.p., t.i.d., at 8 h intervals ) and followed for 16 d. As functional outcome measures, we assessed composite neuroscore, beam-walking, and Morris water-maze performances. Structural outcome was assessed with magnetic resonance imaging (MRI) at 2 d and histology at 16 d post-TBI.

Please cite this article in press as: Pitkänen, A., et al., Effect of lacosamide on structural damage and functional recovery after traumatic brain injury in rats. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.02.001

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Effect of lacosamide on structural damage and functional recovery after TBI

Sham-Veh n=8 Surgery Randomizaon n=41

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Randomizaon Mortality n=2

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Figure 2 Flow chart summarizing the randomization of animals into different treatment groups and their associated mortality.

by the Provincial Government of Kuopio, Finland. All procedures were conducted in accordance with the European Community Council directives 86/609/EEC.

Lateral fluid-percussion induced TBI TBI was induced with lateral fluid-percussion injury (FPI) according to McIntosh et al. (1989). Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg) and magnesium sulphate (127 mg/kg), propyleneglycol (43%), and ethanol (11.7%), then held in a stereotaxic frame (lambda and bregma at the same horizontal level). A midline scalp incision was made, the scalp and temporal muscles were reflected, and a 5 mm craniotomy was created over the left cortex midway between the lambda and bregma, and midway between the sagittal suture and temporal ridge. The dura was left intact and a plastic female Luer-lok connector was secured in the craniotomy with Vetbond adhesive (3 M, St.Paul, MN, USA). The connector was anchored to a screw placed in the skull with dental acrylate, rostral to the bregma. Animals were placed on heating pads whilst anesthetized to maintain normal body temperature. At 90 min following induction of anesthesia, the appropriate level of anesthesia was checked as a response to tail pinch, and animals were attached to the fluid-percussion device (Am Science Instruments, Richmond, VA, USA) to produce moderate TBI. Animals were removed from the device and thereafter the dental cement, screw, and Luer-lok connector was removed before the scalp was sutured. Sham animals underwent surgery but were not injured.

Administration of LCM and vehicle After surgical operation, animals were randomized to the Sham-Veh (vehicle), Sham-LCM, TBI-Veh, or TBI-LCM groups (Fig. 2). LCM was provided by Schwarz Biosciences GmbH and dissolved in 0.9% saline at a concentration of 10 mg/ml. Saline (0.9%, 3 ml/kg, i.p.) served as vehicle. Treatment started at 30 min after the induction of TBI (i.e., 120 min after the induction of anesthesia in the TBI group) or 120 min after induction of anesthesia in the sham-operated rats (corresponding to the time when treatment was started in TBI animals). Treatment was continued at 8 h intervals for 3 d.

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Magnetic resonance imaging MRI was performed with the use of an MRI console (Varian Unity INOVA) interfaced to a 4.7 T horizontal magnet (Magnex Scientific Ltd., Abington, UK). A half-volume coil operated in quadrature mode (diameter 30 mm, Highfield imaging, Minneapolis, USA) was used for signal transmission and reception. At 2 d post-TBI or sham operation, rats were anesthetized (1% halothane, 30% O2 , 69% N2 O) and prepared for MRI. For determination of structural and volumetric alterations, T2 -weighted multi-slice (17 continuous slices) spin-echo images were acquired using TR = 3 s, TE = 70 ms, matrix size of 128 × 256, field of view of 30 mm × 30 mm, and a slice thickness of 1 mm. The third of the trace of the diffusion tensor (Dav ) was quantified from a single slice in the middle of the lesion using a fast-spin-echo sequence with four pairs of bipolar gradients along each gradient axis (slice thickness 1.5 mm, 128 × 256, FOV 40 mm × 40 mm) with three b-values between 0 and 1000 s/mm2 (slice thickness 1.5 mm, TR = 2.5 s). T2 was quantified from the same slice using a multi spin echo sequence (TE = 20, 40, 60, 80 ms, TR = 3.0 s, slice thickness 1.5 mm, 128 × 256, FOV 30 mm × 30 mm). T2 *-weighted images (TE = 15 ms) were acquired to see any intracerebral hemorrhage. The extent of any hemorrhage in the cortex and in the subcortical white matter were visually scored as follows: 0, no blood; 1, small hemorrhage; 2, moderate hemorrhage (either a large area visible in one MRI slice or detectable blood in ≥3 slices); 3, large hemorrhage (wide areas of blood extending rostrocaudally in ≥5 MRI slices). Quantitative T2 and Dav maps were calculated and the regions of interest (ROIs) were analyzed using in house written Matlab-based software (aedes.uef.fi). Regions of interest (ROIs) were manually outlined in T2weighted images (ROIs are indicated in Fig. 4C), the edges of the hyperintense lesion were determined by visual analysis of an expert, and all the voxels within the lesion outlines were included in the count of ‘lesion voxels’.

Behavioral analysis A composite neuromotor score Neuroscore has been used as a standard measure to assess the injury severity and effect of therapy on motor function in the lateral FPI model before (Thompson et al., 2005). Testing was done at 1 d before TBI, and on days 2, 7, and 15 post-TBI (surgery was on day 0). Briefly, animals were given a score from 0 (severely impaired) to 4 (normal) for each of the following 7 indices: (i and ii) left and right (2 indices) forelimb flexion during suspension by the tail, (iii and iv) left and right (2 indices) hindlimb flexion when the forelimbs remain on hard surfaces and the hindlimbs are lifted up and back by the tail, (v and vi) ability to resist a lateral propulsion toward the left and right (2 indices), and (vii) angle board. A composite neuroscore (0—28 points) was generated by combining the scores for each of these seven tests. Beam-walking To evaluate complex motor movements and coordination, beam-walking was performed 1 d prior to TBI and on days 2, 7, and 15 post-TBI according to Ohlsson and Johansson

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(1995). The beam was a 1390 mm long and 21 mm wide wooden bar and was placed 430 mm above the floor. At the right end of the beam there was a black box (250 mm × 200 mm). A wall was placed 30 cm to the left of the beam (rats are more willing to walk when a wall is placed next to the beam). A mirror was behind the beam on the side wall. Starting at 2 d before TBI, rats were habituated to walk on the beam. A rat was put into the box for 1 min. Then, the rat was put onto the beam at a starting distance of 15 cm from the box. The rat was allowed to go to the box and stay there for 1 min. Thereafter, the rat was put on the beam at a starting distance of 35 cm from the box. The rat was allowed to go into the box (and to be there for 1 min). This step was repeated. On the next day, the rat was put into the box for 1 min and then allowed to go to the box starting from 35 cm, followed by 70 cm, and finally from a 100 cm distance from the box. On the testing day the rat was allowed to cross the whole beam three times. Between each run the rat was in the box for 1 min. Scoring was as follows: Score 0 = the rat falls down, Score 1 = the rat is unable to traverse the beam but remains sitting across the beam, Score 2 = the rat falls down during its walk, Score 3 = the rat can traverse the beam, but the affected hindlimb does not aid in forward locomotion, Score 4 = rat traverses the beam with >3 footslips, Score 5 = rat crosses the beam with 1—3 footslips, Score 6 = rat crosses the beam with no footslips. A mean score of three runs for each day was calculated. Morris water-maze Spatial learning and memory performance of rats was tested in the Morris water maze (slightly modified from Halonen et al., 1996). The water-maze test was started at 12 d after the induction of TBI. Over a series of 10 trials on two consecutive days, animals learned the location of a submerged platform using visual cues outside the maze. The time (latency) to reach the hidden platform was recorded for each trial. In addition, path length (swimming distance) and swimming speed were measured. On day 14 following TBI (probe trial), the platform was removed from the maze and rats were allowed to swim for 60 s to allow us to evaluate their memory of the platform location. The time spent in the 4 quadrants of the maze was recorded. The 10 trials per day were averaged for each animal and the mean score was calculated for swimming on days 12 and 13 post-TBI. Differences were calculated across different treatment groups for each swimming day.

cryoprotected in 20% glycerol in 0.02 M potassium phosphate buffer, pH 7.4, for 24 h, frozen in dry ice, and stored at −70 ◦ C. A 1-in-5 series of frozen sections was cut at 30 ␮m thickness in the coronal plane from the rostral end of the amygdaloid complex to the caudal end of the entorhinal cortex using a sliding microtome. Sections were collected into tissue collection solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) and stored at −20 ◦ C until processing. Staining and analysis of sections One series of sections was stained with thionin to characterize the cytoarchitectonic boundaries. A series of adjacent sections was stained using Fluoro-Jade B as described previously to determine the severity and distribution of TBIinduced neuronal damage in the hippocampus and in the cortex around the lesion side, both ipsilaterally and contralaterally (Narkilahti et al., 2003). The severity of ongoing cortical damage was scored in Fluoro-Jade B stained sections as follows: 0 = no damage, 1 = scattered cells, 2 = scattered cells but lesion cavity does not extend through all cortical layers, 3 = scattered cells and lesion cavity extends through all cortical layers, but the dorsoventral extent of the lesion cavity ≤2 times the thickness of the cortex, 4 = scattered cells plus lesion cavity that extends through all cortical layers, and its dorsoventral extent is > 2 times the thickness of the cortex. In the hippocampus, the density of degenerating neurons and fibers was evaluated separately. Neurodegeneration was scored from thionin-stained preparations as follows: 0 = no damage, 1 = <10% cell loss, 2 = 10—50% cell loss, 3 = >50% cell loss (Freund et al., 1992). Ongoing axonal degeneration was scored from Fluoro-Jade B stained preparations: 0 = no Fluoro-Jade B labeled axons, 1 = an occasional labeled axon, 2 = moderate density if labeled axons, 3 = high density of labeled axons (e.g., the score of axonal damage in Fig. 6G was 2). Finally, to quantify the severity of damage to hilar neurons in different treatment groups, we estimated the number of remaining hilar neurons in thionin-stained preparations by using unbiased stereology as described previously in details (see Ndode-Ekane and Pitkänen, 2013). Sampling grid of 120 ␮m × 120 ␮m was laid on the section. For every x—y step, cells were counted using a counting frame of 24 ␮m × 24 ␮m. Counting was performed throughout the section avoiding the neurons that were in focus at the surface of the section. In order to calculate the total number of hilar cells, the following equation was used:

Histological analysis of brain tissue After completion of behavioral testing, that is, 16 d after surgery, rats were perfused for histology to assess the severity of the lesion induced by TBI and the potential neuroprotective effects of LCM treatment. Fixation Rats were deeply anesthetized by an intraperitoneal injection of the anesthesia cocktail described earlier, and intracardially perfused according to the following fixation protocol: 0.9% sodium chloride (30 ml/min) for 2 min, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (30 ml/min) for 30 min. Brains were removed,

Ntot = ˙Q ·

1 1 1 · · ssf asf tsf

where section sampling fraction (ssf) is 1/10, area sampling fraction (asf, the area of counting frame divided by area of sampling grid) 0.04, and tissue sampling fraction (tsf) 1. Statistical analysis Data were analyzed using SPSS for Windows (v. 10.0). Differences between the groups in Morris water-maze, hippocampal and cortical damage, and MRI data were compared with Kruskal—Wallis tests followed by post hoc analysis with Mann—Whitney U tests. Performance in the neuroscore, and

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Figure 3 Functional outcome. (A) Composite neuroscore (mean ± SD). Analysis of performance using R software package (Noguchi et al., 2012) revealed differences between the groups [Wald type statistics (WTS), p < 0.001]. At 2 d post-TBI, TBI-Veh and TBI-LCM groups were similarly impaired. Even though both treatment groups recovered similarly during the first week post-TBI. The TBI-LCM group was more impaired than the TBI-Veh group when assessed at 2 wk post-TBI, (Kruskal—Wallis with Mann—Whitney post hoc analysis, p < 0.05). (B) Beam-walking (mean ± SD). Analysis of performance using R software package (Noguchi et al., 2012) revealed differences between the groups [Wald type statistics (WTS), p < 0.01]. At 2 d post-TBI, both the TBI-Veh and TBI-LCM groups were similarly impaired. Both treatment groups recovered similarly during the first week post-TBI (Kruskal—Wallis with Mann—Whitney post hoc analysis, p > 0.05 on different testing days). At 2 wk post-TBI, performance in the TBI-Veh group did not differ from that in the Sham-Veh group whereas the TBI-LCM group remained more impaired than the Sham-LCM group (p < 0.05). Note a mild non-significant decrease in the performance of the sham-groups both in neuroscore and beam-walking, which probably relates to craniectomy as previously suggested by Cole et al. (2011). (C) Swimming speed in the Morris water-maze (mean ± SEM) did not differ between the group when assessed at 12—14 d post-TBI. (D) Latency to find the hidden platform in the Morris water-maze (mean ± SEM) was impaired both in the TBI-Veh and TBI-LCM groups. Statistical significances: *p < 0.05 as compared to the TBI-Veh group (Mann—Whitney U test); *p < 0.05, p < 0.01, p < 0.001 as compared to the corresponding sham-operated group (Mann—Whitney U test). The number of animals is in parenthesis. Abbreviations: cm, centimeter; LCM, lacosamide; TBI, traumatic brain injury; sec, second.

beam-walking tests was analyzed by using ‘‘R’’ Software Package for ‘‘the Nonparametric Analysis of Longitudinal Data in Factorial Experiments’’ (Noguchi et al., 2012). Further comparison of performance between the different treatment groups at each testing day was done using

Kruskal—Wallis test followed by post hoc analysis with Mann—Whitney U tests. Differences in data at different time points or between the ipsilateral and contralateral sides in the same animal were analyzed by Wilcoxon signed ranks test. A p value of less than 0.05 was considered significant.

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Figure 4 Structural outcome assessed by magnetic resonance imaging (MRI) at 2 d post-TBI. The left column shows a representative animal from the TBI-Veh group and the right column from the TBI-LCM group. (A and B) Thionin-stained coronal sections from a level corresponding to the MRI slices in panels C—H. The location of the cortical injury is indicated with an open arrow. The severity of cortical injury in the TBI-LCM group was comparable to that in the TBI-Veh group (compare representative animals in panels A and B, respectively). (C and D) T2 weighted MR images showing the cortical lesion as hyperintense (note also the enlarged ipsilateral ventricle). Regions of interest are outlined in panel C: lesion (purple), dentate gyrus (black), hippocampus proper (white, includes dentate gyrus), cortex (shown both ipsilaterally and contralaterally, white outline, note that the ROI of the ipsilateral cortex includes the lesion). (E and F) Quantitative T2 maps and (G and H) diffusion (Dav ) maps. Note a similar severity of lesion and similar T2 and diffusion changes in representative cases from both treatment groups. Numerical group averages of the quantitative T2 and Dav analysis are summarized in Table 1. Scale bars: 5 mm in panels A and B, 1 mm in panels C—H. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Effect of lacosamide on structural damage and functional recovery after TBI

Results A Impact severity The pressure used to induce TBI was 3.06 ± 0.07 atm in the TBI-vehicle group and 3.06 ± 0.08 atm in the TBI-LCM group (p > 0.05). The time in apnea was 30.6 ± 21.2 s in the TBI-vehicle group and 27.9 ± 21.1 s in the TBI-LCM group (p > 0.05). Two of 26 injured rats (8%) died within 48 h postTBI (Fig. 2). The cause of death was considered to be related to TBI. Thus, markers of TBI severity did not differ between the vehicle and LCM groups.

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Behavioral analysis Neuroscore Data are summarized in Fig. 3A. Analysis of performance of different treatment groups over the follow-up period using R software package (Noguchi et al., 2012) revealed differences between the groups [Wald type statistics (WTS), p < 0.001]. Further analysis at each time point did not reveal any differences between the groups at baseline (Kruskal—Wallis, p > 0.05). At 2 d after TBI, the Kruskal—Wallis test indicated differences in neuroscore between the groups (p < 0.001). Both the sham-operated (p < 0.05) and TBI groups (p < 0.01) had lowered neuroscores compared to those at baseline, indicating motor deficits induced by the surgery. Both the TBI-Veh and TBI-LCM groups had lower neuroscores than their corresponding sham-operated groups (both p < 0.001), indicating that the lateral FPI caused a more severe motor deficit than sham surgery. LCM did not affect the motor deficit since there was no difference between the TBI-Veh and TBI-LCM groups (p > 0.05). At 7 d and 15 d after TBI, the Kruskal—Wallis test indicated differences in neuroscore between the groups (p < 0.001). Both TBI groups still had a lower neuroscore than their respective sham-operated controls at both time points (p < 0.001). There was no difference between the TBI-Veh and TBI-LCM groups at 7 d post-TBI (p > 0.05). However, at 15 d post-TBI the neuroscore in the TBI-LCM group was lower than that in the TBI-vehicle group (19.9 ± 2.5 vs. 22.1 ± 2.5, p < 0.05). Beam-walking Beam-walking data are summarized in Fig. 3B. Analysis of performance of different treatment groups over the followup period using R software package (Noguchi et al., 2012) revealed differences between the groups (WTS, p < 0.01). Further analysis at each time point did not reveal any differences in beam-walking between the groups at baseline (Kruskal—Wallis, p > 0.05). No difference was found between the Sham-Veh and Sham-LCM groups at any time point. Both the TBI-Veh and TBI-LCM groups were impaired at 2 d post-TBI as compared to corresponding controls (p < 0.001), but there was no difference between the injured treatment groups (p > 0.05). Both the TBI-Veh and TBI-LCM groups improved over the 15-d follow-up. At 15 d post-TBI, the TBI-Veh group performed as well as the Sham-Veh group (p > 0.05), but the TBI-LCM group had not reached the level

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TBILCM

Figure 5 Bar graphs showing the severity of (A) neurodegeneration and (B) axonal injury in the ipsilateral hippocampus for different groups. For scoring, please see ‘‘Materials and methods’’. (C) Total number of neurons in the ipsilateral hilus of the dentate gyrus in different treatment groups estimated by using unbiased stereology. Numbers indicate animals included in each group. Data are shown as mean ± standard deviation. Statistical significances: ***p < 0.001, **p < 0.01 as compared to the ShamVeh group; ### p < 0.01 as compared to Sham-LCM group. There were no differences between the TBI-Veh and TBI-LCM groups in any of the hippocampal subfields analyzed either semiquantitatively (panels A and B) or by using unbiased stereology (panel C). Abbreviations: 1, CA1 subfield; a, CA3a subfield; b, CA3b subfield; c, CA3c subfield; h, hilus; g, granule cell layer; LCM, lacosamide; TBI, traumatic brain injury.

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Figure 6 Structural outcome assessed by histology at 16 d post-TBI. The quantitative analysis is summarized in Fig. 5. (A) A representative thionin-stained coronal section of the hippocampus from a rat in the Sham-Veh group and (B) TBI-Veh group. (C) A representative Fluoro-Jade B-stained section from a rat in the TBI-Veh group, demonstrating neurodegenerative cells (arrows) in the granule cell layer of the dentate gyrus. (D) The severity of neurodegeneration in the granule cell layer in a rat from the TBI-LCM group was comparable to that of a rat from the TBI-Veh group (compare panels C and D). (E) Fluoro-Jade B-positive pyramidal cells (arrows) in the CA3a area of the hippocampus of a rat from the TBI-Veh group. (F) The severity of neurodegeneration in the CA3a subfield of a rat from the TBI-LCM group was comparable to that of a rat from the TBI-Veh group (compare panels E and F). (G) Photomicrograph

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Effect of lacosamide on structural damage and functional recovery after TBI of the Sham-LCM group (4.8 ± 1.4 vs. 5.7 ± 0.3, p < 0.05). However, there was no difference between the TBI-Veh and TBI-LCM groups (p > 0.05). Morris water-maze Morris water-maze data are summarized in Fig. 3C and D. There was no difference in swimming speed between the groups (Fig. 3C). However, there was a difference in latency (time taken to find the submerged platform, Fig. 3D) and path distance (swimming distance traveled by the animals, not shown) between the groups (p < 0.01). Post hoc analysis showed that both TBI groups had longer latencies and swimming distances than their respective sham-operated controls (p < 0.05). There was, however, no difference between the TBI-Veh and TBI-LCM groups. Performance in the probe trial test (% time spent in each quadrant of the maze) did not differ between the groups (p > 0.05, data not shown).

MRI analysis MRI data are summarized in Table 1 and representative images are shown in Fig. 4. Regions of interest were drawn from a representative injured rat on T2 weighted coronal images taken at 2 d post-TBI in Fig. 4C. Lesion volume Lesion volumes did not differ between the TBI-Veh and TBILCM groups (p > 0.05). None of the sham-operated animals had any detectable lesion on MRI. Hemorrhage In 23 of 24 injured rats, T2 * weighted imaging showed intracerebral hemorrhage on the ipsilateral side in the cortex and/or in the subcortical white matter below or adjacent to the impact site (external capsule, corpus callosum, alveus). No difference was found between the TBI-Veh and TBI-LCM groups (p > 0.05). None of the sham-operated animals had any detectable hemorrhage. T2 Ipsilaterally, T2 relaxation time was prolonged in the lesion (24—26 ms longer, p < 0.01) as well as in the ipsilateral cortex (12—16 ms, p < 0.01), dentate gyrus (3—4 ms, p < 0.01), and hippocampus proper (4—5 ms, p < 0.01) in both the TBI-Veh and TBI-LCM groups as compared to corresponding controls. However, there was no difference in T2 between the TBI-Veh and TBI-LCM groups (p > 0.05). Contralaterally, in the cortex, the T2 relaxation time was slightly decreased (1—2 ms) in injured animals as compared to sham-operated animals (TBI-Veh p < 0.01; TBI-LCM p < 0.05). This could relate to a slight difference in physiological conditions between animal groups during anesthesia, causing blood oxygenation

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and blood volume related relaxation changes. It could also reflect the contracup injury effect. Dav Ipsilaterally, Dav was slightly increased in the lesion at 2 d post-injury in both the TBI-Veh and TBI-LCM groups as compared to the corresponding sham-operated groups (Dav increased by 0.08 and 0.07 × 10−3 mm2 /s, respectively, both p < 0.01). There was no difference between the TBI-Veh and TBI-LCM groups (p > 0.05). In the cortex, dentate gyrus and hippocampus the Dav did not differ between the various groups (Table 1).

Histology Data summarizing hippocampal neurodegeneration are shown in Figs. 5 and 6. No Fluoro-Jade B staining was observed in any of the sham-operated animals. Instead, it indicated ongoing degeneration of neurons and axons in all rats with TBI at 15 d after injury. In the traumatized cortex, we found a substantial number of Fluoro-Jade B positive cells with neuronal morphology in all rats. In most rats the band of positive cells extended down to the perirhinal and dorsal aspect of the entorhinal cortex. In the ipsilateral hippocampus, there were typically positive cells with neuronal morphology in the infragranular region and granule cell layer of the dentate gyrus (Fig. 6C and D). In the ipsilateral hippocampus proper, the positive cells were typically located in the CA3a region (Fig. 6E and F). Contralaterally, a few degenerating neurons were found in the granule cell layer and in the infragranular layer. Finally, the ipsilateral thalamus showed a large number of Fluoro-Jade B positive cells. Damaged axons were typically found ipsilaterally in the inner molecular layer, hilus, and CA3a (around pyramidal cells) (Fig. 6G and H). We also found a substantial number of Fluoro-Jade B positive axons with bulbous enlargements in major fiber tracts, including the fimbria-fornix and stria terminalis (data not shown). There was no difference in the severity of cortical damage between the TBI-vehicle and TBI-LCM groups (p > 0.05). Also, the severity of hippocampal neuronal and axonal damage did not differ between TBI-vehicle and TBI-LCM groups in any of the hippocampal subfields (p > 0.05). Estimation of neuronal numbers in the hilus by using unbiased stereology revealed differences between the groups (Fig. 5C, Kruskal—Wallis p < 0.001). Post hoc analysis with Mann—Whitney U test indicated that neuronal numbers did not differ between the Sham-Veh and ShamLCM groups (41 063 ± 8305 vs. 46 071 ± 8516, p > 0.05) or between the TBI-Veh and TBI-LCM groups (22 771 ± 8890 vs. 23 886 ± 5973, p > 0.05). However, neuronal numbers were lower in the TBI-Veh group than in the Sham-Veh (22 771 ± 8890 vs. 41 063 ± 8305, p < 0.01) or Sham-LCM

of Fluoro-Jade B stained sections demonstrating degenerating axons (fluorescent dots indicated with a white arrow) in the CA3a subfield of the hippocampus in a rat from the TBI-Veh group. (H) Degenerating axons in the CA3 subfield of the hippocampus in a rat from the TBI-LCM group. Abbreviations: CA1, CA1 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; g, granule cell layer; h, hilus; ml, molecular layer; o, stratum oriens; p, stratum pyramidale; r, stratum radiatum. The scale bar equals 500 ␮m in A and B and 100 ␮m in C—H. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Quantitative MRI data summarizing T2 relaxation (T2 ) time and 1/3 × the trace of the diffusion tensor (Dav ) in different brain areas, volume of the lesion, and the severity of intracerebral hemorrhage. Brain area

Sham-Veh (8) 3

Lesion volume (mm ) Hemorrhage (Score 0—3) T2 (ms) Ipsilateral side Lesion Cortex Dentate gyrus Hippocampus Contralateral side Cortex Dentate gyrus Hippocampus Dav (10−3 mm2 /s) Ipsilateral side Lesion Cortex Dentate gyrus Hippocampus Contralateral side Cortex Dentate gyrus Hippocampus

0 0

Sham-LCM (7) 0 0

TBI-Veh (12) 38.5 ± 4.5** 2.2 ± 0.6**

55.7 ± 0.5 57.4 ± 0.3 61.5 ± 0.4 61.1 ± 0.2

56.1 ± 0.4 57.2 ± 0.3 62.1 ± 0.6 61.1 ± 0.4

79.7 69.7 64.9 65.4

± ± ± ±

57.5 ± 0.4 62.2 ± 0.6 61.8 ± 0.3

57.7 ± 0.5 61.8 ± 0.5 61.4 ± 0.3

55.8 ± 0.2** 61.9 ± 0.4 61.7 ± 0.3

± ± ± ±

1.8** 1.9** 0.5** 0.6**

0.702 ± 0.013 0.733 ± 0.008 0.728 ± 0.008 0.728 ± 0.006

0.718 ± 0.013 0.731 ± 0.012 0.718 ± 0.022 0.709 ± 0.015

0.786 0.754 0.711 0.714

0.737 ± 0.008 0.706 ± 0.009 0.704 ± 0.007

0.745 ± 0.008 0.705 ± 0.008 0.698 ± 0.008

0.713 ± 0.008 0.713 ± 0.008 0.704 ± 0.006

0.011** 0.011 0.012 0.011

TBI-LCM (12)

Kruskal—Wallis

40.5 ± 3.6** 1.7 ± 0.9**

p < 0.01 p < 0.01

± ± ± ±

p < 0.01 p < 0.01 p < 0.01 p < 0.01

81.8 72.9 65.7 65.5

1.9** 1.5** 0.5** 0.5**

56.3 ± 0.2* 61.9 ± 0.5 62.0 ± 0.3

p < 0.01 — —

± ± ± ±

0.012** 0.010 0.008 0.006

p < 0.01 — — —

0.705 ± 0.006** 0.688 ± 0.006 0.693 ± 0.006

p < 0.01 — —

0.785 0.757 0.700 0.709

Data are presented as mean ± SEM. Kruskal—Wallis indicated differences between the groups in various parameters (right column). Post hoc analysis with Mann—Whitney’s test indicated that there were differences between the TBI and corresponding sham groups (i.e., Sham + Veh vs. TBI + Veh and Sham + LCM vs. TBI + LCM, **p < 0.01; *p < 0.05). There were no differences between the sham-groups or between the TBI + Veh and TBI + LCM groups. In the sham groups, there were no interhemispheric differences. In the both TBI groups, all MRI parameters differed between the ipsilateral (side with TBI) and contralateral sides (p < 0.01, Wilcoxon).

group (22 771 ± 8890 vs. 46 071 ± 8516, p < 0.01). Also, TBILCM group had a reduced number of hilar neurons as compared to that in the Sham-Veh (23 886 ± 5973 vs. 41 063 ± 8305, p < 0.001) or Sham-LCM group (23 886 ± 5973 vs. 46 071 ± 8516, p < 0.001).

Discussion The present study was designed to investigate the neuroprotective effects of a novel antiepileptic drug, LCM, on TBI-induced structural and functional impairments. We had four major findings. Firstly, three days of LCM treatment started at 30 min post-TBI did not cause any motor or cognitive impairments in sham-operated rats. Secondly, LCM treatment did not result in any protection against axonal or neuronal injury after lateral FPI. Third, LCM treatment did not improve post-TBI cognitive recovery or motor recovery. Fourth, LCM did not cause any remarkable impairment in any of the structural and cognitive parameters assessed, even though the motor recovery was delayed. The primary mode of action of LCM is the enhancement of sodium channel slow inactivation (Errington et al., 2008; Sheets et al., 2008). Previous studies have shown mild protection of neurons and axons by several sodium channel blocking AEDs, particularly in status epilepticus or stroke models (see Pitkänen and Kubova, 2004; Pitkänen, 2007). Assessment of T2 and Dav at 2 d post-TBI revealed typical

signs of edema and tissue degeneration, that is, prolongation of T2 time and increased diffusion ipsilaterally in the cortex and the hippocampus in all injured rats. Importantly, there was no difference between the TBI-Veh and TBI-LCM groups, suggesting that LCM had no effect on the severity of vasogenic and cytotoxic edema, or structural damage. Similarly, semiquantitative and quantitative histological analysis at a more chronic time point, 16 d post-TBI, did not reveal any alleviation in the severity of neuronal or axonal degeneration in the LCM group as compared to the vehicle group. The lack of effect after TBI is somewhat unexpected as LCM has shown mild neuroprotective effects in other in vitro and in vivo models of neurodegeneration (see Beyreuther et al., 2007). For example, in hippocampal slice cultures, LCM was anti-apoptotic and protected cells from glutamate-induced excitotoxicity as well as from oxygen-glucose deprivation and staurosporine-induced apoptosis. In an animal model of ischemia that was induced by permanent middle cerebral artery occlusion, LCM reduced infarct volumes when started at the time of induction of ischemia and continued for 4 h, reaching a cumulative dose of 45 mg/kg. LCM has also been known to slightly prolong the survival of superoxide dismutase mutant mice, a genetic model of amyotrophic lateral sclerosis. Furthermore, chronic administration of a slow-release formulation of LCM resulted in the prevention of chemotherapy-related neuropathy in a rat model (Geis et al., 2011). Finally, LCM at a dose of 30 mg/kg twice daily for 3 d (but not at a dose 6 mg/kg) improved motor

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Effect of lacosamide on structural damage and functional recovery after TBI performance in the rotarod test and cognitive function in the Morris water-maze test when administered to mice exposed to closed head TBI of moderate severity induced by a pneumatic impactor. In the same study, LCM also attenuated the post-TBI inflammatory response as assessed by microglial activation at 7 d post-TBI and was neuroprotective. However, neuroprotective effect was not seen if mice were sacrificed at 1 month post-TBI (Wang et al., 2013). It is possible that the injury in the lateral FPI model is more complex and severe than in the other models in which LCM has been tested. Alternatively, other neurodegenerative mechanisms could be more important for post-TBI injury in the lateral FPI model than the ones tackled by LCM. Also, it remains to be explored whether different dosing regimens of LCM, such as more chronic administration and longer follow-up, would improve its efficacy. It also remains to be investigated if there is any dose-dependency in the effect of LCM on recovery after lateral FPI. Recent molecular and cellular studies in a partial cortical undercut model of brain injury in rats showed that administration of LCM at the dose of 100 mg/kg for 7 d, starting on the day after undercut led to reduced injuryenhanced synaptic connectivity related to axonal sprouting (Wilson et al., 2012). The authors linked the effect to inhibition of collapsin response mediator protein 2 (CRMP2) mediated microtubule polymerization, which is required for axonal growth/guidance, but this putative LCM mechanism of action has been questioned recently (Wolff et al., 2012). The authors proposed that the anti-plasticity effect detected in vivo warranted further studies on the potential of LCM as an antiepileptogenic agent after TBI. On the other hand, the observations also raised a concern that LCM might have some unfavorable effects on post-TBI functional recovery requiring axonal sprouting and synaptic plasticity. Our data showed no difference in the beam-walking performance between the LCM and vehicle treated rats with TBI. In composite neuroscore, both groups recovered by the same extent by 1 wk post-injury. During the second week, that is, several days after the discontinuation of LCM, the TBI-LCM group did not, however, reach the score achieved by the TBI-vehicle group. As the half-life of LCM in rats is about 3 h, it is unlikely that the unfavorable delayed recovery could relate to the presence of LCM in the brain (Koo et al., 2011). However, whether a short term LCM treatment would induce long-lasting effects on post-TBI plasticity as was also suggested by Wolff et al. (2012) remains to be explored. Regarding the use of LCM in the treatment of post-TBI co-morbidities clinically, it is not yet clear how LCM compares with other AEDs regarding its neuroprotective and recovery enhancing/compromising properties. Darrah et al. (2011) administered phenytoin, an AED commonly used to treat acute post-TBI seizures, for 1 d starting with a dose of 75 mg/kg at 15 min after controlled cortical impact (CCI)induced TBI in rats. This treatment regimen resulted in neuroprotection, greater plasticity, and improved cognitive outcome. However, continuing phenytoin treatment for 21 d worsened the structural and functional outcome. Previous experimental studies on newer AEDs show that the administration of remacemide at 15 min after parasagittal FPI-induced TBI in rats reduced cortical lesion volume when assessed at 48 h. However, this did not translate into any

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favorable effects on spatial memory (Smith et al., 1997). Hoover et al. (2004) reported that the administration of topiramate at 30 min after lateral FPI improved sensorimotor behavior when assessed at 4 wk post-injury, but had no favorable effects on neuronal survival or learning. Talampanel administered at 30 min after parasagittal FPI reduced neurodegeneration in the contusion area and in the CA1 subfield of the hippocampus when assessed at 7 d after TBI (Belayev et al., 2001). Carisbamate, when administered at 15 min after lateral FPI for 1 d had no effect on brain edema (at 48 h post-injury), lesion size, motor function or learning when analyzed at 4 wk post-injury (Keck et al., 2007). Recently, Dash et al. (2010) reported that administration of 400 mg/kg valproate starting at 30 min or 3 h after CCI-induced TBI and continued for 5 d improved blood—brain-barrier integrity, reduced TBI-associated hippocampal dendritic damage, lessened cortical contusion volume, and improved motor function and spatial memory. This was associated with inhibition of glycogen synthase kinase 3 (GSK-3) and histone deacetylase (HDAC). In summary, even though systematic comparative studies are not available, the present and previous data suggest that the selection of the AED to treat the aftermath of TBI as well as the duration of the treatment may influence subsequent structural and functional recovery.

Acknowledgement We thank Mr. Jarmo Hartikainen for excellent technical assistance.

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Please cite this article in press as: Pitkänen, A., et al., Effect of lacosamide on structural damage and functional recovery after traumatic brain injury in rats. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.02.001