Neuroprotection and anti-seizure effects of levetiracetam in a rat model of penetrating ballistic-like brain injury

Abstract

Purpose: We assessed the therapeutic efficacy of FDA-approved anti-epileptic drug Levetiracetam (LEV) to reduce post-traumatic nonconvulsive seizure (NCS) activity and promote neurobehavioral recovery following 10% frontal penetrating ballistic-like brain injury (PBBI) in male Sprague-Dawley rats.

Methods: Experiment 1 anti-seizure study: 50 mg/kg LEV (25 mg/kg maintenance doses) was given twice daily for 3 days (LEV_3D) following PBBI; outcome measures included seizures incidence, frequency, duration, and onset. Experiment 2 neuroprotection studies: 50 mg/kg LEV was given twice daily for either 3 (LEV_3D) or 10 days (LEV_10D) post-injury; outcome measures include motor (rotarod) and cognitive (water maze) functions.

Results: LEV_3D treatment attenuated seizure activity with significant reductions in NCS incidence (54%), frequency, duration, and delayed latency to seizure onset compared to vehicle treatment. LEV_3D treatment failed to improve cognitive or motor performance; however extending the dosing regimen through 10 days post-injury afforded significant neuroprotective benefit. Animals treated with the extended LEV_10D dosing regimen showed a twofold improvement in rotarod task latency to fall as well as significantly improved spatial learning performance (24%) in the MWM task.

Conclusions: These findings support the dual anti- seizure and neuroprotective role of LEV, but more importantly identify the importance of an extended dosing protocol which was specific to the therapeutic targets studied.

Keywords

Traumatic brain injury post-traumatic seizures neuroprotection motor cognitive function rat

Introduction

Traumatic brain injury (TBI) is a major medical concern of public and military health. According to reports by Centers for Disease Control and Prevention, approximately 2.5 million people in the United States have sustained a traumatic brain injury (“Injury Prevention & Control: Traumatic Brain Injury, Severe Traumatic Brain Injury,” 2014, March 4). Likewise, during recent military conflicts TBI incidence among active duty personnel increased substantially with at least 20% categorized as severe TBI (Centers for Disease Control and Prevention, 2013). It has been established that survivors of severe TBI are at increased risk for early (<7 days post-injury) post-traumatic seizures (Vespa et al., 1999), which contribute to secondary injury processes and worsened neurological outcomes (Kirmani, Mungall, & Ling, 2013; Lu, Mountney, et al., 2013; Vespa et al., 2007), as well as progressive cognitive decline and other associated co-morbidities (Benge, Phenis, Bernett, Cruz-Laureano, & Kirmani, 2013; Bushnik, Englander, Wright, & Kolakowsky-Hayner, 2012). Currently, prompt treatment and prophylactic use of AEDs is standard of care for severe TBI patients; however there are no drugs approved by the United States Food and Drug Administration (FDA) under the category of neuroprotection to improve patients’ neurological function.

Post-traumatic nonconvulsive (silent) seizures (NSC) account for approximately half of all seizures found in civilian severe TBI injuries (Vespa et al., 1999) and represent a large fraction of patients whose seizures have the potential of going undiagnosed. Spontaneously occurring NCS activity is one of the unique characteristics of the penetrating ballistic-like brain injury (PBBI) model occurring acutely, as early as 30 min and resolving within a few days, in approximately 60–75% of all animals subjected to the 10% frontal unilateral PBBI (Lu, Dave, et al., 2013; Lu et al., 2011). In addition, PBBI-induced NCS have been shown to be amenable to traditional AEDs, such as phenytoin and ethosuximide (Mountney, Shear, et al., 2013). The PBBI model also produces significant motor, sensory and cognitive deficits that are evident immediately following the injury and persist up to at least 10 weeks post-PBBI (Davis, Shear, Chen, Lu, & Tortella, 2010; Mountney, Leung, Pedersen, Shear, & Tortella, 2013; Shear et al., 2010; Shear & Tortella, 2013; Shear, Williams, Sharrow, Lu, & Tortella, 2009).

Levetiracetam (LEV) is an adjunctive AED approved by the FDA to treat partial and secondary generalized seizures. Within the past few years, LEV has garnered attention for use in TBI particularly due to its ease of use in critically ill patients, low side effect profile, and unconfirmed potential for neuroprotection (Benge et al., 2013; Dewolfe & Szaflarski, 2013; Kirmani et al., 2013; Rowe et al., 2014). LEV is structurally derived from the nootropic drug piracetam which along with other related compounds are known as cognitive-enhancing modulators of cerebral function (Malykh & Sadaie, 2010). Although the primary clinical use of LEV, unlike its parent molecule, is to control seizures evidence has emerged demonstrating neuroprotection properties of LEV in animal models of TBI (Wang et al., 2006; Zou, Hurwitz, Fowler, & Wagner, 2014, 2015) and cerebral ischemia, (Cuomo et al., 2013; Hanon & Klitgaard, 2001; Komur et al., 2014) as well as improved clinical outcomes in patients (de Groot et al., 2013; Taylor, Heinrichs, Janzen, & Ehtisham, 2011a). Notably, two investigations by Zou and colleagues address the notion that the neuroprotective efficacy of LEV may be dependent on the therapeutic window, and/or dosing duration, of treatment. For example, multiple acute treatments within the first 24 hours post-injury (Zou et al., 2015) were unable to replicate the neuroprotective effects provided by a prolonged, 20 day daily treatment regimen (Zou et al., 2014).

This study aims to evaluate the potential dual function anti-seizure and neuroprotection action of LEV with the premise that such an effective drug could be used as a first-line AED treatment for TBI patients with increased risk of both seizures and cognitive dysfunction. To achieve this objective we used the clinically relevant intravenous (i.v.) route of administration in the penetrating ballistic-like brain injury (PBBI) to evaluate LEV treatment following severe TBI.

2 Methods

2.1 Subjects

Male Sprague-Dawley rats (275–320 g, Charles River Labs, Raleigh, VA) were individually housed in a normal 12 h light/dark cycle. The animal housing facility was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Walter Reed Army Institute of Research. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

2.2 Drug administration

Levetiracetam (Keppra^® Injectable Solution; West-Ward Pharmaceuticals) treatment was administered intravenously via a right jugular vein catheter. For Experiment 1, anti-seizure study, an initial loading dose of 50 mg/kg was given followed by 25 mg/kg maintenance doses given twice daily for 3 days (LEV_3D) (PBBI + vehicle; n = 18 and PBBI + LEV_3D ; n = 15). For Experiment 2, neuroprotection studies, two dosing durations of LEV were used: (1) 50 mg/kg was given twice daily for 3 days (LEV_3D) or (2) 50 mg/kg was given twice daily for 10 days (LEV_10D) (Sham; n = 9; PBBI + vehicle; n = 11; PBBI + LEV_3D; n = 11; and PBBI + LEV_10D; n = 12). In each dosing protocol the daily treatments were delivered at an 8-h interval, 0900 and 1700, with the first injection always administered 30 minutes after the PBBI.

The LEV dose concentrations chosen for the present experiments were adapted from the preclinical literature of LEV (Cuomo et al., 2013; Hanon & Klitgaard, 2001) with modifications based on pilot studies in the PBBI model. Notably, in preclinical and clinical seizure studies, the follow on maintenance doses are often a fraction of the initial loading dose (Bhullar et al., 2014; Hanon & Klitgaard, 2001). In keeping with this, the current anti-seizure Experiment 1 was designed to use a single bolus infusion (50 mg/kg) followed by the reduced twice daily maintenance doses.

2.3 Penetrating ballistic-like brain injury

Unilateral (right) frontal PBBI was performed as previously described (Shear et al., 2010). The PBBI apparatus consists of a computer-controlled ballistic injury device (Mitre Corp, McLean VA) attached to a specially designed PBBI probe. The probe was constructed from a 20 G stainless steel tube with fixed perforations along one end that were sealed by airtight elastic tubing. Animals were anesthetized with 2% isoflurane and positioned in a stereotaxic frame for probe insertion. The scalp was incised along the midline and a 4 mm diameter burr hole was created to expose the right frontal pole of the brain (+4.5 mm antero-posterior, +2 mm medio-lateral from bregma). To induce PBBI, the probe was manually inserted through the cranial window at an angle of 50° from the vertical axis and 25° counter-clockwise from the anterior-posterior axis to a distance of 1.2 cm from the dura surface of the brain. Once the probe was in place, a computer-controlled pulse generator was activated to rapidly inflate/deflate the elastic tubing on the probe to an elliptical-shaped balloon to a size equal to 10% of the total brain volume. All surgical procedures were performed under isoflurane (2% vapor) anesthesia on spontaneously breathing animals. Core body temperature was maintained normothermic (37°C) using a heating blanket (Harvard Apparatus, Holliston, MA). Sham animals were exposed to all the same surgical procedures as PBBI except for the insertion/expansion of the PBBI probe. Following surgery, animals were placed in a warm chamber maintained by a circulatingwater heating system (Gaymar Industries, Orchard Park, NY) until fully recovered from anesthesia.

2.4 Seizure detection and EEG data analysis

For the anti-seizure Experiment 1, the nonconvulsive seizures were detected by 72 h continuous EEG monitoring initiated immediately after the PBBI. The EEG signals were recorded from awake animals using a digital data acquisition system (Harmonie Software, Natus Medical Incorporated, San Carlos, CA) via four skull electrodes (stainless steel screw coupled with a piece of Nichrome wire) and symmetrically implanted over the bilateral parietal region of the brain (+1 mm anterior and +4 mm posterior to bregma,±3.5 mm lateral to midline). Nonconvulsive seizures were defined and identified as previously described (Lu et al., 2011; Lu, Mountney, et al., 2013; Mountney, Shear, et al., 2013) based on the following criteria: 1) repetitive rhythmic spike discharges occurring at a frequency of 1–4 Hz; 2) spike amplitude distinctively greater than background activities; 3) duration of continuous seizure activity for >5 s; and 4) manifested as generalized (bihemispheric) or focal/partial (unihemispheric). Based on these seizure criteria, the following parameters were evaluated for all groups: 1) NCS incidence, number of animals that experienced at least one NCS event; 2) NCS frequency, number of NCS events per animal; 3) NCS episode duration (s), the duration of individual NCS events; 4) NCS total duration (s), the sum duration of all NCS events per animal; and 5) NCS onset latency (h), the time interval between PBBI on the occurrence of the first NCS event per animal. In the PBBI model, the vast majority (99%) of seizures are nonconvulsive in nature, therefore no outward behavioral manifestations are present. All NCS events detected from off-line EEG recordings were confirmed as nonconvulsive by synchronized video recordings.

2.5 Sensorimotor assessments

Neurological deficits were assessed at 30 min post-injury (prior to drug treatment), and again at 24 h, 3d, 7d, 14d and 21d post-injury using the neurological score (NS) as previously described (Bederson et al., 1986; Shear et al., 2010). As the preliminary inclusion/exclusion criteria, PBBI injured animals that do not display a NS ≥6 within 30 min following PBBI are excluded from further study.

The Rotamex-5 rotarod apparatus (Columbus Instruments, Columbus, OH) was used to measure motor coordination and balance as previously described (Shear et al., 2011). Prior to surgery animals were trained to criteria on a fixed-speed version (10 rpm) of the rotarod task, and assessed later for motor performance at 7 and 10 days post-injury. The ability of animals to remain balanced on the rotating rod was assessed at sequential fixed-speed increments of 10, 15, and 20 rpm for a maximum of 60 sec each trial, 2 trials/speed, with a 60 sec inter-trial interval (ITI). Outcome measures were (1) latency (sec) to fall during each fixed-speed increment of 10, 15, and 20 rpm (motor performance) and (2) latency (sec) for both post-injury testing days at the most difficult 20 rpm speed increment (mean motor score).

2.6 Cognitive testing

Cognitive function was assessed at post-injury days 13–17 using the Morris water maze (MWM) task as previously described (Shear et al., 2011). Animals were given 4 acquisition learning trials per day (90 sec/trial with a 30 min ITI) for 5 consecutive days followed by a missing platform trial (probe, a single 60 sec trial given 30 mins following the final acquisition trial) to assess memory retention. Primary outcome measures were operationally defined as (1) latency (sec) to find the hidden platform (acquisition score), (2) percent time of outer perimeter swimming behavior (thigmotaxis) and (3) percent time searching in the target zone (circular area defined solely in the video tracking software that encompasses both the platform area as well as the surrounding annulus) during the missing platform (probe) trial (memory retention score). For visualization of swimming behavior during the missing platform retention trial, single frame pictures of colored traces from video tracking software are included in Fig. 4. Red traces indicate video frames calculating the percentage of time in the target zone, yellow traces indicate video frames used for calculation of percentage of time spent in thigmotaxic perimeter swimming behavior.

2.7 Histopathological assessments

At the indicated post-injury time points, animals were anesthetized and processed for histopathological analysis using the Cavalieri method as previously described (Shear et al., 2010). At 3 days post-injury (anti-seizure study) lesion volume (mm³) wasdetermined by calculating the area of the lesion (mm²) and multiplying the sum of the lesioned areas obtained from each section by the distance between sections (1 mm). Due to the development of large cavitation in the brain and overall hemispheric atrophy at 3 weeks post injury (neuroprotection study), tissue volume loss at this more delayed time point is expressed as a percent of the contralateral (non-injured) hemisphere (Shear et al., 2010).

2.8 Data analysis

All behavioral testing and histological analyses were conducted by trained laboratory personnel who were blinded to the experimental conditions. Statistical significance for anti- seizure effects of LEV treatment was evaluated using nonparametric analysis (Mann-Whitney test). Chi square analysis was used for evaluating a trend in proportions of quantal data (NCS incidence). To control for “floor” effects of NCS, vehicle-treated animals that displayed no NCS events and the corresponding ranked pairs for LEV-treated animals were excluded from statistical analyses, except for the NCS incidence (Lewis-Beck, Bryman, & Liao, 2004; Mountney, Shear, et al., 2013). This exclusion criteria is applied in an unbiased manner across all PBBI injured groups. Histopathological and neurobehavioral outcomes were compared by independent samples t-tests or analysis of variance (ANOVA) followed by Student Newman- Keuls post-hoc and paired t-test analyses when appropriate. All data are presented as the mean±standard error of the mean (SEM), p values <0.05 were considered significant.

3 Results

3.1 Effects of LEV on PBBI-induced NCS

The anti-seizure effect of LEV_3D treatment was evident across all NCS parameters assessed. Spontaneous NCS occurred in 72% of vehicle-treated animals with the first NCS occurring at 19.4 h post-injury (the onset latency). LEV_3D treatment significantly reduced NCS incidence to 33% and delayed the onset latency to 55.4 h post injury (Fig. 1A and B, ^†p < 0.05). On average, vehicle treated animals experienced 11.8±2.2 NCS episode/rat with an average episode duration being 30.5±5.0 sec/event, which yielded an averagecumulative NCS duration of 417.6±115.0 sec/rat. LEV_3D treatment significantly reduced NCS frequency and cumulative duration by 52% and 38% , respectively (Fig. 1C and D, ^†p < 0.05). Although LEV treatment also reduced NCS episode duration, this effect did not achieve statistical significance (data not shown).

3.2 Sensorimotor outcome

The rm-ANOVA comparing neuroscores of sham-operated (normal = 0) and PBBI injured groups revealed significant neurological deficits for all PBBI groups (PBBI + vehicle = 8.6±0.55; PBBI +LEV_3D = 9.1±0.46; and PBBI + LEV_10D = 8.5±0.50). These deficits were sustained out to 3 weeks post-injury regardless of treatment (p < 0.001, data not shown) with no significant between-group differences detected among the three PBBI injured groups at any time point Rotarod Testing: Prior to PBBI, all groups had achieved a stable, baseline level of motor performance (i.e. ability to maintain balance for a min of 50 sec at 10 rpm). The rm-ANOVA (4 groups 3 speed increments) revealed a significant between-group effect at both 7 (p = 0.005) and 10 days post-injury (Fig. 2A, p < 0.05). Similarly, when data is collapsed to show overall means (Fig. 2B) theone-way ANOVA results (3 speeds combined×2 testing days) also showed significant between-group effect (7 days, ^*p = 0.005, and 10 days, ^*p < 0.05, post-injury). Mean latency for PBBI injured groups was significantly lower than sham (Sham 48.6±4.2 sec; PBBI + veh 25.7±4.7 sec; PBBI + LEV_3D 36.4±3.7 sec; PBBI + LEV_10D 33.6±3.5 sec; ^*p < 0.05) at 7 days post-injury with no improvements measured at the 10 day post-injury assessment (Fig. 2B). However, a treatment benefit was detected at the highest rotarod speed tested (20 rpm) where PBBI animals treated with the 10-day dosing regimen of LEV showed a significant improvement in latency to fall (21.7±4.1 sec) as compared to PBBI + veh (9.9±2.1 sec, Fig. 2C, ^†p < 0.001).

3.3 Cognitive outcome

The rm-ANOVA revealed significant between-group differences for latency to find the hidden platform (Fig. 3A, p < 0.001) and thigmotaxic swim patterns (Fig. 3C, p < 0.001). All three PBBI injured groups showed significantly longer latencies (PBBI + veh 63.4±6.3 sec; LEV_3D 65.6±4.8 sec; LEV_10D 48.1±5.2 sec, *p < 0.05) as compared to sham (27.5±1.8 sec). The LEV_10D-treated group displayed significantly reduced escape latencies when compared to the abbreviated dosing regimen PBBI + LEV_3D as well as vehicle-treated PBBI animals (Fig. 3B, # and †, p < 0.05). More specifically, the extended dosing regimen conferred a significant effect of 25% improvement (faster latency than) compared to vehicle treatment alone (Fig. 3B, †p < 0.05) during the spatial acquisition learning paradigm. Post-hoc analysis of the thigmotaxic response revealed significantly greater percentage of time spent circling the outer perimeter of the maze (Fig. 3C) for all injured groups (PBBI + veh 59.6±6.5%; PBBI + LEV_3D 67.6±5.5%; PBBI + LEV_10D 45.6±4.5% , *p < 0.05) as compared to sham (24.2±2.5% ,). However, PBBI animals treated with the extended 10-day duration of LEV treatment showed significant reductions in thigmotaxic behavior vs. animals treated with the 3-day dosing regimen (Fig. 3D, #p < 0.05).

One-way ANOVA results of the missing platform (probe) trial revealed significant between-group differences for percent time spent searching in the target zone (p < 0.05) vs. the thigmotaxic zone (p < 0.05). Post-hoc analysis showed that the PBBI + LEV_3D group and PBBI + veh controls spent significantly less time searching the target (missing platform) zone (Fig. 4B, Sham 3.8±0.42%; PBBI + Veh 1.7±0.42%; PBBI + LEV_3D 1.4±0.49%; ^*p < 0.05) and concomitantly spent a greater percentage of time circling the outer perimeter of the maze (thigmotaxis) (Fig. 4C, Sham 16.0±2.0%; PBBI + veh 37.5±7.8%; PBBI + LEV_3D 47.8±8.3%; ^*p < 0.05). In contrast, the PBBI + LEV_10D-treated group was not significantly different from sham on either of these measures (2.8±0.53% and 21.4±3.8% , respectively). Notably, PBBI animals treated with the 10-day dosing regimen performed significantly better than the PBBI animals that received LEV treatment for only 3 days (Fig. 4B and C, ^#p < 0.05).

3.4 Histopathological assessment

At 3 days post-injury, the brains of animals treated with LEV showed a trend towards reduced lesion volume compared to PBBI + vehicle (Fig. 5A; p = 0.08). However, no therapeutic effects were detected on PBBI-induced hemispheric tissue volume loss at 22 days post-injury (Fig. 5B).

4 Discussion

In this study we report an anti-seizure and neuroprotection profile of LEV in an animal model of penetrating TBI. LEV treatment initiated in at 30 m post-injury, using a 50 mg/kg bolus infusion followed by maintenance doses (25 mg/kg) delivered twice daily for 3 consecutive days, significantly reduced post-traumatic NCS activity. However, this same treatment regimen was not sufficient to confer significant neurobehavioral benefits despite using a higher concentration (e.g. 50 mg/kg twice daily for 3 days). Further work showed that an extended treatment duration of 10 days post-injury was required to see an improvement in motor and cognitive performance following PBBI. Collectively, these data demonstrate unique treatment duration protocols required for LEV to support dual action as a potential anti-seizure and neuroprotection therapy in the acute setting of severe TBI care.

Recent clinical studies have demonstrated LEV’s beneficial cognitive effects for increased verbal memory in glioma patients (de Groot et al., 2013) as well as improved neurological outcomes for TBI patients as compared to treatment standard AEDs (Szaflarski et al., 2007a; Szaflarski, Sangha, Lindsell, & Shutter,2010a, 2010b). Our preclinical PBBI model that induces etiologically relevant, spontaneous NCS (Lu et al., 2011; Mountney, Shear, et al., 2013) and a full complement of neurobehavioral deficits, (Shear et al., 2010, 2011) makes it uniquely qualified to test an AED that has also shown benefits for neurobehavioral outcomes. In terms of its AED pharmacology, LEV is often cited as the AED therapy of choice because of its relative ease of use with no need for blood monitoring due to its broad therapeutic index, linear pharmacokinetics, and nearly 100 % bioavailability (Dewolfe & Szaflarski, 2013; Szaflarski et al., 2007b; Szaflarski, Nazzal, & Dreer, 2014; Taylor, Heinrichs, Janzen, & Ehtisham, 2011b). Since one of our objectives was to determine if acute LEV treatment can also have neuroprotection effects, we chose to use the clinically relevant i.v. route of administration to evaluate LEV treatment following severe penetrating TBI with a particular interest in identifying a treatment dosing duration that confers benefit for anti-seizure and neuroprotective effects.

In the PBBI model, NCS occur in a sporadic and unpredictable nature similar to what has been reported clinically in severe TBI populations (Vespa et al., 2010, 2007). Post-traumatic NCS have been detected by continuous EEG in 18–33% of adult patients with moderate-to-severe traumatic brain injury(Herman et al., 2015a, 2015b). PBBI produces NCS in approximately 60–75% of injured animals, with the majority NCS activity occurring between 18–55 hours post-injury (Lu, Mountney, et al., 2013). We have previously demonstrated that PBBI-induced seizures are amenable to prophylactic treatments with phenytoin (PHT) and ethosuximide (EXT) using a dosing protocol of twice daily i.v. injections for 3 days (Mountney et al., 2013). In the current study we observed similar anti-seizure efficacy of LEV using the same dosing regimen. Importantly, the anti-seizure efficacy was achieved with the added benefit of avoiding the potentially negative effects of these traditional AEDs on cognitive outcomes when dosing is extended beyond the acute phase (Bhullar et al., 2014; Darrah et al., 2011). The demonstration of LEV’s effectiveness to reduce NCS activities indicates that the LEV_3D treatment protocol was sufficient for its intended therapeutic target of seizure protection in the early phase post-injury. In humans, the early post-traumatic seizures, convulsive and nonconvulsive, are defined as seizures occurring within 7 days post injury, and therefore a 7- day AED treatment protocol for seizure prophylaxis is recommended by The Brain Trauma Foundation (Bratton et al., 2007). Notably, a three-day treatment duration of LEV has also been attempted for seizure prophylaxis in patients with subarachnoid hemorrhage (Murphy-Human, Welch, Zipfel, Diringer, & Dhar, 2011). In the Murphy-Human et al. (2011) study, LEV treatment appeared to be less effective compared to the extended treatment recommended by Bratton et al. (2007). Moreover, further analysis showed that this worse effect was largely related to the seizures occurring beyond 3-day LEV treatment. Clinically, these findings underscore the importance of ensuring the duration of LEV treatment is adequately sufficient to encompass the acute spectrum of post-TBI high seizure risk periods.

Several preclinical investigations in rodent models of TBI have demonstrated LEV’s potential for neuroprotection. For example, one study reported compelling results showing that a single, acute post-injury 54 mg/kg dose of LEV resulted in reduced hippocampal cell death and significant improvement in for motor decrements resulting from a closed head injury (CHI) model (Wang et al., 2006). An earlier study also demonstrated neuroprotection measured as a reduction in ischemia infarct volume with 44 mg/kg^–1of LEV administered as a bolus pretreatment followed by 24 h slow infusion 10.2 mg/kg (Hanon & Klitgaard, 2001). More recently others have demonstrated significant improvements in functional outcome following an extended 20-day LEV dosing regimen (Zou et al., 2014). Furthermore, in a follow up study these investigators reported that animals treated with an abbreviated LEV dosing regimen failed to display significant improvement (Zou et al., 2015). Collectively, these findings are supportive of our current findings indicating that LEV’s role in neuroprotection may depend upon the extent to which treatment is delivered beyond the acute post-injury phase.

Critically, negative effects of first line standard treatment phenytoin (PHT) on cognitive outcomes have been reported in both pre-clinical and clinical studies (Bhullar et al., 2014; Darrah et al., 2011; Pagni & Zenga, 2006). In the current study, we opted to limit the extended dosing duration to 10 days post-injury in order to ensure that LEV had cleared the animal’s system prior to cognitive testing at post-injury days 13–17. Similar to Zou and colleagues findings (Zou et al., 2014, 2015), the beneficial neuroprotection effects afforded by extended dosing regimen in PBBI model (10 days, twice daily) were not evident in animals treated with the shorter duration dosing regimen (3 days, twice daily). Importantly, the findings of the current study showing beneficial effects of extended (10 day) LEV treatment on the MWM spatial learning task could be interpreted as conflicting with those of Zou et al. (2014). Overall, these results provide further support for exploring dosing regimens that extend beyond the acute post-injury period and more narrowly defines the effective dosing duration required for LEV treatment in the PBBI model to between 3 and 10 days.

More recent preclinical studies using other injury models have emerged supporting the neuroprotective effects of LEV using even higher dose concentrations and/or as ‘pretreatment’ therapies. For example, pretreatment with a single 100 mg/kg dose of LEV prior to permanent middle cerebral artery occlusion lowered ischemic damage by 50% , improved neurological scores, and decreased both occurrence and duration of NCS (Cuomo et al., 2013). Two other studies have demonstrated significantly reduced neuronal apoptosis and therapeutic benefit in the MWM spatial learning acquisition task in rat pups treated with LEV doses up to 200 mg/kg following hypoxic-ischemic brain injury (Kilicdag et al., 2013; Komur et al., 2014). While preclinical models using pretreatment and neonatal rodents carry different implications for clinical relevance in terms of treatments for TBI, these studies reported promising neuroprotection using higher dose concentrations of LEV. Given the already established clinical safety profile of LEV and evidence in the current study that extended treatment is beneficial for neurobehavioral outcomes, its potential for rapid advancement into TBI clinical studies would certainly benefit from a full dose-response evaluation to further define the optimal pharmacodynamics of this exciting TBI therapy.

Putative mechanistic targets of LEV are relevant to brain injury-induced inflammatory responses and blood brain barrier (BBB) extravasation. For example, several reports implicate a role for the inflammatory molecule interleukin1- beta (IL-1β) in neurodegeneration, secondary cell loss, membrane depolarization and related hyperexcitability including proconvulsant actions (De Simoni et al., 2000; Johnson et al., 2015; Vezzani et al., 1999; Zou et al., 2014). LEV has been shown to decrease IL-1β (Haghikia et al., 2008) as well as reduce depolarization of membrane resting potential in proinflammatory stimulated astrocyte and microglial cultures in vitro (Stienen et al., 2011). Twenty days of in-vivo LEV treatment reversed chronic increase of IL-1β in both hippocampal and cortical tissue in the CCI model (Zou et al., 2014). Additionally, blood brain barrier (BBB) disruption is a hallmark of severe TBI, and can be linked to edema, neuroinflammation and seizure development (Cunningham et al., 2014; Shear et al., 2011; Tomkins et al., 2011). In a rat model of cortical dysplasia induced seizures, treatment with LEV improves BBB integrity and functional/structural properties (Ahishali et al., 2010; Gurses et al., 2009). Specifically relevant to our PBBI model, we have previously demonstrated a biphasic pattern of BBB permeability, that begins at 4 h and peaks again between 48–72 h, marked neuroinflammation, and upregulated IL-1β expression persisting to at least 7 days post-PBBI (Johnson et al., 2015; Shear et al., 2011; Wei et al., 2009; Williams, Wei, Dave, & Tortella, 2007). We hypothesize that in our studies the extended 10 day LEV treatment protocol influenced both the elevated phase of IL-1β and impaired BBB permeability thereby contributing as neuroprotection mechanisms of action.

The finding that LEV treatment failed to improve neurological scores or promote brain tissue sparing, while disappointing, is not all that surprising. In fact, we have seen this in previous neuroprotection drug studies conducted using the PBBI model including dextromethorphan (Shear et al., 2009) and simvastatin (Shear et al., 2014) indicating that neurofunctional benefits are not solely dependent on brain tissue sparing in the PBBI model, or other models of TBI (Marklund, Bakshi, Castelbuono, Conte, & McIntosh, 2006). As noted earlier, LEV belongs to the nootroptic family of cognitive enhancers first defined by its structural parent molecule piracetam. Collectively, these and other nootropics have been developed and advanced for treatment of patients with vertigo, mental impairments, stroke, cognitive decline, multiple sclerosis, dementia and other neurodegenerative disease states (Malykh & Sadaie, 2010). The fact that the cognitive enhancing effects of these compounds have been established in disease processes not necessarily associated with physical trauma indicates possible mechanisms of action that are not wholly dependent upon neuronal rescue are in play.

The primary aim of this study was to determine the extent to which LEV might to act as an acute, potent anti-seizure drug and an effective neuroprotection drug in the PBBI model. By demonstrating that an extended treatment regimen of LEV is required for neuroprotection, this study underscores the importance of conducting dose duration studies in addition to dose- response studies in order to provide optimal treatment protocols for defining therapeutic efficacy. Overall, the results of this study have successfully identified treatment regimens that capture dual (anti-seizure and neuroprotective) benefits of LEV for treating severe TBI. Further studies are warranted to evaluate the full dose-response profile for LEV using the extended 10 day dosing regimen, inclusive of serum pK analysis, as the next critical step toward moving this therapy forward into advance development as a treatment for severe TBI.

Disclaimer

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of Department of the Army or Department of Defense.

Declaration of conflicting interests

The author(s) declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Funding

This work was supported by the United States Army [grant number W81XWH-10-1-0623].

Footnotes

Acknowledgments

The authors would like to thank Ms. Ying Cao, Ms. Rebecca Pedersen, Mr. Justin Sun and Mr. William Flerlage for providing outstanding technical and surgical support.

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