Central nervous system infections can underlie the development of epilepsy, and Theiler’s murine encephalomyelitis virus (TMEV) infection in C57BL/6J mice provides a novel model of infection-induced epilepsy. Approximately 50–65% of infected mice develop acute, handling-induced seizures during the infection. Brains display acute neuropathology, and a high number of mice develop spontaneous, recurrent seizures and behavioral comorbidities weeks later. This study characterized the utility of this model for drug testing by assessing whether antiseizure drug treatment during the acute infection period attenuates handling-induced seizures, and whether such treatment modifies associated comorbidities. Male C57BL/6J mice infected with TMEV received twice-daily valproic acid (VPA; 200 mg/kg), carbamazepine (CBZ; 20 mg/kg), or vehicle during the infection (days 0–7). Mice were assessed twice daily during the infection period for handling-induced seizures. Relative to vehicle-treated mice, more CBZ-treated mice presented with acute seizures; VPA conferred no change. In mice displaying seizures, VPA, but not CBZ, reduced seizure burden. Animals were then randomly assigned to acute and long-term follow-up. VPA was associated with significant elevations in acute (day 8) glial fibrillary acidic protein (astrocytes) immunoreactivity, but did not affect NeuN (neurons) immunoreactivity. Additionally, VPA-treated mice showed improved motor performance 15 days postinfection (DPI). At 36 DPI, CBZ-treated mice traveled significantly less distance through the center of an open field, indicative of anxiety-like behavior. CBZ-treated mice also presented with significant astrogliosis 36 DPI. Neither CBZ nor VPA prevented long-term reductions in NeuN immunoreactivity. The TMEV model thus provides an etiologically relevant platform to evaluate potential treatments for acute seizures and disease modification.
Viral infections of the central nervous system can underlie the development of chronic epilepsy due to an increased expression of inflammatory cytokines, lowered seizure threshold, and increased risk of status epilepticus. For example, infection with human herpes 6B virus is associated with the development of encephalitis, seizures, and epilepsy (Caserta et al., 1998; Solomon et al., 2007). Human patients with viral infection–induced encephalitis who present with seizures during the acute infection period are up to 22 times more likely to develop spontaneous, unprovoked seizures than the general population (Annegers et al., 1988). More importantly, inflammation represents a significant risk factor for seizure induction and maintenance, with proinflammatory cytokines being highly expressed in various animal seizure models (Pernot et al., 2011; Vezzani et al., 2011; Vezzani and Friedman, 2011) and patients with epilepsy (Kan et al., 2012; He et al., 2013; Hu et al., 2014). In fact, some seizure-induced proinflammatory signaling molecules remain upregulated during epileptogenesis (Voutsinos-Porche et al., 2004; Ravizza et al., 2008; Maroso et al., 2010) and may be essential to the establishment of spontaneous recurrent seizures associated with temporal lobe epilepsy (TLE) (Ravizza et al., 2011). Thus, models that recapitulate the clinical symptoms of encephalitis may ultimately provide a useful platform to identify novel compounds that may modify or prevent acute seizures, as well as epileptogenesis, in this and other acquired epilepsies.
Theiler’s murine encephalomyelitis virus (TMEV) infection–induced epilepsy in the C57BL/6J mouse represents a useful model of TLE (Libbey et al., 2008, 2011a; Stewart et al., 2010a,b). Animals develop subsequent behavioral comorbidities (Umpierre et al., 2014), which are also associated with human TLE (Brooks-Kayal et al., 2013). Moreover, TMEV-infected mice present with many characteristics associated with human viral encephalitis–associated seizures (Misra et al., 2008). Approximately 50–65% of TMEV-infected animals develop acute handling-induced seizures and show significant elevations in inflammatory cytokines and hippocampal cell death (Stewart et al., 2010a,b). Nearly 50% of the animals that survive the initial infection then develop spontaneous, recurrent seizures weeks later. However, it is currently unknown whether acute therapeutic intervention during the infection can reduce the severity or block development of acute handling-induced seizures in TMEV-infected animals. Moreover, it is unknown whether acute treatment confers any long-term effects (positive or negative) on the comorbidities associated with TMEV infection (Umpierre et al., 2014), a question that was one goal of these studies.
Traditional antiseizure drugs (ASDs) may control TMEV-induced seizures through direct suppression of seizure propagation via ion-channel modulation or effects on neurotransmission. Thus, the ASDs, valproic acid (VPA) and carbamazepine (CBZ), were subchronically administered during the acute TMEV seizure phase to determine whether these agents could suppress acute behavioral seizures and whether pharmacological inhibition of seizures is sufficient to alter the development of associated long-term behavioral deficits (Umpierre et al., 2014). VPA can reduce seizure frequency and severity through multiple ion channel–centric mechanisms (White et al., 2007; Barker-Haliski et al., 2014b). Additionally, VPA can induce brain-derived neurotrophic factor activation (Yasuda et al., 2009), as well as potently inhibit histone deactylase and glycogen synthase kinase-3 activity (Rosenberg, 2007; Hoffmann et al., 2008; Chiu et al., 2013), which may altogether contribute to network remodeling underlying epileptogenesis (Cantley and Haynes, 2013; Liu et al., 2013; Vezzani et al., 2013). Moreover, VPA is neuroprotective in models of Alzheimer’s disease (Kilgore et al., 2010), traumatic brain injury (TBI) (Dash et al., 2010), and septic encephalopathy (Wu et al., 2013). Conversely, CBZ possess notable antiseizure efficacy most likely mediated by sodium channel inhibition (White et al., 2007; Barker-Haliski et al., 2014b); however, it has yet to be associated with any direct anti-inflammatory effects or modulatory effects on signaling pathways. Thus, VPA and CBZ represent two very diverse strategies to prevent acute TMEV-induced seizures.
The present investigation therefore sought to determine whether pharmacological treatment during the acute infection phase of the TMEV model of acquired epilepsy could modify the acute behavioral seizure incidence and severity. Additionally, the long-term effects of acute therapeutic intervention on associated anxiety-like behavior (Umpierre et al., 2014) and neuropathology (Stewart et al., 2010a,b) known to develop after the acute viral infection were evaluated. Novel and symptom-specific models are of high value to translational research endeavors (Barker-Haliski et al., 2014a); therefore, characterizing the pharmacological profile of these models with known ASDs is necessary before any novel therapy is likely to advance to the clinic. The TMEV model of viral encephalitis–induced epilepsy thus provides an etiologically relevant platform to evaluate compounds with the potential for acute seizure-suppressive, and possibly disease-modifying, effects.
Materials and Methods
Animal Handling and Drug Dosing.
Male, C57BL/6J mice (4–5 weeks old; The Jackson Laboratory, Bar Harbor, ME) were randomly divided into three treatment groups (n = 28/treatment group). ASD-treated or vehicle-treated control groups were subchronically dosed (b.i.d.) for days 0–7 of the acute viral infection based on the study design detailed in Fig. 1. Twice-daily doses of VPA (200 mg/kg) and CBZ (20 mg/kg) were based on the ED50 of these drugs in male mice in the maximal electroshock seizure test (Bialer et al., 2004; Rowley and White, 2010). For reference, a correlative analysis between mouse maximal electroshock ED50 values and human steady state plasma levels has previously been described (Bialer et al., 2004). Investigational compounds were made fresh daily in vehicle (0.5% methylcellulose in water). Body weights were recorded daily during the acute infection period (Fig. 1), and then weekly until behavioral testing (data not shown). Mice were evaluated twice per day for days 3–7 for the presence and severity of handling-induced behavioral seizures by experimenter-conducted visual observation during each observation session (Libbey et al., 2008). Mice were group-housed (9–11 mice/cage) for the duration of the subchronic dosing and behavioral testing periods, that is, up to 36 days postinfection (DPI). Following the acute infection period on day 8, randomly selected cohorts of mice from each treatment group that presented with and without seizures (n = 4/seizure condition/treatment condition; n = 8 total/treatment group) were euthanized for brain collection for immunohistochemical assessment of glial fibrillary acidic protein (GFAP) and NeuN immunoreactivity. All of the surviving animals from each treatment group were then allowed to recover for an additional 4 weeks to assess long-term changes in behavioral performance in the open field (OF) activity monitor. Those mice were then euthanized 36 DPI, and brains were collected for immunohistochemical assessment of GFAP and NeuN immunoreactivity.
Surgical Preparation and TMEV Injections.
One hour following the first ASD dose on day 0, mice were injected intracerebrally with 20 µl TMEV (titer concentration of 2.5 × 105 plaque-forming units) under isoflurane anesthesia, as previously described (Libbey et al., 2008, 2011b; Stewart et al., 2010a,b; Umpierre et al., 2014). All injection procedures were performed under sterile conditions. Following TMEV injection, the animals were monitored until they had recovered from anesthesia. All animal care and use was approved by University of Utah Institutional Animal Care and Use Committee.
Assessment of Handling-Induced Seizures.
ASD or vehicle (0.5% methylcellulose) (VEH) was administered each morning and afternoon (b.i.d.). In an effort to demonstrate the utility of this model as a novel drug-testing platform, all attempts were made to maximize the throughput for testing. To address this, video electroencephalogram (vEEG) monitoring was not implemented so as to reduce the time for surgical implantations and increase the number of mice per drug treatment group that were able to be feasibly monitored by experienced technicians. Animals were evaluated twice per day, both before and after drug dosing, on days 0–7 for clinical assessment of TMEV-induced seizures and severity, as well as general assessment of disease score through the experimenter-conducted observation of motor functions by an experimenter blinded to treatment condition (Rauch et al., 1987; Tolley et al., 1999; Tsunoda et al., 2001; Libbey et al., 2008). The presence and severity of handling-induced seizures were scored according to the Racine scale (Racine, 1972). We have previously implemented this experimenter-conducted observation of motor functions to evaluate animals with TMEV-induced acute seizures (Stewart et al., 2010b; Umpierre et al., 2014). Because physical handling was required to perform the twice-daily drug dosing, which could also provoke a handling-induced seizure, animals were also monitored during the drug-dosing session for the presence or absence of any provoked, for example, handling-induced, seizures (i.e., dosing). Twice-daily handling sessions occurred 30 minutes after drug dosing; this time best suited the pharmacokinetic profile of the ASDs under evaluation [in mice, VPA T1/2 = 30 minutes (Ben-Cherif et al., 2013); CBZ T1/2 = 60 minutes (Nishimura et al., 2008)]. Each drug-dosing session was separated by at least 7 hours, occurring daily at 9:00 AM and 4:00 PM. Mice were not evaluated at any other point during the acute infection period, nor any point thereafter, for the presence of spontaneous seizures either by visual assessment or by 24-hour vEEG.
Tissue Collection for Immunohistochemical Analysis.
Twenty four–hour observations were not included in this study to definitively determine which mice experienced behavioral and/or nonconvulsive seizures during the acute infection period. For this reason, it is possible that a mouse that did not present with behavioral seizures during the twice-daily experimenter-performed handling sessions may have presented with spontaneous convulsive or nonconvulsive seizures at another point during the acute infection period. Additionally, 24-hour vEEG recordings were not included to monitor for subconvulsive electrographic seizures in an attempt to develop this moderate-throughput drug-testing platform. Therefore, all mice were candidates for subsequent analyses of both acute and long-term immunohistochemical changes. Immunostaining, image acquisition, and analysis for acute and long-term cohorts were processed separately upon collection of each cohort. For both acute and long-term collections, animals from each treatment group were sacrificed by decapitation, and the brain was quickly removed into ice-cold 4% paraformaldehyde/0.9% phosphate-buffered saline. Brains were fixed for 24 hours, and then dehydrated and cryoprotected in 30% sucrose/0.9% phosphate-buffered saline for 48 hours before processing for downstream analysis (Friend and Keefe, 2013). Coronal sections of dorsal hippocampus (bregma; −3.3 AP) were cut at 40-µm thick on a freezing-stage microtome (Leica, Wetzlar, Germany) and mounted onto slides before immunostaining. Two adjacent dorsal hippocampal sections from each mouse in each treatment group were processed with Cy3-conjugated GFAP (C9205; Sigma-Aldrich, St. Louis, MO) and fluorescein isothiocyanate–conjugated NeuN (MAB377X; Millipore, Billerica, MA), with 4′,6′-diamidino-2-phenylindole nuclear counterstain (Life Technologies, Carlsbad, CA), as previously described (Friend and Keefe, 2013). Our prior work has demonstrated that TMEV infection is associated with acute and long-term neurodegeneration most predominately in area CA1 of dorsal hippocampus (Stewart et al., 2010a); therefore, images from dorsal hippocampus (CA1) from both contra- and ipsilateral sites of viral infection were collected at 20× magnification on an Axiovision A.1 fluorescence microscope (Zeiss, Jena, Germany). Images were analyzed by ImageJ (NIH, Bethesda, MD) denistometric analysis for percent field area with signal, as previously described (Barker-Haliski et al., 2012). An investigator blinded to treatment condition conducted all image analysis.
Assessment of Motor Function by Rotarod.
There was no further drug administration (VEH or ASD) after 7 DPI. Animals were tested 15 DPI, 8 days after the last administration of VEH or ASD, for their ability to maintain balance on a rotating rod (6 rpm) for 1 minute over three consecutive trials, as previously described (Dunham and Miya, 1957; Rowley and White, 2010; Umpierre et al., 2014). The average latency (seconds) to fall off the rotarod was calculated from the three trials, with the group means calculated for each study under investigation.
OF Assessment of Anxiety-Like Behavior 4 Weeks Postinfection.
As detailed above, following the acute infection period (days 0–8; see Table 1 for study timeline), all animals that were not euthanized for acute immunohistochemical assessments were allowed to recover for 4 weeks before testing in the OF activity monitor (5 weeks postinfection), a known platform to evaluate anxiety-like behavior in rodents (Prut and Belzung, 2003). As stated above, prior research has only monitored vEEG with cortical electrodes (Stewart et al., 2010a); therefore, it remains possible that subconvulsive electrographic seizures originating in other structures may occur in mice infected with TMEV (Stewart et al., 2010a). For these reasons, all mice were candidates for subsequent behavioral testing. On the testing day, one animal from each treatment group (n = 17 CBZ; n = 21 VPA; n = 13 VEH) was randomly monitored in an OF Plexiglas chamber measuring 40L × 40W × 30H cm (AccuScan, Salt Lake City, UT), with a total of eight mice run in parallel for each OF monitoring session (7 sessions total). Animals were allowed to explore the OF for 10 minutes, with measurements of total distance traveled, vertical, horizontal activity, rest time, and stereotypy time recorded in the center and periphery of the OF, adapted from testing methods described previously (Umpierre et al., 2014). This testing window is appropriate to identify anxiogenic effects of the OF (Prut and Belzung, 2003).
Daily body weights were analyzed by repeat measures analysis of variance and post hoc Tukey’s t test. Latency to first seizure Kaplan-Meier curves was determined with a log-rank (Mantel-Cox) test. Effect of treatment on seizure burden and average number of stage 4/5 seizures were evaluated by Kruskal-Wallis test. Latency to fall off the rotarod, OF measures, and immunohistochemical densitometric results were analyzed using one-way analysis of variance and post hoc Tukey’s t test. The proportion of severe seizures in ASD-treated and vehicle-treated animals was determined with a standard score analysis. All statistical analyses were conducted with Prism v.6.0 (GraphPad Software, La Jolla, CA), with P < 0.05 considered statistically significant.
Overall Animal Health Is Reduced during the Acute Viral Infection Period.
Body weights were recorded daily throughout the acute infection period (days 0–7; Fig. 2A), then weekly until completion of the long-term behavioral studies (data not shown). During the acute seizure period (day 0–7), there was a significant main effect of time postinfection on the percent change in body weight for all TMEV-infected animals, regardless of treatment [Fig. 2A; F(7,546) = 276.3, P < 0.0001]. Post hoc analysis, however, revealed no significant differences between VEH- and CBZ-treated mice at any time point. Conversely, VPA- (200 mg/kg) treated mice lost significantly more weight than VEH-treated mice 3–6 DPI (Fig. 2A; day 3, P < 0.03; day 4, P < 0.02; day 5, P < 0.05; day 6, P < 0.01), and than CBZ-treated mice on day 5 (P < 0.03). There were no other observable or significant health differences between treatment groups during the acute viral infection period. However, there was some attrition in the long-term survival for all treatment groups after 8 DPI (period after 1 week postinfection; Fig. 2C). There were four mice from the CBZ treatment group, one mouse from the VPA-treatment group, and eight mice from the VEH treatment group failing to survive to 36 DPI (Fig. 2C). VPA-treated mice demonstrated significantly improved long-term survival relative to VEH- (Fig. 2C; χ2 = 7.35, P = 0.007) and CBZ- (χ2 = 3.90, P < 0.05) treated mice, but VEH- and CBZ-treated mice were no different in their long-term survival (χ2 = 0.57, P > 0.4). Importantly, when body weights from surviving animals were measured weekly after the viral infection period, there was no significant time × treatment interaction in the percent weight change from day 0 values [F(6, 138) = 1.19, P > 0.3; data not shown]. All surviving animals recovered from the viral infection with similar rates of weight gain relative to day 0 weights, completing the study 36 DPI at similar body weights relative to each other (VEH, 115% ± 2.0; CBZ, 111.5% ± 1.1; VPA, 114% ± 2.2; data not shown). Thus, the acute effects of viral infection did not significantly alter long-term health outcomes for the surviving mice in each treatment group, despite some adverse effects on overall long-term survival.
Acute Treatment with Prototype Antiseizure Drugs Produces Varied Effects on Viral-Induced Behavioral Seizures.
The overall latency to first seizure was evaluated using the Kaplan-Meier survival curve, wherein the outcome measure evaluated was the time in which an animal enrolled in the study remained free from seizures of any Racine stage (Fig. 2B). The proportion of animals that presented with and without seizures of any Racine stage was also determined (Fig. 3, A–C).
VPA and CBZ were administered at therapeutically relevant doses (Bialer et al., 2004) to determine whether standard ASDs can affect postsymptomatic seizures associated with TMEV infection. Interestingly, the latency to first seizure of any Racine stage was found to be no different between VEH- and VPA-treated mice (Fig. 2B; χ2 = 0.804, P > 0.3), whereas CBZ-treated mice showed a significant decrease in latency to first seizure versus VEH-treated mice (χ2 = 4.094, P < 0.05). Additionally, the overall proportion or animals presenting with and without seizures (Fig. 3, A–C) was no different between VEH- and VPA-treated mice (z = 0.26, P > 0.5), whereas significantly more CBZ-treated mice presented with seizures than either VEH (z = 2.36, P < 0.01) or VPA (z = 2.61, P < 0.5) treatment groups. Thus, acute CBZ treatment resulted in a substantially greater proportion of animals presenting with at least one handling-induced seizure, whereas VPA treatment did not significantly alter the proportion of animals with seizures relative to VEH-treated mice.
The Likelihood of Presenting with a Stage 5 Seizure Is Increased by Acute CBZ Treatment in Mice with Handling-Induced Seizures.
In addition to analysis of the effect of treatment on the average seizure burden and seizure severity in mice that presented with at least one handling-induced seizure, we sought to determine the likelihood that an animal that presented with a seizure of any score would present with a stage 5 seizure at any point during the acute TMEV infection period (both dosing and handling sessions; Fig. 3D). Odds ratio (OR) calculation revealed that there was a significant difference between VEH- and VPA-treated, TMEV-infected mice that presented with at least one handling-induced seizure. VPA treatment significantly reduced the OR that an animal, which had a seizure of any scale, would present with stage 5 seizures [OR = 2.0, 95% confidence interval (CI) = 0.92–3.05] relative to VEH-treated mice (OR = 4.33, 95% CI = 5.59–3.08). CBZ treatment, however, led to a profound increase (OR = 23.0, 95% CI = 21.00–25.00) in the likelihood that an animal would have a stage 5 seizure relative to both VEH- and VPA-treated, TMEV-infected mice with seizures. Thus, VPA treatment significantly reduced the likelihood that TMEV-infected mice, which presented with a seizure at any point, would present with a stage 5 seizure. Conversely, CBZ treatment significantly increased the likelihood that an animal, which presented with a seizure, would have a stage 5 seizure at any observation point during the acute TMEV infection period.
Drug Dosing Affects Seizure Response Profile Regardless of ASD Treatment.
In addition to assessment of number of animals in each study presenting with and without seizures, the average seizure burden and average number of stage 4/5 seizures for any animal that experienced at least one handling-induced seizure during the dosing or handling sessions were determined (Fig. 4). Seizure burden, which represents the sum seizure score of all observed seizures throughout the specific observation session for any animal that presented with a seizure, provides an important evaluation of frequency and severity of seizures. We conducted this in-depth evaluation of the seizure burden and severity during the observation sessions pre- and postdrug administration (dosing and handling, Fig. 4, G and H, respectively), theoretically when the ASDs under investigation were at minimal and peak plasma concentrations, respectively (Nishimura et al., 2008; Ben-Cherif et al., 2013). This analysis sought to determine whether the therapeutic doses of these ASDs could alter seizure burden before and after drug administration (see Fig. 1A for study protocol). Assessments of the maximum observed seizure stage of each animal that experienced a seizure were made for morning and afternoon sessions (9:00 AM; 4:00 PM) prior to drug dosing (Fig. 4, A–C, dosing), and then 30 minutes after drug administration (Fig. 4, D–F, handling). For all studies, the maximum observed Racine seizure score was determined for each animal during the study days 0–7 and represented as the proportional distribution of all maximum seizure scores observed. Thus, the dosing and handling sessions were separated to determine the types of seizures in each session throughout the acute seizure period.
When the predrug administration (dosing) sessions were analyzed, neither the CBZ-treatment group (95%; Fig. 4B) nor the VPA treatment group (73%; Fig. 4C) demonstrated significantly altered stage 5 seizure incidence relative to the VEH treatment group (86.7%; Fig. 4A). However, VPA treatment did significantly reduce the proportion of animals with a maximum stage 5 seizure relative to the CBZ treatment; 95% of CBZ-treated mice exhibiting seizures experienced a stage 5 seizure, whereas only 73% of VPA-treated mice exhibiting seizures presented with stage 5 during the dosing observation windows of day 0–7 (Fig. 4C; z = 1.81, P < 0.05). Simultaneously, none of the CBZ-treated mice presented with stage 1–3 seizures, whereas 13% of VPA-treated mice presented with stage 1–3 seizures during the dosing sessions (z = 1.68, P < 0.05). Thus, all treatment groups were not significantly different from VEH-treated controls when seizures were observed during the dosing session, but there were significant differences in seizure severity between VPA and CBZ treatment groups long after drug administration (i.e., at least 7 hours).
When mice were then examined for handling-induced seizures 30 minutes after drug administration (handling), a significant reduction in seizure severity occurred only in the VPA-treatment group (Fig. 4, D–F). Although over 88% of VPA-treated mice demonstrated sustained freezing behavior in response to handling (>5 seconds) 30 minutes after drug administration, only one VPA-treated mouse (5.9%) demonstrated a Racine stage 5 seizure at any point in the study (day 0–7; Fig. 4F). As such, the proportion of VPA-treated mice with freezing behavior was significantly different from VEH-treated (0%; z = −4.79, P < 0.0001) and CBZ-treated mice (8.7%; z = −5.03, P < 0.0001). These results are in stark contrast to the effect of VEH or CBZ treatment, wherein the majority of animals (>92% VEH; >86% CBZ) that presented with a seizure during the handling sessions experienced at least a stage 4 Racine seizure 30 minutes after intraperitoneal drug administration (Fig. 4, D and E). These results further suggest that VPA treatment truly protected against further seizure expression when drug was on board (handling), and not simply an effect of a refractory period to seizures due to an event 30 minutes prior during the dosing session.
The seizure burden and average number of stage 4/5 seizures for any animal that experienced at least one handling-induced seizure were determined for the dosing or handling sessions (Fig. 4, G–J). There was no significant effect of any ASD treatment during the dosing session (Fig. 4G; H = 3.22, d.f. = 2, P = 0.2). However, acute ASD treatment significantly affected seizure burden during the handling session (Fig. 4H; H = 28.5, d.f. = 2, P < 0.0001). Post hoc analysis of the handling session revealed that both CBZ and VPA treatment resulted in significant reductions in average seizure burden relative to VEH (Fig. 4H; VPA, P < 0.0001 and CBZ, P < 0.01, respectively). The average number of stage 4/5 seizures in any animal that presented with at least one handling-induced seizure was not significantly altered in the dosing session (Fig. 4I; H = 3.87, d.f. = 2, P = 0.14). However, acute ASD treatment significantly reduced the number of stage 4 or 5 seizures during the handling sessions (Fig. 4J; H = 21.5, d.f. = 2, P < 0.0001). Post hoc analysis revealed, however, that only VPA treatment significantly reduced the number of stage 4 or 5 seizures relative to VEH (P < 0.0001). The number of stage 4/5 seizures in CBZ- and VEH-treated mice was not different from each other (P > 0.05).
Rotarod Assessment of Motor Behavior after Prototypical ASD Treatment.
Our previous work has demonstrated a substantial effect of TMEV-induced seizures on motor coordination on the rotarod 17 DPI (Umpierre et al., 2014). Thus, we performed an evaluation of the effect of acute treatment with prototypical ASDs on motor coordination and performance 15 DPI. Animals did not receive further drug administration after 7 DPI; thus, any effects on the rotarod were due to the acute effects of treatment or disease progression during the TMEV infection period. Because continuous vEEG recordings were not included in this study to identify all mice that presented with behavioral or nonconvulsive seizures during the acute infection period, all mice (seized/nonseized) were subjected to the rotarod testing, as well as other behavioral and immunohistochemical assessments. We have previously demonstrated that VEH-treated, TMEV-infected mice fall off a rotarod after approximately 43 seconds, whereas sham-infected C57BL/6J mice remain on the rotarod for over 57 seconds (Umpierre et al., 2014). Interestingly, ASD treatment during the acute seizure period conferred significant overall improvements in motor coordination relative to VEH-treated, TMEV-infected mice, as measured by latency to fall off a rotarod [Fig. 5A; F(2,53) = 3.16, P = 0.05]. On average, VPA-treated mice remained on the rotarod for 54.0 ± 2.2 seconds, which was significantly improved relative to VEH-treated mice (43.2 ± 3.8 seconds; P < 0.04). Conversely, CBZ-treated mice (44.5 ± 3.8 seconds) were not significantly better on this task than VEH-treated mice (P > 0.5); however, they did not achieve a significant difference from VPA-treated mice (P = 0.09). Thus, our present and previous data would suggest that VPA treatment during the acute infection period can improve motor performance on the rotarod task relative to VEH-treated mice at 15 DPI, further indicative of reduced disease severity in animals acutely treated with VPA. In contrast, acute CBZ treatment did not improve motor coordination relative to VEH-treated mice at 15 DPI, indicating that CBZ treatment during the TMEV infection may not be beneficial to long-term disease outcomes.
Effect of Treatment on OF Activity, a Measure of Anxiety-Like Behavioral Comorbidity.
Mice were allowed to recover to 36 DPI before assessment of OF behaviors. OF is a useful means to evaluate anxiety-like behaviors in rodents (Prut and Belzung, 2003). We have previously demonstrated that TMEV-infected mice demonstrate substantial reductions in time spent in the center of an OF (Umpierre et al., 2014), an effect that is suggestive of an anxiety-like phenotype. None of the drug treatment regimens conferred any significant improvement on the amount of time spent in the center of the OF environment [Fig. 5B; F(2,48) = 2.56, P = 0.088]. However, when the distance traveled in the center was similarly compared, there was a significant overall effect of treatment [Fig. 5C; F(2,48) = 6.41, P = 0.003]. CBZ-treated mice traveled significantly less total distance through the center of the OF relative to VEH-treated mice (P < 0.01), but were not significantly different from VPA-treated mice (P > 0.05). VPA-treated animals showed robust improvements relative to VEH-treated mice in acute behavioral seizures during day 0–7 (Figs. 3 and 4) and motor coordination as evaluated on the rotarod 15 DPI (Fig. 5A). However, VPA-treated mice were not significantly better in OF behavioral end points than VEH-treated mice (Fig. 5, B and C; P > 0.05). Thus, acute CBZ treatment actually resulted in a greater reduction in the distance traveled in the center of the OF versus VEH-treated mice, with center distance traveled being a long-term measure of anxiety-like behavior in TMEV-infected mice 36 DPI. As summarized in Table 1, there was a significant overall effect of treatment on horizontal activity counts [F(2,47) = 2.06, P = 0.046], with CBZ-treated, TMEV-infected mice having a significant increase in horizontal activity counts throughout the entire OF (center + perimeter; P < 0.05) relative to VEH-treated, TMEV-infected mice. There was no such significant difference between VEH- and VPA-treated mice (P > 0.05), and horizontal activity counts were the only OF measure for center + perimeter zones that had any significant effect of drug treatment (Table 1). Such observations further suggest that acute CBZ treatment during the viral infection is associated with potentiated adverse long-term outcomes in this model.
Assessment of Acute and Long-Term GFAP and NeuN Immunoreactivity in Dorsal CA1.
Behavioral differences in both acute seizure severity and long-term postinfection-associated comorbidities provided the rationale to examine the potential for neuroanatomical differences in ASD- versus VEH-treated mice (Fig. 6). TMEV infection results in significant hippocampal pathology and sclerosis, particularly within area CA1 of the dorsal hippocampus (Stewart et al., 2010a; Umpierre et al., 2014). Samples from the long-term cohorts (36 DPI) from this study also demonstrated substantial hippocampal sclerosis (Fig. 6A, white arrows). Thus, we sought to semiquantitatively assess whether pharmacological intervention during the acute infection period would alter short-term (8 DPI; Fig. 6B) or long-term neuropathology (36 DPI; Fig. 6C).
To maximize the numbers of animals available for subsequent behavioral assessments, analysis of 8 DPI immunoreactivity was limited to eight animals/treatment group, with an equally divided number of mice with and without seizure per treatment condition. As detailed in Materials and Methods, it is possible that mice infected with TMEV may have presented with subconvulsive electrographic seizures during the acute infection period, which would have been missed by an experimenter only performing twice-daily observations of motor seizures. Because continuous vEEG recordings were not included in this study to definitively identify those mice that presented with behavioral or nonconvulsive seizures during the acute infection period, all mice (seized/nonseized) were candidates for subsequent immunohistochemical or behavioral evaluations. Acute treatment with VPA and CBZ during the viral infection period did not result in a significant improvement in NeuN labeling in CA1 [Fig. 6A, left, and Fig. 6B; F(2,15) = 1.96, P < 0.1]. However, there was a significant overall effect of treatment on the extent of GFAP immunoreactivity after TMEV infection [F(2,15) = 4.38, P < 0.04], with a Tukey’s post hoc test revealing that VPA was associated with a significantly greater expression of GFAP when compared with CBZ treatment (P < 0.05). However, VPA treatment did not achieve a statistically significant difference in GFAP expression from VEH-treated mice. To examine the potential effect of a single drug arm versus VEH treatment, a two-tailed t test between VEH- and VPA-treated mice alone revealed a significant difference between treatment groups (t = 2.29, P = 0.045; not graphically illustrated). When this two-tailed t test was similarly used to only compare CBZ- versus VEH-treated mice, there was no such significant difference between groups (t = 0.449, P > 0.6). Thus, acute VPA treatment actually increased the level of reactive astrogliosis 8 DPI (Fig. 6B). Conversely, CBZ treatment did not significantly alter the extent of reactive astrogliosis relative to VEH-treated mice 8 DPI (Fig. 6B).
Following completion of all behavioral testing weeks after the initial infection, the surviving animals were sacrificed for assessment of long-term changes in NeuN and GFAP immunolabeling 36 DPI (Fig. 6A, right panels, and Fig. 6C). In all treatment groups, the mice that survived to 36 DPI consisted of 63–66% of TMEV-infected mice with seizures during the acute infection period. Thus, there were no significant differences in the immunolabeling due to the distribution of mice with and without acute seizure presentation. Importantly, there was notable hippocampal damage observed in some mice that did not present with seizure during a handling-induced observation session. Thus, all animals enrolled in the study were evaluated for changes in NeuN and GFAP immunolabeling 36 DPI. Additionally, as for the acute cohort, all surviving mice were included for evaluation of long-term immunoreactivity after TMEV. Similar to the 8 DPI results, neither VPA nor CBZ treatment resulted in effects on NeuN immunolabeling relative to VEH-treated, TMEV-infected mice 36 DPI [Fig. 6C; F(2,24) = 0.86, P > 0.4]. Interestingly, animals from each treatment group presented with notable CA1 sclerosis (Fig. 6A, right panel, white arrows). However, when GFAP staining was subsequently assessed, there was a significant overall treatment effect [Fig. 6C; F(2,24) = 3.43, P < 0.05]. Post hoc assessment revealed that CBZ-treated mice showed significantly elevated long-term GFAP immunostaining relative to VPA-treated mice (P < 0.05), but were not different from VEH-treated mice (P > 0.05). Moreover, VPA treatment did not result in significant changes relative to VEH-treated, TMEV-infected mice (P > 0.05). When treatment groups were similarly compared by two-tailed t test as in the 8 DPI studies, there were no significant differences between any ASD treatment group and VEH (CBZ versus VEH, t = 1.59, P = 0.13; VPA versus VEH, t = 0.77, P = 0.45). Thus, the present results suggest that neither CBZ nor VPA treatment was associated with significant long-term protective effects on astrogliosis or neurodegeneration relative to VEH-treated mice.
The potential benefit of 8-day treatment with VPA or CBZ to modify short- and long-term outcomes associated with central administration of TMEV was evaluated in C57BL/6J mice. These results demonstrate the utility of the TMEV model for drug-testing purposes in an etiologically relevant, syndrome-specific animal model of encephalitis-induced seizures. Interestingly, long-term evaluation of anxiety-like behavior and histopathology suggests that whereas acute reductions in seizure burden can be achieved with ASDs, long-term measures of disease outcome are not similarly improved, indicating that this model may identify novel therapies.
Treatment with therapeutic doses of VPA and CBZ during the acute seizure period conferred different effects on behavioral seizures. VPA reduced the acute seizure burden. Conversely, CBZ exacerbated the consequences associated with TMEV infection. This is supported by the observation that more mice in the CBZ-treatment group presented with seizures than VEH-treated, TMEV-infected mice. Additionally, the OR to have a stage 5 seizure was greatest in CBZ-treated mice, suggesting an increased susceptibility to disease in the CBZ-treated mice because more animals presented with seizures associated with the viral infection. Furthermore, the latency to first seizure presentation in the CBZ-treatment group was significantly accelerated relative to VEH- and VPA-treated mice. These observations are an important clinical consideration for the management of encephalitis-induced seizures. CBZ can be immunosuppressive and induce hypogammaglobulinaemia in humans (Sorrell and Forbes, 1975; Spickett et al., 1996), and has even been attributed to recurrent seizures in Herpes simplex encephalitis (Rice et al., 2007). Thus, the effect of CBZ on immunocompetence may have contributed to the presently observed increase in the numbers of TMEV-infected mice with acute seizure.
The observed effects of drug administration on acute histopathology suggest that VPA treatment may have potentiated astrogliosis at the early time point (Fig. 6B). Such results are intriguing given that VPA treatment significantly improved short-term (15 DPI) measures of recovery in rotarod performance relative to VEH-treated controls. This may suggest that acutely elevated astrogliosis may promote disease recovery in this model. As astrocytes can be both neuroprotective and neurodegenerative in the context of neurologic insult (Sofroniew, 2005), further investigations into the temporal contributions of astrocyte reactivity and disease outcomes in this model are necessary.
Approximately 65% of mice that present with acute seizures during the viral infection subsequently develop epilepsy (Stewart et al., 2010a), and the population of TMEV-infected mice that exhibit acute seizures can go on to develop behavioral impairments weeks later (Umpierre et al., 2014). Thus, we attempted to evaluate the effect of ASD treatment on measures of disease progression 36 DPI, when viral clearance is complete (Libbey et al., 2008) and behavioral deficits are apparent (Umpierre et al., 2014). CBZ, but not VPA, treatment increased the proportion of mice with handling-induced seizures. Not surprisingly, CBZ-treated mice demonstrated significantly worse anxiety-like behavior 36 DPI; for example, decreased distance traveled in the OF center (Fig. 5C). Additionally, CBZ-treated mice had significantly greater horizontal activity throughout the OF, indicative of greater spatial memory deficits. Hippocampal lesions tend to enhance measures of hyperactivity due to the disruption of memory formation, thereby causing animals to ambulate to a greater extent throughout an OF (Praag et al., 1994; Walker et al., 2011). That acute CBZ treatment exacerbated long-term reductions in center distance traveled and increased horizontal activity may suggest greater hippocampal damage in CBZ-treated mice relative to VEH-treated mice. CBZ-treated mice also presented with astrogliosis 36 DPI (Fig. 6, A and C), despite no difference in astrogliosis 8 DPI relative to VEH-treated mice. As TMEV infection is associated with both hippocampal sclerosis and deficits in OF activity (Stewart et al., 2010a,b; Umpierre et al., 2014), the observations that CBZ potentiated behavioral impairments and astrogliosis further suggest that CBZ may not be the preferred treatment in instances of viral encephalitis–induced seizures. Therefore, the TMEV model not only has the potential to identify promising treatments for encephalitis-induced epilepsy, but it may also be useful for identifying treatments to be avoided.
VPA possesses known effects on transcription factors (Rosenberg, 2007; Yasuda et al., 2009; Chiu et al., 2013), which may underlie disease modification (Liu et al., 2013). However, VPA (200 mg/kg b.i.d.) did not improve long-term behavioral measures relative to VEH-treated mice, suggesting that this dose may have been insufficient to target pathways necessary for long-term disease modification (Ravizza et al., 2011; Vezzani et al., 2011; Liu et al., 2013). VPA is neuroprotective in models of Alzheimer’s disease (Kilgore et al., 2010), TBI (Dash et al., 2010), and septic encephalopathy (Wu et al., 2013), but our present results failed to demonstrate any neuroprotection. Relative to VEH-treated mice, VPA-treated mice were no different in OF activity (Fig. 5; Table 1), showed no short- or long-term differences in NeuN immunoreactivity, and displayed hippocampal sclerosis 36 DPI (Fig. 6A, right). Our present results thus demonstrate that, whereas seizure burden was improved during VPA treatment, there was no long-term disease modification as a consequence of VPA treatment at this dose and time point tested.
Our present observations are reminiscent of clinical trials designed to evaluate the prophylactic efficacy of ASDs for the treatment and prevention of seizures following TBI or febrile seizures (Temkin, 2001) and support the utility of the TMEV model as a preclinical drug screening platform. These clinical studies suggested that, whereas ASDs may block acute provoked seizures following a neurologic insult, most treatments failed to prevent subsequent epileptogenesis (Temkin, 2001). In our present study, VPA, but not CBZ, conferred notable beneficial effects on seizure burden, but there were few positive effects on long-term associated comorbidities. In fact, CBZ potentiated much of the effects of TMEV infection alone, making long-term behavioral deficits more severe for CBZ-treated mice. Future studies using vEEG monitoring may confirm a correlation between the behavioral comorbidities examined in this work and TMEV infection-induced epileptogenesis (Stewart et al., 2010a); such work would clarify whether our present results align with clinical ASD prophylaxis efforts (Temkin, 2001). However, the present results suggest that the TMEV model has the potential to identify compounds possessing both acute seizure-suppressive effects and long-term disease-modifying potential. It is difficult to evaluate an antiepileptogenic effect of ASD treatment in post-traumatic or infection-induced epilepsy in clinical trials due to the low incidence of acquired epilepsy (Annegers et al., 1988, 1998; Barker-Haliski et al., 2014a), most likely a cause of many clinical failures of antiepileptogenesis trials (Mani et al., 2011). Moreover, prior attempts at antiepileptogenesis with ASDs have come with considerable adverse effects on cognition and general health outcomes that have limited the complete evaluation of any antiepileptic effect (Dikmen et al., 1991). For these reasons, the TMEV mouse model offers a novel platform for investigator-controlled studies on preclinical ASD efficacy for disease modification.
TMEV infection in mice elevates inflammatory mediators known to contribute to seizure induction and maintenance (Wilcox and Vezzani, 2014). Moreover, this cytokine response promotes the development of acute seizures in TMEV-infected C57BL/6J mice (Libbey et al., 2011a,b; Cusick et al., 2013). There is thus potential for combined acute seizure control with traditional ASDs and subsequent disease modification with compounds possessing alternative mechanisms of action that may modify or prevent the disease, for example, anti-inflammatory compounds (Schmidt, 2012; Loscher et al., 2013). As Temkin (2001) and others (White and Loscher, 2014) have suggested, it is feasible that entirely different classes of compounds will be needed to prevent the development of epilepsy than those that effectively suppress the seizures once the process has progressed into epilepsy. In fact, other models of TLE after insult have suggested that anti-inflammatory agents may be disease-modifying (Maroso et al., 2010, 2011; Vezzani et al., 2013). Furthermore, these agents represent a clinically unexploited mechanism to prevent or modify epilepsy. As postencephalitic epilepsy is often pharmacoresistant (Cruzado et al., 2002), the TMEV model provides a novel and valuable drug development platform. Moreover, the development of acute seizures in TMEV-infected mice can be prevented with the repurposed anti-inflammatory agent, minocycline (Libbey et al., 2011a,b; Cusick et al., 2013), highlighting the potential to evaluate mechanistically novel agents in this model. However, long-term follow-up (>15 DPI) to evaluate treatment effects on behavioral comorbidities was not included in these studies (Libbey et al., 2011a,b; Cusick et al., 2013). Future efforts to characterize any subsequent effects of therapeutic intervention with other agents on disease course, or biomarkers thereof, are thus warranted in this model.
In this study, we demonstrate the utility of the TMEV model of infection-induced seizures and subsequent behavioral comorbidities as a platform to evaluate potentially novel therapeutic approaches for acute seizure control and disease modification. Treatment with VPA during the infection significantly improved acute seizure burden. However, neither ASD treatment prevented long-term behavioral comorbidities associated with TMEV infection. Nevertheless, these results should not detract from the obvious utility of the TMEV model of infection-induced encephalitis and subsequent behavioral comorbidities as an etiologically relevant drug-testing platform. This model may have the potential to identify innovative and mechanistically novel seizure-suppressive, and possibly disease-modifying, therapies for epilepsy.
The authors thank Dr. Robert Fujinami for purified TMEV titers.
Participated in research design: Barker-Haliski, Wilcox, White.
Conducted experiments: Barker-Haliski, Dahle, Pruess, Heck, Vanegas.
Performed data analysis: Barker-Haliski, White.
Wrote or contributed to the writing of the manuscript: Barker-Haliski, Wilcox, White.
- Received December 31, 2014.
- Accepted March 6, 2015.
This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant R01-NS065434 (to K.S.W. and H.S.W.) and Contract HHSN271201100029C (to H.S.W.)].
Part of this work was presented as follows: Barker-Haliski ML, Heck TD, Dahle E, Pruess TH, Wilcox KS, and White H (2014) Treatment of acute behavioral seizures in the Theiler's murine encephalomyelitis virus model of acquired epilepsy disrupts long-term, but not acute, histopathology. Society for Neuroscience Meeting; 2014 Nov 16; Washington, D.C. Abstract 201.01.
- antiseizure drug
- days postinfection
- glial fibrillary acidic protein
- open field
- odds ratio
- traumatic brain injury
- temporal lobe epilepsy
- Theiler’s murine encephalomyelitis virus
- video electroencephalogram
- vehicle (0.5% methylcellulose)
- valproic acid
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics