Evaluating an etiologically relevant platform for therapy development for temporal lobe epilepsy: effects of carbamazepine and valproic acid on acute seizures and chronic behavioral comorbidities in the Theiler's murine encephalomyelitis virus mouse model

Abstract

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.

Fig. 1.

Timeline of acute treatment period and long-term behavioral assessment period. (A) Male C57BL/6J mice were randomly assigned to treatment groups of VEH, CBZ, or VPA on day 0. Immediately after the first drug or VEH treatment, mice were injected intracerebrally with Theiler’s murine encephalomyelitis virus (2.5 × 10⁵ plaque-forming units). Animals were then subchronically dosed (b.i.d.) with CBZ, VPA, or VEH for day 0–7 of the acute infection period. On day 8, animals were randomly assigned to acute or long-term follow-up studies. (B) Animals assigned to long-term follow-up studies were evaluated on the rotarod 15 DPI. All animals that survived to 36 DPI were evaluated for anxiety-like behavior in an OF activity monitor, followed by immunohistochemical (IHC) assessment of NeuN and GFAP immunoreactivity.

Fig. 2.

Body weight change during the acute infection period, latency to first seizure curve, and long-term group survival. (A) Acute TMEV infection leads to significant weight loss in all treatment groups (P < 0.0001). There were no significant differences between VEH- and CBZ-treated mice at any time point. Conversely, VPA- (200 mg/kg) treated mice were significantly different from VEH-treated mice 3–6 DPI (day 3, P < 0.03; day 4, P < 0.02; day 5, P < 0.05; day 6, P < 0.01), and significantly different from CBZ-treated mice on day 5 (P < 0.03). *Significantly different from VEH-treated, TMEV-infected control mice, P < 0.05; ^#significantly different from CBZ-treated, TMEV-infected mice, P < 0.05. (B) The latency to first seizure was compared for each treatment group. Interestingly, there was no significant difference between the latency to seizure onset in VPA-treated versus VEH-treated mice (χ² = 0.804, P > 0.3). Relative to VEH-treated mice, however, CBZ-treated mice showed a profound decrease in latency to first seizure (χ² = 4.094, P < 0.05). *Significantly different from VEH-treated, TMEV-infected mice, P < 0.05. (C) TMEV-infected mice not selected for acute immunohistochemistry were monitored weekly for 5 weeks (36 DPI) for any effects of acute treatment on long-term survival rates. All mice selected for acute immunohistochemistry were omitted from the Kaplan-Meier plot. Long-term survival beyond 8 DPI (1 week postinfection) is only significantly improved by acute treatment of TMEV-infected mice with VPA. Long-term survival was no different between CBZ- and VEH-treated mice after 8 DPI (1 week postinfection). *Significantly different from VEH-treated, TMEV-infected control mice, P < 0.05; ^#significantly different from CBZ-treated, TMEV-infected mice, P < 0.05.

Fig. 3.

Acute CBZ treatment (20 mg/kg b.i.d.) increases the proportion of mice infected with TMEV that present with handling-induced seizures and increases the likelihood that mice with seizure will experience a stage 5 seizure. (A–C) The overall proportion of animals presenting with and without seizures was no different between VEH- and VPA-treated mice (z = 0.26, P > 0.5), whereas significantly more CBZ-treated mice presented with a seizure than either VEH (z = 2.36, P < 0.01) or VPA (z = 2.61, P < 0.5) treatment groups. *Significantly different from VEH-treated, TMEV-infected control mice, P < 0.01; ^#significantly different from VPA-treated, TMEV-infected mice, P < 0.05. (D) OR assessment of all animals that presented with a seizure at any point in the study (dosing or handling) demonstrates that the likelihood of presenting with a stage 5 seizure, if any seizure is observed, is significantly reduced with VPA treatment relative to both VEH- and CBZ-treated mice. Conversely, CBZ treatment concurrent with acute TMEV infection actually increased the likelihood that any CBZ-treated mouse with seizure would present with a stage 5 seizure relative to VEH-treated, TMEV-infected mice. *Significantly different from VEH-treated, TMEV-infected mice; ^#significantly different from VPA-treated, TMEV-infected mice.

Fig. 4.

Time-dependent effect of drug dosing on seizure severity and seizure burden. Male C57BL/6J mice (4–5 weeks old) were randomly divided into treatment groups (n = 28/treatment group) and subchronically dosed for days 0–7 following TMEV infection. The presence and severity of handling-induced seizures and seizure severity were scored according to the Racine scale. Twice-daily handling (Handling) sessions occurred 30 minutes after drug dosing (Dosing) to accommodate the pharmacokinetic profile of VPA [T_1/2 = 30 minutes (Ben-Cherif et al., 2013)] and CBZ [T_1/2 = 60 minutes (Nishimura et al., 2008)]. Each dosing session was separated by at least 7 hours, occurring daily at 9:00 AM and 4:00 PM. (A–C) The maximum seizure severity recorded for any animal that presented with a seizure at any point in the dosing period. ^#Significantly different from CBZ, P < 0.05. (D–F) The maximum seizure severity recorded for any animal that presented with a seizure at any point in the handling period. Handling period occurred 30 minutes after administration of (D) VEH (0.5% MC); (E) CBZ (20 mg/kg); or (F) VPA (200 mg/kg). *Significantly different from VEH, P < 0.05; ^#significantly different from CBZ, P < 0.05. (G) Average seizure burden for any mouse that presented with at least one handling-induced seizure during the dosing session is not significantly different between treatment groups prior to ASD admiration. (H) Average seizure burden in mice that presented with at least one handling-induced seizure during the handling session is significantly reduced by acute ASD treatment 30 minutes prior. Significantly different from VEH, **P < 0.01 and ****P ≤ 0.0001. (I) Average number of stage 4/5 seizures for any mouse that presented with at least one handling-induced seizure during the dosing session is not significantly different between treatment groups prior to ASD administration. (J) Average number of stage 4/5 seizures for any mouse that presented with at least one handling-induced seizure during the handling session is significantly reduced by acute VPA treatment 30 minutes prior. ****Significantly different from VEH, P ≤ 0.0001.

Fig. 5.

Rotarod assessment and OF activity after infection with TMEV. (A) When mice were challenged to maintain balance on a rotating rod (6 rpm) for 60 seconds 15 DPI, only VPA-treated mice demonstrated significant improvements in motor coordination relative to VEH-treated, TMEV-infected mice. *Significantly different from VEH, P < 0.05. (B) Long-term (36 DPI) evaluation of anxiety-like behavior in the OF activity monitor demonstrates that no ASD treatment improved total time (seconds) in the center of an OF, a measure of anxiety-like behavior. (C) Conversely, CBZ-treated mice demonstrated further reduced total distance traveled in the center of the OF, suggesting greater anxiety relative to VEH-treated, TMEV-infected mice. *Significantly different from VEH, P < 0.05.

Fig. 6.

Immunohistochemical assessment of acute (8 DPI) and long-term (36 DPI) changes in GFAP and NeuN expression in area CA1 of dorsal hippocampus of TMEV-infected mice treated with and without acute ASD treatment. (A) Representative photomicrographs of GFAP (red) and NeuN (green) immunoreactivity with 4′,6′-diamidino-2-phenylindole nuclear counterstain (blue) from TMEV-infected mice treated with VEH (0.5% methylcellulose) or CBZ or VPA, 8 days postinfection or 36 days postinfection. White arrows in 36 DPI images indicate areas of sclerosis in area CA1 of dorsal hippocampus. The locations of stratum pyramidale (SP) and stratum radiatum (SR) layers are indicated for reference, as best as possible despite notable hippocampal pathology. (Scale bar = 50 µm, applies to all images.) (B) Immunolabeling for NeuN (8 DPI) demonstrates that no acute treatment protected against TMEV-induced neurodegeneration. VPA-treated mice demonstrated significant elevations in GFAP immunoreactivity relative to CBZ-treated mice, but did not achieve a statistically significant difference from VEH by one-way analysis of variance. *Significantly different from CBZ-treated, TMEV-infected mice, P < 0.05. (C) Long-term (36 DPI) immunolabeling for NeuN demonstrates that no drug treatment protected against TMEV-induced neurodegeneration. CBZ-treated mice demonstrated significant elevations in GFAP immunoreactivity relative to VPA-treated mice, but the elevations were not statistically different from VEH by one-way analysis of variance. Acute and long-term cohorts were processed and analyzed separately for immunohistochemical evaluations. *Significantly different from VPA-treated, TMEV-infected mice, P < 0.05.