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. Author manuscript; available in PMC: 2017 Dec 1.
Published in final edited form as: Epilepsia. 2016 Oct 14;57(12):1958–1967. doi: 10.1111/epi.13577

Acute Treatment with Minocycline, but not Valproic Acid, Improves Long-Term Behavioral Outcomes in the Theiler’s Virus Model of Temporal Lobe Epilepsy

Melissa L Barker-Haliski 1,2,3, Taylor D Heck 1, E Jill Dahle 1, Fabiola Vanegas 1, Timothy H Pruess 1, Karen S Wilcox 1, H Steve White 1,2
PMCID: PMC5154893  NIHMSID: NIHMS816628  PMID: 27739576

Summary

Objective

Infection with Theiler’s murine encephalomyelitis virus (TMEV) in C57Bl/6J mice induces acute seizures and development of spontaneous recurrent seizures and behavioral comorbidities weeks later. The present studies sought to determine whether acute therapeutic intervention with an anti-inflammatory–based approach could prevent or modify development of TMEV-induced long-term behavioral comorbidities. Valproic acid (VPA), in addition to its prototypical anticonvulsant properties, inhibits histone deacetylase (HDAC) activity, which may alter expression of the inflammasome. Minocycline (MIN) has previously demonstrated an antiseizure effect in the TMEV model via direct anti-inflammatory mechanisms, but the long-term effect of MIN treatment on the development of chronic behavioral comorbidities is unknown.

Methods

Mice infected with TMEV were acutely administered MIN (50 mg/kg, b.i.d. and q.d.) or VPA (100 mg/kg, q.d.) during the 7-day viral infection period. Animals were evaluated for acute seizure severity and subsequent development of chronic behavioral comorbidities and seizure threshold.

Results

Administration of VPA reduced the proportion of mice with seizures, delayed onset of symptomatic seizures, and reduced seizure burden during the acute infection. This was in contrast to the effects of administration of once-daily MIN, which did not affect the proportion of mice with seizures nor delay onset of acute symptomatic seizures. However, VPA-treated mice were no different from VEH-treated mice in long-term behavioral outcomes, including open field activity and seizure threshold. Once-daily MIN treatment, despite no effect on the maximum observed Racine stage seizure severity, was associated with improved long-term behavioral outcomes and normalized seizure threshold.

Significance

Acute seizure control alone is insufficient to modify chronic disease comorbidities in the TMEV model. This work further supports the role of an inflammatory response in the development of chronic behavioral comorbidities and further highlights the utility of this platform for the development of mechanistically-novel pharmacotherapies for epilepsy.

Keywords: Acquired epilepsy, valproic acid, minocycline, seizure threshold, anxiety-like behavior, temporal lobe epilepsy

Introduction

Epilepsy is an under-recognized long-term complication of CNS infection1. Higher incidence of epilepsy in less-developed countries (over 75% of 50 million people worldwide) may be attributable to greater prevalence of neurotropic viruses in these regions1. For example, CNS infection in Ecuador accounts for approximately 14.8% of new epilepsy diagnoses2, whereas CNS infection accounts for only 3% of US cases3. The mechanisms by which viral encephalitis contributes to epilepsy are unclear4, but the risk for developing epilepsy after viral encephalitis with seizures increases 22-fold5. It is likely that viral infection of the CNS underlies the development of chronic epilepsy due to increased expression of inflammatory cytokines, lowered seizure threshold, and increased risk of status epilepticus6–8. In both the developed and developing world, infection-induced encephalitis can promote the onset of temporal lobe epilepsy (TLE5). There is thus significant need to identify therapeutic interventions that can attenuate acute seizure presentation in the context of viral encephalitis.

The Theiler’s murine encephalomyelitis virus (TMEV) model exhibits many characteristics of human encephalitis-induced acute seizures4, as well as the chronic behavioral comorbidities9–11 associated with TLE10. Approximately 50–65% of TMEV-infected animals develop handling-induced seizures after the infection and show significant elevations in inflammatory cytokines12; 13. Animals survive the initial infection, and a proportion of mice also later develop spontaneous recurrent seizures (SRS). Mice demonstrate chronic deficits in learning and memory, increased anxiety-like behavior, and neurodegeneration9; 12. This model is thus an etiologically-relevant platform to evaluate novel therapeutic intervention strategies for TLE11.

Anti-inflammatory therapies represent one hypothesized approach to modify the course of TLE due to the role that inflammation plays in seizure induction and maintenance14; 15. However, it is unknown whether anti-inflammatory agents can also disrupt the development of associated chronic comorbidities of TMEV infection (e.g. reduced seizure threshold and increased anxiety-like behavior9; 11; 13). Whether administration of anti-inflammatory agents during the infection can modify the chronic disease is of great translational value to identifying preventative treatments for TLE14, especially considering the difficulty in conducting similar long-term studies in human patients with encephalitis-induced epilepsy4.

Two pharmacological strategies were presently employed to interrogate the TMEV-induced inflammatory response. The first approach was administration of MIN to directly suppress microglial activation and overexpression of inflammatory cytokines16. Existing evidence suggests that MIN may attenuate the severity of TLE after a neurological insult. For example, administration of MIN to rats following pilocarpine-induced SE may prevent onset of SRS17. MIN may reduce acute seizure incidence in the TMEV model through IL-6-dependent mechanisms18; 19. However, it is unknown whether acute MIN administration can also modify the chronic behavioral deficits and neuroinflammation associated with TMEV infection9. The second approach presently employed sub-therapeutic administration of VPA to modulate the expression of inflammasome-associated transcription factors. VPA indirectly suppress inflammation through the modulation of histone deacetylase (HDAC) activity and transcription factor regulation20; 21. Moreover, administration of VPA at sub-therapeutic levels22 is neuroprotective in other post-insult models of epilepsy, including TBI23 and septic encephalopathy24. Our previous work with acute administration of therapeutic (e.g., anticonvulsant) doses of VPA suggested no significant effects of such administration on chronic comorbidities (e.g. anxiety-like behavior) in the TMEV model11, however it is possible that the mechanisms for neuroprotection were not appropriately engaged with the dose tested. Moreover, VPA was selected because it is an FDA-approved antiseizure drug (ASD) that may be utilized in the clinical management of acute seizures that arise following an infection-induced encephalitis event. The present studies thus defined whether administration of anti-inflammatory compounds during the acute TMEV infection could both reduce the incidence and severity of acute behavioral seizures, as well as attenuate associated long-term behavioral deficits. These studies also provided critical evaluation of chronic comorbidity biomarkers of TLE to further validate this model as a relevant platform for drug development for infection-induced epilepsy.

Methods and Materials

See Supplemental Information. For experimental design, see Figure S1.

Results

Animal health during the acute infection

In the high titer study, there was a significant time-dependent effect on daily body weight of TMEV-infected animals (0–9 days post-infection (DPI) Figure 1A; F(7,538)=38.31, p<0.0001). Post-hoc analysis demonstrated that TMEV-infected mice lost more weight than sham-infected mice during the acute infection period9; 12. MIN-treated mice (50 mg/kg, b.i.d.) experienced substantial reductions in body weight relative to vehicle (VEH)-treated mice (p<0.0001), as well as diarrhea and overall morbid appearance. The catabolic effect of MIN treatment was so extensive that all mice in the study were euthanized by 9 DPI.

Figure 1.

Figure 1

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Treatment groups were monitored twice per day during the acute infection period for changes in body weight (A and B), presentation of acute behavioral seizures (C and D), and changes in seizure burden (E–H). A) Mice infected with TMEV show significant reductions in body weight relative to sham-infected mice (n=15); treatment with twice-daily VEH (0.5% methylcellulose, i.p.) or MIN (50 mg/kg, i.p.) did not improve the TMEV-induced body weight loss. Sham-infected animals received VEH during the 7-day acute infection period. MIN (b.i.d.) in TMEV-infected mice was catabolic; by 9 DPI all mice were euthanized. * indicates significantly different from VEH, p<0.05; # indicates significantly different from sham, p<0.05. C) Once-daily administration of VEH, MIN (50 mg/kg, i.p), or VPA (100 mg/kg, i.p.) affected body weight during the TMEV infection (2.5 × 105 PFU). TMEV infection was associated with reduced in bodyweight, which was not improved by once-daily administration of MIN. However, once-daily administration of VPA attenuated TMEV-induced body weight loss. * indicates significantly different from VEH, p<0.05; # indicates significantly different from MIN, p < 0.05. C) TMEV (3.0 × 105 PFU) infection leads to a reduction in the latency to first seizure in both VEH- and MIN-treated mice. However, MIN-treated mice had significantly increased latency to first seizure relative to VEH. * indicates significantly different from VEH, p<0.05; # indicates significantly different from sham, p<0.05. D) Latency to first seizure following TMEV infection was significantly improved by once-daily administration of VPA, but not MIN. VPA-treated mice (74%) survived the acute infection period without experiencing a behavioral seizure during any observation session. * indicates significantly different from VEH, p<0.05. Average seizure burden in the morning and afternoon sessions was also evaluated (0–7 DPI). E) Cumulative seizure burden increased during the acute infection. Twice-daily administration of MIN reduced the burden, albeit animals were markedly lethargic, moribund, and demonstrated significant reductions in body weight. F) Once-daily VPA administration was associated with early and sustained reductions in cumulative seizure burden, whereas MIN was only associated with late reductions in cumulative seizure burden. G) Administration of MIN (50 mg/kg, b.i.d.) significantly reduced the overall seizure burden relative to VEH-treated mice. Sham-infected mice did not present with any seizures during the acute period. H) Reducing the viral titer from that used in (G), as well as reducing the frequency of MIN administration still promoted a reduction in the average seizure burden of MIN-treated mice relative to VEH. Additionally, once-daily administration of VPA (100 mg/kg, i.p.) also significantly reduced the average seizure burden during both morning and afternoon observation sessions. * Indicates significantly different from VEH, p<0.05.

Due to this toxicity associated with twice-daily MIN administration, a reduced dose of MIN (50 mg/kg, q.d.) was administered to mice infected with the less severe medium viral titer in conjunction with the evaluation of VPA (100 mg/kg, q.d.). There was a significant time-dependent change in body weight in TMEV-infected animals treated with VPA or MIN (Figure 1B, F(7,553)=188.8, p<0.0001). Post-hoc analysis revealed that VPA (100 mg/kg, q.d.) treatment was actually associated with improved body weight relative to VEH treatment (p≤0.01, all sessions). Once-daily MIN treatment still resulted in 30% reduction in Day 1 body weight, which differed significantly from VEH 4–7 DPI (Day 4, p<0.05; Day 5, p<0.001; Day 6, p<0.01; Day 7, p<0.0001). There were no other notable health differences between treatment groups.

Effect of treatment on viral-induced behavioral seizures

The latency to first seizure was evaluated with a survival curve (Figure 1C and 1D). Administration of high viral titer and 50 mg/kg, b.i.d. MIN significantly increased the latency to first seizure (Figure 1C; χ2=5.62, p<0.02). However, twice-daily MIN-treated mice were markedly sicker than VEH-treated mice (Figure 1C). Only treatment of mice with once-daily VPA, but not once-daily MIN, significantly increased the latency to first seizure (Figure 1D; VPA, χ2=7.70, p<0.01; MIN, χ2=7.75, p<0.01). However, treatment with MIN (50 mg/kg, q.d.) did not improve the latency to first seizure relative to VEH-treated mice infected with TMEV (χ2=0.029, p>0.8; Figure 1D). Seizure burden provides an important measure of frequency and severity of seizures. Cumulative seizure burden was markedly attenuated with twice-daily MIN administration (Figure 1E), but once-daily MIN only affected seizure burden late in the infection period (Figure 1F). However, VPA exerted an early and sustained attenuation of seizure burden (Figure 1F). These results suggest a dose-dependent effect of MIN on overall health, such that twice-daily MIN administration may have induced significant morbidity and lethargy that masked seizure generalization.

Seizure burden also did not appear to be affected by compound pharmacokinetics (Figure 1G and 1H). Both MIN regimens significantly reduced the average seizure in both AM and PM observation sessions (Figure 1E and 1F). Once-daily administration of VPA also effectively reduced average seizure burden both 30 min after drug administration (AM), and also in the afternoon handling session (Figure 1F), a time likely after the drug had been metabolized and cleared from the circulation25.

The proportion of animals in each group with seizures was also determined (Table 1). There was no significant difference in the proportion of VEH- or twice-daily MIN-treated mice with seizures (z=1.43, p=0.08). However, significantly fewer VPA-treated mice exhibited seizures relative to VEH-treated controls (z=2.29, p=0.01) and mice that received once-daily MIN (z=2.29, p=0.01). Furthermore, there was no difference in the proportion of mice with seizures between the VEH- and once-daily MIN-treated mice (Table 1).

Table 1.

Proportion of mice with and without seizures. Distribution of maximum observed Racine seizure stage.

Treatment Group Size (n) Seizure Observed (n, %) No Seizure Observed (n, %) Maximum Stage 3 (n, %) Maximum Stage 4 (n, %) Maximum Stage 5 (n, %)
VEH (3 × 105 PFU) 25 13 (52%) 12 (48%) 0 (0%) 2 (15%) 11 (84.6)
MIN (50 mg/kg, b.i.d.) 25 8 (32%) 17 (68%) 2 (25%)* 1 (12.5%) 5 (62.5%)
VEH (2.5 × 105 PFU) 28 18 (64.3%) 10 (35.7%) 1 (5.5%) 1 (5.5%) 16 (88.9%)
MIN (50 mg/kg, q.d.) 28 18 (64.3%) 10 (35.7%) 3 (16.7%) 1 (5.5%) 14 (77.8%)
VPA (100 mg/kg, q.d.) 27 9 (33.3%)* 18 (66.7%) 1 (14.2%) 0 (0%) 6 (85.7%)
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*

indicates significantly different from the endpoint measurement of the VEH group in same study, p < 0.05.

The distribution of the maximum observed seizure severity was evaluated (Table 1) to define whether any treatment condition may have modified the insult, e.g. reduced the maximum seizure severity. This is an important differentiating analysis that may indicate whether a treatment can attenuate acute seizure severity. Twice-daily MIN did not significantly reduce the number of mice with Stage 5 seizures (62.5%) relative to VEH-treated mice (84.6%; z=1.55, p=0.06). Once-daily MIN did not attenuate maximum seizure severity; the proportion of MIN-treated mice with Stage 5 seizures was no different from VEH (z=1.22, p>0.2). Moreover, VPA was also without significant effect on Stage 5 seizures. That no treatment significantly reduced the proportion of TMEV-infected mice with Stage 5 handling-induced seizures suggests that the severity of the acute insult was unchanged, regardless of treatment. It is, however, important to note that the once-daily dose of VPA utilized in this study was below the mouse maximal electroshock (MES) median effective dose (ED50). It is thus unlikely that a reduction in the proportion with seizures (Table 1) is due to direct anticonvulsant effects of VPA.

Effect of treatment on open field activity, a measure of anxiety-like behavior

Mice were assessed 46 DPI in an open field (OF). Both percent of total time in the center (F(2,24)=3.56, p=0.044) and percent of total distance traveled in the center (F(2,24)=3.89, p=0.035; Figure 2A and 2B) were significantly affected by treatment. Notably, only MIN-treated mice exhibited improvements in time spent (p<0.05) and distance traveled (p<0.05) in the center of the OF relative to VEH-treated mice. VPA-treated mice, despite showing improvements in acute seizures, did not differ significantly from VEH-treated mice (p>0.05). Furthermore, additional measures of OF activity (Figure 2C–G) suggest that only once-daily MIN was associated with significant changes in exploratory behaviors. These data suggest that once-daily MIN may improve chronic anxiety-like behavior of TMEV-infected mice.

Figure 2.

Figure 2

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Evaluation of motor behavior and coordination 46 DPI in mice treated with once-daily MIN or VPA. A) The time spent in the center of an open field is significantly increased for mice treated with once-daily MIN that exhibited seizure; there is no such increase for mice treated with once-daily VPA * indicates significantly different from VEH-treated, TMEV-infected mice in same seizure history group, p<0.05; # indicates significantly different from VEH-treated mice with seizure, p<0.05. B) Similarly, TMEV-infected mice treated with once-daily MIN show significant increases in the amount of distance travelled in the center of an open field relative to VEH-treated, TMEV-infected mice in the same task. There is also no similar improvement in open field testing for once-daily VPA-treated mice at this time point. * indicates significantly different from VEH-treated, TMEV-infected mice in same seizure history group, p<0.05. C) Activity of TMEV-infected mice in an open field environment is significantly reduced with acute MIN-treatment. * indicates significantly different from VEH measurement in the same seizure history group, p < 0.05. D) The time spent at rest for MIN-treated mice with seizure is significantly different from VEH- and VPA-treated mice. * indicates significantly different from VEH, p<0.05; # indicates significantly different from VPA, p<0.05; E) Horizontal activityis significantly reduced in TMEV-infected mice treated with MIN, suggestive of improved cognitive function. * indicates significantly different from VEH in the same seizure history group, p<0.05 F) There is no significant difference between any group in the vertical activity counts. G) there is no significant difference between any treatment group in the time spent in stereotypical behaviors (i.e. grooming). H) Importantly, there is no significant difference in motor coordination between any groups when evaluated on the rotarod at 46 DPI.

Motor behavior on a rotarod is unaffected weeks after viral infection

Mice were challenged on the rotarod 46 DPI to rule out any confounds of motor coordination at chronic testing periods (Figure 2H). Latency to fall off the rotarod was not affected by treatment (Figure 2H; F(2,54)=0.078, p>0.9).

Seizure threshold is significantly improved by treatment with MIN

A cohort of sham-infected, age-matched C57Bl/6J mice (n=7) were also evaluated to define baseline seizure threshold for this strain (Figure 3A). Importantly, VEH-treated mice had reduced threshold relative to sham-infected mice. The amount of PTZ (mg/kg) necessary to elicit twitch and clonus in drug-treated mice was then compared to VEH-treated mice. Acute MIN-treatment was associated with significantly increased threshold; i.e., more PTZ was necessary to elicit twitch (Figure 3B; t(14)=2.22, p=0.04). The change in threshold following acute treatment with VPA failed to reach statistical significance, albeit fewer mice with seizures were available for testing (n=6; t(12)=2.13, p=0.054). The threshold for clonic seizure was similarly affected. MIN-treatment was associated with significant improvements in threshold 56 DPI (Figure 3B, t(14)=2.14, p=0.050), whereas VPA-treatment was not (Figure 3C, t(12)=2.11, p=0.057).

Figure 3.

Figure 3

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Once-daily MIN, but not VPA, attenuates TMEV-induced reductions in seizure threshold as measured by i.v. PTZ (Metrazol) seziure threshold test 42 DPI in mice with a history of acute seizure. A) For reference, sham-infected, age-matched C57/Bl6J mice were included in the evaluation. # indicates significantly different from sham-infected mice, p < 0.05. * indicates significantly different from VEH-treated mice, p < 0.05. B) Acute adminsitration of MIN significantly increased the amount of PTZ required to induce first twitch and clonus, p < 0.05. C) Acute adminsitration of VPA did not result in a statistical increase in the amount of PTZ required to elicit first twitch or clonus.

Relative to age-matched, sham-infected mice, significantly more VEH-treated mice experienced tonic-extension seizure (TE) during the PTZ test (Table 2); i.e., 75% of VEH-treated, TMEV-infected mice had TE whereas only 14.3% of sham-infected mice experienced TE (χ2=5.53, p=0.019). The proportion of MIN-treated mice displaying TE was significantly reduced relative to VEH- and VPA-treated mice (χ2=5.53, p=0.019). In fact, MIN-treatment reduced susceptibility to TE, such that only 25% of mice experienced TE and there was no statistical difference from sham (χ2=0.27, p>0.5). Conversely, VPA-treated mice were significantly different from sham-infected mice (χ2=6.20, p=0.013) and were not significantly improved relative to VEH-treated mice (χ2=0.14, p>0.3). These data demonstrate that acute treatment with MIN, but not VPA, was associated with significantly attenuated seizure severity in the PTZ threshold test.

Table 2.

Once-daily administration of MIN (50 mg/kg, q.d. i.p.), but not VPA (100 mg/kg, q.d. i.p.), during the acute infection period significantly reduced the proportion of mice with tonic extension seizure and increased the rate of survival after i.v. PTZ infusion 42 DPI. For reference, sham-infected, age-matched C57/Bl6J mice were included in the evaluation. TMEV titer was 2.5 × 105 PFU. Importantly, mice in the MIN treatment group were not significantly different from age-matched, Sham-infected C57Bl/6J mice.

Treatment Group % with Tonic Extension Seizure % Survival Post-i.v. PTZ
Sham-infected (n = 7) 14.3% 85.7%
VEH, TMEV-infected (n = 8) 75% # 0% #
MIN (50 mg/kg, q.d.), TMEV-infected (n = 8) 25%* 62.5%*
VPA (100 mg/kg, q.d.), TMEV-infected (n = 6) 83.3% # 17.7% #
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*

Indicates significantly different from VEH-treated, TMEV-infected mice, p < 0.05.

#

Indicates significantly different from Sham-infected mice, p < 0.05.

Survival following i.v. PTZ was also affected by treatment during the acute infection. Sham-infected mice survived the infusion-induced seizures, whereas all VEH-treated mice died (χ2=10.5, p=0.001; Table 2). MIN-treated mice exhibited significantly improved survival relative to VEH-treated mice (χ2=6.56, p=0.010), which was not different from sham (χ2=1.03, p>0.3). VPA survival in the PTZ test was not different than VEH-treatment (χ2=1.26, p>0.2). In fact, mortality was increased in VPA-treated mice relative to sham-infected mice (χ2=6.20, p=0.013). The dose of PTZ required to elicit both outcomes, i.e., first twitch and clonus, demonstrated a robust protective effect only in favor of acute treatment with MIN (Figure 3B). Moreover, only MIN-treated mice demonstrated a reduced incidence of TE seizures and increased survival (Table 2). These results suggest that treatment with MIN, but not VPA, improves chronic seizure threshold after TMEV infection, despite very few effects on acute symptomatic seizures.

Discussion

Infection-induced encephalitis can lead to TLE due to numerous factors, least of which is enhanced inflammation within the CNS. The TMEV model clearly recapitulates many clinical characteristics of this condition. Herein, we demonstrate that acute administration of VPA, at a dose that is too low to normally confer anti-seizure effects, could surprisingly improve acute disease outcomes. This included preservation of body weight during the acute infection period, delayed presentation of acute symptomatic seizures, reduced proportion of mice with seizures, and demonstrated early and sustained reductions in seizure burden. However, this acute effect did not translate to long-term improvements in TMEV-induced behavioral deficits or reductions in seizure threshold. Conversely, administration of once-daily MIN did not reduce the proportion of mice expressing seizures or delay the onset of acute seizures, albeit mice did show reduced seizure burden late in the acute infection period. MIN-treated mice showed surprising improvements in chronic seizure threshold and behavioral performance. This differential response to VPA and MIN highlights the utility of the TMEV mouse model for drug discovery. More so, these studies suggest that acute seizure control by VPA may be insufficient to modify long-term disease outcomes. Instead, our data suggests that direct suppression of the inflammatory response may more efficiently modify chronic disease course in this model of acquired TLE.

MIN and VPA exert distinctly different anti-inflammatory mechanisms of action. MIN inhibits microglial activation through IL-1B and TNF-α17; 19; 26; 27. VPA targets multiple cellular signaling cascades essential to induction of the inflammasome, e.g. HDACs20. However, low-dose, once-daily VPA was not associated with improvements in chronic behavioral comorbidities, suggesting that this agent did not prevent the development of TLE (or behaviors associated therewith9; 10). These results contrast with work in a model of septic encephalopathy, which suggested the potential of low-dose VPA to modify chronic disease24. How the discrete pathways of the neuroinflammatory processes contribute to seizure induction and maintenance in the TMEV model clearly requires further investigation.

Acute seizure control with conventional ASDs in the TMEV model does not modify chronic disease. Low-dose VPA prevented the onset of seizures in a majority of infected mice, reduced the proportion of mice with handling-induced seizures, and reduced the seizure burden of mice with expressed seizures. It is likely then that the presently observed effects on acute seizures with sub-therapeutic (i.e. below MES ED5022) doses of VPA could be due to modulatory and/or anti-inflammatory factors unrelated to any direct effects on ion channels, i.e. VPA is known to modulate multiple cellular targets, including histone deacetylase (HDAC), extracellular-regulated kinase 1/2 (ERK 1/2), and Wnt/β-catenin28. However, this dose of VPA, despite reductions in seizures, did not significantly alter long-term disease metrics (i.e. OF exploratory behavior or seizure threshold). Our prior work has indicated that anticonvulsant doses of VPA or carbamazepine administered during the acute infection period attenuates acute seizure burden, but there is no associated improvement in chronic TMEV-associated reductions in OF exploratory behaviors11. Thus, acute reductions in seizure presentation with prototypical ASDs has yet to confer chronic disease-modifying effects in the TMEV model of TLE. Clinical use of these ASDs in the context of an infection-induced encephalitis event may therefore require alternative strategies to attenuate the long-term risk for epilepsy in at-risk individuals29.

It is plausible that MIN may be disease-modifying despite little effect on acute seizures. MIN has demonstrated an antiepileptogenic effect in the two-hit model of early life seizures26. In the present study, acute once-daily MIN treatment presently resulted in long-term improvements in behavioral comorbidities known to be associated with TMEV infection9, despite minimal effects on acute handling-induced seizures. These chronic improvements include increased exploratory behavior in the center of the OF, reduced total distance travelled within the entire OF, increased time at rest in this test, and reduced seizure severity and improved survival following PTZ infusion. Importantly, these effects appeared unrelated to any potential insult modification associated with sub-chronic MIN treatment; e.g. there was no reduction in the proportion of MIN-treated mice with seizures, nor were there any changes in seizure severity. While it is possible that this twice-daily treatment paradigm of MIN was indeed playing an ameliorative role on TMEV-induced seizures, as previously reported18; 19, our present data would alternatively suggest that such a reduction may have been more likely due to a decline in general health, rather than direct seizure suppression per se. Therefore, it is therefore possible that seizure generalization was not possible because of the overt decline in muscle tone and overall animal health. In fact, treatment with twice-daily MIN caused substantial worsening in the general health of all mice infected with TMEV to the point that they had to be euthanized by 9 DPI. These findings contrast with prior work with MIN in the TMEV model that did not report adverse health effects18; 19. However, our present results are in general agreement with results from other models of CNS injury or inflammation suggesting mixed effects of MIN administration on overall health of C57/Bl6 mice 30; 31. Our present results further highlight the need to carefully and rigorously evaluate dose-dependent effects of investigational and repurposed compounds in etiologically-relevant models of a disease by multiple independent groups32. However, these present results for disease-worsening with twice-daily MIN, coupled to prior observations with either carbamazepine (CBZ)11 or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA)-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX)33, would suggest that the TMEV model may identify not only treatments that can improve acute outcomes, but also those that could be disease-worsening. That the beneficial effects of acute once-daily MIN treatment on chronic outcomes were not associated with acute improvements during the viral infection period clearly suggest that MIN conferred a disease-, rather than insult-, modifying effect14; a finding in direct contrast to that of VPA.

Reductions in seizure threshold in TMEV-infected mice highlight its potential as a useful, and technically-feasible, biomarker of epileptogenesis; this would also provide a more cost-effective surrogate for chronic vEEG recordings for moderate-throughput drug studies. Changes in threshold that lead to symptomatic seizures are often associated with increased expression of inflammatory markers in animal seizure models and human patients7; 8; 34. For example, transgenic mice that overexpress TNF-α and IL-6 demonstrate neurological dysfunction including cell loss, decreased seizure threshold, and SRS35; 36. Rat pups exposed during infancy to a systemic pro-inflammatory challenge, which is insufficient to produce brain damage or seizures, also demonstrate long-term decreases in threshold, as well as learning and memory deficits, and anxiety-like behaviors well into adulthood37. Based on the proposal by Bröer and colleagues that receiver-operating characteristic (ROC) curves may identify relevant biomarkers of TMEV infection38, threshold to clonus in the present study was found to be a “good” biomarker of VEH-treated, TMEV-infected mice with early seizures (ROC area under the curve (AUC)=0.857±0.98, p=0.021; data not shown). Moreover, decreases in seizure threshold of post-pilocarpine SE rats, as measured by PTZ infusion, directly correlated with onset of SRS weeks after SE39, further supporting the potential that seizure threshold is an etiologically-relevant biomarker of SRS. Furthermore, cerebral malaria in C57Bl6 mice promotes increased susceptibility to myoclonic and tonic-clonic seizures induced by low-dose PTZ up to 45 days after infection40. Indeed, TMEV-infected mice develop SRS and reductions in seizure threshold weeks after infection12; 13. That MIN treatment during the acute seizure phase can mitigate chronic proconvulsant effects associated with infection suggests that seizure threshold is a useful biomarker of TMEV-induced disease progression.

Whether these results demonstrate antiepileptogenic or antiepileptic effects per se cannot be presently defined. MIN may merely delay the presentation of TLE-associated behavioral comorbidities, as evidenced by changes in seizure threshold and OF activity. In this example, the time window of evaluation (56 DPI) may have been too short to fully examine chronic effects of acute treatment. Perhaps the latent phase in this model was extended by the acute intervention. Untreated, TMEV-infected mice can present with unprovoked seizures up to 7 months post-infection12. While longer duration studies would be costly and labor-intensive, they would clearly define the benefit of acute treatment on long-term behavioral deficits in the TMEV model. Moreover, future studies with long-term vEEG monitoring after the infection period would provide a substantial cost-benefit over clinical studies evaluating prophylactic treatment of CNS infections. These clinical studies are clearly warranted, but highly unfeasible due to the low clinical incidence of infections, difficulty in collecting patients in appropriately established clinical settings, and ethical complications of controlling treatment arms. Future preclinical studies should attempt to extend the intervention time frame to clinically-relevant windows, e.g. after the first behavioral seizure. Additionally, long duration monitoring of behavioral comorbidities coupled with evaluation of SRS will definitively address whether acute treatment with anti-inflammatory agents during the acute infection period can prevent the onset of TMEV-induced epilepsy. The TMEV model clearly represents a platform to potentially identify mechanistically novel approaches to modify the disease, rather than the symptoms, of epilepsy.

Supplementary Material

Supp info
epi13577-sup-0001-Study Design_08262016

Key Points Box.

  • MIN or VPA was administered to mice infected with TMEV during the acute infection and mice were evaluated for potential disease-modifying effects.

  • VPA administered at sub-therapeutic levels increased the latency to seizure onset, reduced seizure burden, and reduced the proportion of mice with seizures.

  • VPA treatment was not associated with improvements in long-term behavioral comorbidities.

  • Once-daily MIN reduced the average seizure burden, but did not alter the latency to seizure onset, nor did it reduce the proportion of mice expressing seizures.

  • Once-daily MIN treatment was associated with normalized seizure threshold 56 days post-infection and reduced TMEV-induced anxiety-like behavior.

Acknowledgments

The authors wish to thank Dr. Robert Fujinami for purified TMEV titers. This work was supported by NIH RO1 NS065434 (KSW and HSW) and NIH Epilepsy Therapy Screening Program (ETSP) contract HHSN 271201100029C (HSW).

Biography

Dr. Melissa L. Barker-Haliski is a senior research scientist at the University of Washington.

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Footnotes

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

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