Neuroinflammation in epileptogenesis: insights and translational perspectives from new models of epilepsies

Abstract

Animal models have provided a wealth of information on mechanisms of epileptogenesis and comorbidogenesis and have significantly advanced our ability to investigate the potential of new therapies. Processes implicating brain inflammation have been increasingly observed in epilepsy research. Here, we discuss the progress on animal models of epilepsies and comorbidities that inform us on the potential role of inflammation in epileptogenesis and comorbidity pathogenesis in rodent models of West syndrome and the Theiler’s murine encephalomyelitis virus (TMEV) mouse model of viral encephalitis-induced epilepsy.

Rat models of infantile spasms were generated in rat pups after right intracerebral injections of pro-inflammatory compounds (lipopolysaccharides with or without doxorubicin, or cytokines) and were longitudinally monitored for epileptic spasms and neurodevelopmental and cognitive deficits. Anti-inflammatory treatments were tested after the onset of spasms. The TMEV mouse model was induced with intracerebral administration of TMEV and prospective monitoring for handling-induced seizures or seizure susceptibility, as well as long-term evaluations of behavioral comorbidities of epilepsy.

Inflammatory processes are evident in both models and are implicated in the pathogenesis of the observed seizures and comorbidities. A common feature of these models, based on the data so far available, is their pharmacoresistant profile.

The presented data support the role of inflammatory pathways in epileptogenesis and comorbidities in two distinct epilepsy models. Pharmacoresistance is a common feature of both of these inflammation-based models. Utilization of these models may facilitate the identification of age-specific, syndrome- or etiology- specific therapies for the epilepsies and attendant comorbidities, including the drug resistant forms.

Introduction

Animal models of epilepsy have provided a significant impetus in advancing our knowledge about pathogenic factors and processes involved in seizures and epilepsies, and developing new therapies for human seizures and epilepsies. A recurrent theme in studies of epileptogenesis has been the underlying brain inflammation that may control seizure susceptibility and epileptogenesis in various types of seizures and epilepsies. This understanding has led to a number of attempts to target these processes in an effort to develop new therapies.^{1, 2} Since 2010, when the last Immunity and Inflammation in Epilepsy meeting took place in Milan, Italy,³ a significant progress has been made in developing and characterizing new animal models, including those that shed light in the role of inflammation in early life epileptic encephalopathies, such as early life West syndrome, and post-viral infectious epileptogenesis. In this review, we present a summary of the findings on these new models.

Inflammation in infantile spasms epileptogenesis: recent progress in animal models

Aristea S. Galanopoulou

Infantile spasms (IS) is the signature seizure of West syndrome, an infantile epileptic encephalopathy that presents with IS, hypsarrhythmia (chaotic, multifocally epileptic, and high amplitude disorganized EEG background) and intellectual disabilities. Two of the three criteria are sufficient for the diagnosis of West syndrome, albeit IS is a necessary diagnostic criterion present in all patients. IS occur in 1 of 3,000 live births and carry a grave prognosis: mortality ranges between 7–49% depending on study and follow-up time, intellectual disabilities in ~80% of the affected infants, persisting and often drug-resistant epilepsy in 60–70%, and increased risk for autism spectrum disorders in the infants with IS due to structural etiology.

Exploration of the pathogenic role of the known or suspected etiologies of IS and West syndrome is necessary to identify novel potential therapy targets and determine their antiepileptogenic and disease modifying potential. At least 70 discrete genes have been linked so far to IS through genetic association studies, while additional chromosomal abnormalities, and diverse metabolic and structural brain abnormalities at cortical and/or subcortical sites have also been implicated in the patients with identifiable causes of IS (see recent review in⁴).

Several observations have supported a role for neuroinflammation in IS pathogenesis. The current treatment of IS [adrenocorticotropic hormone (ACTH), glucocorticoids, vigabatrin, ketogenic diet] exhibit anti-inflammatory effects,^{5, 6} even though these are not necessarily their main mode of action. Some key genes implicated in diseases associated with IS are controlled by or regulate downstream inflammatory signaling pathways.⁴ Prominent examples are genes involved in the mammalian target of rapamycin (mTOR) pathway which is regulated by cytokines; and have been associated with IS of genetic etiologies or with focal cortical dysplasias.^{4, 7–10} In addition, IS may be due to infectious etiologies, autoimmune causes, hypoxia/ischemia, all of which involve activation of inflammatory processes.^11–13 Furthermore, there are several studies that demonstrate abnormal levels of cytokines in the serum or cerebrospinal fluid of infants with IS or of lymphocyte subtypes in their blood and normalization of some of these parameters in infants treated with ACTH.^14–16 The role of inflammation, however, may be complex, as suggested by paradoxical spontaneous remission of IS after viral infections or acute febrile illness.^17–19

We have developed animal models of IS that provide insights on the role of neuroinflammation in the pathogenesis and treatment of IS. In the multiple-hit rat model of IS due to structural lesion,²⁰ lipopolysaccharides (LPS), a component of the outer membrane of Gram-negative bacteria, is one of the inducing agents, suggesting that pro-inflammatory factors may contribute to the expression of IS. In brief, this model is generated by right intracerebral doxorubicin (cytotoxic) and right intracortical LPS given to rat pups postnatal day 3 (PN3), followed by intraperitoneal p-chlorophenylalanine (PCPA) on PN3. The PN3 inducing agents are sufficient to generate the phenotype of epileptic spasms, which are first recorded on PN4 and last until ~PN13. The multiple-hit model of IS due to structural lesions is refractory to ACTH and partially/transiently responsive to vigabatrin.²⁰ Of interest, this model predicted a better efficacy-tolerability profile of CPP-115, a vigabatrin analogue with higher affinity for GABA aminotransferase and lower risk for retinal toxicity, at significantly lower doses than vigabatrin.²¹ Indeed, it was rewarding that a recently published case report of an infant with refractory IS treated with CPP-115 corroborated these data in the clinical setting.²²

LPS is known to trigger the release of pro-inflammatory cytokines and generate further inflammation and gliosis. Indeed, elevated expression of cytokines like interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNFα) as well as astro- and micro-gliosis are seen in the perilesional regions of pups treated with the multiple-hit rat model protocol.²³ Although this model is not induced by genetic manipulations, we observed a pathological overactivation of the mTOR pathway, as indicated by the increase of phosphorylated S6 ribosomal protein (pS6) in cortical neurons (expressed preferably in NeuN-positive, parvalbumin-negative neurons) and microglia (OX42-positive).²³ This cortical mTOR overactivation is seen during the period of spasms but resolves when spasms resolve. The mTOR pathway is known to be activated by cytokine signaling presenting as an interesting downstream link with neuroinflammation. It is also a very relevant candidate target for IS pathogenesis, since it is dysregulated in the tuberous sclerosis complex (TSC) patients who carry a 38% risk of manifesting IS,²⁴ while 5–10% of patients with IS have TSC.^24–27 Indeed, using the multiple-hit rat model of IS, we demonstrated that a pulse treatment with high doses of rapamycin, an mTOR complex 1 (mTORC1) inhibitor, between PN4–6 and initiated after the onset of spasms, suffices to stop spasms and partially improves visuospatial learning in the Barnes maze testing (PN16–19), suggesting partial disease modification.²³ In further studies, we confirmed that the dual mTORC1/mTORC2 inhibitor torin 1 given after spasms’ onset also suppresses spasms in the multiple-hit model,²⁸ further strengthening a role of the mTOR pathway in the pathogenesis of IS. Given the significant evidence in favor of the contribution of inflammation in IS pathogenesis, we have extended our studies to anti-inflammatory/antioxidant compounds. Among these, celastrol, a pentacyclic triterpenoid compound that inhibits NF-kB (nuclear factor kappa light chain enhancer of activated B cells) was able to rapidly (within the first hours) reduce behavioral and electroclinical spasms in the multiple-hit rat model of IS.²⁹ Such findings provide converging evidence that neuroinflammation is a significant contributor to IS pathogenesis in this rat model of IS due to structural lesion, which is refractory to ACTH and partially responsive to vigabatrin and its analogs.^{20, 21}

To provide a direct proof that inflammation suffices to trigger spasms, we pursued direct right intracortical infusions of LPS (RicLPS) in PN3 rat pups. RicLPS triggered spasms, starting at PN4, which lasted for several days after induction, confirming that inflammatory triggers can directly cause IS.³⁰ To further delineate the downstream inflammatory pathways that trigger spasms, we then tested a simpler experimental protocol: right intracortical infusion of interleukin-1β (RicIL-1β).³¹ Indeed, RicIL-1β induced spasms in a dose-dependent manner when infused in PN5 rat pups and spasms lasted for several days post-induction.³¹ As expected, parallel co-infusion of IL-1 receptor antagonist (IL-1ra) in the RicIL-1β model reduced spasms acutely, providing further confirmation that the inducing effect was via IL-1R activation.³¹ Both the RicLPS and RicIL-1β models demonstrate ictal electrographic correlates (electrodecremental responses) and epileptic background. Interestingly, while the multiple-hit rat model of IS shows significant neurodevelopmental and cognitive deficits - partially due to the underlying induced lesion and partially due to reversible disease processes, as the disease modifying effects of pulse rapamycin indicate²³ - the RicIL-1β model has minimal behavioral deficits.³¹ The lack of neurodevelopmental and cognitive deficits following RicIL-1β induced spasms is not necessarily incompatible with the clinical phenotype, but may represent the milder – and rarer - phenotype of IS, whereby such cognitive deficits may not be prominent. As such, it dissociates, to a certain extent, the pathogenic processes underlying IS and those of the related neurodevelopmental and cognitive comorbidities.

In summary, our studies provide evidence that inflammation is a component of the underlying pathology in our animal models of IS, can be effectively targeted to suppress spasms after they appear, and may be sufficient to initiate IS in the first week of rat life. As indicated by the RicIL-1β model, induction of spasms does not necessarily result in significant neurodevelopmental and cognitive deficits and may explain some of the milder forms of IS in humans. Our data do not necessarily suggest that neuroinflammation plays no role in controlling the expression of IS-related comorbidities.

Future directions

Ongoing research addresses whether interactions with other signaling pathways or the specific networks affected by neuroinflammation may be the important parameters that determine the effects of inflammatory triggers on cognitive and neurodevelopmental outcomes. The availability of different models of an epilepsy syndrome induced with different methods and exhibiting distinct pathologies will enable the elucidation of the specific roles of each candidate pathogenic process in epileptogenesis but also in the associated comorbidities. It will therefore enable us to determine and confirm efficacy of novel therapeutics across multiple models and pathologies. The advantage of using such chronic models of epilepsies and associated comorbidities, as well as comparing them to milder forms of the same syndromes will allow us to investigate in more detail the full spectrum of efficacy and tolerability of novel therapeutics against a large range of clinically relevant endpoints and outcomes.³²

Epileptogenesis in a murine model of viral encephalitis

H. Steve White, Melissa L. Barker-Haliski and Wolfgang Löscher

Approximately 19,000 individuals per year are hospitalized in the United States with a diagnosis of viral encephalitis.³³ Epilepsy itself is considered an under-recognized long-term complication of CNS infection.³⁴ Higher incidence of epilepsy in less-developed countries (over 75% of the 65 million people worldwide with epilepsy) may, in part, be attributable to an increased incidence of CNS infections in these regions.³⁴ In fact, CNS infection accounts for from approximately 14.8% of newly diagnosed epilepsy in Equador³⁵ whereas in the US, CNS infection likely accounts for approximately 3% of the newly diagnosed epilepsies.³⁶ Patients with viral encephalitis that present with seizures are at substantially higher risk (up to 22 times) for developing spontaneous recurrent seizures (SRS) post infection.^{37, 38} It is not clear whether blocking acute symptomatic seizures will modify the epileptogenic process and delay disease onset. Having access to an etiologically relevant model to evaluate the pathophysiology of viral encephalitis and test novel therapies for the prevention and treatment of encephalitis-induced epilepsy would be an important advance.

In recent years, the Theiler’s murine encephalomyelitis virus (TMEV) mouse model has emerged as a novel “hit and run” model that begins to address this matter.^39–41 C57BL/6 mice, but not SJL mice exposed to TMEV intracerebrally display many of the characteristics of encephalitis-induced human temporal lobe epilepsy (TLE), including neuroinflammation,^{42, 43} neurodegeneration,^42–44 and behavioral comorbidities such as anxiety-like behavior and cognitive deficit.^43–45 Moreover, mice infected with TMEV that present with acute symptomatic seizures display reduced seizure threshold^{41, 45} and an increased liability for the development of abnormal EEG activity and SRS weeks to months following intracerebral inoculation with TMEV.⁴⁰ There is neuropathology consistent with TLE, including hippocampal sclerosis and reactive gliosis (astrogliosis and microgliosis). Of note, the reactive microgliosis observed following TMEV infection substantially differentiates this model from another, more established mouse model of chronic network hyperexcitability, the corneal kindled mouse,⁴² which is frequently employed in the evaluation of novel anticonvulsants.^{46, 47} Thus, TMEV infection in C57Bl/6J mice represents a mechanistically novel and etiologically relevant model of TLE that recapitulates many of the clinical characteristics of human epilepsy. Considering that infection-induced epilepsy in human patients is typically pharmacoresistant,⁴⁸ it reasons that the TMEV mouse model may assist in the identification of potentially transformative therapies for the approximately 30% of patients with pharmacoresistant epilepsy.⁴⁹

The utility of the TMEV model is that it is both suitable for acute, anticonvulsant efficacy studies, as well as for long-duration studies to identify disease modifying and/or antiepileptogenic therapies. Our laboratory has pioneered the use of the TMEV model to investigate novel therapies for both the treatment of acute symptomatic seizures, as well as the prevention of epilepsy and associated behavioral comorbidities.^{43, 45} Given that the pathology of TMEV involves an acute inflammatory storm associated with a marked increase in the expression of the cytokines, TNFα and IL6,⁵⁰ it reasons that anti-inflammatory agents may indirectly modify the disease course and/or reduce disease severity; i.e., be disease modifying.⁵¹ However, it is also entirely plausible that anti-inflammatory agents will exert little, if any, acute anticonvulsant activity. In contrast, compounds with direct antiseizure activity (e.g. ion-channel modifying antiseizure drugs) may reduce the presentation of symptomatic seizures, but not necessarily modify disease onset or seizure expression.^{43, 45}

The TMEV model can indeed demonstrate a robust ability to differentiate the seizure-reducing abilities of investigational agents administered during the acute infection period. Barker-Haliski and colleagues have shown that mice treated twice-daily with anticonvulsant doses of valproic acid (VPA, 200 mg/kg) demonstrated decreased expression and severity of symptomatic seizures during the acute period following virus inoculation.⁴³ In contrast, administration of twice-daily anticonvulsant doses of the antiseizure drug, carbamazepine (CBZ), was actually associated with acute disease worsening, including increased frequency and severity of handling-induced seizures.⁴³ Administration of prototype antiseizure drugs (ASDs) using this drug-testing approach,^{43, 45} including our unpublished work with levetiracetam (LEV; Figure 1), further suggest that symptomatic seizures associated with the TMEV model demonstrate a unique pharmacological response profile that is quite different from other models more extensively used for the early evaluation of investigational antiseizure drugs.⁵² For example, once daily administration of LEV (50 mg/kg, p.o., n=7) or vehicle (VEH; 0.5% methylcellulose, n = 6) for days 3–7 post-infection 30 min prior to testing did not significantly modify the incidence or seizure burden of handling-induced seizures (Figure 1A, t=0.219, p>0.8) associated with TMEV infection (2.5 x 10⁵ PFU, i.c.). In contrast, once-daily LEV administration during days 3–7 post-infection significantly increased the cumulative seizure burden (Figure 1B; F_(1,11)=8.84, p=0.021) and observation session total seizure burden (Figure 1C; F_(1,11)=12.78, p=0.004), albeit the proportion of mice with seizures was not significantly different (83% LEV-treated; 50% VEH-treated, z=1.391, p=0.07). It should be noted that this dose of LEV was selected based on the median effective dose (i.p.) necessary to exert an anticonvulsant effect in the 6 Hz mouse model of limbic seizures^{53, 54} and the plasma concentrations obtained following single oral dosing in mice.⁵⁵ Thus, the TMEV model is well-suited for acute anticonvulsant evaluations in a moderate-throughput drug screening capacity.

Figure 1.

Once-daily oral administration of the anticonvulsant, levetiracetam (LEV; 50 mg/kg, p.o.) during days 3–7 post-infection with Theiler’s murine encephalomyelitis virus (TMEV) does not significantly improve seizure burden during the acute symptomatic seizure period. This study was conducted in 4–5 week old male C57Bl/6J mice (Jackson Labs, Bar Harbor, ME) according to our previously published protocols for acute drug evaluation studies in the TMEV model of infection induced seizures, with a viral titer of 2.5 x 105 PFU,^{45, 71} and a slight modification to include a clinically-relevant dosing paradigm of ASD treatment after the viral infection has occurred (e.g. days 3–7 post-infection). A) The average (Avg) seizure burden of mice with seizures observed during the total 5-day observation period (days 3–7 post-infection) for mice treated with LEV was not significantly different from mice treated with vehicle (VEH; 0.5% methylcellulose (MC); t=0.219, p>0.8). B) However, the cumulative seizure burden of mice treated with LEV was significantly worsened relative to VEH-treated mice (main effect of treatment, F_(1,11)=8.84, p=0.021). C) Finally, the sum seizure burden for each observation session was also significantly worsened in mice treated with once-daily LEV (main effect of treatment, F_(1,11)=12.78, p=0.004). These data, together with our previously published work with anticonvulsant doses of the prototype ASDs VPA and CBZ, illustrate that the TMEV model is a suitable platform for acute evaluations of anticonvulsant efficacy of investigational drugs. Moreover, this model exhibits seizures that are resistant to prototypical ASDs, thereby making it a valid model of pharmacoresistant seizures.

The TMEV model also affords a unique ability to identify compounds that may be disease-modifying or even antiepileptogenic. Results from TMEV-infected mice receiving subchronic, low-dose VPA (100 mg/kg, q.d. i.p.) at levels known to inhibit histone deacetylase (HDAC) activity⁵⁶ were compared to results from TMEV-infected mice that received a low daily dose of the anti-inflammatory agent, minocycline (MIN).⁴⁵ Interestingly, administration of low-dose, once-daily VPA reduced the proportion of mice with seizures, delayed onset of symptomatic seizures, and reduced seizure burden during the acute infection.⁴⁵ In contrast, MIN, did not affect the proportion of mice displaying seizures nor delay onset of acute symptomatic seizures.⁴⁵ Collectively, these results suggest that the TMEV model is a useful platform to differentiate the acute anticonvulsant efficacy of investigational therapies during the encephalitis-induced seizure period.

People with epilepsy often suffer from a number of comorbidities including anxiety, depression and cognitive decline.⁵⁷ The identification of therapies that can modify the expression or reduce the impact of these and other comorbidities would therefore represent an important therapeutic advance. Indeed, a major goal of the National Institute of Neurological Disorders and Stroke Working Group 2015 was to identify therapies that could be disease modifying or antiepileptogenic.⁵⁸ As such, we evaluated the impact of acute treatment with MIN (50 mg/kg, q.d.) and low-dose VPA (100 mg/kg, q.d.) on long-term (e.g. up to 56 days post-infection) behavioral biomarkers of the epileptogenesis process following TMEV infection: open field activity as a measure of anxiety-like behavior, and seizure threshold as a biomarker of epileptogenesis.⁵⁹ Despite marked reductions in the proportion of mice with seizures, VPA-treated mice were not different from VEH-treated mice when long-term anxiety-like behavioral outcomes and seizure threshold were considered.⁴⁵ In contrast, once-daily MIN treatment was associated with improved long-term anxiety-like behavioral outcomes and normalized intravenous pentylenetetrazol seizure threshold. This observation is of particular interest given that acute administration of MIN did not significantly alter acute seizure expression or severity.⁴⁵ Importantly, these results suggest that acute seizure control alone is insufficient to modify chronic disease comorbidities in the TMEV model. As previous clinical trials with prophylactic administration of prototypical ASDs for the prevention of traumatic brain injury-induced epilepsy have demonstrated little to no antiepileptogenic effect,^{60, 61} it was not surprising to find that acute administration of conventional ASDs (VPA low- ⁴⁵ and high-dose⁴³, and CBZ⁴³) was without effect on long-term indices of TLE (e.g. open-field behavior, neuropathology and seizure threshold). Overall, 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 the prevention, and not simply the symptomatic management, of epilepsy.

Ultimately, the TMEV model provides a valid preclinical platform for drug discovery applications in an investigator-controlled setting that aims to mimic human clinical trials and clinical outcomes. Moreover, this model allows for the short- and long-term monitoring of a myriad of behavioral endpoints. For example, treatment with twice-daily MIN caused substantial worsening in the general health of all mice infected with TMEV such that these mice were unavailable for long-term behavioral monitoring.⁴⁵ Furthermore, we have also demonstrated a disease-worsening effect with CBZ,⁴³ as measured by clinical odds ratio analysis, as well as other outcome metrics. This disease worsening effect has also been detected with 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).⁶² From this perspective, the TMEV model can serve as an important platform to identify agents that are acutely anticonvulsant and/or disease modifying, and agents that may be disease-worsening. In this regard, the TMEV model is further validated as a valuable tool in the early screening of novel treatments for infection-induced epilepsy.

The TMEV model is unique among preclinical models of epilepsy in that it is capable of being both a model of pharmacoresistant epilepsy and a model that is sensitive to disease worsening (e.g. increased seizure burden). This is an important consideration for preclinical drug discovery. Indeed, human patients with discrete epilepsy syndromes can exhibit both disease worsening in response to acute ASD treatment as well as adverse side effects following ASD treatment. These two clinical responses are not mutually exclusive. For example, sodium channel-blocking ASDs can exacerbate seizure severity and burden in human patients with Dravet syndrome ⁶³ and these agents are also frequently associated with cognitive adverse effects liabilities in humans patients with temporal lobe epilepsy. ⁶⁴ Furthermore, carbamazepine administration to human patients may exacerbate herpes simplex encephalitis-induced seizures, ⁶⁵ consistent with our findings with carbamazepine in the TMEV mouse model. ⁴³ Pharmacoresistant seizures in preclinical models may be identified as a failure to respond to an acute treatment within a protective index (PI; median behaviorally toxic dose/median effective dose) range of <1–1.5, which is to say that the dose necessary to suppress seizures in that model cannot be achieved without behaviorally-impairing adverse effects (i.e. motor impairment and/or sedation). As an example, Barton and colleagues demonstrated that the 6 Hz 44 mA assay is highly resistant to ASDs at non-behaviorally impairing doses, ⁵⁴ positioning the 6 Hz 44 mA assay as a core component of the NINDS Epilepsy Therapy Development Program’s screening pipeline for pharmacoresistant epilepsy. ⁶⁶ Conversely, worsening of seizure burden would not be quantified using PI, but would instead be defined by longitudinal evaluations in preclinical models with chronic seizures, such as the TMEV mouse model. Indeed, we have previously reported changes in seizure burden in the TMEV model using ASD doses that are otherwise acutely anticonvulsant, ^{43, 45} albeit ineffective in the 6 Hz assay. ⁵⁴ The TMEV mouse model thus represents a model that exhibits acute, handling-induced seizures that may be pharmacologically suppressed with ASDs and investigational agents, but at doses that could induce motor impairment, e.g. pharmacoresistant seizures. Additionally, seizure burden can be longitudinally tracked in this model to identify compounds that could worsen acute and chronic disease course. Further evaluations with the TMEV model are thus necessary to define the median effective doses of ASDs against the acute, infection-induced seizures versus the long-term effect of such treatment on seizure burden and chronic disease. Subsequent studies may also demonstrate whether the pharmacology of acute symptomatic (early) seizures associated with viral encephalitis is predictive for drug effects on difficult-to-treat spontaneous recurrent (late) seizures.

Recently, the finding that infection of C57BL/6 mice with TMEV induces acute symptomatic seizures and epilepsy with hippocampal damage (reminiscent of mesial TLE) has been replicated by W. Löscher’s group.⁶⁷ The latter group characterized the TMEV model in two C57BL/6 substrains and seizure-resistant SJL/J mice by using three TMEV (sub)strains (BeAn-1, BeAn-2, DA). The idea behind this approach was to study what is and what is not necessary for development of acute and late seizures after brain infection in mice. Receiver operating characteristic (ROC) curve analysis was used to determine which virus-induced brain alterations are associated with seizure development. In C57BL/6 mice infected with different TMEV virus (sub)strains, the severity of hippocampal neurodegeneration, amount of MAC3-positive microglia/macrophages, and expression of the interferon-inducible antiviral effector ISG15 were almost perfect at discriminating seizing from non-seizing mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. However, intense microglia/macrophage activation and some hippocampal damage were also observed in SJL/J mice. Overall, the TMEV model provides a unique platform to study virus and host factors in ictogenesis and epileptogenesis.

Future directions

Only time will tell whether a model that displays an acute inflammatory response, such as the TMEV model, will successfully identify and differentiate antiepileptogenic vs. antiseizure treatments and advance a new class of disease-modifying therapies for the patient at risk for epilepsy. Importantly, it will be essential to utilize the TMEV model for drug evaluation studies using the most clinically-meaningful and translationally relevant approach. Based on the above-described studies, it reasons that the TMEV model is well-suited to evaluate and differentiate acute anticonvulsant agents. Given that we have demonstrated that the TMEV model lacks response to two or more ASDs, it is likely a valid model of pharmacoresistant epilepsy.^{43, 45} However, the TMEV model may also gain significant utility as a moderate-throughput approach for the evaluation of potential disease-modifying and/or antiepileptogenic therapies. This is because this model is suitable for rapid, long-term evaluations of chronic disease outcomes in large numbers of animals at risk for developing SRS and attendant comorbidities. By using the chronic changes in seizure threshold and behavioral outcomes, we have been able to demonstrate long-term disease modification of acutely administered agents. Indeed, a reduction in the threshold to pentylenetetrazol-induced tonic-clonic seizures is also observed following rescue from cerebral malaria,⁶⁸ suggesting that seizure threshold is a useful biomarker of the disease process. W. Löscher’s group utilizes the i.v. pentylenetetrazol assay as a biomarker of spontaneous recurrent seizures following status epilepticus,⁵⁹ thus further supporting this assay as an appropriate means to clearly demonstrate disease-modifying and/or antiepileptogenic outcomes following TMEV infection. The TMEV model provides a relevant and technically-feasible approach to acute and chronic drug evaluation studies using technically-feasible biomarkers of the epileptogenic process. In this regard, a moderate-throughput screening approach using the TMEV model is both achievable and feasible. The presence of both reactive astro- and microgliosis in the TMEV model,^{42, 43} which is not observed in the corneal kindled mouse model, a model that is frequently utilized in the anti-seizure drug discovery process,⁶⁹ allows for the identification of novel therapeutic mechanisms and approaches that may lead to the identification of histopathological biomarkers of disease progression.

Importantly, the availability of the TMEV model provides an avenue for evaluating the therapeutic potential of investigational approaches that might include mechanistically novel drugs to prevent the often therapy resistant seizures and attendant comorbidities associated with TLE that results from viral encephalitis and potentially other brain insults.

Conclusions

There is an increasing awareness of the urgency to develop new treatments that will fill the gaps in epilepsy care, including antiepileptogenic, disease modifying therapies, and treatments for drug resistant seizures as well as epilepsy syndromes with limited or in need of safer and more effective treatments, such as the epileptic encephalopathies.⁷⁰ The two new quite distinct epilepsy models discussed here represent unique models for studying West syndrome in infants and post-infectious epilepsy in adults, and support the important role of brain inflammation in the pathogenesis of different types of epilepsy and associated comorbidities. They also provide solid examples of the potential for using such emerging chronic models for the investigation of epileptogenesis and drug-resistance as a function of age- and etiology-specific epilepsy syndromes and differentiation of the pathogenic processes involved in epileptogenesis and associated comorbidities. They also offer suitable platforms to test new therapeutics in a clinically relevant manner and against a wide variety of efficacy and tolerability endpoints and outcomes that may eventually translate into clinical trials.³² An emerging pool of anti-inflammatory drugs that is currently being tested in these and other models may offer therefore a hope for the development or re-purposing therapies that could ameliorate and improve the clinical care of people with epilepsy.

Bullet points.

• Rodent models of West syndrome and the Theiler’s murine encephalomyelitis virus may help to understand the pathogenic role of inflammation in epileptogenesis.

• Antiinflammatory treatments improve seizures and comorbidities in these models.