Elsevier

Experimental Neurology

Volume 279, May 2016, Pages 57-74
Experimental Neurology

Research Paper
Brain inflammation, neurodegeneration and seizure development following picornavirus infection markedly differ among virus and mouse strains and substrains

https://doi.org/10.1016/j.expneurol.2016.02.011Get rights and content

Highlights

  • Brain infection of C57BL/6 (B6) mice with Theiler's virus (TMEV) induces epilepsy.

  • We examined the response of two B6 substrains to three different TMEV (sub)strains.

  • Receiver operating characteristic (ROC) curve analysis was used for correlations.

  • Early and late seizures were observed with DA and a BeAn substrain of TMEV virus.

  • Markers of hippocampal inflammation and neurodegeneration correlated with ictogenesis.

Abstract

Infections, particularly those caused by viruses, are among the main causes of acquired epilepsy, but the mechanisms causing epileptogenesis are only poorly understood. As a consequence, no treatment exists for preventing epilepsy in patients at risk. Animal models are useful to study epileptogenesis after virus-induced encephalitis and how to interfere with this process, but most viruses that cause encephalitis in rodents are associated with high mortality, so that the processes leading to epilepsy cannot be investigated. Recently, intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 (B6) mice was reported to induce early seizures and epilepsy and it was proposed that the TMEV mouse model represents the first virus infection-driven animal model of epilepsy. In the present study, we characterized this model in two B6 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 B6 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 B6 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.

Introduction

Following viral encephalitis, the risk of seizures, particularly acute symptomatic (“early”) seizures, is increased more than 20% over the risk of seizures in the general population (Getts et al., 2008, Libbey and Fujinami, 2011, Vezzani et al., 2016). In addition, viral encephalitis increases the risk of developing epilepsy with late unprovoked seizures; 4–20% of viral encephalitis survivors go on to develop epilepsy (Getts et al., 2008, Misra et al., 2008). Over 100 different neurotropic viruses cause encephalitis in humans, and of these, several different viruses have been suggested to play a role in the development of seizures and epilepsy (Misra et al., 2008, Singhi, 2011, Vezzani et al., 2016). Viruses from the herpes virus group are prominent among these. In animals, viral encephalitis also leads to seizures; the best known example is canine distemper (Spitzbarth et al., 2012). However, the processes leading from viral encephalitis to early and late seizures are poorly understood. Animal models are useful to study these processes, but most viruses that cause encephalitis in rodents are associated with high mortality, so that the processes leading to epilepsy cannot be investigated (Libbey and Fujinami, 2011).

An exception is Theiler's murine encephalomyelitis virus (TMEV), a non-enveloped, positive-sense, single-stranded RNA virus of the Picornaviridae family and Cardiovirus genus, which is a naturally occurring enteric pathogen of the mouse (Libbey and Fujinami, 2011). Intracerebral infection of SJL/J mice with TMEV results in an acute encephalitis which, due to virus persistence and virus spread, is followed by a chronic inflammatory demyelinating disease predominantly in the spinal cord, so that TMEV-infected SJL/J mice are widely used as an animal model of multiple sclerosis (Drescher and Sosnowska, 2008, Libbey and Fujinami, 2011). In contrast, infection of C57BL/6 (B6) mice with TMEV also causes acute encephalitis but, unlike SJL/J mice, B6 mice can clear the virus during the first weeks following infection, so that this mouse strain is considered resistant to the TMEV-induced demyelinating disease (Libbey and Fujinami, 2011). More recently, the groups of Robert Fujinami and Steve White at the University of Utah have recognized the importance of B6 mice as a model of viral encephalitis-induced early and late seizures. They described that approximately 50–75% of B6 mice (male and female) infected with the Daniel's (DA) strain of TMEV developed acute behavioral (early) seizures (Libbey et al., 2008). Typically, seizures were first observed on day 3 post-infection (pi), the highest seizure activity was on day 6 pi and no seizures were observed after day 10 pi. The development of seizures appeared to be specific for B6 mice as no SJL/J (male and female), FVB/N (male) or BALB/c (male) mice developed seizures after intracerebral infection with the DA strain of TMEV (Libbey et al., 2008). The development of seizures was not specific to the DA strain of TMEV, but other TMEV strains and mutants were also able to induce seizures to various degrees; e.g. 40% of B6 mice exhibited acute seizures after infection with the BeAn 8386 (BeAn) strain (Libbey et al., 2011b).

The relationship between viral infection with acute symptomatic seizures and the subsequent development of epilepsy was examined in this model by studying B6 mice infected with the DA strain of TMEV (Stewart et al., 2010a). Monitoring by using long-term video–EEG at 2, 4 and 7 months pi of the TMEV-infected B6 mice that had experienced acute seizures demonstrated that a significant proportion (65%) of the mice developed spontaneous recurrent epileptic seizures following a latent period in which no behavioral seizures were observed (Stewart et al., 2010a). In addition, those animals with epilepsy had hippocampal damage characterized by neuronal cell loss in the CA1/CA2 pyramidal cell layers and gliosis, reminiscent of hippocampal sclerosis in patients with infection-induced temporal lobe epilepsy (Stewart et al., 2010a). Based on these findings, the TMEV-induced seizure model was proposed to represent the first infection-driven animal model for epilepsy (Libbey and Fujinami, 2011).

The present study had various aims. First, because all previous experiments on the novel TMEV model of infection-induced epilepsy have been performed by the same group of researchers, we wanted to replicate their findings. This was initiated by the fact that we had used the BeAn strain of TMEV in thousands of SJL/J and B6 mice over the last ~ 10 years in studies on mechanisms involved in virus-induced demyelination but never observed any seizures in B6 mice (Ulrich et al., 2006, Ulrich et al., 2008, Jafari et al., 2012, Prajeeth et al., 2014). Thus, we hypothesized that either the substrain of B6 mice or the BeAn substrain used in our experiments may have been responsible for the lack of seizures. This hypothesis was addressed by comparing two B6 and two BeAn substrains, including the mouse and virus substrains used in the original studies of Fujinami and White. In addition, we compared the potency of the BeAn and DA TMEV strains to induce seizures and epilepsy in mice. In all experiments, the severity of inflammation and hippocampal damage was examined and correlated with the development of seizures. Finally, because interleukin (IL)-6 and interferon (IFN)-β production of macrophages is thought to play a crucial role in the development of seizures following TMEV infection (Cusick et al., 2013, Moore et al., 2013), the expression of IFN-inducible antiviral effectors was examined in the hippocampus.

Section snippets

Animals

Overall, 198 mice were used in this study. Three-week-old female SJL/J and B6 mice were purchased from Harlan Laboratories (Eystrup, Germany) and from Charles River (Sulzfeld, Germany) and kept in groups of five to eight animals in isolated ventilated cages (Tecniplast, Hohenpeißenberg, Germany) under controlled environmental conditions (22–24 °C; 50–60% humidity; 12/12 h light/dark cycle) with free access to standard rodent diet (R/M-H; Ssniff Spezialdiäten GmbH, Soest, Germany) and tap water.

We

BeAn-1 TMEV

In all BeAn-1-infected mice, a transient mild increase of the TCS was observed on the first day after surgery in infected compared to mock-infected animals (Fig. 2A, B & C; P < 0.05). In accordance with previous studies (Ulrich et al., 2006, Ulrich et al., 2010), clinical signs related to the chronic phase started to occur in TMEV-infected SJL/J mice at day 42 pi. Clinical signs in affected animals mainly consisted of gait abnormalities and comprised ataxia, reduction of righting reflex, and

Discussion

The present study is the first to confirm and significantly extend the data of Robert Fujinami's and Steve White's groups (cf., Libbey and Fujinami, 2011, Vezzani et al., 2016), substantiating that TMEV infection in B6 mice provides an animal model of infection-induced acute and late seizures with a variety of interesting features. While most experiments of Fujinami, White and colleagues used intracerebral infection of B6J mice with the DA strain of TMEV, we used three (sub)strains of virus and

Acknowledgments

We thank Robert S. Fujinami for providing the BeAn-2 and DA strains of TMEV and helpful discussion and advice during preparation of this study. We also thank H. Steve White and Karen S. Wilcox for discussion on the TMEV model. We thank D. Waschke, B. Buck, K. Schöne, P. Grünig, C. Schütz, G. Bougamra, B. Tegtmeyer, N. Weegh, I. Waltl, C. Brandt, K. Töllner, and P. Hampel for skillful technical assistance. The authors declare no competing financial interests. The study was supported by the

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    Present address: Boehringer Ingelheim Veterinary Research Center, Hannover, Germany.

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