Early Seizures

4.1.2.1. Visual observations

While performing the experiments in order to understand the role of the immune system or its modulation in development of early seizures (Cusick et al., 2013; Howe et al., 2012), animals

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were observed visually relatively for a short period of time (2h/day) for 7 – 14 days after infection to confirm the presence of seizures and evaluate the effects of therapeutic modulation on early seizure occurrence (Libbey et al., 2008; Waltl et al., 2018). During visual observations, we noticed early behavioural seizures in 60% of infected animals (Bröer et al., 2016), which is in line with initially reported 50 – 55% animals with early seizures (Libbey et al., 2008; Stewart et al., 2010b). The seizures could see seizures 1 dpi after infection, although they occurred mostly between 3dpi to 7dpi with maximum seizure activity occurring on day 6 post infection (Libbey et al., 2008). This narrow time period of 3-7dpi could be crucial in terms of underlying inflammatory and virus associated pathological responses (Stewart et al., 2010a). During this period, a significant upregulation of chemokines, cytokines (e.g. IL-6 and TNF-α), adhesion molecules in infected C57BL/6 mice has been observed, causing a damage to the blood brain barrier and enhancing the CNS infiltration of peripheral leukocytes (de Vries et al., 1996). We have shown recently in another study that the incidence of early seizures in TMEV-infected mice could be as high as 84% (Waltl et al., 2018). The incidence of early seizures in human patients with encephalitis after CNS infection is between 2% – 67% (Singhi, 2011), while the chances of developing epilepsy are maximum within the 1^st 5 years after encephalitis, and might occur up to 20 years of life (Singhi, 2011).

4.1.2.2. Characterization of EEG recording during early time point

Owing to the short period of visual observation it was hypothesized that probably many early seizures could have been missed, advocating for a continuous vEEG recording (Stewart et al., 2010a). Thus 24/7 vEEG recording with cortical screws revealed that seizure incidence in recorded animals was 77% which is in line with the previously described 75% (Stewart et al., 2010a). Keeping in view the hippocampal sclerosis, the generation of hippocampal focal seizures could not be ruled out, which are difficult to record with cortical screws, so deep electrodes implanted in the dentate gyrus of the dorsal hippocampus showed 68% seizure incidence (Patel et al., 2017). The incidence and frequency of early seizures per mouse during acute phase after infection are higher (14.5 ± 10.5, mean ± SD) as compared to late seizures, 4 months post infection (0.75 ± 0,mean ± SD), (Stewart et al., 2010a). Whereas, in our study the average seizure frequency/week during early phase recording was (5.5 ± 1.7, mean ± SD) significantly higher as compared to seizures (0.998 ± 0.39, mean ± SD) during late phase (16-98 dpi) of recording.

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We could record early seizures in vEEG as early as on 0 dpi few hours after infection, although the maximum seizure frequency (43%) occurred on 4-5 dpi, similar to pervious findings (Stewart et al., 2010a). This suggests that possible pharmacological interventions to counter epileptogenesis shall be initiated as early as possible after the acute insult, since early seizures itself are a risk factor for epilepsy development (Wilson et al., 2018). As expected, a considerable proportion (54.7%) of seizures were focal or partial (Racine scale, I-III), while (45.3%) were generalized seizures (Racine scale, IV-V), with vEEG recording during early phase of recoding (0-7dpi). We could record focal seizures with cortical electrodes, although it is considered difficult, especially when brain regions like hippocampus is believed to be involved in generating seizures (DePaula-Silva et al., 2017; Stewart et al., 2010a). The average length of the focal seizures was much shorter than generalized seizures. Moreover, we observed animals pausing or becoming immobile during video analysis of focal seizures, suggesting that these were different from electrographic seizures where usually no behavioural correlates are observed in videos in another animal model of epilepsy (Twele et al., 2017). However, the depth hippocampal electrode recordings from dentate gyrus (DG) region (Patel et al., 2017), showed that electrographic activity was recorded before appearance of behavioural components in many generalized seizures and continued even after the behavioural seizures subsided. Patel et al., (2017) could not correlate any specific observable behaviours with recorded electrographic activity and classified them as focal (electrographic) seizures. The recording for such focal seizures from depth hippocampal electrodes is suggestive of the fact that there is extensive epileptiform activity and neuronal hyperexcitability taking place in hippocampus post TMEV infection (Patel et al., 2017).

The average duration of seizures also increased as the seizure stage changed, from type – I, 16.41 ± 9.84 sec (mean ±SD) to type-V, 35.88 ± 18.02 sec. The average seizure length recorded was 25.67 ± 15.83 sec, whereas the average seizure length recorded during previously reported studies for focal seizures was 24 ± 13 sec and 50 ± 17 sec for stage IV-V seizures (Stewart et al., 2010a). There has been contradicting results after treatment with anti-seizure drugs during acute phase after infection in this model (Barker-Haliski et al., 2017).

Valproic acid (VPA) administration resulted in a decrease in severity and frequency of early seizures, whereas treatment with Carbamazepine (CBZ) worsened the disease with increased severity and incidence of early seizures (Barker-Haliski et al., 2015). Testing of a number of antiepileptic drugs with varying results suggests the robustness of this model to screen for seizure-reduction effects of pharmacological compounds (Barker-Haliski et al., 2017).

However there are no acute phase studies with vEEG recording extensively discussing the

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quantitative and qualitative aspects of EEG tracing after drug administrations. The last seizure recorded in vEEG was on 8dpi by us as compared to 7dpi by (Stewart et al., 2010a) with cortical electrodes. However, with depth electrode recordings, focal seizures could be recorded in many animals after 8dpi, while the generalized seizures were largely subsided. In our recorded data after 8dpi the animals did not show any seizures for several days before the start of late seizures or epilepsy. In between the early and late seizures occurrence, there is a so called asymptomatic seizure free latent period (Monteyne et al., 1997) of unknown duration, requiring a long term continuous vEEG recording for its determination. Long term vEEG recordings from weeks to months, though laborious, are recommended for rigorous epilepsy studies (Gu & Daltone, 2017).