Herein we report that a proportion of rats given LFPI display evidence of PTE at 6 months postinjury. Rats were divided into epileptic and nonepileptic groups in order to investigate whether structural, functional, and behavioral changes were predictive of PTE. HDM-LD analysis based on T2-weighted MRI revealed that the epileptic and nonepileptic rats displayed minor but significant differences with regard to changes in the ipsilateral hippocampus at 1-week postinjury relative to baseline, with epileptic rats showing an increased thickness in the lateral region and nonepileptic rats showing thinning in the medial-ventral region. In addition, a multivariate logistic regression model including the serial 18F-FDG-PET parameters from the ipsilateral hippocampus was able to significantly predict the epileptic outcome in all of the LFPI cases. With the exception of these findings, the detailed serial MRI, PET, and behavioral analyses for 6 months postinjury found no other differences between epileptic and nonepileptic rats.
The validity and limitations of LFPI as an animal model of PTE
Previous studies have reported inconsistent findings regarding the use of LFPI as an animal model of PTE. Although D'Ambrosio et al. (2004, 2005) report PTE in 92–100% of rats in chronic stages after severe LFPI, Kharatishvili et al. (2006) report incidence rates of 43–50%. The variability in these findings is likely a result of key methodologic differences (i.e., age and injury parameters), as well as the differences in the definition of what is a seizure between these studies. The determination of what is a seizure in rodent-acquired epilepsy models is a highly controversial area, with no generally accepted criteria (D'Ambrosio & Miller, 2010; Dudek & Bertram, 2010). In this study we have used a rigorous definition that had been used in previous publications on experimental posttraumatic epilepsy in the LFPI model by Kharatishvili and colleagues (Kharatishvili et al., 2006, 2007) and subsequently applied in our previous work (Bouilleret et al., 2009, 2011). The determination was done by two independent experienced reviewers who were blinded to the experimental groups, with 44 of 45 of the seizures identified by both of our blinded reviewers and no seizures identified in sham-injury animals. Therefore, it is unlikely that the identified events are normal oscillations or electrical noise or genetically determined absence seizures. Because our methods and definition of PTE closely align with those of Kharatishvili et al. (2006), it is reassuring that our finding that 30–52% of rats given severe LFPI displayed either spontaneous seizures and/or epileptic discharges are similar to those reported by this group (Kharatishvili et al., 2006). This is also consistent with rates of PTE in patients following TBI (Englander et al., 2003; Frey, 2003; Christensen et al., 2009), and supports the use of LFPI in rats to model and study PTE. However, it is important to acknowledge that some workers in the field would question the relevance of brief seizures, even when lasting longer than 5 s, recorded in rodents postexperimental TBI to human posttraumatic epilepsy (Dudek & Bertram, 2010). The median duration of seizures recorded in this study was 14.6 s, with a range of 6–676 s.
There are also limitations with the current study related to the LFPI–PTE model that must be considered when interpreting the present results. Here we conducted 2 weeks of continuous video-EEG recordings at 6 months post-LFPI to detect epileptic rats. This was done because previous studies had demonstrated that the majority of animals given LFPI who develop PTE do so by 6 months (Kharatishvili et al., 2006), 2 weeks of continuous video-EEG recording was the most detailed analysis at 6 months post-LFPI previously reported (Kharatishvili et al., 2006), and compatibility issues associated with the EEG-recording electrodes and MRI scans meant that the EEG electrodes had to be implanted following completion of the serial MRI acquisitions. However, we acknowledge that post-LFPI epileptic animals can experience prolonged seizure-free intervals, and initial seizures may take >6 months to occur (Kharatishvili et al., 2006). Therefore, we cannot exclude the possibility that our “nonepileptic” group included epileptic animals that experienced seizures outside of the monitoring period, or may have gone on to develop seizures had the study continued to a later time point. This may have contributed to the lack of differences between epileptic and nonepileptic animals on many of the measures, and, as such, the remaining discussion should be interpreted in light of this concern. Furthermore, it is important for future studies investigating PTE to consider these limitations and incorporate the most detailed seizure analysis that is feasible.
Neuroimaging and behavioral predictors of PTE
Here, using advanced analysis of MRI and PET parameters, we were able to identify subtle changes that may be predictive of PTE after LFPI in the rat. Specifically, MRI-based HDM-LD hippocampal morphometry analysis identified significant surface changes in the ipsilateral hippocampus that differed between epileptic and nonepileptic rats, and a multivariate logistic regression model that incorporated the serial PET parameters from the ipsilateral hippocampus at 1 week and 1 and 3 months postinjury was able to significantly predict the epileptic outcome in each of the cases. Although these subtle findings reached statistical significance, we interpret them cautiously given that no other measures in the study comparing the epileptic and nonepileptic groups identified differences. Nonetheless, the strength of HDM-LD and multivariate logistic regression analyses is their ability to detect subtle findings (Csernansky et al., 1998; Hogan et al., 2009). Therefore, the differences reported herein may indicate hippocampal abnormalities that occur at various time points post-TBI that are involved in PTE. Indeed, numerous other LFPI studies have implicated hippocampal abnormalities with PTE (Pitkänen et al., 2009), and some suggest that hippocampal changes identified by MRI-based analyses could serve as a surrogate marker for PTE (Kharatishvili et al., 2007). Furthermore, other studies have found that 18F-FDG-PET hypometabolism in the hippocampus, similar to that observed in the epileptic rats here, may be associated with epileptogenesis (Jupp et al., 2012). However, whether the findings reported herein are truly related to the epileptogenic process will require larger and more detailed studies, perhaps focusing histopathologic and biochemical examinations on the regions that showed significant HDM-LD changes, as well as more temporally sensitive PET imaging and EEG analysis.
Despite the use of serial T2-weighted MRI and 18F-FDG-PET imaging to assess structural and functional changes for 6 months post-LFPI, we found no other significant differences between epileptic and nonepileptic rats. The lack of major structural changes associated with PTE reported herein is consistent with previous reports that cortical damage assessed with serial T2-weighted MRI was not related to seizure susceptibility in rats given LFPI (Kharatishvili et al., 2007). Taken together with the lack of functional differences detected by the serial 18F-FDG-PET, the ROI techniques used here were unable to detect any major structural and metabolic changes post-TBI predictive of PTE, limiting their use as predictive tools and suggest that PTE occurs independent of the major structural and functional changes induced by TBI. However, although the ROI techniques used here may lack the capability (i.e., spatial resolution) necessary to solely detect more subtle abnormalities involved in PTE, other neuroimaging modalities, such as diffusion or functional MRI, may be more useful (Kharatishvili et al., 2007).
The behavioral measures also failed to detect significant changes related to epileptic status, indicating that these are not reliable indicators of PTE. However, it is important to note that although no differences were found between epileptic and nonepileptic rats, as a whole the rats given LFPI did display significant imaging and behavioral abnormalities relative to sham-injured rats (see Jones et al., 2008a; Liu et al., 2010; Figs. 4-6). Therefore, although behavioral and imaging abnormalities would be predicted to occur in epilepsy (Golarai et al., 2001; Jones et al., 2008b; Pitkänen et al., 2011), similar changes induced by TBI may have confounded the sensitivity of our measures. Furthermore, the behavioral testing may have occurred at time-points not sensitive to changes related to PTE, and the behavioral tasks were not comprehensive to the entire spectrum of behaviors potentially associated with PTE. For example, the water maze task used in this study was specific to spatial learning and memory and failed to identify significant group differences, whereas other cognitive tasks (Saucier et al., 2008), or variations of the water maze (Shultz et al., 2009), could be used to assess whether other learning and memory abnormalities may serve as PTE biomarkers. Therefore, future studies that employ more detailed techniques may be able to detect slight differences between epileptic and nonepileptic rats post-TBI that were not observed here.
In conclusion, here we examined rats given LFPI using serial MRI, PET, and behavioral assessments for 6 months postinjury. Based on video-EEG monitoring, rats were identified as either epileptic or nonepileptic, and comparisons were made to examine structural, functional, and behavioral changes related to PTE. A subtle difference was identified between epileptic and nonepileptic rats in hippocampal morphometry, and a multivariate logistic regression model that included the serial PET parameters from the ipsilateral hippocampus was able to accurately predict epileptic outcome. Although these findings provide cautious optimism and might direct future studies, unfortunately no other readily identifiable differences between the epileptic and nonepileptic rats were detected. Taken together, these findings indicate that PTE may not be related to major structural, functional, and behavioral changes, and suggest that other more subtle changes or mechanisms may be involved.