In this study, we applied serial rs-fMRI and DTI in a rat model of refractory focal neocortical epilepsy to longitudinally characterize functional connectivity, global network configuration and white matter integrity associated with chronic epilepsy.
By acquiring whole brain connectivity data at multiple time points after epilepsy induction, we gained new insights in the temporal profile of interictal network topology. Our main findings are that (a) graph-based network properties γ and λ increase in the interictal state, indicating a more regular brain network configuration; (b) interhemispheric functional connectivity in epileptic brain decreases, whereas intrahemispheric functional connectivity increases in both hemispheres; and (c) concomitantly, structural white matter integrity is disrupted, not restricted to bundles in close vicinity to the epileptogenic focus, but including the main commissural structure, the corpus callosum.
The importance of network organization for seizure spread in epilepsy has been emphasized in multiple modeling studies 
and confirmed with EEG 
, magnetoencephalography (MEG) 
, rs-fMRI 
and DTI 
. In this study we hypothesized that the focal epileptic brain, during seizure-free periods, would have a state of increased susceptibility to seizure generation and spread, which has been proposed to be associated with a more random network organization 
. This hypothesis is largely based on the previously reported shift towards a more ordered network configuration during seizures, as compared to the interictal states in temporal lobe and absence epilepsy syndromes 
. Interictal network topology in cortical focal epilepsy, however, has until now not been directly compared to the healthy control network state. Our study in a neocortical focal epilepsy model demonstrates that the interictal epileptic brain is characterized by a more ordered configuration, with higher γ and λ as compared to the healthy brain. In contrast with previous global network epilepsy studies, we assessed the network topology serially. This provided unique insights in the unknown interictal neuronal network dynamics. Most importantly, the affected network topology recovers within a time span of ten weeks. This recovery coincides with the reduced seizure frequency. This substantial alterations in network topology could be one of the explanations of the conflicting results found in previous cross-sectional studies. We speculate that the increased intrahemispheric functional connectivity is related to local neuronal sprouting instead to distant functional interactions in the interictal brain status. However, future research is required to address this issue using longitudinal (immuno) histopathological experiments with stainings for both myelination and axonal integrity, preferably at multiple time points after focal epilepsy induction.
The longitudinal changes in global network properties closely matched with the patterns of the intrahemispheric functional connectivity. We suppose a relationship between the increase in both γ and intrahemispheric functional connectivity as γ is a measure of the degree to which functional nodes tend to cluster together. The increased γ corroborates with previous seizure-free network findings in patients with absence epilepsy 
and temporal neocortical epilepsy 
, where higher interictal γ was most pronounced in the EEG and MEG delta bands. This increase is in line with the idea that neural disturbances are correlated with changes in functional network organization 
and probably occur in a wide range of epilepsy syndromes. On the other hand, a different temporal lobe epilepsy (TLE) study reported interictal functional networks with lower λ 
. This dissimilarity with our findings may be explained by differences between location in focus (temporal versus primary motor cortex), duration of epilepsy (more than 13 years versus weeks), use of antiepileptic drugs, and differences between network organization in humans and rats.
λ is a measure of the ability to rapidly combine specialized information from distributed brain areas 
. The observed increase in interictal λ in our study was accompanied by a decrease in interhemispheric functional connectivity, which points toward a relationship between these parameters. The largest deviation in both measures was found at day 21, which subsequently normalized towards the latest time point. Although functional network paths represent sequences of statistical associations, making an analogy with the structural network difficult, modeling has shown that functional resting-state networks largely overlap with the underlying structural network 
. The disturbed corpus callosum integrity may be held responsible for the decrease of both λ and interhemispheric connectivity. The potential relationship between increased λ and reduced interhemispheric functional connectivity is also in agreement with a recent study, that reported a striking loss of interhemispheric low-frequency blood oxygenation level-dependent (BOLD) signal correlations after corpus callosotomy, while intrahemispheric networks were preserved 
. However, whether the integrity of the connecting white matter between the two hemispheres is truly related to the decreased interhemispheric functional connectivity needs further study, for example using computational models 
Knowledge of the status of the epileptic brain's structural connections is important as the above described global network alterations could be caused by white matter abnormalities, such as disruption of association fibers that may underlie the presumed long-distance functional connections. Our structural analyses add to the previous DTI epilepsy work by comparing controls to drug-naïve subjects, longitudinally. The temporal pattern of white matter FA changes resembled the temporal change in λ, suggesting a close relationship: widespread abnormalities at day 21 and 49, and recovery at ten weeks. The TBSS results indicated that the corpus callosum was substantially affected, which was confirmed by specific ROI analysis.
The diffuse structural abnormalities found in both hemispheres with DTI are in agreement with previous partial epilepsy DTI studies (for overview see: 
). Possible explanations for both the structural damage and associated changes in functional network organization include synaptic changes, neuronal death or glial cell damage. Synaptic alterations have been observed during the process of secondary epileptogenesis 
, suggesting that the anatomically distant areas undergo a physiological change consequent to neuronal alterations at the primary epileptogenic zone 
In addition, experimental studies have shown that repeated seizures produce neuronal damage and cell death in the hippocampus 
. Despite the lack of histological studies examining the relationship between recurrent seizures and extrahippocampal remote damage, we know from hippocampal studies that axonal demyelination, formation of axonal spines, increase in interstitial fluid volume due to edema, replacement of axons with glial cells, and astrocyte proliferation may all be associated with the damage caused by seizure activity 
Although rodent epilepsy models may differ from human epilepsy, they allow us to study specific pathophysiological mechanisms that are associated with the development and progression of epilepsy in a detailed and controlled manner. The tetanus toxin model is relative mild as compared, for example, to the lithium-pilocarpine 
and kainate 
TLE models, that require a prolonged status epilepticus inducing diffuse damage. The functional and structural changes that we found in the tetanus toxin model are therefore more likely to result from frequent seizure propagation alone, rather than a direct effect of tetanus toxin-induced brain damage. This idea is strengthened by the temporal relationship we found between seizure frequency and the changes in graph properties, functional connectivity and fractional anisotropy.
A limitation of our animal study is the necessity to use anesthesia. Isoflurane anesthesia is known to suppress overall functional connectivity in a dose-dependent manner 
. Although we have demonstrated that low-frequency BOLD fluctuations are largely preserved under light to mild isoflurane anesthesia 
, the correlation of spontaneous BOLD fluctuations during resting-state fMRI acquisition and therefore the strengths of the graph edges and ROI-based functional connectivity may have been lower than under awake conditions.
The TBSS method we used has some disadvantages 
. TBSS allows, similarly to voxel-based morphometry 
, the comparison of whole-brain maps on a group level, but it is more suited for FA analysis as no spatial smoothing is required. Nonetheless, partial volume effects may still exist 
. TBSS may also result in wrong estimates in regions with multiple, crossing fiber populations 
. Another potential drawback of TBSS is the thinning preprocessing step. The thinning procedure causes the statistical analysis to focus on voxels with highest FA only. White matter changes in the lower FA regions of white matter bundles are therefore ignored. These drawbacks may apply to our data as well, although we believe they have a minor impact. The white matter bundles we analyzed do not contain areas with significant crossing fibers. In addition, the major white matter bundles we analyzed in rats are thin structures by nature (i.e., external capsule, corpus callosum, internal capsule). The effect of thinning will therefore be modest.
Unfortunately we were not able to measure EEG simultaneous with MRI acquisition, which is technically challenging and can affect the fMRI quality because of potential artifacts that electrodes would cause on the T2*
-weighted images. This prevents us to rule out any effect of spontaneous seizures on the resting-state fMRI BOLD fluctuations. We do however believe that this effect is unlikely to have happened. We anesthetized the rats during the resting-state fMRI acquisition using isoflurane, which is a potent inhibitor of spontaneous seizures 
. Directly after each MRI session in all animals, we acquired EEG at identical isoflurane levels and did not observe spontaneous clinical or electrographical seizures. Interictal epileptic spikes were rare. Seizures started to occur only when isoflurane anesthesia was stopped. Therefore we are convinced that functional connectivity, as measured with resting-state fMRI under the anesthetic protocol used in this study, reflects interictal functional connectivity and is not related to frequent spontaneous seizures.
Lastly, although the neocortical tetanus toxin rat model is not related to neuronal cell death 
, direct correlations between gray and white matter MRI measures and histologically measured microstructural integrity are needed. In particular a direct correlation between functional and structural MRI parameters and adaptations at the cellular level will be useful in the characterization of the plasticity process that likely plays a role in brain tissue prone to recurrent spontaneous seizures.
Taken together, frequent focal seizures induce global abnormalities of white matter and of functional brain networks, characterized by increased functional network segregation and ipsilateral functional connectivity, decreased interhemispheric functional connectivity, and concomitantly increased shortest path lengths, for which spontaneous recurrent seizures may be held responsible. We speculate that increased global network segregation and decreased integration may contribute to cognitive dysfunction in patients with focal epilepsy.