Probability of developing PTE
Video-ECoG monitoring based on montage A was performed in 24 rpFPI and 20 sham animals for a total of 1,882 and 784 hours, respectively. Montage A allowed for the detection of three different types of late posttraumatic seizures. Grade 1 and 2 seizures were invariably first detected by the perilesional electrode () while grade 3 seizures appeared, at the best of our spatial-temporal resolution, bilateral in their cortical onset (). The cumulative probability that rpFPI rats developed epilepsy reached 100% at 9 weeks post-injury (). Age-matched shams were recorded at the same time points after surgery for a comparable number of hours and showed no epileptiform ECoG events up to 5.5 months of age (4.5 months post-surgery), consistent with our previous report (D'Ambrosio et al., 2003
). In this study we extended the temporal analysis and found that ~33% of control animals manifested recurrent non-convulsive idiopathic generalized epilepsy at 7−8 months of age. These events, typically 2−10 second long and bilateral in onset, were characterized by a sharp-wave pattern. They were easily distinguished from posttraumatic grade 3 seizures because they were significantly larger in amplitude in the parietal-occipital cortex (), and were therefore disregarded. At 27−28 weeks post-injury these idiopathic seizures represented just 3.6% of all cortical discharges.
Probability of unprovoked seizures following severe lateral FPI
The cumulative probability that FPI rats developed each grade of posttraumatic seizures was also computed (). The probability of developing grade 1 seizures increased over time post-injury in a manner identical to the time course of the epileptic condition itself (), and could be well fit with a single exponential with half time τ1=1.1 weeks. The probability of developing grade 2 or 3 seizures was lower at all times and fit a single exponential with half time τ2=3.0 and τ3=2.8 weeks, respectively. This suggests different mechanisms mediate the genesis of grade 1 vs grade 2 and 3 seizures.
Remission from PTE
During these ECoG studies based on montage A, we identified an animal that presented 6 grade 1 seizures 13 days after injury, but no abnormal ECoG activity thereafter. We considered this animal a case of remission from PTE; it was examined for pathology but was not included in the studies of seizure frequency and duration.
Temporal evolution of electrical and behavioral seizures
We previously determined that grade 2 seizures increase in proportion over the first 2 months post-FPI, while grade 3 seizures do not (D'Ambrosio et al., 2003
). To determine if FPI-induced PTE continues evolving at later time points after injury, we examined the time-dependence of 1) the proportion of each seizure grade, and 2) the behavioral seizure severity. The first electrophysiological parameter was examined in 8 epileptic animals that were recorded weekly from 2 to 28 weeks post-injury (). At 2−3 weeks postinjury, grade 1 seizures accounted for 91±4.4% of all seizures. This proportion progressively decreased over time, reaching 7.8±6.1% at 27−28 weeks postinjury (p<<0.001). At 2−3 weeks postinjury grade 2 seizures were only 8.3±5.4% and increased over time peaking at 36±9.1%% at 14−15 weeks postinjury (p=0.026). Grade 3 seizures were also rare at 2−3 weeks postinjury, being only 5.3±3.1%, and increased over time to 54±11% and 70±13%, at 14−15 and 27−28 weeks postinjury, respectively (p<<0.001 vs
2−3wks; statistics with paired t-test).
Temporal evolution of the FPI-induced posttraumatic epileptic syndrome
Ictal behavior was examined in the same 8 animals. The most common behavioral correlates of electrical seizures during the early weeks post-FPI were stereotyped freeze-like pauses, that were sometimes followed by facial automatisms, and during which the animal did not lose body posture. However, a different behavioral manifestation of electrical seizures became increasingly common over time post-injury. The animal would suddenly interrupt its normal exploratory or grooming behavior, crawl on the bottom of the cage and stop with its head propped on its forelimbs, staying motionless for 1 to 10 seconds, after which grade 3 electrical seizures and sometimes facial automatisms or dystonic posturing of the left hindlimb would appear. We interpret this electro-clinical syndrome as being a complex partial seizure not initiating in the frontal-parietal neocortex and starting during the phase of crawling. We also observed rarer cases of ictal atonia, during which an animal engaged in normal grooming behavior would suddenly fall head-down to the bottom of the cage, remain motionless for several seconds, and after which righting would be hindered by prolonged atonia of the forelimbs. These events were also consistent with partial seizures. At 7 months post-injury three animals exhibited prolonged (≥30 minutes) sequences of electrical seizures during abnormally quiet behavior and without ECoG sleep activity. These events were similar to human non-convulsive partial status epilepticus. The behavioral seizure severity associated with electrical seizures was 1.1±0.2 (range 0−4) at 2−3 weeks post-injury. It increased to 3.1±0.7 (range 1−6) at 17−18 weeks post-injury (p=0.04) and to 4.1±1.0 (range 1−8) at 27−28 weeks post-injury (p=0.02; with Mann-Whitney U test vs 2−3 weeks).
Localization of epileptic foci initiating different seizure types
We previously proposed that grade 1 and grade 2 seizures originated from the frontal-parietal neocortex, while a second epileptic focus was responsible for grade 3 seizures (D'Ambrosio et al., 2003
). To better determine the location of this second focus we acquired 336 hours of paired epidural and depth-electrode video-recordings using montages B and C in 6 animals at 2−4 weeks post-FPI (192hr) and in another 6 animals 6.5−7 months post-FPI (144hr). We classified all epileptic events by the location of the first detected epileptic activity. In the late group (), we observed discharge of the frontal-parietal neocortex in absence of abnormal hippocampal activity (), and of the hippocampus in absence of abnormal neocortical activity (), demonstrating that independent neocortical and hippocampal epileptic foci co-exist in the post-FPI epileptic brain chronically after injury. In addition, grade 1 and 2 seizures, always first detected in the frontal-parietal cortex, sometimes recruited the hippocampal focus (). Grade 3 seizures were always detected in the presence of either leading () or simultaneous hippocampal discharge (not shown), but were never observed to precede it, indicating that grade 3 seizures are never initiated by the frontal-parietal focus, but often by the hippocampus. Epileptiform activity detected in the hippocampus was mostly first detected in the anterior hippocampus (), with or without trailing discharge of posterior hippocampus ().
Independent firing and interaction of the frontal-parietal neocortical and hippocampal epileptic foci as observed by paired epidural and depth-electrode recordings 6.5−7 months post-FPI.
Independent firing and crosstalk of the frontal-parietal neocortical and antero-hippocampal epileptic foci
Temporal changes in cortical discharge rate and duration
We examined the time course of the rate and duration of cortical discharge as detected by montage A. The total cortical discharge rate, as computed as the count, in epileptic animals, of all seizure grades was 2.1±0.51 event/hour (9 rats; range 0.05−4.4 events/hour) at 2−4 weeks and progressively increased to 6.0±1.8 (8 rats; range 0.22−16.4 events/hour) by 27−28 weeks post-injury (p=0.044; ). The rate of grade 1 seizures decreased from 1.79±0.49 events/hour at 2−4 weeks to 0.22±0.1 events/hour at 17−18 weeks post-injury (p=0.008), and persisted at 0.20±0.095 events/hour at 27−28 weeks post-injury (p=0.018; ). The rate of grade 2 seizures rose from 0.24±0.10 events/hour at 2−4 weeks to 1.84±0.78 events/hour at 17−18 weeks (p=0.015) and 2.4±1.29 events/hour at 27−28 weeks post-injury (p=0.12; ). Because grade 1 and 2 electrical seizures appear to originate from the same neocortical epileptic focus, we also evaluated their combined rate as an assessment of the overall activity of that neocortical focus. The combined frontal-parietal seizure rate was 2.04±0.49 events/hour at 2−4 weeks and did not significantly change for the duration of the study, being 2.05±0.86 events/hour at 17−18 weeks (p=0.68) and 2.6±1.53 events/hour at 27−28 weeks post-injury (p=0.24; ). Conversely, the rate of grade 3 seizure progressively increased from 0.06±0.027 events/hour at 2−4 weeks to 3.7±0.76 at 27−28 weeks post-injury (p=0.017; ; statistics with Wilcoxon Signed Rank test vs 2−4 weeks).
Temporal evolution of rate of occurrence and duration of cortical and hippocampal discharge
The duration of all cortical discharges combined was 4.8±0.30 seconds (range 0.5 to 57s) at 2−4 weeks post-injury (n= 389), that increased to 8.3±0.43 seconds (range 0.5 to 89s) at 17−18 weeks (n= 470; p<<0.001), and to 11.9±0.78 seconds (1 to 88s) at 27−28 weeks post-injury (n= 280; p<<0.001; ). Grade 1 seizures lasted 4.2±0.26 seconds at 2−4 weeks post-injury (n= 322), and persisted at 2.84±0.50 seconds at 17−18 at weeks (n=29; p=0.11), and at 4.17±0.97 seconds at 27−28 weeks post-injury (n= 9; p=0.42). Grade 2 seizures lasted 8.7±1.3 seconds at 2−4 weeks post-injury (n=55), persisted at 9.13±0.71 seconds at 17−18 at weeks (n=162; p=0.13), and increased to 12.7±1.5 seconds at 27−28 weeks post-injury (n=101; p=0.02; ). The duration of frontal-parietal seizures (grades 1 and 2 combined) was 4.84±0.31 seconds at 2−4 weeks (n=377), and increased to 8.17±0.63 seconds at 17−18 weeks (n=191; p<<0.001), and to 12.0±1.4 seconds at 27−28 weeks post-injury (n= 110; p<<0.001; ). Similarly, grade 3 seizures lasted 2.46±0.44 seconds at 2−4 weeks post-injury (n=12), and increased in duration to 8.43±0.58 seconds at 17−18 weeks (n=279; p<0.001), and to 11.79±0.89 seconds at 27−28 weeks post-injury (n=170; p<0.001; ; all statistics with Mann-Whitney U test vs 2−4 weeks).
Temporal changes in hippocampal discharge rate and duration
We examined the occurrence of hippocampal discharge as detected by montages B and C. The number of animals showing hippocampal seizures, as defined as those in which the hippocampus fired first or alone, was ~33% (2 out of 6) at 2−4 weeks post-FPI, and increased to 100% (6 out of 6) at 26−27 weeks post-injury, demonstrating progressive MTLE. We then examined the temporal changes in rate and duration of hippocampal discharge as detected by 312hr of recording from epileptic animals with montages B and C. The rate of hippocampal seizures was 0.033±0.02 event/hour (5 rats) at 2−4 weeks and increased to 1.24±0.60 event/hour (6 rats) at 26−27 weeks (; p=0.004). Seizures appearing simultaneously in hippocampus and cortex () have undefined origin, but some or all of these may originate from the hippocampus. Their inclusion brought our estimate of hippocampal seizure rate to 0.1±0.05 event/hour at 2−4 weeks and to 2.75±1.18 at 26−27 weeks (; p=0.004, both statistics with Mann-Whitney U test).
The duration of hippocampal discharge during hippocampal seizures was 5.9±1.3 seconds (n=8; range 2 to 10 s) at 2−4 weeks and 6.8±0.36 seconds (n=147; range 2 to 28 s) at 26−27 weeks (; p=0.55). The inclusion of seizures appearing simultaneously in hippocampus and cortex brought our estimate of the duration of hippocampal discharge to 4.1±0.7 seconds (n=20) at 2−4 weeks and to 6.6±0.24 seconds (n=339) at 26−27 weeks (; p<0.001, both statistics with Mann-Whitney U test).
Structural substrates of the progression of posttraumatic epilepsy
We examined time-dependent changes in brain pathology in 21 FPI and 8 sham animals. Coronal sections obtained from the early group
(2−4 weeks post-FPI) and stained for cresyl violet showed remarkable neuronal loss and calcifications in the ipsilateral thalamus. The ipsilateral hippocampus and temporal neocortex presented either no or mild shrinkage without loss of laminar features (not shown). GFAP immuno-reactivity was markedly increased in the ipsilateral thalamus, hippocampus, and frontal-parietal cortex in all FPI animals, while a focus of glial reactivity was apparent in the temporal cortex of epileptic animals (D'Ambrosio et al., 2003
). Coronal sections obtained from the late group
(27−28 weeks post-FPI) and stained for cresyl violet showed remarkable neuronal depletion and calcifications in the thalamus of all FPI animals (). The ipsilateral hippocampus () and temporal neocortex () presented varying degrees of shrinkage among animals, ranging from negligible to pronounced with loss of laminar features (). Atrophic hippocampi were characterized by atrophy of CA1 and CA3 subfields (). Numerous small nuclei, presumably of glial cells, were observed in ipsilateral hippocampus and temporal cortex (, inset). We quantitatively assessed the temporal changes in asymmetry of hippocampus and temporal neocortex. In the early group
, FPI animals, epileptics and non-epileptics, presented either no (~43%) or mild (~57%) hippocampal asymmetry (, left panel), and either no (~43%) or mild (~57%) asymmetry in the temporal cortex (, left panel). In the late group
, FPI animals presented varying degrees of hippocampal asymmetry (, right panel) ranging from negligible (~14%), mild (~36%), moderate (~36%) to pronounced (~14%), as well as different degree of temporal cortex asymmetry (, right panel), ranging from negligible (~21%), mild (~29%), moderate (~29%) to pronounced (~21%). The differences in hippocampal and temporal cortex asymmetry between the early and late time points were statistically significant (p=0.02, and p<0.04 respectively; one-tailed Mann-Whitney U test). The degree of temporal cortex asymmetry correlated with the degree of hippocampal asymmetry (R=+0.76; p<0.005).
Progressive hippocampal and temporal cortex pathology in posttraumatic epileptic rat