Article Figures & Data
Figures
- Figure 1.
Parameters of experimental FSs lasting 24 or 64 min. A, Duration of seizures on 30 or 70 min of hyperthermia (left y-axis) and threshold temperatures in both paradigms (right y-axis). B, Rats were weighed before and after 30 or 70 min hyperthermia epochs; average weights before and after the seizures differed by <3%. This indicates that the rats did not suffer from dehydration. C, Average core temperatures throughout the 30 or 70 min of hyperthermia fluctuated between 39.5 and 40.8°C. Error bars indicate SEM. *p < 0.05.
- Figure 2.
A 64 min FS increased the severity of the resulting epilepsy compared with a 24 min seizure. A, The percentage of rats developing limbic epilepsy increased by 30% after a 64 min FS compared with a 24 min FS. The duration of FSs affected the duration and the severity of the resulting spontaneous seizures: Mean duration of seizures was significantly longer (136.7 ± 25 s; n = 18) after a 64 min FS compared with a 24 min FS (7.8 ± 0.3 s; n = 57; median durations, 91 vs 7 s). B, C, Typical spontaneous electrographic seizures recorded from hippocampal and cortical bipolar electrodes in adult rats that had sustained 24 (B) or 64 min (C) FS. The arrows point to onset and end of epileptiform discharges. Note the clear development of epileptiform cortical discharges in seizures arising after a 64 min FS, compared with suppression of cortical EEG amplitude observed in seizures that arose after a 24 min FS. Calibration: 1 s. D, Still images from a video of a spontaneous seizure resulting from a 64 min FS: The seizure consisted of alternating and bilateral clonus accompanied by facial automatisms. *p < 0.05. Error bars indicate SEM.
- Figure 3.
Comparison of the interictal activity recorded from hippocampal electrodes in adult rats that sustained either a 24 min or a 64 min FS. A, Examples of interictal epileptiform activity (arrowheads), defined as unilateral or bilateral spikes or sharp-waves series after 64 min FS. These events were never found in control rats. Calibration: 1 s. B, A 64 min FS promoted more discrete epileptiform events per hour of recording compared with a 24 min FS: 0.17 versus 0.9. The mean duration of interictal events after a 64 min FS was drastically longer: 117.5 ± 7.6 s (n = 111 spike series) vs 3.5 ± 0.1 s (n = 136 polyspikes or sharp-waves discharges). C, Average total time (in seconds per hour) spent in interictal activity was not different in rats that developed limbic epilepsy compared with those that did not, after a 24 or a 64 min FS. *p < 0.05. Error bars indicate SEM.
- Figure 4.
Long (64 min) FS induced enduring IL-1β production in hippocampus. A, A representative Western blot. B, Quantitative Western blot analyses of IL-1β protein: OD of IL-1β-immunoreactive bands was normalized to that of actin for each lane and revealed a significant increase 24 h after long FS. IL-1β levels returned to control levels at 48–72 h. *p < 0.05. Error bars indicate SEM.
- Figure 5.
Long (64 min) FS promoted prominent activation of astrocytes and mild activation of microglia within 24 h after the seizures. Left panels, Astrocytosis denoted by the hypertrophic cell body and long and thick processes of the astrocytes was evoked 6 and 24 h after the seizures (C–F and their insets) compared with the control (A, B, and their insets) in CA3 hippocampal area (C, E, vs A) and in the hilus of the dentate gyrus (D, F, vs B). Middle panels, OX-42 immunoreactivity was detected in resting microglia with small cell body and scant ramification in control rats (A′, B′, and their insets). Scattered mildly activated microglial cells (moderate hypertrophic processes) were evident 6 h (C′, D′, and their insets) and 24 h (E′, F′, and their insets) after the onset of FSs. Right panels, IL-1β immunoreactivity was undetectable in control rats (A″, B″) and was robust in glial cells 6 h (C″, D″) and 24 h (E″, F″) after seizures. Double immunostaining (yellow in insets) demonstrated that IL-1β (green) protein expression was localized to astrocytes (GFAP; red). Scale bars: A–F, A′–F′, B″, D″, F″, 25 μm; A″, C″, E″, 50 μm; A–F, insets, A′–F′, 12 μm; C″–F″, insets, 10 μm.
- Figure 6.
Hippocampal IL-1β protein levels were increased in rats that developed epilepsy after a 64 min FS compared with those that did not. A, A representative Western blot showed enhanced levels of IL-1β protein in rats with epilepsy (n = 5) compared with those with interictal activity only (n = 6). B, Quantitative Western blot analyses of IL-1β from CA1 hippocampal area: OD of IL-1β-immunoreactive bands was normalized to that of actin for each lane, and the data were expressed as percentage of control. The analyses demonstrated a significant increase of IL-1β protein levels in rats that developed epilepsy. *p < 0.05. Error bars indicate SEM.
- Figure 7.
Neuronal density and counts in hippocampal areas most affected in limbic epilepsy did not reveal significant cell loss in epileptic and nonepileptic rats that had experienced a 64 min FS early in life. A, The schematic demonstrates the areas of analysis: CA1 and CA3 pyramidal cell layers and the hilus of dentate gyrus. B, D, F, Neuronal densities in hippocampal areas CA1 and CA3 and in the hilus of the dentate gyrus of the epileptic rats did not differ from the densities of the nonepileptic and control rats (n = 9) (one-way ANOVA, CA1, p = 0.93; CA3, p = 0.33; and hilus, p = 0.4). C, E, G, In the three regions examined, numbers of neurons were not significantly different in epileptic, nonepileptic, and control rats (one-way ANOVA, CA1, p = 0.82; CA3, p = 0.83; and hilus, p = 0.96). These data suggested that significant loss of neurons was not a prerequisite for the epileptogenic process that followed prolonged FSs. Error bars indicate SEM.
- Figure 8.
MRI changes did not predict epilepsy. A, Examples of T2-weighted images obtained 1 month after a 64 min FS: increased T2 signal (arrows) in hippocampus of a subset of rats that experienced long FSs (right panels) compared with control rats (left panels). B, T2 intensities were quantified by calculating T2 relaxation time values (in milliseconds). T2 values for the right and left hippocampi of each experimental rat were calculated, and the difference from the mean of control rats was determined. This T2 value difference (Δ) was more robust in the right hippocampus versus the left one, indicating an asymmetry. The plain lines depict rats with increased T2 relaxation time compared with controls. C, Correlation of hippocampal T2 values with the total time spent in interictal activity for individual experimental rats: The highest T2 values in the right hippocampus were observed in rats with maximal times spent in interictal activity (Spearman's r = 0.7; p = 0.04). There was no significant correlation for the left hippocampus. D, Right hippocampal T2 values were significantly higher in three of four epileptic rats and in three of five nonepileptic rats compared with the control rats, but did not differ between epileptic and nonepileptic rats (ANOVA). E, There was no significant correlation between left hippocampal T2 values and IL-1β protein levels (Spearman's r = 0.48; p = 0.19). Error bars indicate SEM. *p < 0.05.
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