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. 2014 Jun 25;34(26):8672-84.
doi: 10.1523/JNEUROSCI.4806-13.2014.

A novel, noninvasive, predictive epilepsy biomarker with clinical potential

ManKin Choy  1 , Celine M Dubé  2 , Katelin Patterson  2 , Samuel R Barnes  3 , Pamela Maras  2 , Arlin B Blood  4 , Anton N Hasso  5 , Andre Obenaus  4 , Tallie Z Baram  6
Affiliations

A novel, noninvasive, predictive epilepsy biomarker with clinical potential

ManKin Choy et al. J Neurosci. .

Abstract

A significant proportion of temporal lobe epilepsy (TLE), a common, intractable brain disorder, arises in children with febrile status epilepticus (FSE). Preventative therapy development is hampered by our inability to identify early the FSE individuals who will develop TLE. In a naturalistic rat model of FSE, we used high-magnetic-field MRI and long-term video EEG to seek clinically relevant noninvasive markers of epileptogenesis and found that reduced amygdala T2 relaxation times in high-magnetic-field MRI hours after FSE predicted experimental TLE. Reduced T2 values likely represented paramagnetic susceptibility effects derived from increased unsaturated venous hemoglobin, suggesting augmented oxygen utilization after FSE termination. Indeed, T2 correlated with energy-demanding intracellular translocation of the injury-sensor high-mobility group box 1 (HMGB1), a trigger of inflammatory cascades implicated in epileptogenesis. Use of deoxyhemoglobin-sensitive MRI sequences enabled visualization of the predictive changes on lower-field, clinically relevant scanners. This novel MRI signature delineates the onset and suggests mechanisms of epileptogenesis that follow experimental FSE.

Keywords: MRI; biomarker; epilepsy; febrile seizures; inflammation; predictive.

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Figures

Figure 1.
Figure 1.
Brain T2 is reduced 2 h after FSE. A–C, Representative color-coded quantitative T2 maps 2 h after the end of hyperthermia from a normothermic (A), hyperthermic (B), and FSE (C) rat. Note that the reduced T2 values occur across the entire brain in the FSE rat. D, Regions of interest are highlighted on a rat atlas. E, T2 values time course after FSE and in normothermic 10-d-old control rats. A reduction of T2 values over the experimental period, characteristic of development, were observed in controls. T2 decreased significantly in a time-dependent manner in FSE rats (n = 19) compared with controls (n = 14, F = 4.11, p = 0.035). The largest effect was observed 2 h after FSE (p < 0.001) and a significant effect was also observed at 18 h (p = 0.007). Blue indicates normothermic control (NT-C) rats and red the FSE rats. Statistical significance was determined by repeated-measures ANOVA followed by post hoc analyses with t tests that were adjusted for multiple-comparisons with Bonferroni's correction. F, Seizures, not hyperthermia, led to the most robust T2 reduction. T2 values for the FSE group were significantly lower than those of either normothermic (n = 22, p < 0.001) or hyperthermic (n = 14, p = 0.014) control groups, indicating that brain T2 reductions in FSE rats resulted from hyperthermia-induced seizures and not from hyperthermia. Statistical significance was determined by ANOVA followed by post hoc analysis with Tukey's honestly significant differences (HSD) test. G, A subset of FSE rats had significantly lower brain T2 values compared with the normothermic controls, which raised the possibility that the reduced T2 values might predict epileptogenesis. T2 values in 9 of 19 FSE rats lay more than 2 SDs from the normothermic controls. Dotted line indicates 2 SDs from mean of NT-C rats (n = 22). Circles indicate individual rats; blue circles are NT-C rats and red circles are FSE rats. Data are presented as mean ± SEM. HT-C, Hyperthermic control; BLA, basolateral amygdala; MEA, medial amygdala; MThal, medial thalamus; DH, dorsal hippocampus; VH, ventral hippocampus. *Statistically significant at p < 0.05.
Figure 2.
Figure 2.
Severity and duration of the inciting FSE and the resulting epileptogenesis. Within the time frame of the experimental FSE, neither the duration of hyperthermia nor the duration of the seizures (Ai) or the number of stage 5 seizures during FSE (Aii) predicted which rats progressed on to epilepsy. Statistical significance was determined by independent samples t test; nonepileptic n = 13, epileptic n = 6. Data are presented as mean ± SEM. B, Epilepsy was found in six of 19 rats and representative EEG traces of spontaneous seizures from each of the six epileptic rats are shown (see also Table 1). Areas in the boxes are shown in expanded time scales. C, Representative EEG traces from three epileptic rats with inter-ictal spike trains. These were observed in five epileptic rats. D, Hippocampal pyramidal cells from epileptic rats had reduced dendritic arborization compared with FSE rats not exhibiting spontaneous seizures (NonEpi) and with normothermic controls (NT-C). Shown are representative Golgi-impregnated sections (Di), traces of reconstructed dorsal hippocampus CA3b neurons (Dii), Sholl analysis of apical dendrites (Diii), and total dendritic length (Div). n = 3–4 rats per treatment group. Data are presented as mean ± SEM. NonEpi, nonepileptic; Epi, epileptic.
Figure 3.
Figure 3.
Amygdala T2 values 2 h after FSE distinguish rats that progressed on to epilepsy. A–F, Representative color-coded quantitative T2 maps of a normothermic control, a nonepileptic, and an epileptic FSE rat. Basolateral amygdala is highlighted in the black box. Scale bar indicates 1 mm. T2 values in the basolateral amygdala (A–G) distinguished the epileptic group from the nonepileptic group, indicating that amygdala T2 values were predictive of epilepsy (epileptic group vs normothermic controls p < 0.001, epileptic group vs nonepileptic group p = 0.017). H, Similar results were found for medial amygdala (p < 0.001 and p = 0.043). I, Hippocampal T2 did not distinguish epileptic from nonepileptic groups (dorsal hippocampus p < 0.001, p = 0.71, ventral hippocampus p < 0.001, p = 0.99). J, To exclude general or systemic effects, amygdala T2 values were normalized to total brain values. The normalized values, reflecting local changes in the basolateral amygdala, separated the epileptic rats from nonepileptic and control animals (epileptic vs nonepileptic rats p = 0.02, epileptic vs control p = 0.024; black line signifies the mean of the controls and dotted line the SEM of controls). K, ROCs for limbic structures and brain T2 values from the FSE rats indicated that amygdala and medial thalamus predicted epilepsy (BLA: AUC = 0.91 ± 0.08, p = 0.005, MEA 0.82 ± 0.10, p = 0.028, MThal 0.78 ± 0.11, p = 0.05). L, Neither hippocampal regions nor brain T2 predicted epilepsy any better than chance (dorsal hippocampus: 0.67 ± 0.17, p = 0.25; ventral hippocampus: 0.49 ± 0.14, p = 0.93; brain: 0.59 ± 0.15, p = 0.54). Ideally, a predictive marker will have a high sensitivity (near 1.0; y-axis) and specificity ([1-specificity] near zero; x-axis). Dotted line indicates AUC of 0.5, a reference to chance. Statistical significance was determined by ANOVA followed by post hoc analysis with Tukey's HSD. Data are presented as mean ± SEM. Values were compared among the sides with lower T2 values in all rats and all groups. NT-C, Normothermic control; NonEpi, nonepileptic; Epi, epileptic; BLA, basolateral amygdala; MEA, medial amygdala; MThal, medial thalamus; DH, dorsal hippocampus; VH, ventral hippocampus. Normothermic controls n = 14, nonepileptic group n = 13, epileptic group n = 6. *Statistically significant at p < 0.05.
Figure 4.
Figure 4.
Reduced T2 values after FSE correlate with increased deoxyhemoglobin levels. A, T2 values were likely reduced because of a paramagnetic effect of deoxyhemoglobin and increased levels of unsaturated (deoxy)hemoglobin after FSE. This paramagnetic effect increases as a function of magnetic field strength. Significant correlation of reduced brain (B) and muscle (C) T2 values with increased blood deoxyhemoglobin levels (−0.73, p = 0.001; −0.47, p = 0.057, respectively). Values were compared among the sides with lower T2 values in all rats and all groups. Shown is a scatter plot of individual rats, with blue circles indicating normothermic control rats (n = 7) and red circles the FSE rats (n = 11).
Figure 5.
Figure 5.
Translocation of HMGB1 from the nucleus to the cytoplasm in amygdala neurons correlates with lowered T2 signal on MRI. A, Representative photomicrographs of immunocytochemistry for HMGB1. Top, Identification of basal amygdala. Bottom, Differences in HMGB1 localization between normothermic control (NT-C) and experimental FSE rats. Arrowheads indicate nuclear HMGB1 and arrows indicate cytoplasmic (translocated) HMGB1. B, Quantitative analysis of HMGB1 expression and localization at 3 h after the end of FSE showed that, whereas the number of HMGB1 expressing cells was not influenced by the occurrence of FSE, the cytoplasmic localization of HMGB1 was significantly higher in sections from FSE compared with NT-C subjects (left graph; NT-C n = 3, FSE n = 3). Similar results were obtained when data were represented as a percentage of cells with translocated HMGB1 over total HMGB1-positive cells (right graph). C, Time course of HMGB1 translocation after FSE demonstrates the transient nature of this early inflammatory process: the localization pattern in amygdala sections from FSE rats resembled that of NT-C sections by 8 h after the end of FSE. Blue indicates NT-C, n = 3; magenta is FSE at the time points indicated (n = 3 per time point). D, Significant correlation of reduced amygdala T2 values with increased HMGB1 translocation (NT-C, in blue, n = 3; FSE, in red, n = 9; p = 0.02). Values were compared among the sides with lower T2 values in all rats and all groups. Data are presented as mean ± SEM. Scale bars, 100 μm. *Statistically significant at p < 0.05.
Figure 6.
Figure 6.
Lower-field, more clinically relevant MRI scanners identify the same epilepsy-predictive changes as high-field MRI. A–F, Representative color-coded quantitative T2 map acquired at 11.7 T and T2* map acquired at 4.7 T in a normothermic control and a FSE rat. C–F, Note the decreased T2 at 11.7 T in the basolateral amygdala, with a corresponding reduction in T2* at 4.7 T in the FSE rat compared with the normothermic control rat. Significant relationships between the T2 and T2* were observed in amygdala and thalamus, regions predictive of epileptogenesis. These data indicate that either method suffices to identify rats destined to become epileptic. G, Basolateral amygdala (n = 11, r = 0.751, p = 0.008). H, Medial thalamus (n = 15, r = 0.846, p < 0.001). Shown is a scatter plot of individual rats. Blue circles indicate normothermic control rats and red circles the FSE rats. Values were compared among the sides with lower T2 values in all rats and all groups. Scale bar, 1 mm.
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