In vivo imaging of glia activation using 1H-magnetic resonance spectroscopy to detect putative biomarkers of tissue epileptogenicity.

  Long-lasting activation of glia occurs in brain during epileptogenesis, which develops after various central nervous system (CNS) injuries. Glia is the cell source of the biosynthesis and release of molecules that play a role in seizure recurrence and may contribute to epileptogenesis, thus representing a putative biomarker of epilepsy development and severity. In this study, we set up an in vivo longitudinal study using (1) H-magnetic resonance spectroscopy (MRS) to measure metabolite content in the rat hippocampus that could reflect the extent and the duration of glia activation. Our aim was to explore if glia activation during epileptogenesis, or in the chronic epileptic phase, can be used as a biomarker of tissue epileptogenicity (i.e., a measure of epilepsy severity).   (1) H-MRS measurements were done in the adult rat hippocampus every 24 h for 7 days after status epilepticus (SE) and in chronic epileptic rats, using a 7 T Bruker Biospec MRI (magnetic resonance imaging)/MRS scanner. We studied changes in metabolite levels that reflect astrocytes (myo-inositol, mIns; glutathione, GSH), microglia/macrophage activation and the associated neuronal cell injury/dysfunction (lactate, Lac; N-acetyl-aspartate, NAA). (1) H-MRS results were validated by post hoc immunohistochemistry using cell-specific markers. Data analysis was done to determine whether correlations exist between the metabolite changes and spontaneous seizure frequency or the extent of neuronal cell loss.   The analysis of (1) H-MRS spectra showed a progressive increase in mIns and GSH levels after SE, which was maintained in epileptic rats. Lac signal transiently increased during epileptogenesis being undetectable in chronic epileptic tissue. NAA levels were chronically reduced from day 2 post-SE. Immunohistochemistry confirmed the activation of microglia and astrocytes and the progressive neuronal cell loss. GSH levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures, whereas S100β levels in epileptic tissue were positively correlated with this outcome measure. A negative correlation was also found between GSH or mIns levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats.   (1) H-MRS is a valuable in vivo technique for determining the extent and temporal profile of glia activation after an epileptogenic injury. S100β levels measured in the epileptic tissue may represent a biomarker of seizure frequency, whereas GSH levels during epileptogenesis could serve as a predictive marker of seizure frequency. Both mIns and GSH levels measured before the onset of spontaneous seizures predict the extent of neuronal cell loss in epileptic tissue. These findings highlight the potential of serial (1) H-MRS analysis for searching epilepsy biomarkers for prognostic, diagnostic, or therapeutic purposes.

Filibian M ，Frasca A ，Maggioni D ，Micotti E ，Vezzani A ，Ravizza T ... -  《-》

In vivo MRS and histochemistry of status epilepticus-induced hippocampal pathology in a juvenile model of temporal lobe epilepsy.

Childhood status epilepticus (SE) initiates an epileptogenic process that leads to spontaneous seizures and hippocampal pathology characterized by neuronal loss, gliosis and an imbalance between excitatory and inhibitory neurotransmission. It remains unclear whether these changes are a cause or consequence of chronic epilepsy. In this study, in vivo MRS was used in a post-SE juvenile rat model of temporal lobe epilepsy (TLE) to establish the temporal evolution of hippocampal injury and neurotransmitter imbalance. SE was induced in P21 rats by injection of lithium and pilocarpine. Four and eight weeks after SE, in vivo (1) H and γ-aminobutyric acid (GABA)-edited MRS of the hippocampus was performed in combination with dedicated ex vivo immunohistochemistry for the interpretation and validation of MRS findings. MRS showed a 12% decrease (p<0.0001) in N-acetylaspartate and a 15% increase (p=0.0226) in choline-containing compound concentrations, indicating neuronal death and gliosis, respectively. These results were confirmed by FluoroJade and vimentin staining. Furthermore, severe and progressive decreases in GABA (-41%, p<0.001) and glutamate (Glu) (-17%, p<0.001) were found. The specific severity of GABAergic cell death was confirmed by parvalbumin immunoreactivity (-68%, p<0.001). Unexpectedly, we found changes in glutamine (Gln), the metabolic precursor of both GABA and Glu. Gln increased at 4 weeks (+36%, p<0.001), but returned to control levels at 8 weeks. This decrease was consistent with the simultaneous decrease in glutamine synthase immunoreactivity (-32%, p=0.037). In vivo MRS showed gliosis and (predominantly GABAergic) neuronal loss. In addition, an increase in Gln was detected, accompanied by a decrease in glutamine synthase immunoreactivity. This may reflect glutamine synthase downregulation in order to normalize Gln levels. These changes occurred before spontaneous recurrent seizures were present but, by creating a pre-epileptic state, may play a role in epileptogenesis. MRS can be applied in a clinical setting and may be used as a noninvasive tool to monitor the development of TLE.

van der Hel WS ，van Eijsden P ，Bos IW ，de Graaf RA ，Behar KL ，van Nieuwenhuizen O ，de Graan PN ，Braun KP ... -  《-》

Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy.

Ravizza T ，Gagliardi B ，Noé F ，Boer K ，Aronica E ，Vezzani A ... -  《-》

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity.

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Frasca A ，Aalbers M ，Frigerio F ，Fiordaliso F ，Salio M ，Gobbi M ，Cagnotto A ，Gardoni F ，Battaglia GS ，Hoogland G ，Di Luca M ，Vezzani A ... -  《-》

Manganese-enhanced MRI reflects seizure outcome in a model for mesial temporal lobe epilepsy.

The neurobiological processes resulting in epilepsy, known as epileptogenesis, are incompletely understood. Manganese-enhanced MRI (MEMRI) can potentially aide in this quest as it provides superior tissue contrast, particularly of the hippocampal subregions. This longitudinal study aims to characterise the changes in the hippocampus of the post kainic acid-induced status epilepticus (KASE) rat model of mesial temporal lobe epilepsy using MEMRI in vivo. Serial acquisition of T(1)-weighted MEMRI images were taken before, 2 days and 6 weeks after KASE (10-30 mg/kg, i.p.) in 14 rats and in 11 control rats, while a second cohort of control (N=6) and epileptic animals (N=10) was imaged at 2 months post KASE only. MnCl(2) (50 mM, 10 μl) was administered in the right lateral ventricle 1 day before scanning. Regions of interest were drawn around the hippocampus and several subregions of the hippocampus (CA1, CA3 and dentate gyrus). Markers of epilepsy such as spontaneous recurrent seizures, hippocampal neuronal loss and mossy fiber sprouting were quantified. A persistent increase in MEMRI signal intensity was found in the hippocampus, CA1 and dentate gyrus in the KASE group compared to the control group (ANOVA P<0.05). The intensity signal in the hippocampus and subregions correlated inversely with the frequency of spontaneous recurrent seizures in the chronic epileptic phase, however there was no relationship observed between histopathological changes such as cell loss and mossy fiber sprouting with seizures. This study demonstrates that MEMRI is able to detect imaging changes in the hippocampus during the course of epileptogenesis relevant for seizure expression. These data strongly indicate a relationship between manganese enhancement and spontaneous seizure outcome, suggesting that MEMRI could provide a preclinical biomarker for the severity of epileptogenesis in vivo in animal models.

Dedeurwaerdere S ，Fang K ，Chow M ，Shen YT ，Noordman I ，van Raay L ，Faggian N ，Porritt M ，Egan GF ，O'Brien TJ ... -  《-》