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

Purpose: 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 1H-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). Methods: 1H-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). 1H-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. Key Findings: The analysis of 1H-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 S100b 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. Significance: 1H-MRS is a valuable in vivo technique for determining the extent and temporal profile of glia activation after an epileptogenic injury. S100b 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 1H-MRS analysis for searching epilepsy biomarkers for prognostic, diagnostic, or therapeutic purposes.