INTRODUCTION

Alterations of cellular and neurochemical mediators involved in epileptic seizures can worsen or prolong seizure activity, and may result in excitotoxicity.^1,2 Epileptic seizures might result from an increase in glutamate-mediated excitation or a reduction in gamma-aminobutyric acid (GABA)-mediated inhibition.³ The present study aimed to provide images of epileptic seizure-induced regional glutamate (Glu) contrast maps (asymmetric magnetization transfer ratio [MTR_asym] at 3.0-ppm) and neurochemical changes in the rat brain, using in vivo chemical exchange saturation transfer (GluCEST) imaging and proton magnetic resonance spectroscopy (¹H-MRS).

METHODS

Fifteen male Wistar rats were divided into two groups (control-[CTRL]-group: n = 7; kainate-induced-[KAI]-group: n = 8). Kainic acid was administered intraperitoneally, using a dose of 15 mg/kg, to induce epileptic seizures. All MR imaging assessments were carried out using a Bruker-7T scanner. Imaging and spectroscopic data were acquired at seven time points (every hour). A T2-weighted slice was selected for GluCEST imaging, with parameters as follows: Turbo-RARE-pulse-sequence, TR/TE = 4,200/36.4 ms, echo-spacing = 6.1 ms, RARE-factor = 16, average = 1, field-of-view = 30 × 30 mm², matrix = 96 × 96, slice-thickness = 1 mm, and inter-pulse-delay = 0.01 ms. CEST datasets with 31 frequency offsets (S₀ and -5 ~ +5 ppm @ 0.33-ppm intervals) were acquired with a continuous-wave RF saturation pulse (power/time=5.6-μT/1-s). All Z-spectra and MTR_asym curves were estimated from B₀ and B₁ corrected images. The GluCEST contrast maps were generated by a relative change in percentage units as follows: GluCEST(%) = 100×(S_−ve – S_+ve)/S_−ve. Where S_-ve and S_+ve in the equation are B₀ corrected signals at –3 and +3-ppm from bulk water, respectively. All procedures were performed based on ROI or pixel-by-pixel methods. Three ROIs from the cortex, hippocampus, and thalamus regions were carefully drawn on a control image (S₀ image). After GluCEST imaging, all animals were assessed for ¹H-MRS data. The VOI (2 × 2 × 3 mm³) position was targeted to the hippocampal region. Water-suppressed in vivo ¹H-MRS spectra were obtained using a PRESS-sequence (TR/TE = 5000/16.3 ms, spectral-width = 5,000 Hz, average = 128, and number of data points = 2,048). Raw data acquired in vivo were analyzed using LCModel with a simulated basis-set. Comparisons between CTRL and KAI groups were made using nonparametric ANOVA (Friedman) as well as Wilcoxon signed-rank and Mann-Whitney U-tests. Due to the non-normal distribution of neurobiological variables, relationships between the neurochemical concentrations and GluCEST imaging contrast values were examined using Spearman's rank correlation coefficient.

RESULTS AND DISCUSSION

In the kainic acid-induced model, we observed different signal intensities and Glu contrasts calculated from Z-spectra and MTR_asym curves in the cortex, hippocampus, and thalamus (Fig. 1). According to results comparing CTRL and KAI groups (Fig. 2), Glu signals in the hippocampal region in KAI rats were significantly higher than those in the CTRL rats at 2-3 hours (during the fourth and fifth scans) following injection (all p<0.05). Notably, the Glu signals in post-injection scans gradually increased compared to those in the baseline scan (all p<0.05; cortex: at fourth and sixth scans; hippocampus: at fourth and fifth scans; and thalamus: at fourth scan), and then decreased after the fourth or fifth scans. Our results and previous studies^4-6 suggest that significant enhancement of Glu concentrations or signals may indicate increased release of Glu, resulting in increased electrical irritability, thereby promoting excitotoxicity. In the in vivo ¹H-MRS of the hippocampal region, 15 neurochemical signals were clearly observed (Fig. 3). In the results comparing CTRL and KAI groups (Fig. 4), Glu, myo-inositol (mIns), and glycerophosphocholine plus phosphocholine (GPC+PCh) concentrations in KAI rats were significantly higher than in CTRL rats according to time after injection, in the hippocampal region (Glu: at fifth and sixth scans, all p<0.05; mIns: fourth [p<0.05], fifth [p<0.01], and sixth scans [p<0.05]; and GPC+PCh: fourth [p<0.005] and fifth scans [p<0.05]). Moreover, the Glu and mIns concentrations in post-scan data of KAI rats were significantly higher than in baseline scans (all p<0.05; Glu: at the fifth scan; and mIns: fourth and fifth scans). Based on our results and previous studies, significantly increased mIns and GPC+PCh concentrations may be implicated in intracellular signaling and osmoregulation,^4,7 as well as transient membrane breakdown induced by seizure activity.^8,9

CONCLUSION

The present study demonstrates that in vivo GluCEST imaging and ¹H-MRS data provide remarkable neurobiological information for interpreting time-dependent influences in the hippocampal region in a rat model of KAI epileptic seizures. Our main findings, which exhibited significant alterations in GluCEST contrast signals and neurochemical concentrations in a comparison between CTRL and KAI rats, suggest that the results can be utilized as key markers of epileptic seizure status.

Acknowledgements

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea [NRF-2015R1C1A1A02036526] and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute [HI14C1090], funded by the Ministry of Health & Welfare, Republic of Korea.

References

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