Differences in the hippocampal frequency of creatine inclusions between the acute and latent phases of pilocarpine model defined using synchrotron radiation-based FTIR microspectroscopy

Abstract

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. Of the animal models developed to investigate the pathogenesis of TLE, the one with pilocarpine-induced seizures is most often used. After pilocarpine administration in animals, three distinct periods—acute, latent, and chronic—can be distinguished according to their behavior. The present paper is the continuation of our previous study which has shown an increased occurrence of creatine inclusions in rat hippocampal formations from the acute phase of pilocarpine-induced status epilepticus (SE) and positive correlation between their quantity and the total time of seizure activity within the observation period. In this paper, we tried to verify if anomalies in hippocampal creatine accumulation were the temporary or permanent effect of pilocarpine-evoked seizures. To realize this purpose, male Wistar rats in the latent phase (3 days after pilocarpine administration) were examined. The results obtained for the period when stabilization of animal behavior and EEG occurs were afterwards compared with ones obtained for the acute phase of pilocarpine-induced SE and for naive controls. To investigate the frequency of creatine inclusions within the hippocampal formation as well as in its selected areas (sectors 1–3 of Ammon’s horn (CA1–CA3), dentate gyrus (DG), and hilus of DG) and cellular layers (pyramidal, molecular, multiform, and granular cell layers), synchrotron radiation-based Fourier-transform infrared microspectroscopy was used. The applied technique, being a combination of light microscopy and infrared spectroscopy, allowed us to localize microscopic details in the analyzed samples and provided information concerning their chemical composition. Moreover, the use of a synchrotron source of IR radiation allowed us to carry out the research at the diffraction-limited spatial resolution which, because of the typical size of creatine inclusions (from a few to dozens of micrometers), was necessary for our study. The comparison of epileptic animals in the latent phase with controls showed statistically significant increase in the number of creatine inclusions for most of the analyzed hippocampal regions, all examined cellular layers, as well as the whole hippocampal formation. Moreover, for the hilus of the DG and CA3 area, the number of creatine deposits was higher in the latent than in the acute phase after pilocarpine injection. In light of the obtained results, an anomaly in the hippocampal accumulation of creatine is the long-term effect of pilocarpine-evoked seizures, and the intensity of this phenomenon may increase with time passing from the primary injury.FigureThe comparison of baseline corrected IR spectra recorded in selected creatine inclusion and nervous tissue (A). The microscopic view of the DG hippocampal area from the animal representing the SE72H group; creatine deposits are visible as darker points (B). The distributions of selected creatine bands: 2,800 cm−1 (C), 1,398 cm−1 (D), and 1,304 cm−1 (E) in the rectangular tissue ...

The biochemical changes in hippocampal formation occurring in normal and seizure experiencing rats as a result of a ketogenic diet

“…14,18,20 The anomalies included conformational changes of proteins (increased ratio of absorbances at 1634 and 1658 cm −1 ), modifications of the unsaturation level (3012 cm −1 /2924 cm −1 and 3012 cm −1 /2800-3000 cm −1 ) of lipids, as well as changes in accumulation of biomolecules which were detected as decreased intensity of bands occurring at 1740, 1240 and 1080 cm −1 . 14,18,20 The anomalies included conformational changes of proteins (increased ratio of absorbances at 1634 and 1658 cm −1 ), modifications of the unsaturation level (3012 cm −1 /2924 cm −1 and 3012 cm −1 /2800-3000 cm −1 ) of lipids, as well as changes in accumulation of biomolecules which were detected as decreased intensity of bands occurring at 1740, 1240 and 1080 cm −1 .…”

“…12, ketogenic diet fed normal animals (K) showed a higher frequency of creatine deposits in the CA3 hippocampal area compared to rats fed with a stan- Fig. 18,20 Animals representing KSE and NSE groups did not show any significant differences with respect to creatine accumulation. The cellular layers for which statistically significant changes were observed at the confidence level of 95% are marked with (*) whilst those significant at the confidence level of 90-95% with (#).…”

“…18,20 Therefore, we decided to verify how the ketogenic diet modifies the frequency of creatine inclusions within examined hippocampal areas. 18,20 Therefore, we decided to verify how the ketogenic diet modifies the frequency of creatine inclusions within examined hippocampal areas.…”

“…See DOI: 10 alternative energy substrates to glucose. [13][14][15][16][17][18][19][20] Our earlier research showed, inter alia, that excitotoxicity, mossy fibers sprouting, iron catalyzed oxidative stress and decreased enzymatic activity of creatine kinase should be taken into account as important phenomena involved in neurodegenerative changes of hippocampal formation and spontaneous seizure activity in the chronic phase of the pilocarpine model of seizures. 12 In this paper, the mechanisms of ketogenic diet action in epileptic hippocampal formation were examined.…”

The influence of the ketogenic diet on the elemental and biochemical compositions of the hippocampal formation

“…For the analysis of distribution and accumulation of selected elements within the hippocampal formation, X-ray fluorescence microscopy (XRFM) was used, while biochemical evaluation of particular hippocampal areas was done by applying synchrotron Fourier transform infrared microspectroscopy (FTIRM). Both techniques have been successfully applied in our previous studies concerning the mechanisms leading to epilepsy which were carried out mostly on the pilocarpine model of seizures [22][23][24][25][26][27][28][29]. Examining seizure-induced elemental changes of the hippocampal formation, we were able to follow the processes of excitotoxicity and mossy fiber sprouting occurring in this brain area [18][19][20].…”