Synchrotron radiation Fourier-transform infrared and Raman microspectroscopy study showing an increased frequency of creatine inclusions in the rat hippocampal formation following pilocarpine-induced seizures

Abstract

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm−1 in IR spectra and around 1056, 908 and 834 cm−1 in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5488-z) contains supplementary material, which is available to authorized users.

FTIR Imaging of Brain Tissue Reveals Crystalline Creatine Deposits Are an ex Vivo Marker of Localized Ischemia during Murine Cerebral Malaria: General Implications for Disease Neurochemistry

“…6−8, 16 In the results reported herein, the creatine deposits were observed only in the molecular and granular layers of the cerebellum, and no deposits were observed within the inner white matter, which is consistent with the distribution of cellular creatine receptors and, therefore, the likely distribution of creatine. 44 A representative example of images of a crystalline creatine microdeposit observed at the boundary of the granular and molecular tissue layers of a cerebellar tissue section from a control mouse is presented in Figure 1.…”

“…5−8, 16 Indeed, in the results reported herein and in the previous work of others, the deposits were observed in the tissue region most affected by the disease (i.e., cerebellum during CM, hippocampus during epilepsy and AD, and brain stem during ALS).…”

“…6−8, 16 The creatine deposits reported herein did not show any correlation to cellular morphology across the molecular or granular layers, as observed by examination of the optical visible light image, FTIR functional group images, K-means cluster images, or histological (H&E) optical images. However, due to the significant difference in cellular layout and density within the molecular and granular layers of the cerebellum, a difference in the morphology of the deposits would be expected if they were intracellular.…”

“…6−8, 16 As the deposits have now been observed in samples collected from four different neurodegenerative diseases, and also in healthy control tissue for each of these studies, it is clear that creatine deposits are not unique markers of a disease and it would seem unlikely that a specific chemical pathway is responsible for the formation of the deposits. A more likely cause of the deposits is a generic biochemical event, common to neurodegeneration.…”

“…5,8,16 Likewise, studies by the same authors have verified the existence of similar deposits postmortem in the brain tissue of human patients who suffered from Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS). 6,7 In these studies, it was hypothesized that the observations are disease specific and may reflect an increase in the Cr/Cr P ratio due to a decrease in tricarboxylic acid (TCA) cycle activity and decreased oxidative metabolism, or a decrease in creatine kinase function during epilepsy, AD, and ALS.…”