Journal of Chemical Neuroanatomy 40 (2010) 140–147 Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures J. Chwiej a,*, J. Dulinska a, K. Janeczko b, P. Dumas c, D. Eichert d, J. Dudala a, Z. Setkowicz b a AGH-University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland Jagiellonian University, Institute of Zoology, Department of Neuroanatomy, Krakow, Poland c SOLEIL, St Aubin, France d ELETTRA, Trieste, Italy b A R T I C L E I N F O A B S T R A C T Article history: Received 29 January 2010 Received in revised form 26 March 2010 Accepted 26 March 2010 Available online 1 April 2010 In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10 mm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammon’s horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958 cm 1) as well as conformational changes of proteins (b-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657 cm 1 as well as the ratio of the absorbance at 1548 and 1657 cm 1) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures. ß 2010 Elsevier B.V. All rights reserved. Keywords: Pilocarpine-induced epilepsy SRFTIR micro-spectroscopy and imaging Biochemical analysis Lipid peroxidation Relative secondary structure of proteins 1. Introduction The term epilepsy involves diverse convulsive disorders, which can lead to an interruption of the functions of the central nervous system. They can be characterized by abnormal motor, sensory and physical phenomena. Although the mechanism of epileptic seizures has been a subject of intensive investigations during the past decades, the etiology of the disease is still to be revealed and epilepsy so far is constituting a serious clinical problem (Mello et al., 1993; Ceru et al., 2005). As it is known, the nervous system is extremely vulnerable to the oxidative stress due to its polyunsaturated fatty acids content (Skaper et al., 1999). Omega-3 fatty acids are indeed crucial to the proper development and function of cell membranes in the brain. Seizures induce rapid accumulation of membrane lipid-derived fatty acids at the synapses, what may be an evidence of maladaptative connectivity (Cole-Edwards and Bazan, 2005). * Corresponding author. Fax: +48 12 634 00 10. E-mail address: jchwiej@novell.ftj.agh.edu.pl (J. Chwiej). 0891-0618/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2010.03.008 Moreover, neurodegenerative diseases such as Parkinson’s disease, amyotrophic lateral sclerosis, prion diseases seem to have common cellular and molecular mechanisms including protein aggregation and the formation of inclusions. Although the aggregates consist of different protein (or proteins), all these misfolded proteins demonstrate similarities in their secondary structure. Typically, the aggregates are formed by fibers containing misfolded protein with b-sheet structure, termed amyloid (Ross and Poirier, 2004). An increased expression of neuronal b-Amyloid precursor protein was observed in post-mortem samples from patients with the temporal lobe epilepsy (Sheng et al., 1994). Likewise, the protein aggregation is supposed to be a possible cause of Unverricht-Lundborg disease—the type of genetic epilepsy (Ceru et al., 2005). The following work is the continuation of our research concerning the role of trace metals in the pathogenesis and progress of epileptic seizures. In frame of it the differences in the distributions of main biomolecules, saturation level of phospholipids as well as the relative secondary structure of proteins between epileptic and control rat hippocampus tissues were examined. Pilocarpine was used in order to induce seizures in rats. J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 Since possible changes in the distribution of biomolecules may occur at the level of single cell, it is necessary to use a characterization technique which combines high spatial resolution (micrometer scale) with high biochemical (spectral) sensitivity. SRFTIR micro-spectroscopy fulfills these requirements (Dumas and Miller, 2003; Miller and Smith, 2005) and allows us to monitor the eventual changes in the nucleic acids, lipids (fatty acids) and proteins distributions together with the abnormalities in protein relative secondary structure. The FTIR micro-spectroscopy has been used to study anomalies occurring in the nervous tissue in case of different neurodegenerative disorders for quite a long time. The method was successfully applied for the analysis of protein conformational changes in case of Alzheimer’s disease and prion diseases (Choo et al., 1996; Gallant et al., 2006; Gough et al., 2005; Kneipp et al., 2002; Kretlow et al., 2006). Within the previous studies of our group it was employed in the research under the mechanisms leading to the atrophy and death of neurons in case of Parkinson’s disease and amyotrophic lateral sclerosis (Szczerbowska-Boruchowska et al., 2005; Szczerbowska-Boruchowska et al., 2007; Kastyak et al., 2010). 2. Epilepsy induction and behavioral observations The rats received a single intraperitoneal (i.p.) injection of pilocarpine (300 mg/kg, Sigma P6503) on day 60 of their postnatal development. Scopolamine methyl bromide (1 mg/kg, Sigma S8502) was injected i.p. 30 min prior to pilocarpine in order to reduce its peripheral effects. Pilocarpine was injected between 9 and 10 a.m. to avoid circadian changes in seizure vulnerability. After that, during the 6-h period following the pilocarpine injection, the animals were placed under continuous clinical observation. This monitoring provided additional data on general parameters of the status epilepticus: (1) the latency of the first motor sign, (2) the time when it occurred within the 6-h observation period, and (3) the time when the acute period ended. The experimental procedure has already been applied in our previous studies on the pilocarpine model of epilepsy (Setkowicz and Mazur, 2006; Setkowicz and Ciarach, 2007). 3. Sample preparation In the present study seven epileptic animals and five naive control rats were examined. Six hours after epilepsy induction, all animals were perfused with physiological saline solution of high analytical purity. The brains were excised, frozen in liquid nitrogen and cut using a cryomicrotome. Typically, three slices including the dorsal part of the hippocampal formation were prepared from each brain (Paxinos and Watson, 1989). First slice with the thickness of 141 10 mm was dedicated to the histopathological analysis. The next one of the same thickness was placed on MirrIR low-e microscope slide (Kevley Technologies) and was used in SRFTIR microspectroscopy study. The last 15-mm-thick neighboring slice was deposited on Ultralene foil and subjected mainly to elemental analysis by applying X-ray fluorescence microscopy. The samples dedicated to the biochemical and elemental analysis were then freeze-dried at 70 8C and stored in this temperature till the measurements (typically from one to three months). Two areas of hippocampal formation were analyzed per sample, they were sector 3 of Ammon’s horn and the dentate gyrus. 4. IR data collection The experiments were carried out at the following synchrotron facilities experimental end-stations: SMIS beamline at SOLEIL and at SISSI beamline of ELETTRA. In first case the IR spectra were collected in transreflection mode using an infrared microscope Continuum XL coupled to a FTIR spectrometer ThermoNicolet 5700. The IR microscope was equipped with a motorized sample stage and a 50 mm MCT detector. The 10-mm thick samples deposited on MirrIR slides were probed with the IR beam of 10 mm in diameter. The spectral resolution was set to 6 cm 1 and acquisitions were performed using 32 scans per spectrum. In this case data acquisition and processing were performed using OMNIC software (Version 7.3). At SISSI beamline, a Bruker IFS 66v/S interferometer coupled to Bruker Hyperion 2000 microscope (fully automated microscope with MCT detector) was used for mapping of the hippocampal tissue. The 15-mm thick sections on Ultralene foil were analyzed in transmission mode with a spatial resolution defined by the apertures sizes set to 10 mm by 10 mm. The spectra were collected for the wavenumber range from 1000 to 4000 cm 1 with a spectral resolution of 4 cm 1 and 128 scans were coadded per spectrum. The data collection and analysis was performed with the OPUS software (Version 6.5). 5. Results Analysis were carried out for the areas of hippocampal formation in which significant differences in elemental composition were noticed between epileptic and control groups during our previous studies, i.e. for the sector 3 of Ammon’s horn (CA3) and the dentate gyrus (DG) (Chwiej et al., 2008). In Fig. 1 the location and typical size of the scanned CA3 and DG areas for selected control sample were presented. The analysis of the distribution of main organic compounds in the tissue was achieved from the mapping of functional groups Fig. 1. Visible light microscope picture of the hippocampal formation (unfixed and non-stained tissue). Squares show the size and location of the scanned areas within CA3 and DG regions; mo, mu, g, p—molecular, multiform, granular and pyramidal layers, respectively. 142 J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 Fig. 2. Comparison of the spectra (baseline corrected, normalized to the maximum of amide I band) collected for different cellular layers of the control CA3 (A) and DG (B) areas as well as ones recorded for the pyramidal (C) and granular (D) cell layers from selected epileptic and control samples. (chemical mapping) present in the recorded IR absorption spectra and characteristic for the specific biomolecules. The data treatment was conducted as following: 1. Identification of the absorption bands present in the spectra collected for tissue samples of CA3 and DG areas (see Fig. 2 and Table 1). Table 1 Tentative assignments of the bands frequencies present in the IR absorption spectra measured for tissue samples of the rat hippocampal formation. Frequency (cm 3400–3200 3300 3080 2960 2930 2850 1750–1720 1655 1545 1460 1380 1300 1260–1220 1170 1085 1 ) Assignment OH str N–H str (protein, amide A) N–H str (protein, amide B) CH3 asym str (lipids) CH2 asym str (lipids) CH2 sym str (lipids) C5 5O str (ester, lipids) Amide I: CO str, CN str, NH bend vib (protein) Amide II: NH bend vib, CN str (protein) CH2 sciss vib; CH3 asym bend vib (lipids) CH3 sym bend vib (lipids) Amide III: CN str, NH bend, CO str, O5 5C–N bend vib (protein) PO2 asym str (nucleic aids) CO–O–C asym str (lipid) PO2 sym str (nucleic aids) str: stretching; asym: asymmetric; sym: symmetric; bend: bending; vib: vibration; sciss: scissoring. 2. Chemical mapping of functional groups meaning two-dimensional imaging of the intensities of selected absorption bands. 3. Two-dimensional imaging of the ratios of selected bands intensities. 4. Delineation of the cellular layers in the obtained chemical maps of CA3 and DG areas through their comparison with the microscopic views of the scanned tissue sections. 5. Calculation of the mean bands intensities or mean ratios of intensities for the cellular layers in the analyzed tissue samples of CA3 and DG areas. The mean values were calculated from 25 pixels randomly chosen from an appropriate layer. 6. Comparative analysis of the results obtained for three different layers constituting the examined CA3 and DG areas in different animal populations, epileptic and control. 7. Statistical analysis of the obtained results in order to confirm the significance of the differences between analyzed animal groups. For the comparison of the examine populations U Mann– Whitney test was used. The U Mann–Whitney test is a nonparametric test that can be used in place of an unpaired t-test to test the null hypothesis that two samples come from the same population, i.e. have the same median. It assumes that the two distributions are similar in shape but not necessarily normal and it is typically used when the abundance of population is small. Because of non-normal distribution of our data as well as small abundance of population (seven epileptic and five control cases examined) U Mann–Whitney test was chosen to analyze statistical significance of differences between epileptic and control group. J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 143 Fig. 3. The chemical maps obtained for the tissue sample of CA3 area from the epileptic rat shown in the bottom right corner. For abbreviations see Fig. 1. In Fig. 2 the typical spectra collected for different layers of control CA3 and DG areas as well as ones recorded for pyramidal and granular cells from selected epileptic and control samples were compared. In Table 1 the absorption bands present in the IR spectra of the hippocampal formation were assigned to their corresponding molecular groups. Several differences between spectra from different cellular layers and also between epileptic and control pyramidal or granular cells can be observed in Fig. 2 for the spectral regions in which the absorption by lipids, proteins and nucleic acids occurs. A detailed analysis of these differences was performed by generating specific chemical cartographies of selected functional groups (see Figs. 3–6). Intensities of the following absorption bands and the following ratios of intensities were considered: 1. The absorption bands around 1084 and 1226 cm 1 were used for the analysis of the distribution of nucleic acids. Absorption in the range of wavenumbers between 1000 and 1300 cm 1 may be associated with the presence of compounds carrying phosphate group (P5 5O double bond stretching modes) as well as with distinct ring vibrations of carbohydrates. Because of it, the mentioned region contains information about nucleic acids, carbohydrates, and phosphorylated lipids and proteins in the tissue. Variations in IR absorption bands between 1000 and 1300 cm 1 may indicate changes in the amount of nucleic acids or phospholipids as well as different degrees of phosphorylation of carbohydrates or glycoproteins (Kneipp et al., 2000). Even though a number of absorption bands in this region remain unassigned, the bands around 1240 and 1080 cm 1 are known to contain significant contributions from asymmetric and symmetric stretching modes, respectively, of phosphodiester groups in nucleic acids and are used for mapping of these molecules (Diem et al., 1999; Liquier and Taillandier, 1996; Szczerbowska-Boruchowska et al., 2007). 2. The amide I absorption band intensity was used for 2D imaging of the accumulation of proteins. Fig. 4. The chemical maps obtained for the tissue sample of CA3 area from the control, non-epileptic rat shown in the bottom right corner. For abbreviations see Fig. 1. 144 J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 Fig. 5. The chemical maps obtained for the tissue sample of DG area from the epileptic rat shown in the bottom right corner. Fig. 6. The chemical maps obtained for the tissue sample of DG area from the control, non-epileptic rat shown in the bottom right corner. 3. The ratio of the absorption at around 1631 and 1657 cm 1 as well as the ratio of absorbance at 1548 and 1657 cm 1 were applied for the analysis of the relative secondary structure of proteins. The first parameter has been used previously to examine protein structural changes in case of b-amyloid deposits in Alzheimer’s disease as well as prion-infected tissues (Miller et al., 2006; Kretlow et al., 2006; Kneipp et al., 2003). The changes of protein secondary structure from a-helix to b-sheet 145 J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 Table 2 The median values of the analyzed parameters obtained for epileptic and control group. CA3 1084 1226 1631/1657 1548/1657 2921/2958 DG 1084 1226 1631/1657 1548/1657 2921/2958 a ** Epilepsy Control Multiform Pyramidal Molecular Multiform Pyramidal Molecular 10.50a 4.70 0.64 0.56 1.83 12.10 7.90 0.43 (p = 0.10)** 0.52 (p = 0.05) 2.09 (p = 0.05) 19.50 11.70 0.48 0.53 1.96 14.30 7.90 0.39 0.52 1.99 15.80 8.20 0.35 0.49 1.87 15.20 8.00 0.41 0.52 1.89 Multiform Granular Molecular Multiform Granular Molecular 10.00 5.90 0.53 (p = 0.08) 0.57 (p = 0.08) 2.20 (p = 0.08) 8.00 4.60 0.55 0.52 1.50 14.00 8.10 0.42 (p = 0.05) 0.54 2.10 (p = 0.05) 14.00 8.70 0.38 0.50 1.90 12.00 9.60 0.34 0.47 1.70 14.00 10.40 0.36 0.49 1.80 Epilepsy Control Intensity in [a.u.]. Statistically significant differences between control and epileptic group were marked, in the parenthesis the p-values of U Mann–Whitney test were shown. are often associated with the shift of the maximum of amide I peak in the direction of lower wavenumbers. This shift is followed by the decrease of the absorbance at 1657 cm 1 and the same by the increase of the ratio of the absorbance at 1548 and 1657 cm 1. Because of it the ratio of the absorbance at 1548 and 1657 cm 1 was used as an additional marker of protein conformational changes. It is necessary to mention that statistically significant correlations (Spearman’s rank correlation coefficient was used as a non-parametric measure of statistical dependence between the variables) between this parameter and the ratio of the absorption at around 1631 and 1657 cm 1 were found both within the following investigation and in our previous research concerning the biochemical anomalies occurring in human substantia nigra as a result of Parkinson’s disease (Szczerbowska-Boruchowska et al., 2007). 4. The absorption in the range of wavenumber from around 2820 to 2996 cm 1 (lipid massif) was used for the determination of lipids distribution. 5. The ratio of the absorption at 2921 and 2958 cm 1 was used for the analysis of the mean saturation level of phospholipids. According to Petibois et al. the following absorption ratio provides a direct measurement of the level of fatty acyl chain peroxidation after oxidative stress (Petibois and Deleris, 2005, 2006). For all the analyzed samples and all the examined cellular layers the mean intensities of selected absorption bands and the average ratios of the intensities of selected absorption bands (ratios of absorbances at selected wavenumbers) were calculat- ed. The mean values were extracted from 25 pixels randomly chosen from the appropriate layers. Afterwards, in order to compare the epileptic group with the control one the medians of these values were found and statistical significance of the differences between them was tested using non-parametric U Mann–Whitney test at the significance level of 0.1. The motivation supporting the choice of the used statistical test was described earlier. In Table 2 the median values of the analyzed parameters calculated for epileptic and control group were presented. As one can notice from Table 2 an elevated saturation level of phospholipids in pyramidal cells of CA3 area (p = 0.05) as well as in multiform (p = 0.08) and molecular (p = 0.05) layers of DG area was noticed. For the same cellular layers we also observed changes in the relative secondary structure of proteins with an increase of the contribution of b-sheet structure as monitored by an increased ratio of the absorbance at around 1631 and 1657 cm 1. In pyramidal layer of CA3 and multiform layer of DG these anomalies were followed by the increased ratio of the absorbance at 1548 and 1657 cm 1. In order to present the mentioned abnormalities, in Fig. 7 (part A), the amide region of the spectra recorded in epileptic and control multiform layer of DG hippocampal area were compared. Additionally, in Fig. 7B the second derivatives of these spectra were presented. As it is possible to notice, significant shift of the spectrum for epileptic case in the direction of lower wavenumbers is a result of an increased, in comparison with control case, absorption at around 1628 cm 1 and decreased at around 1658 cm 1. Fig. 7. The comparison of IR absorption spectra (amide region) from DG multiform layer representing epileptic and control case (A); second derivative of the spectra (B). 146 J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 6. Discussion The main goal of this study was to investigate the influence of pilocarpine-induced epileptic seizures on the distribution of main organic components, and especially to assess the saturation level of phospholipids and the relative secondary structure of proteins in the rat hippocampal formation tissue. To achieve these points, topographic and semi-quantitative biochemical differences between epileptic and control animals were investigated using SRFTIR micro-spectroscopy which provided information on molecular chemistry at micrometer scale. The use of synchrotron source for infrared radiation which is 100–1000 times brighter than conventional one allows smaller regions to be probed with acceptable signal-to-noise ratio (Dumas and Miller, 2003; Miller and Smith, 2005; Miller and Dumas, 2006). In this work 10-mm IR beam was used to analyze biochemical composition of DG and CA3 hippocampal areas. The mentioned sectors of the hippocampal formation were chosen based on our previous studies concerning the elemental abnormalities occurring in the brain tissue as a consequence of pilocarpine-induced seizures (Chwiej et al., 2008). The lipid massif from around 2820 to 2996 cm 1 was used to analyze seizure-induced changes in lipid distribution as well as in saturation level of phospholipids. Statistically significant increase in the ratio of the absorption at around 2921 and 2958 cm 1 was observed for the pyramidal cells of CA3 area as well as for multiform and molecular layers of DG in case of epileptic rats. The analyzed ratio of intensities of the CH2 asymmetric stretching to CH3 asymmetric stretching (n(CH2):n(CH3)) provides information on mean saturation level of phospholipids which can be a measure of the level of lipid peroxidation as a result of oxidation stress (Petibois and Deleris, 2005, 2006). Lipid peroxidation means unspecific oxidation of polyunsaturated fatty acids. Polyunsaturated fatty acids are present in phospholipids of biological membranes and are highly susceptible to be oxidized by reactive oxygen species (ROS) (Waldbaum and Patel, 2009). Oxidative stress meaning an imbalance in oxidant and antioxidant homeostasis is rarely monitored by direct measurements of ROS, due to their transient and unstable nature and their main localization into specific cellular compartments (Waldbaum and Patel, 2009). Indirect markers of oxidative stress have thus to been investigated among which are lipid and protein oxidation or activity of free radical scavenging systems. An increased saturation level of phospholipids and an elevated relative content of proteins with b-type secondary structure were observed within selected cellular layers of CA3 and DG areas of the hippocampal formation. Such results point to possible involvement of the reactive phenomena in the mechanisms responsible for neurodegenerative changes occurring as a consequence of the oxidative stress following pilocarpine-induced seizures in the analyzed brain areas. Alterations in brain phospholipid composition as well as lipid peroxidation level have been previously reported in models of brain injury, hypoxia, schizophrenia as well as epilepsy (Kikuchi et al., 2006; du Bois et al., 2005; Freitas et al., 2005, 2009; Freitas, 2009; Tejada et al., 2006, 2007). An excessive free radical formation and an increase in lipid peroxidation have also been observed in different brain areas during status epilepticus induced by pilocarpine and in the lithium-pilocarpine model (Freitas et al., 2005, 2009; Freitas, 2009; Tejada et al., 2006, 2007; Peternel et al., 2009). It is necessary to mention that all these experiments were performed on homogenized samples and therefore provided only an ‘‘averaged’’ view of lipid oxidative damage in the investigated areas of rat brain. In our investigation, SRFTIR micro-spectroscopy was used to analyze abnormalities in lipids and other organic tissue compounds with 10-mm spatial resolution which has allowed us to probe biochemical changes occurring in selected cellular layers of the hippocampal formation. As it was shown only in some of them, statistically significant differences were revealed between epileptic and control areas. Besides abnormalities in lipids, the changes in the relative secondary structure of proteins were detected for pilocarpinetreated animals. These observations were made based on the features appearing in the IR spectra of the nervous tissue around 1500–1700 cm 1. The amide I band arising primarily from the C5 5O stretching vibrations of the amide groups of the protein backbone and the amide II band arising from amide N–H bending vibrations are known to be sensitive to protein backbone conformation (Fabian and Mantele, 2002). In this work the ratio of the absorbance at around 1631 and 1657 cm 1 as well as the ratio of the absorption at the wavenumbers 1548 and 1657 cm 1 were calculated to detect conformational changes of proteins. The mentioned parameters presented higher values for pyramidal layer of CA3 area as well as in multiform and molecular layers of DG. Such a result indicates that pilocarpine-induced seizures evoke local changes in the relative secondary structure of proteins towards b-sheet structure. It has to be noticed that there is a good correlation between these abnormalities and increased lipid peroxidation which might suggest a common mechanism inducing both types of anomalies—oxidative stress. As known, the proteins exposed to free radical action may exhibit altered primary, secondary and tertiary structure as well as may undergo spontaneous fragmentation and manifest increased proteolysis susceptibility (Waldbaum and Patel, 2009). Seizure activity is associated with region-specific cell death and lasting synaptic reorganization of hippocampal circuitry (Bausch and McNamara, 1999). These changes require new protein synthesis and are associated with a rapid upregulation of immediate-early gene products, among which are several classes of transcription factors whose activity is thought to mediate the long-term changes in synaptic plasticity associated with pathological seizure activity (Sloviter, 1994). Interestingly biochemical changes in CA3 and DG areas were limited only to some of the analyzed cellular layers, namely the pyramidal or molecular and multiform layers, respectively. The hippocampal neurons of pyramidal layers and CA3 express many transcripts related to glucose metabolism (Datson et al., 2004; Lein et al., 2004). It is worth to mention that these neurons are very vulnerable to damage caused by seizure. Moreover, in CA3 neurons levels of transcripts encoding proteins involved in synaptic function and transmitter release are elevated (Greene et al., 2009). More detailed studies which may allow us to detect the changes in elemental composition with the same spatial resolution as in the following studies are necessary. Such experiments are in progress and the next step in our research will be to correlate the elemental and biochemical changes occurring as a consequence of pilocarpine-induced seizures. 7. Conclusions In the present work SRFTIR micro-spectroscopy was for the first time used for the topographic analysis of brain tissue affected by epileptic seizures. The use of synchrotron source of infrared radiation allowed us to detect biochemical abnormalities in hippocampal tissue with the spatial resolution of 10 mm. An increased saturation level of phospholipids and an elevated relative content of proteins with b-type secondary structure were observed for selected cellular layers of CA3 and DG hippocampal sectors. Such results indicate that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures. The works will be continued. The correlations between biochemical and elemental J. Chwiej et al. / Journal of Chemical Neuroanatomy 40 (2010) 140–147 anomalies of nervous tissue as well as the progress of epilepsy and histopahological changes will be examined. Additionally, the influence of neuroprotective agents on the analyzed biochemical parameters will be investigated. Acknowledgements This work was supported by Polish Ministry of Science and Higher Education and the following grants: 1. The European Community-Research Infrastructure Action under FP6 ‘‘Structuring the European Research Area’’ Programme (through the Integrated Infrastructure Initiative ‘‘Integrating Activity on Synchrotron and Free Electron Laser Science’’). 2. The European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 226716. 3. SOLEIL experimental grants: 20060063 and 20080451. 4. ELETTRA experimental grants: 2008127 and 20085318. 5. The Ministry of Science and Higher Education, grants N303 052 31/1626 and DWM/94/IA-SFS/2007. The first author is also grateful for support from the Foundation for Polish Science (START Programme). References Bausch, S.B., McNamara, J.O., 1999. Experimental partial epileptogenesis. Curr. Opin. Neurol. 12, 203–209. Ceru, S., Rabzelj, S., Kopitar-Jerala, N., Turk, V., Zerovnik, E., 2005. Protein aggregation as a possible cause for pathology in a subset of familial UnverrichtLundborg disease. Med. Hypotheses 64, 955–959. 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