Journal of Chemical Neuroanatomy 40 (2010) 140–147
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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.
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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.
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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
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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).
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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).
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