ORIGINAL PAPER

Variations in elemental compositions of rat hippocampalformation between acute and latent phases of pilocarpine-inducedepilepsy: an X-ray fluorescence microscopy study

J. Chwiej • J. Dulinska • K. Janeczko •

K. Appel • Z. Setkowicz

Received: 6 December 2011 / Accepted: 7 March 2012 / Published online: 24 March 2012

� SBIC 2012

Abstract There is growing experimental evidence that

tracing the elements involved in brain hyperexcitability,

excitotoxicity, and/or subsequent neurodegeneration could be

a valuable source of data on the molecular mechanisms trig-

gering or promoting further development of epilepsy. The

most frequently used experimental model of the temporal lobe

epilepsy observed in clinical practice is the one based on

pilocarpine-induced seizures. In the frame of this study, the

elemental anomalies occurring for the rat hippocampal tissue

in acute and silent periods after injection of pilocarpine in rats

were compared. X-ray fluorescence microscopy was applied

for the topographic and quantitative elemental analysis. The

differences in the levels of elements such as P, S, K, Ca, Fe,

Cu, and Zn between the rats 3 days (SE72) and 6 h (SE6) after

pilocarpine injection as well as naive controls were examined.

Comparison of SE72 and control groups showed, for specific

areas of the hippocampal formation, lower levels of P, K, Cu,

and Zn, and an increase in Ca accumulation. These results as

well as further analysis of the differences between the SE72

and SE6 groups confirmed that seizure-induced excitotoxicity

as well as mossy fiber sprouting are the mechanisms involved

in the neurodegenerative processes which may finally lead to

spontaneous seizures in the chronic period of the pilocarpine

model. Moreover, in the light of the results obtained, Cu seems

to play a very important role in the pathogenesis of epilepsy in

this animal model. For all areas analyzed, the levels of this

element recorded in the latent period were not only lower than

those for controls but were even lower than the levels found in

the acute period. The decreased hippocampal accumulation of

Cu in the phase of behavior and EEG stabilization, a possible

inhibitory effect of this element on excitatory amino acid

receptors, and enhanced seizure susceptibility in Menkes

disease (an inherited Cu transport disorder leading to Cu

deficiency in the brain) suggest a neuroprotective role rather

than neurodegenerative and proconvulsive roles of Cu in

pilocarpine-induced epilepsy.

Keywords Metal determination � X-ray microprobe �Neurochemistry

Introduction

Epilepsy still constitutes a serious clinical problem, and there

is an urgent need for a more effective therapeutic strategy.

Studies on epileptogenesis or testing new antiepileptic drugs

require the use of adequate animal models of epileptic sei-

zures [1, 2], and an ideal model should have a behavioral

pattern most closely resembling clinical symptoms in humans

[3]. Chronic models of acquired (symptomatic) epilepsy

include models in which epilepsy or epilepsy-like conditions

are induced by electrical (kindling model) or chemical

(pilocarpine and kainite models) methods in previously

healthy animals, mostly rats [1].

Among the models of temporal lobe epilepsy, the pilo-

carpine model is the most frequently used (for 25 years).

J. Chwiej (&) � J. Dulinska

Department of Medical Physics and Biophysics,

Faculty of Physics and Applied Computer Science,

AGH University of Science and Technology,

Krakow, Poland

e-mail: jchwiej@novell.ftj.agh.edu.pl

K. Janeczko � Z. Setkowicz

Department of Neuroanatomy,

Institute of Zoology,

Jagiellonian University,

Krakow, Poland

K. Appel

Deutsches Elektronen-Synchrotron (DESY),

Hamburg, Germany

123

J Biol Inorg Chem (2012) 17:731–739

DOI 10.1007/s00775-012-0892-1

Administration of pilocarpine in rats evokes sequential

behavioral and electrographic changes that can be divided

into three distinct periods: (1) an acute period that builds up

progressively into a limbic status epilepticus (24 h), (2) a

silent (latent) period with progressive normalization of the

EEG and behavior (from a few to a few dozen days), and (3)

a chronic period with spontaneous recurrent seizures [4].

This article is a continuation of our previous studies

which analyzed changes in the accumulation of metals

resulting from seizures, mechanical brain injury, and the

use of the neuroprotective agent FK-506 [5–7]. We try to

verify whether the compositional changes of Ca, Cu, and

Zn [5], observed before for the acute period of pilocarpine-

induced status epilepticus, were temporary or permanent

irreversible effects. To achieve this, rats classified in the

second period of the pilocarpine model of epilepsy are

examined. The results obtained for the animals 3 days after

pilocarpine injection (SE72) are compared with data

recorded previously for epileptic rats 6 h after treatment

with pilocarpine (SE6) as well as for naive control animals.

Similarly, as in our previous work, the topographic and

quantitative elemental analysis of tissues was done using

X-ray fluorescence microscopy, and measurements were

done at HASYLAB beamline L.

Materials and methods

Animals and seizure induction

All animal-use procedures were approved by the Bioethical

Commission of Jagiellonian University in accordance with

international standards. Adult Wistar rats were obtained from

an animal colony of the Institute of Pediatry, Collegium

Medicum, Jagiellonian University. To exclude the influence

of sex hormones on the brain, only males were used in the

experiment. During their whole life the rats were maintained

under conditions of controlled temperature (20 ± 2 �C) and

illumination (12-h light,12-h dark cycle). A solid diet (Lab-

ofeed) and water were available ad libitum.

On the 60th postnatal day, the rats received single

intraperitoneal injections of pilocarpine (300 mg/kg;

Sigma P6503). Scopolamine methyl bromide (1 mg/kg;

Sigma S8502) was injected intraperitoneally 30 min before

pilocarpine to reduce its peripheral effects. Pilocarpine was

injected between 9 and 10 a.m. to avoid circadian effects of

seizure vulnerability.

Tissue preparation

Six hours (SE6 group) or 3 days (SE72 group) after

pilocarpine injections, the rats were deeply anesthetized

with sodium pentobarbital (Vetbutal; Polfa, Poland) and

perfused transcardially with 0.9 % NaCl of high analytical

purity. After removal of the brain from the skull, the

medulla was dissected, and the brain was frozen in liquid

nitrogen and cut frontally in a cryomicrotome into 15-lm-

thick slices. The specimens of the dorsal part of the hippo-

campus [8] were mounted on Ultralene foil and freeze-dried.

There were six, five, and five rats, respectively, in the SE6,

SE72, and control groups.

Measurements

Experiments were performed at beamline L at HASYLAB,

with X-ray radiation originating from a bending magnet at

the DORISIII storage ring. A polycapillary half-lens was

used to focus the X-ray beam to a spot with a diameter of

around 15 lm. The excitation energy was set to 17 keV

using a double multilayer monochromator. The samples

were mounted in an air atmosphere and positioned at an

angle of 45� with respect to the incident beam. An energy-

dispersive Vortex silicon drift detector from SII Nano

Technology USA was used for fluorescence detection of

the elements, and the exit angle was 45�. Tissue samples

were mapped in two dimensions with 10-s acquisition

times for single spectra. For calibration of the spectrometer

and calculation of elemental sensitivities, reference mea-

surements were performed on NIST Standard Reference

Materials SRM 1833 and SRM 1832 as well as on

MICROMATTER GaP and KCl X-ray fluorescence cali-

bration standards.

The analysis of single X-ray fluorescence spectra and

the batch processing of large data sets were done using the

program PyMca. A detailed description of the algorithms

used in this program can be found in [9]. The program

enables the correction of the spectral background using

polynominal analytical functions. The relative intensity

ratio of the Ka and Kb lines of a specific element is applied

in the program to avoid poor estimation of the area under

the Ka line caused by overlapping with the Kb line of

another element. In PyMca these ratios are obtained by

multiplying the transition probabilities by an absorption

correction term including the X-ray attenuation in all layers

and windows between the sample surface and the active

area of the detector.

Results

The main purpose of the present studies was a comparison

of elemental anomalies occurring in acute and silent peri-

ods after injection of pilocarpine into rats. X-ray fluores-

cence microscopy was applied for elemental analysis of rat

brain tissue. The following elements were detected in the

brain areas analyzed: P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br,

732 J Biol Inorg Chem (2012) 17:731–739

123

Rb, and Sr. This is demonstrated in Fig. 1, which shows a

cumulative spectrum recorded for selected hippocampal

formation tissue.

The masses per unit area of the elements detected in the

nervous tissue were calculated from the intensities of the

Ka lines and the elemental sensitivities obtained from

measurements of reference materials. Raster scanning of

the samples allowed elemental distribution maps to be

obtained for the tissue areas analyzed. In Fig. 2, one can

see the results of topographic analysis done for the hip-

pocampal formation tissue from an epileptic rat in the

silent period after pilocarpine injection. Additionally, the

detailed maps recorded for sector 3 of Ammon’s horn

(CA3) and the dentate gyrus (DG) are presented in Figs. 3

and 4.

The two-dimensional elemental composition maps were

compared with microscopic views of the scanned tissues,

which allowed us to identify areas for further quantitative

analysis, namely, sector 1 of Ammon’s horn (CA1), CA3,

DG, the hilus of the DG, and the neocortex. Additionally,

on the basis of the detailed elemental maps for the DG (see

Fig. 3), we found a strong positive correlation between K

and Fe accumulations. Both elements showed increased

levels for granular and molecular layers, whereas the mass

per unit area of Zn was higher in granular and multiform

layers than in the molecular layer. The source of relatively

high, in comparison with the other elements, amounts of Zn

within the multiform layer is large terminals of mossy

fibers of dentate granule cells that contain the highest

amounts of Zn in the brain.

In contrast to the DG, for CA3 (see Fig. 4) positive

correlation was observed in case of Fe and Zn accumula-

tions, and these elements showed higher levels in pyra-

midal and multiform layers than in the molecular layer.

For CA1, CA3, DG, the hilus of the DG, and the neo-

cortex, the mean masses per unit area of elements analyzed

were calculated. In the calculations, areas of 300 9 300 lm2

were taken into account.

Fig. 1 Cumulative spectrum

for hippocampal tissue from a

rat representing the group of rats

3 days after pilocarpine

injection (SE72 group)

Fig. 2 Comparison of elemental maps obtained for hippocampal

tissue from a selected epileptic rat in the silent period after

pilocarpine injection with the microscopic view of the scanned tissue

area. The scales display masses per unit area of the elements in

micrograms per square centimeter

J Biol Inorg Chem (2012) 17:731–739 733

123

For comparison of the rat groups examined (SE72, SE6,

and control), median values of the mean masses per unit

area were calculated and evaluated (Fig. 5).

The statistical significance of differences between

medians was tested with the nonparametric U (Mann–

Whitney) test at a significance level of 0.05 (see the results

in Table 1). The Mann–Whitney test is a nonparametric

alternative to the t test for independent samples. It assumes

that the variables analyzed were measured on at least an

ordinal scale, and the calculation is based on rank sums.

This statistical test is the most sensitive nonparametric

alternative to the t test, and in some cases may have even

greater power than the t test to reject the null hypothesis

[10]. However, the choice of this particular test was mainly

Fig. 3 Distribution of K, Fe, and Zn in the dentate gyrus from a

selected epileptic hippocampal formation. Pixel size 15 9 15 lm2.

For all the maps the minimal values of the masses per unit area are

marked in black and the maximal values are in color. g granular cell

layer, mo molecular cell layer, mu multiform cell layer

Fig. 4 Distribution of K, Fe, and Zn in sector 3 of Ammon’s horn

from a selected epileptic hippocampal formation. Pixel size

15 9 15 lm2. For all the maps the minimal values of the masses

per unit area are marked in black and the maximal values are in color.

p pyramidal cell layer, mo molecular cell layer, mu multiform cell

layer

734 J Biol Inorg Chem (2012) 17:731–739

123

a result of the small size of the populations examined (from

five to six rats per group) which did not allow us to test the

normality of the distributions of the data analyzed.

The comparison of epileptic rats in the silent phase after

pilocarpine administration with the control group showed

more anomalies in elemental compositions than were pre-

viously observed for the acute period of pilocarpine-

induced status epilepticus. The abnormalities which were

found concern elements such as P, K, Ca, Cu, and Zn. For

all of them except Ca, lower levels were observed for the

SE72 group. In the case of Ca, a statistically significant

increase of mass per unit area was noticed in the DG but a

trend was also observed for CA1, for which the p value was

0.09.

The mass per unit area of K for the SE72 group was,

compared with controls, significantly lower in CA3 and the

hilus of the DG as well as in the neocortex. In turn, a

decrease of the Cu level was observed in all the hippo-

campal areas analyzed. Moreover, in most of them (with

the exception of the DG), the levels of this element were

lower than those detected for the SE6 group.

The comparison of SE72 and SE6 groups demonstrated,

moreover, a decreased level of P in the neocortex as well as

an increased mass per unit area of Zn in the DG.

Discussion

A great deal of our knowledge about epileptic disorders is

derived from animal models, which are a perfect tool for

modern experimental medicine. Temporal lobe epilepsy is

the most common type of partial complex seizure in

adulthood [11, 12]. One of the most often used animal

models of temporal lobe epilepsy is that based on pilo-

carpine. The injection of pilocarpine induces status epi-

lepticus characterized by tonic–clonic generalized seizures,

and after status epilepticus animals go spontaneously into

the seizure-free latent period. Although pilocarpine-treated

animals usually show normal behavior and EEG in the

latent period, several pathophysiological phenomena rela-

ted to epileptogenesis may occur. Among these, the most

widely cited are mossy fiber sprouting, interneuron loss,

rewiring of synaptic circuits, glial cell activation, and

ectopic cell proliferation [13, 14].

In the work reported here, X-ray fluorescence micros-

copy was applied to compare elemental anomalies occur-

ring as a result of pilocarpine-induced seizures in acute and

latent periods after administration of a proconvulsive agent.

A comparison of epileptic rats in the latent period with

controls showed many more anomalies than previously

noticed for rats in the acute phase of pilocarpine-induced

status epilepticus. The abnormalities concerned accumu-

lation of P, K, Ca, Cu, and Zn. Calcium was the only

element with a higher content in the SE72 group than in

controls. The anomalies in the Ca level were recorded for

the DG (p \ 0.01) and CA1 (p = 0.09). Median masses

per unit area of Ca in the DG and CA1 from the SE72

group were almost 50 % higher than those in the control

group.

In the normal brain, Ca2? ions participate in many cel-

lular processes, such as neuronal differentiation and growth,

membrane excitability, exocytosis, and synaptic activity

[15]. The abundance of Ca2? ions determines the physio-

logical status of the neurons; therefore, deregulation of their

homeostasis underlies pathogenic mechanisms of various

neurodegenerative diseases. Activation of postsynaptic

N-methyl-D-aspartate (NMDA) receptors in neurons,

induced by excessive presynaptic glutamate release during

seizures, results in excessive influx of Ca2? into neurons

[16]. The increased concentration of Ca2? ions promotes, in

turn, a high release of glutamate, inducing status epilepti-

cus. Glutamate, acting on a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionate (AMPA)/kainite receptors, allows Na?

and Ca2? ions to enter the cell. As a consequence, Mg2?

ions, which block the NMDA receptors, are removed,

inducing activation of these receptors by glutamate. This

allows further influx of Ca2? into postsynaptic cells,

inducing excitotoxicity and cell death [17]. At the same

time, epileptiform activity induces a prolonged increase in

astrocytic Ca2? excitability that prolongs the period of

epileptiform activity [18]. When compared with seizure-

prone pyramidal neurons in CA1 or CA3 or with hilar

neurons, granular neurons in the DG are relatively more

resistant to seizure-induced excitotoxicity. These granular

neurons sprout mossy fibers, forming aberrant synapses on

dendrites of neighboring granular neurons [19, 20]. The

newly formed excitatory circuit might support recurrent

epileptiform activity during the postseizure period. It could,

therefore, be reflected in the prolonged elevation of intra-

cellular Ca2? levels within the DG recorded in the present

study 72 h following pilocarpine administration.

Taken together, the phenomena might be the source of

the observed increase of the Ca level in the rats 72 h after

pilocarpine administration.

As has already been shown by McNamara et al. [21],

activation of ionotropic and metabotropic glutamate

receptors as well as the TrkB neurotrophin receptors can

promote epileptogenesis. These receptors are present in

membranes of the dendritic spine of glutamatergic neurons

of the DG, whose activation generates increased Ca2? ion

concentrations. This may activate Ca2?-regulated enzymes

implicated in epileptogenesis, such as calcium/calmodulin-

dependent protein kinase II, calcineurin, and protein tyro-

sine kinases Src and Fyn [21]. Therefore, it is possible that

recurrent epileptic seizures after a silent period are mal-

adaptative consequences of these changes.

J Biol Inorg Chem (2012) 17:731–739 735

123

For all other elements with abnormal hippocampal

accumulations in comparison with the control group, lower

masses per unit area were detected. Such a result is prob-

ably an effect of their outflow to the periphery as a result of

blood–brain barrier damage. It is well known that epileptic

seizures produce regional blood–brain barrier openings that

are usually reversible and confined to anatomically limited

brain areas [22–25], and changes in cerebrovascular per-

meability may cause alterations in the ionic environment of

central neurons and glia [23, 25].

The results obtained for Cu seem especially important.

The levels of this element for all areas analyzed were lower

in the SE72 group than in the controls. Comparison of

epileptic rats from latent and acute periods showed similar

relations, and the only region for which the level of Cu did

not differ between the SE72 and SE6 groups was the DG.

Copper, as a key structural element of many proteins, is

a cofactor of many enzymes critical for proper brain

functioning. These enzymes are involved in cellular res-

piration and antioxidant defense as well as in other

Fig. 5 Median values of mean masses per unit area for the areas

analyzed and groups of rats. Statistically significant differences

between the group 72 h after pilocarpine injection (SE72) and the

control group (N) as well as between the SE72 group and the group

6 h after pilocarpine injection (SE6) are marked with green stars and

blue stars, respectively. C neocortex, CA1 sector 1 of Ammon’s horn,

CA3 sector 3 of Ammon’s horn, DG dentate gyrus, H hilus of dentate

gyrus

736 J Biol Inorg Chem (2012) 17:731–739

123

processes required for growth, development, and mainte-

nance of the nervous system [26]. Moreover, Cu ions may

behave as signaling molecules and act on proteins regu-

lating neuronal excitability [27, 28]. Copper can modulate

receptors for both fast excitatory (NMDA and AMPA/ka-

inite receptors) and fast inhibitory (GABAA) transmission

in the central nervous system [29–31]. It is a potent

inhibitor of NMDA receptors and blocks AMPA/kainite

receptors. The possible inhibitory effect of Cu on excit-

atory amino acid receptors and the fact that processes that

finally lead to spontaneous seizures (in the chronic phase)

occur in conditions of a diminished Cu level, as was found

in this study, seem to suggest a neuroprotective role rather

than neurodegenerative and proconvulsive roles of this

element in the case of pilocarpine-induced epilepsy. Such a

result is in agreement with observations of patients with

Menkes disease (an inherited Cu transport disorder leading

to Cu deficiency in the brain). This inherited Cu transport

disease leads to Cu deficiency in the brain, and epilepsy is

its major clinical outcome [32].

Comparison of Zn levels recorded for SE72 and control

groups showed lower masses per unit area only for the CA3

hippocampal area. The accumulation of Zn in the DG was

higher for rats in the latent period than in the acute period

after pilocarpine administration. Such a result is probably

an effect of mossy fiber sprouting, which involves the

formation of new asymmetrical synaptic contacts between

mossy fiber terminals and dendrites of granule cells and

inhibitory interneurons in the inner molecular layer of the

DG [33–35]. Many reports suggest a contributory role of

aberrant mossy fiber sprouting to hyperexcitability and

seizures both in humans with temporal lobe epilepsy and in

animal models of this disorder, including kindling and

pharmacological treatment with convulsants [34, 36–40].

Large terminals of mossy fibers of dentate granule cells

contain the highest amounts of Zn in the brain and can be

visualized by histochemical sulfide–silver staining, called

Timm staining [41]. Increased Timm staining in aber-

rantly sprouted mossy fibers has been previously reported

in different experimental models of epilepsy, including

kindling-, kainate-, and pilocarpine-induced seizures

[42–45]. Moreover, research by Mitsuya et al. [46] on a

intrahippocampal kainate mouse model of mesial tempo-

ral lobe epilepsy showed that during the first 2 weeks after

kainite administration Timm staining in the hippocampus

increases.

Conclusions

X-ray fluorescence microspectroscopy was used to analyze

differences in the topography and accumulation of selected

elements between acute and latent periods in rats after

pilocarpine administration. In light of the results obtained,

Cu seems to play a very important role in the pathogenesis

of epilepsy in the pilocarpine model. For all areas analyzed,

the levels of this element recorded in the latent period were

not only lower than those in controls, but were even lower

Table 1 Statistically significant differences in elemental compositions between analyzed rat groups

Element SE72 vs. N SE72 vs. SE6

C CA1 CA3 DG H C CA1 CA3 DG H

P ;

(0.03)

;

(0.04)

S

K ;

(0.03)

;

(0.02)

;

(0.05)

Ca :

(0.05)

Fe

Cu ;;

(p \ 0.01)

;

(0.03)

;;

(p \ 0.01)

;

(0.02)

;;

(p \ 0.01)

;;

(p \ 0.01)

;

(0.02)

;; (p \ 0.01) ;

(0.02)

Zn ;

(0.02)

::

(p \ 0.01)

Single arrows represent a decease (;) or increase (:) in mass per unit area of the element at 0.01 \ p \ 0.05. Double arrows represent a decrease

(;;) or increase (::) in mass per unit area of the element at p \ 0.01

The p value from the U test is given in parentheses

SE72 group 72 h after pilocarpine injection, SE6 group 6 h after pilocarpine injection, N control group, C neocortex, CA1 sector 1 of Ammon’s

horn, CA3 sector 3 of Ammon’s horn, DG dentate gyrus, H hilus of dentate gyrus

J Biol Inorg Chem (2012) 17:731–739 737

123

than the levels found in the acute period. The decreased

hippocampal accumulation of Cu in the phase of behavior

and EEG stabilization, a possible inhibitory effect of this

element on excitatory amino acid receptors, and enhanced

seizure susceptibility in Menkes disease suggest a neuro-

protective role rather than neurodegenerative and procon-

vulsive roles of Cu in the case of pilocarpine-induced

epilepsy.

Acknowledgments This work was partially supported by the Polish

Ministry of Science and Higher Education and its grant for scientific

research IUVENTUS PLUS no. JP2010005370. The research leading

to these results has received funding from the European Community’s

Seventh Framework Programme (FP7/2007-2013) under grant

agreement no. 226716 and was realized in the frame of experimental

grants DESY-D-II-20080009 EC and I-20110056 EC. The authors

wish to express their appreciation to Henryk Figiel for valuable dis-

cussions and comments in the preparation of the manuscript.

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