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NeuroImage 38 (2007) 57–66

In vivo mapping of temporospatial changes in manganeseenhancement in rat brain during epileptogenesis

Silje Alvestad,a Pål Erik Goa,b Hong Qu,a Øystein Risa,a Christian Brekken,b Ursula Sonnewald,a

Olav Haraldseth,b Janniche Hammer,c Ole Petter Ottersen,c and Asta Håbergb,⁎

aDepartment of Neuroscience, Norwegian University of Science and Technology (NTNU), N-7489 Trondheim, NorwaybDepartment of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), N-7489 Trondheim, NorwaycCentre for Molecular Biology and Neuroscience, and Department of Anatomy, University of Oslo, N-0317 Oslo, Norway

Received 21 February 2007; revised 31 May 2007; accepted 20 July 2007Available online 7 August 2007

Mesial temporal lobe epilepsy is associated with structural andfunctional abnormalities, such as hippocampal sclerosis and axonalreorganization. The temporal evolution of these changes remains to bedetermined, and there is a need for in vivo imaging techniques that canuncover the epileptogenic processes at an early stage. Manganese-enhanced magnetic resonance imaging may be useful in this regard.The aim of this study was to analyze the temporospatial changes inmanganese enhancement in rat brain during the development ofepilepsy subsequent to systemic kainate application (10 mg/kg i.p.).MnCl2 was given systemically on day 2 (early), day 15 (latent), and11 weeks (chronic phase) after the initial status epilepticus. Twenty-four hours after MnCl2 injection T1-weighted 3D MRI was performedfollowed by analysis of manganese enhancement. In the medialtemporal lobes, there was a pronounced decrease in manganeseenhancement in CA1, CA3, dentate gyrus, entorhinal cortex andlateral amygdala in the early phase. In the latent and chronic phases,recovery of the manganese enhancement was observed in all thesestructures except CA1. A significant increase in manganese enhance-ment was detected in the entorhinal cortex and the amygdala in thechronic phase. In the latter phase, the structurally intact cerebellumshowed significantly decreased manganese enhancement. The highlydifferentiated changes in manganese enhancement are likely torepresent the net outcome of a number of pathological and patho-physiological events, including cell loss and changes in neuronalactivity. Our findings are not consistent with the idea that manganeseenhancement primarily reflects changes in glial cells.© 2007 Elsevier Inc. All rights reserved.

Keywords: Kainic acid; Kainate; Epilepsy; Mossy fiber sprouting; Neurons;Glia; CA1; Dentate gyrus; CA3; Entorhinal cortex; Amygdala; Cerebellum

⁎ Corresponding author. Fax: +47 73997708.E-mail address: asta.haberg@ntnu.no (A. Håberg).Available online on ScienceDirect (www.sciencedirect.com).

1053-8119/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.neuroimage.2007.07.027

Introduction

Mesial temporal lobe epilepsy (MTLE) is one of the mostcommon forms of epilepsy in adults and is often refractory tomedication. Both in humans and animal models, MTLE involves ahyperexcitable neural network in mesial temporal lobe structuresand is often associated with hippocampal sclerosis characterized byneuronal loss, gliosis, and axonal reorganization. The structuralchanges may be detected as atrophy and hyperintensity in T2-weighted magnetic resonance imaging (MRI) (Jackson et al., 1990),and as a reduced N-acetyl aspartate signal in proton MR spectro-scopy (Connelly et al., 1994). However, the temporal evolution ofthe changes in MTLE remains to be determined, and there is a needfor noninvasive in vivo imaging techniques that can uncover theepileptogenic processes at an early stage. This, in turn, may offer thepossibility of identifying patients at risk for epilepsy and resolvewhether the structural changes are causes or consequences ofMTLE.

Manganese-enhanced magnetic resonance imaging (MEMRI)has rapidly gained interest in the field of neuroscience as a high-resolution imaging method of brain structure and function. Thisinterest stems from manganese’s dual property as an MRI contrastagent and as a substrate for divalent ion transporters in the brain.Mn2+ may enter excitable neurons via voltage-gated Ca2+ channelsand possibly also via the divalent metal transporter DMT1 (Drapeauand Nachshen, 1984; Narita et al., 1990; Pautler et al., 1998; Takeda,2003). Systemic Mn2+ administration has mostly been used forstructural imaging since it enhances the brain cytoarchitecture (Silvaet al., 2004). Recent studies with animals receiving specific stimuliduring the Mn2+ exposure have shown a strong correlation betweenlocal brain activity and manganese enhancement after systemicallyadministrated Mn2+ without opening of the blood–brain barrier (Yuet al., 2005; Kuo et al., 2006). Furthermore, neuronal uptake ofMn2+

is directly correlated to neuronal activity (Takeda et al., 1994;Watanabe et al., 2004).

Several studies have demonstrated that MEMRI may be usefulin the assessment of brain damage. Watanabe et al. (2004) used

58 S. Alvestad et al. / NeuroImage 38 (2007) 57–66

systemic administration of Mn2+ to assess brain damage in a mouse8 days after injection of kainic acid (KA), a potent neurotoxinactivating the kainate subclass of ionotropic glutamate receptors(Lerma, 1998). The hippocampus showed reduced and more dif-fusely distributed manganese enhancement compared with con-trols. These changes were interpreted as being secondary toneuronal death and subsequent Mn2+ uptake in remaining neuronsand/or activated glia (Watanabe et al., 2004). A recent study usingdirect injection of Mn2+ into the entorhinal cortex 2 weeks afterKA administration reported an increased number of manganeseenhanced pixels in the dentate gyrus (DG) and CA3 subregions ofhippocampus (Nairismagi et al., 2006). It was proposed that thesealterations reflected mossy fiber sprouting.

Systemic administration of KA causes limbic seizures andstatus epilepticus (SE) in rats, followed by a latent phase with noapparent seizure activity and then a chronic phase with spon-taneous recurrent seizures (Ben-Ari, 1985). This animal modelreproduces many of the clinical and histopathological features ofhuman temporal lobe epilepsy (Ben-Ari, 1985), and allows for thestudy of epileptogenic processes during the latent phase.

The aim of this study was to investigate changes in the manga-nese enhancement at three different time points after KA inducedSE. The time points were: early phase (2 days after KA injection),latent phase (15 days after KA injection), and chronic phase(11 weeks after KA injection). To this end we used subcutaneousinjection of Mn2+ coupled to T1-weighted 3D MRI (24 h later),followed by detailed analysis of manganese enhancement in severalregions of interest in the entire brain.

Materials and methods

Animal procedures

This study was approved by the Norwegian Animal WelfareCommittee, and animals were treated in accordance with the guide-lines set by the Norwegian Animal Research Authority. Aftertransportation, male Sprague–Dawley rats (Taconic M&B, Copen-hagen, Denmark) were allowed to recover for 1 week before theexperiment started. The animals had free access to food and water,and were kept under standard laboratory conditions of 22 °C roomtemperature, 66% humidity, and a 12:12 h light/dark cycle.

Kainic acid (KA) from Sigma–Aldrich Co (MO, USA) wasdissolved in saline with pH adjusted to 7.0. Twenty-one rats (297±15 g) received an intraperitoneal injection of 10 mg KA/kg in avolume of 1 ml/100 g. Ten control rats received 9% saline (1 ml/100 g) intraperitoneally.

The rats were placed in separate cages immediately after KA orsaline administration and were monitored for at least 4 h formanifestation of seizures. Seizures were scored according to theRacine score (Racine, 1972): Stage 1, facial clonus; Stage 2,nodding; Stage 3, forelimb clonus; Stage 4, forelimb clonus withrearing; Stage 5, rearing and falling. Status epilepticus (SE) wasdefined as continuous limbic seizures scored as class 4 or 5seizures and lasting for more than 90 min. Only animals thatdeveloped SE, comprising 18 out of the 21 rats injected with KA,were included in the study.

During the whole experimental period the animals weremonitored for weight and general condition and fed with soakedchow in case of weight loss or lack of weight gain. Three out ofthe 18 rats experiencing SE were assigned to the chronic group.These animals were video recorded 6–12 h/day, 7 days/week

from week 3 after KA injection in order to monitor theoccurrence of spontaneous recurrent seizures (SRS). All threeanimals displayed SRS and were considered to have developedchronic epilepsy.

The animals were divided into three experimental groups: earlyphase, i.e. 2 days after KA injection (n=7); latent phase, i.e.15 days after KA injection (n=8); and chronic phase, i.e. 11 weeksafter KA injection (n=3). MnCl2 (1 M) was diluted in bicinesolution (100 mM in deionized water) with pH adjusted to 7.4using NaOH. Final concentration of MnCl2 was 100 mM. Freshlyprepared MnCl2 (80 mg/kg) was injected subcutaneously into theneckfold 24 h prior to MRI.

Magnetic resonance imaging

MRI was performed at 7.0 Tesla using an actively shieldedBruker Biospec 70/20 (Bruker Biospin, Ettlingen, Germany) with a72-mm volume coil for transmission, and an actively decoupledquadrature rat head surface coil for receive-only. Water-cooledBGA-12 (400 mT/m) gradients were used.

MRI was performed on the three KA groups: early (n=7), latent(n=8) and chronic (n=3), and on control animals (n=10). Theanimals were anesthetized with isoflurane during MRI. Anesthesiawas induced with 3.5% isoflurane in 30/70% O2/N2 in a closedchamber. During imaging the rats lay in the prone position in adedicated animal bed heated with circulating air at 37 °C, and 1.5–2.5% isoflurane in 30/70% O2/N2 was delivered to the sponta-neously breathing animals through a snout mask using a smallanimal ventilator (Harvard Apparatus, MA, USA).

Sagittal gradient-echo images were acquired with TR=246 msand TE=11 ms in 16 slices with FOV=5×5 cm and matrix=128×128. Subsequently a 3D volume set was obtained using a T1-weigthed 3D low flip angle gradient-echo sequence (FLASH) withTR=15 ms and TE=2.9 ms, flip angle of 20° and a volume ofview of 5 cm×3 cm×4 cm in the rostral–caudal/dorsal–ventral/left-to-right directions. The acquisition matrix was 256×128×192voxels, reconstructed to a voxel resolution of 195×234×156 μm3.Four averages were used and the total acquisition time was 25 min.The MRI of three animals (one in control group and two in earlygroup) were disregarded from the analysis due to poor imagequality, so the final number of animals in each group was asfollows: early (n=5), latent (n=8), chronic (n=3), and control(n=9).

Data analysis

The T1-weighted 3D images were analyzed using in-housesoftware developed under the Matlab 7.1 environment (Math-Works, Natick, MA, USA, 2005). The sensitivity of the surfacecoil used for reception decreases with the distance from the coil. Topartly compensate for this effect, the following procedure wasused: Measurements on a water phantom was used to fit a singleexponential function along the axis defined by the normal to thereceiver coil plane. The MRI intensity of the animal measurementswas then divided by the same function. To further reduce theinfluence of spatially varying coil sensitivity, we used an intensitymeasure based on the relative contrast between the tissue of interestand reference muscle tissue close by:

CME ¼ IROI � Imuscle

IROI þ Imuscleð1Þ

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where IROI is the mean intensity in the region of interest (ROI),Imuscle is the mean intensity in the reference muscle tissue, and CME

is the resulting manganese enhancement contrast. In the followingwe will refer to CME as “manganese enhancement”.

The manganese enhancement was evaluated in various brainregions, located based on the Swanson and Paxinos and Watson ratatlases (Paxinos and Watson, 2005; Swanson, 2004; see Fig. 1).Two axial and four coronal levels were chosen for analysis. Theaxial levels were located 4.8 mm and 6.3 mm ventral to bregmaaccording to Paxinos and Watson (1998), whereas the coronallevels were located at bregma and 2.5 mm, 6.0 mm and 8.0 mm

Fig. 1. Anatomical localization of region of interest (ROI) in rat brain. Top panel: Sachosen for analysis. (A–F) Chosen slices with ROI's drawn and numbered. The linThe ROIs correspond to the following brain regions: (1) DG, (2) CA1, (3) CA3, (4)(9) CA3, (10) entorhinal cortex, (11) preoptic area, (12) septal nucleus, (13) cingstriatum, (17) hypothalamus, (18) nucleus reuniens, (19) thalamus, (20) CA1, (21)interpeduncular nucleus, (26) pituitary gland, (27) superior colliculus, (28) CA1, (

posterior to bregma according to Swanson (2004). A total of 32different ROIs were defined in these slices together with referencemuscle tissue regions. Some brain structures were covered by morethan one ROI, for instance, the various subregions of thehippocampus (see Fig. 1 for details). In every 3D MRI cube,image slices corresponding to the chosen levels were selected basedon visual landmarks. In each slice, two sets of ROIs were manuallydefined (left and right hemisphere) together with muscle tissueROIs positioned as close as possible to the brain regions in question(Fig. 1). A single manganese enhancement value for eachnumbered ROI in each animal was assessed by first calculating

gittal view showing the location of the two horizontal and four vertical levelses on the left indicate the position of the various slices relative to each other.cerebellum, (5) dorsal striatum, (6) somatosensory cortex, (7) DG, (8) CA1,ulate cortex, (14) somatosensory cortex, (15) dorsal striatum, (16) ventralCA3, (22) motor cortex, (23) medial amygdala, (24) lateral amygdala, (25)29) CA3, (30) inferior colliculus, (31) visual cortex, (32) entorhinal cortex.

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the enhancement independently for the left and the right hemi-spheres and then taking the mean of these two measurements.

The statistical analysis, performed using SPSS 13.0 (Chicago,IL, USA, 2004), was focused on the differences between the groupmeans of the manganese enhancement in each brain region. Therewere four groups: early, latent, and chronic phases of epilepsy, andcontrol. Multiple comparison using Bonferroni correction wasapplied in order to compare manganese enhancement across groupsof animals. Pb0.01 was considered significant. All results aregiven as mean±SEM.

Results

MRI signal enhancement from Mn2+ was detected throughoutthe brain 24 h after MnCl2 injection, as seen in the image of thecontrol rat in Fig. 2. Within the hippocampal formation, the typicalV-shaped dentate gyrus (DG) was enhanced, together with a bandcorresponding to the pyramidal cell layer of cornu ammonis (CA).Structural pathology was obvious at all time points after KAinjection (Fig. 2). In the early phase after KA injection there was ageneral swelling of the brain followed by beginning atrophy in thelatent phase. In the chronic phase the general atrophy was morepronounced. Measurement of manganese enhancement wasperformed in 32 regions of interest (ROIs; Fig. 1) and the resultsare shown in Table 1 and Fig. 3.

Hippocampal formation

In early, latent, and chronic phases after KA injection,manganese enhancement was significantly decreased compared tocontrol in the ROIs located in the temporal CA1 (ROIs 8 and 28). Inthe septal pole of CA1 (ROI 20), manganese enhancement declinedsignificantly in the chronic phase only. In the temporal part of CA3,there was an initial significant decline in manganese enhancement inthe early phase after KA injection, but the reduction did not reachstatistical significance in the latent and chronic phases (ROI 9).However, in ROI 29, which covered more of the temporal and some

Fig. 2. T1-weightedMEMRI in the different stages of epileptogenesis. Top panels: Aof the coronal slices in the bottom panels and vice versa. In the early phase, a generaand shrinkage of the cortex are seen.

of the septal CA3, there was a significant reduction compared tocontrols at all times after KA injection. The septal pole of CA3 (ROI21) was at control level throughout. In both septal and temporal DG,manganese enhancement was significantly reduced compared tocontrol in the early and latent, but not in the chronic, phases.

Other limbic structures

In the entorhinal cortex, manganese enhancement was signifi-cantly decreased in the early phase. In the latent and chronic phases,the signal returned to control level in ROI 10 of the entorhinal cortex,and was significantly increased in ROI 32 in the chronic phase.There were no changes in manganese enhancement in the medialamygdala following KA injection. In the lateral amygdala, manga-nese enhancement was significantly decreased in the early phase,returned to control in latent phase, and then increased significantlycompared to all other time points in the chronic phase.

Subcortical structures and cerebellum

In the preoptic area, ventral striatum and interpeduncularnucleus there was a significant decline in manganese enhancementin both early and chronic phases. In the thalamus and septalnucleus, manganese enhancement was significantly reduced in thechronic phase only. There was no significant change in manganeseenhancement following KA injection in the nucleus reuniens andhypothalamus. In dorsal striatum and cerebellum, manganeseenhancement was significantly reduced in the chronic phase. In thesuperior colliculi, the signal was significantly reduced in the earlyand latent phases compared to control, and fell to an even lowerlevel in the chronic phase. In the inferior colliculi, the signal wassignificantly reduced in the early phase compared to control, butreturned to control levels in the latent phase. In the chronic phase,there was a pronounced drop in manganese enhancement to a levelsignificantly below that measured in all other groups. In thepituitary gland, manganese enhancement remained at control levelsin all groups.

xial views, lower panels: coronal views. Lines in top panels indicate locationl swelling of the brain is observed, while at the later stages enlarged ventricles

Table 1Manganese enhancement in regions of interest (ROIs) in rat brain in early, latent, and chronic phases after systemic kainic acid or saline administration

Slice ID ROI ID Anatomical region Control (n=9) Early (n=5) Latent (n=8) Chronic (n=3)

Hippocampal formationA 1 DG 4.12±0.08 3.22±0.10C 3.58±0.08C 3.65±0.20B 7 DG 3.50±0.07 2.56±0.05C 3.04±0.08C,E 3.08±0.06E

A 2 CA1 3.10±0.08 2.24±0.05C 2.55±0.09C 2.58±0.20B 8 CA1 2.47±0.04 1.54±0.80C 1.91±0.80C,E 1.83±0.70C

D 20 CA1 3.20±0.09 3.07±0.07 2.89±0.10 2.32±0.27C,E,L

E 28 CA1 2.76±0.06 2.06±0.06C 2.25±0.05C 1.80±0.08C,L

A 3 CA3 3.70±0.10 3.02±0.10C 3.28±0.10 3.56±0.22B 9 CA3 3.23±0.05 2.45±0.08C 2.91±0.07E 2.98±0.10E

D 21 CA3 3.50±0.07 3.58±0.10 3.41±0.10 3.45±0.18E 29 CA3 3.62±0.04 2.73±0.05C 2.95±0.06C 2.79±0.12C

Other limbic structuresB 10 Ent Cortex 2.30±0.05 1.74±0.09C 2.19±0.08E 2.23±0.21F 32 Ent Cortex 2.49±0.07 2.05±0.07C 2.35±0.07 3.46±0.06C,E,L

D 23 Med Amyg 2.92±0.06 2.34±0.13 2.71±0.08 2.76±0.26D 24 Lat Amyg 2.35±0.10 1.51±0.06C 1.98±0.07E 3.67±0.39C,E,L

Subcortical structures and cerebellumD 19 Thalamus 2.32±0.04 2.38±0.10 2.22±0.06 1.37±0.11C,E,L

D 18 Nuc Reun 2.27±0.08 2.50±0.16 2.25±0.07 1.59±0.21E

C 12 Sept Nuc 3.81±0.06 3.46±0.14 3.42±0.06 2.23±0.38C,E,L

C 11 Preopt Area 2.65±0.06 2.09±0.09C 2.43±0.13 1.72±0.07C,L

D 17 Hypothal 3.00±0.04 2.92±0.07 2.98±0.06 2.96±0.26C 16 Ven Striatum 1.29±0.07 0.76±0.06C 1.09±0.06 0.29±0.17C,L

E 25 IntPed nuc 4.38±0.04 3.60±0.18C 3.98±0.09 3.37±0.28C

A 5 Dor Striatum 2.56±0.06 2.53±0.09 2.53±0.07 2.05±0.05C,E,L

C 15 Dor Striatum 2.95±0.06 2.65±0.13 2.71±0.03 1.50±0.28C,E,L

E 27 Sup Coll 3.13±0.05 2.52±0.05C 2.70±0.06C 1.49±0.15C,E,L

F 30 Inf Coll 3.33±0.07 2.69±0.05C 3.03±0.05 2.09±0.15C,E,L

A 4 Cerebellum 2.50±0.08 2.39±0.10 2.40±0.08 1.43±0.13C,E,L

E 26 Pit Gland 6.24±0.08 5.86±0.11 6.14±0.05 6.07±0.16

Cortical areasC 13 Cing Cx 3.05±0.05 2.89±0.13 2.91±0.04 1.74±0.25C,E,L

F 31 Vis Cx 2.81±0.06 2.49±0.05 2.65±0.08 2.68±0.12D 22 Motor Cx 2.48±0.06 2.90±0.03C 2.68±0.05 2.52±0.14E

A 6 SomSens Cx 1.65±0.07 1.63±0.10 1.73±0.05 1.61±0.11C 14 SomSens Cx 2.20±0.06 2.05±0.14 2.14±0.04 1.71±0.10

For ROI locations, see Fig. 1 which gives the slice ID and ROI ID.The manganese enhancement contrast values were multiplied with 10. All values are mean±SEM.Superscripts C, E, L indicate significant difference (Bonferroni corrected) on the Pb0.01 level as compared to the control, early and latent phases, respectively.MnCl2 was injected subcutaneously 2 days (early), 15 days (latent), or 11 weeks (chronic) after systemic kainic acid administration. MRI was performed 24 hafter manganese administration.Abbreviations: CA, cornu ammonis; Cing, cingulated cortex; Coll, colliculus; Cx, cortex; DG, dentate gyrus; Dor, dorsal; Ent, entorhinal cortex; Hypothal,hypothalamus; Inf, inferior; IntPed, interpeduncular; Lat, lateral; Med, medial; Nuc, nucleus; Pit, Pituitary; Preopt, preoptic; Reun, reuniens; Sept, septal;SomSens, somatosensory; Sup, superior; Ven, ventral; Vis, visual.

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Cortical areas

In visual and somatosensory cortices, manganese enhancementremained at control levels. In contrast, the signal was significantlyincreased in the motor cortex in the early phase, but returned tocontrol level in the latent and chronic phases. In the anterior cingu-late cortex, manganese enhancement was significantly reduced inthe chronic phase.

Discussion

The present study revealed specific temporospatial changes inmanganese enhancement in rats during the course of epileptogen-

esis. In the early phase, ROIs in the medial temporal lobe showedthe greatest decrease in manganese enhancement. Most of theseROIs exhibited a partial recovery in the latent phase, and two areas(the entorhinal cortex and lateral amygdala) ultimately revealed asignificantly increased signal in the chronic phase. In almost allother brain regions, including the cerebellum, manganese enhance-ment was significantly lower in the chronic phase than in thecontrols.

The factors that underlie changes in manganese enhancementremain to be established. Several alternative hypotheses have beenproposed encompassing both cellular and electrophysiologicalmechanisms (Van der Linden et al., 2002; Aoki et al., 2002, 2004).As stated in the Introduction, Mn2+ uptake has been demonstrated

Fig. 3. Changes in manganese enhancement induced by systemic kainateadministration in percent relative to control (100%) in selected brain regions.Early is 3 days, latent 15 days, and chronic 11 weeks after kainateadministration. The lateral amygdala corresponds to ROI 24; entorhinalcortex to ROI 32; CA3 to ROI 9, DG to ROI 7; CA1 to ROI 8; thalamus toROI 19; cerebellum to ROI 4. For statistical significance, see Table 1.Abbreviations: CA, Cornu Ammonis; DG, dentate gyrus.

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to be directly coupled to neuronal calcium uptake and correlated tolocal neuronal activity (Takeda et al., 1994; Watanabe et al., 2004;Yu et al., 2005; Kuo et al., 2006). In situations with pathologicallyincreased calcium influx into neurons, such as focal ischemia,manganese enhancement was increased in the irreversibly damagedneurons in the hyperacute phase (Aoki et al., 2003, 2004). Theexpression of transferrin receptors and DMT1, proteins transport-ing Mn2+ into brain cells (Aschner et al., 1999; Malecki et al.,1999), may also influence intracellular Mn2+ concentrations.Further, changes in the concentration of the mitochondrial enzymemanganese-binding superoxide dismutase (Mn-SOD) could affectmanganese enhancement. The latter enzyme is predominantlyneuronal, but is also found in astrocytes (Lindenau et al., 2000).

Manganese is also taken up into glial cells. Astrocytes areendowed with ligand- and voltage-gated Ca2+ channels. In fact,under physiological conditions, astrocytes contain approximately80% of total brain Mn2+ (Takeda, 2003) because Mn2+ binds to theenzyme glutamine synthetase that is found exclusively in astrocytes(Norenberg andMartinez-Hernandez, 1979). In astrocytes, increasedexpression of transferrin receptors andDMT1 has been demonstratedto increasemanganese uptake (Erikson andAschner, 2006). It shouldalso be noted that recent evidence points to activated microglia as asink for extracellular Mn2+. A strong positive correlation betweenmanganese enhancement and microglia activation has been demon-strated in a rat model of perinatal hypoxia–ischemia (personalcommunication, Dr. Marius Widerøe, NTNU, Norway).

Manganese is neurotoxic if applied in high doses over aprolonged period, causing neurodegeneration especially in thebasal ganglia (Dobson et al., 2004). The acute effects of manganeseon brain cells are not well known, but one study has demonstratedthat manganese administration leads to calcium sequestration inglial mitochondria (Tjalkens et al., 2006).

MEMRI is a relatively new method, used in a variety of ways bydifferent groups, depending on the hypothesis in question. Often, thecontrast-to-noise ratio (CNR) is usedwhen comparing differentMRIsignal intensities. However, because the sensitivity of the surfacecoil used for reception decreases with the distance from the coil,untreated CNR values collected from different regions of interests(ROI) are not comparable. To compensate for this effect, we chose acombined method based on a partial compensation of the coilsensitivity and the use of an additional local reference for thecontrast measurements. This method does not guarantee fullcompensation of the varying sensitivity effect, which remains as apossible confounding factor. Spatial variations in the excitation field(B1) can act as an additional confounder. These problems are likelyto be of limited significance when comparing the manganeseenhancement of the same brain region in different animals. Thus,both B1 and the receiver sensitivity are expected to be similar indifferent studies given that the animals are reproducibly positionedin the scanner. However, caution should be exercised whencomparing the manganese enhancement in different brain regions.

In the following we will discuss the changes in manganeseenhancement that occurred in the different ROIs in the present study,and correlate these results to the extensive histological and electro-physiological work that has been published on this animal model overclose to three decades (Schwob et al., 1980; Ben-Ari et al., 1980).

Early phase

Histologically, unspecific necrosis, perivascular hemorrhages,and cytotoxic edema predominate in the hippocampal formation in

the acute phase after kainate injections (Sperk et al., 1983; Zuckeret al., 1983). The CA3 and CA1 pyramidal cells and hilar inter-neurons of the DG are susceptible to cell death after KA, whereasthe CA2 neurons and the dentate granule cells are relatively spared(Nadler, 1979; Ben-Ari, 1985; Sperk, 1994). It should be noted thatthe ROI termed “dentate gyrus” also includes parts of the hilarregion.

In the early phase, manganese enhancement was reduced toabout the same extent in all hippocampal subregions, except theseptal pole of CA1 and CA3, which showed no clear reduction.Histological studies have demonstrated that acute cell death ismore extensive in septal than in temporal hippocampus after KAinduced SE (Brandt et al., 2003; Hammer, unpublished results).Thus, the observed reduction in manganese enhancement cannot beexplained by neuronal loss alone, but must reflect overall reductionof neuronal activity in the hippocampus. Supporting this notion,the mitochondrial enzyme, Mn-SOD, has been shown byimmunocytochemistry to decline before neuronal loss is present(Kim et al., 2000), and glucose metabolism is decreased in thehippocampus in the early stage (Ben-Ari et al., 1981). Similarly tothe temporal poles of the hippocampus, manganese enhancementwas initially decreased in the entorhinal cortex and lateralamygdala, which are limbic structures with extensive reciprocalconnections to the hippocampus.

There was no change in manganese enhancement in subcorticalstructures such as the thalamus, hypothalamus, and striatum or inthe cerebellum, indicating no overt acute effects of KA on relevantelectrophysiological and biochemical parameters. In somatosen-sory and visual cortices, manganese enhancement remained atcontrol level, but a significant increase was detected in theprimary motor cortex in the early phase. It is conceivable thatstatus epilepticus induced changes in the motor cortex which, inturn, led to elevated manganese uptake. However, all these cortical

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regions have minimal cell death in the KA model (Schwob et al.,1980). The superior and inferior colliculi displayed acute andlasting decreases in manganese enhancement. The superior colli-culi appear to be particularly sensitive to KA (Fosse and Fonnum,1986).

In summary, reduced manganese enhancement in the earlyphase can be attributed to decreased neuronal activity subsequentto the acute toxic effects of KA.

Latent phase

Manganese enhancement in CA3 and DG recovered from theearly phase to the latent phase. Throughout the latent phase,hippocampal neurons undergo progressive cell death accompaniedby gliosis (Sperk et al., 1983). Reactive astrocytes in the glioticareas could conceivably increase their Mn2+ uptake via mechan-isms described above. In fact, the level of Mn-SOD is increased inastrocytes on days 3 and 7 in the KA model, mainly in the CA1subregion (Kim et al., 2000), but here manganese enhancementwas consistently decreased. Also, the expression of manganese-dependent glutamine synthetase is increased in astrocytes in thelatent phase of KA-induced epilepsy (Hammer, 2006). Sincegliosis is observed throughout the entire hippocampus (Jabs et al.,1997) while changes in manganese enhancement vary along thetemporal–septal axis and between different anatomical subregions,increased astrocytic Mn2+ uptake cannot explain all changes inmanganese enhancement. Indeed, gliosis has been shown to bemore extensive in CA1, where manganese enhancement wassignificantly decreased in the present study, than in the dentategyrus (Altar and Baudry, 1990), where manganese enhancementincreased with time.

As mentioned above, there are reports of a positive correlationbetween microglia activation and increased manganese enhance-ment. When KA is injected systemically, activated microglia can bedetected in both CA1 and CA3 from days 3 to 7 (Morita et al., 2006)as well as on day 14, but they decrease in number after 2 months(personal communication, Hammer). Accordingly, the observednormalization of manganese enhancement in CA3 and the decreasein CA1 cannot be explained by microglial activation. It follows thatthe changes in neuronal properties must be at the heart of the changesin manganese enhancement during epileptogenesis.

The increase in manganese enhancement in DG and CA3 fromthe acute to the latent phase could be explained by increasedneuronal excitability leading to increased Mn2+ uptake in theremaining neurons in these regions. Here, increased Mn2+ uptakemay reflect increased neuronal Ca2+ influx. However, as adivalent cation, manganese may conceivably also replace zinc.Zinc is a neuromodulator abundant in the boutons of the mossyfibers (Haug, 1967; Frederickson et al., 1983). Mossy fibersprouting is a prominent feature in epileptogenesis, but its causalrole in epilepsy is debated. Already 2 days after systemic KAinjection, mossy fiber sprouting occurs and it progresses with time(Cantallops and Routtenberg, 1996; Wuarin and Dudek, 2001).Sprouting of mossy fibers from dentate granule cells results inrecurrent excitation in DG and CA3 (Wenzel et al., 2000; Cavazoset al., 2003; Siddiqui and Joseph, 2005). Mn2+ may possiblyreplace both Zn2+ and Ca2+, thereby leading to increased Mn2+

concentration in DG and CA3, reflecting increased density ofmossy fiber boutons and increased excitation, respectively.Nairismagi et al. (2006), using direct MnCl2 injection into thecaudal entorhinal cortex, reported a positive correlation between the

number of Mn2+-enhanced pixels and mossy fiber sproutingindependent of neuronal activity in the CA3 and DG in the latentphase. The manganese signal enhancement was significantlyincreased in these structures and in the dorsal thalamus, 3 and5 days after Mn2+ injection. There was also a non-significantincrease observed in CA1. These findings are at variance with theresults in the present study. The combination of local injection andthe longer duration from time of injection to MRI will primarilydepict local Mn2+ uptake, axonal transport and trans-synapticuptake in the major local pathways. The input function for brainuptake of systemically administrated Mn2+ will depend on factorssuch as the concentrations of manganese in blood over time, blood–brain barrier permeability and uptake into cerebrospinal fluid. Thenlocal uptake into the brain cells will be affected by factorsmentioned previously, such as cell activity for neurons, the presenceof manganese uptake mechanisms and other biological propertiesspecific for each cell type. Thus, systemic administration ofmanganese would be expected to depict individual cell properties(both neurons and glia) more than local neuronal network activity asin the Nairismagi et al. (2006) study. Moreover, Sprague–Dawleyrats were used in the present study and Wistar rats by Nairismagi etal. (2006). KA-induced neurotoxicity is strongly influenced by age,sex and strain of the rats (Kesslak et al., 1995; Veliskova et al.,2000). For instance, there is a difference in the vulnerability of CA3and CA1 pyramidal cells in Sprague–Dawley and Wistar rats. In thelatter, CA1 is most vulnerable, in the former, CA3a (Covolan andMello, 2000; Brandt et al., 2003).

Chronic phase

Among the different hippocampal subregions, CA1 was uniquein exhibiting a decrease in manganese enhancement from the latentto the chronic phase. This could be explained by progressiveneuronal loss without the restorative effect of mossy fibersprouting. It has been shown that axonal sprouting also occursbetween the CA1 pyramidal cells (Esclapez et al., 1999; Lehmannet al., 2000; Smith and Dudek, 2002; Cavazos et al., 2004). Thissprouting is less extensive than the mossy fiber sprouting andstains poorly with Timm’s procedure, thus reflecting low zinccontent (Nakajima et al., 1991; Smith and Dudek, 2002; Siddiquiand Joseph, 2005). In CA3 and the dentate gyrus, manganeseenhancement increased significantly with time. The majorstructural feature differentiating CA3 and the dentate gyrus fromCA1 is the presence of mossy fiber sprouting in the formerstructures. Mossy fiber sprouting may thus be an important factorfor manganese uptake after systemic as well as local Mn2+

administration (Nairismagi et al., 2006). The present resultssuggest that the normalization of manganese enhancement in DGand CA3 during the course of epileptogenesis could be attributedto increased neuronal excitability, coupled to the establishment ofaberrant pathways and local increase in zinc content. In CA1,neuronal loss may be the major factor contributing to the observeddecline in manganese enhancement.

Only two structures, the lateral amygdala and the entorhinalcortex, displayed a significant increase in manganese enhancementin the chronic phase. Most likely, this increase reflects theestablishment of an epileptic circuit. Sprouting of novel CA3–entorhinal fibers in late epileptogenesis may contribute. Thesefibers can possibly serve as a route for direct transmission ofepileptogenic activity from the hippocampal formation to corticalareas (Siddiqui and Joseph, 2005). It has also been proposed that

64 S. Alvestad et al. / NeuroImage 38 (2007) 57–66

the amygdala may act as a relay station for epileptic dischargespreading to extra-limbic regions (Lothman et al., 1985). The directconnections between the amygdala and the hippocampus are likelyto constitute an important pathway for establishing an epilepticcircuit (Tremblay et al., 1983). Indeed, the entorhinal cortex andthe amygdala have been reported as epileptogenic foci in bothMTLE patients and in animal models (Hudson et al., 1993;Spencer and Spencer, 1994; Bertram, 1997).

Thus, increased manganese enhancement in the lateral amyg-dala and the entorhinal cortex may reflect reorganization of theneuronal pathways and increased activity, heralding a step in thetransition to an ictal state with behavioral seizure manifestations.

The fact that manganese enhancement was increased in theentorhinal cortex and the lateral amygdala and not in the hippo-campal subregions (where epileptic seizures most often originate)may seem paradoxical, but can possibly be explained by greatercell death in the hippocampus than in other structures of theepileptic circuit. In both humans and animals, progressive neuronalloss occurs in the entorhinal cortex, but is mostly restricted to onecortical layer (layer III) (Du et al., 1995). In the amygdala, moreextensive cell death has been reported in the lateral than in themedial portions (Wolf et al., 1997; Tuunanen et al., 1999). Becausemanganese enhancement increased in the lateral, but not in themedial, amygdala, activity appears to be an important denominatorfor manganese enhancement. In the amygdala, increased glucosemetabolism and pathological electrical activity are presentthroughout the latent phase (Ben-Ari et al., 1981), consistent withthe notion that manganese enhancement is connected to neuronalactivity.

In the cingulate cortex and the majority of subcortical nucleistrongly connected to limbic structures, such as the thalamus, septalnucleus, preoptic area, ventral striatum, and interpeduncularnucleus, decreased manganese enhancement was detected. It iswell known that prolonged limbic motor seizures cause cell damagein thalamic nuclei (Schwob et al., 1980; Gale, 1992; Cassidy andGale, 1998; Brandt et al., 2003). The reduction in manganeseenhancement paralleled the reported thalamic hypometabolism inhuman temporal lobe epilepsy (Henry et al., 1993; Juhasz et al.,1999) and may reflect progressive or delayed cell death secondaryto seizure activity and/or decreased neuronal activity in theseregions.

Also, regions not strongly connected to the hippocampus orother limbic structures had significant reductions in manganeseenhancement. In the dorsal striatum and the cerebellum, manganeseenhancement was significantly decreased only in the chronic phase.There are no known acute toxic effects of KA on the cerebellum,still manganese enhancement was reduced in the chronic phase.Interestingly, the cerebellum is known to enter a hypometabolicstate in patients with temporal lobe epilepsy (Savic et al., 1996;Nelissen et al., 2006). This observation points to the importance ofneuronal activity in the manganese enhancement.

Conclusion

This study demonstrates that the epileptogenic process initiatedby systemic KA application is associated with a highly differ-entiated temporospatial pattern of manganese enhancement whichcan be distinguished using MEMRI. With reference to the well-established pattern of structural and functional changes in the KAmodel, we can safely conclude that the MEMRI signal cannotreflect a single pathological or pathophysiological event. The

changes in manganese enhancement are likely to represent the netoutcome of a number of processes, including changes in theactivity level of neuronal populations. Obviously, neuronal cellloss will tend to obscure the effects of increased neuronal activity.This may explain why the MEMRI signal was maintained close tocontrol levels in the hippocampus—a structure known to becritically involved in the epileptic circuitry that developssubsequent to kainic acid administration. The significant loss ofmanganese enhancement in the structurally intact cerebellumsupports the notion that manganese enhancement is stronglydependent on neuronal activity. Although the detailed mechanismswill have to be worked out, manganese-enhanced magneticresonance imaging represents a promising tool for the analysis ofthe structural and pathophysiological changes that underlie thedevelopment of chronic epilepsy.

Acknowledgments

Supported by FUGE MIC (Trondheim), the NorwegianResearch Council (Storforsk), and the European Union BiomedProject GRIPANNT.

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