Angiogenesis is associated with blood^brain
barrier permeability in temporal lobe epilepsy
Vale ¤rie Rigau,1,2,3,* Me ¤lanie Morin,1,2,* Marie-Claude Rousset,1,2Fre ¤ de ¤ric de Bock,1,2Aurore Lebrun,1,2
Philippe Coubes,1,2,4Marie-Christine Picot,4Michel Baldy-Moulinier,4Joe «l Bockaert,1,2Arielle Crespel1,2,4
and Mireille Lerner-Natoli1,2
1Centre National de la Recherche Scientifique UMR5203,Universite ¤ Montpellier1,Universite ¤ Montpellier 2, F34094
Montpellier,2Institut de la Sante ¤ et de la Recherche Medicale U661, F34094 Montpellier,3CHU, Laboratoire d’Anatomie et
Cytologie Pathologiques, F34295 Montpellier and4CHU,Unite ¤ d’Epileptologie, F34295 Montpellier
*These authors contributed equally to this work.
Correspondence to: Mireille Lerner-Natoli PhD, Institut de Ge ¤ nomique Fonctionnelle, 141 rue de la Cardonille, 34094
Montpellier Cedex 5, France
Previous studies from our group, focusing on neuro-glial remodelling in human temporal lobe epilepsy (TLE),
have shown the presence of immature vascular cells in various areas of the hippocampus.Here, we investigated
angiogenic processes in hippocampi surgically removed from adult patients suffering from chronic intractable
TLE, withvarious aetiologies.We comparedhippocampi fromTLEpatients to hippocampiobtained after surgery
or autopsy from non-epileptic patients (NE).We quantified the vascular density, checked for the expression of
angiogenic factors and their receptors andlooked for any blood^brain barrier (BBB) leakage.We used a relevant
model of rat limbic epilepsy, induced by lithium-pilocarpine treatment, to understand the sequence of events.
In humans, the vessel density was significantly higher inTLE than in NE patients.This was neither dependent on
the aetiology nor on the degree of neuronal loss, but was positively correlated with seizure frequency. In the
whole hippocampus, we observed many complex, tortuous microvessels. In the dentate gyrus, when the gran-
ular layer was dispersed, long microvessels appearedradially orientated.Vascular endothelial factor (VEGF) and
tyrosine kinasereceptors were detectedin differenttypes ofcells. Animpairmentofthe BBBwas demonstrated
by the loss of tight junctions and by Immunoglobulines G (IgG) leakage and accumulation in neurons. In the rat
model of TLE,VEGF over-expression and BBB impairment occurred early after status epilepticus, followed by a
progressive increase in vascularization. In humans and rodents, angiogenic processes and BBB disruption were
stillobviousinthe chronic focus, probably activatedbyrecurrent seizures.We suggestthatthe persistent leakage
of serum IgG in the interstitial space and their uptake by neurons may participate in hypoperfusion and in neu-
ronal dysfunction occurring inTLE.
Keywords: temporal lobe epilepsy; angiogenesis; vascular endothelial growth factor; blood-brain barrier disruption; IgG
Abbreviations: AVM¼arterio-venous malformation; CRYPTO¼cryptogenetic; DG¼dentate gyrus; DGL¼dentate gran-
ular layer; DNET¼dysembryoplastic neuroepithelial tumour; DYSP¼ focal dysplasia; Ext-TLE¼external temporal lobe
epilepsy; FH¼fissura hippocampi; GG¼ganglioglioma; HA¼hippocampal atrophy; HS¼hippocampal sclerosis;
ISCH¼ischemia; SE¼status epilepticus; SGL¼subgranular layer; SVZ¼sub-ventricular zone;TLE¼ temporal lobe epilepsy;
VEGF¼vascular endothelial growth factor; ZO-1¼zonula occludens-1.
Received December 29 , 2006. Revised April12, 2007 . Accepted April 30, 2007
The most common form of partial epilepsy, temporal lobe
epilepsy (TLE) is often refractory to antiepileptic drugs, but
can be treated by surgical resection of the focus (Engel,
2001). Mesial temporal lobe epilepsy (MTLE), which
represents more than 60% of TLE, is associated with
hippocampal sclerosis (HS) characterized by severe neuro-
nal loss and intense gliosis. In a previous study on tissue
remodelling in MTLE, we described abundant neural
progenitors in three areas, the sub-granular layer (SGL),
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the subventricular zone (SVZ) and the fissura hippocampi
(FH) (Crespel et al., 2005). Surprisingly, we observed
numerous vascular progenitor-like cells in the same areas.
Moreover, in the dispersed granular layer many long
microvessels, originating from the SGL, paralleled the
radial astrocytic scaffolding (Crespel et al., 2002). These
observations suggested that a vascular remodelling occurred
in MTLE. At the beginning of 20th century, vascular
dysfunction was proposed to be a causal factor for MTLE
or for HS (Bratz, 1899; Spielmeyer, 1927), but this
hypothesis was later ruled out, due to the increased local
functional impairments of the vasculature and the blood–
brain barrier (BBB) were reported in MTLE, such as a
deficit in the control of homoeostasis by aquaporin (Eid
et al., 2005) or leakage of serum albumin (Van Vliet et al.,
2007). The latter was shown to be epileptogenic (Seiffert
et al., 2004; Ivens et al., 2007). Nevertheless, all studies on
human epileptic tissue concerned exclusively MTLE. The
vascularization was not examined in TLE patients with
aetiologies other than HS, even though these forms do not
clearly differ from MTLE in terms of semiology and
sensitivity to anti-epileptic drugs. They all present similar
variations of cerebral blood flow and metabolism (Duncan,
1997; Oommen et al., 2004), suggesting that haemodynamic
changes are related to the epileptic activity per se and not to
the neuropathological substrate.
The present study in adult patients suffering from
intractable TLE with various aetiologies was designed to
investigate: (i) vascular remodelling, (ii) the expression of
angiogenic factors and tyrosine kinase receptors, (iii) a
possible impairment of the BBB. In parallel, we looked for
similar modifications during the development of epilepsy in
the rat model of lithium-pilocarpine-induced status epilep-
ticus (SE) which generates a chronic limbic epilepsy (Turski
et al., 1989).
(Gibbs, 1934). Recently,
Subjects, materials and methods
Subjects and clinical data
This study was in accordance with French Ethical Committee and
received the approval of the Comite ´ National Informatique et
Liberte ´s. All patients (or their families) were informed of
additional studies performed on surgical tissue and provided a
written consent. Tissue was obtained and used in a manner
compliant with the Declaration of Helsinki.
All patients suffered from intractable partial complex seizures
and the epileptic focus was localized on the temporal lobe, as
revealed by neurological examination, long-term EEG-video-
monitoring and morphological MRI to detect HS, defined by T2
hyper signal and decrease of hippocampal volume. They had a
right or left classical TLE, except for one patient who showed an
external temporal lobe focus. All patients underwent blood flow
studies with 99mTc-HMPAO-single photon emission computed
tomography (SPECT), during periictal and interictal periods.
All clinical data are detailed in Table 1. Surgery consisted of
anterior temporal lobectomy with amygdalo-hippocampectomy
made by the same neurosurgeon for all epileptic patients. Several
samples of each hippocampus were either rapidly frozen in liquid
nitrogen or fixed by immersion in 10% buffered formalin and
processed into liquid paraffin for histological evaluation and
immunohistochemistry. The temporal poles were directly frozen in
Two hippocampal control specimens were obtained during
tumour surgery of non-epileptic patients and were processed in a
similar way to the surgical specimens mentioned earlier. These
patients had no history of epileptic seizures but suffered from
either an anaplastic oligoastrocytoma (WHO grade III, n¼1) or
pilocytic astrocytoma (WHO grade I, n¼1) adjacent to the
tumour cell invasion or other neuropathological alterations
within the hippocampal formation. Samples of hippocampi were
rapidly frozen or fixed by immersion in 10% formalin.
In addition, three specimens from autopsied adult patients
without neurological disorders were used as controls. The post-
mortem intervals ranged from 24 to 48h. The tissues were
immersion-fixed for at least 1 month in 10% formalin and
samples were embedded in paraffin. None of these patients had
clinical evidence of neurological disease and the brains were
normal as confirmed by a thorough neuropathological examina-
tion. For the Western blot study, we selected a high-grade brain
tumour (glioblastoma) as a positive control for angiogenesis.
Rat model of limbic epilepsy
All animal procedures were conducted in accordance with the
European Communities Council Directive of November 24, 1986
(86/609/EEC) and approved by the French Ministry of Agriculture
(authorization no. 34178, M.L-N).
Surgery: 60 male Sprague–Dawley rats (Janvier, Le Genest-St-
Isle, France), weighing 200–250g at surgery were anaesthetized
intraperitoneally (i.p.) with 3ml.kg?1of Equithesin and prepared
for surgery, using a David Kopf stereotaxic apparatus. A deep
bipolar electrode (made of 2 strands of 100mm nickel chrome
insulated wires twisted together) was implanted in the right
hippocampus with the following coordinates: anterior to lamda: 4,
lateral to lambda: 2.5, inferior to lambda: 2.5). Two extradural
screws were inserted bilaterally in the parietal bone and one in the
frontal bone (ground electrode). Deep electrodes and screws were
linked to a microconnector fixed to the skull with acrylic cement.
One week post-surgery, rats were injected with lithium
(3meq.kg?1i.p., Sigma, Saint-Louis, MO). Approximately 18h
later, methylscopolamine bromide (1mg.kg?1i.p., Sigma, Saint-
Louis, MO) was administered to limit the peripheral effects of the
convulsant. Thirty minutes later, status epilepticus (SE) was
induced by injecting pilocarpine hydrochloride (30mg.kg?1, i.p.,
Sigma, Saint-Louis, MO) in 42 rats. Animals were put into
individual boxes and their microconnectors were connected to an
EEG preamplifier box (Reega mini8, Alvar, Montreuil, France).
The electrical activity, recorded by deep and extradural electrodes,
was filtered by a computer equipped with Dasylab software
For each animal, limbic seizures were recorded and observed
15–20min after pilocarpine administration. They progressed
rapidly to SE, characterized by continuous discharges on the
EEG and partial as well-generalized seizures. Two hours after SE
onset, we reduced the severity of convulsions with 2mg.kg?1
diazepam i.p. (Roche, Neuilly, France), but limbic discharges
Page 2 of15Brain (2007)V. Rigau et al.
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persisted on the EEG for several hours and rats exhibited partial
seizures, similarly to what was reported by Andre et al. (2000).
Eighteen control rats received the same treatment with lithium
and methylscopolamine, but we used saline instead of pilocarpine.
In previous experiments we and others (Andre et al., 2000)
compared EEG, behaviour and histology of naive and lithium-
treated animals and we found no difference.
Rats were sacrificed at various time-points after the onset of SE,
after an i.p. injection of 5mg.kg?1diazepam (Roche, Neuilly,
France). Except for rats sacrificed in the acute period, others were
recorded twice a week and we checked for interictal spikes or
spontaneous seizures. Seizure recording on EEG was the only
criterion to distinguish silent and chronic periods. Eleven rats died
in the first week following SE. The number of animals used in this
study was: for acute period, between 1 and 12h after SE (n¼13),
for silent period, from 4 to 14d after SE (n¼11); for chronic
period, between 21 and 28d (n¼7). Sham-injected rats were
sacrificed at similar time-points: controls for acute period: 1–3h
(n¼4); controls for silent period: 7–14d (n¼7); controls for
chronic period: 21–28d (n¼7).
Histology, histochemistry and
Human samples: formalin-fixed, paraffin-embedded 4-mm thick
hippocampal sections were deparaffinized, and re-hydrated before
histological evaluation and immunohistochemistry. For particular
protocols without aldehyde fixation, frozen tissue was cut in a
cryostat in 15mm coronal sections.
Animal model: at various time-points after SE or sham
injection, rats were sacrificed by decapitation after i.p. adminis-
tration of 4mg.kg?1diazepam (Roche, Neuilly, France) to induce
T able1 Clinical data
Age at epilepsy
onset (y or m)
L EXT .TLE
Note: F or M: female or male.L or RTLE: left or right temporal lobe epilepsy.Ext TLE: external temporal lobe epilepsy.Delay FPS-S: delay
between first partial seizure and surgery. Aetiologies: HS: hippocampal sclerosis; HA: hippocampal atrophy; DNET: dysembryoplastic
neuroepithelial tumour; DYSP: focal dysplasia; GG: ganglioglioma, AVM: arterio-venous malformation, ISCH: ischaemia,CRYPTO: crypto-
genetic. Engel’s classification:1a: Fully seizure free since surgery.1d: Free disabling seizures but generalized convulsion antiepileptic drug
withdrawal. 2a: Initially free of disabling seizures but rare seizures now. 2b: Rare disabling seizures since surgery. 3: Worthwhile improve-
ment. 4a: No worthwhile improvement, some seizure reduction.
Angiogenesis in temporal lobe epilepsyBrain (2007)Page 3 of15
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relaxation. For standard morphological and immunohistochemical
studies, brains were fixed by immersion in 4% paraformaldehyde
in 0.1M phosphate buffer and cut with a vibratome (30mm
coronal sections). For other studies, hemispheres were separated
after decapitation, one was directly frozen in nitrogen, then cut
with a cryostat (15mm coronal sections) and the other was
dissected to collect the hippocampus which was frozen for protein
extraction (see Western blot protocol).
For all patients, haematoxylin-eosin (HE) staining, performed on
four sections at different levels of anteriority, allowed to confirm
the aetiologies. All hippocampi from patients with HS diagnosed
by MRI corresponded to the histological definition of HS:
shrinkage, almost complete loss of neurons from the CA1, CA3
and end folium subfields, with relative sparing of CA2 and dentate
granule cells and astrocytosis in lesioned subfields (HS, n¼13).
The histological diagnosis in the other patients included focal
hippocampal atrophy corresponding to a loss of volume (not
diagnosed by T2 on MRI) with partial neuronal loss in CA1 and
CA4 subfields (HA, n¼3), dysembryoplastic neuroepithelial
tumour (DNET, n¼3), ganglioglioma (GG, n¼1), focal dysplasia
(DYS, n¼2), arterio-venous malformation (AVM, n¼1), para-
hippocampal ischaemia (ISCH, n¼1) and cryptogenetic with
normal histology (CRYPTO, n¼4).
For rats, Nissl or HE staining provided a way of detecting
neuronal loss and granule cell dispersion.
Histochemicaldetection of vessels
Three techniques were used to easily stain the vasculature of
human or rat hippocampi: (i) direct incubation of fixed sections
in 30,30diaminobenzidine which reveals the endogenous peroxidase
of red blood cells; (ii) direct staining of human or rat IgGs with
species-specific antibodies (Table 2) conjugated either to fluo-
rochrome or peroxidase; this technique allows to stain both
microvessels and IgG accumulation in the parenchyma in case of
BBB disruption; (iii) incubation with the biotin-conjugated
Bandeira simplicifolia isolectin B4, revealed either by Texas Red-
conjugated avidinor avidin–peroxidase
30,30diaminobenzidine as a chromogen, sections were counter-
stained with Nissl, haematoxylin or HE.
For human sections, after quenching of endogenous peroxidase,
an antigen retrieval was performed by immersion in citrate buffer,
pH¼6 and heating (40min at 100?C). The primary antibodies
and their dilutions are listed in Table 2. Biotinylated secondary
antibodies were raised against rabbit, goat or mouse IgGs at 1/500.
All immunoperoxidase reactions were performed using avidin–
biotin method and 30,30diaminobenzidine as chromogen. For
human tissue, this method was performed with the Venting
automatic immunoassaying system (Ventana Nexes, AR). For
double labelling, a second staining was performed with avidin-
alcaline phosphatase and revealed with Fast Red (Ventana Nexes,
AR). Sections were finally counterstained with haematoxylin.
For immunofluorescence in human and animal tissue, we used
secondary antibodies raised against rabbit, goat or mouse IgGs,
conjugated to Cy3 (Jackson Immunoresearch, West Grove, PA)
Alexa488 or Alexa647 (Molecular Probes, Eugene, OR), at 1/2000
For the detection of Zonula Occludens-1 (ZO-1) a marker of
tight junctions, a specific protocol was necessary in human and
rats: without aldehyde fixation, 15mm frozen sections were fixed
in cold methanol for 5min at 4?C. Then a double fluorescence
labelling with ZO-1 antibody and lectin was performed.
Microscope observation and image
Nissl or HE staining, immunohistochemistry revealed by perox-
idase or fluorescence were observed with a Leitz DMRB
microscope (Leica, Wetzlar, Germany) equipped for epifluores-
cence (with tight filter bands centred on the peaks of emission of
Alexa Fluor488, Cy3/Texas Red, Alexa Fluor350) and digitized by
a 1392?1040 resolution cooled CCD camera (Cool Snap,
Princeton Instrument, Trenton, NJ) on a computer using Cool
Snap program and transferred to Adobe Photoshop (version 7) for
Rat double-labelled sections were observed using a confocal
microscope (Zeiss 510 Meta, Go ¨ttingen, Germany) equipped with
an ?25 objective (multi-immersion, numeric opening 0.8) and an
?63 objective (oil, numeric opening 1.4). We used an argon laser
(excitation 488, emission 505–530nm) for Alexa488, a helium
laser (excitation 543, emission 585–615nm) for Texas Red and a
krypton-argon laser (excitation 647nm, emission 660–700nm) for
Alexa647. Images were collected sequentially to avoid cross-
contamination between fluorochromes. Series of 15 optical
sections were projected onto a single image plane and scanned
at 1024?1024 pixel resolution.
temporal poles were frozen in nitrogen and stored at ?80?C.
Samples were dissected, mechanically dissociated and homoge-
nized in lysis buffer containing Tris (50mM), EGTA (1mM),
sucrose (250mM), non-ionic detergent Igepal (0.05%, Sigma
Aldrich, St Quentin Fallavier, France), ionic detergent sodium
deoxycholate (0.25%, Sigma Aldrich, St Quentin Fallavier, France)
and protease inhibitor mixture (Roche Diagnostics, Meylan,
France). After 10min incubation at room temperature, samples
underwent centrifugation for 15min at 14 000rpm at 4?C.
Rat tissue: Hippocampi were frozen and stored at ?20?C
immediately after dissection. They were mechanically dissociated
and homogenized in a lysis buffer containing Tris (50mM), EGTA
sodium fluoride and 1mM orthovanadate) and protease inhibitor
2300rpm at 4?C. The supernatant was centrifuged at 37000rpm
at 4?C for 30min. Then the supernatant with soluble proteins was
stored and membrane proteins in pellet were re-suspended in
solubilization buffer containing Tris (50mM), EDTA (1mM),
phosphatase inhibitors (10mM sodium fluoride and 1mM
Diagnostics, Meylan, France).
Protein concentration was determined by using a BCA-Kit
assay (Sigma, Saint-Louis, MO). Samples of 50mg of protein
acrylamide gel, separated electrophoretically and transferred to
Page 4 of15Brain (2007)V. Rigau et al.
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polyvinyldifluoridine membranes (Hybond-C-extra, Amersham
Biosciences, UK). Membranes were incubated 2h in Tris buffered
salineþTween 5% (TBST) containing 5% skimmed milk, then
overnight at 4?C with the following antibodies diluted in
TBSTþmilk: VEGF, Flk-1, Tie-2 or Actin (see Table 2 for
source and dilution). After washes in TBST, the secondary
incubation was performed during 2h at room temperature with
peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies
(Jackson ImmunoResearch Ann Arbor, ME) 1/4000 or rabbit anti-
goat antibody 1/5000 (Chemicon Temecula, CA). After three
of peroxidase reaction products. To quantify VEGF expression
in the rat model, Western blots were analysed by densitometry
using Photoshop and ImageJ.
Data analysis and statistics
Vascularization of human hippocampi from three groups of
patients (NE n¼5, TLE without HS n¼9, TLEþHS n¼8) was
quantified as follows: for each patient, three sections cut at
different levels of the septo-temporal axis were immunostained
with an anti-Von Willebrand factor antibody. On the three
sections, images centred either on pyramidal or granular layers
were acquired at ?20 magnification in each area: CA1/2, CA3/4
and DG. The density of microvessel network was quantified
T able 2 Primary antibodies and vascular markers
Primary antibodiesSource IsotypeClone or
DilutionSupplier IHC WB
Glial fibrillary acidic
San Fransisco CA
Growth Factor (VEGF)
CD31 (PECAM1)Mouse monoclonal
Vimentine Porcine monoclonalIgG1 V91/6 Ready-
Von Willebrand Factor Mouse monoclonalIgG1
Ki67Mouse monoclonal IgG1
Neurofilaments Mouse monoclonalIgG1
Flk-1 Rabbit polyclonalIgGSc-504 1/200
lectinBiotinylated Isolectin B4L-37591/200SIGMA-ALDRICH
Human IgGMouse biotinylated
?Goat Alexa488 coupled
?Rabbit biotinylated polyclonal
Angiogenesis in temporal lobe epilepsyBrain (2007)Page 5 of15
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according to the point-counting method (de Paz and Barrio,
1985) which takes into account the number, size and tortuosity of
all vessels. A 5?5 grid (total field: 0.08mm2) was superposed to
the digitized image, the number of labelled vessels crossing the
grid was counted by an independent experimenter and expressed
as vascular density (in arbitrary units) for a 0.08mm2area. For
each patient, the values obtained from the three areas (CA1/2,
CA3/4 and DG) in the three sections were pooled to obtain a
mean value for the whole hippocampus. Individual data were
represented on a histogram.
The values of vascular density in the three groups of patients
were compared using the non-parametric Kruskall–Wallis test with
Bonferroni correction. The Spearman correlation coefficient was
used to check for relationship between vascular density and other
clinical parameters: age, gender, duration of epilepsy and seizure
In order to evaluate the progression of the vascular network in
the rat model, we quantified vascular density in the hippocampus
of pilocarpine-treatedrats and
sacrificed during acute, silent or chronic periods. Coronal sections
were collected at three levels of the septo-temporal axis and
stained with 30,30diaminobenzidine and haematoxylin. For each
animal, four images at ?1.25 magnification of the hippocampus
(anterior, median, postero-dorsal, postero-ventral) were digitized.
The surfaces of the hippocampus and of the surrounding lateral
ventricle were measured using Photoshop and ImageJ (Fig. 6B).
The ratio of surfaces: hippocampus/(hippocampusþventricle) was
used as an index of hippocampal shrinkage and ventricle
dilatation of dorsal and ventral hippocampus. At each level
(anterior, median, postero-dorsal, postero-ventral), images centred
on CA1median, CA1lateral, CA3, CA4 and DG were acquired at
?5 magnification and digitized (Fig. 6B). The point-counting was
made with a 5?5 grid (total field: 1mm2) by two independent
experimenters. The score was expressed in arbitrary units of
vascular density for a 1mm2area. The values of vascular density
in the four areas (CA1medianþlateral, CA3, CA4 and DG) were
weighed using the ratio described earlier to take into account the
We looked for putative differences in vascularization of the
anterior and posterior parts of the hippocampus by measuring
anterior and posterior densities in each animal, compared between
the different groups by the paired-sample Wilcoxon rank test.
To reveal differences in vascular density between pilocarpine-
treated rats and age-matched controls all individual data were
pooled (for each area and for the whole hippocampus) and
compared at each period, using the Kruskal–Wallis test and
For animal studies, we quantified Western blot analysis by
measuring optical density and compared control and pilocarpine-
Vascularization of the hippocampus
An increase in the density of the vascular network was
observed in TLE patients, whatever their aetiology: the size
of large vessels remained unchanged, but the microvessels
appeared longer, more tortuous and numerous than in NE
subjects in all hippocampal areas, particularly in the layers
containing neuronal cell bodies (Fig. 1A). Moreover, in
patients with TLEþHS, the microvessels appeared radially
orientated in the dispersed granular layer. The histogram in
Fig. 1B shows individual values of vascular density for the
whole hippocampus. The comparison of groups of patients
revealed significant differences between epileptic and non-
epileptic subjects (NE versus TLE and NE versus TLEþHS,
P50.01). There was no difference between either groups of
epileptic patients (TLE versus TLEþHS) and no correlation
was found between the vascular density and the intensity of
damage (ranging from cryptogenetic TLE to TLEþHS). We
found that the vascular density was correlated neither to
age and gender of patients, nor to duration of epilepsy.
Fig.1 Vascularization of the hippocampus in NE and TLE patients.
(A) Immunohistochemical detection of Von Willebrand factor in
CA1area and in dentate gyrus (DG). Anti-F8 antibody,
Haematoxylin counterstaining. NE: non-epileptic patient,TLE
(crypto): cryptogenetic temporal lobe epilepsy;TLEþHS: temporal
lobe epilepsy with hippocampal sclerosis, pl: pyramidal layer, gl:
granular layer, sgl: subgranular layer, m: dentate molecular layer.
Scale bar: 50mm. (B) Vascular density in the hippocampus was
evaluatedby the pointcountingmethod and expressedin arbitrary
units for five NE patients, nineTLE patients with other aetiologies
than HS and eight TLE patients with HS.Crypto: cryptogenetic,
AVM: arterio-venous malformation, DNET: dysembryoplastic
neuroepithelial tumour,GG: ganglioglioma, HA: hippocampal
atrophy, DYS: focal dysplasia. Arrowheads show twoTLE patients
who had a very low seizure frequency.For each group of patients,
mean and SEM were compared by Kruskall^Wallis test with
Page 6 of15 Brain (2007)V. Rigau et al.
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Nevertheless, it was significantly correlated with the
P50.05). It is worth noting that two patients with less
than one seizure per month (see arrowheads on Fig. 1B)
showed a low density of vessels, while other patients with
1 to 20 seizures per month displayed similar increases in
Expression of the vascular endothelial
We evaluated the expression of VEGF in the hippocampi of
all TLE and NE patients. A basal level of VEGF expression
was observed in neurons of control hippocampi, but in one
NE patient (autopsy after suicide) the VEGF staining was
very strong in CA1 pyramidal neurons. This demonstrates
that these neurons are extremely sensitive to anoxia
and that VEGF induction is rapid. Therefore, only for
VEGF expression, the hippocampi obtained after ablation
of para-hippocampal tumours under the same surgical
conditions as for TLE patients, were used as controls.
In patients with TLE, pyramidal neurons and granule
cells were strongly immunopositive for VEGF. Other cell
types were also stained, but this expression seemed to
depend on aetiology: in cases of TLE secondary to
ischaemia or arterio-venous malformation, VEGF was
highly expressed in vascular cells, whereas in all patients
with HS, VEGF was present in reactive astrocytes of the
CA1 area (Fig. 2A).
Western blot analysis was used to evaluate the expression
of different isoforms of VEGF by their molecular weight.
In human, we observed two isoforms, one at 46kDa
corresponding to the long form of VEGF165 and the other
at 21kDa corresponding to the short form of VEGF121
(Tee and Jaffe, 2001). A high grade tumour (glioblastoma)
was used as a positive control for a maximal expression of
VEGF isoforms. These results confirmed the slight basal
level of VEGF in the hippocampus of NE patients and its
Fig. 2 VEGF expression in humanTLE. (A) Immunostaining of VEGF (brown) and vimentin (red) in CA1and dentate gyrus from a
non-epileptic patient (NE) and from two patients withTLE, either secondary to an ischaemic event (isch) or associated with hippocampal
sclerosis (HS). Higher magnification: astrocyte positive for VEGF and vimentin. Haematoxylin counterstaining. pl: pyramidal layer, gl:
granular layer, m: dentate molecular layer. Scale bar: 50mm. (B) Detection on Western blot of the different isoforms of VEGF. NE:
hippocampus surgically removed from a non-epileptic patient.TLE patients with various aetiologies; hippocampal sclerosis (HS) or
hippocampal atrophy (HA) or focal dysplasia (Dy). A tumour tissue (glioblastoma) was used as a positive control for angiogenesis and
actin antibody as a control of experimental variations.
Angiogenesis in temporal lobe epilepsyBrain (2007)Page 7 of15
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over-expression (both isoforms) in different TLE patients
with either HS or other aetiologies (Fig. 2B). Patients with a
low seizure frequency and a ‘normal’ vascularization had a
moderate expression of VEGF, higher than NE patients (see
the lane ‘HA’ on western blot).
Expression of tyrosine kinase receptors
The VEGF receptor 2 (VEGF-R2 or Flk-1) was expressed in
very few vascular cells in the hippocampus of NE subjects;
in TLE patients, Flk-1 immunostaining was obvious in all
hippocampal areas, mainly on short, branched and sprout-
ing collaterals of microvessels (Fig. 3A).
angiopoietin receptor Tie-2, which is also crucial in
angiogenesis and BBB integrity (Zhang and Chopp, 2002;
Eklund and Olsen, 2006). Tie-2 was rarely detected in
control tissues, whereas many Tie-2-positive cells were
obvious in the hippocampi of all TLE patients, in vascular
cytoplasmic Tie-2 staining was present in numerous
single, small, round cells. They seemed to derive from
highly vascularized areas (SVZ, FH) and to invade all
hippocampal regions. In the vicinity of microvessels, some
of them were grouped in clusters along the vascular walls
(Fig. 3B). These Tie-2-positive cells were immunoreactive
for CD31 (Fig. 3C) and lectin (not shown). Like for VEGF,
the expression of both receptors did not differ depending
on the aetiology of TLE (Fig. 3D).
Disruption of the blood^brain barrier
To evaluate the putative damage of the BBB, we looked for
a leakage of serum IgGs using immunohistochemistry.
In NE patients, IgGs were restricted to the inside of vessels
(not shown), whereas in TLE subjects we observed an
extravascular IgG staining in different areas. IgGs formed
halos with a concentration gradient around vessels in the
dentate granular layer and the hilus. In CA1/CA3 areas,
Surprisingly, the cytoplasm and dendrites of numerous
pyramidal neurons were clearly immunoreactive for IgGs
(Fig. 4A). Additionally, we checked for the expression of
ZO-1, a marker of tight junctions, in only one patient
with TLE; the immunostaining required a specific fixative
protocol, we therefore could not use other samples.
We observed an irregular and discontinuous expression of
ZO-1 in microvessels identified by lectin (Fig. 4B).
Angiogenic processes in extra-hippocampal
structures of the epileptogenic network
To address the question of vascular changes in extra-
hippocampal structures, we looked for angiogenic factors
and receptors in the temporal pole of TLE patients with
different aetiologies. The levels of VEGF isoforms, of Flk-1
and of Tie-2, evaluated byWesternblotting, were
comparable to those measured in the hippocampus,
(Fig. 5A). Therefore, angiogenic factors seem to be highly
expressed in extra-hippocampal structures which participate
in the epileptogenic network. It is worth noting that
haemodynamic variations affect the temporal pole as well as
the hippocampus. These variations do not depend on
damage, as illustrated by SPECT images of cerebral blood
flow, in a patient with a cryptogenetic TLE (Fig. 5B).
Neuronal loss, shrinkage and
neo-vascularization in the animal
model of TLE
In rats, the lithium-pilocarpine injection induced a limbic,
secondarily generalized status epilepticus. Neuronal loss
affected many structures: hippocampus, amygdala, piriform
and entorhinal cortices, thalamic nuclei and substantia nigra.
3–4 weeks (chronic period). At this time, neuronal loss was
evident in pyramidal layers of CA1, CA3 and CA4, frequently
associated with a severe dilatation of lateral ventricles
surrounding the shrunken dorsal hippocampus. The surface
ratio hippocampus/(hippocampusþventricle) decreased gra-
dually, mainly in dorsal hippocampus, reflecting a progressive
shrinkage (around 10% at 1 week post-SE and 520% during
the chronic period).
As shown in Fig. 6A, the vascular network increased in
epileptic rats. There was no obvious difference in the density
of vessels between rats sacrificed during SE and controls.
During the silent period, microvessels appeared more
numerous in the SVZ, FH and SGL; in the chronic period,
their number, size and complexity were greatly increased in
these areas and in the pyramidal and granular layers.
Quantification with the point-counting method revealed
that rats showed an increase in vascular density during
silent and chronic periods compared to age-matched
Moreover, individual values were stronger in anterior
than in posterior hippocampus, even after correction by
the shrinkage ratio (P50.05).
Angiogenic factors in the animal
model of TLE
VEGF expression was studied in rat hippocampi during
acute, silent and chronic periods (Fig. 7A). In control rats,
neurons and astrocytes expressed a basal level of VEGF.
During SE, some neurons showed a high level of VEGF
likely stored in vesicle-like structures. During the silent
period, both neurons and astrocytes strongly expressed
VEGF in large vesicles. During the chronic period, the main
source of VEGF was the high amount of hypertrophic,
reactive astrocytes. Western blot analysis revealed two
bands corresponding to long forms of VEGF165 (46kDa)
and VEGF121 (35kDa) (Fig. 7B). The levels increased
Page 8 of15 Brain (2007)V. Rigau et al.
by guest on June 5, 2013
significantly in SE rats during the acute period compared to
controls (P50.05), decreased to basal levels in the silent
period and slightly increased again during the chronic
period (Fig. 7C).
BBB disruption in the animal model of TLE
The detection of extra-vascular IgGs (Fig. 8A) revealed a
rapid BBB impairment after SE and, as for human TLE, a
particular cytoplasmic staining in neurons. IgGs were first
found in interneurons of the stratum oriens and the hilus
during the acute period, then in numerous pyramidal
neurons of CA1, CA3 and CA4, intensely during the silent
period and at a lower level during the chronic period.
counterstained with HE, we superposed the brightfield
image of IgGs and the fluorescent image of eosin.
Fig. 3 Expression of receptors of angiogenic factors in HumanTLE. Non-epileptic (NE, left) and epileptic patients (TLE, right).
(A) Immuno-detection of Flk-1 (VEGF receptor-2) in thin sprouting collaterals, in CA1area or in the vicinity of the fissura hippocampi (FH)
fromTLE patients. Arrowheads indicate stained microvessels observed at higher magnifications. pl: pyramidal layer, ml: hippocampal
molecular layer, m: dentate molecular layer. (B) Immunostaining of Tie-2 (angiopoietin receptor) in microvessels of the granular layer (gl) of
the dentate gyrus (DG) and of the CA1area fromTLE patients.Tie-2 is also expressed by small round cells, either single or arranged in
clusters close to vessels. Scale bar: 50mm for A and B (DG), 25mm for B (CA1). Haematoxylin counterstaining. (C) Immunofluorescent
detection of Tie-2 (left),CD31 (middle) and merge (right), suggesting that singleTie-2 positive cells are endothelial immature cells. Scale
bar: 25mm. (D): Western blot with antibodies against Flk-1and Tie-2.TLE patients with various aetiologies; hippocampal sclerosis (HS)
or hippocampal atrophy (HA) or focal dysplasia (Dy). NE: hippocampus surgically removed from a non-epileptic patient. A tumour tissue
(glioblastoma) was used as a positive control for angiogenesis and actin antibody as a control of experimental variations.
Angiogenesis in temporal lobe epilepsyBrain (2007)Page 9 of15
by guest on June 5, 2013
We observed that most IgG-positive neurons were eosino-
philic during silent and chronic periods. The loss of tight
junctions was assessed by ZO-1 staining at all time-points.
Hippocampal vessels of control rats showed a uniform
staining of ZO-1at inter-endothelial junctions, whereas 3h
after the beginning of SE and at later stages, the vascular
ZO-1 staining was discontinuous (Fig. 8B).
This study demonstrates for the first time an aberrant
angiogenesis in chronic temporal lobe epilepsy, whatever
the history of the disease or its morphological substrate.
Using a relevant animal model of limbic epilepsy, we were
able to provide evidence of some key events involved
progressively in this vascular remodelling: (1) a transient
up-regulation of VEGF in neurons after seizures, (2) the
expression of tyrosine kinase receptors in microvessels,
(3) an angiogenic sprouting, (4) the disruption of the BBB
with a leakage of IgGs, (5) their uptake by neurons.
Causes of angiogenesis
Are seizures the trigger?
We postulate that angiogenesis observed in human TLE is
not a consequence of excitotoxic processes or inflammatory
reactions, but is induced by seizures per se. Our hypothesis
is based on: (i) the massive up-regulation of VEGF in
neurons after acute, short or long lasting seizures, described
here and by others (Newton et al., 2003; Croll et al., 2004),
(ii) the comparable increase in vascular density and the
similar levels of angiogenic factors in TLE, whatever the
aetiology or the extent of damage, (iii) the similar
expression of VEGF and its receptors in the hippocampus,
Fig. 4 Disruption of the blood^brain barrier in humanTLE. (A) IgG leakage (anti-Human IgG antibody, Haematoxylin counterstaining).
IgGs form halos around microvessels inside and under the granular layer (GL). In CA1, IgG staining is diffuse in the interstitial space and
dense in the cytoplasm of pyramidal neurons. Scale bar: left and middle 200mm, right 50mm. (B) Loss of tightjunctions.Immunostaining of
zonula-occludens-1 (ZO-1, green) and labelling of vessels with isolectin B4 (IB4) in CA1area. Scale bar:15mm.
Fig. 5 Angiogenic processes and haemodynamic changes in
extra-hippocampal structures of the epileptogenic network.
(A) Expression of angiogenic factors and receptors in the temporal
pole evaluated by Western blot with antibodies against VEGF
(differentisoforms),Flk-1and Tie-2. Aetiologies were: hippocampal
sclerosis (HS), focal dysplasia (Dy) or ganglioglioma (GG). A
tumour tissue (glioblastoma) was used as a positive control and
actin antibody as a control of experimental variations.
(B) Cerebral blood flow measured by 99mTc-HMPAO-SPECT
during interictal and ictal periods in a patient with cryptogenetic
TLE. Arrows show the hippocampus and arrowheads the
temporal pole: both are hyperperfused during seizures and
hypoperfused in interictal periods.
Page10 of15Brain (2007)V. Rigau et al.
by guest on June 5, 2013
the primary focus and in the temporal pole cortex, a
structureinvolved in the
(Chabardes et al., 2005).
Once initiated, the angiogenic processes increase pro-
gressively, even in the absence of seizures as observed
during the silent period, in the present study, or after single
short seizures induced by electro-convulsive shock (Newton
et al., 2003; Hellsten et al., 2005; Newton et al., 2006).
In the chronic period, the high expression of VEGF and
tyrosine kinase receptors suggest that angiogenesis is either
chronic or recurrently activated by spontaneous seizures.
To support the second hypothesis, we found a positive
correlation between seizure frequency and vascular density.
Up-regulation of VEGF and Flk-1
We suggest that the first step in the course of angiogenic
events is the induction of VEGF by seizures. In rats, an
up-regulation of VEGF in neurons and glia was described
Fig. 6 Increased vascularization of the hippocampus in a rat model of limbic status epilepticus (SE) induced by lithium-pilocarpine.
(A) Vascularization in different regions of the hippocampus in a control rat and rats sacrificed during acute, silent and chronic periods;
upper images show the subventricular zone (svz) and the pyramidal layer (pl) of CA1, middle images the fissura hippocampi (fh) and the
molecular layer (ml) of CA1, lower images the subgranular layer (sgl) the granular layer (gl) and the stratum moleculare (m) of the dentate
gyrus. Detection of endogenous peroxidase by di-aminobenzidine, Nissl counterstaining, scale bar:100mm. (B) Schema superposed on a
digitized image of the hippocampus, showing the hippocampal (yellow) and ventricular (pink) surfaces which were measured to evaluate
the hippocampal shrinkage; white frames represent the position of the areas selected to quantify the density of vessels, as described in
methods. (C) Means and SEM of vessel density in the hippocampus of rats sacrificed at different periods (acute, silent and chronic) after
pilocarpine-induced SE and of age-matched controls.?P50.02,??P50.01, Kruskall^Wallis test, Bonferroni correction.
Angiogenesis in temporal lobe epilepsy Brain (2007) Page11of15
by guest on June 5, 2013
after SE by Croll et al. (2004). These authors proposed that
hypoxia was the triggering factor, since the VEGF promoter
contains a responsive element to the hypoxia-inducible
factor (HIF) (Semenza, 2000). This is likely to be the case
during a long-lasting SE. Nevertheless, VEGF can be also
induced in response to intracellular signals of metabolic
stress or to extracellular stimuli such as cytokines (Pages
and Pouyssegur, 2005), that are known to occur in human
and experimental seizures (Kann et al., 2005; Vezzani and
In the chronic focus, we observed a strong VEGF
expression by neurons in all cases of TLE. Glial or vascular
expression of VEGF was rare and probably correlated with
the aetiology: VEGF was observed in astrocytes in TLE with
HS and in vascular cells in TLE associated with extra-
hippocampal ischaemia or vascular malformation. In the rat
model of TLE with neuronal loss and gliosis, we confirmed
a progressive over-expression of VEGF by astrocytes. The
levels of both isoforms of VEGF in the hippocampus or
temporal pole of TLE patients compared to a high grade
brain tumour suggested that the chronic focus is persis-
tently exposed to a high concentration of VEGF. Even if
Fig. 7 VEGF expression in rats after limbic status epilepticus (SE).
(A) Confocal images of immunostaining of VEGF (green), NeuN
(red) and GFAP (blue) in the CA1area of control and pilocarpine-
treated rats sacrificed during acute, silent and chronic periods.The
pyramidal layer is strongly labelled with the specific neuronal
marker NeuN. Higher magnifications show neurons or astrocytes
expressing VEGF in vesicle-like structures (green spots). Scale bar:
50mm. (B) Western blot showing the different isoforms of VEGF
at 3h,1week and 3 weeks after pilocarpine-induced SE (P3h, P1w,
P3w) and at 3h or 3 weeks for control animals (C3h,C3w).The
actin antibody was used as a control of experimental variations.
(C) Quantification of Western blot analysis, expressed in optical
density, comparing theVEGF expression in the hippocampus of
pilocarpine-treated rats and age-matched controls at the three
periods (n¼2 for each condition).?P50.05. Kruskall^Wallis test,
Fig. 8 Disruption of the blood^brain barrier in rats after limbic
status epilepticus (SE). (A) IgG leakage and uptake by neurons at
various time-points after SE.Ct and SE 4d: confocal images of IgG
staining in vessels and in neurons in the CA3 area from a control
rat and a rat sacrificed during the silent period (4 days after SE).
Box: higher magnification of a CA3 pyramidal neuron double-
labelled for IgG (green) and NeuN (red). SE 6h: confocal image of
the CA1pyramidal layer (pl) and stratum oriens (so) of a rat
sacrificed during the acute period (6h of SE), stained for IgG
(green) and NeuN (red); arrowheads indicate double-labelled
interneurons. SE1w: immunoperoxidase detection of IgG in the
hilus of a rat sacrificed during the silent period (1week
post-SE) and superposition of the fluorescence emission of eosin;
the arrowhead shows an interneuron which is IgG-positive and
eosinophilic. (B) Confocal images of tight junctions, revealed by
immuno-staining of zonula-occludens-1 (ZO-1, green) and labelling
of vessels withisolectin B4 (IB4, red) in a controlrat and atvarious
time-points after SE. Arrowheads indicate the loss of tight
Page12 of15Brain (2007) V. Rigau et al.
by guest on June 5, 2013
there are other factors released during seizures that
participate in the neo-vascularization of the focus, the
local concentration of VEGF is a pivotal actor that
induces either a physiological or an aberrant angiogenesis
(Ozawa et al., 2004).
VEGF activates tyrosine kinase receptors expressed by
different types of cells which promote both angiogenesis
and neuroprotection (Storkebaum et al., 2004). The
angiogenic effects are mediated mainly by two receptors.
VEGF-R2 (Flk-1) plays a crucial role in endothelial cell
whereas VEGF-R1 modulates proliferation. The neuronal
Flk-1 seems to be the main effector of VEGF-induced
neuroprotection through the activation the PI-3 kinase/Akt
survival pathway (Kilic et al., 2006). In adult rats, VEGF-R1
and VEGF-R2 are found on the surface of endothelial cells
at very low levels. These receptors are both up-regulated
at the surface of neurons and astrocytes after stroke
(Lennmyr et al., 1998). Here, we observed a specific
staining of Flk-1 in sprouting microvessels only in TLE.
We detected no Flk-1 in neurons or glia. This suggests that
this expression is a transient protective mechanism after an
ischaemic or epileptic event.
Up-regulation of Tie-2 receptor
Angiopoietin 1 and 2 (Ang1, Ang2) are known to play a
role in physiological or post-ischaemic angiogenesis in
synergy with VEGF (Zhang and Chopp, 2002). We detected
no angiopoietins, but we were able to observe an intense
expression of their Tie-2 receptor in chronic TLE. When
activated by Ang1, Tie-2 terminates angiogenesis and
improves vascular integrity, mainly via the PI-3 kinase
pathway (Eklund and Olsen, 2006). On the contrary, Ang2
behaves as an antagonist (Bogdanovic et al., 2006). Tie-2
staining was very scarce in NE patients and strong in short
microvessels of all hippocampal areas in TLE patients,
confirming the angiogenic sprouting. Moreover, Tie-2 was
highly expressed by single cells, sometimes grouped in
clusters along the vascular walls. Their localization and their
phenotype is reminiscent of the recruitment of endothelial
progenitor cells from peripheral circulation, which migrate
to re-vascularize an ischemic tissue (Kopp et al., 2006).
Therefore, the high quantity of Tie-2 positive cells provides
further evidence of the persistence of angiogenic processes
in the chronic focus.
What are the consequences of
angiogenesis in the epileptic focus?
Generally, the rapid secretion of growth factors after
seizures is considered as a protective mechanism. This is
the case for VEGF release, since its anti-epileptogenic and
neuroprotective properties were recently demonstrated both
in vitro and in vivo (McCloskey et al., 2005).
Another effect of VEGF, that could be beneficial during
seizures, is the fast adaptation of blood flow to the
metabolic demand of neurons. VEGF, via the Flk-1/Akt
pathway, activates the endothelial nitric oxide synthase
(eNOS) which promotes immediate vasodilatation through
NO release (Ahmad et al., 2006). We previously showed
that eNOS was over-expressed in microvessels of rat
epileptic focus and that inhibiting NO synthesis dramati-
SE (Rondouin et al., 1993; Lerner-Natoli et al., 1994).
Finally, the increased density of microvessels in the
adaptation to improve perfusion during seizures. The ictal
damage, is well-documented in humans (as illustrated in
Fig. 5) and in different animal models (Lerner-Natoli et al.,
1983; Pinard et al., 1987; Pereira de Vasconcelos et al.,
2002; Van Paesschen, 2004).
If, after an acute event such as a stroke, angiogenesis
contributes to tissue revascularization, persistent angio-
genic processes are considered to be deleterious for brain
tissue, mainly due to BBB permeability (Hayashi et al.,
2006). This is likely to be the case in chronic TLE and in
different acute or chronic animal models of epilepsy.
Single short seizures disrupt BBB (Goldman et al., 1987;
Devanand et al., 1994; Oztas et al., 2003) and seizure
frequency is correlated to the degree of leakage (Van Vliet
et al., 2007). VEGF is known to play a main role in
vascular permeability by activating matrix metalloproteases
which break vessel walls (Hayashi et al., 2006) and degrade
tight junctions (Zhang and Chopp, 2002). Our study on
the rat model showed that IgG leakage and ZO-1 decrease
were obvious early after SE, but also during latent
and chronic periods. Since angiogenic factors are recur-
would persist and contribute to the chronic impairment
of the BBB.
The extravasation of IgGs and other serum proteins which
accumulate in the cytoplasm of neurons has already been
reported in the case of acute BBB breakdown, auto-immune
or neurodegenerative diseases (Kitz et al., 1984; Fabian and
Petroff, 1987; Mori et al., 1991; Loberg et al., 1993; Orr
et al., 2005; Hallene et al., 2006). We observed a neuronal
uptake of IgGs in chronic TLE and the rat model showed
that neurons were progressively affected in function of
their vulnerability to seizures (first interneurons, then
CA3/CA1 pyramidal neurons). An immune effect of IgGs
on neuronal function has never been demonstrated in TLE,
but the role of auto-antibodies in different forms of
epilepsy is currently a matter of debate (McNamara, 2002;
Watson et al., 2004; Roubertie et al., 2005). Nevertheless,
Angiogenesis in temporal lobe epilepsyBrain (2007)Page13 of15
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other serum proteins with no immune function are
epileptogenic (Seiffert et al., 2004). Recently, accumulation
of albumin was described in the cytoplasm of neurons and
astrocytes in human epilepsy or after an experimental BBB
disruption (Van Vliet et al., 2007). Its epileptogenic effect
was demonstrated by Ivens et al. (2007) who showed that
albumin uptake by astrocytes altered the potassium inward
rectifying currents. Whether the accumulation of serum
proteins in the interstitial space modifies oncotic pressure
that contributes tovasogenic
hypoperfusion, remains to be elucidated.
The vascular remodelling described in this study is a
common substrate for different forms of drug-refractory
TLE and does not depend on their aetiologies. The
epileptogenic network which show regional variations of
blood flow are affected by neo-vascularization. Even if
angiogenesis accounts for neuroprotective mechanisms
during seizures, angiogenesis may be involved in the
chronic permeability of the BBB with severe consequences,
such as neurovascular uncoupling, inflammation and
excitability. Future studies should focus on factors and
signalling pathways involved in adult angiogenesis. By
understanding the complex cross-talk between neurons, glia
and endothelial cells, we may hope to develop novel
therapeutic approaches of intractable epilepsy.
Supplementary Data is available at BRAIN online.
The authors are grateful to: Dr F. Bertaso for critically
reading the manuscript, Dr G. Alonso for constructive
L. Charvet for iconography and N. Lautredou-Audouy at
the Centre Regional d’Imagerie Cellulaire for confocal
microscopy. The technical assistance of L. Dumas, S.
Lescure and D. Pierre in human histology was deeply
appreciated. This study was supported by the French
Foundation for Research on Epilepsy and the Foundation
The ´re `se and Rene ´ Planiol for Research on Human Neuro-
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