Neuroscience Letters 497 (2011) 163–171

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Review

Anti-epileptogenesis in rodent post-traumatic epilepsy models

Asla Pitkänen a,b,∗ , Tamuna Bolkvadze a , Riikka Immonen c
a
Department of Neurobiology, Epilepsy Research Laboratory, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland
b
Department of Neurology, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland
c
Department of Neurobiology, Biomedical NMR Group, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Post-traumatic epilepsy (PTE) accounts for 10–20% of symptomatic epilepsies. The urgency to understand
Received 9 August 2010 the process of post-traumatic epileptogenesis and search for antiepileptogenic treatments is emphasized
Received in revised form 25 January 2011 by a recent increase in traumatic brain injury (TBI) related to military combat or accidents in the aging
Accepted 15 February 2011
population. Recent developments in modeling of PTE in rodents have provided tools for identification of
novel drug targets for antiepileptogenesis and biomarkers for predicting the risk of epileptogenesis and
Keywords:
treatment efficacy after TBI. Here we review the available data on endophenotypes of humans and rodents
Antiepileptic drug
with TBI associated with epilepsy. Also, current understanding of the mechanisms and biomarkers for
Biomarker
Controlled cortical impact
PTE as well as factors associated with preclinical study designs are discussed. Finally, we summarize the
Lateral fluid-percussion attempts to prevent PTE in experimental models.
Traumatic brain injury © 2011 Elsevier Ireland Ltd. All rights reserved.
Magnetic resonance imaging

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
2. Risk factors for PTE in human and experimental TBI – how do they match? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.1. Human PTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.2. Experimental PTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3. In search of treatment targets – molecular and network reorganization in PTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4. Biomarkers and surrogate markers for PTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
5. Challenge of designing antiepileptogenesis trials to prevent PTE in rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
6. Disease-modification treatment approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
6.1. Antiepileptogenesis after TBI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
6.2. Co-morbidity modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

1. Introduction
[48]. After TBI, the 30-year cumulative incidence of epilepsy is 2.1%
for mild, 4.2% for moderate, and 16.7% for severe injuries [2], and TBI
The rate of hospital admissions for traumatic brain injury (TBI)
is estimated to cause 10–20% of all symptomatic epilepsies [2]. It
across Europe is estimated to be 235/100 000/year [5]. In the
can be estimated that in the USA and the European Union (EU), with
USA, the incidence of emergency department visits for TBI is
a total population of about 800 million, at least 0.5 million surviv-
403/100 000/year and that of hospital admissions 85/100 000/year
ing individuals live with post-traumatic epilepsy (PTE). Data on the
economic burden of PTE are unavailable. Some idea is provided by
the average lifetime cost of TBI, which in the USA is about $200 000
∗ Corresponding author at: A.I. Virtanen Institute for Molecular Sciences, Univer-
per case when scaled to 2004 price levels [5]. Thus, in addition to the
sity of Eastern Finland,
P.O. Box 1627, FIN-70211 Kuopio, Finland. Tel.: +358 50 517 2091;
subjective burden, the economic burden caused by PTE is substan-
fax: +358 17 163025. tial, and means to prevent post-traumatic epileptogenesis would
E-mail address: asla.pitkanen@uef.fi (A. Pitkänen). benefit both the individual and society.

0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2011.02.033
164 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171

TBI refers to a brain injury caused by an external mechan- Table 1

Comparison of risk factors for human post-traumatic epilepsy (PTE) and rat PTE in
ical force such as a blow to the head, concussive forces,
lateral fluid percussion (LFP) injury model.
acceleration–deceleration forces, blast injury or a projectile missile
such as a bullet [53]. Epilepsy is a disorder of the brain character- Risk factor for human PTE Presence in LFP injury
ized by an enduring predisposition to generate epileptic seizures Severe Moderate
and by the neurobiological, cognitive, psychological, and social con-
Penetrating injury No No
sequences of this condition. The definition of epilepsy requires the Skull fracture/wounds with Craniectomy, dura left Craniectomy, dura left
occurrence of at least one epileptic seizure [25]. After TBI, the occur- dural penetration intact intact
rence of seizures has been categorized as immediate (<24 h), early Biparietal contusions No No
(1–7 d), or late seizures (>1 wk after TBI) [27]. The immediate and Acute intracerebral Detectable in 15/15a Detectable in 15/15a
hematoma Score 3–4 in 7/15 Score 3–4 in 3/15
early seizures are considered to reflect the severity of the injury Score 4 in 5/15 Score 4 in 1/15
itself, whereas the late seizures result from maturation of epilep- Intracranial hemorrhage Yes (15/15)b Yes (15/15)b
togenic pathology. Thus, TBI associated with one unprovoked late Multiple or bilateral No No
seizure qualifies for diagnosis of PTE. contusions
Multiple intracranial n.d. n.d.
Currently, there is no universally accepted definition for epilep-
procedures
togenesis or anti-epileptogenesis. This complicates the analysis Lesion location (frontal, Yes n.a.
and interpretation of data obtained in preclinical and clinical temporal)
antiepileptogenic trials. Often “epileptogenesis” or “latency period” Evidence of greater than 1.5–3% increase in the 1.5–3% increase in the
are used synonymously as operational terms that refer to a 5-mm midline shift width of ipsilateral width of ipsilateral
hemisphere (11/15) hemisphere (6/15)
time period between the insult and the occurrence of the first
Loss of consciousness for No No
unprovoked seizure. Evidence is accumulating to show that neu- >24 h
robiological changes that occur during the latency period continue Prolonged length of n.d. n.d.
to progress even after epilepsy diagnosis [67,70]. Therefore, we post-traumatic amnesia
Coma duration >24 h n.a. n.a.
recently proposed a revised definition for epileptogenesis. The
Low GCS (<10) n.a. n.a.
major difference from the previous definition is that epileptogen- Old age n.d. n.d.
esis extends from the latency period to include the epilepsy phase Early PTS (within first week) Yes n.d.
[72]. Thus, the term “epileptogenesis” is defined as the development Epileptiform EEG activity 1 n.d. n.d.
and extension of tissue capable of generating spontaneous seizures, month post-TBI

including (a) development of an epileptic condition and (b) pro-
gression after the condition is established. To describe the effect of
treatments, we suggest the term “disease or syndrome modification”
which refers to a process that alters the development or progres-
sion of a “disease”, in this case epilepsy (either epileptic disease
or epilepsy syndrome). Disease or syndrome modifying interven-
tions may be “antiepileptogenic”, which refers to a process that
counteracts the effects of epileptogenesis, including (a) prevention
(lower percentage of subjects develop epilepsy), (b) seizure modi-
fication (e.g., shorter and less frequent seizures), and (c) cure. These
interventions could also modify comorbidities by reducing or pre-
venting deleterious nonepileptic functional changes in the brain
(e.g., memory, emotional behavior).
The number of reports demonstrating the disease modifying
effects of various treatments on the TBI aftermath in experimen-
tal models is abundant [55,68]. In particular, these studies show
favorable PTE-related co-morbidity modifying effects, including
enhanced recovery of memory, motor performance, or emotional
behavior, whereas evidence for antiepileptogenesis is meager.
Surprisingly, more information is available from humans than
experimental models, which could relate to the relatively recent
recognition of the occurrence of epilepsy in injured animals. To take
the field forward and conduct a study that focuses on prevention
of PTE after TBI, we need valid models that are reproducible and
easy to use, treatments that are designed to counteract the mech-
anisms of post-TBI epileptogenesis, biomarkers and/or surrogate
markers for predicting treatment efficacy, and optimized preclini-
cal study designs that reveal true positive data. We will next discuss
each one of these aspects and outline challenges that we are facing
when trying to tackle post-traumatic epileptogenesis.
2. Risk factors for PTE in human and experimental TBI –
how do they match?
TBI is a heterogeneous disorder with different forms of clinical
presentation. Identification of endophenotypes in injured subjects
with a high risk for PTE can guide attempts to model PTE in clinically
Abbreviations: EEG, electroencephalogram; GCS, Glasgow Coma Scale; n.a., not appli-
cable; n.d., no data; PTS, post-traumatic seizure. For references, see the text. Some
of the changes in LFP injury listed in the table are demonstrated in Fig. 1.
a
Number and size of intracerebral hematomas were scored from 0 (not present)
to 4 (several large hematomas).
b
Includes both microbleed in white and/or grey matter as well as hematomas.
relevant ways, and optimize the selection of animals for pre-
clinical antiepileptogenesis studies. Identification of epileptogenic
endophenotypes is also relevant for the identification of epilepto-
genic mechanisms and presentation of biomarkers (or surrogate
markers) at different phases of the post-traumatic epileptogenic
process. We will next compare the human and experimental data.
2.1. Human PTE
After the first late seizure, 86% of patients were reported
to develop a second seizure within 2 years, suggesting the
establishment of an epileptogenic network [31]. Further, recent
magnetoencephalography (MEG) studies revealed that some
patients without reported seizures after mild TBI have epileptiform
spikes when assessed at 12–140 months post-TBI [49]. Whether
some of these individuals would later develop epilepsy remains to
be assessed. Some studies have also investigated the persistence of
seizures after TBI. For example, Eftekhar et al. [21] reported that not
all, but 75% of patients with penetrating TBI during Iraq–Iran war
continue to have seizures after a 21-year follow-up, even when data
were adjusted for AED treatment. The risk was associated with >3
pieces of shrapnel and with sphincter disturbances. Interestingly,
no association was found between the persistence of epilepsy and
lesion size. Some important questions arising from these observa-
tions are: what are the mechanisms of post-TBI epileptogenesis,
and what mechanisms can counteract the persistence of epilepsy
in a subpopulation of patients with TBI?
Table 1 summarizes the risk factors for PTE obtained from epi-
demiological studies conducted in civilian or military populations.
Many of the identified risk factors reflect the severity of brain
injury, and show that the risk for PTE increases with the severity
 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171 165

of injury. However, data about the association of the type of injury

Reference
with epileptogenesis are meager. A recent study investigating the

[14,15]
[43,44]

[36,37]
civilian population with TBI that included MRI analysis performed

[29]

[81]

[82]

[10]
[7]

[8]
1–360 months (median 12 months) post-TBI reported an associ-
ation of PTE with contusions but not with an isolated diagnosis
of diffuse axonal injury (DAI) [74]. This study found no association
with microbleeds and PTE, which challenges the data from previous

Seizure susceptibility ↑ to ECS-induced sz at

epidemiologic studies (see Table 1).

Sz threshold as adult ±0; Sz threshold for

80% had epileptiform spiking; Increased
Increased susceptibility to PTZ-induced

Increased susceptibility to PTZ-induced

Increased susceptibility to PTZ-induced

One possible epileptogenic factor occurring during the post-TBI
susceptibility to PTZ-induced seizures aftermath is acute symptomatic status epilepticus (SE), which has
been reported to be associated with epilepsy in 41% of cases [33].
88% had epileptiform spiking
Vespa et al. [88] monitored 20 patients with severe non-penetrating
minimal clonic sz as adult ↓

TBI using cortical EEG while they received therapeutic levels of

phenytoin. Ten patients had seizures and seven of these patients
had nonconvulsive SE lasting on average 41 min during the first
week post-injury.

7 d post-TBI
Other recently reported clinical risk factors for PTE in civilian

Abbreviations: CCI, controlled cortical impact; ECS, electroconvulsive shock; FPI, flud-percussio injury; n.d., no data; sz, seizure; P, postnatal day; PTZ, pentylenetetrazol.
seizures

seizures

seizures
populations are a history of depression and three or more chronic
Other

medical conditions at the time of injury [24]. Data on genetic risk

–

factors for outcome from TBI is emerging, but very few studies have
assessed the linkage of genes to PTE [16]. One study found that
ApoE4 allele is associated with a 2.4-fold increased risk of late post-
traumatic seizures after moderate to severe TBI [19]. However, this
Ictal episode ≤10

∼90 (behavioral

was not confirmed by another study [1] which also reported no

Sz duration (s)

assessment)

association with haptoglobin phenotype.

Epileptogenesis occurs in parallel to post-injury recovery [71].
45–60

Recent data suggests that epilepsy has a negative effect on gen-

104
n.d.

n.d.

n.d.
91
50

eral functional outcome when tested using the Glasgow Outcome

Scale (GOSE) [74]. Whether this implies that the severely injured
brain recovers poorly and consequently, is prone to epileptoge-
Sz frequency

nesis remains to be studied. Other testable hypotheses are that

epileptogenic mechanisms counteract those resulting in favor-
0.23/d
0.3/d

0.1/d

able functional recovery, or that epilepsy-related psychological

n.d.

n.d.

n.d.

n.d.

n.d.
n.d

and work-related consequences compromise the outcome when

assessed using GOSE.
spontaneous sz
Latency to

2.2. Experimental PTE

6 months

6 months
4–11 wk

42–71 d
∼2 wk

260 d
n.d.

n.d.

n.d.

A large number of animal models have been developed and used

to investigate the molecular and cellular mechanisms of post-TBI
injury in rodents and pigs, and to test the efficacy of novel treat-
ments on functional recovery [60]. However, it has only recently
20%(mild) to 36%

been pointed out that a significant percentage of injured rodents

(severe injury)

develop epileptiform activity and epilepsy in long-term follow-up

Animals with
epilepsy (%)

1 of 8 (13%)

1 of 17 (6%)

(Table 2). Whether the risk factors for epileptogenesis in a sub-

Epileptogenesis after experimental traumatic brain injury in rodents.

group of animals that develop PTE after TBI correspond to that

∼92%
50%
n.d.

n.d.

n.d.

in humans is little understood. Experiments in the lateral fluid-

9%

percussion (LFP) injury model provide some data. First, as evidence

so far suggests, the development of PTE requires a severe external
impact, that is, about 3 atm when “severity” is calculated based on
Mouse (CD-1) Adult

Mouse (CD-1) Adult

Species, age (strain)

Mouse (B6) Adult

Mouse (B6) Adult

the mortality (>30%) within the injury group. This correlates with
the observations in humans showing that the risk of PTE increases
Rat P32–35

Rat P16–18

with the severity of TBI. Second, the location of the injury in the
Rat Adult

Rat Adult

Rat P17

posterior aspects of the cortex including the perirhinal, postrhinal

and entorhinal cortices is associated with rat PTE [46]. Patient data
suggests that parietal or temporal location of injury is associated
with PTE [35]. However, comparison of the injury location between
rodents and humans is difficult because of the different brain ori-
Closed skull traumatic

entation relative to the skull as well as a lissencephalic appearance

of the rodent brain. Third, as LFP injury causes both grey and white
Parasagittal FPI

brain injury

matter damage, it is of interest that also CCI, in which the damage

Weight-drop

is more confined to the grey matter, can trigger epileptogenesis

Lateral FPI

Lateral FPI

[8,36]. Whether this suggests that, as seen in patients, white mat-

Model
Table 2

ter damage in rodents is not required for epileptogenesis remains

CCI

CCI
CCI

CCI

a testable hypothesis.
166 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171

Fig. 1. Examples of T2 maps (A and B) and T2*-weighted MR images (C and D) of rats with severe (impact force >3.0 atm; left column) or moderate (1.9–2.1 atm; right
column) lateral fluid-percussion (LFP) injury. (A) At 3 d after severe TBI, several hematomas (yellow arrows), a series of microhemorrhages in white matter structures (orange
arrows), and ipsilateral cortical edema (white arrowheads) were observed. In a subgroup of animals with severe TBI, edema was associated with an increased (about 2%)
width of the ipsilateral hemisphere (indicated with white dashed line). (B) After moderate injury, MRI findings were milder than after severe TBI, particularly, the extent of
intracortical hematomas. However, there was significant overlap between the rats with severe or mild injury. For example, 5 of 15 rats with severe and 1 of 15 rats with
moderate injury has the score 4+ (most extensive) hematoma (p = 0.068, Chi-Square test). Swollen ipsilateral hemisphere was found in 11 of 15 rats with severe and 6 of 15
rats with moderate LFP injury (p = 0.065). This resulted in a 1.5–3% increase in the width of the ipsilateral hemisphere when compared to that of the contralateral hemisphere
in the slice taken from the injury epicenter. Also, as we noted in the original description, cortical lesion volume assessed by MRI was 23.9 ± 16.8 mm3 (range 0–69.5 mm3 )
in the moderate group and 31.8 ± 10.9 mm3 (range 12.9–48.4 mm3 ) in the severe group [45]. (C) A T2*-weighted MR images from a rat with severe and (D) with a moderate
injury. The arrow points to the microbleeds in the external capsule that is associated with microglial and astrocytic accumulation (see panel E and also [96]). Panels (E and F)
show the histology at 18 d post-TBI from the same rats. The black arrowhead indicates the cortical lesion. Note the remarkable neurodegeneration that happened in 2–3 wk
post-TBI. The orange arrow indicates gliosis at the site of white matter microhemorrhage detected in MRI Images 2 wk earlier (panels A–D). Scale bar in A equals 1 mm and
in E 1 mm.

We re-analyzed our recent dataset from rats with LFP-induced
TBI, which were imaged with quantitative MRI at 3 d post-injury
[44]. The goal was to assess whether some of the parameters known
to associate with a high risk of PTE in humans could also be found
in rats with severe but not with moderate injury. The parameters
assessed included the occurrence of hematoma(s), hemorrhage,
and early hemispheric swelling on MRI. Our data show that a sub-
group of rats with severe impact, and thus more likely to develop
epilepsy, had a tendency to a larger number of hematomas and
more apparent brain swelling as compared to rats with moderate
injury (Table 1; Fig. 1).
The effect of age and genetic background on experimental
post-TBI epileptogenesis has been assessed recently. CCI injury
was induced at P16–18 and rats were followed-up for up to 2.5
months. No spontaneous seizures were detected, but CCI injury
resulted in permanent lowering of the threshold for clonic seizures
[81]. Interestingly preliminary data from CCI-injured B6 mice sug-
gest that the occurrence of epilepsy is lower than in CD-1 mice,
implying that genetic background affects post-TBI epileptogene-
sis (Table 1) [8,36,37]. Genetic background has also been shown
to the affect the development of co-morbidities, including motor
and cognitive decline after experimental TBI when C57BL/6, FVB/N,
and 129/SvEMS mice were compared [26]. The effects on post-
injury neurodegeneration have been negligible when C57BL/10J
and C57BL/6J mice with subtle differences in genetic background
were compared [94]. So far, no data about single gene mutations
and the risk of PTE in rodents have been published. As some data
from patients suggest, there is an inverse association between the
occurrence of epilepsy and behavioral recovery. It is, therefore, of
interest to note that in the CCI model rats receiving two electrocon-
vulsive seizures (ECSs) within the first 24 h post-contusion showed
accelerated recovery of beam-walking ability, reduced volume of
necrosis and less spontaneous activity compared to animals receiv-
ing only contusions. The effect was even more pronounced after
 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171
7 ECSs [22]. Moreover, daily injection of PTZ (25 mg/kg) started at
24 h post-TBI induced by central FPI in rats and continued for about
4 wk improved the recovery of water-maze performance despite
of strengthening of the severity of behavioral seizures during the
follow-up, that is, induction of kindling [32]. Finally, we have not
observed SE in any of our injured animals which could have com-
promised the interpretation of the cause of epileptogenesis after
TBI.
3. In search of treatment targets – molecular and network
reorganization in PTE
After TBI, the primary injury occurring within minutes to hours
is related to the impact itself, and includes shearing of white mat-
ter tracts, focal contusions, hematomas, (micro)hemorrhages, and
diffuse swelling. At the cellular level this is seen as microporation
of membranes, leaky ion channels, and conformational changes
in proteins. Secondary damage develops over hours to days,
and includes neurotransmitter release, free-radical generation,
calcium-mediated damage, gene activation, mitochondrial dys-
function, and inflammatory responses [53]. Many of the secondary
changes have previously been associated with epileptogenesis
caused by other etiologies like SE [70]. However, causality and the
details of the overlap between molecular pathways underlying each
change in various etiologies need to be explored.
Several large scale molecular profiling studies are available from
mice and rats [11–13,23,39,40,47,50,51,57,73,90,93,95] and one in
humans with TBI [59]. In most of the studies the timing of sampling
has been within 1 wk post-injury, and therefore, involves only the
early post-injury period. Post-traumatic epileptogenicity of identi-
fied genes has not been further analyzed.
One of the prevailing hypotheses for epileptogenesis is acquired
channelopathy which appears as alterations in the expression of
ion channel subunits resulting in altered receptor composition
and increased excitability (for review, see [70]). Interestingly, we
recently showed that in the LFP injury model there are changes in
GABAA receptor subunits in the dentate granule cell layer which
last for several months [66]. Also, Gibson et al. [28] demonstrated
changes in the ␣1, ␣2, ␣3, and ␥2 subunits of the GABAA recep-
tor when assessed within 24 h after central FPI. Changes in GABAA
receptor subunit expression have a temporal association with
decreased phasic and/or tonic inhibition in the dentate granule cells
[54,61,66]. Mao et al. [54] recently showed a 3-fold increase in the
expression of the Nav 1.6 subunit in the hippocampal neurons dur-
ing the 72 h post-LFP injury in rats, but its association with post-TBI
hyperexcitability needs to be explored.
Even though the data on pathologic changes after TBI in humans
is abundant, little data are available that indicate a connection
between the cellular changes and PTE in patients. The data available
show that hippocampal pathology includes mossy fiber sprouting
as well as a 50–70% neuronal loss in pyramidal cell layers, which
have been described in TLE with other etiologies [56,83]. How-
ever, there is no available comparison of whether these changes
would differ between TBI patients with and without PTE. Recently
Tomkins et al. [86] compared the extent of blood–brain bar-
rier (BBB) disruption with MRI in TBI patients with and without
epilepsy. They found larger perilesional BBB disruption in PTE
patients as compared to TBI patients without epilepsy. Interest-
ingly, there was no difference in lesion size between the groups.
In animal models, mossy fiber sprouting has been described
both in rats and mice after CCI and/or LFP injury [36,43]. In the
LFP model, there is also hilar cell loss that varies from 20% to 75%
depending on the time point investigated as well as on the count-
ing method used [30,43,52,80]. In addition, there is a 10–20% loss
of principal cells, particularly in the CA3a region [30]. We have
167
recently shown that hilar cell loss includes over a 50% reduction
in the total number of parvalbumin positive cells which associates
with a decrease in phasic inhibition in granule cells [38,66]. Other
cell types lost in the hilus include mossy cells (35% remaining),
cholecystokinin immunoreactive (IR) cells (44%), and somatostatin-
IR cells [87]. In addition, neurogenesis, gliosis, dendritic plasticity,
and axonal injury have been demonstrated in several models of
TBI associated with PTE but their contribution to epileptogenesis is
unexplored (see [69]). The cortex and different nuclei of the thala-
mus also undergo remarkable neurodegeneration, but there are no
data detailing the cell types involved [80].
One of the major challenges for future work is to find means to
localize the ictogenic focus/network in post-traumatic brain. This
would help to specify which one of the observed molecular and
cellular changes are relevant for post-traumatic epileptogenesis,
and consequently, guide targeting of therapies to the right locations
in antiepileptogenesis trials.
4. Biomarkers and surrogate markers for PTE
The search for biomarkers that predict post-TBI recovery is
active and includes both serum and CSF analyses as well as the
use of various imaging modalities [18]. Again, however, PTE has
not been included as an outcome measure in these studies. We
recently performed quantitative MRI in the hippocampus ipsilat-
eral to LFP injury. We found that the diffusion trace (Dav ) measured
at both early (3 h) and chronic (23 d, 2, 3, 6, 7 and 11 months)
post-injury time points correlated with the number of spikes or
epileptiform discharges quantified in the EEG recorded during a
pentylenetetrazol (PTZ) seizure susceptibility test at 12 months
post-TBI. Correlations were also found between the Dav and the
density of mossy fiber sprouting that is a typical plastic response of
granule cell axons found in the hippocampus both in experimental
and human temporal lobe epilepsy [44]. These results suggest that
quantitative diffusion MRI could serve as a tool to facilitate predic-
tion of increased seizure susceptibility in experimental models of
PTE.
5. Challenge of designing antiepileptogenesis trials to
prevent PTE in rodents
Table 2 summarizes the models of TBI in which epileptogene-
sis has been described. We focus on those models that are used in
the trauma field [60]. A common feature for most of the models is
that ≤50% of animals develop epilepsy, latency is weeks to months
rather than days, and seizure frequency is low. Further, the occur-
rence of seizures can vary substantially within the study population
(Fig. 2). One study reported the occurrence of epileptiform spiking
activity in 80% of rats after LFP injury, occurring also in rats with no
detected seizures [43].
One caveat in the data obtained is that even the longest video-
EEG monitoring studies cover only a small fragment of the entire
epileptogenesis period, and in fact, the duration of the delay
between the insult and the occurrence of first late seizures is not
known. In some models such as weight-drop or closed skull trau-
matic brain injury, only hyperexcitability has been demonstrated,
and the occurrence of spontaneous seizures remains to be shown
[29]. These data on the phenotype of experimental PTE makes us
consider the value of PTE models in antiepileptogenesis studies rel-
ative to that of, for example, status epilepticus (SE) models. As well
described, after chemically or electrically induced SE typically ≥80%
of rats develop epilepsy within 1 month, and the seizure frequency
can be up to 50/d (for review, see [41]), making the SE models sub-
stantially less labor-intensive than PTE models. However, there is
no data that would qualify SE models to replace the PTE models
168 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171
Fig. 2. Examples of the occurrence of spontaneous late seizures in 3 rats (#73, #2, #46) during a 52-wk follow-up (unpublished data from [43]). The grey bars show the
timing of continuous video-EEG monitoring (1 or 2 wk at a time). Seizures were detected relatively infrequently, and each rat had a variable appearance of seizures.
when assessing the molecular and cellular consequences leading
to PTE after TBI. Also, there is no evidence that any treatment mod-
ifying the epileptogenic process after SE would have a similar effect
after TBI.
In all models of TBI analyzed so far, the susceptibility to induced
seizures has been shown to be increased (Table 2). These include
LFP injury, CCI, and weight-drop models in rats as well as closed
skull TBI, LFP injury, and CCI models in mice. Seizure susceptibil-
ity has been reported for kainate, PTZ or ECS induced seizures.
Whether increased seizure susceptibility to induced seizures can
be used as a surrogate marker for ongoing epileptogenesis, and as
an indicator for pinpointing individual animals that will eventually
develop epilepsy, is under dispute. The little available literature
suggests that perhaps it can. Rats with spontaneous seizures trig-
gered by kainic acid or pilocarpine-induced SE at 30–45 d before
a PTZ test showed enhanced susceptibility to PTZ-induced gener-
alized convulsions and SE [6,89]. Further, the latency to the first
generalized convulsion was reduced from 1280s (controls) to 389 s
(post-SE animals)[6]. Interestingly, also after pilocarpine-induced
SE the rats that did not develop SE acutely, and consequently
had no spontaneous seizures during a 45 d follow-up, still showed
increased seizure susceptibility to PTZ even though it was less
than that of rats with spontaneous seizures [6]. It was recently
shown that the non-SE rats after pilocarpine injection also actu-
ally develop infrequent spontaneous electrographic seizures (0.1–1
seizures/wk) within 8 ± 2 months post-pilocarpine [64]. These data
have some similarity to PTE models, in which injured rats do not
show spontaneous seizures or have very infrequent seizures, but
have enhanced response to chemoconvulsants or electroshock (ES),
and show enhanced in vitro excitability [20,29,36,37,61]. Finally,
we recently demonstrated that animals with TBI and spontaneous
seizures show the highest seizure susceptibility in the PTZ test (see
Fig. 7C in [44]).
Currently, the PTE rodent models are laborious to use. One way
to facilitate epileptogenesis and increase the proportion of animals
that develop epilepsy within a reasonable time frame would be to
induce TBI in animals with an epileptogenesis-prone genetic back-
ground, or select animals with the highest risk based on post-TBI
imaging. Another issue to be further explored is to assess whether
a decrease in seizure susceptibility is a valid outcome measure in
antiepileptogenesis studies.
6. Disease-modification treatment approaches
As discussed earlier (see Section 1), we divide the disease
modification after TBI into antiepileptogenesis and co-morbidity
modification.
6.1. Antiepileptogenesis after TBI
Table 3 summarizes the treatment approaches to suppress
epileptogenesis after experimental TBI. The Soltez group investi-
gated the effect of a cannabinoid 1 receptor antagonist, SR141716A
(Rimonabant® ) on epileptogenesis [20]. Interestingly, SR141716A
is a proconvulsant that has been demonstrated to lower the
threshold for kainate-induced seizures [9]. They induced epilepto-
genesis by lateral fluid-percussion induced TBI, and administered
SR141716A (1 mg/kg or 10 mg/kg, i.p.) as a single injection at
2 min post-injury. The threshold for kainate-induced seizures was
assessed at 6 wk post-TBI. The reduction in the latency to kainate-
induced seizures was prevented by SR141716A. Also, the total
time spent in seizures after kainate administration was reduced
in the SR141716A group as compared to the vehicle group. Impor-
tantly, no positive effect was found if SR141716A was administered
20 min post-TBI. Another study by Chrzaszcz et al. [10] used
the closed-skull midline impact model in mice, and adminis-
tered minozac 3 or 6 h after injury. Minozac is a suppressor of
pro-inflammatory cytokine up-regulation. Its discovery and devel-
opment was made using a de novo compound discovery platform
interfaced with hierarchal biological screens for oral bioavailabil-
ity, toxicity, brain penetrance, and stability, and it has been tested
for efficacy in animal models of brain disorders [62]. After 1 wk
post-TBI, minozac-treated mice showed less susceptibility to ES-
induced seizures than sham-operated animals. Whether minozac
treatment prevented the long-term increase in seizure susceptibil-
ity and occurrence of late seizures remains to be explored. The effect
of ketogenic diet on post-TBI epileptogenesis was investigated by
Schwartzkroin et al. [75]. They triggered LFP injury to 8-wk-old rats
and administered ketogenic diet for 3 wk post-injury. Seizure sus-
ceptibility to fluorothyl was not any different from that in rats on
standard diet when assessed at 3 and 6 wk after discontinuation of
ketogenic diet. It should be noted, however, that TBI had no effect
on seizure susceptibility to fluorothyl (seizure threshold, seizure
 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171
Table 3
Treatment approaches to suppress epileptogenesis after TBI in experimental models.
Treatment Mechanism of Action Model
Rimonabant ®
CB1 receptor antagonist Lateral FPI (rat)
(SR141716A)
Minozac® Reduction of proinflammatory Closed skull TBI
cytokine production by activated glia (CD-1 mouse)
Ketogenic diet Unknown (likely multiple) Lateral FPI (rat)
Hypothermia Reduction of brain temperature Parasagittal FPI (rat)
Abbreviations: CB1, cannabinoid receptor 1; ECS, electroconvulsive shock; FPI, fluid percussion injury; PTZ, pentylenetetrazol; TBI, traumatic brain injury.
duration) when injured and sham-operated animals on standard
diet were compared. Finally, Atkins et al. [3] induced moderate
parasagittal FP injury in adult rats. Animals were kept under nor-
mothermic or moderate hypothermic temperatures for 4 h starting
at 30 min post-injury. Seizure susceptibility was tested at 12-wk
post-TBI by using PTZ. Behavioral analysis of data indicated reduced
number of induced seizures during the 60 min post-PTZ injection.
The behavioral severity of seizures was not affected
The list of antiepileptogenic treatment attempts in animal mod-
els is substantially shorter than that in human PTE which includes
several antiepileptic drugs as monotherapy or polytherapy (car-
bamazepine, phenytoin, valproate, phenobarbital), glucocorticoids,
and magnesium sulphate [84,85,92]. Clinical studies have been
conducted with relatively little or no evidence for antiepileptoge-
nesis in animal models. Many of these compounds were, however,
shown to have neuroprotective effects after experimental TBI. In
particular, MgSO4 was shown to be one of the most efficient neuro-
protectants both in acute and chronic experiments after LFP injury
[58,78], and still when given to patients, no effect was seen, rather
they worsened [84]. As a note, we use doses of MgSO4 in our anes-
thesia cocktail when inducing LFP injury that are comparable to
that used in neuroprotection studies, and in our hands 50% of the
rats developed epilepsy in 1 year follow-up [43]. It should be noted
that even though there are reports on the disappearance of seizures
in 25–45% of patients after established PTE (see [21] and refer-
ences therein), there is no evidence that the AEDs used to treat
the condition in these patients had any contribution to remission.
6.2. Co-morbidity modification
The list of compounds that have been tested to enhance post-
TBI recovery in experimental models is extensive and includes
AEDs, excitatory amino acid modulators, Ca-channel modulators,
ROS scavengers, anti-inflammatory drugs, neurotrophic factors,
caspase inhibitors, calpain inhibitors, endocrinological modulators,
and modulators of neurotransmission; furthermore, administra-
tion routes range from systemic pharmacotherapy to cell therapy,
gene therapy, and vagal nerve stimulation [55,68,77]. Even though
many of these therapies have shown favorable effects on co-
morbidities (motor impairment, cognitive and emotional decline)
which associate with PTE in these models, a change in seizure sus-
ceptibility or occurrence of seizures have never been included as
outcome measures.
To control immediate and early seizures the use of AEDs is indi-
cated. During the past years several laboratories have assessed
whether AEDs are neuroprotective, and whether they affect post-
TBI recovery in experimental models. Even though epileptogenesis
has not been assessed, these data give information on the effects
of AEDs on other components of disease or syndrome modifi-
cation, that is, on co-morbidity modification. It has been shown
that the administration of remacemide, which has an affinity for
the NMDA receptor ionophore and Na+ channels, 15 min postin-
jury reduced cortical lesion volume when assessed at 48 h. There
were no favorable effects on spatial memory [79]. Topiramate is
169
Effect Reference
Seizure susceptibility to kainate-induced seizures at 6 wk post-TBI ↓ [20]
Seizure susceptibility to ECS-induced seizures at 7 d post-TBI ↓ [10]
No effect on fluorothyl-induced seizure susceptibility at 3 or 6 wk [75]
post-TBI
Seizure susceptibility to PTZ-induced seizures at 12 wk post-TBI ↓ [3]
a structurally-novel broad-spectrum anticonvulsant that modu-
lates the activity of several ligand- and voltage-gated ion channels
in neurons. These include an inhibitory effect on the AMPA and
kainate subtypes of glutamate receptors, mixed modulatory effects
(usually positive) on some types of GABAA receptors, negative
modulatory effects on some types of voltage-gated Na+ and Ca2+
channels, and a positive modulatory effect on at least one type
of K+ channel [76]. Hoover and colleagues [34] reported that the
administration of topiramate 30 min after lateral FPI improved
sensorimotor behavior when assessed 4 wk postinjury, but had
no favorable effects on neuronal survival or learning. Talampanel,
a non-competitive AMPA antagonist, administered 30 min after
parasagittal FPI reduced neurodegeneration in the contusion area
as well as the hippocampal CA1 subfield when assessed 7 d after
TBI [4]. We recently reported that lacosamide, when administra-
tion was started at 30 min post-LFP injury and continued for 3 d,
had no effect on lesion size or motor impairment when assessed
2 wk postinjury [65]. Also carisbamate, when administered at
15 min post LFP injury for 1 d, had no effect on brain edema (48 h
post-injury), lesion size, motor function or learning when ana-
lyzed 4 wk post-injury [42]. Recently, Dash et al. [17] reported
that administration of 400 mg/kg valproate starting at 30 min or
3 h post-CCI induced injury and continued for 5 d improved BBB
integrity, reduced TBI-associated hippocampal dendritic damage,
lessened cortical contusion volume, and improved motor function
and spatial memory. This was associated with inhibition of glyco-
gen synthase kinase 3 (GSK-3) and histone deacetylase (HDAC).
Interestingly, these behavioral improvements were not observed
when SAHA (suberoylanilide hydroxamic acid), a selective HDAC
inhibitor, was administered. Taken together, there is meager evi-
dence pointing to significant neuroprotective effects of any AEDs
after TBI in animal models which could associate with co-morbidity
modification. However, AEDs like valproate that have mechanisms
of action other than just those on receptor or ligand gated ion
channels, including epigenetic modulatory effects on histone acety-
lation and DNA methylation, may warrant further studies.
7. Conclusions
The first article published on TBI included in PubMed is from
1894 [63] and that on PTE from 1947, more than 50 years later
[91]. Thereafter, about 430 articles have been listed in PubMed on
“posttraumatic epilepsy”. In animal models of TBI that have been
commonly used in the trauma field, epileptogenesis was detected
less than 10 years ago [16,36,43]. Not surprisingly, comparisons of
endophenotypes between patients and animals with PTE are still
in their infancy. Similarly, few attempts have been made to test the
effects of candidate antiepileptogenic treatments in models of PTE
are few. To move further in the search for a cure for PTE, more work
is needed to understand the specific clinical, molecular, and net-
work characteristics of the injured brain in humans and rodents,
which lead to PTE at different ages. Application of novel imaging
techniques can be expected to help in localization of the developing
epileptogenic focus/network in post-traumatic brain, and conse-
170 A. Pitkänen et al. / Neuroscience Letters 497 (2011) 163–171
quently, guide the investigations to the right anatomic target(s).
Such data would have implications for the model development as
well as understanding of the mechanisms of post-traumatic epilep-
togenesis, identification of biomarkers and surrogate markers, and
optimization of preclinical trial designs.
Acknowledgements
This study was supported by the Academy of Finland, the Sigrid
Juselius Foundation, and CURE. We thank Nick Hayward, M.Sc., for
revising the language of the manuscript.
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