Epilepsy is a chronic disorder characterized by the recurrence of unprovoked seizures. The consequences of epilepsy are not only neurobiological but also cognitive, physiological and social.1 Epilepsy affects roughly 50 million people worldwide.2 One third of all affected persons suffer from intractable seizures. With the exception of surgical resection, in most instances treatment is only symptomatic.3
A single seizure is not yet epilepsy; it may be a symptom of infection, trauma, stroke, metabolic derangements etc., and may never repeat.4
The crucial process leading to repeated seizures is epileptogenesis; a process from initial brain damage to the first seizure and beyond, consisting of a sequence of chemical and structural alterations resulting in transformation of normal to epileptogenic brain tissue. Epileptogenesis may last years.5 The initial event can be a seizure per se.6,7
Indices of inflammation in epilepsy have been repeatedly demonstrated but there is no clear answer as to whether inflammation is the cause or a consequence of the disease.8, 9 There is evidence that a single seizure promotes a surge of cytokine production in brain tissue.10,11 Repeated seizures tend to be accompanied by a higher level of brain cytokine production.12 Elevated peripheral blood levels of cytokines, mostly interleukin-6 (IL-6) and antagonist of IL-1 Receptor (IL-1Ra) have been reported after secondarily generalized13 and focal epileptic seizures.14,15 Blood perfusion changes have so far been extensively studied in patients with acute ischemic stroke16,17, and there have also been studies in patients with epilepsy.18,19 In animal studies, early brain perfusion changes after status epilepticus have been suggested to be of prognostic value in relation to future hippocampal shrinkage and degeneration.20,21 Vascular perfusion changes were not localized but spread throughout wide brain areas; in a model of prolonged focal seizures, the effect was explained by diffuse changes in neurovascular coupling.22,23 Changes in cerebral blood perfusion related to seizures have also been attributed to higher metabolic demand24 and leucocyte-endothelial interaction.25 In humans, assessment of perfusion changes after a single seizure26 or status epilepticus27 has been reported to be helpful in localizing the epileptic focus.
It was our aim to check for postictal brain inflammation indices by studying MR perfusion and blood cytokines, and to determine whether the type and number of index seizures, postictal blood concentrations of cytokines and parameters of MR perfusion are predictive for the seizure burden over a one year follow-up.
We
The study was approved by the National Medical Ethics Committee.
Written consent was signed; a full history was recorded, including smoking status. Patients were given an Epilepsy Diary with instructions for its use. A minimum 8 and maximum 24 hours after the last seizure, 2 ml of venous blood in EDTA buffer were taken and transported for immediate processing. C-reactive protein (CRP) was checked on a Point of Care device. Brain MRI with contrast was performed. Routine 32-channel EEG with scalp electrodes was recorded. Patients with reported »hyperperfusion« after visual MRI analysis were invited for a control blood and MRI examination after three months. One year after the index event, patients were contacted by phone and relevant information about disease activity was collected.
Individual researchers (clinicians, immunologist and radiologist) were unaware of the results of investigations performed by other researchers in the course of the study.
The severity of the disease in the year prior to the study was assessed by the National Hospital Seizure Severity (NHS) 3 score28; if two types of seizures were reported, the »lesser« one (including aura) being chosen first. The frequency of seizures in the year prior to the study was defined as: 1. Rare (R) < 1 seizure per year, 2. Often (O) > 1 seizure per year and 3. Very often (V) > 1 seizure per month.
EEG findings were categorized as: 1. Normal, 2. Focal changes (location included), 3. Generalized changes and 4. Nonspecific abnormalities. Syndromological classification was performed based on International League Against Epilepsy (ILAE) criteria.1,29 The category “probable focal symptomatic” was used if the semiology of the seizures strongly suggested focal onset (
The concentrations of interleukins: 4 (IL-4), 6 (IL-6), 10 (IL-10) and 12 (IL-12), antagonist of IL-1 receptor (IL-1Ra), tumour necrosis factor-
Two ml of blood were taken in a test tube recoated with ethylenediaminetetraacetic acid (EDTA), immediately centrifuged and stored until processing at -20°C. A chemoluminiscent immunometry assay based on tagged monoclonal antibodies was performed. In brief, the system adds serum and reagent (cytokine specific antibody conjugated with alkaline phosphatase) into a test tube containing the carrier covered with cytokine specific antibody. The whole sample is then incubated until complex
All MRI images were obtained on a 1.5T MRI scanner (Philips Achieva Nova, Netherlands). Morphological brain studies were obtained using the following sequence protocol: T1 weighted spin echo (SE), T2 weighted fast field echo ( FFE) and diffusion weighted imaging (DWI) in transversal plane; fluid attenuated inversion recovery (FLAIR), T1 weighted inversion recovery (IR) echoplanar imaging (EPI) and T2 weighted fast spin echo (FSE) in coronary and transversal planes; T1 weighted three dimensional (3D) in sagittal plane. MR perfusion was performed by fast field echo (FFE) echo planar imaging (EPI) sequence.
Each subject obtained a 16 G cubital
First, two experienced neuroradiologists independently visually interpreted the morphological MRI sequences and then the functional colour maps of perfusion parameters. An automatic deconvolution program (Neuro T2* perfusion package, Philips Achieva Nova, Netherlands), which is a part of the MRI scanner software, was used. The program automatically calculated the perfusion parameters: negative integral,
The commercially available SPSS version 21.0 program was used. All values are given as absolute (N) and percentage (%). Mean values are expressed as median (Me) and/or mean (M) with standard deviation (SD). The normality of the data was tested by Kolmogorov-Smirnov and Shapiro-Wick tests. Since most of the data was not normally distributed, nonparametric statistical methods were used. Correlation was tested with Pearson’s correlation and Eta coefficient. For group comparison, the Wilcoxon, Cruscal-Wallis, Chi- square and Mann-Whitney U tests were used.
Demographical and clinical data are summarized in Table 1, EEG and syndrome data in Table 2.
Demographical and clinical data AED = antiepileptic drugs; CBZ = carbamazepine; DPH = diphenylhydantoin; F = female; Follow up = seizure burden in one year after study inclusion; F = frontal; FS = focal symptomatic; G = generalized; IGE = idiopathic generalized epilepsy; L = left; LAM = lamotrigine; LEV = levetiracetam; M = male; MCA = middle cerebral artery; N = index; number of index seizures; NHS3 score = National Hospital Seizure Severity 3 score; N = normal; O = often (1/month); PFS = probable focal symptomatic; PREG = pregabalin; R = right, R = rare(1/year); Type/NHS3 = number of types/National Hospital Seizure Severity 3 score; T = temporal; TPM = topiramate; V = very often (> 1/month); VA = valproate
Sex age,
Smoking status
Illness Duration (yr)
syndrome
EEG
FREQUENCY
N-INDEX
AED
TYPE /NHS3
Structural MRI
Follow up 1
F 65
/
7
PFS
G
V
4
LAM, LEV
2(5/10)
Leucopathy
0 2
F 38
5
7
PFS
G
R
1
LEV*
2(2/10)
N
0 3
M 26
/
3
/
N
R
1
LEV*
2(2/12)
N
0 4
F 65
/
/
/
G
1st
1
/
1(11)
N
0 5
F 23
/
15
FS
F
V
1
TPM,
2(2/12)
Resection RF
>100 6
M 35
/
20
FS
F
R
1
LAM
2(2/10)
N
0 7
M 42
/
1
Fs
F(RT)
1st
1
LEV*
1(18)
N
5 8
F 20
10
7
IGE
G
O
1
VA
2(2/13)
N
0 9
F 24
/
1
/
N
2nd
1
/
1(12)
N
0 10
M 21
20
6
PFS
N
0
1
LEV*
1(13)
N
0 11
M 29
/
12
IGE
G
O
1
VA
2(2/14)
N
0 12
M 46
/
/
FS
F(LT)
1st
1
LEV*
1(13)
Encephalomalacy
0 13
F 57
/
/
/
F(RF)
1st
1
/
14
M 27
/
7
FS
F(LT)
R
1
LEV*
1(18)
N
6 15
F 54
/
8
PFS
N
O
1
LEV
1(11)
N
0 16
M 44
10
5
FS
F(LF)
O
3
LAM
1(12)
N
0 17
F 45
/
20
PFS
N
R
1
LAM
1(15)
N
0 18
M 30
10
12
FS
F(RF)
O
1
LAM
1(18)
Gliosis RF
5 19
M 63
/
4
FS
F(RT)
O
5
LEV
2(2/18)
MTS R
12 20
M4 0
/
20
FS
F(RT)
O
1
CBZ
2(2/15)
N
12 21
M 41
/
20
FS
F(LT)
V
5
CBZ,VA
1(10)
N
50 22
M 23
/
8
IGE
G
V
3
LAM
1(18)
N
47 23
F 29
/
11
FS
F(RT)
V
2
TPM,LAM
1(20)
Cortical. Dysplasia
5 24
M 27
/
12
IGE
G
O
7
VA*
1(9)
N
2 25
M 21
10
6
IGE
G
O
6
VA
2(2/8)
N
0 26
F 50
20
34
FS
F(LT)
R
1
/
1(8)
Aneurysm L MCA
0 27
F 43
/
25
PFS
N
V
3
LAM
1(11)
Gliosis RF
0 28
M 50
/
40
IGE
G
V
1
LAM,LEV
1(19)
Cerebellar
18 29
M 39
/
32
/
G
R
1
/
1(8)
N
0 30
M 19
10
5
/
G
R
1
CBZ*
1(9)
N
0
EEG and syndrome FS = focal symptomatic; F = frontal; IGE = idiopathic generalized epilepsy; L = left; O = often; PFS = probable focal symptomatic; R = rare; R = right; T = temporal; V = very often
EEG and syndrome
N (%) Frequency
R
8 (27)
O
11(37)
V
6(20) Syndrome
PFS
6(20)
FS
12(40)
IGE
6(20)
undetermined
6(20)
4(13)
12(40)
5(17)
6(20)
3(10) Focus
RT
4(33)
LT
5(41)
RF
2(16)
LF
1(8)
In four patients (13.3%) the index seizure was their first seizure; patient 3 had only had one seizure in the past.
None of the 30 patients had hypoperfusion on MRI, 25 had normal MRI perfusion parameters (»normoperfusion« patients); five (17%) (Patients 2, 3, 7, 15 and 25) had hyperperfusion (»hyperperfusion« patients) (Figure 1). All »hyperperfusion« patients had normal structural MRI; 9 of the remaining 25 patients with normal perfusion parameters had structural abnormalities. Two had more diffuse structural abnormalities: patient 1 had leucopathy, patient 28 had cerebellar atrophy. Patient 5 had had resection of the right frontal lobe in childhood; patient 26 had an aneurysm of the left medial cerebral artery. We did not find associations between structural MRI abnormalities and either postictal cytokine level or MRI perfusion parameters. Patients 7 and 15 also had hyperperfusion also on the control MRI examination after three months (»control hyperperfusion«). During this period, both patients were seizure free (
increased blood flow (arrow) in the region of the left medial cerebral artery; signal changes adjacent to left frontal bone (asterix) are the artefact (patient 15)Figure 1
MRI parameters after index seizure(s) are shown in Table 3.
MRI perfusion parameters after index seizures (grey matter) statistically significant difference (p < 0,001) statistically significant difference (p < 0,001) BF = blood flow; BV = blood volume; M = mean; Me = median; MTT = mean transit time; N = »negative« (“normoperfusion”); P = »positive« patients (“hyperperfusion”); SD = standard deviation; TTP = time to peak
N
M
Me
SD BF
25
101,7
93
40,8 BV
25
8
7
3,9 MTT
25
6,9
6,9
2,9 TTP
25
13,9
13,8
2,3 BF
5
265,85
234
56,3 BV
5
25,5
27,6
4,7 MTT
5
5,7
5
1,2 TTP
5
14,5
13,5
4,5
Blood samples were drawn 8 hours after the last seizure in most patients, except in patient 1 (24 h), patient 9 (13 h), patient 14 (15 h) and patient 16 (20 h). The blood sample of patient 3 was accidentally lost.
The tested cytokines, including concentrations of IL-6, showed marked variability.
Cytokine levels after the index seizure(s) are summarized in Table 4. Median concentrations of cytokines in the three subgroups of patients (»normoperfusion«, »hyperperfusion«, and »control hyperperfusion«) are provided in Table 5.
Blood cytokine concentration after index seizure(s) patient 3 is not included patient 3 is not included patient 3 is not included patient 3 is not included patient 3 is not included patient 3 is not included patient 3 is not included patient 3 is not included M = mean; Me = median; N = number; Ref = reference values; SD = standard deviation
N
M
Me
SD
Ref.
No (%) above/below reference 29
0.9
0.6
1.3
≤ 1
7(24) 29
5.7
3.7
5.1
≤ 3.9
15(52) 29
42.8
11.4
10.2
≤ 10.9
16(55) 29
66
58.5
40.3
≤ 204
29(100) 29
8.5
7.6
4
4.7-12.4
1(3) 29
27.5
15
54.4
≤ 1.2
7(24) 29
1302.7
676
1797
168-744
14(48) 29
2.9
1.1
4,5
< 3
29(100)
Median concentrations of cytokines (pg/ml) and hs CRP(mg/l) in three subgroups of patients (see text for details) Statistically significant difference (p < 0. 05) Statistically significant difference (p < 0. 05) Statistically significant difference (p < 0. 05) Due to a small number of patients (2) the significance was not tested. H kateri tabeli spada ta podnapis. Nisem opazil superskripta 1. Statistična pojasnila k tabelam označujete z zvezdico in bi tudi to opombo.
IL-4
IL-6
IL-10
IL-12
TNF-α
IFN-
IL-1Ra
hsCRP Normoperfusion
0,6
4,3
11
60,1
7,3
15,1
677
0,9 Hyperperfusion
0,3
1,5
63,7
44,5
9,7
14
407
1.3 Control hyperperfusion
0,5
6,0
5,1
110,4
10,6
27,7
868
1,6
Postictal (1st) and control levels of cytokines in patients with hyperperfusion after the index seizure(s) are summarized in Table 6. The number of index seizures significantly negatively correlated with concentrations of IL-10, IFN-
Basic and control concentrations of cytokines (pg/ml) in patients with hyperperfusion after index seizure(s) Patient demonstrated hyperperfusion also on control MRI after three months. Patient demonstrated hyperperfusion also on control MRI after three months.
Patient
IL-4
IL-6
IL-10
IL-12
TNF-α
IFN-γ
IL-1Ra
hsCRP 0,08
<2
116
54,5
8,19
13,08
352
1,5
0,3
<2
134
99,6
8,97
9,23
822
1,7 /
/
/
/
/
/
/
/
1,77
<2
12,6
127
11,5
108
1479
0,9 0,41
2,47
10,02
24,8
11,7
44,56
676
1,9
0,35
<2
29,8
41,8
12,1
31
361
1,8 0,61
9,49
0,22
196
9,57
10,81
1060
1,4
0,00
<2
0,58
150
14,1
14,7
593
0,7 0,38
<2
11,51
34,6
11,1
14,97
462
1,1
1,01
<2
12,0
42,5
20,2
16,2
660
1,4
Correlation between cytokine concentrations and number of index seizures weak correlation; moderate correlation weak correlation;
Cytokine concentration
Pearson’s coefficient of correlation ρ 0,07 0,23 -0,37 0,24 -0,41 -0,39 -0,22 -0,16
There was a significant correlation among concentrations of the following cytokines: IL-6 and Il-12 (ρ = 0.37, p < 0.05); TNF-α and hsCRP (ρ= 0.71, P < 0.01); TNF-α and IL -1Ra (ρ = 0.51, p < 0.01), and IL-1Ra and IFN-
Taking into account the whole cohort of patients, cytokine concentrations did not correlate with postictal MRI perfusion parameters. Postictal MRI perfusion parameters were furthermore not correlated with any other tested variable (age, gender, smoking status, number of index seizures, antiepileptic drug, epileptic syndrome, EEG, structural MRI or seizure frequency in the previous year).
Analysis was then performed for the two subgroups of patients - »normoperfusion« (25 patients), and »hyperperfusion« (5 patients). There were a slightly higher proportion of smokers in the »hyperperfusion« group: (60%
In the »hyperperfusion« group, concentrations of most of the cytokines were lower but the difference reached significance only for IL-4 (Z = -1.98; p < 0.05); concentrations of TNF-α were higher (Z = -2, 18; p < 0.05) and also hsCRP but the latter did not reach significance.
Patients 7 and 15 (»control hyperperfusion«) had the highest median concentrations of IL-6, IL-12, TNF-α, IFN-
The number of seizures during the follow up year correlated significantly only with the number of antiepileptic drugs (ρ = 0.60, p < 0.01).
Patients with a
Elevated cytokine levels have been demonstrated after epileptic seizures in brain tissue10,11 and in peripheral blood.13-15 Similarly, our study found an abnormal blood level of IL-6 in 14 out of 29 patients 8–24 hours after index seizure(s).
Alapirtti
In another study (in the emergency setting), proinflammatory cytokines were elevated postictally in half of the subjects; however, patients with brain infections, cerebral venous thrombosis and brain tumour were included34; such patients were excluded in our study.
We found a highly significant correlation not only between proinflammatory cytokines IL-6 and IL-12; TNF-
In contrast to previous reports10,14,36,37, we found an inverse correlation between the number of index seizures and the level of proinflammatory cytokines IL-10, TNF-
MRI “normo-” and “hyperperfusion” groups of patients demonstrated different concentrations of cytokines IL-4 and TNF-
There are experimental38 and clinical24 evidences that long lasting blood-brain barrier disruption is linked to epileptogenesis. Increased postictal blood levels of proinflammatory cytokines - as also demonstrated in our study - have been attributed to postictal blood-brain barrier affection. DSC MRI is a noninvasive surrogate biomarker of brain inflammation, capable of demonstrating endothelial dysfunction or increased blood-brain barrier permeability.35 In patients with high frequency seizures, Pizzini
MRI perfusion parameters from our group taken as a whole did not correlate with any tested variable (including seizure frequency or syndrome). Hyperperfusion on MRI - perfusion with a 12-hour delay after a 4-hour pilocarpine induced status epilepticus in rats - correlated with histological signs of neurodegeneration in certain brain areas (not seen on T2 MRI).20
Looking at the clinical data, cytokine blood levels and DSC MRI parameters, we failed to find prognostic factors for the course of disease over a one-year follow up. The main limitation of our study is the smallness and heterogeneity of our group. We propose, however, that the trends shown in our study are worth following up.
In conclusion, our study revealed a subgroup of patients with brain postictal hyperperfusion and higher plasma cytokine values, indicating a probable long lasting blood-brain barrier alteration.
Larger studies are needed to validate the hypothesis that post seizure brain tissue inflammation is a crucial factor of epileptogenesis in selected patients and to determine the diagnostic and prognostic values of the various parameters studied.