Radiol Oncol 2017; 51(3): 277-285. doi:10.1515/raon-2017-0031 277 research article Dynamic susceptibility contrast enhanced (DSC) MRI perfusion and plasma cytokine levels in patients after tonic-clonic seizures Tatjana Filipovic1, Katarina Surlan Popovic2, Alojz Ihan3, David Bozidar Vodusek1 1 Division of Neurology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 3 Institute of Immunology, Medical School, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2017; 51(3): 277-285. Received 17 April 2017 Accepted 14 June 2017 Correspondence to: Filipović Tatjana, M.D., Division of Neurology, University Medical Centre Ljubljana, Zaloška cesta 7a, SI-1000 Ljubljana, Slovenia. Phone: +386 5 223 195; E-mail: tatjana.filipovic@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Inflammatory events in brain parenchyma and glial tissue are involved in epileptogenesis. Blood con- centration of cytokines is shown to be elevated after tonic-clonic seizures. As a result of inflammation, blood-brain barrier leakage occurs. This can be documented by imaging techniques, such is dynamic susceptibility contrast enhanced (DSC) MRI perfusion. Our aim was to check for postictal brain inflammation by studying DSC MRI perfusion and plasma level of cytokines. We looked for correlations between number and type of introducing seizures, postictal plasma level of cytokines and parameters of DSC MRI perfusion. Furthermore, we looked for correlation of those pa- rameters and course of the disease over one year follow up. Patients and methods. We prospectively enrolled 30 patients, 8–24 hours after single or repeated tonic-clonic seizures. Results. 25 of them had normal perfusion parameters, while 5 had hyperperfusion. Patients with hyperperfusion were tested again, 3 months later. Two of 5 had hyperperfusion also on control measurements. Number of index seizures negatively correlated with concentration of proinflammatory cytokines IL-10, IFN-ϒ and TNF-α in a whole cohort. In pa- tients with hyperperfusion, there were significantly lower concentrations of antiinflammatory cytokine IL-4 and higher concentrations of proinflammatory TNF-α. Conclusions. Long lasting blood- brain barrier disruption may be crucial for epileptogenesis in selected patients. Key words: seizure; blood-brain barrier; cytokines; DSC MRI Introduction Epilepsy is a chronic disorder characterized by the recurrence of unprovoked seizures. The conse- quences 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, meta- bolic derangements etc., and may never repeat.4 The crucial process leading to repeated seizures is epileptogenesis; a process from initial brain dam- age to the first seizure and beyond, consisting of a sequence of chemical and structural alterations resulting in transformation of normal to epilepto- genic 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 an- swer 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 Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures278 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 fo- cal 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 neu- rovascular coupling.22,23 Changes in cerebral blood perfusion related to seizures have also been attrib- uted 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 lo- calizing the epileptic focus. It was our aim to check for postictal brain in- flammation 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. Patients and methods Patients We prospectively enrolled 30 patients present- ing in the Emergency Service of the Division of Neurology, University Medical Centre Ljubljana, Slovenia after one or multiple seizures, from 1. 6. 2011 to 31. 12. 2014. Inclusion criteria were: one or more witnessed seizures in the last 24 hours, age 18–65 years, in- formed written consent for study enrolment and informed written consent for the contrast MRI examination. Exclusion criteria were: brain injury, haemorrhage or stroke in the last 6 months, pri- mary or secondary brain tumour any time in the past, known autoimmune disease or any immune modifying therapy in the last 6 months, pregnancy or lactation, alcohol or drug abuse, liver or kidney failure, clinical signs of infection or CRP > 20 (mg/l) and inability to obtain informed written consent (dementia, mental retardation, altered conscious- ness, psychosis). The study was approved by the National Medical Ethics Committee. Study protocol Written consent was signed; a full history was re- corded, 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 buff- er were taken and transported for immediate pro- cessing. 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 in- formation about disease activity was collected. Individual researchers (clinicians, immunolo- gist and radiologist) were unaware of the results of investigations performed by other researchers in the course of the study. Assessment of clinical and EEG data 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 (e.g., “déjà vu”, head turn) in the presence of generalized or unspecific EEG changes. Generalized changes were categorized either as IGE (e.g. multiple spikes/ waves) or nonspecific generalized (e.g. short gener- alized delta/theta rhythm). Immunological methods The concentrations of interleukins: 4 (IL-4), 6 (IL-6), 10 (IL-10) and 12 (IL-12), antagonist of IL-1 recep- tor (IL-1Ra), tumour necrosis factor- α (TNF-α) and Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures 279 interferon –γ (IFN-γ) were measured. The concen- tration of high sensitivity CRP was used addition- ally to rule out acute infections and autoimmune disorders. Two ml of blood were taken in a test tube recoat- ed with ethylenediaminetetraacetic acid (EDTA), immediately centrifuged and stored until process- ing at -20°C. A chemoluminiscent immunometry assay based on tagged monoclonal antibodies was performed. In brief, the system adds serum and re- agent (cytokine specific antibody conjugated with alkaline phosphatase) into a test tube containing the carrier covered with cytokine specific anti- body. The whole sample is then incubated until complex cytokine specific antibody-cytokine-alkaline phosphatase conjugated cytokine specific antibody is formed. The sample is then rinsed (to remove the non-bound reagent) and the luminogenous sub- strate is added. Alkaline phosphatase bound via cytokine to a carrier splits the substrate into an un- stable luminescent intermediary. The level of lu- minescence corresponds to the serum level of cy- tokine. The test was performed using the commer- cial Immulite 1000 and Immulite 2000 XPI systems (Siemens, Germany; detailed description available in: N CEL 19 and N CEL-18 Siemens Manual). Controls were performed with the commercial IMMULITE Cytokine Control Module (Siemens). The concentration of cytokine is calculated by a calibration curve generated by the manufacturer for each lot of reagents and saved at the bar code of the test kit. Reference values are given by the manufacturer for each cytokine and soluble recep- tor. MRI Methods 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 im- aging (EPI) and T2 weighted fast spin echo (FSE) in coronary and transversal planes; T1 weighted three dimensional (3D) in sagittal plane. MR perfu- sion was performed by fast field echo (FFE) echo planar imaging (EPI) sequence. Each subject obtained a 16 G cubital i.v. line. Gadolinium-based contrast agent (Gadovist; Bayer HealthCare Pharmaceuticals, Germany) was used in the standard dose (0.1 mmol/kg), with flow ve- locity 4 ml/s. At the end of the contrast bolus, 20 ml of normal saline with the same flow was given. Contrast agent and normal saline were given by automatic injector. First-pass dynamic susceptibil- ity-weighted contrast-enhanced perfusion-weight- ed imaging (DSC-MRI) was then performed. DSC- MRI was done using the planar echo 3D method: the signal from the whole k-plane was acquired after a single 90° radiofrequency pulse in order to reach maximum study speed. Data acquisition was done before, during and after contrast bolus (20 slices with 5 mm thickness, matrix size 128 x 128 and size of examined field 224 mm). The rep- etition time (TR) was 1501 ms, the acquisition time for each dynamic volume was 1.6 s. The echo time (TE) was 40 ms, flip angle 75°. Each perfusion se- quence consisted of 40 dynamic images, lasting 66 s for each first-pass. First, two experienced neuroradiologists inde- pendently visually interpreted the morphologi- cal MRI sequences and then the functional colour maps of perfusion parameters. An automatic de- convolution program (Neuro T2* perfusion pack- age, Philips Achieva Nova, Netherlands), which is a part of the MRI scanner software, was used. The program automatically calculated the perfu- sion parameters: negative integral, i.e., blood vol- ume (BV), mean transit time (MTT), time to peak (TTP) and an index representing blood flow (BF). The deconvolution method is based on a predeter- mined arterial input function (AIF). AIF was set on the medial cerebral artery (MCA) at the side of the brain with larger diameter and more prominent flat course. According to methods previously de- scribed30, 31, we manually chose and marked two regions of grey matter with either higher blood flow or higher blood volume, so called regions of interest (ROIs), and calculated the relative values of perfusion parameters. Statistical methods The commercially available SPSS version 21.0 pro- gram was used. All values are given as absolute (N) and percentage (%). Mean values are expressed as median (Me) and/or mean (M) with standard de- viation (SD). The normality of the data was tested by Kolmogorov-Smirnov and Shapiro-Wick tests. Since most of the data was not normally distribut- ed, 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. Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures280 Results Demographical and clinical data are summarized in Table 1, EEG and syndrome data in Table 2. In four patients (13.3%) the index seizure was their first seizure; patient 3 had only had one sei- zure 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 (»hyper- perfusion« patients) (Figure 1). All »hyperperfu- sion« patients had normal structural MRI; 9 of the remaining 25 patients with normal perfusion pa- rameters 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 asso- ciations between structural MRI abnormalities and either postictal cytokine level or MRI perfusion parameters. Patients 7 and 15 also had hyperper- fusion also on the control MRI examination after three months (»control hyperperfusion«). During this period, both patients were seizure free (i.e., the date of the last seizure was the date of enrolling in the study). MRI parameters after index seizure(s) are shown in Table 3. 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 (»nor- moperfusion«, »hyperperfusion«, and »control hy- perperfusion«) are provided in Table 5. 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 correlat- ed with concentrations of IL-10, IFN-γ and TNF-α (Table 7). There was a significant correlation among con- centrations 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-γ (ρ = 0.39, p < 0.05). Taking into account the whole cohort of patients, cytokine concentrations did not correlate with pos- tictal MRI perfusion parameters. Postictal MRI per- fusion parameters were furthermore not correlated with any other tested variable (age, gender, smok- ing 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 sub- groups of patients - »normoperfusion« (25 pa- tients), and »hyperperfusion« (5 patients). There were a slightly higher proportion of smokers in the »hyperperfusion« group: (60% vs. 40% in the »normoperfusion« group). The percentage of idio- pathic generalized epilepsy was identical between the two groups (20%). Overall, 12 patients had fo- cal EEG abnormalities but there was only one of these in the »hyperperfusion« group (left temporal changes). In the »hyperperfusion« group, concentrations of most of the cytokines were lower but the differ- ence reached significance only for IL-4 (Z = -1.98; FIGURE 1: 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) Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures 281 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-γ, IL-1Ra and hsCRP among the three groups (»normoperfusion«, »hyperperfu- sion«, »control hyperperfusion«). Conversely, their concentration of IL-10 was the lowest among the three groups. These differences, however, did not reach statistical significance (Table 6). The number of seizures during the follow up year correlated significantly only with the number of antiepileptic drugs (ρ = 0.60, p < 0.01). TABLE 1. Demographical and clinical data 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 (RF) V 1 TPM, LAM, DPH 2 (2/12) Resection RF >100 6 M 35 / 20 FS F (RF) R 1 LAM 2(2/10) N 0 7 M 42 / 1 FS F(RT) 1 ST 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* PREG** 1(13) Encephalomalacy LT 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 RT 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 atrophy 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 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 Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures282 Patients with a first seizure were analysed sepa- rately. They had a lower number of seizure types, lower NHS3-1 score, lower number of index sei- zures and lower number of antiepileptic drugs; but differences were not statistically significant. Discussion 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 et al.32 found an increased level of IL-6 only in the group of patients with temporal lobe epilepsy, whereas in patients with extra-temporal foci cytokine the level remained unchanged. This was not the case in our patient group, in which only four (out of 30) subjects had temporal lobe epilepsy and the level of IL-6 was elevated in 14/29 patients. However, it has previously been reported that patients with active epilepsy, regardless of ep- ilepsy type, duration or frequency of seizures, have elevated levels of IL-6.33 Lehtimaki et al.14 found an increased level of IL-6 in all but two patients with partial seizures, with the greatest magnitude in pa- tients who suffered repeated tonic - clonic seizures; but since patients with alcohol withdrawal and tumour provoked seizures were included in their study group, the interpretation of their results is difficult. In another study (in the emergency setting), proinflammatory cytokines were elevated postic- tally 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-α and hsCRP but also between proin- flammatory cytokines (TNF-α and IFN-γ) and IL- 1Ra. This is consistent with the hypothesis of the inflammatory damage mitigation role of IL-1Ra.35 In contrast to previous reports10,14,36,37, we found an inverse correlation between the number of in- dex seizures and the level of proinflammatory cy- tokines IL-10, TNF-α and IFN-γ. We suggest that this may be a consequence of the design of our study; we tested a single cytokine level and not cy- tokine level dynamics. Two patients with idiopath- ic generalized epilepsy had the highest reported number of index seizures in our group. Since we relied solely on eyewitness observational data, we did not measure the auras, exact number of index seizures or their duration, which may be a crucial factor in measurement of the postictal cytokine lev- el. Our study was performed at emergency settings where video-EEG was not available. MRI “normo-” and “hyperperfusion” groups of patients demonstrated different concentrations TABLE 2. EEG and syndrome 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) EEG Normal 4 (13) Focal 12 (40) Generalized 5 (17) IGE 6 (20) undetermined 3 (10) Focus RT 4(33) LT 5(41) RF 2(16) LF 1(8) 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 TABLE 3. MRI perfusion parameters after index seizures (grey matter) N M Me SD N BF 25 101,7 93 40,8 N BV 25 8 7 3,9 N MTT 25 6,9 6,9 2,9 N TTP 25 13,9 13,8 2,3 P BF 5 265,8** 234 56,3 P BV 5 25,5** 27,6 4,7 P MTT 5 5,7 5 1,2 P TTP 5 14,5 13,5 4,5 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; ** statistically significant difference (p < 0,001) Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures 283 of cytokines IL-4 and TNF-α: patients with hyper- perfusion had significantly lower concentrations of IL-4 and a higher concentration of TNF-α. The two »control hyperperfusion« patients had the highest concentrations of TNF-α and other pro- inflammatory cytokines - IL-6, IL-12, IFN-γ, IL- 1Ra and hsCRP. It is tempting to speculate that »hyperperfusion« and »control hyperperfusion« patients had the highest intensity of brain inflam- mation but further studies will have to check this hypothesis. TABLE 4. Blood cytokine concentration after index seizure(s) N M Me SD Ref. No (%) above/below reference IL-4(pg/ml) 29* 0.9 0.6 1.3 ≤ 1 7(24) IL-6 (pg/ml) 29* 5.7 3.7 5.1 ≤ 3.9 15(52) IL-10 (pg/ml) 29* 42.8 11.4 10.2 ≤ 10.9 16(55) IL-12 (pg/ml) 29* 66 58.5 40.3 ≤ 204 29(100) TNFα (pg/ml) 29* 8.5 7.6 4 4.7–12.4 1(3) IFN-γ (pg/ml) 29* 27.5 15 54.4 ≤ 1.2 7(24) IL-1Ra (pg/ml) 29* 1302.7 676 1797 168–744 14(48) Hs CRP (mg/l) 29* 2.9 1.1 4,5 <3 29(100) M = mean; Me = median; N = number; Ref = reference values; SD = standard deviation; *patient 3 is not included TABLE 5. Median concentrations of cytokines (pg/ml) and hs CRP(mg/l) in three subgroups of patients (see text for details) IL-4 IL-6 IL-10 IL-12 TNF-α IFN-γ 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 hyperperfusion1 0,5 6,0 5,1 110,4 10,6 27,7 868 1,6 * Statistically significant difference (p < 0. 05) TABLE 6. Basic and control concentrations of cytokines (pg/ml) in patients with hyperperfusion after index seizure(s) Patient IL-4 IL-6 IL-10 IL-12 TNF-α IFN-γ IL-1Ra hsCRP 2 (1st) 0,08 <2 116 54,5 8,19 13,08 352 1,5 (control) 0,3 <2 134 99,6 8,97 9,23 822 1,7 3 (1st) / / / / / / / / (control) 1,77 <2 12,6 127 11,5 108 1479 0,9 7* (1st) 0,41 2,47 10,02 24,8 11,7 44,56 676 1,9 (control) 0,35 <2 29,8 41,8 12,1 31 361 1,8 15* (1st) 0,61 9,49 0,22 196 9,57 10,81 1060 1,4 (control) 0,00 <2 0,58 150 14,1 14,7 593 0,7 25 (1st) 0,38 <2 11,51 34,6 11,1 14,97 462 1,1 (control) 1,01 <2 12,0 42,5 20,2 16,2 660 1,4 * Patient demonstrated hyperperfusion also on control MRI after three months. 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 dem- onstrated in our study - have been attributed to postictal blood-brain barrier affection. DSC MRI is a noninvasive surrogate biomarker of brain inflam- mation, capable of demonstrating endothelial dys- function or increased blood-brain barrier perme- ability.35 In patients with high frequency seizures, Pizzini et al.26 demonstrated periictal hyperperfu- Radiol Oncol 2017; 51(3): 277-285. Filipovic T et al. / DSC MRI and cytokines after seizures284 sion (up to 5 hours after seizure) and in patients with low frequency seizures postictal hypoper- fusion (up to 15-28 hours) on both DSC MRI and non-contrast arterial spin labelling (ASL) MRI. No patient in our study had hypoperfusion. Because we chose a minimum of 8-hour postictal delay for MRI imaging (in order to avoid postictal perfu- sion changes that may represent ongoing epileptic activity)18,19 we may have missed contralateral hy- poperfusion that has proposed to be a consequence of widespread dissociation of cerebral neurovascu- lar coupling.22 We did demonstrate postictal hyper- perfusion in a subgroup of patients. MRI perfusion parameters from our group tak- en as a whole did not correlate with any tested var- iable (including seizure frequency or syndrome). Hyperperfusion on MRI - perfusion with a 12-hour delay after a 4-hour pilocarpine induced status epi- lepticus 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 prog- nostic 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. Conclusions In conclusion, our study revealed a subgroup of patients with brain postictal hyperperfusion and higher plasma cytokine values, indicating a prob- able long lasting blood-brain barrier alteration. 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