Dynamic susceptibility contrast enhanced (DSC) MRI perfusion and plasma cytokine levels in patients after tonic-clonic seizures

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 stroke^16,17, 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.

Demographical and clinical data are summarized in Table 1, EEG and syndrome data in Table 2.

Table 1

Demographical and clinical data

Table 2

EEG and syndrome

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 (i.e., the date of the last seizure was the date of enrolling in the study).

Figure 1

MRI parameters after index seizure(s) are shown in Table 3.

Table 3

MRI perfusion parameters after index seizures (grey matter)

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.

Table 4

Blood cytokine concentration after index seizure(s)

Table 5

Median concentrations of cytokines (pg/ml) and hs CRP(mg/l) in three subgroups of patients (see text for details)

Postictal (1^st) 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-γ and TNF-α (Table 7).

Table 6

Basic and control concentrations of cytokines (pg/ml) in patients with hyperperfusion after index seizure(s)

Table 7

Correlation between cytokine concentrations and number of index seizures

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-γ (ρ = 0.39, p < 0.05).

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% vs. 40% in the »normoperfusion« group). The percentage of idiopathic generalized epilepsy was identical between the two groups (20%). Overall, 12 patients had focal 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 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-γ, IL-1Ra and hsCRP among the three groups (»normoperfusion«, »hyperperfusion«, »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).

Patients with a first seizure were analysed separately. They had a lower number of seizure types, lower NHS3-1 score, lower number of index seizures and lower number of antiepileptic drugs; but differences were not statistically significant.

Elevated cytokine levels have been demonstrated after epileptic seizures in brain tissue^10,11 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 epilepsy 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 patients 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 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-α and hsCRP but also between proinflammatory 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 reports^10,14,36,37, we found an inverse correlation between the number of index seizures and the level of proinflammatory cytokines 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 cytokine level dynamics. Two patients with idiopathic 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 level. Our study was performed at emergency settings where video-EEG was not available.

MRI “normo-” and “hyperperfusion” groups of patients demonstrated different concentrations of cytokines IL-4 and TNF-α: patients with hyperperfusion 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 proinflammatory 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 inflammation but further studies will have to check this hypothesis.

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 et al.²⁶ demonstrated periictal hyperperfusion (up to 5 hours after seizure) and in patients with low frequency seizures postictal hypoperfusion (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 perfusion changes that may represent ongoing epileptic activity)^18,19 we may have missed contralateral hypoperfusion that has proposed to be a consequence of widespread dissociation of cerebral neurovascular coupling.²² We did demonstrate postictal hyperperfusion in a subgroup of patients.

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.