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Blood-brain barrier disruption in post-traumatic epilepsy

Tomkins, O; Shelef, I; Kaizerman, I; Eliushin, A; Afawi, Z; Misk, A; Gidon, M;Cohen, A; Zumsteg, D; Friedman, A

Tomkins, O; Shelef, I; Kaizerman, I; Eliushin, A; Afawi, Z; Misk, A; Gidon, M; Cohen, A; Zumsteg, D; Friedman,A (2008). Blood-brain barrier disruption in post-traumatic epilepsy. Journal of Neurology, Neurosurgery, andPsychiatry, 79(7):774-777.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Neurology, Neurosurgery, and Psychiatry 2008, 79(7):774-777.

Tomkins, O; Shelef, I; Kaizerman, I; Eliushin, A; Afawi, Z; Misk, A; Gidon, M; Cohen, A; Zumsteg, D; Friedman,A (2008). Blood-brain barrier disruption in post-traumatic epilepsy. Journal of Neurology, Neurosurgery, andPsychiatry, 79(7):774-777.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Neurology, Neurosurgery, and Psychiatry 2008, 79(7):774-777.

Blood-brain barrier disruption in post-traumatic epilepsy

Abstract

BACKGROUND: Traumatic brain injury (TBI) is an important cause of focal epilepsy. Animalexperiments indicate that disruption of the blood-brain barrier (BBB) plays a critical role in thepathogenesis of post-traumatic epilepsy (PTE). OBJECTIVE: To investigate the frequency, extent andfunctional correlates of increased BBB permeability in patient with PTE. METHODS: 32 head traumapatients were included in the study, with 17 suffering from PTE. Patients underwent brain MRI (bMRI)and were evaluated for BBB disruption, using a novel semi-quantitative technique. Cortical dysfunctionwas measured using electroencephalography (EEG), and localised using standardised low-resolutionbrain electromagnetic tomography (sLORETA). RESULTS: Spectral EEG analyses revealed significantslowing in patients with TBI, with no significant differences between patients with epilepsy and thosewithout. Although bMRI revealed that patients with PTE were more likely to present with intracorticallesions (p = 0.02), no differences in the size of the lesion were found between the groups (p = 0.19).Increased BBB permeability was found in 76.9% of patients with PTE compared with 33.3% of patientswithout epilepsy (p = 0.047), and could be observed years following the trauma. Cerebral cortex volumewith BBB disruption was larger in patients with PTE (p = 0.001). In 70% of patients, slow (delta band)activity was co-localised, by sLORETA, with regions showing BBB disruption. CONCLUSIONS:Lasting BBB pathology is common in patients with mild TBI, with increased frequency and extent beingobserved in patients with PTE. A correlation between disrupted BBB and abnormal neuronal activity issuggested.

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Blood-Brain Barrier Disruption in Post-Traumatic Epilepsy

Corresponding author: A Friedman Departments of Physiology and Neurosurgery, Zlotowski Center for Neuroscience, Ben-Gurion University 84105 Beer-Sheva, Israel Tel: +972 8 6479879/884 Fax: +972 8 6479883 Email: alonf@bgu.ac.il

O Tomkins, I Shelef*, I Kaizerman, A Eliushin, Z Afawi, A Misk, M Gidon, A Cohen, D Zumsteg, A Friedman

* Equal contribution Oren Tomkins, Departments of Physiology and Neurosurgery, Soroka Medical Center and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Ilan Shelef, Department of Neuroradiology, Soroka Medical Center, Beer-Sheva, Israel. Idan Kaizerman, Department of Physiology Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Andrei Eliushin, Department of Neurosurgery, Soroka Medical Center, Beer-Sheva, Israel. Zaid Afawi, Department of Neurology, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel. Adel Misk, Department of Neurology, Shaare Zedek Medical Center, Jerusalem, Israel and Al-Quds University, Jerusalem, Israel. Michael Gidon, Department of Neurosurgery, Soroka Medical Center, Beer-Sheva, Israel. Avi Cohen, Department of Neurosurgery, Soroka Medical Center, Beer-Sheva, Israel. Dominik Zumsteg, Department of Neurology, University Hospital Zurich, Zurich, Switzerland. Alon Friedman, Departments of Physiology and Neurosurgery, Soroka Medical Center and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel and Institute for Neurophysiology, Charité University Medicine, Berlin, Germany. Key Words: Blood-Brain Barrier, Post-Traumatic Epilepsy, Magnetic Resonance Imaging, Electroencephalography. Word count manuscript: 1498 Word count abstract: 229 The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in JNNP and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp.bmj.com/ifora/licence.pdf).

JNNP Online First, published on November 8, 2007 as 10.1136/jnnp.2007.126425

Copyright Article author (or their employer) 2007. Produced by BMJ Publishing Group Ltd under licence.

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ABSTRACT Background: Traumatic brain injury (TBI) is an important cause of focal epilepsy. Animal experiments indicate that disruption of the blood-brain barrier (BBB) plays a critical role in the pathogenesis of post-traumatic epilepsy (PTE). Objective: To investigate the frequency, extent and functional correlates of increased BBB permeability in PTE patients. Methods: 32 head trauma patients were included in the study, with 17 suffering from PTE. Patients underwent brain magnetic resonance imaging (bMRI) and were evaluated for BBB disruption, using a novel semi-quantitative technique. Cortical dysfunction was measured using electroencephalography (EEG), and localized using standardized low resolution brain electromagnetic tomography (sLORETA). Results: Spectral EEG analyses revealed significant slowing in TBI patients with no significant differences between epileptic and non-epileptic patients. While bMRI revealed that PTE patients were more likely to present with intracortical lesions (p=0.02), no differences in the size of the lesion were found between the groups (p=0.19). Increased BBB permeability was found in 76.9% of PTE compared to 33.3% of non-epileptic patients (p=0.047), and could be observed years following the trauma. Cerebral cortex volume with BBB disruption was larger in PTE patients (p=0.001). In 70% of patients, slow (delta band) activity was co-localized, by sLORETA, with regions showing BBB disruption. Conclusions: Lasting BBB pathology is common in mild TBI patients, with increased frequency and extent in PTE patients. A correlation between disrupted BBB and abnormal neuronal activity is suggested.

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Traumatic brain injury (TBI) is considered a major risk factor for post-traumatic epilepsy (PTE).[1] The mechanisms underlying the development of PTE and its prevention remain unknown.[2] Recent animal experiments show that opening the Blood-Brain Barrier (BBB) leads to network changes, long-lasting epileptiform activity and eventual neurodegeneration.[3,4] While previous clinical studies show that altered permeability is observed in neurological patients, including those with TBI,[5,6,7] no data exists regarding the frequency, extent, and significance of BBB disruption in PTE. The aim of this study was to examine BBB permeability following TBI and correlate it to EEG abnormalities and the presence of PTE. METHODS Patient Selection The study protocol was approved by the Soroka Medical Center Helsinki Committee. 17 patients diagnosed with PTE were included. All except one reported repeated partial seizures (with secondary generalization in 10 patients). Control group included 15 non-epileptic TBI patients who reported headaches (86.7%), cognitive impairment (6.7%) or motor dysphasia (6.7%) (Table 1). Patients groups were similar in age (27.9 ± 4.2 and 25.3 ± 2.8 years, respectively, p=0.97) and gender (7 women and 10 men, and 4 women and 11 men, respectively, p=0.47). All patients were healthy prior to TBI without history of neurological or psychiatric disorders. Most patients (90.6%) suffered mild TBI according to a Glasgow Coma Score (GSC) of >13 upon admission. Of all the TBI patients 13 were unconscious for several minutes and 3 for several days with no differences between the groups. Electroencephalography EEG recordings were made by a clinical 128 channel digital EEG acquisition unit (CEEGRAPH IV, Bio-logic Systems Corp., Mundelein, Illinois, sampling rate of 256 Hz) as previously reported from our laboratory.[7] The EEG was interpreted by a physician unaware of the study. Average power for the discrete frequency bands was normalized to each subject’s own total power (1.5-40 Hz). Recordings of 13 healthy adult volunteers (aged 34.8 ± 2.8 years), with no history of brain injury or neurological disease, served as controls. For localization sLORETa was used. [7,8] Magnetic Resonance Imaging and BBB Integrity Evaluation MRI scans were performed using a 1.5 tesla machine (Intera, Philips Medical Systems, Best, the Netherlands). Scans were only performed after 24 hours without seizures. For the evaluation of BBB integrity, images were collected before and after peripheral administration of the contrast medium Magnetol (Gadolinium-DTPA (Gd-DTPA) 0.5M, 0.1mmol/kg) (Soreq Radiopharmaceuticals, Israel). BBB permeability was estimated as previously reported. [5,6] In short, axial T1-weighted spin-echo images (582/15/1 [TR/TE/NEX], section thickness, 5mm; intersection gap, 1mm; matrix, 256 x 256) were obtained. Matching brain images from before and after Gd-DTPA administration were paired and analyzed for statistically significant changes in signal intensity. For lesion volume measurements, T1 MRI images before contrast agent administration were used. The intracerebral lesion was identified by a physician and the number of pixels within the lesion was counted in all slices. Disruption volume was calculated for the same slices by counting all pixels with significant enhancement. Localization of the cortical and BBB lesions according to Brodmann areas was performed by manual anatomical registration to the digitized Talairach brain atlas.

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Table 1. Patients characteristics. Thirty-two patients with TBI were enrolled in this study. Seventeen patients presented with seizures at our outpatient clinic, and 15 presented with other general complaints such as headaches. Patients were tested on average 33.09± 9.99 months after the trauma. sLORETA localization to Brodmann areas of abnormal EEG activity and enhancement is presented in parentheses. MVA- moving vehicle accident.

# Sex Age Time after Trauma Time after Trauma (m) Etiology Symptoms Treatment Abnormal Enhancement1 M 24 4m 4 MVA Cognitive Impairment Valporic acid Rt. Frontal (47)2 M 37 18y 216 MVA Headache Clonazepam No Disruption3 M 49 4d 0.13 MVA Headache - No Disruption4 M 19 6m 6 Blunt Trauma Headaches - -5 M 25 1m 1 Blunt Trauma Headaches - No Disruption6 M 27 2m 2 MVA Headaches - No Disruption7 F 17 4m 4 MVA Headaches - No Disruption8 F 25 5m 5 MVA Headaches Phenytoin No Disruption9 M 17 2m 2 Blunt Trauma Headaches - No Disruption10 M 22 4y 48 MVA Headaches Phenobarbital No Disruption11 F 46 3m 3 MVA Headaches - Rt. Parietal (38)12 M 13 1m 1 Fall Headaches Valporic acid Rt. Parieto-Occipital (18)13 F 23 10y 120 MVA Headaches - -14 M 13 3y 36 MVA Headaches - -15 M 22 5d 0.16 Blunt Trauma Motor Aphasia, Rt. Central Facialis - Lt. Parietal (9)16 M 34 1m 1 Blunt Trauma Behavioral Changes, Susp. Temporal seizures Phenytoin Rt. Temporal (21)17 F 29 16y 192 MVA Seizures Carbamazepine -18 F 12 1m 1 Fall Seizures Valporic acid -19 F 23 7y 84 Fall Seizures - Lt. Parietal (40)20 M 62 1m 1 MVA Seizures Valporic acid Lt. Parietal (2)21 M 15 10d 0.33 MVA Seizures Phenytoin Rt. Parietal (40)22 F 10 4m 4 Blunt Trauma Seizures Phenobarbital No Disruption23 M 18 3y 36 MVA Seizures - Rt. Temporo- Occipital (37)24 M 18 5m 5 Fall Seizures Phenytoin Rt. Parietal (40)25 M 37 11y 132 MVA Seizures Valporic acid Rt. Temporal (47)26 M 68 1m 1 Fall Seizures+ Rt. Hemiparesis Valporic acid Lt. Parietal (3)27 M 17 5y 60 Blunt Trauma Seizures - No Disruption28 M 15 3m 3 Blunt Trauma Seizures Valporic acid -29 F 49 18m 216 Fall Seizures Carbamazepine No Disruption30 M 28 10d 0.33 Blunt Trauma Seizures Phenytoin Lt. Parietal (40)31 F 17 3y 36 MVA Seizures Phenytoin Lt. Fronto- Parietal (8)32 F 23 3y 36 Fall Seizures Lamotrigine -

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RESULTS EEG recordings were performed on 22 patients, 10 days – 11 years (median= 5.5 months) after the trauma. Apparent slowing or interictal epileptiform activity was observed in 78.6% of PTE patients and in 12.5% of non-epileptic patients (p=0.006, χ

2 Pearson’s test) (Table 1). Spectral analysis showed that the delta (1.5-4 Hz) power in both PTE (3.68 ± 0.28 %) and non-epileptic groups (3.76 ± 0.29 %) was significantly higher than that of healthy controls (2.83 ± 0.15 %, p<0.01, Mann-Whitney U test). Only the PTE group showed significantly elevated theta and reduced alpha (2.28 ± 0.14 vs. 1.93 ± 0.09 %, p=0.04 and 1.99 ± 0.14 vs. 2.83±0.15 %, p=0.01, respectively, Mann-Whitney U test) compared to controls. No significant differences were found between the PTE and non-epileptic groups (data not shown). The MRI scans of all patients were evaluated for intracerebral lesion and BBB disruption volumes. In 14 (56%) TBI patients brain regions were identified with significant enhancement indicating increased BBB permeability (fig 1A). In 13 patients (86.7 %) the disrupted BBB was in close proximity to cortical regions around old haemorrhagic contusions. Only one patient displayed BBB disruption with no concomitant lesion. BBB disruption was identified months or years (21.8 ± 10.6, median = 2 months) after the trauma (Table 1). Parenchymal lesions were found in all cortical regions (Table 1). The average lesion volume was 6.0 ± 1.7 cm3 and the average volume of cortex with abnormal BBB was 5.9 ± 1.6 cm3. Although PTE patients were more likely to have a lesion on their MRI scans (80%) than non-epileptic patients (30.8 %, p= 0.02, χ2 Pearson’s test, fig 3A), there was no significant difference in the size of the lesion (6.6 ± 1.9 % vs. 5.3 ± 2.8 cm3, p=0.19, Mann-Whitney U test, fig 1B). PTE patients were more likely to have BBB disruption (76.9%) than non-epileptic TBI patients (33.3%, p=0.047, χ2 Pearson’s test), and the volume of BBB disruption was significantly larger (9.8 ± 2.6 vs. 1.7 ± 0.6 cm3, p=0.001, Mann-Whitney U test, fig 1B). The spatial relationship between BBB disruption and abnormal cortical function was assessed by localizing the cortical sources of the observed abnormal delta activity using sLORETA [8]. In 7 of 10 patients with BBB disruption, sLORETA localized the source for abnormal delta to the same Brodmann area as the BBB disruption (Table 1). Furthermore, of the 3 PTE patients with normal EEG two had no BBB disruption, while the third had not undergone an MRI scan. The volume of cortex localized with abnormal slow activity correlated linearly with the size of the BBB disrupted region (correlation coefficient= 0.53, p=0.04), but not with the size of the cortical lesion (correlation coefficient=0.34, p=0.19, data not shown). Figure 1C-D demonstrates data from PTE patient #16 showing abnormal rhythmic slowing maximally recorded in right temporal electrodes and localized to the anterior part of the middle temporal gyrus (Brodmann area 21). BBB analysis showed focal pathological enhancement in the same region (Brodmann area 21). DISCUSSION Using quantitative EEG analysis and a semi-quantitative method for detecting abnormal BBB permeability following mild TBI we found: (1) Increased EEG slowing in the delta-theta range; (2) Increased BBB permeability in 56% of patients; (3) Spatial correlation between focal enhanced BBB permeability and EEG delta activity; (4) Correlation between the size of the BBB disrupted region, but not that of the anatomical lesion, and the extent of cortical dysfunction and, (5) Patients with PTE were more likely to show abnormal BBB permeability, and in larger cortical areas, compared to non-epileptic TBI patients.

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Consistent with earlier studies,[7,9] TBI patients commonly had abnormal EEG. Interestingly, spectral analyses did not reveal significant differences between our two groups. Abnormal EEG slowing reflects dysfunction of the cortical network and probably neuronal hypersynchronization. While animal models for brain injury consistently reveal electrophysiological evidence for neuronal hyperexcitability and hypersynchronicity,[4,3,10,11] behavioral manifestations of seizures have been rarely reported.[12] This raises the possibility that neuronal hypersynchrony may not necessarily manifest as convulsions,[13] possibly depending on the cortical region involved. Similarities found in EEG analysis between our PTE and non-epileptic patients may be due to a common path, i.e. similar pathologic neuronal dysfunction presenting with different phenotypes. We note that all patients were referred to our outpatient clinic due to significant symptomatology. Further investigation of the differences between these groups and asymptomatic TBI patients is necessary to elucidate this supposition. We used sLORETA [8] to calculate the distribution of the delta band within the cerebral cortex gray matter. The proximity of the cortical region with maximal delta activity to the MR-defined cortical lesion is suggestive of a causative relationship. We found no correlation between the volume of cortex with abnormal delta activity and the size of the anatomical lesion. This may reflect that the area of contusion undergoes neuronal necrosis and gliosis and hence displays no neuronal activity. However, the surrounding brain tissue remains functional and may be subjected to conditions that impact its normal function. The size and co-localization of abnormal EEG activity and BBB disruption supports the supposition that lasting BBB disruption may be causally related to the emergence of neocortical dysfunction. An alternative hypothesis is that seizure activity leads to BBB opening. We believe this is a less likely explanation in these cases, as increased BBB permeability was observed in patients despite apparent complete medical control of seizures, and no seizures were reported at least 24 hours before the brain scans. In addition, recent animal experiments directly demonstrated potential molecular pathways linking BBB leakage and increased neuronal excitability.[3] Due to the retrospective nature of our study and the given evidence of PTE patients with lingering BBB disruption lasting up to several years, we can not exclude that both conditions exist, where early disruption leads to seizure activity, which in turn perpetuates the leakage. Prospective human studies are needed to further elucidate the temporal and spatial relationship between early breakdown of the BBB and development of delayed brain dysfunction, including epilepsy.

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FIGURE LEGENDS Figure 1. (A) Statistically significant enhancement of T1 MRI scan in the region surrounding the cortical lesion in patient #21, 10 days following the trauma. (B) Among PTE patients, the volume of BBB disruption was significantly larger than that of non-epileptic patients. (C) A 34-year-old PTE patient one month following mild TBI. Power spectrum (0.5-11 Hz) showing a marked increase in power at 1.125 Hz, taken from an EEG recording demonstrating abnormal slowing maximal at right frontal and temporal electrodes (inset). (D) sLORETA localizing the pathological signal to the anterior parts of the right middle temporal gyrus (Brodmann area 21, Left), and MRI signal enhancement indicating increased BBB permeability localized to the same region (Right).*= p<0.05.

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