BBB & epilepsy

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  doi:10.1093/brain/awl318 Brain (2007), 130 , 521–534 Blood–brain barrier leakage may lead toprogression of temporal lobe epilepsy E. A. van Vliet, 1 , 2 S. da Costa Arau´jo, 2 S. Redeker, 3 R. van Schaik, 2 E. Aronica 3 and J. A. Gorter 1 , 2 1 Epilepsy Institute of The Netherlands (SEIN), Heemstede, 2 Swammerdam Institute for Life Sciences, Center forNeuroscience and 3 Academic Medical Center, Department of (Neuro)Pathology, University of Amsterdam,Amsterdam, The NetherlandsCorrespondence to: Dr J. A. Gorter, Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The NetherlandsE-mail: Leakage of the blood–brain barrier (BBB) is associated with various neurological disorders, includingtemporal lobe epilepsy (TLE). However, it is not known whether alterations of the BBB occur duringepileptogenesis and whether this can affect progression of epilepsy. We used both human and rat epilepticbrain tissue and determined BBB permeability using various tracers and albumin immunocytochemistry.In addition, we studied the possible consequences of BBB opening in the rat for the subsequent progressionof TLE. Albumin extravasation in human was prominent after status epilepticus (SE) in astrocytes andneurons, and also in hippocampus of TLE patients. Similarly, albumin and tracers were found in microglia,astrocytes and neurons of the rat. The BBB was permeable in rat limbic brain regions shortly after SE, butalso in the latent and chronic epileptic phase. BBB permeability was positively correlated to seizure frequencyin chronic epileptic rats. Artificial opening of the BBB by mannitol in the chronic epileptic phase induced apersistent increase in the number of seizures in the majority of rats. These findings indicate that BBB leakageoccurs during epileptogenesis and the chronic epileptic phase and suggest that this can contribute to theprogression of epilepsy.Keywords : albumin; seizure; fluorescein; Evans Blue; mannitol; status epilepticus Abbreviations : BBB ¼ blood–brain barrier; FJB ¼ fluoro-jade B; TLE ¼ temporal lobe epilepsy; SE ¼ status epilepticus Received June 23, 2006. Revised September 26, 2006. Accepted October 9, 2006. Advance Access publication November 22, 2006. Introduction Due to its unique structure, the blood–brain barrier (BBB)is capable of limiting the penetration of a variety of substances from the blood into the brain. The BBB plays animportant role in the homeostasis and is generally seen as adefence mechanism that protects the brain against variousmolecules that may enter the BBB. The BBB is composed of endothelial cells which form a diffusion barrier, due tothe presence of tight junctions that firmly connectendothelial cells (Kettenmann and Ransom, 2005). Inaddition to this, efflux transporters of the ATP bindingcassette family (e.g. P-glycoprotein and multi-drugresistance-associated proteins), located at the luminal sideof endothelial cells, may restrict further entry of substancesinto the brain. To provide the brain with essential nutrientsand remove excreted substances, endothelial cells alsocontain numerous membrane transporters (  for review see  Lee et al  ., 2001) involved in the influx/efflux of essentialsubstrates such as glucose, amino acids, electrolytes andnucleosides or removal of xenobiotics.Due to the development of small molecular weight tracersthat enter the damaged BBB, disruption was found to beassociated with various neurological disorders such asmigraine (Dreier et al  ., 2005), postconcussion syndrome(Korn et al  ., 2005), multiple sclerosis (Minagar andAlexander, 2003) and epilepsy (Roch et al  ., 2002; Ballabh et al  ., 2004; Neuwelt, 2004; Seiffert et al  ., 2004). BBBdisruption has been shown both in human (Mihaly andBozoky, 1984) as well as in animal studies after acute # The Author  ( 2006 ). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email:    b  y  on S  e p t   em b  er  5  ,2  0 1  0 h  t   t   p:  /   /   b r  ai  n. ox f   or  d  j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   seizures (Nitsch and Klatzo, 1983; Zucker et al  ., 1983;Lassmann et al  ., 1984; Ruth, 1984; Saija et al  ., 1992; Pont et al  ., 1995; Ilbay  et al  ., 2003; Leroy  et al  ., 2003; Oztas et al  .,2003) and has been associated with abnormal EEG patterns(Tomkins et al  ., 2001; Korn et al  ., 2005; Pavlovsky  et al  .,2005). Moreover, Friedman and colleagues showed that focalopening of the BBB by direct cortical application of albumin-containing solution can lead to the generation of an epileptic focus in rats (Seiffert et al  ., 2004). However, it isnot known how the BBB integrity changes duringepileptogenesis and whether alterations in BBB permeability can contribute to spontaneous seizure progression. To getmore insight into the role of BBB disruption in epilepto-genesis and progression of epilepsy, we determined BBBpermeability in epileptic rats and humans and studied thepossible consequences of a compromised BBB for thesubsequent seizure progression. Since previous studies haveshown that albumin can serve as an indicator of compromised BBB function in a variety of pathophysiolo-gical conditions (Cornford and Hyman, 1999; Seiffert et al  .,2004), we used albumin and albumin-binding dyes tovisualize BBB leakage. Material and methods Albumin immunoreactivity (IR) in human brain To determine BBB permeability in human epileptic brain, albuminextravasation was studied by immunocytochemistry. Brain materialwas obtained from the files of the departments of neuropathology of the Academic Medical Center (University of Amsterdam).Patients underwent resection of the hippocampus ( n  = 6) formedically intractable epilepsy. To reduce metabolic injury, thefissural blood supply was kept intact until removal of the hippo-campus. The surgical material is directly fixed after dissection andtherefore in optimal condition. In addition, autopsy material wasused of two epilepsy patients that died during an acute statusepilepticus (SE). These patients had a long history of epilepsy (Table 1) and died before pharmacological treatment was started tostop the SE. Pathological examination excluded encephalitis ormeningitis. This material was compared to normal-appearinghippocampi of five autopsy specimens from patients withouthistory of seizures or other neurological diseases. Tissue wasobtained and used in a manner compliant with the Declaration of Helsinki. Table 1 summarizes the clinical features of all patients.Brain tissue was fixed in 10% buffered formalin, paraffinembedded, sectioned at 6 m m and mounted on organosilane-coated slides (Sigma, St Louis, MO, USA). Two hippocampalsections of each patient were processed for immunocytochemistry.Sections were deparaffinated in xylene, rinsed in ethanol (100, 95and 70%) and incubated for 20 min in 0.3% hydrogen peroxidediluted in methanol. Slides were then washed with phosphate-buffered saline (PBS; 10 mM, pH 7.4) and incubated overnight inanti-albumin (rabbit anti-human albumin; 1 : 20000; DakoCyto-mation, Glostrup, Denmark) at 4  C. Hereafter, sections werewashed in PBS and stained with a polymer based peroxidaseimmunocytochemistry detection kit (PowerVision Peroxidasesystem, ImmunoVision, Brisbane, CA, USA). After washing,sections were stained with 3,3 0 -diaminobenzidine tetrahydrochloride(50 mg DAB, Sigma-Aldrich, Zwijndrecht, The Netherlands) and 5 m l 30% hydrogen peroxide in a 10 ml solution of Tris–HCl. Sectionswere counterstained with haematoxylin, dehydrated in alcohol andxylene and coverslipped. Sections incubated without anti-albuminor with preimmune serum were essentially blank. Experimental animals To evaluate whether changes of BBB permeability occurred duringepileptogenesis, the SE rat model for temporal lobe epilepsy (TLE)was used (Gorter et al  ., 2001).Adult male Sprague–Dawley rats (Harlan CPB Laboratories,Zeist, The Netherlands) weighing 400–550 g were housedindividually in a controlled environment (21 6 1  C; humidity 60%; lights on from 08:00 a.m. to 8:00 p.m.; food and wateravailable ad libitum) . The study was approved by the University Animal Welfare committee. Electrode implantation Rats were anaesthetized with ketamine (57 mg/kg; Alfasan,Woerden, The Netherlands) and xylazine (9 mg/kg; Bayer AG, Table 1 Summary of the clinical and neuropathological data of the patients with epilepsy Patient/age(year)/genderClinical/pathologicaldiagnosisSeizurefrequency/monthsAge atonset(year)Durationepilepsy(year)SeizuretypeAEDs Follow-up(year)Engelclass1/17/M TLE/HS (W3) < 5 11 6 CPS PHT/CBZ/PB 6 I A2/21/M TLE/HS (W3) > 20 2 19 CPS/SGS PHT/CBZ/PB 1 I A3/19/F TLE/HS (W3) 10 6 13 CPS CBZ/PB/VPA 1 I A4/33/F TLE/HS (W3) 10–30 15 18 CPS/SGS CBZ/PB/VPA 2 I A5/27/M TLE/HS (W3) 10–20 10 17 CPS CBZ/PB/VPA 3 I A6/48/M TLE/HS (W3) 5–10 17 31 CPS/SGS PHT/CBZ/PB 1 I A7/18/M* TLE/HMEG 30 < 1 18 CPS/SGS/SE CBZ/PB — — 8/23/M* Epilepsy/ND 10–20 9 14 CPS/SGS/SE CBZ/LEV — — AEDs = antiepileptic drugs; CBZ = carbamazepine; CPS = complex partial seizures; HMEG = hemimegalencephaly; HS = hippocampalsclerosis; LEV = levetiracetam; ND = pathology not defined at autopsy; PB = phenobarbital; PHT = phenytoin; SE = status epilepticus;SGS = secondary generalized seizures; TLE = temporal lobe epilepsy; VPA = valproate; W = Wyler grading system.*Autopsy patients. 522 Brain (2007), 130 , 521–534 E. A. van Vliet et al.   b  y  on S  e p t   em b  er  5  ,2  0 1  0 h  t   t   p:  /   /   b r  ai  n. ox f   or  d  j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   Leverkusen, Germany) and placed in a stereotactic frame. In orderto record hippocampal EEG, a pair of insulated stainless steelelectrodes (70 m m wire diameter, tips were 0.8 mm apart) wereimplanted into the left dentate gyrus under electrophysiologicalcontrol as previously described (Gorter et al  ., 2001). A pair of stimulation electrodes was implanted in the angular bundle. SE induction Two weeks after electrode implantation, each rat was transferred toa recording cage (40 · 40 · 80 cm 3 ) and connected to a recordingand stimulation system (NeuroData Digital Stimulator, CygnusTechnology Inc., Delaware Water Gap, NJ, USA) with a shieldedmulti-strand cable and electrical swivel (Air Precision, Le PlessisRobinson, France). A week after habituation to the new condition,rats underwent tetanic stimulation (50 Hz) of the hippocampus inthe form of a succession of trains of pulses every 13 s. Each trainhad a duration of 10 s and consisted of biphasic pulses(pulse duration 0.5 ms, maximal intensity 500 m A). Stimulationwas stopped when the rats displayed sustained forelimb clonusand salivation for minutes, which usually occurred within 1 h.However, stimulation never lasted > 90 min. Behaviour wascontinuously monitored during electrical stimulation and severalhours thereafter. Immediately after termination of the stimulation,periodic epileptiform discharges (PEDs) occurred at a frequency of 1–2 Hz and were accompanied by behavioural generalized seizuresand EEG seizures SE. The total PED duration was considered as thetotal SE duration. In 34 rats a SE was electrically evoked, whichlasted from at least 3 h, up to 13 h. Electrode implanted control ratswere handled and recorded identically, but did not receive electricalstimulation. EEG monitoring  Differential EEG signals were amplified (10 · ) via a FET transistorthat connected the headset of the rat to a differential amplifier(20 · ; CyberAmp, Axon Instruments, Burlingame, CA, USA),filtered (1–60 Hz), and digitized by a computer. A seizure detectionprogram (Harmonie, Stellate Systems, Montreal, Canada) sampledthe incoming signal at a frequency of 200 Hz per channel. All EEGrecordings were visually screened and seizures were confirmed by trained human observers. All rats were monitored continuously from the SE onwards, until the first spontaneous seizure appeared.Hereafter some rats were disconnected from the set-up. All ratswere connected again 4 months later and continuous EEGrecordings (24 h/day) were started to determine seizure frequency and duration. As previously described (Gorter et al  ., 2001; van Vliet et al  ., 2004), a stable baseline of seizure frequency is normally reached in chronic epileptic rats at this time-point, and no seizureclusters occur. Rats were monitored for at least 1 week andexperiments were not started before a stable baseline was reached. Albumin IR in rat brain Albumin extravasation in the rat was studied by fluorescentalbumin immunocytochemistry. A subset of free-floating sectionsthat was used in Evans Blue (EB) tracer experiments (see below)were washed (2 · 10 min) in 0.05 M PBS and then incubated withanti-albumin (rabbit anti-albumin, 1:100, DakoCytomation,Glostrup, Denmark). After 24 h, the sections were washed in PBS(3 · 10 min) and incubated for 1.5 h in anti-rabbit Alexa Fluor 488(1 : 200, Molecular Probes). Following three additional washes inPBS, sections were mounted on slides (Superfrost Plus, Menzel,Braunschweig, Germany) and coverslipped with mounting mediumfor fluorescence, containing 4 0 ,6-diamidino-2-phenylindole, whichlabels cell nuclei (Vectashield with DAPI, Vector Laboratories,Burlingame, CA, USA). Images were acquired using a confocal-laserscanning microscope and Adobe Photoshop. Quantification of BBB permeability Since the detection of extravasated albumin by immunocytochem-istry is not an accurate measurement to determine whether the BBBwas permeable at a specific time-point, additional experiments wereperformed in rats. In order to quantify BBB permeability duringepileptogenesis and relate changes in permeability directly toseizure activity, rats were injected with two different fluorescenttracers that do not enter the brain under normal circumstances(except for the circumventricular organs). In addition, these tracersbind to albumin, so that a comparison could be made withimmunocytochemical data. Fluorescein (FSC) was used to quantify BBB permeability microscopically, while EB was used in a limitednumber of rats to detect BBB permeability macroscopically and toconfirm the distribution of BBB permeability microscopically asdetected by FSC. These tracers were intravenously (i.v.) adminis-tered via the tail vein (EB, 50 mg/kg i.v., Sigma-Aldrich, Steinheim,Germany; FSC; 100 mg/kg i.v., Merck, Darmstadt, Germany) underisoflurane anaesthesia (4 vol%). EEG recordings were discontinuedduring anaesthesia, which never lasted longer than several minutes.Rats were injected in the acute seizure period (1 day after SEinduction; EB n  = 2; FSC n  = 5; and 2 days after SE; FSC n  = 4), inthe latent period (1 week after SE; EB n  = 1; FSC n  = 5) and in thechronic seizure period (4 months after SE; EB n  = 2; FSC n  = 7),when rats display spontaneous seizures (average seizure frequency 0.3 seizures/h). In addition, electrode implanted control rats thatwere not stimulated were included as well (EB n  = 2; FSC n  = 5).Rats were disconnected from the EEG recording set-up 2 h aftertracer injection and deeply anaesthetized with pentobarbital(Nembutal, intraperitoneally (i.p.), 60 mg/kg). The animals wereperfused through the ascending aorta with 100 ml of physiologicalsalt solution, followed by 300 ml 4% paraformaldehyde/0.2%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brains werepost-fixed in situ  overnight at 4  C, dissected and cryoprotected in30% phosphate-buffered sucrose solution, pH 7.4. After overnightincubation at 4  C, the brains were frozen in isopentane ( À 30  C)and stored at À 80  C until sectioning. Sagittal sections (40 m m)were cut using a sliding microtome. Sections were collected in 0.1 Mphosphate buffer and processed for immunocytochemistry. Detection of EB and FSC To detect extravasation of the fluorescent albumin-binding dyes EBand FSC, sagittal sections were mounted on slides (Superfrost Plus,Menzel, Braunschweig, Germany) and coverslipped with mountingmedium for fluorescence (Vectashield, Vector Laboratories,Burlingame, CA, USA). Tracers were detected using a confocal-laser scanning microscope (Zeiss LSM510) with appropriate filtersettings (EB excitation 546 nm, emission 611 nm; FSC excitation488 nm, emission 520 nm). Images were made using Zeiss software(Zeiss LSM Image browser) and Adobe Photoshop. A quantifica-tion of FSC sections was made for each rat using three differentsections: 2.4, 3.4 and 4.6 mm lateral to bregma (Paxinos andWatson, 1998). Tracers were analysed in limbic brain regions that BBB leakage during progression of epilepsy Brain (2007), 130 , 521–534 523   b  y  on S  e p t   em b  er  5  ,2  0 1  0 h  t   t   p:  /   /   b r  ai  n. ox f   or  d  j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   are thought to be involved in the generation and/or spread of seizure activity, and also in the cerebellum. The following brainregions were analysed: hippocampus (granule cell layer), entorhinalcortex (layer II/III), anterior piriform cortex (layer II/III), amygdala(basolateral amygdala nucleus), thalamus (ventral postero-medial/lateral nucleus) and cerebellum. The confocal grid (271 · 271 m m 2 ,15 · 15 squares) was placed on the selected brain region and thenumber of squares that contained a FSC signal was counted. Theintensity of the FSC signal was evaluated using the histogramfunction in Adobe Photoshop. The average signal intensity measured in control rats (which was close to zero in all analysedregions), was used for the background correction. We constructed a‘permeability index’ (number of squares that contained a FSCsignal · FSC intensity) and all data were expressed as mean 6 SEM.Statistical analysis on the permeability index was performed usingANOVA, followed by the Student’s t  -test. Differences with P  < 0.05were considered significant. A correlation between two ordinalvariables was calculated using a Spearman’s rank correlation test( P  < 0.05). Colocalization study To confirm that EB and FSC bind to albumin and to determinewhether EB and FSC colocalized with specific cell types, doublelabelling was performed on a subset of sections (at least twosections/rat, 2.4, 3.4 and 4.6 mm lateral to bregma) with anti-albumin (rabbit anti-albumin, 1:100, DakoCytomation, Glostrup,Denmark), the microglial marker anti-OX-42 [mouse anti-ratCD11b/c (OX-42), 1:100, PharMingen, CA, USA], the astrocyticmarker anti-glial fibrillary acidic protein (mouse anti-GFAP,1:1000, DakoCytomation, Glostrup, Denmark) and the neuronalmarker anti-NeuN (mouse anti-NeuN, 1:1000, Chemicon, UK).Free-floating sections were washed (2 · 10 min) in 0.05 M PBS,followed by washing (1 · 60 min) in PBS + 0.4% bovine serumalbumin (BSA). BSA was omitted in all solutions for albuminstaining. Sections were then incubated in primary antibodies. After24 h of incubation with the primary antibody, the sections werewashed in PBS (3 · 10 min) and incubated for 1.5 h in Alexa Fluor568 (FSC sections; goat anti-mouse IgG, 1:200, Molecular Probes)or Alexa Fluor 488 (EB sections; goat anti-mouse IgG Alexa, 1:200,Molecular Probes). Following three additional washes in PBS,sections were mounted on slides (Superfrost Plus, Menzel,Braunschweig, Germany) and coverslipped with mounting mediumfor fluorescence (Vectashield, Vector Laboratories, Burlingame, CA,USA). Images were acquired using a confocal-laser scanningmicroscope and Adobe Photoshop. Fluoro-Jade B staining To evaluate whether tracer/albumin containing cells were dege-nerating cells, a Fluoro-Jade B (FJB) staining was performed asdescribed previously (Schmued and Hopkins, 2000) on a subset of sections (at least two sections/rat, 2.4, 3.4 and 4.6 mm lateral tobregma) of rats that were injected with EB. Sections were mountedon coated slides (Superfrost Plus, Menzel, Braunschweig, Germany)and dried overnight at room temperature. They were immersed inabsolute alcohol for 3 min. followed by 70% ethanol for 1 min. anddistilled water for 1 min. The slides were transferred to 0.06%potassium permanganate for 15 min. After rinsing with distilledwater (1 min), the slides were transferred to a 0.001% polyanionicFSC derivative solution (FJB, Histo-Chem Inc., Jefferson, AR, USA)made in 0.1% acetic acid. Slides were rinsed in water, dried,immersed in xylene and coverslipped with mountant for histology (DPX, Sigma-Aldrich, Zwijndrecht, The Netherlands). Images wereacquired using a confocal-laser scanning microscope and AdobePhotoshop. Artificial opening of the BBB To investigate whether alterations in BBB permeability couldinfluence seizure activity, the BBB was opened with mannitol(1.5 g/kg i.v., 25% solution, once daily for 3 consecutive days)under isoflurane anaesthesia (4 vol%) in both control rats ( n  = 5)and in chronic epileptic rats with a stable seizure frequency ( n  = 8).EEG recordings were discontinued during anaesthesia, which neverlasted longer than several minutes. We confirmed that this protocolresulted in BBB extravasation of FSC (data not shown) and thatshort isoflurane anaesthesia combined with physiological saltadministration, does not influence daily seizure activity (van Vliet et al  ., 2006). Rats were under continuous EEG monitoring and thenumber of seizures and the seizure duration were evaluatedbefore, during and after mannitol treatment. Statistical analysiswas performed using the paired Student’s t  -test. Differences with P  < 0.05 were considered significant. ResultsAlbumin IR in human and rat brain Alterations in BBB permeability, resulting in albuminextravasation, were detected using immunocytochemistry.In the human hippocampus of autopsy controls ( n  = 5),no albumin extravasation was observed (Fig. 1A and B).In contrast, in resected hippocampi of patients with TLE( n  = 6) strong albumin IR was present in parenchymathroughout the hippocampus, next to blood vessels (Fig. 1E).Neurons and astrocytes located around these vessels werealso albumin positive (Fig. 1F). Most albumin extravasationwas observed in autopsy material of patients that haddied during SE ( n  = 2). Very strong albumin IR was seenaround all blood vessels within the hippocampus andcortex (Fig. 1C). In addition, many neurons and astrocyteswere also highly immunoreactive (Fig. 1D). No albuminextravasation was observed in the cerebellum of thesepatients.In control rats no albumin could be detected in limbicbrain regions (e.g. hippocampus, Fig. 2A). However, in theacute (1–2 days after SE) and latent phase (1 week after SE)albumin extravasation was evident in the hippocampus(Fig. 2B and C), entorhinal cortex, piriform cortex, thalamus,amygdala and olfactory bulb. In chronic epileptic ratsalbumin was present, but not as widespread as acutely afterSE. Albumin was detected especially in the piriform cortex,but the hippocampus (Fig. 2D), entorhinal cortex, thalamusand amygdala were also immunoreactive for albumin. BBB permeability during epileptogenesisin rats To assess leakage of the BBB, rats were sacrificed in the acuteseizure period (1–2 days after SE), in the latent period when 524 Brain (2007), 130 , 521–534 E. A. van Vliet et al.   b  y  on S  e p t   em b  er  5  ,2  0 1  0 h  t   t   p:  /   /   b r  ai  n. ox f   or  d  j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om 
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