FULL-LENGTH ORIGINAL RESEARCH
Blood–brain barrier leakage after status epilepticus in rapamycin-treated rats I: Magnetic resonance imaging *Erwin A. van Vliet, †‡Willem M. Otte, §Wytse J. Wadman, *§¶Eleonora Aronica, #Gijs Kooij, #Helga E. de Vries, †Rick M. Dijkhuizen, and §Jan A. Gorter Epilepsia, 57(1):59–69, 2016 doi: 10.1111/epi.13246
SUMMARY
Dr. Erwin A. van Vliet is a postdoctoral researcher at the Academic Medical Center, Amsterdam.
Objective: The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this effect via restoration of blood– brain barrier (BBB) dysfunction, contrast-enhanced magnetic resonance imaging (CEMRI) was used to determine BBB permeability throughout epileptogenesis. Methods: Imaging was repeatedly performed until 6 weeks after kainic acid–induced status epilepticus in rapamycin (6 mg/kg for 6 weeks starting 4 h after SE) and vehicletreated rats, using gadobutrol as contrast agent. Seizures were detected using video monitoring in the week following the last imaging session. Results: Gadobutrol leakage was widespread and extensive in both rapamycin and vehicle-treated epileptic rats during the acute phase, with the piriform cortex and amygdala as the most affected regions. Gadobutrol leakage was higher in rapamycintreated rats 4 and 8 days after status epilepticus compared to vehicle-treated rats. However, during the chronic epileptic phase, gadobutrol leakage was lower in rapamycin-treated epileptic rats along with a decreased seizure frequency. This was confirmed by local fluorescein staining in the brains of the same rats. Total brain volume was reduced by this rapamycin treatment regimen. Significance: The initial slow recovery of BBB function in rapamycin-treated epileptic rats indicates that rapamycin does not reduce seizure activity by a gradual recovery of BBB integrity. The reduced BBB leakage during the chronic phase, however, could contribute to the decreased seizure frequency in post–status epilepticus rats treated with rapamycin. Furthermore, the data show that CE-MRI (using step-down infusion with gadobutrol) can be used as biomarker for monitoring the effect of drug therapy in rats. KEY WORDS: Rapamycin, Blood–brain barrier, Status epilepticus, Temporal lobe epilepsy, Contrast-enhanced magnetic resonance imaging, Epileptogenesis.
The blood–brain barrier (BBB) separates the brain from the bloodstream and protects the brain against various potentially harmful substances in the blood. Clinical and experi-
mental data have implicated an important role for BBB dysfunction in the development and progression of epilepsy. BBB disruption may occur in epilepsy patients1–5 and in
Accepted October 9, 2015; Early View publication December 22, 2015. *Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; †Biomedical MR Imaging and Spectroscopy Group, Image Sciences Institute, University Medical Center Utrecht, Utrecht, The Netherlands; ‡Department of Pediatric Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands; §Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands; ¶Stichting Epilepsie Instellingen Nederland, Heemstede, The Netherlands; and #Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Address correspondence to Erwin A. van Vliet, Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. E-mail:
[email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
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Key Points
Gadobutrol leakage was more extensive during the latent phase in rapamycin (RAP)–treated post-SE rats compared to vehicle (VEH) –treated post-SE rats Spontaneous seizures and gadobutrol leakage were reduced during the chronic phase in RAP-treated rats compared to VEH-treated rats Contrast-enhanced MRI can be used as biomarker for monitoring the effect of drug therapy in rats
experimental seizure or epilepsy models.2,6–10 BBB leakage is associated with brain inflammation, transcriptional changes, and lasting effects on astrocytes (e.g., astrogliosis and impaired potassium buffering), leading to enhanced excitability and progression of epilepsy.2,5,10–14 Therefore, protection or restoration of BBB function may have therapeutic potential and may be antiepileptogenic in patients at risk. We recently showed that treatment with the immunosuppressant rapamycin (RAP) during 6 weeks following status epilepticus (SE), reduced BBB albumin leakage and the development of recurrent spontaneous seizures.15 Although this suggests that modulation of BBB permeability can modify epileptogenesis, we cannot exclude that the observed decreased BBB permeability is just a result of a reduced number of seizures during the chronic epileptic phase, caused by modulation of RAP-mediated antiepileptogenic or antiseizure effects other than recovery of the BBB. The aim of the present study is to determine whether RAP mediates antiepileptogenic effects via restoration of BBB dysfunction during epileptogenesis. This requires longitudinal assessments within the same animal. We recently showed that noninvasive assessment of BBB permeability in an experimental epilepsy rat model can be visualized using contrastenhanced in vivo magnetic resonance imaging (CE-MRI).16 An identical CE-MRI approach was used in the current study to map the effects of chronic RAP treatment on BBB permeability throughout the brain during epileptogenesis. Five relevant time points after SE were assessed to characterize the relationship between RAP treatment and epilepsy: during the acute phase (1 day after SE), latent phase (4 and 8 days after SE), and chronic epileptic phase (3 and 6 weeks after SE). We expected that RAP (applied at a clinically relevant time point, i.e., 4 h after SE) would limit BBB leakage, leading to reduction or prevention of recurrent seizures at a later stage.
Methods Animals In total, 20 adult male Sprague-Dawley rats (Harlan Netherlands, Horst, The Netherlands), weighing 350– 450 g, were used. The rats were housed in a controlled Epilepsia, 57(1):59–69, 2016 doi: 10.1111/epi.13246
environment (21 1°C; humidity 60%; lights on 08:00 a.m. to 8:00 p.m.; food and water available ad libitum). All experiments were approved by the animal welfare committee of the University of Amsterdam and Utrecht University. Epilepsy model SE, which potentially leads to spontaneous temporal lobe seizures several days later, was evoked in 12 rats by hourly sequential intraperitoneal injections of kainic acid (KA; 10 mg/ml; first dose 14 mg/kg; subsequent injections 7 mg/kg, Cayman Europe, Tallinn, Estonia). Behavior was observed during KA injections and several hours thereafter, until SE had subsided. Seizure activity was rated behaviorally according to the Racine scale.17 KA injections were continued until class IV and V seizures were elicited for >4 h. Seizures were not terminated pharmacologically. SE severity was classified as follows: score 1 = mild SE, Racine scale I and II seizures and exploratory behavior, which is different from their natural exploratory behavior, since the rats do not walk in a continuous pace; score 2 = moderate SE, head bobbing, periods of immobility and occasional generalized seizures (stage IV or V); and score 3 = severe SE, continuous behavioral stage IV–V seizure activity. Video recordings were performed on the day of the MRI session and used to verify that rats did not have a behavioral seizure at least 2 h prior to each MRI session. To avoid BBB disruption by implanting electrodes and to avoid MRI artifacts, EEG recordings were not performed. Previous continuous video-EEG recordings in similar-treated kainic acid post-SE rats indicated that the latent phase was around 9 days,18 on the basis of which we defined the time points of MRI in the present study. Rapamycin treatment A stock solution of rapamycin (RAP) was prepared in 100% ethanol (150 mg/ml) and stored at 20°C until use. Prior to use, RAP was diluted with vehicle (VEH) solution (5% Tween 80 + 5% polyethylene glycol [PEG] 400) to a 4% ethanol solution. In the treatment group RAP was administered intraperitoneally (6 mg/kg) starting 4 h after status epilepticus (SE), once daily for 7 days (n = 6), and thereafter RAP was administered every other day until rats were killed 7 weeks post-SE. This protocol was based on our previous study in epileptic rats, in which RAP was shown to effectively reduce seizure activity and BBB permeability.15 Different control groups were included. Rats without SE and treated with RAP (n = 4); rats without SE and treated with vehicle (n = 4); and rats with SE and treated with vehicle (n = 6). The vehicle solution contained 5% Tween 80 + 5% PEG 400 + 4% ethanol. Rapamycin blood levels Blood samples for RAP concentration measurements were taken from the tail vein 24 h after the last RAP
61 Effects of Rapamycin on BBB Leakage injection, and collected in 500 ll K2 ethylenediaminetetraacetic acid (EDTA) microtainers (Becton Dickinson, Breda, The Netherlands). Fifty microliters of blood or RAP standards and 50 ll of 0.1 M zinc sulphate were pipetted into microtubes (Sarstedt, Etten-Leur, The Netherlands) and processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously.15 Anesthesia and monitoring of physiologic parameters during magnetic resonance imaging See supplementary methods in Data S1. Magnetic resonance imaging Magnetic resonance imaging was conducted on a 4.7 T horizontal bore Varian MR System (Palo Alto, CA, U.S.A.). A Helmholtz volume coil (9 cm diameter) and an inductively coupled surface coil (2.5 cm diameter) were used for radiofrequency excitation and signal detection, respectively. Rats (VEH n = 4; RAP n = 4) were repeatedly scanned at different time points during epileptogenesis: 7 days before SE (baseline), 1 day after SE (the acute phase), 4 and 8 days after SE (the latent phase), and 3 and 6 weeks after SE (the chronic phase). In addition, two VEH-treated and two RAP-treated epileptic rats were included in the chronic group (6 weeks after SE). These rats were also scanned before SE (baseline), but not at any other time point. Four VEH-treated control rats and 4 RAP-treated control rats were scanned at baseline and scans were repeated 7 weeks later. For image registration purposes, T2*-weighted images (repetition/echo time, 6/2.28 msec; flip angle, 40 degrees; field of view, 40 9 40 9 40 mm3; acquisition matrix, 192 9 192 9 192; voxel resolution, 0.2083 9 0.2083 9 0.3125 mm3) were acquired before contrast-enhanced imaging. BBB permeability was determined with T1-weighted MRI, in which contrast-induced signal changes were calculated from scans before and after tracer infusion, as has been described previously.16 T1-weighted MR images (gradient echo; repetition/echo time, 160/4 msec; flip angle, 90 degrees; acquisition matrix, 256 9 128 9 19; voxel resolution, 0.25 9 0.125 9 1 mm3) were acquired before and 45 min after the start of a 20 min step-down infusion with a T1-shortening contrast agent (gadobutrol). Infusion of contrast agent We previously showed that BBB permeability after kainic acid–induced SE can be quantified using a step-down contrast agent infusion protocol.16 Therefore, this protocol was also used in the present study. Gadobutrol (0.2 M diluted in saline; Gadovist, Schering, Kenilworth, NJ, U.S.A.) was injected via the tail vein using a syringe pump (Model 944; Harvard Apparatus, South Natick, MA, U.S.A.) programmed to reach the highest possible stable blood concentration during 20 min.16
Data analysis T2*-weighted images were matched to a three-dimensional model of a rat brain atlas.19 This involved alignment of the images with a reference image (that is, an average of all T2*-weighted images from the present study) using affine registration. This was followed by nonlinear registration to the rat brain atlas to optimize the alignment between atlas and images. The atlas matching has been described previously.16 The following regions of interest (ROIs) were projected from the atlas on the individual images: the amygdala, entorhinal cortex, hippocampus, piriform cortex, and thalamus. These brain regions are thought to be involved in the generation and/or spread of seizure activity. The motor cortex was added as a nonlimbic region. Regional gadobutrol leakage (Gd-leakage map) was calculated as the relative signal enhancement induced by gadobutrol accumulation: precontrast T1-weighted images were subtracted from postcontrast images and divided by precontrast images. The regional leakage maps were thresholded at 0.2, similarly as described previously.16 Voxels with relative signal enhancement above the threshold were delineated manually by a researcher blinded to group assignments and were projected onto the map. The median value within these voxels was calculated in all ROIs. We never detected a significant difference between the values in the left and right hemispheres. Therefore, the data from the left and right hemisphere of each ROI were pooled. As prolonged treatment with RAP is known to reduce body weight and brain volumes,20 we also determined changes in brain volume during epileptogenesis. Video monitoring In addition to the video recordings that were made 2 h prior to each MRI session (to exclude that rats had seizures shortly before MRI), video recordings were made for each animal 24 h/day during 1 week following the last MRI session (in week 7). This allowed quantification of the seizure frequency during the last week as well as identification of the occurrence of the last seizure. Thereafter, rats were injected with the BBB permeability tracer fluorescein (see following paragraph) and perfusion fixed. This procedure allowed postmortem validation of the in vivo MRI-based BBB permeability measurements. Assessing BBB leakage using fluorescein and (immuno) histochemistry See supplementary methods in Data S1. Statistical analysis Data are reported as mean and standard error of the mean. Statistical analysis of Gd-leakage maps was performed using linear mixed modeling (R package nlme) with group, time, and group 9 time interaction as factors, while correcting for repeated measures within rats. Epilepsia, 57(1):59–69, 2016 doi: 10.1111/epi.13246
62 E. A. van Vliet et al. Brain volume and body weight were analyzed using the factorial repeated-measures analysis of variance, followed by the Fisher’s least significant difference post hoc test. Correlation of Gd-leakage with seizure activity and with fluorescein leakage was done with the Pearson correlation. Statistical analysis of fluorescein leakage, Nissl-stained sections, and the number of seizures was performed using the Mann-Whitney test. Group differences and correlations were considered significant if p < 0.05.
Results
at all time points after SE. Gd-leakage was higher in both VEH- and RAP-treated epileptic rats (p < 0.01) as compared to measurements that were performed before SE in all ROIs (except for the motor cortex), indicating persistent BBB leakage. The comparison of BBB leakage between VEH- and RAP-treated epileptic rats during epileptogenesis will be described for each measured time point in the following paragraphs. Before SE Before SE, there was no Gd signal in any ROI (Figs. 1A and 2A).
Status epilepticus induction using kainic acid Four of six VEH-treated rats received one KA injection (14 mg/kg) and two of six received three KA injections (14 mg/kg, followed by 7 mg/kg). Five of six RAP-treated epileptic rats received one KA injection and one of six received three injections. The mean total dose of KA was not different between VEH-treated epileptic rats (18 3 mg/kg) and RAP-treated epileptic rats (16 2 mg/kg). SE onset was not different between VEH (154 30 min) and RAP-treated epileptic rats (113 23 min). Furthermore, SE duration and behavioral SE severity were not different between groups (both VEH and RAP, mild SE, score 1, duration range 7–9 h).
One day after SE At one day after SE, Gd-leakage was significantly higher in the amygdala, entorhinal cortex, hippocampus, motor cortex, piriform cortex, and thalamus in both VEH- and RAP-treated rats, compared to before SE (p < 0.01). There was no difference between the VEH- and RAP-treated postSE rats (Fig. 3).
Rapamycin produces low seizure frequency in chronic epileptic phase Video recordings revealed that VEH-treated epileptic rats exhibited 4.0 1.0 seizures/day in the week before they were killed. The last detected seizure occurred on average at 14 7 h before perfusion-fixation. RAP-treated epileptic rats had fewer seizures as compared to VEH-treated epileptic rats and experienced on average
