Background
Epilepsy is a common neurological disease characterized by spontaneous recurrent seizures, affecting more than 50 million people worldwide. It is defined by any of the following conditions: (1) at least two unprovoked (or reflex) seizures occurring > 24 h apart. (2) One unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years. (3) Diagnosis of an epilepsy syndrome [
1]. Temporal lobe epilepsy (TLE) is one of the most common forms of focal epilepsy in adults [
2,
3], which is frequently associated with hippocampal sclerosis [
4]. The neuropathological processes associated with human TLE include neuronal loss, aberrant axonal growth and neurogenesis in the hippocampus, neuroinflammation, gliosis, reorganization of the extracellular matrix (ECM), and blood–brain barrier (BBB) dysfunction [
5‐
9].
Matrix metalloproteinases (MMPs) constitute a class of proteases responsible for the remodeling of the ECM. Under normal physiological conditions various MMPs are involved in ECM homeostasis and synaptic plasticity in the brain [
10‐
13]. Under pathological conditions, MMPs can be activated by a variety of stimuli including pro-inflammatory cytokines [
14]. The pro-inflammatory cytokine IL-1β can be produced in the central nervous system (CNS) by activated astrocytes in response to tissue damage, increased neuronal activity or cellular stress [
15‐
18]. Astrocytes contribute to chronic neuroinflammation in epilepsy not only as the major source of IL-1β [
17], but also due to their role in K
+ buffering, uptake of extracellular glutamate, glutamine supply for presynaptic terminals [
19] as well as the ability to control synaptogenesis [
20]. The deregulation of MMP expression and activity has been also associated with TLE, where it may contribute to altered neuronal excitability, acute and chronic neuroinflammation, neurodegeneration, gliosis and a compromised BBB [
14,
21‐
25]. In the electrical post-status epilepticus (SE) rat TLE model, a large-scale transcriptome study revealed that the expression of MMP2, MMP3, MMP9 and MMP14 in the brain was increased and dynamically regulated at different stages of epileptogenesis [
26]. The role of MMP9 in epileptogenesis has been extensively studied in various animal models [
22] with the focus on the modulation of synaptic plasticity associated with seizures [
27]. MMP3 has also been implicated in neurodegenerative disorders [
28‐
31] and shown to contribute to the increased BBB permeability and apoptosis [
32,
33]. Increased expression of MMP3 expression was previously demonstrated in the hippocampus after kainic acid-induced seizures in mice [
34] and after pilocarpine-induced status epilepticus in rats [
35]. In summary, accumulating evidence indicates that increased expression and/or activity of MMPs after an insult can contribute to epileptogenesis. Therefore, reducing MMP expression or activity has been suggested as a strategy for the prevention and/or modulation of epileptogenesis.
The increased gene expression under pathological conditions can be modulated by miRNAs. miRNAs are small non-coding RNAs capable of regulating target gene expression at post-transcriptional level [
36‐
38]. miRNAs have been shown to be involved in the regulation of various biological processes within the CNS [
39,
40] and have been implicated in neurological disorders, such as epilepsy [
41,
42]. miRNAs can regulate gene expression directly through complementary binding to multiple messenger RNA (mRNA) transcripts and indirectly through modulating intracellular signaling pathways associated with the target genes. Several miRNAs have been shown to mediate inflammation in the brain [
40]. This includes miR-146a, which inhibits inflammation in astrocytes [
43‐
45]. Another inflammatory miRNA, miRNA-155, has also been shown to be expressed in astrocytes and been implicated in various CNS pathologies [
46], including epilepsy [
47‐
49]. Since the activation of MMP expression, especially MMP3 and MMP9, has been linked to pro-inflammatory signaling, miR-155 might modulate their expression through the regulation of inflammation. Indeed, miR-155 was previously demonstrated to be involved in the regulation of MMP3 under inflammatory conditions in synovial fibroblasts [
50,
51].
The aim of this study was to investigate whether increased MMP expression under inflammatory conditions can be attenuated by inhibition of miR-155. Therefore, we investigated MMP expression in cultured human fetal astrocytes after IL-1β stimulation and transfection with the antagomiR of miR-155. Furthermore, we characterized MMP and miR-155 expression in resected brain tissue of patients with TLE as well as during epileptogenesis in a rat TLE model.
Methods
Cell cultures
Primary fetal astrocyte-enriched cell cultures were derived from human fetal brain tissue (14–20 weeks of gestation) obtained from medically induced abortions. All material was collected from donors from whom a written informed consent for the use of the material for research purposes was obtained by the Bloemenhove clinic. Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code provided by the Medical Ethics Committee. Tissue samples were collected in DMEM/HAM F10 (1:1) medium (Gibco/ThermoFisher Scientific, Waltham, MA, USA), supplemented with 1% penicillin/streptomycin and 10% fetal calf serum (FCS). Primary cell cultures of astrocytes were prepared as previously described [
49]. The culture medium was subsequently refreshed twice a week. Cultures reached confluence after 2–3 weeks. Astrocytes were used for analyses at passages 2–5. More than 98% of the cells in primary culture, as well as in the successive 12 passages were strongly immunoreactive for the astrocytic marker glial fibrillary acid protein (GFAP) and S100β as previously reported [
45].
Transfection and stimulation of cell cultures
Cells were plated in poly-L-lysine coated plates (5 × 10
4 cells/well in 12-well plates for RNA analysis or 2 × 10
5 cells/well in 6-well plates for protein analysis) and were transfected with miR-146a or miR-155 mimic pre-miRNA (mirVana miRNA mimics, Applied Biosystems, Carlsbad, CA, USA), or antisense locked nucleic acid (LNA) oligonucleotides against miR-155-5p (Ribotask ApS, Odense, Denmark). Oligonucleotides were delivered to the cells using Lipofectamine 2000 transfection reagent (Life Technologies, Grand Island, NY, USA) in a final concentration of 50 nM for a total of 24 h before the stimulation of astrocytes. Astrocytic cultures were stimulated with human recombinant (r)IL-1β (10 ng/ml; Peprotech, Rocky Hill, NJ, USA) for 24 h (for RNA analysis) or for 48 h (for protein analysis) before harvesting the cells. Viability of human cell cultures was not influenced by the stimulation with IL-1β, as shown previously [
52].
Human brain tissue
The cases included in this study were obtained from the archives of the department of (Neuro)Pathology of the Amsterdam UMC, Amsterdam, The Netherlands. A total of 16 brain specimens were examined from patients undergoing surgery for drug resistant TLE. Tissue was obtained and used in accordance with the Declaration of Helsinki and the Research Code provided by the Medical Ethics Committee. All cases were reviewed independently by two neuropathologists, and the classification of hippocampal sclerosis was based on analysis of microscopic examination as described by the International League Against Epilepsy (HS type 1,
n = 12; HS type 2,
n = 4) [
53]. Control material was obtained during autopsy of people without a history of seizures or other neurological diseases (
n = 10). Brain tissue was fixed in 10% buffered formalin and embedded in paraffin.
Experimental animals
Adult male Sprague-Dawley rats (Harlan Netherlands, Horst, The Netherlands) were used in this study which was approved by the University Animal Welfare committee. The rats were housed individually in a controlled environment (21 ± 1 °C; humidity 60%; lights on 08:00 AM–8:00 PM; food and water available ad libitum).
Electrode implantation and status epilepticus induction
Rats were anesthetized with an intramuscular injection of ketamine (74 mg/kg; Alfasan, Woerden, The Netherlands) and xylazine (11 mg/kg; Bayer AG, Leverkusen, Germany), and were placed in a stereotactic frame. In order to record hippocampal EEG, a pair of insulated stainless steel electrodes (70 μm wire diameter, tips 800 μm apart) was implanted into the left dentate gyrus (DG) under electrophysiological control, as described previously [
54]. A pair of stimulation electrodes was implanted in the angular bundle. Two weeks after recovery from the operation, each rat was transferred to a recording cage (40 × 40 × 80 cm) and connected to a recording and stimulation system (NeuroData Digital Stimulator, Cygnus Technology Inc., Delaware Water Gap, PA, USA) with a shielded multi-strand cable and electrical swivel (Air Precision, Le Plessis Robinson, France). A week after habituation to the new condition, rats underwent tetanic stimulation (50 Hz) of the hippocampus in the form of a succession of trains of pulses every 13 s. Each train was of 10 s duration and consisted of biphasic pulses (pulse duration 0.5 ms, maximal intensity 700 μA). Stimulation was stopped when the rats displayed sustained forelimb clonus and salivation for several minutes, which usually occurred within 1 h.
EEG monitoring
To determine seizure frequency, continuous EEG recordings (24 h/day) were made in all rats. Hippocampal EEG signals were amplified (10×) via a field effect transistor that connected the headset to an amplifier (20×; CyberAmp, Axon Instruments, Burlingame, CA, USA), band-pass filtered (1–60 Hz) and digitized by a computer. A seizure detection program (Harmonie, Stellate Systems, Montreal, Canada) sampled the incoming signal at a frequency of 200 Hz per channel. All EEG recordings were visually screened and seizures were confirmed by trained human observers. Seizures were characterized by synchronized high-voltage amplitude oscillations and were scored when the amplitude increased more than 2-fold and lasted for at least 10 s.
Tissue preparation
For in situ hybridization, rats were deeply anesthetized with pentobarbital (Euthasol, AST Farma, Oudenwater, The Netherlands, 60 mg/kg i.p.) and perfused via the ascending aorta (300 ml 0.37% Na
2S/300 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Rats were perfused at three different time points after SE, each corresponding to the phases of epileptogenesis: the acute phase (1 day post-SE,
n = 5), the latent phase (1 week post-SE, absence of electrographic seizures,
n = 3), and the chronic phase (3–4 months post-SE, recurrent spontaneous electrographic seizures are evident,
n = 6) [
55]. Control rats (
n = 4) that were implanted with EEG electrodes but not stimulated were also included. The brains were post-fixated overnight, dissected and paraffin embedded. Tissue was sectioned at 6 μm and mounted on pre-coated glass slides (Star Frost, Waldemar Knittel, Braunschweig, Germany).
For RT-qPCR analysis, rats were decapitated 1 day after SE (acute phase, n = 5), 1 week after SE (latent phase, n = 6) or 3–4 months after SE (chronic phase, n = 5). Control rats (n = 10) included young rats (n = 5) and age-matched controls for the chronic stage (n = 5). The brain was dissected and the parahippocampal cortex (PHC), which includes mainly the entorhinal cortex and parts of the perirhinal and posterior piriform cortex, was removed by incision at the ventro-caudal part underneath the rhinal fissure until approximately 5 mm posterior to bregma, as well as the hippocampus. The hippocampus was sliced into smaller parts (200–300 μm) and the DG and Cornu Ammonis (CA1) regions were cut out of the slices in 4 °C saline solution under a dissection microscope. All material was frozen on dry ice and stored at − 80 °C until use.
RNA isolation and real-time quantitative PCR analysis
For RNA isolation, cell cultures, frozen human brain tissue, or frozen rat brain tissue were homogenized in 700 μl Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). Total RNA, including small RNAs, was isolated using the miRNeasy Mini kit (Qiagen Benelux, Venlo, The Netherlands) according to manufacturer’s instructions. The concentration and purity of RNA were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). To evaluate mRNA expression, 250 ng of cell culture-derived total RNA was reverse-transcribed into cDNA using oligo dT primers. The primers used for the study are listed in (Additional file
1: Table S1). The geometric mean of elongation factor 1α (EF1α) and chromosome 1 open reading frame 43 (C1orf43) expression levels was used for the normalization of RT-qPCR in human tissue and cell cultures; the geometric mean of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-box-binding protein (TBP) expression levels was used for the normalization of RT-qPCR in rat tissue. The PCR mix and cycling conditions were used as previously described [
56].
The expression of miR-155-5p and U6B small nuclear RNA (RNU6B) was analyzed using TaqMan microRNA assays (Applied Biosystems, Foster City, CA, USA). cDNA was generated using TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The PCRs were run on the Roche LightCycler 480 (Roche Applied Science, Basel, Switzerland) with a 384-multiwell format.
Quantification of data was performed using LinRegPCR in which the baseline correction and window-of-linearity are determined for each sample separately, followed by a linear regression analysis on the Log (fluorescence) per cycle to fit a straight line through the PCR data set. The slope of this line is used to determine the PCR efficiency of each individual sample. The mean PCR efficiency per amplicon and the Ct value per sample are used to calculate a starting concentration N0 per sample, which is expressed in arbitrary fluorescence units [
57,
58]. The starting concentration N0 of each specific product was then divided by the geometric mean of the starting concentrations N0 of the reference genes, and this ratio was compared between groups. This value was further normalized to the corresponding control condition. The control condition used for RT-qPCR experiments in primary cultures was the condition stimulated by IL-1β due to undetectable/very low expression of MMP3 and MMP9 in untreated control.
Western blot analysis
Cells were harvested at 48 h after treatment. The cells were washed with ice-cold PBS and homogenized in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM of NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemented with protease inhibitor (EDTA-free protease mixture inhibitor and phosphatase inhibitor (Roche Diagnostics, Almere, the Netherlands)) by incubating on ice for 10 min and collected using a cell scraper. The homogenates were centrifuged at 12,000 x
g for 10 min and the supernatant was used for further analysis. Protein content was determined using the bicinchoninic acid method [
59]. Equal amounts of proteins (5 μg/lane for culture samples or 20 μg/lane for tissue samples) were separated using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Merck, Darmstadt, Germany) for 90 min at 100 V, using a wet electroblotting system (BioRad, Hercules, CA, USA). Blots were blocked for 1 h in 5% non-fat dry milk in Tris-buffered saline-Tween (TBS-T; 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5). Blots were incubated overnight with primary antibodies anti-MMP3 (1:200 mouse monoclonal, clone SL-1 IIIC4, EMD Millipore, Temecula, CA, USA) or anti-β-actin (1:50,000 monoclonal mouse, clone C4, Merck, Darmstadt, Germany). After several washes in TBS-T/ 5% non-fat dry milk, blots were incubated with secondary antibodies goat anti-mouse IgG2b (for MMP3) or goat anti-mouse IgG1 coupled to horseradish peroxidase (both 1:2500; Dako, Glostrup, Denmark) for 1 h. After several washes in TBS-T, immunoreactivity was visualized using ECL PLUS Western blotting detection reagent (GE Healthcare Europe, Diegen, Belgium). Expression of β-actin was used as loading control. Chemiluminescent signal was detected using ImageQuant LAS 4000 analyzer (GE Healthcare, Eindhoven, the Netherlands). Precision Plus Protein Dual Color Standards (Bio-Rad, Richmond, CA, USA) was used to determine the molecular weight of the proteins. For the quantitative analysis of the blots and in-situ micrographs the band intensities were measured densitometrically using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).
In situ hybridization on human and rat brain tissue
Paraffin-embedded brain tissue was deparaffinized in xylene and rinsed in ethanol (2 × 100%, 70%) and sterile water. Antigen retrieval was performed using a pressure cooker in sodium citrate buffer, pH 6.0, at 121 °C for 10 min. The oligonucleotide probe for miR-155 (Additional file
1) contained LNA modification, 2-o-methyl modification and digoxygenin (DIG) label (RiboTask ApS, Odense, Denmark). Sections were incubated with the probe (1:750 dilution) in hybridization mix (600 mM NaCl, 10 mM HEPES, 1 mM EDTA, 5x Denhardts, 50% Formamide) for 1 h at 56 °C. Sections were washed with saline-sodium citrate for 2 min, 0.5x for 2 min, 0.2x for 1 min (in agitation). After washing with sterile PBS, sections were blocked for 15 min with 1% BSA, 0.02% Tween 20 and 1% normal goat serum. Hybridization was detected with alkaline phosphatase (AP) labeled with anti-DIG (Roche Applied Science, Basel, Switzerland). Nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) was used as chromogenic substrate for AP (1:50 diluted in NTM-T buffer (100 mM Tris, pH 9.5; 100 mM NaCl; 50 mM MgCl
2; 0.05% Tween 20)). Negative control assays were performed without probes (sections were blank).
For double-staining, the sections were first processed for in situ hybridization, followed by immunohistochemistry. Slides were washed with PBS and incubated for 1 h at room temperature with primary antibodies in PBS; mouse anti-GFAP (1:4000, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-NeuN (1:2000, MAB377, Chemicon, Temecula, CA, USA), mouse anti-CD34 (1:600, Immunotech, Monrovia, CA, USA) and mouse anti-CR3/43 (1:100, Agilent, Santa Clara, CA, USA) or rabbit anti-IBA-1 (1:2000, Wako Chemicals, Neuss, Germany). After washing with PBS, sections were stained with a polymer-based peroxidase immunohistochemistry detection kit (Brightvision plus kit, ImmunoLogic, Duiven, The Netherlands) according to the manufacturer’s instructions. Signal was detected using the chromogen 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO, USA).
Evaluation of in situ hybridization
Expression of miR-155 was quantitatively analyzed in the DG and CA1 of human brain tissue and in the DG, CA1 and entorhinal cortex of rat brain tissue by measuring the optical density using ImageJ. Since the quantitative analysis does not discriminate between different cell types, a semi-quantitative analysis was also performed in which miR-155 expression was assessed in neurons, cells with glial morphology and blood vessels. The intensity of the staining was evaluated using a scale of 1–4 (1: no; 2: weak; 3: moderate; 4: strong staining). The score represents the predominant cell staining intensity found in each case. Additionally, the relative number of positive cells (0: no; 1: single to 10%; 2: 11–50%; 3: > 50%) was also evaluated. The in situ reactivity score (IRS) was calculated by multiplying the intensity score by the relative number score. The analysis was performed by two researchers that were blinded to group assignments.
Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics 21. Comparisons between multiple groups were done using the Kruskal-Wallis test, Mann-Whitney U test was used to compare two groups. A value of p < 0.05 was assumed to indicate significant difference.
Acknowledgements
We thank Anand Lyer for his contribution. We acknowledge the HIS Mouse Facility of the Amsterdam UMC, Amsterdam and the Bloemenhove Clinic (Heemstede, The Netherlands) for providing fetal tissues.