Animals

Male Wistar rats (Charles River, L’Arbesle, France) were used for the rapid kindling and cytokine-dosage experiments. We used P13 and P74 rats at the time of the PIC intrahippocampal injection. Primary microglial and macrophage cell cultures were prepared from the neocortex of OF1 mice (Charles River, L’Arbesle) and 2-month-old OF1 male mice (Charles River). Animals were housed in standard laboratory conditions with controlled temperature/humidity, a 12:12-hour light/dark cycle, and free access to food and water. Studies were approved by the animal ethical institutional review committee (Bichat-Robert Debré ethical committee, Paris, France, #2015072801547679) and met stipulations of the guide for the care and use of laboratory animals (NIH, Bethesda, Maryland, USA), as well as recommendations of reduction, refinement, and replacement (known as the 3 Rs) [24].

Drugs

PIC and minocycline (Sigma, Lyon, France) were dissolved in saline for the in vivo experiments and in phosphate buffered saline (PBS) for the in vitro experiments. PIC (10 μg/rat) was injected stereotaxically into the ventral hippocampus 24 h before rapid kindling at the following coordinates: P13, 3.0 mm posterior, 3.9 mm left, 4.2 mm ventral; P74, 4.8 mm posterior, 5.3 mm left, 6.5 mm ventral, in relation to bregma. The injection of PIC into the hippocampus aims to mimic encephalitis, which is characterized by brain inflammation, rather than meningitis, which is inflammation in the meninges and the cerebrospinal fluid (CSF). Minocycline (50 mg/kg) was administered as 3 consecutive intraperitoneal (i.p.) injections 48, 24, and 4 h before the start of the kindling protocol. Table 1 summarizes the in vitro and in vivo experiments.

Table 1 Experimental plan summarizing treatments and experiment (exp.) numbers included in the study Full size table

Microglia primary cell cultures

Primary mixed glial cell cultures were prepared from the neocortices of P0-P1 OF1 mice, as described previously [25, 26]. Briefly, the cortices were dissected and the meninges were removed in 0.1 M PBS with 6% glucose and 2% penicillin–streptomycin (PS, Gibco, Cergy Pontoise, France). The tissue was subsequently mechanically dissociated and spun at 1000 rpm for 5 min at 4 °C. The pellet was resuspended in low-glucose Dulbecco’s modified Eagle’s minimum essential medium (DMEM, 31885, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.01% PS. The cells were then plated in poly-D-L-ornithine-coated T75 flasks. Microglia were isolated from the primary mixed glial cultures on day in vitro 14 by shaking for 20 min. The supernatant was collated and spun at 1800 rpm for 5 min at 4 °C. Microglia were resuspended in DMEM supplemented with 0% FBS in 6-well culture plates and cultured for 1 day before treatment.

Microglia were exposed for 4 h to PBS or PIC (1 μg/ml). Supernatants were collected and stored at −80 °C until cytokine levels were measured. Cells were harvested and RNA was extracted for gene expression analysis.

Peritoneal macrophage primary cell cultures

Primary peritoneal macrophage cultures were prepared from 2-month-old OF1 male mice, as described previously [27]. Macrophages were isolated by peritoneal lavage with ice-cold PBS. After 400 × g centrifugation, cells were resuspended in DMEM/F12 medium (Gibco, Cergy Pontoise, France) supplemented with 10% FBS (Gibco) and 0.01% PS (Gibco) in 6-well culture plates. After 1 h, non-adherent cells were removed by washing and adherent cells were found to be ~95% pure based on morphological criteria. Cells were cultured for 1 day before treatment.

Similar to microglia, macrophages were exposed to PBS or PIC (4-hour; 1 μg/ml). Supernatants were collected and stored at −80 °C until cytokine level measurements. Cells were harvested and RNA was extracted for gene expression analysis.

RNA extraction and quantitative PCR

Total RNA from primary microglial cell cultures was extracted using the RNeasy mini kit according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France). Total RNA (500 ng) was subjected to reverse transcription based on equal amounts of RNA using the iScript™ cDNA synthesis kit (Bio-Rad, Marnes-la-Coquette, France). Quantitative PCR was then performed in duplicate for each sample using the SYBR Green Supermix (Bio-Rad) for 40 cycles with a two-step program (5 s of denaturation at 96 °C, and 10 s of annealing at 60 °C). The primers used are summarized in Table 2. The relative expression of genes of interest was compared with that of the reference gene, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Analyses were performed using Biorad CFX Manager 3.0 software.

Table 2 List of PCR primers used in the study Full size table

Multiplex cytokine assay

Freshly excised hippocampi from P14 and P75 rats (24 h after ventral hippocampal injection) were homogenized and total protein was extracted in PBS supplemented with protease inhibitors (Roche Diagnostics, Meylan, France). After a 12500-rpm centrifugation for 30 min, supernatants were collected.

IL-1β, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and interleukin 10 (IL-10) levels were measured in microglia supernatants and hippocampal protein extracts using a Bio-plex 200 and a 96-well magnetic plate assay according to the manufacturer’s instructions (Biorad Laboratories, Marnes la Coquette, France). All samples were run in duplicate, and data were analyzed using Bio-Plex Manager software. For hippocampal measurements, cytokine levels were expressed relative to total protein levels (pg of cytokine/mg of total protein).

Immunohistochemistry

Twenty-four hours after the hippocampal PIC injection, P14 and P75 rats (n = 5 for each experimental group) underwent transcardiac perfusion with 4% paraformaldehyde. Coronal 30-μm-thick free-floating sections were immunolabeled overnight with rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000, Wako) and mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:500, Sigma) antibodies and revealed by Alexa Fluor 488- and Cy3-conjugated anti-rabbit and anti-mouse secondary antibodies (1:500, Invitrogen).

Sections of the ventral hippocampus at the PIC or saline injection sites were imaged using a fluorescence microscope (Zeiss Axio Observer Z1). Immunopositive cells were quantified by a cell count in six different areas distant from the injection site in each animal (square; Fig. 3) using ImageJ software.

Rapid kindling protocol

Animals were anesthetized with isoflurane, stereotaxically injected with 10 μg of PIC (1 μl in the left hippocampus), and implanted with a bipolar stimulating electrode (Plastics One Inc., Roanoke, VA) into the left ventral hippocampus. The coordinates in relation to bregma were as follows: P13, 3.0 mm posterior, 3.9 mm left, 4.2 mm ventral; P74, 4.8 mm posterior, 5.3 mm left, 6.5 mm ventral. A tripolar recording electrode was wrapped around the skull screws above the right hemisphere. Electrodes were fixed to the skull using Integrity composite resin (Densply, York, PA, U.S.A.).

Twenty-four hours after surgery, the animals were connected to a DS8000 electrical stimulator via DLS100 stimulus isolators (World Precision Instruments, Sarasota, FL, U.S.A.). Electroencephalograms (EEGs) were acquired using the MP100/EEG100B acquisition system and AcqKnowledge software (BIOPAC, Santa Barbara, CA, U.S.A.). The electrical stimulus used for the assessment of the ictogenesis parameters and the kindling protocol was a square wave biphasic electrical stimulus with the following characteristics: 10-second train duration, 20 Hz, 1-ms pulse duration, delivered every 5 min. At 24 and 48 h post-injection, afterdischarge threshold (ADT) and afterdischarge duration (ADD) were assessed using 0.1 mA incremental currents delivered every 5 min. An AD was defined as paroxysmal epileptiform activity lasting at least 5 s with peak-to-peak amplitude of at least twice the baseline background EEG observed after the end of the hippocampal electrical stimulation.

The kindling protocol consisted of 60 trains delivered every 5 min with a current of 0.1 mA over the ADT with parameters otherwise similar to those described for afterdischarge detection. Animals were video-recorded during the kindling procedure. Behavioral seizures were scored using Racine’s scale: 1, motor arrest and twitching vibrissae; 2, chewing, head bobbing; 3, forelimb clonus; 4, forelimb clonus and rearing; 5, rearing and falling. Epileptogenesis was analyzed by calculating the numbers of kindling trials needed to first reach a stage 4–5 seizure and the total numbers of stage 4–5 seizures during the kindling procedure. Ictogenesis was analyzed by calculating changes in afterdischarge properties 24 h after the kindling procedure compared to baseline afterdischarge properties. We also determined the severities of behavioral seizures in response to the threshold stimulation in the kindled animals [28–30].

Statistical analysis

Data were analyzed using Prism 5 software (Graphpad, San Diego, CA, U.S.A.). Data were expressed as mean ± standard errors of the mean. Statistical analyses were performed using Kruskal-Wallis followed by Dunn’s posthoc tests and the Mann-Whitney test to compare two groups.