We used 28–30-day old male C57BL6/N mice. All procedures involving animals and their care were conducted in accordance with the principles set out in laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law (D.lgs 26/2014; Authorisation n.19/2008-A issued March 6, 2008 by Ministry of Health); Mario Negri Institutional Regulations and Policies (Quality Management System Certificate—UNI EN ISO 9001:2008—Reg. N° 8576-A); the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE). Experiments were reviewed and approved by the intramural Animal Care and Use Committee, and by the Italian Ministry of Health.

Mice were killed by cervical dislocation. Brain was rapidly removed from the skull and horizontal brain slices (350 μm) from both hemispheres were cut with a vibratome (Leica VT 1000S) in ice-cold modified artificial cerebrospinal fluid (aCSF, mM): 87 NaCl, 2.5 KCl, 1 NaH₂PO₄, 75 sucrose, 7 MgCl₂, 24 NaHCO₃, 11 mM D-glucose, and 0.5 mM CaCl₂. Then, slices were transferred into the incubating chamber and submerged in aCSF containing (mM): 130 NaCl, 3.5 KCl, 1.2 NaH₂PO₄, 1.3 MgCl₂, 25 NaHCO₃, 11 D-glucose, 2 CaCl₂, and constantly bubbled with 95% O₂ and 5% CO₂ at room temperature. Slices were incubated in this condition for at least 1 h before starting the experiment.

Recordings were performed using CMOS-microelectrode array (MEA) BioCamX (3Brain GmbH, Lanquart, Switzerland) at room temperature and slices were continuously perfused with oxygenated aCSF at a rate of 2 ml/min.

The recording array allowed simultaneous extracellular recordings from 4,096 electrodes at a sampling rate of 10 kHz per channel. The channels were arranged in a 64 × 64 array configuration and each square pixel measured 21 × 21 μm. The size of the recording area on the chip was suitable for recording from the entire hippocampal/cortical slice. Once the slice was positioned on the chip, it was held in place with a custom-made anchor of platinum wire and nylon mash.

The epileptiform activity was triggered by slice perfusion for 40 min with aCSF containing zero (0) Mg²⁺ and 100 μM 4-aminopyridine (4-AP) (17, 18), and consisted of synchronized field potentials (FPs) occurring at different frequency rates. Slices were preincubated for 30 min with aCSF alone or with the addition of 10 μg/ml lipopolysaccharide (LPS) before perfusion with the ictogenic cocktail. LPS was washed out for 5 min with aCSF before slice perfusion with 0 Mg²⁺ + 100 μM 4-AP (Supplementary Figure 1A). The tested drugs were added to the perfusion solution as shown in Supplementary Figures 1B, C (see below). Activity was recorded during 10 min sessions (T1 = 0–10 min; T2 = 20–30 min from the start of 0 Mg²⁺ + 100 μM 4-AP perfusion). FPs were detected using BrainWave5 software (3Brain) as follows: high and low threshold were set at +200 μV and −200 μV, energy window 40 ms, refractory period 5 ms and maximum wave duration 500 ms.

We classified the epileptiform activity evoked 0 Mg²⁺ + 100 μM 4-AP into three major categories: (1) ictal events consisting of synchronous and repetitive field potentials (FPs) discharges (>5 s) with frequency >1 Hz (see tracing in Figure 3A); (2) status epilepticus (SE)-like events consisting of repetitive FPs with frequency ranging between 0.8–1.3 Hz and lasting >5 min (see Figure 3A); (3) interictal events consisting of synchronous single or repetitive FPs (<5 s). FP bursts were identified by a minimum of three FPs/burst with an interval between FPs ≤1 s.

At the beginning of the experiment, a digital image of the slice was taken trough a stereomicroscope. During the post-hoc analysis of the epileptiform activities, the digital image was overlayed on the activity map to identify the active areas in the slice. For quantification of epileptiform activity (FP frequency, amplitude and burst duration; incidence of ictal and SE events), we focused the analysis on the most active area in the whole hippocampal/cortical slice, as determined by activity map and the corresponding raster plot (e.g., Figures 2A, B). FPs measures were reckoned by averaging the values from each electrode in the active area. Post-hoc analysis showed that the most active areas were randomly distributed among hippocampus and temporal cortex in the various experimental groups.

Slices were incubated for 30 min in aCSF or LPS (+5 min wash-out), then 0 Mg²⁺ + 100 μM 4-AP perfusion was started. After recording recurrent epileptiform events for at least 10 min, slices were perfused with the selected ASMs (dissolved in the ictogenic cocktail) at their maximal therapeutic plasma concentration in humans (4) for 40 min: phenytoin 50 μM, phenobarbital 100 μM, sodium valproate 800 μM (Sigma-Aldrich, USA), midazolam 100 μM [Accord Helathcare Italia srl (19); n = 5–7 slices/drug from 3 to 5 mice]. The experimental protocol is described in Supplementary Figure 1B. For testing the immunomodulatory drugs, slices were pre-incubated with LPS in aCSF (30 min + 5 min washout), then perfused for 15 min with aCSF ± anakinra [22 μg/ml; 1.3 μM; Swedish Orphan Biovitrum AB (Sobi), Stockholm, Sweden] or chimeric mouse/rat anti-IL-6R antibody (14.7 μg/ml; 100 μM; Genetech Inc., San Francisco, CA, USA). Then, 0 Mg²⁺ + 100 μM 4-AP was perfused (± drugs) for 40 min (n = 6 slices /drug from 3 to 5 mice).

We used an independent set of slices to determine the neuroinflammatory response to 10 μg/ml LPS. After 30 min LPS incubation, slices were washed out in aCSF for either 30 min or 60 min (n = 7–10 slices/time point). At the end of washout, slices were collected and snap-frozen in liquid nitrogen, then stored at −80°C until analysis. Tissue was homogenized in Qiazol Lysis Reagent (Qiagen, Hilden, Germania) and total RNA isolated using the miRNeasy Mini kit (Qiagen) according to manufacturer's instructions. The concentration and purity of RNA were determined at 260/280 nm using a high-speed microfluidic UV/VIS spectrophotometer QIAxpert (Qiagen) and the integrity and quality of RNA were evaluated by 4200 Tapestation (Agilent Technologies, Santa Clara, CA, USA). cDNA was synthesized from 800 ng RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, Massachusetts, USA) following the manufacturer's protocol (Applied Biosystems). RT-qPCR experiments were run in triplicate for each sample using 384-well reaction plates and an automatic liquid handling station (epMotion 5075LH, Eppendorf, Hamburg, Germany) on an Applied Biosystems 7900HT System (Applied Biosystems). mRNA expression was analyzed using QuantiFast SYBR Green PCR Master Mix (Qiagen). The designed primers are reported in Table 1. Data were normalized using geometric mean of 2 independent house-keeping genes (s2Vb and Actb). Cycle threshold (CT) values were obtained using manual threshold.

Statistical analysis was performed by GraphPad Prism 8 (GraphPad Software, USA) for Windows using raw data. Data are presented as bargrams with individual values, and mean ± SEM. Non-parametric tests were chosen due to the low power of normality tests when the sample size is small. In each experiment, statistical analysis is reported in the respective figure legend. Differences between groups were considered significant for values of p < 0.05. Sample size was a priori determined based on literature data and previous experience with the 0 Mg² +100 μM 4-AP model.

We refined an in vitro model of epileptiform activities in hippocampal/temporal cortex slices from naive mice induced by 0 Mg²⁺ + 4-AP. The aim was to mimic the unresponsiveness of epileptiform events to ASMs (17), as observed in FIRES patients. We added the prototypical immune-inflammatory agent LPS to reproduce the cytokine storm that precedes seizure precipitation in FIRES.

Figure 1 shows a significant induction of ictogenic cytokines mRNA, namely Il1b (A), Tnf (D) and Cxcl1 (E) in brain slices after 30 min incubation with 10 μg/ml LPS. This induction was evident after 30 min LPS washout (p < 0.05; p < 0.01 vs. aCSF) and persisted after 60 min washout (p < 0.01 vs. aCSF). Il1rn (B) was also induced at both time points (p < 0.05; p < 0.01 vs. aCSF) although to a minor extent as compared to Il1b transcript. Accordingly, Il1b/Il1rn ratio (C) was significantly increased (p < 0.05; p < 0.01) compared to aCSF incubated slices, thus denoting a predominance of proinfammatory vs. antiinfammatory cytokines.

Epileptiform activity (activity map and raster plot are depicted in Figures 2A, B, D, E) was quantified during 10 min recording at two sequential time points (T1= 0–10 min, Figures 2A, B and T2 = 20–30 min, Figures 2D, E) from the start of 0 Mg²⁺ + 4-AP perfusion. Pre-incubation for 30 min with LPS exacerbated epileptiform activity in slices (Figures 2B, E vs. Figures 2A, D): bargrams show an increased frequency of FPs (p < 0.05 vs. 0 Mg²⁺ + 4-AP alone) and FP burst duration (p < 0.05) at T1 (Figure 2C) and T2 (Figure 2F), without affecting the amplitude of recorded events. Moreover, the incidence SE events (Figure 3A; see Section 2.2 for definition) observed in 0 Mg²⁺ + 4-AP bathed slices was significantly increased by four-fold on average in LPS pre-incubated slices (Figure 3B; p < 0.05).

We tested the effect of specific ASMs that classically fail in FIRES patients, namely 800 μM sodium valproate, 50 μM phenytoin, 100 μM phenobarbital and 100 μM midazolam on 0 Mg²⁺ + 4-AP-evoked epileptiform activities in LPS-pre-exposed vs. naïve slices (Figure 4). Epileptiform events were quantified at T2 = 20–30 min after the beginning of drug perfusion (protocol in Supplementary Figure 1B). In slices exposed to 0 Mg²⁺ + 4-AP (Figure 4A), both phenytoin (p < 0.05), phenobarbital (p < 0.01) and midazolam (p < 0.05) partially reduced the frequency of FPs with residual epileptiform activity still detected, while sodium valproate was ineffective. In LPS-preincubated slices exposed to 0 Mg²⁺ + 4-AP (Figure 4B), only phenobarbital and midazolam partially reduced the frequency of FPs (p < 0.05; p < 0.01) while both phenytoin and sodium valproate were ineffective. The respective activity maps and raster plots are depicted in Figures 4D, E.

Since FP frequency analysis encompassed all epileptiform activities, we specifically analyzed ictal events and SE events (see Section 2.2 for definition) during 20–40 min perfusion. Notably, we found that while ASMs fully inhibited these activities in slices exposed to 0 Mg²⁺ + 4-AP (64% incidence in aCSF vs. 0% with ASMs), the same ASMs were only partially effective in LPS-pre-exposed slices (Figure 4C). In particular, ictal and SE events occurred in 86% of LPS-pre-exposed slices, in 57% of sodium valproate slices (4/7), in 33% of phenytoin and midazolam slices (2/6) and in 20% of phenobarbital slices (1/5 slices; Figure 4C).

These results indicate that seizure resistance to ASMs is exacerbated in LPS pre-treated slices.

We investigated the effect of anakinra and the anti-IL-6R antibody on epileptiform activity in LPS-treated slices. To this aim, we modified the experimental protocol to take into account the time that the immunomodulatory drugs may require to counteract neuroinflammation. Thus, we perfused slices with aCSF containing the immunomodulatory drug for 15 min prior to switching to 0 Mg²⁺ + 4-AP perfusion solution. As for ASMs, the immunomodulatory drugs were perfused for further 40 min (Supplementary Figure 1C), and epileptiform events were measured during 20–30 min (T2). As depicted in Figures 5A, B, both immunomodulatory drugs showed similar effects by significantly delaying the time to onset of the first FP (p < 0.05; p < 0.01 vs. LPS/0 Mg²⁺ + 4-AP) and by reducing FP frequency (p < 0.05; p < 0.01 vs. LPS/0 Mg²⁺ + 4-AP). The respective activity maps and raster plots are depicted in Figure 5C. Importantly, ictal events and SE events in LPS/0 Mg²⁺ + 4-AP (83% incidence; Figure 4C) were abolished by both anakinra and the anti-IL-6R antibody (0% incidence: depicted in Figure 5C, HPC).