According to the ARRIVE guidelines, procedures were carried out to minimize discomfort and pain to treated rats, in compliance with National (D.L. 116 Suppl 40/1992 and D.L. 26/2014) and International guidelines and laws (2010/63/EU Legislation for the protection of animals used for scientific purposes). The experimental protocols were approved by the Ethics Committee of the Fondazione IRCCS Istituto Neurologico C. Besta and by the Italian Ministry of Health (protocol numbers: BR1/2012 and 961/2016-PR). Rats were housed in groups of two or three per cage under controlled conditions (22 ± 2°C and 12:12 light–dark cycle, lights on at 7 a.m.) until pilocarpine-SE induction. From this moment onwards, they were individually housed in cages and they were kept separated for all the duration of the trial. The animals were given free access to food and water.

Pregnant Sprague-Dawley rats (n = 4; Charles River, Calco, Italy) received two intraperitoneal (ip) doses of MAM (15 mg/kg maternal body weight, in sterile saline, MRI Global, Kansas City, Missouri, USA) 12 h apart at embryonic day E15, as previously reported (Colacitti et al., 1999). Twenty-eight young adult litters (280–350 g, 2–3 months old) of MAM-treated dams were used for inducing SE with pilocarpine (270 mg/kg ip), as previously described (Colciaghi et al., 2011). Thirty minutes before pilocarpine, rats received N-methylscopolamine (1 mg/kg, ip) to minimize peripheral cholinergic activation (Clifford et al., 1987).

Pilocarpine treated rats were monitored by two researchers that rated pilocarpine-induced symptoms. Briefly, behavioral seizures were graded as non-convulsive seizures (NCS) and convulsive motor seizures [CMS; tonic-clonic, tonic, and clonic; (Williams et al., 2009)] according to a modified Racine’s scale (Sharma et al., 2018; Colciaghi et al., 2019). SE onset was defined by the time of pilocarpine injection to the occurrence of the first CMS (stage 3–5) followed by continuous seizure activity: once rats reached stage 3–5, the behavioral activity fluctuated between NCS (stage 1–2) to CMS (stage 3–5). Rats that experienced either one isolated stage 3–5 seizure or stage 1–2 NCS only and that did not develop continuous seizure activity were excluded from the study (n = 6 rats, 22% of pilocarpine-treated rats). Diazepam (10 mg/kg ip) was administered 90 min after SE onset to alleviate seizures and decrease mortality rate. Death occurring during SE (n = 6 rats, 27% of MP rats) could not be anticipated on the basis of external monitoring. MAM-pilocarpine treated rats were continuously video-recorded using an infrared camera system (24h/day for 2 months) to detect the onset of spontaneous seizures and to quantify SRS occurrence. SRS were graded as follows: stage 0 through 5, as outlined by Racine (1972); stage 6, cluster of multiple stage 5 seizures; stage 7, violent jumping and running; stage 8, stage 7 plus tonic hindlimb extension and tail rigidity (Pinel and Rovner, 1978a; Pinel and Rovner, 1978b). Two researchers blinded to rats’ treatment observed the videos in a fast-forward mode. In case of any seizure-like activity, the videos were stopped, reversed and watched at normal speed. Since we could just rely on video-monitoring, we chose to restrict seizure quantification to generalized convulsive motor seizures (stages 4-7). Stage 8 seizures were never observed during video-monitoring of chronic epileptic rats. Total amount of generalized convulsive motor seizures were quantified for 50 days of video-recording/each rat. However, during video-monitoring analysis, any event suggestive of focal seizure (Racine stage 1-3, such as orofacial automatisms, head nodding, anterior limb clonus with lordotic posture) together with wild running/jumping behavioral not eventually evolving in CMS, were noted for each rat.

Randomly chosen epileptic MP rats surviving SE were treated immediately after the onset of the first spontaneous seizure with a daily dose of 300 mg/kg (bwt) CBZ formulated in food pellets for two-months (n = 10, hereafter referred to as MP-CBZ rats), according with protocol previously established by Ali and colleagues (Ali et al., 2012). Since the amount of food-intake for the maintenance of body weight of an adult rat is about 60 g/kg bwt/day, CBZ was formulated in food pellets as 5 mg CBZ per 1 g pellet (Mucedola Srl, Milano, Italy). CBZ-containing food (60 g pellet/kg bwt) was supplied in a single feeding each day at 10 a.m. for a total of 60 days (60 administrations). CBZ-treated rats were weighed every 3–5 days to determine any possible effect of CBZ-containing pellet on body weights. Further, the residual pellet possibly left was weighed every day in order to calculate the exact amount of pellet and CBZ dose taken by the individual rat/each day. In order to estimate the bioavailability of the drug, the CBZ serum level was estimated in each individual rat the last day of treatment using the Carbamazepine assay on the ARCHITECT c Systems™ (iCARB-Enzyme immunoassay, Abbott Laboratories), according to product insert and routinely used to monitor CBZ serum level in patients.

Remaining MP rats (n = 6) were used as proper epileptic untreated rat group and received normal pellet (hereafter referred as MP-untreated rats). Additional n = 4 age-matched naïve MAM rats, neither experiencing SE nor SRS, were used as untreated non-epileptic controls (MAM-CTR). At the end of the two-months-trial, all rats were sacrificed for histological/morphometric analysis (see below).

Morphologic analysis was performed as previously described (Colciaghi et al., 2019). Briefly, animals were killed with overdose of chloral hydrate and then perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline at pH 7.2. Brains were removed from the skull, post-fixed overnight, and cut with vibratome (Leica Biosystem, Wetzlar, Germany) into 40 to 50 µm thick coronal sections that were collected in serial order. As morphologic/neuroprotective read-outs of chronic CBZ-therapy, we evaluated the cortical thickness in thionine stained sections (see data analysis section, below) and the Fluoro-Jade (FJ; Histo-Chem Inc., Jefferson, AR, USA) labeling in cortex and hippocampus (Nobili et al., 2015). One series of coronal sections (1 out of 7 sections) was counterstained with 0.1% thionine and at least 6 sections per rat (from -0.3 to -5.8 mm from bregma) were processed for FJ analysis as previously reported (Nobili et al., 2015).

Latency to SE onset and SE behavioral evolution were analyzed in MP-untreated vs MP-CBZ rat group and statistically compared by means respectively of Mann-Whitney non parametric U-test and Student’s t-test. For CMS (stage 4–7) quantification, since MP rats exhibited spontaneous seizure activity with a non-normal distribution (regardless of control or CBZ treatment), a Mann-Whitney U-test was used to compare the total number of seizures between the two experimental groups.

Cortical thickness was measured in 3 serial thionine-stained coronal sections from i) the rostral cortex (anterior commissure, at ~ −0.3/−0.8 mm from bregma), ii) the somatosensory frontoparietal cortex (−2.8/−3.8 mm from bregma), iii) the temporal or posterior cortex (−4.8/−5.8 mm from bregma) (Paxinos and Watson, 1982). Selected sections were digitized by means of Aperio CS2 slide scanner (Leica Biosystems Nussloch GmbH) at 20x magnification and cortical thickness was measured in each section at 0° (1 mm lateral to the midline), 45° and 90° from the midline, as previously described (Colciaghi et al., 2011; Colciaghi et al., 2014) and indicated in Figure 2A . The three measures per section were averaged to a single value and the obtained measures from the 3 serial sections from each area were averaged again to a single value to obtain the mean cortical thickness of rostral, sensorimotor and posterior cortex for each rat. N = 4 MAM-CTR, n = 8 MP-CBZ and n = 6 MP-untreated rats were analyzed. Differences among groups were statistically analyzed for each neocortical area by means of one-way analysis of variance (ANOVA) followed by Tukey HSD as post-hoc comparison test.

All measurements were performed independently by two operators blind the to the animal treatment; data were expressed as mean ± SD (or SEM when indicated) and differences were considered significant with p < 0.05. The sample size of rats necessary to detect a difference of 15% with a power of 80% and alpha 0.05 (Machin et al., 1997) between MP untreated and MP-CBZ groups was estimated using variance values obtained in previous similar cortical thickness determinations (Colciaghi et al., 2014; Nobili et al., 2015) and data of video-monitoring seizure analysis in chronic epileptic pilocarpine/kainic rats treated with CBZ reported by other groups (Chakir et al., 2006; Polli et al., 2014).

MP rats were randomly assigned to untreated- or CBZ-experimental group: a retrospective analysis of SE evolution of single rats was conducted to verify possible difference of SE severity between the two groups ( Figures 1A, B ). SE onset time ranged between 16–70 min after pilocarpine injection. We did not observed differences in regards to latency to SE onset between MP-untreated and MP-CBZ rat groups (respectively 30 ± 8.25 min vs 46 ± 23.77 min, p = 0.242 Figure 1A ). All rats were scored every 15 min after the onset of the first CMS (stage 3–5, Time 0 in Figure 1B ): no differences in seizure development and intensity according to the behavioral scoring of CMS were observed between the CBZ-treated and untreated-rats ( Figure 1B ). The estimated SRS onset was not significantly different between the two groups: 6.43 ± 1.50 and 11.15 ± 4.30 (day after SE ± SD), respectively for MP-CBZ and MP-untreated rats. The day after SRS onset, rats assigned to MP-CBZ experimental group (n = 10) entered the two-months CBZ trial. Two out of 10 MP-CBZ treated rats died due to stage 7 seizures, respectively 3 and 5 days after SRS onset. Data obtained in the CBZ trial (n = 8 rats) are reported in Figure 1C and can be summarized as follows: all rats ate the CBZ-containing food throughout the 24 h day irregularly but continuously, with feeding episodes concentrated in the first 2 h after providing the CBZ pellet (10–12 a.m.) and in the first 2–3 h of the dark period (7–10 p.m.). The total amount of food consumed per day corresponded to a normal caloric diet (food-intake, Figure 1C ), and no difference was detected in the progressive increase of body weight among the treated rats during CBZ treatment (data not shown). Mean CBZ dose taken/day per rat was 252.64 ± 29.78 mg/kg/day (mean ± SD) while serum CBZ level in treated rats was 4.36 ± 0.60 μg/ml, within the suggested therapeutic range for humans [4–12 μg/ml; (Patsalos et al, 2018)].

The quantification of stage 4–7 convulsive motor seizures (Pinel and Rovner, 1978a; Pinel and Rovner, 1978b) during 2 months of video recording was not significantly different between MP-untreated and MP-CBZ rats ( Figure 1D ). SRSs occurred within clusters followed by seizure-free period for both groups; no differences were found between treated vs untreated rats [mean SRS cluster interval (days) ± SD: MP-untreated: 1.84 ± 1.13; MP-CBZ: 2.35 ± 1.80; n.s. p = 0.56; mean inter-cluster interval (days) ± SD: MP-untreated: 15.30 ± 5.8; MP-CBZ: 10.11 ± 5.96; n.s. p = 0.429]. However, CBZ treatment affected stage 4–7 CMS in 2 out of 8 MP-CBZ rats (encircled dots in panel D), hereafter referred to as MP-CBZ/responders. SRS onset time of the two MP-CBZ/responders was respectively 5 and 6 days post-SE.

It’s noteworthy that all rats, regardless of the treatment, daily exhibited events suggestive of focal seizures, such as orofacial automatisms, head nodding and anterior limb clonus with lordotic posture, together with periods of spontaneous and intense wild running or jumping-like behavior without generalized convulsion mainly concentrated in the dark period. In the absence of electroencephalography (EEG) monitoring these events were not included in seizures’ quantification.

We previously showed that the occurrence of SE plus SRS affects the cytoarchitecture of the chronic epileptic MAM/pilocarpine rat brain, significantly reducing both cortical thickness and hippocampal volume after 3 months of SRS (Colciaghi et al., 2011; Colciaghi et al., 2014). We therefore evaluated the effect of recurrent seizures on brain morphology by comparing the cortical thickness of non-epileptic naïve MAM (MAM-CTR), MP-untreated and MP-CBZ rats ( Figure 2 ). Representative serial coronal thionine stained sections from each experimental group are reported in Figures 2A-L . The mean cortical thickness was significantly reduced in both MP-untreated and MP-CBZ rats when compared to non-epileptic MAM-CTR rats, mainly affecting the posterior temporal and entorhinal cortex ( Figure 2M ). In contrast, no significant difference was obtained between MP-CBZ and MP-untreated rats.

To evaluate the localization and extent of degenerating neurons, FJ labeling was performed in MP-CBZ/responder and non-responder rats ( Figure 3 ). We did not detect any FJ⁺ neuron in CBZ-responders ( Figures 3B, D, F ), while CBZ/non-responder rats ( Figures 3A, C, E ) exhibited a FJ⁺ pattern very similar to that previously demonstrated in chronic epileptic MP rats (Nobili et al., 2015). In particular, evident labeling was obtained in deep cortical layer (arrowheads, Figure 3A ) paralleled by intense staining of hippocampal CA pyramidal neurons (arrows Figures 3A, E ) and by the presence of diffuse necrosis in entorhinal cortex (yellow arrowheads, Figure 3C ).

The findings of this study have to be seen in light of some limitations. The primary concerns the use of video-monitoring for spontaneous seizure assessment. In fact, since we could not perform video-EEG monitoring to indisputably identify focal seizures (Racine 1–3 seizure stage) that could be confused with non-epileptic movements related to normal rat activity, the anti-seizure efficacy of CBZ was restricted to the quantification of generalized CMS (stage 4–7). Similarly, activities suggestive of a seizure, such as wild-running behavior without falling, lordosis, tail erection were included in our quantification only when evolving into convulsive tonic-clonic seizures. The exclusion of <3 Racine seizures (and the consequent under-sampling of seizures) is likely the main reason for the relative low number of reported seizures in our limited setting (without video-EEG). The analysis of CMS by video-monitoring was the most reliable readout available to us. Thus, we believe that under-sampling in our experimental conditions was a necessity. However, taking into account that CBZ-in-food-protocol (300 mg/kg/day) was totally effective in suppressing any event suggestive of a seizure in epileptic kainic acid rat model (Ali et al., 2012), that CMS frequency obtained in untreated-MP rat model is similar to what reported by other groups in long-term (50-60 days) video-monitoring of chronic epileptic pilocarpine and kainic rats (Capella and Lemos, 2002; Chakir et al., 2006; Polli et al., 2014), and that early-chronic treatment with CBZ (120 mg/kg/day) in naïve pilocarpine rats was effective in suppressing Racine stage 4-5 CMS (Chakir et al., 2006), we believe that, although preliminary, the present data could offer an experimental paradigm to investigate mechanisms of resistance to specific AED.

The matter of the drug-resistant epilepsy associated with cortical dysplasia has been broadly addressed in the last years in experimental models, including MAM rats, by assessing the efficacy of conventional AEDs (Smyth et al., 2002; Jellett et al., 2015; Battaglia et al., 2017). In general, most of the studies showed a basic medically refractoriness of MAM rats to these conventional AEDs but virtually nothing is known about chronic AED treatment on seizure expression and seizure-related brain damage in epileptic malformed rats. In-vitro experiments showed that epileptiform activity induced by potassium channel blocker 4-Aminopyridine (4-AP) in hippocampal slices from MAM rats was relatively refractory to phenobarbital, CBZ, valproate (VPA), ethosuximide (ESM) and lamotrigine. Accordingly, in vivo experiments demonstrated that VPA was not effective in prolonging latency to SE in MAM animals after acute administration of kainic acid, in contrast to what observed in control animals (Smyth et al., 2002) or when seizures were induced by flurothyl inhalation (Jellett et al., 2015). In 2004, Serbanescu and colleagues, by combining prenatal MAM exposure with postnatal treatment with cholesterol biosynthesis inhibitor AY-9944, generated the two-hit MAM-AY rat model of chronic atypical absence seizures (Serbanescu et al., 2004). ECoG analysis of slow spike-and-wave discharge (SWD) duration did not show any difference between naïve-AY and MAM-AY rats but revealed medical refractoriness of MAM-AY rats to conventional anti-absence drugs (namely ESM and VPA). However, the analysis was restricted to a single drug administration and SWD quantification was limited to 1h recording period.

As CD patients are characterized by pharmaco-resistant epilepsy, the most valid experimental model, where screening long-term efficacy of AEDs, should rely on chronic and spontaneous seizure activity associated with brain malformation. In our opinion, this is a central issue, never addressed so far. In fact, as properly suggested by Potschka (2012), whether disease-associated alterations occur as consequences of repeated seizures, that—in turn—could contribute to therapeutic failure, should be verified in chronic model with SRS and not merely in acute seizure model, e.g., as latency to seizure onset.

To the best of our knowledge, this is the first study testing the outcome of early-chronic AED administration on both seizure activity and brain damage in CD model. As previously reported by Dudek’s group, the once-per-day non invasive CBZ-in-food administration protocol used in the present study offered the evident advantage of minimizing the handling of epileptic rats (Grabenstatter et al., 2007; Ali et al., 2012), thus avoiding the stress and pain unavoidably associated with ip injections, which in turn could induce seizures (Jöels, 2009) thus resulting in a very practicable approach for preclinical drug screening. As pilot, the present study does not claim to get deep into the possible mechanisms of AED-resistance associated with cortical dysplasia, rather it was originally established to verify whether and to what extent SRS could be controlled in MAM/pilocarpine rats, and to dissect the eventual effect of SE from that of SRS on brain damage. Additional trials, on larger cohort of animals treated with different AEDs, will be necessary to address the last issue. In this regard, preliminary unpublished data obtained from few MP rats (n = 4) from our lab show that spontaneous seizure activity of MP-CBZ non-responder rats was not affected either when rats entered in a subsequent trial with clobazam (CLB)-in food (40 mg/kg/day, for 4 weeks). Taken together these data indicate that MAM/pilocarpine rat can be considered a model of medically-intractable seizure or, at least, very poor response to CBZ.

The dysfunction of blood brain barrier (BBB) and the transporter hypothesis have been proposed as possible contributing factors of such a drug resistance in MAM rats. In fact, an intrinsic BBB leakage is a typical feature of the heterotopic regions of MAM rat brain while no changes were observed in tissue with normal cytoarchitecture (Marchi et al., 2006). Further, the occurrence of pilocarpine-induced SE in MAM rats worsens the BBB damage and induces the up-regulation of the multidrug resistance P-glycoprotein 1 (P-gp) in the perivascular astrocytes (Marchi et al., 2006), similarly to what described in human brain specimens of FCD patients (Sisodiya et al., 1999; Sisodiya et al., 2001; Sisodiya et al., 2002). Additional experiments will be necessary to verify whether dynamic changes in BBB function may determine the pharmacological responses of the epileptic malformed brain to AEDs and whether the BBB is preserved in AED-responder rats.

The question whether repeated seizures might be associated with progressive alterations of the brain has been long debated and as yet unresolved. In the current opinion, that a prolonged seizure damages the brain is widely accepted (Sloviter et al., 2006; Sloviter et al., 2007; Noè et al., 2019; Zhang et al., 2012), whereas the hypothesis that seizure recurrence is associated with cumulative brain damage is much more debated, even if supported by recent MRI studies and meta-analysis of MRI morphometry studies in pharmaco-resistant temporal lobe epilepsy patients [TLE; (Bernhardt et al., 2009; Caciagli et al., 2017; Galovic et al., 2019)]. The analysis of convulsive epilepsy models in which seizures are prevented by appropriate treatments would provide a more reliable evaluation of the effect of SE on the brain cellular and molecular features.

MAM/pilocarpine rats represent a convulsive model of chronic epilepsy, since the development of SE is the necessary pre-requisite to determine the later onset of SRS. In this model, therefore, as in most post-SE epilepsy models currently in use, it is difficult to dissect the pathologic effect of SE on the cellular and molecular features of the malformed brain from that of subsequent SRS.

The comparison of brain damage, through already developed morphologic and functional read-outs (Colciaghi et al., 2011; Nobili et al., 2015) revealed that CBZ-responder rats, that experienced frank SE but not stage 4–7 CMS, were similar to non-epileptic MAM-CTR rats. Indeed, FJ⁺ degenerating neurons were never detected in MP-CBZ/responder rats in any region considered. Conversely, the degree of brain damage in CBZ-non responders, that fully developed spontaneous convulsive seizures after SE, was similar to MP-untreated rats but significantly different from MAM-CTR rats. The small number of MP-CBZ responder rats (n = 2) did not allow a statistical comparison of seizure-induced brain damage vs other groups. We are aware that the lack of evident brain damage observed in the 2 CBZ-rats could be interpreted as a fortuitous event. However, we believe it’s worth pointing out since it may also suggest that even infrequent spontaneous CMS, and not merely the occurrence of SE, could eventually contribute to brain damage.