Inflammatory markers in the hippocampus after audiogenic kindling

Journal Pre-proof Inflammatory markers in the hippocampus after audiogenic kindling ´ ´ Junia Lara de Deus, Mateus Ramos Amorim, Procopio Cleber Gama de Barcellos Filho, Jose´ Antonio Cortes de Oliveira, Marcelo ˜ Norberto Garcia-Cairasco, Evelin Capellari Eduardo Batalhao, ´ ˜ Luiz Guilherme Siqueira Branc, Carnio, Ricardo Maur´ıcio Leao, Alexandra Olimpio Siqueira Cunha

PII:

S0304-3940(20)30100-2

DOI:

https://doi.org/10.1016/j.neulet.2020.134830

Reference:

NSL 134830

To appear in:

Neuroscience Letters

Received Date:

22 November 2019

Revised Date:

30 January 2020

Accepted Date:

6 February 2020

Please cite this article as: de Deus JL, Ramos Amorim M, de Barcellos Filho PCG, de Oliveira ˜ ME, Garcia-Cairasco N, Capellari Carnio ´ ˜ RM, Siqueira Branc LG, JAC, Batalhao E, Leao Cunha AOS, Inflammatory markers in the hippocampus after audiogenic kindling, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134830

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Inflammatory markers in the hippocampus after audiogenic kindling.

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Júnia Lara de Deus, 2Mateus Ramos Amorim, 1Procópio Cleber Gama de Barcellos Filho, 1José

Antonio Cortes de Oliveira, 3Marcelo Eduardo Batalhão, 1Norberto Garcia-Cairasco, 3Evelin Capellari Cárnio, 1Ricardo Maurício Leão, 2Luiz Guilherme Siqueira Branco, 1*Alexandra Olimpio Siqueira Cunha

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Ribeirão Preto-SP. Brazil. 2

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Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo.

Department of Morphology, Physiology and Basic Pathology, School of Dentistry of Ribeirão

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Preto, University of São Paulo, Ribeirão Preto-SP. Brazil.

Department of General and Specialized Nursing, School of Nursing of Ribeirão Preto,

*Corresponding author

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Departamento de Fisiologia

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University of São Paulo, Ribeirão Preto-SP, Brazil

Faculdade de Medicina de Ribeirão Preto

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Av. Bandeirantes, 3900

– Ribeirão Preto – SP. Brazil

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Tel: 5516 33153019 [email protected]

Highlights -

Cytokines levels are not altered after repeated audiogenic seizures Seizures spread to limbic areas is independent of hippocampal inflammation Higher levels of cytokines and oxidative stress correlate to more severe seizures

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Abstract

Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the

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hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the

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audiogenic kindling protocol with a loud broad-band noise. We measured pro and antiinflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our

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results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure

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severity, we did not observe differences in the pro inflammatory cytokines IL-1β, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant

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animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of

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cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate. Keywords: Cytokines, Seizures, Audiogenic seizures, WAR

1.0. Introduction

Audiogenic seizures are generalized tonic-clonic reflex seizures caused by the hyperactivation of brainstem auditory areas in response to a high intensity acoustic stimulus [1,2]. Behavioral and electroencephalographic characterization, as well as c-Fos expression after acute audiogenic seizures have shown the involvement of brainstem structures; inferior colliculus, superior colliculus and dorsal periaqueductal grey [2,3,4,5]. Interestingly, the

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repetition of acoustic stimulus may lead to the spread of epileptic activity to limbic areas possibly by the enhancement of colliculus-limbic pathways. This phenomenon is referred to as

audiogenic kindling and is been reported in rats susceptible audiogenic seizures, initially by

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Marescaux et al [6] and subsequently reproduced in the genetically epilepsy-prone rat (GEPR) [7], in Wistar Audiogenic Rats (WARs) [8,9,10,11,12] and in Krushinski-Molodkina rats [13].

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In WARs, as kindling progresses, epileptiform activity initially constrained to brainstem structures, spread to amygdala, hippocampus and auditory cortex [9,10]. Moreover, some

cortices were described [11].

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degree of cell loss in WARs during kindling in amygdala, hippocampus, perirhinal and piriform

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Although the focus of most studies in epilepsy are neurons, networks and synapses, recent investigations have shown that inflammatory pathways can trigger or sustain seizures in a wide

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variety of experimental models of epilepsy [14]. In this respect, inflammatory markers are often reported in brain samples of patients or animal models of acquired epilepsy [15,16]. In

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fact, seizures can lead to inflammation by the direct increase in glial pro-inflammatory cytokines [15] and inflammatory cytokines can trigger seizures through the loss of ionic or neurotransmitter homeostasis indicating a complex bidirectional relationship between seizures and inflammation. In the search for the mechanisms underlying seizure spread in repetitive audiogenic stimulation, the aim of this study was to investigate the role of non-neuronal inflammatory

pathways in the hippocampus during the audiogenic kindling and check for correlations between expression of these markers and severity of observed seizures.

2. Material and Methods Experimental procedures involving animals were designed according to rules for animal research from the National Council for Animal Experimentation Control (CONCEA) and from the Commission for Ethics in Animal Experimentation (CEUA) of the School of Medicine of

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Ribeirão Preto (015/2013).

2.1. Animals

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Animals in the WAR colony are selected according to Doretto et al., [17]. Only animals with high seizure scores in response to audiogenic stimulation are used to mate. Due to the small

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number of animals in the colony, in these experiments, we used only female WARs (n = 12, 6080 days) and seizure resistant Wistar females (n = 7, 60-80 days). In our experiments and

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elsewhere, audiogenic seizures severity in male and female WARs are similar [17,18]. In fact, many articles show that variability of experiments using females is similar to males [19].

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Rats were kept in Plexiglas cages (2-3 animals per cage), food and water ad libitum, 12-h

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dark/light cycle (lights on at 7:00 a. m.) and controlled temperature (22 °C).

2.2. Audiogenic kindling protocol

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We used the protocol previously described by Dutra-Moraes and colleagues [9] for

repeated high-intensity sound stimulation (HISS). Briefly, rats were placed in an acrylic, acoustically isolated arena with a loudspeaker on the top of the arena. After one minute of acclimation, a 110-dB broadband noise stimulus (1-16 kHz) was delivered for one minute or until the occurrence of tonic-clonic seizures. The sound intensity at the arena interior was checked and calibrated regularly with a decibel meter. After stimulation, the animals were

kept in the stimulation chamber for one minute and returned to their home cages. We repeated this procedure 21 times, twice a day, for 11 days. Seizures were scored from recordings by a blind trained observer using a brainstem seizure severity index [8], modified by Rossetti and colleagues [20] (Table S1) or a Racine index for limbic seizures [21] (Table S2). Animals were considered kindled when at least three limbic seizures were observed after the sound stimulus. We also calculated the frequency of limbic seizures by dividing the number of observed limbic seizures by the total number of

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stimulations since the first limbic seizure was observed. The HISS protocol was applied to Stimulated WARs (WAR-HISS, n=7) and Seizure resistant Wistar rats (Wistar-HISS, n=7). Control

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WARs (WAR-Sham, n = 5) were placed in the chamber during the 11 days, without sound.

2.3. Brain and Preparation of tissues

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In the morning of the last stimulation, thirty minutes after the last seizure in response to the last sound stimulus (21st), animals were decapitated. Brains were rapidly collected, frozen

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in dry-ice cold isopentane and stored at -80 °C until sample collection. In a cryostat, punches from dorsal hippocampus thick slices (1200 µm) from both right and left sides were collected

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using a punch needle (1.5 mm diameter).

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2.4. Determination of hippocampus Nitrate Concentration Total nitrate was determined using the Sievers Nitric Oxide Analyzer system (Sievers 280

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NOA; Sievers, Boulder, CO) according to Soriano and colleagues [22]. Briefly, hippocampus samples were deproteinized using cold absolute ethanol and injected into a reaction vessel containing vanadium trichloride (VCl3), which converts nitrate to NO. The NO produced was detected by ozone induced by chemiluminescence. Peak values of NO in hippocampal samples were determined using a standard curve constructed with sodium nitrate (Sigma-Aldrich Brazil, São Paulo, Brazil).

2.5. Measurement of pro and anti-inflammatory cytokines in the hippocampus Punches of hippocampus were thawed and homogenized in 0.5 mL of PBS solution in mM (150 NaCl, 6.5 Na2HPO4, 1.8 NaH2PO4; pH=7.4) plus protease inhibitor cocktail (leupeptin and aprotinin) using a sonicator, Sonics Vibra-Cell VCX-130PB (Sonics and Materials, INC) and then centrifuged at 13.000 rpm for 20 min at 4°C (Centrifugue 5804- Eppendorf). We measured concentrations of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) and anti-

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inflammatory cytokine (IL-10) by a multiplex assay (R&D System, Minnesota, USA) in tissue supernatants using Luminex® Magpix™ technology (Austin, TX, USA). ). Interleukins estimates in samples were normalized by protein concentrations, assessed by the Bio-Rad protein assay

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based on the Bradford assay (#5000205, Bio-Rad Laboratories, USA).

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2.6. Data analysis and statistics

We used descriptive statistics and reported data as means ± SEM. Comparison between

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variables was performed using Student t-test, with a significance level below 5% (P≤0.05). We also used non-parametric Spearman correlation to check if cytokine levels correlated with

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brainstem seizure severity or limbic seizure frequency. All analyses were performed with

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GraphPrism software version 8.0 (GraphPad Software, USA).

3.0 Results

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3.1. Audiogenic Kindling

WARs presented brainstem seizures that first increased and then lowered in severity

(Figure 1), as limbic seizures started to appear, in accordance to previous data [8, 9,10,11,23,24]. All WARs exhibited at least 3 limbic seizure and 85% of them had at least 3 score-5 seizures that consisted of tonic-clonic generalized convulsions with loss of postural control. Limbic

seizures always appeared after an episode of wild running. In all rats, the first limbic seizures always consisted of head and forelimb myoclonus with a mean severity score of 2. Since brainstem seizures co-existed with limbic seizures, some animals presented tonic-clonic seizures after limbic seizures and a second episode of wild running.

3.2. Resistant and susceptible animals have similar expression of cytokines in the hippocampus

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Our data showed no differences in pro and anti-inflammatory cytokines concentrations in the hippocampus of WAR-HISS and Wistar-HISS (Figure 2). Cytokines levels are (in pg/mg

protein): TNF-α (WAR-HISS: 434.7±55.9; Wistar-HISS: 372±37; p=0.55, Figure. 2.A), IL-6 (WAR-

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HISS: 563.5±82.6; Wistar-HISS: 499±54.6, p=0.66, Figure. 2.B), IL-1β (WAR-HISS: 82.3±13.0;

Wistar-HISS: 68.8±8.3, p=0.55, Figure. 2.C), IL-10 (WAR-HISS: 70.3±10.7; Wistar-HISS: 63.5±5,

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p=0.72, Figure. 2.E) and IFN-γ (WAR-HISS: 2077±299.5; Wistar-HISS: 1763±175, p=0.53, Figure. 2.E).

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These results suggest that sound stimulation affects similarly cytokine expression in the

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hippocampus and audiogenic seizures do not affect cytokine expression in the hippocampus.

3.3. Seizures do not change expression of cytokines in the hippocampus of susceptible

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animals

Next, we compared cytokine expression in the hippocampus of WARs submitted to

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seizures with WAR-Sham and our data showed similar concentrations of TNF-α (WAR-Sham: 363.5±44; WAR-HISS: 434.7±55.9, p= 0.37, Figure. 3.A), IL-6 (WAR-Sham: 433.2±58.3; WARHISS: 563.5±82.6, p= 0.26, Figure. 3.B), IL-1β (WAR-Sham: 60.9±8.56; WAR-HISS: 82.3±13.0, p=0.24, Figure. 3.C), IL-10 (WAR-Sham: 54.6±7.4; WAR-HISS: 70.2±10.7; p=0.3, Fig. 3. D) and IFN-γ (WAR-Sham: 1645±244; WAR-HISS: 2077±299.5, p= 0.32, Figure. 3.E).

These data show that audiogenic seizures do not induce increase in cytokines in the hippocampus of susceptible animals.

3.4. Seizure score, frequency and cytokines levels Brainstem seizure scores were positively correlated with pro inflammatory IL-6 (r= 0.8, p= 0.03) and IL-1β (r= 0.81, p= 0.038). Animals with higher severity score tended to have higher concentration of these cytokines (Figure 4.A). Correlations between brainstem seizures scores

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and cytokines were: TNF-α (r= 0.57; p= 0.18), IL-10 (r=0.6; p=0.19) and IFN-γ (r= 0.58; p= 0.18). Limbic seizure frequency of kindled animals, in turn did not correlate with none of

measured cytokines (Figure 4.B). In this case, correlation values were: TNF-α (r= 0.51; p= 0.24),

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IL-6 (r= 0.44; p= 0.3), IL-10 (r= 0.56; p= 0.24), IL-1β (r= 0.44; p= 0.31) and IFN-γ (r= 0.51; p=

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0.24).

3.5. Nitrate content is higher in animals with higher seizure scores

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Nitric oxide (NO) is a gas (or gaseous molecule) present in the CNS and is related to physiological and neuropathophysiological processes [25]. We did not observe significant

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differences in the nitrate content in the hippocampus, when compared animals submitted to HISS (WAR-HISS: 154.5±34.8 and Wistar-HISS: 97.8±22.6, p =0.19) (Figure 5.A). Also, our data

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show that kindled animals presented nitrate content measures similar to WAR-Sham (145.6 ± 34, p=0.60) (Figure 5.B).

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Interestingly, nitrate content strongly correlated with midbrain seizure severity (r = 0.95,

p= 0.004), but not with limbic seizure frequency (r = 0.18, p= 0.72) (Figure 5.C and 5.D). Our data indicate that oxidative stress may play a role in seizure spread that occur during repeated audiogenic stimulation.

4.0. Discussion

Here we show that audiogenic kindling, a phenomenon that plastically expands audiogenic seizures neuronal network from the brainstem to the forebrain [6,7,9], progresses independent on increase of hippocampal inflammatory cytokines. We used audiogenic susceptible animals, in which kindling produces epileptiform paroxysms in the EEG and cell proliferation in the dorsal hippocampus, and limbic seizures with similar semiology to those described by Racine [21] in the amygdala electrical kindling [8,10,11]. The mechanisms by which seizure circuitry expands in audiogenic kindling are largely

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unknown. As brainstem seizures become more severe after stimulation of forebrain structures, there might be a bilateral enhancement of auditory-limbic pathways [26]. Although inflammatory markers are highly increased in many brain regions after the amygdala electrical

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kindling as well as in naïve DBA/2J strain of mice [27], we did not detect differences in our experiments.

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In our study, we found positive correlations in between brainstem seizure severity and expression of IL-6 and IL-1β, indicating that in susceptible animals, the higher the expression of

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these cytokines in the hippocampus the more severe, brainstem seizures will be. Neuroinflammation is characterized by the presence of cytokines and inflammation

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mediators in the central nervous system, produced by cells of the immune system as well as cells of microglia, astrocytes and neurons [14]. The association of seizures and inflammation

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has been investigated in a variety of models and tissues from patients [28]. Seizures can trigger inflammation [14] and seizure predisposition is considerably increased by systemic and CNS

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inflammatory processes [28,29]. After the induction of Status Epilepticus (SE) by the administration of kainic acid or

pilocarpine, “waves of inflammation” mediated by astrocytes and neurons, were reported during the period preceding the onset of spontaneous seizures [30,31]. These experiments revealed increases in pro-inflammatory cytokines IL-1β, TNF-α, IL-6, as well as the TLR in the

hippocampus, followed, more chronically, by increases in chemokines and their receptors, both in the latent period after SE, where circuit re-organization takes place [30,31,32] Pereira and colleagues [33] demonstrated the expression of Bradykinin receptors, B1 and B2, after repetitive audiogenic stimulations in male WARs is increased, but the concentrations of IL-1β or the anti-inflammatory cytokine IL-10 are not. However, since seizure scores were not previously analyzed, it is hard to compare our data. Oxidative stress plays an important role as modulator in epilepsy, in humans and

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experimental animals [25]. Previous studies that showed that several experimental epilepsy models can be modulated by NO and the blockade of NO synthase was not able to induce audiogenic seizures in Wistar resistant animals [35]. Although we did not find differences in

had higher nitrate content in the hippocampus.

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nitrate concentration, we showed that animals that exhibited higher midbrain seizure score,

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In conclusion, our findings indicate that in our model, audiogenic seizures of WARs will spread and recruit the hippocampus, without the increase in the expression of inflammation or

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oxidative stress markers. This suggests that the development of audiogenic seizures in these animals could be a neuronal/network process. Recent data from our group showed that

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GABAergic inhibition is reduced in the hippocampus of naïve WARs, indicating possible alterations in inhibition/excitation balance in hippocampus [24].

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Our findings indicate that WARs could be use as models for a better understanding of neuronal network plastic changes that occur during the progression of some epileptic

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disorders.

Credit Author Statement Júnia Lara de Deus: Study Design, Data Analysis, Kindling Experiments, Manuscript draft and revision. Mateus Ramos Amorim: Tissue preparation, Cytokine measurements, Manuscript drafts. Procópio Cleber Barcellos Gama Filho: Tissue preparation, Cytokine measurements.

José Antonio Cortes de Oliveira: Audiogenic Strain Selection and Mantainance. Marcelo Eduardo Batalhão: Nitrate Estimates. Evelin Capellari Cárnio: Nitrate Content Analysis, Manuscript Revision. Norberto Garcia-Cairasco: Audiogenic Strain Selection and Mantainance, Seizure Analysis, Manuscript Revision. Ricardo Maurício Leão: Manuscript Draft and Revision. Luiz Guilherme Siqueira Branco: Cytokine Measurement Analysis, Experiment Supervision Manuscript Draft and Revision. Alexandra Olimpio Siqueira Cunha: Study Design, Data

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Analysis, Seizure Analysis, Experiment Supervision, Manuscript Draft and Revision.

Conflicts of Interest

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Authors declare no conflicts of interest.

Acknowledgements

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Authors thank José Fernando Aguiar for excellent technical assistance. Funding from FAPESP: grant #2015/22327-7 (A.O.S.C.); grant #2017/09878-0 (M.R.A); grant #2016/17681-9

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(E.C.C.); grant #2007/50261-4; INCT-Translational Medicine grant #2007/50261-4 (N.G.C.); grant #2016/17681-9 (L.G.S.B.); grant #2016/01607-4 (R.M.L.).

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J.L.D holds CNPq PhD student fellow. E.C.C., N.G.C., L.S.B. and R.M.L. hold CNPq Research

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Fellowships. A.O.S.C. holds FAPESP Research Fellowship.

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Figure Legends

Figure 1. Midbrain and limbic seizures during kindling. A and B. Mean midbrain and limbic seizure severity score of WARs (n=7) along stimulation sessions. Wistar rats (n=7). Data represent mean ± SEM.

Figure 2. Inflammation markers in the hippocampus after repeated HISS. Interleukin levels in

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the hippocampus of the seizure susceptible (WAR-HISS, n=7) and seizure-resistant (WISTARHISS, n=7) rats. A) TNF-α, B) IL-6, C) IL-1β, D) IL-10 and E) IFN-γ. Data represents mean ± SEM.

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Student t-test, p≥0.05.

Figure 3. Inflammation markers in the hippocampus of seizure-susceptible rats. Interleukin

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levels in the hippocampus of non-stimulated WARs (WAR-Sham, n=5) and after repeated HISS

Student t-test, p≥0.05.

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(WAR-HISS, n=7). A) TNF-α, B) IL-6, C) IL-1β, D) IL-10 and E) IFN-γ. Data represents mean ± SEM.

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Figure 4. Midbrain seizure scores and limbic seizure frequency as a function of cytokine production. A) TNF-α, IL-1β, IL-6, IL-10 and IFN-γ concentration in the hippocampus. B) TNF-α,

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IL-1β, IL-6, IFN-γ and IL-10. Dots represent each animal (n=7). Non-parametric Spearman

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correlations considering p<0.05 as significant.

Figure 5. Nitrate content in the hippocampus. A. Nitrate content in punches of hippocampus from Wistar-HISS (n=7) and WAR-HISS (n=7). B. WAR-Sham (n=5) compared to WAR-HISS. Data represent mean ± SEM. Student t-test p≥0.05. C. Mean midbrain seizure score and D. Limbic seizure frequency as a function of nitrate content. Linear regression lines and 95% confidence

intervals are shown. Dots represent each animal. Non-parametric Spearman correlations

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