Journal of the Neurological Sciences 308 (2011) 35–40

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Journal of the Neurological Sciences

j ourna l homepage: www.e lsev ie r.com/ locate / jns

The involvement of Na+, K+-ATPase activity and free radical generation in thesusceptibility to pentylenetetrazol-induced seizures after experimental traumaticbrain injury

Luiz Fernando Almeida Silva c, Maurício Scopel Hoffmann a, Leonardo Magno Rambo b,Leandro Rodrigo Ribeiro b, Frederico Diniz Lima c, Ana Flavia Furian a, Mauro Schneider Oliveira a,Michele Rechia Fighera d, Luiz Fernando Freire Royes a,c,⁎a Centro de Ciências da Saúde, Laboratório de Neurotoxicidade e Psicofarmacologia, Departamento de Fisiologia, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazilb Programa de Pós-graduação em Ciências Biológicas: Bioquímica, Universidade Federal de Santa Maria, 90035-003 Porto Alegre, RS, Brazilc Centro de Educação Física e Desportos, Departamento de Métodos e Técnicas Desportivas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazild Instituto do Cérebro (InsCer), Pontifícia Universidade Católica do Rio Grande do Sul— PUC, Porto Alegre,90619-900 Brazil

⁎ Corresponding author at: Departamento deMétodosde Educação Física e Desportos, Universidade Federal deMaria, RS, Brazil. Fax: +55 55 3220 8031.

E-mail address: nandoroyes@yahoo.com.br (L.F.F. Ro

0022-510X/$ – see front matter. Crown Copyright © 20doi:10.1016/j.jns.2011.06.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 December 2010Received in revised form 10 June 2011Accepted 16 June 2011Available online 6 July 2011

Keywords:Na+,K+-ATPaseFluid percussion brain injuryOxidative damageSeizuresProtein carbonylationTBARS

Although the importance of brain trauma as risk factor for the development of epilepsy is well established, themechanisms of epileptogenesis are not well understood. In the present study, we revealed that the injection of asubthreshold dose of PTZ (30 mg/Kg, i.p.) after 5 weeks of injury induced by Fluid Percussion Brain Injury (FPI)decreased latency for first clonic seizures, increased the time of spent generalized tonic–clonic seizures andelectrocorticographic (EEG)waveamplitude. In addition, statistical analysis revealed thatN-acetylcysteine (NAC)(100 mg/kg) supplementation during 5 weeks after neuronal injury protected against behavioral andelectrographical seizure activity elicited by subthreshold dose of PTZ. The supplementation of this antioxidantcompound also protected against theNa+,K+-ATPase activity inhibition and concomitant increase in the levels ofoxidative stress markers (protein carbonylation and thiobarbituric acid-reactive substances-TBARS) in site andperi-contusional cortical tissue. In summary, the current experiments clearly showed that FPImodel induces earlyposttraumatic seizures and suggest that an alteration in the lipid/protein oxidation,membrane fluidity, and Na+,K+-ATPase activitymay be correlatedwith neuronal excitability, a significant component of the secondary injurycascade that accompanies TBI.

e Técnicas Desportivas, CentroSanta Maria, 97105-900 Santa

yes).

11 Published by Elsevier B.V. All rights res

Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction

Traumatic brain injury (TBI) is a devastating disease thatcommonly causes disability and strongly affects the quality of life ofpatients [1,2]. TBI also exacerbates seizure severity in individuals withpreexisting epilepsy [28], being one example of the process ofepileptogenesis [3]. In this context, it has been demonstrated thatearly lesion in Central Nervous System (CNS) alter the transportdynamic of the blood–brain barrier (BBB) and deteriorate the balanceof inhibitory and excitatory neurotransmitter system [4]. Thisneuronal dysfunction predisposes to subsequent development ofspontaneous recurrent seizures in the presence of prior subtle brainmalformation [5]. Nevertheless, there is great need for the identifi-cation of biomarkers that provide quantitative measures of the

process of post-traumatic epileptogenesis and predict which TBIpatients are likely to develop epilepsy.

In this context, oxidative stress, an imbalance between oxidantsand antioxidants, contributes to the pathogenesis of TBI [6,1]. Recentstudies have demonstrated that the oxidative damage mediated byreactive oxygen species (ROS) and nitrogen species (RNS) is asignificant component of the secondary injury cascade that accom-panies TBI [7–11]. Although several evidences supporting the ideathat any cellular constituent may be a target for free radical damage[12], the inhibition of some selected targets such as Na+,-K+-ATPasemay play an important role in the hyperexcitability induced by TBI[13]. Na+,K+-ATPase is a membrane bound enzyme known to play apivotal role in cellular ionic gradient maintenance and is particularlysensitive to reactive species [14,15]. Several studies have suggestedthat ROS inhibit the activity of Na+, K+-ATPase by oxidation of SHgroups and alteration of the membrane fluidity [16–19]. Recently, ithas demonstrated that experimental Fluid Percussion Injury (FPI)induces impairment in animal performance in a spatial learning,increase of the oxidative stress markers, and decrease in Na+,K+-ATPase activity suggesting that cognitive impairment following TBI

erved.

36 L.F.A. Silva et al. / Journal of the Neurological Sciences 308 (2011) 35–40

may result, at least in part, from an increase of oxidative stressmarkers that occurs concomitantly with a decrease in Na+,K+-ATPaseactivity [20].

It is well known that the overproduction of free radicals may leadto the development of epileptic focus (neurons) by the disruption ofthe antioxidant activity [21,22] and by the oxidative inactivation ofmembrane Na+,K+-ATPase. Thesemechanismswould be a key reasonbehind enhanced neuronal excitability and seizuremanifestation [23],since Na+,K+-ATPase activity is inhibited in brain of epileptic patientspost-mortem[24] and mutation in the Na+,K+-ATPase α subunit genehas been associated with epilepsy in humans [25]. Therefore, thepresent study was performed to investigate the effect of the freeradicals generation and Na+,K+-ATPase activity in the process ofpost-traumatic epileptogenesis.

2. Materials and methods

2.1. Animal and reagents

Adult male Wistar rats (270–300 g) were maintained undercontrolled light and environment (12:12 h light/dark cycle, 24±1 °C, 55% relative humidity), with standard laboratory chow andwater ad libitum. Animal utilization reported in this study has beenconducted in accordance with the policies of the National Institute ofHealth Guide for the Care and Use of Laboratory Animals (NIHPublications No. 80-23) revised in 1996, and with the approval of theEthics Committee for Animal Research of the Federal University ofSanta Maria (23081.018516/2006-57).

2.2. Traumatic brain injury and drug treatment

FPI was carried out as described previously [26,27]. Briefly, animalswere anesthetized with a single i.p. injection of Equithesin (6 ml/kg),a mixture containing sodium pentobarbital (58 mg/kg), chloralhydrate (60 mg/kg), magnesium sulfate (127.2 mg/kg), propyleneglycol (42.8%), and absolute ethanol (11.6%) and placed in a rodentstereotaxic apparatus. A burr hole of 3 mm in diameter was drilled onthe right convexity, 2 mm posterior to the bregma and 3 mm lateral tothe midline, taking care to keep the dura mater intact. A plastic injurycannula was placed over the craniotomy with dental cement. Whenthe dental cement hardened, the cannula was filled with Chloram-phenicol, closed with a proper plastic cap, and the animal wasremoved from the stereotaxic device and returned to its homecage.After 24 h, the animals were anesthetized with Halothane, and hadthe injury cannula attached to the fluid percussion device and placedin a heatpad maintained at 37±0.2 °C. TBI was produced by a fluid-percussion device developed in our laboratory. A brief (10–15 ms)transient pressure fluid pulse (4.05±0.17 atm) impact was appliedagainst the exposed dura. Pressure pulses were measured extracra-nially by a transducer (Fluid Control Automação Hidráulica, BeloHorizonte, MG, Brazil) and recorded on a storage oscilloscope (GouldLtd., Essex, UK). Sham-operated animals underwent an identicalprocedure, with the exception of FPI.

In order to evaluate the combination between electrographicseizures, oxidative and neurochemical alterations in cerebral cortexafter TBI, immediately after FPI procedure, a subset of animals weresupplemented with NAC (100 mg/kg) or its vehicle (distillated H2O)by intragastric gavage during 5 weeks after FPI procedure.

2.3. Seizure evaluation

After 4 weeks of FPI procedure, all animals were deeply anesthe-tized and two screw electrodes were placed bilaterally over theparietal cortex, along with a ground lead positioned over the nasalsinus as described by [22]. The experiments for electrocorticographic(EEG) recordings were performed 5 days after surgery as described by

[23]. Briefly, the rat was connected to the lead socket in a swivel insidea Faraday's cage, and the EEG was recorded using a digitalencephalographer (Neuromap EQSA260, Neurotec LTDA, Brazil).EEG signals were amplified, filtered (0.1 to 70.0 Hz, bandpass),digitalized (sampling rate 256 Hz) and stored in a PC for off-lineanalysis. Routinely, a 10 min baseline recording was obtained toestablish an adequate control period. After baseline recording, thesham or TBI animals received an injection of saline (0.9% NaCl,1 ml/kg,i.p) and/or subconvulsant dose of PTZ (30 mg/Kg, i.p.) [41]. Theanimals were observed for the appearance of generalized tonic–clonicconvulsive episodes for 20 min according to Ferraro et al. [28], whodescribe generalized convulsive episodes as generalized whole-bodyclonus involving all four limbs and tail, rearing, wild running andjumping, followed by sudden loss of upright posture and autonomicsigns, such as hypersalivation and defecation. PTZ-induced general-ized convulsions typically lasted between 30 and 60 s, and werefollowed by a quiescent period. During the 20-min observation period,the latency for generalized tonic–clonic convulsions was measured.EEG recordings were visually analyzed for seizure activity, whichwere defined by the occurrence of the following alterations in therecording leads: isolated sharp waves (≥1.5×baseline); multiplesharp waves (≥2×baseline) in brief spindle episodes (≥1 s≥5 s);multiple sharpwaves (≥2×baseline) in long spindle episodes (≥5 s);spikes (≥2×baseline) plus slow waves; multispikes (≥2×baseline,≥3 spikes/complex) plus slowwaves; major seizure (repetitive spikesplus slow waves obliterating background rhythm, ≥5 s). For quanti-tative analysis of EEG amplitude, we averaged EEG amplitude over the20 min of observation.

2.4. Tissue processing for neurochemical analyses

Immediately after the EEG and behavioral evaluation, the animalswere killed by decapitation and their brain was exposed by removingthe parietal bone. The brains were quickly removed and a coronalsection (7 mm) of the injured hemisphere corresponding to theimpact site of injury was dissected (see Fig. 1). The cortical tissuessurrounding the injured core were homogenized in cold 10 mM Tris–HCl buffer (pH 7.4) and used for determination of carbonyl content,TBARS and Na+,K+-ATPase activity.

2.5. Measurement of thiobarbituric acid-reactive substances (TBARS)content and protein carbonyl

For the TBARS assay, the cortical tissues surrounding the injuredcore were homogenized in ultra-pure water and mixed with the TBAreagent (15% of trichloroacetic acid, 0.375% of thiobarbituric acid and2.5% v/v of HCl). After 30 min of incubation, samples were centrifuged(3000 ×g, 15 min) and then TBARS levels were measured at 532 nmaccording to Ohkawa et al.[29]. For the protein carbonyl assay, thecortical tissues surrounding the injured core were homogenized incold 10 mM Tris–HCl buffer (pH 7.4) and carbonyl protein contentwas determined by Yan et al.[30] adapted for brain tissue by Lima etal.[13]. Briefly, homogenates were diluted to 750–800 μg/ml ofprotein in each sample, and 1 ml aliquots were mixed with 0.2 ml of2,4 dinitrophenylhydrazine (10 mM DNPH)or 0.2 ml HCl (2 M). Afterincubation at room temperature for 1 h in a dark ambient, 0.6 ml ofdenaturing buffer (150 mM sodium phosphate buffer, pH 6.8 contain-ing 3% SDS),1.8 ml of heptanes(99.5%) and 1.8 ml of ethanol (99.8%)were added sequentially, andmixedwith vortex agitation for 40 s andcentrifuged for 15 min. Next, the protein isolated from the interfacewas washed two times with 1 ml of ethyl acetate/ethanol 1:1 (v/v)and suspended in 1 ml of denaturing buffer. Each DNPH sample wasread at 370 nm in a Hitaschi U-2001 spectrophotometer against thecorresponding HCL sample (blank), and total carbonylation calculatedusing a molar extinction coefficient of 22,000 M−1 cm−1.

37L.F.A. Silva et al. / Journal of the Neurological Sciences 308 (2011) 35–40

2.6. Protein determination

Protein content was measured colorimetrically by the method ofBradford [31] using bovine serum albumin (1 mg/ml) as standard.

2.7. Statistical analysis

Behavioral data were analyzed by the Kruskall–Wallis test(nonparametric one-way analysis variance), and are presented asmedian and interquartile ranges. Pos hoc analyses were carried out byDunn's multiple comparison test. The amplitude of EEG recordingsand neurochemical parameters were carried out by one- or two-wayanalysis of variance (ANOVA) and only F- values of Pb0.05 arepresented. All data are expressed as mean±S.E.M.

3. Results

In the present study we revealed that the injection of PTZ at30 mg/kg caused only minor behavioral and EEG alterations in shamgroup (Fig. 1A and B). However, this subeffective dose caused theappearance of generalized tonic–clonic seizures characterized bymultispikes plus slow waves and major seizure activity characterizedby 2–3 Hz high-amplitude activity in the recording leads (Fig. 1C andD). In addition, statistical analysis showed that the subeffective dose ofPTZ decreased latency for first clonic seizures [H(1)=4.29; pb0.05Fig. 1I] and increased the time of spent generalized tonic–clonicseizures [H(1)=4.84; pb0.05; Fig. 1J]. Statistical analysis also revealedthat FPI had no effect on the baseline EEG amplitude but increased in

Fig. 1. Representative EEG recordings obtained from sham-PTZ group (A), TBI-PTZ group (C)EEG recordings outlined by boxes are shown in B, D, F and H, respectively. The arrowhead inmyoclonic jerk (I), time spent in generalized tonic–clonic seizure (J) and EEG amplitude (K).n=8-12 (K). * Pb0.05 compared to all other groups.

EEG wave amplitude after subthreshold dose of PTZ [F(3,34)=20.28;pb0.05; Fig. 1K].

In order to evaluate the association between posttraumatic seizureselicited by PTZ with oxidative damage in this model of neuronal injury,we analyzed effect of NAC supplementation (100 mg/Kg; p.o) onbehavioral and EEG alterations induced by PTZ (30 mg/kg, i.p.). EEGrecordings showed that supplementation of NAC (100 mg/Kg; p.o.)during 5 weeks caused only minor behavioral and EEG alterations insham group (Fig. 1E and F). On the other hand, the supplementation ofNAC protected against PTZ-induced seizures characterized by latencyfor myoclonic jerks [H(1)=4.54; pb0.05; Fig. 1I], increased the time ofspent generalized tonic–clonic seizures [H(1)=4.84; pb0.05 Fig. 1J]and EEG wave amplitude [F(3,34)=4.81;pb0.05; Fig. 1K]. Of note, EEGrecordings confirmed the increase in seizure latency induced by NACsupplementation (Fig. 1G and H). Moreover, EEG recording confirmedthe decreased the time spent in convulsions, since there is a cleardecrease in electrographical seizure activity in these experimentalgroups (Fig. 1G and H).

Statistical analysis revealed that 5 weeks after FPI, TBARS [F(1.70)=19.84; pb0.05] and protein carbonyl content [F(1.70)=26.79;pb0.05] increased in the ipsilateral cortex of FPI group whencompared with the sham group. In addition, the injection ofsubeffective dose of PTZ, which had no effect per se, increasedTBARS [F(7.70)=33.34; pb0.05] and protein carbonyl content [F(7.70)=14.38; pb0.05] in the ipsilateral cortex of FPI animals incomparison with sham group. In the present study we also showedthat supplementation of NAC (100 mg/Kg; p.o.) during 5 weeks hadno significant effect on TBARS and protein carbonyl content per se,however protected against increase of TBARS [F(7.70)=10.56;

, sham-PTZ/NAC group (E) and TBI-PTZ/NAC group (G). The expanded waveforms fromdicates PTZ administration (30 mg/kg, i.p.). Effect of TBI on PTZ-induced latency for firstData are median±interquartile ranges for n=8-12 (I and J). Data are mean+S.E.M. for

38 L.F.A. Silva et al. / Journal of the Neurological Sciences 308 (2011) 35–40

pb0.05] and protein carbonyl content increase [F(7.70)=15.64;pb0.05] of FPI animals injected with PTZ (Table 1).

Considering that Na+,K+-ATPase enzyme plays a pivotal role incellular ionic gradient maintenance and is particularly sensitive toreactive species [14,15], we decided to evaluate the involvement ofthis enzyme in the progression and manifestation of seizure elicitedby PTZ in this post-traumatic model of epilepsy. Statistical analysisrevealed a decrease in Na+,K+-ATPase activity in the ipsilateralcerebral cortex of FPI animals [F(7.70)=37.90; pb0.05] whencompared with sham animals. Post hoc analysis also revealed thatthe injection of subeffective dose of PTZ decreased Na+,K+-ATPaseactivity in sham animals when compared with the control group(sham-NaCl group), and that the decrease in the activity of this en-zyme was larger in FPI animals. Statistical analysis also revealed thatNAC supplementation (100 mg/Kg; p.o) protected against Na+,K+-ATPase inhibition in the ipsilateral cerebral cortex of FPI [F(7.70)=18.72; pb0.05] and FPI-PTZ group [F(7.70)=5.63; pb0.05].

4. Discussion

The sequelae of TBI, including posttraumatic epilepsy, represent asocietal problemwhere the importance of brain trauma as a risk factorfor the development of epilepsy is well established [32,33]. Never-theless, clinical trials aiming the prevention of epilepsy following TBIhave failed [34] due to the multiple pathophysiologic epileptogenicprocesses that are likely activated simultaneously or sequential bybrain trauma [35]. Another limiting factor is the heterogeneity ofpatient populations. For instance, it is difficult to match both theseverity and locations of TBI between placebo and treatment groups.Thus, significant resources are required to develop a better under-standing of the pathophysiology mechanism as targets for potentialprophylactic therapies. In line of this view, most of the dataconcerning the progression of damage have been collected duringthe first 1–2 months post-injury, which also corresponds to the timeperiod when most of the neuronal alterations, implying increasedexcitability of injured tissue, have been performed [36,37].

In the present study we revealed that a simple FPI episode in ratparietal cortex decreases Na+,K+-ATPase activity with a concomitantincrease in the levels of oxidative stressmarkers (protein carbonylationand TBARS), 5 weeks after the injury. Our results also revealed that theinjection of subeffective dose of PTZ (30 mg/Kg, i.p.) caused earlyposttraumatic seizures suggesting that an alteration in the lipid/proteinoxidation, membrane fluidity, and Na+,K+-ATPase activity may becorrelatedwith posttraumatic epilepsy development. In fact, the resultspresented in this report showed that N-acetylcysteine (NAC) supple-mentation, a powerful antioxidant both in the peripheral tissues andcentral nervous system (CNS) [38,39], was effective in preventing ofbehavioral and electrographic seizures induced by PTZ in this post-traumatic model of epilepsy. The supplementation of this compoundalso protected against the increase in the levels of oxidative stressmarkers (protein carbonylation and TBARS) and Na+,K+-ATPase

Table 1Effect of FPI on Na+,K+-ATPase activity, protein carbonyl content and TBARS content.

Na+,K+-ATPase activity(nmol Pi/mg protein/min)

Protein ca(nmol/mg

Treatment Sham TBI Sham

NaCl 93.90±4.56 51.44±2.35a 5.13±0.6PTZ 68.06±5.38a 39.73±4.06a,c 6.59±0.4NAC 104.0±3.62 103.45±4.4b 5.48±0.5NAC/PTZ 106.7±4.49c 99.09±4.06d 7.00±0.4

Data are mean±SEM for n=8–12 in each group.a pb0.05 compared with NaCl-Sham group.b pb0.05 compared with NaCl-TBI group.c pb0.05 compared with PTZ-Sham group.d pb0.05 compared with PTZ-TBI group.

activity inhibition. These experimental findings suggest that a dysregu-lation of both inhibitory and excitatory neurotransmission followingtraumatic injury is responsive to antioxidant treatment. Furthermore, itis plausible to propose that these alterations play an important role inneuronal cell loss following TBI, and thus contribute to the pathophys-iology of cerebral damage following brain injury [40].

These results are in full agreement with the view that free radicalscontribute to neural injury following cerebral ischemia [41] and TBI[20]. In addition, oxidative stress in CNS is involved in thedegeneration of neurons in several rodent models of experimentalepilepsy and seizure, such as the amygdale kindling model [42], thekainic acid model [43], the PTZ kindling model [44], and the acutePTZ-induced seizure [45]. In line of this view, a substantial body ofevidence has suggested that a cascade of biological events, includingreactive species generation, underlies the development and propaga-tion of epilepsy [46–48,45]. In addition, the total activity of superoxidedismutase (SOD) and the content of the antioxidant α-tocopherolhave been found to be reduced in rat brain homogenates after acutePTZ-induced convulsion [49]. Previous studies from our group havedemonstrated that the injection of PTZ-induced convulsive activity isaccompanied by ROS generation and inhibition of local Na+,K+-ATPase activity [50,51,13,22,52]reinforcing the assumption that theinhibition of some selected targets for free radicals increases cellularexcitability [53–55].

Nevertheless, since there is no information available describingwhether posttraumatic seizures are an epiphenomenon of braininjury or whether they exacerbate damage for a given injury severity,there is still some debate about specific mechanisms that initiate orsustain epileptogenesis. While the weight-drop model of TBI wasfound to show a persistent susceptibility to PTZ-evoked behavioralseizures for at least 15 weeks with no spontaneously seizures [56],spontaneous chronic seizures in rats originated from the neocortex atthe side of injury have been observed [26]. Such seizures have beenalso found to be progressively worsened and spread over timecorrelating with hyperexcitability in the injury cortex [27]. Inaddition, increasing neuronal excitability by negative modulation ofan inhibitory neurotransmitter system (GABA) after TBI has showedto present a positive effect on the recovery of cognitive function [57].Although the determining factor for such a discrepancy is unknown,these apparent contradictions may be explained by differentexperimental conditions (species, tissue analyzed and type of modelTBI), however, there are caveats to consider when interpreting theexperimental data available [37].

On the other hand, the results presented in this report suggest thata significant increase of protein carbonylation and TBARS that occursconcomitantly with a decrease in Na+,K+-ATPase activity mayinfluence in the membrane electrophysiology by altering the actionpotential production, decreasing, this way, the threshold to seizuresinduced by sub effective dose of PTZ (30 mg/Kg; i.p). In concordancewith this view, the results presented in this report demonstrated thatNAC supplementation protected against electroencephalographic

rbonyl contentprotein)

TBARS content(μmol MDA/mg protein)

TBI Sham TBI

2 8.50±0.34a 0.140±0.75 0.336±0.09a

0 12.09±0.95a,b 0.240±0.10a 0.530±0.20a,b,c

5 6.36±0.65b 0.158±0.02 0,161±0.03b

2 6.75±0.56d 0.145±0.04 0,149±0.07d

39L.F.A. Silva et al. / Journal of the Neurological Sciences 308 (2011) 35–40

seizures and oxidative damage in this model of TBI. Furthermore, theobservation available from in vitro slice studies support the idea thatcortical and hippocampal excitability are increased in a variety of TBImodels [58–60]. In addition, a considerable body of evidence hassuggested that oxidative damage, mediated by reactive oxygen andnitrogen species, including the superoxide ion, hydrogen peroxide,hydroxyl radical, nitric oxide, and peroxynitrite, is well recognized asa significant component of the secondary injury cascade thataccompanies TBI [7]. In fact, while free-radical-induced damage hasbeen proposed to be involved in traumatic cell injury and cell death,free radical scavengers, such as catalase and glutathione peroxidase(GSH-Px) are associated with partial amelioration of traumatic injury[61–63].

Another point to be addressed is that the neurotoxicity effectexerted by subeffective dose of PTZ (30 mg/kg, i.p) in FPI animals aswell as the effective protection exerted by NAC supplementationsuggests that convulsive activity and neurochemical parameters areclosely linked events in this model of TBI. In addition, it is plausible topropose that important changes in the brain after FPI may affect thesusceptibility to seizure or events followed by epileptiform activityinduced by PTZ. With respect to the effects of subconvulsant dose ofPTZ (30 mg/kg,i.p) in cerebral cortex after FPI , data from our studysuggest that changes in this structure could be related to the specificfunction of PTZ as selective blocker of the chloride ionophore complex[64,65] or to cellular thiol homeostasis [45]. Furthermore, the PTZ-induced excitability proposed in this report is in accordance withpreviously report data showing that single convulsive dose of PTZresults in significant changes in many parameters such as GABAA

receptor density and function [64,65], whole brain hydroxyl radicals[49], free fatty acids, and glutathione peroxidase activity in specificbrain areas [48]. However, this explanation remains speculative innature, and further studies are necessary to determine thismechanism.

In conclusion, the present study reports that a simple FPI episodein rat parietal cortex decreases Na+,K+-ATPase activity with aconcomitant increase in the levels of oxidative stressmarkers (proteincarbonylation and TBARS), 5 weeks after the injury. In addition, theearly posttraumatic seizures caused by injection of subeffective doseof PTZ (30 mg/Kg, i.p.) and the significant protection elicited byantioxidant compound (NAC) suggest that failure of some selectedtargets, such as Na+,K+-ATPase elicited by ROS generation, mayincrease cellular excitability and facilitate the appearance and/orpropagation of convulsions after TBI.

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