Leonardo Magno Rambo

Na+,K+-ATPase activity impairment after experimental traumatic brain injury: relationship to spatial learning deficits and oxidative stress.

Traumatic brain injury (TBI) is a devastating disease that commonly causes persistent mental disturbances and cognitive deficits. Although studies indicate that oxidative stress and functional deficits occurring after TBI are interrelated events, the knowledge of the mechanisms underlying the development of such cognitive deficits has been limited. Thus, in the present study, we investigated the effect of fluid percussion brain injury (FPI) on a spatial learning task and levels of oxidative stress markers, namely, protein carbonylation and thiobarbituric acid-reactive substances (TBARS) and Na+,K+-ATPase activity 1 or 3 months after FPI in rats. Statistical analysis revealed that FPI increased the scape latency and mean number of error in Barnes maze test 1 and 3 months after FPI. We also found that protein carbonylation and TBARS content increased in the parietal cortex 1 and 3 months after FPI. In addition, 3 months after FPI, protein carbonylation levels increased both in ipsilateral and contralateral cortices of FPI animals. Indeed, statistical analysis revealed a decrease in Na+,K+-ATPase activity in the cerebral cortex of 1 month FPI animals. Furthermore, the decrease in enzyme activity found 3 months was larger, when compared with 1 month after FPI. These results suggest that cognitive impairment following TBI may result, at least in part, from increase of two oxidative stress markers, protein carbonylation and TBARS that occurs concomitantly to a decrease in Na+,K+-ATPase activity.

Swimming training prevents pentylenetetrazol-induced inhibition of Na+, K+-ATPase activity, seizures, and oxidative stress

Purpose:  In the present study we decided to investigate whether physical exercise protects against the electrographic, oxidative, and neurochemical alterations induced by subthreshold to severe convulsive doses of pentyltetrazole (PTZ).

Adaptation to oxidative challenge induced by chronic physical exercise prevents Na+,K+-ATPase activity inhibition after traumatic brain injury

Physical exercise is likely to alter brain function and to afford neuroprotection in several neurological diseases. Although the favorable effects of physical exercise on traumatic brain injury (TBI) patients is well known, little information is available regarding the role of free radicals in the improvement induced by physical exercise in an experimental model of TBI induced by fluid percussion injury (FPI). Thus, we investigated whether 6 weeks of swimming training protects against oxidative damage (measured by protein carbonylation and thiobarbituric acid-reactive substances-TBARS) and neurochemical alterations represented by immunodetection of alpha subunit and activity of Na(+),K(+)-ATPase after FPI in cerebral cortex of rats. Statistical analysis revealed that physical training protected against FPI-induced TBARS and protein carbonylation increase. In addition, physical training was effective against Na(+),K(+)-ATPase enzyme activity inhibition and alpha(1) subunit level decrease after FPI. Pearsons correlation analysis revealed that the decrease in levels of catalytic alpha(1) subunit of Na(+),K(+)-ATPase induced FPI correlated with TBARS and protein carbonylation content increase. Furthermore, the effective protection exerted by physical training against FPI-induced free radical correlated with the immunocontent of the catalytic alpha(1) subunit maintenance. These data suggest that TBI-induced reactive oxygen species (ROS) generation decreases Na(+),K(+)-ATPase activity by decreasing the total number of enzyme molecules, and that physical exercise protects against this effect. Therefore, the effective protection of selected targets, such as Na(+),K(+)-ATPase induced by physical training, supports the idea that physical training may exert prophylactic effects on neuronal cell dysfunction and damage associated with TBI.

Additive anticonvulsant effects of creatine supplementation and physical exercise against pentylenetetrazol-induced seizures.

Although physical activity and creatine supplementation have been a documented beneficial effect on neurological disorders, its implications for epilepsy are still controversial. Thus, we decided to investigate the effects of 6 weeks swimming training, creatine supplementation (300 mg/kg; p.o.) or its combination seizures and neurochemical alterations induced by pentylenetetrazol (PTZ). We found that 6 weeks of physical training or creatine supplementation decreased the duration of PTZ-induced seizures in adult male Wistar rats, as measured by cortical and hippocampal electroencephalography and behavioral analysis. Importantly, the combination between physical training and creatine supplementation had additive anticonvulsant effects, since it increased the onset latency for PTZ-induced seizures and was more effective in decrease seizure duration than physical training and creatine supplementation individually. Analysis of selected parameters of oxidative stress and antioxidant defenses in the hippocampus revealed that physical training, creatine supplementation or its combination abrogated the PTZ-elicited increase in levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonylation, as well as decrease in non-protein-thiols content, catalase (CAT) and SOD activities. In addition, this protocol of physical training and creatine supplementation prevented the PTZ-induced decrease in hippocampal Na+,K+-ATPase activity. Altogether, these results suggest that protection elicited physical training and creatine supplementation of selected targets for reactive species-mediated damage decrease of neuronal excitability and consequent oxidative damage elicited by PTZ. In conclusion, the present study shows that physical training, creatine supplementation or its combination attenuated PTZ-induced seizures and oxidative damage in vivo, and provide evidence that combination between creatine supplementation and physical exercise may be a useful strategy in the treatment of convulsive disorders.

The involvement of Na+, K+-ATPase activity and free radical generation in the susceptibility to pentylenetetrazol-induced seizures after experimental traumatic brain injury.

Although the importance of brain trauma as risk factor for the development of epilepsy is well established, the mechanisms of epileptogenesis are not well understood. In the present study, we revealed that the injection of a subthreshold 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 and electrocorticographic (EEG) wave amplitude. In addition, statistical analysis revealed that N-acetylcysteine (NAC) (100mg/kg) supplementation during 5 weeks after neuronal injury protected against behavioral and electrographical seizure activity elicited by subthreshold dose of PTZ. The supplementation of this antioxidant compound also protected against the Na(+),K(+)-ATPase activity inhibition and concomitant increase in the levels of oxidative stress markers (protein carbonylation and thiobarbituric acid-reactive substances-TBARS) in site and peri-contusional cortical tissue. In summary, the current experiments clearly showed that FPI model induces early posttraumatic seizures and suggest that an alteration in the lipid/protein oxidation, membrane fluidity, and Na(+),K(+)-ATPase activity may be correlated with neuronal excitability, a significant component of the secondary injury cascade that accompanies TBI.

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases.

Prostaglandin E2 (PGE2) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na+,K+‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE2 decreases Na+,K+‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE2. Na+,K+‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE2 (0.1–10 μM) for 30 min decreased Na+,K+‐ATPase activity in a concentration‐dependent manner. However, PGE2 did not alter Na+,K+‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE2 on Na+,K+‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na+,K+‐ATPase. We found that the inhibitory effect of PGE2 (1 μM) on Na+,K+‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE2‐induced decrease of Na+,K+‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE2. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE2‐induced decrease of Na+,K+‐ATPase activity. Accordingly, PGE2 increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE2 decreases Na+,K+‐ATPase activity in vivo. The results presented here imply Na+,K+‐ATPase as a target for PGE2‐mediated signaling, which may underlie PGE2‐induced increase of brain excitability.

Differential effects of atorvastatin treatment and withdrawal on pentylenetetrazol‐induced seizures

Purpose:  Statins are selective inhibitors of 3‐hydroxyl‐3‐methyl‐glutaryl coenzyme A (HMG‐CoA) reductase, the rate‐limiting enzyme of the mevalonate pathway for cholesterol biosynthesis. Increasing evidence indicates that statins, particularly atorvastatin, are neuroprotective in several conditions, including stroke, cerebral ischemia, traumatic brain injury, and excitotoxic amino acid exposure. However, only a few studies have investigated whether statins modulate seizure activity. In the current study we investigated whether atorvastatin or simvastatin alters the seizures induced by pentylenetetrazol (PTZ), a classical convulsant.

Modulation of pentylenetetrazol-induced seizures by prostaglandin E2 receptors

There is evidence that prostaglandin E2 (PGE2) facilitates the seizures induced by pentylenetetrazol (PTZ), but the role of PGE2 receptors (EPs) in the development of seizures has not been evaluated to date. In the current study we investigated whether selective EP ligands alter PTZ-induced seizures in adult male Wistar rats by electrographic methods. Selective antagonists for EP1 (SC-19220, 10 nmol, i.c.v.), EP3 (L-826266, 1 nmol, i.c.v.) and EP4 (L-161982, 750 pmol, i.c.v.) receptors, and the selective EP2 agonist butaprost (100 pmol, i.c.v.) increased the latency for clonic and generalized tonic-clonic seizures induced by PTZ. These data constitute pharmacological evidence supporting a role for EPs in the seizures induced by PTZ. Although more studies are necessary to fully evaluate the anticonvulsant role these compounds and their use in the clinics, EP ligands may represent new targets for drug development for convulsive disorders.

Prostaglandin E 2 potentiates methylmalonate-induced seizures

Purpose:  Methylmalonic acidemias are inherited metabolic disorders characterized by methylmalonate (MMA) accumulation and neurologic dysfunction, including seizures. It is known that metabolic crises in affected patients are precipitated by infections. Although growing evidence supports that inflammation facilitates seizures, it is not known whether inflammatory mediators facilitate MMA‐induced seizures. Therefore, in this study we investigate the involvement of cyclooxygenase‐2 (COX‐2) and prostaglandin E2 (PGE2) in MMA‐induced seizures.

Creatine increases hippocampal Na+,K+-ATPase activity via NMDA–calcineurin pathway

Achievements made over the past few years have demonstrated the important role of the creatine and phosphocreatine system in the buffering and transport of high-energy phosphates into the brain; however, the non-energetic processes elicited by this guanidine compound in the hippocampus are still poorly understood. In the present study we disclosed that the incubation of rat hippocampal slices with creatine (10mM) for 30 min increased Na(+),K(+)-ATPase activity. In addition, intrahippocampal injection of creatine (5 nmol/site) also increased the above-mentioned activity. The incubation of hippocampal slices with N-methyl-d-aspartate (NMDA; MK-801, 10 μM) and NMDA Receptor 2B (NR2B; ifenprodil, 3 μM) antagonists but not with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate antagonist (DNQX, 10 μM) and nitric oxide synthase inhibitor (NOS; l-NAME, 100 μM), blunted the effect of creatine on Na(+),K(+)-ATPase activity. Furthermore, the calcineurin inhibitor (cyclosporine A, 200 nM) as well as the Protein Kinase C (PMA, 100 nM) and Protein Kinase A (8-Br-cAMP, 30 μM) activators attenuated the creatine-induced increase of Na(+),K(+)-ATPase activity. In addition, the incubation of hippocampal slices with creatine (10mM) for 30 min increased calcineurin activity. The results presented here suggest that creatine increases Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway, proposing an putative underlying non-energetic role of this guanidine compound. However, more studies are needed to assess the contribution of this putative alternative role in neurological diseases that present decreased Na(+),K(+)-ATPase activity.