A dual action of alpha-lipoic acid in the brain: an electrophysiological evaluation

Dupla ação do ácido alfa-lipoico no encéfalo: uma avaliação eletrofisiológica

Abstracts

Oxidative stress causes metabolic and structural abnormalities during reperfusion. In an animal model of electrophysiological evaluation of cerebral ischemia-reperfusion, alpha-lipoic acid effect on the oxidative stress was studied by mean absolute amplitude of EEG spectra evaluation. The left carotideal infusion of 3.03 mM alpha-lipoic acid in Wistar rats after cerebral ischemia and reperfusion caused initial reduction and partial final recuperation of the various EEG spectral frequency mean absolute amplitudes (p<0.05). The left intracarotideal infusion of 6.06 mM alpha-lipoic acid significantly reverted the induced depression of mean absolute amplitude of theta and delta spectra. Nevertheless there was an increasing pattern of ischemia demonstrated by mean absolute amplitude depression of almost all EEG spectra with 60.6 mM alpha-lipoic acid infusion. These observations suggest that, depending on the administered concentration, alpha-lipoic acid may act in a dual way, protecting from ischemia at lower concentrations and worsening this process at higher doses.

alpha-lipoic acid; anti-oxidant; pro-oxidant; cerebral ischemia; electroencephalogram (EEG); spectral mean absolute amplitude

Estresse oxidativo causa anormalidades metabólicas e estruturais durante reperfusão após isquemia. Em um modelo animal de avaliação eletrofisiológica de isquemia-reperfusão, o efeito da infusão do ácido alfa-lipóico sobre o estresse oxidativo foi estudado por meio da avaliação da média das amplitudes absolutas dos espectros eletrencefalográficos. A infusão intracarotídea esquerda de ácido alfa-lipoic 3,03 mM em ratos Wistar após isquemia-reperfusão cerebral causou significante redução inicial e recuperação parcial final da média das amplitudes absolutas dos vários espectros eletro - encefalográficos (p<0,05). A infusão intracarotídea esquerda de ácido alfa-lipoic 6,06 mM significantemente reverteu a depressão induzida da média das amplitudes absolutas médias dos espectros teta e delta. Houve, contudo, maior depressão da média das amplitudes absolutas de quase todos os espctros com infusão de ácido alfa-lipóico 60,6 mM. Estas observações sugerem que, dependendo da concentração administrada, o ácido alfa-lipoic tem duplo efeito, protegendo da isquemia em baixas concentrações e piorando este processo em doses altas.

ácido alfa-lipóico; anti-oxidante; pró-oxidante; isquemia cerebral; eletrencefalograma (EEG); amplitude absoluta média espectral

A dual action of a-lipoic acid in the brain: an electrophysiological evaluation

Dupla ação do ácido a-lipoico no encéfalo: uma avaliação eletrofisiológica

Otoni Cardoso do Vale^I; Daniel Sá Roriz Fonteles^II; Francisco Romero Cabral^III; Manassés Claudino Fonteles^IV

Clinical Research Unit of the University Hospital Walter Cantidio, Department of Pharmacology and Clinical Medicine, Federal University of Ceará and Ceará State University Fortaleza CE, Brazil

^IProfessor Adjunto de Neurologia e pesquisador, Departamento de Medicina Clínica, Faculdade de Medicina, Universidade Federal do Ceará

^IIPsicólogo e bolsista

^IIIFarmacêutico-Bioquímico, bolsista

^IVProfessor titular de Farmacologia e Reitor da Universidade Estadual do Ceará, pesquisador

ABSTRACT

Oxidative stress causes metabolic and structural abnormalities during reperfusion. In an animal model of electrophysiological evaluation of cerebral ischemia-reperfusion, a-lipoic acid effect on the oxidative stress was studied by mean absolute amplitude of EEG spectra evaluation. The left carotideal infusion of 3.03 mM a-lipoic acid in Wistar rats after cerebral ischemia and reperfusion caused initial reduction and partial final recuperation of the various EEG spectral frequency mean absolute amplitudes (p<0.05). The left intracarotideal infusion of 6.06 mM a-lipoic acid significantly reverted the induced depression of mean absolute amplitude of theta and delta spectra. Nevertheless there was an increasing pattern of ischemia demonstrated by mean absolute amplitude depression of almost all EEG spectra with 60.6 mM a-lipoic acid infusion. These observations suggest that, depending on the administered concentration, a-lipoic acid may act in a dual way, protecting from ischemia at lower concentrations and worsening this process at higher doses.

Key words: a-lipoic acid, anti-oxidant, pro-oxidant, cerebral ischemia, electroencephalogram (EEG), spectral mean absolute amplitude.

RESUMO

Estresse oxidativo causa anormalidades metabólicas e estruturais durante reperfusão após isquemia. Em um modelo animal de avaliação eletrofisiológica de isquemia-reperfusão, o efeito da infusão do ácido a-lipóico sobre o estresse oxidativo foi estudado por meio da avaliação da média das amplitudes absolutas dos espectros eletrencefalográficos. A infusão intracarotídea esquerda de ácido a-lipoic 3,03 mM em ratos Wistar após isquemia-reperfusão cerebral causou significante redução inicial e recuperação parcial final da média das amplitudes absolutas dos vários espectros eletro - encefalográficos (p<0,05). A infusão intracarotídea esquerda de ácido a-lipoic 6,06 mM significantemente reverteu a depressão induzida da média das amplitudes absolutas médias dos espectros teta e delta. Houve, contudo, maior depressão da média das amplitudes absolutas de quase todos os espctros com infusão de ácido a-lipóico 60,6 mM. Estas observações sugerem que, dependendo da concentração administrada, o ácido a-lipoic tem duplo efeito, protegendo da isquemia em baixas concentrações e piorando este processo em doses altas.

Palavras-chave: ácido a-lipóico, anti-oxidante, pró-oxidante, isquemia cerebral, eletrencefalograma (EEG), amplitude absoluta média espectral.

We have previously studied the electroencephalographic effects on mean absolute amplitudes of electroencephalogram (EEG) waves in Wistar rats submitted to global cerebral ischemia due to common carotid arteries ligation, arterial hypotension and reperfusion¹. There are only few experimental reports in the literature concerning the value of the EEG as a marker of induced cerebral ischemia in animals^2-4. According to Siesjö and Siesjö⁵ two mechanisms are responsible for secondary cerebral involvement after brain ischemia. The first mechanism involves cellular signal transduction modifications from glutamate and growth factor receptors stimulation with intracellular signal modulation, deleterious calcium enzyme activation and genetic protein synthesis expression abnormalities⁶. The second one is related to oxygen supply during reperfusion and involves oxygen reactive species and free radicals production. Oxygen reactive species and free radicals could structurally compromise various macromolecules by oxidation and reduction reactions causing, among others, DNA strand braking⁷, with products formation such as 8-hydroxy-2'-deoxyguanosine⁸, lipid peroxydation products^9,10 and carbonyl group formation¹¹.

Otherwise, one of the most important aims in the treatment of cerebral ischemia, during the acute phase, is to minimize or even avoid oxygen reactive species structural lesions, and consequently broaden the therapeutic window in order to implement thrombolytic therapy. Although without beneficial clinical evidence, many experimental procedures suggest that pharmacological entities, blocking cytotoxic cascade events during ischemic process or acting as free radical or reactive oxygen species scavengers, could reduce infarct volume due to focal ischemia or late effects of global ischemia with reperfusion.

Thioctic acid, 1,2-dithiolane-3-pentanoic acid or a-lipoic acid and its reduced form, di-hydrolipoic acid, are natural components of biologic membranes, acting as mytochondrial lipoamid co-factor of a-ceto-acid de-hydrogenase^12,13. Its participation in oxy-reduction state from external sources has been focused in various recent studies. It is easily absorbed and taken by cells, being finally reduced to di-hydrolipoic acid with participation of the systems NADH and NADPH¹⁴. The mitochondria are the main site of a-lipoic acid anti-oxidant action. Within the intracellular space the di-hydrolipoic is produced from a-lipoic acid and acts as anti-oxidant in the extracellular space¹⁵.

The a-lipoic acid also increases glutathione intracellular levels^16,17, and as a result, it could be an important component of the therapeutic arsenal of cerebral vascular ischemic diseases. Other a-lipoic acid and di-hydrolipoic acid actions in ischemia and reperfusion have been described. Ion Fe⁺² chelation by di-hydrolipoic acid¹⁸, and nitric oxide¹⁹ and other oxygen reactive species scavenging, as well as E vitamin and other anti-oxidant regeneration²⁰ are important a-lipoic acid roles. Using our previous idealized model¹, we decided on studying the electrophysiological effects of intracarotideal a-lipoic acid infusion.

METHOD

Male Wistar rats, weighing 300-500g at the time of surgery were used in the present study. The animals were fasted with free access to water during 24 hours before the experimental procedure. Three paired groups of animals (n=6, each paired group) were used in the first part of this study. Intracarotid a-lipoic acid infusion (different concentrations: 3.03 mM, 6.06 mM and 60.6 mM) started 30 minutes after both common carotid arteries occlusion. The animals of group 1 were infused with 3.03 mM a-lipoic acid, the animals of group 2 with 6.06 mM a-lipoic acid. The animals of group 3 received 60.6 mM a-lipoic acid infusion. In the second parf of this study, three other groups of six animals each were studied in order to determine brain tissue reduced glutathione (GSH) contents by spectrophotometry: one made of rats with both common carotid arteries occlusion, one with both common carotid arteries occlusion plus 6.06 mM a-lipoic acid intracarotid infusion and the last one with both common carotid arteries occlusion plus 60.6 mM a-lipoic acid intracarotid infusion. The mean weight of the animals was 339.3 ± 50.6 g. The weights of the animals of all studied groups were subject to Kolmogorov-Smirnov statitistical evaluation and considered as a normal distibution. Under intraperitoneal urethane anesthesia (1.5g/Kg), all the animals were submitted to surgical procedures that briefly consisted of cervical median incision with exposure of the trachea; identification and occlusion of the common carotid arteries; insertion and fixation of a polyethylene cannula (PE 50) into the left internal carotid artery in order to infuse a-lipoic acid solution; insertion and fixation of a polyethylene cannula into right internal carotid artery in order to record the intracarotideal blood pressure. The carotid blood pressure was monitored by using a Narco Bio-system physiograph. The right carotid cannula was connected to a P23 Statham transducer, being the carotideal blood pressure registered by a physiograph.

The animals were kept under continuous electrocardiographic and electroencephalographic monitoring. This system consisted of a preamplifier, an amplifier with adjustable time constants, an AD converter (12 bits), a 486 PC compatible microcomputer and Braintech^® software to study the spectral frequencies and the absolute amplitude average of the chosen events of the EEG. This software analyses the frequency components with precision of 0.35 Hz and uses epochs of 2.84 seconds. Subcutaneous electrodes were inserted in right frontal (F4), left frontal (F3), right parietal (P4), left parietal (P3), vertex (Cz) regions with reference to bilateral auricular (A1 and A2) surface electrodes. The ground electrode was subcutaneously inserted in the nose region. Surface electrodes kept on anterior paws were used for electrocardiographic monitoring. The technical details of the system are similar to those presented in a recent review²¹. The electroencephalographic and electrocardiographic activities of each animal were monitored during about 30 minutes before and 30 minutes after the beginning of a-lipoic acid infusion into distal segment of left common carotid artery. The right intracarotid blood pressure was monitored during all the experiments. Two minutes periods from each ten minutes of EEG and EKG monitoring were saved on hard disk for future analysis. The mathematical study of the average of the EEG spectral amplitudes alfa (8.09-12.66 Hz), beta₁ (13.01-19.69 Hz), beta₂ (20.84-26.02 Hz), beta₃ (26.37-32.70 Hz), theta (4.22-7.73 Hz) and delta (0.35-3.87 Hz) was done by choosing 2.84 s epochs (about 30 epochs from each animal group) of the saved EEG on hard disk. Epochs with artifacts were rejected. The rate of 3.03 mM a-lipoic acid infusion was 41.91 µl/100g/min.With 3.03 mM a-lipoic infusion, we considered initial, intermediate and final infusion periods and ANOVA was used to evaluate statistical significant differences (p<0.05) of the absolute amplitudes of the EEG main spectra. When the normality test failed, variance of repeated measures (Friedman) was considered.

Two other paired groups were included in this study: 6.06 mM a-lipoic acid infusion (45.87 µl/100g/min; n=6) and 60.6 mM a-lipoic acid infusion (44.84 µl/100g/min; n=6); paired t-test was used to evaluate statistical significance. We also performed spectrophotometric analysis of GSH in brain tissue fragments after both common carotid arteries occlusion, and after both common carotid arteries occlusion plus carotid infusion of a-lipoic (6.06 mM and 60.6 mM). The infusion of a-lipoic acid began 30 minutes after both common carotid arteries occlusion and it was done during 30 minutes.

Six cortical fragments from each rats were removed after craniectomy and kept on liquid nitrogen before their preparation for spectrophotometric analysis by modified Beutler et al.²² method. Briefly, brain tissue fragments were weighted, homogenized and put into tubes with 6% trichloroacetic acid (TCA) solution (50 mg of brain tussue into 1 ml of 6 % TCA). After centrifugation (4000 rpm), 500 µl of each tube float solution were added to 500 µl of 6 % TCA; 1 ml of 6 % TCA acted as a blank tube. After that, 4 ml of 4.3% NaHPO₄ solution plus 500 µl of 5,5-dithio (2´-nitrobenzoic) acid (DTNB) (40 mg of DTNB/100 ml of 1 % sodium citrate) were added to each tube. Each tube was analized by spectrophotometry at 412 nm of wavelength and the absorbance was registred. From previous determination of GSH standard curve, it was possible to know GSH tissue content in the brain fragments. The groups of the second part of this study were used to this analysis. The choice of those groups was based on the electrophysiological analysis of the first part of this study.

All the animals were euthanazed with lethal dosis of urethane after the end of the experiment.

RESULTS

We observed that intracarotideal 3.03 mM a-lipoic acid infusion induced initial reduction (phases I and II) and final (phase III) partial reversal of mean absolute amplitude EEG waves (Table 1, Fig 1, Fig 2 - Panel A). Many mean absolute amplitude differences of EEG spectra between paired sub-groups reflecting initial reduction (control and initial or intermediate a-lipoic infusion phase) and final reversal (intermediate and final a-lipoic infusion phase) were statistically significant (p<0,05). More details about such mean EEG spectra absolute amplitude differences are presented in Tables 1 and 2, and Fig 2 - Panel A. The least significant differences were observed with left and right parietal alpha, right parietal delta and left and parietal theta spectra.

This pattern of EEG spectra mean absolute amplitude variation with 3.03 mM a-lipoic acid intracarotideal infusion was not observed when we used other a-lipoic acid concentrations. With 6.06 mM a-lipoic acid infusion, there was a significant mean absolute amplitude increase (p<0.05) of right parietal delta, and left and right frontal theta spectra (Table 3, Fig 2B).

With 60.6 mM a-lipoic acid infusion, we observed a significant (p<0.05) mean absolute amplitude decrease of all EEG studied spectra with exception of right beta₃ and left frontal delta spectra (Table 4, Fig 3). The greatest statistical differences were found with right frontal (p=0.0024) and left parietal beta₂ (p=0.0064), right (p=0.0052) and left parietal beta₁ (p=0.0054), right frontal (p=0.0083) and left parietal alpha (p=0.0066), right frontal theta (p=0.0098), and right (p=0.0030) and left parietal delta spectra (p=0.0066).

There was a statistical significant increase of the brain tissue level of GSH evaluated by spectrophotometric method after infusion of 6.06 mM a-lipoic acid by intracarotid route. So, we found (0.372±0.021 µg of GSH/ml)/mg of brain tissue in rats with both common carotid arteries occlusion; in rats with both common carotid arteries occlusion plus 6.06 mM a-lipoic acid infusion, we found (0.444 µg of GSH/ml)/mg of brain tissue (p=0.024). With 60.6 mM a-lipoic acid infusion, the variations of GSH brain tissue contents were not significantly different (p>0.05).

DISCUSSION

In a previous observation, we demonstrated that computerized study of EEG spectral brain activity could be a valuable electrophysiological tool for evaluating drug cytoprotective action¹. We also demonstrated that intracarotid GSH infusion could significantly revert to the previous wave amplitude the wave depression imposed by ischemia² and this effect was atributed to its physiological protection against oxidative stress. This fact has driven us to study a-lipoic acid intracarotideal infusion in Wistar rats with bilateral carotid arteries occlusion as previously described.

Previous studies have demonstrated a-lipoic acid cytoprotective action against reperfusion injury in animal models of global^23,24 and focal²⁵ cerebral ischemia.

Our demonstrations that 3.03 mM a-lipoic acid infusion promotes initial reduction and final partial EEG spectra mean absolute amplitude recuperation, allowed us to speculate that, in this condition, this substance has an initial pro-oxidant and a late anti-oxidant action. As we can see in Tables 1 and 2 and in Fig 2 – Panel A, many amplitude reductions and some amplitude final augmentations (left frontal alpha, theta and delta) are statistically significant. Some anti-oxidants, in some circumstances, produce eventually more oxidative stress , acting as pro-oxidant. Di-hydro-lipoic acid (reduced form from a-lipoic acid) reduces Fe⁺⁺⁺ to Fe⁺⁺, activating Fenton reaction with OH^. and OH^- production as a pro-oxidant mechanism²⁶. The di-hydro-lipoic acid also originates sulfur free radicals, and so, compromises some proteins such as 1- antiproteinase, with previous ^.OH radicals generation²⁷. The exogenous a-lipoic acid is rapidly reduced to di-hydrolipoic acid; in mitochondria the enzyme lipoate de-hydrogenase induces reduction of a-lipoic acid using NADH as co-factor²⁸; in the cytosol the enzyme glutathione reductase actively participates of this reduction using NADPH as co-factor²⁹. We speculate that such a-lipoic acid reductions could act as a possible cytotoxic mechanism. Roy and colleagues³⁰ suggested that lipoic acid could reduce NADH levels, using it as a co-factor during its reduction stress; during oxidative stress, both a-lipoic acid and di-hydrolipoic acid act as free radicals scavengers. It is possible that, at least, in a-lipoic acid infusion initial phases, the cerebral metabolic conditions of our experimental model behaved as it was in reduction stress. The final oxidative stress justifies the observed a-lipoic acid anti-oxidant activity.

This pattern of pro and anti-oxidant effect was not observed when we used 6.06 mM a-lipoic acid infusion. There was a significant mean absolute amplitude increase of right parietal and left and right frontal theta EEG spectra. So, we suppose that this anti-oxidant effect is more prominent in generating theta activity structure (the hippocampus). We experimentally observed by spectrophotometric method a significant increase of GSH in Wistar rat cerebral parenchyma with both common carotid arteries occlusion after 6.06 mM a-lipoic acid intracarotideal infusion (data not yet published). The anti-oxidant effect of 6.06 mM a-lipoic acid in our experiments could be due to GSH regeneration, after its reduction to di-hydrolipoic acid. Di-hydro-lipoic acid could actively participate of GSH regeneration mechanisms according to the chemical reaction: di-hydro-lipoic acid + oxidized glutatione (GSSG)® reduced glutathione (2GSH) + a-lipoic acid²⁰.

With our present experimental model, the intracarotideal infusion of more concentrated (60.6 mM) a-lipoic acid induced significant mean absolute amplitude reduction of all EEG spectra in almost all studied regions (Fig 3, Table 4). Therefore our data suggest that intracarotideal direct infusion of 60.6 mM a-lipoic acid in rats with both common carotid arteries occlusion has pro-oxidant effect. This could result of a counter regulatory effect on GSH production. Sen and colleagues³¹, studying the intracellular thiol regulation by a-lipoic acid, verified that high concentrations of this acid produced cytotoxic lesions of stimulated mitogenic lymphocytes, in which thiol depletion and DNA fragmentation were detected. It was also verified that a-lipoic acid increases caspase-3 activation, having a role in cellular apoptosis³². Therefore, apoptotic events may be occurring with the higher concentrations used herein.

In the present experimental model high concentrations of a-lipoic acid was directly infused into the common carotid artery and rapidly gained access to cerebral parenchyma without systemic enzymatic reduction. So, the a-lipoic acid could be reduced directly in brain cells by glutathione reductase NADPH-dependent action with concomitant decreasing of GSH contents, and de-hydrogenase NADH-dependent action. Decreased contents of GSH could explain the pro-oxidant activity of higher concentrations of a-lipoic acid in our experimental model.

In a recent review Moini et al.³³ emphasized the antioxidant and prooxidant activities of a-lipoic and dihidrolipoic acid. According to these researchers, These are many evidences that both a-lipoic and dihidrolipoic acid exhibit direct free radical scavenging propreties and that there are only in vitro evidence of their prooxidant propreties. Our data suggest by first time the presence of this phenomena in an in vivo preparation induced by a-lipoic acid.

Acknowledgment – The authors would like to thank Dr. Domingos Barreto da Silva and Maria Sílvia Helena Freire de França by their valuable technical assistance.

Received 4 December 2002

Received in final form 26 March 2003

Accepted 10 April 2003

O presente estudo experimental foi financiado pelo CNPq

Dr.Otoni Cardoso do Vale - Rua Vicente Linhares 614/700 - 60135-270 Fortaleza CE - Brasil.

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Publication Dates

• Publication in this collection
  12 Nov 2003
• Date of issue
  Sept 2003

History

• Accepted
  10 Apr 2003
• Reviewed
  26 Mar 2003
• Received
  04 Dec 2002