BRAIN RESEARCH

E L S E V I E R Brain Research 717 (1996) 184-188

Short communication

o -Lipoic acid protects against reperfusion injury following cerebral ischemia in rats

Manas Panigrahi b, Yarasi Sadguna a, Bangalore R. Shivakumar a, Sastry V.R. Kolluri b, Sashwati Roy c, Lester Packer c, Vijayalakshmi Ravindranath a.*

a Department of Neurochemisto', National Institute of Mental Health and Neurosciences, Bangalore 560 029, butia b Department of Neurosurgeo', National Institute of Mental Health and Neuroseiences, Bangalore 560 029, India

c Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA

Accepted 20 December 1995

Abstract

Ischemic-reperfusion injury in humans occurs in conditions such as stroke, cardiac arrest, subarachnoid hemorrhage or head trauma. Maximal tissue damage is observed during reperfusion, which is primarily attributed to oxidative injury resulting from production of oxygen free radicals. One of the major consequences of such damage is the depletion of the cellular antioxidant, glutathione (GSH) leading to oxidation of protein thiols to disulfides and the loss of activity of critical enzymes having active thiol group(s). Thus, the maintenance of thiol homeostasis is an important factor in cell survival. The effect of thiol antioxidants like c~-lipoic acid and the isopropyl ester of GSH was examined on the morbidity and mortality of rats subjected to reperfusion following cerebral ischemia induced by bilateral carotid artery occlusion and hypotension. While the GSH isopropyl ester had no significant protective effect; after pretreatment of rats, c~-lipoic acid was detected in the rat brain and it dramatically reduced the mortality rate from 78% to 26% during 24 h of reperfusion. The natural thiol antioxidant, c~-lipoic acid is effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia.

Keywords: Cerebral ischemia; Reperfusion; Brain; Oxidative stress; Glutathione; o~-Lipoic acid

Reactive oxy-free radicals are formed during ischemia- reperfusion injury of the brain [5,18]. The source of oxy- free radicals may include stimulation of the xanthine- xanthine oxidase system in the cerebral vessels [2], elec- tron leakage and redox state alterations of electron trans- port components of the ubiquinone-ubiquinol-oxidoreduc- tase in the mitochondria, arachidonic acid metabolism or release of excitatory amino acids [1]. The oxy-free radicals formed can initiate lipid peroxidation [6] and mediate oxidative damage of cellular proteins.

The inhibition of free radical production and lipid per- oxidation using polyethylene glycol conjugated-superoxide dismutase and catalase [9], allopurinol [10], dimethyltb- iourea [10] or desferrioxamine [4,12] have been examined as potential therapeutic agents to attenuate reperfusion

* Corresponding author. Dept. of Neurochemistry, NIMHANS, Hosur Road, Bangalore, 560 029, India. Fax: (91)(80)663-1830.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0006-8993(96)00009-1

injury following cerebral ischemia, but with limited suc- cess.

Among the various animal models, reduction of mean arterial blood pressure to 50 mm Hg (systemic hypoten- sion) combined with bilateral carotid artery occlusion in the rat has been used as model for severe ischemia [20]. In this model of iscbemia, cerebral blood flow is reduced to 5% of the normal and it produces consistent damage leading to high mortality rate (greater than 70%) among the animals, within 24 h of reperfusion. This model has been used in our laboratory for the study of the glutathione (GSH) and protein thiol (PrSH) homeostasis in brain re- gions during reperfusion following severe ischemia [19].

Earlier studies from our laboratory [19] and others [17] have demonstrated that reperfusion injury following cere- bral ischemia in the rat results in loss of GSH. The depletion of GSH is incidentally accompanied by increase in the levels of thiobarbituric acid reactive substances (TBARS) and reactive oxygen species. However, the ma-

M. Panigrahi et al. / Brain Research 717 (1996) 184-188 185

jor consequence of GSH loss during oxidative stress in the brain is the formation of protein glutathione mixed disul- fides (PrSSG) and loss of protein thiols [15]. This is unlike the liver or lung, wherein the depleted GSH is essentially converted to GSSG, which is effiuxed out of the cell preventing modification of protein thiols [16]. The loss of GSH and formation of PrSSG in the brain results in dysfunction of various membrane functions in cells, such as inhibition of Na+K+-ATPase activity and mitochondrial enzyme activities. Thus, rapid restoration or maintenance of thiol homeostasis in the brain during reperfusion may help the brain to recover from ischemia-reperfusion injury. Therefore, the effect of two thiol antioxidants, c~-lipoic acid (lipoate) and GSH isopropyl ester, on the mortality of rats during reperfusion following ischemia was examined.

Lipoamide functions as a cofactor in the multienzyme complexes that catalyze the decarboxylation of c~-keto acids, such as pyruvate, c~-ketoglutarate and branched chain c~-keto acids. More recently, the antioxidant func- tions of lipoate and its reduced form dihydrolipoic acid (DHLA) have been recognized [1 l].

Lipoate was a kind gift from Dr. H.J. Tritschler of ASTA Medica, Frankfurt, Germany. Glutathione isopropyl ester was obtained from Dr. K. Noguchi of Yamanouchi Pharmaceutical Co. Ltd., Japan. All other chemicals were obtained from Sigma Chemical Co., USA or from Quali- gens, India.

Male Sprague-Dawley rats (aged 3 -4 months and weighing 200 to 250 g) were obtained from the Central Animal Research Facility of the National Institute of Men- tal Health and Neurosciences and used for all experiments. Animals had free access to pelletted diet (Lipton India Ltd., Calcutta, India) and water, ad libitum.

Anesthesia was induced with ether and maintained with halothane and ether mixture in 70% nitrous oxide and 30% oxygen using Boyle's apparatus. A catheter was introduced into the femoral artery for monitoring blood pressure (Dynascope 100) and for withdrawal of blood. The saphe- nous vein was used for blood infusion. Body temperature was maintained at 37°C by external heating. Common carotid arteries were exposed through a midline skin inci- sion. Cerebral ischemia was produced by the occlusion of both the common carotid arteries using aneurysm clips and withdrawal of blood (5-6 ml) to decrease the mean arterial pressure (MAP) to 50 mm Hg for 30 min duration [20]. At the end of the ischemic period, carotid arteries were declamped and the phlebotomized blood was reinfused into the animals and reperfusion was allowed. Sham oper- ated controls were used in all experiments. The pCO2 and pO 2 levels were monitored before and after the induction of ischemia.

The drugs, c~-lipoate or GSH isopropyl ester were administered intravenously (through the saphenous vein) to two different groups of animals just before initiation of ischemia at a dosage of 25 mg of c~-lipoate/kg body weight or 100 mg of glutathione isopropyl ester /kg body

weight in 1 ml of sodium bicarbonate (7.5% w/v) . The control group received vehicle alone. The animals were grouped as follows: Group 1-bilateral carotid artery occlu- sion with hypotension; Group 2-c~-lipoate pretreated rats; Group 3-glutathione isopropyl ester pretreated and Group 4-sham operated controls.

All animals which survived for 24 h were anesthetized with ether and perfused transcardially with cold normal saline (0.9%, w / v ) to remove blood from the cerebral tissue. Animals were decapitated and the brain was dis- sected into different regions (cortex, hippocampus and striatum). The brain regions were flash frozen in liquid nitrogen and processed immediately. The samples were weighed and homogenized in 10 vols. sulfosalicylic acid (0.5%, w / v ) containing 1 mM EDTA. An aliquot of the acid supernatant was used for the assay of total GSH using the method described by Tietze [21].

In order to determine the uptake of c~-lipoate into the brain, the drug was administered intravenously (through the saphenous vein) to rats at a dosage of 25 m g / k g body weight in 1 ml of sodium bicarbonate (7.5%, w/v) . Con- trol animals received vehicle alone. Animals were sacri- ficed 1 h and 24 h after administration of c~-lipoate. The animals were anaesthetized with ether and perfused tran- scardially with ice-cold normal saline (30 ml), prior to decapitation and removal of the brain. The cortex was dissected out and flash frozen in liquid nitrogen. The cortex was weighed and homogenized for 1 min on ice with 0.4 ml of a mixture of water/methanol/acetonitrile ( 5 5 / 2 0 / 2 5 ) containing monochloroacetic acid (1%, v /v ) . The mixture was centrifuged and analyzed by HPLC using an electrochemical detector with a gold mercury electrode [7]. A microsorb C-18 column (3 /xM, Rainin Instruments) was used. The mobile phase consisted of water /methanol / acetonitrile ( 5 5 / 2 0 / 2 5 ) containing monochloroacetic acid (1%, v / v ) and the flow rate was 1 ml /min . Animals were similarly treated with c~-lipoic acid and sacrificed after 1, 3 and 24 h and the brain regions were dissected and ana- lyzed for GSH content as described above.

All analyses were carried out in duplicate. The test of significance was carried out using Chi-square test, Student's t-test or ANOVA followed by 'S-N-K' test (as appropriate) and the values were considered to be statisti- cally different from controls, if P < 0.05.

Post-ischemic recovery was studied in all the groups of animals. The behavior of the animals in Group 1 and 3 were similar. They usually remained hypoactive. Those animals which survived for more than 6 -8 h adopted a 'hunchback' posture and walked with extended paws. The animals were hyperexcitable and reacted to sound or han- dling with forceful jerks. Animals belonging to Group 2 (c~-lipoic acid pretreated) were more active and there was no incidence of seizures. They were also less excitable than other groups.

The mean pO 2 levels, before and after the onset of ischemia were 103 _+ 8.2 and 106.3 __ 25.6 mm Hg, respec-

186 M. Panigrahi et al. / Brain Research 717 (1996) 184-188

100

5o _J

0

_A A

o o

J

i i i i i I

4 8 12 16 20 24

REPERFUSION TINE ( h r )

Fig. 1. Mortality rate in different groups of animals during 24 h reperfusion following bilateral carotid artery occlusion with hypotension for 30 min. Rats were pretreated with vehicle alone ( • - • ), oz-lipoic acid (O -Q )o r glutathione isopropyl ester (O-O) , as described in 'Methods'. Asterisk indicates values significantly (P < 0.001) different from vehicle-treated controls.

tively whereas pCO 2 levels before and after the onset of ischemia were 37.2 _+ 9.0 and 36.8 _+ 8.2 mm Hg, respec- tively. The mean blood glucose level was 109 m g / 100 ml (data not shown).

During 24 h of reperfusion (Fig. 1), the number of animals surviving in Group 1 were four out of a total of nineteen animals (79% mortality), in Group 2, nine out of twelve animals (25%) and three out of ten animals sur- vived in Group 3 (70% mortality). Experiments (Group 1 and 2) were repeated with larger number of animals. In the animals subjected to ischemia and pretreated with vehicle alone (Group 1), only seven out of thirty-two rats survived (78% mortality), whereas seventeen animals out of twenty three survived in the a-lipoate pretreated rats (Group 2, 26% mortality).

I-1CONTROL

2 . 5 [ ] ISCHEMIA

• ISCHEMIA + ALA

~ 1.0

Q.5

0 C T H P S T

Fig. 2. Glutathione levels in rat brain regions after reperfusion tbr 24 h following 30 min ischemia. Rats were subjected to bilateral carotid artery occlusion with hypotension for 30 rain followed by reperfusion for 24 h ( [ ] ) . Rats were also treated with o~-lipoic acid prior to onset of ischemia ( • ) . Sham operated controls (blank bars, []) were incorporated during experimentation. Data are expressed as percent of control levels. GSH levels in control cortex (CT), striatum (ST) and hippocampus (HP) were 1.45_+0.12, 1.46+0.17, and 1.44_+0.09 /zmol/g tissue, respectively. Values are mean + S.D. (n = 3). Asterisks indicate values that are signifi- cantly different from controls (* P < 0.05).

GSH levels were assayed in the brain regions of rats that survived 24 h of reperfusion following ischemia (Fig. 2). In vehicle treated animals subjected to ischemia/reper- fusion, the GSH levels in cortex, striatum and hippocam- pus were typically 50.5%, 43.3% and 44% of the corre- sponding sham-operated controls. However, in animals that were pretreated with o~-lipoate, the GSH levels were 89%, 92% and 88% of the corresponding controls in cortex, striatum and hippocampus, respectively. This was accompanied by decrease in the levels of thiobarbituric acid reactants (TBARS). The TBARS levels in brain re- gions of animals subjected to ischemia-reperfusion injury for 24 hr without any pretreatment were 293%, 292% and 386% of controls in the cortex, striatum and hippocampus, respectively. In animals treated with o~-lipoate and sub- jected to ischemic reperfusion injury, TBARS levels were 148%, 176% and 156% of controls in cortex, striatum and hippocampus, respectively (data not shown).

o~-Lipoate was not detected in brain samples of animals that received vehicle alone. In the cortex of animals sacri- ficed 1 h after administration of the drug, the mean (S.D.) o~-lipoate levels was 2.14 (0.77) nmol /g wet tissue. The c~-lipoate levels in animals sacrificed 24 h later was 0.5 (0.44) nmol /g wet tissue (data not shown). In the animals which were administered with o~-lipoate only (without ischemia) GSH levels showed a small but significant ele- vation (11% increase over controls) in the cortex, 3 h after the i.v. injection. At the end of 24 h after administration of o~-lipoate, the GSH levels in the cortex were not signifi- cantly different from controls. A small but significant increase (10.4, 12.9 and 9.4% respectively)in the GSH levels was seen in the hippocampus at 1, 3 and 24 h after the administration of c~-lipoate, while no significant change was observed in the GSH levels in the striatum (data not shown).

o~-Lipoic acid is a naturally occurring antioxidant which

M. Panigrahi et al. / Brain Research 717 (1996) 184-188 187

can be readily absorbed from the diet. It has the capacity of reaching both the aqueous and lipid environments of cells [13]. Pretreatment with a single dose of a-lipoate dramatically reduced the mortality of rats subjected to reperfusion injury following cerebral ischemia, from 78% to 26%. This was accompanied by attenuation of brain GSH loss. The present study demonstrates for the first time that ce-lipoate administeied intravenously is able to cross the b lood-bra in barrier and is detectable in the brain. Since the administration of a-lipoic acid has no detectable toxicological side effects, this makes c~-lipoic acid an attractive antioxidant.

Earlier studies have demonstrated that administration of DHLA but not a-lipoic acid reduced the size of the infarct produced after middle cerebral artery occlusion in mice [14]. In a recent study, adult gerbils were treated with a-lipoic acid twice a day for 7 days and thereafter fore- brain ischemia was induced for 5 min. The prolonged pretreatment with c~-lipoic acid effectively protected the animals against ischemia injury as observed by locomotor activity and morphological examination of CA1 hippocam- pal pyramidal neurons after 5 days of recovery [3]. How- ever, the present study demonstrates that a-lipoic acid, the oxidized form, administered as single dose intravenously, offers effective protection against reperfusion injury fol- lowing cerebral ischemia in rats. This observation has potential therapeutic applications, a-Lipoic acid could be used as a neuroprotectant prior to procedures that place the brain at risk, such as by-pass surgery.

Decrease in GSH levels as a result of post-ischemic reperfusion in cortex, striatum and hippocampus of ani- mals treated with vehicle alone compared to the sham operated animals are in accordance with our earlier obser- vation where reperfusion injury following cerebral is- chemia in this animal model resulted in loss of GSH [19]. c~-Lipoic acid appeared to prevent the ischemia/reperfu- sion induced loss of GSH from cortex, striatum and hip- pocampus of brain. The protective effect of c~-lipoic acid in the present study could be due to (i) its free radical (e.g. hydroxyl, peroxyl or singlet oxygen) scavenging capacity; (ii) its competition for free transition metals as a chelator and /o r ; (iii) augmentation of the intracellular thiol (e.g., GSH) pool. Thiols like 2-mercaptoethanol and N-acetyl- L-cysteine have also shown to increase neural cell survival rate [8]. However, administration of GSH-isopropyl ester had no effect on the mortality rates during reperfusion, in contrast to the dramatic effects of a-lipoic acid. This may probably be due to inadequate levels of GSH-isopropyl ester reaching the brain. GSH levels were not significantly increased in the brain following administration of the ester (Ravindranath, unpublished observations).

The remarkable protective effects of ce-lipoic acid ob- served in the present study need to be substantiated in other animal models as it may offer a novel mode of attenuating reperfusion injury following cerebral ischemia.

Acknowledgements

The authors thank Dr. Klaus Wessel, ASTA Medica for helpful discussion.

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