ORIGINAL PAPER

Alpha Lipoic Acid Alleviates Oxidative Stress and PreservesBlood Brain Permeability in Rats with Subarachnoid Hemorrhage

Mehmet Ersahin • Hale Z. Toklu • Sule Cetinel •

Meral Yuksel • Can Erzik • M. Zafer Berkman •

Berrak C. Yegen • Goksel Sener

Accepted: 22 September 2009 / Published online: 13 October 2009

� Springer Science+Business Media, LLC 2009

Abstract The neuroprotective effect of alpha lipoic acid

(ALA; 100 mg/kg, po), a dithiol antioxidant, on experi-

mentally induced subarachnoid hemorrhage (SAH) was

assessed in Wistar albino rats. Neurological examination

scores recorded at the 48th h of SAH induction were

increased in SAH groups, which were accompanied with

significant increases in the formation of reactive oxygen

species, DNA fragmentation ratios, malondialdehyde levels

and myeloperoxidase activity, while significant decreases

in the brain glutathione content and Na?, K?-ATPase

activity were observed. On the other hand, ALA treatment

reversed all these biochemical indices as well as SAH-

induced histopathological alterations. Increased brain edema,

impaired blood-brain-barrier permeability and neurological

scores were also improved by ALA treatment. The results

demonstrate that ALA exerts neuroprotective effects via

the enhancement of endogenous antioxidant enzyme activity,

the inhibition of neutrophil accumulation and free radical

generation, suggesting a therapeutic potential in reducing

secondary injury after SAH in patients.

Keywords Alpha lipoic acid � Antioxidant �Cerebral vasospasm � Subarachnoid hemorrhage �Lipid peroxidation

Introduction

Despite sophisticated medical management and neurosur-

gical techniques, subarachnoid hemorrhage (SAH) from

ruptured intracranial aneurysm is associated with high

morbidity and mortality rates. Experimental and clinical

studies have shown that the acute symptoms of SAH are

attributed to increased intracranial pressure, decreased

cerebral perfusion pressure and the resulting ischemia [1,

2]. In the clinical setting, most deaths occur within hours

after onset of SAH [3], and those patients who survive the

initial hemorrhage and overcome vasospasms frequently

suffer from persistent cognitive deficits, psychosocial

impairments, and a decreased quality of life because of

acute brain injury [4].

A growing body of evidence suggests that the potential

vasoconstrictors released from activated platelets, as well as

from mechanically damaged erythrocytes, participate in the

acute vasospastic/ischemic response following SAH [5].

Experimental studies indicate that the generation of reactive

oxygen species (ROS) is one of the mechanisms playing a

crucial role in the acute brain injury which leads to the

delayed cerebral vasospasm following SAH [6–8] and that

M. Ersahin � M. Z. Berkman

Department of Neurosurgery, Haydarpasa Numune Education

and Research Hospital, Istanbul, Turkey

H. Z. Toklu � G. Sener (&)

Department of Pharmacology, School of Pharmacy, Marmara

University, Tıbbiye Cad., 34668 Istanbul, Turkey

e-mail: gsener@marmara.edu.tr; gokselsener@hotmail.com

S. Cetinel

Department of Histology & Embryology, School of Medicine,

Marmara University, Istanbul, Turkey

M. Yuksel

Vocational School of Health Related Professions, Marmara

University, Istanbul, Turkey

C. Erzik

Department of Medical Biology, School of Medicine, Marmara

University, Istanbul, Turkey

B. C. Yegen

Department of Physiology, School of Medicine, Marmara

University, Istanbul, Turkey

123

Neurochem Res (2010) 35:418–428

DOI 10.1007/s11064-009-0072-z

these radicals are casually related to the expression of cere-

bral ischemia [9, 10]. Thus, the use of free radical scavengers

in animal models of SAH [11, 12] and some clinical trials

[13, 14] have demonstrated beneficial effects of these agents

on ischemic neurological deficits due to SAH-induced

vasospasms. We have recently shown that melatonin, which

is a potent free radical scavenger, reversed SAH-induced

histopathological and biochemical alterations [15].

Alpha lipoic (ALA) acid, a dithiol antioxidant, is an

important co-factor in pyruvate dehydrogenase and

a-ketoglutarate dehydrogenase in the mitochondria [16].

The metabolic antioxidant lipoic acid is a low molecular

weight substance that is absorbed from the diet and crosses

the blood-brain barrier. Although the metabolic role of

ALA has been known for more than 50 years, its effects

when given exogenously have become known recently.

Several studies have provided evidence that ALA supple-

mentation decreases oxidative stress and restores reduced

levels of other antioxidants in vivo [17]. ALA influences a

number of cell processes, including direct radical scav-

enging, recycling of other antioxidants, accelerating glu-

tathione (GSH) synthesis, and modulating transcription

factor activity, especially that of nuclear factor (NF)-kappa

B, which may account for its dramatic effects in oxidative

stress conditions [18].

Several studies have shown that ALA exerts multiple

pharmacological actions that prevent nerve degeneration in

experimental in vitro models of diabetes [19], Parkinson

[20], and Alzheimer diseases [21]. Similarly, ALA has

been shown to exert a neuroprotective effect in several in

vivo models of neurologic injury, such as traumatic brain

injury [22], cerebral ischemia [23], seizures [24] and

autoimmune encephalomyelitis [25].

In the light of these findings, the aim of the current study

is to determine the effects of alpha lipoic acid on SAH-

induced oxidative brain injury and neurological symptoms

using biochemical, neurological and histopathological

approaches.

Materials and Methods

Animals and Experimental Design

All experimental protocols were approved by the Marmara

University Animal Care and Use Committee. Male Wistar

albino rats (300–350 g) were housed in a temperature-

controlled room (22 ± 2�C) with standardized light/dark

(12/12 h) cycles, where the relative humidity (65–70%)

was kept constant. Rats were fed with standard rat pellets

and tap water ad libitum.

Rats were divided into three groups as: (1) sham-

operated and vehicle-treated control group (n = 18), (2)

vehicle-treated SAH group (n = 18), (3) ALA (100 mg/kg/

day, po)-treated SAH group (n = 18). The active ingredi-

ent ALA (99% purity, (R)-a-lipoic acid) was supplied by

Mikrogen Pharmaceuticals (Istanbul, Turkey) and it was

dissolved in 10% DMSO (v/v in corn oil). Either ALA or

DMSO was administered through an orogastric tube

immediately after the induction of SAH and was continued

daily for 2 days.

At the 48th h of surgery, neurological examinations

were performed in all groups. Then, subgroups of the rats

in each group were decapitated to obtain brain tissue

samples for the biochemical analyses (n = 6), for Evans

blue assay and edema evaluation (n = 6). The remaining

rats (n = 6) in each group were given the fixative perfusion

for histological preparation and analysis.

Induction of SAH

In the present study, the rat basilar artery vasospasm model

was used according as previously described [26]. In the

anesthetized rats injected intraperitoneally (ip) with keta-

mine (100 mg/kg) and chlorpromazine (2 mg/kg), a small

incision was made in the area of the occipitocervical

junction to expose the atlanto-occipital membrane. After

the animals were placed on the stereotaxic frame, the cis-

terna magna was tapped with a 27-gauge needle, and

0.3 ml of cerebral spinal fluid was gently aspirated. Freshly

drawn blood (0.3 ml) taken from the femoral artery was

then injected aseptically into the cisterna magna within a

2-min period. Immediately after the injection of blood, the

puncture site was sealed with glue to prevent the formation

of a fistula. To permit blood distribution around the basal

arteries, each rat was tilted at an angle of 20� for 30 min

with its head lowered.

Neurological Examination

Since functional scoring is important in testing neuropro-

tective drugs, a simple set of commonly used neurological

tests were used to assess the normal and abnormal function

following SAH in rats. The neurological examination scores

were conducted according to Bederson’s modified neuro-

logical examination test [22, 27] by a ‘blinded’ investigator.

A twenty-point neuroscore was used to assess motor and

behavioral deficits. The sequence of testing animals by a

given task was randomized for the animals. Briefly, the

consciousness, performance in a smooth climbing platform,

extremity tonus, walking and postural reflexes, circling and

response to the nociceptive stimuli were assessed. For

walking and posture, rats were allowed to move about freely

on the floor, while they were observed. In the circling test,

the rats were held gently from the tail, suspended one meter

above the floor, and observed for forelimb flexion, where

Neurochem Res (2010) 35:418–428 419

123

normal rats are expected to extend both forelimbs toward

the floor. The rotation degree and time were measured.

Finally, the responses to the nociceptive stimuli were

assessed by tail-immersion test in 56�C water.

Evans Blue Assay for the Evaluation of Blood Brain

Barrier Permeability

To evaluate the blood brain barrier (BBB) integrity, Evans

blue dye (EB) was used as a marker of albumin extrava-

sation [22]. Briefly, EB (2% in saline, 4 ml/kg) was

injected via the jugular vein at the 48th h of the SAH

induction and it was allowed to remain in circulation for

30 min. Then, chests were opened and the rats were per-

fused transcardially with 250 ml of saline at a pressure of

110 mm Hg for approximately 15 min. After decapitation,

the brain was removed and dissected into cerebral cortex

and cerebellum, which were then weighed separately for

the quantitative measurement of EB-albumin extravasation.

Brain samples were homogenized in 2.5 ml phosphate-

buffered saline and mixed by vortexing for 2 min after the

addition of 2.5 ml of 60% trichloroacetic acid to precipitate

the protein. Samples were cooled and then centrifuged for

30 min at 1000g. The supernatant was measured at 620 nm

for the absorbance of EB using a spectrophotometer

(Shimadzu UV1208, Japan). EB was expressed as lg/mg of

brain tissue against a standard curve.

Evaluation of the Brain Edema

Brain edema was evaluated by the gravimetric method

based on the measurement of the water content of brain

[22]. The whole brain was weighed and then dried for

48 h at 100�C, afterwards re-weighed. The percentage of

water was calculated according to the following formula:

% H2O = [(wet weight - dry weight)/wet weight] 9 100.

Chemiluminescence (CL) Assay

To assess the role of reactive oxygen species in SAH-

induced brain damage, luminol and lucigenin chemilumi-

nescences were measured as indicators of radical forma-

tion. Lucigenin (bis-N-methylacridiniumnitrate) and

luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) were

obtained from Sigma (St Louis, MO, USA). Measurements

were made at room temperature using Junior LB 9509

luminometer (EG&G Berthold, Germany). Tissue samples

cut into small pieces were put into vials containing PBS-

HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH

7.2). ROM were quantitated after the addition of the

enhancers, lucigenin or luminol, for a final concentration

of 0.2 mM. Luminol detects a group of reactive species,

such as •OH, H2O2, HOCl radicals, while lucigenin is

selective for O2-. Counts were obtained at 1 min intervals

and the results were given as the area under curve (AUC)

for a counting period of 5 min. Counts was corrected for

wet tissue weight and expressed as relative light units (rlu)

per milligram of tissue [28].

DNA Fragmentation Assay

The percentage of DNA fragmentation in the brain tissue

was determined as an indicator of cell death, including

apoptosis. Brain tissue samples were homogenized in 10

volumes of a lysis buffer (5 mM Tris–HCL, 20 mM ethyl-

ene diamine tetraacetic acid [EDTA], 0.5% (v/v) t-octyl-

phenoxypolyethoxyethanol [Triton-X 100]; pH = 8.0).

Two separate samples of 1 ml were taken from the mucosal

samples and centrifuged at 25,000g for 30 min to separate

the intact chromatin in the pellet from the fragmented DNA

in the supernatant [29]. The supernatant was taken out to be

saved and the pellet was re-suspended in 1 ml of Tri-EDTA

buffer (pH = 8.0), 10 mM:1 mM, respectively. Both the

supernatant and the re-suspended pellet were assayed then

for the DNA content by diphenylamine reaction described

by Burton [30].

Malondialdehyde and Glutathione Assays

Brain samples were homogenized with ice-cold 150 mM

KCl for the determination of malondialdehyde (MDA) and

glutathione (GSH) levels, indicating lipid peroxidation and

intracellular antioxidant status, respectively. The MDA

levels were assayed for the products of lipid peroxidation

and results are expressed as nmol MDA/g tissue [31]. GSH

was determined by a spectrophotometric method based on

the use of Ellman’s reagent and results are expressed as

lmol GSH/g tissue [32].

Measurement of Myeloperoxidase Activity

Myeloperoxidase (MPO) activity, which is accepted as an

indicator of neutrophil infiltration in tissues, was measured

by a procedure similar to that described by Hillegas et al.

[33]. Brain tissue samples were homogenized in 50 mM

potassium phosphate buffer with a pH of 6.0, and centrifuged

at 41,400g for 10 min. The pellets were then suspended in

50 mM PB containing 0.5% hexadecyltrimethylammonium

bromide (HETAB). After three freeze and thaw-cycles, with

sonication between cycles, the samples were centrifuged at

41,400g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml

of reaction mixture containing 50 mM PB, o-dianisidine,

and 20 mM H2O2 solution. One unit of enzyme activity was

defined as the amount of MPO present that caused a change

in absorbance, measured at 460 nm for 3 min. MPO activity

was expressed as U/g tissue.

420 Neurochem Res (2010) 35:418–428

123

Na?, K?-ATPase Activity

Since the activity of Na?, K?-ATPase, a membrane-

bound enzyme required for cellular transport, is very

sensitive to free radical reactions and lipid peroxidation,

reductions in this activity can indicate membrane damage

indirectly. Measurement of Na?, K?-ATPase activity is

based on the measurement of inorganic phosphate

released by ATP hydrolysis during incubation of brain

homogenates with an appropriate medium containing

3 mM ATP as a substrate. The total ATPase activity was

determined in the presence of 100 mM NaCl, 5 mM KCl,

6 mM MgCl2, 0.1 mM EDTA, 30 mM Tris–HCl (pH

7.4), while the Mg2?-ATPase activity was determined in

the presence of 1 mM ouabain. The difference between

the total and the Mg2?-ATPase activities was taken as a

measure of the Na?, K?-ATPase activity [34]. The

reaction was initiated with the addition of the homogenate

(0.1 ml) and a 5-min preincubation period at 37�C was

allowed. Following the addition of Na2ATP and a 10-min

re-incubation period, the reaction was terminated by the

addition of ice-cold 6% perchloric acid. The mixture was

then centrifuged at 3500g, and Pi in the supernatant

fraction was determined by the method of Fiske and

Subarrow [35]. The specific activity of the enzyme was

expressed as nmol Pi mg-1 protein h-1. The protein

concentration of the supernatant was measured by the

Lowry method [36].

Histopathological Preparation and Analysis

Anesthetized (ip ketamine and chlorpromazine) rats were

perfused transcardially with a solution of 2.5% glutaral-

dehyde in 0.1 M PBS (pH 7.4). To obtain basilar artery

sections, anterior midline of the brainstem was removed.

For light microscopic analysis, perfused tissue specimens

were fixed in 10% formaldehyde, dehydrated in alcohol

series, clearing in toluene end embedding in paraffin.

Paraffin sections (5 lm) were stained with hematoxylin

and eosin (H&E) and examined under a photomicroscope

(Olympus BH 2, Tokyo, Japan).

For the electron microscopic evaluation, tissues

obtained from perfused rats were post-fixed with 1% OsO4,

dehydrated in routine alcohol series and embedded in

Epon-812 resin. Semi-thin (1 lm) sections, stained with

toluidine blue, and thin (60 nm) sections, stained with 1%

lead citrate and uranyl acetate, were examined under a

JEOL 5200 JSM (Japan) electron microscope.

Statistical Analysis

Statistical analysis was done using a GraphPad Prism 3.0

(GraphPad Software, San Diego; CA; USA). All data are

expressed as means ± SEM. Groups of data were com-

pared with an analysis of variance (ANOVA) followed by

Tukey’s multiple comparison tests. Values of P \ 0.05

were considered as significant.

Results

Neurological examination score recorded at the 48th h of

surgery was significantly higher in the vehicle-treated SAH

group when compared with the control group (P \ 0.001),

while the score was significantly reduced in the ALA-

treated SAH group (P \ 0.05; Table 1). Similarly, brain

water content and EB content were also significantly higher

than the control group, indicating brain edema and BBB

permeability (P \ 0.05 and P \ 0.001). On the other hand,

ALA treatment reduced both parameters.

Luminol and lucigenin CL in the brain tissues of the

vehicle-treated SAH group were significantly higher than

those of the control group (P \ 0.01 and P \ 0.05), indi-

cating enhanced generation of ROS in the tissue while

these increases were significantly suppressed by ALA

treatment (P \ 0.05; Fig. 1).

In the vehicle-treated SAH group, as compared with the

control group, the MDA level in the brain samples showed

a marked increase, indicating enhanced lipid peroxidation

(P \ 0.001), while ALA administration abolished the ele-

vation in the brain MDA level (Fig. 2a). In accordance

with that, SAH caused a significant depletion in brain GSH

Table 1 Neurological examination scores, brain water content, Evans blue (EB) content, luminol and lucigenin chemiluminescence (CL) values

in the brain tissues of rats in the vehicle- or ALA-treated sham-operated control (C) and subarachnoid hemorrhage (SAH) groups

C SAH

Vehicle-treated ALA-treated Vehicle-treated ALA-treated

Neurological score 1.1 ± 0.9 1.3 ± 0.3 8.33 ± 3.5*** 3.50 ± 2.8?

Brain water content (%) 77.5 ± 0.4 75.9 ± 0.3 81.0 ± 1.4* 78.7 ± 0.5

EB content (mg/g tissue) 2.0 ± 0.1 2.1 ± 0.2 3.0 ± 0.2*** 2.5 ± 0.1?

Each group consists of six rats

* P \ 0.05, *** P \ 0.001, compared to control group; ? P \ 0.05, compared to vehicle-treated SAH group

Neurochem Res (2010) 35:418–428 421

123

content when compared to control group (P \ 0.01), while

in the ALA-treated SAH group, brain GSH content was

found to be preserved (Fig. 2b).

In the brain samples of the vehicle-treated SAH group,

DNA fragmentation was significantly increased as com-

pared to the control group (P \ 0.001; Fig. 2c), while ALA

treatment abolished the SAH-induced increase in DNA

fragmentation.

Myeloperoxidase activity was significantly elevated in

the brain tissues of the vehicle-treated SAH group as

compared to that of the control group (P \ 0.001; Fig. 3a).

However, ALA treatment reversed the SAH induced

increase in brain MPO level to normal. On the other hand,

in the vehicle-treated SAH group Na?, K?-ATPase activity

was significantly reduced, implicating an impaired mem-

brane transport function, while the measured Na?, K?-

ATPase activity in the brain tissue of the ALA-treated SAH

group was not different than that of the control rats

(Fig. 3b).

Light microscopic evaluation revealed that basilar

arteries obtained from sham-operated control rats had a

regular alignment with endothelial cells and elastic lamina

(Fig. 4a), while in the vehicle-treated SAH group a

prominent vasospasm of the basilar arteries was clearly

demonstrated, and the continuity of both endothelium and

elastic lamina was distrupted, indicating a severe degen-

eration (Fig. 4b). In the basilar arteries of the rats treated

with ALA, arterial vasospasm was decreased providing the

integrity of the endothelium (Fig. 4c).

On transmission electron microscopic analysis, compared

to control (Fig. 5a) group, SAH injury in the vehicle-treated

animals (Fig. 5b) was observed as severe degeneration in

the endothelium, elastic lamina and connective tissue of the

basilar artery with intense edema and vacuolizations in the

cytoplasm of both endothelial and muscle cells. Elastic

lamina showed detachments and has lost its continuity in

most of the observed areas. The nuclei of both endothelial

0

10

20

30

**

+

C SAH

ALA-treated

vehicle-treated(a)

Lum

inol

CL

(rlu

/mg)

0

5

10

15

20

25 *(b)

C SAH

+

Luci

geni

n C

L (r

lu/m

g)

Fig. 1 a Luminol and b lucigenin chemiluminescence (CL) levels in

the vehicle- or ALA-treated sham-operated control (C) and subarach-

noid hemorrhage (SAH) groups. Each group consists of six rats.

Values are represented as mean ± SEM. * P \ 0.05, ** P \ 0.01,

compared to control group; ? P \ 0.05, compared to vehicle-treated

SAH group

0

10

20

30

40

50

60 ***

+++

(a)

C SAH

ALA-treated

vehicle-treated

MD

A (

nmol

/g)

0.0

0.5

1.0

1.5

2.0

**+

(b)

GS

H (

µmol

/g)

0.0

0.1

0.2 ***

++

(c)

DN

A fr

agm

enta

tion

%C SAH

C SAH

Fig. 2 a Malondialdehyde (MDA), b glutathione (GSH) levels, and

c DNA fragmentation ratio (%) in the vehicle- or ALA-treated sham-

operated control (C) and subarachnoid hemorrhage (SAH) groups.

Each group consists of six rats. Values are represented as mean ±

SEM. ** P \ 0.01, *** P \ 0.001, compared to control group;? P \ 0.05, ?? P \ 0.01, ??? P \ 0.001, compared to vehicle-

treated SAH group

422 Neurochem Res (2010) 35:418–428

123

and muscle cells were contracted due to the spasm of the

artery. In the ALA-treated SAH group, the injury was found

to be ameliorated (Fig. 5c), while the cytoplasmic vacuoli-

zations and edema were no longer seen and the contraction

of the basilar artery was reduced.

Discussion

The results of the present study demonstrate that alpha

lipoic acid with its free radical scavenging properties sig-

nificantly reduced formation of ROS in the brain tissue,

delimitated neutrophil accumulation, preserved antioxidant

GSH levels, decreased lipid peroxidation and neuronal

injury at the 48th h after SAH. Moreover, the marked

neurological deficit observed after SAH, which may have

resulted from edema formation and oxidative brain injury,

was significantly improved after the administration of

ALA, as BBB integrity was preserved.

The major consequence of SAH is delayed, persistent

vasospasm of intracranial conduit arteries, which results in

the disability of post-SAH patients. Dysfunction of the

endothelium resulting in enhanced production of vaso-

constrictors, phenotypic changes of the receptors in endo-

thelium and smooth muscle cells, increased sensitivity of

0

1

2

3

4

5

***

+++

(a)

ALA-treatedvehicle-treated

C SAH

MP

O a

ctiv

ity (

U/g

)

0

5

10

15

20(b)

*

C SAH

Na+

, K+-A

TP

ase

activ

ity( µ

mol

/mg/

h)

Fig. 3 a Myeloperoxidase (MPO), and b Na?, K?-ATPase activities in

the vehicle- or ALA-treated sham-operated control (C) and subarachnoid

hemorrhage (SAH) groups. * P \ 0.05, *** P \ 0.001, compared to

control group; ??? P \ 0.001, compared to vehicle-treated SAH group

Fig. 4 Light microscopy. a Control group; regular endothelium

(arrow) with elastic lamina (arrowhead) and nucleus of muscle cell

(*). b Vehicle-treated SAH group; arterial vasospasm with contracted

elastic lamina (arrowhead), nucleus of endothelium (arrow) note the

distrupted endothelium, interstitial edema (*) and the loss of elastic

lamina (curved arrow). c ALA-treated SAH group; the integrity was

provided in endothelium (arrowhead), nucleus of muscle cell (*) and

elastic lamina (arrow). HE staining, 9200, insets 9400

Neurochem Res (2010) 35:418–428 423

123

vascular smooth muscle cells to vasoconstrictors, release of

spasmogens from lysed blood clot and inflammatory

response of the vascular wall have been proposed as

pathological mechanisms participating in the development

of post-SAH spasm [1]. Although the causative factors

underlying the development of cerebral vasospasm are

poorly understood, it is well known that cerebrovascular

effects of ROS contribute to disease progression following

SAH [37]. Since SAH-related cerebral ischemia still has a

substantial impact on mortality while the prognosis of the

surviving patients remains poor, extensive experimental

and human studies are currently performed to aid in

designing advanced therapeutic strategies for better pro-

tection of the cerebral tissues. ALA and its reduced form,

dihydrolipoic acid (DHLA) are capable of quenching ROS

such as hydroxyl radicals, peroxyl radicals, superoxide,

hypochlorous acid and peroxynitrite and they prevent sin-

glet oxygen-induced DNA damage, exhibit chelating

activity, reduce lipid peroxidation and increase intracellu-

lar glutathione levels [17]. Therefore, ALA and DHLA

have the potency to improve SAH-induced oxidative

injury. Previously, Panigrafi et al. [38] have shown that the

natural thiol antioxidant ALA is effective in improving

survival and protecting the rat against reperfusion injury

following cerebral ischemia.

Although the major mechanism of action of ALA

appears to be due to its ability to substitute for glutathione,

ALA was shown to increase NO biosynthesis in cultured

endothelial cells and improve endothelial NO-dependent

vasomotor function in a variety of conditions [39, 40] via

amelioration of endothelial GSH status [41]. Moreover,

ALA was also shown to reduce plasma levels of IL-6 and

plasminogen activator inhibitor-1, suggesting that mecha-

nisms other than antioxidant effects may be involved [42].

ALA down-regulates the expression of redox-sensitive pro-

inflammatory proteins including TNF and inducible nitric

oxide synthase. As a naturally occurring cofactor for the

mitochondrial enzymes pyruvate dehydrogenase and alpha-

ketoglutarate dehydrogenase, ALA has been shown to have

a variety of properties which can interfere with the path-

ogenesis or progression of neurodegenerative diseases [43].

It was found that ALA inhibits alpha-ketoglutarate dehy-

drogenase-mediated generation of superoxide and hydro-

gen peroxide [44].

Various experimental studies demonstrated that disrup-

tion of the BBB is the primary cause of SAH-induced brain

edema [45–47]. Doczi et al. [48] have reported that early

cerebral edema, one of the major determinants of mortality

following SAH, develops due to BBB breakdown, while

increased BBB permeability has been correlated with

delayed cerebral ischemia and poor clinical outcome.

Ischemia of the brain tissue causes destruction of the

functional and structural integrity of the BBB, which is

responsible for regulating the transport of fluids and soluble

Fig. 5 Transmission electronmicroscopy: a1 Control group;

endothelial cell (arrowhead)

and elastic lamina (arrow) and

the nuclei of muscle cells (n).

b1 Vehicle-treated SAH group;

detachment of elastic lamina

(arrow), severe edema of

interstitium (white doubleheaded arrow) contracted nuclei

of muscle (**) and degeneration

of endothelial cell

(arrowheads). c1 ALA-treated

SAH group; reduction of edema

and vacuolizations and

maintaining the regular

morphology of endothelial cell

(arrowhead), sarcoplasm (s) and

elastic lamina (arrows)

424 Neurochem Res (2010) 35:418–428

123

substances [46]. In this sense, Na?, K?-ATPase, which is

responsible for the maintenance of neuronal excitability and

the control of cellular volume in the central nervous system

[49], is an important parameter to investigate SAH-induced

brain damage. Inhibition of the activity of this crucial

enzyme is associated with various neuropathological con-

ditions, including cerebral ischemia, neurodegenerative

disorders and spinal cord edema, which also provide evi-

dence for the vulnerability of Na?, K?-ATPase to free

radical attacks [50–52]. The decrease in Na? and K? gra-

dients under ischemic conditions, resulting in reduced

uptake and thereby increased extracellular concentration of

different neurotransmitters, may further exacerbate the

ischemic conditions [53]. Similarly, in the present study,

decreased Na?, K?-ATPase activity due to SAH, indicating

membrane damage and deterioration of membrane fluidity,

is in accordance with impaired BBB, increased lipid per-

oxidation and increased brain water content. Decreased

plasma membrane transporter in the brain of animals sub-

jected to SAH may also be due to overall cell death with the

consequent reduction in Na?, K?-ATPase activity, which is

further supported by increased DNA fragmentation. On the

other hand, ALA treatment, via its powerful antioxidative

properties, decreased brain edema and lipid peroxidation,

preserved BBB, increased Na?, K?-ATPase activity and

improved SAH-induced injury. Our results are in agreement

with the studies which have shown that lipoic acid preserves

the integrity of the BBB, while alleviating the oxidative

stress-induced changes in different models of brain injury

[22, 54].

It is generally accepted that cerebral vasospasm after

SAH is produced by the compounds released from the

subarachnoid clot, which decrease blood flow to the

affected brain regions, leading to ischemia and generation

of ROS, causing a secondary injury and brain edema that

result in neurological deficits [55]. It has been shown that

SAH causes an imbalance between vasoconstrictors and

vasodilators, which is mainly responsible for the neuro-

logical deficits, is closely related to the generation of ROS

[56]. Several experimental and human studies have shown

that inhibition of ROS by several antioxidant compounds

decreased SAH-induced cerebral vasospasm and ischemia

[14, 15] In the present study, luminol Cl levels, which

detect •OH, H2O2, HOCl radicals, as well as lucigenin CL

levels detecting O2- were significantly increased, impli-

cating that SAH causes increased ROS production in the

cerebral tissue and ALA treatment significantly inhibited

SAH-induced ROS production. The CL results of the cur-

rent study are in agreement with a number of studies which

show that ALA and its reduced form DHLA directly

scavenge reactive oxygen and nitrogen species [16–18].

It has been demonstrated that SAH-induced free radical

generation leads to neuronal damage by promoting lipid

peroxidation, protein breakdown and DNA damage, which

in turn lead to cellular apoptosis, endothelial injury and

increases BBB permeability [57]. Thus, lipid peroxidation

plays an important role in the genesis of chronic vasospasm

and associated neurological outcomes in SAH. In the

present study, lipid peroxidation is increased significantly

while the antioxidant GSH level is decreased in the cere-

bral tissues, indicating oxidative damage in the brain of

SAH-induced rats. It is well known that GSH, a major

intracellular antioxidant molecule, and GSH-related anti-

oxidant enzymes protect tissues from the damaging effects

of free radicals and constitute an important mechanism

against oxidative stress [58]. In the current study, as ALA

reduced the oxidative injury of cellular structures, the level

of the intracellular antioxidant GSH, which is otherwise

oxidized when inactivating free radicals, was not changed.

Thus, it appears that the anti-oxidative effect of ALA on

lipid peroxidation does not directly involve the expenditure

of tissue GSH stores, but the antioxidant pool is further

supported by the action of ALA. Several other studies have

demonstrated that ALA scavenges ROS and reactive

nitrogen species, prevents DNA damage, exhibits chelating

activity, reduces lipid peroxidation and increases intracel-

lular GSH levels [17]. Besides its antioxidant properties

through scavenging the ROS, the specific stimulatory effect

of ALA on the intracellular GSH levels [59, 60] further

suggests that it may be an important component of the

treatment regimen of the cerebrovascular ischemic

diseases.

It has been demonstrated that SAH damages the BBB,

and then the blood cells such as neutrophils and macro-

phages accumulate in the brain and further sustain the

cerebral inflammatory cascade [46]. Neutrophils could

greatly exacerbate the pathological sequelae of brain

trauma by altering vascular permeability, contributing to

oxidative damage, inducing further neuronal damage via

the secretion of lysosomal enzymes and cytokines or by

altering cerebral vascular blood flow [61, 62]. Activity of

myeloperoxidase (MPO) enzyme, which is extensively

used for the quantitative indication of inflammation, was

increased in the brain tissue at the 48th h of subarachnoid

hemorrhage, implicating that SAH-induced cerebral

ischemic damage is a neutrophil-dependent inflammatory

condition. Similarly, polymorphonuclear leukocyte

recruitment has been correlated with increased cerebral

edema [63] and neutrophil depletion has been reported to

attenuate increases in brain water content and to decrease

infarct size following ischemic brain injury [64]. On the

other hand, ALA treatment in the current study offered

neuronal protection by a neutrophil-dependent mechanism,

as it was previously shown to exert mucosal protection

through the inhibition of neutrophil recruitment in the

gastric and colonic tissues [65, 66]. The current results are

Neurochem Res (2010) 35:418–428 425

123

in agreement with similar studies, which have shown that

increased MPO activity plays a role in SAH-induced

ischemic brain damage and the antioxidant drugs prevent

the damage by inhibiting neutrophil infiltration [15, 67].

Alpha lipoic acid treatment, in the present study, sup-

pressed the high percentage of DNA fragmentation in the

brain, while oxidative brain injury was improved. Simi-

larly, it was demonstrated that application of the antioxi-

dant ALA in animal and cell culture models decreases

oxidative stress and supports the endogenous antioxidant

systems potently- and apoptosis-related cell death in tissues

exposed to oxidant injury [68, 69].

Despite sophisticated medical management and neuro-

surgical techniques, subarachnoid hemorrhage still results

in a high mortality rate. Considering that oxidative stress

plays important roles in the pathogenesis of acute brain

injury after SAH [70, 71], reducing oxidative stress,

thereby activating the survival pathway could be a thera-

peutic target for acute brain injury after SAH in a clinical

situation. The results of the current study demonstrate that

ALA has a significant neuroprotective effect mediated via

enhancing the activity of endogenous antioxidant enzymes,

inhibiting neutrophil accumulation and free radical gener-

ation. Since ALA is known to have various metabolic

antioxidant properties, can cross the blood-brain barrier,

and does not exhibit any serious side effects, it is possible

to speculate that ALA has the potential to be a novel

therapeutic agent for the reduction of secondary injury after

SAH in patients.

References

1. Kozniewska E, Michalık R, Rafałowska J et al (2006) Mecha-

nisms of vascular dysfunction after subarachnoid hemorrhage. J

Physiol Pharmacol 57(Suppl 11):145–160

2. Takao A (1999) Oxyhemoglobin as the principal cause of cere-

bral vasospasm: a holistic view of its actions. Crit Rev Neurosurg

9:303–318

3. Broderick JP, Brott TG, Duldner JE et al (1994) Initial and

recurrent bleeding are the major causes of death following sub-

arachnoid hemorrhage. Stroke 25:1342–1347

4. Hutter BO, Kreitschmann-Andermahr I, Gilsbach JM (2001)

Health-related quality of life after aneurysmal subarachnoid

hemorrhage: impacts of bleeding severity, computerized tomog-

raphy findings, surgery, vasospasm, and neurological grade. J

Neurosurg 94:241–251

5. Peyrot F, Ducrocq C (2008) Potential role of tryptophan deriva-

tives in stress responses characterized by the generation of

reactive oxygen and nitrogen species. J Pineal Res 45:235–246

6. Nishizawa S, Yamamoto S, Yokoyama T et al (1997) Dysfunc-

tion of nitric oxide induces protein kinase C activation resulting

in vasospasm after subarachnoid hemorrhage. Neurol Res

19:558–562

7. Imperatore C, Germano A, d’Avella D et al (2000) Effects of the

radical scavenger AVS on behavioral and BBB changes after

experimental subarachnoid hemorrhage. Life Sci 66:779–790

8. Ostrowski RP, Tang J, Zhang JH (2006) Hyperbaric oxygen

suppresses NADPH oxidase in a rat subarachnoid hemorrhage

model. Stroke 37:1314–1318

9. Asano T, Takakura K, Sano K et al (1996) Effects of a hydroxyl

radical scavenger on delayed ischemic neurological deficits fol-

lowing aneurysmal subarachnoid hemorrhage: results of a mul-

ticenter, placebo-controlled double-blind trial. J Neurosurg

84:792–803

10. Haley EC Jr, Kassell NF, Apperson-Hansen C et al (1997) A

randomized, double-blind, vehicle-controlled trial of tirilazad

mesylate in patients with aneurysmal subarachnoid hemorrhage: a

cooperative study in North America. J Neurosurg 86:467–474

11. Karaoglan A, Akdemir O, Barut S et al (2008) The effects of

resveratrol on vasospasm after experimental subarachnoidal

hemorrhage in rats. Surg Neurol 70:337–343

12. Aladag MA, Turkoz Y, Ozcan C et al (2006) Caffeic acid

phenethyl ester (CAPE) attenuates cerebral vasospasm after

experimental subarachnoidal haemorrhage by increasing brain

nitric oxide levels. Int J Dev Neurosci 24:9–14

13. Green AR, Ashwood T (2005) Free radical trapping as a thera-

peutic approach to neuroprotection in stroke: experimental and

clinical studies with NXY-059 and free radical scavengers. Curr

Drug Targets CNS Neurol Disord 4:109–118

14. Munakata A, Ohkuma H, Nakano T et al (2009) Effect of a free

radical scavenger, edaravone, in the treatment of patients with

aneurysmal subarachnoid hemorrhage. Neurosurgery 64:423–428

(discussion 428-9)

15. Ersahin M, Toklu HZ, Cetinel S et al (2009) Melatonin reduces

experimental subarachnoid hemorrhage-induced oxidative brain

damage and neurological symptoms. J Pineal Res 46:324–332

16. Packer L, Witt EH, Tritschler HJ (1995) a-Lipoic acid as a bio-

logical antioxidant. Free Radic Biol Med 19:227–250

17. Moini H, Packer L, Saris NE (2002) Antioxidant and prooxidant

activities of alpha-lipoic acid and dihydrolipoic acid. Toxicol

Appl Pharmacol 182:84–90

18. Packer L (1998) Alpha-lipoic acid: a metabolic antioxidant which

regulates NF-kappa B signal transduction and protects against

oxidative injury. Drug Metab Rev 30:245–275

19. Vincent AM, Stevens MJ, Backus C et al (2005) Cell culture

modeling to test therapies against hyperglycemia-mediated

oxidative stress and injury. Antioxid Redox Signal 7:1494–

1506

20. Bharat S, Cochran BC, Hsu M et al (2002) Pre-treatment with R-

lipoic acid alleviates the effects of GSH depletion in PC12 cells:

implications for Parkinson’s disease therapy. Neurotoxicology

23:479–486

21. Abdul HM, Butterfield DA (2007) Involvement of PI3K/PKG/

ERK1/2 signaling pathways in cortical neurons to trigger pro-

tection by cotreatment of acetyl-Lcarnitine and alpha-lipoic acid

against HNE-mediated oxidative stress and neurotoxicity:

implications for Alzheimer’s disease. Free Radic Biol Med

42:371–384

22. Toklu HZ, Hakan T, Biber N et al (2009) The protective effect of

alpha lipoic acid against traumatic brain injury in rats. Free Radic

Res 25:1–10

23. do Vale OC, Fonteles DS, Cabral FR et al (2003) A dual action of

alpha-lipoic acid in the brain: an electrophysiological evaluation.

Arq Neuropsiquiatr 61(3B):738–745

24. Freitas RM (2009) The evaluation of effects of lipoic acid on the

lipid peroxidation, nitrite formation and antioxidant enzymes in

the hippocampus of rats after pilocarpine-induced seizures.

Neurosci Lett 455:140–144

25. Jones RE, Moes N, Zwickey H et al (2008) Treatment of

experimental autoimmune encephalomyelitis with alpha lipoic

acid and associative conditioning. Brain Behav Immun 22:538–

543

426 Neurochem Res (2010) 35:418–428

123

26. Delgado TJ, Brismar J, Svendgaard NA (1985) Subarachnoid

haemorrhage in the rat: angiography and fluorescence microscopy

of the major cerebral arteries. Stroke 16:595–602

27. Bederson JB, Pitts LH, Tsuji M et al (1986) Rat middle cerebral

artery occlusion: evaluation of the model and development of a

neurological examination. Stroke 17:472–476

28. Haklar G, Yuksel M, Yalcın AS (1998) Chemiluminescence in

the measurement of free radicals: theory and application on a

tissue injury model. Marmara Med J 11:56–60

29. Wyllie H (1980) Glucocorticoid induced thymocyte apoptosis is

associated with endogenous endonuclease activation. Nature

284:555–556

30. Burton K (1956) A study of the conditions and mechanism of the

diphenylamine reaction for the colorimetric estimation of

deoxyribonucleic acid. Biochem J 62:315–323

31. Beuge JA, Aust SD (1978) Microsomal lipid peroxidation.

Methods Enzymol 53:302–311

32. Beutler E, Duron O, Kelly BM (1963) Improved method for the

determination of blood glutathione. J Lab Clin Med 61:882–888

33. Hillegas LM, Griswold DE, Brickson B et al (1990) Assessment

of myeloperoxidase activity in whole rat kidney. J Pharmacol

Methods 24:285–295

34. Reading HW, Isbir T (1980) The role of cation activated ATPase

in transmitter release from the rat iris. Q J Exp Physiol 65:105–

116

35. Fiske CH, Subbarow Y (1925) The colorimetric determination of

phosphorus. J Biol Chem 66:375–400

36. Lowry OH, Rosenbrough NJ, Farr AL et al (1951) Protein

measurements with the folin phenol reagent. J Biol Chem

193:265–275

37. Paravicini TM, Sobey CG (2003) Cerebral vascular effects of

reactive oxygen species: recent evidence for a role of NADPH-

oxidase. Clin Exp Pharmacol Physiol 30:855–859

38. Panigrahi M, Sadguna Y, Shivakumar BR et al (1996) Alpha-

lipoic acid protects against reperfusion injury following cerebral

ischemia in rats. Brain Res 717:184–188

39. Lee WJ, Lee IK, Kim HS, Kim YM et al (2005) Alpha-lipoic acid

prevents endothelial dysfunction in obese rats via activation of

AMP-activated protein kinase. Arterioscler Thromb Vasc Biol

25:2488–2494

40. Sena CM, Nunes E, Louro T (2007) Effects of alpha-lipoic acid

on endothelial function in aged diabetic and high-fat fed rats. Br J

Pharmacol. doi:10.1038/sj.bjp.0707474 (e-pub ahead of print 1

October 2007)

41. Smith AR, Hagen TM (2003) Vascular endothelial dysfunction in

aging: loss of Akt-dependent endothelial nitric oxide synthase

phosphorylation and partial restoration by (R)-alpha-lipoic acid.

Biochem Soc Trans 31:1447–1449

42. Sola S, Mir MQ, Cheema FA et al (2005) Irbesartan and lipoic

acid improve endothelial function and reduce markers of

inflammation in the metabolic syndrome: results of the Irbesartan

and Lipoic Acid in Endothelial Dysfunction (ISLAND) study.

Circulation 111:343–348

43. Maczurek A, Hager K, Kenklies M et al (2008) Lipoic acid as an

anti-inflammatory and neuroprotective treatment for Alzheimer’s

disease. Adv Drug Deliv Rev 60:1463–1470

44. Ambrus A, Tretter L, Adam-Vizi V (2009) Inhibition of the

alpha-ketoglutarate dehydrogenase-mediated reactive oxygen

species generation by lipoic acid. J Neurochem 109(Suppl

1):222–229

45. Germano A, d’Avella D, Cicciarello R et al (1992) Blood-brain

barrier permeability changes after experimental subarachnoid

hemorrhage. Neurosurgery 30:882–886

46. Del Zoppo GJ, Hallenbeck JM (2000) Advances in the vascular

pathophysiology of ischemic stroke. Thromb Res 98:73–81

47. Doczi TP, Joo F, Adam G et al (1986) Blood-brain barrier

damage during the acute stage of subarachnoid hemorrhage, as

exemplified by a new animal model. Neurosurgery 18:733–739

48. Doczi TP, Joo F, Balas I (1995) Atrial natriuretic peptide (ANP)

attenuates brain oedema accompanying experimental subarach-

noid haemorrhage. Acta Neurochir (Wien) 132:87–91

49. Streck EL, Feier G, Burigo M et al (2006) Effects of electro-

convulsive seizures on Na(?), K(?)-ATPase activity in the rat

hippocampus. Neurosci Lett 404:254–257

50. Grisar T (1984) Glial and neuronal Na?–K? pump in epilepsy.

Ann Neurol 16(Suppl):S128–S134

51. de Souza Wyse AT, Streck EL, Worm P et al (2000) Precondi-

tioning prevents the inhibition of Na?, K?-ATPase activity after

brain ischemia. Neurochem Res 25:971–975

52. Yang YB, Piao YJ (2003) Effects of resveratrol on secondary

damages after acute spinal cord injury in rats. Acta Pharmacol Sin

24:703–710

53. Stys PK (1998) Anoxic and ischemic injury of myelinated axons

in CNS white matter: from mechanistic concepts to therapeutics.

J Cereb Blood Flow Metab 18:2–25

54. Morini M, Roccatagliata L, Dell’Eva R et al (2004) Alpha-lipoic

acid is effective in prevention and treatment of experimental

autoimmune encephalomyelitis. J Neuroimmunol 148:146–153

55. Kolias AG, Sen J, Belli A (2009) Pathogenesis of cerebral

vasospasm following aneurysmal subarachnoid hemorrhage:

putative mechanisms and novel approaches. J Neurosci Res

87:1–11

56. Ayer RE, Zhang JH (2008) Oxidative stress in subarachnoid

haemorrhage: significance in acute brain injury and vasospasm.

Acta Neurochir Suppl 104:33–41

57. Lewen A, Matz P, Chan PH (2000) Free radical pathways in CNS

injury. J Neurotrauma 17:871–890

58. Reiter RJ, Tan DX, Acuna-Castroviejo D et al (2000) Melatonin:

mechanisms and actions as an antioxidant. Curr Top Biophys

24:171–183

59. Busse E, Zimmer G, Schopohl B et al (1992) Influence of alpha-

lipoic acid on intracellular glutathione in vitro and in vivo.

Arzneimittelforschung 42:829–831

60. Han D, Tritschler HJ, Packer L (1995) Alpha-lipoic acid

increases intracellular glutathione in a human T-lymphocyte

Jurkat cell line. Biochem Biophys Res 207:258–264

61. Wedmore CV, Williams JT (1981) Control of vascular perme-

ability by polymorphonuclear leukocytes in inflammation. Nature

289:646–650

62. Tonnesen MG, Worthen GS, Johnston RB (1988) Neutrophil

emigration, activation, and tissue damage. In: Clark RAE, Hensen

PM (eds) The molecular and cellular biology of wound repair.

Plenum, New York, pp 149–184

63. Schoettle RJ, Kochanek PM, Magargee JM et al (1990) Early

polymorphonuclear leukocyte accumulation correlates with the

development of post-traumatic cerebral edema in rats. J Neuro-

trauma 7:207–217

64. Matsuo Y, Onodera H, Shiga Y et al (1994) Correlation between

myeloperoxidase-quantified neutrophil accumulation and ische-

mic brain injury in the rat: effects of neutrophil depletion. Stroke

25:1469–1475

65. Kolgazi M, Jahovic N, Yuksel M et al (2007) Alpha-lipoic acid

modulates gut inflammation induced by trinitrobenzene sulfonic

acid in rats. J Gastroenterol Hepatol 22:1859–1865

66. Sehirli O, Tatlidede E, Yuksel M et al (2008) Antioxidant effect

of alpha-lipoic acid against ethanol-induced gastric mucosal

erosion in rats. Pharmacology 81:173–180

67. Wilson JX, Gelb AW (2002) Free radicals, antioxidants, and

neurologic injury: possible relationship to cerebral protection by

anesthetics. J Neurosurg Anesthesiol 14:66–79

Neurochem Res (2010) 35:418–428 427

123

68. Dincer Y, Telci A, Kayali R et al (2002) Effect of alpha-

lipoic acid on lipid peroxidation and anti-oxidant enzyme

activities in diabetic rats. Clin Exp Pharmacol Physiol 29:

281–284

69. Vincent AM, McLean LL, Backus C et al (2005) Short-term

hyperglycemia produces oxidative damage and apoptosis in

neurons. FASEB J 19:638–640

70. Matz PG, Copin J-C, Chan PH (2000) Cell death after exposure to

subarachnoid hemolysate correlates inversely with expression of

CuZn-superoxide dismutase. Stroke 31:2450–2458

71. Matz PG, Fujimura M, Lewen A et al (2001) Increased cyto-

chrome c-mediated DNA fragmentation and cell death in man-

ganese-superoxide dismutase-deficient mice after exposure to

subarachnoid hemolysate. Stroke 32:506–515

428 Neurochem Res (2010) 35:418–428

123