ORIGINAL PAPER

SCH58261 the Selective Adenosine A2A Receptor BlockerModulates Ischemia Reperfusion Injury Following BilateralCarotid Occlusion: Role of Inflammatory Mediators

R. A. Mohamed • A. M. Agha • N. N. Nassar

Received: 22 June 2011 / Revised: 18 October 2011 / Accepted: 28 October 2011 / Published online: 10 November 2011

� Springer Science+Business Media, LLC 2011

Abstract In the present study, the effects of SCH58261, a

selective adenosine A2A receptor antagonist that crosses the

blood brain barrier (BBB) and 8-(4-sulfophenyl) theophyl-

line (8-SPT), a non-selective adenosine receptor antagonist

that acts peripherally, were investigated on cerebral ische-

mia reperfusion injury (IR). Male Wistar rats (200 - 250 g)

were divided into four groups: (1) sham-operated (SO), IR

pretreated with either (2) vehicle (DMSO); (3) SCH58261

(0.01 mg/kg); (4) 8-SPT (2.5 mg/kg). Animals were anes-

thetized and submitted to occlusion of both carotid arteries

for 45 min. All treatments were administered intraperito-

neally (i.p.) post carotid occlusion prior to exposure to a

24 h reperfusion period. Ischemic rats showed increased

infarct size compared to their control counterparts that

corroborated with histopathological changes as well as

increased lactate dehydrogenase (LDH) activity in the

hippocampus. Moreover, ischemic animals showed habit-

uation deficit, increased anxiety and locomotor activity. IR

increased hippocampal glutamate (Glu), GABA, glycine

(Gly) and aspartate (ASP). SCH58261 significantly

reversed these effects while 8-SPT elicited minimal change.

IR raised myeloperoxidase (MPO), tumor necrosis factor-

alpha (TNF-a), nitric oxide (NO), prostaglandin E2 (PGE2)

accompanied by a decrease in interleukin-10 (IL-10),

effects that were again reversed by SCH58261, but 8-SPT

elicited less changes. Results from the present study point

towards the importance of central blockade of adenosine

A2A receptor in ameliorating hippocampal damage follow-

ing IR injury by halting inflammatory cascades as well as

modulating excitotoxicity.

Keywords Adenosine � Ischemia reperfusion injury �Hippocampus � Neurotransmitters � NO � TNF-a

Abbreviations

BBB Blood brain barrier

DAG Diaceyl glycerol

DMSO Dimethyl sulfoxide

GABA c-aminobutyric acid

Glu Glutamate

Glycine Gly

IL-10 Interleukin10

IP3 Inositol triphosphate

IR Ischemia reperfusion

MPO Myeloperoxidase

NO Nitric oxide

PGE2 Prostaglandin E2

TNF-a Tumor necrosis factor-alpha

8-SPT 8-(4-Sulfophenyl) theophylline

SCH58261 7-(2-phenylethyl)-5-amino-2-(2-furyl)-

pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-

c]pyrimidine

SO Sham operated

Introduction

Adenosine acts at four receptor subtypes, A1, A2A–B and

A3 [1], where the A2A receptor is considered as the main

‘‘inhibitory’’ signal of the immune response in the

R. A. Mohamed � A. M. Agha � N. N. Nassar (&)

Department of Pharmacology and Toxicology,

Faculty of Pharmacy, Cairo University,

Kasr El-Aini Street, Cairo 11562, Egypt

e-mail: nnagah@yahoo.com; nnassar@cu.edu.eg

123

Neurochem Res (2012) 37:538–547

DOI 10.1007/s11064-011-0640-x

periphery [1]. However, centrally adenosine receptors

exert opposing effects to those of the systemic ones [1].

Therefore, special interest is directed towards adenosine

which is markedly elevated following ischemia and

reperfusion (IR) [1]. Adenosine exerts an important tonic

modulation of synaptic transmission in different brain

regions such as the hippocampus, striatum and olfactory

cortex [1]. Studies show that A2A receptor and its mRNA

are abundantly expressed in the hippocampus (CA1, CA3

and dentate gyrus) [2]. Contrary to A1 receptors, the A2A

are excitatory and their stimulation results in calcium

(Ca2?)-dependent release of glutamate (Glu) [1], thus

being one factor that initiates Glu induced excitotoxicity

[1, 3]. Among the deleterious effects of Glu release is the

accumulation of Ca2? in the cytosol, which further

exacerbates inflammatory processes ultimately leading to

neuronal death [4]. In the brain, prolonged ischemia leads

to considerable neuronal death and infarction [5] which is

further aggravated by reperfusion injury [4]. Endothelial

damage and recruitment of neutrophils with subsequent

release of proinflammatory cytokines represent leading

events in the late phase of ischemia reperfusion injury

[4, 6].

Evidence suggests that following transient or perma-

nent bilateral carotid occlusion, activated peripheral

immune cells and platelets become mobilized and infil-

trate into the brain parenchyma [4]. Brain inflammation

has been implicated in the development of brain edema

and secondary brain damage in ischemia [6]. During

ischemia, intracellular adenosine concentrations are ele-

vated owing to the imbalance between ATP degradation

and re-synthesis [7]. Metabolic stress associated with

hypoxia, ischemia, and excessive neuronal firing elicits

large increases in the concentration of extracellular

adenosine which controls subsequent tissue damage [7].

The activation of A2A receptor which appear to manifest

at a delayed fashion are responsible for the injurious

effects [7]. Selective adenosine A2A receptor antagonists

were reported to reduce cerebral damage induced by

global ischemia [8] which is mainly due to the inhibition

of Glu release [9]. Moreover, A2A receptor knockout mice

have been shown to be protected against ischemic brain

damage [10]. SCH58261, the selective A2A receptor

blocker that crosses the blood brain barrier (BBB) was

found to guard against neurological deficit as well as

possessing antidepressant and antiparkinsonian activity

[1]. However, many mechanisms underlying the protec-

tive role of A2A receptor blockade in ameliorating damage

induced by IR remain elusive. Accordingly, it became the

objective of the current investigation to delineate the role

of adenosine A2A receptor blockade in halting IR injury

via modulating excitatory as well as inflammatory

mediators.

Methods

Animals

Adult male Wistar rats weighing (200–250 g) were

obtained from the National Research Center Laboratory,

Cairo, Egypt. Rats were kept under controlled environ-

ment, at a constant temperature (23 ± 2�C), humidity

(60 ± 10%) and light/dark (12/12 h) cycle. Animals were

singly housed and acclimatized for 1 week before any

experimental procedures and were allowed standard rat

chow and tap water ad libitum. Animal handling and

experimental protocols were approved by the Research

Ethical Committee of the Faculty of Pharmacy, Cairo

University (Cairo, Egypt), and comply with the Guide for

the Care and Use of Laboratory Animals (ILAR 1996).

Groups and Treatments

Animals were randomly allocated into 4 groups (n = 30;

each group). In group 1 both carotid arteries were exposed

without occlusion to serve as sham operated (SO) group

receiving dimethyl sulfoxide (DMSO) as vehicle. The

remaining 3 groups were subjected to 45 min. ischemia

followed by 24 h reperfusion to serve as either (1) IR group

only, (2) IR ? SCH58261 (Sigma-Aldrich, CA, USA;

0.01 mg/kg, i.p.) [11, 12] or (3) IR ? 8-SPT (Sigma-

Aldrich, CA, USA; 2.5 mg/kg, i.p.) [13]. Both SCH58261

and 8-SPT were received following removal of carotid

occlusion at the beginning of the 24 h reperfusion period.

Induction of Cerebral Ischemia Reperfusion

Rats were anaesthetized with thiopental (50 mg/kg; i.p.).

Temperature was maintained at 37�C during surgery using a

heating pad [14]. A midline incision was made and both

carotid arteries were exposed then occluded for 45 min using

artery clamps [15]. Following the occlusion, clamps were

removed and the wound was sutured and reperfusion was

allowed for 24 h [16]. Following surgical procedure, rats

were housed individually and received an intramuscular

injection of 30,000 U of penicillin G in aqueous suspension

(Durapen; GC Hanford, New York, NY, USA) and a sub-

cutaneous injection of buprenorphine hydrochloride (30 lg/

kg Buprenex; Hospira, Inc., Lake Forest, IL, USA).

Brain Infarct Size

At the end of 24 h reperfusion period, animals (n = 4)

were intracardiacally perfused with isotonic saline and

sacrificed by spinal dislocation. Brains were then sliced

into 2 mm coronal sections and incubated with 1% tri-

phenyltetrazolium chloride (TTC) at 37�C in 0.2 M Tris

Neurochem Res (2012) 37:538–547 539

123

buffer (pH 7.4) for 20 min. While viable cells stain bright

red when TTC is converted to red formazone pigment by

NAD and lactate dehydrogenase, infracted cells lose the

enzyme as well as cofactor and thus remain unstained or

stain dull yellow. The brain slices were placed over glass

plate and the infarcted areas were traced by a 100 squares

in 1 cm2 transparent plastic grid. In each brain slice, the

average infarcted area of both sides as well as the non

infarcted area were determined. Infarcted area was

expressed as a percentage of total brain area [17, 18].

Histopathological Investigation

Following 24 h of reperfusion, brains were collected and

immediately fixed in 10% phosphate buffered formalin.

Subsequently, brains were embedded in paraffin, and 5 lm

sections were prepared and stained with haematoxylin and

eosin (H&E) and examined microscopically (940, 9100).

Behavioral Tests

Open Field Test (OFT)

The test was performed under white light in a quiet room

and testing was monitored by an overhead camera [19].

Rats were placed singly in the central area of the open field

box and monitored for 10 min. The open field box was

wiped clean between each test. During the 10 min moni-

toring period, the following parameters were recorded [20]:

(1) ambulation, (2) grooming, and (3) rearing frequencies,

(4) latency time (s) as well as (5) habituation deficit [19].

For calculating the habituation deficit, the frequency of

square entries was recorded during the initial (baseline) and

final 5 min testing periods, where each animal was utilized

as its own control. Activity score was calculated as percent

change in the second 5 min from baseline using the for-

mula provided by [19]. Accordingly habituation and

activity score are inversely proportional, where a low score

indicates increased habituation.

Biochemical Parameters

Tissue Collection

Twenty four hours post ischemia; all animals were eutha-

nized by cervical dislocation. Brains were removed imme-

diately and both hippocampi were dissected on ice cold

plates. In one set of animals, the hippocampi were homog-

enized in ice-cold saline and used for the determination of

lactate dehydrogenase (LDH), nitric oxide (NO), tumor

necrosis factor-alpha (TNF- a), prostaglandin E2 (PGE2) and

interleukin 10 (IL-10) contents. In another set, the hippo-

campi were divided into two portions for the determination

of myeloperoxidase activity (MPO) and neurotransmitter

(Glu, ASP, c-aminobutyric acid (GABA) and Gly) contents.

All measured parameters were normalized to protein con-

tent, measured according to Bradford assay [21].

Determination of Brain Amino Acids

Hippocampus was homogenized in 70% high performance

liquid chromatography (HPLC) methanol (1/10 weight/

volume) and was used for the estimation of Glu, ASP,

GABA and Gly using a fully automated high-pressure

liquid chromatography system (HPLC; Perkin-Elmer, MA,

USA). The phenylisothiocyanate derivatization technique

described by Heinrikson and Meredith [22] was adopted in

the current investigation. Hippocampal tissues were dried

under vacuum following reconstitution with 2:2:1 mixture

(v) of methanol:1 M sodium acetate trihydrate:triethyl-

amine. The derivatization procedure using a 7:1:1:1 mix-

ture (v) of methanol:triethylamine:double-distilled deionized

water:phenylisothiocyanate, was performed for 20 min at

room temperature then re-subjected to vacuum until dry-

ness. Subsequently, derivatized amino acids were recon-

stituted with sample diluent consisting of 5:95 mixture

(v) of acetonitrile:5 mM phosphate buffer (pH = 7.2).

Samples were then sonicated and filtered (0,45 lm; Mil-

lipore, USA). A Pico-Tag physiological free amino acid

analysis C18 (300 mm 9 3.9 mm i.d) column from Waters

(MA, USA) and a binary gradient of Eluents 1 and 2

(Waters) were used, the column temperature was at set

46 ± 1�C. A constant flow rate of 1 ml/min was main-

tained throughout the experiment. 20 ll of samples were

injected and the absorbance of the derivatized amino acids

was measured at 254 nm. All amino acids standards were

prepared in double-distilled deionized water, except for

GABA standards, which were prepared in polyethylene

vials to prevent adhesion to glass.

Lactate Dehydrogenase (LDH) Estimation

Homogenates were centrifuged at 14,000g, 4�C, where the

activity of LDH was estimated in the supernatant using

Stanbio LDH (Texas, USA) kit, according to the manu-

facturer procedure at 340 nm.

Nitric Oxide (NO) Estimation

Nitric oxide was assayed according to the method of

Miranda et al. [23], where hippocampal homogenates were

deproteinated with zinc sulphate for 48 h at 4�C, and then

centrifuged at 12,000g for 15 min at 4�C. To an aliquot of

the supernatant vanadium trichloride (0.8% in 1 M HCl)

was added for the reduction of nitrate to nitrite, followed

by the rapid addition Griess reagent consisting of

540 Neurochem Res (2012) 37:538–547

123

N-(1-naphthyl) ethylenediamine dihydrochloride (0.1%)

and sulfanilamide (2 in 5% HCl), incubated for 30 min at

37�C, cooled and the absorbance at 540 nm was measured.

Myeloperoxidase (MPO) Estimation

A slight modification of the method described by Krawisz

et al. [24] was used for the estimation of MPO (EC

1.15.1.1) activity (U/mg protein). Hippocampus was

homogenized in hexadecyltrimethylammonium bromide

(1%) in potassium phosphate buffer (100 mM, pH 6), then

subsequently subjected to 3 freeze and thaw cycles and

sonicated for 10 s followed by centrifugation at

10,000g for 15 min at 4�C. To the supernatant, a reaction

mixture of o-dianisidine hydrochloride (0.167%) and H2O2

(0.0005%) in potassium phosphate buffer (50 mM, pH 6)

was added. The change in absorbance was monitored at

1 min intervals at 460 nm for 4 min.

Tumor Necrosis factor (TNF)-a), Interleukin (IL)-10,

and Prostaglandin (PG)E2 Estimations

Hippocampal TNF-a, IL-10, and PGE2 were measured by

ELISA kits. TNF-a kit was purchased from Invitrogen

(California, USA) while IL-10 and PGE2 kits were pur-

chased from R&D Systems (USA). All the procedures of

the used kits were performed following the manufacturer’s

instructions.

Statistical Analysis

Data are expressed as mean ± SEM. Statistical compari-

sons were carried out using one-way analysis of variance

(ANOVA) followed by Tukey–Kramer Multiple Compar-

isons Test. All analysis utilized GraphPad Prism 5.0 sta-

tistical package for Windows (La Jolla, CA, USA). Non-

parametric One-way analysis of variance test (Kruskel-

Wallis Test) followed by Dunn’s Multiple Comparisons

Test were used for estimation of ambulation, rearing, and

grooming frequencies, as well as habituation deficit. The

minimal level of significance was identified at P \ 0.05.

Results

Effect of SCH58261 and 8-SPT on Infarct size,

Histological Changes and LDH Following IR Injury

IR induced approximately a 40% infarct size compared to

control SO rats (Fig. 1) that paralleled occurrence of

pyknotic nuclei in the CA1, CA3 and hilus of the hippo-

campus (Fig. 2). Moreover, the hilus showed vaculated

cells that reflect degeneration that was reflected on

enhanced LDH (Fig. 3) activity. SCH58261, on the other

hand reduced infarct size (10%) compared to IR and

reverted the histopathological changes as well reducing

LDH and preventing cellular necrosis after IR. However,

8-SPT did not decrease the infarct size (approximately

40%) or revert the histological changes. Moreover, 8-SPT

did not alter the IR-induced increase in LDH.

Effect of SCH58261 and 8-SPT on Activity Score

(habituation), Latency, Ambulation, Rearing

and Grooming

In the open field test, IR increased locomotor activity

during the 10 min test period. Notably, hyperactivity was

observed during both initial and final 5 min of testing.

Although both IR and SO rats showed habituation relative

to baseline, however, IR animals displayed a habituation

deficit as indicated by a higher H1 score compared to SO

animals (Fig. 4a). Moreover, in the IR group there was a

significant increase in ambulation (Fig. 4b), rearing

(Fig. 4c) and grooming frequencies (Fig. 4d) compared to

SO indicating an overall increase in anxiety and locomotor

activity while latency time (Fig. 4e) showed nearly no

difference between both groups. On the other hand,

Fig. 1 A representative photograph of brain coronal sections (n = 4)

(a) coronal sections showing the infarct areas (in white) in control (A),

ischemia/reperfusion brain (B) and the protection afforded by

SCH582621 (C) versus 8-SPT (D) pretreatment. Infarct area was

determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

b Summary of the quantitative analysis of infarct areas. Values are

expressed as mean ± SEM (n = 6), *, #, @ P \ 0.05 compared to

control, IR or SCH58261 group

Neurochem Res (2012) 37:538–547 541

123

SCH58261 treatment resulted in an increased habituation

indicated by a significant lowering in H1 score. By the

same token, a significant decrease in ambulation and

rearing frequencies was observed for SCH58261. Mean-

while, only SCH58261 could reduce grooming frequency.

On the other hand, 8-SPT had no effect on H1 score,

ambulation, rearing and grooming frequencies and failed to

reduce the IR-associated hyperactivity.

Effect of SCH58261 and 8-SPT on Hippocampal Nitric

Oxide (NO) Content and Neurotransmitter

Concentrations Induced by Ischemia Reperfusion (IR)

Injury

NO production was reduced by SCH58261 (124%) but not

8-SPT (162%) treatment compared to SO group following

IR in hippocampal homogenate (Fig. 5a). Rats subjected to

IR showed a significant increase in Glu (195%; Fig. 5b),

aspartate (ASP; 165%, Fig. 5c), c-amino butyric acid

(GABA; 196%; Fig. 5c), and glycine (GLY; 189%;

Fig. 5e) compared to SO group. SCH58261 reduced the

former amino acid concentrations: Glu (111%), ASP

(75%), GABA (125%) and Gly (101%) compared to SO

group.

Effect of SCH58261 and 8-SPT on Generation

of Inflammatory Mediators Induced by IR Injury

Following 24 h of reperfusion, MPO was significantly

increased (225%, Fig. 6a) indicating enhanced neutrophil

activation. This finding corroborated with the elevation

of the inflammatory cytokine, TNF-a (198%; Fig. 6b)

Fig. 2 Representative

photomicrographs depicting

histopathological changes in

CA3 (b), hilus (c) and CA1

(d) areas of the hippocampus.

Control animals show normal

arcitecture of different areas of

the hippocampus. While IR

induced nuclear pyknosis

(arrow) of CA3, hilus and CA1

areas and induced cellular

vaculation (v) suggestive of

cellular degeration, SCH58261

unlike 8-SPT pretreatment

preserved hippocampal cellular

structure (9 40,100)

542 Neurochem Res (2012) 37:538–547

123

production and the marked decline in the anti-inflammatory

cytokine IL-10 (54%; Fig. 6c). Furthermore, the inflam-

matory mediator PGE2 (333%; Fig. 6d) was elevated by

the insult. On the other hand, SCH58261 ameliorated MPO

(82%), TNF- a (86%), IL-10 (111%) and PGE2 (157%)

compared to SO. While treatment with 8-SPT, the

peripherally acting adenosine receptor antagonist resulted

in no protective effect on the former parameters.

Discussion

The selective adenosine A2A receptor antagonist

SCH58261 guards against global cerebral IR as evidenced

by: (1) decrease in cerebral infarct size which corroborated

with histopathological findings (2) attenuating anxiety,

hyperactivity and habituation deficit associated with IR; (3)

ameliorating excitotoxic damage illustrated by a reduction

in Glu, Gly as well as ASP concentrations in the hippo-

campus; (4) decrease in neutrophil recruitment; (5)

amending inflammatory mediators as well as LDH and (6)

boosting the anti-inflammatory cytokine IL-10. However,

treatment with non-selective adenosine 8-SPT, exerted

partial protection against increased Glu, Gly and TNF-aconcentrations.

In the present study, anxiogenic-like behavior accom-

panied by a habituation memory deficit and enhanced

locomotor activity were observed in animals subjected to

Fig. 3 Effect of IR alone or IR treated with either SCH58261

(0.01 mg/kg, i.p.), 8-SPT (2.5 mg/kg, i.p.) following removal of

carotid occlusion at the onset of the 24 h reperfusion period on lactate

dehydrogenase (LDH) activity. Data represent the means of 10

experiments ± SEM; *, #,@ P \ 0.05 compared to control, IR or

SCH58261 group, respectively using One-way ANOVA followed by

Tukey–Kramer multiple comparisons test for latency time

Fig. 4 Effects of IR alone or IR

treated with either SCH58261

(0.01 mg/kg, i.p.), 8-SPT

(2.5 mg/kg, i.p.), following

removal of carotid occlusion at

the onset of the 24 h reperfusion

period on a activity score,

b ambulation, c rearing,

d grooming and e latency. Data

represent the means of 10

experiments ± SEM;

*, #,@ P \ 0.05 compared to

control, IR or SCH58261 group,

respectively using One-way

ANOVA followed by Tukey–

Kramer multiple comparisons

test for latency time. Non-

parameteric One-way ANOVA

(Kruskel–Wallis test) followed

by Dunn’s multiple

comparisons test for % of

activity score, ambulation,

grooming and reering

Neurochem Res (2012) 37:538–547 543

123

Fig. 5 Effects of IR alone or IR

treated with either SCH58261

(0.01 mg/kg, i.p.), 8-SPT

(2.5 mg/kg, i.p.), following

removal of carotid occlusion at

the onset of the 24 h reperfusion

period on a NO, b glutamate

(GLU), c aspartate (ASP),

d c-aminobutyric acid (GABA),

and e Glycine (Gly). Data

represent the means of 10

experiments ± SEM;

*, #,@ P \ 0.05 compared to

control, IR and SCH58261

group, respectively using

One-way ANOVA followed by

Tukey–Kramer multiple

comparisons test

Fig. 6 Effects of IR alone or IR

treated with either SCH58261

(0.01 mg/kg, i.p.), 8-SPT

(2.5 mg/kg, i.p.), following

removal of carotid occlusion at

the onset of the 24 h reperfusion

period on a myeloperoxidase

(MPO), b tumor necrosis factor-

alpha (TNF-a), c interleukin-10

(IL-10) and d prostaglandin E2

(PGE2). Data represent the

means of 10

experiments ± SEM;

*, #,@ P \ 0.05 compared to

control, IR and SCH58261

group, respectively using One-

way ANOVA followed by

Tukey–kramer multiple

comparisons test

544 Neurochem Res (2012) 37:538–547

123

ischemia followed by 24 h reperfusion. These findings are

in agreement with a report by Milot and Plamondon [19].

Evidence exist that global cerebral ischemia results in

hypermotility owing to the inability of animals to habituate

to a novel testing environment [25]. This effect may be

attributed to cell loss in the hippocampus [3]. Indeed in the

current study, we report increased infarct size in IR group

(Fig. 1b), which corroborated with vaculations and pky-

notic nuclei upon histopathological examination (Fig. 2).

Moreover, the IR group displayed increased LDH con-

centration compared to SO group which reflects enhanced

necrosis (Fig. 3). Neuronal depolarization and massive

release of excitatory amino acids and consequent excito-

toxicity play an important role in excitotoxic cell death

[3, 4]. Such effect is in line with the observed increase in

Glu and ASP in the hippocampus of IR rats in the current

study (Fig. 4b, c). In addition, in the present investigation

we report an increase in Gly (Fig. 4e) concentration fol-

lowing IR. Kleckner and Dingledine [26] noted that Gly the

co-agonist facilitates Glu induced NMDA receptors acti-

vation. Furthermore, ischemia induces an increase in cel-

lular adenosine, which further activates A2A receptor that

enhances Glu outflow [12, 27]. Meanwhile, adenosine,

directly, through activation of DAG/IP3 pathway increases

intracellular Ca2? [28] resulting in cellular toxicity to CA1

neuron as seen from histopathological findings (Fig. 2) and

disrupting hippocampal function (such as spatial mapping)

leading to hypermotility as documented in the current

study.

The protective effect afforded by selective A2A receptor

blockade by SCH58261reported in this study is in line with

other previous reports, in different other models of IR

[7, 11, 12]. SCH58261 by virtue of its ability to reduce Glu

and ASP concentrations, as seen in this work, through

blockade of central A2A receptor [9, 11] and possibly via

the present reduction of Gly, might reverse behavioral

effects induced by IR injury. Paradoxically, in the current

study, the non-selective blocker, 8-SPT, that does not cross

BBB attenuated the IR induced increase in Glu and Gly.

These central effects imply a change in blood brain barrier

(BBB) permeability following IR as reported by Knight

et al. [29] thus enhancing the penetration of 8-SPT into the

brain to a certain extent. Notably, adenosine A1 presyn-

aptically decrease Glu release while A2A increases it [30,

31]. This modulation of Glu release is particularly impor-

tant within the hippocampus [32]. Adenosine released upon

ischemia is in a range that activates A2A receptor [33], thus

blocking it provides protection against ischemic induced

injury. On the contrary, blockade of A1 receptor accentu-

ates ischemic damage [34]. Thus, 8-SPT via blocking all

adenosine receptors non-selectively, induced opposite

effects resulting in mild amelioration of IR induced chan-

ges seen in this study.

In the present study, interestingly an increase in GABA

concentration was observed rather than decreased levels

following IR. The results of the current study are in line

with other reported studies [11, 35, 36]. Notably, this

paradoxical increase in GABA concentration might be

mediated through Glu–glutamine cycle, which induces the

production of GABA from Glu [35]. Moreover, the

increase in intracellular adenosine following IR has been

shown to enhance the release of a plethora of neurotrans-

mitters, including GABA [1]. However, one might argue

that the increase in GABA would be expected to ameliorate

the behavioral changes induced by IR. A plausible reason

for the observed hyperactive phenomenon could be attrib-

uted to the desensitization of GABAA receptor following

elevation of TNF-a [37]. Certainly, this study corroborates

an increase in TNF-a with increase in GABAA level in IR

rats. Another plausible explanation is the decline in GABA

concentrations by SCH58261, which coincided with

reduced anxiety in open field test. By the same token, lack

of selectivity with 8-SPT partially reduced TNF-a, thus

indicating that a certain reduction in the cytokine level is

required to attenuate GABA concentration.

In the present study, following IR, we demonstrate an

enhanced MPO activity, thus reflecting increased neutro-

phil infiltration consistent with the report of Anaya-Prado

et al. [38]. Neutrophils are one source for cytokine pro-

duction [39] and the increase in TNF-a seen in the present

study has been previously shown to exert excitotoxicity

via interaction with presynaptic AMPA receptors, hence

increasing Ca2? influx with subsequent release of Glu

[40]. Interestingly, adenosine A2A receptor activation has

been shown to increase the release of proinflammtory

cytokines [41]. Moreover, we report an increase in TNF-aaccompanied with a decline in its counter partner IL-10

which is in agreement with the findings of Abdallah et al.,

[42]. IL-10 is known to halt the devastating effects of

proinflammatory cytokines [43]. These alterations in

cytokines were held in check by pretreatment with the

selective A2A antagonist (SCH58261) as manifested by a

decline in MPO which further results in a decrease in

TNF-alpha. Interestingly the inflammatory process in the

brain relies on contribution of inflammatory cells, mainly

microglia, that are not normally found in the periphery

[1], which are the primary source of TNF-a [44]. Fur-

thermore, A2A receptors are upregulated on microglial

cells following IR [33]. Since 8-SPT lacks selectivity to

A2A receptor and induced no change in MPO activity,

reported in this study, a finding that offers plausible

explanation for the partial attenuation of TNF-alpha with

8-SPT versus complete protection by SCH58261 that

decreased MPO activity significantly.

Interestingly, TNF-a has been shown to induce the

expression of nitric oxide synthase (NOS) [45] which in

Neurochem Res (2012) 37:538–547 545

123

turn induces cellular damage [46].This effect might afford

one explanation to the increased NO levels recorded in

this study which corroborates with the findings of Leker

and Shohami [47] after IR. Such an increment was

reversed by A2A antagonism (SCH58261), where a plau-

sible explanation for this phenomenon might be attributed

to the increase in intracellular Ca2? induced by elevated

levels of excitatory amino acids following IR observed in

the present investigation. The deleterious effects of NO

resides in its rapid reaction with superoxide produced in

excess during reperfusion to form peroxynitrite [48]

contributing to cell death as seen with vaculations and

pkynotic nuclei upon histopathological examinations.

Moreover, the IR group displayed increased LDH con-

centration compared to SO group which reflects enhanced

necrosis.

Apart from glutamatergic synaptic activity that upreg-

ulates COX-2 activity [40, 49], A2A receptor activation

induces its expression and the PGE2 production, which

might indicate a pro-inflammatory role of A2A receptor

[41]. In addition, TNF-a increases expression of COX-2,

while excitotoxicity increases arachidonic acid release

[49]. These events could thus explain increased concen-

trations of PGE2 in the hippocampi of IR rats, in the current

study, which is in agreement with previous studies [42, 50].

Following brain injury in the hippocampus, these events

trigger a central inflammatory reaction [51, 52]. Very

effectively, SCH58261, normalized PGE2 level beside its

anti-excitotoxic activity, thus providing an add on benefit

to the efficacy of A2A receptor blockade in a model of IR

injury.

Although the non-selective A2A antagonist,8-SPT,

afforded partial protection against the IR induced increase

in Glu, Gly as well as TNF-a, however, it did not alter the

infarct size/increased LDH, as well as histopathological

and behavioral changes induced by IR. These events imply

that (1) although ischemia alters BBB permeability and

allows passage of 8-SPT, nevertheless, its concentration

might not be enough to completely block A2A receptor

compared to SCH58261 as reflected on behavioral changes

and infarct size. Moreover, blockade of other adenosine

protective receptor (A1) by non-selective 8-SPT might

induce opposite effects resulting in mild amelioration of IR

induced changes seen in this study; (2) the ability of

SCH582061 to ameliorate TNF-a compared to incomplete

protection offered by 8-SPT is suggestive of inability to

modulate neutrophil TNF-a release as evident by its

inability to correct MPO. Taken all together, the present

investigation highlights a potential therapeutic utility for

SCH58261 for being a selective A2A receptor blocker in IR

brain injury via modulating excitotoxic as well as inflam-

matory mediators.

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