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: [email protected]; [email protected]
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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