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Research Report

Pentoxifylline attenuates TNF-α protein levels and brain

edema following temporary focal cerebral ischemia in rats

Abedin Vakili a,⁎, Somye Mojarrad a , Maziar Mohammad Akhavan b , Ali Rashidy-Pour c
a
Laboratory of Cerebrovascular Research, Department and Research Center of Physiology, School of Medicine,
Semnan University of Medical Sciences, Semnan, Iran
b
Skin Research Center—Laboratory of Protein and Enzyme, Shahid Beheshti University (M.C.), Shohada-e Tajrish Hospital,
Shahrdari St., 1989934148 Tehran, Iran
c
Laboratory of Learning and Memory, Research Center and Department of Physiology, Semnan University of Medical Sciences, Semnan, Iran

A R T I C LE I N FO AB S T R A C T

Article history: Cerebral edema is the most common cause of neurological deterioration and mortality
Accepted 3 January 2011 during acute ischemic stroke. Despite the clinical importance of cerebral ischemia, the
Available online 8 January 2011 underlying mechanisms remain poorly understood. Recent studies suggest a role for TNF-α
in the brain edema formation. To further investigate whether TNF-α would play a role in
Keywords: brain edema formation, we examined the effects of pentoxifylline (PTX, an inhibitor of TNF-
Pentoxifylline α synthesis) on the brain edema and TNF-α levels in a model of transient focal cerebral
Focal cerebral ischemia ischemia. The right middle cerebral artery (MCA) of rats was occluded for 60 min using the
Blood–brain barrier permeability intraluminal filament method. The animals received PTX (60 mg/kg) immediately, 1, 3, or 6 h
Brain edema post-ischemic induction. Twenty-four hours after induction of ischemic injury,
TNF-α permeability of the blood–brain barrier (BBB) and brain edema were determined by in situ
Rat brain perfusion of Evans Blue (EB) and wet-to-dry weight ratio, respectively. TNF-α protein
levels in ischemic cortex were also measured at 1, 4, and 24 h after the beginning of an
ischemic stroke by using an enzyme-linked immunosorbent assay method. The
administration of PTX up to 6 h after occlusion of the MCA significantly reduced the brain
edema. Moreover, PTX significantly reduced the concentration of TNF-α in ischemic brain
cortex up to 4 h post-transient focal stroke (P < 0.002). Finally, treatment by PTX led to a
significant decrease in EB extravasations (P < 0.001). Our data demonstrate that PTX
administration up to 6 h after ischemia can reduce brain edema in a model of transient
focal cerebral ischemia. The beneficial effects of PTX may be mediated, at least in part,
through a decline in TNF-α production and BBB breakdown.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction because of disturbances in cell membrane (Zador et al., 2009).

Vasogenic cerebral edema is the most common type of brain
Brain edema is the most common cause of neurological edema, resulting from a disruption in blood–brain barrier (BBB)
deterioration and mortality during acute ischemic stroke (Strbian et al., 2008; Zador et al., 2009). Cerebral edema usually
(Zador et al., 2009). Ischemic brain edema is initially cytotoxic begins to develop shortly after the onset of ischemia and

⁎ Corresponding author. Fax: +98 231 3354186.

E-mail address: Abvakili@yahoo.com (A. Vakili).

0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2011.01.001
120 BR A I N R ES E A RC H 1 3 7 7 ( 2 01 1 ) 1 1 9 –12 5
increases intracranial pressure (ICP) and potentially leads to
brain ischemia, herniation, and finally death (Manley et al.,
2004). Despite its clinical importance, pathophysiology of
cerebral edema remains poorly understood. Currently, the
clinical treatment of brain edema is based mainly on injection
of hyperosmolar agents (e.g. mannitol, hypertonic saline) in
the attempt to draw water out of brain tissue and decrease ICP
(Manley et al., 2004). However, new therapeutic strategies are
needed to more effectively treat brain edema following
neurological disorders.
Recently, several lines of evidence support a role for
tumor necrosis factor-α (TNF-α) in the pathophysiology of
brain edema (Yang et al., 1999; Gregersen et al., 2000; Kimura
et al., 2003; Hosomi et al., 2005). TNF-α has been demon-
strated to participate in opening of the BBB and edema
formation, which follows cerebral ischemic injury (Yang
et al., 1999; Hosomi et al., 2005), brain trauma (Shohami
et al., 1997), sepsis (Tsao et al., 2001), and bacterial meningitis
(Ramilo et al., 1990).
Pentoxifylline (PTX), a phosphodiesterase inhibitor, rapidly
penetrates into the BBB following systemic administration
(Watkins et al., 2003; Fujimoto et al., 1976) and efficiently
inhibits TNF-α mRNA synthesis (Nataf et al., 1993; Hong et al.,
1995; Shohami et al., 1996; Hoie et al., 2004). Our previous studies
showed that PTX has a neuroprotective effect in a model of focal
cerebral ischemia (Nekoeian et al., 2005; Vakili and Zahedi
Khorasani, 2007a). An earlier study has demonstrated no effect
of PTX on brain edema 6 h after the middle cerebral artery
occlusion (MCAO) in hypertensive rats (Johansson and Olsson,
1989). In addition, one study has reported that PTX significantly
lowered cerebral edema, along with the reduced levels of TNF-α
by 80%, in closed head injury in rats (Shohami et al., 1997).
To the best our knowledge, only a few studies have
addressed the effects of the PTX on cerebral edema following
Fig. 1 – Regional cerebral blood flow (rCBF; % from baseline) before and during MCAO and after reperfusion in the saline or PTX
(60 mg/kg) treated groups.
experimental brain injuries and, to date, no study has investi-
gated its effects on BBB integrity. Hence, we investigated the
influence of PTX on brain edema, BBB permeability, and TNF-α
protein levels in focal cerebral ischemic stroke that was induced
by temporary occlusion of the middle cerebral artery in rats.
2. Results
2.1. Cerebral blood flow
Regional cerebral blood flow (rCBF) was reduced to less than of
20% of baseline in both saline and PTX treated groups. PTX did
not significantly change rCBF during MCAO or reperfusion as
compared with the control group (Fig. 1).
2.2. Time-course effects of PTX on brain water content
Measurement of brain water content (BWC) was used to
evaluate brain edema. BWC in the left and right hemispheres
of the sham-operated rats was 78.8% ± 0.13% and 78.7% ± 0.14%,
respectively. Induction of focal cerebral ischemia in the saline
group significantly increased BWC of ischemic hemisphere
(right) to 82.99% ± 0.35% (P < 0.001). As shown in Fig. 2A, PTX
administration at beginning (80.6% ± 0.24%), 1 h (80.8% ± 0.3%),
3 h (81.03% ± 0.25%), or 6 h (80.78% ± 0.28%) after MCAO signif-
icantly reduced post-ischemic increase of BWC as compared
with the saline group (P < 0.001). In addition, the repetitive
injections of PTX immediately, 1 h, 3 h, 6 h after MCAO,
significantly reduced the percentage of BWC (edema) (81.5% ±
0.32%) as compared with the control group (82.99% ± 0.35%)
(P < 0.001). Furthermore, no significant difference in BWC was
found in non-ischemic hemisphere between the groups
(Fig. 2B).
 BR A I N R ES E A RC H 1 3 7 7 ( 2 01 1 ) 1 1 9 –1 25 121

Fig. 3 – Evans Blue extravasation (μg/g brain tissue) in the

sham-operated and saline or PTX (60 mg/kg) treated groups
24 h after MCAO. #P < 0.001 versus sham group; *P < 0.001
versus saline groups.

sphere (non-ischemic) in the sham (0.18 ± 0.02 μg/g tissue),

saline (0.20 ± 0.01 μg/g tissue), and PTX treated (0.16 ± 0.04 μg/g
tissue) groups.

2.4. Effect of PTX on TNF-α protein levels in ischemic

cortex

Induction of focal cerebral ischemia increased TNF-α protein

levels significantly at 1 h (5.45 ± 0.60 pg/mg protein), 4 h (14.48±
2.1 pg/mg protein), and 24 h (6.63± 0.81 pg/mg protein) following
ischemia in the right ischemic cortex in the saline treated
groups as compared with the sham-operated group (19 ±
0.28 pg/mg protein) (P < 0.001; Fig. 4). TNF-α protein levels in
cortical ischemic tissue of the saline groups were significantly
increased 1 h after MCAO, reached to peak at 4 h post-
Fig. 2 – Brain water content in ischemic hemisphere (A) and ischemia, and reduced to baseline 24 h after MCAO. Systemic
non-ischemic hemisphere (B) in the saline or PTX treated and administration of PTX at 1 h (2.60± 0.66 pg/mg protein) and 4 h
sham-operated groups. The animals received either single or
multiple injections of PTX (60 mg/kg). PTX administrated
immediately, 1, 3, or 6 h following ischemia induction. For a
single injection, each animal received PTX only in one of
these time points. For multiple treatments, the animals
received PTX (60 mg/kg) at all time points. #P < 0.001 versus
the sham group; *P < 0.001 versus the saline groups.

2.3. Effect of PTX on BBB permeability

Evans Blue (EB) extravasation was used as a marker of BBB

breakdown following cerebral ischemia. Induction of cerebral
ischemia significantly increased the EB dye content of the
ischemic hemisphere (right) (P < 0.001) in the saline group (1.2 ±
0.16 μg/g tissue) as compared with the sham group (0.16 ±
0.02 μg/g tissue; P < 0.001). Administration of PTX at beginning Fig. 4 – Concentrations of TNF-α (pg/mg protein) in the
of ischemia significantly reduced the EB extravasations (0.67 ± sham-operated, saline and PTX (60 mg/kg ip) treated groups
0.04 μg/g tissue; P < 0.001) into ischemic brain as compared with at 1 h, 4 h, and 24 h following MCAO in cortical ischemic
the saline group (1.2 ± 0.16 μg/g tissue; Fig. 3). There are no brain. #P < 0.001 versus sham group; *P < 0.001 versus saline
significant difference between the EB content in left hemi- groups.
122 BR A I N R ES E A RC H 1 3 7 7 ( 2 01 1 ) 1 1 9 –12 5
(3.27± 0.71 pg/mg protein) but not 24 h (4.15± 1.27 pg/mg pro-
tein) after MCAO significantly reduced the TNF-α amount in
cortical ischemic tissue as compared with the saline group
(P = 0.002; Fig. 4). These results indicate that PTX reduced TNF-α
production in cortical ischemic tissue up to 4 h after MCAO.
3. Discussion
Our results indicated that systemic injection of PTX (60 mg/kg)
up to 6 h following focal stroke reduces brain edema. This
interesting finding indicates that the anti-edematous effects
of PTX persisted for at least 6 h after post-transient focal
stroke. This result is in accordance, to some extent, with our
recent work showing a neuroprotective effect of PTX on cortical
brain ischemic damage, which lasted for at least 3 h post-
transient focal stroke in rats (Vakili and Zahedi Khorasani,
2007a). Furthermore, our results are consistent with other study
showing an anti-edematous effect of PTX (20 mg/kg) against
brain edema following close head injury in rats (Shohami et al.,
1996). However, our present finding is in disagreement with an
earlier report demonstrating no beneficent effect of PTX on
brain edema in a permanent model of focal ischemia (Johansson
and Olsson, 1989). Although the reason for this discrepancy is
not clear, it may be related to the experimental design and
methodology. In their study, PTX continuously infused with a
dose of 0.30 mg/kg per minute starting 30 min after MCAO and
brain edema was measured by specific gravity 6 h later in
spontaneously hypertensive rats. We measured, however, the
effects of PTX (60 mg/kg) on brain edema 24 h after transient
MCAO. We chose this time because previous studies have
showed that brain edema reached its maximum 24 h after
ischemia (Lin et al., 1993; Slivka et al., 1995) and brain trauma
(Louin et al., 2006).
The duration of PTX action is limited by its short half-life
(less than 1 h) (Fujimoto et al., 1976; Rocci et al., 1987). Thus,
we tested whether multiple treatments of PTX can reduce
brain edema more than a single injection. The animals were
treated with PTX immediately, 1, 3, and 6 h after MCAO. The
findings revealed no significant changes on the percentage of
brain edema following multiple injections of PTX, suggesting
that a single injection is sufficient enough to effectively reduce
brain edema.
An enhancing effect of PTX on the cerebral blood flow has been
reported in humans with cerebrovascular disorder (Bowton et al.,
1989). Nevertheless, animal studies have failed to observe an
effect of PTX on regional cerebral blood flow during ischemia or
reperfusion in an experimental animal model (Johansson, 1986;
Toung et al., 1994). In this study, we did not observe any effect of
PTX administration at initial of ischemia on regional cerebral
blood flow before during MCAO. Thus, we can exclude the
possibility that the observed anti-edematous effect of PTX is due
to an increased cerebral blood flow during MCAO.
To determine the mechanism of the anti-edematous action
of PTX, TNF-α concentration was measured 1 h, 4 h, and 24 h
after MCAO in the saline and PTX treated animals. We found a
transient increase in TNF-α concentration 1 h after ischemia,
reached to a peak during 4 h, and returned to baseline at 24 h
post-ischemia. This finding is consistent with a report that
demonstrated that TNF-α reached to a peak 4 h after close
head injury (Shohami et al., 1997). Moreover, we found that
PTX attenuates TNF-α activity in ischemic cortex, suggesting a
role for TNF-α in the formation of brain edema in acute phase
of transient focal stroke. In support of this result, other studies
demonstrated that TNF-α is involved in brain edema following
cerebral ischemia (Megyeri et al., 1992; Yang et al., 1999;
Gregersen et al., 2000).
PTX is currently used for the treatment of some vascular
disorders (Jacoby and Mohler, 2004). In recent years, PTX has
evoked new interest because it has been found to attenuate
the synthesis of TNF-α and other pro-inflammatory cytokines
such as interleukin-1 (IL-1), IL-6 in vivo and in vitro (Han et al.,
1990; Kambayashi et al., 1995; Lauterbach et al., 1995;
Marcinkiewicz et al., 2000). Our results showed an attenuation
of brain edema by PTX along with decrease of BBB permeabil-
ity and TNF-α level in ischemic cortex. Several evidence
demonstrated that TNF-α is activated during brain ischemia
(Gregersen et al., 2000; Maddahi and Edvinsson, 2010). It
causes damages to cerebral endothelial cells (Kimura et al.,
2003) and increases BBB permeability (Megyeri et al., 1992;
Yang et al., 1999) and thus contributes to brain edema
formation. Therefore, we propose that the anti-edematous
effects of PTX are more likely due to an inhibition of TNF-α
synthesis and, consequently, the protection of the BBB against
disruption.
Recent studies reported that pro-inflammatory cytokines
interleukin-1β (IL-1β) and IL-6 and activation of NF-κB play
important roles in the pathogenesis of brain edema (Bemeur
et al., 2010; Rama Rao et al., 2010). The fact that PTX is able to
decrease the levels of other inflammatory cytokines such as
IL-1 and IL-6 as well as the activation of NF-κB (Han et al., 1990;
Kambayashi et al., 1995; Lauterbach et al., 1995; Marcinkiewicz
et al., 2000) suggests that the beneficial effect of PTX, at least in
part, may result from the inhibition of production of these
cytokines and, consequently, their signaling pathways. In
addition, several evidences indicated that free radicals and
oxidants have important roles in the development of edema
and BBB disruption in experimental cerebral ischemia (Kondo
et al., 1997; Heo et al., 2005). The fact that PTX exhibits
antioxidant and free radical scavenger activities (Bhat and
Madyastha, 2001) suggests that these properties may under-
line the anti-edematous effect of PTX. Further experiments are
needed to clarify these possibilities.
In conclusion, our study showed that PTX treatment could
reduce up to 6 h after the onset of focal cerebral ischemia brain
edema. This effect may be mediated via the inhibition of TNF-
α synthesis and, consequently, the protection of the BBB
disruption. Potential therapeutic applications of PTX in stroke
patients need further studies.
4. Experimental procedures
4.1. Animals
Adult male Wistar rats (320 ± 10 g) were obtained from breeding
colony of Semnan University of Medical Sciences (SUMS),
Semnan, Iran. All rats were housed in cages in a 12-h light/dark
cycle at 22–24 °C, with food and water ad libitum. All
procedures were conducted in agreement with the National
 BR A I N R ES E A RC H 1 3 7 7 ( 2 01 1 ) 1 1 9 –1 25
Institutes of Health Guide for Care and Use of Laboratory
Animals.
4.2. Middle cerebral artery occlusion and laser Doppler
flowmetry
MCAO was induced by intraluminal filament method as
described previously (Vakili et al., 2005). Briefly, animals
were anesthetized with chloral hydrate (400 mg/kg ip) and
the right common carotid (CCA) and external carotid arteries
were exposed through a midline neck incision. A nylon thread
(3-0) inserted into the internal carotid artery and gently
advanced until laser Doppler flowmetry (LDF; Moor Instru-
ments DRT4, England) showed a sharp decrease in the
ipsilateral cerebral blood flow to less than 20% of baseline,
indicating adequate occlusion of the MCA. After 60 min of
MCAO, reperfusion was done by withdrawing the intraluminal
filament for 23 h.
To monitor the regional CBF during cerebral ischemia, a laser
Doppler flowmetry probe (Moor Instruments DRT4, England)
was positioned in direct contact with the right temporal bone
after a limited dissection of the temporalis muscle at the middle
distance between the eye and the ear (Harada et al., 2005; Lecrux
et al., 2007). To fix and prevent the displacement of LDF probe, a
burr hole (1 mm diameter) was drilled 5 mm lateral and 1 mm
posterior to the bregma. Local right cortical CBF was continu-
ously monitored before, during the occlusion, and up to 15 min
after the reperfusion.
4.3. Brain water content
PTX was administrated at a dose of 60 mg/kg as the most
effective dose. This dose was chosen based on our pilot study
using different doses of the PTX (15, 30, 60, and 120 mg/kg) in
focal cerebral ischemia model.
Cerebral edema was evaluated by determining brain water
content (BWC) (Vakili et al., 2007b). For therapeutic window
study, animals were randomly assigned six different groups.
Group 1 was sham-operated (n = 8); Group 2 (n = 8) was the
control group which received saline (1 mL/kg) as control; and
four groups that received PTX at the beginning (n = 8), 1 h (n = 8),
3 h (n = 8), and 6 h (n = 8) after MCAO, respectively. Because half-
life of PTX is less than 1 h (Fujimoto et al., 1976; Rocci et al.,
1987), one additional group was injected (PTX) repetitively at
the beginning, 1 h, 3 h, and 6 h after MCAO. The investigator
who performed animal surgery was blinded to the treatment of
groups. Twenty-four hours after MCAO, all rats were killed and
the brains were removed. Then, the pons and olfactory bulb
were removed and the brains were weighted to obtain their wet
weight (WW). Subsequently, brains were dried at 110 °C for 24 h
to determine their dry weight (DW). BWC was calculated by
using the following formula: (WW − DW) / WW × 100.
4.4. Evaluation of blood–brain barrier permeability by
using Evans Blue
BBB permeability was measured at 24 h after MCAO in 3
different groups: Group 1 was sham-operated (n = 6), Groups 2
and 3 (n = 8 in each group) received saline or PTX at beginning
of MCAO, respectively. EB (2% in saline, 1 mL/kg) was injected
123
from tail vein at beginning of reperfusion. In all of these
groups, BBB permeability was evaluated by measuring EB
extravasations (Kucuk et al., 2002; Vakili et al., 2007b). Briefly,
24 h after ischemia, rats were deeply anesthetized, chest wall
was opened, and transcardially perfused with 250 mL saline
solution to clear cerebral circulation of EB. After decapitation,
the brains were removed and hemispheres were separated
and weighed. Each hemisphere was placed in the 2.5 mL
phosphate buffer saline, was homogenized, and then 2.5 mL of
60% trichloroacetic acid (Merck, Germany) was added to
precipitate protein. Samples were centrifuged for 30 min at
3500 rpm. The supernatants were measured at 610 nm for
absorbance of EB using a spectrophotometer (UV–visible–
single beam, PD-303UV; Apel, Japan). The results were
expressed as microgram per gram tissue of wet weight of
brain tissue calculated against a standard curve.
4.5. Determination of TNF-α in cerebral cortex
Animals were randomly divided into seven groups. Group 1
was sham-operated (n = 6) and Groups 2–4 were controls that
received saline (1 mL/kg) at the beginning of ischemia and the
levels of TNF-α in their ischemic cortex tissue were measured
at 1 h (n = 6), 4 h (n = 6), and 24 h (n = 6) after MCAO, respectively.
Groups 5–7 (n = 6 in each group) received PTX (60 mg/kg ip) at
the beginning of ischemia and the levels of TNF-α in their
ischemic cortex tissue were measured at 1 h, 4 h, and 24 h
after MCAO, respectively.
The levels of TNF-α in the ischemic cortex tissue was
detected using an enzyme-linked immunosorbent assay
(ELISA) test as described previously (Hang et al., 2004). Briefly,
animals were killed 1 h, 4 h, and 24 h after MCAO. The brains
were removed and cerebral cortex, dissected. The cortex was
immediately frozen and kept at −70 °C. The frozen brain tissue
was homogenized using a homogenizer in 500 μl buffer contain-
ing phosphate buffered saline (PBS) pH 7.2 (1 mmol/L phenyl-
methylsulfonyl fluoride (PMSF), 1 mg/L pepstatin A, 1 mg/L
aprotinin, 1 mg/L leupeptin) and centrifuged at 12,000g for
20 min at 4 °C. Afterwards, supernatant was collected and total
protein was determined by Micro BCA Protein Assay Kit. The
level of TNF-α in ischemic cortex tissue supernatant was
measured using an ELISA kit (Diaclone, France) specific for rat
TNF-α. The measurement of TNF-α was performed step-by-step
based on the protocol booklet of ELISA kit. The TNF-α contents
were expressed as pg TNF-α/mg total protein (Hang et al., 2004).
4.6. Statistical analysis
Data are presented as mean ± SEM. Data were analyzed by
non-parametric Kruskal–Wallis ANOVA on ranks followed by
a Dunn's test. Differences were considered significant at
P < 0.05 (SigmaStat 2.0; Jandel Scientific, Erkrath, Germany).
Acknowledgments
This work was financially supported (Grant No. 243) by
Semnan University of Medical Sciences, Semnan, Iran. We
would like to thank Dr. P. Kokhaei for his critical reading of the
manuscript.
124 BR A I N R ES E A RC H 1 3 7 7 ( 2 01 1 ) 1 1 9 –12 5
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