European Journal of Neuroscience

European Journal of Neuroscience, Vol. 36, pp. 2632–2639, 2012 doi:10.1111/j.1460-9568.2012.08174.x

NEUROSYSTEMS

Experience-dependent brain plasticity after stroke: effect of

ibuprofen and poststroke delay

Jan A. Jablonka,1,2 Malgorzata Kossut,2 Otto W. Witte3 and Monika Liguz-Lecznar 2

1
Department of Animal Physiology, Faculty of Biology, Warsaw University, Warsaw, Poland
2
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland
3
Hans Berger Department of Neurology, Jena University Hospital, Jena, Germany

Keywords: 2DG, inflammation, plasticity, rat, somatosensory cortex

Abstract
Despite indications that brain plasticity may be enhanced after stroke, we have described impairment of experience-dependent
plasticity in rat cerebral cortex neighboring the stroke-induced lesion. Photothrombotic stroke was centered behind the barrel cortex
in one cerebral hemisphere of rats. Plasticity of cortical representation of one row of vibrissae was induced by sensory deprivation of
all surrounding whiskers for 1 month, and visualized with [14C]-2-deoxyglucose autoradiography. In control rats deprivation resulted
in an enlargement of functional cortical representation of the spared row of vibrissae. After a focal stroke neighbouring the barrel
cortex, no plasticity of the spared row representation was found. Investigation of plastic changes with deprivation initiated 1 week and
1 month after stroke have shown that later poststroke onset of deprivation resulted in a partial recovery of cortical plasticity in the
barrel field. Western blot analysis of proinflammatory enzyme cyclooxygenase-2 (COX-2) expression revealed its strong upregulation
in the barrel cortex 24 h after stroke. When chronic treatment with the anti-inflammatory drug ibuprofen (10 mg ⁄ kg or 20 mg ⁄ kg)
accompanied deprivation, plasticity was restored. Ibuprofen applied before the ischemia also prevented the poststroke upregulation
of COX-2. The results strongly suggest that poststroke impairment of experience-dependent cortical plasticity is caused by stroke-
induced inflammatory reactions that subside with poststroke delay and can be at least partially ameliorated by pharmacological
treatment.

Introduction
Poststroke recovery requires rewiring of the brain by restorative neural
plasticity. Unfortunately, the mechanisms underlying this plasticity are
only poorly understood. Several results indicate that the poststroke
brain environment might be supportive for plasticity (Hagemann
et al., 1998; Que et al., 1999; Redecker et al., 2002; Blicher et al.,
2009; Butefisch, 2004). It has been suggested that a favourable time
window for ‘stroke-induced brain plasticity’ opens shortly after stroke
(Biernaskie et al., 2004; Maulden et al., 2005).
In contrast to the hypothesis suggesting facilitation of plasticity
after stroke (see for example Hsu & Huang, 1997; Murphy & Corbett,
2009) we have shown that, following a cortical lesion, experience-
dependent plasticity in the somatosensory cortex (S1) of rat is not
increased, but shows a pronounced depression (Jablonka et al., 2007).
In these experiments, plasticity of cortical representation of vibrissae
was evoked in the barrel field by clipping all but one row of vibrissae.
In control rats this treatment results in enlargement of functional
cortical representation of the spared row, as visualized by [14C]-2-
deoxyglucose (2DG) autoradiography. After a focal photothrombotic
stroke neighbouring the barrel cortex, no experience-dependent
Correspondence: Malgorzata Kossut, as above.
E-mail: kossut@nencki.gov.pl
Received 17 November 2011, revised 27 April 2012, accepted 30 April 2012
rearrangement of the spared row representation could be observed
(Jablonka et al., 2007). It is evident that in the poststroke cortex there
must be factors that counterbalance the plasticity-supporting environ-
ment. It is also clear that these factors subside with time, as brain
plasticity after stroke is well documented.
Brain infarct causes inflammatory responses at the injury site as
well as in the surrounding area (Schroeter et al., 2002; Wang et al.,
2007; Di Filippo et al., 2008). We hypothesized that such inflamma-
tory responses are a reason for the impairment of brain plasticity
following stroke. Indeed, Candelario-Jalil (2008) showed that sup-
pression of the inflammatory responses supports the spontaneous
recovery after stroke. Inflammation in the brain begins within a few
hours after stroke and persists for at least several weeks (Barone &
Feuerstein, 1999; Tan et al., 2003; Wang et al., 2007). It was recently
reported that poststroke impairment of sensory learning is reduced by
treatment with the anti-inflammatory drug ibuprofen (Greifzu et al.,
2011). Also, we have found that experience-dependent cortical
plasticity is rescued by inhibition of metalloproteases (Cybulska-
Klosowicz et al., 2011).
The present experiments were designed to evaluate the time-related
dynamics of poststroke plasticity impairment and to examine whether
suppression of the inflammatory response in the poststroke brain can
improve experience-dependent plasticity. In order to suppress inflam-
matory responses after stroke, we chronically applied ibuprofen, a

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

nonselective cyclooxygenase (COX) inhibitor that crosses the blood–
brain barrier (Parepally et al., 2006). To verify the stroke-induced
inflammatory response we tested the expression of cyclooxygenase-2
(COX-2) in the barrel cortex. COX-2 is a pro-inflammatory enzyme
that participates in glutamate excitotoxicity and cytotoxicity associated
with inflammation after stroke (Cherubini et al., 2005; Bidmon et al.,
2000). The experiments with delayed onset of the deprivation after
stroke were to test whether avoiding the period when inflammation is
strong would enhance the plastic change.
Materials and methods
The experimental protocol was approved by the First Ethical
Commission in Warsaw, Poland, and was in accordance with the
European Communities Council Directive of 24 November 1986
(86 ⁄ 609 ⁄ EEC).
Male Wistar rats (230 g) received a photothrombotic stroke in the
somatosensory cortex caudally and medially to the barrel field
representation (Fig. 1). Sensory deprivation was performed by trim-
ming all the whiskers contralateral to the stroke except for row B. The
trimming was repeated every second day for 1 month.
Surgical procedure
Photothrombotic stroke was inflicted as described previously (Jab-
lonka et al., 2007) under anaesthesia induced by a mixture of 3%
isoflurane in air. The body temperature was controlled and sustained at
36.5 ± 0.5 C. A catheter (0.75 mm) was inserted into the rat’s
femoral vein. An incision was made down the midline of the head and
the skull surface exposed and cleaned. A fibre-optic bundle mounted
on a cold light source was placed stereotaxically on the exposed skull
bone, with its light centre (Ø = 1.5 mm) 4.5 mm posterior to bregma
and 4 mm lateral to the midline (aperture E ⁄ 2; 2750K, KL 1500 LCD;
Schott, Germany). Rose Bengal (0.4 mL, 10 mg ⁄ mL; 330000; Sigma-
Fig. 1. Localization of stroke-induced lesion, 30 days after stroke. Tangential
section across cortical layer IV stained for cytochrome oxidase activity. Arrow
marks the stroke-induced lesion. Arrowheads, rows of barrels; A, anterior; P,
posterior. Scale bar, 1 mm.
ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 36, 2632–2639
Effect of ibuprofen on poststroke plasticity 2633
Aldrich, St Louis, MO, USA) was injected intravenously through a
catheter implanted into the femoral vein and the skull surface was
illuminated for 20 min.
Experimental groups
The following groups were used:
(1) Animals with ibuprofen (a-methyl-4-(isobutyl)phenylacetic acid;
I1892; Sigma-Aldrich, St Louis, MO, USA) intraperitoneal
injections just before the stroke and every day for 1 month, and
whisker deprivation starting immediately after the stroke. Two
different doses of ibuprofen were applied – 10 mg ⁄ kg (n = 6) and
20 mg ⁄ kg. (n = 7).
(2) Animals with deprivation starting with different delays after the
stroke – immediately after stroke (n = 7), 1 week after (n = 4) and
28 days after (n = 5).
(3) Control groups:
intact + whisker deprivation (n = 7);
intact + whisker deprivation + 20 mg ⁄ kg ibuprofen injections
(n = 4);
stroke only (n = 7).
(4) Animals for testing COX-2 expression in the brain
intact (n = 4)
stroke only (n = 5)
stroke + single ibuprofen injection (10 mg ⁄ kg) before stroke
(n = 4)
stroke + single ibuprofen injection (20 mg ⁄ kg) before stroke
(n = 5)
Animals were kept separately in plastic cages in a 12-h light–dark
cycle, at a temperature of  20 C, with free access to food and water.
2DG mapping
After a month of vibrissae deprivation, we performed 2DG functional
brain mapping. The animals were put in a restrainer and the whiskers
of the non-deprived side, except for row B, were cut close to the skin.
2DG, which was labelled with 14C (7 lCi ⁄ 100 g, specific activity
55 mCi ⁄ mmol; American Radiolabeled Chemical, St Louis, MO,
USA), was injected intramuscularly. The whiskers of rows B on both
sides of the snout were stroked in a rostrocaudal direction twice per
second with a hand-held mechanical stimulator. After 30 min of
stimulation, the rats were killed with an i.p. Vetbutal (0.4 mL ⁄ 100 g;
Biovet, Pulawy, Poland) injection and perfused with 4% paraformal-
dehyde (Sigma-Aldrich, St Louis, MO, USA) for 3 min. Then the
brain was removed and the cortex of both hemispheres flattened
between glass slides, frozen in heptane at )70 C and stored at this
temperature. Hemispheres were cut on a cryostat at )20 C into 40-
lm tangential sections, which were collected alternately on specimen
slides and cover slips. The sections on cover slips were immediately
dried and exposed on Kodak Mammography film for 2 weeks with a
set of [14C] standards (American Radiolabeled Chemicals, St. Louis,
MO). The remaining sections were stained for cytochrome oxidase
activity to identify the barrel field.
COX-2 expression analysis
The expression of COX-2 was analysed using the Western blot
method in barrel cortex homogenates 24 h after stroke induction The
tissue samples were taken from both hemispheres according to the
method described by Strominger & Woolsey (1987). The protein was
2634 J. A. Jablonka et al.
isolated using a Total Protein Extraction Kit (Millipore Billerica, MA,
USA) and protein concentration was measured in each probe with
Pierce BCA Protein Assay Kit (Thermo Scientific Rockford, IL,
USA). Ten micrograms of total protein from each sample was loaded
onto 9% SDS-PAGE gel and run at 100 V for 3 h. Proteins were
transferred onto Immobilon P membrane (Millipore Billerica, MA,
USA). Nonspecific binding was blocked by incubating membranes in
10% nonfat milk for 2 h at room temperature and then membranes
were incubated overnight at 4 C with polyclonal epitope-specific
rabbit antibody against COX-2 (no. DLN-15526; 1 : 200; Dianova,
Hamburg, Germany) and mouse antiactin monoclonal antibody
(MAB1501; 1 : 1000; Millipore Billerica, MA, USA). After washing
in Tris-buffered saline with Tween, membranes were incubated with
biotinylated antirabbit secondary antibody (1 : 200; Vector Labora-
tories, Burlingame, CA, USA) and biotinylated antimouse secondary
antibody (1 : 200; Vector Laboratories, Burlingame, CA, USA),
followed by horseradish peroxidase-linked avidin (Vector). Blots were
visualized enzymatically with diaminobenzidine as a substrate [SIG-
MAFAST 3,3¢-diaminobenzidine (DAB) tablets; Sigma].
Data analysis
The infarct volume was estimated 1 month after stroke in a separate
group of rats (n = 7), on sections cut in the coronal plane stained for
cytochrome oxidase and examined with a Nikon Eclipse 80i
microscope equipped with a digital camera (Evolution VF; MediaCy-
bernetics Bethesda, MD, USA). In order to obtain the lesion volume,
the lesion area was measured in every third section (40-lm sections).
The lesion volume was calculated by multiplying the lesion area by
the distance between the sections and summing the results.
The autoradiograms were analyzed by a computer image analysis
program (MCID Res Inc., Ontario, Canada). The software enables
display of an image of a stained section (from which the autoradio-
gram was obtained) and an adjacent cytochrome oxidase-stained
section which was superimposed on the autoradiogram so that the
relations of the barrel field and the position of the stroke could be
accurately determined (Fig. 1). The width of the 2DG-labelled cortical
representation of row B whiskers was measured on every section at
three locations across the row. Pixels with 2DG uptake intensity
exceeding the mean of the surrounding cortex by > 15 % were
considered labelled representations. Results, tested first for relatively
equal widening within layers, were averaged for all sections (layers II–
VI) of one hemisphere. The significance of the differences in labelling
of row B representation between the brain hemispheres was calculated
with paired two-tailed Student’s t-tests. Comparisons of row B
representation width between experimental groups were made sepa-
rately for control and experimental hemispheres with one-way anova.
The statistical analysis for control hemispheres did not revealed any
significant differences between experimental groups (anova – F7,39 =
1.76, P = 0.12). Statistical analysis of differences between experimental
hemispheres revealed significant effects of experimental situation on the
width of the row B cortical representations (anova – F7,39 = 14,
P = 0.003). Results of post hoc comparisons (Tukey–Kramer multiple
comparisons test) between respective groups are presented in the results.
Western blots were densitometrically quantified using ImageJ
software (NIH, Bethesda, MD, USA). Absolute optical density (OD)
was normalized to the ODs of the corresponding bands of actin
loading control and expressed as relative abundance in arbitrary units.
For the statistical analysis, ratios of COX-2 band intensities in
experimental hemispheres to respective control hemispheres were
calculated and compared between experimental groups. The statistical
ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
analysis for comparison of the experimental groups was carried out
with Kruskal–Wallis one-way anova (sigma plot software; Systat
Software, San Jose, CA, USA). For post hoc pairwise comparison the
Tukey–Kramer test was used.
Results
Stroke-induced lesion
The extent of cortical lesion 1 month after the stroke was defined on
cytochrome oxidase-stained sections. The lesion was situated pos-
teromedially to the barrel field (Fig. 1; marked with arrow) in the
posterior parietal cortex and extended through all cortical layers,
tapering off in layer VI. The mean lesion volume in animals killed
1 month after the stroke was 0.8 ± 0.3 mm3 and the mean distance of
the stroke-induced lesion edge from the B1 barrel edge was
1200 ± 208 lm. The infarct was confined to the cortical tissue and
in any case it did not included the subcortical white matter. Ibuprofen
had no influence on the volume of the lesion core as the volume of the
lesion in treated animals was 0.6 ± 0.3 mm3 vs. 0.8 ± 0.3 mm3 in the
stroke group without the treatment (unpaired t-test – t6 = 1.39,
P = 0.18).
The lesion was reflected by the reduced intensity of 2DG uptake
(Fig. 2; marked with asterisk). We estimated the extent of the dip in
metabolic activation. No significant differences were observed
between groups. The region of lowered metabolism was more
extensive than the lesion and amounted to 1.2 ± 0.3 mm3 in rats
without pharmacological treatment, 1 ± 0.4 mm3 in rats with
10 mg ⁄ kg ibuprofen and 1 ± 0.5 mm3 in rats receiving 20 mg ⁄ kg
ibuprofen.
2DG labelling of row B cortical representations
Bilateral stimulation of row B vibrissae during 2DG brain mapping
enhanced isotope incorporation in the barrel fields of both hemi-
spheres. The functional representation of row B whiskers was visible
as a band of increased OD situated over histological row B barrels in
layer IV, but discernible on the autoradiograms from layer II to VI
(Jablonka et al., 2007). Unilateral deprivation of all vibrissae except
row B resulted in significant (50%) widening of the spared row B
representation in the contralateral hemisphere, as compared to control
row B representation in the non-deprived hemisphere (1196 ± 119 vs.
798 ± 34 lm, paired t-test – t6 = 8.67, P = 0.0001; Figs 2 and 5).
The stroke did not influence the width of row B representations in
non-deprived animals, either in the lesioned hemisphere or in the
intact (806 ± 74 and 803 ± 67 lm respectively; paired t-test –
t6 = 0.14, P =0.9).
Effect of poststroke delay on deprivation-induced plasticity
Whisker deprivation that started immediately after the stroke did not
result in significant widening of the spared row B representation
(856 ± 107 vs. 889 ± 67 lm; paired t-test – t3 = 8.58, P = 0.34;
Figs 2 and 5). When we postponed the deprivation until 1 week after
the stroke, the spared row B whiskers cortical representation was
significantly wider than the non-deprived side row B representation
(20%; 961 ± 37 vs. 798 ± 24 lm; paired t-test – t6 = 8.67, P = 0.003;
Fig. 5A). However, it did not differ significantly from that in either
non-deprived animals after stroke or rats deprived immediately after
stroke (961 ± 37, 806 ± 74 and 898.8 ± 67 lm respectively; Tukey–
Kramer test – F7,39 = 14, P = 0.23; Fig. 3) Thus, 1 week after the
European Journal of Neuroscience, 36, 2632–2639
 Effect of ibuprofen on poststroke plasticity 2635

Fig. 2. Effect of stroke upon experience-dependent plasticity. Digitized pseudocoloured autoradiograms of tangential cortical sections at the level of layer IV
presenting the 2DG labelling following stimulation of row B vibrissae in intact controls, in rats with all vibrissae except row B removed for 30 days (deprivation),
and in rats with stroke and deprivation. Arrows point to labelling corresponding to stimulated row B representations. Asterisk marks the stroke-induced lesion; Au,
auditory cortex; x, air bubble artefact.

Fig. 3. High magnification of 2DG-labelled row B representation in the poststroke groups, on digitized autoradiograms of tangential sections of the cortex at
different poststroke time points. Asterisk marks the stroke-induced lesion.

stroke, the plasticity-impeding factors were less effective but still
present. The extent of plastic change in that group was smaller than in
the deprived controls without stroke (20% vs. 50%) and the width of
spared-row representations amounted to 961 ± 37 and
1196 ± 119 lm respectively (Tukey–Kramer test – F7,39 = 14,
P = 0.009; Fig. 5A).
When deprivation started 1 month after stroke the spared-row
representation was enlarged by 25% (1001 ± 153 lm in the deprived
vs. 797 ± 88 lm in the non-deprived hemisphere; paired t-test –
t4 = 4.93, P = 0.008; Fig. 5A). This did not differ from deprivation in
the group delayed by 1 week (1001 ± 153 and 961 ± 37 lm respec-
tively; unpaired t-test – t7 = 0.51, P = 0.63; Fig. 3) and still differed
significantly from that observed in deprived rats without stroke (the
widths of spared-row representations were 1001 ± 153 and
1196 ± 119 lm, respectively; Tukey–Kramer test – F7,39 = 14,
P = 0.006; Fig. 5A). Thus, even after a long poststroke delay the
extent of the plasticity change in cortical representation did not reach
the control value.
Effect of ibuprofen
Ibuprofen by itself did not influence plasticity in healthy animals. Rats
without stroke, subjected to deprivation and treated with ibuprofen
(20 mg ⁄ kg), had the same enlargement of spared whiskers represen-
tation (54%) as control animals (50%). The width of the spared row B
representation was 1140 ± 41 vs. 740 ± 23 lm in the non-deprived
hemisphere (paired t-test – t3 = 39.91, P = 0.00003; Fig. 5B).
After stroke, daily ibuprofen injections at a dose of 10 mg ⁄ kg
resulted, in whisker-deprived rats, in a statistically significant increase
in the width of the spared-row cortical representation compared to the
contralateral hemisphere (17% of widening; 1029 ± 63 vs.
877 ± 86 lm; paired t-test – t5 = 5.79, P = 0.002; Fig. 4). However,
the width of labelling was not significantly different from that in the
group with stroke but without ibuprofen. The extent of the plastic
change was smaller than in the deprived rats without stroke
(1029 ± 63 vs. 1196 ± 120 lm; Tukey–Kramer test – F7,39 = 14,
P = 0.03; Fig. 5B).
Injections of a higher dose (20 mg ⁄ kg) of ibuprofen had a stronger
influence on cortical remodelling, as the spared-row representation
widened by 33% (1067 ± 63 vs. 805 ± 70 lm; paired t-test –
t6 = 11.3, P = 0.00003; Fig. 4). The width of spared-row representa-
tion in that group did not differed significantly from the width of
spared-row representation in the control group with deprivation
(1067 ± 63 and 1196 ± 120 lm; Tukey–Kramer test – F7,39 = 14,
P = 0.12; Fig. 5B) and was significantly greater than in the group with
stroke and without ibuprofen (1067 ± 63 vs. 806 ± 74 lm; Tukey–
Kramer test – F7,39 = 14, P = 0.001; Fig. 5B).
Poststroke expression of COX-2
The Western blot analysis revealed a single immunoreactive band of
70 kDa which corresponded with COX-2 protein (Fig. 6). In control
intact animals COX-2 expression was low and no interhemispheric
differences were found, giving a value of 1.14 ± 0.26 for the
interhemispheric COX-2 expression ratio. In stroke animals without
ibuprofen treatment the expression of COX-2 in the hemisphere
contralateral to the stroke was similar to that in control tissue, whereas
in the affected hemisphere COX-2 expression increased compared to
the contralateral barrel cortex, giving a stroke to control hemisphere
ratio > 2· higher than in control animals (2.99 ± 0.32 vs. 1.14 ± 0.26,
respectively; anova – F17,3 = 33.92; P = 0.004). A single injection of
the lower dose of ibuprofen (10 mg ⁄ kg) before the stroke induction did

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 36, 2632–2639
2636 J. A. Jablonka et al.
Fig. 4. High magnification of digitized autoradiograms of tangential sections
of the cortex showing the effect of ibuprofen on plasticity of row B
representation in the poststroke groups.
not change the COX-2 expression in barrel cortex neighbouring the
infarct area. The interhemispheric ratio of COX-2 expression was
similar to that in the stroke group (2.81 ± 0.35 vs. 2.99 ± 0.32; anova
– F17,3 = 33.92; P = 0.001). However, increasing the ibuprofen dose
to 20 mg ⁄ kg significantly attenuated the augmentation of COX-2
expression in the injured hemisphere to give a ratio value of 1.5 ± 0.39,
which did not differ significantly from the control values (Fig. 6).
Discussion
Our results demonstrate that, following stroke, experience-dependent
plasticity of cortical vibrissae representations was impaired. This
impairment of plasticity decreased when the poststroke onset of
deprivation was delayed. The plasticity may be restored by application
of ibuprofen, showing that the poststroke impairment of brain
plasticity is caused at least partially by stroke-induced inflammatory
reactions in the brain. This thesis is supported by the results of COX-2
expression Western blot analysis, which revealed an elevation in
ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
Fig. 5. Effect of deprivation on cortical representation plasticity
(mean ± SD). (A) Influence of the onset of induction of poststroke plasticity.
(B) Influence of different doses of ibuprofen on the poststroke plasticity. The
differences between hemispheres are marked with asterisks (*). Dots (•) mark
differences in relation to experimental hemisphere of intact group with
deprivation. Diamonds (¤) refer to the relations between experimental
hemispheres of particular groups. •P < 0.05; **,••,¤¤P < 0.01; ***,•••P < 0.001.
COX-2 levels in somatosensory cortex 24 h after ischemia, an effect
that was abolished with application of ibuprofen prior to the stroke
induction. We excluded the possibility that the ibuprofen by itself
influenced plasticity (Fig 5).
Partial whisker deprivation leads to enlargement of the cortical
representation of the spared whiskers at the expense of the
representation of the removed whiskers. This cortical map reorga-
nization involves CaMKII and CRE and requires NMDA receptor-
dependent induction of long-term potentiation (Glazewski et al.,
2000; Barth et al., 2000). It also involves depression which is similar
to long-term depression and weakening of intracortical synaptic
connections (Drew & Feldman, 2009). Activity of intracortical
inhibitory circuits is altered in the whisker-deprived representation
(Li et al., 2009). Loss and growth of dendritic apical branches is
enhanced, and their spines undergo rearrangement (Tailby et al.,
2005). Within days, axons on both excitatory and inhibitory neurons
elongate in the direction of the neighbouring cortex deprived of
sensory input (Marik et al., 2010). We found that stroke strongly
affected this type of cortical plasticity. With a focal stroke in the
vicinity of the barrel field, we observed a complete loss of use-
dependent remodelling of cortical representations in the afflicted
hemisphere (Figs 2 and 3). The effect was not due to impairment of
stimulation-driven 2DG uptake in the lesioned hemisphere because
we found previously that, 1 month after stroke, the 2DG labelling of
cortical row B representation in non-deprived animals was the same
in the hemisphere with and without stroke; it also did not differ from
the extent of labelling in controls without the stroke (Jablonka et al.,
2007).
European Journal of Neuroscience, 36, 2632–2639

Fig. 6. Influence of unilateral photothrombotic stroke and ibuprofen treatment
upon the COX-2 expression in adjoining rat barrel cortex. Top panel shows
examples of the blots. The position of protein markers (kDa) is indicated at the
left of the panel. A unique immunoreactive band, corresponding to a protein of
70 kDa, was detected with anti-COX-2 antibody in all homogenates. Lower
bands of 43 kDa show immunostaining for actin, used as a loading control.
Bottom panel shows quantification of COX-2 expression changes in somato-
sensory cortex after photothrombotic stroke and ibuprofen treatment. Bars
represent the mean ± SD ratio of COX-2 expression in somatosensory cortex of
experimental (with infarct) hemisphere to control hemisphere. **P < 0.01;
***
P < 0.001.
The position of the lesion may be important for creating a pro-
plastic milieu at a distant but strongly connected site (Carmichael
et al., 2005). We did not examine this aspect in our experiments. The
lesioned region is very sparsely connected with the barrel field (Fabri
& Burton, 1991). We concentrated here on diffuse non-directed effects
of stroke on cortical plasticity, expecting a facilitatory effect (Dohle
et al., 2009). Instead, we found a strong suppression of experience-
dependent plasticity in a region neighbouring the stroke, suggesting a
generalized reduction of ability to modify cortical representations in
the vicinity of cortical damage. Recently, influence of the same kind of
stroke on the ocular dominance plasticity in mice has been observed
(Greifzu et al., 2011). Ocular dominance plasticity shares mostly the
same cellular mechanisms as the S1 experience-dependent remodel-
ling (Buonomano & Merzenich, 1998). It can be assumed that Greifzu
et al. (2011) observed the same phenomenon, the impairment of
experience-dependent cortical representation plasticity after focal
stroke in the vicinity of the visual cortex, as we observed in the
barrel field of rats and mice (Jablonka et al., 2007; Cybulska-
Klosowicz et al., 2011).
In contrast with the results of Greifzu et al. (2011), we found that
the changes in brain physiology appearing immediately after the stroke
ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 36, 2632–2639
Effect of ibuprofen on poststroke plasticity 2637
are the most detrimental to the experience-dependent plasticity. When
the deprivation began 7 days after the stroke, the plastic change in the
representation of the spared whiskers was partially re-established,
although still significantly smaller than in the rats without stroke.
Greifzu et al. (2011) did not observed any beneficial effect of
poststroke delay upon monocular deprivation-induced ocular domi-
nance shift. However, in the same animals, a poststroke delay of 1 or
2 weeks improved visual acuity and contrast sensitivity which had
been impaired by the stroke.
Our previous results, which demonstrated considerable remapping of
cerebral cortex after the stroke, indicated that the heightened activation
of many cortical regions, activity not evoked by stimulation of vibrissae,
persisted up to 4 weeks after stroke onset, while 2 months after stroke
the interhemispheric equilibrium stabilized (Jablonka et al., 2010). We
observed here that 3 weeks difference in the onset of deprivation
between the groups (1 week to 1 month delay) influenced the experi-
ence-dependent plasticity improvement by just a few per cent, similarly
to the effect of the 1-week delay. Apparently, the factors impeding
plasticity after stroke recede partially before the 7th poststroke day and
thereafter until the 28th day do not ameliorate remarkably. One may
therefore assume that disorganized brain activity disturbs the proper
brain response to the plasticity-inducing experience. Perhaps even
longer poststroke delay of deprivation would restore the plasticity of
cortical representation to the control value.
On the other hand, the immediate pathologies, such as the poststroke
blood supply restriction and reperfusion (Schaller & Graf, 2004), energy
failure and decrease in ion pump activity (Hossmann, 1999), spreading
depression (Dietrich et al., 1994), metabolic changes and oedema
(Marmarou, 2007) are detrimental to plasticity. Stroke-induced inflam-
mation, by itself or by some indirect influences (Wang et al., 2008; Di
Filippo et al., 2008), may also impair experience-dependent cortical
reorganization. Our results showed that 24 h following injury a
proinflammatory enzyme COX-2 was highly upregulated in the
somatosensory cortex of the lesioned hemisphere. Earlier, Ahmad
et al. (2009) showed increased expression of COX-2 protein 4 and 24 h
after middle cerebral artery occlusion, and Bidmon et al. (2000)
observed a similar effect after cerebral cortical photothrombosis. Also,
similarly to the above-mentioned papers, the increase we observed was
confined to the hemisphere which had suffered the stroke. Based on the
literature data we tested COX-2 expression 24 h after induction of
ischemia, as at later survival times it was found to return to control
values (Bidmon et al., 2000; Strauss et al., 2000).
The anti-inflammatory drug ibuprofen partially re-established the
poststroke plasticity. Western blot analysis revealed that ibuprofen
also had a strong effect upon COX-2 expression. A single application
of the higher ibuprofen dose (20 mg ⁄ kg) prevented the COX-2
upregulation in the vicinity of the infarct 24 h after the stroke. The
lower dose (10 mg ⁄ kg) was ineffective in attenuating COX-2
expression. However, with chronic application of ibuprofen at the
lower dose, a small increase in plasticity of the peri-infarct barrel
cortex was observed when the results from deprived and control
hemispheres were compared. This change was not large enough to be
significantly different from that observed in rats who had undergone
stroke without ibuprofen. The higher ibuprofen dose was sufficient to
restore experience-dependent plasticity in the barrel cortex.
Ibuprofen might interact directly as a nonselective inhibitor of
cyclooxygenases or by influencing transcription factors – nuclear
receptor protein peroxisome proliferator-activated receptors (PPARs;
Kojo et al., 2003). Both cyclooxygenases and PPARs may influence
the expression of MMP-9 metalloproteinase (Wang et al., 2008; Yin
et al., 2011), which is important both for the integrity of the blood
brain barrier and for structural plasticity mechanisms participating in
2638 J. A. Jablonka et al.
the reorganization of dendritic spines (Wang et al., 2008). The MMP-
9-induced impairment of poststroke plasticity changes has recently
been demonstrated in mice, in the same experimental model of
photothrombotic stroke and experience-dependent plasticity changes
(Cybulska-Klosowicz et al., 2011). Thus, one of the potential
mechanisms for the beneficial effect of ibuprofen on poststroke
plasticity might involve counteracting the unfavourable effects of
MMP-9 upregulation after stroke.
Recent results on mice show no influence of ibuprofen on the size
of focal photothrombotic stroke and no influence on decreased ocular
dominance plasticity after stroke, but a pronounced improvement in
visual acuity and contrast sensitivity in ibuprofen treated mice
(Greifzu et al., 2011). The discrepancy between our results concerning
the effect of ibuprofen on cortical experience-dependent remapping in
the barrel cortex, and those shown by Greifzu et al. (2011) in the
visual cortex, despite the similar model of the focal photothrombotic
stroke, may well be due to the difference in drug treatment onset
(30 min before in our model and just after the stroke in the Greifzu
et al. experiment) and ⁄ or the ibuprofen dose we used (the highest
dose we used was 20 mg ⁄ kg whereas Greifzu et al. (2011) treated
mice with 60 mg ⁄ kg). Also, in our brain mapping experiments the rats
were not anesthetized, while deep anaesthesia was used by Greifzu
et al. (2011) for optical recording of ocular dominance changes. As
anesthesia dampens the brain’s metabolic response (Shapiro et al.,
1978), small increases in ocular dominance shift might have been
missed by the experimenters. Some other factors might also have been
of some importance – the experimental animals (mice vs. rats), cortical
area tested (S1 vs. V1), different durations of deprivation (4 weeks for
whiskers vs. 2 weeks monocular).
It is important to stress here that our experimental design did not
address the issue of recovery of function. We examined only cortical
remodelling in response to changed sensory input. Our results
demonstrate a decrease in experience-dependent plasticity in the
cortex neighbouring a focal cortical stroke and its partial reestablish-
ment after application of an anti-inflammatory drug, ibuprofen. In the
acute phase, stroke reduces plastic remodelling of the cortex; with
longer poststroke interval plasticity is partially restored. We probably
observed a sequential action of several factors interfering with
plasticity that diminished with increasing poststroke survival time.
The injection of the anti-inflammatory drug may act on one or several
of these factors; this requires further studies. However, even when the
deprivation was applied 1 month after the stroke, the plastic change in
the cortical representation was not more extensive than in the control
rats without stroke. No pro-plastic effect of stroke was observed in our
experience-dependent plasticity model.
Acknowledgements
The research was supported by the Polish–German Cooperation Program in
Neuroscience grant no. S006 ⁄ -P-N ⁄ 2007 ⁄ 01 and Polish Ministry of Science and
Higher Education grant no. 0133 ⁄ P01 ⁄ 2010 ⁄ 70. We thank Renata Zakrzewska
Marcin Kazmierczak and Ewa Nosecka for excellent technical assistance.
Abbreviations
14
2DG, C 2-deoxyglucose; COX, cyclooxygenase.
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