Blood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain.

Abstract

The slow time course of neurodegeneration after brain ischemia/reperfusion opened a realistic time window for the application of protective therapies to prevent spreading of brain damage. In this work, we studied the ability of micromolar concentrations of this flavonoid in the blood to protect against brain damage induced by transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H-E) staining, 'in situ' terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL), 'in situ' metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining. Intravenous injections of kaempferol, at a dose of 10-15 mumol/L of blood 30 min before the induction of a 60 min ischemia-episode and just after reperfusion, led to >90% and 70-80% (TTC, H-E, TUNEL) decrease of brain damage in the temporal-frontal areas of neocortex and striatum, respectively, but only 40-50% decrease of brain damage was observed in the hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also produced a similar reduction of metalloproteinase activation and loss of anti-laminin staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly complete protection against the increase of protein nitrotyrosines, and also afforded strong protection against the increase of apoptotic cell death (TUNEL) and biochemical markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase degradation. On these grounds, a potential new therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested.

Full text

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Research Report
Blood micromolar concentrations of kaempferol afford
protection against ischemia/reperfusion-induced
damage in rat brain
Carmen López-Sáncheza, Francisco Javier Martín-Romerob, Fei Sunc, Laura Luisc,
Alejandro K. Samhan-Ariasb, Virginio García-Martíneza, Carlos Gutiérrez-Merinob,⁎
aHuman Anatomy and Embryology, Faculty of Medicine, University of Extremadura, PO Box 108, 06080 Badajoz, Spain
bDepartment of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06071-Badajoz, Spain
cMinimally Invasive Surgery Centre (CCMI), 10071 - Cáceres, Spain
A R T I C L E I N F OA B S T R A C T
Article history:
Accepted 30 August 2007
Available online 21 September 2007
The slow time course of neurodegeneration after brain ischemia/reperfusion opened a
realistic time window for the application of protective therapies to prevent spreading of
brain damage. In this work, we studied the ability of micromolar concentrations of this
flavonoid in the blood to protect against brain damage induced by transient focal cerebral
ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery
occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium
chloride (TTC) staining, hematoxylin–eosin (H–E) staining, ‘in situ’ terminal
deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL),
‘in situ’ metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining.
Intravenous injections of kaempferol, at a dose of 10–15 μmol/L of blood 30 min before the
induction of a 60 min ischemia-episode and just after reperfusion, led to N90% and 70–80%
(TTC, H–E, TUNEL) decrease of brain damage in the temporal–frontal areas of neocortex and
striatum, respectively, but only 40–50% decrease of brain damage was observed in the
hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also
produced a similar reduction of metalloproteinase activation and loss of anti-laminin
staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected
against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly
complete protection against the increase of protein nitrotyrosines, and also afforded
strong protection against the increase of apoptotic cell death (TUNEL) and biochemical
markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase
Keywords:
Kaempferol
Brain ischemia/reperfusion
Protection against brain oxidative
stress
Brain protection against apoptosis
Transient ischemic attack
Animal models of human disease
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⁎ Corresponding author. Fax: +34 924 289419.
E-mail address: carlosgm@unex.es (C. Gutiérrez-Merino).
Abbreviations: AcLEHD-AFC, acetyl-LEHD-7-amido-4-(trifluoromethyl)coumarin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; H–E, hematoxylin
and eosin staining; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; IC50, concentration needed to produce 50% of the
biological effect; i.v, intravenous; MMP, extracellular matrix metalloproteinase; PARP, poly-(ADP-ribose) polymerase; PBS, phosphate
buffered saline; PBS-T, PBS supplemented with 0.05% Tween-20; POD, peroxidase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis; S.E, standard error; TCA, tricholoroacetic acid; TTC, 2,3,5-Triphenyltetrazolium chloride; TUNEL, terminal deoxyribonu-
cleotidyl transferase-mediated dUTP-fluorescein nick end labeling; Tween-20, polyethylene glycol sorbitan monolaurate
0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2007.08.087
available at www.sciencedirect.com
www.elsevier.com/locate/brainres

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degradation. On these grounds, a potential new therapeutic role of kaempferol to acute
treatment of ischemic stroke is suggested.
© 2007 Elsevier B.V. All rights reserved.
1. Introduction
Neurodegeneration induced by ischemic brain insults has
been shown to follow characteristic spatio-temporal patterns,
and this has lead to the identification of areas displaying rapid
necrotic death surrounded by areas undergoing slowly devel-
oping cell death (Hossmann, 1994; Lehrmann et al., 1997;
Iadecola, 1997). Extracellular matrix degradation upon activa-
tion of matrix metalloproteinases (MMP) has been shown to
take place in transient focal cerebral ischemia, which likely
plays a major role in the spatial spreading of neurodegenera-
tion in brain, largely due to the activation of MMP-2 and MMP-
9 (Romanic et al., 1998; Heo et al., 1999; Asahi et al., 2000; Gu et
al., 2002, 2005; Chang et al., 2003). These experimental
observations are consistent with the occurrence of different
types of neuronal death after brain ischemia/reperfusion
insults developing with very different time windows, like
glutamate-mediated excitotoxicity (Szatkowski and Atwell,
1994) or apoptosis (Saito et al., 2004; Copin et al., 2005; Gu et al.,
2005). This is particularly relevant to protect against brain
degeneration after ischemia/reperfusion, because the slow
time course of neurodegeneration opened a realistic time
window for the application of protective therapies. Basal
ganglia, particularly the striatum and the hippocampus, and
cortical neurons have been shown to be the brain areas most
sensitive to ischemia/reperfusion (Lehrmann et al., 1997; Gu et
al., 2005). Caspases involved in apoptotic cell death in
experimental models of focal and global brain ischemia have
been reported to follow different temporal profiles of activa-
tion in rat brain (Cao et al., 2002; Cho et al., 2003). For instance,
while caspase-3 peaked in the penumbral cortex at 6–12 h
following ischemia, significant induction and levels of pro-
teolytically activated caspase-9 can be better seen after 24 h
post-ischemia (Cho et al., 2003). Moreover, administration of
the caspase-9 inhibitor Z-Leu-Glu(Ome)-His-Asp(Ome)-FMK
after focal cerebral ischemia has been reported to reduce the
infarct volume by 49% and to improve neurological outcome
(Mouw et al., 2002). This experimental observation is consis-
tent with the proposal of caspase-9 as a mediator of oxidative
stress-induced apoptosis after focal cerebral ischemia (Saito
et al., 2004).
On the other hand, it has been shown that peroxynitrite
and other reactive oxygen species play a major role in brain
damage associated with ischemia/reperfusion (Chan, 1996;
Iadecola, 1997; Gu et al., 2002; Saito et al., 2004). Plant
flavonoids are peroxynitrite scavengers of low toxicity for
mammals found in many vegetables commonly used in
human nutrition (Manach et al., 1996; Hollman and Katan,
1997). Several studies have shown protective effects of
different flavonoids against ischemia/reperfusion brain dam-
age after transient focal cerebral ischemia or global cerebral
ischemia in model experimental animals, e.g. Crataegus
flavonoids in gerbils (Zhang et al., 2004), epigallocatechin
gallate in rats (Choi et al., 2004) and gerbils (Lee et al., 2004),
ginsenosides in rats (Tian et al., 2005; Zhou et al., 2006),
isoliquiritigenin in rats (Zhan and Yang, 2006), Scutellaria
baicalensis Georgi flavonoids in rats (Shang et al., 2006) and
resveratrol in rats (Tsai et al., 2007). In a previous study, we
showed that micromolar concentrations of kaempferol
afforded full protection against the apoptosis of rat cerebellar
granule neurons in culture induced by K+-deprivation in the
extracellular medium (Samhan-Arias et al., 2004), a well
established model for neuronal apoptosis (D'Mello et al.,
1993; Nardi et al., 1997; Martin-Romero et al., 2000, 2002).
Other flavonoids, such as apigenin and quercetin, were found
to be much less effective than kaempferol (Samhan-Arias et
al., 2004). In contrast, kaempferol protection against gluta-
mate-induced excitotoxicity to neurons in culture is relatively
low at micromolar concentrations of this flavonoid (Ishige et
al., 2001; Kim et al., 2001).
Transient focal cerebral ischemia in rats can be readily
attained by middle cerebral artery occlusion (Longa et al.,
1989; Belayev et al., 1996). We have recently developed a new
surgical approach, which we called “transfemoral selective
‘intraluminal wiring’ technique” (Sun et al., 2005), by
selective endovascular placement of a guidewire into a target
vessel under fluoroscopic guidance. Transfemoral selective
‘intraluminal wiring’ is a minimum invasive technique that
minimized subarachnoid hemorrhage (Sun et al., 2005),
which has been reported to occur with the intraluminal
thread model of middle cerebral artery occlusion in rats
(Longa et al., 1989; Schmid-Elsaesser et al., 1998).
The major aim of this work is to evaluate the efficiency of
acute administration of micromolar concentrations of kaemp-
ferol in blood as neuroprotector agent against brain degener-
ation after transient focal ischemia/reperfusion elicited by
middle cerebral artery occlusion in rats.
2.Results
2.1.
brain damage after ischemia/reperfusion
Protection by micromolar kaempferol in blood against
The low toxicity of kaempferol to Wistar rats was ascertained
before running experiments with transient focal cerebral
ischemia. None of the adult Wistar rats (n=6) treated during
3 days with intraperitoneal injections of 11 mg kaempferol per
day died within a 2 week observation period, and this was
consistent with the lack of toxicity of this flavonoid noticed
previously (Manach et al., 1996; Hollman and Katan, 1997).
Taking into account that kaempferol affords nearly complete
protection of cerebellar granule neurons in culture with a
concentration needed to produce 50% of the effect (IC50) of 8±
2 μmol/L (Samhan-Arias et al., 2004), we decided to treat rats
with intravenous injections of kaempferol a short time prior
the ischemic insult, such that upon dilution within the overall
rat blood volume, approximately 6 mL/100 g for adult rats
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(Hauptman et al., 1989; Sakai et al., 2004), the blood concen-
tration of kaempferol varied between (IC50/2) and (2·IC50)
determined for neurons in culture. This was be attained with
two i.v. injections of 0.7 mL of 50, 100 and 200 μmol/L
kaempferol in PBS, i.e. 0.01, 0.02 and 0.04 mg kaempferol per
injection, to get kaempferol concentrations in the blood close
to (IC50/2), IC50and (2·IC50), respectively. Kaempferol solutions
in PBS used for i.v. injections were prepared from 500-fold
concentratedstocksolutionsofkaempferolinDMSO,suchthat
thevolumeofthevehicle(DMSO)administeredwasminimized
to 1.4 μL per i.v. injection.
A group of 36 experimental rats were subjected to two
injections of 0.7 mL of PBS containing 50 (n=9), 100 (n=18) or
200 (n=9) μmol/L kaempferol slowly administered through a
catheter inserted intothe tail vein, 30 min before focal cerebral
ischemia (injection 1) and just after reperfusion (injection 2).
Another 18 control rats were subjected to the same treatment
only with the vehicle for kaempferol administration, namely
1.4 μL of DMSO in 0.7 mL PBS. The lack of toxicity of these
treatments was assessed with additional controls rats (n=9)
treated with kaempferol without the ischemia/reperfusion
surgical intervention, which were kept under observation for
two days. None of these control animals died, nor showed
neurological deficits, evaluated as indicated in Experimental
procedures.
Rats treated with the IC50of kaempferol, i.e. with two i.v.
injections of 100 μmol/L, showed a large reduction of the brain
infarct area monitored by TTC and also by H–E staining (Fig. 1).
The results pointed out that the treatment with kaempferol
producedanalmostcompletereductionoftheinfarctareainthe
brain neocortex (i.e. higher than 90% reduction), as dense white
areas cannot be seen in TTC staining. However, the decrease of
TTC staining observed at the temporal-frontal regions of the
striatum (slice at 6 mm from the front), pointed out that damage
is more restricted to scattered groups of cells. Therefore, in order
to quantitatively evaluate the extent of protection afforded by
kaempferolinthemajorbrainregionsofthisslice(neocortexand
striatum),TTC-stainedsampleswereexcised,homogenatedand
lysed in PBS-T as indicated in Experimental procedures. The
results of the absorbance readings at 490 nm for striatum and
neocortex of slices taken at 6 mm from the forefront edge of the
brainareshowninFig.2,andindicatedthatkaempferolafforded
more than 75% protection against the ischemia/reperfusion
brain damage monitored by TTC in these areas. In the temporal–
Fig. 1 – Kaempferol protected against ischemia/reperfusion-induced brain damage as monitored by TTC. Serial 2-mm rat brain
coronal slices stained with TTC are shown for control (DMSO-treated) and rats treated with i.v. injections of 100 μmol/L
kaempferol, panels A and B, respectively. The position of the slices within the rat brain is schematically indicated over the
picture of the rat brain shown at the left (see also Table 1). I and C mean ischemic and control hemispheres, respectively. The
images shown in panels A and B are representative of the results obtained with n=6 control and n=6 kaempferol-treated rats.
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caudal brain sections (coronal slices from 8 to 12 mm from the
front pole of the rat brain), the reduction of the infarct area was
estimated from the reduction of the size of dense white areas,
and indicated that damage by ischemia/reperfusion at caudal
striatum and hippocampus is not completely protected by
kaempferol.
Fig. 3 and Table 1 show that the reduction by kaempferol
i.v. injections of infarct brain areas monitored by TTC-
staining is dose-dependent. We have selected images of the
brain slice at 6–8 mm from the front pole of the rat brain to
show the dose-dependence protection of kaempferol against
brain damage by the ischemia/reperfusion insult in Fig. 3. In
Table 1, we have summarized the extent of reduction of
brain damage monitored by TTC-staining afforded by differ-
ent kaempferol doses in different coronal brain slices. These
Table 1 – Reduction of the brain damage by kaempferol as
assessed by TTC
Slice # (mm
forefront)
Extent of brain damage (% total
ischemic hemisphere area)a
DMSO
i.v.
I.v. injections
of kaempferol
(n=6)50 μmol/
L
(n=3)
100 μmol/
L
(n=6)
200 μmol/
L (n=3)
2 (2–4)
3 (4–6)
4 (6–8)
5 (8–10)
6 (10–12)
7 (12–14)
15±5
35±5
50±5
45±5
30±5
15±5
5±2
18±2
25±2
30±5
15±2
7±2
n.d.
10±2
15±2
18±2
b5
n.d.
n.d.
8±2
12±2
15±2
b5
n.d.
The results shown in this table are the mean±S.E. of the results
obtained with the number of rats indicated in the text for each
experimental condition.
aCalculatedfromtheaverageofthepercentageofdamagedsurface
area at both sides of each 2 mm-thick slice, weighed by the
absorbance changes at 490 nm for TTC staining (see text for
further details).
Fig. 2 – Quantitation of the protection afforded by kaempferol
against rat brain damage at the slice located 6 mm from the
front pole of the brain, as monitored by the absorbance at
490 nm of TTC-stained slices homogenized as indicated in
Experimental procedures. The results shown are the
means±S.E. of n=6 control rats and n=6 rats treated with
100 μmol/L i.v. injections of kaempferol. Abbreviations of the
abscissae axis: C and I, control and ischemic brain
hemisphere, respectively; number, distanceof the slice to the
forefront brain edge (in mm); last letters Str or Cx, striatum or
cortical neurons, respectively, and when it is not indicated,
the values given correspond to the average of both striatum
and cortical damaged areas within the same slice. The
absorbance at 490 nm of slices non-stained with TTC (blanks)
was lower than 0.03, i.e. same as that of control sample I6.
Asterisks mean statistically significant difference with
respect to DMSO i.v. (P<0.05).
Fig. 3 – The protection by kaempferol against brain damage after ischemia/reperfusion was dose-dependent. The images shown
point out the reduction of the extent of damage monitored by TTC staining and DQ-gelatin staining (MMP) in the matching faces of
vicinalcoronalsliceslocatedatadistanceof6–7mmfromthefrontpoleofthebrain(areaindicatedintheinsetbrainpictureusingthe
samecodenumberofFig.1).IandCmeanischemicandcontrolhemispheres,respectively.Forabetterdirectvisualcomparison,the
brightness of the control hemispheres has been equalized in all the fluorescence microscopy images shown in this figure. The
images shown are representative of those obtained with n=6 control rats (DMSO-treated), n=6 rats treated with 100 μmol/L
kaempferoli.v.andn=3ratstreatedwiththeotheri.v.injectionsassayed,i.e.50and200μmol/Lkaempferol.RedarrowsintheMMP
image of vehicle DMSO i.v. point to areas showing a larger decrease of fluorescence intensity in kaempferol-treated rats.
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results indicated that the protection against brain damage
afforded by 200 μmol/L kaempferol i.v. injections was only
slightly higher than that afforded by 100 μmol/L kaempferol
i.v. injections, therefore, pointing out that this latter dose
was almost saturating. Furthermore, these results also
indicated that 50 μmol/L kaempferol i.v. treatment is close
to the IC50dose of kaempferol.
2.2.
and extracellular laminin matrix degradation induced by the
ischemia/reperfusion insult
Kaempferol i.v. injections attenuated MMP activation
MMP activation was monitored in situ in coronal tissue slices
using DQ-gelatin as substrate with rats treated with the vehicle
DMSO i.v. (n=6), 50 μmol/L kaempferol i.v. (n=3), 100 μmol/L
kaempferol i.v. (n=6) and 200 μmol/L kaempferol i.v. (n=3), as
indicated in Experimental procedures. The increase of green
fluorescence intensity staining after transient focal cerebral
ischemia in the infarct brain areas monitored by TTC staining of
vicinal coronal slices confirmed MMP activation in rats treated
with vehicle DMSO i.v. (Fig. 3). The strong fluorescence signal in
the ventricles is due to the liquid filling this space as products of
degradation of DQ-gelatin are water soluble. Areas showing
highergreenfluorescenceintensityaremarkedbyarrowsinFig.3.
In addition, both the inter-hemisphere connections and callous
body are heavily stained, but in these latter cases there is no
significantdifferencebetweenischemicandcontrolhemispheres
strongly suggesting that this is likely due to tissue damage by the
physical stress associated to brain cutting into coronal slices.
Kaempferol i.v. injections decreased the intensity of the fluores-
cence staining associated with MMP activation in the ischemic
brain hemisphere, as it is also shown in Fig. 3. Digital intensity
analysisoftheimageswithWCIFImageJ softwareyielded values
of 2.2±0.2 (n=6), 1.6±0.1 (n=3), 1.3±0.1 (n=6) and 1.1±0.1 (n=3) for
the ratio (ischemic hemisphere/control hemisphere) of the
fluorescence intensity at the (striatum+neocortex) areas marked
by arrows in Fig. 3 in rats treated with DMSO i.v., 50, 100 and
200 μmol/L kaempferol i.v., respectively. The decrease of the
fluorescence intensity ratio (ischemic hemisphere/control hemi-
sphere) afforded by kaempferol i.v. was statistically significant
(Pb0.05). In addition, we noticed that the brain tissue was more
loosely packed in damaged areas, as it should be expected if
extracellular matrix proteins are extensively degraded.
Laminin is one of the extracellular matrix proteins that is a
substrate of MMP (Gu et al., 2005). Thus, anti-laminin staining is
expected to decrease in brainareas where MMP is activated. The
intensity of anti-laminin staining has been monitored by
fluorescence microscopy of rat brain slices using a fluorescent-
labeled secondary IgG antibody. The intensity of anti-laminin
staining in the infarct brain areas monitored by TTC- and H–E-
staining decreased with respect to the fluorescence intensity of
control hemisphere in rats treated with DMSO i.v. as shown in
Fig. 4. Moreover, the areas displaying lower anti-laminin
fluorescence staining are matched by those showing enhanced
MMP activity staining. The difference in fluorescence intensity
between ischemic and normal brain hemispheres is largely
decreasedinthecorrespondingbrainsliceofkaempferol-treated
rats (n=3) after staining with anti-laminin (Fig. 4). Digital
intensity analysis of the images of coronal slices 6 mm away
from the front pole of the brain with WCIF Image J software
yielded values of 0.55±0.1 (n=3) and 0.85±0.1 (n=3) for the
fluorescenceintensityratio(ischemichemisphere/controlhemi-
sphere) at the marked (striatum+cortical) regions in rats treated
with vehicle DMSO i.v. and with 100 μmol/L kaempferol i.v.,
respectively. These results were also confirmed with 200 μmol/L
kaempferol i.v.-treated rats (n=3, data not shown), which were
similar to those shown in Fig. 4 for 100 μmol/L kaempferol i.v.-
treated rats. The difference between kaempferol- and vehicle
DMSO-treated rats was statistically significant (Pb0.05). A closer
looktothedetailsshowninFig.4revealsthatkaempferolclearly
protects again smoothing of the surface of brain slices at the
striatum and neocortex, being this protective effect more potent
infronto-temporalareasthaninmorecaudalcoronalbrainslices
near the hippocampal area. Therefore, kaempferol treatment
producedastrongprotectionagainsttheproteolyticdegradation
of laminin induced by ischemia/reperfusion, again except at the
hippocampal region and caudal cortical areas located nearby.
2.3.
of nitrotyrosines produced by the transient focal
ischemia/reperfusion insult
Kaempferol i.v. injections blocked the increase
Ischemia/reperfusionproducesalargeincreaseofoxidativestress
inbrain,withanenhancedproductionofperoxynitrite,whichisa
major agent in neuronal degeneration induced by ischemia/re-
perfusion episodes (Iadecola, 1997). Peroxynitrite is formed from
the rapid reaction between superoxide anion and nitric oxide
generated within the brain either by endothelial or glial cells or
neurons (Iadecola, 1997; Bolaños et al., 1997). On the other hand,
kaempferol is a flavonoid with ROS scavenger properties (Bors
etal.,1994;Pannalaetal.,1997),anditalsoinhibitsthegeneration
ofsuperoxideanioninneurons(Samhan-Ariasetal.,2004).Owing
to the short life of peroxynitrite in mammalian tissues, nitrotyr-
osines formed upon its reaction with proteins have been widely
used as a fingerprint for peroxynitrite generation in tissues
(Murphy et al., 1998), and in ischemic brain insults in particular
(Osuka et al., 2001; Zhu et al., 2004). The generation of
peroxynitrite in inflammation is now well established (Bao and
Liu, 2002) and inflammation of the cortical areas of the ischemic
hemisphere shown later to be damaged by TTC-staining can be
observed in our animals after the ischemia/reperfusion insult.
The level of protein nitrotyrosines has been quantified by dot
blot, using the same total protein loaded per dot, using samples
from n=3 vehicle DMSO i.v.-treated rats and n=3 rats treated
with 100 μmol/L kaempferol i.v. Samples were taken from the
areasrevealedtobedamagedbyTTCstainingusingvicinalbrain
slicesandalsofromthesameregionalareasofthenon-ischemic
brain hemisphere. It is shown in Fig. 5A that there is a large
increase of protein nitrotyrosines in the ischemic brain hemi-
spherewithrespecttothenon-ischemichemisphereofthesame
brain slice, on average, nearly 5-fold and 3.5-fold for brain slices
at5–6and8–9mmfromthefrontbrainedge,respectively.Fig.5B
shows that kaempferol prevented the increase of nitrotyrosines
in the ischemic brain hemisphere, yielding a ratio of protein
nitrotyrosines between ischemic and control (non-ischemic)
brain hemispheres is close to 1 in rats treated with kaempferol.
Consistent with the results of TTC- and H–E-staining and
metalloproteinases activation, only for the caudal section of
thestriatumtheratioofproteinnitrotyrosinesbetweenischemic
and control brain hemispheres remained slightly higher than 1.
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To assess the extent of lipid peroxidation in infarct brain
areas, 1 mm-thick coronal slices at 4–6 and 8–10 mm from the
front pole of the brain were cut from rats treated with vehicle
DMSO i.v. and with 100 and 200 μmol/L kaempferol i.v. (n=3
rats of each condition). After TTC staining of the middle 2 mm
coronal slice at 6–8 mm from the front pole, cortical and
striatum areas of the 1 mm-thick slices vicinal to the infarct
areas highlighted by TTC staining in the 2 mm slice were
carefully excised and separately homogenated for TBARS
assay as indicated in Experimental procedures. TBARS assay
pointed out that rats treated with vehicle DMSO i.v. injections
had only a weak increase of lipid peroxidation after the
ischemia/reperfusion insult used in this study, as MDA
measurements yielded values 0.30±0.05 (n=8) and 0.22±0.05
(n=8) nmol MDA/mg protein in the heavily damaged cortical
and striatum areas of the ischemic hemisphere and their
mirror areas of the control hemisphere, respectively. Kaemp-
ferol treatment prevented this increase of lipid peroxidation,
since rats treated with 100 or 200 μmol/L kaempferol i.v.
injections showed values of 0.20±0.05 (n=8) nmol MDA/mg
protein in both hemispheres.
2.4.
of apoptosis induced by ischemia-reperfusion
Kaempferol i.v. injections produced a large reduction
In order to ascertain the effect of kaempferol the extent of
apoptosis induced by the ischemia/reperfusion insult, we
performedTUNELhistochemistry,becauseTTCandH–Estaining
can monitor cell death both by apoptosis and necrosis. Fig. 6
shows that the ischemia/reperfusion insult promoted extensive
cell death through apoptosis both in cortical areas and in basal
gangliaoftheischemic-hemisphere,apointwhichwenoticedin
Fig. 4 – Kaempferol i.v. injections afforded partial protection against the decrease of brain anti-laminin staining by
ischemia/reperfusion. The ability of 100 μmol/L kaempferol i.v. injections to afford protection against the decrease of anti-laminin
stainingwasexperimentallyassessedatthefollowingdistancesfromthefrontpoleoftheratbrain:6–7mm(interfacebetweenslices
3and4)and9–10mm(interfacebetweenslices5and6).Thepositionofthesliceswithintheratbrainisschematicallyindicatedover
thepictureoftheratbrainshownattheleft,see Table1fortranslationofslicenumberintodistancetothefrontpoleofthebrain.For
each case, the following images of vicinal coronal slices are included: H–E-stained image (H–E, left), DQ-gelatin-stained fluorescence
microscopy image as in situ marker of MMP activity (MMP, center) and fluorescence images of the slices stained with a
FITC-secondary antibody against anti-laminin (right). I and C mean ischemic and control hemispheres, respectively. For a better
direct visual comparison, the brightness of the control hemispheres has been equalized in all the fluorescence microscopy images
showninthisfigure.Highermagnificationimagesoftheanti-lamininstainingoftheratstriatumatthesliceslocatedat6–7mmfrom
the front pole of the brain are also shown on the right side to better highlight the protectionof 100 μmol/L kaempferol i.v. injections
againsttheobserveddecreaseofanti-lamininstaininginthisbrainarea.Theanti-laminin-stainedfluorescencemicroscopyimages
shown are representative of those obtained in experimental triplicates, n=3 control rats and n=3 kaempferol-treated rats. For
comparative purposes, microscopy fluorescence images were obtained with the Nikon fluorescence microscope with the same
exposuretimeandcameragainofthemicroscope.Brainslicesnotshowninthefigurewereusedtofixtheexposuretimeandcamera
gain of the microscope before taking the images shown in panels A and B, to avoid FITC-photobleaching by repetitive Xenon
lamp shots. Red arrows in the anti-laminin images point to areas showing larger increase of fluorescence intensity in
kaempferol-treated rats.
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a previous study (Sun et al., 2005). Control hemispheres are not
shown since their positive staining was negligible. As it is also
shown in Fig. 6, kaempferol treatment produced a marked
decrease of the number of apoptotic cells in the fronto-temporal
neocortex and striatum. The decrease of apoptotic cells in
kaempferol-treated rats was higher than 90% in cortical areas
of the ischemic hemisphere, i.e. apoptotic cells are hardly seen.
However, a large number of apoptotic cells can still be seen in
some regions of the basal ganglia and the hippocampus of
kaempferol-treated rats. TUNEL-POD-stained images were
inverted with WICF Image J software to make groups of stained
cells appear as bright spots, and histograms of pixels staining
Fig. 6 – Kaempferol treatment protected against apoptoticcell death as monitored byTUNEL processingof brain slices. Protectionby
treatment with 100 μmol/L kaempferol i.v. injections is shown in vicinal coronal sections at 6–7 mm (left) and 8–9 mm (right) from
theforefrontedgeofthebrainstainedwithH–EandTUNELasindicatedinExperimentalprocedures.Theapproximatepositionofthe
slices within the rat brain is schematically indicated over the picture of the rat brain shown at the left, see Table 1 for translation of
slice number into distance to the front pole of the brain. The arrows in TUNEL-POD images indicate brain areas (neocortex and
striatum) where kaempferol treatment afforded the largest decrease of positive apoptotic cells stained by TUNEL technology. The
images shown are representative of those obtained in experimental triplicates, n=3 control rats and n=3 kaempferol-treated rats.
Fig. 5 – Kaempferol treatment protects rat brain against the increase of protein nitrotyrosines by ischemia/reperfusion.
Representative dot blots of experimental triplicates are shown for rats treated with vehicle DMSO i.v. injections (A) and treated with
100 μmol/L kaempferol i.v. injections (B). The samples were excised from neocortex (Cx) and striatum (Str) areas of the ischemic
hemisphere shown to be damaged by TTC staining of the vicinal brain slices, and from the symmetrical areas of the control
(non-ischemic) brain hemisphere. The amount of protein loaded in each dot was kept constant for the samples taken from both
hemispheres. The ratio values given are the means obtained for measurements done with samples excised from DMSO (n=3) and
kaempferol-treated rats (n=3). The differences between the ratio values obtained for paired samples of DMSO and
kaempferol-treated rats are all significant (P<0.05).
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werebuiltupforthestriatumandalsofornon-stainedareas,the
latter for background signal correction. Thereafter background
was subtracted, and measurements with WCIF Image J yielded
that treatment with 100 μmol/L kaempferol i.v. decreased the
intensity of TUNEL-POD staining by 65±5% (n=3, Pb0.05) in the
striatumatcoronalslices5–6mmawayfromthefrontpoleofthe
rat brain and 45±5% decrease (n=3, Pb0.05) in the hippocampal
area of coronal slices 8–9 mm away from the front pole of the rat
brain. While the treatment with 200 μmol/L kaempferol i.v.
produced a 10% further decrease of TUNEL-POD staining, the
treatment with 50 μmol/L kaempferol i.v. reduced the attenua-
tion of TUNEL-POD staining afforded by kaempferol to approx-
imatelyhalfofthemaximalattenuationattainedwith100μmol/L
kaempferol i.v. injections (data not shown).
Since the activation of caspase-3 in focal and global brain
ischemia is transient, peaking in penumbral cortex at 6–12 h
followingischemia(Choetal.,2003),wedecidedtomonitorthe
decrease of the levels of poly-(ADP-ribose) polymerase (PARP)
inthe brainsamplescollected24h following transientcerebral
ischemiainourratmodel,asadelayedfingerprintofcaspase-3
activation. Furthermore, PARP degradation is a widely accept-
ed marker for apoptotic processes in neuronal cell cultures
(Zhang et al., 1994; Nardi et al., 1997). Thus, we have used PARP
degradation to further support the occurrence of apoptosis in
the major areas shown by TTC to be damaged in brain slices,
namely, striatum, the hippocampus and cortical regions in
temporal–frontal and temporal–caudal coronal slices. To
better highlight the occurrence of apoptosis within these
areas, samples for PARP analysis were excised from vicinal
areas of neighbor brain slices of 1 or 2 mm thickness, such that
one slice was used for TTC staining and the next for PARP
detection by Western blotting. A total number of n=3 vehicle
DMSO i.v.-treated rats and n=3 rats treated with 100 μmol/L
kaempferol i.v. were used forPARP analysis. Fig.7Ashows that
thelevelsofPARParemarkedlydecreasedintheischemicwith
respect to the control brain hemisphere. The intensities of the
116 kDa PARP bands of Western blots were measured as
indicated in Experimental procedures, yielding the following
intensity ratios between ischemic and control brain hemi-
spheres (means±S.E. of triplicate experiments): 0.17±0.02 and
0.29±0.02 for slices at 5–6 and 8–9 mm from the forefront edge
ofbrain,respectively. Onthecontrary,kaempferol-treatedrats
showed no significant decrease of PARP in the ischemic with
respect to the control brain hemisphere (Fig. 7B), yielding
intensity ratios between ischemic and control brain hemi-
spheres close to 1.0±0.2 (means±S.E. of triplicate experi-
ments). These results demonstrated that kaempferol
treatment was also highly efficient to block apoptotic path-
ways mediated by PARP-degradation in brain after transient
focal ischemia/reperfusion insults.
Caspase-9 activation has been shown to take place after
transientcerebralischemiainratbrain,beingitsinductionand
proteolytic activation clearly noticeable 24 h following the
ischemic insult (Cao et al., 2002, 2003). Moreover, it has been
reportedtobeamediatorofoxidativestress-inducedapoptosis
after focal cerebral ischemia (Saito et al., 2004). On these
grounds, wehavemeasured caspase-9activityinlysatesofthe
striatum and neocortex infarct areas which were excised from
coronal slices in rats treated with vehicle DMSO i.v. (n=3), and
with 50 μmol/L (n=3), 100 μmol/L (n=3) and 200 μmol/L (n=3)
kaempferol i.v. injections. In all the cases, a vicinal coronal
slice was stained with TTC before cutting in order to minimize
the presence of non-damaged tissue in the selected areas, as
this will attenuate differences between ischemic and control
brain hemispheres activity measurements. Homogenization
and caspase-9 activity measurements of the excised pieces of
the coronal brain slices were performed as indicated in
Experimental procedures. Fig. 8 shows that kaempferol
treatment largely attenuated the increase of caspase-9 activity
in the ischemic hemisphere. The results point out that the
Fig. 7 – Kaempferol treatment protected against PARP proteolysis in rat brain after ischemia/reperfusion. Representative Western
blots of experimental triplicates are shown for vehicle DMSO i.v.-treated rats (A) and rats treated with 100 μmol/L kaempferol i.v.
injections(B).Thesampleswereexcisedfromneocortexandstriatumareasoftheischemichemisphere(I)showntobedamagedby
TTC staining of the vicinal brain slices, and from the symmetrical areas of the control (non-ischemic, C) brain hemisphere. For
kaempferol-treatedrats(B),samplesexcisedfromneocortex(Cx)andstriatum(Str)wereanalyzedseparatelyandlabeledasICxand
CCx, for ischemic and control hemisphere neocortex samples, and IStr and CStr, for ischemic and control hemisphere striatum
samples (1 and 2 mean duplicate samples). The same amount of protein of each hemisphere was loaded per lane.
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power of kaempferol as blocking agent against the increase of
caspase-9-like activity in the ischemic brain hemisphere
displays regional heterogeneity. From higher to lower kaemp-
ferol protection: cortical areasNstriatum (frontal)Nstriatum
(caudal) and hippocampus. In addition, our results indicated
thatcaspase-9activationwasstrongerin themid-brainareaof
the ischemic brain hemisphere (Fig. 8 and results not shown),
where the extension of the infarct area monitored by TTC
staining reached its peak.
3. Discussion
The regional brain damage after transient focal ischemia/
reperfusion in our rat model shows that basal ganglia, mostly
the striatum, the hippocampus and close areas of the
neocortex are by far the most sensitive areas to this insult, in
excellent agreement with earlier works (Lehrmann et al., 1997;
Gu et al., 2005). In addition, damage at the corpus callosus was
alsonoticed,althoughinthiscaseitislikelythatthereisalarge
contribution coming from the slice processing, as revealed by
MMP activity. The extension of damage within the striatum
can be more clearly seen in temporal–frontal slices (5–6 mm
from the forefront brain edge) than in temporal–caudal slices
(8–10 mm from the forefront brain edge), being the damage at
the hippocampus readily appreciated in the latter slices. The
extent of damaged brain regions detected with TTC after the
standard ischemia/reperfusion treatment with the vehicle
DMSO i.v. injections varied within the 18 rats analyzed in this
workbetweenthatshowninourpreviouspaper(Fig.2ofSunet
al., 2005) and that shown in Fig. 1. The more significant
variation observed was the extent of spreading of the infarct
areainthebrainneocortexoftheischemichemisphere.Infarct
volume estimations were done as indicated previously (Sun et
al., 2005), and for DMSO-treated rats yielded values ranging
from 30 to 40% of total ischemic hemisphere volume.
There is a close overlap between the brain infarct areas
monitored by TTC and those displaying MMP activation
monitored by in situ fluorescent staining using DQ-gelatin
and by loss of anti-laminin staining, in good agreement with
the reported activation of MMP-2 and MMP-9 in transient focal
cerebral ischemia (Romanic et al., 1998; Heo et al., 1999; Asahi
et al., 2000; Chang et al., 2003; Copin et al., 2005; Gu et al., 2005;
Tsuji et al., 2005). It has been shown that cell death through
apoptosis makes a significant contribution to brain damage in
ischemia/reperfusion (Hossmann, 1994; Chan, 1996; Gu et al.,
2005), and H–E and TTC staining monitor cell death, but not
only apoptotic cell death. Due to this reason, the regional
spreading and incidence of apoptosis in our experimental
model of ischemia/reperfusion have been visualized using
TUNEL, as also done in a previous publication (Sun et al., 2005).
On the other hand, neurodegeneration through apoptosis can
follow several biochemical pathways converging to the
activation of executive caspases, and the high relevance of
caspase-3 and caspase-9 activation to cell death through
apoptosis observed after ischemia/reperfusion in rat brain is
now well established (Krajewski et al., 1999; Cao et al., 2001,
2002; Mouw et al., 2002; Cho et al., 2003; Carboni et al., 2005; Li
and Gong, 2007). Since caspase-3 activity has been shown to
peak between 6–12 h following ischemia (Cho et al., 2003), as a
fingerprint of its activation within brain areas showing
enhanced cell death by TTC and H–E staining, we have
measured thedecreaseof thelevels ofPARP, a wellestablished
substrate of caspase-3 in neuronal apoptosis in vitro (Zhang et
al., 1994; Nardi et al., 1997).
Intravenous injection of 0.7 mL of 100 or 200 μmol/L
kaempferol through a catheter inserted into the tail vein,
30 min before focal cerebral ischemia, and another identical
injection just after reperfusion have proven to be very efficient
in protection against rat brain damage by ischemia/reperfu-
sion. Intravenous injections of 0.7 mL of 50 μmol/L kaempferol
were less effective in protection against brain damage. As an
adult Wistar rat weighting 350 g contains a total blood volume
of approximately 20 mL (Hauptman et al., 1989; Sakai et al.,
2004), these results pointed out that kaempferol concentra-
tions in blood between 10 and 15 μmol/L are highly effective to
afford protection against brain damage induced by transient
focal cerebral ischemia and reperfusion in our animal models,
since it has been shown that blood clearance of flavonoids is a
slow process developing with half-times of several hours
(Hackett, 1986, Manach et al., 1996, Hollman and Katan, 1997).
Fig. 8 – Dose-dependent protection by kaempferol i.v.
injections against caspase-9 activation in rat brain after
ischemia/reperfusion. The ratio between the caspase-9 activity
of mirror areas of ischemic (I) and control (C) hemispheres is
plotted for rats treated with the vehicle (DMSO i.v. injections,
white bars), and rats treated with 50 μmol/L (light gray), 100
μmol/L (dark gray) and 200 μmol/L (black bars) kaempferol i.v.
injections.Theactivityofcaspase-9wasmeasuredasindicated
in Experimental procedures in homogenates of excised cortical
(Cx) and striatum (Str) areas of brain coronal slices located at
5–6 mm and 9–10 mm from the front pole of the brain and close
to those shown to be damaged by TTC staining in vicinal
1 mm-thick brain slices. In addition, the corresponding mirror
areas of the control hemisphere were also carefully excised,
homogenatedandtheircaspase-9activitymeasured.Abscissae
codes for samples: the first letter I or C stands for ischemic or
control hemisphere; the number 5 or 9 in the middle means
distanceofthe1mm-thickbraincoronalslicetothefrontpoleof
the rat brain; and the last letters Cx or Str mean cortical or
striatumareas,respectively.Theresultsshownarethemeans±
S.E. of triplicate experimental rats. Asterisks mean statistically
significant difference with respect to DMSO i.v. (P<0.05).
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Moreover, our results allow to estimate an IC50≈3–4 μmol/L in
blood for kaempferol protection against rat brain infarct by
transient cerebral ischemia induced by middle cerebral artery
occlusion.
The effects of the intravenous injections of kaempferol on
brain damage and biochemical markers used in this study are
summarized in Table 2. The blockade by kaempferol of the
increase of protein nitrotyrosines in the ischemic hemisphere
indicated that it provided a potent antioxidant protection
against nitric oxide-derived radicals during the brain insult
associated withtransientfocalcerebralischemia.Noteworthy,
only a weakincrease of thiobarbituric acid reactivespeciescan
be detected in lysates of brain areas shown to be damaged by
TTC staining, approximately 0.1 nmol MDA/mg protein. The
low increase of levels of MDA production measured in our
study is in excellent agreement with the data reported in Choi
et al. (2004), i.e. 0.19±0.03 and 0.41±0.05 nmol/mg wet tissue
for control sham and the ischemic brain, respectively, and
close to those reported in Shang et al. (2006), i.e. 0.40±0.03 and
0.50±0.05 nmol/mg protein for cortical areas of the control
sham and ischemic hemisphere, respectively. Although
higher values for MDA production have been reported by
other authors, it should be noted that addition of butylated
hydroxytoluene before heating of the samples for TBARS is
critical to prevent lipid oxidation during samples processing
(Asakawa and Matsushita, 1979). This is particularly relevant
for these samples, because of the strong pro-oxidant proper-
ties of nitric oxide through peroxynitrite (see above). In
addition, our vehicle DMSO i.v. injections may also attenuate
brain lipid peroxidation in our model of transient focal
cerebral ischemia/reperfusion, as DMSO is a well-known
potent hydroxyl radical scavenger. Therefore, at most only
scarce groups of brain cells have suffered significant lipid
peroxidation in rats treated with our transient focal cerebral
ischemia protocol. Because of the small increase observed in
MDA production in infarct areas of the ischemic hemisphere,
the protection afforded by kaempferol in this parameter is
only weakly significant (P≥0.05).
In a previous paper (Samhan-Arias et al., 2004), we have
shown that kaempferol efficiently protects, IC50=8±2 μmol/L,
cerebellar granule neurons in culture against cell death
through oxidative stress-mediated apoptosis. In this work,
we show by TUNEL histochemistry that blood concentrations
of 10–15 μmol/L kaempferol largely decrease apoptosis after
transient focal cerebral ischemia/reperfusion in rat brain. As
shown in Table 2, there is a large attenuation of the increase of
PARP degradation and of caspase-9 activity in the ischemic
brain hemisphere with respect to the control (non-ischemic)
hemisphere in kaempferol-treated rats, and this points out
that kaempferol treatment efficiently blocks ischemia/reper-
fusion-induced apoptotic pathways mediated by these bio-
chemical markers. Moreover, Table 2 highlights the close
values obtained for the overall attenuation afforded by
kaempferol in TUNEL staining, PARP degradation and cas-
pase-9 activation after ischemia, thus, indicating that the
predominant apoptotic biochemical pathways in neocortex
and striatum induced by transient focal ischemia in rat brain
involve activation of both caspase-3 and caspase-9. This
conclusion is in goodagreementwith theconclusionsattained
in a previous work of another laboratory (Cao et al., 2002). In
addition, apoptotic cell death seems to be the major cell death
pathway in the neocortex and striatum after the transient
focal cerebral ischemia insult was applied in this work,
because the values obtained for attenuation of TTC staining
are at most 10% higher than those obtained from attenuation
of TUNEL staining (Table 2). On these grounds, we conclude
that the kaempferol treatment applied in this work is very
efficient in protecting against rat brain damage through
apoptosis after transient focal cerebral ischemia/reperfusion.
Table 2 also show that the efficiency of the kaempferol
treatment to prevent rat brain damage after transient focal
ischemia is higher for neocortical areas than for basal ganglia,
and in temporal–frontal areas of the neocortex and striatum
than in more caudal areas. It is to be noted that the stronger
protection by the kaempferol treatment has been noticed at
cortical areas, where a nearly complete protection (N90%) can
Table 2 – Extent of protection of 100 μmol/L kaempferol i.v. injections against ischemia/reperfusion-induced brain damage as
monitored by the different cell death and biochemical markers used in this work
Attenuation of the changes of marker (%)a
Distance to the front pole:5–6 mm8–10 mm
MarkerNeocortexStriatumNeocortexStriatumb
TTC staining
H–E staining
DQ-gelatin staining (MMP)
Decrease of anti-laminin
Increase of nitrotyrosines
TUNEL staining
PARP degradation
Increase of caspase-9 activity
N90
N80
N90
N90
N95
N90
N90
80–90
75–85
70–80
65–75
60–70
N95
60–70
N90
75–85
80–90
60–70
75–85
60–70
N95
N90
80–90
80–90
50–60
40–50
35–45
50–60
85–95
40–50
n.e.c
45–55
See Results for details of calculations. The attenuation ranges listed in the table are the average of those obtained from at least three
independent experiments with triplicate measurements for each condition (n≥6).
aPb0.05 with respect to DMSO i.v. injections for all the data listed in this table.
bIncludes caudal regions of the striatum and hippocampus.
cn.e., not measured.
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be observed except for caudal cortical areas close to the
hippocampus. These results are fully consistentwith a leading
role for peroxynitrite-mediated oxidative stress and MMP-9
activation in cortical neuronal apoptosis, as proposed in (Gu
et al., 2002). The efficiency of protection afforded by kaemp-
ferol treatment was slightly lower (70–80%) in the temporal-
frontal areas of the striatum, i.e. in coronal slices 5–6 mm
away from the front pole of the rat brain (Table 2). However, it
is to be noted that heterogeneity of the extent of protection
within this complexstructural region of the brain can be easily
observed in TTC, anti-laminin and TUNEL-stained images. A
differential ability of kaempferol treatment to protect distinct
basal ganglia structures against cell death by transient middle
cerebral artery occlusion in rats is clearly indicated by the
much lower efficiency of protection observed in this brain
region in coronal slices 8–10 mm away from the front pole of
the rat brain (40–50% on average, Table 2). A more in dept
analysis of this point requires further experimental studies
focussed at a more ultrastructural and cellular level which is
beyond the scope of this work. The death observed after
kaempferol treatment in internal basal ganglia very near to
the point where the wire was inserted to induce focal cerebral
ischemia is likely due to local damage during the surgical
intervention, such as local small blood vessels breakdown
upon mechanical handlings. However, this cannot be the case
for the hippocampal region. Glutamate-excitotoxic neuronal
death is involved in neuronal hippocampal death in ischemia/
reperfusion (Benveniste et al., 1984; Szatkowski and Atwell,
1994), and it has been shown that kaempferol shows only a
limited ability to protect against excitotoxic neuronal death
(Ishige et al., 2001).
Owing to the low toxicity of kaempferol and another
flavonoids for humans (Manach et al., 1996; Hollman and
Katan, 1997) and to the slow renal clearance of flavonoids in
mammals (Hackett, 1986), our results suggest that diet- or
pharmaceuticals-supplementation with this flavonoid may be
helpful to attenuateischemic/reperfusion-induced braindam-
age. In this regard, it is to be noted that micromolar
concentrations of flavonoids can be reached in the blood-
stream through oral ingestion (Abd El Mohsen et al., 2002).
4.Experimental procedures
Adult male Wistar rats weighting 350–400 g were used in this
study. Animal care and all experimental procedures were
carried out in accordance with guidelines of the European
Communities Council Directive (86/609/EEC). Protocols were
approved by the Ethics Committee for Animal Research of the
local government.
4.1.
“intraluminal wiring”
Focal cerebral ischemia by transfemoral selective
The animals were anesthetized with ketamine (50 μg/g,
intramuscular), diazepam (2.5 μg/g, intramuscular) and atro-
pine (0.05 μg/g, intramuscular). Rats were mounted in the
pronepositiononastereotaxicinstrument(KopfInstruments).
After a cranial midline incision, a burr hole (1 mm in diameter)
was made with a microdrill 2 mm posterior and 3.5 mm left to
bregma. A truncated 21-gauge needle was fixed on the hole
with two miniature screws and dental cement. Once the
wound was sutured, the animal recovered from anesthesia
and fasted for 24 h. To induce focal cerebral ischemia, the rats
were again anesthetized and maintained over a hot-plate at
37.0±0.5 °C. The flexible plastic tip (0.5 mm in diameter, PF319,
Perimed) of a master laser-Doppler probe (PF418, Perimed) was
introduced through the cranial 21-gauge needle until the tip of
the probe gently contacted the brain surface, thus measuring
the parietal cortical perfusion in the territory of the middle
cerebral artery by means of a laser-Doppler flowmeter (PF4001
Master, Perimed). The signal was digitalized (PF472 A/D
converter, Perimed) and sent to an IBM® PC compatible
computer for continuous recording, storage and later analysis.
Middle cerebral artery occlusion was induced by means of
transfemoral selective intraluminal wiring as we have de-
scribed in detail in a previous paper (Sun et al., 2005), and
assessed by the sudden fall in cortical perfusion measured by
the laser-Doppler probe (Fig. 1 online). The guidewire was left
in place for 60 min, and then removed to allow for 24 h of
reperfusion. The left femoral artery was tied, the wound was
closed and the animal was allowed to recover from anesthesia
in a chamber with room air at an ambient temperature of
25 °C. Neurological evaluation was carried out 24 h after
ischemia using the forepaw-outstretching test, as we have
indicated in a previous work (Sun et al., 2005). Surgical
interventions showing subarachnoid hemorrhage or incorrect
localization of thewire, as assessedby onlya smalldecreaseof
parietal perfusion in Doppler recordings, were considered
failed and these rats were discarded. Only rats showing
neurological deficits were selected for this study, 54 rats in
total after discarding surgical failures.
4.2.
different procedures and assays
Experimental animals distribution in groups used for
The 18 DMSO-treated rats and the 36 kaempferol-treated rats
were distributed randomly such that each type of experimen-
tal determination was done, at least, with n=3 animals. This
was achieved as follows:
Group I 18 rats were used for whole brain analysis by TTC
stainingafter cuttinginto 2 mm-thick coronal slices:
6 rats of vehicle DMSO i.v., 6 rats of 100 μmol/L
kaempferol i.v., 3 rats of 50 μmol/L kaempferol i.v.
and 3 rats of 200 μmol/L kaempferol i.v.
Group II 12 rats brains were fixed and embedded in paraffin
as indicated below and used for H–E, anti-laminin
staining and TUNEL-POD. This group is the sum of 4
subgroupsof 3 rats of each of the treatments applied
in this work (vehicle DMSO i.v., 50 μmol/L kaemp-
ferol i.v., 100 μmol/L kaempferol i.v. and 200 μmol/L
kaempferol i.v.).
Group III 12 rats were used for DQ-gelatin staining, nitrotyr-
osines and PARP levels measurements. This group is
the sum of 2 subgroups of the following treatments: 6
rats of vehicle DMSO i.v. and 6 rats of 100 μmol/L
kaempferol i.v. All of these brains were cut as follows
(distance in mm with respect to the front pole of the
brain given in the parenthesis): one 2 mm-thick
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coronal slice (2 mm), six 1 mm-thick coronal slices (4
to 9 mm) and one 2 mm-thick coronal slice (10 mm).
The 2 mm-thick slices and one of the central 1 mm-
thick slices were routinely stained with TTC. The rest
of1mm-thicksliceswerealternatedrandomlyforuse
in DQ-gelatin staining or (nitrotyrosines plus PARP
levels determinations).
Group IV 12 rats were used for DQ-gelatin staining, caspase-9
activity measurements and TBARS.This group is the
sumof4subgroupsof3ratsofeachofthetreatments
applied in this work (vehicle DMSO i.v., 50 μmol/L
kaempferol i.v., 100 μmol/L kaempferol i.v. and 200
μmol/L kaempferol i.v.). All of these brains were cut
as follows (distance in mm with respect to the front
pole of the brain given in the parenthesis): one
2 mm-thick coronal slice (2 mm), six 1 mm-thick
coronal slices (4 to 9 mm) and one 2 mm-thick
coronalslice(10mm).The2mm-thickslicesandone
of the central 1 mm-thick slices were routinely
stained with TTC. The rest of 1 mm-thick slices
were alternated randomly for use in DQ-gelatin
staining or (caspase-9 activity measurements plus
TBARS).
4.3.
damaged regions (TTC, MMP, Anti-laminin, TUNEL and H–E)
Dissection of brain slices and assessment of brain
The brains were carefully removed from the skull, washed
in ice-cold PBS for 5 min and sectioned by using a tissue
slicer (Stoelting, Woodale, IL, USA). Routinely, 2 mm or
1 mm thick coronal sections from the frontal to the
occipital pole were collected. Thereafter, selected slices
were processed for 2,3,5-triphenyltetrazolium chloride
(TTC) or MMP or anti-laminin staining as indicated below.
In all the cases, slices were selected pair-wise, such that for
any slice stained with MMP or anti-laminin, a vicinal slice
was stained with TTC. In addition, areas shown to be
damaged by TTC staining were pooled for protein nitrotyr-
osines and PARP quantification using anti-nitrotyrosine and
anti-PARP, respectively, as indicated below.
Slices for TTC staining were incubated in PBS containing
2% TTC (Sigma) at 37 °C for 15 min and photographed. For
quantitative absorbance readings at 490 nm, TTC-stained
samples were homogenized and lysed in 0.7 mL phosphate
buffered saline (PBS) supplemented with 0.05% Tween 20
(PBS-T), first in a glass potter homogenizer and then by 40
passes through the tips of a P100 micropipette followed by 10
passes through the a 25-gauge needle. Protein concentration
of lysates was calculated by the method of Bradford (1976),
using the Bio-Rad protein assay reagent and bovine serum
albumin as standard.
MMP staining of freshly excised brain slices was performed
by incubation during 45–60 min at 37 °C with 200 μL of Locke's
buffer (154 mmol/L NaCl, 4 mmol/L NaHCO3, 5 mmol/L KCl,
2.3 mmol/L CaCl2, 1 mmol/L MgCl2, 5 mmol/L glucose and
10 mmol/L 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid (HEPES), pH 7.4) supplemented with 0.1 mg DQ-gelatin/
mL, the fluorogenic substrate of MMP (Molecular Probes
Handbook of Fluorescence Probes and Research Products, 10th
ed.). Degradation of DQ-gelatin by MMP results in a large
enhancement of fluorescein green fluorescence intensity. A
mild wash step with 200 μL of Locke's buffer was applied to
brain slices 5 min before image acquisition. Sections were
visualized and photographed under a Nikon stereo-fluores-
cence microscope.
For anti-laminin immunohistochemistry, after cutting
one 1 mm-thick central coronal slice between 6 and 8 mm
from the front pole for TTC staining, the remaining anterior
and posterior pieces of the brain were fixed in paraformal-
dehyde 4%, dehydrated, embedded in paraffin wax and
sectioned in coronal sections at 10 μm. Thereafter, the slides
were washed in 1% bovine serum albumin (BSA) in PBS,
blocked in 1.5% sheep serum, and incubated with rabbit anti-
laminin (Sigma) at 1/30 in blocking solution overnight at 4 °C.
Anti-rabbit IgG fluorescein isothiocyanate (Sigma), 1/300 in
1% BSA/PBS, was used as secondary antibody. Sections were
visualized and photographed under a Nikon stereo-fluores-
cence microscope.
For TUNEL technology, the sections were treated as in-
structed (Cheng et al., 2002) with an in situ cell death detection
kit, POD (Roche). Apoptotic cells were observed under micros-
copy using a Vector VIP substrate kit (peroxidase detection;
Vector Laboratories) as in (Sun et al., 2005). Sections were
compared to the unaffected hemispheres, which were used as
control.
Vicinalsectionswerecounter-stainedwithhematoxylin–eosin.
4.4.
and nitrotyrosine detection by dot-blot
PARP proteolysis measurements by Western blotting
Samples used for immunoblots were taken from 1 mm-width
brain coronal slices using the tip of a Pasteur pipette, and
rapidly frozen in liquid N2. Samples homogenization was
performed in 0.5 mL of 1% Nonidet P40, 0.5% sodium
deoxycholate, 1% sodium dodecyl sulfate in PBS, pH 7.4,
supplemented with COMPLETE™ protease inhibitor cocktail.
Samples were homogenized with several passes through a 25-
gauge needle at 4 °C, and then incubated 30 min on ice.
Homogenates were centrifuged at 10,000×g for 10 min to
remove non-lysated material, and the supernatant of this
centrifugation was frozen at −80 °C until use. SDS-PAGE was
performed with 20–30 μg protein of lysate loaded per lane, as
we have indicated in a previous publication (Martin-Romero
et al., 2000). After electrophoresis, gels were electroblotted to
nitrocellulose membranes, and membranes were blocked
with 10% (w/v) non-fat dry milk in PBS-T and gently shaking
for 1 h at room temperature. Membranes were then incubated
for 1 h at room temperature with the primary antibody
diluted in PBS-T. After extensive washing in PBS-T, horserad-
ish peroxidase-conjugated secondary antibody diluted in PBS-
T was added and incubated for 1 h at room temperature.
Finally, luminol substrate (Supersignal, Pierce) was added and
membranes were exposed to chemiluminescence imaging
screens. Screens were scanned using a Molecular Imager FX
System from Bio-Rad. Signals were quantified by volumetric
integration using the Quantity One Software. Anti-PARP rabbit
polyclonal antibody (H-250, Santa Cruz Biotechnology, Inc.)
was used as the primary antibody, and a secondary horse-
radish peroxidase-labeled anti-rabbit IgG antibody was used
for PARP determination. The good performance of our
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Western blots for the detection of PARP degradation after
caspase-3 activation was tested with low-K+-induced apopto-
sis of cerebellar granule neurons in culture (results not
shown). The culture of cerebellar granule neurons was
performed as indicated in previous publications from our
laboratory (Martin-Romero et al., 2000, 2002).
Immunodetection of tyrosine nitration was performed by
dot blot using 20 μg protein/dot from total lysates as described
above. After sample addition, nitrocellulose membranes were
incubated at room temperature for 30 min, dried at 52 °C for
15 min in oven and blocked with 10% (w/v) non-fat dry milk in
PBS-T for 1 h at room temperature before the application of
mouse anti-nitrotyrosine monoclonal antibody (Calbiochem
CC22.8C7.3) at 1:2000 dilution. After incubation for 3 h at room
temperaturewiththeanti-nitrotyrosineantibody,thesamples
were washed with PBS-T and then incubated for 1–2 h with
secondary horseradish peroxidase-labeled anti-mouse IgG
antibody. Finally, luminol substrate (Supersignal, Pierce) was
added and membranes were exposed to chemiluminescence
imaging screens. Screens were scanned using a Molecular
Imager FX System from Bio-Rad. The whole dot signals were
quantified by volumetric integration using the Quantity One
Software.
4.5.Caspase-9 activity measurements
Caspase-9 activity measurements in homogenates were done
using the caspase-9 fluorescence substrate Ac-LEHD-AFC in
the assay buffer recommended by the manufacturer (Calbio-
chem): 50 mM HEPES, 1 mM EDTA, 100 mM NaCl, 10 mM
dithiotreitol and 0.1% Chaps (pH 7.4), and the reaction was
started by addition of 50 μM Ac-LEHD-AFC (from a DMSO 50-
fold concentrated stock solution). At different times of
incubation at 37 °C (up to 2 h), the fluorescence intensity
was recorded with excitation and emission wavelength of 400
and 505 nm, respectively. Parallel samples were run to record
the increase of fluorescence in the presence of 0.5 μM
caspase-9 inhibitor (Calbiochem), which was found to be
lower than 10% of the increase of fluorescence recorded in its
absence, and this was subtracted for caspase-9 activity
calculations. The conversion of fluorescence increase into
caspase-9 activity was performed using the fluorescence
increase produced by assays done with different dilutions of
a stock solution of 1 unit/μL human recombinant caspase-9.
We have obtained a caspase-9 activity of control hemispheres
gave values of 0.55±0.05 units/h/mg protein. Fluorescence
intensity readings were taken with a Perkin Elmer (mod. 650–
40) fluorescence spectrophotometer equipped with thermo-
stated cuvette holder. In all the cases, the samples were
diluted before fluorescence readings to ensure that the sum of
the absorbances at 400 and 505 nm (excitation and emission
wavelengths, respectively) was lower than 0.1 in order to
avoid artifactual results due to inner filter effects of the
solution (Lakowicz, 1983).
4.6.Thiobarbituric acid reactive substances (TBARS) assay
Samples were immediately frozen and stored at −80 °C, and
thawn just before processing for measurements. TBARS assay
to measure malondialdehyde (MDA) has been performed
following the protocol described by Buege and Aust (1978),
with the modifications introduced by Asakawa and Matsush-
ita (1979). Briefly, 1–2 mg of brain homogenates prepared as
indicated above for caspase-9 measurements was diluted to
0.5 mL in PBS-T, mixed under vortex with 1 mL solution
containing 15% TCA, 0.375% 2-thiobarbituric acid and
0.25 mol/L HCl and supplemented with 15 μL of 0.1 mol/L
butylated hydroxytoluene in ethanol to prevent lipid perox-
idation during later steps of samples processing in this assay
(Asakawa and Matsushita, 1979). Thereafter, samples were
heated at 100 °C during 15 min and insoluble material was
removed by centrifugation at 1000×g during 15 min. The
supernatants were collected and supplemented with 0.25%
sodium sulfite to minimize interference from other aldehydes
(Asakawa and Matsushita, 1979), and their absorbance was
read at 535 nm and lipid oxidation was expressed as MDA
production, calculated using an extinction coefficient for the
MDA-coloured derivative of 1.56˙105M−1cm−1(Buege and
Aust, 1978).
4.7.Statistical analysis
Alldatawereobtainedinatleastthreeindependentexperiments
with replicates of three or more for each condition. Results are
expressed as means±standard error (S.E.). Statistical analysis
was carried out by Mann–Whitney non-parametric test. Signif-
icant difference was accepted at the Pb0.05 level.
4.8.Chemicals
Bovine serum albumin, butylated hydroxytoluene, Chaps,
dimethyl sulfoxide (DMSO), EDTA, HEPES, kaempferol, para-
formaldehyde, sodium deoxycholate, sodium dodecyl sulfate,
sodium sulfite, 2-thiobarbituric acid, TCA, TTC and Tween-20
were supplied by Sigma, St. Louis, MO, USA. DQ-gelatin was
from Invitrogen (Molecular Probes), Eugene, OR, USA. COM-
PLETE, Nonidet P40 and Cell death detection kit POD were
purchased from Roche, Mannheim, Germany. Ketamine was
from Pfizer, Madrid (Spain). Diazepam and atropine were
obtained from B. Braun, Rubí-Barcelona (Spain). Ac-LEHD-
AFC, caspase-9 inhibitor and caspase-9 human recombinant
were supplied by Calbiochem, Darmstadt, Germany. Vector
VIP substrate kit was from Vector Laboratories, Burlingame,
CA, USA.
Acknowledgments
Professor J. Usón, Scientific Director of the CCMI (Cáceres, Spain)
for helpful discussions. Supported by grants no. SAF2003-08275
from the Spanish MEC and SCSS0633 and 3PR05A078 from Junta
deExtremadura.AKSAholdsapredoctoralfellowshipofJuntade
Extremadura.
Appendix A. Supplementary data
Supplementary data associated with thisarticle can be found, in
the online version, at doi:10.1016/j.brainres.2007.08.087.
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