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JPET #139618
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Vascular protection with candesartan after experimental acute stroke in
hypertensive rats: A dose-response study
Wieslaw Kozak, Anna Kozak, Maribeth H. Johnson, Hazem F. Elewa and Susan
C. Fagan
Program in Clinical and Experimental Therapeutics, College of Pharmacy,
University of Georgia (AK, HFE, SCF), Institute of General and Molecular
Biology, Nicolas Copernicus University, Torun (Poland) (WK); Departments of
Neurology (SCF), Biostatistics (MHJ), Medical College of Georgia, Specialty
Care Service Line, Veterans Administration Medical Center, Augusta, GA (AK,
HFE, SCF)
JPET Fast Forward. Published on June 17, 2008 as DOI:10.1124/jpet.108.139618
Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: Hypertension before and after stroke and candesartan
Corresponding Author:
Susan C. Fagan, Pharm.D.
UGA College of Pharmacy
CJ-1020 Medical College of Georgia
1120 15th Street
Augusta, GA 30912-2450
phone: (706) 721-4915; fax: (706) 721-3994
Number of text pages: 14
Number of tables: 3
Number of figures: 7
Number of references: 26
Number of words in abstract: 236
Number of words in introduction: 365
Number of words in discussion: 691
Nonstandard abbreviations: SHR = spontaneously hypertensive rat
Recommended section: Cardiovascular
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Abstract
We have shown that candesartan decreases the acute stroke-induced elevation
of mean arterial blood pressure (MAP) in Wistar rats and improves functional
outcome. The aim of the present study was to determine whether the same
benefit could be achieved in spontaneously hypertensive rats (SHR).
METHODS. Animals were subjected to middle cerebral artery occlusion (MCAO)
or sham for 3 hours followed by reperfusion. Either candesartan 0.1 mg/kg, 0.3
mg/kg, 1.0 mg/kg or saline were administered. MAP of the rats was monitored
by means of telemetry and neurologic function was assessed. Infarct size,
edema formation and hemoglobin content in the ischemic hemisphere were
evaluated 24 h after the stroke. RESULTS. MAP of SHR increased immediately
upon MCAO from 135 (baseline) to 189 mmHg, and remained elevated until
reperfusion. Candesartan decreased MAP in a dose-dependent manner, with a
drop below baseline after a dose of 1.0 mg/kg. SHRs were experienced greater
BP lowering effects of candesartan after stroke compared with a sham condition
(p<0.0001). Neurologic deficit after stroke was reduced in candesartan-treated
animals revealing a dose-dependent effect (p<0.01). Infarct size, edema
formation and hemoglobin content were all reduced by candesartan at doses of
0.1 and 0.3 mg/kg (p<0.05 for all). Candesartan 1 mg/kg was not different from
saline. CONCLUSION. Low doses of candesartan provide neurovascular
protection after stroke in SHRs. Caution is warranted since acute stroke
increases the sensitivity to BP lowering, increasing the likelihood of overshooting.
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Introduction
Hypertension is present in almost 80% of acute stroke patients and is known to
increase the occurrence and severity of ischemic stroke (Li et al., 2005). The
newest clinical guidelines for management of patients with acute ischemic stroke,
recommend only treating the most severely elevated pressures in the first 24
hours and then only decreasing MAP by 15% (Adams et al., 2007). For patients
with preexisting hypertension, there is support for restarting antihypertensive
medications, but only after 24 hours (Adams et al, 2007).
The consequences of hypertension before and after stroke can also be
demonstrated in animals. Spontaneously hypertensive rats (SHR) exhibit more
severe neurological deficits and edema formation (Slivka et al, 1995) and more
frequently demonstrate extensive cerebral infarct damage than normotensive
animals following permanent focal cerebral ischemia (Coyle, 1986; Barone et al,
1992). However, the studies using SHR have yielded contradictory results
regarding the influence of blood pressure on ischemic injury and prognosis. It
has been postulated that antihypertensive drugs may reduce the pressure-
dependent cerebral blood flow to the ischemic penumbra, or conversely, post-
stroke hypertension may be deleterious and facilitate edema formation in the
ischemic brain tissue (Harms et al, 2000). It is hypothesized that in the SHR,
stimulation of brain and cerebrovascular angiotensin II (Ang II) systems
contributes to vasoconstriction, increased expression of proinflammatory factors,
and increased microvessel permeability (Ito et al, 2001). In support, it has been
demonstrated that Ang II AT1 receptor blockade reduces blood pressure,
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normalizes the brain microcirculation and decreases the vulnerability to stroke in
chronically hypertensive rats (Ito et al., 2002; Ando et al., 2004; Zhou et al.,
2005). Acute and prolonged Ang II AT1 receptor blockade prior to the onset of
experimental stroke may contribute to the protection against brain ischemia and
inflammation in the SHR (Nishimura et al., 2000; Zhou et al., 2006). However,
this protection with AT1 receptor inhibition as a pretreatment is not directly
correlated with blood pressure reduction (Ito et al., 2002).
Recent studies from our laboratory showed that candesartan, an AT1
receptor blocker, administered after reperfusion in acute ischemic stroke
normalizes blood pressure, reduces neurovascular damage and improves
outcome in normotensive male Wistar rats (Fagan et al., 2006). The purpose of
the present study was to assess whether or not candesartan at reperfusion will
be similarly protective in SHRs, a model that is likely to be more clinically
relevant.
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Methods
Animals
Male spontaneously hypertensive rats (SHR) 9-week-old, weighing ca. 200
g, were purchased from Charles River Breeding Company (Wilmington,
Massachusetts, USA). After shipping, the animals were housed in individual
plastic cages for 5 - 6 days prior to the surgery and experimentation. The rats
were provided food and water ad libitum, a 12:12-h light/dark cycle at 24°C, and
30–40% humidity. Rats were weighed and randomly assigned to each
experimental group. When the rats reached body weight of ca. 240 g, they were
instrumented for telemetry blood pressure monitoring, and when they further
gained body mass to 275–300 g they were subjected to the experimental
protocol. Usually, the rats were at 12–13 week of age at the time of the
experimental cerebral ischemia or sham.
The Institutional Animal Care and Use Committee (IACUC) of the Charlie
Norwood VA Medical Center in Augusta, Georgia approved all procedures, in
accordance with the Guide for the Care and Use of Laboratory Animals from the
National Institutes of Health.
Blood pressure telemetry
Telemetry transmitters (Data Sciences International, St. Paul, Minnesota,
USA) were implanted according to the manufacturer’s specifications as described
elsewhere (Fagan et al., 2006). Briefly, the rats were anesthetized with sodium
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pentobarbital (65 mg/Kg, i.p.; Abbott Laboratories, Chicago, Illinois, USA) and a
midline incision was performed to expose the abdominal aorta. The exposed
vessel was shortly occluded to allow insertion of the transmitter catheter into the
abdominal aorta. Using tissue glue, the catheter was secured in place and the
incision (abdominal muscle and skin) was sutured. Rats returned to their
individual cages and were allowed to recover from surgery for 10 days. By
placing rats on top of the telemetry receivers arterial pressure waveforms were
continuously recorded throughout the study. Data were recorded every 10
minutes for several days before the stroke (or sham) and until the sacrifice at 24
hours after the onset of stroke.
Experimental cerebral ischemia
Animals were anesthetized with 2% isoflurane via inhalation. Cerebral
ischemia was induced using the intraluminal suture middle cerebral artery
occlusion (MCAO) model (Zea Longa et al., 1989). The right middle cerebral
artery (MCA) was occluded with 19-21 mm 3-0 surgical nylon filament, which was
introduced from the external carotid artery lumen into the internal carotid artery to
block the origin of the right MCA. The animals were kept under anesthesia for
only 10 minutes for the surgical procedure. The suture was removed after 3
hours of occlusion and the animals were returned to their cages.
Prior to reperfusion, the animals were subjected to the test for assessment
of neurological function. Immediately after reperfusion, either saline or doses of
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candesartan cilexitil (Astra-Zeneca; 0.1 mg/Kg, 0.3 mg/Kg, and 1.0 mg/Kg), as
indicated in the figures, were administrated intravenously by tail vein at an
injection volume of 1 ml/Kg. A group of 12 SHRs and 12 Wistars were shams for
the same doses as above.
Perimed Laser Doppler Perfusion Imaging hardware and software system
(PeriScan PIM 3 System; Perimed AB, Stockholm, Sweden) was used to
document reduction of flow due to MCAO. In a subset of animals (n=5), the skin
was reflected, the bone cleaned and scans were performed at baseline, 5
minutes after the MCAO, 5 minutes after reperfusion (before drug administration)
and prior to sacrifice at 24 hours. All using the same scale, images were saved
and average perfusion units were documented in both hemispheres at each time
point. All endpoints were assessed in a blinded fashion.
Neurological assessment
Neurological function was measured prior to reperfusion and at 24 hours
(just before sacrifice) using the Bederson score (Bederson et al., 1986). An
animal with no apparent deficits obtained a 0; the presence of forelimb flexion =
1; decreased resistance to push = 2; and circling = 3. A score of 3 is consistent
with a middle cerebral artery occlusion. Only animals with a score of 3 prior to
reperfusion were included in the analysis of infarct size, hemoglobin, and
neurological function. In addition to the Bederson score, several other behavioral
tests such as beam walk, paw grasp and elevated body swing test were
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performed and neurobehavioral deficit was assessed as described elsewhere
(Wahl et al., 1992; Borlongan and Sanberg, 1995).
Assessment of infarct size, edema and hemoglobin content
At 24 hours after the onset of MCAO, anesthesia was performed with
Ketamine 44 mg/Kg and Xylazine 13 mg/Kg administered intra-muscularly,
animals were then perfused with saline, sacrificed, and their brains were
removed. The brain tissue was sliced into seven 2 mm-thick slices in the coronal
plane and stained with a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC)
(Sigma Chemical Co., St. Louis, Missouri, USA) for 15-20 minutes. Images of
the stained sections were taken. Using image analysis software (Zeiss-KS300,
Oberkochen, Germany), infarction zones were measured and percentage infarct
size corrected for edema was calculated. Edema was quantified as the
difference in area between the hemispheres, expressed as a percent of the
contralateral hemisphere. The ischemic and non-ischemic hemispheres of the
slices for the enzyme-linked immunosorbent assay (ELISA) were separated and
processed, using the non-ischemic side as a control. After homogenizing the
TTC-stained slices in the core of the infarct and collecting the supernatants,
ELISA was performed to measure the hemoglobin content of the brain tissue
(Hilali et al., 2004).
Statistical analysis
For blood pressure data, the average of all measurements prior to MCAO
was the pre-stroke value. Values obtained during the 3 hours of MCAO were
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averaged for the estimate of BP during stroke, the values for the first 2 hours
post-reperfusion were averaged for an estimate of the immediate effects of the
drugs, and all values 5 hours after the onset of ischemia were averaged for the
post-stroke value. For assessment of the effect of the stroke on the response to
BP lowering, a two strain (Wistar vs SHR) by 2 condition (stroke vs. no stroke)
factorial analysis of covariance was used to analyze differences in MAP within
the time periods above, using baseline MAP as a covariate. A 2-way mixed
model repeated measures analysis of variance (ANOVA) was used to determine
treatment (saline, candesartan 0.1mg/Kg, candesartan 0.3mg/Kg and
candesartan 1 mg/Kg), time (pre-stroke, MCAO, 2hr post-perfusion, post-stroke),
and treatment by time differences in mean arterial pressure (MAP). The area
under the curve (AUC) for the entire duration of the experiment was also
calculated for each rat where baseline was considered to be zero. Maximum
(MAX) and minimum (MIN) MAP were also determined. Differences among
different treatments and control were determined by one-way ANOVA for AUC,
MAX, MIN, average infarct size, edema, hemoglobin content, and post-perfusion
values of the Bederson, beam walk, and paw grasp scores. A Tukey-Kramer
adjustment for multiple comparisons was used for all post-hoc mean
comparisons. All analyses were performed using SAS 9.1.3 (SAS Institute Inc.,
Cary, North Carolina, USA). Statistical significance was measured at an alpha
level of 0.05. All values are represented as mean ± standard deviation (SD).
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Results
Changes of blood pressure in the SHR upon brain ischemia, reperfusion
and candesartan treatment
Changes in mean arterial pressure (MAP; 1-h averages) over time by
treatment are illustrated in Figs 1 and 2. Of note, we tested the effectiveness of
candesartan in reduction of MAP in the sham SHR. Data shown in Figure 1
demonstrates that all three doses of candesartan used (0.1, 0.3, and 1 mg/Kg)
reduced MAP within two hours of injection from a baseline (approximately 140
mmHg) to below baseline. Reduction was dose-dependent, with the most
pronounced and long-lasting effect seen for a dose of 1.0 mg/Kg. MAP of
animals treated with doses of 0.3 mg/Kg and 1.0 mg/Kg of candesartan returned
to the pre-injection levels after 48 h and 72 h, respectively, whereas MAP of the
rats injected with a dose of 0.1 mg/kg returned to baseline within 24 h of the
treatment. SHRs experienced greater BP lowering effects of candesartan after
stroke compared to the sham situation (p<0.0001) and their Wistar counterparts
(p=0.014) (Wistar data not shown).
Within 1 h of ischemia, MAP of the rats increased from baseline (ca. 140
mmHg) to approximately 190 mmHg, and remained at that level until reperfusion
(Fig. 2). Reperfusion provoked a drop of MAP, and it was dramatically enhanced
by candesartan treatment. The repeated measures ANOVA showed a significant
interaction between treatment and time (p<0.0001). There were no significant
differences among the groups for the pre-stroke and the MCAO values (Table 1).
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For the two hours post-reperfusion the candesartan 0.3 mg/Kg and candesartan
1 mg/Kg groups lowered BP the most whereas candesartan 0.1mg/Kg and the
saline groups were not different. For the 19 hours during the post-stroke period
the candesartan 1 mg/Kg group had MAP that was significantly lower than saline
and the candesartan 0.3 mg/Kg group was intermediate. The post-stroke means
for candesartan 0.1mg/Kg and the saline groups were significantly higher than
their pre-stroke values (all p<0.001). There was a significant difference among
the groups for AUC (p=0.0007) and MIN (p=0.0036) (Table 2). The candesartan
1 mg/Kg group was significantly lower than the saline group and the lowest dose
of candesartan but not different from the candesartan 0.3 mg/Kg group. A dose
of 0.3 mg/kg of candesartan following reperfusion caused a decrease of the MAP
to the pre-stroke baseline, while a dose of 1.0 mg/kg provoked a drop of MAP
below baseline. Post-reperfusion MAP following the injection of a dose of 0.1
mg/Kg of candesartan also decreased, however to a lesser extent and remained
above baseline until sacrifice. It did not differ significantly from that seen in saline
group (control), except during the first hour following reperfusion. There were no
differences among the groups for the MAX BP values during the duration of the
experiment.
Infarct size, hemispheric edema and behavior
Infarct size (Fig. 3A) and brain edema (Fig. 3B) were diminished in the SHR
treated with candesartan. This reduction, however, was dose-related within a
narrow range of candesartan doses (a range between 0.1 – 0.3 mg/Kg in our
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study). The greatest effects on infarct size and edema were observed in the rats
treated with a dose of 0.3 mg/kg of candesartan. Infarct size in the saline-treated
group (n = 16) was 62.3% compared to 50.7% of 0.3 mg/Kg candesartan group.
Calculated edema revealed a similar reduction (23.3% in saline group vs. 18.3%
in 0.3 mg/kg candesartan group). Table 3 shows that there are significant
differences among the groups for infarct size (p=0.0004) and edema (p=0.023).
The candesartan 0.3 mg/Kg group had the lowest infarct size and edema, and
was significantly different from the control group. Neither of the other two
candesartan doses were different from the saline group.
Animals receiving candesartan showed significantly better neurologic
function prior to sacrifice, assessed in the Bederson score (Fig. 4) as well as in
beam walk (Fig. 5A) and paw grasp (Fig. 5B) tests. In the elevated body swing
test, all animals subjected to ischemia, regardless of the treatment upon
reperfusion, revealed a consistent left-oriented circling. There were significant
differences among the groups for post-stroke values of Bederson scores
(p=0.0012) and paw grasp (p=0.005). Both the candesartan 0.3 mg/Kg and 1
mg/kg groups had Bederson and beam walk scores that were significantly better
than the control group.
Effect of candesartan on hemoglobin content in the ischemic brain tissue
Figure 6 shows the effect of three doses of candesartan on hemoglobin
content in the ischemic brain tissue collected 24 h after the onset of stroke. As
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can be seen, doses of 0.1 mg/Kg and 0.3 mg/Kg of candesartan reduced
hemoglobin content in a dose-dependent manner (8.3 ng/mg and 4.0 ng/mg for
doses of 0.1 and 0.3 mg/kg of candesartan, respectively, compared to 9.7 ng/mg
in saline-treated rats). Only the 0.3 mg/kg group was significantly different from
control (p=0.0092). Hemoglobin content in the ischemic brain tissue of rats
treated with a dose of 1.0 mg/Kg of candesartan, however, did not differ from that
seen in the saline-treated group (10.0 ng/mg in candesartan-treated vs. 9.7
ng/mg in saline-treated rats).
Cerebral Perfusion
In all 5 animals tested, MCAO resulted in consistent reductions in average
perfusion units + standard error (493.5 + 25.7 baseline versus 352 + 23.4 after
occlusion) in the ischemic hemisphere, which ranged from 41.1 to 68% of the
contralateral hemisphere five minutes after occlusion. Perfusion was restored to
relative hyperemia (521 + 25.8; average of 13.5% higher than contralateral ) in
the five minutes after reperfusion (Figure 7A). Hyperemia was bilateral prior to
sacrifice in the saline treated (no asymmetry) but asymmetry was maintained in
the candesartan 0.3 mg/kg animals (Figure 7B).
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Discussion
The optimal approach to management of elevated blood pressure during
the acute stroke period is unclear (Adams et al., 2007). Data presented here
indicate that partial lowering of the pressure during reperfusion is beneficial in the
SHR. It is represented by the improvement in neurologic score, reduction of
infarct size and brain tissue damage, as demonstrated by decreased hemoglobin
and edema formation in rats treated with candesartan at doses of 0.1 and 0.3
mg/kg immediately after reperfusion. On the other hand, reduction of blood
pressure below baseline in the SHR treated with a dose of 1.0 mg/kg eliminated
this protective effect. In fact, the animals treated with candesartan at a dose of
1.0 mg/kg revealed neurological and vascular damage resembling that seen in
rats treated with saline. It is of particular importance that hypertensive rats
appeared more sensitive to candesartan after stroke than normotensive rats,
since normotensive Wistar rats given a dose of 1.0 mg/kg immediately after
reperfusion decreased MAP to the baseline, and showed improvement in
neurologic outcome (Fagan et al., 2006).
Our data suggest that in the hypertensive rats, lowering acutely increased
blood pressure may be beneficial. However, extension of the decrease of
pressure to below SHR baseline values with candesartan can be deleterious. It
indicates that post-stroke recovery and homeostatic defense processes against
post-ischemic brain damage are indeed pressure-dependent. Thus, our present
results add to the discussion regarding the management of blood pressure after
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stroke. Presumably, there is a feedback and fine inter-relationship among blood
pressure, processes limiting the infarct size, and mechanisms involved in the
recovery of ischemic tissue.
It is possible, however, that the improved outcomes with candesartan
occurred despite of reducing blood pressure. It is known that apart from blood
pressure reduction, Ang II AT1-receptor antagonism also inhibits inflammation
(Ando, 2004), normalizes cerebrovascular autoregulation (Nishimura, 2000),
promotes angiogenesis (Forder et al., 2005), reduces oxidative damage
(Sugawara et al., 2005) and prevents apoptosis (Lou et al., 2004). Although
these effects have been studied under chronic conditions, they may be beneficial
in the event of an acute stroke. There may also be non-AT1-mediated
mechanisms involved in the protective effects of candesartan. Of note, it has
been postulated that stimulation of the Ang II AT2 receptor may be protective in
focal cerebral ischemia (Iwai et al., 2004).
In line with the above, Engelhorn et al. (2004) found that post-ischemia
treatment with candesartan (clinically relevant procedure similar to that applied in
our present studies) at a dose that had no significant effect on blood pressure,
reduced infarct size and improved neurological score. Also, early administration
of candesartan (3 h after onset of ischemia) to normotensive rats has been
shown to be neuroprotective, but only when excessive BP lowering is avoided
(Brdon et al., 2007). We are the first group to add the additional benefit of a
reduction in vascular damage and hemorrhage to the benefits of candesartan in
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SHRs as well as identifying an interaction between preexisting hypertension and
stroke in the response to BP lowering with candesartan. An interesting finding in
our study of improved neurologic function with candesartan 1 mg/kg despite no
histologic neuro- or vascular protection, suggests an additional beneficial effect
of the drug on recovery and perhaps brain plasticity.
Our findings are limited by the nature of the model (mechanical versus
embolic; SHR versus other models of hypertension), the short duration of follow-
up (24 hours), and the inability to determine the mechanism of the protective
effects seen. It is most likely that the protective effects of candesartan are
multimodal, partly due to BP lowering and partly due to pleiotropic vascular
protective effects. It is clear that blood pressure lowering after reperfusion
protects the vasculature and is neuroprotective (Elewa et al., 2007). Translating
this to human stroke patients is complicated by the fact that premorbid
conditions, such as vascular disease due to chronic hypertension, changes the
sensitivity to a given dose of antihypertensive and may make it more likely to
overshoot baseline values and eliminate the benefit. Restarting blood pressure
medications in patients with acute stroke, as has been recently recommended
(Adams et al., 2007), may be perilous if the same pre-stroke doses are used.
More research is necessary to identify patients most likely to benefit from blood
pressure lowering with candesartan after stroke.
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Acknowledgments Candesartan Cilexitil was provided by Astra Zeneca
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Footnotes
Financial support provided by the American Heart Association Southeast Affiliate
– Grant in Aid (SCF) and VA Merit Review (SCF)
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Legends for Figures Figure 1. Changes of mean arterial pressure (MAP; mmHg) of non-stroke SHR injected
intravenously (arrow) in the separate groups with 3 doses of candesartan (n=4
per group). MAP values are 1-h means ± SEM. Black horizontal bars indicate a
night-time in the 12:12 h light/dark cycles.
Figure 2.
Mean arterial blood pressure (MAP; mmHg) after acute stroke in the SHR. MAP
was recorded every 10 min (telemetry) prior to stroke (baseline between 6 and 9
AM), during the onset of ischemia (at 10 AM; left arrow), reperfusion (at 1 PM;
right arrow) and then during the following 21 hrs until sacrifice next day. At
reperfusion, the animals were intravenously injected with saline (n=8) and/or with
candesartan at the doses as shown. The black horizontal bar indicates night-
time in the light/dark cycles. Values shown are 1-h averages ± SEM.
Figure 3.
Effect of candesartan on ischemia-induced infarct size (panel A) and edema
(panel B) in the SHR. Doses of candesartan, or saline as control, were injected
intravenously at the time of reperfusion during MCAO, as shown in Fig. 2. (* = p
< 0.05 compared to control)
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Figure 4.
Effect of candesartan on Bederson score in the SHR subjected to MCAO. The
animals were tested before reperfusion and treatment (white bars), and only
animals with a score of 3 were further examined. Dashed bars represent the
scores (means ± SEM) assessed 24 h after the reperfusion and injections, prior
to sacrifice. (* = p < 0.05 compared to control)
Figure 5.
Tests of beam walk (panel A) and paw grasp (panel B) of the SHR as described
in Fig. 4. Rats were tested prior to sacrifice. (* = p < 0.05 compared to control)
Figure 6.
Effect of candesartan on hemoglobin content (Hb; ng/mg of protein) in the
ischemic hemispheres of SHR. (* = p < 0.05 compared to control)
Figure 7.
Laser Doppler scan images (PIM 3) of tissue perfusion (top) and orientation
(bottom). (A ) A representative animal where perfusion is symmetric at baseline,
reduced (dark blue = 3.6 perfusion units) in the ischemic hemisphere at 5
minutes after MCAO (5’MCAO) and returns to relative hyperemia (dark red =
1295 perfusion units) in the ischemic hemisphere 5 minutes after reperfusion (5’
after reperfusion). (B) The animal treated with saline demonstrated bilateral
hyperemia prior to sacrifice and this was accompanied by a large infarct (white)
with hemorrhage. In the animal treated with candesartan (cand) 0.3 mg/kg, the
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hyperemia in the ischemic hemisphere was still present at 24 hours and this was
associated with a smaller infarct size. The animal treated with candesartan
(cand) 1 mg/kg had symmetric, lower perfusion prior to sacrifice and a larger
infarct.
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Table 1. MAP for each group at different time points – Mean (SD)
Treatment N Pre-stroke MCAO 2hr post-perfusion * Post-stroke *
Saline 7 135.3 (7.5) 188.6 (7.3) 173.5 (10.0) a 160.2 (6.4) a
Candesartan 0.1 7 146.2 (8.2) 187.4 (8.3) 166.2 (9.8) a 160.6 (12.5) a
Candesartan 0.3 7 146.0 (7.5) 189.4 (8.1) 151.2 (10.6) b 148.2 (12.7) a,b
Candesartan 1 7 139.1 (8.2) 186.3 (8.0) 145.6 (7.6) b 133.2 (10.4) b
* Means with the same letter are not significantly different
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Table 2. AUC, MIN and MAX values for each group – Mean (SD)
Treatment
N AUC * MIN, mmHg * MAX, mmHg Change pre
to post, 6-9
am *
Saline 7 4340 (145) a 128.0 (6.4) a 193.2 (7.5) 22.7 (9.5) a
Candesartan 0.1 7 4382 (286) a 138.8 (7.6) a 192.2 (8.2) 7.2 (11.1) b
Candesartan 0.3 7 4123 (293) a, b 134.1 (10.5) a,b 192.8 (9.0) -1.4 (8.7) b,c
Candesartan 1 7 3804 (228) b 120.3 (9.6) b 189.5 (8.1) -9.3 (10.0) c
p-value 0.0007 0.0036 0.84 <0.0001
* Means with the same letter are not significantly different
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Table 3. Infarct size and edema for each group – Mean (SD)
Treatment N Infarct size * Edema *
Saline 16 62.3 (6.3) a 23.3 (3.0) a
Candesartan 0.1 7 58.0 (3.5) a,b 21.2 (2.4) a,b
Candesartan 0.3 11 50.7 (6.7) b 18.3 (5.0) b
Candesartan 1 11 61.8 (6.8) a 22.8 (5.5) a,b
p-value 0.0001 0.023
* Means with the same letter are not significantly different
† Analysis performed on the ranks
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100
110
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160
170
180
6:00
8:00
10:0
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4:00
Time (h)
MA
P (
mm
Hg
)Candesartan 0.1mg/kg (n=4)
Candesartan 0.3mg/kg (n=4)
Candesartan 1mg/kg (n=4)
Figure 1
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190
200
6:00
7:00
8:00
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5:00
6:00
7:00
8:00
9:00
10:0
0
Time (h)
MA
P (m
mH
g)
Saline (n=8)
Candesartan 0.1mg/kg (n=7)
Candesartan 0.3mg/kg (n=7)
Candesartan 1mg/kg (n=7)
MCA
Reperfusion
Figure 2
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0
10
20
30
40
50
60
70
80
Saline (n=16) Candesartan 0.1mg/kg(n=7)
Candesartan 0.3mg/kg(n=11)
Candesartan 1mg/kg(n=11)
Infa
rct
size
ave
rag
e (%
)
*
Figure 3A
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*
0
5
10
15
20
25
30
Saline (n=16) Candesartan 0.1mg/kg(n=7)
Candesartan 0.3mg/kg(n=11)
Candesartan 1mg/kg(n=11)
Ed
ema
(% c
han
ge
fro
m c
on
tral
ater
al)
*
Figure 3B
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**
0
0.5
1
1.5
2
2.5
3
3.5
Saline (n=16) Candesartan 0.1mg/kg(n=7)
Candesartan 0.3mg/kg(n=11)
Candesartan1mg/kg(n=11)
Bed
erso
n s
core
(0-
3)before reperfusion
before sacrificing
* *
Figure 4
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** *
0
0.5
1
1.5
2
2.5
3
Saline (n=8) Candesartan 0.1mg/kg(n=7)
Candesartan 0.3mg/kg(n=11)
Candesartan 1mg/kg(n=8)
Bea
m w
alk
(0-3
po
ints
)
*
*
Figure 5A
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0
0.5
1
1.5
2
2.5
3
Saline (n=8) Candesartan0.1mg/kg (n=7)
Candesartan0.3mg/kg (n=11)
Candesartan 1mg/kg(n=8)
Paw
gra
sp (
0-3
po
ints
) *
Figure 5B
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0
2
4
6
8
10
12
14
Saline (n=16) Candesartan0.1mg/kg (n=7)
Candesartan0.3mg/kg (n=11)
Candesartan 1mg/kg(n=11)
Hb
(n
g/m
g o
f p
rote
in)
*
Figure 6
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