jpet fast forward. published on june 17, 2008 as doi:10...

JPET #139618

1

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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 17, 2008 as DOI: 10.1124/jpet.108.139618

at ASPE

T Journals on N

ovember 19, 2020

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET #139618

2

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

[email protected]

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

3

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.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

4

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,


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

5

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.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

6

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

7

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

8

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

9

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

10

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).


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

11

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).


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

12

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

13

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

14

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).


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

15

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

16

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

17

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.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

18

Acknowledgments Candesartan Cilexitil was provided by Astra Zeneca


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

19

References Adams HP, del Zoppo G, ALberts AJ, Bhatt DL, Brass L, Furlan A, Grubb RL,

Higashida R, Jauch EC, Kidwell C, Lyden PD, Morgenstern LB, Qureshi AI,

Rosenwasser RH, Scott PA, Wijdicks EFM (2007) Guidelines for the early

management of adults with ischemic stroke. From the AHA/ASA Stroke Council.

Stroke 38(5): 1655-1711

Ando H, Zhou J, Macova M, Imboden H, Saavedra JM, MD (2004) Angiotensin II

AT1 receptor blockade reverses pathological hypertrophy and inflammation in

brain microvessels of spontaneously hypertensive rats. Stroke 35: 1726-1731.

Barone FC, Price WJ, White RF, Willette RN, Feuerstein GZ (1992) Genetic

hypertension and increased susceptibility to cerebral ischemia. Neurosci.

Biobehav. Rev. 16: 219-233.

Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bratowski J (1986) Rat

middle cerebral artery occlusion: evaluation of the model and development of a

neurologic examination. Stroke 17: 472-6.

Brdon J, Kaiser S, Hagemann F, Zhao Y, Culman J, Gohlke P (2007)

Comparison between early and delayed systemic treatment with candesartan of

rats after ischemic stroke. J Hypertens 25: 187-196.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

20

Borlongan CV, Sanberg PR (1995) Elevated body swing test: a new behavioral

parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. J.

Neurosci. 15(7 Pt 2):5372-5378.

Coyle P (1986) Different susceptibilities to cerebral infarction in spontaneously

hypertensive (SHR) and normotensive Sprague-Dawley rats. Stroke 17: 520-525.

Elewa HF, Kozak A, Johnson MH, Ergul A, Fagan SC (2007) Blood pressure

lowering after experimental cerebral ischemia provides neurovascular protection.

J Hypertens 25: 855-859.

Engelhorn T, Goerike S, Doefler A, Okorn C, Forsting M, Heusch G, Schultz R

(2004) The angiotensin II type 1-receptor blocker candesartan increases cerebral

blood flow, reduces infarct size, and improves neurologic outcome after transient

ischemic in rats. J Cerebr Blood Flow Metab 24: 467-474.

Fagan SC, Kozak A, Hill WD, Pollock DM, Xu L, Johnson MH, Ergul A, Hess DC

(2006) Hypertension after experimental cerebral ischemia: candesartan provides

neurovascular protection. J. Hypertens. 24: 535-539.

Forder JP, Munzenmaier DH, Greene AS (2005) Angiogenic protection from focal

ischemia with angiotensin II type 1 receptor blockade in the rat. Am J Physiol

Heart Circ Physiol 288: H1989-H1996.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

21

Harms H, Wiegand F, Megow D, Prass K, Einhaupl KM, Dirnagl V (2000) Acute

treatment of hypertension increases infarct sizes in spontaneously hypertensive

rats. Neuroreport 11: 355-359.

Hilali HM, Simpkins AN, Hill WD, Waller JL, Knight RA, Fagan SC (2004) Single

slice method for quantification of hemorrhagic transformation using direct ELISA.

Neurol Res 26: 93-8.

Ito H, Takemori K, Suzuki T (2001) Role of angiotensin II type 1 receptor in the

leucocytes and endothelial cells of brain microvessels in the pathogenesis of

hypertensive cerebral injury. J. Hypertens 19: 591–597.

Ito T, Yamakawa H, Bregonzio C, Terrón JA, Falcón-Neri A, Saavedra JM (2002)

Protection against ischemia and improvement of cerebral blood flow in

genetically hypertensive rats by chronic pretreatment with an angiotensin II AT1

antagonist. Stroke 33: 2297-2303.

Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T,

Horiuchi M (2004) Possible inhibition of focal cerebral ischemia by angiotensin II

type 2 receptor stimulation. Circulation 110:843-848.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

22

Li C, Engstrom G, Hedblad B, Berglund G, and Janzon L (2005) Blood pressure

control and risk of stroke: a population-based prospective cohort study. Stroke

36: 725-730.

Lou M, Blume A, Zhao Y, Gohlke P, Deuschl G, Herdegen T, Culman J. (2004)

Sustained blockade of brain AT1 receptors before and after focal cerebral

ischemia alleviates neurologic deficits and reduces neuronal injury, apoptosis,

and inflammatory responses in the rat. J Cerebr Blood Flow Metab 24: 536-547.

Nishimura Y, Ito T, Saavedra JM (2000) Angiotensin II AT1 blockade normalizes

cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously

hypertensive rats. Stroke 31: 2478–2486.

Slivka A, Murphy E, Horrocks L (1995) Cerebral edema after temporary and

permanent middle cerebral artery occlusion in the rat. Stroke 26: 1061-1065.

Stokes J, IIIrd, Kannel WB, Wolf PA, D'Agostino RB, Cupples LA (1989) Blood

pressure as a risk factor for cardiovascular disease. The Framingham Study--30

years of follow-up. Hypertension 13: 13-18.

Sugawara T, Kinouchi H, Oda M, Shoiji H, Onae T, Mizoi K (2005) Candesartan

reduces superoxide production after global cerebral ischemia Neuroreport 16:

325-328.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

23

Wahl Fl, Allix M, Plotkine M, Boulu RG (1992) Neurological and behavioral

outcomes of focal cerebral ischemia in rats. Stroke 23: 267-272.

Zea Longa E, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle

cerebral artery occlusion without craniectomy in rats. Stroke 20: 84-91.

Zhou J, Ando H, Macova M, Dou J, Saavedra JM (2005) Angiotensin II AT1

receptor blockade abolishes brain microvascular inflammation and heat shock

protein responses in hypertensive rats. J Cereb Blood Flow Metab 25: 878–886.

Zhou J, Pavel J, Macova M, Yu ZX, Imboden H, Ge L, Nishioku T, Dou J,

Delgiacco E, Saavedra JM (2006) AT1 receptor blockade regulates the local

angiotensin II system in cerebral microvessels from spontaneously hypertensive

rats. Stroke 37: 1271-1276.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

24

Footnotes

Financial support provided by the American Heart Association Southeast Affiliate

– Grant in Aid (SCF) and VA Merit Review (SCF)


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

25

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)


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

26

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

27

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.


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

28

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


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

29

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



at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


JPET #139618

30

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


† Analysis performed on the ranks


at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


100

110

120

130

140

150

160

170

180

6:00

8:00

10:0

012

:00

14:0

016

:00

18:0

020

:00

22:0

00:

002:

004:

006:

008:

0010

:00

12:0

014

:00

16:0

018

:00

20:0

022

:00

0:00

2:00

4:00

6:00

8:00

10:0

012

:00

14:0

016

:00

18:0

020

:00

22:0

00:

002:

004:

006:

008:

0010

:00

12:0

014

:00

16:0

018

:00

20:0

022

:00

0:00

2:00

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

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on June 17, 2008 as D

OI: 10.1124/jpet.108.139618

at ASPET Journals on November 19, 2020 jpet.aspetjournals.org Downloaded from


110

120

130

140

150

160

170

180

190

200

6:00

7:00

8:00

9:00

10:0

011

:00

12:0

013

:00

14:0

015

:00

16:0

017

:00

18:0

019

:00

20:0

021

:00

22:0

023

:00

0:00

1:00

2:00

3:00

4:00

5:00

6:00

7:00

8:00

9:00

10:0

0

Time (h)

MA

P (m

mH

g)

Saline (n=8)



Candesartan 1mg/kg (n=7)

MCA

Reperfusion

Figure 2




JPET


OI: 10.1124/jpet.108.139618



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




JPET


OI: 10.1124/jpet.108.139618



*

0

5

10

15

20

25

30




Ed

ema

(% c

han

ge

fro

m c

on

tral

ater

al)

*

Figure 3B




JPET


OI: 10.1124/jpet.108.139618



**

0

0.5

1

1.5

2

2.5

3

3.5



Candesartan1mg/kg(n=11)

Bed

erso

n s

core

(0-

3)before reperfusion

before sacrificing

* *

Figure 4




JPET


OI: 10.1124/jpet.108.139618



** *

0

0.5

1

1.5

2

2.5

3




Bea

m w

alk

(0-3

po

ints

)

*

*

Figure 5A




JPET


OI: 10.1124/jpet.108.139618



0

0.5

1

1.5

2

2.5

3

Saline (n=8) Candesartan0.1mg/kg (n=7)

Candesartan0.3mg/kg (n=11)


Paw

gra

sp (

0-3

po

ints

) *

Figure 5B




JPET


OI: 10.1124/jpet.108.139618



0

2

4

6

8

10

12

14

Saline (n=16) Candesartan0.1mg/kg (n=7)

Candesartan0.3mg/kg (n=11)


Hb

(n

g/m

g o

f p

rote

in)

*

Figure 6




JPET


OI: 10.1124/jpet.108.139618




at ASPE

T Journals on N

ovember 19, 2020


ownloaded from


jpet fast forward. published on june 17, 2008 as doi:10...

Documents