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Introduction
Stroke is the third leading cause of death, only preceded
by heart disease and cancer in many
countries[1]. According to recent estimates published by the World Health
Organization, about 15 million people per year fall victim to
stroke worldwide. Therefore, prevention is the only
possible way to curb the stroke
pandemic[2]. Blood pressure level is one of the most consistent and powerful predictors
for stroke. As a result, blood pressure control is an
important way to reduce the morbidity of
stroke[3_5].
Recently, the importance of combination therapy has
been well recognized in the treatment of
hypertension. Clinically, combination therapy against hypertension using
2 or more drugs from different classes can produce better
drug efficacy[6,7]. Both β-adrenergic blockers and
dihydro-pyridine calcium antagonists are widely used in the
treatment of hypertension. Atenolol and amlodipine are
prototypes of the long-acting drugs from these 2 classes. Several
works within our Department (Second Military Medical
University, Shanghai, China) have demonstrated that there
is significant synergism between atenolol and amlodipine in
lowering and stabilizing blood pressure. For an acute setting,
the synergistic effect is at its highest when the dose
proportion of the 2 drugs is 10:1 in hypertensive
rats[8, 9].
A number of reports have shown that the combination
therapy of antihypertension has overt effects on organ
protection in hypertensive rats[10]. However, to the best of our
knowledge, little information is available regarding the
impact of the combination therapy of atenolol and amlodipine
on the prevalence of stroke. Given that stroke-prone
spontaneously hypertensive rats (SHR-SP) are the most widely
used animal models for experimental
stroke[11], this study employed this animal model to investigate the synergism
between atenolol and amlodipine on blood pressure
reduction and stroke prevention.
Materials and methods
Drugs and animals Amlodipine was purchased from
Nanjing Pharmaceutical Co (Nanjing, China) and atenolol
was purchased from Shanghai Second Pharmaceutical Co
(Shanghai, China). SHR-SP of either sex were provided by
the Animal Center of our University. They were housed with
controlled temperature (23_25 °C) and lighting (8:00_20:00
light, 20:00_8:00 dark), and with free access to standard food
and drinking water. All the animals used in this experiment
received humane care and the study was in compliance with
the institutional guidelines for the health and care of
experimental animals.
Blood pressure measurements Systolic blood pressure
(SBP), diastolic blood pressure (DBP), and heart period were
continuously recorded using the previously described
technique[12,15]. Briefly, the rats were anesthetized with a
combination of ketamine and diazepam. A polyethylene catheter
was inserted into the lower abdominal aorta via the left
femoral artery for blood pressure measurement and another
catheter was inserted into the left femoral vein for phenylephrine
administration. A third catheter was placed into the stomach
via a mid-abdominal incision for drug administration. After a
2 d recovery period, the animals were placed in individual
cylindrical cages. The aortic catheter was connected to a
blood pressure transducer via a rotating swivel that allowed
the animals to move freely in the cage. After habituation, the
blood pressure signal was digitized by a microcomputer. SBP,
DBP, and heart period values were determined online. The
mean values and standard deviation of SBP and DBP were
calculated. The standard deviation of both values obtained
was denoted as the quantitative parameter of variability, that
is, systolic blood pressure variability (SBPV) and diastolic
blood pressure (DBPV) for each rat.
Determination of baroreflex sensitivity In the
earlier-mentioned blood pressure recording condition, baroreflex
sensitivity (BRS) was measured in the conscious rat, by
using the previously described
method[14,15]. Briefly, a bolus injection of phenylephrine was used to induce a blood
pressure elevation. The dose of phenylephrine (5_10 µg/kg) was
adjusted to raise the SBP to about 30 mmHg. The slope with
the largest correlation coefficient (r) of heart period/SBP was
expressed as BRS (ms/mmHg). The mean of the 2
measurements with the proper dose was taken as the final result.
Stroke symptom observation and morphological
examination To detect the stroke symptoms, the movement of
limbs, respiration, diet, fur, and consciousness of all
SHR-SP, they were observed twice daily (at 8:00 and 18:00). When
the rats died, the brains were removed, and signs of
hemorrhage, edema, or infarction were examined and then
photographed.
Probability sum test To determine whether the drugs
were acting synergistically, we used the probability sum test
according to previous reports[10,16]. Briefly, compared with
the mean values from the control rats, the treated rats with a
decrease in blood pressure >20 mmHg were defined as
responders and rats with a decrease in blood pressure
³20 mmHg were defined as non-responders. For SBPV or DBPV,
the criterion was 2 mmHg. The formula used is as follows:
q=PA+B/(PA
+PB_PA×
PB). Here, A and B indicate drug A and
drug B and P (probability) is the percentage of responders in
each group. PA+B is the real percentage of responders and
(PA+PB_P
A×PB) is the expected response rate.
PA+PB is the sum of the probabilities when drug A and drug B were used
alone.
PA×PB is the probability of rats responding to both
drugs when they were used alone. When q<0.85, the
combination was antagonistic, when q>1.15, the combination was
synergistic, and when q was between 0.85 and 1.15, the
combination was additive.
Experimental protocols
Acute studies The experiment was performed in
8-month-old, female SHR-SP. They were randomly divided
into 3 groups (n=8 in each group) and received 10 mg/kg
atenolol, 1.0 mg/kg amlodipine, and a 10+1.0 mg/kg
combination of these 2 drugs (intragastrically) respectively. The
drugs were dissolved in 0.8% carboxymethylcellulose
sodium. After about 4 h habituation (from 8:00 to 12:00),
blood pressure was recorded during a period of 60 min (from
12:00 to 13:00) and BRS was measured using the earlier-mentioned methods. These values were taken as baseline
before administration. After 30 min habituation, a single dose
of these drugs was given via an intragastric catheter. About
30 min after drug administration, blood pressure was recorded
for another 60 min (from 15:00 to 16:00 ) and BRS was
determined again. These values were defined as after
administration values.
Chronic studies The experiment was performed in
6-week-old, SHR-SP of either sex. Forty male and 40 female
rats were randomized into 4 groups: the control group and 3
groups treated with 10
mg·kg-1·d-1 atenolol, 1.0
mg·kg-1·d-1 amlodipine, and a 10+1.0
mg·kg-1·d-1 combination of these 2
drugs (intragastrically), respectively (n=20 in each group, 10
male and 10 female). The drugs were mixed into the food and
administrated consecutively. The survival time was recorded
(from birth to death).
Statistical analysis The investigators were blind to the
procedures during blood pressure and heart period recording,
BRS determination, and morphological examination.
Statistical analysis data are expressed as the mean±SD.
Comparisons between pre- and post-drug were made by paired
t-tests. Comparisons among 4 or 3 groups were made by
one-way ANOVA. In the chronic experiment, the
Kaplan-Meier analysis was used to estimate survival probabilities.
Log-rank testing was used to evaluate the equality of
survival curves. P<0.05 was considered statistically significant.
Results
Effects of atenolol and amlodipine on blood pressure,
heart period, and BRS The effects of a single dose of atenolol
and amlodipine, either alone or in combination, on blood
pressure, heart period, and BRS in conscious SHR-SP are
shown in Figure 1. Compared with the baseline value, SBP
was significantly decreased in all 3 groups (atenolol:
171±33.2 vs 190±27.9 mmHg,
P<0.01; amlodipine: 175±56.8
vs 189±51.5 mmHg, P<0.05; and in combination: 156±23.4
vs 189±23.4 mmHg, P<0.01). Compared with the baseline, DBP also
significantly decreased in 2 groups (P<0.05), but not in the
amlodipine group (P>0.05). The mean heart period value
after administration was markedly higher only in the rats
treated with atenolol alone. Compared with the baseline
value, BRS did not exhibit any significant changes in all the
groups tested.
Effects of atenolol and amlodipine on SBPV and DBPV
Compared with the baseline value, there was a significant
decrease in SBPV and DBPV in the combination group
(P<0.05), but not in the groups of atenolol and amlodipine alone
(P>0.05; Figure 2).
Synergistic interaction of atenolol and amlodipine on
blood pressure and blood pressure variability Based on the
results presented in Figure 1, the effectiveness of the
decrease in blood pressure was calculated for the rats
individually. Compared with the baseline, the rats with a
decrease in blood pressure >20 mmHg were defined as
responders and those with a decrease in blood
pressure £20 mmHg were defined as non-responders. The results of
probability testing are presented in Table 1. We arrived at
q-values of 1.17 for SBP and 2.67 for DBP for the combination
of atenolol and amlodipine. Compared with the baseline value,
the rats with a decrease in BPV >2 mmHg were defined as
responders. According to this criterion, the
q-values were
2.48 for SBPV and 2.10 for DBPV for the combination of
atenolol and amlodipine.
Effects of atenolol and amlodipine on survival time
Among the 80 rats studied, all the animals that died from
stroke were confirmed to display neurological symptoms of
stroke and/or brain pathological examination. The survival
time expressed by the Kaplan-Meier survival curve is shown
in Figure 4. A significant difference was detected between
the control and the 3 drug treatment groups (Log-rank
testing χ2=17.34, P<0.001). Compared with the control group,
the lifespan of SHR-SP in either sex was both significantly
increased by atenolol and amlodipine alone and in
combination (male: 328±46.7 d, 357±109 d, and 399±152 d
vs 262±85.7 d; female: 569±127 d, 576±89.4 d, and
608±121vs 397±149 d). In the male rats, survival time was 1.25-fold greater in the
atenolol group, 1.36-fold greater in the amlodipine group,
and 1.50-fold greater in the combination treatment group. In
the female rats, similar results were obtained. Compared with
the control group, the survival time was 1.43-fold greater in
the atenolol group, 1.45-fold greater in the amlodipine group,
and 1.53-fold greater in the combination group.
Discussion
Blood pressure level is an important determinant for the
prevalence of stroke. The risk of stroke is increased by about
25% with each 10 mmHg increase in SBP[3,
4]. Therefore, the use of antihypertensive drugs is an important way of
reducing the morbidity of stroke. Combination treatment of
different antihypertensive drugs may be an ideal and more
effective method for blood pressure control and stroke prevention.
The proper combination of different drugs may produce
synergy of drug responses, for example, improved blood
pressure control and compliance compared with using a single
drug alone[17]. In this work, we selected atenolol and
amlodi-pine as the combination with a proportion of 10:1 according
to our previous report[9]. Our results confirmed that the
combination between atenolol and amlodipine was synergistic
on blood pressure control in SHR-SP rats. An obvious
decrease in SBP and DPB was found in SHR-SP treated by this
combination, with a maximal decrease of SBP (33
mmHg) and DBP (27 mmHg). These values were significantly higher than
the treatment of atenolol and amlodipine alone. Meanwhile,
the q-values for SBP and DBP after administration were higher
than 1.15, the threshold value for synergistic effects.
Both β-adrenergic blockers and dihydropyridine calcium
antagonists are widely used in antihypertensive therapy.
The combination of a β-adrenergic blocker and a
dihydro-pyridine calcium antagonist is a logical
choice[18]. Theore-tically, calcium antagonists are vasodilators
and tend to increase plasma rennin levels, which may be offset by
β-blockers[19]. A combination of these compounds can also
neutralize the side-effects of both, for example, the initial
heart rate decreases induced by atenolol, and the rise in
peripheral resistance elicited by some
β-blockers[20]. Our results confirmed these benefits. In the atenolol group, the
heart period was significantly increased, but not in the
combination treatment group.
Elevated blood pressure is not the unique factor
determining the occurrence of stroke in hypertension. It has been
proposed that BPV may play an important role in
determining the prognosis of hypertension. Parati et al found that for
patients with similar mean hypertension levels for the 24 h
after treatment, those whose BPV levels were lower had less
severe end-organ damage than those with higher BPV
levels[21]. Our previous study also confirmed this in hypertensive
rats and proposed that BPV might be a new strategy for the
treatment of hypertension[22,23], even including its most
important complication, stroke. Therefore, it is very important
to emphasize the role of BPV in antihypertensive therapy
and stroke prevention. In the present work, we found that in
SHR-SP, BPV was not influenced by treatment with atenolol
or amlodipine alone, but was markedly reduced when they
were used in combination. The q-values for SBPV and DBPV
were 2.48 and 2.10, respectively. These findings
demonstrate that the combination has an overt synergistic effect
on stabilizing BPV in SHR-SP rats.
The most important aspect of this study may be the
significantly prolonged lifespan following combination therapy
of atenolol and amlodipine, as opposed to either atenolol or
amlodipine alone. In the setting of the acute studies, we
found that the combination therapy displayed a maximal
decrease of SBP and DBP associated with a significant
stabilization in blood pressure, which suggests that a decrease of
blood pressure and BPV may contribute concurrently to
stroke-preventive action in SHR-SP.
In conclusion, atenolol and amlodipine in combination
have a synergistic effect in lowering and stabilizing blood
pressure in SHR-SP. Combination therapy is likely to be the
optimal way of preventing stroke in hypertension.
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