Download Morphine reduces the threshold of remote ischemic preconditioning

中青年优秀论文评选
危重医学临床与基础研究
陆姚 男
1985 年 7 月出生
Morphine reduces the threshold of remote ischemic preconditioning
against myocardial ischemia and reperfusion injury in rats: role of
opioid receptors
Yao Lu1, Chun-shan Dong1*, Jun-ma Yu1, Lei Li1, Hong Li2
1 Department of Anaesthesiology, Third affiliated hospital of Anhui Medical
University, Hefei 230061, China; 2 Department of Anaesthesiology, Second affiliated
hospital of The Third Military University, Chongqing 230061, China.
*Corresponding author: Department of Anaesthesiology, Third affiliated hospital of
Anhui Medical University, Hefei 230061, China. Email: [email protected]
Phone: +865512183386
Abstract
Aims: Opioid receptors mediate the cardioprotection of remote ischemic
preconditioning (RIPC). We test the hypothesis that mophine reduces the threshold of
cardioprotection produced by RIPC.
Main methods: Anesthetized, open chest, male Sprague-Dawley rats were
randomly assigned to 1 of 7 treatment groups. RIPC1 and RIPC3 were respectively
induced by 1 or 3 cycles of 5 minutes femoral artery ischemia interspersed with 5
minutes reperfusion. Mophine (MOR, 100 ug/kg) and opioid receptors antagonist
naloxone (NAL, 6 mg/kg) were respectively used to administered 30 minutes before
sustaining ischemia. The combination of MOR and RIPC1 in the absence or presence
of NAL before coronary artery occlusion. Ischemia and reperfusion injury was then
induced by 30 minutes of left coronary artery occlusion followed by 120 minutes of
reperfusion. Infarct size, as a percentage of the area at risk, was determined by
2,3,5-triphenyltetrazolium staining.
Key findings: RIPC3 and the combination of MOR and RIPC1 groups reduced the
infarct size compared with control. RIPC1, morphine and naloxone didnot affect
infarct size. Naloxone pretreatment reversed cardioprotection of the combination of
MOR and RIPC1 treatments.
Significance: Mophine reduces the threshold of remote ischemic preconditioning,
and opioid receptors mediate this augmentative effects.
Keywords: morphine; remote ischemic preconditioning; myocardial ischemia;
opioid receptor
Introduction
Przyklenk et al.1 firstly found that brief episodes of ischemia in one coronary bed
render remote virgin myocardium resistant to infarction, this phenomenon was named
“remote ischemic preconditioning (RIPC)”. Subsequent studies confirmed the
existence of this protective effect of remote preconditioning in other organs, such as
kidney2, small intestine3 or skeletal muscle4. Although the mechanisms of remote
ischemic preconditioning are incompletely understood, some similarities to ischemic
preconditioning have been discovered in the conveyance of external signals to
intracellular targets that ultimately lead to protection. Remote ischemic
preconditioning also can be elicited via opioid, bradykinin and adenosine5. It was
demonstrated that δ1 and κ opioid receptors are mediated in the cardioprotection of
remote ischemic preconditioning6,7. Morphine, a μ receptor agonist with δ and κ
receptor agonist properties8, has been shown to mimic ischemic preconditioning in the
intact and isolated rat hearts9,10. Previous studies indicate that morphine enhances the
cardioprotection induced by volatile anesthetics (eg, isoflurane and helium)
preconditioning11-13. The addition of morphine infusion to remote ischemic
postconditioning during reperfusion could confer greater percentage of ST-segment
resolution and lower peak TnI levels in patients submitted to primary percutaneous
coronary intervention (PCI) 14. In this study, we hypothesized that morphine reduces
the threshold of cardioprotection induced by remote ischemic preconditioning. We
further hypothesized that this protective effect is mediated by activation of opioid
receptors in rats.
Materials and Methods
This study was conducted in accordance with our institutional guidelines on the
use of live animals for research and the experimental protocol was approved by the
Animal Care and Use Committee of Anhui Medical University. Male SpragueDawley rats, weighing between 280 and 300 g were used for this study.
Surgical procedures - ischemia and reperfusion injury model
Rats were anesthetized by intraperitoneal administration of pentobarbitone (50
mg/kg) maintained by repeat doses of 25 mg/kg every 60-90 min as necessary. All of
the animals underwent tracheotomy and tracheal intubation. Mechanical ventilation
was provided with a Harvard Apparatus Rodent Respirator (Harvard Apparatus,
Boston, MA), and the rats were ventilated with room air at 70-80 breaths per minute.
Body temperature was monitored and maintained at 37 ± 1°C (mean ± SD) using a
heating pad. The right carotid artery was cannulated for direct blood pressure
monitoring via a pressure transducer and a lead-II electrocardiogram monitored heart
rate via subcutaneous stainless steel electrodes connected to a PowerLab monitoring
system (ML750 PowerLab/4sp with MLT0380 Reusable BP Transducer; AD
Instruments, Colorado Springs, CO). Hemodynamic values including heart rate (HR)
and mean arterial blood pressure (MAP) were recorded at baseline, at the end of the
treatment period and the end of the ischemia and reperfusion periods respectively for
comparison. The left femoral vein was cannulated for drug administration. A left
thoracotomy was performed to expose the heart at the fifth intercostal space. After
removing the pericardium, a 6-0 Prolene loop, along with a snare occluder, was
placed at the origin of the left coronary artery in preparation for inducing ischemia
reperfusion injury. Regional ischemia was induced by pulling the snare and securing
the threads with a mosquito hemostat. Ischemia was confirmed by
electrocardiographic changes, a substantial decrease in mean arterial pressure and
cardiac cyanosis. Rats were omitted from further data analysis if severe hypotension
(arterial mean blood pressure less than 30 mmHg) or intractable ventricular
fibrillation occurred. After surgical preparation, the rat was allowed to stabilize for 15
min.
Study groups and experiments protocol
Rats were randomly assigned to receive 1 of 7 treatments (figure 1). All animals
were subjected to 30 minutes of ischemia by occlusion of the left coronary artery
followed by 2 hours of reperfusion by release of the occlusion: RIPC1 and RIPC3
group were respectively induced by 1 or 3 cycles of 5 minutes right femoral artery
ischemia interspersed with 5 minutes reperfusion before coronary occlusion. A non
specific opioid receptors antagonist naloxone (NAL) (Sigma Chemical Co.) was used
to evaluate the involvement of opioid receptors. While MOR and NAL group were
respectively induced by intravenous morphine (David Bull Laboratories, 100 ug/kg13)
or naloxone (6 mg/kg13) administered 30 minutes prior to sustaining ischemia. MOR
+ RIPC1 and NAL + MOR + RIPC1 groups of rats received 1 cycle of 5 minutes right
femoral artery ischemia interspersed with 5 minutes reperfusion plus morphine (100
ug/kg) in the absence or presence of naloxone (6 mg/kg) before ischemia and
reperfusion injury. As negative control (CON), a group merely received ischemia and
reperfusion injury.
Infarct size determination
The hearts were excised and transferred to a Langendorff apparatus on completion
of the reperfusion period and immediately perfused with normal saline for 1 min at a
pressure of 100 cm H2O to flush out residual blood. The snare was securely
retightened and 0.25% Evan blue dye injected to stain the normally perfused region of
the heart. This procedure allowed visualization of the normal, non ischemia region
and the area at risk (AAR). The hearts were then frozen, and cut into 2 mm slices.
Thereafter, the slices were stained by incubation at 37°C for 20 min in 1% 2, 3,
5-triphenyltetrazolium (Sigma Chemical Co.) in phosphate buffer at pH 7.4. This was
followed by immersion in 10% formalin for 20 min to enhance the contrast of the
stain. The areas of infarct and risk zone for each slice were traced and digitized using
a computerized planimetry technique (SigmaScan 4.0, Systat Software Inc.,
Richmond, CA). The volumes of the left ventricles, infarct size (IS), and area at risk
were calculated by multiplying each area with slice thickness and summing the
product. The infarct was expressed as a percentage of the area at risk (IS/AAR) and
this ratio was used to compare the differences among the groups.
Statistical Analysis
Data are expressed as mean ± standard deviation and data analysis was performed
with a personal computer statistical software package (Prism v4.0; GraphPad
Software, San Diego, CA). The hemodynamic data were analyzed using two-way
analysis of variance, with the Bonferroni correction applied for multiple comparisons
if significant F ratios were obtained. Risk areas and infarct sizes and expressed as a
percentage of the area at risk (IS/AAR), were analyzed between groups using one way
analysis of variance with a Student-Newman-Keuls post hoc test for multiple
comparisons. Statistical differences were considered significant if the P value was less
than 0.05.
Results
A total of 45 rats were instrumented to obtain 42 rats in the study. 3 rats were
omitted from further data analysis for severe hypotension or intractable ventricular
fibrillation. There was one each from those receiving RIPC3, NAL and MOR + RIPC1
treatments.
Hemodynamics
Hemodynamic values including heart rate, mean arterial blood pressure and
rate-pressure product (RPP) at baseline, after treatment, 30 min after left coronary
artery occlusion, and after 2 hours of reperfusion, were collected (table1). There were
no significant differences between groups at baseline, after treatment, 30 min of
occlusion, and at 2 h of reperfusion. Compared with baseline, only after MOR +
RIPC1 treatment reduced MAP and RPP. There was a significant drop of MAP and
RPP at 30 min of ischemia and 2 h of reperfusion in all rats, confirming the successful
induction of ischemia and repersuion injury model.
Myocardial infarct size
The AAR ranged from 0.41 ± 0.04 cm3 to 0.46 ± 0.06 cm3 and there was no
difference in AAR between the control and treatment groups. As shown in figure 2
the IS/AAR of CON was 54.7 ± 6.0%, 3 cycles of remote ischemic preconditioning
(RIPC3) and the combination 1 cycle of remote ischemic preconditioning and
morphine (MOR + RIPC1) markedly reduced IS/AAR to 28.3 ± 4.9% and 29.0 ± 7.0%
respectively (P < 0.01 versus control). One cycle of remote ischemic preconditioning
(RIPC1) and intravenous morphine (MOR, 100 ug/kg) or naloxone (NAL, 6 mg/kg)
prior to sustaining ischemia had no effect on infarct size (IS/AAR: RIPC1 51.1 ± 7.4%;
MOR 53.8 ± 6.7%; NAL 54.4 ± 6.9%; P > 0.05 versus control). However, naloxone
could reverse the cardioprotective effect produced by MOR + RIPC1 treatment
(IS/AAR: NAL + MOR+ RIPC1 55.2 ± 6.5%; P < 0.01 versus MOR + RIPC1 ).
Disscussions
The results from this study confirm previous findings showing that 3 cycles of 5
minutes femoral artery ischemia interspersed with 5 minutes reperfusion before
coronary occlusion produce protective effect against myocardial ischemia and
reperfusion injury. Then 1 cycle of 5 minutes femoral artery ischemia interspersed
with 5 minutes reperfusion and pretreament with morphine (100 ug/kg) alone before
sustaining ischemia could not show this protection. However, the combination of this
low dose of morphine and a single 5 minutes cycle of femoral artery ischemia and
reperfusion reduced myocardial infarct size to a similar extent to 3 cycles of femoral
artery ischemia and reperfusion, indicating that morphine reduces the threshold of
remote ischemic preconditioning. Naloxone abolished the protective effect induced by
the combination of subthreshold doses of morphine and remote ischemic
preconditioning, impling that opioid receptors mediate this additive effect.
Opioid receptor agonists including morphine9,10, fentanil15 and remifentanil16,
were shown to induce cardioprotection when administered before and after prolonged
ischemia. Pretreatment with morphine at a dose of 300 ug/kg prior to ischemia
reduced myocardial infarct size12. Patel et al.17 firstly founded that naloxone, the non
specific opioid receptor antagonist, was capable of cancelling the myocardial
infarct-limiting effects produced by remote intestinal preconditioning in rats. They
demonstrated that the involvement of opioid signalling in remote ischemic
preconditioning. Dickson et al.18 revealed that administration of a reverse-phase
concentrate generated from the coronary effluent of preconditioned rabbit hearts
evokes significant protection in mesenteric tissue, which mediated by opioid receptors
stimulation and KATP channel activation. Subsequent experimental studies have
showed the role of δ1 opioid receptor in remote limb preconditioning of skeletal
muscle in pigs and in remote infrarenal aortic preconditioning of the rat hearts4,6.
Nevertheless, Zhang et al.7 failed to detect the involvement of the δ1 opioid receptor
and instead showed the κ opioid receptor in remote preconditioning by femoral artery
ischemia stimulus to reduce myocardial infarct size in rat hearts. It has been
documented that endogenous opioids generated by the preconditioning stimulus in the
remote organ or tissue enter the blood stream where they act directly on the
myocardium to produce cardioprotective effects19.
Ludwig et al.12 indicated that combined administration of morphine and isoflurane
produces a significant reduction in myocardial infarct size that greatly exceeds the
protection induced by either drug alone in rats. Then investigation proceed to show
that morphine lowers the threshold of helium preconditioning and also implies that
opioid receptors mediate helium preconditioning and its augmentation by morphine in
rabbits13. A single-center, parallel-group, randomized study demonstrated that both
remote ischemic postconditioning alone and in combination with intravenous infusion
of an opioid receptor agonist, just before and at the onset or reperfusion to acute
ST-segment elevation myocardial infarction patients achieved higher proportion of
full resolution of ST-segment deviation 30 minutes after primary PCI, However, the
addition of morphine infusion to remote ischemic postconditioning did not confer an
improvement as far as the primary efficacy measure (full ST-segment resolution) was
concerned, but was associated with greater percentage of ST-segment resolution and
lower peak TnI levels14.
There are several limitations to this study. Firstly, the results obtained are
restricted to rats; there may be relevant species differences in severity and duration of
the ischemia response and its modulation by these treatments. Secondly, as observed
in the current investigation, remote ischemic preconditioning previously was shown to
be dependent on the number of brief exposure episodes induced before left coronary
artery ischemia and reperfusion. Whether exposure episodes shorter than 5 minutes
will also reduce myocardial infarct size in rats was not examined in this study and
remains to be demonstrated. Thirdly, the study design does not allow conclusion of
the specific opioid receptor of the observed effect.
In summary, our findings have demonstrated that remote preconditioning by
femoral artery occlusion prior to sustaining ischemia produces protective effects
against myocardial ischemia and reperfusion injury in rats. It was furtherly
demonstrated that mophine reduces the threshold of the remote ischemic
preconditioning and also indicates that opioid receptors mediate this augmentative
effects.
Refferences
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Figure and Table Legends
Figure 1
Bar graphs depicting the experimental protocol. CON = control; RIPC3 = 3 cycles of 5
minutes right femoral artery ischemia interspersed with 5 minutes reperfusion; RIPC1
= 1 cycle of 5 minutes right femoral artery ischemia interspersed with 5 minutes
reperfusion; MOR = intravenous morphine (100 ug/kg); NAL = intravenous naloxone
(6 mg/kg).
Figure 2
Myocardial infarct size (IS) as a percentage of the are of the area at risk (AAR) for the
different groups. CON = control; RIPC3 = 3 cycles of 5 minutes right femoral artery
ischemia interspersed with 5 minutes reperfusion; RIPC1 = 1 cycle of 5 minutes right
femoral artery ischemia interspersed with 5 minutes reperfusion; MOR = intravenous
morphine (100 ug/kg); NAL = intravenous naloxone (6 mg/kg).
Values are presented as mean ± SD. * P < 0.01 versus CON; # P < 0.01 versus MOR +
RIPC1.
Table 1
Hemodynamic parameters for different groups. Baseline = 15 min before surgery; 30
min occlusion = 30 minutes after regional ischemia; 2 h reperfusion = 2 hours after
reperfusion; HR = heart rate (beats per min); MAP = mean arterial blood pressure
(mmHg); RPP = rate-pressure product (mmHg/min per 1000). CON = control; RIPC3
= 3 cycles of 5 minutes right femoral artery ischemia interspersed with 5 minutes
reperfusion; RIPC1 = 1 cycle of 5 minutes right femoral artery ischemia interspersed
with 5 minutes reperfusion; MOR = intravenous morphine (100 ug/kg); NAL =
intravenous naloxone (6 mg/kg).
Values are presented as mean ± SD. ‡ P < 0.05, † P < 0.01 versus baseline
Figure 1
Figure 2
Table 1
Group
HR
CON
RIPC3
RIPC1
MOR
MOR+ RIPC1
NAL
NAL+ MOR+ RIPC1
MAP
CON
RIPC3
RIPC1
MOR
MOR+ RIPC1
NAL
NAL+ MOR+ RIPC1
Hemodynamic Parameters ( ± s)
n
Baseline
Treatment
Ischemia
Reperfusion
6
6
6
6
6
6
6
392 ± 27
393 ± 25
376 ± 12
390 ± 11
385 ± 16
392 ± 18
392 ± 21
379 ± 21
384 ± 25
371 ± 15
381 ± 13
371 ± 13
381 ± 21
387 ± 12
348 ± 25†
342 ± 25†
341 ± 21†
351 ± 12†
339 ± 11†
342 ± 21†
351 ± 24†
326 ± 17†
330 ± 30†
321 ± 22†
334 ± 14†
315 ± 20†
324 ± 23†
328 ± 28†
6
6
6
6
6
6
6
112 ± 17
111 ± 20
110 ± 18
105 ± 22
106 ± 9
108 ± 19
114 ± 22
106 ± 16
106 ± 21
105 ± 15
99 ± 28
93 ± 13‡
102 ± 22
103 ± 18
79 ± 13†
73 ± 18†
73 ± 5†
64 ± 15†
72 ± 8†
70 ± 16†
73 ± 12†
86 ± 7†
83 ± 12‡
78 ± 14†
77 ± 15‡
80 ± 5†
83 ± 14‡
84 ± 10†
RPP
CON
6 44 ± 8
40 ± 8
27±6†
27 ± 1†
RIPC3
6 43 ± 8
41 ± 8
25±7†
27 ± 4†
RIPC1
6 41 ± 7
39 ± 6
25±2†
25 ± 5†
MOR
6 41 ± 9
38 ± 11
23±6†
26 ± 6†
MOR+ RIPC1
6 41 ± 5
34 ± 5‡
25±3†
25 ± 16†
NAL
6 42 ± 8
39 ± 9
24±5†
27 ± 5†
NAL+ MOR+ RIPC1 6 44 ± 8
40 ± 7
25±3†
29 ± 4†
HR = heart rate (beats/min); MAP = mean arterial blood pressure (mmHg); RPP =
rate-pressure product (mmHg/min per 1000).
‡ P < 0.05, † P < 0.01 vs. baseline