Ye Zhang

Remifentanil Preconditioning Protects against Ischemic Injury in the Intact Rat Heart

Background:Opioid receptors mediate cardiac ischemic preconditioning. Remifentanil is a new, potent ultra-short-acting phenylpiperidine opioid used in high doses for anesthesia. The authors hypothesize that pretreatment with this drug confers cardioprotection. Methods:Male Sprague-Dawley rats were anesthetized and the chest was opened. All animals were subjected to 30 min of occlusion of the left coronary artery and 2 h of reperfusion. Before the 30-min occlusion, rats received either preconditioning by ischemia (ischemic preconditioning, 5-min occlusion, 5-min reperfusion × 3) or pretreatment with remifentanil, performed with the same regime (3 × 5-min infusions) using 0.2, 0.6, 2, 6, or 20 μg·kg−1·min−1 intravenously. The experiment was repeated with naltrindole (a selective Δ-opioid receptor antagonist, 5 mg/kg), nor-binaltorphimine (a selective &kgr;-OR antagonist, 5 mg/kg), or CTOP (a selective μ-opioid receptor antagonist, 1 mg/kg) administered before remifentanil-induced preconditioning or ischemic preconditioning, respectively. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Results:There was a dose-related reduction in infarct size/area at risk after treatment with remifentanil that was similar to that seen with ischemic preconditioning. This effect was prevented or significantly attenuated by coadministration of a μ, &kgr;, or Δ-opioid antagonist. The infarct-sparing effect of ischemic preconditioning was abolished by blockade of &kgr;-opioid receptors or Δ-opioid receptors but not by μ-opioid receptors. Conclusion:Remifentanil mimics cardioprotection via all three opioid receptors. This differs from ischemic preconditioning, which confers cardioprotection via &kgr;- and Δ-, but not μ-opioid receptors. Part of the protective effect of remifentanil may be produced by μ-agonist activity outside the heart.

Remifentanil Preconditioning Confers Cardioprotection via Cardiac κ- and δ-Opioid Receptors

Background:Remifentanil preconditioning (RPC) reduces the infarct size in anesthetized rat hearts, and this effect seems to be mediated by all three types of opioid receptors (ORs). Because there is evidence of only &kgr;- and &dgr;- but not &mgr;-ORs in the rat heart, the authors investigated whether RPC confers cardioprotection via cardiac &kgr;- and &dgr;-OR as well as via extracardiac &mgr;-OR agonist activity. The authors also investigated the involvement of signaling mechanisms, namely protein kinase C and mitochondrial adenosine triphosphate–sensitive potassium (KATP) channels. Methods:The hearts of male Sprague-Dawley rats weighing 190–210 g were removed, mounted on a Langendorff apparatus, and perfused retrogradely at 100 cm H2O with Krebs-Ringer’s solution. All hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The study consisted of three series of experiments on the effect of ischemic preconditioning or RPC (10, 50, and 100 ng/ml remifentanil) after blockade of OR subtypes (&dgr;-OR antagonist naltrindol, &kgr;-OR antagonist nor-binaltorphimine, and &mgr;-OR antagonist CTOP). The involvement of protein kinase C or the KATP channel in the cardioprotection of RPC was also investigated using specific blockers in each group. RPC was produced by three cycles of 5-min perfusion of remifentanil in Krebs-Ringer’s solution interspersed with a 5-min reperfusion with Krebs solution only. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Results:Infarct size as a percentage of the area at risk was significantly reduced after RPC from 51.9 ± 5.0% (control, n = 8) to 36.2 ± 10.0% (100 ng/ml RPC, n = 8, P < 0.01). This effect was stopped by pretreatment with naltrindol (52.3 ± 5.2%) and nor-binaltorphimine (43.5 ± 6.0%) but not CTOP (37.1 ± 6.0%). Chelerythrine and GF109203X, both protein kinase C inhibitors, abolished the effects of RPC or ischemic preconditioning on infarct size as a percentage of area at risk. 5-Hydroxydecanoate (a selective mitochondrial KATP channel blocker) also abolished the cardioprotection of RPC and IPC, but HMR-1098 (a selective inhibitor of the sarcolemmal KATP channel) did not. Conclusion:Cardiac &dgr;- and &kgr;- but not &mgr;-ORs mediate the cardioprotection produced by RPC. Both protein kinase C and the mitochondrial KATP channel were involved in this effect.

Intrathecal Morphine Preconditioning Induces Cardioprotection via Activation of Delta, Kappa, and Mu Opioid Receptors in Rats

BACKGROUND: Small doses of intrathecal morphine provide cardioprotection similar to that conferred by IV morphine. However, the extent of intrathecal morphine preconditioning (IT-MPC) relative to that resulting from ischemic preconditioning (IPC) is unknown. Further, it is uncertain whether IT-MPC is mediated by opioid receptor dependent pathways. In this study, we compared the extent of cardioprotection conferred by IT-MPC with IPC and investigated the role of opioid receptors in this effect. METHODS: Eighty anesthetized, open-chest, male Sprague-Dawley rats were assigned to 1 of 13 groups (n = 6–7) after successful intrathecal catheter placement. Rats in the IPC group were subjected to three 5-min cycles of myocardial ischemia (induced by occlusion of the left main coronary artery) interspersed with 5 min of reperfusion. After IPC, myocardial ischemia and reperfusion injury was induced by 30 min of left main coronary artery occlusion followed by 2 h of reperfusion. In the IT-MPC groups, the rats were given 3 consecutive 5 min intrathecal morphine infusions (0.03, 0.3, 3.0, or 30.0 &mgr;g/kg, respectively) interspersed with 5 min infusion-free periods, before myocardial ischemia reperfusion injury. In 2 other groups either 300&mgr;g/kg of IV morphine or 10 &mgr;L of intrathecal normal saline were given. The selective delta, kappa, and mu opioid receptor antagonists naltrindole, nor-binaltorphimine, and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), respectively, were given to groups of animals receiving IT-MPC to evaluate the relative role of the specific opioid receptor subtypes in IT-MPC preconditioning. Myocardial infarct size (IS), as a percentage of the area at risk (AAR), was determined by 2,3,5-triphenyltetrazolium staining. RESULTS: Intrathecal morphine 0.3 to 30 &mgr;g/kg reduced myocardial IS compared with intrathecal normal saline control animals. The IS/AAR were 33% ± 10% (0.3 &mgr;g/kg), 29% ± 10% (3 &mgr;g/kg) and 29% ± 16% (30 &mgr;g/kg), versus 53% ± 8% for the control group (P < 0.01). The reduction in IS/AAR with IT-MPC was similar to that produced by IV morphine (33% ± 6%, P = 0.84) and IPC (22% ± 4%, P = 0.41). Myocardial preconditioning due to IT-MPC was attenuated by co-administration of any one of the opioid receptor antagonists (IT-MPC + naltrindole 50% ± 9%, IT-MPC + nor binaltorphimine 43% ± 6%, IT-MPC + CTOP 53% ± 9%, P = 0.14). CONCLUSIONS: IT-MPC produced comparable cardioprotection to myocardial IPC and IV morphine. Myocardial preconditioning from intrathecal morphine seems to involve delta, kappa, and mu opioid receptors.

Intracerebroventricular administration of morphine confers remote cardioprotection-Role of opioid receptors and calmodulin

The current study aimed to delineate the mechanism of remote preconditioning by intracerebroventricular morphine (RMPC) against myocardial ischemia-reperfusion injury. Male Sprague-Dawley rats were given an intracerebroventricular morphine injection before myocardial ischemia and reperfusion injury. Ischemia-reperfusion injury was achieved by 30min of left coronary artery occlusion followed by 120min of reperfusion. The effects of remote preconditioning by intracerebroventricular morphine preconditioning were also determined upon selective blockade of the δ, κ or μ-opioid receptors, or calmodulin (CaM). The infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Remote preconditioning by intracerebroventricular morphine reduced infarct size in the ischemic/reperfused myocardium, and the effect was abolished by the selective blockade of any one of the three δ, κ and μ opioid receptors or CaM. Furthermore, remote preconditioning by intracerebroventricular morphine increased the expression of CaM in the hippocampus and the plasma level of calcitonin gene-related peptide (CGRP). The results of the present study provide evidence that the cardioprotection of remote preconditioning by intracerebroventricular morphine involves not only all three types of opioid receptors in the central nervous system, but also CaM, which releases CGRP, one of the mediators of remote preconditioning.

Morphine preconditioning confers cardioprotection in doxorubicin-induced failing rat hearts via ERK/GSK-3β pathway independent of PI3K/Akt.

Preconditioning against myocardial ischemia-reperfusion (I/R) injury can be suppressed in some pathological conditions. This study was designed to investigate whether morphine preconditioning (MPC) exerts cardioprotection in doxorubicin (DOX)-induced heart failure in rats and the mechanisms involved. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (ERK) and glycogen synthase kinase (GSK)-3β pathways were examined. Normal and DOX-induced failing rat hearts were subjected to I/R injury using a Langendorff perfusion system with or without MPC or ischemic preconditioning (IPC). The PI3K inhibitor (wortmannin) or ERK inhibitor (PD98059) was infused before MPC. In normal hearts, both MPC and IPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by I/R injury. Pretreatment with wortmannin or PD98059 abrogated the protective effects of MPC and suppressed the phosphorylation of Akt, ERK and GSK-3β. In failing rat hearts, however, MPC retained its cardioprotection while IPC did not. This protective effect was abolished by PD98059 but not wortmannin. MPC increased the level of p-ERK rather than p-Akt. The phosphorylation of GSK-3β induced by MPC was reversed by PD98059 only. IPC did not elevate the expression of p-ERK, p-Akt and p-GSK-3β in failing rat hearts. We conclude that MPC is cardioprotective in rats with DOX-induced heart failure while IPC is not. The effect of MPC appears to be mediated via the ERK/GSK-3β pathway independent of PI3K/Akt.

Remote ischemic preconditioning improves spatial learning and memory ability after focal cerebral ischemia-reperfusion in rats.

Objective: Using a rodent model of middle cerebral artery occlusion (MCAO), we investigated the effect of remote ischemic preconditioning (RIPC) on spatial learning and memory ability after focal cerebral ischemia-reperfusion (I/R). Method: Thirty-six male Sprague-Dawley rats were randomly allocated into 3 groups (n=12, each): sham group, rats were subjected to sham operations without MCAO and RIPC; I/R group, rats were subjected to 1h of MCAO followed by 3 days of reperfusion; I/R+RIPC group, rats were subjected to RIPC and 1h MCAO followed by 3 days of reperfusion. The spatial learning and memory ability of the rats was measured by the Morris water maze task.The activity of cholineacetyltransferase (ChAT) in the hippocampus CA1 region was observed by an immunohistochemistry method. Results: In the Morris water maze task, MCAO elicited a significant decrease of the ability of spatial learning and memory in contrast to the sham group. Meanwhile, RIPC induced a significantly shorter prolongation of the escape latency (p<0.05); greater number passing through the platform (p<0.05) and less time for exploring the target quadrant (p<0.05) as compared with values for the I/R group. Furthermore, the number of ChAT positive cells in the CA1 region in the I/R+RIPC group was strikingly more than those of the I/R group (p<0.05). Conclusions: RIPC could improve the ability of spatial learning and memory after focal cerebral ischemia-reperfusion probably due to its protection of the cholinergic neurons in the hippocampal CA1 region.

Remifentanil preconditioning protects rat cardiomyocytes against hypoxia-reoxygenation injury via δ-opioid receptor mediated activation of PI3K/Akt and ERK pathways

Remifentanil preconditioning has been demonstrated to reduce myocardial ischemia reperfusion injury in rat hearts, while the mechanisms are not fully understood. This study investigated the protective effects of remifentanil against hypoxia-reoxygenation injury in adult rat cardiomyocytes and the mechanisms involving opioid receptors and downstream phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinase (ERK) signaling pathways. Adult rat cardiomyocytes were pretreated with remifentanil at different concentrations and then subjected to 90min hypoxia followed by 120min reoxygenation. The δ- (naltrindole), κ- (nor-binaltorphimine), or μ-opioid receptor antagonist (CTOP), as well as ERK inhibitor (PD98059) or PI3K inhibitor (wortmannin) was added before remifentanil preconditioning, respectively. Remifentanil showed significant protective effects against hypoxia-reoxygenation injury by increasing cell survival (Trypan blue staining) while reducing LDH activity and cell apoptosis (Hoechst staining). These effects were markedly reversed by naltrindole and were partially blocked by nor-binaltorphimine. Pretreatment of either PD98059 or wortmannin also abolished the protective effects of remifentanil. Following remifentanil preconditioning, the phosphorylation level of Akt reached peak at 10min of reoxygenation. ERK phosphorylation, however, was subsequently enhanced at 120min of reoxygenation. The phosphorylation levels of Akt and ERK were both blocked by naltrindole, but not nor-binaltorphimine or CTOP. Wortmannin inhibited the phosphorylation of both Akt and ERK, whereas PD98059 suppressed the phosphorylation of ERK only. In conclusion, our results suggested that remifentanil protected adult rat cardiomyocytes from hypoxia-reoxygenation injury and its effects appears to be dependent on the δ-opioid receptor mediated activation of PI3K/Akt and subsequent ERK signaling pathways.

Dexmedetomidine Combined with General Anesthesia Provides Similar Intraoperative Stress Response Reduction When Compared with a Combined General and Epidural Anesthetic Technique.

BACKGROUND:Epidural anesthesia may attenuate the sympathetic hyperactivity and stress response from surgery. In this study, we compared the stress response, hemodynamic variables, and recovery profiles of patients undergoing total IV anesthesia (TIVA) and intraoperative dexmedetomidine with those receiving epidural anesthesia and TIVA. METHODS:Ninety patients undergoing elective open gastrectomy under TIVA were recruited. The dexmedetomidine group (group D, n = 30) received IV dexmedetomidine 0.6 &mgr;g/kg before the induction of general anesthesia, followed by dexmedetomidine 0.4 &mgr;g/kg/h until peritoneal closure. The control group (group C, n = 30) received volume-matched normal saline infusion as placebo. The epidural group (group E, n = 30) received epidural anesthesia with 0.375% ropivacaine combined with TIVA. The hemodynamic variables and recovery characteristics during emergence were evaluated. Blood samples for norepinephrine (NE), epinephrine (E), cortisol (Cor), and cytokines (tumor necrosis factor-&agr; [TNF-&agr;], interleukin [IL]-6, and IL-10) were obtained before the administration of dexmedetomidine or epidural anesthesia (baseline), immediately after tracheal intubation, upon incision, at the time of celiac exploration, and at tracheal extubation. RESULTS:Compared with group E, there were no differences in the plasma concentration levels of NE, E, Cor, and cytokines (TNF-&agr;, IL-6, and IL-10) in group D at all time points. The levels of NE and E in groups D and E were significantly lower than that in group C, at all time points following induction (all P < 0.0001 except at incision which were P = 0.001 and P = 0.004), and the level of Cor in groups D and E was significantly lower than that in group C at celiac exploration (P = 0.017 and P = 0.019) and immediately after tracheal extubation (P < 0.0001). The levels of TNF-&agr;, IL-6, and IL-10 increased after the celiac exploration in the 3 groups. The levels of plasma TNF-&agr;, IL-6, and IL-6/IL-10 ratio were higher in group C than in groups D and E at celiac exploration and tracheal extubation (all P < 0.0001 except at celiac exploration which were P = 0.005 and P =0.038 for TNF-&agr; and P = 0.049 and P = 0.038 for IL-6/IL-10 ratio). In group D, the heart rate was significantly slower after commencing dexmedetomidine and remained significantly slower throughout the operative course (all P < 0.0001 except at tracheal extubation which was P = 0.032). The number of patients who required intervention because of intraoperative hypotension was significantly higher in group E (36.7%) compared with groups D and C (13.3% and 10.0%) (P = 0.037, P = 0.015). The times to eye opening and tracheal extubation were similar in all groups. There were fewer incidences of agitation in group D (6.7 %) than in group C (26.6%) (P = 0.038). CONCLUSIONS:When used in conjunction with TIVA, intraoperative dexmedetomidine blunts surgical stress responses to an extent comparable to combined epidural and general anesthesia without compromising hemodynamic stability and with minimal adverse effects during the intraoperative period.

Cardiac μ-opioid receptor contributes to opioid-induced cardioprotection in chronic heart failure

Background The therapeutic potential of cardiac &mgr;‐opioid receptors in ischaemia‐reperfusion (I/R) injury during opioid‐modulating diseases, such as heart failure, is unknown. We aimed to explore the changes of cardiac &mgr;‐opioid receptor expression during heart failure, and its role in opioid‐induced cardioprotection. Methods Rats received doxorubicin (DOX) or were subjected to coronary artery ligation to induce heart failure, or received normal saline (NS) as control. Hearts from NS or DOX rats were isolated and subjected to myocardial ischaemia and reperfusion in an in vitro perfusion system. The opioid [D‐Ala,2 N‐MePhe,4 Gly‐ol]‐enkephalin (DAMGO), with a high &mgr;‐opioid receptor specificity, morphine, and remifentanil were administrated before I/R with or without opioid receptor antagonists, or an extracellular signal‐regulated kinase (ERK) inhibitor. Results Cardiac &mgr;‐opioid receptor mRNA concentrations were 3.2 times elevated in DOX‐treated rats compared with NS rats, while cardiac &mgr;‐opioid receptor protein concentrations showed 6.1‐ and 3.5‐fold increases in DOX‐treated and post‐infarcted rats, respectively. DAMGO reduced I/R‐caused infarct size, expressed as the ratio of area at risk, from 0.50 (0.04) to 0.25 (0.03) in failing rat hearts, but had no effect on infarct size in control hearts. DAMGO promoted phosphorylation of ERK and glycogen synthase kinase (GSK)‐3&bgr; only in failing hearts. DAMGO‐mediated cardioprotection was blocked by an ERK inhibitor. The &mgr;‐opioid receptor antagonist D‐Pen‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) prevented morphine‐ and remifentanil‐induced cardioprotection and phosphorylation of ERK and GSK‐3&bgr; in failing hearts. In contrast, &dgr;‐ and &kgr;‐opioid receptor selective antagonists were less potent than CTOP in the failing hearts. Conclusions Cardiac &mgr;‐opioid receptors were substantially up‐regulated during heart failure, which increased DAMGO‐induced cardioprotection against I/R injury.

Specific MicroRNAs comparisons in hypoxia and morphine preconditioning against hypoxia-reoxgenation injury with and without heart failure

Aims Ischemia reperfusion (I/R) injury is an inevitable event arising during the cardiovascular diseases development and the process of potent surgical treatments. microRNAs (miRNAs) are critical regulators of multiple cell processes including I/R injury. The present study aims to quantify miRNA alterations and regulated genes upon hypoxia‐reoxygenation (H/R) injury in a rat heart failure model comparing with normal cardiomyocytes. Main methods Chronic heart failure was established by injecting doxorubicin (2 mg/kg/week) for 6 weeks, then H/R was performed on primary cultured cardiomyocytes isolated from normal and failed heart. Cellular injury was evaluated by detecting LDH release levels, cell variability and apoptotic rate. Dysregulated miRNAs in control, hypoxia preconditioning (HPC) and morphine preconditioning (MPC) groups under two conditions were quantified by microarray analysis. Fas protein expression was analyzed using Western Blotting analysis. Key findings Chronic heart failure was confirmed with lower ejection fraction (EF), and significant cellular injury. HPC could reverse the injury induced by H/R in normal heart rather than failed heart, otherwise, MPC significantly attenuated cellular injury dose dependently in both conditions. There was 12 miRNAs significantly altered after doxorubicin injection, 7 downregulated and 5 upregulated. miR‐133b‐5p, miR‐6216, miR‐664–1–5p and let7e‐5p were differentially expressed after HPC and MPC treatments. The direct interaction between miR‐133b‐5p and target gene Fas were established. The Fas protein expression was manipulated by MPC not HPC affording protective effect against H/R injury. Significance We investigated that miR‐133b‐5p might play a particularly important role in the cardioprotective effect of MPC by regulating the target gene Fas.