Neuroprotective effects and mechanisms of fentanyl preconditioning against brain ischemia

Introduction

Fentanyl is an artificial agonist of opioids, and belongs to the μ-opioid receptor agonists. During cardiopulmonary bypass (CPB), a large dose of fentanyl was administered (usually achieved 50–100 μg/kg, loading dose exceeded 50 μg/kg, maintenance dose was 20–30 μg/min). Since the advent of CPB, cerebral complications from overt stroke to subtle cognitive dysfunction after CPB for cardiac surgery have been well documented; the postoperative incidence of dysfunction of cognition exceeded 20–70%. The etiology of these injuries are probably associated with cerebral microemboli, global or regional ischemia, inflammation, cerebral temperature modulation and metabolic abnormality. There are disputes about the effects of large doses of μ-opioid receptor agonist; furthermore, evidence about the effect of large doses of fentanyl on brain injury during CPB have not been reported. Some researchers documented that μ-opioid receptor agonists (including morphine or fentanyl) had neuroprotective effects, and predominantly by the activation of δ1 opioid agonist. While reports that μ-opioid receptor agonists have no neuroprotective effect, even causing brain injury can also been seen.

Objective

There have been some disputes about the clinical application of μ-opioid receptor agonists, and it is paramount to explore the effects and mechanisms of fentanyl on brain ischemia.

Methods

In this study, we investigated the effects of fentanyl on cerebral ischemia during the perioperative period, and the mechanisms of neuroprotective effects modulated by subtype opioid receptors or the molecular signaling pathway were explored. This study consisted of three models: moderate hypothermia cardiopulmomary bypass in the rat, brain slices with oxygen-glucose deprivation (OGD), and PC12 cell lines with OGD. Immunohistochemistry, terminal deoxynucleotidyl transferase dUTP-bition nick end labeling (TUNEL) staining, electron microscopy, RT-PCR, western blot, TTC staining, LDH release, Flow CytoMeter and Hoechst33258 staining were used.

Results

This protective effect of fentanyl preconditioning is U-shaped as demonstrated in the dose–response curves. It was found that micromolar concentrations of fentanyl are antiapoptotic, whereas with increasing concentrations fentanyl lacks apparent protective effects. Clinical concentrations of fentanyl preconditioning have neuroprotective effects against cerebral ischemia injury, and antiapoptosis is one of the underlying mechanisms. In detail, we demonstrated that moderate hypothermic cardiopulmonary bypass in the rat can induce hippocampal c-fos, bcl-2 and bax mRNA expressions and protein expressions, increase neuronal apoptosis, and worsen histology injury detected by electron microscope. F50 (fentanyl: LD = 50 μg/kg, MD = 2 μg/kg/min) or F800 (fentanyl: LD = 800 μg/kg, MD = 32 μg/kg/min) can attenuate brain ischemia injury induced by CPB, especially in the dose of 50 μg/kg. Furthermore, the same results were demonstrated in brain slices or PC12 cells with OGD injury. Either 50 ng/ml or 500 ng/ml fentanyl (the peak concentration or plateau concentration, respectively, of fentanyl with loading dose 50 μg/kg) decreases neuronal apoptosis and inhibits the release of LDH so it can attenuate brain slice injury, Moreover, fentanyl preconditioning can strengthen the protein expression of Bcl-2 while Bax protein expression was inhibited. Compared with fentanyl 500 ng/ml, fentanyl 50 ng/ml played a stronger role in brain slices or PC12 cells with OGD injury.

In addition, we determined that δ-opioid receptors are unique and have a specific role in neuroprotection against OGD injury by activating the MAPK pathway, specifically through δ1-opioid receptors. In detail, fentanyl preconditioning had a protective effect on brain slices with OGD injury. The effect was mainly mediated by the δ1 subtype opioid receptor because the role of fentanyl preconditioning was blocked or attenuated by naloxone (a nonselective opioid receptor antagonist), naltrindole (nonselective δ-opioid receptor antagonist) and BNTX δ1-opioid receptor antagonist), respectively. However, naltriben (δ2-opioid receptor antagonist), β-funaltrexamine (μ-receptor antagonist) or norbinaltor-phimine (κ-opioid receptor antagonist) cannot inhibit the protective effect of fentanyl preconditioning. Phosphorylated ERK1/2 was up-regulated by fentanyl preconditioning. BNTX or U0126 can block the ERK1/2 phosphorylation induced by fentanyl preconditioning, while the neuroprotective effect of fentanyl preconditioning was also abolished.

Conclusion

Collectively, we found that fentanyl preconditioning played a role in neuronal protection against hypoxic ischemia, attenuated ischemia injury and inhibited apoptosis of neurons. δ-opioid receptors were unique and had a specific role in neuroprotection against OGD injury by activating the MAPK pathway, mainly through δ1-opioid receptors. μ-opioid receptor agonist can be safely applied to brain ischemia during CPB.