Hibernation-like state induced by an opioid peptide protects against experimental stroke

Abstract

Background: Delta opioid peptide [D-ala2,D-leU5]enkephalin (DADLE) induces hibernation in summer ground squirrels, and enhances preservation and survival of isolated or transplanted lungs and hearts. In the present study, we investigated the protective effect of DADLE in the central nervous system.

Results: Adult Sprague-Dawley rats were pretreated with DADLE (4 mg/kg every 2 h x 4 injections, i.p.) or saline prior to unilateral occlusion of the middle cerebral artery (MCA). Daily behavioral tests revealed that ischemic animals treated with DADLE did not show any significant behavioral dysfunctions compared with saline-treated ischemic animals. Opioid antagonists only transiently inhibited the protective effect of DADLE, indicating the participation of non-opioid mechanisms in DADLE neuroprotection. Histological examination using triphenyltetrazolium chloride (TTC) revealed that brains from ischemic animals treated with DADLE, either alone or with adjuvant opioid blockers, exhibited almost completely intact striata. In contrast, brains from ischemic animals that received saline showed significant infarction in the lateral striatum. Analyses of apoptotic cell death revealed a significant increase in the p-53 mRNA expression in the striatum of ischemic animals that received saline, while those that received DADLE exhibited near normal striatal p-53 expression. This protective effect was accompanied by significant increments in protein levels of glial cell line-derived neurotrophic factor in the striatum of DADLE-treated ischemic animals.

Conclusion: These results indicate that DADLE protected against necrotic and apoptotic cell death processes associated with ischemia-reperfusion injury. The present study demonstrates that delta opioids are crucially involved in stroke, suggesting that the opioid system is important in the study of brain injury and protection.

Figures

Figure 1

[D-ala2,D-leU5]enkephalin (DADLE) attenuates ischemia-induced asymmetrical motor deficits. Ischemic animals pretreated with saline (S), naltrexone alone (N) or naloxone methiodide alone (NM) displayed significant biased swing activity (panel A), postural bias (panel B), spontaneous rotational behavior (panel C), and forelimb akinesia (panel D). In contrast, ischemic animals pretreated with DADLE + naltrexone (D + N) or DADLE + naloxone methiodide (D + NM) exhibited significant dysfunctions in the same behavioral tests only at 0 to 24 h post occlusion and reperfusion surgery of the middle cerebral artery (MCAor) (days 0 and 1), and thereafter showed near normal behaviors at 48 h and 72 h post MCAor (days 2 and 3). In contrast, ischemic animals pretreated with DADLE alone (D) displayed near normal behaviors throughout the post-MCAor test period.

Figure 2

[D-ala2,D-leU5]enkephalin (DADLE) reduces necrotic and apoptotic (panel B) cell death associated with ischemia. (A) Triphenyltetrazolium chloride (TTC) staining at 72 h post occlusion and reperfusion surgery of the middle cerebral artery (MCAor) revealed that the striata from ischemic animals exposed to ischemia/reperfusion surgery and pretreated with saline showed dehydrogenase-deficient tissue (negative TTC stains). In contrast, the striata from ischemic animals pretreated with DADLE, DADLE + naloxone methiodide, or DADLE + naltrexone did not reveal any dehydrogenase deficiency. (B) Ischemic animals exposed to ischemia/reperfusion injury and treated with saline showed a significant increase in mRNA expression of p-53 in their ischemic striata (65% increment compared with intact striatum of normal, control animals) at 24 h after stroke surgery. In contrast, ischemic animals treated with DADLE exhibited only a small increment in p-53 mRNA expression in their ischemic striata at the same time period (28% increment compared with intact striatum of normal, control animals; not significantly different from control values). Comparisons of ischemic striata between these two groups showed a marked reduction in p-53 mRNA expression in DADLE-treated ischemic animals compared with saline-treated ischemic animals. (C) Immunohistochemical analyses of phenotypic markers of apoptosis revealed that DADLE significantly reduced caspase-3- (panels a, b, c, d) and Fas-positive cells (panels e, f, g, h) in DADLE-treated ischemic animals compared to saline-treated ischemic animals. Quantitative data shown in bar graphs and represent means ± S.E.M. Asterisks correspond to statistical significance at P <0.05. To better capture the necrotic and apoptotic cells in saline-treated ischemic animals (panel d), higher magnification images are generated from propidium iodide (a) and caspase-3 (b) immunofluorescently labeled striatal cells, respectively. Scale bar = 50 μm (a), 100 μm (b).

Figure 3

[D-ala2,D-leU5]enkephalin (DADLE) increases expression of glial cell line-derived neurotrophic factor (GDNF) in brain tissues. Enzyme-linked immunosorbent assays (ELISA) revealed that the levels of GDNF proteins, but not nerve growth factor (NGF), were significantly higher in striatal and cortical tissues harvested from ischemic animals treated with DADLE compared with those treated with saline. Data are expressed as mean percent of control ± S.E.M. Asterisk indicates P <0.05. n = 6 samples for each neurotrophic factor examined.

Figure 4

Schematic diagram summarizing timeline of experimental procedures in this study.