Remote limb ischemic postconditioning protects against neonatal hypoxic-ischemic brain injury in rat pups by the opioid receptor/Akt pathway

Abstract

Background and purpose: Remote ischemic postconditoning, a phenomenon in which brief ischemic stimuli of 1 organ protect another organ against an ischemic insult, has been demonstrated to protect the myocardium and adult brain in animal models. However, mediators of the protection and underlying mechanisms remain to be elucidated. In the present study, we tested the hypothesis that remote limb ischemic postconditioning applied immediately after hypoxia provides neuroprotection in a rat model of neonatal hypoxia-ischemia (HI) by mechanisms involving activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

Methods: HI was induced in postnatal Day 10 rat pups by unilateral carotid ligation and 2 hours of hypoxia. Limb ischemic postconditioning was induced by 4 conditioning cycles of 10 minutes of ischemia and reperfusion on both hind limbs immediately after HI. The opioid antagonist naloxone, phosphatidylinositol-3-kinase inhibitor wortmannin, or opioid agonist morphine was administered to determine underlying mechanisms. Infarct volume, brain atrophy, and neurological outcomes after HI were evaluated. Expression of phosphorylated Akt, Bax, and phosphorylated ERK1/2 was determined by Western blotting.

Results: Limb ischemic postconditioning significantly reduced infarct volume at 48 hours and improved functional outcomes at 4 weeks after HI. Naloxone and wortmannin abrogated the postconditioning-mediated infarct-limiting effect. Morphine given immediately after hypoxia also decreased infarct volume. Furthermore, limb ischemic postconditioning recovered Akt activity and decreased Bax expression, whereas no differences in phosphorylated ERK1/2expression were observed.

Conclusions: Limb ischemic postconditioning protects against neonatal HI brain injury in rats by activating the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

Figures

Figure 1

Representative pictures of 2,3,5-triphenyltetrazolium chloride monohydrate-stained coronal brain slices from each group (A) and quantitative analysis of infarct volume (B) in the HI group and groups treated with limb ischemic postconditoning (HI+PostC), naloxone+postconditioning (naloxone+HI+PostC), wortmannin+postconditioning (WM+HI+PostC), morphine (HI+morphine), and wortmannin+morphine (WM+HI+morphine) at 48 hours after HI; n=8 to 10 in each group. Limb ischemic postconditioning and morphine treatment resulted in significant reduction in infarct volume, respectively. Pretreatment with naloxone or wortmannin abolished the reduction in infarct volume induced by postconditioning. Wortmannin also blocked morphineinduced infarct sparing effect. Values are the mean±SEM; *P<0.05 versus HI group; #P<0.05 versus postconditioning-treated group; @P<0.05 versus morphine-treated group.

Figure 2

Evaluation of neurological outcomes in the modified grip-traction test (A), forelimb placement test and back pressure test (B) in groups of sham operation (sham), HI or treated with limb ischemic postconditoning (HI+PostC) at 4 weeks after HI; n=8 to 10 in each group. Animals in the HI group exhibited severe neurological deficits. Limb ischemic postconditioning significantly improved neurological outcomes. Values are the mean±SEM; *P<0.05 versus HI group; #P<0.05 versus sham-operated group.

Figure 3

Representative pictures (A) and quantification of brain tissue loss (B) in groups of sham operation (sham), HI or treated with limb ischemic postconditoning (HI+PostC) at 1 week and 4 weeks after HI; n=7 to 10 in each group. Limb ischemic post-treatment did not reduce brain tissue loss. Values are the mean±SEM; #P<0.05 versus sham-operated group.

Figure 4

Representative Western blots (A) and quantitative analysis of pAkt (B), Bax (C), and pERK (D) expression in groups of sham operation (sham), HI or treated with limb ischemic postconditoning (HI+PostC); n=5 to 6 in each group at 24 hours after HI. The HI brain injury induced decrease in pAkt levels. Limb ischemic postconditioning resulted in a recovery of pAkt expression and reduction in Bax expression (A–C). No significant differences of pERK expression were observed among these groups (D). Values are the mean±SEM; *P<0.05 versus HI group; #P<0.05 versus sham-operated group.

Figure 5

Representative Western blots (A) and quantitative analysis of pAkt (B) expression in groups of sham operation (sham), HI or treated with limb ischemic postconditoning (HI+PostC), naloxone+postconditioning (naloxone+HI+PostC), and morphine (HI+morphine); n=6 in each group at 48 hours after HI. The pAkt expression remained decreased in the HI group at 48 hours after HI. Limb ischemic postconditioning and morphine treatment restored the Akt activity. Naloxone blocked this postconditioning-mediated effect. Values are the mean±SEM; *P<0.05 versus HI group; #P<0.05 versus sham-operated group.