Protective effect of delayed remote limb ischemic postconditioning: role of mitochondrial K(ATP) channels in a rat model of focal cerebral ischemic reperfusion injury

Abstract

Delayed remote ischemic postconditioning (DRIPost) has been shown to protect the rat brain from ischemic injury. However, extremely short therapeutic time windows hinder its translational use and the mechanism of action remains elusive. Because opening of the mitochondria K(ATP) channel is crucial for cell apoptosis, we hypothesized that the neuroprotective effect of DRIPost may be associated with K(ATP) channels. In the present study, the neuroprotective effects of DRIPost were investigated using adult male Sprague-Dawley rats. Rats were exposed to 90 minutes of middle cerebral artery occlusion followed by 72 hours of reperfusion. Delayed remote ischemic postconditioning was performed with three cycles of bilateral femoral artery occlusion/reperfusion for 5 minutes at 3 or 6 hours after reperfusion. Neurologic deficit scores and infarct volumes were assessed, and cellular apoptosis was monitored by terminal deoxynucleotidyl transferase nick-end labeling. Our results showed that DRIPost applied at 6 hours after reperfusion exerted neuroprotective effects. The K(ATP) opener, diazoxide, protected rat brains from ischemic injury, while the K(ATP) blocker, 5-hydroxydecanote, reversed the neuroprotective effects of DRIPost. These findings indicate that DRIPost reduces focal cerebral ischemic injury and that the neuroprotective effects of DRIPost may be achieved through opening of K(ATP) channels.

Figure 1

Schematic of the design and changes in cerebral blood flow (CBF) in animals for part 1 and part 2 of the experiment. All animals underwent 90 minutes of ischemia (dark bar) and 72 hours of reperfusion (open bar) (n=8 for each). (A) In part 1, rats were subjected to delayed remote ischemic postconditioning (DRIPost) at 3 or 6 hours after reperfusion, and the DRIPost protocol was three cycles of bilateral femoral artery occlusion 15 seconds/5 minutes/8 minutes ischemia (I)/15 seconds/5 minutes/8 minutes reperfusion (R). (B) In part 2, except for delayed 6 hours/5 minutes DRIPost, the K_ATP channel blocker 5-hydroxydecanote (5-HD) or opener diazoxide (DIAZ), as well as the vehicle physiologic saline and dimethyl sulfoxide (DMSO), was injected through the caudal vein 20 minutes (for 5-HD and saline) or 30 minutes (for DIAZ and DMSO) before DRIPost to investigate the role of K_ATP channels in DRIPost-induced neuroprotection against focal cerebral ischemia–reperfusion injury. (C, D) Changes in CBF in animals subjected to 90 minutes ischemia followed by 72 hours reperfusion. Right common carotid artery (CCA) occlusion reduced CBF to ∼50% of the baseline, and additional middle cerebral artery (MCA) occlusion further decreased CBF to ∼20%. CCAo, CCA occlusion; MCAo, MCA occlusion; MCAr, MCA release.

Figure 2

Neurologic scores and infarct volume after 90 minutes of transient middle cerebral artery occlusion in control (CON) and delayed remote limb ischemic postconditioning (DRIPost) groups in part 1 (n=8 for each). DRIPost was performed with bilateral femoral artery occlusion/reperfusion at 3 or 6 hours after reperfusion. Neurologic scores are presented as the median (range); Data for infarct volumes are expressed as the mean±s.d. Neurologic scores were evaluated at 24 hours (A), 48 hours (B), and 72 hours (C) after reperfusion using the Garcia scoring system. (D) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 hours after reperfusion. (E) Statistical analysis of the percentage of infarct volume was determined for each study group. 3 h/6 h-15 s, 5 min and 8 min groups: DRIPost performed at 3 or 6 hours after reperfusion. The occlusion duration varied from 15 seconds, 5 minutes to 8 minutes, and bilateral femoral arteries were released for the same duration and repeated for three cycles. ^#P<0.05 versus Con group. ^*P<0.05 versus 6 h-5 min group.

Figure 3

Neurologic scores and infarct volume after 90 minutes of transient middle cerebral artery occlusion in the control (CON), delayed remote limb ischemic postconditioning (DRIPost), 5-HD (K_ATP blocker, 20 mg/kg, intravenously), DIAZ (K_ATP opening, 5 mg/kg, intravenously), 0.9% (w/v) saline, dimethyl sulfoxide (DMSO) groups in part 2 (n=8 for each). DRIPost was performed with bilateral femoral artery occlusion/reperfusion at 6 hours after reperfusion. Neurologic scores are presented as the median (range); Date for infarct volumes are expressed as the mean±s.d. Neurologic scores were evaluated at 24 hours (A), 48 hours (B), and 72 hours (C) after reperfusion using the Garcia scoring system. (D) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 hours after reperfusion. (E) Statistical analysis of the percentage of infarct volume was determined for each study group. ^#P<0.05 versus Con group. ^*P<0.05 versus DRIPost group. DIAZ, diazoxide; 5-HD, 5-hydroxydecanote; NS, normal saline.

Figure 4

(A) Representative sections of nuclear DNA fragmentation staining performed by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) in the CA₁ region of the hippocampus from rats that randomly received 5-HD (20 mg/kg, intravenously) or DIAZ (5 mg/kg, intravenously) after 90 minutes of ischemia and 72 hours of reperfusion (Con). In the presence and absence of 5-HD or DIAZ pretreatment, or with a combination of DRIPost (three cycles of 5 minutes occlusion/reperfusion at 6 hours after reperfusion) and 5-HD or DIAZ. (a) Control (Con); (b) 5-HD; (c) DIAZ; (d) DRIPost; (e) DRIPost+5-HD; (f) DRIPost+DIAZ. (B) Quantitative analysis of the number of TUNEL-positive neurons in the CA₁ region of the hippocampus. Data are presented as the mean±s.d. Scale bar, 20 μm. ^#P<0.05 versus Con group. ^*P<0.05, versus DRIPost group. DIAZ, diazoxide; 5-HD, 5-hydroxydecanote.