Combined preconditioning with hypoxia and GYKI-52466 protects rats from cerebral ischemic injury by HIF-1α/eNOS pathway

Abstract

Cerebral ischemic injury has been the leading cause of death and long term disability in the world because of the lack of successful therapies to it, leading to neurological and behavioral deficits. The present study aims to investigate the effects of combined preconditioning (PC) with hypoxia and GYKI-52466 (GYKI) on cerebral ischemic injury and to explore the mechanism. The results showed that combined preconditioning with hypoxia and GYKI-52466 increased the survival rate of cerebral ischemia rats, alleviated the neurological deficit, increased the object recognition and social recognition memory of rats and suppressed the inflammatory reaction induced by cerebral ischemia. Further experiments found that preconditioning with hypoxia and GYKI-52466 significantly increased the HIF-1α and eNOS expression as well as eNOS activity, while inhibitors of HIF-1α and eNOS abolished the protective effects of hypoxia+GYKI PC on neurological deficit. Taken together, these results indicate that combined preconditioning with hypoxia and GYKI-52466 is effective to prevent cerebral ischemia injury, while HIF-1α and eNOS may be involved in the mechanism.

Keywords: Cerebral ischemic injury; GYKI-52466; HIF-1α; eNOS; hypoxia; preconditioning.

Figure 1

Experimental design. Rats received preconditioning with hypoxia or GYKI-52466 or hypoxia+GYKI-52466 for four constitutive days before MCAO surgery, then rested for one day. Next, rats received MCAO or sham surgery, then recovered for another day. Some rat then received behavioral or memory tests and sacrifice; some rats were sacrificed for biochemical assays.

Figure 2

Effect of hypoxia and/or GYKI preconditioning on rat neurological deficit after 90-min cerebral ischemia. Rats received preconditioning with hypoxia or GYKI-52466 or hypoxia+GYKI-52466 for four constitutive days and 90-min cerebral ischemia surgery, then tested for neurological deficit. Ischemia significantly increased the neurological deficit score compared to Sham rats (p<0.05). Hypoxia PC or GYKI PC significantly decreased the neurological deficit scores (p<0.05). Combined PC with hypoxia and GYKI-52466 further decreased the neurological deficit score. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.

Figure 3

Changes in object recognition and social recognition memory. Object recognition and social recognition memory capacity was indicated by object recognition index and social recognition index, respectively. Ischemia significantly decreased object recognition index and social recognition index. Hypoxia PC or GYKI PC increased the object recognition index and social recognition index; hypoxia+GYKI PC increased them more dramatically. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.

Figure 4

Changes in escape latency and travel length in Morris water maze. (A) Shows the representive traces of rats traveled in different groups. (B) Shows the trends of escape latency in Morris water maze study and (D) demonstrates the area under the curve (AUC) calculated from (B). (C) Shows the trends of travel length and (E) demonstrates the AUC calculated from (C). Escape latency andtravel length was significantly increased in Ischemia group, but decreased by hypoxia PC or GYKI PC. Hypoxia+GYKI PC decreased it to a significantly lower level. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.

Figure 5

Changes in the proinflammtory factors (IL-6, IL-1β and TNF-α) in the brain. Rats received preconditioning with hypoxia or GYKI-52466 or hypoxia+GYKI-52466 for four constitutive days before MCAO surgery, then rested for one day before they were sacrificed for proinflammtory factor assays in the brain. Cerebral ischemia caused significant increase in cerebral proinflammatory factors, which was reversed by treatment of hypoxia PC, GYKI PC or hypoxia+GYKI PC. The levels of proinflammatory factors in the hypoxia+GYKI PC group was significantly lower thanhypoxia PC or GYKI PC group. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.

Figure 6

Changes in the expression of HIF-1α and eNOS and eNOS activity in the brain. Rats received preconditioning with hypoxia or GYKI-52466 or hypoxia+GYKI-52466 for four constitutive days before MCAO surgery, then rested for one day before they were sacrificed for Western blot and eNOS activity assays in the brain. The expression of HIF-1α was significantly increased by hypoxia PC and hypoxia+GYKI PC, but not changed by ischemia or GYKI PC. The expression of eNOS and eNOS activity was only increased by hypoxia+GYKI PC. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.

Figure 7

Changes in neurological deficit scores by inhibitors of HIF-1α and eNOS. We treated rats with preconditioning with hypoxia+GYKI PC for four constitutive days and HIF-1α inhibitor PX-478 or eNOS inhibitor L-NIO before MCAO surgery, then re-measured the neurological deficit score. Both HIF-1α inhibitor PX-478 and eNOS inhibitor L-NIO abolished the effects of hypoxia+GYKI PC on neurological deficit score, while treatment of PX-478 or L-NIO alone had no impact on neurological deficit score. PC: preconditioning; GYKI: GYKI-52466. #: p<0.05. N=10 per group.