Remifentanil Postconditioning Improves Spatial Learning And Memory Ability Following Global Cerebral Ischemia-reperfusion In Rats And Its Mechanism

Background: In the clinical setting, many surgeries or anaesthia techniques such as severe craniocerebral trauma operation, coronary artery bypass grafting, carotid artery transplantation,deliberated controling blood pressure, can cause global cerebral ischemia, which lead to the deficiency or loss of blood perfusion to brain tissue. Although the active treatment or surgery restored the brain blood supply, the restoration of blood flow to the ischemic brain will aggravate the damage of brain and cause cerebral dysfunction, this phenomenon is well known as cerebral ischemia-reperfusion injury(CIRI). Some patients will leave severe or long-term complications after CIRI, such as cognitive impairment, which affect the quality of life of patients after surgery. It is an urgent challenge for scholars to reduce the damage of cerebral ischemia and to protect the brain function, in particular, cognitive function, so as to promote the rehabilitation of patients, ultimately improve the patients’ quality of life and reduce the burden of society and family.Early studies suggested that transient ischemic preconditioning could enhance the organs tolerance to the subsequent longer period of ischemia, which had a protective effect on the ischemic tissues and cells. But ischemic preconditioning is not universal to use in clinical because it has potential risks to cause damage of important organs such as the heart and brain. In recent years, anesthetic preconditioning was implemented to simulate ischemia preconditioning. Studies have found that anesthetic preconditioning had the same protective effect as ischemic preconditioning on ischemic organs and cells.Recently, researchers have demonstrated that anesthetics postconditioning was used during reperfusion stage can also reduce ischemia-reperfusion injury, they confirmed that anesthetics postconditioning also offered organ protective effects. The difference between preconditioning and postconditioning lies in the "processing" time: preconditioning occurs before ischemia, aims to make organs to adapt and tolerate the ischemia; postconditioning aims to maximizely reduce reperfusion injury. Since postconditioning and the clinical treatment for patients have temporal consistency, postconditioning has a broad prospects in the setting of clinical apply.Remifentanil is the unique opioid that has characteristic of direct elimination by non specific plasma esterase, small volume of distribution, fast redistribution and short half-life time of elimination. Remifentanil preconditioning has been demonstrated to possess universal protective effects on ischemic reperfusion injury of heart, brain and other organs. Whetheter remifentanil postconditioning can reduce the injury induced by global cerebral ischemia-reperfusion and the cerebral protective mechanism of remifentanil postconditioning is not clear. Recent studies have found that remifentanil preconditioning offered cerebral protection via activation of TNF- α /TNFR1, JNK signaling pathway during cerebral ischemia-reperfusion. Thus, we hypothesize that remifentanil postconditioning plays a protective role following cerebral ischemiareperfusion, thereby improves spatial learning and memory ability after global cerebral ischemia-reperfusion in rats. To prove this hypothesis, we use the global brain ischemia model to investigate whether remifentanil postconditioning(RP) could improve spatial learning and memory ability after global cerebral ischemia- reperfusion in rats and to explore its mechanism of this effect. Methods Forty healthy, adult, male SD rats were selected for our first part study. Rats were randomly devided into five groups: sham group, control group, RP1 group, RP2 group, RP3 group. Global cerebral ischemia-reperfusion model was built with the occlusion of four vessels. We observed remifentanil postconditioning on cerebral ischemia-reperfusion injury in rats. We study the protective effect of remifentanil postconditioning on hippocampal neurons in rats. We also investigate whether remifentanil postconditioning could improve spatial learning and memory ability after global ischemia reperfusion in rats. Remifentanil was immeditely injected through tail vain during five minutes at the beginning of reperfusion, the spatial learning and memory ability of rats was measured by the Morris water maze test at 3-7 days after the global cerebral ischemia-reperfusion. We observed the survival neurons in hippocampal CA1 density by Hematoxylin-Eosin(HE) staining and detected the change of acetyl transferase(ChAT) content using immunohistochemical method in hippocampal CA1 region. We also investigated the relationship between remifentanil postconditioning reduced the death of cholinergic and pyramidal neurons in hippocampal CA1 area after global cerebral ischemia reperfusion and remifentanil postconditioning improved the spatial learning and memory ability after global cerebral ischemia reperfusion. On this basis, we investigated whetheter the inhibition of the apoptosis in hippocampal CA1 region took part in the protection of remifentanil postconditioning after global cerebral ischemia reperfusion or the PI3 K pathway was involved in the effect of remifentanil postconditioning. Sixty healthy, adult, male SD rats were selected for our second part study. Rats were randomly devided into five groups: sham group, model group, R group, LY+R group, LYgroup. We gave the PI3 K inhibitor LY294002 10 min before global cerebral ischemia in rats. We observed remifentanil postconditioning on the Morris water maze test at 3-7 days after global cerebral ischemia reperfusion injury in rats.After finishing the Morris water maze test, we detected the apoptosis of neurons in hippocampal CA1 area using the Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) method. We observed the expression of apoptosis related gene Bcl-2 and Bax in hippocampal CA1 region using reverse transcription-polymerase chain reaction(RT-PCR) method and the expression of apoptosis related gene Bcl-2 and Bax in hippocampal CA1 region using western blot method. We investigated that remifentanil postconditioning could improve spatial learning and the memory ability after global cerebral ischemia reperfusion through PI3K/AKt signaling pathway to inhibit the apoptosis of neurons in hippocampal CA1 region. Results: Our experiments showed that: ① The spatial learning and memory ability was significantly impaired(P<0.05), the number of hippocampal CA1 pyramidal neuronal necrosis and choline acetyltransferase positive cell number was significantly decreased following global cerebral ischemia reperfusion(P< 0.05). High, middle dose of remifentanil postconditioning(RP) significantly reduced the number of necrotic pyramidal neurons induced by cerebral ischemia reperfusion(P< 0.05) and significantly increased choline acetyltransferase(Ch AT) expression in hippocampus CA1 area(P< 0.05). We found that high, middle dose of remifentanil postconditioning significantly improved patial learning and memory ability after global cerebral ischemia reperfusion(P< 0.05), there were no difference in two groups about the number of hippocampal CA1 pyramidal cells, Ch AT content and Morris water maze test(P>0.05). However, the number of hippocampal CA1 pyramidal cells, the content of ChAT and Morris water maze test in low dose remifentanil postconditioning were the same as global ischemia reperfusion group(P> 0.05). Compared with the sham group, the spatial learning and memory ability was significantly impaired(P< 0.05), the number of apoptotic cells in hippocampus CA1 area was increased significantly(P< 0.05), the ratio of Bcl/Bax and Bcl-2 m RNA/Baxm RNA was significantly decreased(P< 0.05) in the global cerebral ischemia reperfusion group. High, middle dose of remifentanil postconditioning significantly improved spatial learning and memory ability(P< 0.05), reduced the number of apoptotic neurons in hippocampal CA1 region(P< 0.05), up regulationed the antiapoptotic gene and protein Bcl-2 and inhibited the apoptosis promoting gene and protein Bax(P< 0.05).However, pretreatment with PI3 K inhibitor LY294002 completely inhibited the protective effect of remifentanil postconditioning. Conclusion ① Remifentanil postconditioning has significantly improved spatial learning and memory ability through it reduced the death of cholinergic and pyramidal neurons in hippocampal CA1 area following global cerebral ischemia reperfusion in rats. ②Remifentanil postconditioning improved spatial learning and memory ability after global cerebral ischemia reperfusion in rats by inhibiting neuronal apoptosis in hippocampus CA1 region through PI3K/Akt signaling pathway.