The Role Of Neuroglobin In The Neuroprotection Of Limb Ischemic Preconditioning In Rats And Its Mechanisms

Ischemic neuropathy is a leading cause of morbidity, mortality and disability rate. Activation of endogenous brain protection is a novel approach to prevent brain ischemia/reperfusion injury, and which has attracted extensive attention. Ischemic preconditioning is a phenomenon that brief episode of sub-lethal ischemia protects organs or tissues against a subsequent lethal ischemic insult. Brain ischemic preconditioning was first demonstrated by Kitagawa and colleagues. They showed in the gerbil that short periods of global cerebral ischemia can protect the brain against subsequent prolonged ischemia. Recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning. The finding opened up vast vista of the study to increase the resistance of important vital organs such as brain to ischemia with transient ischemic preconditioning in non-vital organs. Our recent study demonstrated that limb ischemic preconditioning (LIP) could attenuate delayed neuronal death (DND) or apoptosis of pyramidal neurons in the CA1 hippocampus induced normally by serious brain ischemia/reperfusion. However, mechanisms underlying the neuroprotection of LIP have not been well understood.Neuroglobin (Ngb) first described in Nature by Burmester at 2000 is a globin with high affinity for oxygen and is widely expressed in neurons of central and peripheral nervous systems in mouse and human. Since then, Ngb have also been recognized in rat and gerbil, suggesting that Ngb is present in a broad range of vertebrate species. Recent studies suggested that Ngb plays an important role in the protection of brain neurons from ischemic and hypoxic injuries, and apoptosis. For example, Ngb overexpression can protect neurons against hypoxic/ischemic insults, and knocking down Ngb expression increases hypoxic neuronal injury in vitro and ischemic cerebral injury in vivo. Ngb acts as an endogenous neuroprotectant, which has provided a new strategy to the study of brain hypoxic/ischemic insults. Our preliminary study using immunohistochemistry staining showed that LIP up-regulated the Ngb expression in the rat CA1 hippocampus, but whether Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusion is still unclear and need to be clarified.Mitochondria, besides playing a key role in energy metabolism within the cell, are involved in a cohort of other processes like integration of dead signal and determination of cell fate. Mitochondrial-mediated apoptosis pathway is considered to be important to neuronal death after cerebral blood flow arrest. Recent investigations have shown that mitochondrial-centered mechanisms are important mediators in promoting development of the preconditioning response. In the brain, ischemic preconditioning diminishes mitochondrial dysfunction induced by ischemia and confers neuroprotection. Targeting mitochondria may be a useful strategy to reduce ischemic brain injury. Bcl-2 gene families are identified as apoptosis regulating genes. Inhibiting effect of anti-apoptotic proteins Bcl-2 on apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax, inhibiting their oligomerization, and thus inhibiting mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Whether mitochondrial mechanisms are involved in neuroprotection of Ngb induced by LIP remain unclear.Therefore, the present study was primarily undertaken to investigate the role of Ngb in the neuroprotection of LIP against global brain ischemia by observing changes in expression of Ngb in the CA1 hippocampus after LIP in rat global brain ischemic model, influences of up- or down-regulating Ngb expression on the neuroprotection of LIP, and the involvements of mitochondria in the process. The present study also compared Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it between young and aged rats, and in normal rats. The results obtained provided new and more direct evidence for illustrating the role of Ngb in the neuronal protection of LIP and new clues for the study in prevention and therapy of ischemic neuropathies.1 Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusionUsing four-vessel occluding global brain ischemia model, the role of Ngb in the neuroprotection of LIP against severe ischemic insult was investigated by observing the influence of LIP on the expression of Ngb in the rat CA1 hippocampus, and the influence of Ngb inducer Hemin and Ngb (antisense oligodeoxynucleotides, AS-ODNs) on the neuroprotection of LIP.1.1 LIP up-regulates Ngb expression in rat CA1 hippocampus and induces brain ischemic toleranceMethods: Seventy male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 5 groups:①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.④Sham of LIP+cerebral ischemia group: The bilateral femoral arteries were exposed for 60 min but not clipped, and then global brain ischemia was induced as those in cerebral ischemia group.⑤LIP+cerebral ischemia group: Global brain ischemic insult for 8 min was induced as those in cerebral ischemia group immediately after LIP as in LIP group. Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blot), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia, or LIP, or cerebral ischemia in corresponding group. The profile of pyramidal neurons DND in the CA1 hippocampus was evaluated under light microscope by determining the neuronal density (ND) and histological grade (HG). The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The HG was divided into 4 grades according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, death of many neurons; gradeⅢ, death of almost complete neurons.Results: RT-PCR and Western blot analysis showed that basal expression of Ngb mRNA and protein in the CA1 hippocampus could be observed in sham group of cerebral ischemia. Compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously up-regulated after LIP (P<0.05), while significantly down-regulated after cerebral ischemia (P<0.05). LIP significantly up-regulated the expressions of Ngb mRNA and protein in LIP+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while sham operation of LIP before the cerebral ischemia was without effect on the down-regulation of Ngb mRNA and protein expressions induced by cerebral ischemia.Thionine staining showed that pyramidal neurons in the CA1 hippocampus in sham group of cerebral ischemia and LIP group were normal and lined up in order, and no DND was found. There were obvious DND of pyramidal neurons in cerebral ischemia group, and HD increased and ND decreased significantly compared with sham group of cerebral ischemia and LIP group (P<0.05). In LIP+cerebral ischemia group, the DND normally induced by cerebral ischemia was effectively prevented, which represented with increase in ND and decrease in HG compared with cerebral ischemia group (P<0.05), while, the sham operation of LIP (in sham of LIP+cerebral ischemia group) had no effect on the DND normally induced by cerebral ischemia.The above results indicated that LIP could up-regulate the expression of Ngb, and induce brain ischemic tolerance in the rat CA1 hippocampus.1.2 Hemin up-regulates Ngb expression in the rat CA1 hippocampus and induces brain ischemic toleranceMethods: Eighty and four male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.③Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.④Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group. According to doses of Hemin used, this group was divided into 3 subgroups: 10 mg/kg/d, 20 mg/kg/d and 40 mg/kg/d groups.Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blotting analysis), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia or cerebral ischemia in corresponding group. Results: RT-PCR and Western blot analysis showed that compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously down-regulated after cerebral ischemia (P<0.05). The administration of Hemin dose-dependently up-regulated the expressions of Ngb mRNA and protein in Hemin+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while pretreatment with vehicle of Hemin had no influence on the Ngb mRNA and protein expressions.Thionine staining showed that the outline of the pyramidal neurons was kept intact, and no neuronal loss was found in the CA1 hippocampus in sham group of cerebral ischemia. Obvious destruction of the CA1 hippocampus was found in cerebral ischemia group, the value of ND was decreased, and HG was increased compared with that in sham group of cerebral ischemia (P<0.05). Intraperitoneal injection with Hemin before cerebral ischemia showed a significant protective effect against the DND induced by cerebral ischemia, which was represented with dose-dependent increase in ND and decrease in HG after administration of Hemin, especially in large doses (P<0.05). Whereas, pretreated with Hemin vehicle before the cerebral ischemia had no significant effect on the DND normally induced by cerebral ischemia.The above results indicated that Hemin could up-regulate the Ngb expression and induce brain ischemic tolerance.1.3 Ngb AS-ODNs inhibit brain ischemic tolerance induced by LIPMethods: Fifty and five male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:①ACSF+LIP+cerebral ischemia group: ACSF (artificial cerebrospinal fluid) was intraventricularly injected 30 min prior to LIP. LIP was preformed by occlusion of bilateral femoral arteries of the rats for 10 min 3 times in an interval of 10 min, and then the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia immediately after the LIP.②S-ODNs+LIP+cerebral ischemia group: Ngb (sense oligodeoxynucleotides, S-ODNs) in a dose of 1.6 nmol was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group.③AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group. According to doses of Ngb AS-ODNs used, this group was divided into 3 subgroups: 0.4 nmol, 0.8 nmol, and 1.6 nmol groups.Eleven rats were included in each group, and assigned to 2 observations:①Ngb protein expression (n=5()Western blotting analysis), and②Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observation at time point of 6 h and 7 d after the cerebral ischemia in corresponding group.Results: Western blot analysis showed that compared with ACSF+LIP +cerebral ischemia group, the expression of Ngb protein in AS-ODNs+LIP+ cerebral ischemia group significantly down-regulated in a dose-dependent manner (P<0.05). While there was no influence on the Ngb protein expression induced by LIP after administration Ngb S-ODNs.Thionine staining showed that pyramidal neurons in the CA1 hippocampus arranged in order, and the structure of most them was clear in ACSF+LIP+cerebral ischemia and S-ODNs+LIP+cerebral ischemia groups. Administration of Ngb AS-ODNs dose-dependently induced obvious DND of pyramidal neurons in the CA1 hippocampus, which was represented with the decrease in the value of ND, and increase in HG compared with ACSF+LIP+ cerebral ischemia group (P<0.05). The administration of Ngb S-ODNs had no effect on the histological characteristic of the CA1 hippocampus.The above results indicated that Ngb AS-ODNs partly inhibited brain ischemic tolerance induced by LIP via inhibiting the expression of Ngb protein.1.4 LIP and Hemin reduce the increase of the serum Ngb protein level induce by cerebral ischemiaMethods: Thirty male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 6 groups:①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.④LIP+cerebral ischemia group: LIP was preformed as those in LIP group, and then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the LIP.⑤Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.⑥Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.Five rats were included in each group, and the serum Ngb protein level was observed by ELISA at 6 h after the sham operation of cerebral ischemia, or LIP, or cerebral ischemia.Results: The level of serum Ngb protein in sham group of cerebral ischemia and LIP group was low, and no significant differences between the two groups. Compared with the two groups, the level of serum Ngb was significantly increased in cerebral ischemia group (P<0.05). The increase in the level of serum Ngb after brain ischemia was significantly prevented by LIP or administration of Hemin prior to cerebral ischemia (P<0.05), while vehicle of Hemin had no evident effect on the level of serum Ngb protein. The above results indicated that LIP and Hemin could significantly prevent the increase of the serum Ngb protein level induced by cerebral ischemia.2 Ngb particapates in LIP-induced brain ischemic tolerance via mechanisms involving in mitochondriaTo investigated whether Ngb particapates in LIP-induced brain ischemic tolerance via mitochondria mechanisms, the study observed the effect of up-regulated Ngb expression in the rat CA1 hippocampus on the mitochondria trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure in rat CA1 hippocampus.Methods: One hundred and twenty-six male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into 7 groups:①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.③LIP+cerebral ischemia group: LIP was preformed by occlusion of bilateral femoral arteries of rats for 10 min 3 times in an interval of 10 min, then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the last occlusion of the femoral arteries.④AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP+cerebral ischemia group.⑤S-ODNs+LIP+cerebral ischemia group: Ngb S-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP + cerebral ischemia group.⑥Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.⑦Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.Eighteen rats were included in each group, and assigned to 4 observations:①mitochondria trans-membrane potential at 24 h (n=3),②Na+-K+-ATPase activity of mitochondria at 6 h and 24 h (n=10),③Bcl-2 and Bax mRNA expression at 24 h (n=3), and④neuronal and mitochondrial ultrastructure of the rats CA1 hippocampus on 3 d (n=2), in which the rats were, respectively, sampled at the corresponding time point after the sham operations of cerebral ischemia, or cerebral ischemia in each group.Results: Flow cytometry analysis showed that mitochondrial trans- membrane potential significantly decreased in cerebral ischemia group compared with that of sham group of cerebral ischemia (P<0.05). The decrease was significantly ameliorated by LIP prior to cerebral ischemia insult (P<0.05). However, Pretreatment with Ngb AS-ODNs before LIP significantly inhibited the ameliorating effect of LIP on mitochondrial trans-membrane potential, which was represented with the decrease in the potential in AS-ODNs+LIP+cerebral ischemia group compared with that in LIP + cerebral ischemia group (P<0.05). In contrast, pretreatment with Ngb S-ODNs before LIP was without effect on the potential. Hemin significantly increased mitochondrial trans-membrane potential compared with that in cerebral ischemia group (P<0.05), whereas, Hemin vehicle administrated had no evident effect on it.Results of Na+-K+-ATPase activity at 6 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, but significantly decreased after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the enzyme activity was further decreased in LIP + cerebral ischemia group (P<0.05). Pretreated with Ngb AS-ODNs before LIP significantly increased the enzyme activity when compared to LIP + cerebral ischemia group (P<0.05). However, administration with Ngb S-ODNs before LIP was without effect upon the enzyme activity. Compared with cerebral ischemia group, Hemin significantly decreased the enzyme activity (P<0.05), and no difference of the enzyme activity was found at Hemin vehicle group. Results of Na+-K+-ATPase activity at 24 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, which significantly decreased in cerebral ischemia group (P<0.05). The enzyme activity was significantly increased by LIP, when compared to cerebral ischemia group (P<0.05). Compared to LIP + cerebral ischemia group, the application of Ngb AS-ODNs before LIP significantly decreased the enzyme activity (P<0.05), but Ngb S-ODNs had no influence. When the animals were pretreated with the Hemin before the lethal ischemic insult, the enzyme activity was significantly increased compared with that in cerebral ischemia group (P<0.05), and no evident effect was observed in Hemin vehicle + cerebral ischemia group.RT-PCR analysis showed that Bcl-2 and Bax mRNA are less in the rat CA1 hippocampus in the sham group of cerebral ischemia, which was significantly up-regulated after cerebral ischemia (P<0.05). Pretreated with LIP before cerebral ischemia dramatically up-regulated the expression of Bcl-2 mRNA, but down-regulated the expression of Bax mRNA (P<0.05). Administration of Ngb AS-ODNs before LIP+cerebral ischemia significantly inhibited Bcl-2 mRNA expression and increased Bax mRNA expression (P<0.05). However, Ngb S-ODNs had no effect on the Bcl-2 and Bax mRNA expression. Ngb inducer Hemin could also significantly up-regulate the expression of Bcl-2 mRNA, and down-regulate the expression of Bax mRNA (P<0.05). While, pretreated with vehicle of Hemin before cerebral ischemia had no effect on the Bcl-2 and Bax mRNA expression.Transmission electron microscopic examination showed that the structure of neurons and mitochondria in sham group of cerebral ischemia was normal. Cerebral ischemia for 8 min induced obvious destruction of the neuronal and mitochondrial ultrastructure. The neuron was swollen and cellular organs were significantly decreased; meanwhile, the mitochondria were swollen or pyknosis and its cristae were broken, dissolved and disappeared. But in LIP + cerebral ischemia group, the above injury changes were prevented clearly. When the animals were pretreated with Ngb AS-ODNs prior to LIP + cerebral ischemia, the protective effect of LIP was prevented, while, Ngb S-ODNs had no effect on them. Ngb inducer Hemin significantly inhibited destruction of the neuronal and mitochondrial ultrastructure induced by cerebral ischemia, but Hemin vehicle had no effect on the cerebral ischemia induced destruction of the neuronal and mitochondrial ultrastructure.The above results indicated that the up-regulation of Ngb expression participated in the brain ischemic tolerance induced by LIP via the mechanisms involving in modulated mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure.3 The expression of Ngb in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged ratsUsing four-vessel occluding global brain ischemia model, the role of age on the Ngb expression and the LIP neuroprotection against severe ischemic insult was investigated by observing the Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged rats.Methods: Sixty male Spague-Dawley rats aged 3 months (thirty) and 21-23 months (thirty) with permanently occluded bilateral vertebral arteries were randomly divided into cerebral ischemic group and LIP + cerebral ischemic group, respectively. The expression of Ngb mRNA and protein in the CA1 hippocampus were investigated by RT-PCR and Western blot methods. The profile of DND of pyramidal neurons in the CA1 hippocampus was evaluated by using thionin staining.Results: RT-PCR and Western blot analysis showed that the expression of Ngb mRNA and protein after cerebral ischemia for 8 min in aged rats was decreased compared with that in the young rats which suffered a identical cerebral ischemia with the aged rats (P<0.05). LIP upregulated Ngb mRNA and protein expression in both young and aged rats suffered cerebral ischemia (P<0.05). However, the upregulation of Ngb expression in aged rats was significantly less than that in young rats (P<0.05).Neuropathological evaluation showed that obvious DND of pyramidal neurons was found in young and aged rats after cerebral ischemia. Although LIP effectively protected the pyramidal neurons in the CA1 hippocampus against DND normally induced by ischemic insult (P<0.05), the neuroprotection of LIP for aged rats was less effective than that for young rats.The above results indicated that the expression of Ngb and the up-regulating effect of LIP on the expression in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia.4 LIP up-regulate the expression of Ngb in the normal rat CA1 hippocampusTo investigate whether LIP could induce the up-regulation of Ngb expression in CA1 hippocampus of normal rats, the study observed the time courses of the Ngb expression induced by LIP in the normal rat CA1 hippocampus.Methods: Forty male Wistar rats were randomly divided into 2 groups:①Sham group (n=8): The bilateral femoral arteries of the rats were dissected out and exposed for 60 min, but not clamped. ②LIP group (n=32): The bilateral femoral arteries of the rats were dissected out and clamped for 10 min 3 times in an interval of 10 min.The expression of Ngb mRNA in the CA1 hippocampus was measured by RT-PCR analysis, according to the timing of reperfusion after LIP, rats in LIP group were divided into 4 subgroups: 30min, 1h, 3h and 6h. The expression of Ngb protein was measured by Western blot analysis, rats in LIP group were divided into 4 subgroups: 1h, 6h, 12h and 1d.Results: RT-PCR analysis showed that basal expression of Ngb mRNA in the CA1 hippocampus could be observed in sham group. The Ngb mRNA expression was significantly up-regulated by LIP. The expression of Ngb mRNA peaked at 30 min (P<0.05), then gradually decreased at 1 h and 3 h, but still higher than that of sham group (P<0.05). The expression of Ngb mRNA returned to sham level at 6h after LIP.Western blot analysis showed that compared with sham group, the Ngb protein expression was significantly up-regulated after LIP, which peaked at 1h (P<0.05), then gradually decreased at 6 h and 12 h, but still higher than that of sham group (P<0.05). The expression of Ngb protein at 1d after LIP was still higher than that of sham group, but no statistic difference when compared to sham group.The above results indicated LIP could induce up-regulation of Ngb expression in CA1 hippocampus of normal rats.5 Conclusions(1) LIP and Ngb inducer Hemin could significantly up-regulate the expression of Ngb in the CA1 hippocampus in cerebral ischemic rat, meanwhile protect the pyramidal neurons in the area against cerebral ischemia insults and decreased the serum Ngb protein level. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. These results indicated that the up-regulation of the Ngb expression played an important role in the brain ischemic tolerance induced by LIP. (2) LIP effectively improved the deterioration in the mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neuronal and mitochondrial ultrastructure induced by cerebral ischemic insult. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. Ngb inducer Hemin could mimic the above effects of LIP. The results suggested that LIP plays neuronal protection, at least, through improving structure and function of mitochondria.(3) The expression of Ngb and the up-regulating effect of LIP on it in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia. These differences might be one of underlying reasons why the aged rats exhibited severe DND after cerebral ischemia and why the neuroprotective effect of LIP was less in the aged rats than that in the young rats.(4) LIP could induce up-regulation of Ngb expression in CA1 hippocampus in normal rats.