Our findings provide the first evidence that activation of Notch signaling contributes to EA pretreatment-induced cerebral ischemic tolerance. EA pretreatment before ischemia significantly increased Notch1,Notch4 receptors and Jag1 ligand mRNA in the striatum of adult rats and did not affect the Notch1 NICD and Hes1 mRNA levels, suggesting no activation of Notch pathway. However, after ischemia/reperfusion, Hes1 mRNA and Notch1 NICD protein level in EA group reached the peak at 2 h and 24 h, respectively, while both of them reached the peak at 72 h in CON group. The earlier activation of Notch signal may be caused by the higher expression of Notch1, Notch4 and Jag1 before ischemia in the EA pretreatment group. Although EA pretreatment can not increase the activation of Notch pathway before ischemia/reperfusion, it prepared the essential materials for the following Notch pathway activation after ischemia/reperfusion. Intraventricular injections with MW167 can block the cleavage of all four Notch receptors and lead to inactivation of Notch pathway in the central nervous system . The γ-secretase inhibitors may prove to be more superior agents than that target a single cell type, because they may target pathogenic events in multiple cell types (neurons, lymphocytes and microglia) involved in cerebral ischemic injury. Our current data demonstrated that the neuroprotective effect of EA preconditioning was abrogated in the presence of intraventricular injections with MW167, suggesting that γ-secretase-mediated Notch signaling is required for the cerebral ischemic tolerance induced by EA pretreatment. Thus, the activated Notch signaling at the early stage after reperfusion is beneficial to the survival of neurons in the ischemic cerebral tissues.
Although the pivotal roles that Notch signaling takes part in the cellular differentiation of the developing central nervous system (CNS) have been proved, the functions of the signaling pathway, especially under pathological conditions in the adult CNS, remain to be illuminated. A line of data indicates that the expressions of notch receptors were up-regulated after MCAO in the brain of rat. [5, 7, 13]. The same phenomenon was observed in our study which demonstrated that focal cerebral ischemia/reperfusion can enhance Hes1 mRNA and Notch1 NICD protein level in striatum of adult rat. However, different to our results, Kawai et al. found that the mRNA of Hes5, an important downstream gene of Notch signal, was decreased on days 1 and 3 after global ischemia in rats, suggesting that the Notch signaling may be inhibited after transient forebrain ischemia . The differences in animal strain, stroke model or treatment regimen may account for the discrepancy. So the phenomenon that ischemia-reperfusion can modulate Notch pathway in the CNS has been widely accepted.
In this study, we found that intraventricular injections with the γ-secretase inhibitor MW167 without EA pretreatment could reduce brain damage and ameliorated functional outcome in a rat model of stroke, suggesting that the inhibition of Notch signaling activation after ischemia/reperfusion could lessen the cerebral injury. In accordance with our results, it has been reported that Notch signaling increase the vulnerability of neurons to apoptosis. And γ-secretase inhibition improves functional outcome and reduces brain damage in ischemic stroke in mice by activating microglial cells and facilitating the infiltration of proinflammatory leukocytes . However, some results support the idea that the activation of Notch pathway exerts the neuroprotective effect after ischemia. Gastric lavage with soybean isoflavone decreased the ischemia-induced apoptosis in the cerebral cortex and this effect of soybean isoflavone was associated with activation of the Notch signaling in the cerebral cortex . Notch receptor activation inhibits cell death in neural stem cells in vitro rapidly, and this survival effect of Notch ligands was antagonized by a γ-secretase inhibitor. A more delayed activation of Notch signaling, which was induced by infusing the Notch ligand Dll4 into the cerebral lateral ventricle of adult rats, had no effect on improving cerebral infarct size but improved the motor skills over a period of 45 days . A possible explanation for these apparently contradicting results is the different time frame. Our data showed that the earlier activation of Notch signal after ischemia/reperfusion induced by EA pretreatment provided protective effects.
However, our results raise the issue of why the earlier activation of Notch signaling induced by EA can change its characteristic from possible damaging effect to protective effect. The cerebral infarction induced by MCAO in rats and mice can be thought as a sphere in which cells in the inner core die rapidly by necrosis, in minutes to a few hours. However the cells in the outer “penumbral regions” die slowly, in a period of days to weeks. The evidence that neurons in the cerebral ischemic penumbra undergo apoptosis was first provided by Linnik and coworkers . Whether or not a neuron survives when exposed to potentially lethal injury is likely to depend upon which of the antiapoptotic and proapoptotic pathways prevails. Studies of stroke in animal models support the hypothesis that the brain activates antiapoptotic signaling pathways involving neurotrophic factors and cytokines as responds to ischemic insults. The ischemia-related insults induce the rapid increased expression of several neurotrophic factors and cytokines in the rodent brain, leading to activation of signaling pathways that result in suppressing oxygen radical production and stabilizating cellular calcium homeostasis [17, 18]. The effect of Notch pathway depends on the integration with other cellular signals, the degree of Notch activation, and the time point when the Notch pathway is activated during the cell cycle in development . Earlier activation of Notch signaling in the striatum induced by EA pretreatment probably enhanced more survival signals. Later activation of Notch pathway resulted from ischemia/reperfusion in CON induced more neurodegenerative pathways.
Previous in vitro studies suggest that the cleavage of Notch activates the survival cascade downstream of the insulin receptor involving the serine/threonine kinase Akt and mammalian target of rapamycin, and that Ser727 phosphorylation of STAT3 also mediates the survival effects induced by Notch activation in neural stem cells . There is a crucial physiologically crosstalk between the two major signal transduction pathways — Notch–Hes and JAK–STAT in the central nervous system . It is demonstrated that the neuroprotective effect of EA pretreatment may be relevant to activation of PI3K/Akt and JAK/STAT signals [21, 22]. These results imply that the activation of Notch signaling may link the EA pretreatment with some survival signal transducers. Notch pathway is also a useful paradigm to demonstrate the complexity of cross-talk pathway with nuclear factor-kB, transforming growth factor-β and Wnt signalings in ischemia/reperfusion [23, 24].
In conclusion, the current study provides novel insights into the neuroprotection induced by EA pretreatment: EA preconditioning induces neuroprotection by controlling the timing of Notch pathway activation. However, further studies are demanded to delineate the downstream factors of Notch signaling for understanding the mechanisms of EA pretreatment-induced brain ischemia tolerance.