The Role Of Notch Signaling Pathway In The Neuroprotection Induced By HBO Preconditioning

Stroke is a common cerebral vascular accident in clinical, it has been identified as"the number one killer"to human health by the World Health Organization. Nowadays, there are no effective measures for prevention and treatment of it. In recent years, it has been found that a number of different non-ischemic preconditioning can induce a similar effect of ischemic tolerance, thus we speculate that these different pretreatments activated some common pathway signal and ultimately induced protective effect through the basic regulatory mechanisms. Notch signaling pathway which has important role in embryonic development has been shown involved in the responses of cerebral ischemia in vivo or vitro. Hyperbaric oxygen preconditioning, due to the advantage of safe, practicability and validity, has been confirmed effectively in inducing neuroprotection by a large number of studies. However, it has not been reported whether the Notch signaling pathway is involved in the neuroprotective effect induced by HBO preconditioning. So this study used middle cerebral artery occlusion model in rats to explore the effect of Notch signaling on the neuroprotection induced by hyperbaric oxygen preconditioning.Part I Effect of repeated hyperbaric oxygen preconditioning on Notch signaling pathwayMethods1. Confirmation of the effect of neuroprotection induced by HBO preconditioning30 Male SD rats were randomly divided into three groups: sham group, ischemia-reperfusion (I/R) group and HBO+I/R group. Sham-operated rats underwent an identical surgery, except that the suture was not inserted, in I/R group and HBO+I/R group animals were preconditioned with normal air or hyperbaric oxygen. HBO preconditioning was performed according to our published methods (Xiong L et al., 2000).Twenty-four hours after the last treatment, the animals of I/R group and HBO+I/R group underwent middle cerebral artery occlusion (MCAO) with 3-0 nylon monofilament for 120 min. The neurological outcome was evaluated at 24, 48 and 72h after reperfusion. Then at the time point of 72h after reperfusion, the animals were killed and the infarct volume was assessed by TTC staining.2. Effect of repeated hyperbaric oxygen preconditioning on Notch signaling pathway2.1 The variation of Notch signaling activation after cerebral ischemia/ reperfusion48 male SD rats were randomly divided into two groups: sham group (n=12) and I/R group (n=36). The focal cerebral ischemia was induced by MCAO for 120 min in I/R group. At the time points of 2h, 24h and 72h after reperfusion, we measured the levels of NICD, HES-1 in the right cortical and striatum tissue samples by immunofluorescence, western blotting and RT-PCR in brain tissue samples.2.2 The variation of Notch signaling activation after hyperbaric oxygen preconditioning96 Male SD rats were randomly divided into four groups: sham group (n=12), I/R group (n=36), HBO only group (n=12) and HBO+I/R group (n=36). Sham-operated rats underwent an identical surgery, except that the suture was not inserted. 12 animals were killed at 24h after the last pretreatment. The other 72 animals in I/R group and HBO+I/R group were subjected to MCAO 24h after last pretreatment for 2h and were killed at 2h, 24h and 72h after reperfusion. Immunofluorescence, western blotting and RT-PCR were used to detect the levels of NICD, HES-1 in the right cortical and striatum tissue samples.Results1. Confirmation of the effect of neuroprotection induced by HBO preconditioningIn HBO+I/R group, the infarct volume was significantly less than that in I/R group (P<0.05), and the neurologic deficit scores was improved compared to I/R group (P<0.05).2. Effect of repeated hyperbaric oxygen preconditioning on Notch signaling pathway2.1 Western Blotting In striatum tissue samples: the levels of NICD in sham-operated animals were very low by western blotting, while that in I/R group 24h after reperfusion were significantly elevated(*P<0.05 vs. sham) and kept rising to 72h after reperfusion ($P<0.05 vs. I/R 24h).After hyperbaric oxygen preconditioning, the levels of NICD were significantly elevated in HBO only group(#P<0.05 vs. sham), and peaked at 24h of reperfusion, then returned to basal levels 72h after ischemia-reperfusion. At the point of 2h after ischemia-reperfusion, the levels of NICD in HBO+I/R group were higher than that I/R group (#P<0.05), but at the points of 24h, 72h after ischemia-reperfusion, they were less than that in I/R group (#P<0.05)In cerebral cortex:the levels of NICD in sham-operated animals were very low by western blotting, while that in I/R group 24h after reperfusion were significantly elevated(*P<0.05 vs. Sham) and kept rising to 72h after reperfusion ($P<0.05 vs. I/R 24h). After hyperbaric oxygen preconditioning, the levels of NICD were significantly elevated at 2h of reperfusion in HBO+I/R group(&P<0.05 vs. sham), and peaked at 24h of reperfusion, then returned to basal levels 72h after ischemia-reperfusion. At the point of 2h after ischemia-reperfusion, the levels of NICD in HBO+I/R group were higher than that I/R group (#P<0.05).2.2 Real Time-PCRThe levels of HES-1 mRNA in striatum tissue samples and cerebral cortex samples are alike as show below:The transcription of HES-1 gene in sham-operated group is weak, and was elevated 2h after reperfusion (*P<0.05 vs. sham); while the transcription of HES-1 gene significantly reduced at 24h after reperfusion, from 24h to 72h after reperfusion, HES-1 transcription showed a rising trend, with the highest HES-1 transcription 72h after reperfusion($P<0.05 vs. I/R 2h). After hyperbaric oxygen preconditioning, the levels of HES-1 transcription were significantly elevated in HBO only group(#P<0.05 vs. sham), and peaked at 2h of reperfusion, then returned to basal levels 72h after ischemia-reperfusion. But at the point of 2h, 24h after ischemia-reperfusion, the levels of HES-1 transcription in HBO+I/R group were higher than that I/R group (#P<0.05).2.3 ImmunofluorescenceA few of NICD (Notch intracellular domain) positive cells was detected in sham-operated animals. But a large number of NICD positive cells were detected in the animals subjected to MCAO 24h after reperfusion. The numbers of NICD positive cells kept rising to 72h after reperfusion.After HBO preconditioning, a large number of NICD positive cells were detected in HBO only group and peaked at 24h of reperfusion after HBO pretreatment. However, the numbers of NICD positive cells were significantly decreased at the point of 72h after ischemia-reperfusion in HBO+I/R group.Conclusions1. Notch signaling pathway was activated after focal cerebral ischemia reperfusion in SD rats.2. Notch signaling pathway was activated after HBO preconditioning, and the peak expression of its related molecules NICD, HES-1 was advanced, indicating its participation in the neuroprotective effect induced by HBO pretreatment. PartⅡThe effect ofγ-secretase inhibitor (DAPT) on the induction of ischemic tolerance by HBO preconditioningMethods1. Confirmation of the effect of DAPT12 Male SD rats were randomly divided into three groups: sham group, HBO only group and DAPT+HBO group, in which animals were preconditioned with normal air or hyperbaric oxygen.Animals in DAPT+HBO group were intracerebroventricularly infused with 50μm/L 5μL DAPT 3h before each HBO treatment for 5 days. Twenty-four hours after the last treatment, all groups of animals were killed and the levels of NICD were detected by western blotting in striatum tissue samples.2. Effect ofγ-secretase inhibitor (DAPT) on the induction of ischemic tolerance by HBO preconditioning60 Male SD rats were divided into six groups: sham group, I/R group, DAPT+I/R group, HBO+I/R group, Vehicle+HBO+I/R group and DAPT+HBO +I/R group.Animals in DAPT+HBO+I/R group were intracerebroventricularly infused with DAPT 3h before each HBO treatment for 5 days. Twenty-four hours after the last treatment, MCAO was performed for 120 min. The neurological outcome was evaluated at 24, 48 and 72h after reperfusion. Then at the time point of 72h after reperfusion, the animals were killed and the infarct volume was assessed by TTC staining. Results1. Confirmation of the effect of DAPTCompared to sham and HBO only groups, the levels of NICD was significantly decreased in DAPT+HBO group (#P<0.05).2. Effect ofγ-secretase inhibitor (DAPT) on the induction of ischemic tolerance by HBO preconditioningTreatment with 50μm/L 5μL DAPT by intracerebroventricularly infusion 3h before each HBO treatment for 5 days, the infarct volume was significantly bigger than that in HBO+I/R group (#P<0.05), and the neurologic deficit scores at 24, 48 and 72h after reperfusion were significantly less compared to HBO+I/R group (#P<0.05). But there were no significant difference between the HBO+I/R and DMSO+HBO+I/R groups.ConclusionsThe administration of Notch signaling inhibitor DAPT can partially reverse the neuroprotective effect induced by hyperbaric oxygen preconditioning. This further confirmed that Notch signaling was involved in the neuroprotection effect induced by hyperbaric oxygen preconditioning.Summary1. Notch signaling pathway was activated after focal cerebral ischemia reperfusion in SD rats.2. Notch signaling pathway was activated after HBO preconditioning, and the peak expression of its related molecules NICD, HES-1 was advanced, indicating its participation in the neuroprotective effect induced by HBO pretreatment.3. The administration of Notch signaling inhibitor DAPT can partially reverse neuroprotective effect induced by hyperbaric oxygen preconditioning. This further confirmed that notch signaling was involved in the neuroprotection effect induced by hyperbaric oxygen preconditioning.