| Neonatal ischemic stroke is defined as a cerebrovascular event resulting from focal or generalized interruption of blood supply manifested by neurologic dysfunction. A group of heterogenous primary disorders have been acknowledged as risk factors for ischemic stroke in neonates, including asphyxia, congital heart disease, systemic infection, prothrombotic state as well as severe blood loss/anemia. In recent years, increased morbidity of neonatal ischemic stroke has been documented partially due to the improvements in neuroimaging techniques. However, it still remains a tough challenge to protect the immature brain from ischemic insults and improve the outcome. Long-term neurologic defects like motor disabilities, congnitive deficits or epilepsy are commonly presented in patients with neonatal stroke history. The pivotal strategy of therapy is to preserve or restore the neuronal population and function in ischemic region as much as possible, which is a multi-faceted maneuver regulated by cellular signaling network in the brain.Along with other well-studied pathways mediated by death receptor, protein kinase, Toll-like receptor, PI3K-Akt/ERK or Nuclear factor-κB, Notch signaling, an evolutionarily conserved pathway, has been recognized for its important role in ortchestrating embryonic development and after-birth homeostasis in central nervous system. After binding with ligands expressed on the membrane of juxtaposed cells, Notch receptors are cleavaged to release its active intracellular domain (Notch intracellular domain, NICD) which acts as a transcriptional regulator of several target genes. Evidences of Notch’s involvement in neural progenitor regulation, synaptic plasticity, cell migration and cell fate dertermination rationalize the hypothesis that Notch pathway may underly the cellular response to brain injuries including ischemia. Studies from adult stroke have pointed out a context-dependent effect of Notch signaling. The immature brains possese totally different developmental features, and the role of Notch signaling in neonatal ischemic stroke is much less known to date.In order to investigate the specific role of Notch signaling in the immature brain challenged by ischemia, two different models of neonatal stroke were developed and subject to acute blockage of canonical Notch signaling pathway by DAPT, a potent y-secretase inhibitor. Changes in post-ischemia survival rate, infarct size in the brain as well as long-term impairment of motor function were evaluated to demonstrate the effects of acute Notch signaling inhibition on a systemic level. Furthermore, cell apoptosis, proliferation, differentiation preference and angiogenic activity in ischemia-affected brain regions were detected and assessed, to determine the cellular mechnisms by which Notch signaling regulates an adapted response to ischemic attack.Meanwhile, microarray detection of neurogenesis-related gene expression was performed to compare the differences in transcriptional profile caused by Notch signaling regulation. Objective:To establish practical rat model of neonatal ischemic stroke, and observe the changes in Notch signaling function induced by ischemia. To assess the effects and mechanisms of acute blockage of Notch signaling by DAPT (N-[N-(3, 5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester) on the outcome of neonatal ischemic stroke. Methods:1. Animal model and drug intervention:Male Sprague-Dawley rats at postnatal age of 10 days (P10) were ramdomly assigned to two different models of ischemia:1)Hypoperfusion:bilateral common carotid arteries were permanently ligated; 2) Transient focal ischemia:induced by intraluminal suture occlusion of middle cerebral artery for 1 hour then the suture removed. Rats of hypoperfusion or transient focal ischemia were further divided into two groups, respectively:(1) DAPT:DAPT (100mg/kg) was injected intraperitoneally 1 hour after the occlusion, (2) ischemia: normal saline in equral volume to DAPT injection was given intraperitoneally at 1 hour after the occlusion. Rats in sham control group received sham surgery without ligation or occlusion of any artery. Rats were weighted and evaluated for general condition at P11, P13, P15, P17, P24, P21, and P28 after surgery. Western blot was performed to quantify the production of some key moslecules (NICD, Hesl and Hes5) in Notch signaling pathway using rat brain tissue harvested on P15.2. Evaluation of outcome:The short-term survival rate was obtaind at P15. The infarct size was measured by 2,3,7-triphenyltetrazolium (TTC) stain method at 1 day after ischemia. Rotarod test was carried out 4 weeks after surgery to evaluate the long-term motor function.3. Cellular mechenisms:brain slices were obtained from tissue samples collected on individual time points after ischemia.24 hours after ischemia, cells undergoing apoptosis were detected in penumbra area by means of in situ terminal deoxynucleotidyl treansferase-mediated dUTP-biotin nick end labeling (TUNEL). For checking of new born cells, rats were given consecutive injection of biomodeoxyuridine (BrdU) for 3 days after stroke (50mg/kg,1 hour after occlusion and daily for the next 2 days) and sacrificed on the day 5. Immunohistochemistry using diaminobenzidine (DAB) staining system was applied to detect the accumulation of BrdU+cells in ischemic cortex and ipsilateral subventricular zone (SVZ). Meanwhile, double labeling of BrdU and cell-type specific markers using immmunofluorescence staining method was applied to identify the phenotypes of these newly proliferated cells in penumbra on day 5. Immunofluorescence of Laminin was also checked in penumbra on day 14 after ischemia for semiquantitive evaluation of angiogenesis. cDNA microarray was used to screen neurogenesis-related genes with differential expression under DAPT intervention and reverse transcription quantitative real-time PCR was used to verify the results from microarray detection.Results:1. P10 SD rat models of neonatal ishchmic stroke were established in two different ways:hypoperfusion models and focal ischemia models, by permanent ligation of bilateral common carotid arteries and transient intraluminal suture occlusion of left middle cerebral artery respectively. Body weight loss was most significant within the 1 week following ischemia surgery and the motor function was impaired 4 weeks later.2. Notch signaling was activated after ischemia and the activation was effectively inhibited by acute treatment of DAPT. NICD, Hesl and Hes5 were uniformly downregulated in the DAPT group on day 5 compared with ischemia group with sinifigance (p<0.001).3. DAPT promoted survival on day 5 after ischemia in both two models (Hypoperfusion model:77.8%:33.3%, Focal ischemia model:72.7%:33.3%, compared with ischemia group respectively).4. Under focal ischemia, DAPT reduced 22.26% infarct volume on day 1 after surgery compared with vehicle-treated ischemic rats (p<0.05).5. DAPT ameliorated the long-term motor function impairment caused by ischemia.6. DAPT reduced the total number of apopotic cells in penumbra on day 1 after surgery in comparison with that of ischemia group (p<0.001).7. DAPT further promoted accumulation of new born cells in peri-infarct region 5 days after both hypoperfusion and transient focal ischemia (p<0.05), and reversed the reduction of new born cells in SVZ region after stroke (p<0.05).8. DAPT modulated the phenotypes of new born cells in penumbra of focal ischemia by increasing Nestin+/BrdU+ colocalization ratio as well as reducing Ibal+/BrdU+ fraction in comparison with that in vehicle-treated ischemic group (p<0.05).9. DAPT increased the vascular density in penumbra on day 14 after focal ischemia (p<0.05).10. DAPT regulated the gene profiles associated with neurogenesis and neural stem cell differentiation in penumbra after focal cerebral ischemia. Expressions of 7 new genes were induced by DAPT and 4 genes were silenced.2 genes’ expression was further promoted by DAPT compared with ischemic condition, while 11 genes’ expression was down-regulated after DAPT treatment. The genes exhibiting significant changes in expression under DAPT treatment were functionally associated with cell proliferation, migration and synaptic plasticity.Conclusion:1. Ischemic injury activated the function of Notch signaling pathway in neonates’ immature brains. Acute application of DAPT, the y-secretase inhibitor, effectively inhibited this activation effect.2. Acute blockage of Notch signaling pathway by DAPT recuced the infarct size in brain, promoted short-term survival as well as long-term motor function recovery after neonatal ischemic stroke.3. DAPT’s blockage of Notch signaling induced neuroprotective effects by inhibiting apoptosis, promoting cell proliferation and accumulation as well as angiogenesis in penumbra and regulating the differentiation of new born cells.4. Acute blockage of Notch signaling pathway by DAPT regulated the expression of several genes associated with neurogenesis and neural stem cell differentiation. |
PDF Full Text Request