| Backgrounds:It has been well established that adult neurogenesis occurs throughout life in the subventricular zone (SVZ) of the lateral ventricle and in the subgranular zone (SGZ) of dentate gyrus in mammals.Newly generated cells in the adult brain can migrate, differentiate into functional mature neurons and integrate into neuronal networks, including those involved in cognitive function. However, during aging, there is an age-related decrease in adult neurogenesis; but the cause of this phenomenon is poorly understood. Notch signaling pathway defines an essential pathway controlling cell fate. Notch signaling pathway plays important roles in the maintenance, proliferation, and differentiation of neural stem cells (NSCs) in both developing and adult brains. After membrane-bound Notch receptor binds to ligands which present on an adjacent cell, Notch extracellular and trans-membrane domains are sequentially cleaved, then Notch intracellular domain (NICD) is released and trans-located into nucleus, where it regulates transcription. Animal studies have shown that Notch signaling pathway is required for the maintenance of the reservoir of young-adult mice SVZ and SGZ stem cells, whereas inhibiting Notch signaling pathway could reduce the maintenance and proliferation of NSCs. However, its roles in aged neurogenesis are still unknown.Ischemic stroke stimulates the proliferation of neuronal stem/progenitor cells (NSCs) located in the SVZ and SGZ of the adult mammal brain. The ischemia-induced newborn cells migrate into ischemically injured brain regions, where they differentiate into mature neurons and replenish the damaged ones, then restore the brain functions at some extent. However, molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Animal studies have shown thatthe same Notch signaling pathway involved in neurogenesis under physiologic conditions also contributes to the neurogenesis-promoting effect of ischemia in young-adult brain: Notch signaling pathwayis activated by ischemia in vitro and in vivo;Activating Notch signaling pathway could reduce the death of NSCs, increase neurogenesis and improve motor deficits after ischemia, whereas inhibiting Notch signaling pathway blocks ischemia-induced neurogenesis. In addition, the astroglial response of the SVZ to injury is also accompanied by activation of the Notch signaling pathway. But its effects on ischemia-induced neurogenesis in aged brain have not been investigated yet.AimsTo clarify the roles of Notch signaling pathway in neurogenesis in the SVZ of aged rat brain in health and after ischemic stroke, in the present study we performed the following investigations:1. We examined the changing expressions of Notch signaling pathway core components in the SVZ of aged rat brain and young-adult rat brain; and the cell type expressions of Notchsignaling pathway core components in the SVZ of normal aged rat brain in vivo; then investigated the effects of manipulating Notch signaling pathway on the cell proliferation and neurogenesis in the SVZ of normal aged rat brain in vivo.2. We examined the time course of Notch signaling pathway activities in the SVZ of aged rat brain after ischemic stroke; then we assessed the effects of forced activation or blockade of Notch signaling pathway on the cell proliferation and neurogenesis in the SVZ of aged rat brainand observed the subsequent functional outcomes after stroke in vivo.MethodsNotch signaling pathway activator or inhibitor were loaded into osmotic mini-pumps and administrated into lateral ventricle of aged rat brain. Permanent focal cerebral ischemia was induced by electrocoagulation of distal middle cerebral arterial. The Notch signaling pathway expressions were determined by immunohistochemistry and Western blot analysis. The cell type expression of Notch signaling pathway and effects of manipulating Notch signaling pathway on cell proliferation and neurogenesis were assessed by double-or triple-label immunofluorescence. Functional outcomes were examined by a battery of behavioral tests, such as Bederson score, elevated body swing, Rotarod, cylinder, limb placing and staggered ladder rung walking tests.ResultsPart Ⅰ Expressions of Notch signaling pathway core components in the SVZ of aged and young-adult rat brain andeffects of altering Notch signaling pathway on the cell proliferation and neurogenesis in the SVZ of aged rat brain in vivoNotch signaling pathway activator and inhibitor were prepared as described previously in our lab and administrated into left lateral ventricle by using osmotic mini-pumps. Western blot analysis showed that expressions of Notch signaling pathway core components, such as Notch1、Notch4receptors, Jagged1ligands and Notch intracellular domain (NICD), were all significantly decreased in the SVZ of normal aged rat brain, compared with young-adult rat. Consistent with Western blot results, the numbers of Notchl-, Notch4-, Jaggedl-, NICD-positive cells were also decreased in the SVZ with aging as detected by immunohistochemistry. Triple-label immunofluorescence results showed that Notch1, NICD and Hes1immunoreactivities were mainly co-localized with the neuronal marker DCX-positive cells, whereas Jagged1immunoreactivity was co-expressed with the astroglial marker, GFAP-positive cells in the SVZ of aged rat brain. Double-label immunofluorescence results showed that the numbers of newly generated cells (BrdU-positive), neural precursor cells (DCX-positive) and newly generated neural precursor cells (BrdU-DCX-co-positive) in the SVZ of normal aged rat brain were all increased after intracerebroventricular (ICV) administration of the Notch signaling pathway activator for3d, whereas Notch signaling inhibitor reduced the cell proliferation and neurogenesis. These findings suggest that the expressions of Notch signaling pathway core components decrease with aging and are mainly co-localized in neural precursor cells. Enhanced Notch signaling pathway expression increases cell proliferation and neurogenesis in the SVZ of normal aged rat brain in vivo.Part Ⅱ Time course of Notch signaling pathway activities and effects of forced activation or blockade of Notch signaling pathway on neurogenesis and functional outcomes in aged rats after ischemic strokeWestern blot results showed that Notch1and NICD expressions increased at1d after ischemic stroke. Double-label immunofluorescence results showed that Notch signaling pathway inhibitor attenuated the ischemia-induced increase in BrdU-, DCX-and BrdU-DCX-labeling cells, whereas Notch signaling pathway activator promoted cell proliferation and neurogenesis after ischemic stroke. Behavioral tests showed that Notch signaling pathway activator improves functional outcomes of aged rats at both1d and3d after stroke, while Notch signaling pathway inhibitor exacerbate the outcomes. Thus, enhanced Notch signaling pathway is also beneficial to aged rat brain after ischemic stroke. Conclusions1. The expressions of Notch signaling pathway core components decrease with aging. Notch1, NICD and Hesl are localized to neural precursor cells, whereas Jagged1is expressed in astrocytes, in the SVZ of aged rat brain. Enhanced Notch signaling pathway expression increases cell proliferation and neurogenesis in the SVZ of normal aged rat brain in vivo.2. Notch signaling pathwayis activated byischemia in the SVZ of aged rat brain. Enhanced Notch signaling pathway promotes neurogenesis and improves functional outcomes in aged rats after stroke. |
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