| BackgroundAdult neurogenesis exists in specific areas of the brain such as subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus all lifelong. The proliferation and differentiation towards specific phenotypic of adult neural stem cells are determined by the characteristics of its niche in the brain. There are plenty of evidences showing that microglial cells, astrocytes cells, ependymal cells and endothelial cells are part of niche of ANSCs on the cell level. The neuroimmune system has been proposed as the composition of its niche which impacts on the proliferation, differentiation and survival of ANSCs. The interaction of the brain immune cells (especially in microglia cells and astrocytes cells), peripheral immune cells (especially T cells and macrophages), neurons and adult ANSCs maintains the brain homeostasis and self-renewing. Especially in the innate immune response, the immune network mediated by microglia was regarded as the most important component of ANSCs niche.Microglia is also called macrophages settled in the substantial brain, which is originated from mesoderm. Unlike other classic glial cells originated from ectoderm, it is derived from the mononuclear cell which invaded the developing brain. In recent years, Professor Yuan Lin proposed a serial of hypotheses that the human body has the supporting-storing system and self monitoring system. It holds that after the mesenchyme of mesoderm differentiated into various organs and systems, it leaves a part to form the fascia connective tissue network throughout the body. The network is centered on stem cells, providing support and store for the regeneration of other functions cells, which is called supporting-storing system. Participated with the activity of nervous system and immune system, it maintains the stability and normal function of internal environment, forming the self-monitoring system. Microglia is support cell and niche cell for ANSCs. It is dominant in neuroimmune network, interacts with ANSCs, maintaining self-renewal of the ANSCs and determining the signal of regulating and controlling neurogenesis. This kind of microglia exists in the niche of ANSCs, forming microglia immune network which interacts with ANSCs. It’s lack of classic M1microglia phenotype but possesses unique characteristics of M2microglia phenotypes (promoting neurogenesis phenotype). As support cell and niche cellof ANSCs, it maintains the homeostasis and self-renewal of the brain.When ischemia damages the brain, microglia is the earliest involved in inflammation. In recent years, it has been the most intense debating topic in neuroscience that whether microglia plays the promoting neurogenesis/neuroprotec-tion phenotype role or neurotoxic phenotype role in the brain ischemia damage. As is well-known, microglia under different conditions plays dual role of neuroprotection or neurotoxin. The exact role is depended on the activated status and functional phenotype. Although former research has shown that microglia is involved in suppressing neurogenesis, underlying mechanism is mediated by proinflammatory factors and chemokine produce like IL-β1, TNF alpha, IL-6, IL-18, monocytes chemotaxis protein1and all kinds of hydrogen and nitrogen reaction. But now more and more evidences show that microglia is contributed to neurogenesis of different stages as proneurogenesis phenotype in certain physiological and pathological condition. It can promote the neurogenesis by expressing major histocompatibility complex Ⅱ antigen, IFN-γ, IL-4, TGF-(31and produce neurotrophic factor IGF-1, NGF, BDNF, NT-3, and GDN.Neurogenesis exsits in SVZ and DG zone all the time before and after birth of neonatal rat, which reaches to the peak7days after birth. With the time goes on, ability of neurogenesis proliferation is getting weak. However, it grows again after the hypoxic ischemic brain damage. The mechanism is not clear whether the apoptosis of new neurons and ischemic damage can activate the ANSCs or not after birth. Some researchers thought that apoptosis of new neurons is caused by insufficiency support of neurotrophic factors in neurogenesis zone, however, pathological damage can upregulate the expression of neurotrophic factor, which can increase the neurogenesis. In this experiment, we established the transient ischemic model with bilateral common carotid artery occlusion (BCCAO) for10minutes in7-day neonatal rat, to induce neurogenesis in DG and SVZ. We sought to observe the ultrastructural changes of microglia in DG area after early ischemia, and to observe the change of proliferation neurotrophic factor IGF-1and ANSCs in DG and SVZ after ischemia at different time points. Furthermore, we used the IGF-1receptor blockers, blocking the expression of IGF-1, to observe the change of ANSCs proliferation in DG and SVZ, which may provide a new strategy to clinical treatment on cerebral vascular disease.ObjectiveTo study the effect of IGF-1mediated by activation of microglia to ANSCs proliferation by transient forebrain ischemic induced neurogenesis in postnatal day7rats, which may provide new targets for endogenous repair of ANSCs and cell replacement therapy in brain ischemia damage and neurodegenerative diseases. It can also provide new theory of supporting-storing system with experimental base.1. To observe the early ultrastructural change of microglia in the DG after ischemia injury by transmission electron microscope (TEM).2. To detect the expression of neurotrophic factor IGF-1in DG and SVZ of neonatal rat after ischemia injury at different time points with immunohistochemical method.3. To detect the proliferation of ANSCs of ischemia injury in DG and SVZ of neonatal rat with immunohistochemical method at different time points.4. Block the expression of IGF-1with IGF-1receptor blockers (JB1), detect the expression of neurotrophic factor IGF-1in DG and SVZ of neonatal rat after ischemia injury with immunohistochemical method. 5. After IGF-1receptor blockers (JB1) blocked the expression of IGF-1, detect the proliferation of ANSCs in DG and SVZ of neonatal rat after ischemia injury with immunohistochemical method.Methods1. Make transient forebrain ischemic model with postatal7rats, separating subcutaneous tissue, common carotid artery and the vagus nerve, clipping the bilateral common carotid artery for10min with artery clip, as transient forebrain ischemic model (BCCAO). Operating the same procedure but not clipping artery as sham group (SS).2.70postatal7rats were randomly divided into ischemia (BCCAO) group (n=27), sham (SS) group (n=27), ischemia plus antagonist (BCCAO+JB1) group (n=8) and ischemia plus saline (BCCAO+SS) group (n=8). Continue subcutaneous injecting IGF-1receptor blockers (JB1) for7days after ischemia as (BCCAO+JB1) groups, the same procedure but to inject same volume of saline as (BCCAO+SS) group.3. The ultrastructure of microglia in the DG of BCCAO group (n=3) and SS group (n=3) was observed on the next day of ischemia with transmission electron microscope (TEM).4. The cell proliferation and IGF-1in the DG and SVZ of BCCAO group (n=24) and SS group (n=24) were observed on the1,4, or7days after ischemia with immunohistochemical staining.(subcutaneous inject BrdU mark on proliferation of ANSCs at6h,4h and2h before infusion for ischemia).5. The cell proliferation and IGF-1in the DG and SVZ of BCCAO+JB1group (n=8) and BCCAO+SS group (n=8) were detected after7days of continuous treatment, with JB1or not.(subcutaneous injection of BrdU mark of proliferation of ANSCs at6h,4h and2h before infusion for ischemia after7days).Results1. The rodlike structure of resting microglia in DG in SS group changed into activated serrated structure early in BCCAO group after ischemia in neonatal rat. 2. The number of BrdU+cells and IGF-1+cells of BCCAO group significantly increased in the DG and SVZ1,4, or7days after ischemia compared with that of the same day in SS group respectively (P<0.05)3. After the administration of JB1, the IGF-1expression was blocked, and the IGF-1+cells in the DG and SVZ were absent in BCCAO+JB1group, while the IGF-1in the DG and SVZ expressed normally in BCCAO+SS group.4. The number of BrdU+cells in the DG and SVZ in BCCAO+JB1group sharply decreased compared with BCCAO+SS group (P<0.05)7days after ischemia.ConclusionsIschemia-reperfusion injury activated the immuno-neuro-endocrine network (INEN) in neonatal rat. The first activated is the innate immune cells of the central nervous system that is also called microglia, especially in the hippocampus which is sensitive to ischemia. This experiment shows that a great quantity of microglia exsit in the specific zone of DG1day after ischemia. The morphology changes from the rodlike structure of resting microglia state into activated serrated structure, and it is lack in lysosome structure of classic Ml microglia phenotype. It shows that microglia is participated in neurogenesis in early ischemia. The change of microglia’s morphological structure explains that proneurogenesis is neuroprotection phenotype of microglia. The expression of IGF-1was upregulated by activated microglia of its own expression, or the interaction with astrocytes, neurons endotheliocyte or peripheral T cell that can contribute to proliferation of ANSCs in the DG and SVZ. The proliferation of ANSCs decreases dramatically after the usage of the JB1, suggesting that IGF-1mediated by microglia with proneurogenic phenotype contributes to the recovery of transient forebrain ischemia damage.In conclusion, immuno-neuro-endocrine network mediated by microglial has interaction with ANSCs, maintaining the stability of brain’s homeostasis and self-renew, which can give new ideas for clinical treatment of cerebrovascular diseases and central neurodegenerative diseases. It also provides experiment support for the supporting-storing system theory of fasciology. |
PDF Full Text Request