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Effects Of Intermittent High Glucose On Adult Rat Neural Stem Cells In Hypoxic-ischemic Injury As Well As The Expression Of Mitogen-activated Protein Kinase Signaling Pathway

Posted on:2013-10-03Degree:MasterType:Thesis
Country:ChinaCandidate:J ChenFull Text:PDF
GTID:2234330395961629Subject:Neurology
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In the field of basic and epidemic research, it has been confirmed that there were great internal relations between hypoxic-ischemic brain injury and abnormal glucose metabolism including diabetes mellitus, impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). Glucose is the most important source of energy for the brain, and hence when the glucose is too low or high, it may influence nerve function and result in brain injury. Both hypoglycemia and hyperglycemia are important risk factors for ischemic stroke. They are of equal importance in the prevention and treatment of stroke. Intensive control of glucose fluctuation may have the benefit of alleviating cerebral injury caused by ischemia reperfusion.Recent advances reveal that endogenic and transplanted neural stem cells (NSCs) can be activated by cerebral ischemia and partly take part in the regeneration of neural function. However, low survival and insufficient neuronal differentiation of NSCs within ischemic core and peri-infarct regions, hampers the efficacy of the therapy. It has proved that the glucose level is a crucial factor for self-renewal and multipoietic activity of NSCs following ischemia, whereas few studies have explored the effect of intermittent high glucose on the NSCs or progenitor cells following an oxygen glucose deprivation/reperfusion (OGD/R) insult. To the best of our knowledge, most previous researches focused on the effect of constant high glucose on proliferation, survival and apoptosis in neural stem cells, which revealed that moderate high glucose facilitated the NSCs survival following OGD/R. However, in clinical practice the glucose level in patients with stroke always frequently fluctuates, because the progress of acute stroke patients are very unstable. Therefore previous studies didn’t totally reflect the injury influence of intermittent high glucose in diabetes patients on neural stem cells. In this study, we tried to examine the effect of high glucose and intermittent high glucose on the survival and proliferation of in vitro ischemia-exposed NSCs, which partially mimic glucose fluctuations in patients with stroke. Experiment measures, such as CCK-8assay, EdU incorporation assay, immunostaining, flow cytometry and western blot were used in this study. We also further examined the regulatory effect of intermittent high glucose on the activation of mitogen-activated protein kinases (MAPKs) signaling molecules. These findings may have important implications for the intensive glycemic control in stroke patients, and provide a further understanding for the fate of NSCs following cerebral ischemia.Part I Establishment of rat model of hypoxic-ischemic injury in adult neural stem cells in vitroObjectives:To establish a simple, stable and reliable model of hypoxic-ischemic injury in adult neural stem cells in vitro, provide a tool for understanding the fate of NSCs following cerebral ischemia.Methods:1.Cell culure and identification:The ANSCs cell line from adult Fisher344rats were cultured in serum-free medium and identified using Nestin and DAPI immunofluorescent double staining.2.Methods to induce oxygen glucose deprivation/reoxgenation (OGD/R) of adult neural stem cell:ANSCs were washed with an Earle’s balanced salt solution without glucose for two times. Then were incubated for a different period (2,4,6,8and10h) in a tri-gas incubator with an atmosphere of1%O2,5%CO2and94%N2,98%humidity at37℃.And then these cells were removed from the anaerobic incubator, washed, and added DEME/F12containing bFGF supplement. A normoxic-normoglycemic control group was employed.3. Morphological assessment, viability assay and apoptosis assay: Morphological assessment of ANSCs was made by light microscopy after reoxgenation for24hours. CCK-8colorimetric method was used to determine the survival and proliferation of ANSCs, and flow cytometry was used to detect the apoptosis of ANSCs.Results:1. The great majority (94.6%) of cells expressed Nestin, indicating most cells keep in the stem and/or progenitor state.2. We found that the OD value and survival rate in the OGD cells increased as compared with those in control group after the setting of oxygen glucose deprivation for2hours. While the morphological damage of ANSCs aggravated gradually and the OD value decreased in OGD cells following the prolongation of times. Under the setting of oxygen glucose deprivation for6h,the OD value in OGD cells obviously decreased as compared with that in the control group(P<0.05). Under the setting of oxygen glucose deprivation for6h, the survival rate obviously decreased and the apoptosis rate significantly increased in OGD cells as compared with that in the control group (P<0.05).Under the setting of oxygen glucose deprivation for6h, the apoptosis rate of NSCs excessed to50%. Conclusions:1. We have established a simple, stable and reliable model of OGD/R of ANSCs in vitro successfully by means of tri-gas incubator and Earle’s balanced salt solution without glucose inducing oxygen glucose deprivation.2. Oxygen glucose deprivation for6hours may be suitable for inducing a model of OGD/R in NSCs in vitro.Part Ⅱ Effect of intermittent high glucose on proliferation of hypoxic adult neural stem cells(ANSCs) in vitroObjectives:To explore effects of constant or intermittent high glucose on viability and proliferation of ANSCs following in vitro ischemia.Methods:1. Methods to induce oxygen glucose deprivation/reoxgenation (OGD/R) of adult neural stem cell:We induced oxygen glucose deprivation/reoxgenation (OGD/R) of adult neural stem cell to mimic a hypoxic-ischemic injury insult.2.Groups:Different concentrations of D-glucose were added to the nutritive medium after1%O2for6h:normal control group containing17.5mmol/l glucose; constant high glucose H1group containing27.75mmol/l glucose; constant high glucose H2group containing41.75mmol/l glucose; Intermittent high glucose group containing17.5mmol/l glucose and alternating with41.75mmol/l glucose. Mannitol was used as a control to exclude a possible effect of osmolality on cell viability.3. Viability and proliferation assays:CCK-8colorimetric method was used to determine the viability of ANSCs, and EdU incorporation assay and flow cytometry were used to detect the proliferation of ANSCs.Results:1.Using CCK-8assay, we found that the OD value in all groups obviously increased in48hours as compared with that in the control group(P<0.05). In addition, the OD value of constant high glucose group H1was higher than all other three group after hypoxia for12hours. The OD value in all groups decreased obviously as compared with that in the control group following the prolongation of time.The OD value of intermittent high glucose group was lower than other three groups obviously (P<0.05).2. There was no significant difference between high glucose group and osmolality control groups.The influence of osmolality on cell viability was excluded.3. EdU uptake was reduced both in high glucose and constant high glucose treated cells, indicating less proliferation. We also used flow cytometry to determine the effect of intermittent high glucose on the cell cycle of NSCs after OGD. We found that PI and SPF significantly decreased in cultures exposed to high glucose compared with that in control group.Iintermittent high glucose treatment consistently inhibited DNA duplication and cell division of NSCs in vitro ischemia.Conclusions:1. Glucose supplement timely after hypoxia may protection NSCs from hypoxia-ischemic injury.2. Mild elevated glucose after hypoxia may protect NSCs against hypoxia. While higher elevated glucose or intermittent high glucose may exacerbate the hypoxia injury.3. Intermittent high glucose does more harm to NSCs in hypoxia-ischemic injury compared with constant high glucose.4. Both Intermittent high glucose and constant high glucose inhibits NSCs proliferation by delaying cell G1-S transition.Part Ⅲ The regulatory effect of intermittent high glucose on the activation of mitogen-activated protein kinases (MAPKs) signaling molecules Objectives:To examine the activation of three members of MAPKs pathway:extracellular signal regulated kinase1/2(ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38kinase (p38).Methods:1. Methods to induce oxygen glucose deprivation/reoxgenation (OGD/R) of adult neural stem cell:We induced oxygen glucose deprivation/reoxgenation (OGD/R) of adult neural stem cell to mimic a hypoxic-ischemic injury insult.2. Groups:Different concentrations of D-glucose were added to the nutritive medium after1%O2for6h. Western blot was used to examine the activation of three members of MAPKs pathway:extracellular signal regulated kinase1/2(ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38kinase (p38).Results:1. The total-ERK2, total-JNK2and total-p38were constantly expressed in NSCs and no significant change was found in all groups2. We found that the expression levels of P-ERK decreased at constant high glucose and intermittent high glucose group. While the expression of P-JNK and P-p38increased obviously as compared with that in the control group.Conclusions:Constant high glucose or intermittent high glucose inhibits the viability and proliferation of neural stem cells by activating JNK/p38MAPK and inhibiting ERK pathways.
Keywords/Search Tags:Intermittent high glucose, Neural stem cells, Hypoxia and ischemia, Proliferation, Mitogen-activated protein kinase signaling pathway(MAPK)
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