The Study Of Activated Glial α7-nAChR On The Inhibition Of β Amyloid Mediated Neural Stem Cell Disability

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive impairment of congnitive function and affective disorder. The AD morbidity shows a fast increase in recent years and the amount of AD patients will increase to115.4million in2050. However, there is no effective drug or method to prevent and treat AD so far.There are many hypotheses about the pathogenesis of AD, and the inflammation hypothesis has aroused much attention. The accumulated Aβ activate glial cells and induce inflammation, which cause the inhibition and apoptosis of nerve cells. In the central nervous system, the microglia and astrocyte secrete a lot of cytokine, chemokin and complement, which induce inflammatory cells infiltration and chronic inflammation. The IL-1, IL-6, TNF-a, MCP-la secreted by microglia damage nerve cells and induce the apoptosis of neurons. The NO and chemokine secreted by astrocyte induce the apoptosis of neuron and assemble microglial cells around Aβ plaque, which leading to the microglia secretes more inflammatory mediators in return. Therefore, the Aβ-mediated chronic inflammation arouse neurons apoptosis and leading to neurodegenerative disorders. Study reports that the inherent neural stem cells (NSCs) in the brain could differentiate to neurons and maintain the number of neurons in the adult. The neurogenesis of adult plays an important role in the maintaining of cognitive function, and the newborn neurons could improve cognitive and behavioural functions. The proliferation and differentiation of NSC in adult is based on a good microenvironment, the pro-inflammation cytokines, chemokines in the microenvironment all could inhibit neurogenesis. Therefore, only the CNS neuroinflammation of AD is weakened, can the inherent NSC proliferate and differentiate into nerve cells.There are reports suggesting that α7nicotinic acetylcholine receptor (a7-nAChR) activation could inhibit inflammation. The nAChR is a member of the ligand gated ion channel and widely expressed in peripheral nervous and CNS. We have confirmed that the microglia and astrocyte have the expression of a7-nAChR, there mainly take function in the transmission of cholinergic neurotransmitter and has a high affinity with P-amyloid (AP). Tracey found that macrophage a7-nAChR activation reduce the synthesis and secretion of inflammatory mediators. The microglia is macrophage-like cell in the CNS and the neuroinflammation might be weakened as a7-nAChR activation. In this study, the transwell co-culture system is build, in which the NSC and glial cells are cultured in the upper layer and lower layer respectively. The Aβ is added to activate the glial cells and to simulate the inflammation microenvironment of AD, and then, the liquichip technology is used to detect the cytokine and chemokine concentration, the flow cytometry is used to measure the NSC apoptotic rate. The nicotine is added to activate a7-nAChR to study the effects of microglial α7-nAChR activation on the inflammatory mediator secretion and on the protection of NSC. What’s more, the AD inflammation models are prepared by injection of Aβ1-42through stereotaxic technique. The rat glial cell α7-nAChR is activated by nicotine, and then the BrdU-labeled NSCs are injected to hippocampi. The survival and differentiation of transplanted NSC are observed with immunnofluorescence methods. Methods1. NSCs culture and identificationHippocampi were dissected from the cerebrum of new-born rats within24hours after birth, and the tissue was digested with trypsin and centrifuged. The cells were cultured with the culture medium, which contains EGF, bFGF, B27, after that, the cells were cultured in the flask. Nestin staining was used to identification of NSCs.2. Microglial cells culture and identificationThe hippocampi of newborn Sprague-Dawley rats were isolated for microglial cell cultures. The cells were suspended in10%FBS and plated at a density of5×105cells/ml onto poly-L-lysine-coated75cm2flasks. The culture medium was renewed2days after plating. The purified microglial cells were collected and suspended in10%FBS and then added to poly-L-lysine-coated24-well plates at a density of5×105cells/ml for identification or co-culture.OX42staining was used to identification of microglia.3. Astrocyte culture and identificationHippocampi were dissected from the cerebrum of new-born rats within24hours after birth, and the tissue was digested with trypsin and centrifuged. Subsequently, the supernatant was discarded, and dissociated cells were plated in culture flasks. After8-10days, the culture flasks were shaken at37℃for4hours in a rotator, and then the adherent cells were collected for further culture. To assess astrocytic purity, GFAP expression was detected.4. The building of co-culture system and the grouping of in vitro study.The NSC and glial cell were co-cultured in the upper and lower layer of transwell plate respectively, the ratio of NSC and microglia and astrocyte was1:1:1.A. control group (Astrocyte+Microglia+NSCs):the NSCs were cultured in the upper layer, and the microglia and astrocyte were culture in the lower layer. B. astrocyte group (Astrocyte+NSCs+Aβ1-42):the NSCs were cultured in the upper layer, and the astrocytes were cultured in the lower layer,10umol/L Aβ1-42were added to the astrocytes.C. microglia group (Microglia+NSCs+β1-42):the NSCs were cultured in the upper layer, and the microglial cells were cultured in the lower layer,10μmol/L Aβ1-42were added to the microglial cells.D. co-culture group (NSCs+Microglia+Astroycte+Aβ1-42):the NSCs were cultured in the upper layer, the microglial and astrocyte cells were cultured in the lower layer,10μmol/L Aβ1-42were added to the glial cells.E. a7-nAChR blocked group (NSCs+Microglia+Astrocyte+Aβ1-42+nicotine+bungatotoxin):the NSCs were cultured in the upper layer, the microglial and astrocyte cells were cultured in the lower layer, the bungatotoxin was used to block the glial a7-nAChR, then the nicotine was added to the glial cells1hour before the addition of10μmol/L Aβ1-42.F. a7-nAChR activated group (NSCs+Microglia+Astrocyte+Aβ1-42+nicotine):the NSCs were cultured in the upper layer, the microglial and astrocyte cells were cultured in the lower layer, the nicotine was added to the glial cells1hour before the addition of10μmol/L Aβ1-42.5. The determination of cytokines/chemokinesFollowing treatment of cultured glial cells with10μmol/L Aβ1-42for48hours, supernatants were collected to detect concentration of cytokines and chemokines. Cytokines/chemokines were measured simultaneously using a LiquiChip work station, which employed bead-based xMAP (flexible multi-analyte profiling) technology, according to manufacture instructions.6. Flow cytometry for analysis of apoptosisThe level of apoptosis in NSCs was measured using a Fluorescein FragELTM DNA Fragmentation Detection Kit according to the manufacturer’s instruction. After Aβ1-42treatment for48h, NSCs were collected and washed with PBS twice. Next,100μl Binding Buffer and10μl FITC-labeled Annexin-V (20μg/ml) were added to each sample, and the samples were incubated in the dark for30min; then,5μl PI was added. Cell apoptosis was analyzed using a FACScan flow cytometer (Becton-Dickinson, USA). The data were analyzed using CellQuest3.0software (Becton-Dickinson, USA). The negative controls were processed using the same procedure but without Annexin V-FITC and PI.7. Aβ1-42and NSCs injection.The Aβ1-42and NSCs were injected into hippocampi by stereotaxic technique.A. NSC group (NSC):the NSCs were injected into hippocampiB. Aβ group (Aβ):the Aβ1-42was injected into hippocampi.C. NSC inflammation model group (Aβ1-42+NSCs):the NSCs were injected into hippocampi after the Aβ1-42was injected.D. Nicotine group (Aβ1-42+NSCs+nicotine):the NSCs were injected into hippocampi after the Aβ1-42was injected, and nicotine was given by subcutaneous injection during the period.8. Immunofluorescence for analysis of the proliferation and differentiation of NSCs.The brain slices were fixed and incubated with a blocking buffer for2h. Next, brain slices were incubated with a primary antibody at4℃overnight, followed by incubation with a secondary antibody for90min, and staining with DAPI.9. ELISA for IL-6, MCP-1Ｐ, MIP-1α and TNF-a Quantifination.The concentration of IL-6, MCP-1α, MIP-1α and TNF-a in the hippocampi was quantified by ELISA kit. Samples were detected according to the manufacturer’s instruction. A standard curve was performed in duplicate, and the levels of IL-6, MCP-la, MIP-1α and TNF-a were evaluated by use of the standard curve as reference.10. Spontaneous alternation in the Y Maze.Immediate spatial working memory performances were assessed by recording spontaneous alternation behavior in a single-session Y-maze test. The maze apparatus consisted of three identical arms spaced at120°from each other and made of white plastic. During the test, each mouse was placed at the end of one arm and allowed to move freely in the entire maze for10min. An arm entry was recorded when a mouse moved all4feet into the arm. Measured parameters were the total number of arms entries and the sequence of entries into the three arms.11. Statistical analysisData were analyzed using SPSS16.0statistical software (SPSS, Chicago, IL, USA) and expressed as the mean±S.E.M. Statistical analysis was completed using a one-way ANOVA with Fisher’s LSD post hoc tests for multiple comparisons of the means. Differences were considered to be statistically significant with*P<0.05.Result1. The culture and identification of NSCs, microglia and astrocytes.The microglia, astrocyte and NSCs were identified by immunofluorescence. The microglial cells were CD11b/c positive and had a platode and orbicular-ovate body. The astrocytes were GFAP positive as the picture showed and the cells were bold and had many branches. The NSCs were nestin positive and had a bright cellular spheres. The purity of NSCs, microglia and astrocyte is over95%.2. Microglial and astrocytes cells α7-nAChR reduce the secretion of IL-6, MIP-1α and RANTES in the co-culture system in vitro study.With the stimulation of Aβ1-42for48h, microglia or astrocyte secreted numerous IL-6, MIP-1α and RANTES alone. In the co-culture group, the levels of IL-6, MIP-1α and RANTES were12.8,12.4,9.5times to the control group, however, it’s just3.7, 4.5,2.7times when the microglia and astrocyte a7-nAChR were activated by nicotine. To make sure that nicotine activated α7-nAChR and reduced the inflammatory factor levels, the bungarotoxin was used in this research. The results showed that the concentration of IL-6, MIP-la and RANTES increased to10.6,11.5and9.7times to the control group when the a7-nAChR was blocked. The results suggested that microglia and astrocyte had a combination effect in the Aβ-mediated inflammation. The inflammatory factor levels of co-culture group were significantly higher than the microglia or astrocyte group. Microglia and astrocyte α7-nAChR activation reduced inflammatory factors secretion and weakened inflammation.3. Microglia and astrocyte a7-nAChR activation protects NSCs.With the stimulation of Aβ,microglia and astrocyte secreted various kinds of inflammatory factors and induced NSCs apoptosis. The NSCs apoptotic rates of microglia group and astrocyte group were20.32%and21.06%, respectively. However, the apoptotic rate increased to33.34%when the microglia and astrocyte co-cultured. When nicotine was added to activate a7-nAChR, the NSCs apoptotic rate decreased to13.91%, compared with25.46%in the a7-nAChR blocked group.4. Microglia and astrocyte a7-nAChR activation reduce rat hippocampi IL-6, MCP-la, MIP-1α and TNF-α levels in vivo study.In the NSC group, the IL-6, MCP-la, MIP-1α and TNF-a concentration was31.4±7.0,67.6±12.1,132.8±9.9,64.8±6.8pg/mg, and in the Aβ group, the IL-6, MCP-la, MIP-1α and TNF-α levels raised significantly, were57.8±9.5,175.4±8.2,307.7±11.6,214.0±12.2pg/mg. In the NSC inflammation models group, the inflammatory mediator levels were also very high, however, when the rats were gave nicotine, the IL-6, MCP-la, MIP-1α and TNF-α concentration decreased to37.2±3.4,92.7±6.8,193.1±12.8and101.8±8.5pg/mg, compared with the NSC inflammation models group, the inflammatory mediators of nicotine group decreased significantly.5. Glial cells a7-nAChR activation improves the proliferation and differentiation of NSCs in AD inflammation models.Glial cells α7-nAChR activation improved the proliferation of NSCs, and increased the ratio of NSCs differentiated to neuron, cholinergic neuron and astrocyte, while the microglia ratio had no change.6. Glial cells a7-nAChR activation improves rat learning and remembering function.Y maze was used to detect the rat learning and remembering function, compared with the NSC inflammation models group, the learning and remembering function of nicotine group was significant improved.Conclusions1. The co-culture system of NSCs and astrocyte and microglial cells are building in this research.2. Aβ1-42activates glial cells and exacerbates neuroinflammation, which increases the NSCs apoptosis rate.3. Glial a7-nAChR activation decreases the inflammatory mediator levels and weakens Aβ1-42-mediated inflammation.4. Glial a7-nAChR activation weakens Aβ1-42-mediated inflammation on the inhibition of proliferation and differentiation of NSCs, and promotes the implanted NSCs differentiating to neurons, cholinergic neuron and astrocyte.5. Glial a7-nAChR activation and NSCs implant improve the learning and remembering function of AD rat models.