| Parkinson’s disease(PD) is a common progressive and neurodegenerativedisease in central nervous system.The primary pathology of PD is selective lossof dopaminergic neurons in the substantia nigra pars compacta.Previous studiesmainly focused on the mechanisms of dopaminergic neurona apoptosis.Axonaldegeneration of dopaminergic neurons was thought to be accompanied withneurons’ loss for a long time.Recently,some findings have demonstrated thatsimply inhibiting process of dopaminergic neurons apotosis could not postponethe onset and development of PD efficiently. Dopaminergic axonal degenerationmight be a key feature and target for PD.Although we have been graduallyconvinced of the important role of axonal degeneration in PD, the mechanismsand the role of axonal degeneration remain unknown. And,we have little specialmethods to treat the axonal degeneration in PD.So the studying of mechanismsand the role of axonal degeneration in PD will not only bring us new theories,butalso bring us new strategies and target to treat PD.Part1. Establishment of a new cell model of dopaminergic axonal degeneration for Parkinson’s disease in vitroObjective To investigate dopaminergic axonal degeneration induced by MPP~+in mouse ventral mesencephalic culture and establish a new cell model for PDresearch in vitro. Methods C57BL/6mouse embroys of14d were used to makeventral mesencephalic dissociated culture. And it was divided into the control andMPP~+-treated groups. Different final concentrations (0.1,0.5,1.0and10.0μM) ofMPP~+were added into the MPP~+-treated groups on the7th day and then incubated.By application of anti-tyrosine hydroxylase (TH) monoclone antibodyimmunostaining, we observed morphology,axons length,axons number andamount of dopaminergic neurons at the time of2,4,6,8,12,24h. TUNEL was usedto identify the apoptotic dopaminergic neurons. Results Followed by MPP~+induced injury, TH-ir cells decreased and morphology changed obviously.Although most cell bodies existed, axons length and number decreased. Cellularinterlaced axons were sparse and unsmooth with a string of beads swelling orsegments. A few of TH-ir cells showed axon existed but cell body loss. Thesephenomena including TH-ir cells,axons length and number decreased,which weredependent on dose and time of MPP~+-treated. We found that decrease of cellamounts,axons length and number by10.0μM MPP~+for24h had statisticalsignificance. At this time, TH-ir cells, axons length and number was (254±11)/perwell,(158.99±12.13)μm/per cell,(1.82±0.30)/per cell in control groups,and(126±16)/per well,(64.18±19.06) μm/per cell,(1.17±0.35)/per cell in MPP~+-treated groups. TH-ir cells, axons length and number had decreased50.39%,59.8%,59.8%. Amount of apototic TH-ir cell accounted for90.7%in all losscells,which indicated that apotosis was the main mode of cells loss in thisexperiment system. Conclusion MPP~+with different final concentrations(0.1,0.5,1.0and10.0μM) could cause injury related morphological changs to TH-ir cells,axon length and number.It was the best method that10.0μM MPP~+treated dopaminergic neurons for24h for the model.Part2. Identified dopaminergic axonal degeneration in Parkinson’s diseaseObjective To study axon degeneration of MPP~+-treated dopaminergic neurons inPD by detecting amyloid precursor protein (APP)and microtube proteinβ-Tubulin Ⅲ to observe the fact ofaxonal transport defect and microtubedisassembled. Methods It was divided into control and MPP~+-treated groups.MPP~+with final concentration(10.0μM) were added into the MPP~+-treated groupson the7th day,and incubated for24h.Control groups were treated by nothing.Double staining with TH and APP immunofluorescence detected the expressionof APP in dopaminergic neurons. To detect the expression of β-TubulinⅢ in cellextracts, MPP~+-treated groups were fixed with4%paraformaldehyde whichcontained dehydranter (0.1M PB,10mM EGTA,2mM MgCl2,0.2%Triton X-100)to remove dissociative and left polymerization microtube protein.Controlgroups were fixed with4%paraformaldehyde only. Results TH-ir cells withlow expression of APP were normal in control groups. TH-ir axons with highexpression of APP,and APP local accumulation were segments in MPP~+-treatedgroups. Western-blot analysis showed polymerization microtube proteinβ-TubulinⅢ in cell extracts that mean density ratio of MPP~+-treated groups was4.36±0.3,and control groups was10.08±0.6, β-Tubulin Ⅲ expressionin cellextracts of MPP~+-treated groups was decreased56.75%than controlgroups.Conclusion10.0μM MPP~+treated for24h caused dopamineric axondegeneraton, axonal transport defect and microtube disassembled.Part3. Exploring dopaminergic axonal degeneration associated signalpathways in Parkinson’s diseaseObjective To explore dopaminergic axonal degeneration associated signal pathways in PD.Methods Final concentrations of GSK-3β inhibitor lithiumchloride(5、10、20、30μM) and ROCK inhibitor Fasudil (25、50、100、150μM)wereadministrated to the cell model that we had established. It was divided intocontrol and inhibitor-treated groups.10.0μMMPP~+treated the control groups onthe7th day,and incubated for24h.10.0μΜMPP~+and various final concentrationsLiCl or Fasudil were added into inhibitor-treated groups cell culture on the7thday, and incubated for24h. TH immunofluorescence staining was used toobserve axons length and axons number of TH-ir cells.Based on the results of thisexperiment, we chose suitable inhibitor and its final concentrations which hadobvious effects to analyse polymerization β-Tubulin Ⅲexpression in cell extractsof dehydranter(described above) treated control and inhibitor-treated groups.Results①Axon length of control groups was(88.45±17.12)μm, and that of5、10、20、30μM LiCl-treated was (119.03±25.19),(115.92±20.10),(97.21±15.58),(88.31±16.26) μm。 Comparing to the control groups,there were statisticalsignificance at5and10μM LiCl-treated groups,but were not at20and30μMLiCl-treated groups. Axons number of control groups was (1.56±0.32), and of5,10,20,30μM LiCl-treated groups was (1.75±0.41),(1.66±0.33),(1.65±0.3),(1.64±0.38). Comparing to the control groups, there were no statisticalsignificance at various LiCl-treated groups.②A xons length of controlgroups was(88.45±17.12)μm, and of25,50,100,150μM Fasudil-treated groups was(95.97±20.67),(118.30±19.11),(132.39±26.95),(87.01±25.89)μm. Comparing tocontrol groups,there were statistical significance at50and100μM Fasudil-treatedgroups,but were no at25and150μM Fasudil-treated groups. Axons number ofcontrol groups was (1.55±0.29), and of25,50,100,150μM Fasudil-treated groupswas (1.69±0.43),(1.65±0.35),(1.53±0.38),(1.57±0.50). Comparing to controlgroups, there were no statistical significance at various Fasudil-treated groups.③Western-blot analysis showed mean density ratio of β-TubulinⅢ in cellextracts in5μM,10μM LiCl-treated groups was5.68±0.29and5.35±0.22, controlgroups was3.56±0.14. Comparing to control groups,there were statisticalsignificance at5and10μM LiCl-treated groups,but were no at20and30μMLiCl-treated groups. β-Tubulin Ⅲ expression in cell extracts inceasedat5μM and10μM LiCl-treated.④Western-blot analysis showed mean density ratio ofβ-Tubulin Ⅲ in cell extracts in50,100μM Fasudil-treated groups was4.61±0.32and4.45±0.21, control groups was2.76±0.16. Comparing to control groups,therewere statistical significance at50and100μM Fasudil-treated groups,but were noat25and150μM Fasudil-treated groups. β-Tubulin Ⅲexpression in cell extractsinceased at50μM and100μM Fasudil-treated. Conclusion PI3-kinase/Akt/GSK-3β and Rho/ROCK signal pathways associated with dopaminergic axonaldegeneration in PD.Suitable concentrations of LiCl and Fasudil could protectdegenerate dopaminergic axon by its inhibitory effects on signal pathway. |
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