Intranasal LPS Induces Parkinson’s Model In Mice And Intervene In The Rho Kinase Target In This Model

Part Ⅰ Intranasal LPS induces Parkinson’s model in miceObjective:To investigate whether intranasal LPS could induce characteristic behavioral, pathological and neurochemical changes in PD in mice. MPTP served as a positive control in this study.Methods:10-month-old female C57BL/6mice were divided into saline control group, LPS group and MPTP group.10μL LPS (1mg/mL) and MPTP (40mg/mL) were slowly introduced into the right nasal cavity every other day for5months. Motor behaviour was analyzed in an open-field test during the1st,3rd and5th month. At the5th month, adhesive removal test (ART) was performed to evaluate the motor symmetry. Then the mice were sacrificed and the brain was removed for immunostaining and Western blot assay to determine the expression of tyrosine hydroxylase (TH), a-synuclein and choline acetyl transferase (CHAT). DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), DA/HVA, noradrenaline (NE), isoprenaline (Iso),5-hydroxytryptamine (5-HT) and5-hydroxyindoleacetic acid (5-HIAA) released from the striatum were detected using a HPLC-electrochemical detection system.Results:LPS, but not MPTP-administered mice developed a progressive reduction in ambulatory motor activity and obvious motor asymmetry. The mice developed severe loss of TH-immunoreactive (TH-ir) neurons, accumulation of a-synuclein in the right SN and reduction in TH expression and release of neurotransmitters including dopamine (DA) in the striatum5months after LPS and MPTP instillation compared with saline control and contralateral brain.Conclusion:Intranasal instillation of LPS induces Parkinson’s model in mice Part Ⅱ Mechanisms underlying intranasal LPS induces Parkinson’s model in miceObjective:To elaborate the mechanism how LPS induces Parkinson’s model in mice by in vivo and in vitro study.Methods:Splenic mononuclear cells (T cells) in the mice were seperated and were exposed to ectogenic a-synuclein. The systemic inflammation were examined at the5th month after LPS instillation. The brain of the mice was removed for immunostaining and Western blot assay to determine the expression of F4/80and p-NF-KB/p65expression in the substantia nigra (SN), the amount of NF-κB in the brain. Release of IL-1β、IL-6、TNF-a in the SN was determined by ELISA assay. The markers for M1phenotype and M2phenotype of microglia, iNOS and Arginase-1(Arg-1) were determined by Western blot analysis. BV-2cells were exposed to LPS in vitro for48hours, then the release of IL-1β, IL-6, TNF-a, NO into the supernatant (conditioned medium) was tested by ELISA assay. The expression of iNOS, NF-kB, AP-1and the substrates of Rho kinase MYPT and MLC were determined by Western blot analysis. The cell viability and LDH release were determined by MTT and LDH.Results:Splenic mononuclear cells did not respond to the ectogenic a-synuclein stimulation. NF-κB expression was strongly enhanced inside the brain at the5th month after LPS instillation. We also found F4/80and p-NF-KB/p65were greatly up-regulated in LPS-treated right SN compared with contralateral brain and/or saline control. TNF-a, IL-1β was also released from the same area in these mice while IL-6expression was not changed. The expression of Arg-1was decreased while the ratio of iNOS/Arg-1was obviously increased in LPS-treated right brain compared with contralateral brain and/or saline control. In vitro study indicated that LPS could activate BV-2cells to release inflammatory mediator and cytokines including NO, IL-1β, IL-6and TNF-a to the into the supernatant (conditioned medium).The expression of iNOS, NF-kB, AP-1and the phospho-MYPT and MLC were all increased in comparison to the saline control. The conditioned medium from BV-2cells exposed to LPS resulted in reduction in cell viavility and increase in LDH release in PC12cells.Conclusion:The in vivo study showed that intranasal LPS instillation did not influence systemic inflammatory and immune responses, while microglia were activated inside the brain in LPS-treated mice. The activated microglia were induced to the inflammatory M1phenotype, resulting in infalmmatory response. The in vitro study indicated that LPS could also activate BV-2cells to cause infalmmatory response and Rho kinase. The conditioned medium from BV-2cells exposed to LPS resulted in reduction in cell viavility and cell death in PC12cells. Part III Protective role and mechanism of Rho kinase inhibitor Fasudil in LPS-induced PD mice modelObjective:To assay the potential therapeutic effect of Rho kinase inhibitor Fasudil for LPS-induced PD mice model and the possible mechanism.Methods:8-10-week-old female C57BL/6mice were divided into saline control group, LPS group and Fasudil group.10μL LPS (lmg/mL) were slowly introduced into the right nasal cavity every other day for1month, simutaneously Fasudil (40mg/kg) was intraperitoneally injected in Fasudil group. Motor behaviour was analyzed in an open-field test after1month. ART was then performed to evaluate the motor symmetry. Then the mice were sacrificed and the brain was removed for immunostaining and Western blot assay to determine the expression of TH, a-synuclein in the SN and the olfactory bolb (OB), TH expression in the striatum.The expression of NF-κB and the markers for M1phenotype and M2phenotype of microglia, iNOS and Arginase-1(Arg-1) and the substrates of Rho kinase MYPT and MLC were investigated by Western blot analysis.Results:Intranasal LPS phosphalated the substrates of Rho kinase MYPT and MLC inside the brain, while Fasudil could greatly inhibit the phosphalation of MYPT and MLC. Fasudil alleviated intranasal LPS-induced a progressive reduction in ambulatory motor activity, severe loss of TH-immunoreactive (TH-ir) neurons, accumulation of a-synuclein in the right SN, reduction in TH expression in the striatum, and shift of microglia to M1phenotype to activate inflammatory response inside the brain.Conclusion:Fasudil alleviated the severity of PD changes in mice by inhibiting Rho kinase in brain and reversing inflammatory M1phenotype to anti-inflammatory M2phenotype microglia Conclusions1. Intranasal instillation of LPS induces Parkinson’s model in mice.2. Intranasal instillation of LPS induces PD model in mice possibly by inducing microglia to inflammatory Ml phenotype and activating Rho kinase in the brain.3. Fasudil could effctively intranasal instillation of LPS-induced activation of Rho kinase in the brain.4. Fasudil alleviated the behavoral and pathological changes of PD in mice by reversing inflammatory Ml phenotype to anti-inflammatory M2phenotype microglia and inhibiting Rho kinase in brain.