Sleep-restriction Inhibits Neurogenesis Through Decreasing The Infiltration Of CD169~+ Macrophages To Ischemic Brain After Stroke

Part Ⅰ To establish the effect of sleep-restriction on the infiltration of CD169~+ macrophages to the ischemic brain and post-stroke neurogenesis and functional recoveryObjective : To establish the effect of sleep-restriction(SR)on the infiltration of CD169~+ macrophages to the ischemic brain during stroke recovery as well as the effect on neural regeneration and functional recoveryMethods: Adult male C57BL/6 mice were subjected to 60-min transient middle cerebral artery occlusion followed by reperfusion for 28 days.The SR protocol was accomplished by depriving mice of sleep for 20 hours a day for 14 consecutive days starting at 14 days post-ischemia(dpi).We first assessed cell proliferation by treating MCAO mice with bromodeoxyuridine(Brd U)for three times with an 8-hour interval at 27 dpi.The number of Brd U+/DCX+ double positive cells in the ischemic lateral subventricular zone(SVZ)and striatum was analyzed 16 hours after the last injection of Brd U.Mice were sacrificed at 28 dpi to measure the number of CD169~+macrophages in the lateral ventricle CP,the third ventricle CP and cerebrospinal fluid(CSF)by immunofluorescence staining and flow cytometry.The functional tests were performed at 28 dpi,including Pole test,Elevated body swing test and Rotarod test.Results: SR during stroke recovery significantly decreased the number of Brd U+/DCX+ cells in the SVZ and striatum at 28 dpi,compared to that in non-SR MCAO group.Immunofluorescence and flow cytometry analysis showed that the number of CD169~+ macrophages in lateral ventricle CP and the third ventricle CP and CSF was significantly decreased in SR mice at 28 dpi,compared to that in non-SR MCAO group.SR also significantly decreased the number of CD169~+ macrophages in the ischemic brain parenchyma at 28 dpi.Functional analysis results showed that SR significantly increased the times to turn and to reach the floor on the pole test,reduced behavioral asymmetries on the EBST and decreased times until drop on the Rotarod test at 28 dpi.Conclusion: Sleep restriction can significantly reduce the number of CD169~+macrophages in the lateral ventricle CP and the third ventricle CP and the ischemic brain parenchyma,and thus inhibit neural regeneration and neural differentiation during ischemic stroke recovery.Part Ⅱ To elucidate the route and mechanisms of CD169~+macrophages migration to the ischemic brain parenchyma during stroke recoveryObjective: To elucidate the route and mechanisms of CD169~+ macrophages migration to the ischemic brain parenchyma during ischemic stroke recovery.Methods: Adult male C57BL/6 mice were subjected to 60-min transient middle cerebral artery occlusion followed by reperfusion for 28 days.The SR protocol was accomplished by depriving mice of sleep for 20 hours a day for 14 consecutive days starting at 14 days post-ischemia(dpi).At 14 dpi,a cannula was inserted to the left lateral ventricle for i.c.v.injection.The neutralizing antibody to IFN-γ,CX3CL1 or ICAM-1 or control Ab was injected slowly into the left lateral ventricle starting at 1dpi and 7 dpi of sleep-restriction.Mice were sacrificed at 28 dpi to measure the expression of IFN-γ,IFN-γR,CX3CL1,ICAM-1 by western blotting.The number of CD169~+ macrophages in the ischemic lateral ventricle CP,third ventricle CP and cerebral parenchyma(CSF)was measured by immunofluorescence staining.Furthermore,we assessed cell proliferation by treating MCAO mice three times with bromodeoxyuridine(Brd U)with an 8-hour interval at 27 dpi.The number of Brd U+/DCX+ double positive cells in the ischemic lateral subventricular zone(SVZ)and striatum was analyzed 16 hours after the last injection of Brd U.The functional tests were performed at 28 dpi,including Pole test,Elevated body swing test and Rotarod test.Results: SR during stroke recovery significantly decreased the protein expression of IFN-γ,IFN-γR,CX3CL1 and ICAM-1 in the CP at 28 dpi,without significant effects on VCAM-1 expression,compared to non-SR MCAO group.Furthermore,Immunofluorescence results showed that the number of CD169~+ macrophages in the ischemic lateral ventricle CP,third ventricle CP and CSF was significantly decreased after treatment with a neutralizing antibody to IFN-γ,CX3CL1 or ICAM-1 in SR MCAO group at 28 dpi.SR significantly decreased the number of Brd U+/DCX+double positive cells in the SVZ and striatum,compared to that in SR +MCAO group.Functional analysis results showed that the times to turn and to reach the floor on the pole test,and the times until drop on the Rotarod test at 28 dpi were all significantly decreased after blockade of IFN-γ,CX3CL1 or ICAM-1.Conclusion: SR inhibited the infiltration of CD169~+ macrophages from CP and CSF into ischemic lateral cerebral parenchyma through inhibiting IFN-γ/IFN-γR signaling-mediated modulation of chemokines(CX3CL1 and ICAM-1)in the CP.Part Ⅲ To explore the effect of sleep-restriction on Tregs after inhibiting the infiltration of CD169~+ macrophages into the ischemic brain parenchyma after strokeObjective: To explore the effects of sleep-restriction on the percentage of regulatory T cells(Tregs)by reducing CD169~+ macrophages to the ischemic lateral parenchyma,and post-stroke neurogenesis and functional recovery after stroke.Methods: Adult male C57BL/6 mice were subjected to 60-min transient middle cerebral artery occlusion followed by reperfusion for 28 days.The SR protocol was accomplished by depriving mice of sleep for 20 hours a day for 14 consecutive days starting at 14 days post-ischemia(dpi).At 14 dpi,a cannula was inserted to the left lateral ventricle for i.c.v.injection.Splenic Kaede green+ CD169~+ macrophages cells sorted from Kaede transgenic mice by fluorescence-activated cell sorting were directly administered into the cerebral parenchyma(CSF)of C57BL/6 mice at 14 dpi and 21 dpi,then we detected Kaede green+ CD169~+ macrophages cells in the ischemic brain parenchyma at 22 dpi by flow cytometry.The neutralizing antibody(anti-IFN-γ,anti-CX3CL1 or anti-ICAM-1)or Ig G1 isotype control antibody was administered 1and 7 days after SR.Flow cytometry was used to detect the percentage of Tregs in the ischemic lateral cerebral parenchyma at 28 dpi.Results: Flow cytometry analysis showed that Kaede green+CD169~+ macrophages cells were detected in the ischemic brain parenchyma at 22 dpi,indicating that CD169~+ macrophages could migrate from CSF to ischemic brain parenchyma.Compared to non-SR MCAO group,the percentage of CD4+CD25+Foxp3+ Tregs in the ischemic brain parenchyma of SR MCAO group was significantly decreased at 28 dpi.Furthermore,blockade of IFN-γ,CX3CL1 or ICAM-1 with their respective neutralizing Abs significantly decreased the percentage of CD4+CD25+Foxp3+ Tregs in the ischemic brain parenchyma of SR MCAO group.Exogenous administration of Kaede green+CD169~+ macrophages cells significantly increase CD4+CD25+Foxp3+Tregs cells in the ischemic brain parenchyma.Conclusion: The infiltration of CD169~+ macrophages cells into the ischemic lateral cerebral parenchyma could recruit Tregs into ischemic brain during stroke recovery.Sleep restriction inhibited this recruitment of Tregs cells and thus inhibit neurogenesis and functional recovery.