| Objective: The present study with cannabinoid receptor 2(CB2R) agonist and CB2 R knockout mice(CB2RKO) is to explore the crucial role of CB2 R on neuroinflammation and cognitive impairment induced by ?-amyloid protein(A?) in Alzheimer's disease(AD) model mice and to provide more experimental basis for CB2R-targeted anti-inflammation treatment for neurodegenerative diseases including AD.Methods:1 Behavior experiments6- or 8-month APP/PS1 transgenic AD model mice, at early or middle symptomatic stage, were treated with selective CB2 R agonist JWH-015 intraperitoneally at a dose of 0.5mg/kg/day for 8 weeks. Then novel object recognition test(NOR) and Morris water maze(MWM) were used to explore the effects of JWH-015 on non-spatial and spatial cognitive performance of 8-or 10-month APP/PS1 mice. CB2 RKO mice with bilateral intrahippocampal injection of A?1-42(CB2RKO mice/AD model) were used to confirm whether the above effect of JWH-015 was mediated by CB2 R and to evaluate the effects of CB2 R deficiency on non-spatial cognitive performance in AD model mice.2 A? depositionAt the end of behavioral experiments, Congo red staining was used to observe the effects of JWH-015 on cortical and hippocampal A?-contained plaque number in APP/PS1 mice.3 Glial cell immunoreactivityImmunofluorescence experiment was used to test the effects of JWH-015 and CB2 R deficiency on immunofluorescence intensity of microglia marker ionized calcium-binding receptor adapter molecular 1(Iba1) and astrocyte marker glial fibrillary acidic protein(GFAP) in cortex and hippocampus of AD model mice.4 Microglia functional phenotypeQuantitative RT-PCR test was applied to detect the effects of JWH-015 and CB2 R deficiency on mRNA expression of M1 microglia phenotype markers(IL-6, TNF-?, iNOS), M2 microglia marker chitinase-3 like protein(Ym1/2) and CB2 R in prefrontal cortex(PFC) and hippocampus of AD model mice.5 Dendritic complexityGolgi-Cox staining was applied to detect the effects of JWH-015 on dendritic length, branching point and spine density of pyramidal neurons in PFC and hippocampal dentate gyrus(DG) of APP/PS1 mice;6 NeurogenesisProliferating neural stem cells were labeled by bromodeoxyuridine(BrdU), and then immunohistochemistry was applied to evaluate the effects of JWH-015 on hippocampal BrdU positive cell number of APP/PS1 mice.Results: 1 The effects of CB2 R on non-spatial cognitive performanceCompared with control littermates, there were a dramatically decrease of novel object recognition index in 8- and 10-month APP/PS1 mice(P<0.001, P<0.05); And administration of JWH-015 could increase their novel object recognition index significantly(P<0.001, P<0.05); To confirm whether the beneficial effect of JWH-015 was mediated by CB2 R and test the effect of CB2 R deficiency on non-spatial cognitive ability, we carried out novel object recognition test in CB2 RKO mice/AD model. Results showed that with respect to CB2 RKO mice/control group, CB2 RKO mice/AD model had a lower recognition index, which was not altered by administration of JWH-015. It indicates that specific activation of CB2 R by JWH-015 can improve the PFC-dependent non-spatial cognitive ability of AD model mouse. Moreover, compared with WT mice/AD model, recognition index deficit in CB2 RKO mice/AD model was significantly enhanced(P<0.05), suggesting that CB2 R deficiency deteriorates non-spatial cognitive impairment of AD model mice. All of these findings make out that CB2 R plays a crucial regulative role on non-spatial performance in AD model mice.The effects of CB2 R on spatial cognitive performanceIn MWM, both of 8- and 10-month APP/PS1 mice had a longer escape latency(P<0.05, P<0.05)and less crossing platform numbers(P<0.05, P<0.05)than their control littermates, suggesting there is a spatial cognitive impairment in AD model mice; JWH-015 treatment could reverse escape latency of 8-month APP/PS1 mice completely(P<0.05); However, the escape latency of 10-month APP/PS1 mice and times across platform of 8- or 10-month APP/PS1 mice were unaltered by JWH-015 treatment, which indicates that the beneficial effect of JWH-015 on hippocampus-dependent spatial memory is more pronounced in AD model mice at early symptomatic stage rather than middle symptomatic stage. All of these behavior experiments support that the effect of CB2 R on spatial cognitive performance is more apparent than that of non-spatial cognitive performance. To make sure whether the impacts of CB2 R on spatial and non-spatial cognitive ability were related with A?-related pathological changes in affected brain regions, we made some mechanism explorations as followed.2 The effects of CB2 R on A? plaque burdenThere were age-dependent increasing of plaques numbers in cortex and hippocampus of 8- and 10-month APP/PS1 mice, and no plaques were observed in WT mice; Compared with APP/PS1 mice, administration of JWH-015 failed to modify cortical and hippocampal plaque number, suggesting that activation of CB2 R by JWH-015 has no influence on A? deposition of AD model mice.3 The effects of CB2 R on glial cell immunoreactivityIn comparison with control littermates, immunoreactivity of Iba1 and GFAP were significantly increased in cortex and hippocampus area of 8- and 10-month APP/PS1 mice(P<0.05); And JWH-015 treatment significantly reduced cortical but not hippocampal Iba1 immunofluorescence intensity of 8- and 10-month APP/PS1 mice(P<0.05, P<0.01); To confirm whether the regulative role on Iba1 immunoreactivity of JWH-015 was mediated by CB2 R and observe the effects of CB2 R deficiency on microglia immunoreactivity, we detected the immunofluorescence intensity of Iba1 in CB2 RKO mice/AD model. Compared with CB2RKO/control group, immunofluorescence intensity of Iba1 in CB2 KO mice/AD model was significantly elevated(P<0.05),which was unaltered by JWH-015 treatment. All of above support that specific activation of CB2 R by JWH-015 is effective to decrease microglial immunoreactivity of AD model mice. In contrast to WT mice /AD model, there was an elevated tendency of Iba1 immunofluorescence intensity in CB2 RKO mice/AD model; In addition, microglia in CB2 RKO mice/AD model exhibited a relative bigger round soma with numerous thicker and shorter branching than microglia in WT mice/AD model. And there was no obvious difference of GFAP immunofluorescence intensity between groups in AD model mice with intrahippocampal injection of A?1-42, indicating that the cognitive impairment of AD model mice may not be tightly linked to astroglia immunoreactivity. The results of immunofluorescence test showed that CB2 R exerts a vital regulative role on microglia immunoreactivity and morphology in AD model mice. To explore whether altered reactive microglia morphology was accompanied by functional changes, we observed the mRNA expression of M1/M2 microglia phenotype markers.4 The effects of CB2 R on M1/M2 microglia phenotype transformationWith respect to control littermates, mRNA expression levels of M1 microglia phenotype marker IL-6 and TNF-a were significantly increased in PFC and hippocampus of 8- and 10-month APP/PS1 mice(P<0.05); In contrast to APP/PS1 mice, JWH-015 treatment could down-regulate IL-6 and TNF-a mRNA expression, potentiate M2 microglia phenotype marker Ym1/2 mRNA expression, and these above effects of JWH-015 were more apparent in PFC than hippocampus; These results suggest that activation of CB2 R by JWH-015 could promote microglia functional phenotype transformation in different brain regions of AD model mice. To make sure whether these above effects of JWH-015 were mediated by CB2 R and explore the effect of CB2 R deficiency on microglia functional phenotype transformation, we observed mRNA expression level of M1/M2 microglia phenotype markers in CB2 RKO mice/AD model. Compared with CB2 RKO mice/control group, mRNA expression of IL-6, TNF-? and iNOS were significantly up-regulated(P<0.001, P<0.01, P=0.0507) and Ym1/2 mRNA expression was significantly down-regulated(P<0.05) in CB2 RKO mice/AD model, none of which was altered by JWH-015 treatment. It proves that specific activation of CB2 R by JWH-015 contributes to microglia phenotype transformation in AD model mice. Meanwhile, compared with WT mice/AD model, mRNA expression levels of IL-6 and TNF-? in CB2 RKO mice/AD model were more pronounced(P<0.01, P<0.05), hinting that CB2 R deficiency promotes the expression of M1 microglia phenotype in AD model mice. Compared with control littermates, CB2 R mRNA expression in PFC and hippocampus of 8- and 10-month APP/PS1 mice were significantly increased(P<0.05), none of which was altered by JWH-015 treatment. Based on above results, it concludes that CB2 R takes a significant part in regulation of microglial immunoreactivity, reactive morphology and functional phenotype in different brain regions of AD model mice, which is closely related with its ameliorative effect on cognitive deficit.5 The effects of CB2 R on dendritic complexityCompared with WT littermates, there were significant reductions of pyramidal neuronal dendritic length, branching points, spine density in PFC and DG of 8- and 10-month APP/PS1 mice(P<0.001), which indicates the dendritic complexity changes in different brain regions of AD model mice is related with its cognitive impairment. Compared with APP/PS1 mice, JWH-015 treatment can significantly increase dendritic length, branching points, spine density of PFC(P<0.001) but not spine density of DG zone, which indicates that activation of CB2 R exerts a region-dependent ameliorative effect on dendritic complexity in AD model mice; And the mechanism of these effects appear to be related with regulation of neuroinflammation in affected brain regions, but need more explorations.6 The effects of CB2 R on neurogenesisCompared with control littermates,hippocampal BrdU positive cell number of 10-month rather than 8-month APP/PS1 mice was decreased significantly(P<0.05), suggesting there is age-dependent decline of neurogenesis of AD model mice; And JWH-015 treatment counteracted the decreasing of hippocampal BrdU positive cell number of 10-month APP/PS1 mice(P<0.05), which indicates that activation of CB2 R could promote neurogenesis of 10-month APP/PS1 mice; and these effects may also be related with modulatory role of CB2 R on inflammation microenvironment in hippocampus of AD model mice.Conclusions:Specific activation of CB2 R can reverse non-spatial cognitive impairment of AD model mice, which is related with the regulation of microglia-mediated neuroinflammation as well as restoration of neurogenesis and dendrite complexity in affected brain regions;Deletion of CB2 R can deteriorate non-spatial cognitive deficit and notably modify microglia morphological and functional phenotype in AD model mice; And all of these support that CB2 R takes a significant effect on AD progressive neuoinflammation and cognitive competence and exerts a high potential as an anti-inflammation target. |
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