Fig. 6

a Bar graph shows the percentage area of TH+ positive staining in SNpc by comparing the injected ipsilateral to the untreated contralateral side in heterozygous nude rats at four timepoints (2 weeks, 4 weeks, 6 weeks, 8 weeks) injected with AAV9-GFP (n = 3) or AAV9-α-syn (n = 5). A significant difference (Two-way ANOVA p < 0.05; treatment: F (1, 24) = 243.8, p < 0.01; timepoint: F (3, 24) = 55.62, p < 0.01; post hoc analysis: Tukey’s multiple comparison test) of percentage positive cells was observed between the heterozygous nude rats injected with αsyn when compared to GFP injected controls at 4 (30 days), 6 (45 days), and 8 (60 days) weeks timepoint. A progressive loss of dopamine neurons was observed in heterozygous nude rats injected with AAV9-α-syn when compared at 4, 6, and 8 weeks timepoint. b Bar graph shows the area units of the α-syn oligomer expression by staining with the α-syn33 antibody (donation from Dr. Rakez Kayed [29]) of the heterozygous nude rats injected with AAV9-GFP (n = 3) or AAV9-α-syn (n = 5) at four different timepoints. At 2 weeks (14 days) α-syn expression begins to be observed and then increases further at 4 weeks (30 days). No significant difference was observed in the expression level of α-syn oligomers in the heterozygous nude rats treated with AAV9-α-syn normalized to the dopamine neurons in the SNpc region at 4, 6, and 8 weeks timepoint. c Bar graph shows the area units for Iba1 staining for microglia in heterozygous nude rats injected with either AAV9-GFP (n = 3) or AAV9-α-syn (n = 5) in the SNpc region at four different timepoint. Heterozygous nude rats injected with AAV9-α-syn showed increased number of Iba1 cells in SNpc region when compared to GFP injected controls at 4, 6, and 8 weeks (Two-way ANOVA p < 0.0001; treatment: F (1, 24) = 36.01, p < 0.0001; post hoc analysis: Tukey’s multiple comparison test). No significant difference was observed between heterozygous nude rats injected with AAV9-αsyn at 4, 6, and 8 weeks timepoint. d Bar graph shows the area units for MHC II staining for activated microglia in heterozygous nude rats injected with either AAV9-GFP (n = 3) or AAV9-α-syn (n = 5) at four different timepoints. The microglia express MHCII only in the heterozygous nude rats injected with AAV9-α-syn in SNpc region gradually increasing from 4 weeks to 8 weeks (Two-way ANOVA p < 0.001; treatment: F (1, 24) = 60.63, p < 0.0001; timepoint: F (3, 24) = 12.43, p < 0.0001; post hoc analysis: Tukey’s multiple comparison test). The MHCII positive microglia was observed higher in 8 weeks timepoint when compared to the 6 and 4 weeks coinciding with dopamine neuron loss in SNpc. e Bar graph shows the number of CD4 T cells in the injected side of the SNpc region in heterozygous nude rats at four different timepoints. A significant difference in the number of CD4 T cells was observed in heterozygous nude rats injected with AAV9-αsyn when compared to GFP injected controls at 4, 6, and 8 weeks timepoint (Two-way ANOVA p < 0.001; treatment: F (1, 23) = 205, p < 0.0001; timepoint: F (3, 23) = 86.33, p < 0.0001; post hoc analysis: Tukey’s multiple comparison test). Also, a gradual increase in the number of CD4 T cells was observed in heterozygous nude rats injected with AAV9-α-syn as time increases. f Bar graph shows the number of CD8 T cells in the injected side of the SNpc region in heterozygous nude rats at four different timepoints. A significant difference in the number of CD8 T cells was observed in heterozygous nude rats injected with AAV9-αsyn when compared to GFP-injected controls at 4, 6, and 8 weeks timepoint (two-way ANOVA p < 0.001; treatment: F (1, 23) = 396.7, p < 0.0001; timepoint: F (3, 23) = 190.7, p < 0.0001; post hoc analysis: Tukey’s multiple comparison test). Also, a gradual increase in the number of CD8 T cells was observed in heterozygous nude rats injected with AAV9-α-syn as time increases. *p < 0.05, **p < 0.001 when compared to GFP injected controls at a given timepoint; #p < 0.05 when compared to α-syn injected heterozygous nude rats at the previous timepoint. The bars in the graph represent mean ± standard error of the mean. g A representative figure explaining the timeline of dopamine neuron loss (TH) coinciding with microglial MHCII activation and CD4 and CD8 T cells infiltration along with Iba1 microglia and α-syn33 oligomer expression attaining stable expression after 4 weeks of AAV9-α-syn injection. The timeline for each immunohistochemistry staining represented here is percentage of expression attaining 100% either at 0 or 8 weeks (60 days)