Fig. 8

Analysis of neurotoxicity induced by rAAV2-retro and RV-ΔG at injection sites and retrogradely labeled nuclei. (a) Schematic diagram of virus injection and tissue extraction for RNA sequencing. (b) Injection of rAAV2-retro-YFP into the LHA caused alterations in gene expression profiles. (c) Injection of RV-∆G-EGFP into the LHA caused alterations in gene expression profiles. (d) Gene profile alterations in retrogradely labeled mPFC following injection of rAAV2-retro-YFP into the LHA. Red dots represent the genes with significant change, while the black dots represent the genes without significant change. (e) Gene profile alterations in retrogradely labeled NAc following injection of RV-∆G-EGFP into the LHA. (f-g) GO-term pathway analysis of differentially expressed genes at the site of RV-∆G-EGFP injection (LHA) and in retrogradely labeled nucleus (NAc). (h-i) Immunostaining for the microglial marker Iba1 in the rAAV2-retro-YFP group. Iba1-positive cells were observed on the injection site, but barely in the PBS injected (Mock) LHA. (j-k) No activated microglia was detected in the ipsilateral mPFC following injection of rAAV2-retro-YFP or PBS (Mock) into the LHA. (l-m) Intense microglial activation was observed at the injection site when RV-∆G-EGFP was injected into the LHA, while few signals were observed in the PBS injected (Mock) LHA. (n-o) Intense microglial activation was observed in the retrogradely labeled ipsilateral NAc following injection of RV-∆G-EGFP into the LHA, but not in the ipsilateral NAc of Mock control. (p) Quantification of mean fluorescence intensity (Mean±SEM) of Iba1 in LHA after virus injection. rAAV2-retro-YFP: 14.09±0.8373, Mock: 6.059±1.132, rAAV2-retro-YFP vs Mock: P=0.0013; RV-∆G-EGFP: 19.75±1.403, Mock: 7.885±1.265, RV-∆G-EGFP vs Mock: P = 0.0008; rAAV2-retro-YFP vs RV-∆G-EGFP: P = 0.0133; n = 4, n is mice number. (q) Quantification of mean fluorescence intensity (Mean±SEM) of Iba1 in mPFC injected with rAAV2-retro-YFP and PBS. rAAV2-retro-YFP: 6.438±1.114, PBS: 7.713±1.192, P = 0.4639, n = 4. (r) Quantification of mean fluorescence intensity (Mean±SEM) of Iba1 in NAc injected with RV-∆G-EGFP and PBS. RV-∆G-EGFP: 17.20±1.408, Mock: 6.883±0.8132, P = 0.0007; n = 4. (s) Immunostaining for oxytocin in PVN neurons 7 days following the injection of RV-∆G-EGFP into the LHA. (t, u) Boxed areas in S were magnified to show oxytocin-positive neurons in the ipsilateral and contralateral PVN to the virus injection side. (v) Higher-magnification images of the dashed box depicting the ipsilateral PVN in S with green color and merged color. Green, GFP expressed by RV-∆G-EGFP; red, oxytocin immunostaining signal. (w) Quantification of oxytocin-positive neurons (Mean±SEM) in the ipsilateral and contralateral PVN. Contra: 23.50±1.443, Ipsi: 62.00±3.136, P=0.0001, n = 4. MFI, mean fluorescence intensity; Ipsi, ipsilateral; Contra, contralateral. n is mice number. Scale bars =200 μm for S; 100 μm for H-O; 50 μm for T-V. Unpaired t-tests, *P < 0.05, **P < 0.01, ***P < 0.001