Fig. 4. Acute effects of mitochondria transplantation on mitochondrial function and immune response.
a Representative images of reduced 3,3’-diaminobenzidine (DAB), the electron acceptor of COX, in the mPFC of the four experimental groups at two and seven days after transplantation. Darker brown color of reduced DAB represents increased ex-vivo activity of COX. Scale bar: 50 µm. b, c Quantification of COX activity at two (b) and seven (c) days after transplantation. b One-way ANOVA showed significant differences in COX activity between groups two days post-procedure (F(3,9) = 66.4, P < 0.0001). Mitochondria transplantation significantly increased the activity of COX in PM as compared to all other groups (P < 0.0001). c Seven days after surgery, COX activity was similar in all groups (F(3,8) = 1.17, P = 0.241). N = 3 animals/group; 6–8 sections/animal; n = 12 total animals/time point. d f Representative images of oxidized H2DFCDA (red) of the four experimental groups at two (d) and seven (f) days after transplantation. Scale bar: 20 µm. e, g Quantification of ROS production at two (e) and seven (g) days after transplantation. e Two days after transplantation, one-way ANOVA showed a significant difference between groups in ROS production (F(3,8) = 9.54, P < 0.005). ROS production was significantly increased in PV as compared to all other groups, while PM ROS level was reduced to that of SV (PV vs. SV, P < 0.048; PV vs. PM, P < 0.045; PV vs. SM P < 0.003; PM vs. SV, P = 0.999). Mitochondrial transplantation had no significant effect in SM rats (SM vs. SV, P = 0.246). g Seven days after transplantation, one-way ANOVA showed a significant difference between groups in ROS production (F(3,8) = 11.6, P < 0.003). Sustained significant increase in PV as compared to SV (P < 0.039) and normal levels in PM are depicted (PM vs. PV, P < 0.038 and PM vs. SV, P > 0.999). However, in the SM group, a substantial increase was observed as compared to SV and PM groups (P < 0.006). N = 3 animals/group; 6 sections/animal; n = 12 total animals/time point. h–m Transcripts of the pro-inflammatory TNF-α, IL-1β, IL-6, the anti-inflammatory IL-10, TGF-β cytokines, the chemokine CX3CL1 and its receptor CX3CR1 and the inflammatory homeostasis index TNF-α/IL-10 ratio were measured by real-time PCR in the mPFC of two control (Poly I:C and saline offspring) and four experimental groups. h In Poly I:C offspring, a significant decrease was observed in IL-10 (P < 0.043) and an increase in CX3CL1 (P < 0.0008) and TNF-α/IL-10 ratio (P < 0.003) transcript levels as compared to saline rats. i, k Linear discriminant analysis (LDA) of groups’ immune profiles based on the expression of the immune factors (ΔΔCt values) at two (i) and seven (k) days following mitochondria transplantation. For each group, the ellipse represents ±2σ around the group score centroid. i A significant canonical discrimination between the immune profiles of the four experimental groups (PF1 < 0.031) shows an overlap between groups two days after the surgical procedure. j One-way ANOVA showed that the significant discriminating predictor at two days, TNF-α/IL-10 ratio (Wilk’s λ = 0.518; 93.8%), was similar between PM and SV groups (P > 0.05), and between PV and SM groups. The latter groups were significantly lower from both SV (P < 0.021 and P < 0.035, respectively) and PM (P < 0.012 and P < 0.02, respectively). k Seven days after transplantation, a significant canonical discrimination between immune profiles of the four experimental groups (PF1 < 0.002 and PF2 < 0.015) shows relatively disparate groups. l, m Seven days after transplantation, one-way ANOVA showed two significant discriminating predictors IL-1β (Wilk’s λ = 0.675; 100%) (l) which was significantly increased in all groups as compared to SV (at least P < 0.010), and CX3CL1 (Wilk’s λ = 0.554; 100%) (m), which was significantly increased in SM compared to all other groups (at least P < 0.04). Values are means of ΔCt (h) and ΔΔCt (j, l, m) ±95% confidence interval. h–m N = 4 animals/group, assessed in triplicates. n Representative images of immunofluorescence staining of Iba1, a microglial-specific calcium-binding protein, in the mPFC of the four experimental groups seven days after transplantation. o, p Quantification of Iba1+ cells at two and seven days after transplantation. Iba1+ cells were quantified as the sum of Iba1 staining (red) co-localized with Dapi (blue) divided by the tissue area. o Two days after transplantation, no differences were observed between groups (F(3, 8) = 0.799, P = 0.528). p Seven days after transplantation, significant differences were observed between groups in the number of Iba1+ microglia in the mPFC (F(3,8) = 5.56, P < 0.023) due to an increase in microglia number in PM as compared to SV and PV (PM vs. SV, P < 0.036; PM vs. PV, P < 0.030). N = 3 animals/group; 4–5 sections/animal; n = 12 total animals/time point. Scale bar: 100 µm. All values are means ± s.e.m. *P < 0.05; **P < 0.03; ***P < 0.006; ****P < 0.0001.