Fig 1: Peripheral NR2B mediates CPIP-induced c-fos induction and glial activation in SDH. (A) Immunofluorescence showed c-fos induction through acute to chronic phase after CPIP which was abolished by ifenprodil. (B) Quantification of results from A (n = 5 in each). Significant microglia (Iba 1 staining) (C, D) and astrocyte activation (GFAP staining) (E, F) were observed only in chronic but not acute phase. One-way ANOVA, * p < 0.05 versus sham, # p < 0.05 versus CPIP. Abbreviations: SDH, spinal cord dorsal horn; CPIP, chronic post-ischemia pain; NS, normal saline; NMDA, N-methyl-D-aspartate; IFEN, ifenprodil; Iba1, ionized calcium binding adaptor molecule 1; GFAP, glial fibrillary acidic protein.
Fig 2: MC1R disruption amplifies microglia activation and alters Nrf2 response to aSyn overexpression in the nigrostriatal pathway. MC1Re/e and WT mice were injected unilaterally with human WT aSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFa, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t-test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group.I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 3: Focal and proximal microglia differentially upregulate common markers of activation. Focal (red line) microglia (A) colocalized with TGFß1 (B, D), and CD68 (C, D). Proximal (orange line) microglia colocalized with TGFß1, but not CD68. Pearson correlation was used to colocalize IBA1 with TGFß1 (E) and CD68 (F). Images (A–C) were taken at 20× magnification. Insets (D1–D4) were taken at 63× magnification. Groups were analyzed via a mixed linear model. Scale bar: 100 µm. CD68, cluster of differentiation 68; Contra., contralateral; IBA1, ionized calcium-binding adapter molecule 1, Prox., proximal; TGFß1, transforming growth factor beta 1,
Fig 4: LPNP-TLR4 siRNA downregulates the immune response of microglia following LPS stimulation. (A–D) Gene expression of TNF-α, IL-6, IL-1β, and IL-10 in cultured microglial cells treated for 24 h with LPNP-TLR4 siRNA (n = 3) at different concentrations (0, 10, 50, and 100 nM, with 0 nM representing LPNP-scrambled siRNA (100 nM)). Afterward, the cells were treated with LPS (100 ng/mL) for 2 h (in addition to the first PBS group). (E–G) Representative images show Iba1-ir microglial cells near the injection spot (asterisk) at the morphological level in the rat hypothalamus treated with LPNP-TLR4 siRNA 18 h before followed by intravenous LPS (100 μg/kg) 2 h before sacrifice. (H) Quantification of the Iba1-ir microglial cell number and (I) soma size following LPS stimulation (n = 4). The data are presented as means ± SD and were analyzed using one-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: 100 μm in panels (E–G).
Fig 5: Microglia morphology and expression signatures are comparable to wild-type 3 days following in utero electroporation. a, e E17.5 wild-type Iba1 and Cd45 expression in the hypothalamus (a) and cortex (e) show an even distribution of Iba1+ cells throughout the brain parenchyma. b, f E17.5 pCIG2 IUE (E14.5) Iba1 and Cd45 expression in the hypothalamus (b) and cortex (f) show Iba1+ cells are evenly distributed throughout the brain parenchyma, while very few Cd45high cells are present in the brain parenchyma. c, g E17.5 wild-type Iba1 and P2ry12 expression in the hypothalamus (c) and cortex (g) show even distribution of both Iba1+ and P2ry12+ cells throughout the brain parenchyma. d, h E17.5 pCIG2 IUE (E14.5) Iba1 and P2ry12 expression in the hypothalamus (d) and cortex (h) show Iba1+ and P2ry12+ cells are relatively evenly distributed throughout the brain parenchyma. Scale bar represents 250 µm (a-e”). 3V, third ventricle. Dashed lines outline the ventricles and mark the division between the thalamus and hypothalamus. White arrows mark Iba1+ single-positive (a–b, e–f) or Iba1+/P2ry12+ double-positive (c–d, g–h) cells, while white arrowheads mark Iba1+/Cd45high double-positive (b, f) or Iba1+ single-positive (c–d, g–h) cells. i Quantification of Iba1+/Cd45high cells within the parenchyma of the hypothalamus in IUE brains as compared to wild-type (mean ± SEM; wild-type n = 3, IUE n = 3; p = 0.2227). j Quantification of Iba1+/Cd45high cells within the parenchyma of the cortex in IUE brains as compared to wild-type (mean ± SEM; wild-type n = 3, IUE n = 3; p = 0.1551)
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