Fig 1: PK system activation in DRG. a Representative images of PK2 immunofluorescence signal in DRG sections of CTR, BTZ 14 days, BTZ 28 days, and PC1 (BTZ + PC1) mice. Quantitative analysis of PK2 signal (b) was computed as integrated optical density for arbitrary areas (6 sections per animal, 5 animals per group). One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test for multiple comparisons. *p < 0.05, ***p < 0.001 vs CTR; °°°p < 0.001 vs BTZ day 28. c, d mRNA levels of PK-R1 and PK-R2 respectively, measured by real-time qPCR, 14 days after the first BTZ administration (c.d. 2.4 mg/kg) in CTR and BTZ mice and at the end of the BTZ protocol (c.d. 4,8 mg/kg) in CTR, BTZ, and BTZ + PC1. mRNA levels, determined by real-time qPCR, were expressed in relation to GAPDH and presented as fold increases over the levels of CTR animals (at the same time point). Data represent mean ± SD of 6 mice/group. At day 14, statistical analysis was performed by means of t test while at day 28 by one-way ANOVA followed by Bonferroni’s post-test. ***p < 0.001 vs vehicle/CTR (at the same time point); °°p < 0.01 vs BTZ day 28
Fig 2: PK system activation in the sciatic nerve. a Representative images of PK2 immunofluorescence signal in the sciatic nerve sections of CTR, BTZ 14 days (corresponding to c.d. 2.4 mg/kg), BTZ 28 days (corresponding to c.d. 4.8 mg/kg), and PC1 (BTZ + PC1) mice. Cell nuclei were counterstained with DAPI (blue fluorescence). Quantitative analysis of PK2 signal (b) was computed as integrated optical density for arbitrary areas (6 sections per animal, 5 animals per group). One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test for multiple comparisons. ***p < 0.001 vs CTR; °°°p < 0.001 vs BTZ day 28. c, d mRNA levels of PK-R1 and PK-R2 respectively, measured by real-time qPCR, 14 days after the first BTZ administration (c.d. 2,4 mg/kg) and at the end of BTZ protocol (c.d. 4.8 mg/kg, day 28). Data represent mean ± SD of 6 mice/group. Statistical analysis was performed by mean of one-way ANOVA
Fig 3: PK2 serum levels. PK2 serum levels were measured by ELISA 14 days after the first BTZ administration, before starting the PC1 treatment (CTR and BTZ groups), and at the end of the BTZ/BTZ + PC1 protocol (CTR, BTZ, BTZ + PC1 groups). Data represent mean ± SD of 6 mice/group. At day 14, statistical analysis was performed by means of t test while at day 28 by means of one-way ANOVA followed by Bonferroni’s post-test.*p < 0.05, **p < 0.01 vs CTR; °°°p < 0.001 vs BTZ day 28
Fig 4: Effect of PC1 treatment on macrophage activation and cytokine levels in the sciatic nerve. a, b mRNA levels of CD68 and the percentage of CD68 positive area in the sciatic nerve of CTR, BTZ 14 days (day 14 corresponding to c.d. 2,4 mg/kg), BTZ 28 days (day 28, corresponding to c.d. 4,8 mg/kg), and PC1 (BTZ + PC1) mice. Quantitative analysis of CD68 signal was computed as integrated optical density for arbitrary areas (6 sections per animal, 5 animals per group). c Representative immunofluorescence images of CD68 in the sciatic nerve sections of the same experimental groups. One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test for multiple comparisons. **p < 0.01, ***p < 0.001 vs CTR; °p < 0.05 vs BTZ day 28. d Immunofluorescence double-staining shows the colocalization (yellow, arrowhead) of PK2 (green) with CD68 (activated macrophages, red) in the sciatic nerve of CTR, BTZ day 14, BTZ day 28, and BTZ + PC1 mice (PC1). Cell nuclei were counterstained with DAPI (blue fluorescence). e–h The mRNA levels of TLR4 and of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 respectively. i The mRNA of the anti-inflammatory cytokine IL-10. All the measurements were performed 14 days after the first BTZ administration (c.d. 2.4 mg/kg), before starting PC1 treatment (CTR and BTZ groups), and at the end of the BTZ protocol (c.d. 4.8 mg/kg) (CTR, BTZ, BTZ + PC1 groups). mRNA levels, determined by real-time qPCR, were expressed in relation to GAPDH and presented as fold-increases over the levels of CTR animals (at the same time point). (a, e–i) Data represent mean ± SD of 6 mice/group. At day 14, statistical analysis was performed by means of t test while at day 28 by using one-way ANOVA followed by Bonferroni’s post-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle/CTR (at the same time point); °p < 0.05, °°°p < 0.001 vs BTZ day 28
Fig 5: PK system activation in the spinal cord. a Representative images of PK2 immunofluorescence signal in spinal cord sections of CTR, BTZ 14 days (corresponding to c.d. 2,4 mg/kg), BTZ 28 days (corresponding to c.d. 4.8 mg/kg), and PC1 (BTZ + PC1) mice. Quantitative analysis of PK2 signal (b) was computed as integrated optical density for arbitrary areas (6 sections per animal, 5 animals per group). One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test for multiple comparisons. *p < 0.05, ***p < 0.001 vs CTR; °°°p < 0.001 BTZ day 28. c, d mRNA levels of PK-R1 and PK-R2 respectively, measured by real-time qPCR, 14 days after the first BTZ administration (CTR and BTZ) mice and at the end of the BTZ protocol (c.d. 4,8 mg/kg) in CTR, BTZ, and BTZ + PC1. mRNA levels, determined by real-time qPCR, were expressed in relation to GAPDH and presented as fold increases over the levels of CTR animals (at the same time point). Data represent mean ± SD of 6 mice/group. At day 14, statistical analysis was performed by means of t test while at day 28 by means of one-way ANOVA followed by Bonferroni’s post-test. ***p < 0.001 vs CTR; °°°p < 0.001 vs BTZ day 28
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