Angiotensin II type 1 and type 2 receptors modulate TWIK1 channel expression and pain sensitivity in a rat model of neuropathic pain

Figures

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  FIGURE 1. TWIK1 and AT1R/AT2R expression in DRG neurons. (A) Representative ABC/DAB images of TWIK1 expression in whole L4 DRG. Insets show higher magnification views (×40). Scale bar: 250 μm; inset: 50 µm. (B) Quantification of TWIK1 staining. Left: correlation between neuronal cell area and mean cytoplasmic TWIK1 intensity (Spearman’s rs = 0.23, p < 0.0001). Right: percentage of cytoplasmic TWIK1 intensity by neuronal size (small, medium, large). Data are presented as mean ± SEM. (C) Representative immunofluorescence images showing co-localization of TWIK1 with IB4 (non-peptidergic neurons) and TrkA (peptidergic neurons). Correlation analysis shows a negative association with IB4 (rs = −0.19, p = 0.0004) and a positive association with TrkA (rs = 0.62, p < 0.0001). (D) Representative immunofluorescence images showing co-localization of TWIK1 with AT1R and AT2R in L5 DRG. Positive correlations were observed for both receptors (rs = 0.68 and 0.67, respectively; p < 0.0001). Scale bar: 50 µm.

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  FIGURE 2. TWIK1 mRNA expression in L4/L5 DRG after CCI and angiotensin receptor blockade. Female Wistar rats were treated with telmisartan, PD123319, or their combination (Telmi + PD). TWIK1 mRNA levels were normalized to GAPDH. Data are presented as mean ± SEM (N = 4 per group). TWIK1 expression was increased in the ipsilateral Telmi + PD group compared with the contralateral side (p = 0.0284) and ipsilateral CCI (p = 0.0174). Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test.

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  FIGURE 3. TWIK1 expression in DRG neurons after CCI and pharmacological modulation of angiotensin II receptors. (A) Representative immunofluorescence images of TWIK1 in L4/L5 DRG from contralateral and ipsilateral sides at 3, 7, and 14 days after CCI. (B) Quantification of TWIK1 staining by neuronal size (small, medium, large) at each time point. (C) Representative images of TWIK1 staining after 14 days of treatment with telmisartan, PD123319, or their combination (T + PD). (D) Quantification of TWIK1 staining expressed as percentage of maximum cytoplasmic intensity. Data are presented as mean ± SEM (N = 5 per group). Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test; contralateral vs. ipsilateral comparisons by Mann–Whitney U test. Scale bar: 50 µm.

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  FIGURE 4. Effects of AT1R and AT2R antagonists on neuropathic pain behavior after CCI. Time-course analysis of behavioral responses in contralateral and ipsilateral hind paws (N = 9 per group). (A) Spontaneous foot lifting (SFL; events/5 min). (B) Cold allodynia assessed by Choi’s test (score 0–3). (C) Mechanical sensitivity measured as paw withdrawal threshold (PWT; von Frey test). Data are presented as mean ± SEM. Statistical analysis: two-way repeated-measures ANOVA followed by Dunnett’s post hoc test.

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  FIGURE 5. AT1R and AT2R expression in L4/L5 DRG neurons after CCI and angiotensin receptor modulation. (A,B) Representative confocal images of AT1R staining in normal, contralateral and ipsilateral DRG at 3, 7, and 14 days after CCI. (C) Quantification of AT1R staining at each time point. (D) Representative images after 14 days of treatment with telmisartan, PD123319, or T + PD. (E) Quantification of AT1R staining expressed as percentage of maximum cytoplasmic intensity. Data are presented as mean ± SEM (N = 5 per group). Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test; Mann–Whitney U test for contralateral vs. ipsilateral comparisons. Scale bar: 50 µm. (F,G) Representative immunofluorescence images of AT2R staining in normal and also contralateral and ipsilateral DRG at 3, 7, and 14 days after CCI. (H) Quantification of AT2R staining at each time point. (I) Representative images after 14 days of treatment with angiotensin receptor antagonists. (J) Quantification of AT2R staining expressed as percentage of maximum cytoplasmic intensity. Data are presented as mean ± SEM (N = 5 per group). Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test; Mann–Whitney U test for…

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  FIGURE 6. Circulating cytokine levels after CCI and angiotensin receptor antagonism. Plasma levels (pg/mL) of IL-1β (A), TNF-α (B), IL-6 (C), and IL-10 (D) were measured by ELISA at 14 days after CCI. Experimental groups included control, sham, CCI, and treated animals (telmisartan, PD123319, T + PD). Data are presented as mean ± SEM. Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test.

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  FIGURE 7. GFAP expression and TWIK1 mRNA levels after CCI and angiotensin receptor modulation. (A) Representative immunofluorescence images of GFAP staining in L5 DRG from contralateral and ipsilateral sides in sham, 14-day CCI, and treated animals. (B) Quantification of GFAP-positive area (% total area). Data are presented as mean ± SEM (N = 5 per group). Statistical analysis: Kruskal–Wallis with Dunn’s post hoc test; Mann–Whitney U test for pairwise comparisons. Scale bar: 40 µm. (C) TWIK1 mRNA levels in cultured DRG neurons treated for 2 or 3 days with inflammatory cytokines (IL-1β, IL-6, TNF-α; inflammatory soup, IS) or control (Ctrl), normalized to GAPDH. Data are presented as mean ± SEM.