Systemic Neutralization of Granulocyte–Macrophage Colony-Stimulating Factor Attenuates Stellate Ganglion Neuroinflammation and Cardiac Sympathetic Overactivation in Chronic Heart Failure in Rats

Figures

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  Figure 1. GM−CSF neutralization attenuates inflammatory marker expression in the stellate ganglia (SG) during CHF. (A) Representative immunoblots for (a) GM-CSF, (b) IBA1, (c) TNF-α, and (d) pro IL-1β in SG tissue lysates across sham, CHF, and CHF + GM-CSF Ab treatment groups. GAPDH was used as the internal loading control for all proteins. (B–E) Densitometric quantification of (B) GM-CSF, (C) IBA1, (D) TNF-α, and (E) pro IL-1β protein expression normalized to GAPDH. Data are expressed as mean ± SEM (n = 6 per group). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test or Kruskal–Wallis test followed by Dunn’s test where appropriate. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 2. GM−CSF expression in the heart and lungs in all experimental groups. (A) Representative immunoblots of GM-CSF in (a) heart and (b) lung tissue lysates from sham, CHF, and CHF + GM-CSF Ab groups. GAPDH and β-actin served as internal loading controls for heart and lung samples, respectively. (B) Densitometric quantification of GM-CSF protein expression in (a) heart and (b) lung tissues. Heart data were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Lung data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SEM (n = 6 per group). * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 3. Systemic neutralization of GM−CSF reduces circulating GM−CSF levels in CHF rats. Serum GM-CSF concentrations (pg/mL) measured by ELISA in sham, CHF, and CHF + GM-CSF Ab groups. Data are presented as mean ± SEM (n = 6 per group). Statistical significance was evaluated using Welch’s ANOVA followed by Dunnett’s post hoc test for multiple comparisons. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 4. GM−CSF neutralization attenuates the increase in calcium current density in SG neurons during CHF. (A) Representative bright-field micrograph of a sympathetic post-ganglionic neuron from the SG during whole-cell patch-clamp recording. Scale bar = 100 μm. (B) Representative traces of voltage-gated Ca2+ currents recorded from sham, CHF, and CHF + GM−CSF antibody (Ab) groups. Currents were elicited by a voltage step protocol from a holding potential of −80 mV (−60 mV to +60 mV with 5 mV increments, 500 ms duration). (C) Quantitative analysis of mean Ca2+ current density (pA/pF). Data represent currents elicited by a 500 ms test pulse at 0 mV from a holding potential of −80 mV. Values are presented as mean ± SEM (n = 4–6 neurons per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 5. GM−CSF neutralization attenuates increased neuronal excitability in SG neurons of CHF rats. (A) Representative membrane potential tracings from sympathetic post-ganglionic neurons across the sham, CHF, and CHF + GM-CSF Ab groups. (B) Quantitative data for frequency of action potentials (spikes per second) and (C) current threshold (pA) required to elicit an action potential. Data are presented as mean ± SEM (n = 8 neurons per group). Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 6. GM−CSF neutralization reduces the exaggerated CSNA in CHF rats. (A) Representative raw tracings of CSNA recorded at baseline and in maximum response to intracardiac KCl (3 M) injection across (a) sham, (b) CHF, and (c) CHF + GM-CSF Ab groups. (B) Quantitative analysis of CSNA, represented as a percentage of the maximum response (% Max). Data are expressed as mean ± SEM (n = 6 per group). Statistical significance was evaluated using Welch’s ANOVA followed by Dunnett’s post hoc test for multiple comparisons. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.

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  Figure 7. GM−CSF neutralization improves HRV in CHF rats. (A) Representative power spectral density tracings of HRV from (a) sham, (b) CHF, and (c) CHF + GM-CSF Ab groups. Dashed green lines delineate the low-frequency (LF: 0.2–0.75 Hz) and high-frequency (HF: 0.75–2.5 Hz) spectral bands. (B) Quantitative analysis of (a) LF power (ms2), (b) HF power (ms2), and (c) the calculated LF/HF ratio across all experimental groups. Data are presented as mean ± SEM (n = 6 per group). Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. sham, # p < 0.05 vs. CHF.