Role of Central Inflammatory and Oxidative Pathways in the Morphine Exacerbation of Cardiovascular Effects of Sepsis in Rats

Figures

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  Figure 1. Time-related (A–C) and cumulative changes (areas under the curves, AUCs, (B–D)) in mean arterial pressure (MAP) and heart rate (HR) caused by i.v. morphine (3 or 10 mg/kg) in sham-operated and septic (cecal ligation and puncture, CLP) male rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. Data are presented as means ± SEM from 6–8 observations. Statistical significance was validated using the one-way ANOVA (A–C) or repeated measures ANOVA (B–D) followed by the Tukey’s post hoc test. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “sham/morphine-10”, c p < 0.05 vs. “CLP/saline”, d p < 0.05 vs. “CLP/morphine-10”.

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  Figure 2. Time-related (A–C) and cumulative changes (areas under the curve, AUC, (B–D)) in time-domain indices of HRV (SDNN and rMSSD) evoked by i.v. morphine (3 or 10 mg/kg) in sham-operated and septic (CLP) male rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. Values are means ± SEM of 6–8 observations. Statistical significance was validated using the one-way ANOVA (A–C) or repeated measures ANOVA (B–D) followed by the Tukey’s post hoc test. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “CLP/Saline”, c p < 0.05 vs. “CLP/morphine-10”.

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  Figure 3. Time-related (A–C) and cumulative changes (areas under the curve, AUC, (B–D)) in frequency-domain indices of HRV (total power and LF/HF ratio) induced by i.v. morphine (3 or 10 mg/kg) in sham-operated and septic (cecal ligation and puncture, CLP) male rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. Values are means ± SEM of 6–8 observations. Statistical significance was validated using the one-way ANOVA (A–C) or repeated measures ANOVA (B–D) followed by the Tukey’s post hoc test. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “CLP/Saline”, c p < 0.05 vs. “CLP/morphine-10”, d p < 0.05 vs. “sham/morphine-10”.

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  Figure 4. Time-related (A–C) and cumulative changes (areas under the curve, AUC, (B–D)) in the maximum rate of rise of blood pressure waves (dP/dtmax) and isovolumic relaxation constant (Tau, a measure of diastolic function) evoked by i.v. morphine (3 or 10 mg/kg) in sham-operated and septic (cecal ligation and puncture, CLP) male rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. Values are means ± SEM of 6–8 observations. Statistical significance was validated using the one-way ANOVA (A–C) or repeated measures ANOVA (B–D) followed by the Tukey’s post hoc test. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “CLP/saline”, c p < 0.05 vs. “CLP/morphine-10”, d p < 0.05 vs. “sham/morphine-10”. Supplementary Files S1–S5 contain the raw data illustrating the cardiovascular effects of morphine in CLP rats.

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  Figure 5. Effects of prior i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor), DPI (150 μg/5 μL/rat NADPHox inhibitor) or fasudil (70 μg/5 μL/rat, ROCK inhibitor) on AUCs of the response of mean arterial pressure (MAP, (A)), heart rate (HR, (B)), time-domain (SDNN, (C); rMSSD, (D)) and frequency-domain (total power, (E); LF/HF ratio, (F)) indices of HRV, maximum rate of rise of blood pressure waves (dP/dtmax, (G)), and isovolumic relaxation constant (Tau, (H)) to morphine (10 mg/kg) in septic rats. Values are means ± SEM of 6–8 observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. a p < 0.05 vs. “CLP/Saline”, b p < 0.05 vs. “CLP/morphine”.

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  Figure 6. Effects of prior i.v. naloxone (1 mg/kg i.v.) or i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor), DPI (150 μg/5 μL/rat NADPHox inhibitor) or fasudil (70 μg/5 μL/rat, ROCK inhibitor) on morphine-10-evoked rises in protein expressions of MCP1 (A) and TLR4 (B) in hearts of septic rats. Values are means ± SEM of 4–5 observations. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “CLP/saline”, c p < 0.05 vs. “CLP/morphine-10”.

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  Figure 7. Effects of prior i.v. naloxone (1 mg/kg i.v.) or i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor), DPI (150 μg/5 μL/rat NADPHox inhibitor) or fasudil (70 μg/5 μL/rat, ROCK inhibitor) on morphine-10-evoked rises in protein expressions of MCP1 (A) and TLR4 (B) in the rostral ventrolateral medulla (RVLM) of septic rats. Values are means ± SEM of 5 observations. a p < 0.05 vs. “sham/saline”, b p < 0.05 vs. “CLP/saline”, c p < 0.05 vs. “CLP/morphine-10”. Supplementary Files S8–S10 contain the raw data of the protein expression studies.

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  Figure 8. Schematic presentation of possible central signaling pathways involved in the interaction between μ-opioid receptors and inflammatory and oxidative pathways in sepsis. PAMPS: pathogen-associated molecular patterns, TLR4: Toll-Like Receptor-4, MYD88: Myeloid Differentiation primary response 88, NADPHox: Nicotinamide adenine dinucleotide phosphate oxidase, MOR: μ-opioid receptor, Pi3K: Phosphoinositide-3 kinases, AkT: Protein kinase B, PKC: Protein kinase C epsilon type, ROCK: Rho-associated coiled-coil kinase, ROS: Reactive Oxygen Species, MAPK: Mitogen-activated protein kinase, JNK: c-Jun N-terminal Kinase, ERK: Extracellular signal-regulated kinase, NF-κB: Nuclear Factor kappa-B, MCP1: Monocyte Chemoattractant Protein-1, iNOS: Inducible Nitric Oxide Synthase, TNF- α: Tumor Necrosis Factor-alpha, IL-1: Interlukin-1.

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  Figure 9. Diagrammatic representation of the timeline of surgical procedures and drug regimens.