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

Abstract

Background/Objectives: Sepsis remains one of the most serious and possibly fatal complications encountered in intensive care units. Considering the frequent use of narcotic analgesics in this setting, we investigated whether the cardiovascular and peripheral and central inflammatory features of sepsis could be modified by morphine. Methods: Rats were instrumented with femoral and intracisternal (i.c.) indwelling catheters and sepsis was induced by cecal ligation and puncture (CLP). Results: The i.v. administration of morphine (3 and 10 mg/kg) significantly and dose-dependently aggravated septic manifestations of hypotension and impaired cardiac autonomic activity, as reflected by the reductions in indices of heart rate variability (HRV). Cardiac contractility (dP/dtmax) was also reduced by morphine in septic rats. The morphine effects were mostly eliminated following (i) blockade of μ-opioid receptors by i.v. naloxone and (ii) inhibition of central PI3K, MAPK-ERK, MAPK-JNK, NADPH oxidase (NADPHox), or Rho-kinase (ROCK) by i.c. wortmannin, PD98059, SP600125, diphenyleneiodonium, and fasudil, respectively. Further, these pharmacologic interventions significantly reduced the heightened protein expression of toll-like receptor 4 (TLR4) and monocyte chemoattractant protein-1 (MCP1) in brainstem rostral ventrolateral medullary (RVLM), but not cardiac, tissues of CLP/morphine-treated rats. Conclusions: Morphine worsens cardiovascular and autonomic disturbances caused by sepsis through a mechanism mediated via μ-opioid receptors and upregulated central inflammatory, chemotactic, and oxidative signals. Clinical studies are warranted to re-affirm the adverse cardiovascular interaction between opioids and the septic challenge.

Keywords: autonomic function; cardiovascular; inflammation; morphine; oxidative stress; sepsis.

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”.

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”.

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”.

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.

Figure 5

Effects of prior i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPK_ERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPK_JNK 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”.

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, MAPK_ERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPK_JNK 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”.

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, MAPK_ERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPK_JNK 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.

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.

Figure 9

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