Inflammatory and chemotactic signals of the brainstem solitary tract mediate the morphine exacerbation of impaired reflex chronotropism in septic rats

Figures

• 

  FIGURE 1. Diagrammatic representation of the timeline of surgical procedures, drug regimens and experimental groups.

• 

  FIGURE 2. Effect of i.v. morphine (3 or 10 mg/kg) on increases and decrease in MAP elicited by phenylephrine (panel (A)) and sodium nitroprusside (panel (B)) (1–16 μg/kg each), respectively (upper panels) and reciprocal changes in HR (panels (C, D), respectively) in conscious sham operated rats and CLP 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 five to six observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “sham/morphine-10”, dP < 0.05 vs. “CLP/Morphine-10”.

• 

  FIGURE 3. Effect of i.v. morphine (3 or 10 mg/kg) on baroreflex curves generated by phenylephrine (1–16 μg/kg) and baroreflex sensitivity (BRSPE, slopes of the curves) in conscious sham operated rats and CLP rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. In (A), the regression lines represent the following groups: sham/saline (solid line), sham/morphine-3 (dashed line), sham/morphine-10 (dotted line). In (B), the regression lines represent the following groups: CLP/saline (solid line), CLP/morphine-3 (dashed line), CLP/morphine-10 (dotted line), and CLP/naloxone/morphine-10 (dash-dotted line). In (C), BRSPE. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “sham/morphine-10”, dP < 0.05 vs. “CLP/Morphine-10”.

• 

  FIGURE 4. Effect of i.v. morphine (3 or 10 mg/kg) on baroreflex curves generated by sodium nitroprusside (1–16 μg/kg) and baroreflex sensitivity (BRSSNP, slopes of the curves) in conscious sham operated rats and CLP rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. In (A), the regression lines represent the following groups: sham/saline (solid line), sham/morphine-3 (dashed line), sham/morphine-10 (dotted line). In (B), the regression lines represent the following groups: CLP/saline (solid line), CLP/morphine-3 (dashed line), CLP/morphine-10 (dotted line), and CLP/naloxone/morphine-10 (dash-dotted line). In (C), BRSSNP. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “sham/saline”.

• 

  FIGURE 5. Effect of i.c. pretreatment with PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor, panel (A)), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor, panel (B)), wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor, panel (C)), fasudil (70 μg/5μL/rat, ROCK inhibitor, panel (D)) or DPI (150 μg/5 μL/rat NADPHox inhibitor, panel (E)) on baroreflex curves generated by phenylephrine (1–16 μg/kg) and baroreflex sensitivity (BRS curves) in conscious CLP rats treated with morphine (10 mg/kg). In (F), BRSPE. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “CLP/morphine”.

• 

  FIGURE 6. Effect of i.c. pretreatment with PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor, panel (A)), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor, panel (B)), wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor, panel (C)), fasudil (70 μg/5μL/rat, ROCK inhibitor, panel (D))or DPI (150 μg/5 μL/rat NADPHox inhibitor, panel (E)) on baroreflex curves generated by sodium nitroprusside (1–16 μg/kg) and baroreflex sensitivity (BRS curves) in conscious CLP rats treated with morphine-10. In (F), BRSSNP. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “CLP/morphine”.

• 

  FIGURE 7. (A) illustrates the effects of 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/5μL/rat, ROCK inhibitor) on morphine (10 mg/kg)-evoked rises in immunohistochemical protein expressions of MCP1 in the nucleus tractus solitarius (NTS) of septic rats. Representative images of the NTS expression of MCP1 are shown in (B). Values are means ± SEM of five observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “CLP/morphine-10”.

• 

  FIGURE 8. (A) illustrates the effects of 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/5μL/rat, ROCK inhibitor) on morphine (10 mg/kg)-evoked rises in immunohistochemical protein expressions of TLR4 in the nucleus tractus solitarius (NTS) of septic rats. Representative images of the NTS expression of TLR4 are shown in (B). Values are means ± SEM of five observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “CLP/morphine-10”.

• 

  FIGURE 9. (A) is a photomicrograph of a coronal section of the medulla oblongata corresponding to Plate no 136 (−12.36 mm from bregma) in the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986; Nakano et al., 2015; Abuiessa et al., 2020; Sallam et al., 2021b), stained with cresyl violet. The nucleus tractus solitarius (NTS) is outlined on the left side with a dotted red line. On the right side, the area enclosed within the square box represents the region selected for further analysis, which is illustrated in Figures 8 and 9 and shown at a higher magnification in (B) (scale bar represents 500 µm). (B) shows a magnified view of the NTS, highlighting the morphology of its neurons as revealed by cresyl violet staining (scale bar corresponds to 40 µm). (C) depicts an even higher magnification of the area enclosed by the square box in panel B, allowing a more detailed visualization of the neuronal features (scale bar represents 20 µm).