Morphine aggravates inflammatory, behavioral, and hippocampal structural deficits in septic rats

Abstract

Although pain and sepsis are comorbidities of intensive care units, reported data on whether pain control by opioid analgesics could alter inflammatory and end-organ damage caused by sepsis remain inconclusive. Here, we tested the hypothesis that morphine, the gold standard narcotic analgesic, modifies behavioral and hippocampal structural defects induced by sepsis in male rats. Sepsis was induced with cecal ligation and puncture (CLP) and behavioral studies were undertaken 24 h later in septic and/or morphine-treated animals. The induction of sepsis or exposure to morphine (7 mg/kg) elicited similar: (i) falls in systolic blood pressure, (ii) alterations in spatial memory and learning tested by the Morris water maze, and (iii) depression of exploratory behavior measured by the new object recognition test. These hemodynamic and cognitive defects were significantly exaggerated in septic rats treated with morphine compared with individual interventions. Similar patterns of amplified inflammatory (IL-1β) and histopathological signs of hippocampal damage were noted in morphine-treated septic rats. Additionally, the presence of intact opioid receptors is mandatory for the induction of behavioral and hemodynamic effects of morphine because no such effects were observed when the receptors were blocked by naloxone. That said, our findings suggest that morphine provokes sepsis manifestations of inflammation and interrelated hemodynamic, behavioral, and hippocampal deficits.

Figure 1

Time sequence of the new object recognition test.

Figure 2

A Layout out of the procedure for calculating the distance travelled in Morris Water Maze.

Figure 3

Effect of morphine (7 mg/kg, s.c.) on percentage maximum possible effect (%MPE)  (A) 24 h pre-surgery, (B) 6 h pre-surgery, and (C) 24 h post-surgery. Data were expressed as mean ± S.E.M (n = 6–9). One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 4

Effect of morphine (7 mg/kg, s.c.) on hemodynamic responses 24 h after CLP or sham operation. Data were expressed as mean ± S.E.M (n = 6–9). (A) Systolic blood pressure (SBP), (B) heart rate (HR), followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline.

Figure 5

Behavioral and cognitive effects of morphine (7 mg/kg, s.c.) on CLP induced sepsis in rats using Morris Water Maze (A) time spent on platform quadrant, (B) distance travelled, (C) number of crossings, (D) escape latency, and the corresponding Pearson correlation with the systolic blood pressure, (E) time spent of platform quadrant, (F) distance travelled, (G) number of crossings, (H) escape latency. Data were expressed as mean ± S.E.M (n = 6–7). One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 6

Effect of morphine (7 mg/kg, s.c.) on % spontaneous alternation and time spent in novel arm A, arms B and C and in the centre, of Y-maze in sham and CLP rats. The total duration of arm visits was recorded in the 5 min retention test. Data were expressed as mean ± S.E.M (n = 6–12). One-Way ANOVA followed by the Tukey’s post hoc test was employed to measure statistical significance. $ P<0.05 for difference in performance of rats in the novel arm vs. arms B & C and center of the same group. Difference in the performance between septic rats treated with morphine and other groups in the corresponding arm *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 7

Effect of morphine (7 mg/kg, s.c.) on the behaviour of septic male rats in the New Object recognition test (A) discrimination measure, (B) discrimination index, (C) recognition index, and the corresponding Pearson correlation with systolic blood pressure, (D) discrimination measure, (E) discrimination index, (F) recognition index. Data were expressed as mean ± S.E.M. One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline.

Figure 8

Effect of morphine (7 mg/kg, s.c.) on the exploratory functions in new object recognition test (A) distance travelled, (B) horizontal movements, (C) number of line crossings, (D) time spent in platform quadrant, and the corresponding Pearson correlation with the systolic blood pressure. (E) Distance travelled, (F) horizontal movements, (G) number of line crossing, (H) and time spent in central squares in sham and septic rats. Data were expressed as mean ± S.E.M (n = 6–11). One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 9

Effect of morphine (7 mg/kg, s.c.) on serum IL1β levels, in septic rats 24 h after CLP or sham operation. Data were expressed as mean ± S.E.M (n = 6–9). One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 10

Effect of morphine (7 mg/kg, s.c.) on (A) normal neurons, (B) degenerated neurons, (C) glial cells in CA1 region of hippocampus of sham and septic rats. Representative photomicrographs of the rat hippocampus (CA1, HE, × 400) are shown. Data are means ± S.E.M. of observations. Normal neurons (black arrow), misaligned degenerated, shrunken neurons with pyknotic and hyperchromatic nuclei (red arrow) necrotic pyramidal with satellitosis and neuronophagia (green arrow). Data were expressed as mean ± S.E.M. One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance. *P < 0.05 vs. Sham-saline, ⁺P < 0.05 vs. CLP-saline, ^#P < 0.05 vs. Sham-morphine.

Figure 11

Effect of µ-opioid receptor blockade by naloxone (0.5 mg/kg, s.c.) on morphine (7 mg/kg, s.c.) evoked alternations in (A) SBP, (B) plasma IL1β, (C) distance travelled, (D) recognition index, (E) horizontal movements, (F) number of crossings recorded in the new object recognition test in sham rats. Data were expressed as mean ± S.E.M (n = 5–9). One-way ANOVA followed by the Tukey’s post hoc test was used to measure statistical significance *P < 0.05 vs. Saline, ⁺P < 0.05 vs. Morphine group.