Ablation of the Right Cardiac Vagus Nerve Reduces Acetylcholine Content without Changing the Inflammatory Response during Endotoxemia

Abstract

Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a "non-neuronal cardiac cholinergic system" that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX) with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg body weight, intravenously) and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory (IL-1β, IL-6, TNF-α) cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX) led to a marked decrease in heart acetylcholine concentration and an increase in cardiac acetylcholinesterase activity. Without LPS, VGX changed rat hemodynamic parameters, including heart frequency, cardiac output, and end-diastolic volume. In contrast, VGX during endotoxemia did not significantly change the concentration and expression of proinflammatory cytokines in the heart. In conclusion we demonstrate that right cardiac vagal innervation regulates cardiac acetylcholine content but neither improves nor worsens systemic inflammation.

Keywords: endotoxemia; hemodynamic parameters; inflammation; right cardiac vagus nerve resection; sepsis.

Figure 1

Blood cholinesterase (CHE) activities at the end of the experiments. Blood enzyme activity presented as median (IQR = Inter Quartile Range) at 4 h after LPS injection (T5). No significant effect of right cardiac vagus nerve resection (VGX) was observed (p > 0.05). ^§: significant LPS effect for (A) acetylcholinesterase (ACHE, U/g Hb) activity (sham saline versus sham LPS, p = 0.031, and VGX saline versus VGX LPS, p < 0.001) and for (B) butyrylcholinesterase (BuCHE, U/L) activity (sham saline versus sham LPS, p < 0.001, and VGX saline versus VGX LPS, p < 0.001). The circles present outliers.

Figure 2

(A) Acetylcholine (ACH) and (B) acetylcholinesterase (ACHE) in rat heart at the end of the experiments. Data presented as median (IQR = Inter Quartile Range) at 4 h after LPS injection (T5). ^#: significant effect of right cardiac vagus nerve resection (ACH in ng/mg protein: p = 0.038, ACHE in U/mg protein: p = 0.004 (sham saline versus VGX saline) and p = 0.05 (sham LPS versus VGX LPS). ^§: significant LPS effect: (VGX saline versus VGX LPS, p = 0.016). The circles present outliers.

Figure 3

Plasma pro-inflammatory cytokines ((A) IL-1ß, (B) IL-6, (C) TNF-α) at the end of the experiment. Plasma data presented as median (IQR = Inter Quartile Range, in pg/mL) at 4 h after LPS injection (T5). No significant effect of nerve resection (VGX) was obtained (p > 0.05). Significant LPS effect (^§: sham LPS versus sham saline and VGX LPS versus VGX saline, p ≤ 0.001 for both) was detected in all plasma proinflammatory cytokines measured. The circles present outliers.

Figure 4

Heart proinflammatory cytokine concentrations ((A) IL-1ß, (B) IL-6, (C) TNF-α) at the end of the experiments. Heart data presented as median (IQR = Inter Quartile Range, in pg/mg protein) at 4 h after LPS injection (T5). No significant effect of nerve resection was obtained (p > 0.05). Significant LPS effect (^§: sham LPS versus sham saline and VGX LPS versus VGX saline, p ≤ 0.001 for both) was detected in all tissue proinflammatory cytokines measured. The circles present outliers.

Figure 5

Cardiac proinflammatory mRNA expression ((A) IL-1ß, (B) IL-6, (C) TNF-α) at the end of the experiments. Data presented as normalized expression (median, IQR = Inter Quartile Range) at 4 h after LPS injection (T5). No significant effect of right cardiac vagus nerve resection was observed (p > 0.05). Significant LPS effect (^§: sham LPS versus sham saline and VGX LPS versus VGX saline, p ≤ 0.001) was detected in all tissue proinflammatory cytokine mRNA levels measured. The circles present outliers.

Figure 5

Cardiac proinflammatory mRNA expression ((A) IL-1ß, (B) IL-6, (C) TNF-α) at the end of the experiments. Data presented as normalized expression (median, IQR = Inter Quartile Range) at 4 h after LPS injection (T5). No significant effect of right cardiac vagus nerve resection was observed (p > 0.05). Significant LPS effect (^§: sham LPS versus sham saline and VGX LPS versus VGX saline, p ≤ 0.001) was detected in all tissue proinflammatory cytokine mRNA levels measured. The circles present outliers.

Figure 6

Effect over the experimental time in cardiovascular and hemodynamic measurements ((A) mean arterial blood pressure, (B) heart rate, (C) cardiac output, (D) enddiastolic volume, mean ± SD) in control and endotoxemic rats and after right cardiac vagus nerve resection (VGX). Data are represented as means ± SD. Changes during the experimental period are presented: (end of the experiment versus data before LPS): “+”: time effect p ≤ 0.05.

Figure 7

Representative figure of right cardiac vagus nerve preparation. 1 = Nervus vagus dexter, 2 = Cardiac branch of the right vagus nerve, 3 = Truncus brachiocephalicus.