Role Of Ambiguus Nucleus In The Cholinergic Anti-inflammatory Pathway

The cholinergic nervous system, acting via the vagus nerve, serves as one of thehuman body’s natural anti-inflammatory mechanisms to prevent excessive release ofinflammatory cytokines in (for example) infection/sepsis or autoimmune diseases,such as rheumatoid arthritis. The cholinergic anti-inflammatory pathway can inhibitthe activation of macrophage in turn the generation of proinflammatory factors suchas TNF or interleukin (IL).via releasing acetylcholine from vagal efferent nerve. Thenacetylcholine combines specially with the nAchRα7receptors on the surface ofimmune cell to exert anti-inflammatory effect by affecting the,intracellular signaltransduction.Nucleus ambiguus (NA) is an important visceral and somatic motor nucleus whichestablishes widely connections with the central nervous system and peripheral organs.NA, together with dorsomedial vagal solitary complex, ventrolateral medulla andintermediate reticular formation, forms the medullary visceral zone (MVZ). NAcontains a great variety of neurotransmitters including acetylcholine, biogenic amines,neuropept ides and amino acids, and their receptors. NA plays an important role in themodulation of visceral and somatic activities, including respirat ion, cardiovascularactivity, immunologic function, digestion and endocrine functions. NA and the vagalcomplex (DVC) come together to form the visceral zone of medulla oblongata; Theaxons come from the neurons of NA and DMV compose the parasympatheticpreganglionic nerve. What’s more, there are Round-trip fiber connections betweenNA and the nucleus of solitary tract (NTS). In addition, NA is also involved in theregulation of immune function such as affecting cytokines release by the internalneurons e.g.the cholinergic neurons. Our previous works have been shown that c-Fos expression in DVC significantlyincreased in endotoxin-inflamed rats. We also found that the vagal efferent fiberdischarge frequency increased. These results indicate that the DVC involed in thecholinergic anti-inflammatory.Meanwhile some vagus efferent nerve stem from NA,and there is a structural and functional connectivity between NA and DVC. Accordingly wespeculate that NA involed in the cholinergic anti-inflammatory. Studies by Gaykemaet al. G. E. Hermann（2001）showed that the right side of each brain stem containedsomewhat more c-Fos-activated NST cells than the corresponding left side aftersystemic administration of LPS. Simons CT et al. have already shown that hepaticafferents which ascend predominantly within the left cervical vagal trunk to terminatewithin the left NST were the principal pathway by which systemic exposure toendotoxin provoked c-Fos activation of brain stem neurons. We propose the followinghypotheses that if brain lateralization is involved in the regulation of the inflammatoryprocess and two sides of NA are differently involved in the modulation ofinflammatory responses. In the current experiment, the LPS inflamed rat model wasestablished, then electrophysiological techniques and immunohistochemistry stainingtechnique were used to investigate whether NA neurons and astrocytes are involved inthe LPS-induced inflammatory response. Meanwhile, the preganglionic neurons ofefferent vagus nerve originate from DMV as well as NA. If NA is involved in the theLPS-induced inflammatory reaction, then whether it is correlated with the cholinergicanti-inflammatory pathway? In this research, we will explore these issues throughthree-part experiment.The first part: We established LPS inflamed rat model by intraperitoneal injectionof LPS, then using electrophysiological techniques to record discharge of the vagusnerve as well as femoral artery blood pressure; using the immunohistochemistrystaining technique to observe the expression of c-Fos in neurons and the expression ofGFAP in astrocytes of DVC and NA.In addition, the ELISA experimental methodwas used to detect the the changes of serum TNF-α and IL-4levels along with thenumber of white blood cells. Thus, the role of NA in inflammatory response as well as the correlation between NA and the DVC (DMV, NTS and AP) duringinflammatory response were explored. The results showed that: compared with thecontrol group, the vagus nerve discharge frequency of the LPS administrated group,significantly increased while the levels of TNF-alpha and IL-4Showed SignificantIncrease.What’s more, the expression of c-Fos and GFAP in NA and DVC wereobviously increased compared to those of the control group. Therefore, we can inferthat LPS triggers inflammation. Futhermore, astrocytes and neurons in NA can beactivated by LPS, indicating that NA is involved in the LPS-induced inflammation.The second part: After the left NA leision was set up, we recorded the vagus nervedischarge frequency as well as the femoral artery blood pressure. Meanwhile, thechanges of serum TNF-alpha, IL-4levels were detected while the expression of c-Fosand GFAP in the DVC were observed, for further exploring the effect of the left NA inLPS-induced inflammation.The results：Compared with the Sham-operated+LPSadministrated group, the TNF-α level in left NA lesion+LPS administrated groupsignificantly decreased while the IL-4level significantly increased. What’s more, thec-Fos-IR neurons in NTS significantly increased after damaging the left NA.Therefore, these results suggest that the left NA mediates the regulation ofLPS-induced inflammatory response. Moreover, the left NA has modulatory effect onpro-inflammatory cytokines such as TNF-α and IL-4except for activating neurons inthe NTS.The third part: After the right NA was lesioned, the same methods and detectionindex as the part two were adopted to investigate the modulatory effect of right NA onLPS-induced inflammation. The results：Compared with the Sham-operated+LPSadministrated group, the TNF-α level in right NA lesion+LPS group has no significantchanges while the IL-4level was significantly increased. Besides, the expression ofc-Fos and GFAP in DVC have no changes. The results: The right NA inhibit thegeneration of anti-inflammatory cytokine, while have no significant role in thepro-inflammatory cytokines, we can infer that there is different roles of right and left NA in inflammatory response.