Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Since its discovery in the early 2000s, the mechanisms of the CAP have been thoroughly studied. We now have a good picture of the pathway, and in particular, the target organ, the spleen, where NE, memory T cells, Ach, and macrophages work as a very efficient team to down-regulate inflammatory mediators². We have also recently published data on the importance of a functional prostaglandin system in mice, in particular, PGE₂, which is obviously a mandatory component for ACh release in the spleen after activation of the CAP³.

The experimental technique, known as vagus nerve stimulation, can easily be performed in a laboratory, but a few important protocols must be observed regarding the animals and the surgery procedure. We use a sine-wave stimulation signal, which is supposed to be a "charge-balanced" stimulation, in order to avoid building up electrical charges in the nerve that could destroy it (as opposed to monophasic stimulation). While stimulating the nerve, it is not possible to separate the afferent from the efferent signal, which can only be done for other purposes with vagotomy experiments. The duration and frequency used for the experiment are known to have an effect on cytokine release following inflammatory challenge, for instance, but would not affect the heart rate or give obvious side effects in healthy controls.

First of all, the experimenter must always keep in mind that the handling, surgery, and recovery of the animals must be done according to the ethical rules for animal care. The other point is that practicing the surgery is of great importance. Isolating the vagus nerve is a difficult step, which can be lethal for the animal if vascular damage occurs. With experience, the surgery can be done within 15 – 20 min, meaning that the animal does not have to be anesthetized for a long period of time, which helps the recovery.

A discussion can also be raised regarding the choice of a good SHAM-operated animal. In our protocol, we used animals that only underwent surgery at the level of the neck, without placing the electrode under the vagus nerve. The reason for choosing this option was that just placing the electrode under the nerve would mechanically stimulate the nerve to some extent (as has been observed previously)¹⁹, creating the risk of masking potential mechanisms that occur at very low levels of inflammation. If using very high doses of LPS, the best SHAM-operated control would probably be to place the electrode under the nerve, as the effect of the SHAM surgery would not interfere or mask the obvious electrical stimulation effect on the burst of cytokines.

We have developed the current protocol to fit the different issues encountered within our field of research. As discussed in our previous paper, we looked at the possible involvement of the prostaglandin system in the CAP. Due to enzymatic metabolism, we thought that the 1-hr recovery time of the original protocol would not suit our experiment. Therefore, we extended the time lapse after surgery to 6 hr. In so doing, we also had to titrate down the dose of LPS in order to meet the ethical criteria. We chose a recovery time of 6 hr, a delay that we thought long enough for this purpose. We also titrated the LPS from 15 mg/kg (the original protocol) down to 0 mg/kg. At 2 mg/kg, we saw the last effect of the VNS on cytokines (Figure 2), an effect that disappeared at lower doses.

While the main effect of the VNS in the spleen is known and characterized by the activation of memory T cells and thereafter, macrophages-the cells that allow the decrease of cytokines-we also showed here that VNS seems to directly induce the release of mediators (it is still unclear from which cells, necessitating further study) in order to recruit other cell types from the primary response to inflammation. Indeed, while VNS strongly decreased KC/GRO after a strong inflammatory induction (15 mg/kg LPS; i.e., the original protocol), it activates a very fast release of KC/GRO in the absence of inflammation. The chemokine KC/GRO, a IL-8 related protein with strong chemotactic properties, is known to have an important role in leucocyte development (e.g., driving maturation and activation), trafficking (e.g., attraction and recruitment of neutrophils), and function¹⁵. For instance, neutrophils are important members of the phagocytic system of the innate immune system. They act as the first line of host defense against invading pathogens, but they are also important mediators of inflammation-induced injury¹⁶. Interestingly, in our attempt to improve the protocol, we then came across this observation that highlights the direct involvement of other cell types in the spleen in vagus nerve functioning.

All the pioneering work in the field has focused on the spleen, the target organ of the CAP, and on the immunoregulatory effect of the pathway in response to sepsis or peripheral systemic inflammation. Importantly, this work was done using acute stimulation of the vagus nerve, such as the one presented in this paper. This can be used for molecular analysis of specific organs or of the short-term effects seen in functional studies involving chronic animal models of inflammation (24 to 48 hr maximum following the surgery).

However, the wandering course of the vagus nerve also implies that the CAP could be of significant use to treat chronic inflammatory diseases involving the different innervated organs¹³. One of the most-studied organs in this context is the gut, with a focus on inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, but also the postoperative-induced ileus¹⁷. Moreover, the importance of the CAP regulation in many other inflammatory diseases involving the liver, kidney, and lung is also under investigation¹⁸. Nevertheless, the current protocol could not be used in this context, as the long-term effect of vagus nerve activation would require repetitive stimulations that can of course only be done with an in vivo implanted electrode. For this purpose, the animal should undergo a surgery where stimulating cuff electrodes are placed around the vagus nerve²⁰. It is also important to note that the latter technique is much more frequently described in humans or in animals of a larger size. Another way of stimulating the vagus nerve is mechanical, as non-invasive and percutaneous stimulation of the vagus nerve showed an immunosuppressive effect in a murine model of endotoxemia²¹. However, the main concerns of this technique would be the reproducibility of the results and how to ensure that the vagus nerve is stimulated the same way in a long-term evaluation of the animal models. Devices have been developed for humans, such as trans-auricular VNS stimulation or trans-cervical VNS stimulation, that deliver electrical pulses, which are well tolerated by the subjects²¹. They have been used mainly to treat epilepsy and migraines and have showed promising results that could lead to future ambulant and exogenous therapy.

To summarize, particularly because of the extensive innervation of the vagus nerve in the body, the CAP regulation is studied in a wide range of inflammatory diseases in the hope of finding interesting molecular mechanistic properties that could lead to potential future therapeutic targets. Here, we presented an acute method to stimulate the vagus nerve. It allows the study of the CAP in limited inflammatory responses thanks to a moderate inflammatory stimulus combined with a longer time range between the VNS and the analysis (allowing enzymatic metabolism to take place, for instance).

Understanding the molecular mechanisms underlying the vagus nerve stimulation effect, as well as its efficient use in the treatment of inflammatory diseases, is of great importance, particularly from a clinical perspective. Indeed, the advantages of the methodology are broad. Due to the nature of extremely fast nerve signaling, it is fast and effective; an observable effect on cytokine levels, for instance, occurs in less than 1 hr.⁴ Also, mechanical, non-invasive, and percutaneous stimulation of the nerve can be used, giving hope for future ambulant and easily-administered therapies. Finally, contrary to regular treatment, vagus nerve stimulation uses an endogenous pathway. Therefore, unlike in all drug therapies, no new agents are introduced into the body, thereby avoiding any potential side effects.