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

Summary

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Abstract

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses).

The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Introduction

Innate immunity provides an immediate first line of defense against infections and diseases in a wide range of organisms. It not only initiates the primary immune response to eliminate the threat, but it also plays a pivotal role in activating and educating the adaptive immunity that carries out secondary immune responses in a pathogen-specific manner. Inflammation is orchestrated by a plethora of cytokines and chemokines, which in turn have the ability to attract other immune cells to the site of infection and to induce the cardinal signs of inflammation, such as redness, swelling, pain, loss of function, and fever. The duration and intensity of inflammation depend o....

Protocol

All animal experiments were performed according to the guidelines for the care and use of animals approved by the local Ethics Committee at Karolinska Institutet, Stockholm. The local Ethics Committee follows the European Union directives on animal care.

NOTE: The main changes from the original protocol are the recovery time after surgery (6 hr versus 1 hr) and the level of LPS injected (2 mg/kg versus 15 mg/kg). Otherwise, the different settings related to the surgery itself have not been changed.

1. Preparing Material for Stimulation

1. Turn on the computer and the data acquisition sy....

Results

Level of TNFα and Interleukin-1β (IL-1β) after Increasing the Time Lapse after Surgery and Decreasing the Dose of LPS

As shown previously, using the original protocol, VNS decreased the levels of TNFα (169.3 ± 24.9 pg/mg in SHAM versus 39.7 ± 10.8 pg/mg in VNS, p < 0.001) and IL-1β (360.0 ± 40.21 pg/mg in SHAM versus 191.7 ± 27.2 pg/mg in VNS, p < 0.01) in the spleen after intraperitoneal LPS injection (15 mg/kg) (Fig.......

Discussion

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 th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Computer	Toshiba	-	Any computer is actually compatible
MP-150 data acquisition system	Biopac Systems	MP150WSW
Acknowledge software	Biopac Systems
Mice C57Bl/6	Charles River
Anesthetic machine	Simtec Engineering
Medical oxygen bottle	AGA	107563
Medical air bottle	AGA	108639
Vetflurane (1,000 mg/g)	Virbac	137317
LPS	Sigma-Aldrich	L2630
Saline	Merck Millipore	1024060080
PBS 10x	Sigma-Aldrich	P5493	Diluted 10 times for used concentration
Syringe (1 ml)	BD Plastipak	303172
Needles 23 G	KD-FINE	900284	0.6 x 30 mm (blue)
Microdissecting forceps (curved)	Sigma-Aldrich	F4142
Dissecting scissors	Sigma-Aldrich	Z265969
Surgical suture 4-0	Ethicon	G667G
Euthanasia unit	Euthanex Smartbox	EA-32000
Cavilon No Sting Barrier Film	3M Health Care	3346N
TH1/TH2 9-Plex assay, ultrasensitive kit	MesoScale Discovery	K15013C-1
Stimulating electrode device	Biopac Systems	STIMSOC
Aesculap Isis shaver	Agnthos	GT420
R70			Rodent diet from Lantmannen, Stockholm, Sweden