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 on several factors, but resolving the inflammation and restoring homeostasis is a critical step to avoid the onset of chronic inflammatory diseases. Recent advances in the field of neuroscience and immunology have unraveled specific neural mechanisms with immense therapeutic potential to control inflammation both in the central nervous system and in the periphery. One of these mechanisms is the cholinergic anti-inflammatory pathway (CAP), also known as the inflammatory reflex, which is driven by the autonomic nervous system^4,5.

It is currently thought that inflammatory mediators activate sensory nerves and send signals concerning the state of inflammation to the central nervous system. A reflex response is then activated through the efferent vagus nerve. An extensive study on the anatomical details of the CAP has revealed a parasympathetic-sympathetic model composed of two nerves, the vagus nerve and splenic nerve, respectively⁶. In the CAP, the activated cholinergic efferent vagus nerve ends in the celiac-mesenteric ganglion, resulting in the activation of the adrenergic splenic nerve by a mechanism yet to be explored. The splenic nerve, thus activated, is known to innervate in close proximity to immune cells in the white pulp, marginal zone, and red pulp of the spleen, the principal and mandatory organ of the CAP^7,8. Norepinephrine (NE) from the splenic nerve endings binds to the corresponding β₂ adrenergic receptors expressed on splenic T lymphocytes. This induces choline acetyl transferase (ChAT)-mediated acetylcholine (ACh) release, which in turn activates α7 nicotinic acetylcholine receptors (α7nACh) on macrophages, thereby limiting cytokine production and inflammation². Consequently, it is now clear that the nervous system is able to regulate inflammation in peripheral tissues and to restore local immune homeostasis.

As the name of the pathway suggests, the ACh system is of central importance to the functioning of this neuro-immune regulating pathway. Interestingly, the mechanisms involved in the activation of the CAP seem to be different in the periphery and in the central nervous system. While the importance of nicotinic receptors (α7nAChR) in the spleen has been demonstrated earlier⁹, muscarinic receptors (mAChR) are mandatory for the central activation of the pathway^10,11. More recently, peripheral administration of a centrally-acting M1 muscarinic agonist significantly suppressed serum and spleen tumor necrosis factor α (TNFα) during lethal murine endotoxemia, an action that required intact vagus nerve and splenic nerve signaling¹². We have also shown recently that mice lacking prostaglandin E₂ (PGE₂) were not able to respond to vagus nerve stimulation and did not down-regulate the LPS-induced release of cytokines in the serum and spleen³. Therefore, the CAP might also be regulated by systems other than the main ACh pathway.

The vagus nerve has been named as such because of its wandering course in the body, innervating principal organs including the liver, lung, spleen, kidneys, and gut¹³. Considering this large innervation and the very potent immunosuppressive effect of the vagus nerve, the therapeutic potential of the CAP could cover a wide range of inflammatory conditions. The vagus nerve can be electrically (or mechanically) activated, with control over the voltage and frequency, and contrary to conventional treatment, with no drugs added to the body. Trials are currently underway in rheumatic patients, for instance, to test the clinical significance of VNS in treating chronic inflammation¹⁴. Altogether, the neuro-immune communication and regulation of inflammation are currently under investigation, which will provide a possible alternative treatment to conventional therapy. Therefore, analysis of the vagus nerve stimulation effect in the different innervated organs, but also characterization of the potential therapeutic action in animal models of chronic inflammation, would definitely give insights and hope for new potential therapeutic targets.

The original methodology developed by Tracey and colleagues⁴ could not be transposed to our field of research due to overstimulation of the inflammatory response (by a lethal dose of LPS) and a too-short time range between CAP activation and the read-out. In the present paper, we will present the changes made to the original protocol, compare the two different methodologies on cytokine levels, and highlight a new and opposite observation on the target organ (the spleen).

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 system linked to the stimulating electrode (Figure 1A).
2. Enter the Acknowledge program.
3. Prepare a stock solution of LPS at a concentration of 5 mg/ml in 1x PBS, aliquot it, and store it at -20 °C. On the day of the experiment, thaw an aliquot and prepare an adequate sample of LPS (0.5 mg/ml) in order to inject around 100 µl into the animal, according to the weight.

2. Preparing the Animal for Anesthesia

1. Use C57Bl/6 mice. Maintain them under climate-controlled conditions with a 12-hr light/dark cycle, feed them standard rodent chow, and give them water ad libitum.
2. Perform surgery on mice that weigh around 25 g on the day of the experiment.
   NOTE: When inflammatory reactions are induced, it is particularly important to perform regular checkups and observations of the clinical reactions in the animals. Premature euthanasia by CO₂ inhalation is needed if the animal condition does not fulfill the ethical criteria.
3. Set the anesthetic machine.
   1. Make sure that the tubing is properly connected and is not damaged in any way. Make sure that the ventilated area is working properly. Connect the key filter to the isoflurane bottle and fill the vaporizer with a sufficient amount of isoflurane.
   2. Open the gas supply (air and oxygen) and make sure that the bottles contain enough gas for the experiment. A three-way connector is then able to send the isoflurane flow to the induction chamber or the mask.
4. Turn the three-way connector to the induction chamber. Pick one mouse from the home cage and insert the animal into the chamber. Adjust the flow regulator to 1.0 L/min oxygen and 1.0 L/min air. Adjust the isoflurane concentration to 4 - 5%.
5. When the desired level of anesthesia is reached, When the desired level of anesthesia is reached, shave the surgical area and move the animal from the chamber to the mask. Turn the three-way connector to the mask flow. Adjust the flow regulator to 0.25 L/min oxygen and 0.25 L/min air.
   1. Adjust the isoflurane concentration to 1.5 - 2.5%. Check the level of anesthesia by reflex control and respiratory rate before starting the surgical procedure.
6. Fix the legs of the mouse to the work bench using adhesive tape. Make sure that the nose of the animal is still carefully positioned in the mask.

3. Surgery and Stimulation of the Vagus Nerve

1. Disinfect the surgical area with 70% ethanol.
2. Using a scalpel, carefully incise the skin at the level of the neck (incision of around 1 to 1.5 cm).
   Note: In the protocol, the surgery procedure ends here for SHAM-operated animals. Indeed, it has been shown that just touching the nerve with a metal tool already stimulates it to some extent. Therefore, to get a more accurate surgery control animal when using a small amount of LPS, stop the surgery at this step.
3. With the help of a microscope (12.5X objective), isolate the left vagus nerve from the carotid artery using dissecting forceps. First locate the sternocleidomastoid muscle by removing skin and fat layers, and then retract it in order to put the forceps behind both the nerve and the artery.
   Note: The following step is very tricky, as the nerve and artery are closely adhered to each other. Therefore, it is very easy to cut the vessel and kill the animal. However, by placing the forceps very carefully between the nerve and artery, they eventually separate, and it is possible to isolate the nerve.
4. Place the electrode (Figure 1B) under the vagus nerve. The needle electrode is quite long, so even if the nerve moves slightly during the stimulation, it will always be in contact with the electrode.
5. Perform an intraperitoneal (i.p.) injection of LPS (2 mg/kg) with the help of a syringe (for the read-out on cytokine levels; i.e., to measure the down-regulating effect).
6. Wait 5 min before starting the stimulation.
7. Stimulate the vagus nerve for 5 min at 5 V and 1 Hz by pushing the start button in the Acknowledge program.
8. Remove the electrode and suture the wound of the animal with surgical suture thread.
9. Spray a No Sting Barrier Film (NSBF) on the wound in order to improve healing and to protect from infections.

4. Recovery of the Animal

1. After surgery, move the animal back to its home cage for awakening and recovery. Under infra-red light in order to maintain body temperature, make sure to monitor the animal until full consciousness has been regained.
2. Let the animal recover in its cage for 6 hr before sacrifice for analysis.
   Note: The effect of the vagus nerve stimulation is very fast and has also been shown to be long lasting (up to 48 hr), so the recovery time can be set by the experimenter according to the needs of the study.

5. Sacrifice for Further Analysis

1. Place the animal in a cage linked to a CO₂ administration device.
2. Set the device on a 5-min cycle of CO₂ inhalation.
3. When the euthanasia is done, collect the organs of interest and directly freeze them on dry ice for further analysis (e.g., the measurement of cytokine levels in spleen extracts using a mouse TH1/TH2 9-Plex assay)³.

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 intrap...

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

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