The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

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...

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The authors have nothing to disclose.

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...

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 system4,5.
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 CAP7,8. Norepinephrine (NE) from the splenic nerve endings binds to the corresponding &#946;2 adrenergic receptors expressed on splenic T lymphocytes. This induces choline acetyl transferase (ChAT)-mediated acetylcholine (ACh) release, which in turn activates &#945;7 nicotinic acetylcholine receptors (&#945;7nACh) 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 (&#945;7nAChR) in the spleen has been demonstrated earlier9, muscarinic receptors (mAChR) are mandatory for the central activation of the pathway10,11. More recently, peripheral administration of a centrally-acting M1 muscarinic agonist significantly suppressed serum and spleen tumor necrosis factor &#945; (TNF&#945;) 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).

<table border="0" cellpadding="0" cellspacing="0" width="665">
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Computer</td>
			<td style="width:91px;">Toshiba</td>
			<td style="width:136px;">-</td>
			<td style="width:191px;">Any computer is actually compatible</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">MP-150 data acquisition system</td>
			<td style="width:91px;">Biopac Systems</td>
			<td rowspan="2" style="width:136px;">MP150WSW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Acknowledge software</td>
			<td style="width:91px;">Biopac Systems</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Mice C57Bl/6</td>
			<td style="width:91px;">Charles River</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anesthetic machine</td>
			<td style="width:91px;">Simtec Engineering</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical oxygen bottle</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">107563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical air bottle&#160;</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">108639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetflurane (1000mg/g)</td>
			<td style="width:91px;">Virbac</td>
			<td style="width:136px;">137317</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">LPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">L2630</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Saline</td>
			<td style="width:91px;">Merck Millipore</td>
			<td style="width:136px;">1024060080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS 10X</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5493</td>
			<td>Diluted 10 times for used concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe (1 ml)</td>
			<td style="width:91px;">BD Plastipak</td>
			<td style="width:136px;">303172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Needles 23G</td>
			<td style="width:91px;">KD-FINE</td>
			<td style="width:136px;">900284</td>
			<td>0.6 x 30 mm (blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Microdissecting forceps (curved)</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">F4142</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dissecting scissors</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z265969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical suture 4-0</td>
			<td style="width:91px;">Ethicon</td>
			<td style="width:136px;">G667G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Euthanasia unit</td>
			<td style="width:91px;">Euthanex Smartbox</td>
			<td style="width:136px;">EA-32000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Cavilon No Sting Barrier Film</td>
			<td style="width:91px;">3M Health Care</td>
			<td style="width:136px;">3346N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">TH1/TH2 9-Plex assay, ultrasensitive kit</td>
			<td style="width:91px;">MesoScale Discovery</td>
			<td style="width:136px;">K15013C-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

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.

Level of TNF&#945; and Interleukin-1&#946; (IL-1&#946;) 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 TNF&#945; (169.3 &#177; 24.9 pg/mg in SHAM versus 39.7 &#177; 10.8 pg/mg in VNS, p &#60; 0.001) and IL-1&#946; (360.0 &#177; 40.21 pg/mg in SHAM versus 191.7 &#177; 27.2 pg/mg in VNS, p &#60; 0.01) in the spleen after intrap...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

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

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.

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

The overall goal of this surgical intervention is to observe the effect of the vagus nerve stimulation in inflammatory conditions. To analyze and understand the different mechanisms and cells involved in the so-called cholinergic anti-inflammatory pathway. This method was developed in the Kevin Tracey lab in the Feinstein Institute.The method can help answer key questions in the neuroimmunology field. As recent advances establish a close link between the nervous and immune systems. The main advantage with this technique is that it can be easily set up in a laboratory.But important rules have to be followed regarding animal care and the surgery protocol. The wandering course of the vagus nerve throughout the whole body, and its ability to down regulate information in various animal models implies that its stimulation could be useful in the treatment of many inflammatory diseases. Usually individuals new to this technique will struggle to identify and isolate the vagus nerve from the carotid artery under microscopic observation.However, although it is tricky at the beginning it becomes easier with time and practice. Begin by turning on the computer and the data acquisition system linked to the stimulating electrode. Launch the data acquisition and analysis software.Then follow an aliquot of 5 milligrams per milliliter lipopolysaccharide or LPS and dilute it to zero point five milligrams per milliliter with saline. Anesthetize a 25 gram mouse in an induction chamber. When the desired level of anesthesia is reached, move the animal from the chamber to the mask.Turn the three-way connector to feed the mask, and adjust the flow regulator. Check for a surgical plane of anesthesia by loss of the righting reflex and a regular respiratory rate of around 100 breaths per minute before starting the surgical procedure. Once the mouse is properly anesthetized, fix the legs of the mouse to the workbench with adhesive tape.Make sure that the nose of the animal is still carefully positioned in the mask. Properly disinfect the surgical area according to the approved procedures. Then, after making a one to one point five centimeter incision in the skin at the level of the neck, position a stereo microscope with a 12 point five x objective to view the surgical area.Locate the sternocleidomastoid muscle by removing the layers of skin and fat with forceps. Retract the sternocleidomastoid muscle and move the forceps behind both the left vagus nerve and the carotid artery. Isolate the nerve from the artery by placing the forceps very carefully between the nerve and artery.This is a critical step in the procedure, as the nerve and the artery are close to each other, it's very easy to cut the vessels and kill the animals. However by placing very carefully the forceps between the nerve and the artery, the eventually separate and it's possible to isolate the vagus nerve. Now, place the electrode under the vagus nerve.As the needle electrode is quite long, the nerve will always be in contact with the electrode even if it moves slightly. Next perform an intraperitoneal injection of LPS and wait five minutes before starting the stimulation. After five minutes, stimulate the vagus nerve for five minutes at five volts and one hertz by pushing the start button in the acquisition software.When the stimulation period is over, remove the electrode and then stitch the wound of the animal with surgical suture thread. Spray a no-sting barrier film on the wound in order to improve healing and to protect from infections. Finally, move the animal into a clean cage and monitor as it regains full consciousness.The following images demonstrate the effect of reduced LPS concentration and increased latency to sacrifice on levels of TNF alpha and IL one beta following vagus nerve stimulation. Here, it can be seen that the original paradigm significantly decreases both TNF alpha and IL one beta compared to sham operated controls. The protocol demonstrated in this video significantly reduces the levels of TNF alpha but not IL one beta.As seen here, a six-hour recovery without LPS injection does not affect levels of TNF alpha or IL one beta. Once mastered, this technique can be done in approximately 20 minutes if it is performed properly. After its development, this technique has paved the way for researchers in the field of neuroimmunology.To study the connection between the central nervous and the immune system. And also study the vagus nerve innervation of new organs and the effect of the cholinergic anti-inflammatory pathway in chronic inflammatory diseases. After watching this video, you should have a good understanding of how to locate, isolate and stimulate the vagus nerve.And also keep in mind that the level of induced inflammation and the time for recovery of the animal might be important issues to take into account.

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.7K 조회수.  Karolinska University Hospital. The overall goal of this surgical intervention is to observe the effect of the vagus nerve stimulation in inflammatory conditions. To analyze and understand the different mechanisms and cells involved in the so-called cholinergic anti-inflammatory pathway. This method was developed in the Kevin Tracey lab in the Feinstein Institute.The method can help answer key questions in the neuroimmunology field. As recent advances establish a close link between the nervous and immune systems. The...