The authors are grateful for the experimental help in developing the method of intralymphatic immunization in mice from Iris Erdmann, Barbara von Beust, and Julia Maria Mart&#237;nez-G&#243;mez. Thanks also to Maggy Arras and Nikola Cesarovic for letting us use their surgical theatre for this video production.&#160;

1. Anesthesia of Mice
<ol>
	<li>Prepare the anesthetics by mixing ketamine (dissociative anesthetic) and xylazine (sedative and analgesic) in buffered saline. The concentrations of ketamine and xylazine in the final solution are 12.5 and 2 mg/ml, respectively.</li>
	<li>Inject the anesthetics to the mice by intraperitoneal administration using a syringe with a 25-30 G needle. Use 0.1 ml/10 g of body weight.</li>
	<li>Apply an ophthalmic ointment to the mice&#8217;s eyes in order to prevent drying-out of the corneas.</li>
	<li>Assure that the mouse is sufficiently anesthetized by pinching its foot or toe with the forceps. If the mouse does not react with reflexes to the pinch, proceed to surgery. If the mouse reacts to the pinch with reflexes or muscle contractions, wait 1-2 min and repeat the pinch test. If the mouse still reacts, replace the mouse with a new animal and repeat the procedures from step 1.2 onwards.</li>
</ol>
2. Surgical Incision of the Inguinal Area of the Mouse
<ol>
	<li>Place the mouse on its back.</li>
	<li>Wet one inguinal region with disinfecting ethanol (70%). To provide better disinfection, the inguinal region may be shaved to remove fur prior disinfection with ethanol.</li>
	<li>Take the hind leg and bend the hip joint to produce an approximately 90&#176; angle of the hip joint.</li>
	<li>Using a curved microdissecting forceps, take the mouse skin in the inguinal area where the hip joint is bent and pull the wetted skin up slightly.</li>
	<li>While holding the skin up with the forceps, cut a small incision (&#60;5 mm) through the skin using surgical scissors.</li>
	<li>Place the tip of the closed scissors into the incision and further open the incision by opening the scissors while holding it inside the incision. This will cause the skin to tear and should produce a diameter of less than 10 mm. Note: A cold light source with flexible light guides can be used to improve visibility. In older mice, especially males, the identification of the lymph node may be difficult due to much fat tissue in the inguinal region.</li>
</ol>
3. Intralymphatic Injection
<ol>
	<li>Prepare a syringe (0.5 ml or less) with a 28-30 G hypodermic needle; a short bevel may be preferable to a long bevel. Aspirate 10 &#956;l of the vaccine to be injected and assure that the syringe is free of air.</li>
	<li>Localize the inguinal lymph node with help of the curved forceps and the tip of the closed scissor. The lymph node will appear greyish within the more whitish fat tissue and can be further identified through afferent and efferent capillaries entering and leaving the lymph node.</li>
	<li>Immobilize the lymph node by holding it between the branches of the curved forceps.</li>
	<li>Take the syringe and insert the needle into the lymph node with the bevel facing up; making sure the whole bevel enters the lymph node.</li>
	<li>Inject the vaccine (10 &#956;l). If the lymph node is swelling (blowing up), the injection can be assumed successful. If the needle is not placed deep enough into the lymph node, some or the entire vaccine may leak out and not enter the lymph node. If the needle is inserted too deep, the injected vaccine will be released subcutaneously below the lymph node. In case of the latter, repeat the injection or replace the mouse with a new untreated mouse.</li>
</ol>
4. Closing the Incision by Suturing
<ol>
	<li>Open a pack of sterile surgical suture.</li>
	<li>Take hold of the suture needle with a needle holder at the distal portion of the needle body. Tighten the needle holder by squeezing it until the first ratchet catches.</li>
	<li>Take hold of the skin at one side of the incision using forceps (depending on the surgeon's preference, toothed or un-toothed forceps or skin hooks may be used). Insert the needle ca. 2-3 mm from the edge of the incision, entering from the outside of the skin.</li>
	<li>Loosen hold of the skin with the forceps and grasp the opposite side of the incision with the forceps. Insert the needle ca. 2-3 mm from the edge of the incision, entering from the inside of the skin.</li>
	<li>Loosen the needle holder and grasp the needle end with the forceps. Pull the needle with the tread through the skin, leaving a convenient length of the tread outside the first needle-insertion point (if several mice are to be treated, leave a shorter end; 5-10 mice can be operated on with one suture needle/tread).</li>
	<li>Make the preferred knot according to standard surgeon protocols. One stitch is sufficient if the incision is not larger than 10 mm. For larger incisions, make a second stitch.</li>
</ol>
5. Post-operational Treatment
<ol>
	<li>Place the mouse in the cage and cover with tissues to keep warm. When treating multiple mice, place them close to each other to keep warm. Alternatively, place the mice on a warm pad until they have woken up.</li>
	<li>Observe the mice until they wake up.</li>
	<li>Observe the mice daily with regards to wound healing as well as other clinical symptoms such as wound infections. The wound is typically sufficiently closed within two days and healed within seven days of the procedure.</li>
</ol>

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TMK is named as inventor of patents dealing with intralymphatic immunotherapy in man. TMK has been scientific advisor and has received travel expenses from ImVisioN GmbH, Cytos Biotechnology, MannKind Corporation,&#160;and XBiotech USA Inc. PJ has no conflict interest to disclose.

Intralymphatic immunization and immunotherapy have been shown to be appropriate for stimulation of both antibody responses and T-cell responses. As demonstrated in this video-article, the intralymphatic procedure of vaccination is a quick and easy method for stimulating strong immune responses in mice. A trained surgeon can perform the procedure during 3-4 min. The session can also be shared between two surgeons where one is typically doing anesthesia and the suturing and the second surgeon is doing the incision and the ...

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this procedure requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of T- and B-cell responses. In addition, the strength and the type of immune responses induced also depend on the density or frequency of such interactions as well as the microenvironment itself, especially the content of cytokines. As only a small fraction of a vaccine injected into a peripheral tissue reaches a lymph node, vaccinations of mice and humans by direct injection of the vaccine into lymph nodes, the site were immune responses are generated, have been performed. In man, the procedure is guided by ultrasound, a procedure also used for the administration of imaging agents for visualization and diagnosis in the lymphatic system. In mice, the procedure is invasive. Here, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals1, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control; 10 &#956;l is used for first injections and in young mice with small lymph nodes, whereas 20 &#956;l can be injected into lymph nodes of older or already primed mice, which have larger lymph nodes. The incision can be closed with a single stitch using surgical sutures. By this method, mice have vaccinated with plasmid DNA2,3, messenger RNA4, peptide1,3,5,6, protein7-10, particles11, bacteria12 as well as adjuvants7,13, and strong improvement of immune responses against all type of vaccines have been observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited or very costly. In human, the intralymphatic method of immunization has been applied to allergy patients14,15 or to patients with cancer16-21. Although the current notion is that the intralymphatic method is more invasive than other injectable methods such as intramuscular and subcutaneous injections, the pain perception is not higher than after a venous puncture15. It is expected that intralymphatic vaccination will become an alternative or complement to other methods of prophylactic and especially therapeutic vaccinations. This article describes in details how the procedure of intralymphatic vaccination is performed in mice. All procedures described, were approved by the Cantonal Veterinary Agency of Zurich and performed according Swiss federal guidelines and directives on the protection of animals used for scientific purposes.

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Ketamine (Ketasol-100)</td>
			<td style="width:135px;">Graeub AG, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anesthetics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Xylazine (Rompun)</td>
			<td style="width:135px;">Bayer, Germany</td>
			<td style="width:119px;"></td>
			<td>Anesthetics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Viscotears Eye-Gel</td>
			<td style="width:135px;">Novartis, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To keep eyes from drying out during anesthesia.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">BD Micro-Fine 0.5 ml</td>
			<td style="width:135px;">BD Medical, France</td>
			<td style="width:119px;"></td>
			<td>29 G Insulin syringes with permanently attached needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">6-0 Dermalon Monofilament nylon</td>
			<td style="width:135px;">Covidien, MA, USA</td>
			<td style="width:119px;"></td>
			<td>For sutures (0.7 metric, 18G, 45 cm, Blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Curved forceps, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision and holding of lymph node</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Straight surgical scissors, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Needle holder, 5.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To close incision with suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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intralymphatic immunotherapy and vaccination in mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

The procedure of intralymphatic injections in mice, despite the surgical nature, is straight forward and relatively fast. A trained person can perform the procedure in 3-4 min. The incision that is closed with one stitch typically heals within two days (Figure 1)
Intralymphatic vaccination or immunization has been performed with mRNA, plasmid DNA, peptides, proteins, virus and bacteria. Figure 2 illustrates the antibody production after immunization with the p...

Watch this Scientific Journal Video about Intralymphatic Immunotherapy and Vaccination in Mice at JoVE.com

Intralymphatic Immunotherapy and Vaccination in Mice

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

intralymphatic-immunotherapy-and-vaccination-in-mice

Research

JoVE Journal

Immunology and Infection

22.9K Views.  University Hospital Zurich. Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.