The overall goal of this procedure is to stimulate the immune responses of mice by directly injecting vaccine into their lymph nodes. This is accomplished by first anesthetizing the mouse and making a small incision in the inguinal region. Next, locate and immobilize the inguinal lymph nodes.
The final step is to inject vaccine into the inguinal lymph node. Ultimately, intra lymphatic vaccine administration can be evaluated by measuring its response ti and comparing it to traditional delivery methods such as subcutaneous injections. Generally individuals new to this method, which struggle to hit the lymph node with a vaccine.
It may take some training to coordinate the immobilization of the lymph node, the insertion of the needle, and the injection of the vaccine cause the needle easily gets outta position and then the vaccine is injected outside the lymph node. To begin anesthetize the mouse as described in the accompanying text protocol. As soon as possible, apply an ophthalmic ointment to the mouse's eyes in order to prevent the corneas from drying out.
Then place the mouse in a supine position and pinch the mouse's foot with forceps to verify it is sufficiently anesthetized. If no reaction is observed, place a tissue over the mouse's head for protection from light and disinfect one inguinal region with 70%ethanol. Then lift the hind leg and bend the hip joint to a 90 degree angle.
Now with curved micro dissecting forceps, pull up the wedded skin in the bend and cut a small incision through the skin. Then place the tip of the closed scissors into the incision. Open the scissors to tear a diameter of less than 10 millimeters in the inguinal region.
Identify the lymph node by its spherical and grayish appearance against the whiter surrounding fat tissue. Use a 0.5 milliliter syringe with a 28 gauge short bevel hypodermic needle to aspirate 10 microliters of the vaccine to be injected. Now immobilize the lymph node between the branches of the curved forceps.
Then with the bevel facing upward, insert the syringe needle into the lymph node. Make sure the whole bevel enters the lymph node. Inject the vaccine and look for the swelling that indicates a successful injection.
Open a pack of sterile surgical suture with a needle holder. Take hold of the suture needle at the distal portion of the needle body. Then tighten the needle holder by squeezing it until the first ratchet catches and pull the thread out.
Next, take hold of the skin at one side of the incision. Insert the needle from the outside of the skin at the edge of the incision. Now loosen the hold on the skin and grasp the opposite side of the incision with the forceps.
Insert the needle from the inside of the skin about two to three millimeters from the edge of the incision. Again, grasp the needle. End with the forceps and loosen the needle holder.
Pull the needle with the thread through the skin, leaving a convenient length of the thread outside the first needle insertion point. Make the preferred surgical knot. If the incision is larger than 10 millimeters.
Make a second stitch when the incision is sufficiently closed. Transfer the mouse to a warming pad to recover. Monitor the mice daily for wound healing as well as other clinical symptoms such as infection.
These intra lymphatic injections in mice are straightforward and relatively fast. An incision that is closed with one stitch typically heals within two days.Here. Antibody production was measured after immunization with the protein phospholipase A two.
The major B venom allergen. The intra lymphatic root stimulated strong antibody responses with a 0.01 microgram injection of PLA A two. While a 10 microgram subcutaneous injection of PLA A two was required to achieve an adequate response.
In this experiment, intra lymphatic vaccination was used to stimulate cytotoxic T-cell responses. A single subcutaneous injection of TCR 3 1 8 mice with G 33 elicited and expected cytotoxic eight T-cell response. That was amplified approximately six orders of magnitude after an intra lymphatic injection CD four T-cell functions were also tested in a tumor challenge assay.
When the immunized mice were challenged intravenously with NP 52 expressing syngeneic EL four tumor cells, the mice developed systemic lymphoma. Immunization by the intra lymphatic root resulted in much longer survival times than immunization by the subcutaneous root. In humans.
Intra lymphatic vaccine administration is performed with the aid of ultrasound by allocating subcutaneous lymph nodes. Interestingly in humans, radiolabeled protein tracers indicate that subcutaneous injections poorly to lymph nodes biodistribution after direct intra lymphatic injection to the lymphatic system facilitates efficient delivery to immune cells for immune response stimulation. Once mastered, this procedure of int lymphatic vaccination can be performed in approximately two to three minutes per mouse.
