A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

The protocol provides an accurate, safe, and efficient non-surgical alternative to traditional surgical and blind injection methods. It is also the most accurate injection technique for atrophy thymuses in aged or immunodeficient mice. The key advantage is a minimally stressful injection procedure that is very safe and precise, but also rapid, thus enabling large-scale experiments without data compromise from surgical stress-related immunosuppression.

Maintaining alignment of the needle and transducer while advancing it through the chest wall takes some practice. We therefore recommend collaborating with an experienced interventional radiologist to perform injections or provide training. Helping to demonstrate the procedure will be Shaina Anuncio, a technician from my laboratory.

To begin, apply a thin layer of depilatory cream to the anterior chest area of the mice for less than one minute to remove the fur. Use a wet paper towel to remove the cream and loose fur. Confirm the appropriate anesthetic depth by unresponsiveness to the hind paw pinch.

Apply ophthalmic ointment to both eyes to prevent corneal drying. Place one mouse at a time in the supine position on the heated platform of the small animal ultrasound imaging station with the nose cone in place. Secure the mouse to the stage with medical adhesive tape at the hind and fore limbs.

Disinfect the fur-free upper thorax skin using a chlorhexidine gluconate applicator. Activate the highest frequency linear probe available. Typically, the probe with the highest spatial resolution for the size of the animal being imaged.

Activate the probe by tapping the corresponding button after the start up screen. To optimize the ultrasound settings for imaging and injection, adjust the depth of the field of view to an appropriate size for the target animal by adjusting the vertically oriented sliders on the right side of the screen. Adjust the gray scale gain by sliding the button along the horizontal bar on the bottom of the screen.

Then, adjust the focal zone to the anticipated level of the thymus. Apply about one milliliter of ultrasound gel to the transducer surface while it is upright, either resting in the ultrasound machine holder or in the hands of an assistant. To prepare a small sterile field next to the heated platform, empty a sterile probe cover, rubber band, sterile gloves, and sterile ultrasound gel onto the sterile field.

Then, put on the sterile gloves. Carefully place the sterile probe cover over the ultrasound transducer and gel. To maintain sterility, touch only the sterile cover.

Slide the sterile rubber band over the probe cover to keep it in place. Place two to three milliliters of sterile ultrasound gel onto the transducer. Place the ultrasound gel-topped probe vertically on the disinfected portion of the anterior chest wall of the mouse for initial imaging, while maintaining sterility.

View and optimize the ultrasound image as demonstrated earlier. To scan the anterior chest of the mouse in a transverse plane, hold the transducer vertically and move it up and down from the neck to the abdomen in a paintbrush-like or sweeping motion. With the heart centered in the field of view, sweep the transducer slightly toward the neck.

Note the two round paired black structures on either side of the upper chest. Using the ultrasound probe, locate the widest portion of the thymus, which is usually the ideal target site for injection. Anticipate a horizontal needle trajectory in the chosen location, and note where the major blood vessels are located at this site.

These vessels should be avoided during the injection. The blood vessels will be hypoechoic pulsatile structures. If unsure, activate the color Doppler mode by tapping the color button on the screen to check for flow within the vessels.

Hold the transducer in one hand and a 30-gauge insulin needle with 10 microliters of injectate in the other. To begin the injection process, move the transducer laterally so that the thymus is off-center in the ultrasound field of view. Ensure that the other side of the field of view consists of mostly ultrasound gel and nothing else.

Place the tip of the needle in the gel under the transducer, and slowly move the needle until it is visualized adjacent to the skin surface. While continuously imaging the needle under ultrasound, insert the needle into the thymus gland with a percutaneous trajectory away from blood vessels. Use a cross thymus horizontal trajectory to place the needle tip in the thymic lobe contralateral to the entry site.

Once the needle tip is inside the desired portion of the thymus, quickly inject the contents from the 30-gauge syringe while using sonographic visualization. To stabilize the syringe during needle insertion and injection, hold the syringe between the thumb and third finger, and control the syringe plunger with the index finger. Remove the needle after all the contents have been deposited.

In young mice, identifying the thymus gland was straightforward due to the gland's large size. It was more challenging in older or immunodeficient mice. However, it was still very feasible with modern ultrasound equipment.

Successful injection was noted when the needle did not traverse critical structures, such as the heart, aorta, or one of the inferior vena cavae. The needle tip was visualized in its target location during the injection, confirming the intrathymic deposition. The intrathymic location of the injection was confirmed using Luciferin as an injectate into luciferase transgenic mice.

These were evaluated immediately with bioluminescence imaging, confirming the injection's correct location without sacrificing the animal. Alternatively, Trypan Blue was injected as a visual marker of the injection site and injection accuracy using ex vivo with necropsy. Our group has been using this technique for several large studies, including cancer immunotherapy research, based on the injection of genetically modified progenitor cells, and to improve thymic function in immunodeficient mice.