Adult Thymectomy

Removal of the thymus in an adult mouse creates a "status quo" in the immune system: no new T cells will be generated, allowing the analysis and experimental manipulation of existing T cells without any dilution by incoming T cells.

Thymectomy of the adult mouse is similar to that of the neonate; however, as the thymus undergoes considerable enlargement in the first few weeks of life, some adjustments in technique are required as described below. Protocols for young and adult thymectomy in rats are described in unit 15.12. Two procedures are presented: the "suction" method is similar to that described in neonatal thymectomy except a larger cannula is used, while in the "dissection" method, the large adult thymus is manipulated and removed with forceps.

Materials

Mouse (>3 weeks old) 70% ethanol

Dissecting board (~7 x 10-cm) Elastic bands

Iris scissors (10-cm straight) Iris forceps (2 pair of 10-cm half-curved) Sterile 5 x 5-cm gauze sections Suction cannula (support protocol)

Rubber tubing, 6-mm diameter

Clay Adams autoclip applier (#7630-000-900) and 9-mm wound clips (#7631) Incandescent desk lamp

Additional reagents and equipment for anesthesia (unit 1.4) and intraperitoneal injection (unit 1.6)

Anesthetize and dissect the mouse

1. Place scissors and forceps in a small beaker with 70% ethanol.

2. Anesthetize mouse by intraperitoneal injection.

Pentobarbital is recommended for this procedure (see Table 1.4.1). The anesthesia must be deep enough to avoid any spontaneous movement by the animal. As with all surgical procedures, anesthesia is crucial for the success of an operation (see unit 1.4). Mice should have slow, shallow breathing and should not respond to squeezing the hind feet.

3. Place the mouse on the dissecting board in the dorsal position with its head facing the operator. As illustrated in Figure 1.10.3, place a rolled-up gauze pad under the mouse's shoulders to aid in pushing the heart and thymus forward for easier access. Restrain the arms and legs by placing them under parallel elastic bands. Extend the mouse's neck by securing the head with a third rubber band placed in its mouth.

4. Swab neck and upper chest area with 70% ethanol. With scissors, begin a midline longitudinal skin incision over the suprasternal notch. Extend the incision 2 to 3 cm down the chest.

5. Loosen the skin from the underlying muscle using the blunt end of the forceps. Reflect the skin to expose the thoracic cage.

6. Stabilizing the thoracic cage with your free hand, insert scissors under the sternum and cut to the second or third rib.

In some mice the sternum and thus the midline are difficult to identify; in this case, it is prudent first to identify the trachea by freeing and retracting the submaxillary glands. The trachea will mark the midline as well as the point of insertion of the scissors into the chest. The second ribs are easily identified as they form the first prominent "v" from the suprasternal notch where they join the sternum.

Suprasternal Notch Mice
Figure 1.10.3 Adult thymectomy. (A) Position of the adult mouse secured to the operating board with rubber bands. (B) Location of the sternal notch. The incision is made in the midline from the sternal notch to the second or third rib (blowup).

Care and Handling of Laboratory Animals

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