Intrabronchial Instillation of Streptococcus pneumoniae in Immunocompetent Rodent Models

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Anesthetize, Intubate, and Inoculate Animals

NOTE: Specific pathogen-free (SPF) immunocompetent male Sprague-Dawley rats weighing 100 – 120 g are recommended. Provide all animals with food and water ad libitum and house them socially on wood chips or other absorbent bedding with standard 12-hour light-dark cycles.

1. Prior to conducting any experiments, obtain and sterilize a metal cannula and polyethylene tubing of the appropriate size for the animal species. Obtain sterile 1 mL disposable syringes and sterile disposable 25-gauge needles.
   Caution: Always dispose of these syringes and needles in a sharps container.
   1. For rats, purchase or manufacture a metal cannula that is approximately 12 cm in length, has an ID of 0.9 – 1.0 mm and OD of 1.0 – 1.2 mm and is bent to an approximate 45° angle at one end. Cut an 11- to 12-inch length of polyethylene tubing with an ID of 0.4 mm and OD 0.8 mm. (Refer to Figure 1A.)
   2. Place the metal cannula in a glass, screw-cap tube, and autoclave by standard methods to sterilize. Soak and store cut lengths of the polyethylene tubing in a covered beaker containing alcohol.
2. Prepare the work surface and intubation device.
   1. Place a clean disposable mat onto the work surface, close to the water bath. Transfer the metal cannula, in its glass storage tube, to this surface. Remove the cap from the storage tube and slide the "handle" end of the cannula to the open end of the tube.
   2. Using sterile forceps, remove one length of polyethylene tubing from the beaker and insert it through the inside of the sterile metal cannula, making sure it moves freely. Fit a sterile disposable 25-gauge needle (for rats) onto the free end of the polyethylene tubing by sliding the needle several mm into the tubing. (Refer to Figure 1A for rat).
   3. Place guide marks on both the metal cannula and polyethylene tubing to indicate the depth of insertion in the animal by following the intubation procedure described below on a single euthanized animal. Open the chest cavity for observation, then place the metal cannula properly and advance the polyethylene tubing appropriately. Using an indelible pen, mark the metal cannula where it meets the mouth of the animal and the polyethylene tubing where it meets the top of the metal cannula to aid proper placement in the remaining animals.
3. Within a fume hood (or using an appropriate scavenging system), anesthetize up to 6 animals at a time in a closed chamber supplied with ≤5% isoflurane in 1.5 L/min oxygen until the gag reflex is inhibited (approximately 1 min).
   NOTE: Prior to conducting any experiments, establish the safe dose and exposure time for isoflurane under the specific conditions being used (e.g. size/type of chamber and size/species/strain of animal) to prevent inadvertent lethal exposure.
4. Fill a sterile disposable 1 mL syringe (for rats) with sterile saline by placing the sterile tip into the saline and pulling it back on the plunger. Attach the syringe to the needle fitted onto the polyethylene tubing and flush the entire volume of saline through the tubing until the syringe is emptied.
5. Fill the syringe with the agar-based inoculum from the container in the water bath, and flush agar through the tubing until the tubing is primed (e.g. completely filled with the agar).
   1. For rats, remove the 25-gauge needle (fitted onto the polyethylene tubing) from the disposable syringe. Discard the used syringe in a sharps container. Fill a new, sterile disposable 1 mL syringe with the agar-based inoculum from the container in the water bath by placing the sterile tip into the inoculum and pulling back on the plunger. Re-attach the needle (fitted onto the polyethylene tubing) and flush agar through the tubing by depressing the plunger just until the tubing is completely filled with agar.
6. Remove one animal from the anesthetic chamber. Place the animal in a supine position on the disposable mat with the head to the right and tail to the left as shown in Figure 1C.
7. Using the intubation device (i.e. the metal cannula fitted with polyethylene tubing), intubate the animal and insert the cannula into the large lobe of the left lung (see Figure 1D).
   1. Insert the free end of the metal cannula into the animal's mouth. Turn the cannula so that the free end is angled upward and gently advance it into the trachea, carefully bypassing the laryngeal structures with a slight twisting motion. Confirm insertion into the trachea as opposed to the esophagus by gently sliding the cannula slightly forward and back several times while palpating the tracheal rings with the left forefinger as shown in Figure 1C.
   2. When the cannula reaches the bifurcation where the trachea splits into the left and right bronchi, make a slight twisting motion toward the animal's left side to ensure that the cannula is inserted into the left bronchus. Advance the metal cannula until the end is halfway to three-quarters down the left lung, using the previously placed guide mark to confirm that the appropriate depth has been reached.
   3. With the metal cannula in place, advance the polyethylene tubing several mm. Use the previously placed guide mark on the tubing to ensure it is advanced only far enough to exit the end of the metal cannula and not puncture the lung.
8. With both cannulae in place, use the attached syringe to instill 100 µL (for rats) of the agar suspension deep into the large lobe of the left lung (see Figure 1D). Withdraw several mm of the polyethylene tubing, and then gently remove the intact intubation device. Set it back into the glass storage tube and move the animal into a fresh cage to recover.