Development of a Rat Lung Infection Model Using Intratracheal Bacterial Inoculation

Resumo

Source: Nguyen, J. Q., et. al. Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis. J. Vis. Exp. (2017)

This video demonstrates the procedure of intratracheal inoculation in an anesthetized rat, involving catheter placement, bacterial delivery into the trachea, and subsequent lung infection establishment for studying host-pathogen interactions.

Protocolo

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

1. Intratracheal Inoculation

1. Remove the rat from the anesthesia chamber and place it dorsally on the rodent intubation stand. Attach the rat's front teeth to the holder to keep the rat in place.
   NOTE: If the rat starts to awaken during the procedure, the catheter can be removed and the rat returned to the anesthesia chamber to reach a deeper plane of anesthesia.
2. Use the dominant hand to move the tongue to the same side as the support hand with broad-point dressing thumb forceps.
3. Use your support hand and secure the tongue with the laryngoscope against the rat's lower jaw and visualize the trachea and esophagus of the animal. The trachea will open and close as the rat breathes.
4. Insert the catheter containing a 20-gauge blunted needle into the trachea. There may be slight resistance, and insertion into the trachea may be "bumpy" due to the catheter rubbing the tracheal cartilages. Due to moisture or shallow respiration, the trachea can be covered by the epiglottis, which prevents visualization of the trachea. Gently touching the edge of the epiglottis cartilage will cause the cartilage flap to open and uncover the trachea.
5. Remove the blunted needle from the catheter while ensuring the catheter remains in the trachea.
6. Allow a few seconds to pass for the rat to be able to breathe around the catheter inserted into the trachea.
7. Firmly seat the trachea indicator onto the catheter opening. Movement of the aerosol barrier will indicate that the catheter is inserted correctly into the trachea.
8. Ensure that the rat is lying on the intubation stand such that the chest of the animal is facing perpendicular to the plane of the intubation stand.
9. Remove the trachea indicator and deliver 100 µL (10⁷ CFU, colony forming units) of the inoculum containing Francisella tularensis using a 200 µL pipette tip, firmly seating the tip against the catheter opening.
10. Attach a 1 mL slip-tip tuberculin syringe filled with air and deliver 300 µL of air to ensure the inoculum reaches the rat's lungs.
11. Remove the catheter from the trachea and remove the rat from the surgical platform.
12. Allow the rat to awaken and return to the cage. Make sure that the breathing has returned to normal.
13. Determine the CFU in the F. tularensis inoculum to confirm the CFU delivered in the trachea.
14. Repeat steps 1.1 - 1.9 on unvaccinated (naïve) animals with 100 µL PBS in place of the inoculum.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
GreenLine fiber optic blade size 0	Carefusion	5-5231-00	Macintosh American profile
GreenLight system laryngoscope handle	Carefusion	4559GSP
Exel International Safelet I.V. Catheter	EXEL INTERNATIONAL	26743
Slip Tip Sterile Syringes 1mL	BD	309659
Broad Point Dressing Thumb Forceps	Thermo Scientific	76-302
200 μL barrier tip	GeneseeScientific	24-142
1,000 μL pipette tip	Olympus Plastics	24-173
Dremel 3000-2/28 Rotary tool kit	Dremel	3000228
Rodent Intubation Stand	Braintree Scientific	RIS 200
Isoflurane	Butler Schein	NDC 11695-6776-2
Rodent anesthesia machine	Surgivet	VTC302 Classic T3
Rodent Anesthesia chamber	Braintree Scientific	AB 1