Establishing a Mouse Model of Burn Infections with Pathogenic Bacteria

Samenvatting

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)

This video demonstrates how to establish a burn infection in a mouse model by applying a heated brass block followed by pathogenic bacterial inoculum. The bacteria colonize the exposed extracellular matrix and form a biofilm, enabling in vivo studies of host-pathogen interactions.

Protocol

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

1. Mouse Model of Burn Infection Caused by Bioluminescent Acinetobacter baumannii

1. Use adult female BALB/c mice aged 7-8 weeks and weighing 17-19 g. Allow the mice to acclimate to laboratory conditions for at least 3 days before the start of the experiment. Maintain the mice in a 12 h light/dark cycle under a room temperature of 21 ℃ and give them food and water ad libitum.
2. Anesthetize the mice with an intraperitoneal injection of a ketamine-xylazine cocktail (100 mg/kg-20 mg/kg). Lightly touch the palpebra of each mouse with a cotton swab; an absence of the palpebral reflex suggests an appropriate anesthetic depth. Cover the mouse's eyes with vet ointment to prevent them from drying out during anesthesia.
3. Shave the mice on the back to expose as much skin as possible by using a 50-blade hair clipper.
4. Place the lid of a 35 mm Petri dish underneath each mouse's abdomen to keep their backs relatively horizontal.
5. Boil water in a 250 mL beaker (80% full) using a 10" x 10", 220 volts alternating current (VAC) hotplate. Immerse a brass block (1 cm x 1 cm cross section) into the beaker until thermal equilibration with the water is reached. Thermal equilibration usually takes <5 min and is indicated by the re-boiling of the water in the beaker.
6. Prior to creating the burn injury, administer pre-emptive analgesics (a subcutaneous injection of 0.1 mg/kg buprenorphine) for pain relief.
7. Ten minutes after the pre-emptive analgesics, gently press the heated brass block to the shaved area on the back of the mice for 7 s to induce burn wounds. 
   NOTE: To avoid thermal injury to the working personnel, wear thermal-resistant gloves when performing the burning procedure.
8. Administer 0.5 mL of sterile saline through subcutaneous injection to prevent dehydration.
9. Five minutes after the thermal injury is induced, inoculate 50 µL of bacterial suspension containing 5 x 10⁶ colony-forming units (CFU) in phosphate-buffered saline (PBS) onto the mouse burns using a pipette. Then, using a sterile cotton swab in a zigzag motion on the skin, smear the aliquots on the burns to distribute the bacterial cells in the burned area as evenly as possible.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Mouse	Charles River Laboratories, Wilmington, MA	BALB/c	7-8 weeks age, 17-19 g weight
Acinetobacter baumannii	Brooke Army Medical Center, Fort Sam Houston, TX	Clinical isolate	Engineered luminescent strain
Insulin syringes	Fisher Scientific	14-826-79	BD Lo-Dose U-100 Insulin syringes for injection
Sodium chloride	Fisher Scientific	721016	0.9% Sodium Chloride
Phosphate-buffered saline, 1x solution	Fisher Scientific	BP24384	A standard phosphate buffer used in many biomolecular procedures
Inoculating loops	Fisher Scientific	22-363-605	For smearing bacterial inoclum on burn surface of mice
Brass block	Small Parts, Inc., Miami, FL	10 mm by 10 mm	For creation of burns in mice
Extreme Dragon PBI/Kevlar high-heat gloves	Superior Glove Works Ltd, Cheektowaga, NY	PBI83514	Heat resistant gloves
Greiner dishes	Sigma-Aldrich Co. LLC	P5112-740EA	35 mm ×10 mm
Corning digital hot plate	Cole-Parmer Instrument Company, LLC	UX-84301-65	10" x 10", 220 VAC, for boiling water
Mouse/Rat thin line water heated surgical bed	E-Z Systems	EZ-211	Prevents heat loss and hypothermia during surgery
Vet ointment	S&K Pharma, GmbH	Kerato Biciron 5%, Augensalbe	Opthalmic ointment to prevent eye dryness