Bacterial-induced neutrophilic nasal inflammation in mice

Prepare the chosen bacteria

Culture the corresponding bacteria on sheep blood agar plates (BD, Biosciences 254071), according to their specific growth requirements.

In our hands, bacteria strains used were Pseudomonas aeruginosa Vitroids^TM (Sigma-Aldrich, VT000256), Staphylococcus aureus Lenticules^TM (Sigma-Aldrich, CRM06571M) and Streptococcus pneumoniae (ATCC^®, 49619^TM). Overnight culture conditions were 37°C for S. aureus and P. aeruginosa and 37°C in 5% CO₂ for S. pneumoniae.

Prepare the nasal tampon

In our hands, a Merocel pope ear wick (Medtronic, MI, USA) was cut at a dimension of 4 mm x 1 mm x 1 mm and sterilized by dry heat 100ºC during 3h.

Prepare and sterilize your instruments for the surgical procedure

Stereomicroscope (Motic SMZ-171)

1.5-mm microdrill (Medtronic, MI, USA)

Sterile fields (Hartmann Mediset^®)

Temperature monitor (PhysioSuite^® Kent Scientific Corporation)

15-mm scalpel blade

5.0 non-resorbable polypropylene sutures (Monosoft^TM, Covidien)

Scalpel

Forceps

Scissors

Needles

Gloves (CardinalHealth^TM Protexis^TMPI micro surgical gloves)

Eye drops (Ocry-gel, TVM)

Chlorhexidine digluconate (Hibidil^®, Regent Medical).

Tape

IR lamp (IR100 Infrared lamp, MEDISANA^®)

Ultrasonic bath (Clifton)

Surgery platform

Prepare your bacterial solution

From the bacterial cultures, take isolated colonies and prepare serial dilutions up to 10⁹ CFU/ml in NaCl 0.9% and measure it with a spectrophotometer with a reference optical absorbance at 600nm (OD_600nm) = 1.

Administration of anesthesia and analgesia to the mouse

Weight the mouse and administer general anesthesia accordingly: a mix of Xylazin (maximum 15mg/kg) and Ketamin (maximum 80 mg/kg) intraperitoneally.

Administer analgesia: Temgésic (maximum 0,05 mg/kg) subcutneously.

Wait 15-20 minutes for the aesthesia and analgesia to do their effect before starting the surgical intervention.

Shave the snout over the intervention area and fix the mouse on a sterile field (Figure 1, A).

Monitor and maintain the intraoperative body temperature of the mouse during the procedure.

Disinfect the intervention area with Chlorhexidine digluconate and use eye drops to prevent eyes from drying.

Use a 15-mm scalpel blade to make a 8-mm midline incision over the nasal dorsum to the snout. Raise skin flaps laterally to expose the nasal bone (Figure 1, B).

Use a 1.5-mm microdrill to drill the nasal bone over the nasal fossa and remove the upper part of the septum. Be extremely careful not to drill completely the bucco-sinusal bone communication. Clear out any bleeding to prevent aspiration (Figure 1, C).

Inoculate the pre-cut nasal tampon with 10 μL of the corresponding bacterial solution or saline for controls and carefully place it into the nasal cavity (Figure 1, D-F).

Suture skin flaps with 5.0 non-resorbable polypropylene sutures and dettach the mouse from the surgical field (Figure 1, G).

Place the mouse in a cage softly heated laterally by an IR lamp for recovery after surgery. Make sure the lamp is not too close or too far from the cage (Figure 1, H).

Once awake and active, place the mouse into an ABSL-2 facility.

To overcome the possible post-operative pain, administer Temgésic (maximum 0,05 mg/kg) subcutaneously to the mice every 12h for 24h after surgery.

Closely watch the animals every day for at least one week to ensure their well-being. According to humane endpoints, mice reaching a score of non well-being or losing more than 20% of their body weight must be euthanized.