In vivo wound healing assay

6 mm Wound surgery

Subject: Skin excisional wounding of mice

List of materials:

Lubricant eye

Clippers

Veet^® cream

Clean empty mouse cage with appropriate wet food and shredded paper

Scale

25 needle gauge

1ml syringe

Anaesthetic induction chamber

Anaesthetic machine for delivery of Isoflurane^®

Nose cone for isoflurane^®

Permanent marker

Autoclaved forceps and scissors

1 jar with ethanol 70%

Skin disinfectant: ethanol 70%, chlorhexidine

Autoclaved cotton tips

6mm biopsy punch sterile disposable

Autoclaved disposable pads

Towel

Drugs:

Pain Killer:

Buprenorphine

Acetaminophen

Anaesthetic: Isoflurane^®

NB: It is recommended to shave the mice the day before the surgery to minimise the time under anaesthesia.

Depilation

List of materials:

Lubricant eye

Clippers.

Veet^® cream.

Anaesthetic induction chamber

Anaesthetic machine for delivery of Isoflurane^®

Nose cone for isoflurane^®

Clipper to ear-notch mice

heat mat

Drugs:

Anaesthetic: isoflurane®

Pain killer: Acetaminophen

Protocol:

D -1: mice depilation

1. Put heat mat under the cage.

2. Mice are placed in the anaesthetic induction chamber where they will receive Isoflurane^® 4-5% (1L/min oxygen flow rate), utilising a precision vaporiser unit.

3. Put lubricant eye.

4. Under general anaesthesia using 2% Isoflurane^® inhalation using nose cone, animals will be depilated on their back skin using clippers and Veet^® cream. Short term application of Veet^® does not alter the readout given the possible irritation.

If it's not already done: Use the Universal Ear-Notching System or toe-clipping to identify individual animals after surgery.

5. Mice are placed in the cage and monitor until total recovery.

6. Acetaminophen 1mg/ml PO is given to mice in the drinking water.

Wound surgery

List of materials:

Clean empty mouse cage with appropriate wet food and shredded paper

Scale

25 needle gauge

1ml syringe

Anaesthetic induction chamber

Anaesthetic machine for delivery of Isoflurane^®

Nose cone for isoflurane^®

Permanent marker

Autoclaved forceps and scissors

1 jar with ethanol 70%

Skin disinfectant: ethanol 70%, chlorhexidine

Autoclaved cotton tips

6mm biopsy punch sterile disposable

Autoclaved disposable pads

Towel

Drugs:

Pain killer:         Buprenorphine

                         Acetaminophen

Anaesthetic:     Isoflurane^®

Protocol:

D0: surgery

Before wounding, wet food and shredded paper will be placed in cages that will contain 1 animal. 

Skin wounds do not need to be performed in a sterile environment given the natural bacterial colonization of the skin. However, within this procedure, there will be strict adherence to clean and sterile surgical techniques.

Under general anaesthesia using 2% Isoflurane^® inhalation we will perform skin wounds as described below.

Immediately prior to surgery:

7. Weigh the mice.

8. buprenorphine 0.05mg/kg SC (total volume made up to 0.5ml via sterile normal saline dilution) administrated with 1ml syringe and 25G needle. Another dose of buprenorphine (0.05mg/kg SC) will be given to the mice 6 hours after surgery.

9. Mice are placed in the anaesthetic induction chamber where they will receive Isoflurane^® 4-5% (1L/min oxygen flow rate), utilising a precision vaporiser unit.

Anaesthetic maintenance:

10. The mice will be given Isoflurane^® 1-5% via nose cone (0.3-0.5L/min oxygen flow Rate), utilising a precision vaporiser unit.

Surgery:

11. Skin will next be cleaned using an appropriate topical antiseptic agent, in line with standard veterinary practice (70% ethanol, betadine, chlorhexidine, or any condonation of these) in at least three rotation using cotton tips.

12. 6mm wound: longitudinal single dotted line will be drawn on the back to prepare wound placement (from head to tail following the spine) using a permanent pen.

13. The withdrawal reflex is testing by pinching the foot of the mouse with fingers or tweezers to verify adequate depth of anaesthesia. If the mouse does not move, you can proceed to surgery.

14. The animal is positioned into lateral recumbence, the skin over the back is tented away from the body and a single full-thickness wound is made through the folded skin using a 4mm punch biopsy device to cause 2 wounds in mirror image. Biopsy punches are single use only, and discarded between animals. Instruments will be cleaned using 70% ethanol between each surgery (10min contact time).

Post-operative wound management and scoring:

        -D0: buprenorphine 0.05mg/kg SC (total volume made up to 0.5ml via sterile normal saline dilution) will be given to the mice 6 hours after surgery.

        Add Acetaminophen (1mg/ml) in drinking water if it has not been added the day before.

        -D1 to 7-10: the mice will be scored as per the specific scoresheet.

        For score between 1 and 4, the mice will be monitored daily and given acetaminophen 1mg/ml in drinking water.

        For score between 5 to 9, the mice will be monitored twice daily and given Buprenorphine SC twice daily in addition to Acetaminophen 1mg/ml in drinking water. Require vet assistance.

        For score above 9, the mice will be euthanised.

Post-wounding animals are kept as one animal per cage to avoid interfering each other wounds. At day 4 post wounding (or longer in the event of delayed primary scab formation) animals will be transferred to one cage, 3-5 animals per cage.

Comments

We will provide no bandage or antibiotics: skin wounds are normally colonized with bacteria with little risk of infection due to natural “innate” immune response upon wounding. An infection requiring antibiotics usually occurs if wound is occluded and bandage is not changed frequently. The overall duration of this surgery is about 5 minutes, and is kept to the minimum time necessary.

The day following surgery, it is expected that the animals’ behaviour will be unremarkable including normal levels of activity and eating/drinking.