Materials and Reagents

Note: Unless otherwise stated, items are supplied by general laboratory suppliers and veterinary or medical suppliers.

1. Sterilized items (autoclaved or purchased as sterile)
   
   1. 1 ml syringes (Becton Dickenson, catalog number: 309659)
   2. Gauge 27 needles, 3 per animal (Becton Dickenson, catalog number: 305109)
   3. Cotton wool balls (Supermarket home brand)
   4. Surgical gauze swab, cut into 1cm squares (ProVet, GAUZ HS N1)
   5. Small animal surgical drapes with window of approximately 2 cm x 4 cm (Theatrewrap, SA)
   6. Silicon instrument mat (e.g., Medline DYNJHOLD1)
   7. Square restaurant grade linen napkin(s) (e.g., Lunaweddingandeventsupplies.com.au, black linen napkin 50 cm x 50 cm)
   8. Stainless steel instrument dish (e.g., Instrumentation concept, KDY8)
   9. Scalpel blade #11 (ProVet, SCAL HS11) (or to your preference)
   10. Suture thread [Silkam #4 (AMA, BRAC0762130) or #6 (AMA,BRAC0762113) or to your preference]
   11. Surgical gloves (ProVet, GLOV CLF7)
   12. Distilled water (in house, autoclaved in Schott bottles)
   13. Phosphate buffered saline pH 7.2 (PBS) (in house, autoclaved in Schott bottles)
   
   
2. Non-sterile items
   
   1. Permanent marker (Officeworks)
   2. Spray bottles containing 70% ethanol (Spray bottle from hardware store, e.g., Bunnings)
   3. Clean gown (ProVet, GOWN S KS)
   4. Hair cover (e.g., Medline NONBOUF21)
   5. Surgical mask (ProVet, SURG MHS03)
   6. Autoclave bags (Provet, Henry Schen, various sizes, AUTO P HM2; AUTO P HM4)
   7. Bubble wrap cut to size to place around the rat (Officeworks)
   8. Tape (masking tape, Officeworks)
   9. Paper towel (Tork)
   10. Tube rack (Thermo Fisher, 21-200-285)
   11. 50 ml Falcon tube (Thermo Fisher, NUN339652) containing 70% ethanol
   12. Small Petri dishes (Thermo Fisher, Sterilin 121V)
   13. 70% ethanol
   14. Vaseline
   
   
3. Biological material
   Rats [Female Piebald Viral Glaxo (PVG) rats 160 g+ obtained from Animal Resource Centre (ARC), Canning Vale, WA 6970]
   
   
4. Drugs
   Note: All drugs purchased through ProVet (provet.com.au).
   
   1. Ketamine [Ketamil, Troy Laboratories, 50 mg/kg [50 mg/ml] diluted in phosphate buffered saline (PBS)]
      Note: Ketamil is a scheduled poison and should be stored with appropriate levels of security. It should be protected from light and stored at room temperature, observing expiry date on the product label.
   2. Xylazil [Xylazil, Troy Laboratories, 10 mg/kg (10 mg/ml) diluted in PBS (Store at 20-25 °C and observe product expiry date on label.)]
   3. Carprieve (Norbrook Australia, Pty. Ltd.; Victoria, Australia, 5 mg/kg (5 mg/ml in sterile water) (Store protected from light at 2 °C to 8 °C. Once open store refrigerated for up to 28 days.)
   4. Luxyl protective eye drops (Tubilux Pharma SpA Via Costarica 20/22 Pomezia Italy) (Store below 25 °C. Once open store refrigerated for a maximum of 60 days and dispose of immediately should sterility be compromised.)
   5. Betadine (500 ml ProVet, BETA AS) (Store below 25 °C protected from light, observing any expiry information on the label.)

Equipment

1. Schott bottles 50-250 ml
2. Surgical instruments (Kaisers surgical instruments Pty Ltd)
   
   1. Watchmakers forceps (one or two coarse, one or two medium and two fine points)
   2. A medium pair with tips bent inwards towards each other to form a ‘hook’ is also useful
   3. Fine dissecting spring-form scissors (in different sizes)
   4. Needle holder or small Spencer Wells forceps for suturing
   5. Small scissors for cutting suture thread
3. Bench-top autoclave (Siltex, Practica 20L lab steam sterilizer) 
4. Weigh balance suitable for weighing animals (e.g., Mettler Toledo, MW 1002T/00)
5. Small animal clippers (e.g., Wahl KM2)
6. Binocular surgical/dissecting microscope with sufficient working distance (e.g., Nikon, model: SMZ460)
7. Light source to illuminate surgical site (e.g., Schott, catalog number: KL 1500 LED)
8. Clear Perspex animal supports approx. 25 x 10 cm (made in house; also available from Plastic Warehouse)
9. Mouth bar (Made in house through engineering workshop, Figure 1) (An alternative would be to adapt the mouth bar from a stereotaxic frame such as the WPI 502612)
10. Small retort stand and boss head clamp (Labdirect, Kartell retort stand; Technos boss head clamp)
11. Diamond keratotomy knife (Geuder, catalog number: G-19345) 
12. Small animal heat pad (My pet warehouse, Ref 234016)
13. Spare animal cage(s) for use during induction of anesthesia and/or after recovery
14. Anal thermometer
    

Note: Also required is a suitable surgical table or bench space.

Figure 1. Stainless steel mouth bar. A. The mouth bar was machined from a stainless steel rod. (B) Shaped to slip into the mouth and (C) with a hole for the top incisors to go through. B and C. The nose clamp has a curved end to accommodate the nose and is tightened with the use of two screws. One presses the clamp onto the nose and the second applies slight upwards pressure from beneath the nose clamp.

Procedure