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1. Introduction and Definitions:

A stereotaxic instrument is a device which permits the precise location of a moveable object in space. It enables an investigator to place an electrode or cannula accurately in a small brain structure even though that structure is hidden from view, deep inside the brain. This makes it possible to stimulate or disrupt such a structure without directly disturbing overlying brain tissue. The basis of stereotaxic surgical technique is that the various regions of the brain have a definite and predictable location with respect to the surrounding parts of the skull.

Abbreviations: 

Animal Care Services ACS, Principal Investigator PI, subcutaneous SC, intravenous IV, intraperitoneal IP, intramuscular IM, per os PO, per rectum PR, biological safety cabinet BSC, Tamoxifen TAM, Accelerated Hydrogen Peroxide AHP

2. Materials:

• Isoflurane
• Xylocaine cream
• Weigh scale
• Analgesics (as per AUP)
• Sterile syringes (multiple sizes)
• Sterile needles (multiple sizes)
• PPE (cap, gloves, mask, clean lab coat or surgical gown)
• Standard sterile surgical gloves Clippers
• Weigh boats
• 2% chlorhexidine surgical scrub
• Povidone-iodine solution
• 70 % isopropyl alcohol
• Heating pad
• Surgical towels
• Paper towels
• Ophthalmic ointment
• Stereotaxic unit and ear bars
• Drill bits, cannula guide, screwdriver and screws
• Cannulas
• Cannula dummies
• Surgical kit
• Gauze
• Swab applicators
• Periosteal elevator
• Suture material
• 0.9% sodium chloride
• Lactated Ringers solution
• Dental acrylic
• Heat pad
• Antibiotic ointment

3. Procedures

• Animals health needs to be assessed before proceeding with any procedure.
• Use a decontamination spray to clean the drill.
• Anesthetize the rat as per SOP 10.6 Anesthesia in Rats.
• Administer analgesics as per the approved Animal Use Protocol.
• Shave the top of the rats’ head; this should be performed away from the surgical area.
• Administer bupivacaine subcutaneously.
• If the animal is showing signs of regaining consciousness, place back in the induction chamber.
• Apply a small amount of xylocaine gel to the ear bars and position the animal on the ear bars. Alternatively, a small drop of gel can be applied directly in the ears.
• Open the jaw and move the tongue to the side to avoid its teeth from getting caught on the bite bar. Place the bite bar in between the upper and lower jaws.
• Administer sterile ophthalmic ointment and reapply as required.
• Assess corneal/blinking reflex.
• Test for a pedal reflex to ensure the rat has reached a surgical plane of anaesthesia.
• Don standard sterile surgical gloves using aseptic technique.
• Incise the skin over the skull using a scalpel blade and retract skin ensuring that it does not go beyond the start of the eyes.
• Gently remove the periosteum with the dental periosteal elevator
• Retract the membrane in four locations.
• Wipe the top of the skull with a sterile cotton swab until bregma is visualized.
• Ensure that the left arm of the stereotax is at zero degrees.
• Insert the drill bit, ensuring it is tight and attach the drill to stereotax arm.
• Find the anterior-posterior line (vertical) of best fit and take the anterior-posterior measurement (bottom, flat bar).
• Find the medial-lateral line (horizontal) of best fit and take the medial-lateral measurement (top, horizontal bar).
• Make your calculations. Place drill where required.
• Drill the cannula hole(s), and 4 screw holes. Drill only once for the screw holes.
• Instill the screws. Hold them with tweezers or haemostats and use the screwdrivers. DO NOT screw them in fully, leave some head space.
• If filtered air is accessible, use an air hose to dry around the screws.
• Use a straight edge to ensure the cannula guides are straight and at 90 degrees.
• Remove the drill and replace with the cannula guide holder. Put the cannula guides on and recheck for straightness.
• Lower the cannula guides until they are in the center of the cannula hole. Place a drop of sterile saline in the cannula hole – slowly lower down to the skull to check placement of cannula guide (liquid should displace).
• Take dorsal-ventral (DV) measurements (the top, vertical bar).
• Note the initial DV measurement from the surface of the skull to later assess depth.
• Lower the cannula to the calculated depth.
• Mix the dental acrylic in a weigh boat and transfer slowly to a disposable pipette. The solution should not be too runny as that will increase the exothermic reaction and may potentially cause tissue irritation.
• The scalp must be dry and free of any tissue for maximum adherence.
• Apply the acrylic from the pipette and use the small spatula and alcohol moistened cotton tipped applicators to form it into a skullcap.
• Ensure all screws are completely covered, and that the cannulas are not protruding excessively. The cannula should have no more than a 1 mm projection above the skull cap.
• Prepare and add more acrylic cement, as necessary.
• Wait 3-5 minutes, or until the acrylic is firm, then withdraw the cannula guide holder.
• Put the pins in carefully.
• Administer 5 – 10 mL Lactated Ringers subcutaneously.
• Clean around the skull cap with a swab and sterile saline.
• Remove any acrylic cement from the surrounding skin, ensuring no sharp edges have formed.
• Apply a small amount of antibiotic ointment around the skull cap with a sterile swab.
• Remove any excess skin if necessary.
• Remove the rat from the ear bars. Place in a clean cage lined with paper towel over a heat pad (placed under half of the cage) and recover animals as per SOP 10.4 Rodent Post-operative Care (Rat).
• Record the surgical procedure, injections, and rat identity number on the cage card. Appropriately identify the animal (ear punch, tail markings, etc.).
• All rats must be ambulatory with a strong righting reflex before being returned to the rack.