Rodent stereotaxic surgery protocol

A stereotaxic frame holds the skull in a fixed position, so you can reach almost any brain region from its coordinates.³ Labs use it to make targeted lesions, inject tracers or viruses, and implant electrodes, cannulas and microdialysis probes.^3,2 Cetin and colleagues report that viral gene delivery this way works in mice and rats at any postnatal age up to adulthood.³

Careful surgery protects your data as well as the animal. A hidden infection can confound results, and so can the changes in behavior and physiology that pain brings.¹ Rodents hide pain and infection well, and they are most active at night, when few staff are around to see the signs.¹

Every surgical procedure must be approved by your animal care committee, such as an IACUC, or another authority before you start. If approval is in any doubt, do not go ahead.¹ Get training in surgical technique and asepsis first, for the animals’ sake and for your data.¹ Your approved protocol and your veterinary staff set the anesthesia, pain relief and doses, so none are given here.¹

Rodent surgery does not need a dedicated suite. A tidy, well-lit bench out of the flow of traffic will do, cleaned first with a disinfectant.¹ Split the space into three zones, for preparation, surgery and recovery.¹ An assistant can run anesthesia and prepare each animal while you get ready, which shortens its time under anesthesia. The assistant can also watch over recovery.¹

Instruments can serve several animals in a session if you clean and sterilize them between animals. Wiping instruments with alcohol does not sterilize them.¹ Your committee may limit how many animals one pack can serve. Lay the instruments out on a sterile drape in the order you will use them.¹ Wear a cap and mask, and change gloves between animals.¹ The sterile field covers the surgical area, the prepared skin on the animal and the front of the surgeon. Touching anything outside it breaks sterility.¹

Anesthesia is needed for surgery and can be given by inhalation or injection. Ask your veterinary staff for agents and doses.¹ Give pain relief before surgery begins, as your approved protocol directs.¹ Check the depth of anesthesia with a toe pinch before you start.² At a surgical depth, the animal does not pull its foot back. Keep the animal warm on a pad made for surgery, and track its temperature with a rectal thermometer.^2,1

Shave the head from between the ears to just between the eyes.² Pritchett-Corning and colleagues advise shaving an area about three times the size of the incision, so take the patch out to the sides as well.¹ Protect the eyes with lubricating ointment.¹ Then scrub the skin with surgical scrub and alcohol in turns, ending with scrub, and keep the antiseptic out of the eyes.^1,2

1. Place the ear bars

   Slide one ear bar into the ear canal and hold it while you slide in the other. The ear bars are seated when the head can no longer move side to side.²

2. Secure the mouth

   Secure the mouth in the incisor bar at the front of the frame, and check with a ruler that the head looks level.² You level it exactly at bregma and lambda once the scalp is open.

3. Open the scalp

   Cut along the midline of the scalp from lambda to between the eyes, about 1 cm long in mice and 2 cm in rats.² Hold the skin open with hemostats and dry the skull with sterile swabs.²

4. Level the skull

   Touch the cannula tip to bregma, then to lambda, and compare the two heights. Raise or lower the incisor bar until they match, so the skull is flat and atlas coordinates apply.

5. Record bregma

   Put the cannula tip over bregma and write down its front-back (AP) and side (ML) coordinates.²

6. Find the target

   Look up the target’s distances from bregma in a stereotaxic atlas for your species. Move the cannula to them by adding them to, or subtracting them from, your bregma readings.²

7. Mark the drill site

   Lower the cannula tip until it touches the skull, record that depth (DV), and mark the spot with a sterile pencil.²

1. Drill the hole

   Drill at the mark until you are through the skull, without letting the bit go into the meninges or blood vessels below.² Check that the cannula passes straight through the hole without touching the edge. If it touches, widen the hole and check again.²

2. Open the meninges

   Gently pierce the meninges with a sterile needle so the cannula goes in cleanly.²

3. Set anchor screws

   For a head mount, set skull screws in small holes around the site. In rats, Geiger and colleagues use six, two in front of the cannula hole, two to the sides and two behind.²

4. Lower the implant

   Lower the cannula slowly and straight down to the depth you planned, without touching the sides of the hole.²

5. Cement it in place

   Cover the cannula, screws and skull with a thin layer of dental cement, then a thicker one.² As the cement thickens, free the skin from it and shape the cap smooth before it sets, so it does not rub the skin later.²

6. Let it set

   Let the cement dry fully before you take the animal out of the frame.²

Handle tissue gently and keep it moist. Good tissue handling lowers the risk of infection, speeds healing and reduces pain after surgery.¹ Warm any fluids you give, which may help prevent a drop in body temperature. Pritchett-Corning and colleagues note that mouse and rat body temperature is higher than ours, so fluids must be warmer.¹

Recover each animal in its own cage on flat paper bedding, with the cage half on a heating pad so the animal can choose its temperature.^2,1 Return it to the holding room only once it can right itself and move about normally.¹ Check every animal daily, weekends and holidays included, for pain, distress and infection.²

Signs to watch for

• Pain or distress: moving little, crying out when handled, a hunched posture, diarrhea, swelling or discharge at the head mount, and not eating or drinking.² Swelling or discharge can also mean infection.¹ Report any of these signs to your veterinary staff.
• Infection at the incision: redness, swelling, discharge, pain or a wound that opens. The risk is highest in the first 7 days.¹
• A dull or sluggish animal that stops eating needs a check for untreated pain or infection.¹
• A healthy animal is bright, alert and active, eats and drinks, and holds its normal postures.¹

Animals usually lose a little weight after surgery and regain it with pain relief and food. Make food and water easy to reach, with longer sipper tubes or wet food on the cage floor.¹ Weigh each animal before surgery and daily after, so you can see the loss and the recovery. Geiger and colleagues allow at least a week of recovery before testing.² At the end of the study, check placement in brain sections cut along the probe track.²

• The head shifts in the frame. Reseat the ear bars until the head cannot move side to side.²
• Bregma and lambda are at different heights. Adjust the incisor bar and measure again before you set any coordinate.
• The cannula touches the side of the hole. Widen the hole and check again until the cannula passes straight through.²
• Cement catches the skin as you shape the cap. Free the skin and smooth the cap before it hardens, so it will not rub later.²
• An animal eats little after surgery. Rodents can hide pain, so check for untreated pain or infection, and make food easier to reach.¹
• The incision is red, swollen or open. These are signs of infection, so tell your veterinary staff.¹
• Targets miss. Check placement in brain sections at the end of every study, and refine your coordinates from what you find.²