Apr 03, 2025
Mouse Stereotaxic Surgery
Forked from Mouse Stereotaxic Surgery
- Alexandra Nelson1,
- Antonio Falasconi2,3,4,
- Harsh Kanodia2,3,4,
- Silvia Arber2,3,4
- 1University of California San Francisco;
- 2Biozentrum, University of Basel;
- 3Friedrich Miescher Institute for Biomedical Research;
- 4Aligning Science Across Parkinson’s (ASAP)
Protocol Citation: Alexandra Nelson, Antonio Falasconi, Harsh Kanodia, Silvia Arber 2025. Mouse Stereotaxic Surgery. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8eme7l2w/v1
Manuscript citation:
Adapted from: Jonathan S Schor, Isabelle Gonzalez Montalvo, Perry WE Spratt, Rea J Brakaj, Jasmine A Stansil, Emily L Twedell, Kevin J Bender, Alexandra B Nelson (2022) Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in Parkinsonian mice eLife 11:e75253
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2025
Last Modified: April 03, 2025
Protocol Integer ID: 125804
Keywords: Mouse, Surgery, Stereotaxic Surgery, Implants, ASAPCRN, mouse stereotaxic surgery this protocol, mouse stereotaxic surgery, stereotaxic surgery in mice, performing stereotaxic surgery, applicable to intracranial injection, intracranial injection, mouse brain, regions of mouse brain, mouse, mice, placement of implant, implant
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020551
European Union’s Horizon 2020 research and innovation programme
Grant ID: InterAct, grant agreement No 101018151
Swiss National Science Foundation
Grant ID: N/A
Abstract
This protocol describes the steps for performing stereotaxic surgery in mice. It is applicable to intracranial injections (e.g. virus, drug) and placement of implants (e.g. optical fibers, electrode arrays) into targeted regions of mouse brains.
Troubleshooting
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval should be obtained before performing these experiments. Approval was obtained from the Cantonal Veterinary Office Basel-Stadt and performed in compliance with the Swiss Veterinary Law guidelines.
Prepare drugs and viruses
Draw up in syringes all drugs that you will need for the surgery (typically anesthetics, analgesics; also sterile normal saline).
Half an hour before start of surgery, inject mouse with Buprenorphine SC (Temgesic)(0.1mg/kg) subcutaneously.
If using viruses, obtain aliquots from the -80 deg C freezer and dilute with normal saline to the desired titer and place in an ice bucket.
Surgery room setup
Set a clean, empty cage halfway onto the heating pad on the prep area, and turn on the heating pad to medium.
Place your sterile surgical tools (forceps, scalpel, small scissors, hemostat, and surgical clips) on one side of the stereotax.
Place a packet of sterile swabs nearby.
Make sure the small heating pad on the stereotax is on, and cover with folded napkins and tape down.
Mouse preparation.
Anesthesia and incision.
Turn on the oxygen and adjust on the anesthesia machine to a flow rate of 0.4 L/minute.
Once the animal has fallen asleep, place it on the stereotaxic frame by gently opening its mouth and putting its teeth through the bite bar and slipping the nose cone over the snout. Then, turn the isoflurane flow rate to 2%.
Apply lubricant to the eyes bilaterally to prevent corneal damage.
Adjust the ear bars so that the animal’s head is symmetrically held in the stereotax.
Apply Betadine on the shaved scalp using a swab.
Inject mouse with a mixture (50:50) of Lidocaine (10mg/kg) Ropivacain (Naropin) (3mg/kg) in the area of the surgery.
Use the scalpel to cut a midline incision down the long axis of the scalp. Use the small scissors to enlarge the incision to the desired length. Use the surgical clips to grab the midpoint of the skin on each side of the incision to enlarge the surgical field.
Align the skull's bregma and lambda sagitally and balance the brain using knobs of the stereotax.
Drilling and skull preparation.
Place the drill (with a fresh drill bit) onto the stereotaxic arm.
Drill hole at desired coordinates:
- Put the drill bit at Bregma, lift up, and move in AP and ML to the desired coordinates using the stereotax.
- Using foot pedal and stereotax, drill through the skull at this location
Remove the drill from the stereotax.
Injection.
Fill up a 1uL calibrated glass capillary (Drummond Scientific) with the needed amount of viral solution previously prepared
Use the stereotax to position the needle about 1 cm above the skull.
Lower the needle down to and through the drill hole, until it is at the surface of the brain (this is sometimes hard to guess with a drill hole, but do your best). Then proceed to reach your deisred dorsoventral coordinates.
Start the injection connecting your capillary to the air source (Picospritzer Parker,US or Openspritzer).
After the injection has been completed, leave in place another 5 minutes, then withdraw the needle by 0.2 mm and leave another 5 minutes.
Slowly withdraw the needle while watching for fluid coming out of the hole upon withdrawal.
Implants
After all injections, but before implanting any devices, prepare Metabond (dental cement):
- Scoop a small amount of the powder into a small weigh boat.
- Add 3 drops of liquid + 1 drop of catalyst to the weigh boat
- Mix using a Metabond brush inserted in the brush handle.
Use the Metabond brush to paint the resulting white liquid over the exposed skull, trying to avoid all holes. If you cover a hole, use forceps or the end of a 32G needle to remove the dried Metabond.
Apply a small amount of Metabond to the lower 3rd of any ferrule (for optical fiber ferrules) or electrode array connectors. This will dry in 1-2 minutes.
Implant optical fibers by first inserting the fiber-ferrule assembly in a set of several ceramic ferrules and sleeves stuck together (a ferrule “stick”), and placing the stick (fiber down) in one of the grooves on the end of the stereotaxic “wand”, secured with a clamp.
For the first layer, using the back of a swab or another device, drip the dental acrylic over the base of your implant, completely covering the hole it was inserted in, the fiber or electrodes, and extending to the skull screw and/or ground wires.
You want a little of the acrylic to come up to the bottom of the ferrule or the base of the electrode array connector.
Do not touch the mouse or implant until the first layer of acrylic has hardened.
You can test whether it is ready by checking the residual acrylic in your silicone bowl, and if this is hard, probing the edge of the acrylic with your forceps. If it has completely hardened, then you can start the second layer.
In the case of the electrode array implant, you will bend the loop ground wire between the ground wire hole and the implant in a C shape and tuck it against the right side of the implant.
Prepare another batch of dental acrylic and apply to make sure all exposed wire, skull screw is covered, and build a “helmet” of smooth dental cement around the rest of the implant, such that it is a smooth surface all around the implant.
- With optical fiber-ferrules, about ½ to 2/3 of the ferrule should remain exposed above the top of the acrylic, to provide access.
- With electrode arrays, the resin should be completely covered and the plastic connector about ½ covered with acrylic.
Once the implant is secured with solid acrylic, the stick and wand on which it was inserted can be gently removed.
Closing/Suturing
After completion of all injections and implants, the scalp can be sutured.
- For non-implant surgery, this typically involves 3-4 sutures along the length of the incision.
- For implant surgery, this sometimes involves 1 suture (most often posterior), but sometimes no sutures are required as the scalp fits just around the implant. You do not want the scalp to be too tight around the implant.
Open a packet of sterile suture.
Use a hemostat to grab the end of the curved needle that is closest to the thread, and pull the suture out of the stiff backing.
Your first incision should be placed in the middle of the area you desire to close.
- Using the hemostat in your right hand (if you are right handed) and the forceps in your left hand to pick up the edge of the scalp, drive the tip of the needle down through the scalp on the right side of the incision, near the edge.
- Then use the forceps to grab the scalp on the left side of the incision, and drive the tip of the needle up through the scalp.
- Overall the needle should make a right to left “U” trajectory.
- Now let go of the needle with the hemostat, and grab the end that has come through the left side, and pull the needle until just 2 cm of thread remain on the right side.
- Using your forceps in your left hand, and hemostat in the right, grab the thread on the left side, loop it twice around the end of the hemostat, and then use the tip of the hemostat to grab the bit of thread on the right side and pull it through the loops.
- This can be gently tightened, and is your first knot.
- Now use the same technique, but with only one loop, to tie additional knots over the first one.
- After you have a total of 3-4 knots, you can trim the ends to about 3 mm in length.
Repeat this process in front of and behind the first stitch, to close the remainder of the incision.
Remove mouse from stereotax as follow:
- Turn the isoflurane to off
- Remove the ear bars
- Loosen the nose cone
- Gently pull the mouse from the stereotax
- Place the mouse in the recovery cage
Inject mouse with analgesics:
- Buprenorphine (0.05 mg/kg IP)
- Metacam (5 mg/kg SQ).
Monitoring and cleanup
As the mouse is recovering, and before starting the next surgery, clean and sterilize your instruments:
- Wipe tips of all instruments clean with a kimwipe and ethanol
- Place instrument tips briefly in the bead sterilizer
- Return instruments to your surgical area
- Clean the surgical area and replace the napkins.
Postoperative Care
After surgical procedures are completed administer: Buprenophine (0.1mg/kg) subcutaneously 1-2x on the day of the surgery (with a break not exceeding 4-6h) and Meloxicam (Metacam, 5 mg/kg) subcutaneously on the day of surgery, at awakening to assure analgesia.
One day post surgery, check on mice and administer Meloxicam (Metacam, 5 mg/kg) subcutaneously on at an interval of 24 h from last injection.
Day two post surgery, check on mice and administer Meloxicam (Metacam, 5 mg/kg) subcutaneously on at an interval of 24 h from last injection.
Continue checking daily until there are no signs of pain, then at least weekly thereafter.