Jul 23, 2025
Dual Hemisphere Craniotomy for Electrophysiology
- Anna Lakunina1,
- Conor Grasso1,
- Benjamin Barad2,
- Avalon Amaya1
- 1Allen Institute for Neural Dynamics;
- 2Scripps Research Institute
- Allen Institute for Neural Dynamics
Protocol Citation: Anna Lakunina, Conor Grasso, Benjamin Barad, Avalon Amaya 2025. Dual Hemisphere Craniotomy for Electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjoe2lx1/v1
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: July 25, 2024
Last Modified: July 23, 2025
Protocol Integer ID: 104102
Keywords: Neurosurgery, Rodent Neurosurgery, Electrophysiology, Craniotomy, Cranial Implant, Cranial Window, SHIELD, In Vivo, dual hemisphere craniotomy for electrophysiology, injections within the cranial window, dual hemisphere craniotomy, whole hemisphere craniotomy, extension of the whole hemisphere craniotomy, cranial windowing surgery, cranial window over the cortex, craniotomy, cranial window, access to both hemisphere, single hemisphere, complex surgical procedure, stereotactic injection, surgical procedure, hemisphere, steps for injection, cortical damage, variations of stereotactic injection, procedure, electrophysiology, stabilizing headframe, mouse cortex, injection, timing of injection, neural imaging
Abstract
This protocol describes the surgical procedure, instrumentation, and reagents necessary for the installation of a stabilizing headframe and cranial window over the cortex of an adult mouse. This protocol should be utilized for neural imaging and experimentation regarding the mouse cortex.
This procedure accommodates two variations of stereotactic injections (Optional 1 and Optional 2). Specifically, this protocol outlines the steps for injections before and after the craniotomy and durotomy. The timing of injections during cranial windowing surgeries depends on the coordinate location. Injections outside of the cranial window should be performed prior to the craniotomy and durotomy to allow for adequate burr hole sealing. Injections within the cranial window should be performed after the craniotomy and durotomy to minimize cortical damage.
The surgical procedure described in this protocol is an extension of the Whole Hemisphere Craniotomy for Electrophysiology. This surgical procedure should be used in cases where access to both hemispheres of the brain is necessary during experiments. If access to a single hemisphere is sufficient, the Whole Hemisphere Craniotomy should be used instead, as it is a less complex surgical procedure.
Image Attribution
Gabriel Rodriguez, Allen Institute.
Guidelines
Only perform this procedure in accordance with IACUC and veterinary requirements.
This protocol has been written for review purposes only and should not be utilized for training. Users of this protocol should be familiar with common vocabulary and techniques for rodent neurosurgery.
Materials
Anesthesia and other drugs
Ethiqa XR Buprenorphine Extended-Release Injectable Suspension for Mice and Rats 1.3mg/mL, 3mLFidelis PharmaceuticalsCatalog #099114
IsofluranePatterson VeterinaryCatalog #07-890-8115
1 g DexamethasonebiorbytCatalog #orb134330
Ceftriaxone Injection AmerisourceBergen MWI Animal HealthCatalog # 094311
Lactated Ringers Injection, USP, Preservative-Free, BaxterHenry Schein Animal HealthCatalog #059380
1 g AtropinebiorbytCatalog #orb322218
1 g CarprofenbiorbytCatalog #orb321211
Note
Drugs should only be administered in accordance with IACUC and veterinary requirements. Ensure timing, dosage, and route of administration are accounted for.
Surgical tools and supplies
| Tool / Supply | Manufacturer / Supplier | Part Number | |
| Black handle scissors, ToughCut | Fine Science Tools | 14058-11 | |
| Scalpel handle | Fine Science Tools | 10003-12 | |
| Iris forceps | Fine Science Tools | 11064-07 | |
| Dumont #5 45° forceps | Fine Science Tools | 11251-35 | |
| 45° Vannas scissors, 8cm | World Precision Instruments | 500260 | |
| 45° or 90° Durotomy probe | Fine Science Tools | 10066-15 | |
| Plastic sterilization container | Fine Science Tools | 20810-02 | |
| large iris forceps | Fisher Scientific | 13-820-073 | |
| PREempt Disinfectant spray | McKesson Corporation | 21101 | |
| 70% Ethanol (Diluted in-house) | Sigma Aldrich | 459836 | |
| Alchohol wipes | Becton, Dickinson and Company | 326895 | |
| Sterile Surgical Drape, 18x26 | Fisher Scientific | NC9517505 | |
| Sterile Multi-well plate, 24 well | Avantor | 29443-952 | |
| Nair hair removal cream | Arm & Hammer | 40002957 | |
| Betadine Solution 10% | McKesson Corporation | 1073829 | |
| Hemostatic Agent Surgifoam | McKesson Corporation | 403360 | |
| Sterile Gauze, 3x3” squares, (autoclave sterilized) | Patterson Veterinary | 07-893-8587 | |
| Cotton swabs, double ended, (autoclave sterilized) | Avantor | 89133-810 | |
| Sugi Absorption Spears | Fine Science Tools | 18105-01 | |
| Insulin syringes, U-100, 0.3 ml, 31G | Avantor | BD328438 | |
| Insulin syringes, U-100, 1 ml, 31G | Avantor | BD328418 | |
| Luer-Lock Syringe, 20 ml OR | Avantor | 53548-025 | |
| Luer-Lock Syringe, 10ml | Avantor | 75846-756 | |
| 25G 5/8-inch needle | Avantor | 89134-134 | |
| 32 mm Syringe Filter 0.2 µm Supor Membrane | Avantor | 75846-756 | |
| Press ‘n’ Seal | Medline | CLO70441 | |
| Saran Wrap | GLAD | Amazon B015CLAVU | |
| Sterile Drill Bits, 0.5/0.4, FG1/4 AND/OR | NeoBurr | 1734948 | |
| Sterile Drill Bits, 1.4/1.1, FG4 AND/OR | NeoBurr | 1734214 | |
| Sterile Drill Bits 1.0/4.2, EF4 | NeoBurr | 1730012 | |
| Sterile Scalpel blades, #10 OR | Avantor | 21909-378 | |
| Sterile Scalpel blades, #11 | Avantor | 21909-380 | |
| Systane Eye Ointment | Systane | Amazon ALCON293787 | |
| Artificial Cerebrospinal Fluid.V* | Made in-house. Protocol referenced. | http://dx.doi.org/10.17504/protocols.io.besjjecn | |
| C Universal 4-META Catalyst, 0.7 ml | Parkell | S371 | |
| B Quick Base for MetaBond, 10 ml | Parkell | S398 | |
| Radiopaque L-Powder, white, 5 gm | Parkell | S396 | |
| Radiopaque L-Powder, clear 3 gm | Parkell | S399 | |
| CS-5R Coverslips, 5 mm | Warner Instruments | 64-0700 | |
| Vetbond Glue | Patterson Veterinary | 07-805-5031 | |
| Superglue, Singles | Krazy Glue | Amazon PK4 KG58248SN | |
| 3 ml transfer pipette, plastic | Avantor | 52947-970 | |
| Ortho-Jet BCA Liquid | Lang Dental Maufacturing Company | Ortho-Jet BCA Liquid | |
| Black cement (1) = 4 parts of Ortho-Jet BCA Powder (mixture) AND | Lang Dental Manufacturing Company | Ortho-Jet BCA Liquid | |
| Black cement (2) = 1 part of Powder tempura point, black | Jack Richeson & Co | 1# Black 62, Amazon B00JGZ8Q1A | |
| Kwik-Sil Sealant | World Precision Instruments | KWIK-SIL | |
| Kwik-Cast Sealant | World Precision Instruments | KWIK-CAST | |
| Heat-sterilized Glass pipettes AND/OR | Drummond Scientific | 3-000-203-G/X | |
| Heat-sterilized Glass pipettes | World Precision Instruments | 1B120F-4 | |
| “Marker” glass pipette, pulled, broken, and Sharpie mark for measuring coordinates | World Precision Instruments | 1B120F-4 | |
| Microcapillary Pipette tips | Eppendorf | 89009-310 | |
| Parafilm | Avantor | 52858-000 | |
| Lightweight Mineral Oil | Sigma-Aldrich | M8410 | |
| 30 gauge, 2" Backfilling Needle | Drummond Scientific | 3-000-027 | |
| Sterile Bone Wax | Central Infusion Alliance, Lukens | CIA2160287, 901 | |
| Sterilization pouches | Avantor | 89140-804 |
All tools / supplies can be substituted with their equivalent.
Key:
AND = Including the tool/supply in row below.
OR = Can use tool/supply in row below instead.
Autoclaved sterilized = Sterilized in-house.
mixture = Mix with tool/supply in row below.
Equipment
| Equipment | Manufacturer / Supplier | Part Number | |
| Small Animal Stereotactic Instrument | Kopf | 1900 | |
| Adjustable Stage Platform | Kopf | 901 | |
| Stereo Microscope | Leica | M80 | |
| Gooseneck Illumination | AM Scope | LED-6WA | |
| On-axis Illumination | Lecia | KL2500 LED | |
| Bead sterilizer | Sigma-Aldrich | Z378585 | |
| Small Animal Temperature Control System | CWE Inc. | TC-1000 | |
| Large Heat plate/pad | Lectro-Kennel | Outdoor Heated Pet Pad | |
| Dental Drill | NSK | Pana-Max2 M4 | |
| Oxygen Concentrator | Nidek Medical Products | Nuvo Lite Model 525 | |
| Isoflurane with oxygen delivery system | Patterson Scientific | Tec 3 EX | |
| Isoflurane induction chamber | Patterson Scientific | 78933385 | |
| Ear bars | Kopf | 1922 | |
| Ultra Fine Point Sharpie | Sharpie | 37001 | |
| Metabond ceramic mixing dish | Parkell | S387 | |
| Electrode Holder | Kopf | 1970 | |
| Galaxy Mini Centrifuge | Avantor | 76269-066 | |
| P20 Pipettor | Gilson | F123600 | |
| Silver wire | Stoelting | 50880 | |
| Midgard Precision Current Source | Stoelting | 51595 | |
| Nanoject II Variable Volume (2.3 to 69 nL) Automatic Injector OR | Drummond Scientific | 3-000-204 | |
| Nanoject III Programmable Nanoliter Injector | Drummond Scientific | 3-000-207 |
All equipment can be substituted with their equivalent.
Key:
OR = Can use equipment in row below instead.
Materials/Equipment designed/made in-house (CAD available upon request)
| Material | Part Number | |
| Well Cap | 0160-055-09 | |
| Bregma Stylus | 0251-900-04 | |
| Dovetail Clamp | 0111-200-00 | |
| 0⁰ Ear bar Headframe Clamp | 0155-110-00 | |
| Prober Holder | 0155-200-00 | |
| Bilateral SHIELD Implant | 0251-170-01 | |
| SHIELD Bilateral Implant Tracer | 0274-122-00 or equivalent | |
| Bilateral SHIELD Headpost and Well | 0160-075-00 |
All equipment can be substituted with their equivalent.
Personal Protective Procedures (PPE):
| Suggested PPE | |
| Gloves | |
| Disposable lab coat | |
| Disposable face mask | |
| Shoe covers / surgery shoes | |
| Scrubs | |
| Surgical cap | |
| Biohazard sharps disposal container | |
| Biohazard waste disposal container | |
| Blue light blocking glasses |
Utilize PPE in accordance with IACUC and veterinary requirements. Ensure sterility when necessary.
Troubleshooting
Safety warnings
- Personal Protective Equipment (PPE) should be used at all times while operating this protocol.
- Isoflurane Warning: Acute over-exposure to waste anesthetic gases (WAG) may cause eye irritation, headache, nausea, drowsiness, or dizziness. Repeated exposure may cause damage to cardiovascular system and central nervous system. Refer to MSDS for additional information. Consult the surgical workstation guide to ensure all parts of the dispensation rig are functioning properly.
Ethics statement
Research-focused rodent neurosurgery must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s).
This protocol has been approved by the Allen Institute Animal Care and Use Committee (IACUC).
PHS Assurance : D16-00781
AAALAC : Unit 1854
Before start
Reference protocol below for all general setup and takedown procedures for rodent neurosurgery:
Optional 1:
For stereotactic injections prior to performing the craniotomy and durotomy, follow section titled "Optional 1" in addition to the standard sections.
Optional 2, 2.1, 2.2:
For stereotactic injections after performing the craniotomy and durotomy, follow sections titled "Optional 2", "Optional 2.1" and "Optional 2.2" in addition to the standard sections.
Headpost and Headframe will be used interchangeably throughout this protocol.
Graphical Overview of Procedure
Map diagram of protocol workflow.
Expose and Prepare Skull Surface
20m
After hair removal and disinfection, create midline incision with a scalpel blade from approximately behind the eyes to the front of the ears.
Illustration of initial skin incision on mouse head.
Mouse fixed to stereotactic instrument base via bite bar and ear bars. Fur from top of head has been removed and skin has been sterilized (see General Setup and Takedown Procedures).
Using Vannas scissors, cut a round teardrop shape of skin away.
Illustration of skin removal on mouse head.
On the left and right side remove enough skin to see the interface of the skull and cheek muscle. On the posterior side stop as soon as the neck muscle is exposed. Anteriorly, stop at around 1mm past the rostral rhinal vein.
Tear or cut the periosteum and ensure removal at surgical site. Rehydrate with Artificial Cerebrospinal Fluid (ACSF) if needed to finish removal.
Note
Utilize a 10mL or 20mL syringe, 25G 5/8-inch needle, and a syringe filter to store and dispense ACSF.
Detach muscles to allow more surface area for headframe securing.
Illustration of skin removal boundaries and muscle removal extent.
Detach the cheek muscle on the left side of the skull by poking into the interface between the muscle and skull, and then drag the Dumont forceps along the length of the cheek. Detach the neck muscles with Dumont forceps by scraping the muscle attachments.
On the left side of the skull, pull separated muscle and skin away from the skull as shown in the illustration. At the posterior edge, tuck the detached neck muscles underneath the skin.
Seal all along incision site with Vetbond. Use a Sugi Absorption Spear to absorb any excess fluid either prior or during. Extend Vetbond seal 1-2mm past incision site along healthy skin.
Align the Skull
5m
Locate Bregma and Lambda landmarks with dovetail clamp and Bregma Stylus, and use them to level the skull in the anterior-posterior axis within 0.1mm.
Illustration of Bregma and Lambda landmarks.
If Lambda-Bregma offset is greater than 0.1mm in X, use the yaw adjustment on stereotactic alignment system to adjust the yaw to within 0.1mm.
At midline, approximately midway between Bregma and Lambda, measure 2mm laterally on both the left and right hemisphere, ensuring that the skull is level in the medial-lateral axis within 0.15mm.
Optional (1): Perform Stereotactic Injections Outside of the Cranial Window
10m
Optional 1
This optional section accommodates injections prior to performing the craniotomy and durotomy. Typically, this is done for injections that will be outside the craniotomy window, as placement of the headframe can obstruct some coordinates. Injection coordinates within the craniotomy window are performed after the craniotomy and durotomy as it will minimize cortical damage.
Skip this section if you will not be performing injections prior to the craniotomy and durotomy.
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject.
Begin at step 8.2.6 "Mark the Injection Site".
Stop at step 8.5 "Suturing".
Reference protocol Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6 for performing stereotactic injections via iontophoresis.
Begin at step 8.2.6 "Mark the Injection Site".
Stop at step 8.5 "Suturing".
Once completed with the injection, fill burr hole with bone wax with the broken end of a cotton swab.
Note
Do not leave excess wax residue on the skull. Excess wax can contribute to an insufficient seal between the skull and the headframe.
Optional (2): Mark Stereotactic Injection Coordinates for After Craniotomy and Durotomy
7m
Optional 2
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the first section out of three required. This first section is specifically for identifying and marking injection coordinates while the skull is level. These markings will be referenced in the second optional section (Optional 2.1).
Skip this section if you will not be marking injection coordinates for injections after the craniotomy and durotomy.
Considerations:
For injections within the cranial window, it is important to take the injection depth into consideration. Avoid targeting subcortical brain structures, as they cause more brain edema due to the required insertion depth via pipette.
Additionally, ensure you maintain bregma as the fiducial point. Avoid the use of multiple viruses/pipettes as pipettes are not made identically (variance of up to 0.8mm difference).
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject.
Begin at step 8.2.6 "Mark the Injection Site".
Stop before step 8.2.7 "Drilling the Burr Hole".
Reference protocol Stereotaxic Surgery by Iontophoresis V.6 for performing stereotactic injections via iontophoresis.
Begin at step 8.2.6 "Mark the Injection Site".
Stop at step 8.5 "Suturing".
Ensure injection coordinate is marked as precisely as possible. This is best done with a fine point sharpie mark with a notch or point negative made from the marker pipette tip.
Etch Craniotomy and Secure Headframe
20m
Ensure skull is level.
If you have performed an angled injection, return mouse back to level state.
Using the dovetail clamp, zero Bregma stylus over Bregma, and replace with the SHIELD Bilateral Implant Tracer.
Using the SHIELD Bilateral Implant Tracer as a guide, etch the shape of the craniotomy onto the skull using 45° forceps or #11 scalpel blade.
Illustration of SHIELD Bilateral Implant Tracer centered over Bregma.
Note
CAD image for craniotomy shape is available on brain-map.org.
Remove the dovetail clamp from the stereotactic arm with the SHIELD Bilateral Implant Tracer still connected. Do not move the X or Y axis.
Using the FG1/4 or EF4 drill bit, drill a shallow trench over the etch.
Illustration of dual hemisphere craniotomy etched into skull.
Return the dovetail clamp with connected tracer back onto sterotax arm. Confirm size and shape of craniotomy. Repeat steps - Step #21 until the craniotomy etch is accurate.
Note
It is crucial that the craniotomy trace is accurate. The craniotomy shape is specifically designed to avoid disruptions to the sinus confluences.
Secure the Headframe
20m
With the dovetail clamp still centered over Bregma, raise the Z axis and then replace the SHIELD Bilateral Implant Tracer with the Bilateral SHIELD Headpost.
Lower the Z axis until the headframe comes in contact with the skull.
Note
The contact point for this headframe is between the eyes. However, the fixed skin by the eyes will likely prevent the headframe from making direct contact with the skull. This is acceptable, but the headframe should be placed as close to the skull as possible without compressing the fixed skin into the eye or disrupting the level plane of the skull.
Note
Lowering the headframe too far down onto the mouse's skull can potentially crack a cranial suture line. If this occurs, your headframe will not attach to the skull due to cerebrospinal fluid leakage.
Prepare and apply the first round of Metabond.
Apply Metabond inside the headpost and around the outside where it's accessible. Place Metabond so that the majority of the skull within the well remains exposed. Let dry ~ 00:05:00 .
Illustration of Metabond placement for Bilateral SHIELD Headpost.
Turn off the isoflurane, remove the ear bars, and loosen the nose cone slightly.
Carefully detach the headframe from the dovetail clamp.
Prepare another Metabond tray.
Pull back nose cone completely and remove the mouse from bite bar. Either lay the mouse on the heating pad or hold mouse in hand.
Apply Metabond to the outside of the headpost to cover all exposed muscle. Extend Metabond to 1-2mm past the exposed muscle and tissue to create a seal.
Note
Be as efficient and quick as possible to ensure mouse stays anesthetized during this step.
Note
Clean up any Metabond close to the eyes and smooth over any sharp points to avoid irritating the mouse.
Using the 0° earbar headframe clamp, return mouse back onto the stereotax. Ensure headframe is secured in place by ear bar clamp.
Note
Be as efficient and quick as possible to ensure mouse stays anesthetized during this step.
Place headframe plate into the clamp, nose cone to nose, and turn isoflurane on.
Replace saran wrap over mouse body.
Optional (2.1): Align Injection Device for Stereotactic Injections After Craniotomy and Durotomy
7m
Optional 2.1
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the second section out of three required. This second section is specifically for aligning the injection device with the desired injection coordinates that were marked in Optional 2. Since the craniotomy and/or durotomy can obscure the coordinate fiducials we need to align the injection device to the precise X and Y (ML and AP) coordinates. After the craniotomy in Optional 2.2, the Z axis (DV) will be manipulated to perform the injections. If there are multiple coordinates, identify one of them and zero the coordinates creating that point as a fiducial to the remaining coordinates.
Note
Oftentimes Bregma may still be visible after drilling the craniotomy; however, it is still recommended to identify the coordinates prior.
Skip this section if you will not be marking injection coordinates for injections after the craniotomy and durotomy.
Ensure skull is level.
If headframe has been secured, set the roll and pitch of the mouse to zero degrees.
Attach injection device with loaded pipette onto stereotactic arm and center over injection location. This stereotactic coordinate should be pre-marked from Step 17
Place the injection device onto the stereotax arm.
Zero the X and Y and coordinates over marked injection coordinate.
Remove injection device from stereotax arm.
DO NOT move the stereotax arm out of the way. It will stay in this position during the craniotomy and durotomy.
Note
Nanoject/Iontophoresis pipettes should not be changed after setting up the stereotax arm. The variation in pipette straightness can cause the injection location to move up to 0.8mm.
Perform Craniotomy
35m
Using drill and FG1/4 drill bit, drill away any excess Metabond that would interfere with the implant.
Using the drill and the FG1/4 or EF4 return to the trench and drill until you have cracked through all the way around.
Note
The drilling process will take 30-40 minutes.
Specifically for this dual hemisphere craniotomy, you need to fully drill and separate the skull at the sinus confluences. These are located at the deep curves of the craniotomy etch that sit right along the midline.
Note
The sinus confluence sites will contain skull that is significantly thicker than the majority of the skull.
Note
The anterior confluence sits close to the rostral rhinal vein and is surrounded by cranial vessels. Be sure to note the difference between a cranial bleed and the rostral rhinal bleeding during your drilling.
If you need to remove debris or blood during the craniotomy you can rinse with ACSF. Be aware that soaking the skull in ACSF will cause it to become soft and can cause the drill to take off more layers than anticipated.
Rinse the well using copious amounts of ACSF and sterile gauze/Kimwipes to get rid of debris before removing the skull island.
Place Hemostatic Agent Surgifoam within the well if anticipating bleeds.
Note
The dual hemisphere craniotomy borders 3 major blood vessels. It is highly likely that you will require anticipatory Surgifoam ready within the window before removing the skull island.
Add ACSF to the well. Using 45° forceps, gently pry up the skull at multiple points to ensure the skull island is sufficiently separated.
Note
If skull is still attached (i.e. bone bridge, not cracked), remove ACSF and continue drilling. This is especially crucial at the sinus confluences. Rinse before removal.
When the skull island appears ready to be removed (sufficiently detached), use 45° Dumont forceps to carefully remove the skull island, being careful to minimize pressure on the brain (i.e. pressure from skull island or tools).
With 45° Dumont forceps, gently grab the skull island from the edge. Slowly pull the skull upwards. Watch for any unnoticed bone bridges, the adherence of the dura or sagittal sinus to the skull, bleeding, and bending of the skull island at the skull sutures.
Note
Avoid any rapid movements to prevent the skull from dipping down and damaging the cortex or the ripping/tearing of blood vessels or sinuses.
Stop all bleeds with Hemostatic Agent Surgifoam, ACSF rinses and/or sterile Sugi absorbent spears. Make sure there is always ACSF covering the brain surface.
Note
Ensure all bleeds are stopped as rapidly and efficiently as possible. Excess blood loss can result in mouse death.
Perform a Durotomy
35m
Make the initial incision for each of the hemispheres using a durotomy probe.
Use angled Vannas scissors cut the dura back to the bone line. Remove dura either by cutting along the bone line or using 45° Dumont forceps to pull flaps over the bone line. No dura should remain within the window.
Remove dura by using 45° Dumont forceps to hold the dura taught and the angled Vannas scissors to cut.
Use caution when cutting the dura away near the sagittal sinus and large cranial vessels. The dura is connected to the sagittal sinus as well as many large vessels branching off of the sinus. Excess tugging or pressure can compromise the vessels and cause bleeding.
Two separate durotomies will need to be performed as there are two hemispheres.
Stop all bleeds with Hemostatic Agent Surgifoam, ACSF rinses and/or sterile Sugi absorbent spears. Make sure there is always ACSF covering the brain surface.
Note
Ensure all bleeds are stopped as rapidly and efficiently as possible. Excess blood loss can result in mouse death.
Optional (2.2): Perform Stereotactic Injections Within the Cranial Window
10m
Optional 2.2
This optional section accommodates injections that will be performed after the craniotomy and durotomy. This is the last section out of the three required. This second section is specifically for injecting the desired material at the injection coordinates that were marked in Optional 2.
Skip to next section if not performing stereotactic injections within the cranial window.
Ensure mouse's skull is level.
Set the roll and pitch of the mouse to zero degrees.
Note
Since the headframe was placed on a level skull, having the pitch and roll set to zero with mouse in the ear bar clamp creates a level skull.
Reminder: the visual cortex headframe and ear bar clamp have a pitch of -6° and 6° respectively.
Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject.
Begin at step 8.3 " If Injecting with Nanoject II" or 8.4 " If Injecting with Nanoject III".
Stop at step 8.5 "Suturing".
Reference protocol Stereotaxic Surgery by Iontophoresis V.6 for performing stereotactic injections via iontophoresis.
Begin at step 8.4 "Inject Virus with Iontophoresis"
Stop at step 8.5 "Suturing".
Raise and remove injection device from stereotax arm.
Apply and Seal the Implant
10m
Remove custom made Bilateral SHEILD Implant from packaging and rinse in a well of ACSF.
If there are visible air bubbles, rinse again and/or use the ACSF syringe or a sterile swab soaked in ACSF to detach bubbles.
Note
Air bubbles on the implant occasionally arise from surface tension when implant is being soaked and/or removed from ACSF well. This is to prevent the addition of air into/onto the brain.
Place the implant so the inside edge sits over the craniotomy and slowly lower the silicone coated probe onto the center of the implant.
Note
If the implant is placed too forcefully or dropped, cortex damage may occur.
It is best to have a significant amount of ACSF covering the cortex to cushion it's placement.
Gently compress until all edges of the implant lip are contacting the full perimeter of the craniotomy.
Note
Excess pressure on the implant can cause detrimental brain compression.
Apply Vetbond to the perimeter of the implant that is contacting the skull. Be careful not to cover any holes in the implant.
Illustration of pressure tool holding implant in place of craniotomy.
Complete Implant Seal and Attach Well
5m
Carefully raise and detach the pressure tool holding the implant from the stereotax arm.
Secure Bilateral SHIELD well to headframe.
Using Krazy glue or equivalent, apply glue to the top of the headframe ring and place the well in the correct orientation over the headframe, using pressure for 5-10 seconds to allow the glue to adhere.
Reinforce Vetbond seal and cover any additional exposed skull with Metabond.
Apply Metabond within the well halfway up the implant lip with the wooden end of a cotton swab. Be careful not to cover any holes in the implant with Metabond.
Note
If you do get Metabond on the implant, it is best to wipe it away immediately with the cotton end of the swap. The Sorta-Clear easily scuffs.
With the remaining Metabond, cover the connection point between the well and the metal headframe to reinforce the well attachment. Let dry for ~ 00:05:00 .
Apply black cement over top of the white Metabond in the well. Be careful not to cover any holes in the implant. Allow to dry completely ~00:05:00 .
Note
This step is only useful if imaging the animal's brain as it reduces glare and light leak. If only planning on using this animal for electrophysiology, the white Metabond creates a nice contrast to electrodes being lowered into the brain.
5m
Add equal parts black cement powder and Ortho-Jet BCA liquid to an empty well of the 24 well plate.
Use the broken end of a swab to layer the black cement.
Note
You will need to work quickly as black cement hardens rapidly.
Once Metabond and black cement have fully dried, attach a well cap to protect the implant from gathering dirt and debris in cage.
Illustration of complete dual hemisphere surgery.
Proceed to takedown and recovery steps.
Recover Mouse and Takedown
10m
Remove mouse from stereotactic instrument base once Metabond and black cement are fully dry.
Turn off isoflurane.
Detach headframe clamp from headframe carefully.
Remove earbars.
Remove mouse teeth from bitebar by lightly scruffing mouse in a way that raises the teeth out of the bite bar.
Obtain the mouse’s postoperative weight.
Place the mouse back in a recovery cage and put the cage on the 37 °C heat plate.
Reference General Setup and Takedown Procedures for Rodent Neurosurgery protocol for takedown procedures.
Protocol references
SHIELD: Skull-shaped hemispheric implants enabling large-scale electrophysiology datasets in the mouse brain. Bennett, Corbett et al. Neuron, Volume 112, Issue 17, 2869 - 2885.e8 doi: https://doi.org/10.1016/j.neuron.2024.06.015
Allen Institute for Brain Science. (2023). Stereotaxic Injection by Nanoject Protocol V.6. protocols.io. dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6
Allen Institute for Brain Science. (2024). Stereotaxic Injection by Iontophoresis V.6. protocols.io. dx.doi.org/10.17504/protocols.io.14egn8ewzg5d/v6