Headplate and cranial window surgery for two-photon imaging of inferior colliculus in transgenic mice

Headplate and cranial window surgery for two-photon imaging of inferior colliculus in transgenic mice

Materials and tools

• (Titanium) headplate
• Scalpel holder and scalpel blades
• 2 pairs coarse forceps
• fine forceps (Dumont #5 Forceps Biology, Fine Science Tools)
• Coverslip forceps (Dumont #5/45, Fine Science Tools)
• Fine scissors
• Curette
• Sterile saline (0.9% NaCl) or artificial cerebrospinal fluid
• Analgesics
• etching gel, e.g. Etch-Rite (Pulpdent Corporation)
• Dental adhesive, e.g. OptiBond FL (Kerr Dental)
• Dental composite, e.g. Charisma and Charisma Flow (Kulzer GmbH)
• UV curing light
• Dental drill and drill head (Ø 0.3 mm)
• Cover glass (Ø 3 mm) and custom steel rings for cranial window, glued together with optical adhesive such as NOA 78 (Norland Products)
• Cyanoacrylate glue (“superglue”)
• Paper points
• Haemostatic gelatin sponge, e.g. Spongostan (Ferrosan Medical Devices A/S)
• Absorbent sponge, e.g. Sugi (Kettenbach GmbH & Co. KG)

Surgical procedures

1. Headplate Installation
   1. Check vaporizer. Fill isoflurane if needed
   2. Anaethesize mouse with 5% isoflurane
   3. Once unconscious, remove mouse from box and put under face mask and over a heat pad.
      Switch to ~0.8L/min, 2% isoflurane
      Note: Adjust % as required. Usually decreases over the course of surgery.
   4. Apply eye ointment (Duratear)
   5. Inject carprofen (Rimadyl) s.c. (5mg/kg; 0.25mL of 0.5mg/mL solution for a 25g mouse)
   6. Inject buprenorphine (Temgesic) s.c. (0.05 mg/kg; 0.04mL of 0.03mg/mL solution for a 25g mouse)
   7. [for cranial window] Inject 15% D-mannitol i.p. (0.4 mL/35 g mouse) to minimize brain swelling.
      Note: inject equal volume of saline at the end of the surgery.
   8. Wetting fur on scalp with soap water (or lidocaine spray) and shave the scalp with scalpel
   9. Using a scalpel sprayed with lidocaine, cut open skin from caudal ridge of skull to middle of two eyes
   10. Apply lidocaine to incision site with spray
   11. Pull skin apart laterally to expose skull. Cut connective tissue between skin and skull if necessary
   12. Locate anatomical landmarks Bregma and Lambda
   13. Make a small incision caudally along the midline to separate the muscles at the dorsal neck region. Push away the muscles laterally to expose the caudal edge of skull and the attached muscles.
   14. Detach muscle from occipital bone by cutting with fine scissors or scrapping with forceps. Avoid damaging the muscle by keeping the incisions as close to the occipital bone as possible
   15. [Optional] For extra surface in headplate attachment, also detach muscles attached dorsolateral to lambda.
   16. Scratch bones with a curette or the tip of a scalpel to remove periosteum and residual attached muscle. The area should cover the dorsal surface of skull from rostral to Bregma, caudal edge of skull where muscle was detached, and extends laterally
   17. Apply etching gel (phosphoric acid), until bone surface becomes slightly whiter and matt (porous). This should take 15-30 s. Wipe away the etching gel with cotton swab wet with Ringer/Saline and rinse with Ringer/Saline.
   18. Apply bonding primer (Optibond primer) to skull using a microapplicator. Air dry the primer with pressured air, or create air flow with a suction held at a distance 
       Note: Make sure no excessive amount of primer remain on surface, i.e. should not look too “wet”, but can be a bit shiny. However, a desiccated “over-dried” surface will also hamper proper bonding in the next steps.
   19. Apply bonding adhesive (Optibond adhesive) to skull with applicator or paper point in a “scratching” action. 
       IMPORTANT: The scratching action ensures that the adhesive gets into small pores of the bone and prevent these pores from closing up
   20. Apply adhesive to the bottom and side surfaces of the headplate. 
       Note: In principle, all surface expected to be glued should have a small amount of adhesive
   21. Position the headplate on the skull, so that the opening is slightly caudal to Lambda. 
       Positioning depends on dimension of the headplate and purpose of the experiment. A well centered (lateral) headplate makes head-fixation nicer for chronic animals, and helps with standardization of position.
       Make sure the headplate is as close to the skull as possible. Too much adhesive between headplate and skull may prevent short working distance objectives from reaching the brain
   22. Cure the adhesive with UV light.
       o Be careful and make sure the headplate stay in correction position while curing
       o Illumination white light may also cure the adhesive and composite (charisma). Turn down the light intensity or block it with orange (UV) filters if they tend to harden too quickly while handling.
   23. Apply UV-cured dental composite (Charisma) to create a well in the opening and secure the headplate to skull. Cure composite with the UV light
       o Make sure all gaps between central opening of headplate and the skull are filled
       o Make sure the dental composite has solid contact with the back part of the skull (occipital bone), in addition to the top parts of the skull (parietal and interparietal bones). This gives extra-strength to the skull, prevents accidental breaking during head fixation, and helps reduce relative motion between skull and brain during imaging. 
       o For large volume, layers of composite can be applied in multiple rounds. 
       o Fill up the rostral part of skull and let a bit of charisma to “grab onto” the headplate. Be careful not to overdo it as this will increase the imaging distance from the objective
       o TIP: Flowable composites (fluidic ones) can be used to help fill in small gaps and corners
       o TIP: Clean your tools with EtOH before handling charisma so that it will not stick too strongly onto the tools. Preferably use smooth unscratched part of tools.
   24. Close up opening by attaching skin to caudal side of headplate with superglue
        
2. Craniotomy
   1. With a fine drill (Ø 0.3 mm), sequentially thin away the bone in an extended area in the opening of the headplate, layer-by-layer. 
      • Note: The bone above the inferior colliculus seems to be in 3 layers: the middle layer is more vascularized while superficial and deep layers are relatively vessel-free. 
      • TIP: Small bleeding from the bone may be stopped by briefly drilling towards the source. Otherwise, apply slight pressure help stop bleeding. Stop larger bleeding by applying a small piece of pre-wet Spongostan (gelatin sponge) for a brief period (one to a few minutes), and then remove it slowly and gently with plenty of saline.
   2. Once you drill beyond the vascularized middle layer, apply a bit of saline/Ringer to the bone. You should be able to see through the bone and check your position.
      • TIP: For cranial window installation (e.g. steel ring constructs), check position of inferior colliculus and extend the size of the craniotomy around it to accommodate the window.
   3. Continue to thin the bone above the inferior colliculus until it becomes membranous.
      • You can check by very gently pressing down on the bone with forceps or a still drill head. It should become flexible.
      • The whole area should extend rostrolaterally to the sinus and a bit caudomedially to cerebellum
   4. Once the intended area is thinned, remove all debris by rinsing with saline/Ringer and removing with suction or sugi.
      • Make sure any bleeding has been stopped at this point to ensure a blood-free brain surface when the bone is removed.
   5. Apply saline/Ringer on the bone. Use a pair of fine forceps, very carefully chip and lift away the bone above the brain starting from the cerebellum side.
      • Working in solution while removing the bone help prevent damage to dura mater.
      • For larger pieces that adhere strongly to dura, use two pairs of fine forceps: pick up a piece of bone on one side with the bent forceps and very slowly lift it up bit by bit like opening a rooftop trapdoor. The other fine forceps can be used to gently detach the bone from the connective tissue in a way similar to blunt dissection techniques.
      • IMPORTANT:  for prepared cranial windows constructs (e.g. double glass, steel rings) make sure the cranial window fits into the opening BEFORE actually opening up the bones. This will avoid having to drill or chip away more bone at the edge later on, which may add bone shards on the brain surface and interfere with imaging.
   6. The inferior colliculus should be rather white and have a shiny surface, compare to the more gray/dark tone of the cerebellum, and the more vascularized cerebral cortex. The dura mater should be intact at this point, which forms a continuous surface on top of the IC and cerebellum.
       
3. Cranial window installation 
   1. For “custom” glass window
      • Break glass coverslips with forceps
      • Search for a glass piece that fits in the exposed area.
        1. It should completely fit through the bones and not be stuck by edges of the bone.
   2. For pre-prepared cranial window constructs (e.g. double glass, steel rings, etc.)
      • The craniotomy should already fit the size of the cranial window
      • Expand the craniotomy if necessary; avoid debris & bone shards on brain surface.
   3. Fit the glass piece in
      • A blood-free brain surface is highly beneficial to imaging. Try to prevent formation of blood clot by promptly rinse away any blood on IC surface. Try to remove any clotting blood with forceps before putting the glass window in.
   4. While applying pressure and holding the window in place, apply a tiny drop of superglue with the tip of a paperpoint to 2-3 contact points between the window and the bone.  Then, proceed to secure the cranial window with dental adhesive or cement. For UV-cured adhesives, it is advisable to cover the glass area with a small piece of aluminium foil to avoid exposure to UV curing light.
      • Alternatively, the whole window can be fastened with superglue for a removable window.
      • Applying pressure is important to prevent growth of connective tissue between the glass window and the brain. Rule of thumb: If you think you have pressed too much, you are good.
      • Be careful not to put too much glue and cover up the glass window. This will be hard to remove afterward and also in risk of removing the anchoring glue while doing so.
      • Vapor of superglue will condense on the clean surface of the glass window and making it foggy. This is completely normal and very easily removed by gentle scratching with forceps, once the window is fixed. The removal can be done (preferably) at the first imaging session.
   5. [When applicable] Close the lid of the headplate once the glue is dried
   6. [When using D-mannitol] Inject saline i.p. (equal volume to D-mannitol)
       
4. Recovery from surgery
   1. Stop the isoflurane, and wait for the first sign of spontaneous movement.
   2. Let the animal recover under a heat lamp

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