Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

Abstract

Source: Alieva, M. et al. Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy. J. Vis. Exp.  (2019)

This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the brain.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tumor Cell Implantation and Cranial Imaging Window Preparation

1. Surgical Preparation
   1. Use adult mice (>6 weeks old) of any strain or gender. Sedate a mouse by injecting fluanisone [neuroleptic] + fentanyl [opioid]) (0.4 mL/kg) + benzodiazepine sedative (2 mg/kg) at a dose of 1:1:2 in sterile water. Assess the animal's sedation state by toe pinch.
      NOTE: In this protocol, both female and male C57BL/6 mice were used due to the same genetic background of the tumor cell line used in these experiments (GL261). The mouse will stay fully sedated for 1.5 h. Alternatively, use inhalation anesthesia such as isoflurane (1.5%–2% isoflurane/O₂ mixture).
   2. Mount the mouse on a stereotactic frame and secure the head using a nose clamp and two ear bars.
   3. Use a heating lamp to preserve body temperature. Heating lamp should be used with caution, alternatively water recirculating heating pads can be used.
   4. Apply eye ointment to protect the mouse's corneas from drying.
   5. Use sharp scissors to shave the fur on the skull (dorsal area from the mouse's eyes to the base of its skull) and disinfect the exposed skin with 70% ethanol.
   6. Cut the skin in a circular manner with sharp scissors and scrape away the periosteum underneath with a cotton swab. Apply a drop of lidocaine 1% + epinephrine 1:100,000 for 5 min and remove the excess with a cotton swab.
   7. Glue the edges of the skin to the skull with cyanoacrylate glue.
   8. Place the stereotactic frame under a dissection stereo microscope with 4x magnification.
   9. Visualize the skull through the dissection microscope and drill a circular groove of 5 mm in diameter over the right parietal bone. Perform this step carefully and only superficially, avoiding any pressure on the skull.
   10. Apply a drop of cortex buffer (125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES buffer, 2 mM MgSO₄, and 2 mM CaCl₂ [pH 7.4]) and lift the bone flap using thin forceps.
   11. For the following steps, keep the brain surface covered with cortex buffer unless indicated otherwise.
   12. Under the dissection microscope, visualize the brain surface and remove the dura mater using curved, tapered, very fine point tweezers. If bleeding occurs at this stage, use an absorbable gelatin sponge to stop it.

2. Cranial Imaging Window Preparation

1. Remove the cortex buffer and place a drop of silicone oil on the craniotomy site to avoid air bubbles under the window.
2. Seal the exposed brain with a 6 mm coverslip. Apply cyanoacrylate glue between the coverslip and the skull. Gently press the coverslip against the skull with the help of fine tweezers to ensure minimal distance between the brain and the coverslip.

Materials

25G x 16 mm hypodermic needles	BD Microlance
Absorbable gelatin sponge	Pfizer	300600
Coverslips round 6 mm	VWR international		Gelfoam
Cyanoacrylate glue	Pattex	631-0168	Pattex Ultra gel
Dental cement	Vertex Dental		Vertex Self-Curing
Drill	Dremel		Dremel 3000 (dental drill may be more convenient) + 105 Engraving Cutter
Fine curved Tweezers	Dumont
Silicone Oil	Sigma Aldrich	AGT508
Stereotaxic frame	Stoelting	181838
Surgical stereo microscope	Olympus		Lab standard stereotaxic, rat and mouse
Opthalmic ointment	Kela Veterinaria		Duodrops veter kela 10 m
Xylocaine (Lidocaine 1% + Epinephrine 1:100,000) Local anesthetic	Astrazeneca		Xylocaine (Lidocaine 1% + Epinephrine 1:100,000)
Hypnorm	VetaPharma Ltd		Hypnorm (Fentanyl citrate 0.315 mg/mL + Fluanison 10 mg/mL)
Midazolam	Actavis		Midazolam Actavis 5 mg/mL