Small Animal Brain Extraction
Objective:
To extract the entire brain from the cranium.
Procedure:
- Remove the skin, and tease/scrape away muscle and facia from the dorsal and posterior part of the skull. The eyes are removed by several deep snips with small surgical scissors.
- To obtain a stable grasp of the skull during the process, clamp the left or right mandible with a suitable sized hemostat.
- Rongeurs with a 2-3 mm jaw face are used to remove the bone. Beginning at upper edge of the foramen magnum, take small bites of bone progressing upward (dorsally) and laterally. Maintain pressure away from the brain surface to ensure that slippage of the rongeurs will not penetrate the brain.
- Create a channel of removed bone along both lateral surfaces at a level just above the junction of the zygomatic arch. When completed, a flap of bone remains that can be carefully elevated and broken off near the junction of sutures that form the bregma point.
- Remove the remaining bone along the dorsal surface up to the sinus lying between the olfactory bulbs and the frontal pole of cortex. Also remove the thin bone that forms the eye socket.
- Before proceeding with extraction of the olfactory bulbs, the bone over the posterior part of the nasal cavity is crushed with the rongeurs. The location of the crush is several millimeters in front of a line even with the rostral part of the eye socket. The bone lying above the bulbs should be easily broken free and removed at this point.
- The dura mater is likely to still be intact over cortex and the olfactory bulbs. To remove the dura from the top of the olfactory bulbs, use a fine pair of rat-tooth forceps to grasp the dura lying at the junction of the bulbs and the frontal pole of cortex. Elevate the dura slightly and cut it with small (sharp!) scissors, making a slit if possible both rostrally and posteriorly. A cut may be made down the side of the posterior bulb so that a flap of dura can be reflected from each of the bulbs. The rest of the dura over the cortical surface can be reflected and slit along the midline and other areas so that it can be totally brought down along the lateral surface or simply cut off. The dura that lies between the cerebellum and cerebral hemispheres (forming the lateral sinuses) must be elevated, cut and reflected laterally. It is essential that this be removed completely or any that remains will slice (or cleave) the cerebellum from the cerebrum.
- The olfactory bulbs are innervated from the nasal cavity through the cribriform plate that has numerous small holes (or foramenae). Unless the axon fiber bundles passing through the cribriform plate are undercut, the bulbs will tear away from the rest of the cerebrum. To undercut the olfactory nerve bundles use a small tipped weighing spatula or other equivalent implement. Pass the blade of the spatula between the bulbs carefully until contact with bone is felt. While maintaining contact with the bone, gently nudge each of the bulbs laterally about a millimeter.
- Next, move the blade of the spatula to the lateral surface of the bulb, and again make contact with the bone. Slide the blade between the bone and bulb a millimeter or so and gently lift, very slightly (nudge) along 2-3 locations or more along the length of the bulb. Do the same for the other bulb. When the bulbs are free of the attachments formed by the olfactory nerves, each bulb should be obviously loose and movable.
- Moving to the back and side of cerebellum, the bone and tissue that forms the ‘floor’ of the brain stem should be removed. On each side of the brainstem, the attachment of the trigeminal nerve is visualized, and with small blade scissors (4-5mm) the trigeminal nerve is cut near its attachment to the brainstem.
- Using a small bladed spatula, elevate the brainstem and cut any remaining attached blood vessels. Continue to elevate the brainstem until the optic nerves pull free or they can be cut. At this point the olfactory bulbs should slip free of the dura that lies between them. If not, stop and return to gently prying the bulbs free of remaining attachments. Do not use the cerebrum to pull the bulbs away from any firm attachments or the bulbs will be torn off.
- Place the extracted brain in buffer solution.
For more information:
Fixation Methods & Perfusion Solutions
Perfusion Protocol, Pump Calibration
Post-Fixation Buffer Solutions, Storage, Timing
