Preparing Wedge Brain Slices to Visualize Auditory Neurons
Trascrizione
Begin by using a razor blade to cut the skin at the midline of the skull from the nose to the back of the neck. Peel back the skin to expose the skull, and starting at the base of the skull and continuing towards the nose, use small scissors to make an incision in the skull through the midline.
At the lambda suture, make cuts in the skull from the midline laterally towards the ear on both sides to peel back the skull to expose the brain. Starting at the rostral end, use a small lab spatula to gently lift the brain from the skull to allow the optic nerves to be severed.
Continue to gently work the brain backwards, exposing the ventral surface, and use fine forceps to carefully pinch the trigeminal nerves near the ventral surface of the brain stem to cut them. Place the preparation in a glass Petri dish filled with cold slicing solution, and place the dish under a dissecting microscope. Trim the facial nerves close to the brain stem to expose the vestibulocochlear nerves.
Use fine forceps to push the tips into the foramina where the vestibulocochlear nerve exits the skull as far as possible. Pinch to sever the nerves on both sides, leaving the nerve root attached to the brain stem.
Remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body. Then, pinch the remaining cranial nerves and connective tissue to free the brain completely from the skull, taking care to preserve the remaining spinal cord, if possible.
To prepare the surface of the brain for fixture to the stage, place the brain ventral side up, and use a blunt tool to gently immobilize the spinal cord to stabilize the brain tissue. Insert open forceps at an approximately 20-degree angle through the brain to the bottom of the dish so that the tips exit the dorsal surface of the brain caudal to the optic chiasm, and use a razor blade to cut along the forceps.
Next, prepare a one cubic centimeter block of 4% agar and spread a small drop of glue into a rectangle on the stage. Use forceps to carefully lift the brain and gently dab the excess liquid with the edge of a paper towel. Then, place the blocked surface onto the glue so that the ventral surface will be facing the direction of the blade during slicing, and push the agar block gently against the dorsal surface as a support.
To acquire wedge slices with the cochlear nerve root on the thick side and the medial, olivocochlear neurons, and the medial nucleus of the trapezoid body on the thin side, place the magnetic disk with the attached brain onto the stage holder, and place the holder in the slicing chamber of a vibratome with the ventral surface of the brain oriented toward the blade. Fill the chamber with ice-cold slicing solution and lower the blade into the carbogen-bubbled solution.
Cut slices caudal to the region of interest to make sure the slices are symmetrical. Then, shift the stage approximately 15 degrees to one side. Continue slicing carefully until the auditory nerve root is close to the surface on one side and the facial nerve can be seen at the surface of the other side of the slice.
Shift the stage 15 degrees back to the original position, and move the blade away from the tissue. Spin the stage base 90 degrees so that the lateral edge of the thin side is facing the blade, and lower the blade several hundred microns before slowly bringing the blade close to the edge of the tissue. With the blade retracted slightly, lower the blade to the desired thickness of the thin edge of the slice.
Move the blade back from the tissue and spin the stage base back so that the ventral surface faces the stage base. Make a cut to designate the rostral surface of the wedge slice, and transfer the slice to a one-square-centimeter piece of interface paper. caudal surface down.
The facial nerve should be visible on both hemispheres of the slice on the rostral surface. Then, move the slice to a 35-degree Celsius incubation chamber to recover for 30 minutes.
