Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

In this video, we present how to expose and remove the intact pig brain and the intact pituitary by opening the skull and dura. Ethics statement:Animal anesthesia and euthanasia were performed in accordance with Principles of laboratory animal care"and approved by the Danish Council for Animal Research Ethics. Anesthesia was induced in male Gottingen minipigs by an intramuscular injection of midazolam and ketamine.

Five to ten minutes later, when the animals were deeply sedated, an ear vein was cannulated and a lethal overdose of sodium pentobarbital was given intravenously. To secure the animals were completely euthanized, the interdigital pain reflex was tested as previously shown. After proper euthanization, the animals were transcardially perfused with five liters of isotonic saline.

All the procedures demonstrated are, accordingly, performed postmortem, excluding the need for welfare precautions necessary for long term anesthesia and post-procedural survival. Instruments used. Heavy scalpel handle number four, and non-sterile scalpel blades, number 23.

Good for skin incisions, and soft tissue removal. Scalpel handle number seven, and non-sterile scalpel blades, number 11. Optimal for dural incision, and precision work.

Surgical forceps, with a bite that ensures a firm grip. KAIRison Bone Punch. Must be robust, with a bite size of three to five millimeters.

Bone rongeur. Must be robust and have a bite size around five to 15 millimeters, for delicate bone work. Larger bone rongeur.

With a bite size around 25 times 15 millimeters, for removal of thicker bone pieces. Bone chisel. The head should not be larger than 20 millimeters.

A mallet, suitable for the bone chisel. Micro scissors, with a sharp tip.Dissector.Decapitation. The pig is decapitated by making a high circular cervical incision, just below the mandibular angle.

Continue the incision, through the soft tissue of the neck. Cut through larynx, and esophagus, until the bone and spinal column is reached. Approximately at the level of the cranio-cervical junction.

Advance the incision from the anterior side of the cranio-cervical junction, through the anterior atlantooccipital membrane. Whereby the spinal cord is exposed. Simultaneously, an assistant will pull the pig body away from the pig head, as this will ease the access between the skull base and the first cervical vertebra.

Make sure the spinal cord is completely severed, before a forceful extension at the cranio-cervical junction finalizes the decapitation. Skull opening. Place a dorsal longitudinal incision through the skin and underlying soft tissue.

From the back of the snout over the vertex of the head and down through the posterior part of the occipital region. Expose the dorsal and posterior part of the skull by removing soft tissue located lateral to the initial incision. Release the temporal muscle from the skull.

Clean the posterior occipital bone from soft tissue. Use the posterior interest of the foramen magnum to remove the occipital bone in order to expose the dura-covered cerebellum. Return to the anterior side of the skull and select an entry point in the frontal bone, just in front of the eyes.

At this point, use a bone chisel to penetrate the skull, and enter the frontal sinus. Use the extent of the frontal sinus for further removal of the other skull lamina. An inner thin skull lamina covering the cerebrum, is exposed.

Open this lamina gently, to expose the dura covering the anterior part of the brain. Continue the bone removal laterally, through the temporal and parietal bone. In order to release the final part of the skull, located between the already-exposed parts of the cerebrum and cerebellum.

Use the chisel to break open the remaining posterior skull bone, from one side, just like one opens a door. Brain removal. Lift the dura, for gentle incision close to the sagittal sinus.

Use a dural opening for further removal of the dural sac covering the brain. Special care must be taken when removing the dura situated between the cerebrum and the cerebellum. As preservation of this dural leaf will prevent subsequent brain removal.

Finally, the dura covering the cerebellum and the lower brain stem is sectioned. Position the pig head vertically, and release the cerebrum by blunt dissection of the olfactory bulbs from the dura-covered skull base. Section the exposed optic chiasm.

Follow it by subsequent exposure and section of the pituitary stalk, and the oculomotor nerves. Release the brain stem by sectioning of the lower cranial nerves. Use a chisel to release the brain from the skull base.

Pituitary removal. In the skull base, identify the pituitary stalk, with surrounding dural leaf. Cut the dural leaf laterally to the pituitary stalk.

Use a dissector to release the pituitary, and lift it out of the pituitary fossa. Results and conclusion. The described technique enables safe removal of the postmortem pig brain and pituitary, in approximately 15 to 20 minutes.

The obtained tissue may be submitted for microscopic analysis followed by oriented sectioning, into smaller brain slabs. The slabs are suitable for chemical analysis, or further histological processing.