Autopsy Neuropathology: Brain Cutting Guide

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Preparation and Performance of Autopsy Brain Exam

• Review case history for pertinent details prior to cutting brains… Always be ready to answer the question – why am I cutting this brain? Pay particular attention to the following clues in history that will guide examination and tissue sampling
• Trauma – recent vs. remote, extent
• Infection
• Seizures
• Neurodevelopmental abnormalities
• Neurodegenerative brain disease (“dementia”)
• Hypoxic/ischemic brain injury (global vs. localized, recent versus remote)
• Metabolic brain disorders (malnutrition, alcoholism, vitamin deficiency)
• Notation of these (and/or any other relevant) histories allows for proper preparation for case cutting and avoids “cutting blind”

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• Ensure adequate fixation of brain prior to gross examination
• A minimum of two weeks is adequate only if using 20% formalin solution
• For most cases, ~ 4 weeks is preferred, especially if brain is soft, edematous, or immature, or if using 10% formalin solution
• Suspension of brain during fixation is critical
• String threaded under basilar artery and secured under lid is optimal method
• Use of hairnet to secure brain is acceptable, but results in artifact and can be quite messy (hairnet holds brain and is secured under lid). This hairnet method is preferred for very soft and/or immature brains
• Allowing brain to sink to bottom of container during fixation results in “bucket brain”, with significant asymmetry and fixation artifact.
• Print off cassettes prior to brain cutting
• 10 cassettes per case is the starting point for all cases – only rare cases need more caps
• Flip all cassettes (in order) so the bottom of the cap is facing up, use ruler or other straight edge to line up cassettes. Cassettes should advance from first to last cap left to right (like reading a book)
• Use clear office tape (“Scotch tape”) to secure bottom of caps
• Caps can then be easily transported to morgue for cutting and remain separated by case and in order

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Day Before Brain Cutting – Specimen Rinsing

• Brains should be rinsed with clean water beginning the evening prior to or morning of examination
• Maintain specimen identity: Always rinse brain in the original and properly labeled container – specimen identity can be lost at this point if specimens are moved between containers or the container is not labeled (some lab staff only label the lid, which is inadequate as the lid is removed during rinsing)
• Always handle/dispose of formalin properly: Set aside formalin in separate container and return to brain container following brain cutting if entire brain is to be saved
• With brain in bucket, rinse until water remains clear. Allow brain to sit in water overnight to minimize fumes during cutting
• If lab setup allows, rinse brain with constant low flow of water overnight for optimal rinsing and minimal formalin exposure (not done at LLUMC)

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Tools and Set-Up for Brain Cutting

• Brain cutting knife
• A long and broad blade that resists flexing is preferred. The disposable knifes common in surgical pathology are too flexible to be optimal for brain cutting
• Blade should be sharpened with a well-honed double bevel – single bevel knifes will not work
• Round, blunt tip blades are preferred. Your hands will be next to the blade during cutting, and as such, sharp tipped blades should be avoided. The rounded end allows the blade to be used as a functional spatula when needed
• Cutting board
• Large cafeteria tray – used to hold and arrange cut slices during examination
• Small scissors, forceps, and a scalpel with #60B or #70 blades
• Metric ruler
• Clip board and writing utensils for gross notes and cassette key
• Ventilated space for brain cutting – can be ventilated table or grossing station

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Brain Cutting – External Examination

• Perform careful inspection of external surface of brain prior to sectioning
• Symmetry: Are the left and right side the same size, shape, color, and consistency?
• Gyral pattern: Is there a normal adult-type gyral pattern, atrophy, and/or edema?
• Leptomeninges: Are there any hemorrhages, siderosis, exudate, or other abnormalities?
• Blood vessels: Is there any critical stenosis or occlusion of vessels? Aneurysms? Is the circle of Willis intact? Remember to look inside Sylvian fissure, lift up hindbrain, and inspect anterior communicating arteries, as vascular lesions can often “hide” in these places
• Cranial nerves: Perform a quick examination of whatever cranial nerves remain attached to brain
• Trauma: Closely observe areas susceptible to trauma (frontal, temporal, and occipital poles, orbitofrontal region, peri-Sylvian regions for either acute or remote hemorrhages/contusions. Remote contusions (small cavitary defects at gyral crests) can be very small and challenging to identify. Inspect brain very carefully if there is any suspicion of remote trauma
• Herniations: Is there herniation of the medial temporal lobes, cingulate gyrus, or cerebellar tonsils?
• Dura should be inspected for hemorrhage, defects, exudate, or thrombosis

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Brain Cutting – First Cut through Forebrain

• Forebrain is sectioned in coronal plane
• First cut is through the mammillary bodies (often hide under meninges, lie posterior to pituitary stalk and optic commissure)
• Ensure that brain is level (frontal and occipital poles), and that first cut through the mammillary bodies is perpendicular to the longitudinal fissure, straight up and down
• Should divide brain into anterior and posterior halves along coronal plane

Brain Cutting – Removing the Hindbrain

• Next step is to remove hindbrain from the caudal (posterior) half of forebrain
• Support cerebellum with non-cutting hand, with fingers slightly underneath and lifting hindbrain
• Using scalpel in right hand, cut straight and level through midbrain (cut is touching inferior aspect of but not through the mammillary bodies)
• Sweep blade in a flat plane to cut full thickness of midbrain while avoiding cutting into temporal lobes/hippocampi – no rounded cuts or “ice cream scooping” of midbrain!!!
• Left hand holding tension on hindbrain will feel tension release when all midbrain tissues are cut
• Set hindbrain aside for later sectioning

Brain Cutting – Forebrain Sectioning

• Lay rostral forebrain cut surface down on cutting board with temporal lobes facing to left, brain at close edge of cutting board (keeps knuckles and handle from contacting board)
• Support brain (including temporal lobes) with non-cutting hand
• With knife in hand, place back/dull side of blade flat against cutting board, angle the sharp edge up (~ 1 cm off board), and slide toward brain
• When sharp edge contacts brain, lift back of blade while maintaining contact with brain so that blade is parallel with cutting board. These steps ensure that blade is parallel with cutting board, and cut thickness is ~ 1 cm
• Cut through brain utilizing the full length of the blade (long strokes), let the sharp edge cut (don’t “push” the blade)
• Observe the far end of your blade and the relationship with the cutting board. Don’t let the blade tip dip down or rise up, resulting in asymmetric “pie wedge” brain sections
• Both rostral and caudal halves of forebrain are sectioned in this manner

Brain Cutting – Forebrain Sectioning

• Section rostral (anterior) forebrain half first
• All rostral forebrain sections must be flipped to maintain “right on right”. Caudal (posterior) forebrain sections must NOT be flipped (right is already on right)
• Place first section of ROSTRAL forebrain* in middle of tray, then place next section above, working towards the upper left of tray (red arrow)
• Place first unflipped section of CAUDAL forebrain** below first rostral section, then continue to place following sections below, working towards lower right of tray (blue arrow)
• When finished, sections should be placed in order, with frontal pole*** in upper left, occipital pole**** in lower right, right side on right (opposite of neuroradiologic orientation)

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Brain Cutting – Hindbrain Sectioning

• Next step is to detach the brainstem from the cerebellum. This is a critical cut and should be done with great care
• Place hindbrain upside down (midbrain down) on cutting board, cerebellum facing left
• With your left hand, gently push medulla to help expose gap between the brainstem and cerebellum
• With scalpel held at perpendicular to surface of brainstem/cerebellum, cut through the cerebellar peduncles. Blade should only be inserted about 2 cm deep to avoid cutting into the vermis. Cut both right and left cerebellar peduncles
• With left hand, pull brainstem forwards away from cerebellum and gently cut through remaining white matter structures (the flat curved midbrain/pontine tectum is ~ 1 mm thick, and should be carefully cut

Left and right cerebral peduncles are already cut in this image

Brainstem tectum is partially cut by the tip of the scalpel… it doesn’t take much!

• Cut brainstem from rostral (superior) to caudal (inferior) via serial cross sections, each should be ~ 3-4 mm thick
• Keeping right on right, left on left, line up brainstem from top to bottom
• Divide cerebellum into right and left halves (bisect vermis), place on respective sides of cutting board to maintain orientation
• Section cerebellar hemispheres in sagittal plane, line up on cutting board as follows (see image)
• Right hemisphere on right side of brainstem, left side on left
• Midline section on top of tray, lateral section below
• Anterior surface of each hemisphere facing brainstem
• Superior surface of each hemisphere facing up (top of tray)

Hindbrain Sectioning

R half, midline

L half, midline

Lateral slice

Lateral slice

Ant

Post

Sup

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Anterior/Rostral Forebrain - Coronal Section

Cap 1 : Right frontal lobe watershed. Need to see neocortex at sulcal depth ~ 2 cm off midline, plus a small volume of adjacent white matter. This is an excellent section to evaluate for global hypoxic/ischemic brain injury of the neocortex. Also put section of midline dura mater (with superior sagittal sinus) if dura is present for evaluation

Forebrain, Level of Anterior Basal Ganglia - Coronal

Cap 2 : Left superior temporal gyrus. This brain section comprises a “chubby finger”, with neocortex on three sides and central white matter. This is important, as this section will be in the same cap as the right lateral parietal lobe (see following slide). Also put section of right side supratentorial dura mater if dura is present for evaluation

Mid Forebrain, Mammillary Bodies - Coronal

Cap 5 : Right basal ganglia. Section will contain lentiform nucleus, caudate nuclei, amygdala and nucleus basalis, as well as internal, external, and extreme capsules. This section is quite useful given the number of nuclei and tracts present for evaluation. Internal capsule is particularly useful for evaluation of axonal trauma.

Cap 10 : Mamillary bodies and hypothalamus. This will fit alongside the vermis in cap 10. This cap is very useful for evaluation for nutritional deficiency and alcoholism (amongst other diseases)

Mid Forebrain, Thalamus/Subthalamic Nuclei - Coronal

Cap 6 : Left thalamus. The thalamus section also has many critical structures. This section should be taken at the level of the subthalamic nucleus (looks like a small football, asterisk on right side in image). The subthalamic nuclei lie right next to the red nuclei and substantia nigra. It may not be seen on initial sectioning, so area often needs to be trimmed thinner to identify the best level for sampling. This section is typically useful in neurodegenerative disease (i.e., PSP) and neonatal brain injury.

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Coronal Mid Forebrain, Level of Lateral Geniculate Nuclei

Cap 3 : Right hippocampi and right cerebral white matter Right cerebral white matter and left-sided cerebral dura mater (if present) also go in this cap.

Cap 4 : Left hippocampus, corpus callosum, and left cingulate gyrus. Both hippocampi should be taken at level of lateral geniculate nuclei (“Napolean’s hat”, see image above). Take left hippocampus in same manner as right (see Cap 3 discussion). Also submit corpus callosum and cingulate gyrus from this level

Cap 2 : Right parietal lobe, superolateral. This goes with the left temporal lobe in cap 2. The morphology of this section (white outside, gray inside) helps to distinguish from temporal lobe.

Brainstem (Midbrain and Pons)

Cap 7 : Midbrain. Midbrain should be taken at level of cerebellar peduncular decussation (see asterisk). Trim the left side if necessary (typically adults). This level is sometimes referred to as the “Mickey Mouse” level due to the passing resemblance to the murine namesake. Unfortunately, this level is often torn/disrupted due to mechanical autopsy artifact or suboptimal technique when removing the brainstem. Do your best…

Cap 8 : Pons. Take the mid pons (avoid the midbrain and medulla junction regions). This area should look like bacon/StripplesTM due to the crossing white matter tracts and basal pontine nuclei. Trim the left and/or anterior aspect if necessary (typically adults). Will always want to see the locus coerulei (blue dots by the fourth ventricle)

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Medulla Oblongata and Cerebellum

Cap 9 : Medulla and right cerebellar hemisphere. Find the point of closure of the fourth ventricle (“obex”, blue arrow left), and take the medulla one section above that level. As before, avoid “transitions” between adjacent levels of brainstem. Also submit the right cerebellar hemisphere with folia, white matter, and dentate gyrus (rectangular shaped) in the same cap.

Cap 10 : Anterior/superior vermis. A small section of anterosuperior vermian folia go in cap 10 along with mammillary bodies. This cap is the particularly useful for evaluating for brain injury due to alcoholism and/or epilepsy

Other Tissues / Sampling Variation�

• Spinal cord: If cord is grossly normal, section the cord from rostral to caudal and submit alternating sections so that all cord levels are well represented. Take smaller sections of other tissues in caps 1-10, and leaving room for spinal cord sections as follows
• 1-3: Cervical cord, rostral to caudal
• 4-6: Thoracic cord, rostral to caudal
• 7-9: Lumbar cord, rostral to caudal
• 10: Cauda equina
• Dura mater: Submit representative sections as follows:
• 1: Midline dura (with superior sagittal sinus
• 2: Right-sided dura mater
• 3: Left-sided dura mater
• Pituitary gland: Bisect in axial plane and submit half of gland in cap 10
• Discrete lesions: (e.g., infarcts, tumors, contusions, plaques, etc) Selectively sampled with location documented in the cap key. Either submit in caps that have “extra room” or leave less critical areas (e.g., one hippocampal/white matter block, thalamus) unsampled and substitute for discrete lesions
• In lieu of the above recommended sampling method, extra caps may be printed for complex cases or additional tissues. I prefer modifying the contents of the standard 10 caps, and rarely print extra, particularly when examining specimens off-campus).

Cassette 1

Cassette 2

Cassette 3

Cassette 4

Cassette 5

Cassette 6

Cassette 7

Cassette 8

Cassette 9

Cassette 10

Block Key:

_1: Right frontal lobe watershed, midline dura mater

_2: Left superior temporal gyrus, right parietal lobe, left supratentorial dura

_3: Right hippocampus, right cerebral hemispheric subcortical white matter, right supratentorial dura mater

_4: Left hippocampus, left cingulate gyrus, corpus callosum

_5: Right basal ganglia (level of amygdala)

_6: Left thalamus (level of subthalamic nucleus)

_7: Midbrain (trimmed on left)

_8: Pons (with locus coerulei)

_9: Medulla (level above obex), right cerebellar hemisphere with dentate nuclei

_10: Mammillary bodies, anterosuperior cerebellar vermis

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* If pituitary gland is present, bisect and place one half in cassette 10

* Spinal cord may also be included in cassettes 1-10 if smaller pieces of other sites are taken (prox to dist)

* Lesions that require dedicated sampling requires modification of other sections (or printing of extra cassettes)

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