All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
NOTE: The protocol describes a symmetric bihemispheric brain cutting procedure for postmortem brain assessment finalized for neuropathological studies in humans. Detailed descriptions of the apparatuses, instruments, materials, and supplies necessary for human brain cutting will be excluded. Materials and supplies for brain dissections are selected at the discretion of the single investigator and are based on autopsy tools allowed or approved at each research institution. The minimal set of tools and materials required for this procedure is described in the Material/Equipment Table. Specific cutting procedures and precautions for suspected transmissible brain diseases, such as human CJD, are outside the aims of this manuscript and are available from other sources.
1. Symmetric Bihemispheric Brain Cutting
Note: Ensure that the brain has received the necessary tissue fixation (using, for example, neutral-buffered 10% formalin) for a period of two to three weeks, depending on the periagonal, metabolic (i.e., pH), and tissue preservation (i.e., temperature) conditions. However, for imaging-pathology correlation studies, a longer period of fixation (>5.4 weeks) has been suggested.
- Place the brain on a flat surface facing the investigator, with the frontal poles directed in the opposite direction with respect to the investigator.
- Place the brain to allow full and clear visualization of all cortical gyri and sulci of the entire cerebrum (Figure 1a).
- First, look for meningeal anomalies, macroscopic hemispheric asymmetries (possible indicators of focal, lobar, or generalized hemispheric phenomena of atrophy), macroscopic tissue lesions (i.e., tumors or herniation), congenital malformations, vessel abnormalities, and any other possible abnormalities or unusual presentations of the cerebral surface.
NOTE: For detailed descriptions of how to assess a human brain, refer to commercially available neuropathology textbooks and autopsy manuals.
2. Protocol Sequence
- Face frontal poles away from the investigator, with the superficial aspects of the hemispheres (telencephalon) facing the investigator. Take as many digital pictures as necessary in each particular case to document possible macro-anomalies and to account for possible clinico-neuroanatomical and post-cutting considerations. Have a research assistant take digital photographs perpendicularly to the brain to capture the entire cortical surface (Figure 1a - c).
- Mark pre and postcentral cortical gyri using ink or colored needles before cutting the brain (Figure 1b).
NOTE: This procedure facilitates a more immediate recognition of the motor and somatosensory primary cortices after cutting.
- Rotate the brain by 180 degrees while keeping it facing the same direction (i.e., the frontal poles facing away from the investigator). Carefully inspect the base of the brain. Pay special attention to the conditions of cerebrovascular systems (i.e., basilar and vertebral arteries and the circle of Willis) and cranial nerves at their brainstem exit/entrance levels. Manage the olfactory bulbs and tracts with special care to avoid tissue laceration due to their extreme frailty.
- Take as many digital pictures as necessary in each particular case to document possible macro-anomalies and to account for possible clinico-neuroanatomical and post-cutting considerations. Have a research assistant take digital photographs perpendicularly to the brain to capture the entirety of the cortical and brainstem surfaces.
- Facing the base of the brain and using a scalpel, cut the brainstem transversally at the level of the upper portion of the pons (as close as possible to the base of the cerebrum). Carefully inspect the SN (i.e., for pallor) and other neighboring structures. Take note, possibly using an audio recorder device, of any unusual appearance of the brain compared to a normal brain.
- Again, rotate the brain by 180 degrees and, using a sharp knife, separate the two hemispheres by cutting the corpus callosum centrally through the medial longitudinal fissure and following a fronto-occipital direction. Inspect each side of each hemisphere for possible anomalies (e.g., ventricular enlargements, malformations, tissue softening, tumors, etc.). See Figure 2a.
- Take as many digital pictures as necessary in each particular case to document possible macro-anomalies and account for possible clinical-neuroanatomical and post-cutting considerations. Have a research assistant take digital photographs perpendicularly to the brain to capture the entire cortical surface. Note any unusual brain feature compared to a normal brain.
- Place the two hemispheres flat, lying on their medial aspects, with the frontal lobes facing away from the investigator, as shown in Figure 2b. Place them so that their centers touch (also in case of marked hemispheric asymmetry).
- Using a sharp knife, manually cut through both cerebral hemispheres, starting at the frontal poles and moving towards the occipital poles through the entire length of the hemispheres. Obtain two series of 1 cm thick blocks of brain tissue (around 18 slabs for each hemisphere).
- Place the brain slabs in an anatomically organized (fronto-occipital direction) sequence on a separate flat surface. Use a white surface with a ruler printed on it for better contrast when photographing. Display the two series of cerebral slabs in an anatomically symmetric way (fronto-occipital direction), ensuring their coronal surfaces are visible for direct eye inspection and digital photography (Figure 3a). Use cutting surfaces with printed millimetric grids on both sides to localize brain structures, sizes, and possible abnormalities in a more accurate manner.
- Take as many digital pictures as necessary in each particular case to document possible macro-anomalies and to account for possible clinico-neuroanatomical and post-cutting considerations. Have a research assistant take digital photographs perpendicularly to the brain to capture the entire cortical surface. Take notes (possibly using an audio recorder device) of any unusual aspect of the brain compared to a normal brain.
- Using a sharp scalpel, manually dissect smaller rectangular blocks of brain tissue for each established cerebral region. Follow the proposed cerebral region collection scheme described in Table 1.
- Put each tissue block in separately labeled histocassettes.
NOTE: Each block of brain tissue needs to be cut to fit, as much as possible, the standard histocassette maximal volume (30 x 20 x 4 mm3).
- Label the histocassettes using a de-identifying code for each case and using specific neuroanatomical identifiers (do not use random letters or numbers for different brains; rather, always use the same regional anatomical names or corresponding numbers, as shown in Table 1). Create de-identifying codes, for example, by generating random or semi-random numbers for each case (i.e., BRC 130, where 5B stays for Brain, R stays for Resource, C stays for Center and 130 is a progressive accession or AD160001, where AD stands for "Alzheimer's disease study," 16 is the year when the autopsy was performed (2016), and 0001 a progressive accession specimen number).
NOTE: This step is very helpful for future researchers. Keep a legend and specify the hemisphere (L = left hemisphere, R = right hemisphere). Use two different colors of histocassettes, establishing a specific color for each hemisphere.
- Take as many digital pictures as necessary in each particular case to document possible macroanomalies and to account for possible clinical-neuroanatomical and post-cutting considerations. Have a research assistant take digital photographs perpendicularly to the brain to capture the entire cortical surface. Take note of any unusual feature of the brain compared to a normal brain.
- Take digital pictures (as many as necessary or desired) of the entire cut brain and the associated histocassettes.
Table 1. Bihemispheric Cutting Scheme. This table shows the single anatomical area to dissect in both the left and right hemispheres of each brain. The bihemispheric symmetric cutting can be done on fresh and fixed brains.
| Neuroanatomical region | Label | Left Fixed | Right Fixed | Left Frozen | Right Frozen |
| Olfactory bulb | OB | | | | |
| Polar frontal cortex | PFC | | | | |
| Middle frontal gyrus | MFG | | | | |
| Orbito-frontal gyrus | OFG | | | | |
| Parietal cortex lobulus inferior | PCLI | | | | |
| Superior temporal cortex | STC | | | | |
| Middle temporal cortex | MCC | | | | |
| Anterior cingulate cortex | ACC | | | | |
| Posterior cingulate cortex | PCC | | | | |
| Pre-central gyrus (Put a pin before cutting) | PrCRG | | | | |
| Post-central gyrus (Put a pin before cutting) | PoCRG | | | | |
| Occipital gyrus (including Broadmann area 17 and 18) | OC | | | | |
| Amygdala | AMY | | | | |
| Basal ganglia at the level of anterior commissure with basal nucleus of Meynert | BGMY | | | | |
| Thallamus and subthallamus | TH-STH | | | | |
| Corpora mammillary and hypothalamus | MAM+HYPO | | | | |
| Basal forbrane nuclei | BF | | | | |
| Anterior hippocampus | AH | | | | |
| Posterior hippocampus (at corpus geniculate lateral level), including entorhinal cortex and inferior temporal cortex | PH+EC+ITC | | | | |
| Cortex cerebelli and Nucleus dentate | CRBDe | | | | |
| Mesencephalon (including Substantia nigra) | MESSN | | | | |
| Pons including Locus coeruleus (LC) | PONS | | | | |
| Medula oblongata Medula including DMV | MObi | | | | |
| Medula spinalis | MSpi | | | | |
| Spinal cord upper | SC | | | | |
| Dura Madre | DM | | | | |
