Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Summary

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Abstract

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Introduction

Neuropathologists have the scientific privilege, intellectual honor, and diagnostic obligation to assess human brains. For many decades, detailed clinical descriptions of brain diseases and major efforts to individuate their possible neurohistological correlates in human postmortem brains have been undertaken. Historically, those efforts represented the most productive modality by which the medical sciences, and neurology in particular, advanced in the modern era. Thanks to previous eminent neuropathologists and their dedication, determination, scholarship, and astonishing capacity to discriminate between normal and abnormal brain tissues (often using very rudimental tools), we can now investigate and target diseases such as Alzheimer-Perusini's disease (unfairly only called Alzheimer's disease; APD/AD)¹, Parkinson's disease (PD)², Creutzfeldt-Jakob disease (CJD)³, Lou-Gehrig's disease/Amyotrophic Lateral Sclerosis (ALS)⁴, and Guam disease⁵, to mention a few.

Advanced techniques of neuroimaging, such as high-definition computerized tomography (i.e., multisection spiral CT scan; CT angiography), functional and morphological magnetic resonance imaging (i.e., fMRI, diffusion-MRI, tractography-MRI, etc.), Positron Emission Tomography (PET), ultrasound-based imaging, and others, have certainly modified our general approach on how to diagnose and cure neurological and psychiatric patients. Nonetheless, although neuroimaging techniques are capable of visualizing a person's brain when alive, they do not offer the opportunity, at the occurring moment, to directly analyze the highly intricate cellular and subcellular structures of cells, such as neurons; or to visualize, mark, and quantify specific types of intracellular lesions; or to precisely indicate their neuroanatomical or subregional localization at circuital and sub-circuital anatomical levels. For example, neuroimaging techniques cannot identify or localize Lewy Bodies (LB) in pigmented neurons of the Substantia Nigra (SN), a common pathologic feature associated with PD, or neurofibrillary tangles (NFT) in the entorhinal cortex, a classical feature of AD and other brain pathologies. Neuropathological investigations combined with advanced digital microscopy are still unreplaceable for detailed clinicopathological correlations and, thus, for definitive diagnoses.

Due to the peculiar anatomo-functional properties of the human brain, and especially to its anatomical localization (that is, inside the skull, a natural protective system that does not allow the direct examination of its content), the introduction of in vivo neuroimaging techniques have extraordinarily helped clinicians and investigators to find initial answers to some of the mysteries of this complex tissue. However, there is no clinical or neuroimaging methodology that can replace the unique opportunity to directly analyze brain tissue during an autopsy. Only the organized collection, preservation, and categorization of human brains can allow direct and systematic investigations of neuronal and non-neuronal cells, their subcellular constituents, intracellular and extracellular pathological lesions, and any type of abnormality inside the brain to confirm, modify, or redefine clinical diagnoses and to discover new clinicopathological correlations. One of the apparent limitations concerning the assessment of the brain at autopsy has been the fact that this procedure is a cross-sectional methodology. There will always be a delay between an ongoing neuropathological process (clinically manifested or not) and the chance, if any, to define it at the neurohistological level. This is mainly due to the incapacity of the human brain to regenerate itself. It is not currently possible to obtain brain tissue in vivo without creating permanent damage. Consequently, it is not possible to longitudinally and neuropathologically assess the same brain/person. However, standardized brain banking procedures and an increased awareness for brain donation among the general public could greatly contribute to the resolution of brain-autopsy timing issues by consistently increasing the number of cases to collect and analyze. In this manner, more adequate numbers of postmortem brains could be obtained to define constant patterns of pathological origin and progression for each specific type of brain lesion associated with each human brain disease. This would require donation and collection of as many brains as possible from patients affected by any neuropsychiatric disorder, as well as from healthy control subjects across all ages. One possible method could be collecting as many postmortem brains as possible from general and specialized medical centers as a standard routine. The need for brain donations has been recently expressed by those who study dementia and normal aging⁶. The same necessity should be expressed by the neuropsychiatric field as whole.

For the abovementioned and for other reasons, an update of ongoing brain cutting procedures is necessary. Moreover, brain cutting procedures should be universally standardized across different neuropathology research centers around the world, also taking in account the possibility to employ current and future biotechnological techniques to better investigate and, hopefully, to definitively understand, the causes and mechanisms of brain diseases in humans.

Here, mainly for research purposes, we describe a symmetric methodology for postmortem brain cutting in humans. This procedure proposes collecting more cerebral regions than normally done and from both cerebral and cerebellar hemispheres. A symmetric bi-hemispheric brain cutting procedure will fit much better with our current knowledge of human neuroanatomy, neurochemistry, and neurophysiology. This method also allows the possibility to neuropathologically analyze the unique features of the human brain, such as hemispheric specialization and lateralization that are associated with higher cognitive and non-cognitive functions typically or exclusively present in our species. Whether there are specific pathogenetic relationships between hemispheric specialization/lateralization and specific types of brain lesions, or whether a peculiar neuropsychiatric pathogenetic event is initially, prevalently, or exclusively associated with a specific hemisphere and function is not currently known. By describing this symmetric brain cutting procedure, we aim to propose an updated method of human brain dissection that could help to better understand normal and pathological conditions in a highly specialized tissue, the brain. This method also takes into consideration those morpho-functional hemispheric aspects that exist only in humans.

Protocol

Procedures involving postmortem human tissues have been reviewed by the institutional review board and exempted under 45 CFR (Code of Federal Regulations).

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 to perform 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 material 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 sources⁷.

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 suggested⁸.

1. Place the brain on a flat surface facing the investigator, with the frontal poles directed in the opposite direction with respect to the investigator.
2. Place the brain in such a way as to allowing a full and clear visualization of all cortical gyri and sulci of the entire cerebrum (Figure 1a).
3. First look for meningeal anomalies, macroscopic hemispheric asymmetries (possible indicators of focal, lobar, or generalized hemispheric phenomena of atrophy), macroscopic tissutal 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 on how to assess a human brain, refer to commercially available neuropathology textbooks and autopsy manuals^9,10.

2. Protocol Sequence

1. 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).
2. 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.
3. 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 tissutal laceration, due to their extreme frailty.
   1. 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.
4. 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 cerebrum). Carefully inspect the SN (i.e., for pallor)¹¹ and other neighboring structures¹². Take note, possibly using an audio recorder device, of any unusual appearance of the brain in comparison to a normal brain¹³.
5. 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.)¹³. See Figure 2a.
   1. 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 note of any unusual feature of the brain in comparison to a normal brain.
6. 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 in such a way that their centers touch (also in case of marked hemispheric asymmetry).
7. 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).
8. 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), making sure that 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.
   1. 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 in comparison to a normal brain.
9. 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.
   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).
   2. 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 B 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.
   3. Take as many digital pictures as necessary in each particular case to document possible macroanomalies 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 note of any unusual feature of the brain in comparison to a normal brain.
10. Take digital pictures (as many as necessary or desired) of the entire cut brain and the associated histocassettes.
11. Punch (e.g., by Accu-punch) small pieces of tissue for DNA extraction and genetic analyses. Use a punch of 2 - 5 mm in diameter.
    NOTE: For its high content of genomic material, the cerebellum is the preferred choice; however, any other region is fine.
12. Re-immerse all histocassettes containing brain tissue blocks in the same type of fixative solution (e.g., 10% neutral-buffered formalin) as previously used until the next step of tissue processing.
13. Follow standard procedures for human formalin-fixed tissue processing¹⁴.

3. A Special Approach: Alternating Frozen and Fixed Symmetric Bihemispheric Cutting

NOTE: The symmetric bihemispheric brain cutting protocol described in section 2 offers the possibility of cutting tissue slabs from unfixed, fresh brains (when available) in the same systematic and symmetric manner.

1. Place the entire fresh brain upside down (preferably on a semispherical bowl-like plastic surface) for 8 - 10 min in a -80 °C freezer to harden the brain tissue without provoking biochemical damage and to facilitate the manual cutting.
2. Using a sharp knife, cut both hemispheres in an alternate and consecutive manner, following the brain cutting protocol described in Section 2, but freeze and fix every other slab (from both hemispheres and along a fronto-occipital anatomical direction).
   1. At this point, do not try to cut each cerebral region, as described in Table 1. Cut specific fresh brain regions only if required for immediate RNA or protein extraction (i.e., for genomic or proteomic studies)¹⁵.
3. After cutting, immediately freeze, label, and number each fresh tissue. Take digital pictures of the entire slab series; pack each slab in a single plastic bag; collect the slabs in a separate, one-brain-only container; and store the container in a dedicated -80 °C freezer.
   ​NOTE: The freezer should be dedicated to human brain tissue only. Only later will single frozen brain regions be cut as required for each specific experiment.
4. Immerse every other tissue slab chosen for fixation (10% neutral-buffered formalin or another fixative) in separate bags containing a sufficient volume of fixative (3/1 volume of fixative/tissue-block ratio). Label each bag by consecutively numbering them following a fronto-occipital sequence. Seal each bag, take digital pictures, and store them in a plastic container.
5. Open the fixative-containing bags after 2 weeks of tissue fixation and cut each cerebral region as described in Table 1.

4. Histostain and Immunohistochemistry

NOTE: The set of cerebral regions cut based on the proposed scheme (Table 1) are sufficient to satisfy most, if not all, currently established consensus-based pathological criteria for AD¹⁶, PD¹⁷, Dementia with Lewy Bodies (DLB)¹⁸, Frontotemporal Dementia (FTD/MND)¹⁹, Progressive Suprabulbar Palsy (PSP)²⁰, Multiple System Atrophy (MSA)²¹, Chronic Traumatic Encephalopathy (CTE)²², etc.

1. For each brain region and for both hemispheres, perform the following minimum set of histostains: Hematoxylin and Eosin (H&E), cresyl violet (CV; if quantitative morphometric studies are planned, for example), and silver staining (if "exploratory" analyses are needed).
2. For each brain region and for both hemispheres, perform the following minimum set of immunohistochemistry protocols: β-amyloid (βA), phosphorylated-Tau (pTau), phosphorylated α-synuclein (pα-syn), and phosphorylated-TDP43 (pTDP43), as described¹⁴.
   NOTE: The total number of tissue sections in order to assess each brain following this protocol is 46 (if all cerebral regions from both hemispheres are available).

Results

Protocol Length

The time spent for a single symmetric bihemispheric fixed brain cutting procedure is estimated at 1 h (excluding the time spent setting up the dissection table, tools, and cutting surfaces; labeling; etc.). The time required for a single symmetric bi-hemispheric alternating frozen and fixed brain cutting procedure is estimated to take 2 h. It can take at least between 4 - 6 weeks to obtain definitive histol...

Discussion

This brain cutting method can be adapted to the specific needs of each neuropathology lab (for example, by reducing the number of cerebral regions to assess for each hemisphere) while still retaining the bihemispheric symmetric cutting procedure as one of its main features. This proposed protocol could be used for routine procedures (research-oriented neuropathological centers) or only when necessary (specific clinically oriented studies). It can be selectively used only for specific types of investigations (i.e.,

Materials

Name	Company	Catalog Number	Comments
Copy of signed informed consent allowing autopsy and brain donation for research use.
Detailed clinical history of the subject which should include a detailed description of any neurologic and psychiatric symptoms and signs.
Medical or nonmedical video-recordings when available (especially useful in movement disorders field). Next-of-kin’s consent required.
Neuroimaging, neurophysiology, neuropsychiatric and assessment or clinicometric scales.
Genetic and family history data. Genetic reports review, if neurogenetic diseases were diagnosed.
Histology Container	ELECTRON MICROSCOPY SCIENCES	64233-24
Histology Cassettes	VWR	18000-142 (orange)
Histology Cassettes	VWR	18000-132 (navy)
Knife Handles and Disposable Blades	ELECTRON MICROSCOPY SCIENCES	62560-04
Long Blades	ELECTRON MICROSCOPY SCIENCES	62561-20
Disposable Blade Knife Handles	ELECTRON MICROSCOPY SCIENCES	72040-08
Scalpel Blades	ELECTRON MICROSCOPY SCIENCES	72049-22
Accu-Punch 2 mm	ELECTRON MICROSCOPY SCIENCES	69038-02
Polystyrene Containers – Sterile	ELECTRON MICROSCOPY SCIENCES	64240-12
Dissecting Board	ELECTRON MICROSCOPY SCIENCES	63307-30
Formalin solution, neutral buffered, 10%	Sigma-Aldrich	HT501128 SIGMA
Hematoxylin Solution, Gill No. 2	Sigma-Aldrich	GHS280 SIGMA
Eosin Y solution, aqueous	Sigma-Aldrich	HT1102128 SIGMA
anti-beta-amyloid	Covance, Princeton, NJ	SIG-39220	1:500
anti-tau	Thermo Fisher Scientific	MN1020	1:500
anti-alpha-synuclein	Abcam	ab27766	1:500
anti-phospho-TDP43	Cosmo Bio Co.	TIP-PTD-P02	1:2000
Digital Camera	Any
Head Impulse Sealing machine	Grainger	5ZZ35