We thank the thousands of brain donors, patients, families, and neuroscientists around the world who, during the last two centuries and through their generous gifts and intellectual efforts, helped to discover how the human brain works, to understand devastating brain diseases, and to develop treatments thereof. We particularly thank Mrs. Cecilia V. Feltis for editing and reviewing this manuscript.

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 s...

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.,</e...

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&#39;s disease (unfairly only called Alzheimer&#39;s disease; APD/AD)1, Parkinson&#39;s disease (PD)2, Creutzfeldt-Jakob disease (CJD)3, Lou-Gehrig&#39;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Copy of signed informed consent allowing autopsy and brain donation for research use.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Detailed clinical history of the subject which should include a detailed description of any neurologic and psychiatric symptoms and signs.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical or not-medical video-recordings when available (especially useful in movement disorders field). Next-of-kin&#8217;s consent required.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroimaging, neurophysiology, neuropsychiatric and assessment or clinicometric scales.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Genetic and family history data. Genetic reports review, if neurogenetic diseases were diagnosed.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histology Container</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>64233-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology Cassettes</td>
			<td>VWR</td>
			<td>18000-142 (orange)</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology Cassettes</td>
			<td>VWR</td>
			<td>18000-132 (navy)</td>
			<td></td>
		</tr>
		<tr>
			<td>Knife Handles and Disposable Blades</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>62560-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Long Blades</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>62561-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Blade Knife Handles</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>72040-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>72049-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu-Punch 2 mm</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>69038-02&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Containers &#8211; Sterile</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>64240-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Board</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>63307-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128 SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Solution, Gill No. 2</td>
			<td>Sigma-Aldrich</td>
			<td>GHS280&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution, aqueous</td>
			<td>Sigma-Aldrich</td>
			<td>HT1102128&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-beta-amyloid</td>
			<td>Covance, Princeton, NJ</td>
			<td>SIG-39220</td>
			<td>1&#160; 500</td>
		</tr>
		<tr>
			<td>anti-tau</td>
			<td>Thermo Fisher Scientific</td>
			<td>MN1020</td>
			<td>1&#160; 500</td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein</td>
			<td>Abcam</td>
			<td>ab27766</td>
			<td>1&#160; 500</td>
		</tr>
		<tr>
			<td>anti-phospho-TDP43</td>
			<td>Cosmo Bio Co.</td>
			<td>TIP-PTD-P02</td>
			<td>1 2000</td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Head Impulse Sealing machine&#160;</td>
			<td>Grainger</td>
			<td>5ZZ35</td>
			<td></td>
		</tr>
	</tbody>
</table>

symmetric bihemispheric postmortem brain cutting to study healthy and pathological brain conditions in humans

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.

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...

Watch this Scientific Journal Video about Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans at JoVE.com

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

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.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

The overall goal of this procedure is to systematically collect and analyze a constant series of cerebral regions symmetrically dissected from both hemispheres of the human brain in order to assess severity, clinical pathologic staging and hemispheric distribution of various brain diseases. This method can help answer key questions in the neuropathology field providing a systematically organized collection of qualitative and quantitative neuropathologic data for detailed clinical pathologic and imaging pathologic correlations. The main advantage of this technique consists in yielding relevant data related to the pathologic processes per se as well as the histofunctional specialized aspect of each cerebral hemisphere.After fixing post-mortem human brain tissue according to the text protocol, place the brain on a flat surface with the frontal poles directed opposite the investigator. Orient the brain to allow full and clear visualization of all cortical gyri and sulci of the entire cerebrum. Examine the tissue for meningeal anomalies, macroscopic hemispheric asymmetries, macroscopic tissue allegiance, congenital malformations, vessel abnormalities or any other abnormalities or unusual presentations of the cerebral surface.With the frontal poles oriented away from the investigator and the superficial aspects of the hemispheres facing the investigator, take as many digital pictures as necessary to document possible macro anomalies and to account for possible clinic, neural, anatomical and post-cutting considerations. Take digital pictures perpendicularly to the brain to capture the entire cortical surface. Then using ink or colored needles, mark pre and post central cortical gyri.Next, with the frontal poles still facing away, flip the brain 180 degrees then carefully inspect the base of the brain paying special attention to the cerebrovascular systems and the cranial nerves at the brain stem exit and entrance levels. Manage the olfactory bulbs and tracts with special care to avoid tissue laceration due to their extreme frailty. Take digital photographs perpendicularly to the brain to capture the entirety of the cortical and brain stem surfaces.Facing the base of the brain and using a scalpel, cut the brain stem transversely at the upper portion of the pons as close as possible to the base of the cerebrum. Carefully inspect the substantia nigra for pallor and inspect other neighboring structures. Using an audio recording device, take note of any unusual appearance of the brain in comparison to a normal brain.Here, the olfactory bulb and tract of the left and then right hemispheres are being dissected. Flip the brain back over and using a sharp knife, follow a fronto-occipital direction to separate the two hemispheres by cutting the corpus callosum centrally through the medial longitudinal fissure. Inspect each side of each hemisphere for possible anomalies.After taking as many pictures as necessary, including perpendicularly to the brain to capture the entire cortical surface, place the two hemispheres flat laying on their medial aspects with the frontal lobes facing away from the investigator and with their centers touching. Using a sharp knife, manually cut through both cerebral hemispheres starting at the frontal poles and move towards the occipital poles through the entire length of the hemispheres to obtain two series of one centimeter thick slabs of brain tissue. Position the brain slabs in an anatomically organized and symmetric sequence on a flat white surface with a ruler printed on it for photographing.Ensure that the slab's coronal surfaces are visible for direct eye inspection and digital photography. Use cutting surfaces with printed millimetric grids on both sides to localize brain structures, sizes, and possible abnormalities in a more accurate manner. With the coronal surfaces visible, take digital photographs perpendicularly to capture the entire cortical surface.Take notes of any unusual aspect of the brain in comparison to a normal brain. Next, using a sharp scalpel, manually dissect small rectangular blocks of brain tissue for each established cerebral region. Follow the proposed cerebral region collection scheme described in this table.Create the identifying codes to label the histo cassettes by generating random or semi-random numbers for each case where AD stands for Alzheimer's Disease Study, 16 stands for the year the autopsy was performed and 0001 is a progressive accession specimen number. Place each tissue block in separate histo cassettes. Take as many digital pictures as described earlier of the entire cut brain and the associated histo cassettes.After taking a complete set of pictures, punch two to five millimeter pieces of tissue for DNA extraction and genetic analysis. Finally, immerse all histo cassettes containing brain tissue blocks in the same type of fixative solution as previously used and process the tissue according to the text protocol. This table describes some preliminary semi-quantitative data using a symmetric bihemispheric brain cutting procedure performed on a series of human brains collected in the brain bank.Preliminary data show that most control brains from older subjects were actually positive for beta-A neuritic plaques and tau-neurofibrillary tangles and that levels of insoluble of beta-A neuritic plaques were higher in the left entorhinal cortex and hippocampus in comparison to the same brain regions on the right side. Once mastered, this technique can be completed in an hour for fixed brains and in two hours for fresh brains if it's properly performed. While attempting this procedure, it's important to remember following the proposed cutting scheme, collect each cerebral region from both hemispheres based upon precise human anatomical knowledge.Following this procedure, other methods like DNA, RNA or protein extraction can be performed in order to answer additional questions such as what are the activation levels of a specific gene in a specific brain region in a specific hemisphere in a specific brain disease. With each diffusion, this technique will pave the way for researchers in the field of neuropathology, clinical pathology and imaging pathology correlation to explore the hemispheric related pathologic aspects of various neuropsychiatric diseases. After watching this video, you should have a good understanding of how to collect specific anatomical regions from both hemispheres of the human brain to assess severity of the disease, pathologic stage of the disease and hemispheric distribution of different neuropathologic processes.Don't forget that working with the human brain is working with the history of a person with complex neuroanatomical circuits and possibly with different diseases and that blades, scalpels, formalin must be handled with extreme precaution.

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13.8K Views.  Biomedical Research Institute of New Jersey (BRInj). The overall goal of this procedure is to systematically collect and analyze a constant series of cerebral regions symmetrically dissected from both hemispheres of the human brain in order to assess severity, clinical pathologic staging and hemispheric distribution of various brain diseases. This method can help answer key questions in the neuropathology field providing a systematically organized collection of qualitative and quantitative neuropathologic data for detailed clinical pathologic a...