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 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 (&#62;5.4 weeks) has been suggested8.
<ol>
	<li>Place the brain on a flat surface facing the investigator, with the frontal poles directed in the opposite direction with respect to the investigator.</li>
	<li>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).</li>
	<li>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. 
	&#8203;NOTE: For detailed descriptions on how to assess a human brain, refer to commercially available neuropathology textbooks and autopsy manuals9,10.</li>
</ol>
2. Protocol Sequence
<ol>
	<li>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).</li>
	<li>Mark pre&#160;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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>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&#160;cm thick blocks of brain tissue (around 18 slabs for each hemisphere).</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Put each tissue block in separately labeled histocassettes. 
		&#8203;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).</li>
		<li>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&#160;AD160001, where AD stands for &#34;Alzheimer&#39;s disease study,&#34; 16 is the year when the autopsy was performed (2016), and 0001 a progressive accession specimen number). 
		&#8203;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Take digital pictures (as many as necessary or desired) of the entire cut brain and the associated histocassettes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Follow standard procedures for human formalin-fixed tissue processing14.</li>
</ol>
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.
<ol>
	<li>Place the entire fresh brain upside down (preferably on a semispherical bowl-like plastic surface) for 8 - 10 min in a -80 &#176;C freezer to harden the brain tissue without provoking biochemical damage and to facilitate the manual cutting.</li>
	<li>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).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C freezer. 
	&#8203;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.</li>
	<li>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.</li>
	<li>Open the fixative-containing bags after 2 weeks of tissue fixation and cut each cerebral region as described in Table 1.</li>
</ol>
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.
<ol>
	<li>For each brain region and for both hemispheres, perform the following minimum set of histostains: Hematoxylin and Eosin (H&#38;E), cresyl violet (CV; if quantitative morphometric studies are planned, for example), and silver staining (if &#34;exploratory&#34; analyses are needed).</li>
	<li>For each brain region and for both hemispheres, perform the following minimum set of immunohistochemistry protocols: &#946;-amyloid (&#946;A), phosphorylated-Tau (pTau), phosphorylated &#945;-synuclein (p&#945;-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).</li>
</ol>

The authors have nothing to disclose.

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><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 nonmedical video-recordings when available (especially useful in movement disorders field). Next-of-kin&amp;rsquo;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&amp;nbsp;</td><td></td></tr><tr><td>Polystyrene Containers &amp;ndash; 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&amp;nbsp;SIGMA</td><td></td></tr><tr><td>Eosin Y solution, aqueous</td><td>Sigma-Aldrich</td><td>HT1102128&amp;nbsp;SIGMA</td><td></td></tr><tr><td>anti-beta-amyloid</td><td>Covance, Princeton, NJ</td><td>SIG-39220</td><td>1:500</td></tr><tr><td>anti-tau</td><td>Thermo Fisher Scientific</td><td>MN1020</td><td>1:500</td></tr><tr><td>anti-alpha-synuclein</td><td>Abcam</td><td>ab27766</td><td>1:500</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&amp;nbsp;</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 ...

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Research

JoVE Journal

Medicine

13.9K Views.  Biomedical Research Institute of New Jersey (BRInj). 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.

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Neuroscience

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Preparing Free-Floating Slice Cultures from an Adult Human Brain

Source: Fernandes, A., et al. <a href="https://app.jove.com/v/59845">Short-Term Free-Floating Slice Cultures from the Adult Human Brain.</a> J. Vis. Exp. (2019).
This video demonstrates a technique for preparing free-floating slice cultures from the adult human brain. Thin slices are generated from a brain section using a vibratome. These slices are then suspended in a culture medium supplemented with growth factors to maintain tissue viability.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sterilization of materials
NOTE: ...