We wish to dedicate this work to Dr.ssa Michela Mangieri. Before she died prematurely, she conceived and started the Abbiategrasso Brain Bank Project.
We are grateful to our brain donors, who are generously contributing to research donating the noblest organ of their body; without them this research would not be possible.
We are grateful to Valeria Marzagalli for her precious work in the ABB project.
The authors thank Prof. Johannes Attems, Dr. Paolo Fociani and Dr. Giorgio Giaccone for their precious guidance and wise counsel.
We would like to thank Dr.ssa Alice Cirrincione and Miss Giulia Bortone for their precious help throughout the project. 
Many thanks to Mrs. Tere Cassani for her support and to &#8220;Federazione Alzheimer Italia&#8221; for its collaboration. 
The authors are grateful to Dr. Matteo Moretti and Prof. Antonio Marco Maria Osculati, Department of Public Health, Experimental and Forensic Medicine, University of Pavia.

The authors have nothing to disclose.

According to WHO, approximately 50 million people are currently suffering from dementia, a figure that is projected to triple by 2050. Alzheimer&#8217;s Disease is the major cause of dementia, followed by cerebrovascular disease and other age-related neurodegenerative disorders. In 2017, WHO developed the Global Dementia Observatory to raise awareness of dementia and encourage a global action plan against it1. Each individual has their own brain aging trajectory, therefore the search for the cure can be challenging due to the complexity of the pathogenesis of neurodegenerative diseases. Perhaps, each person possesses her or his own pathogenesis written in the brain tissue requiring a personalized approach. Thus, the study of brain tissue will be the key to understanding the mechanisms of neurodegeneration.
Looking back at the history of neuroscience, we realize that the most impressive and groundbreaking discoveries could never have occurred without the direct examination of the human brain. Throughout time, the source of brain tissue to be studied has changed from crude dissections, random &#8216;chance encounters&#8217; and, in some cases, illegal trading, to organized brain collections and strategic modern brain banks. The consideration of many ethical aspects is one of the main factors differentiating modern brain banks from the brain collections of the past. The first real modern Brain Banks (BBs) were instituted in the second half of the 20th century. Nicholas Corsellis and Wallace Tourtelotte can be considered pioneers of modern brain banking. In the UK, Corsellis assembled a collection holding over 1000 well-documented brains affected with various mental and neurological disorders2. Furthermore, Corsellis helped reveal the need to preserve fresh brain tissue in ice for the sake of biochemical testings3. Meanwhile in the USA, Wallace Tourtelotte introduced antemortem brain donation programs to facilitate solicitation of potential brain donors and ensure that the brains collected are accompanied by a complete medical and neurological history4,5. For a historical overview of brain collections and modern BBs, see Carlos et al.6.
So, why do we still need human brains? Brain diseases can only be given a definite diagnosis following neuropathological examination. Neuropathology challenges the clinical diagnosis, and is the key to a correct interpretation of the clinical symptoms and to the discovery of the histological bases of new syndromic variants. Indeed, the diagnosis can be redefined based on the pathological picture. Nonetheless, the autopsy rate has declined in the last decades due to recent development of innovative neuroimaging techniques. Through brain imaging, the morphological, functional and metabolic alterations in the brain, as well as the extent of protein misfolding, can be appraised in vivo. However, in vivo neuroimaging and other biomarker studies can only give an &#8220;estimate&#8221; of the pathological picture as they are unable to detect subtle cellular and molecular changes. Advances in molecular imaging and the discovery of new biomarkers, target molecules, and tracers7 (e.g., amyloid, TAU, microglial tracers) render the human brain even more indispensable to the interpretation of data obtained from clinical evaluations and biomarker testing. Furthermore, the omics technologies (genomics, epigenomics, transcriptomics, metabolomics, proteomics, lipidomics, etc.), performed on fresh and frozen brain tissue, opened up new possibilities for understanding disease mechanisms and discovering risk genes, novel diagnostic and prognostic markers, and potential drug targets8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23.
For these purposes, modern BBs archive well-characterized, high-quality brain tissues making them available for the scientific community3,24. The brains supplied by BBs should be accompanied by a complete clinical history. The activity of a BB includes the following: (1) Recognition and recruitment of diseased and healthy individuals to brain donation programs; the ideal condition would be to achieve a multi-disciplinary follow-up of donors throughout life to obtain a complete clinical, lifestyle and social history, and biomarker profiles; indeed, cognitive reserve and brain structure depend on lifestyle and socio-educational factors25,26, so these information enriches the total data at hand. (2) Acquisition of the brain (consisting of cerebrum, cerebellum and brain stem) and related tissues (e.g., spinal cord, cranial nerves and ganglia, etc.) following the donor&#8217;s demise, all the while observing standardized legal and ethical regulations. (3) Appropriate processing (dissection, fixation, freezing) of the brain, as defined in a standardized operative protocol, to obtain high quality tissue and to allow for future use in multidisciplinary research. (4) Detailed neuropathological characterization providing a final definite diagnosis. (5) Storage and distribution of tissue material to research community27,28.
All BBs store both frozen and formalin-fixed-paraffin embedded tissues. Each BB has its own protocol. Except for particular studies, such as the bihemispheric cutting protocol of the Biomedical Research Institute (New Jersey)29 and the Deramecourt&#8217;s study of cerebrovascular pathology30, the biggest BBs in the world simply cut the cerebrum, cerebellum and brainstem along the midline (sagittal plane). One half is dissected fresh and then frozen for biochemical studies, while the other is fixed in formalin for histopathological assessment. So, biochemical and histopathological analyses are conducted separately on each hemisphere. The decision as to which side is fixed or frozen (laterality), depends on the singular bank31,32,33,34,35. As many neurological diseases are asymmetrical, our BB offers a unique protocol for slicing fresh brains: adjacent sections of the brainstem and each hemisphere are alternately fixed and frozen; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. Through this method, the use of brain tissue is optimized, and a complete histological characterization of all frozen material can be achieved with the possibility to obtain and compare histological and biochemical information from all areas of both hemispheres.
The frame of our brain bank project is the town of Abbiategrasso. Abbiategrasso is a small town about 22 km southwest of the city of Milan, in Northern Italy. It has a population of about 32,600 people. It is home to the Golgi-Cenci (GC) Foundation. GC Foundation is part of a large Rehabilitation Geriatric Hospital (ASP Golgi-Redaelli), and is an institute focusing on research about aging and care of the elderly. Particularly, it focuses on studying mental aging, the social and behavioral factors influencing it, and the biology and pathology underlying age-dependent neurocognitive disorders (NCDs). In 2009, the GC Foundation launched a new longitudinal study with 1321 participants (out of 1644 eligible subjects: initial response rate of 80.3%) born between 1935 and 1939 (aged between 70&#8211;75 years), of Caucasian ethnicity, living in the same small geographic area. The study was called InveCe.Ab (Invecchiamento Cerebrale Abbiategrasso; in English: Brain Aging Abbiategrasso, ClinicalTrials.gov, NCT01345110) and is currently ongoing. InveCe.Ab is planned to obtain a cohort with maximum homogeneity and least variability in order to assess the incidence, prevalence and natural history of dementia, along with its possible risk or protective factors, including behavioral, psychosocial, clinical and biological variables36. The cohort&#8217;s characteristics are shown in Figure 1 and Table 1. The epidemiological data are consistent with the dementia trend in European population37,38 and InveCe.Ab participants have homogeneous genetic and environmental characteristics, representing a good model to study the trajectory from normal aging to neurocognitive disorders. Indeed, homogeneous populations require fewer subjects to reach adequate statistical power. The InveCe.Ab methodology has already been reported elsewhere36 but it is important to underline its multi-dimensional approach through periodic checks (every 2&#8211;3 years) using the same set of evaluations including: blood sampling (metabolic panel, homocysteine and vitamins, DNA extraction to profile Apolipoprotein E (APOE) and other genetic polymorphisms related to cognition and aging), anthropometric measurements (weight, height and waist), Talking While Walking Test (dual task test), an interview to appraise lifestyle (Mediterranean diet adherence, levels of physical activity and cognitive engagement) and social factors (social involvement, loneliness), a neuropsychological evaluation and a clinical general examination. To compare such longitudinal data with postmortem neuropathological data would be crucial for research. Therefore, our team and particularly Dr. Michela Mangieri conceived the neuropathological approach mentioned above. Since 2014 and during the second follow-up, the InveCe.Ab participants were asked to donate their brains, and thus bringing about the birth of the Abbiategrasso Brain Bank (ABB). The core donors of ABB are the InveCe.Ab participants but ABB is now open to other volunteer donors. They are patients from ASP Golgi-Redaelli, home to several patients afflicted with different neurological diseases or adult volunteers who learn about the ABB Project and belong to the same geographic area (Abbiategrasso and surroundings). All donors undergo the same evaluation protocol.
The authors propose a method to track individual trajectories of normal aging and the possible progression to NCDs, and to accurately manage, process and characterize brains acquired from such donors followed longitudinally. Moreover, our aim is to meet and engage individuals in periodic neurological assessments, seminars and educational activities regarding the well-being of the brain and to raise their awareness of brain donation for research purposes.

<table><tbody><tr><td>50ml polypropilene conical tube 30x115mm</td><td>BD</td><td>405253</td><td></td></tr><tr><td>Anti-GFAP</td><td>Dako</td><td>Z0334</td><td>policlonal primary antibody (anti-rabbit); dilution = 1:1000</td></tr><tr><td>Anti-NeuN (A60)</td><td>Chemicon</td><td>MAB377</td><td>monoclonal primary antibody (anti-mouse); dilution = 1:1000</td></tr><tr><td>Anti-phospho TAU (AT8)</td><td>ThermoScientific</td><td>MN1020</td><td>monoclonal primary antibody (anti-mouse); dilution = 1:200</td></tr><tr><td>Anti-phospho TDP-43 (pS409/410-2)</td><td>CosmoBio</td><td>TIP-PTD-P02</td><td>policlonal primary antibody (anti-rabbit) ; dilution = 1:4000; pretreatment : Three step in microwave for 2 min-1 min-2 min with citrate buffer 0.01M pH 6</td></tr><tr><td>Anti-&amp;alpha;-SYN (KM51)</td><td>Novocastra</td><td>NCL-L-ASYN</td><td>monoclonal primary antibody (anti-mouse); dilution = 1:500; pretreatment: 1) Three step in microwave for 2 min-1 min-2 min with citrate buffer 0.01M pH 6 ; 2) 70% formic acid in H2O for 10 min</td></tr><tr><td>Anti-&amp;beta;amyloid (4G8)</td><td>BioLegend</td><td>800703</td><td>monoclonal primary antibody (anti-mouse); dilution = 1:1000; pretreatment : 70% formic acid in H2O for 10 min</td></tr><tr><td>Cutting board</td><td>BD</td><td>352070</td><td></td></tr><tr><td>DMEM High Glucose</td><td>Carlo Erba</td><td>FA30WL0101500</td><td>medium</td></tr><tr><td>Electrical saw with 5d blade</td><td>CEA</td><td>06.06.14/06.00.16</td><td></td></tr><tr><td>Electrode pH measure surface 12mm</td><td>CEA</td><td>70064.250 HHH</td><td></td></tr><tr><td>Electrode pH needle</td><td>Fisher Scientific</td><td>11796338</td><td></td></tr><tr><td>EnVision+System-HRP</td><td>Dako</td><td>K4001</td><td>secondary antibody (anti-mouse); dilution 1:2</td></tr><tr><td>EnVision+System-HRP</td><td>Dako</td><td>K4003</td><td>secondary antibody (anti-rabbit); dilution 1:2</td></tr><tr><td>Ethylether</td><td>SMI</td><td>8401530</td><td></td></tr><tr><td>Feather safety trimming knife blade 14cm</td><td>Fisher Scientific</td><td>11749798</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Carlo Erba</td><td>FA30WS1810500</td><td>medium supplement; dilution = 20%</td></tr><tr><td>Forceps 15cm surgical or anatomical</td><td>Uvex</td><td>500XG</td><td></td></tr><tr><td>Gloves</td><td>CEA</td><td>01.28.14</td><td></td></tr><tr><td>Glue</td><td>Arcobaleno</td><td>2624000800002</td><td></td></tr><tr><td>Head supporter</td><td>Lacor</td><td>60456</td><td></td></tr><tr><td>L-Glutamine (100X)</td><td>Carlo Erba</td><td>FA30WX0550100</td><td>medium supplement; dilution = 1%</td></tr><tr><td>Measuring tape</td><td>CEABIS</td><td>CEATA34</td><td></td></tr><tr><td>Non adsorbable monofilament black polyamide</td><td>UHU Bostik</td><td>8000053131470</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution (100X)</td><td>Life Technologies</td><td>11140050</td><td>medium supplement; dilution = 1%</td></tr><tr><td>Pen/Strept Solution (100X)</td><td>Carlo Erba</td><td>FA30WL0022100</td><td>medium supplement; dilution = 1%</td></tr><tr><td>Spinal needle quincke tipe point 20GA 3.50IN 0.9x90mm</td><td>CEA</td><td>03.06.16</td><td></td></tr><tr><td>Sterile scalpel with n&amp;deg;21 blade</td><td></td><td></td><td></td></tr><tr><td>Surgical basin</td><td>Olcelli Farmaceutica</td><td>A930857255</td><td></td></tr><tr><td>Surgical mallet and surgical cisel</td><td>CEA</td><td>79.68.88</td><td></td></tr><tr><td>Surgical scissor</td><td>CEA</td><td>27.08.45/79.68.64</td><td></td></tr><tr><td></td><td>Feather</td><td>M130RC</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains at JoVE.com

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

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 ...

abbiategrasso-brain-bank-protocol-for-collecting-processing

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

Anatomy of the Central and Peripheral Nervous System

SCIENTISTS IN ACTION

17.2K Views.  Golgi-Cenci Foundation. Als een neurocognitieve aandoening prevalentie voortdurend toeneemt, ons doel is te verkrijgen, karakteriseren en op te slaan kwaliteit hersenweefsel monsters om nauwkeurige diagnose te bereiken en licht werpen op de neurodegeneratieve mechanismen. Gezien de hersensymetrie, onze bemonstering methodologie maakt het mogelijk alle meta studies worden uitgevoerd op histologisch goed gedefinieerde weefsels van beide hemisferen en de vele gegevens kunnen worden verzameld en gecorreleerd door middel...

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

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

Biology

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 ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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.

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 ...

Medicine

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

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 ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

Detection of Neuronal Aging in a Cortical Organoid Section

Source: Shaker, M. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons.

Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. ...

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

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