The collection and processing of human brain tissue for research

To further understand the neuroanatomy, neurochemistry and neuropathology of the normal and diseased human brain, it is essential to have access to human brain tissue where the biological and chemical nature of the tissue is optimally preserved. We have established a human brain bank where brain tissue is optimally processed and stored in order to provide a resource to facilitate neuroscience research of the human brain in health and disease. A donor programme has been established in consultation with the community to provide for the post-mortem donation of brain tissue to the brain bank. We are using this resource of human brain tissue to further investigate the basis of normal neuronal functioning in the human brain as well as the mechanisms of neuronal dysfunction and degeneration in neurodegenerative diseases. We have established a protocol for the preservation of post-mortem adult human brain tissue firstly by snap-freezing unfixed brain tissue and secondly by chemical fixation and then storage of this tissue at -80 degrees C in a human brain bank. Several research techniques such as receptor autoradiography, DNA and RNA analysis, are carried out on the unfixed tissue and immunohistochemical and histological analysis is carried out on the fixed human tissue. Comparison of tissue from normal control cases and from cases with neurodegenerative disorders is carried out in order to document the changes that occur in the brain in these disorders and to further investigate the underlying pathogenesis of these devastating neurological diseases.     

Quantitative receptor autoradiography in the human brain. Methodical aspects

Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed: image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of beta-radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Effect of aging on neurogenesis in the canine brain

An age-dependent decline in hippocampal neurogenesis has been reported in laboratory rodents. Environmental enrichment proved to be a strong trigger of neurogenesis in young and aged laboratory rodents, which are generally kept in facilities with a paucity of environmental stimuli. These data raise the question whether an age-dependent decline in hippocampal cell proliferation and neurogenesis can also be observed in individuals exposed to diversified and varying surroundings. Therefore, we determined rates of canine hippocampal neurogenesis using post-mortem tissue from 37 nonlaboratory dogs that were exposed to a variety of environmental conditions throughout their life. Expression of the neuronal progenitor cell marker doublecortin clearly correlated with age. The analysis of doublecortin-labeled cells in dogs aged > 133 months indicated a 96% drop in the aged canine brain as compared to young adults. Expression of the proliferation marker Ki-67 in the subgranular zone decreased until dogs were aged 85-132 months. In the aging canine brain amyloid-beta peptide deposits have been described that might resemble an early pathophysiological change in the course of human Alzheimer's disease. Comparison of Ki-67 and doublecortin expression in canine brain tissue with or without diffuse plaques revealed no differences. The data indicate that occurrence of diffuse plaques in the aging brain is not sufficient to trigger enhanced proliferation or enhanced neurogenesis such as described in human Alzheimer's disease. In addition, this study gives first proof that an age-dependent decline also dominates hippocampal neurogenesis rates in individuals living in diversified environments.     

Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE

The number of proteomics studies concerning human brain samples has been increasing in recent years, in particular in the discovery of biomarkers for neurological diseases. The human brain samples are obtained from brain banks, which are interested in providing high quality human nervous tissue. In order to provide brain banks as well as scientists working in the proteomics field with measures for tissue quality, the critical factors after death, the effect of post-mortem interval (PMI) and storage temperature on the human brain proteome were investigated. This study was focused on the gray matter of the frontal cortex. The PMI was artificially prolonged from the time of autopsy (2 h after death) by storing samples at 4 degrees C or room temperature over 18, 24, and 48 h. The samples were analyzed by 2-D DIGE using a pH 4-7 gradient, revealing a time course of quantitative protein changes. The degradation of three proteins, peroxiredoxin-1, stathmin, and glial fibrillary acidic protein were further confirmed by Western-blot analysis. Proteins vulnerable to PMI were analyzed by the 2-D DIGE analysis of cortex samples from three donors, and were derived from a variety of functional groups, including metabolic, structural, stress response, antioxidants, synaptosomal, and neuronal proteins.     

Effects of tissue storage and freezing on brain glutamate uptake

Glutamate uptake appears to be stable when measured in rat striatal synaptosomes from tissue stored for up to four hours post-mortem at 25°C. Between four and eight hours storage at room temperature there is a sharp 70% decrease in uptake. Freezing of tissue on dry ice, storage at 4°C for up to 7 days and at ?80°C for 5 days results in 20–30% residual glutamate uptake. Quantitatively similar data can be obtained in eight extrastriatal brain areas. Kinetic analysis of glutamate uptake in stored and frozen tissue reveals the loss of the majority of both sodium-dependent high affinity and temperature-sensitive low affinity sites (v_max-values) while the respective K_m-values are not significantly changed. Pharmacological properties of the high affinity uptake versus a number of specific and metabolic uptake inhibitors remain unaltered by the storage and freezing procedure. The tissue treatment chosen for the present study roughly corresponds with the preparation of human post-mortem brain tissue for enzyme-, receptor-binding- or neuro-transmitter assays. It therefore seems conceivable that meaningful uptake studies can be performed on human autopsy material, thus adding an important parameter to the battery of neurochemical markers already accessible for post-mortem $\text{in}$$\text{vitro}$ examination.     

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity

Recently, the zebrafish (Danio rerio) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.     

A centric/non-centric impact protocol and finite element model methodology for the evaluation of American football helmets to evaluate risk of concussion

American football reports high incidences of head injuries, in particular, concussion. Research has described concussion as primarily a rotation dominant injury affecting the diffuse areas of brain tissue. Current standards do not measure how helmets manage rotational acceleration or how acceleration loading curves influence brain deformation from an impact and thus are missing important information in terms of how concussions occur. The purpose of this study was to investigate a proposed three-dimensional impact protocol for use in evaluating football helmets. The dynamic responses resulting from centric and non-centric impact conditions were examined to ascertain the influence they have on brain deformations in different functional regions of the brain that are linked to concussive symptoms. A centric and non-centric protocol was used to impact an American football helmet; the resulting dynamic response data was used in conjunction with a three-dimensional finite element analysis of the human brain to calculate brain tissue deformation. The direction of impact created unique loading conditions, resulting in peaks in different regions of the brain associated with concussive symptoms. The linear and rotational accelerations were not predictive of the brain deformation metrics used in this study. In conclusion, the test protocol used in this study revealed that impact conditions influences the region of loading in functional regions of brain tissue that are associated with the symptoms of concussion. The protocol also demonstrated that using brain deformation metrics may be more appropriate when evaluating risk of concussion than using dynamic response data alone.     

A new antigen retrieval technique for human brain tissue

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.     

Methods for the identification of differentially expressed genes in human post-mortem brain

Microarrays can be used to monitor the expression of thousands of genes simultaneously. This technique requires high-quality RNA which can be extracted from a variety of tissues and cells including post-mortem human brain. Given the vast amount of information obtained from microarray studies, it is critical to establish valid analysis techniques to identify differentially expressed genes. This technical report describes the basic methodology and analyses used to identify such genes in human post-mortem brain tissue.     

Consensus Brain-derived Protein,Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.     

Distribution of tau proteins in the normal human central and peripheral nervous system

In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.     

Postmortem stability of somatostatin in brain tissue

The stability of somatostatin (SS) in brain tissue was studied in human material obtained post-mortem and in the rat. In both human and rat brain, loss of SS was found to occur in tissue frozen to –70°C. In the rat, this loss varied from 26 to 70 percent depending on the type of tissue processing used. These data suggest that, for the study of SS in postmortem brain, use of frozen material should be avoided.     

Light and electron microscopic immunohistochemical detection of bromodeoxyuridine-labeled cells in the brain: different fixation and processing protocols.

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde-paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.     

In vitro study of the biological activity of RNAs after incubation of hog liver, heart and brain tissue at room temperature

The biological activity of RNA, isolated from tissue which was incubated for 1, 3, or 6 hours at room temperature (simulation of post-mortem conditions), was preserved. However, the different organs used differ from each other. When liver is used, qualitative differences in the in vitro translation products are observed, after one hour incubation at room temperature, whereas when heart and brain are used these differences are not observed. We have also shown that relatively small amounts of post-mortem tissue is sufficient for RNA extraction. When using frozen tissue it is absolutely necessary to add RNase inhibitors during thawing to reduce the loss of biological activity.     

Pitfalls in Membrane Binding Sites Studies in Post-Mortem Human Brain

Abstract

A number of neurotransmitter receptor sites have been characterized biochemically in post-mortem human brain from normal subjects and in several neurological and psychiatric diseases. Such studies are valid, however, only when appropriate pre-mortem and post-mortem conditions are controlled. The effects of age, pre-mortem agonic conditions, drug therapy and post-mortem delay on the characteristics of five binding sites (alpha-1, alpha-2 and beta adrenergic receptors, 5HT-2 serotoninergic receptors, imipramine binding sites) were studied. Age related changes in receptor number were found. Pre-mortem anoxia and hypovolemia had no influence on receptor characteristics. The drugs administered before death, in particular neuroleptics, were found to affect binding to some receptors in post-mortem tissue. A post-mortem delay up to 24 hours after death had no effect on binding sites. Experimental strategies (single point values or saturation curves) were also compared.