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.     

Tryptophan is a marker of human postmortem brain tissue quality

Postmortem human brain tissue is widely used in neuroscience research, but use of tissue originating from different brain bank centers is considered inaccurate because of possible heterogeneity in sample quality. There is thus a need for well-characterized markers to assess the quality of postmortem brain tissue. Toward this aim, we determined tryptophan (TRP) concentrations, phosphofructokinase-1 and glutamate decarboxylase activities in 119 brain tissue samples. These neurochemical parameters were tested in samples from autopsied individuals, including control and pathological cases provided by 10 different brain bank centers. Parameters were assessed for correlation with agonal state, postmortem interval, age and gender, brain region, preservation and freezing methods, storage conditions and storage time, RNA integrity, and tissue pH value. TRP concentrations were elevated significantly ( p  = 0.045) with increased postmortem interval; which might indicate increased protein degradation. Therefore, TRP concentration might be one useful and convenient marker for estimating the quality of human postmortem brain tissue.     

Twenty-first century brain banking: practical prerequisites and lessons from the past: the experience of New York Brain Bank,Taub Institute,Columbia University

Generally accepted methods for processing postmortem brains are lacking, despite the efforts of pioneers in the field, and the growing awareness of the importance of brain banking for investigating the pathogenesis of illnesses unique to humans. Standardizing methods requires compromises, institutional or departmental mindset promoting collaboration, and the willingness to share ideas, information, and samples. A sound balance between competition and institutional interests is needed to best fulfill the tasks entrusted to health care institutions. Thus, a potentially widely accepted protocol design involves tradeoffs. We successfully integrated brain banking within the operation of the department of pathology. We reached a consensus whereby a brain can be utilized for diagnosis, research, and teaching. Thus, routing brains away from residency programs is avoided. The best diagnostic categorization possible is being secured and the yield of samples for research maximized. Thorough technical details pertaining to the actual processing of brains donated for research were recently published. Briefly, one-half of each brain is immersed in formalin for performing the neuropathologic evaluation, which is combined with the teaching task. The contralateral half is extensively dissected at the fresh state to obtain samples ready for immediate disbursement once categorized diagnostically. The samples are tracked electronically, which is crucial. This important tracking system is described separately in this issue. This report focuses on key lessons learned over the past 25 years of brain banking including successful solutions to originally unforeseen problems.     

Supply and use of human tissue for research purposes: survey of BATB affiliated tissue banks

This paper describes a survey undertaken to identify the extent of supply and use of human tissue in research by BATB affiliated tissue banks. Approximately one third of tissue banks registered with the BATB are currently supplying samples that are found to be unsuitable for clinical use, for research. These banks all obtain consent for research and all supply tissue for in-house research. Some tissue is transferred to other public and commercial institutions. A harmonised network approach is proposed as the way forward to meet the increasing demand for human tissue in research.     

Constitutive modelling of brain tissue: Experiment and theory

Recent developments in computer-integrated and robot-aided surgery—in particular, the emergence of automatic surgical tools and robots—as well as advances in virtual reality techniques, call for closer examination of the mechanical properties of very soft tissues (such as brain, liver, kidney, etc.). The ultimate goal of our research into the biomechanics of these tissues is the development of corresponding, realistic mathematical models. This paper contains experimental results of in vitro, uniaxial, unconfined compression of swine brain tissue and discusses a single-phase, non-linear, viscoelastic tissue model. The experimental results obtained for three loading velocities, ranging over five orders of magnitude, are presented. The applied strain rates have been much lower than those applied in previous studies, focused on injury modelling. The stress-strain curves are concave upward for all compression rates containing no linear portion from which a meaningful elastic modulus might be determined. The tissue response stiffened as the loading speed increased, indicating a strong stress-strain rate dependence. The use of the single-phase model is recommended for applications in registration, surgical operation planning and training systems as well as a control system of an image-guided surgical robot. The material constants for the brain tissue are evaluated. Agreement between the proposed theoretical model and experiment is good for compression levels reaching 30% and for loading velocities varying over five orders of magnitude.     

Effects of different correlation metrics and preprocessing factors on small-world brain functional networks: a resting-state functional MRI study

Graph theoretical analysis of brain networks based on resting-state functional MRI (R-fMRI) has attracted a great deal of attention in recent years. These analyses often involve the selection of correlation metrics and specific preprocessing steps. However, the influence of these factors on the topological properties of functional brain networks has not been systematically examined. Here, we investigated the influences of correlation metric choice (Pearson's correlation versus partial correlation), global signal presence (regressed or not) and frequency band selection [slow-5 (0.01-0.027 Hz) versus slow-4 (0.027-0.073 Hz)] on the topological properties of both binary and weighted brain networks derived from them, and we employed test-retest (TRT) analyses for further guidance on how to choose the "best" network modeling strategy from the reliability perspective. Our results show significant differences in global network metrics associated with both correlation metrics and global signals. Analysis of nodal degree revealed differing hub distributions for brain networks derived from Pearson's correlation versus partial correlation. TRT analysis revealed that the reliability of both global and local topological properties are modulated by correlation metrics and the global signal, with the highest reliability observed for Pearson's-correlation-based brain networks without global signal removal (WOGR-PEAR). The nodal reliability exhibited a spatially heterogeneous distribution wherein regions in association and limbic/paralimbic cortices showed moderate TRT reliability in Pearson's-correlation-based brain networks. Moreover, we found that there were significant frequency-related differences in topological properties of WOGR-PEAR networks, and brain networks derived in the 0.027-0.073 Hz band exhibited greater reliability than those in the 0.01-0.027 Hz band. Taken together, our results provide direct evidence regarding the influences of correlation metrics and specific preprocessing choices on both the global and nodal topological properties of functional brain networks. This study also has important implications for how to choose reliable analytical schemes in brain network studies.     

Characterization and localization of 3H-arginine8-vasopressin binding to rat kidney and brain tissue

Anatomic, behavioral and pharmacologic evidence suggests that arginine8-vasopressin (AVP) serves as a CNS neurotransmitter or neuromodulator. We have characterized AVP binding to membrane and tissue slice preparations from brain and kidney, and examined the anatomical distribution of these binding sites. Conditions for the binding assay were optimized using kidney medullary tissue. Binding of 3H-AVP (S.A. = 30-51 Ci/mmol, NEN) to brain and kidney membranes and tissue slices was saturable, temperature dependent, linearly related to protein concentration (or number of tissue slices), reversible, and specific since the ability of cold AVP to displace 3H-AVP from binding was greater than oxytocin and other related peptide fragments. Autoradiographic localization of 3H-AVP binding was restricted to kidney medullary tissue. In brain tissue, 3H-AVP binding was found to occur in concentrated foci. Brainstem areas such as the nucleus tractus solitarius (NTS) showed a high density of AVP binding sites. Since local injections of AVP into the NTS have been shown to influence blood pressure, the present study presents the first anatomical evidence for the presence of AVP specific binding sites which might mediate this effect.     

Dysregulated mitochondrial genes and networks with drug targets in postmortem brain of patients with posttraumatic stress disorder (PTSD) revealed by human mitochondria-focused cDNA microarrays

Posttraumatic stress disorder (PTSD) is associated with decreased activity in the dorsolateral prefrontal cortex (DLPFC), the brain region that regulates working memory and preparation and selection of fear responses. We investigated gene expression profiles in DLPFC Brodmann area (BA) 46 of postmortem patients with (n=6) and without PTSD (n=6) using human mitochondria-focused cDNA microarrays. Our study revealed PTSD-specific expression fingerprints of 800 informative mitochondria-focused genes across all of these 12 BA46 samples, and 119 (+/->1.25, p<0.05) and 42 (+/->1.60, p<0.05) dysregulated genes between the PTSD and control samples. Quantitative RT-PCR validated the microarray results. These fingerprints can essentially distinguish the PTSD DLPFC BA46 brains from controls. Of the 119 dysregulated genes (+/-> or =125%, p<0.05), the highest percentages were associated with mitochondrial dysfunction (4.8%, p=6.61 x 10(-6)), oxidative phosphorylation (3.8%, p=9.04 x 10(-4)), cell survival-apoptosis (25.2%, p<0.05) and neurological diseases (23.5%, p<0.05). Fifty (50) dysregulated genes were present in the molecular networks that are known to be involved in neuronal function-survival and contain 7 targets for neuropsychiatric drugs. Thirty (30) of the dysregulated genes are associated with a number of neuropsychiatric disorders. Our results indicate mitochondrial dysfunction in the PTSD DLPFC BA46 and provide the expression fingerprints that may ultimately serve as biomarkers for PTSD diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of PTSD.     

Effect of antemortem and postmortem factors on [3H]MK-801 binding in the human brain: transient elevation during early childhood

The effect of a number of antemortem and postmortem factors on [3H]MK-801 binding was investigated under equilibrium conditions in the frontal cortex of human brains of 38 controls. Binding values transiently increased during the early postnatal period reaching a maximum at the age of about 2 years. After age 10 years [3H]MK-801 binding sites disappeared at 5.7% per decade. The storage time of brain tissue had a reducing effect on these binding sites. There was no effect of gender, brain weight or postmortem time interval and the binding sites were bilaterally symmetrically distributed in the frontal cortex.     

Hippocampal electrical activity and gamma-aminobutyrate metabolism in brain tissue following administration of homocysteine

Abstract: The concentration of γ-aminobutyrate (GABA) and the activity of glutamate decarboxylase and GABA-transaminase were measured in extracts of mouse brain before the onset and during the course of generalized seizures induced by systemic administration of homocysteine thiolactone. The results indicate that whole brain GABA metabolism is unaffected by subconvulsive and convulsive doses of homocysteine at all stages of the generalized seizure. Electroencephalographic monitoring of rat brain electrical activity via hippocampal electrode implantation allowed the course of homocysteine-induced seizures to be followed and afforded a means of quantifying such seizures.     

Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain

The aim of the present study is to evaluate the oxidative damage in rats of different ages. Weaned rats of 25 g and adults of 300 g were used in groups of 6, a single i.p. dose of morphine sulfate of 3, 6 or 12 mg/kg was administered. All animals were sacrificed to measure GSH and 5-HT levels in brain by liquid chromatography, as well as Na⁺, K⁺-ATPase and total ATPase enzymatic activity. 5-HT levels decreased significantly (p<0.05) in adult animals that received 3 and 6 mg morphine. Na⁺, K⁺-ATPase activity increased significantly (p<0.05) in all groups of weaned animals. In adult animals, Na⁺, K⁺-ATPase and total ATPase partially diminished. GSH levels diminished significantly (p<0.05) both in weaned and in adult groups. The results indicate age-induced changes in cellular regulation and biochemical responses to oxidative stress induced by morphine.     

The diversity of p53 mutations among human brain tumors and their functional consequences

The p53 tumor suppressor is implicated in cell cycle control, DNA repair, replicative senescence and programmed cell death. Inactivation of the p53 contributes to the wide range of human tumors, including glial neoplasms. In this review, we describe the regulation and biochemical properties of p53 protein that may explain its ability to activate various genetic programs underlying cellular responses to stress conditions. The overall spectrum of p53 mutations is rather shared between tumor types indicating that these mutations are not tumor type-specific. However, there is one example of germ-line mutation of p53 gene (the deletion of the codon 236) that is associated with a familiar brain tumor syndrome. We compare the frequency and type of most common mutations among various brain tumours (focusing on glioblastomas) and their consequences on protein functions. Furthermore, we discuss the most promising approaches of potential brain tumor therapy, including an adenovirus-mediated p53 gene transfer. Human glioblastomas are highly sensitive to the effects of p53 activity when the wild-type p53 is introduced ectopically. It suggests that the genetic or pharmacological modulation of the p53 pathway is potentially important strategy in the treatment of human cancers.     

Role of aquaporins in cell migration and edema formation in human brain tumors

The aquaporins (AQPs) are a family of transmembrane water channel proteins widely distributed and play a major role in transcellular and transepithelial water movement. Moreover, recent evidence indicates that AQPs may be involved in cell migration, angiogenesis, and tumor growth. This review article summarizes literature data concerning the involvement of AQP-1 and -4 in human brain tumor growth and edema formation and suggests a potential therapeutic approach by antagonizing their biological activity.     

Evidence that lipolytic peptide B occurs in the ACTH/MSH-cells of the pituitary and in the brain

Summary Several lipid-mobilizing peptides occur in the pituitary, among them -lipotropin and lipolytic peptide A and peptide B. The latter two peptides are distinct from -lipotropin and appear to be chemically related to the neurophysins. Immunohistochemistry has now revealed that the lipolytic peptide B of the pituitary is localized in the ACTH- and MSH-cells. In addition, immunoreactive peptide B was found in axons of the posterior lobe of the pituitary. Immunoreactive peptide B was found also in nerve fibers and nerve cell bodies in the hypothalamus, particularly in the hypothalamo-hypophyseal tract and in the magnocellular neuronal system. Immunoreactive nerve fibers were numerous also in the periventricular nucleus of the thalamus. The antiserum against peptide B cross-reacts with neurophysin I, and hence, it cannot be excluded that at least part of the immunostaining in the brain reflects the presence of the latter component. However, the regional distribution of immunoreactive peptide B and neurophysin was not identical. Therefore, it is possible that authentic peptide B occurs not only in the pituitary but also in the brain.     

Brain banks: benefits,limitations and cautions concerning the use of post-mortem brain tissue for molecular studies

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.     

Characterization and tissue distribution of a novel human cytochrome P450-CYP2U1

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.     

Assessment of pine forest damage by blight based on Landsat TM data and correlation with environmental factors

Landsat TM images were obtained of blight damage to a Japanese red pine forest in the western part of Hiroshima Prefecture, Japan, using a spectral vegetation index; that is, the ratio of the digital number (relative reflectance on the ground surface) of TM Band 4 to Band 3 observed in May 1987, which decreased with the increase in the canopy cover of damaged pine trees measured in the field. The TM images suggested that the areas of damaged forest were concentrated in or near cities, industrial areas and expressways. The correlation between forest damage and environmental factors (air pollution and urban development) around the pine forest was therefore analysed by overlaying the blight damage with the proportion of developed areas obtained from TM data or mesh data of air pollution. The results of analysis indicated a significant correlation between forest damage and environmental factors, and showed that these two environmental factors made nearly equal contributions to the blight damage in the pine forest. This suggests that urban development and air pollution may affect the physiology of pine trees and promote blight by reducing the resistance of trees to the pinewood nematode.     

Perfusion and molecular diffusion-weighted MR imaging of the brain: in vivo assessment of tissue alteration in cerebral ischemia

Summary.  The combined use of perfusion imaging (PI) and diffusion-weighted imaging (DWI) is opening a new window into the processes that occur during the first hours of ischemia. DWI detects changes in molecular diffusion associated with cytotoxic edema. PI characterizes the degree of regional hypoperfusion. Regions showing mismatches between DWI and PI, i.e. hypoperfused areas with normal diffusion behavior are considered potentially salvageable. We present results of 11 patients with an occlusion of the middle cerebral artery stem and spontaneous stroke evolution. Whereas the infarct was clearly visible on initial DWI and PI, surrounding tissue at risk of infarction was marked in all patients by an increased blood volume and transit time, but only in a subgroup (n = 3) where alteration were more pronounced this tissue at risk was progressively infarcted. These human DWI and PI data show alterations in the area of tissue at risk which correlates with infarct progression. Received June 29, 2001 Accepted August 6, 2001 Published online August 20, 2002     

Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2

A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.