Magnetic resonance microscopy at 14 Tesla and correlative histopathology of human brain tumor tissue

Magnetic Resonance Microscopy (MRM) can provide high microstructural detail in excised human lesions. Previous MRM images on some experimental models and a few human samples suggest the large potential of the technique. The aim of this study was the characterization of specific morphological features of human brain tumor samples by MRM and correlative histopathology. We performed MRM imaging and correlative histopathology in 19 meningioma and 11 glioma human brain tumor samples obtained at surgery. To our knowledge, this is the first MRM direct structural characterization of human brain tumor samples. MRM of brain tumor tissue provided images with 35 to 40 μm spatial resolution. The use of MRM to study human brain tumor samples provides new microstructural information on brain tumors for better classification and characterization. The correlation between MRM and histopathology images allowed the determination of image parameters for critical microstructures of the tumor, like collagen patterns, necrotic foci, calcifications and/or psammoma bodies, vascular distribution and hemorrhage among others. Therefore, MRM may help in interpreting the Clinical Magnetic Resonance images in terms of cell biology processes and tissue patterns. Finally, and most importantly for clinical diagnosis purposes, it provides three-dimensional information in intact samples which may help in selecting a preferential orientation for the histopathology slicing which contains most of the informative elements of the biopsy. Overall, the findings reported here provide a new and unique microstructural view of intact human brain tumor tissue. At this point, our approach and results allow the identification of specific tissue types and pathological features in unprocessed tumor samples.     

Large-scale in silico modeling of metabolic interactions between cell types in the human brain

Metabolic interactions between multiple cell types are difficult to model using existing approaches. Here we present a workflow that integrates gene expression data, proteomics data and literature-based manual curation to model human metabolism within and between different types of cells. Transport reactions are used to account for the transfer of metabolites between models of different cell types via the interstitial fluid. We apply the method to create models of brain energy metabolism that recapitulate metabolic interactions between astrocytes and various neuron types relevant to Alzheimer's disease. Analysis of the models identifies genes and pathways that may explain observed experimental phenomena, including the differential effects of the disease on cell types and regions of the brain. Constraint-based modeling can thus contribute to the study and analysis of multicellular metabolic processes in the human tissue microenvironment and provide detailed mechanistic insight into high-throughput data analysis.     

Gene expression‐based drug repurposing to target aging

Aging is the largest risk factor for a variety of noncommunicable diseases. Model organism studies have shown that genetic and chemical perturbations can extend both lifespan and healthspan. Aging is a complex process, with parallel and interacting mechanisms contributing to its aetiology, posing a challenge for the discovery of new pharmacological candidates to ameliorate its effects. In this study, instead of a target‐centric approach, we adopt a systems level drug repurposing methodology to discover drugs that could combat aging in human brain. Using multiple gene expression data sets from brain tissue, taken from patients of different ages, we first identified the expression changes that characterize aging. Then, we compared these changes in gene expression with drug‐perturbed expression profiles in the Connectivity Map. We thus identified 24 drugs with significantly associated changes. Some of these drugs may function as antiaging drugs by reversing the detrimental changes that occur during aging, others by mimicking the cellular defence mechanisms. The drugs that we identified included significant number of already identified prolongevity drugs, indicating that the method can discover de novo drugs that meliorate aging. The approach has the advantages that using data from human brain aging data, it focuses on processes relevant in human aging and that it is unbiased, making it possible to discover new targets for aging studies.     

Differential expression of apolipoprotein d in human astroglial and oligodendroglial cells.

Apolipoprotein D (Apo D) is a secreted lipocalin in the nervous system that may be related to processes of reinnervation and regeneration. Under normal conditions, Apo D is present in the central nervous system in oligodendrocytes, astrocytes, and some scattered neurons. To elucidate the regional and cellular distribution of Apo D in normal human brain, we performed double immunohistochemistry for glial fibrillary acidic protein (GFAP) and Apo D in samples of postmortem human cerebral and cerebellar cortices. Most of the GFAP-positive cells in the gray matter had features of protoplasmic astrocytes and were mainly Apo D-positive. Apo D staining was mostly confined to the cell soma and proximal processes, whereas GFAP extended to a rich and extensive array of processes. The fibrous astrocytes in the white matter were immunoreactive for GFAP but not for Apo D. In the white matter, Apo D was mainly detected in oligodendrocytes and extracellularly in the neuropil. The results of the present study support a specific behavior for each astrocyte type. These findings suggest that Apo D expression may be cell-specific, depending on the particular tissue physiology at the time of examination.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.     

Modelling neurodegenerative diseases with 3D brain organoids

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post‐mortem brain tissue, non‐human primate tissue, or in vitro two‐dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three‐dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.     

Identification of flotillin-1 as an interacting protein for antisecretory factor

Antisecretory factor (AF) also named S5a/Rpn10 was originally identified through its capacity to inhibit intestinal hypersecretion and was later shown to be a component in the proteasome complex. AF is also a potent anti-inflammatory agent and can act as a neuromodulator. In this study we used yeast two-hybrid screens, with yeast strain PJ692A transformed with the bait vector pGBKT7 (AF aa 1-105) against yeast strain Y187 pretransformed with human brain or placenta cDNA libraries, to identify AF-binding proteins. Flotillin-1 was identified as a specific interacting factor with AF. Immunohistochemistry showed co-localization of AF and flotillin-1 in nervous tissue. Flotillin-1 is an integral membrane protein and a component of lipid rafts, a membrane specialization involved in transport processes. Intracellular AF may affect secretory processes by regulating the localization of signal proteins to lipid rafts.     

A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain

A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.     

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.     

Notch receptor expression in human brain arteriovenous malformations

The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well‐understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1–4 expression. We have found that compared to normal brain vascular tissue Notch‐3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch‐1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.     

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.     

A Simplified Method for Isolating Highly Purified Neurons, Oligodendrocytes, Astrocytes, and Microglia from the Same Human Fetal Brain Tissue

Elucidation of the underlying pathogenic mechanisms leading to apoptosis of neurons and oligodendrocytes and activation of microglia and astrocytes in different neurodegenerative and neuroinflammatory disorders remains a challenge in neuroscience. In order to overcome the challenge and find out therapeutic remedies, it is important to study live and death processes in each and every cell type of the brain. Here we present a protocol of isolating highly purified microglia, astrocytes, oligodendrocytes, and neurons, all four major cell types of the CNS, from the same human fetal brain tissue. As found in vivo, these primary neurons and oligodendroglia underwent apoptosis and cell death in response to neurodegenerative challenges. On the other hand, astroglia, and microglia, cells that do not die in neurodegenerative brains, became activated after inflammatory challenge. The availability of highly purified human brain cells will increase the possibility of developing therapies for different neurodegenerative disorders. M. Jana and A. Jana have equal contribution to the work.     

Pilot feasibility study of in vivo intraoperative quantitative optical coherence tomography of human brain tissue during glioma resection

This study investigates the feasibility of in vivo quantitative optical coherence tomography (OCT) of human brain tissue during glioma resection surgery in six patients. High‐resolution detection of glioma tissue may allow precise and thorough tumor resection while preserving functional brain areas, and improving overall survival. In this study, in vivo 3D OCT datasets were collected during standard surgical procedure, before and after partial resection of the tumor, both from glioma tissue and normal parenchyma. Subsequently, the attenuation coefficient was extracted from the OCT datasets using an automated and validated algorithm. The cortical measurements yield a mean attenuation coefficient of 3.8 ± 1.2 mm^?1 for normal brain tissue and 3.6 ± 1.1 mm^?1 for glioma tissue. The subcortical measurements yield a mean attenuation coefficient of 5.7 ± 2.1 and 4.5 ± 1.6 mm^?1 for, respectively, normal brain tissue and glioma. Although the results are inconclusive with respect to trends in attenuation coefficient between normal and glioma tissue due to the small sample size, the results are in the range of previously reported values. Therefore, we conclude that the proposed method for quantitative in vivo OCT of human brain tissue is feasible during glioma resection surgery.     

The regulation of connective tissue metabolism by vasoactive intestinal polypeptide.

Immunohistochemical studies have confirmed the innervation of bone with neuropeptidergic neurons containing vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). In this study, we report effects of VIP on connective tissue cell metabolism. VIP stimulated PGE2 production in human articular chondrocytes, human osteoblast-like cells and human synovial cells, however, stromelysin production was unaffected. VIP also stimulated cAMP production in human osteoblast-like cells, but not in human articular chondrocytes or synovial cells. These findings are suggestive of a role of VIP in connective tissue cell metabolism which may contribute to the inflammatory processes of arthritis.     

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.     

Rapid chemical clearing of white matter in the post-mortem human brain by 1,2-hexanediol delipidation

Three-dimensional (3D) imaging based on chemical tissue clearing in the post-mortem human brain is a promising approach for stereoscopic understanding of central nervous system diseases. Especially, delipidation of lipid-rich white matter (WM) is a rate-determining step in human brain clearing by hydrophilic reagents. In this study, we described the rapid delipidation of WM by a 1,2-hexanediol (HxD)-based aqueous solution. HxD delipidation enabled rapid clearing of a formalin-fixed human brain specimen including the WM. Although harsh HxD delipidation was applied to the brain tissue, conventional pathological staining patterns and various types of antigenicity were sufficiently preserved. Furthermore, HxD delipidation was compatible with 3D imaging of fluorescently-labeled tissue samples. HxD delipidation could be useful in future 3D neuropathological diagnosis.     

Dopamine is necessary to endogenous morphine formation in mammalian brain in vivo

Morphine, the most used compound among narcotic analgesics, has been shown to be endogenously present in different mammalian/invertebrate normal tissues. In this study, we used mice that cannot make dopamine due to a genetic deletion of tyrosine hydroxylase specifically in dopaminergic neurons, to test the hypothesis that endogenous dopamine is necessary to endogenous morphine formation in vivo in mammalian brain. When dopamine was lacking in brain neurons, endogenous morphine was missing in brain mouse whereas it could be detected in brain from wild type rodent at a picogram range. Our data prove for the first time that endogenous dopamine is necessary to endogenous morphine formation in normal mammalian brain. Morphine synthesis appears to be originated from dopamine through L-tyrosine in normal brain tissue. Morphine synthesis is not considered to occur inside the same neuron in normal tissue; released dopamine might be transported into morphinergic neuron and further transformed into morphine. A physiological role for endogenous morphine is suggested considering that dopamine could modulate thermal threshold through endogenous morphine formation in vivo. Thus, dopamine and endogenous opiates/opioid peptides may be interconnected in the physiological processes; yet, endogenous morphine may represent a basic link of this chain.     

Patterning of brain organoids derived from human pluripotent stem cells

The emerging technology of brain organoids deriving from human pluripotent stem cells provides unprecedented opportunities to study human brain development and associated disorders. Various brain organoid protocols have been developed that can recapitulate some key features of cell type diversity, cytoarchitectural organization, developmental processes, functions, and pathologies of the developing human brain. In this review, we focus on patterning of human stem cell-derived brain organoids. We start with an overview of general procedures to generate brain organoids. We then highlight some recently developed brain organoid protocols and chemical cues involved in modeling development of specific human brain regions, subregions, and multiple regions together. We also discuss limitations and potential future improvements of human brain organoid technology.     

Enhancement of asynchronous release from fast-spiking interneuron in human and rat epileptic neocortex

Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca²⁺ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca²⁺ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.