Human Central Nervous System Tissue Research Articles

Apoptosis in measles virus-infected human central nervous system tissues

Apoptosis in measles virus-infected human central nervous system tissues

Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

An adenovirus vector encoding tyrosine hydroxylase activity may enter human cns cells in primary dissociated cultures

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP2) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP2-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.

Myelin/oligodendrocyte glycoprotein is alternatively spliced in humans but not mice.

Myelin/oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed on the oligodendrocyte cell surface and the outermost surface of myelin sheaths. Due to this localization, MOG is a primary target antigen involved in immune-mediated demyelination. We previously reported that MOG is a unique member of the immunoglobulin (Ig) superfamily in that it possesses two large hydrophobic domains. MOG is highly conserved between deduced peptide sequences of rodent and human MOG (approximately 89% identity). We have completed an investigation of alternative splicing within the human and mouse MOG genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total cellular RNA isolated from both fetal and adult human central nervous system (CNS) tissues reveals a complex array of alternatively spliced MOG-specific variants and the presence of two novel exons. Exon 3 encodes a short hydrophilic domain containing multiple in-frame termination codons that would result in truncation of MOG prior to translation of its transmembrane domain. Exon 7 encodes an additional hydrophilic domain that replaces MOG's second hydrophobic domain in one splice variant. We also observed that five of our eight MOG variants exhibited an alternative internal 3' splice acceptor within MOG's terminal exon. Surprisingly, no splicing was observed in a developmental study using mouse brainstem RNA.

An adenovirus vector encoding tyrosine hydroxylase activity may enter human CNS cells in primary dissociated cultures

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP2) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP2-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.

Phenolic and tyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system.

In the present study we investigated the biochemical properties of in vitro phenolic (5'D) and tyrosyl (5D) ring deiodination and the tissue concentrations of T4, T3, and rT3 in adult human central nervous system (CNS) tissue. All samples were obtained from nontumoral tissue at autopsy (n = 6) or neurosurgical operation (n = 5). Both phenolic and tyrosyl ring deiodinase activities were demonstrable in all samples obtained intraoperatively, whereas only tyrosyl ring deiodination was evident in the tissues obtained postmortem. The phenolic ring deiodination pathway corresponded to the type II 5'-deiodinase isoenzyme with regard to its high affinity for T4 and rT3 (Km = 2.2 and 2.4 nmol/L, respectively), its insensitivity to 6-propyl-n-2-thiouracil (PTU), and the sequential reaction mechanism. No PTU-sensitive 5'-deiodination of rT3 was demonstrable. Tyrosyl ring deiodination of both T4 and T3 showed typical type III 5D kinetics (Ka, 6.5 nmol/L for T4 and 3.4 nmol/L for T3) and was PTU insensitive. Nanomolar concentrations of tissue T4, T3, and rT3 were detected in samples obtained both intraoperatively and postmortem. They were very similar to the absolute values of the apparent Km for T4, T3, and rT3 in the phenolic and tyrosyl ring deiodination pathways. In conclusion, we have demonstrated the coexistence of both phenolic and tyrosyl ring deiodinase activities in the human CNS. Their kinetic characteristics, substrate specificity, and reaction mechanisms are very similar to the corresponding type II 5'- and type III 5-iodothyronine deiodinase activities in rat brain. In contrast to the findings in the rat CNS, no PTU-sensitive phenolic ring deiodinase (i.e. type I 5'D) activity was found in the human CNS. This may explain the relatively high tissue concentrations of rT3.

Myelination by transplanted human and mouse central nervous system tissue after long-term cryopreservation

Human and mouse oligodendrocytes were transplanted, after a long period of cryostorage, into newborn mouse brain. Tissue fragments were obtained from brain and spinal cord of 10-week-old human fetuses and from the periventricular zone of embryonic and newborn mouse brains. Samples were stored at -180 degrees C for periods of 3 days to over 5 years. Frozen or fresh fragments were transplanted into the brains of newborn shiverer mutant mice, which are deficient in myelin basic protein (MBP). Normal myelin, produced by grafted oligodendrocytes, was detected by immunohistochemistry with an anti-MBP antiserum. The best results were obtained with isospecific grafts. The timing of myelin appearance did not depend significantly on the species or age of the donor. Myelination obtained with mouse grafts was more profuse when the donor was younger (embryonic versus newborn). Cryopreservation over 5 years did not impede the graft's ability to produce myelin and can be considered for long-term storage of oligodendrocytes in view of cell therapy.

Characterization of nucleoside transport activity in rabbit cortical synaptosomes.

Rabbit central nervous system (CNS) preparations have been used to study the central effects of adenosine, but little is known about the specific uptake mechanisms in rabbit brain involved in the regulation of extracellular adenosine concentrations. The present study assessed the kinetic and pharmacological characteristics of the uptake of [3H]uridine (a poorly metabolized substrate for adenosine transporters) by rabbit cortical synaptosomes, to define the transporter subtypes involved and to evaluate species variability in transporter characteristics. [3H]Uridine transport into rabbit cortical synaptosomes was mediated by two saturable, facilitated diffusion systems with characteristics compatible with the es and ei transporter subtypes identified in other mammalian species. About 65% of the total transport was mediated by the es system, and Km estimates of 320 and 94 microM were determined for [3H]uridine uptake by the es and ei transporter, respectively. These results differ significantly from the subtype ratio and kinetic characteristics reported for rat and guinea pig cortical synaptosomes, where most of the transport was mediated by an ei subtype. Dipyridamole, dilazep, nitrobenzylthioinosine, R75231, soluflazine, and mioflazine were relatively more effective as inhibitors of es-mediated uptake (compared with ei), while the substrates adenosine, cytidine, and guanosine did not distinguish between the es and ei transporters in rabbit cortical synaptosomes. These results highlight the significant species-tissue variability in nucleoside transporter characteristics and subtype expression, and emphasize the need to characterize the transporters in human CNS tissue to allow the rational development of CNS-active therapeutics based on inhibition of nucleoside transport.

Microwave antigen retrieval for immunocytochemistry on formalin‐fixed, paraffin‐embedded post‐mortem CNS tissue

Microwave antigen retrieval methods were assessed for a panel of antibodies on formalin-fixed, paraffin-embedded sections from a range of human central nervous system (CNS) tissues taken at post-mortem. The immunoreactivity of a number of antigens (synaptophysin, PGP9.5, GFAP, carbonic anhydrase II, CD68, ferritin, HLA-DR, alpha beta-crystallin, measles and herpes viruses) was markedly enhanced by this procedure compared with other methods of antigen unmasking, such as trypsinization. Enhancement was noted both by an increased number of positive cells and by the intensity of reactions within individual cells and their processes. Furthermore, the microwave method produced uniform immunostaining over large surface area sections with no loss of morphological detail. Large cryostat sections of CNS tissue can be difficult to obtain with good morphology. The ability to retrieve a wide range of antigen in formalin-fixed, paraffin-embedded CNS tissue will greatly increase the range of investigations that can be carried out on this type of stored material.

The neuropathology of South American mummies.

Few studies of intracranial mummified brain tissue have been undertaken. This is because of the infrequency in which preserved human central nervous system tissue is encountered and the scarcity of available mummies from different parts of the world. This study undertook a systematic analysis of 15 naturally mummified human brains from 1000 B.C. to 1500 A.D. excavated from the deserts of northern Chile. Gross examination revealed relatively well-preserved dura mater, cerebral hemispheres, cerebellum, and spinal cord in several cases. Five cases showed evidence of intracranial disease. Three cases had evidence of external injury. One case revealed subarachnoid and one case revealed intracerebral hemorrhage. Samples of central nervous system tissues were taken for further analysis. The samples were rehydrated and processed for structural analysis by light and electron microscopy. Light microscopy of the brain parenchyma revealed an eosinophilic staining background with vascular structures but few cellular elements present. The dura mater demonstrated normal dural architecture consisting of collagen fibrils. Electron microscopy did not clearly demonstrate individual neurons or axonal processes. Bundles of collagen fibrils with typical periodicity were clearly seen in the dura mater. The examination of ancient human central nervous system tissues reveals normal and abnormal neuroanatomy.

Beware of connective tissue proteins: Assignment and implications of collagen absorptions in infrared spectra of human tissues

Infrared spectra of human central nervous system tissue and human breast carcinoma are presented. The spectra are discussed in terms of the composition of the tissues. It is shown that differences between spectra of white and grey matter can be rationalised on the basis of differences in lipid content. Spectra of the choroid plexus and arachnoid villus of the meninges show a series of absorptions not observed in other CNS tissue. These absorptions are discussed in terms of the connective tissue content of the samples. We demonstrate that the presence of collagen results in the appearance of a series of characteristic absorptions which may be mis-assigned as DNA phosphate absorptions. The implications of the presence of collagen in tissues for the diagnosis of disease states by IR spectroscopic methods, with particular reference to cancer, is discussed.

Infrared spectra of human central nervous system tissue: Diagnosis of alzheimer's disease by multivariate analyses

AbstractWe present an infrared (IR) spectroscopic view of the pathological features of Alzheimer's disease (AD), along with a means of classifying the spectroscopic data nonsubjectively using several multivariate methods. The results demonstrate that IR spectroscopy can potentially be used in the diagnosis of AD from autopsy tissue. Although several spectral features that appear to be related to the disease process can be detected by a visual inspection of the IR spectra of AD grey matter, the appearance of these features was inconsistent. A more successful (consistent) diagnosis is achieved using multivariate analysis of the spectroscopic data. It is shown that correct classification of white and grey matter from brains identified by standard pathological methods as heavily, moderately, and minimally involved can be achieved with success rates of greater than 90% using appropriate methods. Classification of tissue as either control or AD was achieved with a success rate of 100%. © 1995 John Wiley & Sons, Inc.

Media components influence viral gene expression assays in human fetal astrocyte cultures.

In vitro neurovirological studies of viral infectivity or viral gene expression may be confounded by the multiple neural cell types and/or fibroblast contamination present in early passage cultures prepared from dissociated human central nervous system (CNS) tissue. We have developed highly enriched astrocyte cultures for neurovirological study by culturing in a serum-free defined medium, B16, supplemented with basic fibroblast growth factor (FGF-2). Subculture in this medium selects against fibroblast proliferation and favors sustained proliferation of a highly enriched glial fibrillary acidic protein (GFAP)-positive cell population. These astrocytes support productive replication of cytomegalovirus (CMV) and transient expression of transfected CMV and human immunodeficiency virus type 1 (HIV-1) viral promoters. By comparison, CNS cultures developed in standard serum-containing medium initially contain predominantly astrocytes, but show increasing contamination with fibroblasts with sequential passage. These cultures support CMV viral synthesis in both fibroblasts and astrocytes, cell types distinguishable only by immunostaining for cell specific antigen. CMV or HIV-1 promoter activities, quantitated by transient gene expression assays, are distinctly lower in CNS cultures maintained in serum-containing medium.

B7/BB-1 antigen expression on adult human microglia studied in vitro and in situ.

In this study, we have examined the expression and function of B7/BB-1 on individual glial cells, by utilizing surgically resected adult human central nervous system (CNS) tissues, tissues derived from fetal human CNS, and pathology material from cases of multiple sclerosis (MS). Immunofluorescence analysis using enriched adult human derived cultures of microglia and oligodendrocytes, and mixed microglia/astrocyte cultures, demonstrated that B7/BB-1 was expressed on microglia. Adult human-derived oligodendrocytes and astrocytes, and human fetal astrocytes were B7/BB-1 negative under all culture conditions. Flow cytometry studies demonstrated a low basal level of B7/BB-1 expression on microglia that was up-regulated following incubation with interferon-gamma (IFN-gamma). Co-culture of purified fresh allogeneic CD4+ T cells with microglia for 24 h resulted in clustering of T cells around microglia and microglial B7/BB-1 expression. Preincubation of microglia with an anti BB-1 monoclonal antibody (mAb) prior to microglia: CD4+ T cell co-cultures resulted in partial inhibition of the ability of microglia both to present recall antigen to autologous CD4+ T cells and to present antigen to allogeneic CD4+ T cells in primary mixed lymphocyte reaction (1 degree MLR). The CTLA-4 Ig fusion protein inhibited the ability of microglia to present antigen in both antigen presentation assays to an even greater extent than did the anti BB-1 mAb. The BB-1 antibody also inhibited the ability of microglia to stimulate previously activated T cells in a secondary 2 degrees MLR. In sections of multiple sclerosis brain, B7/BB-1 expression was observed on activated microglia in select parenchymal lesions, and on perivascular cells and infiltrating monocytes. B7/BB-1 immunoreactivity was not found in normal appearing white matter from MS brain or from non-inflammatory brain specimens. Our results indicate that the B7/BB-1 molecule plays a functional role in the capacity of microglia to serve as CNS antigen-presenting cells that can both initiate and perpetuate CD4+ T cell activation.

Fc Receptors for IgG on Cultured Human Microglia Mediate Cytotoxicity and Phagocytosis of Antibody-coated Targets

We have utilized surgically resected human central nervous system (CNS) tissue to determine the expression and functions of Fc receptors (Fc gamma R) on individual cell types found within the CNS. We observed all three classes of Fc gamma R on microglial cells in situ and in vitro, but not on astrocytes or oligodendrocytes. Incubation of cultured microglia with immune complexes (antibody-coated red blood cells) induced phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and oxidative bursts. We also found that microglia have the capability to produce T cell stimulatory soluble mediators after Fc gamma R crosslinking. These functional responses were enhanced by pretreatment of the microglia with interferon-gamma (IFN-gamma). Our results implicate microglial effector responses triggered by interaction of Fc gamma R with opsonized antigens as potential mediators of tissue injury within the CNS. Such injury may be particularly applicable to multiple sclerosis, an inflammatory demyelinating disease characterized by intrathecal production of immunoglobulins and cytokines.

Scientific and ethical concerns in neural fetal tissue transplantation.

This report presents a brief overview of the medical and ethical issues involved with the procurement, preparation, safety, efficacy, and subject protection of human fetal central nervous system tissue in the context of neural transplantation. The ethical perspectives from which to view the clinical use of fetal tissue include the following: 1) that fetal tissue from elective abortions is a surgical specimen; 2) that the use of such tissue involves fetal experimentation in which the fetus is a subject; and 3) that fetal tissue is considered as a cadaveric organ specimen, similar to other organs, but with special considerations compared with adult cadaveric tissue. The latter approach appears to be the most applicable and is parallel to the use of cadaveric organs and tissues after a declaration of brain death. Additional issues include the following: 1) the safety and quality of fetal tissue for implantation; 2) the hypothesis that "legitimization" and "redemption" (potentially positive effects of tissue donation in general) may lead to an increase in elective abortion rates; 3) the ethical issues of the validity and value of human experimentation involving neural grafting; and 4) the type of consent to be obtained and the appropriate timing. Elective abortions, however, probably will continue to be the primary source of fetal tissue for grafting for some time, until other tissue sources become available.

Infrared spectroscopic characterisation of multiple sclerosis plaques in the human central nervous system

Fourier transform infrared spectroscopy has been used for the characterisation of white matter, grey matter and multiple sclerosis plaques from human central nervous system tissue. We demonstrate significant differences in the infrared spectra of the three types of tissue, which show that an infrared spectroscopic discrimination of multiple sclerosis plaques from healthy brain tissue is possible in principle. The spectral changes reveal pronounced lipid loss in plaques, consistent with the demyelinating nature of the disease. The chronic plaques studied here can also be distinguished from other non-myelinated areas of the brain, based on difference in water content.

Macrophage colony-stimulating factor in human fetal astrocytes and microglia. Differential regulation by cytokines and lipopolysaccharide, and modulation of class II MHC on microglia.

CSF-1 is a growth factor that selectively promotes the proliferation, survival, and differentiation of cells of the mononuclear phagocyte series. As part of a study on the role of cytokine and hematopoietic growth factors in central nervous system (CNS) development and inflammation, we examined the expression of CSF-1 in dissociated glial cells cultured from human fetal CNS tissue. CSF-1 mRNA and protein were constitutively expressed by astrocytes. The steady state level of CSF-1 mRNA was markedly up-regulated by both IL-1 beta and TNF-alpha in a time- and dose-dependent manner, whereas only a minimal increase was detected after stimulation with LPS. In unstimulated astrocyte cultures, CSF-1 protein levels gradually increased to 3.5-fold base-line values by 96 h and were significantly increased by all three stimulants in the order of IL-1 > or = TNF > LPS. Low levels of CSF-1 mRNA and protein were also detected in unstimulated microglia cultures. In contrast to astrocyte cultures, CSF-1 mRNA and protein increased significantly after stimulation with LPS, but changed only minimally after exposure to TNF-alpha or IL-1 beta. The effect of CSF-1 on cell proliferation, morphology, and class II MHC Ag expression was determined in highly enriched cultures of microglia and astrocytes. Microglia treated with CSF-1 showed a modest level of proliferation and differentiation into rod-shaped cells, whereas neither cell number nor shape was changed in astrocyte cultures. Interestingly, marked inhibition of both basal and IFN gamma-induced class II MHC Ag expression was observed in microglial cells cultured in the presence of CSF-1, whereas no effect was detected in astrocytes. These results suggest the possibility that in situ production of CSF-1 in the CNS may regulate normal glial cell development and contribute to the immunologic status of the CNS through the down-regulation of class II MHC expression.

Species diversity of neuronectin and cytotactin expression patterns in the vertebrate central nervous system

Two extracellular matrix proteins of brain tissue, neuronectin (NEC1) and cytotactin (CT), are disulfide-bonded multimers of Mr 180,000–250,000 subunits. The previously known distribution of these molecules is, however, very different. Human NEC1 is found throughout the white matter of rostral segments of the adult central nervous system (CNS) but not in rostral gray matter or in caudal CNS segments, including the cerebellum. In contrast, CT is absent or expressed at a low level in the adult chicken cerebrum but highly expressed in the cerebellum. Despite these differences in distribution, results obtained with antibodies that recognize NEC1 and CT in several vertebrate species indicate that these molecules are identical or at least closely related: (1) αNEC1 antibodies recognize proteins affinity-purified with CT-binding proteoglycan; (2) proteins recognized by αNEC1 and αCT antibodies in cells constitutively expressing the molecules, cells in which expression is induced by growth factors and phorbol ester and cells treated with tunicamycin (to block glycosylation) are identical in subunit composition and mobility on SDS gels; (3) the removal of NEC1 from culture supernatants by immunoprecipitation removes all molecules reactive with αCT antibodies and vice versa; (4) immunoblots of brain extracts with αNEC1 and αCT antibodies yield identical results. Having demonstrated the structural similarity between NEC1 and CT, we reexamined their distribution in the CNS. Surprisingly, the temporal and spatial distribution pattern of NEC1/CT varied greatly among species. Immunohistochemical and immunoblot experiments with adult human CNS tissues revealed significant levels of NEC1/CT in rostral but not caudal segments. In contrast, in cows and pigs the molecule is found throughout the CNS. Adult rat and mouse brains show regionally restricted expression of NEC1/CT in several areas of the cerebrum — distinct from those showing NEC1/CT in the human — and in the molecular layer of the cerebellum. Tests with fetal and newborn tissues revealed that CNS development in humans, cows and pigs is not accompanied by the marked decline in NEC1/CT levels or the changes in subunit composition found in the chicken CNS. The marked species diversity in temporospatial expression patterns suggests that intrinsic and/or extrinsic elements controlling the expression of NEC1/CT have diverged during vertebrate evolution.

Immunological detection of glutamate receptor subtypes in human central nervous system.

Glutamate receptors are the principal excitatory neurotransmitter receptors in the central nervous system and are involved in a number of normal and pathological neuronal processes. Using subunit-specific antipeptide antibodies developed against the predicted amino acid sequences of several rat glutamate receptor cDNAs, we have identified these proteins in post-mortem human central nervous system tissue. Immunoblotting of dissected brain regions demonstrates that these receptor proteins are differentially distributed. The ability to identify these proteins in post-mortem human tissues should allow examination of the changes in levels of receptor subtypes that occur in a variety of neurological and psychiatric diseases.