Biomedical subjects

N Herschkowitz

Publications and source records attributed to N Herschkowitz.

At least 55 records · Page 3Linked to original sources

PubMed1987

Simultaneous immunofluorescence and autoradiography: a useful technique for investigating neurotransmitter uptake by neurons and glia in primary central nervous system culture.

Previous studies on the localization of radiolabelled neurotransmitters in cultured cells of neural origin have relied on the comparison of cell morphology, as determined by immunocytochemistry, with the patterns of labelling on autoradiograms. We present here a method combining simultaneously autoradiography, following the uptake of tritium-labelled amino acid transmitters, with indirect immunofluorescence using antibodies against both surface and intracellular antigens. Using a fixative containing only a low concentration of glutaraldehyde (4% paraformaldehyde, 0.1% glutaraldehyde), a similar retention of gamma-[3H]aminobutyric acid (GABA) and D-[3H]aspartate was achieved as with the higher concentrations commonly used, with the advantage that the autofluorescence associated with glutaraldehyde fixed tissue was eliminated, and the immunoreactivity of the antigens to be localized was not destroyed. Using this method GABA and D-aspartate accumulating cells, in dissociated mouse central nervous system (CNS) cultures, could be reliably identified as oligodendrocytes, and some multiprocessed astrocytes, by anti-galactocerebroside (GC) and anti-glial fibrillary acidic protein (GFAP) immunofluorescence respectively. GABA-accumulating neuron-specific enolase (NSE) positive neurons could be clearly identified but no D-aspartate accumulating neurons were found. This technique should have a wide application in the investigation of whether selective transport mechanisms coexist with antigens characteristic of a certain cell type or sub-type.

Animals↗

PubMed1987

Oligodendroglial and astroglial heterogeneity in mouse primary central nervous system culture as demonstrated by differences in GABA and D-aspartate transport and immunocytochemistry.

Using simultaneous autoradiography and immunofluorescence we have investigated the functional heterogeneity amongst oligodendrocytes and astrocytes in primary mouse central nervous system (CNS) culture as expressed by differences in their ability to accumulate gamma-[3H]aminobutyric acid [( 3H]GABA) and D-[3H]aspartate. We have used a range of specific antibodies that identify oligodendrocytes and astrocytes, from precursor to fully mature cells, to address the question of whether all neuroglial cells are capable of expressing this function. Our results showing that A2B5-, 03-, and galactocerebroside-positive cells became heavily labelled with these two neuroactive amino acids, whereas cells expressing the myelin proteins 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) did not, demonstrate that this capacity is already present in oligodendrocytes at early developmental stages but may not extend to fully mature cells. Astrocytes in culture exhibited a large degree of variability with respect to their ability to transport GABA and D-aspartate. When grown in either serum-containing or serum-free hormone supplemented culture medium two morphologically distinct of glial fibrillary acidic protein (GFAP)-positive astrocyte were identified, process-bearing and epithelioid. Process-bearing cells became heavily labelled with the amino acids under both growth conditions, whereas, data showed that although epithelioid astrocytes were not, or only lightly, labelled with either amino acid in serum-containing cultures, when grown in serum-free culture medium they became more heavily labelled. Thus the expression, in culture, by epithelioid astrocytes, of one of the functions attributed to these cells is largely dependent on growth conditions.

Animals↗

PubMed1986

Selective uptake of neuroactive amino acids by both oligodendrocytes and astrocytes in primary dissociated culture: a possible role for oligodendrocytes in neurotransmitter metabolism.

CNS glia may be involved in the modulation of neuronal excitability through their capacity to accumulate and metabolize neuroactive amino acids. To investigate the possible role of oligodendrocytes in amino acid neurotransmitter metabolism, we have used light microscopic autoradiography, following the uptake of 3H-labelled amino acids by dissociated cultures of neonatal mouse brain, characterized immunocytochemically using cell-type specific markers. Oligodendrocytes, recognized by their characteristic galactocerebroside membrane staining, rapidly accumulated [3H] gamma-aminobutyric acid (GABA), becoming intensely labelled over cell body and processes after short incubations. In contrast, oligodendrocytes became only lightly labelled with [3H]L-glutamate and aspartate, which preferentially labelled astrocytes. [3H]D-aspartate, a non-metabolized analogue of L-glutamate, was avidly accumulated by oligodendrocytes, labelling cell bodies and processes after short incubations, to a similar extent as GABA. Thus, oligodendrocytes possess a transport mechanism for these excitatory amino acids, but rapidly metabolize them and release the metabolites. Not only the GC-positive cells but also the GC-negative undifferentiated oligodendrocyte precursors accumulated both GABA and D-aspartate, suggesting that this may be a function expressed early in the differentiation of oligodendrocytes. Net uptake of [3H] beta-alanine and [3H]glycine by oligodendrocytes was not observed under any conditions tested. A small number of oligodendrocytes were labelled with [3H]taurine after longer incubations. The uptake of certain neuroactive amino acids is thus a property shared by astrocytes and oligodendrocytes, the latter acting in a protective fashion around neuronal perikarya and axons.

Alanine↗

PubMed1986

Cerebral metabolic studies in situ by 31P-nuclear magnetic resonance after hypothermic circulatory arrest.

Cerebral high energy phosphates were studied in the intact rabbit brain using nuclear magnetic resonance spectroscopy. The effect of hypothermia on degradation kinetics in total ischemia due to circulatory arrest was examined, measuring phosphocreatine, adenosine triphosphate, and inorganic phosphate as a function of time at three different temperatures (35, 24, 21 degrees C). Phosphocreatine- and ATP-decays followed single exponential functions at all three temperatures. The half-life times increased by approximately a factor of three upon lowering the temperature from 35 to 21 degrees C with activation energies of 15-20 kcal/mol, which corresponds to values of Q10 between 2.4 and 3.2. In the temperature range studied, no critical temperature was found below which metabolism would stop completely. We conclude that nuclear magnetic resonance spectroscopy allows, in the intact animal, quantitative assessment of the influence of hypothermia on energy metabolism in the brain. This influence is a major concern in the field of cardiac surgery in infants and children who are often operated in total circulatory arrest under deep hypothermia.

Adenosine Triphosphate↗

PubMed1986

Diagnostic approach in children with severely retarded psychomotor development of unknown origin.

In infants and children with severe motor and/or mental retardation combined with a loss of acquired skills or with characteristic signs, such as e.g. a cherry red spot in the fundus, it is easy to suspect the presence of a hereditary metabolic disorder and to undertake the appropriate investigations. The diagnostic approach is, however, more difficult in retarded children with an uncertain developmental stillstand. Our goal was to devise strategies of how to deal with these patients from a diagnostic point of view. We retrospectively studied 77 children affected by severe developmental lag of unknown origin and without any clear signs of improvement or regression. A diagnosis of a hereditary metabolic disorder could be established in 12 of these children. In 5 other children a chromosomal abnormality or a structural abnormality of cerebral development was detected. Sixty children remained without any aetiological or pathogenetic diagnosis; 55 of them could be reexamined clinically after one year, and 32 of these showed then a clear improvement of their mental and/or motor performance. From the individual analysis of the 77 patients and from literature we conclude that if a child presents multiple minor malformations, investigations should concentrate on chromosomal aberrations and on possible structural brain abnormalities. Metabolic studies should be performed if the same disease already occurs in the family and where characteristic signs are present. In children without such signs the psychomotor development should be reevaluated after one year. If after this year there is an improvement, there is no reason for starting extensive investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗

PubMed1985

Double ligand ELISA technique for the estimation of antibodies to brain tissue antigens in patients with neurological disorders.

A double ligand enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibodies against brain tissue antigens in the sera of patients with neurological diseases. The sera were tested on human white matter homogenate. The technique consists of successive incubations with the human serum to be tested, rabbit immunoglobulin G (IgG) to human immunoglobulins (Ig), alkaline phosphate-labeled protein A and alkaline phosphatase substrate. This procedure has the advantage of increased sensitivity compared to the classical ELISA. Application of this procedure to the sera of patients with neurological diseases showed that the unspecific binding is very low and the results are reliable. Moreover the test allows the detection of antibodies to chemically different antigenic structures that can occur in a variety of neurological diseases.

Adolescent↗

PubMed1985

Utilization of galactose in cultured brain cells of neonatal mice.

Metabolism of galactose was examined in dissociated brain cells from neonatal mice after 10-13 days in culture. Consumption of galactose at levels up to 26 mM was much less than consumption of glucose at corresponding concentrations. Lactate was consumed from the media at all galactose levels, in contrast to experiments with glucose in which lactate was formed and released into the media. Generation of CO2 from 4 mM glucose was 9-fold greater than from an equimolar level of galactose. Relatively low concentrations of glucose could reduce uptake of galactose, whereas galactose at levels up to 11.6 mM failed to inhibit consumption of glucose or formation of lactate. In glucose-deficient states, galactose supplementation of the media led to a marked increase in sulfatide synthesis by oligodendrocytes in the culture with a maximum effect at 2.3 mM. Under these conditions, [1-14C]galactose was incorporated directly into the carbohydrate portion of sulfatide, although most of the label was found in phospholipids and in the nonlipid fraction of the cellular homogenate. These data suggest that galactose is poorly metabolized by brain cells, but does not exhibit toxic effects.

Animals↗

PubMed1984

Uptake of [3H]GABA by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study.

Uptake of [3H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [3H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galactocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [3H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by beta-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [3H]beta-alanine, which labeled only astrocytes. Oligodendroglial [3H]GABA uptake was Na+-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.

Animals↗

PubMed1984

Production and characterization of monoclonal antibodies to the myelin glycolipid sulfatide.

Sulfatide is enriched in the myelin sheath and accounts for 5% of the total lipids in this membrane. In the present work we describe the production and characterization of mouse monoclonal antibodies against sulfatide. The antibodies were detected and characterized in a previously described ELISA test system. The clone AIC3IA2 produced antibodies of the IgG3 class with high specificity for sulfatide. These antibodies showed almost no cross-reactivity with galactocerebroside or with any of the other lipids we tested. When used with the peroxidase antiperoxidase technique the antibodies stained a cell population either in fixed or unfixed brain cell cultures, indicating a surface localization of sulfatide in the respective cell population. In double-staining experiments the stained cell population was identified as myelin basic protein-positive oligodendrocytes.

Animals↗

PubMed1984

Metabolic studies on vaccinia-virus-infected oligodendrocytes in brain cell cultures.

Twelve-day-old cultures of dissociated newborn mouse brain were infected with neurotropic vaccinia virus strain WR. Using the indirect immuno-fluorescence staining technique the total destruction of galactocerebroside (GL) or myelin basic protein (MBP)-positive oligodendrocytes could be detected after 72 h of infection. The activity of the oligodendrocyte-specific enzymes, cerebroside sulfotransferase (CST) and 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was 27% and 17% respectively of the activity in noninfected controls. This reduction was not a result of viral-induced inhibition of host protein synthesis. In cultures treated with puromycin GC- and MBP- positive oligodendrocytes were detectable at a time at which no CST or CNP activity could detected.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1984

Parvalbumin, a neuronal protein in brain cell cultures.

Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000). In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.

Animals↗

PubMed1983

Normal proliferation rate of galactocerebroside positive oligodendrocytes in brain cell cultures of the hypomyelinated mouse mutant jimpy.

Proliferation of oligodendrocytes from the jimpy (jp) hypomyelinated mouse mutant was studied in dissociated brain cell cultures. This was done by combining anti-galactocerebroside (GC) immunostaining (for identifying oligodendrocytes) with [3H]thymidine autoradiography (for identifying proliferating cells). Previously we showed that the expression of GC in culture by jp oligodendrocytes is not altered by the jp mutation. Present results show that in 7-, 14- and 21-day-old jp cultures oligodendrocytes proliferate at a rate similar to that of normal GC+ oligodendrocytes. This indicates that, in jp brain cell cultures, oligodendrocytes which are not affected by mutation in their capability to express GC are also unaffected with regard to their proliferation rate.

Animals↗

PubMed1983

Immunocytochemical investigations of murine leukodystrophies. A study of the mutants 'jimpy' (jp) and 'myelin deficient' (mld).

Sections of the central nervous system of the leukodystrophic mouse mutants 'jimpy' (jp) and 'myelin deficient' (mld), as well as of healthy littermates, were immunostained for glial fibrillary acidic protein (GFA), myelin basic protein (MBP) and myelin-associated glycoprotein (MAG). Adjacent sections were stained conventionally for myelin. In jp, GFA-stained astrocytes were abnormally prominent already at the age of 12 days. A considerable amount of MBP and MAG was present in the vicinity of axons, although no myelin was visible in the conventional stains for myelin. In mld, GFA-staining astrocytes were present in normal numbers. MAG could be demonstrated in its normal localization along axons, but MBP was visible only in the comparatively old animal (85 days). Here, it was demonstrated in an abnormal site--the perikarya and the proximal parts of the processes of oligodendrocytes. Thin myelin sheaths present in this animal could not be stained for MBP.

Animals↗

PubMed1983

Production and characterization of mouse antibodies against the brain lipid sulfatide.

One of the lipids in the myelin sheet is sulfatide, a galactosphingolipid. Our aim was to develop a test system to detect and characterize antibody against this lipid. Mouse anti-sulfatide antibody was estimated by a solid-phase microtube assay with labeled Staphylococcus aureus protein A. With labeled rabbit anti-mouse Ig antibody, there was no difference between mouse anti-sulfatide serum and control sera from animals immunized with unrelated antigens. Results show that BALB/c mice produce antibodies against sulfatide if this low molecular weight compound is injected together with cholesterol, lecithin and bovine serum albumin. The antisera are specific for sulfatide but crossreact with galactocerebroside. However, mouse IgM antibody binds to sulfatide-coated polyvinyl plates non-specifically. Thus, only test procedures which avoid detection of IgM antibody can be used to estimate antibody specific for sulfatide and probably also for other lipophilic compounds.

Animals↗

PubMed1983

Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures.

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.

Animals↗

PubMed1983

Development of immunologically identified brain cells in culture: quantitative aspects.

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.

Animals↗

PubMed1982

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures.

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.

Animals↗

PubMed1982

Hereditary neurovisceral mannosidosis associated with alpha-mannosidase deficiency in a family of Persian cats.

Several kittens in a family of Persian cats had a metabolic storage disease. Clinically the disorder was characterized by hepatomegaly, neurological signs and early death. The microscopic lesions consisted of widespread vacuolation of neurons and glial cells in the central nervous system and in liver cells. Electronmicroscopically the lesions consisted of intracytoplasmic accumulation of membrane-bound "empty" vacuoles. In addition to the storage disease, poor myelination of the cerebral white matter was found. The defect was reproduced in breeding trials. On biochemical analysis of brain tissue, deficient function of the enzyme alpha-mannosidase was detected. The clinical and pathological features of mannosidosis in Persian cats were compared to similar defects in other species.

Animals↗