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PubMedDate not supplied

Development of a new automated enzyme immunoassay for the determination of neuron-specific enolase.

Neuron-specific enolase (NSE) represents the gamma gamma- and alpha gamma- isoforms of the dimeric glycolytic enzyme enolase. NSE is predominantly found in neurons and neuroendocrine cells and has proven to be a marker for tumors derived from these cells. It is widely accepted in the monitoring of patients with small cell lung cancer and is also of value as an aid in diagnosis. Recently it has become of interest in the monitoring of brain damage. Monoclonal antibodies against gamma-enolase were raised in mice and selected for optimal performance on the Cobas Core enzyme immunoassay system. The antibody combination of choice was MAb 18E5 for capturing and MAb 84B10 for detection which is accomplished by using a horseradish peroxidase conjugate and the substrate 3,3',5,5'-tetramethylbenzidine. The resulting assay is a one-step enzyme immunoassay of the sandwich type. It is performed on the fully automated Cobas Core immunoassay analyzer with a total assay time of 45 min. The sample volume is 10 microliters. Calibration is done by a 1-point recalibration using a lot-specific master calibration curve provided with the kit. The dynamic range is 0-200 ng/ml. The analytical detection limit (standard 0 + 2SD) of the Cobas Core NSE EIA II was 0.1 ng/ml. Intra- and interassay coefficients of variation were < 5% and < 6%, respectively. A Hook Effect was not observed up to a concentration of 20'000 ng/ml. Test results correlated closely with the well established polyclonal Cobas Core NSE EIA (r = 0.99). In summary, the Cobas Core NSE EIA II is a rapid, reliable and convenient test for measuring NSE in human serum.

Animals↗
PubMed2006

Baboon/dSmad2 TGF-beta signaling is required during late larval stage for development of adult-specific neurons.

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2-mediated TGF-beta signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult-specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis.

Activin Receptors, Type I↗
PubMed1985

Preparation and characterization of monoclonal antibodies to human neuron-specific enolase.

Neuron-specific enolase (NSE) has been increasingly recognized as a marker for neuroendocrine tumors including small cell carcinoma of the lung (SCCL). To prepare monoclonal antibodies (MAbs) specific for human NSE, we first developed a simple method of purifying NSE by direct chromatofocusing of a crude extract of human brain tissue. BALB/c mice were then immunized with our preparation of NSE, and MAbs against NSE were generated utilizing a hybridoma technique. The antibodies were screened against both NSE and non-neuronal enolase (NNE) by a solid-phase radioimmunoassay (SPRIA). After cloning and subcloning of hybridomas, two groups of anti-NSE MAbs were identified by SPRIA. One group reacted specifically with NSE but not with its isoenzyme NNE, irrespective of whether antigens were glutaraldehyde fixed or unfixed. A second group reacted with both NSE and NNE when the latter were glutaraldehyde fixed, but surprisingly with neither antigen in the absence of fixation. Group I antibodies were further characterized by immunoblotting, and by immunocytochemistry of normal brain and liver sections and sections of SCCL. The results further supported the specificity of group I antibodies for NSE. These MAbs have potential utility in the diagnosis and management of neuroendocrine tumors, and in further understanding the biology of NSE.

Antibodies, Monoclonal↗
PubMed2002

Adenovirus-mediated Bcl-X(L) expression using a neuron-specific synapsin-1 promoter protects against disseminated neuronal injury and brain infarction following focal cerebral ischemia in mice.

The effects of an adenovirus-mediated Bcl-X(L) expression, driven by a neuron-specific human synapsin-1 promoter, on the degree of injury, were examined after transient focal ischemia in mice. Therefore, injections of vehicle, of an adenoviral E1-deleted control vector (Ad-dE1), or a Bcl-X(L) vector (Ad-Syn-Bcl-X(L)) were stereotactically made in the striatum. Seven days later, focal ischemia was induced either by 30 min or 2 h of intraluminal thread occlusion. In line with previous data, 30 min of middle cerebral artery (MCA) occlusion reproducibly resulted in disseminated neuronal injury of the striatum, as revealed by cresyl violet and TUNEL 3 days after ischemia. The degree of cell injury was significantly reduced in Ad-Syn-Bcl-X(L) treated as compared with Ad-dE1 and vehicle-treated animals. On the other hand, 2 h of MCA occlusion produced reproducible infarcts both in vehicle and Ad-dE1 treated animals 24 h after ischemia. The infarct area at the level of the striatum was significantly decreased by Ad-Syn-Bcl-X(L) treatment. The present data demonstrate that an adenoviral Bcl-X(L) expression with a neuron-specific synapsin-1 promoter provides a powerful tool, which not only diminishes disseminated neuronal injury, but also protects against tissue infarction.

Adenoviridae↗
PubMed1999

Neurone-specific enolase and N-acetyl-aspartate as potential peripheral markers of ischaemic stroke.

BACKGROUND: After stroke, brain-specific proteins (including neurone-specific enolase) leak into the blood. The question addressed in the present study was whether N-acetyl-aspartate (amino acid derivative localized in cerebral neurones) could also serve as a peripheral marker of ischaemic damage. N-acetyl-aspartate levels were determined in the blood of stroke patients and related to clinical outcome, volume of infarction and to serum neurone-specific enolase. METHODS: Blood samples from 19 patients (seven women, 12 men, mean age of 73 years, range 56-88 years) were collected during the first 4 days after stroke and analysed for neurone-specific enolase (radioimmunoassay) and/or N-acetyl-aspartate (mass spectrometry). Clinical outcome was assessed using the Glasgow Outcome Score, and volume of infarction was calculated using computerized tomography (CT). Control values of N-acetyl-aspartate, determined in six female and nine male volunteers (mean age 47.4 years; range 28-73 years) were 0.26 +/- 0.02 mumol L-1. RESULTS: The increase in serum N-acetyl-aspartate was highly significant (P < 0.0001) within the first 24 h and at 72 h after stroke and correlated (P < 0.05) with volume of infarction only in patients with a bad prognosis (Glasgow Outcome Score < 5). Serum N-acetyl-aspartate at 24 h and neurone-specific enolase at 72 h were negatively correlated, suggesting that more N-acetyl-aspartate reaches the blood when brain tissue is less irreversibly affected. CONCLUSION: Serum N-acetyl-aspartate appears to be an early peripheral marker of ischaemically affected brain neurones, and the ratio of N-acetyl-aspartate to a protein marker, such as NSE, may serve as an index of irreversibility.

Aged↗
PubMed2003

Malignant fibrous histiocytoma masquerading as germ cell tumor by producing beta-human chorionic gonadotropin and neuron-specific enolase.

Neuron-specific enolase and beta-human chorionic gonadotropin are serum markers frequently found associated with germ cell tumors. To our knowledge, we report the first case of a malignant fibrous histiocytoma producing both markers and discuss the significance of this unusual condition in the differential diagnosis of retroperitoneal tumors.

Biomarkers, Tumor↗
PubMed1993

Neuron-specific enolase (NSE) and non-neuronal enolase (NNE) mRNAs are co-expressed in neurons of the rat cerebellum: in situ hybridization histochemistry.

Using in situ hybridization histochemistry, we analysed the localization of mRNAs for neuron-specific enolase (NSE) and non-neuronal enolase (NNE) in the rat cerebellum at various postnatal developmental stages. Synthetic 45 meric oligonucleotides corresponding to partial sequences of the non-coding region of rat NSE or NNE mRNA were 35S-labeled to approximately the same specific activity and used as hybridization probes. On examination of the adult rat cerebellum, both NSE and NNE signals were detected in all identified and presumed neurons which included Purkinje cells, internal granule cells and presumed stellate/basket cells in the cerebellar cortex and neurons of the dentate nucleus. Examination of the cerebellum during postnatal development also revealed coexistence of NSE and NNE signals in these neurons from early stages. During development, both signals coincidentally increased in Purkinje cells and neurons of the dentate nucleus, while only NSE signals showed a gradual increase in the internal granule cells in which NNE signals remained at the same level from early postnatal to adult stages. The external granule cells showed NNE signals until postnatal day 7 but thereafter the signals became less distinct, especially in cells if the inner zone of the external granule cell layer. Thus, it was shown that NSE and NNE were commonly coexpressed at the mRNA level in various neurons of the cerebellum except for very undifferentiated external granule cells which expressed only NNE mRNA.

Aging↗
PubMed1996

Glutamate-induced efflux of protein, neuron-specific enolase and lactate dehydrogenase from a mesencephalic cell culture.

A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium. Glutamate toxicity was induced in sister cultures by 15 min exposure to 100 mumol/l glutamate in a Ca2+ containing salt solution. Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the neuron-specific enolase content in the cells and in the culture medium. The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of lactate dehydrogenase catalytic activity concentration and neuron-specific enolase mass concentration in the culture medium. It was found that the efflux fraction calculated from estimations of the cells was clearly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium. Calculations of the recovery of lactate dehydrogenase and neuron-specific enolase and experiments designed to study the efflux of lactate dehydrogenase and neuron-specific enolase during incubation and washing showed that higher amounts of neuron-specific enolase are released than lactate dehydrogenase. A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme determinations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers. This indicates that the determination of the neuron-specific enolase efflux fraction (cells) is an accurate and sensitive marker of damaged neurons. The lactate dehydrogenase efflux fraction seems to be less sensitive for the quantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the cell culture.

Animals↗
PubMed2001

Reference values of amniotic fluid neuron-specific enolase.

OBJECTIVE: Enolase is a dimeric cytoplasmic enzyme whose double gamma isoenzyme, neuron-specific enolase, is predominantly found in neuronal and neuroendocrine tissues. Cell injury causes its release into the blood and cerebrospinal fluid (CSF). Neuron-specific enolase has been measured in the serum and CSF of adults and full-term asphyxiated neonates as a marker of neurological injury. We recently observed an elevation of neuron-specific enolase in the amniotic fluid of women whose neonates subsequently developed intraventricular hemorrhage or periventricular leukomalacia. The purpose of our study was to establish reference values of neuron-specific enolase in the amniotic fluid as a function of gestational age. METHODS: A total of 110 amniotic fluid samples, obtained primarily for genetic studies (16-20 weeks, n = 22), for evaluation of preterm labor (21-35 weeks, n = 66) and for fetal lung maturity studies (36-40 weeks, n = 22), were analyzed for neuron-specific enolase. Samples were from women who subsequently delivered term neonates with normal neurological examinations or who delivered preterm neonates with normal neurosonograms up to the 7th day of life. Descriptive statistics and non-parametric correlations were used for analysis. RESULTS: There was no correlation between gestational age and concentration of neuron-specific enolase (Spearman's r = 0.059, p = 0.63). The overall mean neuron-specific enolase value was 2.5 +/- 1.39 microg/l. The highest value obtained was 6 microgl. Of the 110 women, 105 (95.5%) had neuron-specific enolase values of less than 5 microg/l, while five (4.5%) had values ranging from 5 to 6 microg/l. CONCLUSIONS: The amniotic fluid level of neuron-specific enolase does not change as a function of gestational age. These stable levels may have utility in the evaluation of cases with fetal neurological injury.

Adult↗
PubMed1986

Immunohistochemical localization of neurofilaments and neuron-specific enolase in 29 cases of neuroblastoma.

Twenty-nine neuroblastomas have been examined with the use of rabbit antibodies specific for each of the three neurofilament polypeptides, with a monoclonal antibody specific for the NF-L polypeptide, and with a rabbit antibody specific for neuron-specific enolase. When frozen material was used, all neuroblastomas were positive with the neurofilaments antibodies. When alcohol-fixed paraffin-embedded material was used, neurofilament staining was weaker and the fixation procedure appeared to destroy the epitopes recognized by the NF-L antibodies preferentially. Although all neuroblastomas were positive for neurone-specific enolase, so were two rhabdomyosarcomas, suggesting that NSE is not an appropriate marker to distinguish the different small blue cell tumors of children.

Adolescent↗
PubMed1983

Neuron-specific enolase as an index of neuronal regeneration and reinnervation.

Neuron-specific enolase (NSE) is a glycolytic isoenzyme which is located in central and peripheral neurons and neuroendocrine cells. Another enolase isoenzyme, non-neuronal enolase (NNE), occurs in glial cells. The purpose of this study was to follow any changes in NSE and/or NNE in cranial motor neurons after separation of their cell bodies from their axon terminals. One hypoglossal nerve in the rat and the cynomolgus monkey was thus crushed or cut and, after a given period, the brains were perfusion fixed. Immunocytochemistry, using anti-rat NSE and NNE or anti-human NSE and NNE, was performed on Vibratome-sectioned specimens of the hypoglossal nuclei. In the rat, NSE immunostaining decreased in the affected neurons 2 to 10 days following axonal injury. The change was greatest on the 10th day. Twenty days following nerve crush. NSE staining began to recover on the operated side and by the 45th day had returned to normal levels. NSE changes in the monkey were similar to those in the rat. In rats, where the nerve was cut and the proximal stump was translocated to a normally innervated muscle to inhibit re-formation of synaptic contacts, the NSE remained low for 60 days after nerve injury. As NSE levels fell during degeneration, there was a slight increase in NNE in some of the monkey specimens but not in others; the NNE alterations were, therefore, equivocal. The results demonstrate that the content of NSE in neurons serves as a molecular marker of axon injury, regeneration, and target reinnervation.

Animals↗
PubMed1995

Neurotrophic and neuroprotective effects of neuron-specific enolase on cultured neurons from embryonic rat brain.

We previously reported that the gamma gamma-isozyme of enolase, NSE), one of the glycolytic enzymes, promoted the survival of embryonic rat neocortical neurons in culture, but alpha alpha-isozyme (non-neuronal enolase) had no effect. In the present study, the neurotrophic effects of NSE on cultured mesencephalic and spinal neurons from rat embryo were examined. NSE promoted the survival of neurons not only in neocortical cultures but also in mesencephalic and spinal cord cultures. Furthermore, NSE showed neuroprotective action on cultured neocortical neurons in a low-oxygen atmosphere. By contrast, non-neuronal enolase did not show any neurotrophic or neuroprotective activities. To clarify the mechanism of the neurotrophic effect of NSE, the binding of NSE to cultured neurons was determined by radio-receptor assay using 125I-labelled NSE. The specific binding, which was dose-dependent, saturable, and calcium-dependent, could be detected. These results suggest that NSE has neurotrophic and neuroprotective effects on rather a broad spectrum of neurons in the central nervous system. The existence of specific binding of NSE to cultured neurons suggests the possibility that receptor-like or carrier-like molecules on the neuronal surface are involved in the neurotrophic activity of NSE.

Animals↗
PubMed1989

Neuron-specific enolase and retinoblastoma. Clinicopathologic correlations.

Neuron-specific enolase (a glycolytic, ubiquitous, intracellular enzyme) has recently been reported to be detectable in the aqueous humor of eyes containing retinoblastoma. Aqueous humor from 17 patients with histologically proven retinoblastoma was assayed for the presence of neuron-specific enolase (NSE). NSE was detectable in 17 out of 17 patients with levels between 619 and 60,000 ng/ml. A multitude of clinocopathological parameters were examined for statistically significant correlations with levels of aqueous humor NSE. This investigation demonstrated that only two parameters, the presence of tumor invasion into the anterior chamber, and inflammation significantly correlated with aqueous NSE levels. Histological parameters which did not correlate with aqueous NSE levels included tumor necrosis, calcification, Flexner-Wintersteiner rosettes, exophytic/endophytic tumor type, tumor extent relative to the equator, and optic nerve/choroidal invasion. Clinical parameters which showed no correlation included patient sex (M/F), enucleation age, presentation age, family history, laterality, prior treatment, and presence of metastatic disease. Neuron-specific enolase is present in the anterior chamber of eyes enucleated for retinoblastoma, but additional testing is necessary to determine the normal levels of neuron-specific enolase in children's eyes and the levels in eyes with lesions simulating retinoblastoma.

Anterior Chamber↗
PubMed1981

Immunocytochemical localization and developmental profile of neuron specific enolase (NSE) and non-neuronal enolase (NNE) in aggregating cell cultures of fetal rat brain.

In aggregating cultures, neuron specific enolase (NSE) was first detected biochemically at 3 days. NSE levels increased with time in aggregate cultures and at 48 days reached a level which was 33% of that found in adult rat brain in vivo. The level of non-neuronal enolase (NNE) was essentially identical in aggregate cultures and normal rat brain. Immunocytochemically, NSE(+) cells first appeared at 10 days in vitro. Their number increased until 20 days in culture and then remained constant. When the immunocytochemical localization of NSE and NNE was compared in vibratome sections of 25 day aggregates, all identifiable neurons were NSE(+), NNE(-) and glial cells were NSE(-), NNE(+). In 1 micron thick epon sections of 30 day aggregates NSE antiserum stained neuronal cytoplasm intensely. Comparison of NSE staining in 1 micron thick epon sections with the same cell in an adjacently cut thin section provided conclusive evidence that NSE(+) cells were neurons and NSE(-) cells were glia. These results demonstrate that the three-dimensional organization of aggregate cells provides an excellent environment for neuronal differentiation and also emphasize the advantages of this culture system for multidisciplinary studies of brain development.

Animals↗
PubMed2001

Neuron-specific expression of Cre recombinase during the late phase of brain development.

Gene targeting to disrupt gene expression in a temporal and spatial manner in a specific tissue using Cre recombinase-mediated gene inactivation has been proven to be useful to study in vivo gene function. To delete genes specifically in neurons during the late phase of brain development, we have generated transgenic mouse lines that express Cre recombinase under the control of the murine neurofilament-H (mNF-H) gene promoter. In this study, we report that one of these mouse lines expresses Cre recombinase specifically in the neurons of the brain and spinal cord during the late stage of their development. The transgenic line displays specific excision of the loxP-flanked gene in the neurons just after embryonic day 18.5 (E.18.5), which coincides with the later phase of brain maturation including spinal cord and olfactory bulb area. This mNF-H-cre transgenic mouse line will be valuable for studying in vivo functions of neuron-specific genes, particularly, defining their precise roles in the mature nervous system using conditional gene targeting strategies.

Age Factors↗
PubMed1994

Age and differentiation-related differences in neuron-specific tubulin immunostaining of olfactory sensory neurons.

Olfactory sensory neurons (OSNs) are unusual mammalian neurons because they are produced continually throughout adult life and because their production is upregulated after injury. Because OSNs also have an unusual immunological profile and do not bind the commonly used antibody markers for neurons, we sought new antibody markers for studies of OSN regeneration. In this report, we characterize the staining patterns of antibodies to the Class III beta, neuron-specific, tubulin (NST) in rat olfactory tissue sections, to determine if these antibodies specifically label OSNs. In tissue sections from newborn rats, monoclonal antibodies to NST labeled cell bodies and processes of both immature (olfactory marker protein, OMP, -negative) and mature (OMP-positive) OSNs. In tissue sections from adult rats, immature OSNs showed both cell body and dendrite staining with anti-NST, while mature OSNs showed little or no cell body staining. Mature OSNs appeared to have both axonal and dendritic anti-NST staining. Axonal staining was suggested by the complete labeling of the olfactory nerve bundles and the nerve fiber layer of the olfactory bulb. The extent of labeling was judged by comparison with anti-OMP staining. Mature OSN dendritic staining was suggested because a much higher number of dendrites were anti-NST stained in the epithelium than cell bodies. These changes suggest both age and differentiation-related changes in subcellular distribution of NST in OSNs. NST antibodies are thus good markers for all OSNs in the newborn rat, but selective markers for immature OSNs and mature OSN processes in the adult rat. NST antibodies may also be useful probes for beta III tubulin function in neurons.

Age Factors↗
PubMed1997

Serum neuron-specific enolase is a marker for neuronal damage following status epilepticus in the rat.

We determined the serum concentrations of neuron-specific enolase (s-NSE) in rat pups of 1, 2, 3, and 4 weeks of age and in adult rats that were subjected to lithium-pilocarpine status epilepticus (SE). Damage to brain regions was rated on a scale of 0 (no damage) to 5 (> 50% cell loss). Rat pups of 1-2 weeks of age had a higher baseline s-NSE than the adults. Following SE, 1 week old rat pups had no elevation of s-NSE and no histologic evidence of damage. At older ages the increases in NSE ranged from 18.9 +/- 0.8 ng/ml in the 2 week old (vs. 11.5 +/- 0.5 control) to 35.8 +/- 2.1 ng/ml in the 3 week old (vs. 12.1 +/- 0.8 control). In the adult rats s-NSE increased from 5.4 +/- 0.4 in the control animals to 30.4 +/- 1.3 after SE. The different brain regions examined had distinctive ontogenic profiles for SE-induced damage. Elevation of s-NSE after SE correlated with overall histologic evidence for damage.

Animals↗
PubMed1996

Neuron-specific enolase, a marker of acute neuronal injury, is increased in complex partial status epilepticus.

PURPOSE: To determine whether complex partial status epilepticus (CPSE) causes brain injury in humans. Serum neuron-specific enolase (s-NSE) is an accepted marker of acute brain injury, and increases in s-NSE have been correlated with the duration and outcome of generalized convulsive status epilepticus. s-NSE levels in CPSE are unknown. Increase in s-NSE in CPSE would provide new information about the degree of brain injury in CPSE and would help confirm that CPSE is a medical emergency. METHODS: This was a pilot prospective study of serial levels of s-NSE and outcome in CPSE. Eight patients with confirmed CPSE and no acute neurologic deficit were identified prospectively. Results were compared with those of normal and epileptic control groups, and outcome was assessed at hospital discharge or at 7 days with the Glasgow Oucome Scale (GOS). RESULTS: The mean peak s-NSE was 21.81 ng/ml, which for the 8 patients with CPSE was four times higher than that of normal controls (mean s-NSE = 5.36 SD = 1.66, p = 0.0003) and epileptic controls (mean s-NSE = 4.61 SD = 1.74, p. = 0.001). CONCLUSION: The increase in s-NSE provides new evidence that CPSE causes brain injury in humans.

Biomarkers↗