An enzyme immunoassay to quantify neurofilament light chain in cerebrospinal fluid.

Neurofilament light chain is a component of the axonal cytoskeleton. The concentration of the neurofilament light chain in cerebrospinal fluid may reflect axonal damage or the extent of white matter damage. In this study we describe a sensitive immunoassay for the detection of neurofilament light chain in cerebrospinal fluid using commercially available materials. The detection limit of the assay was 5 ng/l and the assay was linear up to 390 ng/l. Mean recovery was 91.5% and inter-assay and intra-assay coefficients of variation were below 18%. Strongly increased levels of neurofilament light chain were observed in patients with cerebrovascular accidents, subarachnoid hemorrhage and severe traumatic brain injury, suggesting the occurrence of axonal damage in these conditions.

Cerebrospinal fluid neurofilament and glial fibrillary acidic protein in patients with cerebral vasculitis.

Few diseases in clinical medicine cause as much diagnostic consternation as central nervous system (CNS) vasculitis because of its varying modes of presentation and frequently overlapping clinical and pathological features. There are no pathognomonic clinical or laboratory findings. The purpose of the present retrospective study was to validate the use of the light subunit of neurofilament triplet protein (NFL) and glial fibrillary acidic protein (GFAP) as markers of CNS tissue damage for patients with systemic or isolated CNS vasculitis. Levels of cerebrospinal fluid (CSF) NFL and GFAP were measured using ELISAs. Both CSF NFL and CSF GFAP concentrations were significantly higher in a patient group diagnosed with CNS vasculitis (P < 0.01 and P < 0.05, respectively) than in a patient group for whom CNS vasculitis was excluded. In the future, analysis of CSF NFL in particular, but also GFAP, may be a useful complement in the difficult clinical task of diagnosing CNS vasculitis.

Selective introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis.

We have developed a novel method in which antisense DNA is selectively electroporated into individual adult neural progenitor cells. By electroporation of antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) we demonstrate that ciliary neurotrophic factor (CNTF) is an instructive signal for astroglial type 2 cell fate specifically mediated via activation of STAT3. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway induced only a transient increase in glial fibrillary acidic protein (GFAP) expression, and inhibition of this signaling pathway did not block the induction by CNTF of glial differentiation in progenitor cells. In addition we show that microelectroporation is a new powerful method for introducing antisense agents into single cells in complex cellular networks.

CSF sulfatide distinguishes between normal pressure hydrocephalus and subcortical arteriosclerotic encephalopathy.

OBJECTIVES: To examine the CSF concentrations of molecules reflecting demyelination, neuronal and axonal degeneration, gliosis, monoaminergic neuronal function, and aminergic and peptidergic neurotransmission in a large series of patients with normal pressure hydrocephalus (NPH) or subcortical arteriosclerotic encephalopathy (SAE), to elucidate pathogenic, diagnostic, and prognostic features. METHODS: CSF concentrations of glycosphingolipid (sulfatide), proteins (neurofilament triplet protein (NFL), glial fibrillary acidic protein (GFAP)), neuropeptides (vasoactive intestinal peptide (VIP), 4-aminobutyric acid (GABA)), and monoamines (homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (HMPG)) were analysed in 43 patients with NPH and 19 patients with SAE. The diagnoses of NPH and SAE were based on strict criteria and patients with NPH were subsequently operated on. Twelve clinical variables, psychometric tests measuring perceptual speed, accuracy, learning, and memory and a psychiatric evaluation were performed in all patients and before and after a shunt operation in patients with NPH. RESULTS: The CSF sulfatide concentration was markedly increased in patients with SAE (mean 766, range 300-3800 nmol/l) compared with patients with NPH (mean 206, range 50-400 nmol/l) (p<0.001). 5-HIAA, GABA, and VIP in CSF were higher in patients with SAE than in patients with NPH. The patients with NPH with cerebrovascular aetiology had higher sulfatide concentrations and a poorer outcome after shunt surgery than patients with NPH with other aetiologies. CONCLUSIONS: The pathogenesis of the white matter changes in NPH and SAE is different and ischaemic white matter changes can be a part of the NPH state. The markedly increased CSF sulfatide concentrations in patients with SAE indicate ongoing demyelination as an important pathophysiological feature of SAE. The CSF sulfatide concentration distinguished between patients with SAE and those with NPH with a sensitivity of 74% and a specificity of 94%, making it an important diagnostic marker.

Bcl-2 expression regulates cell sensitivity to S100beta-mediated apoptosis.

The S100beta protein is overexpressed in the brain of patients with Alzheimer's disease and Down's syndrome and is able to induce apoptosis in neurons at high concentrations. The intracellular events that regulate the apoptotic effect are largely unknown. This study investigates the roles of the bcl-2 proto-oncogene, one of the best-defined apoptotic genes, on cell death induced by S100beta. Human neuronal precursor NT2/D1 cells showed a high degree of cell death by apoptosis after exposure to 2 microM S100beta in serum-free medium. Death was preceded by a down-regulation of the Bcl-2 protein. Gene transfer with a full-length bcl-2 cDNA under the control of a constitutive promoter in NT2 cells elevated Bcl-2 protein levels and repressed S100beta-mediated cell death. When exposed to retinoic acid, the NT2/D1 cells differentiated into a neuronal phenotype. The differentiated cells up-regulated their levels of Bcl-2 and became resistant to S100beta-induced cell death. Downregulation of Bcl-2 by an antisense oligonucleotide in the differentiated cells, however, increased their susceptibility to S100beta-related cytotoxicity. Therefore, apoptosis induced through S100beta signaling is subject to regulation by Bcl-2. A combined alteration such as up-regulation of S100beta together with down-regulation of Bcl-2 may be important in the pathogenesis of Alzheimer's disease and Down's syndrome.

Neurofilament protein levels in CSF are increased in dementia.

The neurofilament is the major cytoskeletal structure of myelinated axons. In this study, CSF levels of the light subunit of the neurofilament protein (NFL) were increased in patients with vascular dementia (VAD), AD, and frontotemporal dementia (FTD) compared with neurologically healthy individuals. Because NFL is localized mainly in myelinated axons, these results suggest that the degeneration of white matter in these disorders causes the increased CSF NFL levels.

Astroglial and neuronal proteins in cerebrospinal fluid as markers of CNS involvement in Lyme neuroborreliosis.

Is Lyme neuroborreliosis, even in its early phase, a parenchymatous disorder in the central nervous system (CNS), and not merely a meningitic process? We quantified cerebrospinal fluid (CSF) levels of four nerve and glial cell marker proteins in Lyme neuroborreliosis patients with pretreatment durations of 7-240 days. All 23 patients had meningoradiculitis, and six had objective signs of encephalopathy. Glial fibrillary acidic protein (GFAp) pretreatment levels in CSF, and the light subunit of neurofilament protein (NFL) levels were related to clinical outcome and declined significantly after treatment (P < 0.001 and P < 0.01, respectively). NFL was detectable in 11 out of 22 patients, and pre- and post-treatment NFL levels were associated with the duration of neurological symptoms within 100 days prior to treatment. Neuron-specific enolase (NSE) concentrations also decreased after therapy (P < 0.001), while CSF levels of glial S-100 protein remained unchanged. The pretreatment duration of disease was related to postinfectious sequelae. GFAp, NSE and NFL levels in CSF are unspecific indicators of astroglial and neuronal involvement in CNS disease. The findings in the present study are in agreement with the hypothesis that early and late stages of Lyme neuroborreliosis damage the CNS parenchyma.

Detection of light subunit neurofilament and glial fibrillary acidic protein in cerebrospinal fluid of Trypanosoma brucei gambiense-infected patients.

Light subunit neurofilament (NFL) and glial fibrillary acidic protein (GFAP) concentrations were determined in cerebrospinal fluid (CSF) of 34 patients with human African trypanosomiasis (HAT), five serologically positive but parasitologically unconfirmed individuals, and four healthy controls without evidence of HAT. In patients with second stage HAT (n = 30), NFL levels were abnormally elevated in 10 cases and GFAP levels in five. The astrogliosis observed in HAT and experimental models of HAT is confirmed in our study by the presence of increased GFAP levels in the CSE The abnormal NFL CSF levels reflect structural damage of nerve cells in 33 % of the second-stage patients studied. To our knowledge, this is the first time neuronal damage in HAT patients is demonstrated by using biochemical markers of brain damage in the CSF.

An acquired sensitivity to H2O2-induced apoptosis during neuronal differentiation of NT2/D1 cells.

Brief exposure of neuronally differentiated human NT2/D1 cells to hydrogen peroxide induced cell death by apoptosis with an ED50 of 30 microM, whereas a 70-fold higher concentration was required to obtain an ED50 effect in undifferentiated NT2/D1 neuronal precursor cells. This enhanced sensitivity in NT2/D1 neurons was correlated with an 8-fold lower level of intracellular glutathione. Pretreatment with N-acetyl-L-cysteine, an agent that is able to raise the levels of intracellular glutathione, promoted the survival of hydrogen peroxide-treated NT2/D1 neurons. Thus, the low glutathione level may contribute to the high sensitivity of NT2/D1 neurones to hydrogen peroxide-induced apoptosis. This study indicates that neuronal susceptibility to oxidative damage is developmentally regulated.

Neurofilament protein in cerebrospinal fluid: a potential marker of activity in multiple sclerosis.

The neurofilament protein is a major structural protein of neurons and a marker for axonal damage. The concentrations of the light subunit of the neurofilament triplet protein (NFL) in CSF were significantly increased in patients with relapsing-remitting multiple sclerosis compared with healthy controls (p<0.001). Seventy eight per cent of patients with multiple sclerosis showed increased NFL concentrations. Significant correlations between the NFL concentration in CSF and clinical indices were discerned for disability, exacerbation rate, and time from the start of the previous exacerbation to the time of the lumbar puncture. The results suggest that axonal damage occurs during relapsing-remitting multiple sclerosis and that the damage contributes to disability and the appearance of clinical exacerbations. The concentration of NFL in CSF is a potential marker of disease activity in multiple sclerosis and might be useful in future clinical trials of multiple sclerosis.

Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases have increased levels of neurofilament protein in CSF.

In the present study we describe an ELISA to quantify the light subunit of the neurofilament triplet protein (NFL) in CSF. The method was validated by measuring CSF NFL concentrations in healthy individuals and in two well-characterized groups of patients with amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). The levels were increased in ALS (1,743 +/- 1,661 ng/L; mean +/- SD) and AD (346 +/- 176 ng/L) compared with controls (138 +/- 31 ng/L; p < 0.0001 for both). Within the ALS group, patients with lower motor neuron signs only had lower NFL levels (360 +/- 237 ng/L) than those with signs of upper motor neuron disease (2,435 +/- 1,633 ng/L) (p < 0.05). In a second study patients with miscellaneous neurodegenerative diseases were investigated (vascular dementia, olivopontocerebellar atrophy, normal pressure hydrocephalus, cerebral infarctions, and multiple sclerosis), and the CSF NFL level was found to be increased (665 +/- 385 ng/L; p < 0.0001). NFL is a main structural protein of axons, and we suggest that CSF NFL can be used to monitor neurodegeneration in general, but particularly in ALS with involvement of the pyramidal tract.

Glial fibrillary acidic protein in the cerebrospinal fluid of patients with dementia.

Glial fibrillary acidic protein (GFAP) is the structural protein of the astroglial intermediate filament that forms the morphological basis of astrogliosis. In the present study, GFAP concentrations in cerebrospinal fluid were measured in patients with various dementia diseases. A significant correlation between GFAP and age was found both in the total dementia group and in the controls. Covariance analysis with GFAP as dependent variable and age and albumin ratio as covariates followed by multiple group comparisons showed that, with regard to GFAP levels, the controls (n = 39) differed significantly from the patients with vascular dementia (n = 20; p < 0.05), senile dementia of the Alzheimer type (n = 29; p < 0.05), and 'pure' Alzheimer's disease (n = 8; p < 0.05), but not from those with frontal lobe dementia (n = 5).

Increased cerebrospinal fluid levels of glial fibrillary acidic protein (GFAp) in Lyme neuroborreliosis.

Glial fibrillary acidic protein (GFAp), the main protein constituent of the intermediate filaments of astrocytes, was analysed in the cerebrospinal fluid (CSF) of 20 patients with Lyme neuroborreliosis as a marker of the astroglial reaction. The mean GFAp level before antibiotic treatment in the study group was significantly elevated (592 pg/ml +/- 596 [SD]) compared to that in 24 healthy controls (121 +/- 87 [SD]) (p < 0.01). The highest CSF-GFAp levels were seen in the patients with the most severe disease, but the levels were also increased in patients with peripheral paresis, such as facial palsy with no or only minor encephalitic symptoms. This implies that the infection was not limited to radix dorsalis or the meningeal tissues, but affected the central nervous system as well. Furthermore, the astroglial reaction seemed to occur early in Lyme neuroborreliosis since CSF-GFAp levels were elevated also in patients with recent (< 3 weeks) onset of disease. After antibiotic treatment, the GFAp levels decreased. It is suggested the CSF-GFAp concentrations might be useful for monitoring CNS involvement in Lyme neuroborreliosis.

Glial fibrillary acidic protein in CSF of multiple sclerosis patients: relation to neurological deficit.

Glial fibrillary acidic protein (GFAp) was analysed in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and healthy controls. Patients with relapsing-remitting course (n = 13) were followed with quantitative neurological examinations and lumbar punctures during a 24-month period. The patient group was a subsample from a randomised, double-blind clinical trial of acyclovir on MS: 7 patients were treated with acyclovir and 6 were placebo controls. CSF was also collected from 5 age-matched healthy individuals with normal quantitative neurological examinations. The CSF assays disclosed increased concentrations of GFAp in MS patients compared to controls (p < 0.01). Furthermore, the GFAp levels correlated significantly with the deficit score (p < 0.01) but not with exacerbation frequency. When the group treated with acyclovir was compared with the placebo group, no significant change of CSF GFAp was observed. In the present study we show that GFAp is increased in CSF of patients with MS and that the levels correlate with the neurological dysfunction. Further work is needed to ascertain whether determinations of CSF GFAp can be used to monitor disease progression in MS or whether the assay may be useful to evaluate therapeutic intervention.

A sensitive ELISA for glial fibrillary acidic protein: application in CSF of adults.

The present study concerns an ELISA for glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF). The application of the method in CSF of children has previously been described in this journal. We have now adapted the technique to allow determination of the much higher GFAP concentrations found in normal and pathological CSF of adults. The assay range was extended to 16,000 pg/ml. CSF levels as high as 170,000 pg/ml could be assayed since dilution experiments indicated immunological identity between purified GFAP and GFAP in CSF. In normal controls the concentrations correlated significantly with age (P < 0.001, Spearman rank correlation test). The concentrations were less than 200 pg/ml before 20 years of age and increased to between 500 and 1300 pg/ml at approximately 75 years of age. This increase of CSF GFAP probably reflects formation of astroglial filaments in the CNS and it must be taken into consideration when determining the pathological CSF GFAP level. The method was validated using CSF samples from patients with either astrogliosis or acute tissue destruction in the CNS. Although augmented levels were observed in both groups it is quite clear that patients with acute tissue destruction may display very high CSF GFAP concentrations, whereas levels in patients with astrogliosis were only modestly increased if at all.

Glial and neuronal marker proteins in the silicone chamber model for nerve regeneration.

In the present study, neuronal and Schwann cell marker proteins were used to biochemically characterize the spatiotemporal progress of degeneration/regeneration in the silicone chamber model for nerve regeneration. Rat sciatic nerves were transected and the proximal and distal stumps were inserted into a bridging silicone chamber with a 10-mm interstump gap. Using dot immunobinding assays, S-100 protein and neuronal intermediate filament polypeptides were measured in different parts of the nerve 0-30 days after transection. In the most proximal nerve segment, all the measured proteins were transiently increased. In the proximal and distal stumps adjacent to the transection, the studied proteins were decreased indicating degeneration of the nerve. Within the silicone chamber, the regenerating nerve expressed the Schwann cell S-100 protein already at 7 days, whereas the neurofilament polypeptides appeared later. These observations are corroborated by previous morphological studies. The biochemical method described provides a new and fast approach to the study of nerve regeneration.

Quantitative alterations of S-100 protein and neuron specific enolase in the rat nervous system after chronic 2,5-hexanedione exposure.

The regional changes in quantities of the glial S-100 protein and the neuron specific enolase in the rat nervous system have been studied after long-term exposure to 2,5-hexanedione. The wet weights of most of the examined nervous tissues were found to be reduced, with an extensive effect seen in the brain stem. Using dot immunobinding assays, the concentrations of S-100 were found to be increased in most of the examined tissues, but unaffected in the brain stem. The total amount of S-100 per tissue was markedly reduced in the brain stem. The content of neuron specific enolase was reduced only in the brain stem. Thus the effects of 2,5-hexanedione on the nervous system varied regionally. The brain stem was severely atrophied with a reduction of neuronal as well as of glial marker proteins. Other brain regions contained increased glial cell marker proteins as signs of progressive astroglial reactions.

Proteolysis of filament proteins in glial and neuronal cells after in vivo stimulation of hippocampal NMDA receptors.

An intrahippocampal injection of N-methyl-D-aspartate induced the appearance of degradation products of both the 68 kiloDalton neurofilament protein and the glial fibrillary acidic protein, as revealed by immunoblot techniques. The degradation of these two filament proteins was maximal at 10 days after the lesion. The degradation patterns were similar to those induced with calpains or calcium in vitro. There were no degradation effects on the 200 kD neurofilament protein as tested with both mono- and polyclonal antibodies. Consequently, the neuronal degeneration after excessive activation of NMDA receptors appears to involve calcium activation of proteolytic enzymes. The effects on the glial proteins are probably secondary to neuronal damage but could be related to calcium dependent processes.

A sensitive ELISA for glial fibrillary acidic protein: application in CSF of children.

In the present study we describe a sensitive ELISA for determination of glial fibrillary acidic protein (GFAP). To validate the method combined determinations of GFAP and S-100 protein were performed in cerebrospinal fluid (CSF) of normal children and children with autism. The GFAP ELISA is of sandwich type and uses the biotin-avidin system. Sensitivity was 16 pg/ml. Between-day precision was 0.079 (coeff. of variance). S-100 protein concentrations were measured using a commercially available ELISA kit. Normal CSF from children and young adults were analysed. The CSF levels of GFAP in normal children were low (16-163 pg/ml). Both GFAP and S-100 protein concentrations correlated with age (P < 0.01 and P < 0.05, respectively), but the GFAP increment was more pronounced, probably reflecting the age-dependent expansion of the fibrillary astrocytes in the central nervous system (CNS). GFAP levels in children with infantile autism were higher than those in normal children of the same age range. S-100 protein concentrations were similar in both groups. High levels of GFAP in combination with normal S-100 protein concentrations in CSF indicates reactive astrogliosis in the CNS. In conclusion, the sensitive ELISA described makes it possible to measure low levels of GFAP present in the CSF of children. Combined assays of GFAP and S-100 protein can be used to discriminate between acute and chronic brain disorders in children.

Quantitative and qualitative alterations of neuronal and glial intermediate filaments in rat nervous system after exposure to 2,5-hexanedione.

The precise mechanism for the neurotoxicity of 2,5-hexanedione is not known, but cross-linking of neurofilament proteins has been suggested as one possibility. In this study the effects of long-term exposure to 2,5-hexanedione were studied in the rat nervous system with special reference to regional changes in the quantities of neuronal and glial intermediate filaments. Using enzyme-linked immunosorbent assays the concentrations of 68- and 200-kDa neurofilament polypeptides were shown to be reduced in all brain regions studied. Similar results were obtained in the sciatic nerve. The concentration of glial fibrillary acidic protein was decreased in the cerebellar vermis and the dorsal cerebral cortex, whereas it was increased in the spinal cord, a result suggesting a regional variation in glial sensitivity. The intermediate filaments of the exposed animals were also immunoblotted using polyclonal antisera against the various neurofilament polypeptides and glial fibrillary acidic protein. In all tissues studied, several aggregates with molecular weights higher than those of the monomeric polypeptides were demonstrated. Contrary to clinical observations, these data indicate pronounced effects in both CNS and PNS and call for further studies on CNS effects in humans.