Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

Granular cell tumors: evidence for heterogeneous tumor cell differentiation

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.     

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.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in para-benzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues. Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues.Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.Abbreviations GFAP glial fibrillary acidic protein - NSE neuron-specific enolase - PBS phosphate-buffered saline - PAP peroxidase anti-peroxidase - FITC fluorescein-isothiocyanate     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

Peripheral distribution of nervous system-specific S-100 protein in rat

S-100 protein, a nervous system-specific protein, was determined in a soluble extract of various rat tissues with a sensitive enzyme immunoassay method, which consisted of a solid-phase with immobilized anti-S-100 antibody and the antibody labeled with beta-D-galactosidase from Escherichia coli. The minimum detectable amount of S-100 protein was 3 pg/assay. Central nervous tissues (cerebrum, cerebellum, and brain stem) contained 1.4 to 2.8 micrograms S-100 protein/mg protein, whereas most of the peripheral tissues contained less than 0.05 microgram/ml of the specific protein. However, the level of S-100 protein was high in adipose tissue (0.5--1.1 micrograms/mg) and in trachea (about 0.5 microgram/mg), which involves cartilage. The S-100 protein levels in several tissues were significantly higher in female rats than in males at ages of 5 to 6 weeks.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Immunohistochemical study of doublecortin and nucleostemin in canine brain

Finding a marker of neural stem cells remains a medical research priority. It was reported that the proteins doublecortin and nucleostemin were related with stem/progenitor cells in central nervous system. The aim of the present immunohistochemical study was to evaluate the expression of these proteins and their pattern of distribution in canine brain, including age-related changes, and in non-nervous tissues. We found that doublecortin had a more specific expression pattern, related with neurogenesis and neuronal migration, while nucleostemin was expressed in most cells of almost every tissue studied. The immunolabeling of both proteins decreased with age. We may conclude that nucleostemin is not a specific marker of stem/progenitor cells in the dog. Doublecortin, however, is not an exclusive marker of neural stem cells, but also of neuronal precursors.Key words: nucleostemin, doublecortin, stem cells, dog brain, aging.     

Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers

Traumatic brain injury (TBI) is an acute event resulting from external force to the brain and is a major cause of death and disability associated with high health care costs in the western world. Additional injuries, originating from the secondary molecular events after the initial intensive care, may be limited by the use of objective biomarkers to provide the best treatment and patient prediction outcome. In this study, hexapeptide ligand libraries (HLL) have been used for the enrichment of suggested protein biomarkers for TBI in cerebrospinal fluid (CSF). HLL have the potential to enrich low abundant proteins and simultaneously reduce the high abundant proteins, rendering a sample with significantly reduced dynamic range. The CSF proteome from two TBI inflicted patients have been extensively mapped using a large initial sample volume obtained by extraventricular drainage. Shotgun proteomics, in combination with isoelectric focusing (IEF) and nano-LC–MS/MS, identified 339 unique proteins (MudPIT scoring p ≤ 0.05) with a protein overlap of 130 between the patients. As much as 45% of the proteins reported in the literature to be associated with degenerative/regenerative processes occurring after a trauma to the head were identified. Out of the most prominent potential protein biomarkers, such as neuron specific enolase, glial fibrillary acidic protein, myelin basic protein, creatine kinase B-type and S-100β, all except myelin basic protein were detected in the study. This study shows the possibility of using HLL as a tool for screening of low abundant protein biomarkers in human CSF.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

MEASUREMENT OF NEURON-SPECIFIC (NSE) AND NON-NEURONAL (NNE) ISOENZYMES OF ENOLASE IN RAT, MONKEY AND HUMAN NERVOUS TISSUE

Four double antibody solid-phase radioimmunoassay systems are described for the measurement of neuron-specific enolase (NSE) and non-neuronal enolase (NNE) from rat, monkey and human brain tissue. NSE and NNE are antigenically distinct, making their respective assays specific. The levels of neuronal and non-neuronal enolase (an enolase recently shown to be localized in glial cells) are determined in various regions of rat, monkey and human nervous system. Both neuronal and glial enolases are major proteins of brain tissue with each representing about 1.5% of total brain soluble protein. NSE levels are highest and NNE levels lowest in brain areas having a high proportion of grey matter, such as the cerebral cortex. The reverse is true for areas high in white matter, such as the pyramidal tract and the corpus callosum. Peripheral nervous system levels of NSE are much lower than those of brain with the spinal cord intermediate between the two. Radioimmunological and immunocytochemical data show that neuron-specific enolase is also present in neuroendocrine cells located in non-nervous tissue, which include pinealocytes, parafollicular cells of the thyroid, adrenal medullary chromaffin cells, glandular cells of the pituitary and Islet of Langerhans cells in the pancreas. Unlike neurons, these cells also contain non-neuronal enolase in high amounts.     

Biochemical changes in the injured brain

Brain metabolism is an energy intensive phenomenon involving a wide spectrum of chemical intermediaries. Various injury states have a detrimental effect on the biochemical processes involved in the homeostatic and electrophysiological properties of the brain. The biochemical markers of brain injury are a recent addition in the armamentarium of neuro-clinicians and are being increasingly used in the routine management of neuropathological entities such as traumatic brain injury, stroke, subarachnoid haemorrhage and intracranial space occupying lesions. These markers are increasingly being used in assessing severity as well as in predicting the prognostic course of neuro-pathological lesions. S-100 protein, neuron specific enolase, creatinine phosphokinase isoenzyme BB and myelin basic protein are some of the biochemical markers which have been proven to have prognostic and clinical value in the brain injury. While S-100, glial fibrillary acidic protein and ubiquitin C terminal hydrolase are early biomarkers of neuronal injury and have the potential to aid in clinical decisionmaking in the initial management of patients presenting with an acute neuronal crisis, the other biomarkers are of value in predicting long-term complications and prognosis in such patients. In recent times cerebral microdialysis has established itself as a novel way of monitoring brain tissue biochemical metabolites such as glucose, lactate, pyruvate, glutamate and glycerol while small non-coding RNAs have presented themselves as potential markers of brain injury for future.     

Elevated S100B and Neuron Specific Enolase Levels in Patients with Migraine-without Aura: Evidence for Neurodegeneration?

Although migraine has mainly been considered as a benign disease, there is cumulative evidence of silent changes in the brain, brainstem, or cerebellum and subtle subclinical cerebellar dysfunction. In this study, in order to investigate a possible neuronal and/or glial damage at the cellular level in migraine, we measured and compared serum levels of S100B which is a protein marker of glial damage or activation, and neuron specific enolase (NSE) which is a marker of neuronal damage, in migraine patients and control subjects. Serum levels of S100B and NSE were measured in blood samples from 41 patients with migraine-without aura taken during a migraine attack (ictal) and in the attack-free period between migraine attacks (interictal) and 35 age- and sex-matched controls. Patients with migraine-without aura had significantly higher ictal serum levels of S100B and NSE (P < 0.05, for both) than control subjects; whereas in the interictal phase, there was a significant increment only in S100B levels (P < 0.05) compared to controls. On the other hand, serum levels of S100B and NSE in ictal and interictal blood samples did not differ significantly. The findings of increased ictal serum S100B and NSE levels together with increased interictal levels of S100B suggested that migraine might be associated with glial and/or neuronal damage in the brain and a prolonged disruption of blood–brain barrier. Increased interictal serum levels of S100B might point out to an insidious and slow damaging process in migraine patients.     

Neuronal and Glial Marker Proteins in the Evaluation of the Protective Action of MK 801

A quantitative dot immunobinding procedure was used to quantify glial [the S-100 protein and the glial fibrillary acidic (GFA) protein] and neuronal (the 68- and 200-kDa neurofilament polypeptides, neuron-specific enolase, and neuronal cell adhesion molecule) markers. A single intraperitoneal administration of 10 mg/kg of MK 801 blocked the increase of glial parameters and the decrease in content of neuronal marker proteins that occurred as the response to an N-methyl-D-aspartate (NMDA) lesion in the rat hippocampus. The degradation products of GFA protein and the 68-kDa neurofilament polypeptide that were induced by the NMDA lesion did not appear after MK 801 treatment. This study shows that brain-specific proteins are a set of precise tools for the evaluation of neuroprotective effects of antagonists to excitatory amino acids.     

Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse

The levels of the S-100 and 14-3-2 proteins were determined in a number of regions of mouse brain at intervals from 1 day to 30 months of age. Both S-100 and 14-3-2 were found in measurable amounts as early as the first day of postnatal age but did not begin to accumulate rapidly in the forebrain, brain stem and cerebellum of the mouse brain until some time between the 7th and 14th days. From days 14 to 28 the levels of S-100 and 14-3-2 in each region continued to increase rapidly with the exception of the forebrain where the rate of accumulation of S-100 appeared to lag considerably behind that in the other regions. The proteins continued to accumulate at a rapid rate until approximately 6 months of age. From 6 to 30 months of age, the levels of 14-3-2 remained relatively stable in cerebellum, hippocampus and hypothalamus and appeared to decrease slightly in striatum and cerebral cortex. In the case of S-100, the level of the protein increased in all regions of brain from 6 to 30 months but the increase was most pronounced in the hippocampus, hypothalamus and striatum. The principal conclusion derived from this study is that the biochemical development and aging of the central nervous system are regionally selective processes.     

FUNCTIONAL PROPERTIES OF NEURONAL AND GLIAL ISOENZYMES OF BRAIN ENOLASE

Two of the major brain enolase (EC 4.2.1.11) isoenzymes exist as cell specific forms. The neuron specific enolase (NSE) is localized in neurons and the non-neuronal enolase (NNE) in glial cells. A third enolase containing one subunit from each of the above species is also present in brain and has been designated hybrid enolase. The stabilities of the brain enolases towards incubation with chloride and bromide salts is markedly different. NNE is rapidly inactivated upon incubation in 0.5 M-KCI or KBr while NSE is minimally effected and the hybrid has an intermediate stability. The inactivation is temperature dependent and reversible by salt removal. Magnesium exerts a stabilizing effect on each enzyme form. The mechanism of the reversible salt inactivation involves dissociation of the enolase subunits with reassociation occurring during reactivation. The brain enolases also display marked stability differences during incubation in 3 M-urea. with the neuronal form again being more stable. The urea inactivation was highly reversible for NNE but only marginally so for NSE. The neuronal enolase is also by far the most stable of the brain enolases at 50°C.