A More Sensitive Radioimmunoassay for Neuron-Specific Enolase Suitable for Cerebrospinal Fluid Determinations

Abstract: Neuron-specific enolase (NSE) and non-neuronal enolase (NNE) have been shown to be highly specific neuronal and glial products respectively and are therefore useful as biochemical markers of the two major cell types in the vertebrate central nervous system. An iodinated radioimmunoassay (RIA) procedure for human NSE (NSE-H) with approximately 50-fold greater sensitivity than the previously available tritiated assay is described. This assay is capable of detecting 100 pg of NSE-H per assay. NSE levels in human cerebrospinal fluid (CSF) which were previously undetectable with the tritiated RIA are now easily measured and have been shown to be approximately 2 ng/ml of CSF. Furthermore, results obtained with the newly described assay procedure on more concentrated brain tissue extracts are comparable to the tritiated RIA. The iodinated NSE RIA is also shown to be capable of accurately detecting added amounts of NSE in human CSF, indicating the potential clinical usefulness of this assay in determining elevated levels of NSE in CSF.     

Neuronal Survival Factor from Bovine Brain Is Identical to Neuron-Specific Enolase

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.     

Brain Levels of Neuron-Specific and Nonneuronal Enolase in Huntington's Disease

Abstract— Levels of the cell-specific brain isoenzymes of enolase were determined in basal ganglia and cerebral cortical tissue of Huntington's disease and age- and sex-matched control brain. Neuron-specific enolase (NSE) levels are decreased an average of 45% in basal ganglia from patients with Huntington's disease whereas the glial-specific form of enolase, nonneuronal enolase (NNE), is not significantly altered. In contrast, levels of NSE in cerebral cortical tissue from Huntington's disease patients remains unchanged in comparison with controls whereas NNE levels are significantly increased. NNE and NSE levels appear to be specific biochemical indicators of glial and neuronal cell number and viability. Levels of these cell-specific isoenzymes may therefore prove useful in quantitating neuropathological changes in various neurological disorders.     

Identification of Neuron-Specific Enolase and Nonneuronal Enolase in Human and Rat Brain on Two-Dimensional Polyacrylamide Gels

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.     

脑脊液腺苷脱氨酶和干扰素-r水平在结核性脑膜炎中的诊断价值

目的：探讨脑脊液腺苷脱氨酶和干扰素-酌水平在结核性脑膜炎中的诊断价值。方法：选取2012 年6 月-2014 年6 月期间于 我院进行治疗的结核性脑膜炎患者50 例作为研究组,同期健康体检者50 例作为对照组1,50 例非结核性脑膜炎者作为对照组 2。检测脑脊液中腺苷脱氨酶和干扰素-酌水平,并进行比较。结果：研究组患者脑脊液中腺苷脱氨酶和干扰素-r水平分别为 （22.45± 4.23）U/L和（36.45± 13.56）ng/L;对照组1 患者脑脊液中腺苷脱氨酶和干扰素-r水平分别为（16.32± 3.24）U/L 和 （12.78± 2.67）ng/L,对照组2 患者脑脊液中腺苷脱氨酶和干扰素-r水平分别为（7.48± 4.01）U/L 和（13.25± 2.89）ng/L,三组比较 差异有统计学意义（P＜0.05）,其中研究组患者脑脊液中腺苷脱氨酶和干扰素-r水平显著高于对照组1 和对照组2,两两比较差 异均有统计学意义（均P＜0.05）。结论：脑脊液腺苷脱氨酶和干扰素-r在结核性脑膜炎患者中具有诊断价值,值得推广应用。     

Determination of Brain Enolase Isozymes with an Enzyme Immunoassay at the Level of Single Neurons

Abstract Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes ( αα , αγ , and γγ forms) were prepared by use of β- d -galactosidase from Escherichia coli as label and the purified rabbit antibodies to αα and γγ enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab'fragments and the antibody F(abapos;)₂-immobilized polystyrene beads could determine amounts as small as 1 amol (10⁻¹⁸ mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the γγ but also the αγ and αα enolases at a level of amol per cell body, although the concentration of γγ was the highest. Immunohistochemical experiments on the cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both α and γ subunits of the enolase were stained intensely in axons.     

Ventricular Cerebrospinal Fluid Monoamine Transmitter and Metabolite Concentrations Reflect Human Brain Neurochemistry in Autopsy Cases

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.     

Homeostasis of Brain and Cerebrospinal Fluid Calcium Concentrations During Chronic Hypo-and Hypercalcemia

Three-week-old rats were made hypocalcemic or hypercalcemic by being fed diets low or high in Ca. Both total and ionized [Ca]s in the plasma decreased about 40% and remained depressed for 4 weeks in rats fed a low-Ca diet. Plasma [Ca]s in rats fed a high-Ca diet increased by 30% and remained elevated for 7 weeks. After 8 weeks on the diets, cerebrospinal fluid (CSF) [Ca] changed by less than 30% whereas brain [Ca] changed by less than 20% of the chronic changes in plasma ionized [Ca]. Assuming a brain extracellular volume of 20% and noting that brain extracellular volume equilibrates freely with CSF, the findings demonstrate only small perturbations in the Ca content of the brain cellular compartment during sustained hypo or hypercalcemia. Partial regulation of CSF and brain extracellular Ca suggests a role for the blood-brain barrier in regulating CNS [Ca] during chronic changes in plasma [Ca].     

Determination of Brain 5-Hydroxytryptamine Turnover in Freely Moving Rats Using Repeated Sampling of Cerebrospinal Fluid

A simple technique is described for repeated sampling of cerebrospinal fluid (CSF) from the freely moving rat and its use in the determinations of 5-hydroxytryptamine (5-HT) turnover validated. A catheter, constructed from polyethylene tubing (PP10) was implanted via a cranial approach into the cisterna magna and x-ray studies confirmed that the catheter avoided the cerebellum. 5-HT turnover was determined from the rate of rise of 5-hydroxyindoleacetic acid (5-HIAA) in both CSF and brain following an injection of probenecid (200 mg/kg i.p.). Concentrations of 5-HIAA, 5-HT and tryptophan were determined by high pressure liquid chromatography. Turnover values for individual rats were obtained using CSF samples. After p-chlorophenylalanine treatment (when brain 5-HT was depleted by 43%) 5-HT turnover values obtained were comparably reduced whether determined from CSF (-67%) or brain (-74%). Thus differences of rat brain 5-HT turnover are proportionately reflected by CSF measurements. The method for sampling of CSF should be applicable in a wide range of pharmacological and physiological situations.     

Erythropoietin in Cerebrospinal Fluid: Age-related Reference Values and Relevance in Neurological Disease

We aimed to establish age-related reference values for Erythropoietin (EPO) in cerebrospinal fluid (CSF) and to evaluate concentrations in neurological diseases. CSF and serum EPO was measured in controls with tension-type headache (CTTH), in patients with ALS, dementia and depression using ELISA technique. Stability experiments showed CSF EPO to be stable for two and a half months and over two thaw/freeze cycles. A positive correlation of CSF EPO with age was found (P < 0.01). We found a CSF/serum EPO concentration ratio of 0.126, pointing towards an intrathecal synthesis of EPO. The ALS group showed significantly lowered CSF EPO compared to age-matched CTTH (P < 0.012), whereas the dementia and depression group showed no significant differences compared to CTTH. The establishment of age-related reference values in a large cohort of controls will improve the interpretation of future CSF EPO evaluations in neurological diseases. The authors have not reported any conflicts of interest.     

实验用猴脑脊液采集技术方法的创新

脑脊液在艾滋病的研究中有着重要的意义。近年来脑脊液的检测逐步成为SIV/SHIV感染猴模型研究和应用中的重要指标。传统的采集方法不易学习和掌握。针对上述情况我们优化了脑脊液的采集方法,优化后的方法明显缩短穿刺时间,显著提高成功率。     

Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D-Related Chronic Hypo- and Hypercalcemia in Rats

Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3.     

Monitoring the Effect of a Tryptophan Load on Brain Indole Metabolism in Freely Moving Rats by Simultaneous Cerebrospinal Fluid Sampling and Brain Dialysis

Rats were given L-tryptophan, 50 mg/kg i.p., and its concentration in the CNS was monitored in individual freely moving animals using repeated sampling of cisternal CSF and concurrent striatal dialysis. The 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid (5-HIAA) was also measured. Results were compared with changes of central tryptophan and 5-HIAA concentrations in brains of rats killed at various times after administration of L-tryptophan, 50 mg/kg i.p. Tryptophan changes in CSF were proportionate to those in whole brain and followed essentially identical time courses. Results for the striatal dialysate and whole striatum also paralleled each other. Similarly, results for 5-HIAA showed proportionality between CSF and brain and between dialysate and striatum. The data obtained were used to determine pharmacokinetic data for individual rats, i.e., areas under curves for both tryptophan and 5-HIAA and half-lives for the decline of tryptophan. Kinetic parameters varied considerably from rat to rat. However, mean half-lives for tryptophan in CSF, brain, dialysate, and striatum were all comparable. Results in general show the value of repeated CSF sampling and intracerebral dialysis for concurrent monitoring of changes of indole metabolism in the whole brain and a specific brain region, respectively. The methods should be suitable for the continuous monitoring of changes of central transmitter metabolism in parallel with observation of behavior following environmental or dietary changes or drug administration. They also should be of use in the investigation of drug kinetics in the CNS.     

Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of 5-Hydroxy Eicosatetraenoic Acid in Cerebrospinal Fluid After Subarachnoid Hemorrhage

Abstract: To detect and identify lipid peroxides in the CFS following subarachnoid hemorrhage (SAH), CSF samples were obtained sequentially from 10 patients who developed typical vasospasm and were analyzed by HPLC and gas chromatography-mass spectrometry. One of the peaks appearing on the 7th day after SAH was identified as 5-hydroxy eicosatetraenoic acid. On HPLC, an identical peak was detected in samples from other SAH patients. The results gave unequivocal evidence that peroxides of arachidonic acid are present in the CSF following SAH, and a correlation between them and the occurrence of vasospasm seemed likely. The hypothesis that lipid peroxides are involved in the genesis of vasospasm deserves further investigation.     

Identification and Determination of Tele-Methylhistamine in Cerebrospinal Fluid by Gas Chromatography-Mass Spectrometry

Abstract: The presence of tele-methylhistamine in human cerebrospinal fluid has been established. The concentration was determined with the use of deuterated tele-methylhistamine. The preparation of the deuterated standard is described. The concentration range in samples from neuropsychiatric patients was 0.1-2.5 ng/ml. The structure of the pentafluoropropionyl derivative used for gas chromatography was studied with the aid of proton nuclear magnetic resonance spectroscopy.     

Increased Levels of Neuron-Specific Enolase in PC 12 Pheochromocytoma Cells as a Result of Nerve Growth Factor Treatment

Abstract: Treatment of PC 12 pheochromocytoma cells with nerve growth factor (NGF) resulted in increased levels of neuron-specific enolase (NSE). Neither insulin, growth hormone, cytochrome c , nor sodium butyrate increased NSE levels. Epidermal growth factor (EGF) did increase NSE levels, although not to the same extent as NGF. As little as 1 ng/ml NGF induced the maximal increase in NSE. As PC 12 cells increased in density, the NSE levels increased even in untreated cells.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.