Chronic Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta) with Cisterna Magna Ports: Update on Refinements

More than 20 y ago, we developed an animal model for chronic and continuous collection of cerebrospinal fluid (CSF) from conscious rhesus macaques. Since our previous publication in 2003, we have successfully implanted 168 rhesus macaques using this approach. Our experience enables us to provide up-to-date information regarding the model, including refinements to our implant design, reductions in maintenance, and new procedures for dealing with contamination. The results of our experiences have reduced the number of surgeries required and helped to increase the longevity of the implant, with some functioning for more than 18 y. Building on our success in rhesus macaques, we attempted to develop similar animal models in the African green monkeys and dogs but have been unable to develop reliable chronic models for CSF collection in these species.Abbreviation: CMP, cisterna magna port; CSF, cerebrospinal fluid

Cerebrospinal (CSF) biomarkers and pharmacokinetics are reliable tools for monitoring the therapeutic effect of compounds used for the treatment of various neurodegenerative diseases. CSF can be collected by using several methods, including lumbar and cisterna magna punctures or implanted devices.^{3,6,8,9,11-13} Each method has its own specific challenges but no matter which technique is used, performing CSF collections safely is imperative to avoiding risks to the animals and to providing the best CSF samples for analysis.¹ To support our research focus on neurocognitive disorders (including Alzheimer disease, Parkinson disease, and sleep disorders), we developed an NHP model of chronic CSF collection (the cisterna magna port [CMP] model) more than 20 y ago.⁴ This model allows safe, repeatable and reliable collections of CSF samples from the cisterna magna in conscious rhesus macaques (Macaca mulatta). The information summarized herein updates this animal model since its introduction in 2003 and reflects our 18 additional years of experience with it. We also provide information regarding our attempts to develop CMP models in African green monkeys and dogs. We recommend that readers review the 2003 article for further information and understanding of the CMP model.⁴     

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。     

Cerebrospinal Fluid Parameters in Healthy Volunteers During Serial Lumbar Punctures

Lumbar punctures were performed on four occasions over a 5-day period (8:30 a.m. on days 1, 3, and 5; 2:30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day-to-day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total gamma-aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5-HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.     

A Novel Newborn Rat Kernicterus Model Created by Injecting a Bilirubin Solution into the Cisterna Magna

Background

Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats.

Methods

On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH₂O (pH = 8.5) was injected into the CM at 10 µg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test.

Results

The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (P<0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (P = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test.

Conclusion

By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.     

A Technique for Collection of Exudate from Pea Seedlings

Ethylenediaminetetraacetic acid (EDTA), at concentrations higher than 1.0 millimolar, is phytotoxic to etiolated seedlings of Pisum sativum. Substantial vascular exudation from pea epicotyls could be obtained without tissue damage at 0.5 millimolar EDTA if the solution was buffered at pH 7.5 with sodium N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid. Treated seedlings exuded 950 micrograms (leucine equivalents) of ninhydrin-positive material per day and 870 micrograms (glucose equivalents) of anthrone-positive material per day. Amino acid analysis showed the exudate to have glutamine as the major amido nitrogen containing compound and sucrose was shown to be the major sugar. Radiolabeled tryptophan and sucrose applied to cotyledons were transferred through the epicotyl and into the collection medium. The pH profile for exudation shows half maximal exudation at pH 7.2, indicating the promotion of exudation by EDTA is probably not due simply to Ca²⁺ chelation.     

Amine Metabolites in the Lumbar Cerebrospinal Fluid of Humans with Restricted Flow of Cerebrospinal Fluid

DETERMINATION of homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5HIAA) in human lumbar cerebrospinal fluid (CSF) is becoming an important tool in the study of the metabolism in the brain of their respective precursors, dopamine and 5-hydroxytryptamine and in the interpretation of the effects of drugs on these substances. The assumption that the concentration of the acidic metabolites HVA and 5HIAA in the lumbar CSF gives a measure of the amount of turnover of the parent amines in the brain is supported by several findings. (1) Amine metabolite concentrations in the lateral ventricular CSF of the dog correlate with their concentrations in adjacent brain areas¹. (2) Peripherally administered HVA only penetrates slightly or not at all to lateral ventricular CSF in the cat² or dog³, similar results being obtained for 5HIAA in the dog⁴. (3) Drugs which alter brain amine turnover in laboratory animals also alter the concentrations of the acidic metabolites in dog³, rabbit⁵ and human⁶ CSF in the appropriate direction. (4) In Parkinsonism and in senile and presenile dementia, conditions in which there is evidence of defective turnover of amines in the brain, low concentrations of HVA and 5HIAA are found in the CSF⁷.     

DSA技术在恒河猴脑脊液动态采集中应用

探索数字减影血管造影(digital subtraction angiography,DSA)插管技术在恒河猴(Macaca mulatta)脑脊液动态采集中的应用。将28只恒河猴作为实验动物,随机分为实验组和对照组,每组14只,经左前肢静脉推注3%戊巴比妥钠溶液全麻后,常规剔除背部脊柱周围体毛、消毒,实验组从穿刺开始就在DSA技术下行腰椎间隙穿刺,对照组直接行腰椎间隙穿刺,将硬膜外麻醉导管经腰椎蛛网膜下腔送至枕大池并埋置泵。对不同穿刺方法的结果数据进行χ2检验;神经功能参照改良Tarlov评分标准,切口采用改良的切口愈合等级评分标准。实验组经腰椎间隙穿刺蛛网膜下腔全部成功;对照组8只穿刺成功,6只穿刺失败;所有动物埋置泵均能从泵中采集到无色透明的脑脊液;术后恒河猴均于当天恢复正常。在DSA技术下经腰椎间隙穿刺蛛网膜下腔并埋置泵,成功率高,操作简便、安全性好,可行性强;通过埋置的泵采集脑脊液,快速省时,可实现在动物清醒时、全生理状态下对脑脊液进行动态多次采集,有助于提高非人灵长类实验动物的福利,由于采样导管置于枕大池,采集到的脑脊液样品质量有保证,可用于研究缺血性脑卒中发生机制,满足检验需要,具有一定的实用价值。     

Prothrombin in Normal Human Cerebrospinal Fluid Originates from the Blood

In spite of the fact that prothrombin is produced by cells within the central nervous system, its presence in the cerebrospinal fluid (CSF) has not been investigated. We determined the concentration of prothrombin in CSF with reference to the concentration in plasma in paired samples from 18 normal control patients and 4 patients with relapsing-remitting type of multiple sclerosis (MS). The newly developed ELISA was very specific (no cross-reactivity with thrombin) and sensitive (detection limit—0.7 ng/ml) with an imprecision of CV = 8.3% (intraseries) and 7.0% (interassay). The mean prothrombin concentration in normal CSF was 0.55 mg/l (CV ± 33%, range: 0.28–0.93 mg/l), in normal plasma 121.8 mg/l ± 21%, resulting in a mean CSF/plasma concentration quotient (Q_Proth)—4.5 · 10^–3 (CV ± 35%, range: 2.1–8.3 · 10^–3) corresponding to a mean albumin quotient in this group of subjects of Q_Alb = 5.8 · 10^–3. Due to the Q_Proth and the molecular weight of prothrombin (72 kDa)—similar to that of albumin—we conclude that prothrombin in normal human CSF originates predominantly (>95%) from blood. The enzymatic activity in CSF is conserved. Comparable results obtained in MS patients with only few small MRI lesions suggest that local chronic inflammatory disease of the central nervous system does not influence prothrombin concentration in the CSF if the blood-CSF barrier function is normal.     

Immunoglobulin Levels in the Cerebrospinal Fluid

The immunoglobulins G, A, M, and D have been measured in the cerebrospinal fluid of 207 patients with neurological disease. Raised levels of IgG, expressed as a percentage of total cerebrospinal fluid (C.S.F.) protein, were found in 62% of 45 cases of multiple sclerosis compared with 14% of 160 cases with various other neurological disorders. Thus measurement of the IgG level is probably a useful confirmatory investigation in multiple sclerosis. IgA and IgM were found only in the C.S.F. of patients with a raised protein level, and IgD was not detected.     

A Sensitive Method for Characterization of Oligoclonal Immunoglobulins in Unconcentrated Cerebrospinal Fluid

Abstract: Although isoelectric focusing has been used to demonstrate the presence of oligoclonal IgG in the CSF, the technique has not allowed detection of oligoclonal IgG in unconcentrated CSF. A new technique is reported, by which unconcentrated CSF is separated by isoelectric focusing, and the IgG bands are then detected by radioimmunofixation. Samples as small as 20 μl may be used.     

Analysis of Brain and Cerebrospinal Fluid from Mouse Models of the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Changes in the Lysosomal Proteome

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Highlights

• •Proteomic analysis of brain and cerebrospinal fluid from mouse NCL models.
• •Disease-associated changes in lysosomal proteins and markers of neuroinflammation.
• •Biomarker candidates identified for further evaluation.

     

Selenium Concentration in Cerebrospinal Fluid Samples from a Paediatric Population

Selenium is an important trace element for brain function. Our objective was to analyse cerebrospinal fluid (CSF) selenium (Se) in 89 paediatric patients. We also studied correlations between Se and other biochemical variables (age, CSF protein concentrations and glutathione peroxidase activity and plasma Se values). Cerebrospinal fluid Se values showed a significant negative correlation with the age of patients (r = −0.476; p < 0.0001), and positive with CSF total protein concentrations and GPX activity (r = 0.446, p < 0.001; r = 0.431; p = 0.001, respectively). No association was observed between plasma and CSF Se concentrations. Median CSF Se values were 32 times lower when compared with those for plasma. In conclusion, CSF Se concentrations depend on age and total CSF protein values. The association observed between CSF Se and GPX activity suggests that Se quantification might be a reflection of some Se-dependent protein function. Cerebrospinal fluid Se values were independent of serum Se concentrations.     

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.     

A Noninvasive Approach in the Hydrodynamics of the Cerebrospinal Fluid in the Spinal Cavity

Biophysics - Abstract—A model of the movement of cerebrospinal fluid in the spinal cavity is proposed, which makes it possible to calculate the velocity profiles and expenditure of the...     

Characterization of Nitrotyrosine-Modified Proteins in Cerebrospinal Fluid

Background

HIV-associated neurocognitive disorders (HAND) has been associated with the up-regulation of various oxidative stress pathways. Previous studies have linked the neuronal damage observed in individuals diagnosed with HAND to increased nitrotyrosine modification of neuronal proteins.

Materials and methods

Tyrosine nitration alters protein structure and function, affects biological half-life, and potentially prevents the phosphorylation of key tyrosine residues involved in signal transduction pathways. Therefore, in this study we employed proteomics-based experimental approaches to investigate nitrotyrosine-modified proteins in pooled cerebrospinal fluid (CSF) of individuals diagnosed with HAND. To identify specific nitrotyrosine-modified proteins in the CSF of individuals diagnosed with HAND, affinity purification and high-performance tandem mass spectrometry are utilized in a “bottom-up” proteomics approach.

Results

From tandem mass spectrometric analysis, we identified major proteins that underwent nitration as a result of nitro-oxidative stress in the CSF of individuals diagnosed with HAND. We also utilized analytical and biochemical techniques to characterize the expression and modification site of in vivo nitrated lipocalin-type prostaglandin-D synthase in HAND CSF.     

Adenovirus Types 3 and 5 Isolated from the Cerebrospinal Fluid of Children

Adenoviruses types 3 and 5 were isolated from the cerebrospinal fluid of two sick children. These agents were thought to be responsible for the neurological state of the children at the phase of illness when they were isolated. The possibility of mixed infections or of superimposed infections was considered since one was isolated following herpes zoster infection in the one child and the other following varicella. The role of adenoviruses as latent agents of potential pathogenicity is discussed.     

Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease

Alzheimer’s disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer’s disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer’s disease patients and non-demented controls to identify potential biomarkers for Alzheimer’s disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer’s disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer’s disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.