SPARC/osteonectin, an endogenous mechanism for targeting albumin to the blood-cerebrospinal fluid interface during brain development

Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin-binding protein SPARC (osteonectin/BM-40/culture-shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real-time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co-localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC-immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood-CSF barrier is developmentally and physiologically regulated.     

Increased transfer ofCa into brain and cerebrospinal fluid from plasma during chronic hypocalcemia in rats

Recent studies have shown regulation of central nervous system [Ca] after chronic hypo- and hypercalcemia. To investigate the mechanism of this regulation , 3-week-old rats were fed diets for 8 weeks that contained low or normal levels of Ca. Plasma [Ca] was 40% less in rats fed the low Ca diet than in animals fed normal diet. Unidirectional transfer coefficients for Ca (K_Ca) and Cl (K_Cl) into cerebrospinal fluid (CSF) and brain were determined from the 10 min uptake of intravenously injected⁴⁵Ca and³⁶Cl in awake animals. K_Ca for CSF was 68% greater in low-Ca rats than in normal rats. Likewise, the values of K_Ca for brain regions with areas adjacent to the ventricles like the hippocampus and pons-medulla were 50% higher than in normal animals. On the other hand, K_Cas for parietal cortex, a brain region distant from the choroid plexus and not expected to be influenced by Ca entry into CSF, were similar between the groups. Comparison of the regional ratios of K_Ca/K_Cl revealed that a selective increase of Ca transport occurred into CSF and all brain regions except the parietal cortex in Ca-deficient rats. The results suggest that Ca homeostasis of CSF and brain [Ca] during chronic hypocalcemia is due to increased transfer of Ca from blood to brain, and that the regulation occurs via the CSF, possibly at the choroid plexus, but not via the cerebral capillaries.     

Magnesium concentrations in blood and cerebrospinal fluid during delirium tremens.

Magnesium in plasma, erythrocytes, and cerebrospinal fluid (CSF) was measured immediately after hospital admission in 9 patients with delirium tremens (DT) and 11 patients with impending DT. Blood samples were taken daily during the acute state; a second lumbar puncture was performed when the patient's condition had improved. Plasma magnesium was low in patients with DT during the first days of the acute state and then spontaneously normalized. Normal plasma magnesium was consistently seen among patients with impending DT. Magnesium in erythrocytes and CSF was normal in both diagnostic categories. Patients with a high blood-alcohol concentration (BAC) at admission had a decreasing plasma magnesium, patients with a low BAC had a moderately increasing plasma magnesium, and patients with a BAC at nil had a more marked increase in plasma toms or with their duration. This finding, combined with the normal CSF magnesium and the lack of correlation between plasma and CSF magnesium, indicates that disturbances in magnesium metabolism do not play a role in the etiology or pathogenesis of DT; but it may be that disturbances in magnesium metabolism contribute to the development of alcoholic encephalopathy.     

Heterogeneous models for blood-cerebrospinal fluid barrier permeability to serum proteins in normal and abnormal cerebrospinal fluid/serum protein concentration gradients

Cerebrospinal fluid (CSF)/serum concentration gradients (Q) of individual proteins (albumin, IgG, α₂-macroglobulin) have been studied in controls and in patients in whom the lumbar CSF flow is altered (medullary compression) or the blood-CSF barrier (BCB) function impaired (acute idiopathic polyneuropathy and acute meningoencephalitis). The analysis of relationships among protein Q has been performed by total and multiple regressions and the actual BCB permeability to individual proteins has been interpreted according to the accepted theoretical porous or vesicular BCB models. The exponential Q-IgG vs. Q-albumin total regression, and the poor Q-α₂-macroglobulin vs. Q-albumin regression found in controls, together with the different multiple regressions among proteins and the high Q-IgG vs. Q-albumin partial regression coefficients found in medullary compression, acute idiopathic polyneuropathy and acute meningoencephalitis, indicated that different permeability mechanisms can be postulated. Heterogeneous, fairly independent permeability BCB mechanisms maintain the normal CSFZ/serum protein concentration gradient. Pinocytotic vesicles or pores of radius exceeding 1000–1500 Å, probably located at the capillary endothelium, account for the main serum-derived CSF protein fraction(s) with large hydrodynamic radius (R). A more selective endothelial vesicular transport with a radius of 250 Å transfers a negligible amount of protein from serum into CSF. Proteins with small R also enter the CSF through a set of selective pores of radius 120 Å, probably at the level of the choroidal epithelium. Pinocytotic vesicles with a radius of 250 Å and increased rate of formation induce the accumulation of proteins below an obstruction of lumbar CSF flow. An increased formation rate of vesicles with a radius of 450 Å can explain the increased capillary permeability in nerve roots in acute idiopathic polyneuropathy. Loss of selectivity was the main feature of BCB in acute meningoencephalitis, and it seemed to be due to pores or vesicles with a radius larger than 1000–1500 Å. The heterogeneity of BCB mechanisms must be taken into account when the intrathecal synthesis of a protein, also derived from serum (for example IgG), has to be measured.     

Molecular forms of cholinesterases in cerebrospinal fluid, blood plasma, and brain tissue of the beagle dog.

In the present study we have investigated the activity of AChE and BuChE in the cisternal CSF of the Beagle dog and have outlined the characteristics of the molecular forms of both enzymes. The same enzymes were also investigated in samples from blood plasma and brain tissue. It is concluded that AChE in CSF originates from the spinal cord and brain tissues as a result of some secretory process. A combined origin from both blood plasma and brain tissue appears to be probable for the BuChE of the CSF.     

Characteristic protein fractions of cerebrospinal fluid disc electrophoretic analysis

The present work was undertaken to determine characteristic proteins of normal cerebrospinal fluid (CSF) by disc electrophoresis in polyacrylamide gel, and to evaluate their usefulness as indicators of blood-brain-CSF barrier disturbance. The technique has been applied to 1280 samples of unconcentrated CSF obtained from 27 reference subjects and 847 neurological patients, with a simultaneous analysis of 361 sera.The pre-albumin content (mean ± S.D. as a percentage of total protein, 11.0 ± 2.3%) was higher than formerly reported. One reason for this is that preliminary concentration was not necessary, and the second is related to the principle of protein resolution. The no. 5 protein band of the post-albumin group (3.9 ± 1.1% was characteristic, though it has not yet been identified. The no. 3 protein band of the post-transferrin group (5.2 ± 1.7%)was highly specific to CSF; it was found to be closely related to transferrin, and may correspond to τ fraction obtained by other methods. Barrier dysfunction was easily recognizable by the appearance of polymers of haptoglobin 2-1 or 2-2, because only haptoglobin 1-1 was detected in normal CSF. The percentage of the main region of the G-zone (13.7 ± 2.6%) was postulated as normal content of CSF immunoglobulins.     

肌萎缩侧索硬化患者的脑脊液蛋白及髓鞘碱性蛋白相关研究

目的探讨肌萎缩侧索硬化(ALS)患者的脑脊液蛋白、髓鞘碱性蛋白(MBP)水平及脑脊液蛋白与临床特征之间的关系。方法回顾性研究行腰穿查脑脊液的29例确诊ALS患者,检测其脑脊液蛋白及MBP水平,并按性别、年龄、病程、起病部位及临床功能评分〔肌萎缩侧索硬化功能分级量表(ALS-FRS)评分〕等不同临床特征分组,分析不同临床特征对脑脊液蛋白水平的影响。结果 29例ALS患者脑脊液蛋白水平为(0.43±0.15)g/L,其中脑脊液蛋白轻度增高患者9例(31%),最高为0.89g/L;不同性别〔男(0.42±0.15)g/L,女(0.45±0.18)g/L,t=0.501,P=0.620〕、年龄〔60岁组(0.43±0.17)g/L,≥60岁(0.44±0.13)g/L,t=0.141,P=0.889〕、病程〔1年组(0.37±0.11)g/L,≥1年(0.49±0.17)g/L,t=-2.23,P=0.054〕、起病部位〔球部起病组(0.38±0.11),肢体起病组(0.45±0.17),t=0.330,P=0.743〕、ALS-FRS评分〔30分组(0.42±0.16)g/L,≤30分组(0.44±0.16)g/L,t=0.092,P=0.928〕分组间比较,脑脊液蛋白水平差异均无统计学意义。29例患者中13例进行了脑脊液MBP检测,MBP水平(1.66±0.78)nmol/L,13例患者MBP水平均增高,最高达3.39nmol/L。MBP水平与脑脊液蛋白水平无相关性(R=0.198,P=0.517)。结论 ALS患者脑脊液蛋白增高多见。部分ALS患者脑脊液MBP水平增高,但与脑脊液蛋白水平无相关性。     

Tau protein concentrations in cerebrospinal fluid of patients with multiple sclerosis

FUNDAMENTALS AND OBJECTIVE: Multiple sclerosis (MS) is the prototype of demyelinating disease, but recently, it has been shown that the existence of axonal lesions contribute to irreversible central nervous system damage in this disease. Tau proteins are considered to be important for maintaining the stability of axonal microtubules involved in the mediation of fast axonal transport of synaptic constituents. There have been reports of increased cerebrospinal fluid (CSF) tau concentrations in patients with MS, and it has been suggested that this could be a marker of axonal damage. The objective of the present study was to elucidate whether CSF tau levels could be a marker of MS activity. PATIENT AND METHODS: We measured tau concentrations in the CSF of 20 patients with MS (nine in the first, seven in the second, one in the fourth exacerbation, and three patients with chronic progressive course) and 32 age- and sex-matched controls, using a specific enzyme-linked immunosorbent assay method. RESULTS: The CSF tau concentrations of patients with MS did not differ from those of controls, and they were not correlated with age at onset and duration of the disease. CONCLUSION: CSF tau concentrations are not a marker of MS activity.     

Developmental profile of plasma proteins in human fetal cerebrospinal fluid and blood

Total protein, alphafetoprotein, albumin, prealbumin, alpha-1-antitrypsin, transferrin and ceruloplasmin levels were measured in samples of human fetal and neonatal cerebrospinal fluid (CSF) (97 cases), obtained between 12 and 41 weeks of gestation. In 39 of these cases (13 to 40 weeks gestation) plasma was also available for comparative analysis. CSF was collected from lateral ventricles in the first half of gestation and from the lumbar region in the second. Since these CSF samples were obtained from different sites, the protein levels in the lateral ventricle (early) samples could not be compared directly with those in the lumbar (later) samples. However, the mean protein levels in the lumbar samples were lower than those in the ventricular samples, which is in accord with the decline in CSF protein levels described in maturing animal fetuses. Despite a wide scatter of results, particularly in the first half of gestation, significant decline in the level of CSF alphafetoprotein was demonstrated during both first and second halves of gestation, and of albumin and prealbumin in the second half. No sex differences were found except for ceruloplasmin in lumbar CSF later in gestation, when males had higher levels than females. In fetal plasma, protein levels increased with increasing gestation apart from alphafetoprotein and prealbumin which both declined progressively. CSF/plasma ratios were dissimilar for different proteins, and changed with increasing gestation. These findings support the concept that the human fetal blood brain barrier matures early.     

Cellular transfer of macromolecules across the developing choroid plexus of Monodelphis domestica

Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1–65 and young adults were injected with biotinylated dextrans (3–70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein-positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid-insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.     

Functional effectiveness of the blood-brain barrier to small water-soluble molecules in developing and adult opossum (Monodelphis domestica)

We have evaluated a small water-soluble molecule, biotin ethylenediamine (BED, 286 Da), as a permeability tracer across the blood-brain barrier. This molecule was found to have suitable characteristics in that it is stable in plasma, has low plasma protein binding, and appears to behave in a similar manner across brain barriers as established by permeability markers such as sucrose. BED, together with a 3000-Da biotin-dextran (BDA3000), was used to investigate the effectiveness of tight junctions in cortical vessels during development and adulthood of a marsupial opossum (Monodelphis domestica). Marsupial species are born at an early stage of brain development when cortical vessels are just beginning to appear. The tracers were administered systemically to opossums at various ages and localized in brains with light and electron microscopy. In adults, the tight junctions restricted the movement of both tracers. In neonates, as soon as vessels grow into the neocortex, their tight junctions are functionally restrictive, a finding supported by the presence of claudin-5 in endothelial cells. However, both tracers are also found within brain extracellular space soon after intraperitoneal administration. The main route of entry for the tracers into immature neocortex appears to be via the cerebrospinal fluid over the outer (subarachnoid) and inner (ventricular) surfaces of the brain. These experiments demonstrate that the previously described higher permeability of barriers to small molecules in the developing brain does not seem to be due to leakiness of cerebral endothelial tight junctions, but to a route of entry probably via the choroid plexuses and cerebrospinal fluid.     

Tau protein concentrations in cerebrospinal fluid of patients with amyotrophic lateral sclerosis

OBJECTIVE: To elucidate whether cerebrospinal fluid (CSF) concentrations of the microtubule-associated tau protein are related to the risk for sporadic amyotrophic lateral sclerosis (SALS). PATIENTS/METHODS: We measured tau concentrations in the CSF of 18 patients with SALS and 75 age- and sex-matched controls, using a specific ELISA method. RESULTS: The mean CSF concentrations of tau protein did not differ significantly between SALS patient and control groups, were not influenced by the clinical form (spinal vs bulbar) of ALS, and were not correlated with age, age at onset, and duration of the disease. CONCLUSIONS: CSF tau concentrations are not a biochemical marker of ALS.     

Blood-brain barrier selectivity and synaptic turnover during delirium tremens and related clinical states. A study of brain and blood proteins in the cerebrospinal fluid

The synaptic membrane protein D2 was measured in the cerebrospinal fluid (CSF) from patients with delirium tremens and related clinical states immediately after admission to the hospital before treatment was initiated and again after recovery. during this period of on average 5 days duration the concentration of D2 increased to double normal value possible indicating increased breakdown of old synapses during the process of readaptation to the alcohol free state. IgG, albumin and alpha 2-macroglobulin were measured in plasma and in CSF and the results indicated a decreased selectivity of blood-CSF-barrier permeability to proteins in the clinically recovered patients; this was probably due to increased micropinocytotic activity also occurring during readaptation to the alcohol free state.     

Oxytocin levels in saliva correlate better than plasma levels with concentrations in the cerebrospinal fluid of patients in neurocritical care

In the converging fields of neuroendocrinology and behavioural neuroscience, the interaction between peripheral secretion and the central release of oxytocin in humans has not yet been comprehensively assessed. Because the human brain is not directly accessible and the collection of human cerebrospinal fluid (CSF) usually requires invasive procedures, easier accessible compartments such as blood or saliva have attracted increasing attention. In the present study, we prospectively determined oxytocin concentrations in the 3 compartments comprising the plasma, CSF and saliva of 50 critically ill patients with neurological and neurosurgical diseases. All samples per patient were collected concomitantly. Oxytocin was measured by a highly sensitive and specific radioimmunoassay. The strength of correlation was assessed by the Spearman rank correlation coefficient. Correlation analyses revealed modest to strong correlations for oxytocin between the saliva and CSF compartments, whereas predominantly weak correlations were found between the CSF and plasma, as well as between the plasma and saliva compartments. In conclusion, we have demonstrated modest to strong correlations between the saliva and CSF compartments suggesting that saliva oxytocin may help to assess CSF oxytocin levels. By contrast, plasma oxytocin failed to correspond well with CSF oxytocin levels because predominantly weak correlations were found between the CSF and plasma, as well as between the plasma and saliva compartments, which are unlikely to have any biological relevance. Further research is needed to clarify to what extent saliva oxytocin may serve as a biomarker reflecting brain oxytocin activity.     

脑脊液与血浆的蛋白、糖、氯化物不同比值鉴别诊断结核性脑膜炎与病毒性脑膜炎的意义

目的 研究脑脊液(CSF)与血浆的蛋白、糖、氯化物含量不同比值鉴别诊断结核性脑膜炎(结脑)与病毒性脑膜炎(病脑)的意义.方法 检测102例结脑和107例病脑患者CSF和血浆的蛋白、糖、氯化物含量,计算CSF与血浆的蛋白、糖、氯化物含量3种不同比值(CSF/血浆蛋白≥0.2、糖≤0.5、氯≤0.7,CSF/血浆蛋白≥0.1、糖≤0.6、氯≤1.1,CSF/血浆蛋白≥0.3、糖≤0.4、氯≤0.6)诊断结脑的敏感性和特异性.结果 结脑组CSF与血浆的蛋白、糖、氯化物含量3种比值的阳性率均显著高于病脑组(P<0.05～0.01);以CSF/血浆的蛋白≥0.2、糖≤0.5、氯≤0.7诊断结脑的敏感性(96.1%,97.1%,99.0%)和特异性(97.2%,98.1%,99.1%)均较高,而CSF/血浆的蛋白>10.1、糖≤0.6、氯≤1.1的特异性(66.4%,69.2%,75.7%)较低,CSF/血浆的蛋白≥0.3、糖≤0.4、氯≤0.6的敏感性(48.0%,44.1%,42.2%)较低.结论 CSF与血浆的蛋白、糖、氯化物含量比值≥0.2、≤0.5及≤0.7对鉴别诊断结脑与病脑具有重要意义.     

Active and total T cells in blood and cerebrospinal fluid during the course of aseptic meningitis

Patients with aseptic meningitis (AM) were examined with the active T cell rosette test, which has been claimed to reflect cell-mediated immunocompetence more accurately than determination of total T cells. Higher percentages of active T cells were demonstrated in CSF compared to blood regardless if specimens were obtained on days 1–4, days 5–10, or later than 20 days after onset of symptoms. Active T cell percentages in CSF decreased when values for specimens obtained on days 5–10 were compared with those taken later than 20 days after onset, while no significant variations of active T cell percentages in blood were observed. The percentages of total T cells were higher in CSF than blood in specimens from days 5–10, and later than 20 days after onset, but no significant fluctuations of total T cells occurred in either CSF or blood over the course of AM.     

Synaptic membrane protein D2 in the cerebrospinal fluid of manic-melancholic patients.

The synaptic membrane protein D2 was measured in the cerebrospinal fluid (CSF) of manic-melancholic patients. The concentration of D2 increased with the age of the patients until about 35 years of age. No difference was found between the D2-concentration in CSF from a control group compared with different manic-melancholic subgroups. The D2-concentration in CSF collected from the patients during depression or mania was compared with CSF collected from the same patients when their moods were normalized. In the case of the depressed patients, we found that the D2-concentration increased slightly when the mood was normalized.     

Comparison of the response to phytohemagglutinin, of peripheral blood monuclear cells and cerebrospinal fluid lymphocytes in multiple sclerosis and other neurological diseases

Proliferative response to phytohemagglutinin (PHA) of cerebrospinal fluid lymphocytes (CSF-L) and peripheral blood mononuclear cells (PMBC) from patients with multiple sclerosis (MS) and other neurological diseases (OND) was tested using either bulk culture conditions or limiting dilution techniques in the presence of additional exogenous IL2. CSF-L response to PHA was found significantly lower than that of PBMC in both MS and OND patients. There was no significant difference in the response of CSF-L between the two groups. The limiting dilution analysis of the frequency of the precursors of PHA-responsive cells showed a strikingly decreased frequency of these precursors among CSF-L.     

The relationship of free and conjugated catecholamines in plasma and cerebrospinal fluid in cerebral and meningeal disease

Summary The concentration of free and conjugated norepinephrine (NE), epinephrine (E) and dopamine (DA) were measured by a modified radioenzymatic assay in the plasma and in the cerebrospinal fluid (CSF) of 45 patients with normal and in 21 patients with disturbed blood-CSF barriers. In patients with an undisturbed blood-CSF barrier the free NE and E in CSF were 128±45 ng/l and 27±20 ng/l (mean values±S.E.), respectively, and represented about 50 % of the average plasma values. Mean DA was not different in plasma (47±22 ng/l) and in CSF (41±19 ng/l). Both in plasma and in CSF, considerable higher free catecholamine (CA) levels were measured in patients with dysfunction of the blood-CSF barrier. In one patient with bacterial meningitis twofold higher concentrations of free NE and DA in CSF as compared with plasma were detectable. Sulfate conjugates of catecholamines are predominant in plasma and CSF.The contribution of conjugated CA to total CA in plasma from patients with normal blood-CSF barrier averaged 69.7 %, 63.1 % and 98.1 % for NE, E and DA, respectively and was significantly lower in the CSF (p<0.001). In patients with disturbed blood-CSF barrier, the increases of conjugated CA were more pronounced in CSF than in plasma. Further, the contribution of conjugated NE and E to total NE and E in CSF was not only increased in patients with bacterial meningitis, but also in patients with renal insufficiency compared to the control patients (p<0.02 and p<0.001 resp.). Free and conjugated NE, E and DA in the plasma and CSF were related significantly (p<0.01 resp.) with stronger correlation for conjugated CA (p<0.001 resp.). These results together with findings in the literature, suggest that there is little or no rostral-caudal gradient in CSF CA conjugate concentrations and that even in patients with intact blood-CSF barrier plasma conjugated CA concentrations influence those in CSF. Thus only free CA levels in CSF may reflect the central adrenergic activity.