High performance collection of cerebrospinal fluid in rats: evaluation of erythropoietin penetration after osmotic opening of the blood-brain barrier.

An important issue to be considered when studying a new drug for treatment of central nervous system (CNS) diseases is its ability to cross the blood-brain barrier (BBB) and distribute throughout the brain. As cerebrospinal fluid (CSF) has demonstrated to be an invaluable reservoir to study CNS availability of therapeutic proteins, we have developed an improved method for CSF sampling from the cisterna magna of rats. The technique enables the simple and rapid collection of adequate quantities (50-75 µl) of blood-free CSF, rendering a high percentage of animal survival (99%) without clinic or neurological consequences. Its success in avoiding blood contamination of CSF lays in the use of a mixture of lidocaine/ephinephrine topically injected in the rat's suboccipital area and neck. Another relevant feature of the methodology is its low cost, since the puncture device can be easily assembled with cheap and available materials and, more importantly, neither expensive stereotaxic equipment nor frame is required. The present method is demonstrated by studying the CSF pharmacokinetics of recombinant human erythropoietin (rhEPO), a well-studied therapeutic candidate for neurological diseases. Moreover, we applied this technique to evaluate a strategy of osmotic disruption of the BBB to achieve a faster delivery of rhEPO into the CNS.

Blood chemistry, hematology and tryptophan level in cerebral malaria children.

BACKGROUND: Cerebral malaria is a deadly complication of P. falciparum infection, yet its pathogenesis remains incompletely understood. OBJECTIVE: The blood chemistry, hematology, protein and tryptophan levels in the cerebrospinal fluid (CSF) of cerebral malaria children were investigated. METHODS: Fifteen children (2.44 +/- 0.25 yr) diagnosed with cerebral malaria were used for this study. The control subjects consist of healthy and malaria-free children (2.50 +/- 0.16 yr). Two ml of blood were collected from each child between 0830 h and 0930 h. Blood chemistry and hematological parameters were analyzed using 2 ml each of Synchron CX5 auto-analyzer. The cerebrospinal fluid (CSF) was collected from the children using the lumbar puncture method, by inserting a sterile needle between the 4th and 5th lumbar vertebrae collected into sterile tubes. The CSF tryptophan, plasma and CSF protein concentrations and CSF protein concentration were determined. RESULTS: There were no significant (p > 0.01) differences in the plasma protein, glucose and CSF glucose levels of the cerebral malaria children as compared with the control. The packed cell volume (PCV) of the cerebral malaria children hemoglobin (Hb) levels were significantly (p < 0.01) lower as compared to control, but were significantly higher in CSF tryptophan and erythrocyte sedimentation rate (ESR) of cerebral malaria children were observed. Results of the study showed that cerebral malaria affected the CSF protein level, ESR, Hb and PCV, but do not affect plasma protein, glucose and CSF glucose concentrations. CONCLUSION: Data of the present study indicate that CSF protein, tryptophan, ESR, Hb and PCV could be used as possible markers in the diagnosis of cerebral malaria.

Serum time course of two brain-specific proteins, alpha(1) brain globulin and neuron-specific enolase, in tick-born encephalitis and Lyme disease.

METHODS: Time courses of the serum concentrations of two brain-specific proteins (BSP), alpha(1) brain globulin (alpha(1)BG, an astroglial marker) and neuron-specific enolase (NSE), were studied in patients with severe tick-born encephalitis (TBE) and Lyme disease (LD; neuroborreliosis). The concentrations were determined on the second day of the acute phase and then on the 7th, 12th, 18th, and 23rd days. Apparent rate constants for the elimination of the BSP from blood (k(e)) were calculated with the non-linear regression. RESULTS: In patients with TBE, the highest serum concentrations of alpha(1)BG and NSE, observed on the second day, were followed by their monotonic decrease to the normal levels reached by the 23rd day. The mean k(e) values for alpha(1)BG and NSE were found to be significantly different (0.086+/-0.003 vs. 0.057+/-0.006 day(-1), respectively; p<0.05). Higher serum levels of both BSP were observed in the more severe clinical cases and in the cases with unfavorable outcomes. Similar profiles were also observed for the serum alpha(1)BG and NSE in LD. CONCLUSIONS: These results suggest that, in the patients examined, the blood-brain barrier was partially impaired; the quantitative parameters of the serum BSP time courses can be indicative of the extents of the neuronal and/or glial lesions.

The effect of adjuvant steroid treatment on serial cerebrospinal fluid changes in tuberculous meningitis.

Three recent studies found that corticosteroids improve clinical outcome and mortality in tuberculous meningitis (TBM), although the exact mechanism of action of the drug remains speculative. A number of reports on the effect of corticosteroids on cerebrospinal fluid (CSF) findings in TBM have been published, often with conflicting results regarding serial cell counts and protein levels. As part of a controlled, randomized trial on the effect of oral prednisone on outcome in childhood TBM at our institution, CSF was collected and analysed weekly during the 1st month of treatment. We found no significant difference in serial CSF cell counts between the steroid and non-steroid groups in the study. However, the steroid group had significantly lower CSF protein and globulin levels after the 1st month of treatment, and a more steady rise in CSF glucose levels than the non-steroid group. Knowledge of the different CSF responses during the course of anti-tuberculosis therapy is important in clinical decision-making.

Significant decreases of ulinastatin-like immunoreactive substance in the cerebrospinal fluid of patients with dementia.

Studies to elucidate changes in the contents of ulinastatin-like immunoreactive substance (UTIRS) and alpha 1-microglobulin-like immunoreactive substance (alpha 1 MIRS) in cerebrospinal fluid (CSF) of patients with dementia were performed. Levels of UTIRS in the dementia group were significantly (p < 0.01) lower than those in dementia-free subjects. In addition, lower levels of UTIRS were registered in subjects afflicted with a higher severity of dementia. However, the levels of alpha 1 MIRS were not affected in the dementia group compared with dementia-free subjects. Neither UTIRS nor alpha 1 MIRS contents were related to the existence and stages of Parkinson's disease. These results suggest that UTIRS levels in the CSF are associated with the severity of dementia.

Total protein and electrophoretic pattern of cerebrospinal fluid in sheep with some common neurological disorders.

Cerebrospinal fluid (CSF) samples were collected from normal sheep and cases of some common ovine neurological diseases and analysed by agarose gel electrophoresis. Protein fraction concentrations determined from clinically normal sheep of mixed ages were used to establish reference values. The CSF albumin and globulin concentrations were significantly increased in meningo-encephalitis cases (P < 0.05) compared to healthy sheep. The group mean CSF albumin percentage for each of the neurological diseases studied was not significantly different from the mean CSF albumin concentration for control sheep (P > 0.05). In the ovine neurological diseases studied in this series, agarose gel electrophoresis of CSF did not add further to the information already gathered from the total CSF protein concentration.

Cerebrospinal fluid composition of cattle with endotoxin-induced mastitis treated with isotonic (0.9%) or hypertonic (7.5%) sodium chloride.

This study examined the safety of intravenous hypertonic saline in cattle with experimental gram-negative endotoxemia. Cerebrospinal fluid (CSF) composition was examined in five control cows and eight treated cows 24 hours after the intramammary infusion of 1 mg of endotoxin. Four of the endotoxin challenged cows were treated intravenously with isotonic (0.9%) sodium chloride and four cows were treated intravenously with hypertonic (7.5%) sodium chloride. Decreased CSF osmolality, and sodium and alpha globulin concentrations and increased CSF concentrations of beta globulin were observed in both endotoxin-challenged saline-treated groups. No CSF compositional differences were observed between endotoxin-challenged cows receiving isotonic or hypertonic saline. Although no cytologic or biochemical evidence of salt poisoning was observed in cows receiving hypertonic saline, significant changes were observed in the CSF composition of both endotoxin-infused saline-treated groups.

Idiopathic lumbosacral plexus neuropathy in two children.

Two children diagnosed as having idiopathic lumbosacral plexopathy are presented. Although an apparently rare condition, it is probably underdiagnosed since it is difficult to recognise. Before such a diagnosis, careful exclusion of compressive and infiltrative lesions of the spinal cord, cauda equina and lumbosacral plexus is of utmost importance.

Zn-binding globulin in human fetal brain and liver: a marker for passive blood/CSF transfer of protein.

The presence of Zn-binding globulin (ZnbG) during human fetal development was studied in cerebrospinal fluid (CSF) and plasma with immunodiffusion methods and in brain, CSF, plasma and liver using immunocytochemical methods. At the earliest stages examined with immunocytochemistry (5-6 weeks gestation) no staining for ZnbG was visible in liver, plasma, CSF or brain. However, the primitive mesenchyme exhibited a prominent staining reaction. In late embryonic and early fetal stages, staining for the protein was most prominent in the spinal cord, brain stem and diencephalon and in the choroid plexuses and marginal and subplate zones in the telencephalon. At the cellular level, synaptic strata and territories were most strongly stained. The distribution of ZnbG in the early developing central nervous system suggests that this protein may be involved in the initial establishment of CNS circuitry. Embryonic brain was positive for ZnbG well before the protein could be detected in CSF, plasma or liver. The early occurrence of ZnbG in brain tissue prior to its presence in liver or plasma also suggests that the protein is synthesized in early fetal brain. At the time when CSF first became positive (17 weeks gestation), the brain staining had largely disappeared. ZnbG in plasma increased throughout gestation to reach 2.6 +/- 0.4 mg/100 ml at term and subsequently increased to an adult value of 6.8 +/- 1.5 mg/100 ml.(ABSTRACT TRUNCATED AT 250 WORDS)

[Significance of oligoclonal proteins and magnetic resonance imaging in the diagnosis of multiple sclerosis]. / Znaczenie bialek oligoklonalnych i magnetycznego rezonansu jadrowego w diagnostyce stwardnienia rozsianego.

The new techniques identifying inactive clinical injury, as well as revealing immunological disturbances connected with the CNS, are very helpful for arriving at the correct diagnosis of multiple sclerosis. In their analysis the authors of the work include six cases and emphasize the great practical significance of magnetic resonance imaging and oligoclonal bands in the cerebrospinal fluid in making the diagnosis of multiple sclerosis.

Steroid hormones and steroid hormone binding globulins in cerebrospinal fluid studied in individuals with intact and with disturbed blood-cerebrospinal fluid barrier.

We measured in simultaneously withdrawn cerebrospinal fluid (CSF) and serum samples from 56 endocrinologically grossly normal patients the concentrations of several lipophilic unconjugated steroids [i.e. dehydroepiandrosterone (DHEA), androstenedione, cortisol, progesterone, testosterone] and their hydrophilic counterparts, i.e. DHEA-sulfate, or hydrophilic binding proteins, i.e. albumin, sex hormone-binding globulin (SHBG) and corticosterone-binding globulin (CBG). CSF levels of total (i.e. free plus protein-bound) DHEA, androstenedione, cortisol, progesterone, and testosterone were found to be in the 0.02-2 nM range and only cortisol reached levels approximately 20 nM. These values were of the same order of magnitude as the reported and calculated free serum levels of these steroids. In patients with disturbed (abnormally leaky) blood-CSF barrier (BCB) function, CSF levels of these steroids were not different from those with intact BCB, in contrast to DHEA-sulfate, CBG and SHBG whose CSF levels were significantly elevated, that is similar (i.e. 2-5) fold as those of albumin. In vitro demonstrated low affinity (micromolar) interactions of steroids with neuronal membrane-bound neurotransmitter receptors should be considered in perspective to the here reported finding that steroids occur in vivo at best in nanomolar concentrations in the CSF. Whether in other extracellular fluid compartments of the brain higher levels of steroids than in CSF can accumulate is as yet not clear. Very probably, pathological production or excessive dosage of steroids that are negligibly bound to SHBG or CBG will produce CSF and brain levels in the near micromolar range.

[Evaluation of cerebrospinal fluid proteins in patients with subacute sclerosing panencephalitis (SSPE)]. / Ocena bialek plynu mózgowo-rdzeniowego u chorych na podostre stwardniejace zapalenie mózgu (SSPE).

CSF proteins of 62 patients with SSPE aged 6 to 26 years were analysed by polyacrylamide gel electrophoresis. The total IgG concentration in the CSF was increased to 16.8 +/- 10 mg/dl and the IgG index was raised to from 2.7 +/- 0.5 to 3.6 +/- 3.0. All SSPE patients had pathological subfractions gamma 4 and gamma 5. The per cent proportion of the gamma 1 subfraction was raised only in the youngest patients. A tendency was observed for higher values of the prealbumin:albumin index and the prealbumin: transferrin index. The youngest SSPE patients had a tendency for greater total protein concentration, lower prealbumin concentration, and highest IgG index values (3.6 +/- 3.0). In early disease period a tendency was noted for higher concentration of total protein, higher total IgG level and higher IgG index. These results suggest that SSPE patients have a constant stimulation of B-cells and variable increase of blood-brain barrier permeability.

Cerebrospinal fluid investigations in tuberculous meningitis.

The results of conventional cerebrospinal fluid (CSF) investigations (CSF cell count, protein and glucose concentrations and Pandy's test for CSF globulin) obtained on admission and sequentially from weekly follow-up lumbar punctures for 4 weeks were evaluated in 99 children (median age 28 months) with stage II (50 children) and stage III (49 children) tuberculous meningitis. On admission, six children (6%) had a CSF cell count greater than 500 x 10(6)/l and nine (9%) a polymorphonuclear predominance. A CSF protein less than 0.8 g/l was found in 17 children (18%) of 97 in whom CSF protein was evaluated. Globulin was either absent or present as a trace only in 26 children (27%). CSF glucose was less than 2.2 mmol/l in 58 cases (60%) and less than 2.5 mmol/l in 67 (69%). In 63 children weekly CSF specimens obtained for the 1st 4 weeks of therapy showed an uninterrupted decline in cell count in 23 (37%), a fluctuating downward trend in 27 (43%) and a fluctuating upward trend in 13 (21%). Sequential CSF protein values in 57 children showed an uninterrupted rise in three (5%), a fluctuating upward course in 19 (33%), an uninterrupted downward trend in seven (12%), and a fluctuating downward course in 28 (49%). Of the 61 children in whom sequential CSF glucose concentrations were available, 11 (18%) experienced fluctuating concentrations, values falling to less than 2.2 mmol/l after being greater than 2.2 mmol/l on admission or after having risen to greater than 2.2 mmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Total protein, albumin quota, and electrophoretic patterns in cerebrospinal fluid of dogs with central nervous system disorders.

Cerebrospinal fluid was analyzed for total protein, albumin quota, and electrophoretic patterns of albumin and alpha-, beta-, and gamma-globulins in 10 healthy dogs and 35 dogs with CNS disorders. Values obtained in healthy dogs were used to establish control data. Samples also were collected from dogs with neurologic diseases that were classified according to clinical and pathologic diagnosis as inflammation, neoplasm, and spinal cord compression. The albumin quota and total CSF albumin values were used as indicators of blood-brain barrier disturbance. Four patterns were observed on agarose electrophoresis that included intrathecal immunoglobulin production, intrathecal immunoglobulin production combined with blood-brain barrier disturbance, blood-brain barrier disturbance, and unaltered CSF. These patterns correlated with the observed clinical and pathologic conditions. Seemingly, agarose electrophoresis of CSF is a simple and reliable technique that aids in the diagnosis of CNS disorders of dogs.

Low molecular weight plasma proteins in the cerebrospinal fluid of children with hematological malignancies.

The concentration of beta-2-microglobulin (beta 2-m) and of post gamma globulin (P gamma G) was examined in serum and cerebrospinal fluid from children with acute lymphatic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Data were analysed in order to determine whether concentration of beta 2-m or P gamma G during remission would be of value in predicting relapse or eventual outcome. Mean serum concentration of beta 2-m was similar in good and poor prognosis patients with ALL in remission and was not significantly altered in CNS or marrow relapse. Mean CSF concentration in NHL was also similar in both prognostic groups, and in poor prognosis patients was not significantly altered in relapse. The same pattern was seen when P gamma G was measured in CSF (serum concentration of this protein being too low for accurate determination). High within patient variability of levels of beta 2-m and P gamma G appeared to relate to chemotherapy rather than the disease process. Concentration of P gamma G was persistently raised in three children with brain damage of differing etiologies. Levels of two other low molecular weight proteins, retinol binding protein and alpha 1-microglobulin, were also determined in order to establish that beta 2-m and P gamma G concentration was not influenced by alteration in permeability of the blood-brain barrier. The beta 2-m and P gamma G concentration, although higher than reported in healthy children [5] does not appear to be of value as a prognostic indicator in ALL and NHL in children.

[Cerebrospinal fluid proteins: II. Normal values of protein fractions obtained by electrophoresis (variations related to race, sex and age)]. / Proteínas do líquido cefalorraqueano: II. Valores normais das frações proteicas obtidas por electroforese (variações ligadas a cor, sexo e idade).

The total protein content and protein fractions, obtained by electrophoresis on cellulose acetate strips, of CSF collected from the cisterna magna of 213 patients (with no neurological diseases) were determined in order to verify variations related to race, sex and age, as well as to establish the proteinogram normal limits. No differences between caucasians and coloured persons were observed with respect to CSF total protein and protein fractions. Children (5 months to 11 years old) do not present differences related to sex or age, on their proteinogram. Children's CSF total protein, relative values of pre-albumin, alpha 1--and beta-globulins, absolute values of albumin, alfa 2--, beta--, tau --and gamma-globulins, differ from those found in adults. Differences between males and females in the normal CSF proteinogram were found in adults. As a consequence of these findings, the CSF proteinogram normal limits for children, males and females were separately established. In adults, statistically significant positive correlation between age and the 7 protein fractions when expressed in mg/100 ml were observed, as well as between age and total protein. Comparison of the results obtained in this research with those found in some publications was carried out and is briefly discussed.

Post-gamma globulin. II. Radioimmunoassay determination of levels of post-gamma globulin and beta 2-microglobulin.

Post-gamma globulin previously isolated and partially sequenced in this laboratory was used for production of polyclonal and monoclonal (hybridoma) antibodies. A radioimmunoassay method was developed for quantitation of post-gamma globulin with either antibody. The titration curves obtained were treated statistically and found practically indistinguishable. The sensitivity of the method adopted for the quantitation of post-gamma globulin in a variety of biological fluids was 0.13 ng/ml and the upper limit of precision was 2.5 ng/ml. The following results were obtained (mean +/- 1 SD): normal sera, 0.96 +/- 0.20 microgram/ml; pregnancy sera, 1.08 +/- 0.28 micrograms/ml; cord blood 2.08 +/- 0.33 micrograms/ml; hospitalized patient's sera, 1.3 +/- 0.54 micrograms/ml; geriatric subjects' sera 2.26 +/- 1.10 micrograms/ml; cerebrospinal fluid, 5.37 +/- 3.36 micrograms/ml; saliva, 1.22 +/- 0.67 micrograms/ml; synovial fluid, 1.27 +/- 0.41 micrograms/ml and urine, 0.11 +/- 0.125 microgram/ml. To shed light on the catabolism of post-gamma globulin the levels of beta 2-microglobulin were also measured radiometrically. Correlative statistical analysis of all the data have shown that renal handling of post-gamma globulin and beta 2-microglobulin may be very similar but not necessarily identical.