Chemical and cytological analysis of cerebral spinal fluid after intrathecal injection of hypodense fluorescein / Análise química e citológica do líquor após injeção intratecal de solução hipodensa de fluoresceína

ABSTRACT INTRODUCTION: Intrathecal fluorescein has been effective for topographic diagnosis of rhinoliquorrhea. Nonetheless, there are no reports on the study of cerebral spinal fluid (CSF) after use of intrathecal fluorescein. OBJECTIVE: A prospective study attempting to evaluate CSF through chemical and cytological analysis, after injection of fluorescein. METHODS: Prospective analysis of 24 samples of CSF after intrathecal injection of fluorescein for topographic diagnosis of CSF fistulae, collected at the time of puncture and after 24 and 48 h, divided by cellularity: Group 1, up to five cells, and Group 2, with more than five cells. RESULTS: The yellow-greenish color of CSF remained after 48 h in 36%, evidencing permanence of fluorescein. No changes in protein and glucose levels were observed between 0-24 h and 0-48 h. In group 2, an increase in cell count was observed between 24 h and 48 h (p = 0.019). In both groups, there was an increase of neutrophils between 0 and 48 h (p = 0.048) and a decrease between 24 and 48 h (p = 0.05). CONCLUSION: Intrathecal fluorescein provoked discreet meningeal reactions, such as an increase of cells between 24 and 48 h and an increase of neutrophils at 24 h, with a subsequent decrease at 48 h with no correlation with symptomatology.

RESUMO Introdução: A fluoresceína intratecal tem sido efetiva no diagnóstico topográfico da rinoliquorréia. Entretanto, não há estudos no líquor após o uso de fluoresceína intratecal. Objetivo: Estudo prospectivo visando avaliar o líquor, através de análise química e citológica, após injeção de fluoresceína. Método: Análise prospectiva de 24 punções após injeção intratecal de fluoresceína para diagnóstico topográfico de fístula liquórica, coletado no momento da punção, 24 e 48 horas, divididos pela celularidade: grupo 1, com até 5 células e grupo 2 com mais de 5 células. Resultado: A coloração amarelo-esverdeada do líquor permaneceu após 48 horas em 36%, evidenciando permanência de fluoresceína. Observou-se ausência de mudanças no nível de proteína e glicose entre 0-24 horas e 0-48 horas. No grupo 2, um aumento na contagem celular foi observado entre 24 e 48 horas (p = 0,019). No dois grupos juntos, observou-se um aumento de neutrófilos entre 0 e 48 horas (p = 0,048) e uma diminuição entre 24 e 28 horas (p = 0,05). Conclusão: Fluoresceína intratecal provocou discretas reações meníngeas, como o aumento de células entre 24 e 48 horas e aumento dos dos neutrófilos em 24 horas com uma subsequente dimi nuição em 48 horas sem correlação com sintomas.

Chemical and cytological analysis of cerebral spinal fluid after intrathecal injection of hypodense fluorescein.

INTRODUCTION: Intrathecal fluorescein has been effective for topographic diagnosis of rhinoliquorrhea. Nonetheless, there are no reports on the study of cerebral spinal fluid (CSF) after use of intrathecal fluorescein. OBJECTIVE: A prospective study attempting to evaluate CSF through chemical and cytological analysis, after injection of fluorescein. METHODS: Prospective analysis of 24 samples of CSF after intrathecal injection of fluorescein for topographic diagnosis of CSF fistulae, collected at the time of puncture and after 24 and 48h, divided by cellularity: Group 1, up to five cells, and Group 2, with more than five cells. RESULTS: The yellow-greenish color of CSF remained after 48h in 36%, evidencing permanence of fluorescein. No changes in protein and glucose levels were observed between 0-24h and 0-48h. In group 2, an increase in cell count was observed between 24h and 48h (p=0.019). In both groups, there was an increase of neutrophils between 0 and 48h (p=0.048) and a decrease between 24 and 48h (p=0.05). CONCLUSION: Intrathecal fluorescein provoked discreet meningeal reactions, such as an increase of cells between 24 and 48h and an increase of neutrophils at 24h, with a subsequent decrease at 48h with no correlation with symptomatology.

Pre-analytical factors influencing the stability of cerebrospinal fluid proteins.

Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4h, 24h or 14 days at -20°C, 4°C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an -80°C environment. The results were obtained using immunoassays for amyloid-beta 1-42 (Aß42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling. Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. Aß42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage. We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at -80°C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions.

Identifying pharmacodynamic protein markers of centrally active drugs in humans: a pilot study in a novel clinical model.

Recognizing specific protein changes in response to drug administration in humans has the potential for significant utility in clinical research. In spite of this, many methodological and practical questions related to assessing such changes are unanswered. We conducted a series of clinical studies to assess the feasibility of measuring changes in proteins related to drug administration using a mass-spectrometry proteomics technique capable of quantifying hundreds of proteins simultaneously in cerebrospinal fluid (CSF) and plasma. Initially, the normal variability of proteins in these compartments was characterized in 16 healthy volunteers over a 2-week period. Drug-associated changes were subsequently assessed in the plasma and CSF proteomes of 11 subjects given atomoxetine, which served as a selective, centrally active probe to test the model. Protein levels in the CSF and plasma were unchanged between visits in the normal variability study. In contrast, statistically significant changes were detected in the CSF protein pattern after drug treatment. These studies suggest that identification of changes in the CSF proteome associated with the administration of centrally active drugs is feasible, and may be of value in the development of new drugs, as well as broader clinical research.

Angiostrongylus cantonensis: efficacy of albendazole-dexamethasone co-therapy against infection-induced plasminogen activators and eosinophilic meningitis.

Angiostrongyliasis is one of the common causes leading to eosinophilic meningitis. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may play a role in the pathogenesis. Administration of steroid drugs has been reported to possibly relieve the symptoms of eosinophilic meningitis. This study evaluates the curative effects of albendazole-dexamethasone co-therapy on eosinophilic meningitis in BALB/c mice. Assay indicators for the therapeutic effect include worm recovery, histopathological score of meningitis, tPA, uPA, total protein, and leukocyte counts. The results show that the albendazole-dexamethasone co-therapy significantly decreased (P<0.05) these factors after treatment on day 5 post-inoculation (PI), in contrast to treatment on 15 PI. Thus, the timing of medication is important and is closely related to the anthelmintic efficacy of a drug. At the same dosage and days post-infection, the earlier administration shows better results. This study showed that albendazole-dexamethasone co-therapy is an effective approach for the treatment of parasitic meningitis.

Proteomics analysis of prefractionated human lumbar cerebrospinal fluid.

Cerebrospinal fluid (CSF) is produced by the chorioid plexus in the ventricles. It surrounds the brain and bone marrow, and reflects several different disorders of the central nervous system (CNS). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and to elucidate the basic molecular mechanisms that either cause, or result from, CNS disorders. However, some disease-associated proteins are of low-abundance and are difficult to detect. A low total-protein concentration, a high amount of albumin and immunoglobins, and a wide dynamic range (several orders of magnitude) of protein concentration cause several difficulties in the identification of low-abundance CSF proteins. In this study, advantage was taken of the range of different hydrophobic properties of CSF proteins, and a reversed-phase solid-phase extraction (SPE) cartridge was used to prefractionate human lumbar CSF proteins into three separate fractions prior to two-dimensional gel electrophoresis resolution of the proteome. A portion of the high-abundance CSF proteins were removed from two (eluted with 35% and 50% acetonitrile) of the three fractions. Some trace CSF proteins were preferentially enriched in the two fractions, and many proteins were detected in the two-dimensional (2-D) gels of the two fractions. Among the novel proteins identified, sixty-two protein spots that represent forty-two proteins were characterized. Most of the proteins have not been annotated in any previous 2-D map of human CSF, and several have been implicated in CNS diseases. The prefractionation of CSF proteins with SPE, followed by proteomics analysis, provides a new method to explore low-abundance, disease-specific CSF proteins.

The clinical, cerebrospinal fluid, and histopathologic effects of epidural ketorolac in dogs.

OBJECTIVE: To evaluate the clinical, cerebrospinal fluid (CSF), and histopathologic effects of epidural ketorolac. STUDY DESIGN: Blinded, randomized, placebo controlled study. ANIMALS: Twenty-two adult mixed breed dogs with 16 treatment and 6 control dogs, weighing 14.4 to 29.8 kg. METHODS: Dogs were anesthetized and epidural catheters were placed at the lumbosacral space. Catheter placement was evaluated fluoroscopically. Ketorolac (0.4 mg/kg) or placebo (5% ethanol) was administered epidurally over a 52-hour period, with 5 injections given at 12-hour intervals. At 1, 2, 4, or 8 hours after the first and last injection of ketorolac, dogs were anesthetized and CSF was obtained. Control dogs had CSF sampled 1 hour after the first and last ethanol injection. Neurologic function and pain responses were evaluated before and during the study. Selected dogs were then killed and necropsies performed. RESULTS: None of the dogs exhibited any clinical or neurologic abnormalities during the study. No statistical difference was noted in pain response or CSF analysis between treatment and control dogs. Gross necropsy revealed gastrointestinal ulceration of varying degrees in all treatment dogs. Histopathologic analysis of the spinal cord and meninges revealed minimal focal leptomeningeal phlebitis in 2 of 8 treatment dogs and minor subdural inflammation in 1 control dog. No changes to the neural structures were noted in any dogs. CONCLUSIONS: Epidural administration of ketorolac did not cause clinical signs, alteration in CSF values, or pathologic changes to the spinal cord when used for short duration. Gastrointestinal ulceration was common when ketorolac was administered epidurally at 0.4 mg/kg every 12 hours for 5 treatments. CLINICAL RELEVANCE: This study documented the neurologic safety of epidural ketorolac in dogs before an efficacy trial can be performed. Gastrointestinal ulceration may limit use to short duration or a single injection.

Cerebrospinal fluid protein changes in multiple sclerosis after dental amalgam removal.

A relationship between multiple sclerosis (MS) and dental silver-mercury fillings has been suggested by some investigators, but never proven. This study documents objective biochemical changes following the removal of these fillings along with other dental materials, utilizing a new health care model of multidisciplinary planning and treatment. The dramatic changes in photolabeling of cerebrospinal fluid (CSF) proteins following these dental interventions suggest CSF photolabeling may serve as an objective biomarker for monitoring MS. The clear-cut character of these changes should also encourage more research to better define this possible association between dental mercury and MS.

Magnetic resonance imaging contrast media in the subarachnoid space. A comparison between gadodiamide injection and gadopentetate dimeglumine in an experimental study in pigs.

RATIONALE AND OBJECTIVES: The authors studied the neural tolerance and contrast enhancement of a nonionic, gadodiamide injection (gadolinium [Gd]-DTPA-BMA), and an ionic, gadopentetate dimeglumine (Gd-DTPA), contrast medium in the subarachnoid space of the pig. METHODS: Sixteen experiments were performed in eight pigs. Lumbar and lateral C1-C2 punctures were performed. Ten milliliters of Gd-DTPA-BMA or Gd-DTPA with Gd concentrations varying from of 500 mmol/L to 0.625 mmol/L were injected, in four experiments via the lumbar route and in 12 experiments via the C1-C2 puncture. RESULTS: Four pigs injected via the C1-C2 puncture with a Gd concentration of 500 mmol/L had signs of somatomotor irritation and all were paretic after 24 hours. No somatomotor effects were observed in the other experiments, where lower concentrations of Gd were used. Marked enhancement of the cerebrospinal fluid with no visible signal differences was obtained with concentrations from 10 to 0.625 mmol/L. CONCLUSIONS: Both Gd-DTPA-BMA and Gd-DTPA are remarkably well tolerated in the subarachnoid space. In doses relevant for imaging purposes no adverse effects were seen.

Effect of thalidomide on the inflammatory response in cerebrospinal fluid in experimental bacterial meningitis.

In experimental bacterial meningitis in rabbits, the inflammatory process is largely mediated by cytokines such as IL-1 and TNF-alpha. Since thalidomide has been shown to inhibit TNF-alpha production, experiments were carried out to determine whether the drug can modulate the inflammatory response to either lysates of H. influenzae (gram negative) or heat killed S. pneumoniae (gram positive) in rabbits. The introduction of a lysate of H. influenzae into the CSF of rabbits causes a very acute inflammatory response, as indicated by a rapid increase in TNF-alpha levels in the CSF and a concomitantly rapid leukocytosis. In contrast, the introduction of heat killed S. pneumoniae, induces a more indolent inflammatory response which also wanes more slowly. Thalidomide treatment reduces TNF-alpha production in both experimental systems, but has a greater effect on the more indolent gram positive inflammatory response in which peak TNF-alpha levels in the CSF are reduced by > 50%. Also, a sustained inhibition of leukocytosis is observed in the inflammatory response to heat-killed gram positive bacteria. In meningeal inflammation induced by the Gram negative lysate, treatment with thalidomide results in only a 29% inhibition of TNF-alpha release into the CSF. In contrast to the drug effect on TNF-alpha, thalidomide treatment does not significantly affect IL-1 levels in these models of rabbit bacterial meningitis.

Mercury and proteins in cerebrospinal fluid in subjects exposed to mercury vapor.

Mercury (Hg) and protein levels in cerebrospinal fluid (CSF), and mercury in plasma (P), erythrocytes (Ery), and urine (U), were determined in 10 workers exposed to mercury vapor for 2-28 (median 13) years and in 16 occupationally unexposed referents. CSF-Hg was analyzed using radiochemical neutron activation analysis and P-Hg, Ery-Hg, and U-Hg were analyzed using cold-vapor atomic absorption spectrometry. P-Hg and U-Hg were significantly higher, but Ery-Hg was similar in the exposed workers (37 nmole/liter, 16 nmole/mmole creatinine, and 56 nmole/liter, respectively) compared with the referents (7.1 nmole/liter, 1.9 nmole/mmole creatinine, and 52 nmole/liter, respectively). CSF-Hg was correlated to P-Hg, and in workers with current high exposure (P-Hg > 50 nmole/liter), the CSF-Hg was significantly higher than in the reference group (1.08 versus 0.35 nmole/liter; P = 0.002). In two individuals, studied after ceased occupational exposure, a decrease of CSF-Hg was seen. There were no indications of changes in the CSF protein pattern in the exposed workers.

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.

Abnormal proteins in the cerebrospinal fluid of a patient with Creutzfeldt-Jakob disease following administration of human pituitary growth hormone.

A Brazilian case of Creutzfeldt-Jakob disease in a hypopituitary patient who had received cadaver-derived human pituitary growth hormone between 1968 and 1977 is reported. The clinical diagnosis was confirmed during his lifetime by the demonstration of two abnormal 30-kDa proteins in the cerebrospinal fluid by two-dimensional gel electrophoresis. These proteins, characteristic of Creutzfeldt-Jakob disease, present isoelectric points of 5.1 and 5.2. Furthermore, both proteins migrate as doublets, each one displaying a molecular weight variant of about 29-kDa. This is one of 16 cases of the disease associated to therapy with cadaver-derived human growth hormone and one of the few examples among such cases of confirmation of the clinical diagnosis by biochemical characterization of abnormal proteins in the cerebrospinal fluid.

Haloperidol induced CSF protein variations in schizophrenic patients: as studied by two-dimensional electrophoresis.

High resolution two-dimensional electrophoresis of cerebrospinal fluid (CSF) from 10 schizophrenic patients demonstrated a 21% average difference in the number of proteins which could be detected in patients undergoing haloperidol therapy when compared with CSF from the same patients after withdrawal from neuroleptic treatment. Proteins affected were trace proteins, as we found no significant variation in either the total CSF protein content or the integrated protein density on each electrophoretic gel. Three mechanisms which might account for these observations are: (1) a small change in liver protein synthesis or degradation would have little if any visible effect on the concentration of major blood or CSF proteins, such as albumin, but it could significantly alter trace proteins, such as alpha 2-haptoglobin, since their concentrations are orders of magnitude less than that of the major proteins, (2) haloperidol might alter the blood-CSF protein filtration system which could affect the visibility of the trace proteins, and (3) proteins synthesized in the Central Nervous System (CNS) or enhanced in the CSF could be differentially affected by haloperidol. While additional research will be required to determine the basis for the effects of haloperidol on CSF proteins, the current studies provide information which may be helpful in delineating disease specific protein alterations from those induced by drug therapy.

Abnormal proteins in the cerebrospinal fluid of a patient with Creutzfeldt-Jakob disease following administration of human pituitary growth hormone

A Brazilian case of Creutzfeldt-Jakob disease in a hypopituitary patient who had received cadaver-derived human pituitary growth hormone between 1968 and 1977 is reported. The clinical diagnosis was confirmed during his lifetime by the demonstration of two abnormal 30-kDa proteins in the cerebrospinal fluid by two-dimensional gel electrophoresis. These proteins, characteristic of Creutzfeldt-Jakob disease, present isoelectric points of 5.1 and 5.2. Furthermore, both proteins migrate as doublets, each one displaying a molecular weight variant of about 29-kDa. This is one of 16 cases of the disease associated to therapy with cadaver-derived human growth hormone and one of the few examples among such cases of confirmation of the clinical diagnosis by biochemical characterization of abnormal proteins in the cerebrospinal fluid

Cerebrospinal fluid proteins in men with chronic encephalopathy after exposure to organic solvents.

Cerebrospinal fluid was examined for 23 patients with chronic toxic encephalopathy after heavy exposure to organic solvents and 23 healthy age-matched referents. No differences were found between the patients and referents with respect to the levels of albumin, immunoglobulin, prealbumin, alpha-1-antitrypsin, beta-2-microglobulin, haptoglobin, or the astroglial cell proteins S100 and glial fibrillary acidic protein in the cerebrospinal fluid. The albumin ratio was normal for both the patients and the referents. The patient group had had heavy exposure to organic solvents, but its members had not been exposed for at least one year before the study. It was concluded that, if exposure to organic solvents affects proteins in cerebrospinal fluid, such effects are probably reversible.