The Blood-Cerebrospinal Fluid Barrier Is Selective for Red Cabbage Anthocyanins and Their Metabolites.

The study aim was to determine whether strongly bioactive hydrophilic red cabbage anthocyanins possess the ability to cross the blood-cerebrospinal fluid barrier (blood-CSF barrier) and whether there is a selectivity of this barrier toward these compounds. To fulfill objectives, red cabbage preparation, containing nonacylated and acylated anthocyanins, was administered to 16 sheep with implanted cannulas into the brain third ventricle, and next, within 10 h, blood, urine, and the cerebrospinal fluid (CSF) were collected and analyzed with HPLC-MS/MS. Though, in blood plasma and urine after red cabbage intake, both, acylated and nonacylated anthocyanins and their metabolites occurred, but only nonacylated derivatives were present in the CSF, and their changes in the profile and concentration in the CSF resulted from the fluctuation of these pigments' concentration and profile in blood, their different abilities to permeate via the blood-CSF barrier, and their transformations in this barrier. Results indicate that the blood-CSF barrier is selective for red cabbage anthocyanins.

Cerebrospinal fluid analysis in 58 ruminants showing neurological disorders / Análise do líquido cefalorraquidiano em 58 ruminantes com distúrbios neurológicos

Ruminants may be affected by a wide variety of central nervous system (CNS) diseases. Cerebrospinal fluid (CSF) analysis forms the basis for ante mortem diagnostic evaluation of ruminants with clinical signs involving the CNS. Despite its importance as a tool to aid diagnosis, data regarding CSF examinations in spontaneous cases of CNS diseases in ruminants from Brazil are limited, and most reports involve experimental studies. Therefore, this study aimed to report the results of CSF analysis in 58 ruminants showing signs of neurological disorders. CSF samples for analysis were obtained from 32 cattle, 20 sheep, and 6 goats by cerebello-medullary cistern (n=54) or lumbosacral space (n=4) puncture. These ruminants showed neurological signs related to viral (n=13), mycotic (n=3), or bacterial (n=15) infections, and toxic (n=21), traumatic (n=4), or congenital disorders (n=2). CSF analysis from ruminants with viral infections presented lymphocytic pleocytosis, even though CSF showed no changes in several cases of rabies. Neutrophilic pleocytosis, cloudiness, presence of fibrin clots, and abnormal coloration were evident in the CSF of most cases of CNS bacterial infection, such as meningoencephalitis, meningitis, abscesses, myelitis, and a case of conidiobolomycosis. On the other hand, CSF was unchanged in most cases of toxic disorders, as botulism and hepatic encephalopathy. Elevated CSF density was observed in 60% of ruminants diagnosed with polioencephalomalacia. Our findings show that evaluation of CSF is a valuable diagnostic tool when used in association with epidemiological, clinical and pathological findings for diagnosis of CNS diseases in ruminants.(AU)

Os ruminantes podem ser afetados por uma grande variedade de doenças do sistema nervoso central (SNC). A análise do líquido cefalorraquidiano (LCR) constitui a base da avaliação diagnóstica ante mortem de ruminantes com sinais clínicos envolvendo o SNC. Apesar de sua importância como ferramenta para auxiliar no diagnóstico, os dados referentes aos exames do LCR em casos espontâneos de doenças do SNC em ruminantes no Brasil são limitados, e, a maioria dos relatos envolve estudos experimentais. Portanto, este trabalho teve como objetivo relatar os resultados da análise do LCR em 58 ruminantes com distúrbios neurológicos. Amostras do LCR foram obtidas de 32 bovinos, 20 ovinos e 6 caprinos por punção da cisterna cerebelo-medular (n=54) ou espaço lombossacro (n=4) para posterior análise. Esses ruminantes apresentaram sinais neurológicos relacionados a infecções virais (n=13), micóticas (n=3) ou bacterianas (n=15), e desordens tóxicas (n=21), traumáticas (n=4) ou congênitas (n=2) A análise do LCR de ruminantes com infecções virais apresentou pleocitose linfocítica, embora, em vários casos de raiva, o LCR não tenha apresentado alterações. Pleocitose neutrofílica, turbidez, presença de coágulos de fibrina e coloração anormal foram evidentes no LCR da maioria dos casos de infecções bacterianas do SNC, como meningoencefalites, meningites, abscessos, mielite e um caso de conidiobolomicose. Por outro lado, o LCR não foi alterado na maioria dos casos dos distúrbios tóxicos, como botulismo e encefalopatia hepática. A densidade elevada no LCR foi observada em 60% dos ruminantes diagnosticados com polioencefalomalácia. Nossos resultados mostram que a avaliação do LCR é uma valiosa ferramenta de diagnóstico, quando usada em associação com os achados epidemiológicos, clínicos e patológicos para o diagnóstico de doenças do SNC em ruminantes.(AU)

Molecular forms of C-type natriuretic peptide in cerebrospinal fluid and plasma reflect differential processing in brain and pituitary tissues.

C-type natriuretic peptide (CNP) is a paracrine growth factor widely expressed within tissues of the central nervous system. Consistent with this is the high concentration of CNP in cerebrospinal fluid (CSF), exceeding levels in the systemic circulation. CNP abundance is high in hypothalamus and especially enriched in pituitary tissue where - in contrast to hypothalamus - processing to CNP-22 is minimal. Recently we have shown that dexamethasone acutely raises CNP peptides throughout the brain as well as in CSF and plasma. Postulating that molecular forms of CNP would differ in central tissues compared to forms in pituitary and plasma, we have characterized the molecular forms of CNP in tissues (hypothalamus, anterior and posterior pituitary gland) and associated fluids (CSF and plasma) using size-exclusion high performance liquid chromatography (SE-HPLC) and radioimmunoassay in control (saline-treated) and dexamethasone-treated adult sheep. Three immunoreactive-CNP components were identified which were consistent with proCNP (1-103), CNP-53 and CNP-22, but the presence and proportions of these different fragments differed among tissues. Peaks consistent with CNP-53 were the dominant form in all tissues and fluids. Peaks consistent with proCNP, conspicuous in hypothalamic extracts, were negligible in CSF whereas proportions of low molecular weight immunoreactivity (IR) consistent with CNP-22 were similar in hypothalamus, posterior pituitary gland and CSF. In contrast, in both plasma and the anterior pituitary gland, proportions of higher molecular weight IR, consistent with CNP-53 and proCNP, predominated, and low molecular weight IR consistent with CNP-22 was very low. After dexamethasone, proCNP like material - but not other forms - was increased in all samples except CSF, consistent with increased synthesis and secretion. In conclusion, immunoreactive forms of CNP in central tissues differ from those identified in anterior pituitary tissue and plasma - suggesting that the anterior pituitary gland may contribute to systemic levels of CNP in some physiological settings.

Selenium speciation in paired serum and cerebrospinal fluid samples of sheep.

This study was performed to characterise selenium (Se) and Se species in cerebrospinal fluid (CSF) of sheep and its relation to the respective Se concentrations in serum. Paired samples from 10 adult sheep were used for the study. Five sheep were fed a diet with a marginal Se concentration of <0.05mg Se/kg diet dry weight (dw, Se(-)), and five animals were fed the same diet supplemented with sodium selenite revealing a concentration of 0.2mg Se/kg diet dw (Se(+)). The feeding strategy was conducted for two years; The results on metabolic effects were published previously. At the end of the feeding period, paired samples of serum and CSF were collected and analysed using ion exchange chromatography inductively coupled plasma-dynamic reaction cell-mass spectrometry (IEC-ICP-DRC-MS) technique for total Se concentration and concentrations of Se species. Albumin concentrations were analysed additionally. The feeding strategy caused significant differences (p<0.01) in serum Se concentrations with 33.1±5.11µg Se/l in the Se(-) group and 96.5±18.3µg Se/l in the Se(+) group, respectively. The corresponding total Se concentrations in CSF were 4.38±1.02µg Se/l and 6.13±1.64µg Se/l in the Se(-) and the Se(+) group, respectively, missing statistical significance (p=0.077). IEC-ICP-DRC-MS technique was able to differentiate the Se species selenoprotein P-bound Se (SePP), selenomethionine, glutathione peroxidase-bound Se (Se-GPx), selenocystine, thioredoxin reductase-bound Se, ovine serum albumin-bound Se (Se-OSA), SeIV and SeVI in ovine serum and CSF. Quantitatively, SePP is the main selenoprotein in ovine serum followed by Se-GPx. The CSF/blood ratio of albumin (QAlbumin) reflected a physiological function of the blood-CSF barrier in all sheep. QSe-species were higher than QAlbumin both feeding groups, supporting the hypothesis of local production of Se species in the brain. Significant positive regression lines for CSF vs. serum were found for albumin and Se-OSA only, suggesting a role of albumin to convey Se across the blood-CSF barrier. The ovine model, together with the IEC-ICP-DRC-MS technique to characterise the Se species, might be a worthwhile model for further studies as repeated sample collection as well as modification of the nutritional status is feasible and effective.

Photoperiod affects the cerebrospinal fluid proteome: a comparison between short day- and long day-treated ewes.

Photoperiod is the main physical synchronizer of seasonal functions and a key factor in the modulation of molecule access to cerebrospinal fluid (CSF) in animals. Previous work has shown that photoperiod affects the transfer rate of steroids and protein hormones from blood to CSF and modulates choroid plexus tight junction protein content. We hypothesized that the CSF proteome would also be modified by photoperiod. We tested this hypothesis by comparing CSF obtained from the third ventricle of mature, ovariectomized, estradiol-replaced ewes exposed to long day length (LD) or short day length (SD). Variations in CSF protein expression between SD- or LD-treated ewes were studied in pools of CSF collected for 48 h. Proteins were precipitated, concentrated, and included in a polyacrylamide gel without protein fractionation. After in-gel tryptic digestion of total protein samples, we analyzed the resulting peptides by nanoliquid chromatography coupled with high-resolution tandem mass spectrometry (GeLC-MS/MS). Quantitative analysis was performed using 2 methods based on spectral counting and extracted ion chromatograms. Among 103 identified proteins, 41 were differentially expressed between LD and SD ewes (with P < 0.05 and at least a 1.5-fold difference). Of the 41 differentially expressed proteins, 22 were identified by both methods and 19 using extracted ion chromatograms only. Eighteen proteins were more abundant in LD ewes and 23 were more abundant in SD ewes. These proteins are involved in numerous functions including hormone transport, immune system activity, metabolism, and angiogenesis. To confirm proteomic results, 2 proteins, pigment epithelium-derived factor (PEDF) and gelsolin, for each individual sample of CSF collected under SD or LD were analyzed with Western blots. These results suggest an important photoperiod-dependent change in CSF proteome composition. Nevertheless, additional studies are required to assess the role of each protein in seasonal functions.

Melanin concentrating hormone (MCH) in the cerebrospinal fluid of ewes during spontaneous oestrous cycles and ram effect induced follicular phases.

The melanin-concentrating hormone (MCH) is a neuropeptide synthesized by neurons of the lateral hypothalamus and incerto-hypothalamic area that project throughout the central nervous system. The aims of the present report were: (1) to determine if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal and the oestrous phase of spontaneous oestrous cycles; and (2) to study if MCH levels in CSF of ewes vary acutely during the follicular phase induced with the ram effect in anoestrous ewes. In the first experiment, CSF was collected from 8 adult ewes during spontaneous oestrous and during the mid-luteal phase (8-10 days after natural oestrus). In the second experiment, performed during the mid non-breeding season, a follicular phase was induced with the ram effect. After isolating a group of 16 ewes from rams, CSF was obtained from 5 of such ewes (control group). Three rams were joined with the ewes, and samples were obtained 12h (n=6) and 24h (n=5) later. In Experiment 1, there were no differences in MCH concentrations in CSF measured during the mid-luteal phase and spontaneous oestrus (0.14 ± 0.04 vs. 0.16 ± 0.05 ng/mL respectively). In Experiment 2, MCH concentrations tended to increase 12h after rams introduction (0.15 ± 0.08 vs. 0.35 ± 0.21 ng/mL, P=0.08), and increased significantly 24h after rams introduction (0.37 ± 0.15 ng/mL, P=0.02). We concluded that MCH concentration measured in the CSF from ewes increased markedly during the response to the ram effect but not during the natural oestrous cycle of ewes.

Independent changes of thyroid hormones in blood plasma and cerebrospinal fluid after melatonin treatment in ewes.

The authors measured the effects of exogenous melatonin treatment on the concentrations of total (T) and free (f) fractions of thyroxine (T4) and triiodothyronine (T3) in cerebrospinal fluid (CSF) and blood plasma as well as the expression of their binding/transporter protein, transthyretin (TTR), in the choroid plexus of ewes from May to August. Melatonin implantation in May and July mainly prevented the decrease in plasma for fT3 and TT3 exhibited in untreated group, and induced a limited decrease in TT4 in June. By contrast, melatonin implantation prevented the decrease in CSF fT3 observed in the untreated group. No effect of melatonin was found on the expression of TTR mRNA in the choroid plexus There were a correlations between blood fT4 and CSF TT4 concentrations in both control and melatonin treated group (r(2)-0.4; P < 0.01 vs. r(2)-0.14; P < 0.05), as well as between blood fT3 and CSF TT3 concentrations but only in the melatonin-treated group (r(2)-0.26; P < 0.02). We conclude that T3, the active form of the hormone within the brain, is regulated by melatonin independently of the peripheral changes within the blood. The lack of correlation between plasma fT3 and CSF TT3 in the control group suggests that an increase in local T3 conversion could contribute as an additional source of T3 in the CSF during the period of increasing day length. These data seem to confirm a local nature for recently discovered connections between the pineal melatonin signal and thyroid-dependent seasonal biology in mammals.

Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid.

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.

Safety of continuous intrathecal midazolam infusion in the sheep model.

UNLABELLED: We investigated the safety of midazolam administered by continuous intrathecal infusion in relevant animal models. Preservative-free midazolam was delivered to sheep and pigs by using implanted infusion systems (SynchroMed pumps plus silicone catheters). Sheep received midazolam 5 mg/d (n = 4) or 15 mg/d (n = 7) or saline (n = 2) for 43 days at 125 micro L/h. One sheep received 10 mg/d. Infusion concentrations ranged from 1.7 to 2.5 mg/mL (5 mg/d) and from 2.5 to 5.0 mg/mL (15 mg/d). Pigs were evaluated for toxicity only and received 15 mg/d (n = 2) or saline (n = 1) for 43 days at 125 micro L/h. Behavior, neurologic function, and vital signs were documented. Serum and cerebrospinal fluid chemistry and cytology were evaluated, and histology was performed on spinal cord tissue. Behavior and neurologic function remained normal in all subjects. Gross and microscopic evaluation of spinal tissue revealed mild inflammation surrounding the catheter tract in both the midazolam-treated and the saline-treated groups. This inflammation was likely attributable to the mechanical presence of the catheter. These data demonstrate that continuous intrathecal infusion of preservative-free midazolam at doses up to 15 mg/d were well tolerated. IMPLICATIONS: We investigated the toxicity of preservative-free intrathecal midazolam delivered continuously via implanted infusion systems in sheep and pigs. Doses of 5-15 mg/d were well tolerated. The lack of neurotoxicity observed suggests that intrathecal midazolam may be an alternative for the treatment of intractable pain that is unresponsive to opioids.

Regulators of the neuropeptide-degrading enzyme, EC 3.4.24.15 (thimet oligopeptidase), in cerebrospinal fluid.

Endopeptidase EC 3.4.24.15 (EP 24.15; thimet oligopeptidase) is a soluble metalloendopeptidase implicated in the metabolism of a number of neuropeptides, including neurotensin, gonadotropin-releasing hormone, and opioid peptides. We have shown previously that thiol reducing agents, such as dithiothreitol, activate EP 24.15 by mediating the conversion of inactive multimeric forms to active monomers and that this conversion involves the disruption of intermolecular disulfide bonds involving cysteine residues 246, 248, and 253. We have identified two components of cerebrospinal fluid that activate recombinant EP 24.15, but have no effect on a thiol-independent cysteine mutant form of the enzyme. The low molecular weight (<10 kDa) component co-elutes with glutathione by reversed-phase HPLC, whereas the high molecular weight component (>50 kDa) is sensitive to digestion with trypsin, suggesting it is proteinaceous in nature. These results suggest that EP 24.15 activity in the brain may be modulated by factors released into cerebrospinal fluid.

Measurement and possible function of GnRH in cerebrospinal fluid in ewes.

Large mammal models are unique in that it is possible to analyse the real-time release of neural factors over several hours or even days in a conscious unstressed state. Until recently, hypophyseal portal blood sampling was the only reliable method available for this purpose. However, development of an alternative approach, in which cerebrospinal fluid (CSF) is collected from specific sites within the cerebroventricular system, has provided another route by which hypothalamic activity can be investigated. Use of this approach, in combination with other methods, such as intracerebroventricular infusion or simultaneous hypophyseal portal blood collection, has yielded exciting novel data that challenge long-held dogma on the pathways of communication in the brain. It is clear that factors in the CSF are released site-specifically and, thus, this fluid is not homogeneous; the concentration of a factor in lumbar CSF may bear no relation to its ventricular concentration. Data also indicate that there is little, if any, transfer of factors between the CSF and the hypophyseal portal system. In addition, there is mounting evidence indicating that factors in CSF may serve as part of a non-synaptic communication system in the brain. Establishing an unequivocal function for CSF-borne factors may prove technically difficult, if not impossible. However, we believe that there is strong evidence supporting a role for one such factor, GnRH in CSF, in sexual behaviour.

Prostaglandin E2 in cerebrospinal fluid of fetal and newborn sheep: central versus peripheral source.

During the perinatal period, prostaglandin (PG) E2 levels show parallel changes in cerebrospinal fluid (CSF) and blood which may be important for the adaptation of the fetus to extrauterine life. It is not known, however, whether PGE2 in the CSF originates from a local or a peripheral source. Experiments were carried out in term fetal and newborn sheep chronically instrumented with a cannula inside the third ventricle and vascular lines. Indomethacin was given intracerebroventricularly (i.c.v.) (50 or 100 micrograms at hourly intervals), alone or in combination with intravenous (i.v.) PGE2 (1 or 1.5 micrograms/kg/min). In the fetus, i.c.v. indomethacin reduced PGE2 levels in both CSF and plasma. Conversely, no significant change was noted at either site when indomethacin was given i.c.v. to the newborn. At both ages, PGE2 increased in the CSF during i.v. infusion of the compound, but this elevation was proportionately smaller than in plasma. We conclude that, in the perinatal period, brain and peripheral circulation function as separate compartments with respect to PGE2, though there is passage of the compound across the blood-brain barrier. Results provide indirect evidence that perinatal brain produces PGE2 in measurable amounts.

High-frequency oscillatory ventilation compared with conventional mechanical ventilation in newborn lambs: effects of increasing airway pressure on intracranial pressures.

We tested the hypothesis that intracranial pressures and cerebral perfusion pressure in the newborn are more seriously affected by increasing airway pressure during high-frequency oscillatory ventilation (HFOV) than during conventional mechanical ventilation (CMV). Mean airway pressure was acutely elevated in stepwise fashion to 25 cm H2O in six anesthetized, paralyzed newborn lambs. Pressure (mean +/- SE) increased similarly during HFOV and CMV in the jugular vein (7 +/- 1 and 8 +/- 1 cm H2O, respectively), the sagittal sinus (6 +/- 1 and 7 +/- 1 cm H2O), and the cerebrospinal fluid of the lateral ventricle (4 +/- 1 and 6 +/- 1 cm H2O). Decreases in arterial blood pressure (-13 +/- 2 and -10 +/- 2 cm H2O) and cerebral perfusion pressure (-17 +/- 2 and -16 +/- 2 cm H2O) were also similar during HFOV and CMV. Intracranial pressure-volume curves were generated by incrementing cerebrospinal fluid volume in eight lambs. Curves generated during HFOV and CMV were similar, reflecting a similar intracranial compliance during the two ventilatory modes. These data indicate that intracranial compliance and the effects of increasing airway pressure upon intracranial pressures are not significantly different between HFOV and CMV.

A technique for serial sampling of cerebrospinal fluid from conscious swine and sheep.

We devised a method for the atraumatic repeated collection of cerebrospinal fluid samples from conscious swine and sheep. Indwelling needles, with injection caps, were secured intracisternally and protected with plastic "crowns" attached to their skulls. The crowns permitted the animals freer movement with minimal risk of damage to the needles. With sheep, cerebrospinal fluid was withdrawn directly by puncturing the self-sealing injection caps attached to the hubs of the fixed needles. With the pigs, which are less amenable to handling, lengths of sterile polyethylene tubing inserted into the fixed needles enable collection without continuously disturbing the pigs. Serial samples were withdrawn from sheep (.10 to .30 ml) for up to 3 weeks with no problems, and from pigs (.05 to .15 ml) for 8 to 12 days, until the cannulae failed.

Distribution of deoxynivalenol in cerebral spinal fluid following administration to swine and sheep.

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi. In evaluating DON as a potent CNS (emetic, anorexic) agent, its cerebral spinal fluid (CSF) and plasma pharmacokinetics were studied in pigs, a species very sensitive to the effects of DON, and sheep, a more tolerant animal. After intravenous administration, DON was detected very rapidly (less than 2.5 min) in the CSF of both species, but whereas peak levels (t-max) occurred at 5-10 min in sheep, in swine it was 30-60 min. It would appear that the very rapid and extensive tissue distribution of DON in swine (Vd gamma = 1.13 1 kg-1) may be slowing the rate of diffusion of the toxin into the CSF compared to sheep (Vd beta = 0.19 1 kg-1) where the toxin is confined essentially to the extracellular compartment. Area under curve calculations indicate approximately 2 1/2 times the amount of toxin eventually reaches the pig CSF compared to sheep CSF. A good relationship between blood-CSF DON levels was apparent in both species, although limitations in detection methods made it impossible to resolve a slow terminal phase (gamma) in swine CSF which was evident in the plasma profile after iv administration. Following oral administration of DON to pigs, a close correlation between plasma and CSF DON levels was observed. The toxin could be detected in CSF for up to 20 hr post-dosing.

Evidence of high concentrations of melatonin in lateral ventricular cerebrospinal fluid of sheep.

In this study we examined the cerebrospinal fluid (CSF) for regional differences in the concentration of melatonin. Cerebrospinal fluid samples were collected at hourly intervals from either the lateral ventricle or the cisterna magna of nine Merino ewes and were compared against jugular plasma. The study revealed that the CSF of the cisterna magna and the lateral ventricle had temporal patterns of melatonin that were similar to those found in jugular plasma. However, the concentrations of melatonin within the CSF obtained from the lateral ventricle were one order of magnitude higher than those of the jugular plasma, as verified by gas chromatography and mass spectrometry, while the melatonin concentrations within the CSF obtained from the cisterna magna were comparable to those of the jugular plasma. The data from this study suggest that there may be regional differences in the concentration of melatonin within the CSF and indicate that this medium is an important route of transport for melatonin from the pineal gland to putative target tissues.