Simple methods for cerebrospinal fluid collection in fetal, neonatal, and adult rat.

BACKGROUND: Cerebrospinal fluid (CSF) collection and its analysis are common medical practices useful in the diagnosis, therapy, and prevention of central nervous system (CNS) disorders. In recent years, several types of research have improved our insight into CSF and its role in health and disease. Yet, many characteristics of this fluid remain to be fully understood. NEW METHODS: Here, we describe how to collect CSF from embryonic, postnatal, and adult stages of the rat. In adults, CSF can be collected through simple stereotaxic surgery to expose the membrane overlying the cisterna magna (CM) of an anesthetized rat and collection of CSF through micropipette puncture through the membrane. In embryos and pups, CSF is aspirated, using a fire-polished micro-capillary pipette, from the CM of animals. RESULTS: Application of these methods provides the maximum volume of pure, uncontaminated CSF (embryonic day 19: 10-15 microliter, postnatal day 5: 20-30 microliter, adults: 100-200 microliter) with a success rate of approximately 95% in every age. COMPARISON WITH EXISTING METHODS: Compared to the existing protocols, these methods obtain considerable volumes of CSF, which may accelerate the measurement of biological markers in this fluid. Also, these techniques do not require surgical skills and according to the practical points mentioned during sampling, the procedures can be performed in rapid fashion. CONCLUSION: We describe simple methods for collecting CSF in live rats. These protocols provide clean, uncontaminated CSF for experiments to understand the exact role of this fluid in the development and maintenance of the CNS health.

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

The beneficial effects of chick embryo extract preconditioning on hair follicle stem cells: A promising strategy to generate Schwann cells.

The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.

The Beneficial Potential of Genetically Modified Stem Cells in the Treatment of Stroke: a Review.

The last two decades have witnessed a surge in investigations proposing stem cells as a promising strategy to treat stroke. Since growth factor release is considered as one of the most important aspects of cell-based therapy, stem cells over-expressing growth factors are hypothesized to yield higher levels of therapeutic efficiency. In pre-clinical studies of the last 15 years that were investigating the efficiency of stem cell therapy for stroke, a variety of stem cell types were genetically modified to over-express various factors. In this review we summarize the current knowledge on the therapeutic efficiency of stem cell-derived growth factors, encompassing techniques employed and time points to evaluate. In addition, we discuss several types of stem cells, including the recently developed model of epidermal neural crest stem cells, and genetically modified stem cells over-expressing specific factors, which could elevate the restorative potential of naive stem cells. The restorative potential is based on enhanced survival/differentiation potential of transplanted cells, apoptosis inhibition, infarct volume reduction, neovascularization or functional improvement. Since the majority of studies have focused on the short-term curative effects of genetically engineered stem cells, we emphasize the need to address their long-term impact.

The blood-CSF-brain route of neurological disease: The indirect pathway into the brain.

The brain is protected by the endothelial blood-brain barrier (BBB) that limits the access of micro-organisms, tumour cells, immune cells and autoantibodies to the parenchyma. However, the classic model of disease spread across a disrupted BBB does not explain the focal distribution of lesions seen in a variety of neurological diseases and why lesions are frequently adjacent to the cerebrospinal fluid (CSF) spaces. We have critically reviewed the possible role of a blood-CSF-brain route as a disease entry pathway into the brain parenchyma. The initial step of this pathway is the transfer of pathogens or immune components from the blood into the CSF at the choroid plexuses, where the blood-CSF barrier (BCSFB) is located. The flow of CSF results in disease dissemination throughout the CSF spaces. Access to the brain parenchyma from the CSF can then occur across the ependymal layer at the ventricular surface or across the pial-glial barrier of the subarachnoid space and the Virchow-Robin spaces. We have reviewed the anatomy and physiology of the blood-CSF-brain pathway and the brain barriers controlling this process. We then summarised the evidence supporting this brain entry route in a cross-section of neurological diseases including neuromyelitis optica, multiple sclerosis, neurosarcoidosis, neuropsychiatric lupus, cryptococcal infection and both solid and haematological tumours. This summary highlights the conditions that share the blood-CSF-brain pathway as a pathogenetic mechanism. These include the characteristic proximity of lesions to CSF, evidence of disruption of the brain barriers and the identification of significant pathology within the CSF. An improved understanding of pathological transfer through the CSF and across all brain barriers will inform on more effective and targeted treatments of primary and secondary diseases of the central nervous system.

Altered folate binding protein expression and folate delivery are associated with congenital hydrocephalus in the hydrocephalic Texas rat.

Hydrocephalus (HC) is an imbalance in cerebrospinal fluid (CSF) secretion/absorption resulting in fluid accumulation within the brain with consequential pathophysiology. Our research has identified a unique cerebral folate system in which depletion of CSF 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is associated with cortical progenitor cell-cycle arrest in hydrocephalic Texas (H-Tx) rats. We used tissue culture, immunohistochemistry, in-situ PCR and RT-PCR and found that the in-vitro proliferation of arachnoid cells is highly folate-dependent with exacerbated proliferation occurring in hydrocephalic CSF that has low FDH but high folate-receptor-alpha (FRα) and folate. Adding FDH to this CSF prevented aberrant proliferation indicating a regulatory function of FDH on CSF folate concentration. Arachnoid cells have no detectable mRNA for FRα or FDH, but FDH mRNA is found in the choroid plexus (CP) and CSF microvesicles. Co-localization of FDH, FRα and folate suggests important functions of FDH in cerebral folate transport, buffering and function. In conclusion, abnormal CSF levels of FDH, FRα and folate inhibit cortical cell proliferation but allow uncontrolled arachnoid cell division that should increase fluid absorption by increasing the arachnoid although this fails in the hydrocephalic brain. FDH appears to buffer available folate to control arachnoid proliferation and function.

In Vitro Effects of Wistar Rat Prenatal and Postnatal Cerebrospinal Fluid on Neural Differentiation and P roliferation of Mesenchymal Stromal Cells Derived from Bone Marrow.

OBJECTIVES: Cerebrospinal fluid (CSF) plays an important role in cortical development during the fetal stages. Embryonic CSF (E-CSF) consists of numerous neurotrophic and growth factors that regulate neurogenesis, differentiation, and proliferation. Mesenchymal stem cells (MSCs) are multi-potential stem cells that can differentiate into mesenchymal and non-mesenchymal cells, including neural cells. This study evaluates the prenatal and postnatal effects of CSF on proliferation and neural differentiation of bone marrow MSCs (BM-MSCs) at gestational ages E19, E20, and the first day after birth (P1). MATERIALS AND METHODS: In this experimental study, we confirmed the mesenchymal nature of BM-MSCs according to their adherence properties and surface markers (CD44, CD73 and CD45). The multi-potential characteristics of BMMSCs were verified by assessments of the osteogenic and adipogenic potentials of these cells. Under appropriate in vitro conditions, the BM-MSCs cultures were incubated with and without additional pre- and postnatal CSF. The MTT assay was used to quantify cellular proliferation and viability. Immunocytochemistry was used to study the expression of MAP-2 and ß-III tubulin in the BM-MSCs. We used ImageJ software to measure the length of the neurites in the cultured cells. RESULTS: BM-MSCs differentiated into neuronal cell types when exposed to basic fibroblast growth factor (b-FGF). Viability and proliferation of the BM-MSCs conditioned with E19, E20, and P1 CSF increased compared to the control group. We observed significantly elevated neural differentiation of the BM-MSCS cultured in the CSF-supplemented medium from E19 compared to cultures conditioned with E20 and P1 CSF group. CONCLUSIONS: The results have confirmed that E19, E20, and P1 CSF could induce proliferation and differentiation of BM-MSCs though they are age dependent factors. The presented data support a significant, conductive role of CSF components in neuronal survival, proliferation, and differentiation.

Fetal Cerebrospinal Fluid Promotes Proliferation and Neural Differentiation of Stromal Mesenchymal Stem Cells Derived from Bone Marrow

ABSTRACT Embryonic cerebrospinal fluid (E-CSF) contains many neurotrophic and growth factors, acts as a growth medium for cortical progenitors, and can modulate proliferation and differentiation of neural stem cells. Mesenchymal stem cells (MSCs) are multipotential stem cells that can differentiate into several types of mesenchymal cells as well as nonmesenchymal cells, such as neural cells. In the present study, the effect of E-CSF on proliferation and neural differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was investigated to test whether E-CSF is capable of driving these cells down the neuronal line. To verify the multipotential characteristics of BM-MSCs, the cells were analyzed for their osteogenic and adipogenic potential. Expression of the neural markers, MAP-2 and β-III tubulin, was determined by Immunocytochemistry. BM-MSCs differentiate into neuronal cell types when exposed to b-FGF. BMMSCs cells cultured in medium supplemented with CSF showed significantly elevated proliferation relative to control cells in media alone. E-CSF (E17-E19) supports viability and stimulates proliferation and, significantly, neurogenic differentiation of BM-MSCs. The data presented support an important role for CSF components, specifically neurotrophic factors, in stem cell survival, proliferation and neuronal differentiation. It is crucial to understand this control by CSF to ensure success in neural stem cell therapies.

Neonatal hydrocephalus is a result of a block in folate handling and metabolism involving 10-formyltetrahydrofolate dehydrogenase.

Folate is vital in a range of biological processes and folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus (HC). 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion for the supply of 1-carbon groups. In previous studies, we found a deficiency of FDH in CSF associated with the developmental deficit in congenital and neonatal HC. In this study, we therefore aimed to investigate the role of FDH in folate transport and metabolism during the brain development of the congenital hydrocephalic Texas (H-Tx) rat and normal (Sprague-Dawley) rats. We show that at embryonic (E) stage E18 and E20, FDH-positive cells and/or vesicles derived from the cortex can bind methyl-folate similarly to folate receptor alpha, the main folate transporter. Hydrocephalic rats expressed diminished nuclear FDH in both liver and brain at all postnatal (P) ages tested (P5, P15, and P20) together with a parallel increase in hepatic nuclear methyl-folate at P5 and cerebral methylfolate at P15 and P20. A similar relationship was found between FDH and 5-methyl cytosine, the main marker for DNA methylation. The data indicated that FDH binds and transports methylfolate in the brain and that decreased liver and brain nuclear expression of FDH is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and neonatal HC. Folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus. 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion. We show that FDH binds and transports methylfolate in the brain. Moreover, we found that a deficiency of FDH in the nucleus of brain and liver is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and neonatal hydrocephalus cells.

Central neural activation following contact sensitivity peripheral immune challenge: evidence of brain-immune regulation through C fibres.

This study tested the hypothesis that peripheral immune challenges will produce predictable activation patterns in the rat brain consistent with sympathetic excitation. As part of examining this hypothesis, this study asked whether central activation is dependent on capsaicin-sensitive C-fibres. We induced skin contact sensitivity immune responses with 2,4-dinitrochlorobenzene (DNCB), in the presence or absence of the acute C-fibre toxin capsaicin (8-methyl-N-vanillyl-6-nonenamide) to trigger immune responses with and without diminished activity of C-fibres. Innovative blood-oxygen-level-dependent functional magnetic resonance imaging data revealed that the skin contact sensitivity immune responses induced with DNCB were associated with localized increases in brain neuronal activity in treated rats. This response was diminished by pre-treatment with capsaicin 1 week before scans. In the same animals, we found expression of the immediate early gene c-Fos in sub-regions of the amygdala and hypothalamic sympathetic brain nuclei. Significant increases in c-Fos expression were found in the supraoptic nucleus, central amygdala and medial habenula following immune challenges. Our results support the idea that selective brain regions, some of which are associated with sympathetic function, process or modulate immune function through pathways that are partially dependent on C-fibres. Together with previous studies demonstrating the motor control pathways from brain to immune targets, these findings indicate a central neuroimmune system to monitor host status and coordinate appropriate host responses.

Fingerprint changes in CSF composition associated with different aetiologies in human neonatal hydrocephalus: inflammatory cytokines.

PURPOSE: Hydrocephalus (HC) has a multifactorial and complex picture of pathophysiology due to aetiology, age at and duration since onset. We have previously identified distinctions in markers of cell death associated with different aetiologies. Here, we examined cerebrospinal fluid (CSF) from human HC neonates for cytokines to identify further distinguishing features of different aetiologies. METHODS: CSF was collected during routine lumbar puncture or ventricular tap from neonates with hydrocephalus, or with no neurological condition (normal controls). Total protein, Fas receptor, Fas ligand, stem cell factor (SCF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin growth factor-1 (IGF-1), tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were measured and compared between 8 unaffected and 28 HC neonatal CSF samples. RESULTS: Total protein was significantly (P < 0.05) raised in late-onset hydrocephalus (LOH). Fas receptor was raised (P < 0.05) in post-haemorrhagic hydrocephalus (PHH) and spina bifida with hydrocephalus (SB/HC), but no difference in Fas ligand was found. SCF was raised (P < 0.05) in SB/HC. HGF was found in all HC and was increased (P < 0.01) in PHH. Increased VEGF was found in PHH (P < 0.01) and SB/HC (P < 0.05). Variable levels of IL-6, TNF-α and IGF-1 were found in all HC groups compared with none in normal. CONCLUSIONS: LOH was unusual with significantly raised total protein indicating an inflammatory state. Increased Fas receptor, VEGF, IGF-1 and HGF suggest anti-apoptotic and repair mechanism activation. By contrast, elevated TNF-α and IL-6 indicate inflammatory processes in these neonatal brains. Taken with our previous study, these data indicate that different pathophysiology, inflammation and repair are occurring in HC of different aetiologies and that additional treatment strategies may benefit these infants in addition to fluid diversion.

CNS inflammation and bone marrow neuropathy in type 1 diabetes.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells.

BACKGROUND: Fetal cerebrospinal fluid (CSF) contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. METHODS: In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and ß-III tubulin) was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. RESULTS: PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. CONCLUSIONS: Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important in vivo for the promotion of normal brain development. There were significant differences in the effects on PC12 cells compared to primary cortical cells. This suggests there is an interaction in vivo between developmental stage of cells and the composition of CSF. The data presented here support an important, perhaps driving role for CSF composition, specifically neurotrophic factors, in neuronal survival, proliferation and differentiation. The effects of CSF on PC12 cells can thus be used to further investigate the role of CSF in driving development without the confounding issues of using primary cells.

The 'scavenger' m7GpppX pyrophosphatase activity of Dcs1 modulates nutrient-induced responses in yeast.

Dcs1, the m7GpppX pyrophosphatase of Saccharomyces cerevisiae, has been reported to 'scavenge' capped 5' end fragments generated by 3'-->5' mRNA degradation. We now show that the absence of Dcs1, and the closely related Dcs2 protein, compromises cellular responses to glucose-deprivation stress as well as to step changes in glucose availability. Dcs1 and Dcs2 form homo- and heterodimers, with the heterodimer appearing as cells enter diauxie. Despite the previously observed increase in abundance of the mRNA encoding the neutral trehalase (Nth1) in the stationary phase, the total enzyme activity of Nth1 decreases in this phase of growth. Changes in trehalase activity are significant because the non-reducing disaccharide trehalose is thought to stabilize cellular components under stress conditions. In the dcs1Delta and dcs1Deltadcs2Delta mutants, normal regulation of trehalase activity is lost. Nutrient stress induces DCS1 and DCS2 transcription via the cAMP-PKA signalling pathway. Dcs1 also becomes phosphorylated as the availability of glucose diminishes, and we test the role of this phosphorylation in the stress response. Further evidence indicates that Dcs1 plays a complementary role to the translation factor eIF4E in preventing capped 5' fragments of mRNA from interfering with translation initiation. We conclude that Dcs1 function influences cellular responses to changes in nutrient avialability, while Dcs2 seems to act as a modulator of Dcs1 function.

The combined application of FTIR microspectroscopy and ToF-SIMS imaging in the study of prostate cancer.

At present. a prognosis for prostate cancer (CaP) is determined by its accurate assessment of disease grade and stage. Histopathological typing using the Gleason grading system is the most universally accepted approach for grading CaP and provides an indication as to the aggressiveness of the tumour at the time of presentation. However, this system is based upon a visual criterion of pattern recognition that is operator dependent and subject to intra- and inter-observer variability, which can result in inappropriate patient management. Thus, there is a need for a molecular based diagnostic technique to grade tissue samples in a reliable and reproducible manner. In this paper we report a prototype diagnostic classifier for Gleason graded CaP tissue, based upon the integration of FTIR microspectroscopy with linear discriminant analysis (LDA). Blind testing of this model demonstrates 80% agreement of FTIR-LDA grade to histology, for the specimens analysed. We also study the effects of connective tissue absorption upon the area ratio of peaks at A1030 cm(-1)/A1080(cm(-1) which we use as a criterion to biospectroscopically map and distinguish areas of benign from malignant tissue. In addition, imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been applied to study freeze-dried, freeze-fractured prostate cancer cells in vitro. Preliminary results demonstrate localisation of various species including K, Ca and Mg within the cytoplasm that are present at millimolar concentrations and vital to cell physiology. The soft ionisation technique employed also permits for molecular information to be obtained and this has been used to evaluate chemically, different fracture planes within the analysis area.