Cerebrospinal Fluid Research

The subcommissural organ (SCO) is a highly conserved brain gland present throughout the vertebrate phylum; it secretes glycoproteins into the cerebrospinal fluid (CSF), where they aggregate to form Reissner's fiber (RF). SCO-spondin is the major constituent protein of RF. Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. The aims of the present investigation were: (i) to identify and partially characterize the SCO-secretory compounds present in the SCO gland itself and in the RF of the Sprague-Dawley rat and non-hydrocephalic hyh mouse, and in the CSF of rat; (ii) to make a comparative analysis of the proteins present in these three compartments; (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods.

The proteins of the SCO secreted into the CSF were studied (i) by injecting specific antibodies into ventricular CSF in vivo; (ii) by immunoblots of SCO, RF and CSF samples, using specific antibodies against the SCO secretory proteins (AFRU and anti-P15). In addition, the glycosylated nature of SCO-compounds was analysed by concanavalin A and wheat germ agglutinin binding. To analyse RF-glycoproteins, RF was extracted from the central canal of juvenile rats and mice; to investigate the CSF-soluble proteins secreted by the SCO, CSF samples were collected from the cisterna magna of rats at different stages of development (from E18 to PN30).

Five glycoproteins were identified in the rat SCO with apparent molecular weights of 630, 450, 390, 320 and 200 kDa. With the exception of the 200-kDa compound, all other compounds present in the rat SCO were also present in the mouse SCO. The 630 and 390 kDa compounds of the rat SCO have affinity for concanavalin A but not for wheat germ agglutinin, suggesting that they correspond to precursor forms. Four of the AFRU-immunoreactive compounds present in the SCO (630, 450, 390, 320 kDa) were absent from the RF and CSF. These may be precursor and/or partially processed forms. Two other compounds (200, 63 kDa) were present in SCO, RF and CSF and may be processed forms. The presence of these proteins in both, RF and CSF suggests a steady-state RF/CSF equilibrium for these compounds. Eight AFRU-immunoreactive bands were consistently found in CSF samples from rats at E18, E20 and PN1. Only four of these compounds were detected in the cisternal CSF of PN30 rats. The 200 kDa compound appears to be a key compound in rats since it was consistently found in all samples of SCO, RF and embryonic and juvenile CSF.

It is concluded that (i) during the late embryonic life, the rat SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. The possibility they are involved in brain development has been discussed.

Diffusion tensor imaging (DTI) is a non-invasive MRI technique that has been used to quantify CNS abnormalities in various pathologic conditions. This study was designed to quantify the anisotropic diffusion properties in the brain of neonatal rats with hydrocephalus (HCP) and to investigate association between DTI measurements and cytopathology.

DTI data were acquired between postnatal day 7 (P7) and P12 in 12 rats with HCP induced at P2 and in 15 age-matched controls. Animals were euthanized at P11 or P22/P23 and brains were processed with immunohistochemistry for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule (Iba-1), and luxol fast blue (LFB) to assess astrocytosis, microglial reactivity and degree of myelination, respectively.

Hydrocephalic rats were consistently found to have an abnormally low (at corrected p-level of <0.05) fractional anisotropy (FA) value and an abnormally high mean diffusivity (MD) value in the cerebral cortex (CX), the corpus callosum (CC), and the internal capsule (IC). Immunohistochemical analysis demonstrated trends of increasing astrocyte and microglial reactivity in HCP rats at P11 that reached statistical significance at P22/P23. A trend toward reduced myelination in the HCP rats was also found at P22/P23. Correlation analysis at P11 for the CC demonstrated statistically significant correlations (or trends) between the DTI measurement (the decreased FA and increased MD values) and the GFAP or Iba-1 rankings. The immunohistochemical rankings in the IC at P22/P23 were also significantly correlated or demonstrated a trend with both FA and MD values.

This study demonstrates the feasibility of employing DTI on the brain in experimental hydrocephalus in neonatal rats and reveals impairments in multiple regions of interest in both grey and white matter. A strong correlation was found between the immunohistochemical results and the changes in anisotropic diffusion properties.

Adjustable shunts are very popular in the management of hydrocephalus and are believed to help in minimizing the number of surgical revisions. The drawback with almost all constructions is that they may be accidentally readjusted in relatively weak magnetic fields (around 30–40 mTesla)

The ProGav Miethke shunt is composed of an adjustable ballon-spring valve unit and an integrated over-drainage compensating gravitational device (known as the shunt assistant). A mechanical 'brake' is intended to prevent changes to the valve's performance level in a strong magnetic field. We evaluated the performance and hydrodynamic properties of a sample of three valves in the UK Shunt Evaluation Laboratory.

All the shunts showed good mechanical durability over the three-month period of testing, and good stability of hydrodynamic performance over a one-month period

The pressure-flow performance curves, operating, opening and closing pressures fell within the limits specified by the manufacturer, and changed according to the programmed performance levels. The operating pressure increased when the shunt assistant was in the vertical position, as specified. The valve has a low hydrodynamic resistance (0.53 mm mmHg ml^-1 min^-1). External programming proved to be easy and reliable. Strong magnetic fields from a 3 Tesla MR scanner were not able to change the programming of the valve.

The ProGAV shunt is an adjustable, low resistance valve that is able to limit posture-related over-drainage. Unlike other adjustable valves, the ProGAV cannot be accidentally re-adjusted by external magnetic field such as a 3T MR scanner.

Prostaglandin E₂ (PGE₂) is involved in the regulation of synaptic activity and plasticity, and in brain maturation. It is also an important mediator of the central response to inflammatory challenges. The aim of this study was to evaluate the ability of the tissues forming the blood-brain interfaces to act as signal termination sites for PGE₂ by metabolic inactivation.

The specific activity of 15-hydroxyprostaglandin dehydrogenase was measured in homogenates of microvessels, choroid plexuses and cerebral cortex isolated from postnatal and adult rat brain, and compared to the activity measured in peripheral organs which are established signal termination sites for prostaglandins. PGE₂ metabolites produced ex vivo by choroid plexuses were identified and quantified by HPLC coupled to radiochemical detection.

The data confirmed the absence of metabolic activity in brain parenchyma, and showed that no detectable activity was associated with brain microvessels forming the blood-brain barrier. By contrast, 15-hydroxyprostaglandin dehydrogenase activity was measured in both fourth and lateral ventricle choroid plexuses from 2-day-old rats, albeit at a lower level than in lung or kidney. The activity was barely detectable in adult choroidal tissue. Metabolic profiles indicated that isolated choroid plexus has the ability to metabolize PGE₂, mainly into 13,14-dihydro-15-keto-PGE₂. In short-term incubations, this metabolite distributed in the tissue rather than in the external medium, suggesting its release in the choroidal stroma.

The rat choroidal tissue has a significant ability to metabolize PGE₂ during early postnatal life. This metabolic activity may participate in signal termination of centrally released PGE₂ in the brain, or function as an enzymatic barrier acting to maintain PGE₂ homeostasis in CSF during the critical early postnatal period of brain development.

Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture.

To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [¹⁴C] adenosine transport across this cellular layer, [¹⁴C] adenosine metabolism inside the cells, and cellular uptake of [¹⁴C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts.

CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 ± 9 Ω cm² at day 8, while permeability towards [¹⁴C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [¹⁴C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion.

However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [¹⁴C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [¹⁴C] adenosine in the donor chamber was mostly present as [¹⁴C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [¹⁴C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.