Michael Mäder

Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

S-100 protein concentration in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Abstract We evaluated S-100 levels in paired cerebrospinal fluid (CSF) and serum samples in a group of 135 patients referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit from June 1993 to May 1995. The patients were seen in a prospective case control study. The diagnosis of probable CJD during life was made in any patient presenting with rapidly progressive dementia of less than 2 years’ duration, typical periodic sharp wave complexes (PSWCs) in the EEG and at least two of the following findings: myoclonus, visual/or cerebellar symptoms, pyramidal and/or extrapyramidal signs and/or akinetic mutism. Patients presenting with the above clinical signs and symptoms but without PSWCs were classified as possible, while those with a dementia of a duration exceeding 2 years and without PSWCs were classified as other. S-100 was determined in paired CSF and serum samples by a commercially available enzyme-linked immunosorbent assay. In a group of 76 patients with definite and probable CJD, S-100 concentration (median 25 ng/ml, range 2–117) in CSF was significantly higher (P < 0.0001) than in 32 patients diagnosed as other (median 4 ng/ml, range 1–19). Serum levels of S-100 were below 0.5 ng/ml in all groups. At a cut-off of 8 ng/ml an optimum sensitivity of 84.2% with a specificity of 90.6% for the diagnosis of CJD by the determination of S-100 in CSF is obtained. S-100 levels exceeding 8 ng/ml in CSF support the diagnosis of CJD in any patient presenting with rapidly progressive dementia.

Matrix metalloproteinase-9 is elevated in serum of patients with amyotrophic lateral sclerosis

SMatrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), were analysed by enzyme-linked immunosorbent assay (ELISA) and by zymography in serum and cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). In contrast to patients with inflammatory diseases, MMP-9 levels were not elevated in CSF of ALS patients. In serum, however, compared to healthy donors, MMP-9 was significantly (p = 0.0003) increased up to levels as high as those of viral meningoence-phalitis (VM) or bacterial meningitis (BM) patients. MMP-9 levels remained elevated during long-term observation of ALS patients. In the absence of an inflammatory response, the results indicate that the increase of MMP-9 in serum of ALS patients might be caused by upregulation of MMP-9 in denervated muscles or in degenerating peripheral nerves following motor neurone loss.

Choroid plexus: the major site of mRNA expression for the β-trace protein (prostaglandin D synthase) in human brain

Expression of beta-trace protein (beta-trace), recently identified as glutathion-independent prostaglandin D synthase (prostaglandin-H2 D-isomerase; EC 5.3.99.2), was localized in paraffin sections of the human brain by in situ hybridization using digoxigenin-labeled antisense cRNA probes. The mRNA for beta-trace was predominantly found in the epithelial cells of the choroid plexus. Hybridization signals were also obtained in some oligodendrocytes, particularly in the white matter. In the leptomeninges, specific signals were found in meningeal macrophages and in single cells of the arachnoid barrier layer. The cells exhibiting hybridization signals with the antisense cRNA probes for beta-trace were identified by counterstaining with antibodies directed against specific cell markers. Additionally, beta-trace mRNA was localized in tubular epithelial and basal cells of the human epididymis and in different cell types within the seminiferous epithelium of the testis.

Matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF): elevated levels are primarily related to CSF cell count

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P<0.0001), but not with CSF/serum albumin ratio (Q(Alb)) (r=0.212; P=0.057), a measure for BBB impairment. Moreover, the CSF/serum MMP-9 ratio (Q(MMP-9)) did not correlate with Q(Alb)(r=0.192; P=0.100). By use of a Boyden chamber, in which granulocytes migrated through a reconstituted basement membrane, it was demonstrated that the MMP-9 concentration in the lower chamber correlated very significantly with the number of accumulated cells (r(2)=0.7692; P<0.0001). The meaning of the increase of MMP-9 in CSF is critically discussed.

Choline acetyltransferase-like immunoreactivity in small diameter neurones of the rat dorsal root ganglion.

In the rat choline acetyltransferase (ChAT)-like immunoreactivity (ChAT-LI) was demonstrated in the dorsal root ganglion (DRG), in the superficial spinal cord and in the subepithelial layer of the ureter using immunohistochemical techniques. In the L1 DRG, 66% of the neurones were ChAT-LI. They did not express neurofilament immunoreactivity (RT97 negative) but could also contain calcitonin gene-related peptide-like immunoreactivity (CGRP-LI). In the superficial spinal cord and in the subepithelial plexus of the ureter--both areas where high numbers of fine afferent fibres have been demonstrated--CGRP-LI and ChAT-LI were co-distributed, indicating that ChAT can be found in the peripheral and central endings of small afferents. The data provide morphological evidence that a substantial proportion of afferent fibres are cholinergic.

Interferon-β-1 b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis

Abstract.Recent reports have shown that matrix-metalloproteinases (MMPs) facilitate T-cell migration into the CNS and play a role in disruption of the blood-brain-barrier and myelin break-down. An increase of MMP-9 serum levels predicts disease activity in relapsing remitting multiple sclerosis (RRMS). Interferon-β (IFN-β), which is an established treatment for RRMS, inhibits T-cell migration in vitro in parallel with the downregulation of MMP expression. Only limited data are available for primary progressive multiple sclerosis (PPMS) which differs in demographic and immunological aspects as well as in MRI criteria from RRMS. In this study, 19 patients with laboratory-supported definite PPMS were treated with 8 x 106 IU IFN-β1b (Betaferon®) subcutaneously every other day. Serum was collected before treatment and on months 1, 2, 3, 6 and 9 during treatment. Levels of MMP-9 and of its natural inhibitor known as tissue-inhibitor of matrix-metalloproteinase-1 (TIMP-1) were quantified by ELISA. In addition MMP-2 serum levels were determined by zymography. 19 healthy volunteers served as controls. Before treatment serum levels of MMP-9 were elevated in patients with PPMS compared with controls, whereas there was no difference in TIMP-1 serum levels. During treatment with IFN- β1b the concentration of MMP-9 in the serum of 18 out of 19 PPMS patients decreased,whereas serum levels of MMP-2 and TIMP-1 remained nearly unaffected. Our results demonstrate that the MMP-9 to TIMP-1 ratio in patients with PPMS is elevated in comparison with healthy controls. The suppression of MMP-9 by IFN-β1b indicates that this drug is immunomodulatory active in PPMS patients. Further studies are necessary to test if IFN-β exerts a beneficial effect in PPMS.

Beta-trace protein concentration in cerebrospinal fluid is decreased in patients with bacterial meningitis

Although meninges represent a major site of biosynthesis, beta-trace protein (beta-trace) has not been studied in the cerebrospinal fluid (CSF) of meningitis patients. We measured beta-trace in lumbar CSF of normal controls (n = 27) and in patients with various neurological diseases (n = 92) by an immunonephelometric assay. The mean concentration of beta-trace in CSF of control patients was 16.6+/-3.6 mg/l. In bacterial meningitis (n = 41), CSF beta-trace was significantly decreased (8.7+/-3.9 mg/l; P< 0.001), whereas in spinal canal stenosis it was elevated (29.2+/-10.3 mg/l; P= 0.002). In viral meningoencephalitis (n = 12), beta-trace CSF concentrations were normal. Beta-trace concentrations remained below the normal range even after curing of bacterial meningitis, and normalisation of CSF leucocytes and blood-CSF barrier function. Beta-trace may be a useful tool for studying the pathophysiology of bacterial meningitis.

Purification and N-terminal sequence of β-trace, a protein abundant in human cerebrospinal fluid

beta-Trace, a protein that represents a major constituent of human cerebrospinal fluid with unknown function has been purified to apparent homogeneity by gel chromatography and electrophoresis. After sodium dodecylsulfate electrophoresis on polyacrylamide gels (SDS-PAGE) and Western blotting, the N-terminal sequence (28 amino acids) has been determined. The high degree of identity with the corresponding sequences of prostaglandin D synthetase from rat (68%) and human (93%) suggests a strong relationship if not identity with this enzyme. Thus, 30 years after its discovery beta-trace might unravel as a well-known protein of the prostaglandin metabolism.

Experimental pneumococcal meningitis in rabbits: the increase of matrix metalloproteinase-9 in cerebrospinal fluid correlates with leucocyte invasion

Gelatinolytic activity of matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, was studied by quantitative zymography in a rabbit model of bacterial meningitis during 24 h after inoculation with Streptococcus pneumoniae. In cerebrospinal fluid (CSF), MMP-2 was constitutively present and its level did not change during the experiment. In contrast, MMP-9, hardly detectable in CSF of healthy animals, increased dramatically. The increase of MMP-9 was correlated with both, an increase of CSF cell count and of total protein concentration. Intrathecal production of MMP-9 and MMP-2 was demonstrated by zymography of equal amounts of total protein from CSF and serum. Homogenates, prepared from various cortical regions of infected rabbits did not show increase of MMP activities. On the other hand, leucocytes isolated from CSF expressed high levels of MMP-9 suggesting a significant contribution of these cells to the elevation of MMP-9 activity in this body fluid.