Wolfgang Beuche

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.

Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on silica-sensitive,bg/bg-negative and Fc-positive monocytes

Previous experiments with nerves enclosed in millipore diffusion chambers had shown that myelin degradation during Wallerian degeneration depends on invasion by non-resident cells. The present study was aimed at a more precise identification of the invading cell population. Monoclonal antibody studies of degenerating nerves showed many cells with the Fc marker; cells having the Lyt-1, Lyt-2, Ia or Mac-1 markers were sparse or absent. Nerves transplanted into mice of the Chediak-Higashi bg/bg strain were invaded by cells lacking the bg/bg marker (giant lysosomes), while cotransplanted muscle tissue was invaded by cells with the bg/bg marker. Blocking monocytes with silica reduced both cell invasion and myelin degradation in degenerating nerves. These observations show that Wallerian degeneration of peripheral nerve fibers involves a subset of monocytes which are silica-sensitive and have Fc receptors but no bg/bg giant lysosomes.

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.

A New Approach Toward Analyzing Peripheral Nerve Fiber Populations. I. Variance in Sheath Thickness Corresponds to Different Geometric Proportions of the Internodes

The thickness of the myelin sheath is known to increase with axon caliber, but there is also a superimposed, slight variation in sheath thickness depending on whether a fiber of a given caliber has very long or very short internodes. This relationship between myelin sheath thickness and the geometric proportion of the internode has been shown in subserial sections of isolated nerve fibers. It allows a prediction of sheath thickness from the quotient internode length/axon caliber, or conversely, a prediction of internode foreshortening from sheath thickness. We applied this new approach to the analysis of sciatic fiber populations of frogs, mice, rats and cats. The geometric proportions of these fibers were defined by the quotient internode length/fiber caliber. This quotient was compared with minor variation in sheath thickness as determined with a computer-assisted technique measuring large numbers of fibers in low-power electron micrographs. The method also calculated fiber shrinkage and recalculated all data for circular fiber profiles. The data obtained confirmed previous electron microscopic measurements showing that there is a slight reduction in sheath thickness when a fiber of a given caliber has relatively short internodes, and vice versa. A population of very thin, thinly myelinated fibers was also revealed. Sheath thickness and the geometric proportions of internodes in frogs differed markedly from those in mammals.

Intracortical excitability in the hand motor representation in hand dystonia and blepharospasm

We sought to determine the activity of inhibiting and facilitating cortical circuits in areas surrounding a hand muscle motor representation in focal dystonia and in controls. In 15 patients with hand dystonia, 16 patients with blepharospasm, and age‐matched controls, we applied suprathreshold transcranial magnetic stimuli with a figure‐eight coil over the optimal representation of the relaxed abductor digiti minimi muscle of the dominant hand. Additional conditioning stimuli were given through a second figure‐eight coil that was held either above the test coil or 2 cm or 4 cm apart in the anterior, posterior, lateral, or medial direction. We measured intracortical excitability in each of the nine positions of the conditioning coil. Intracortical inhibition was reduced in both patient groups at all conditioning coil positions. With both coils centered, the intracortical facilitation did not differ between patients and controls. After shifting the conditioning coil, the intracortical facilitation tended to be less diminished in patients than in controls, this difference between patients and controls was significant for the anterior, posterior, and medial 4‐cm conditioning coil shift. Our results demonstrate decreased intracortical inhibition in the cortical hand muscle representation not only in patients with hand dystonia, but also in patients with blepharospasm. In addition, our findings in both patient groups show a trend toward a relatively increased intracortical facilitation in surrounding motor areas.

A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness.

The new approach used in this study is based on the concept that axon caliber is not the only factor affecting the thickness of the myelin sheath. It is necessary to consider the entire geometric proportions of the internode, since sheath thickness corresponds to the relationship between axon caliber and the length of the internode. This type of analysis was applied to the regenerated internodes in rat sciatic nerves. Survival periods of 4, 9, 18 and 36 weeks were studied after lesions had been placed in young adult rats. The data show significantly thinner sheaths for regenerated fibers as compared with normal nerves, consistent with previous observations. This reduction in sheath thickness, however, corresponded quantitatively to the degree of foreshortening of internodes in the regenerated nerves. An average reduction of 10 in the quotient internode length/fiber caliber corresponded to a reduction of about 0.015 in the relative thickness of the sheath (quotient axon diameter/fiber diameter). This means that regenerated myelin sheaths are not truly hypoplastic; rather, they are adapted to the reduced internode length, and have the same relationship found for normal fibers. In partially damaged nerves there was a clear distinction in terms of sheath thickness between regenerated fibers and undamaged fibers. Demonstration of this phenomenon by scatter diagrams opens new possibilities for the quantitative assessment of neuropathies.

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.

Riluzole does not have an acute effect on motor thresholds and the intracortical excitability in amyotrophic lateral sclerosis

Intracortical excitability in amyotrophic lateral sclerosis (ALS) is impaired. The effectiveness of the glutamate antagonist riluzole (Rilutek®, Rhône-Poulenc Rorer) in ALS has been shown in clinical studies. In healthy subjects it modifies intracortical excitability in a frequently used double-stimulus paradigm of transcranial magnetic stimulation (TMS). Under riluzole intracortical inhibition is enhanced in healthy individuals, although not always significantly, whereas intracortical facilitation has been described as reduced [10, 11]. We wanted to find out whether riluzole affects and potentially rebalances impaired intracortical excitability in ALS. We, therefore, enrolled 13 patients with clinically and electromyographically confirmed ALS into this study. Five patients had to be excluded because motor thresholds were too high to get reliable motor evoked potentials (MEPs). In the remaining 8 patients, mean age was 59.9±11.9 years (± standard deviation) and mean symptom duration 9.6±2.5 months. Intracortical excitability was assessed before and 1.5 hours after the first intake of a loading dose of 100 mg of riluzole using a conventional paired-pulse TMS paradigm with interstimulus intervals (ISI) ranging from 1–30 ms and intensities adjusted to yield MEPs of 1.0 mV for test pulses and of 90% active motor threshold for conditioning pulses. Patients’ baseline results were compared to those of 9 age-matched, healthy control subjects. Before drug intake, motor thresholds did not differ between groups, but there was significantly less intracortical inhibition in the ALS patient group. Riluzole intake did not significantly alter motor thresholds or intacortical excitability in the ALS patients. We conclude that riluzole does not immediately influence intracortical excitability in ALS. Our results are in contrast to the findings of Stefan et al (1998) [14] where a partial normalization of intracortical inhibition in ALS was observed after at least 5 days of drug intake. The difference between that study and our result may indicate a delayed onset of riluzole’s influence on intracortical excitability.

Decrease of S100 beta protein in serum of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin characterized by loss of upper and lower motor neurons and concomitant astrogliosis. We have investigated the S100 beta protein levels in serum as a marker for astroglia of patients with ALS (n = 41) in comparison to a control group (n = 32). Additionally we have investigated 12 patients at different follow-up time points (minimum 6 months). We could not observe a significant difference of S100 beta protein in patients with ALS in comparison to our control group (P = 0.11) but we could clearly see a decrease of S100 beta levels in the further course of the disease. As S100 beta is also seen as a protein with nerve growth factor activity we assume that the fall of serum levels may reflect the loss of nerve growth stimulation in patients with ALS and suppose that repetitive measurements of S100 beta in serum can be used as an objective marker for disease progression.

Chronic Enteral Poisoning Caused by Potassium Permanganate: A Case Report

To our knowledge, this is the first case report of a multiple, low dosage ingestion of manganese. A 66-year-old male patient is presented, who ingested 125 ml of a 8% solution of potassium permanganate (10 g) within 4 weeks. As early as 2 weeks after the beginning of poisoning, psychological alterations were noted. Neurological examination revealed disturbances of many subsystems of the CNS. Visually evoked potentials showed prolongation of the P2-latency, not reported in earlier publications. Levels for manganese were elevated in peripheral blood as well as in hair samples. Treatment with calcium trisodium pentetate decreased serum levels and increased urine excretion of manganese. Nine months after poisoning, the first signs of progressive Parkinson disease became evident. The time-course of neurological symptoms seems to depend on a critical dose of manganese.