Henk Veldman

Immunofluorescence microscopy of a myopathy: α-Actinin is a major constituent of nemaline rods

A biopsy of skeletal muscle taken from a child with the clinical symptoms of congenital nemaline myopathy was studied. Light and electron microscopy revealed rod-like structures within the muscle fibres, and thus confirmed the clinical diagnosis. Indirect immunofluorescence, using specific antibodies against actin and desmin (both derived from chicken gizzard) as well as against α-actinin and tropomyosin (both from porcine skeletal muscle) revealed that the rods consist of massive accumulations of α-actinin. Desmin seems to be peripherally associated with the rods. Anti-actin and anti-tropomyosin did not stain the rods; however, a masking effect could not be ruled out. These findings support previous hypotheses that nemaline rods can be taken to be lateral-polymers of normal Z-disks.

Insulin-like and fibroblast growth factors in spinal cords, nerve roots and skeletal muscle of human controls and patients with amyotrophic lateral sclerosis

Insulin-like growth factors (IGF-I and IGF-II) and fibroblast growth factors [acidic FGF (aFGF) and basic FGF (bFGF)] are trophic for motor neurones in vitro and (in laboratory animals) in vivo. An immunohistochemical investigation was performed on the distribution of these factors in the neuromuscular system of control patients and patients with amyotrophic lateral sclerosis (ALS). Comparisons were made with rat tissue. IGF-I immunoreactivity (IGF-I-IR) was seen in motor neurone cell bodies and axons, astroglia and Schwann cells, and in muscle fibres. IGF-II-IR was weak in all these cells. aFGF-IR was present in motor neurone cell bodies and axons, oligodendroglia and muscle fibres, but was not demonstrable in Schwann cells. bFGF-IR was present in motor neurone cell bodies and axons, and in astroglia, but was not seen in Schwann cells or muscle fibres. The distribution of the IGFs and FGFs in material from motor neurone disease (MND) and controls was similar. A role for any of these factors in the etiology of MND is, therefore, unlikely. IGF-I-IR and aFGF-IR were stronger in type II than in type I muscle fibres and were increased in denervated fibres. Species differences were found for IGF-I and bFGF. The function of these factors is apparently not entirely similar in humans and rats.

PRE- AND POSTSYNAPTIC NEUROMUSCULAR JUNCTION ABNORMALITIES IN MuSK MYASTHENIA

Autoantibodies to muscle‐specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre‐ and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti‐MuSK antibodies reduce the stability of muscle–nerve contact. Muscle Nerve, 2010

Oxidant treatment causes a dose-dependent phenotype of apoptosis in cultured motoneurons

Evidence is growing that reactive oxygen species (ROS), by‐products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose‐dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose). J. Neurosci. Res. 54:778–786, 1998.

Rimmed vacuoles and the added value of SMI-31 staining in diagnosing sporadic inclusion body myositis.

Problems in diagnosing sporadic inclusion body myositis may arise if all clinical features fit a diagnosis of polymyositis, but the muscle biopsy shows some rimmed vacuoles. Recently, immunohistochemistry with an antibody directed against phosphorylated neurofilament (SMI-31) has been advocated as a diagnostic test for sporadic inclusion body myositis. The aims of the present study were to define a quantitative criterion to differentiate sporadic inclusion body myositis from polymyositis based on the detection of rimmed vacuoles in the haematoxylin-eosin staining and to evaluate the additional diagnostic value of the SMI-31 staining. Based on clinical criteria and creatine kinase levels in patients with endomysial infiltrates, 18 patients complied with the diagnosis of sporadic inclusion body myositis, and 17 with the diagnosis of polymyositis. A blinded observer counted the abnormal fibres in haematoxylin-eosin-stained sections and in SMI-31-stained sections. The optimal cut-off in the haematoxylin-eosin test was 0.3% vacuolated fibres. Adding the SMI-31 staining significantly increased the positive predictive value from 87 to 100%, but increased the negative predictive value only to small extent. We conclude that (1) patients with clinical and laboratory features of polymyositis, including response to treatment, may show rimmed vacuoles in their muscle biopsy and that (2) adding the SMI-31 stain can be helpful in differentiating patients who respond to treatment from patients who do not.

Nerve growth factor receptor immunostaining in the spinal cord and peripheral nerves in amyotrophic lateral sclerosis.

SummaryIn animal experiments, nerve transection is followed by expression of nerve growth factor receptors (NGFR) on Schwann cells of both motor and sensory nerve fibres distally to the site of the lesion. To determine whether denervated Schwann cells in amyotrophic lateral sclerosis (ALS) similarly express NGFR, a study was made of post-mortem material of peripheral nerves and ventral roots from ALS cases and age-matched controls, using immunolabelling methods. Dorsal roots and spinal cords were also examined for the presence of NGFR. In all the ALS cases and controls, NGFR immunostaining was seen in the outer layer of vessel walls, perineurial sheaths, connective tissue surrounding fascicles in nerve roots and in the substantia gelatinosa of the spinal cord. In ALS, NGFR staining was also present in the Schwann cells of degenerated nerve fibres in mixed peripheral nerves, in ventral roots and, to a lesser extent, in dorsal roots. NGFR immunoreactivity was also seen in elongated cells extending from the perifascicular connective tissue into the nerve fascicles. It is concluded that denervated Schwann cells in ALS express NGFR and that NGFR immunostaining on Schwann cells may be used as an indicator of axonal degeneration. The NGFR labelling in the dorsal roots supports the notion that ALS is not a pure motor syndrome.

Absence of characteristic features in two patients with inclusion body myositis

According to recently published criteria a diagnosis of definite sporadic inclusion body myositis is made if the typical histopathological abnormalities (rimmed vacuoles and abnormal accumulations of proteins, in addition to mononuclear cell infiltrates) are present. The two women described here presented with myositis which was unresponsive to treatment. Patient 1 had features of non-progressive sporadic inclusion body myositis clinically, whereas patient 2 had a very slowly progressive limb girdle syndrome. The cryostat sections of the first biopsies did not show rimmed vacuoles, even in retrospect. Only a repeated biopsy, 12 years after presentation in one patient and 18 years after presentation in the other, disclosed the typical features of sporadic inclusion body myositis. The initial absence of abnormal fibres probably represents a real absence or scarcity rather then a sampling error due to a multifocal nature of the histological abnormalities. It is of importance for the clinician to realise that some patients with myositis unresponsive to treatment, even if both clinical and histological features do not suggest sporadic inclusion body myositis, may prove to have the disease on repeated histopathological examination.

Sequence of motor nerve terminal involvement in acrylamide neuropathy.

SummaryAcrylamide neuropathy is characterized by distal multifocal axonal degeneration. In this condition long and large myelinated fibers are affected more than short and thin fibers. The purpose of the present study was to investigate the sequence of nerve terminal involvement. The study was limited to axons that belonged to one type of neuron, of approximately equal diameter but differing in length. Axon terminals from α motor neurons were investigated in five muscles from rats. The results show that the initial motor nerve terminal degeneration is widespread and not restricted to terminals of the longest axons with the largest volumes. It is suggested that the variation in degree of involvement of the motor nerve terminals is determined both by differences between endplates and the regenerative capacity of neurons.

Collagen IV deposits do not prevent regrowing axons from penetrating the lesion site in spinal cord injury.

Scarring is suggested to impede axon regrowth across the lesion site in the injured adult mammalian central nervous system. Collagen Type IV, as a major component of the scar formed after injury, is an impediment for successful axonal regeneration and a decrease in its amount is a prerequisite for regrowing axons to cross the lesion in the postcommissural fornix in the injured adult rat (Stichel et al. [1999] Neurosci. 93:321–333). The aim of the present study was to analyze the relationship between collagen IV deposits and regrowing axons at various times after dorsal hemi‐section of the adult rat spinal cord. Immunohistochemical double staining revealed that penetrating neurofilament‐positive axons and collagen IV deposits were co‐localized in the lesion site in the initial stages of axonal sprouting (between 7 and 14 days post‐operatively) and were still present 1 and 2 months post‐operatively. Interestingly, collagen IV‐immunoreactive areas located around cystic cavities formed at the site of injury 1 month post‐operatively, were devoid of axons. In conclusion, our observations indicate that collagen IV deposits after spinal cord injury do not prevent neurofilament‐positive regrowing axons from penetrating the lesion site. J. Neurosci. Res. 62:686–691, 2000.

Calcitonin gene-related peptide-like immunoreactivity, in botulinum toxin-paralysed rat muscles

Changes in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) at the motor endplates of botulinum toxin-paralysed rat muscles were investigated using immunohistochemistry. One day following toxin injection, a dramatic increase in CGRP-LI was detected at the motor endplates and within preterminal axons of the soleus and gastrocnemius muscles. The upregulation of CGRP-LI persisted throughout the period during which muscle fibres were paralysed and new neuromuscular junctions were being formed by the growing sprouts. Decline of CGRP-LI at the motor endplates coincided with clinical recovery. Both up- and down-regulation of CGRP-LI took place earlier in the soleus than in the gastrocnemius muscle. Up-regulation of CGRP-LI was also detected in a subpopulation of motor axons in the sciatic nerves and in the spinal motor neurons innervating the paralysed muscles. These results indicate that levels of CGRP are regulated, at least partly, by changes in the target innervation. They also suggest an important role for CGRP in the regenerative processes following muscle paralysis.