Neuronal involvement in cisplatin neuropathy: prospective clinical and neurophysiological studies.

Although it is well known that cisplatin causes a sensory neuropathy, the primary site of involvement is not established. The clinical symptoms localized in a stocking-glove distribution may be explained by a length dependent neuronopathy or by a distal axonopathy. To study whether the whole neuron or the distal axon was primarily affected, we have carried out serial clinical and electrophysiological studies in 16 males with testicular cancer before or early and late during and after treatment with cisplatin, etoposide and bleomycin at limited (<400 mg/m2 cisplatin), conventional (approximately 400 mg/m2 cisplatin) or high (>400 mg/m2 cisplatin) doses. At cumulative doses of cisplatin higher than 300 mg/m2 the patients lost distal tendon and H-reflexes and displayed reduced vibration sense in the feet and the fingers. The amplitudes of sensory nerve action potentials (SNAP) from the fingers innervated by the median nerve and the dorsolateral side of the foot innervated by the sural nerve were 50-60% reduced, whereas no definite changes occurred at lower doses. The SNAP conduction velocities were reduced by 10-15% at cumulative doses of 400-700 mg/m2 consistent with loss of large myelinated fibres. SNAPs from primarily Pacinian corpuscles in digit 3 and the dorsolateral side of the foot evoked by a tactile probe showed similar changes to those observed in SNAPs evoked by electrical stimulation. At these doses, somatosensory evoked potentials (SEPs) from the tibial nerve had increased latencies of peripheral, spinal and central responses suggesting loss of central processes of large dorsal root ganglion cells. Motor conduction studies, autonomic function and warm and cold temperature sensation remained unchanged at all doses of cisplatin treatment. The results of these studies are consistent with degeneration of large sensory neurons whereas there was no evidence of distal axonal degeneration even at the lowest toxic doses of cisplatin.

Comparative electrophysiological, functional, and histological studies of nerve lesions in rats.

The aim of this study was to establish a nerve lesion model to compare serial electrophysiological and functional outcome measures with histological findings. The relative significance of the parameters in lesions of diverse severity, the time course of recovery, and the tools for serial longitudinal studies after nerve lesions were studied in rats. We compared weekly electrophysiological and functional studies for 100 or 150 days in rats after crush or section/suture of the sciatic nerve at midthigh level. Finally, tibial nerves were taken for histology. We confirmed that recovery was faster and more complete in nerves regenerating after crush than after section, irrespective of method of evaluation. Furthermore, continuous maturational changes occurred in control nerves, and such continuous growth-related changes should be taken into account when evaluating maturational changes during nerve regeneration. A lack of correlation between evaluation methods supports that functional, morphological, and physiological parameters show different aspects of the recovery process after nerve lesions, and that these outcome measures should be included separately in therapeutic studies.

Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice.

Amyotrophic lateral sclerosis (ALS) is mainly a sporadic neurodegenerative disorder characterized by loss of cortical and spinal motoneurons. Some familial ALS cases (FALS) have been linked to dominant mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Transgenic mice overexpressing a mutated form of human SOD1 with a Gly93Ala substitution develop progressive muscle wasting and paralysis as a result of spinal motoneuron loss and die at 5 to 6 months. We investigated the effects of neurotrophic factor gene delivery in this FALS model. Intramuscular injection of an adenoviral vector encoding cardiotrophin-1 (CT-1) in SOD1G93A newborn mice resulted in systemic delivery of CT-1, supplying motoneurons with a continuous source of trophic factor. CT-1 delayed the onset of motor impairment as assessed in the rotarod test. Axonal degeneration was slowed and skeletal muscle atrophy was largely reduced by CT-1 treatment. By monitoring the amplitude of the evoked motor response, we showed that the time-course of motor impairment was significantly decreased by CT-1 treatment. Thus, adenovirus-mediated gene transfer of neurotrophic factors might delay neurogenic muscular atrophy and progressive neuromuscular deficiency in ALS patients.

Axonal elongation through long acellular nerve segments depends on recruitment of phagocytic cells from the near-nerve environment. Electrophysiological and morphological studies in the cat.

The distal nerve stump plays a central role in the regeneration of peripheral nerve but the relative importance of cellular and humoral factors is not clear. We have studied this question by freezing the tibial nerve distal to a crush lesion in cat. The importance of constituents from the near-nerve environment was assessed by modification of the contact between the tibial nerve and the environment. Silicone cuffs, containing electrodes for electrophysiological assessment of nerve regeneration, were placed around the tibial nerve distal to the crush site. The interaction between long acellular frozen nerve segments (ANS) and the near-nerve environment was ascertained by breaching the silicone cuff to allow access of cellular or humoral components. Tibial nerves were crushed and frozen for 40 mm and enclosed in nerve cuffs with 0.45-microm holes or 2.0-mm holes to allow access of humoral factors or tissue ingrowth, respectively. In a second set of experiments, tibial nerves were crushed and either frozen for 20+20 mm, leaving a 10 mm segment with viable cells in the center (stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable nerve segment. The regeneration was monitored electrophysiologically by implanted electrodes and after 2 months the nerves were investigated by light and electron microscopy. The results indicate that soluble substances in the near-nerve environment, such as nutrients, oxygen or tropic substances did not exert any independent beneficial effect on the outgrowing axons. However, phagocytic cells entering the acellular segment from the near-nerve environment were crucial for axonal outgrowth in long ANS.

Spinal muscular atrophy: present state.

Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disease caused by homozygous deletions or mutations in the SMN1 gene on Chr.5q13. SMA spans from severe Werdnig-Hoffmann disease (SMA 1) to relatively benign Kugelberg-Welander disease (SMA 3). Onset before birth possibly aggravates the clinical course, because immature motoneurons do not show compensatory sprouting and collateral reinnervation, and motor units in SMA 1, in contrast to those in SMA 3, are not enlarged. Genetic evidence indicates that SMN2, a gene 99% identical to SMN1, can attenuate SMA severity: in patients, more SMN2 copies and higher SMN protein levels are correlated with milder SMA. There is evidence that SMN plays a role in motoneuron RNA metabolism, but it has also been linked to apoptosis. Several mouse models with motoneuron disease have been successfully treated with neurotrophic factors. None of these models is, however, homologous to SMA. Recently, genetic mouse models of SMA have been created by introducing human SMN2 transgenes into Smn knockout mice or by targeting the Smn gene knockout to neurons. These mice not only provide important insights into the pathogenesis of SMA but are also crucial for testing new therapeutic strategies. These include SMN gene transfer, molecules capable to up-regulate SMN expression and trophic or antiapoptotic factors.

Diagnostic challenges in combined multiple sclerosis and centronuclear myopathy.

The first case of combined centronuclear myopathy and multiple sclerosis is reported. The difficulties of diagnosing multiple sclerosis in patients with muscular disorders associated with the central nervous system involvement are discussed.

Post reinnervation maturation of myelinated nerve fibers in the cat tibial nerve: chronic electrophysiological and morphometric studies.

The extent to which the long-term recovery of nerve fibers differs according to the cause of Wallerian degeneration is not clear, although outgrowth of axons is better after lesions with continuity of basal lamina of the Schwann cell tubes (nerve crush) compared with lesions with interruption of basal lamina (nerve section). Post-reinnervation maturation of myelinated nerve fibers of the cat tibial nerve was followed in chronic electrophysiologic studies after crushing, sectioning, and section+freeze lesions, and compared with morphometric analysis of the same nerves. The amplitudes of the compound nerve action potentials (CNAPs) recovered to a much lesser extent after sectioning than after crushing the nerve. This difference could be related to a smaller number of large fibers, a greater degree of sprouting after sectioning than after crushing, or less synchronization of conduction in regenerated fibers. In comparison, the compound muscle action potentials (CMAPs) recovered to a greater extent than the CNAP after sectioning and section+freeze, though not to the same degree or as fast as after crushing. The difference between the recovery of the CNAP and the CMAP could be due to better regeneration of motor fibers, to differences in the size of motor units or to a better summation of motor unit action potentials. The maximal conduction velocities (CV) in mixed nerve and in motor fibers increased faster after crushing than after sectioning and section+freeze to 60%-70% of control values. The diameters of the largest myelinated fibers increased in all lesions to about 80% of controls. The relation between fiber diameter and CV was influenced by remodeling of myelin during maturation. Hence, long-term functional recovery is influenced by the nature of the nerve lesion, and a smaller proportion of fibers recovered functionally after nerve section than after crush.

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles.

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.

Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy.

Cardiotrophin-1 (CT-1), an IL-6-related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1-treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1-treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.

Muscle phosphoglycerate mutase deficiency with tubular aggregates: effect of dantrolene.

A patient with muscle phosphoglycerate mutase deficiency (PGAMD) and exercise-induced muscle cramps had tubular aggregates in muscle and increased muscle Ca2+-adenosine triphosphatase and calcium content. Two ischemic forearm exercise tests induced contractures in the patient. On dantrolene treatment, the patient became asymptomatic, and the ischemic test was performed without contracture. These findings suggest that cramps in muscle PGAMD are caused by a high calcium release from the sarcoplasmic reticulum relative to calcium re-uptake capacity.

Histology and platinum content of sensory ganglia and sural nerves in patients treated with cisplatin and carboplatin: an autopsy study.

Cisplatin is a valuable antineoplastic drug which as a dose-limiting side-effect causes sensory neuropathy, and which therefore is often combined with less neurotoxic carboplatin. It has not been possible to reproduce cisplatin neuropathy in experimental animals, and the neurotoxic mechanism in man is disputed. We investigated post-mortem material from 12 patients and 15 control subjects. Half of the fibres with diameters of > or = 9 microns, or more than 15% of all fibres (P < 0.02), had disappeared in the sural nerves of patients. Signs of axonal regeneration were lacking. The dorsal root ganglia D12 and L2 of some but not of all patients contained necrotic neurons and nodules of Nageotte. The mean volume of the somata was reduced by 18% (P < 0.03). A relation between cumulated doses, treatment free interval and changes in nerve or ganglia was not found. The platinum content was high in all tissues except in the spinal cord when the patient had died shortly after treatment, and it decreased with increasing interval, least so in liver, sensory ganglia and sural nerves. The results support the hypothesis that cisplatin neuropathy is a neuroneopathy rather than a dying-back axonopathy.

Therapeutic benefit of ciliary neurotrophic factor in progressive motor neuronopathy depends on the route of delivery.

Ciliary neurotrophic factor (CNTF) has demonstrated therapeutic effects in several mouse mutants with motoneuronal degeneration. However, the poor bioavailability and toxic side effects of recombinant CNTF protein have complicated its use in patients with amyotrophic lateral sclerosis. CNTF gene transfer strategies were developed but faced the question of whether CNTF should be delivered to motoneuron cell bodies or to their axons or muscle targets. To address this issue, we have used an adenoviral vector (AdCNTF) coding for a secretable form of CNTF and compared different routes of its administration in the mouse mutant progressive motor neuronopathy (pmn). Intramuscular, intravenous, and intracerebroventricular injections of AdCNTF or the control vector AdlacZ resulted in transgene expression in skeletal muscle fibers, hepatocytes, and ependymal cells, respectively, as determined by histochemistry and reverse transcription-polymerase chain reaction. AdCNTF intramuscularly treated and intravenously treated pmn mice showed a 25% increase in mean life span and a reduced degeneration of phrenic myelinated nerve fibers, which correlated with elevated CNTF serum bioactivities. In contrast, intracerebroventricular AdCNTF administration did not affect the mean life span or motor axonal degeneration of pmn mice. The differential efficacy of peripheral and central CNTF vector administrations might be of interest for future studies in human motor neuron diseases.

Adenovirus-mediated transfer of the neurotrophin-3 gene into skeletal muscle of pmn mice: therapeutic effects and mechanisms of action.

Several neurotrophic factors (CNTF, BDNF, IGF-1) have been suggested for the treatment of motor neuron diseases. In ALS patients, however, the repeated subcutaneous injection of these factors as recombinant proteins is complicated by their toxicity or poor bioavailability. We have constructed an adenovirus vector coding for neurotrophin-3 (AdNT-3) allowing for stable and/or targeted delivery of NT-3 to motoneurons. The intramuscular administration of this vector was tested in the mouse mutant pmn (progressive motor neuronopathy). AdNT-3-treated pmn mice showed prolonged lifespan, improved neuromuscular function, reduced motor axonal degeneration and efficient reinnervation of muscle fibres. NT-3 protein and also adenovirus vectors, when injected into muscle, can be transported by motoneurons via retrograde axonal transport to their cell bodies in the spinal cord. Using ELISA and RT-PCR analyses in muscle, spinal cord and serum of AdNT-3-treated pmn mice, we have investigated the contribution of these processes to the observed therapeutic effects. Our results suggest that most if not all therapeutic benefit was due to the continuous systemic liberation of adenoviral NT-3. Therefore, viral gene therapy vectors auch as adenoviruses, AAVs, lentiviruses and new types of gene transfer not based on viral vectors that allow for efficient in vivo liberation of neurotrophic factors have potential for the future treatment of human motor neuron diseases.

A new mitochondrial tRNA(Met) gene mutation in a patient with dystrophic muscle and exercise intolerance.

A 30-year-old woman with a novel heteroplasmic U4409C mtDNA mutation in the tRNA(Met) gene presented with growth retardation, muscle weakness, severe exercise intolerance, and lactic acidosis. Muscle biopsy showed unusually severe dystrophic features. The mutation was not present in maternal relatives or 25 healthy subjects. Single-fiber PCR-RFLP analysis of mtDNA showed higher proportion of the mutation in COX-negative than in COX-positive muscle fibers.

Axonal elongation through acellular nerve segments of the cat tibial nerve: importance of the near-nerve environment.

Peripheral nerve regeneration is considered to be influenced by structural, cellular and humoral factors in the distal nerve stump. Axonal elongation was, however, not affected by the presence of a 20 mm acellular nerve segment (ANS) distal to a crush lesion in a cat tibial nerve which was shielded from the environment by a silicone cuff [K. Fugleholm, H. Schmalbruch, C. Krarup, Early peripheral nerve regeneration after crushing, sectioning, and freeze studied by implanted electrodes in the cat, J. Neurosci., 14 (1994) 2659-2673]. In the present study axons were challenged to regenerate through crush lesions combined with 30-, 40-, 50-, 60- and 70-mm ANSs. For 30- and 40-mm ANSs, the nerves were shielded by impermeable silicone cuffs containing electrodes for electrophysiological evaluation of axonal elongation. All nerves were examined histologically by light microscopy 9 weeks after the lesion. The elongation through the shielded 30-mm ANS was slower than through a shielded nerve segment with viable cells. In the isolated 40-mm ANS, incomplete Wallerian degeneration and lack of blood vessels were observed, and axonal elongation was severely impaired. Regeneration across 40-70 mm non-shielded ANSs was intact and there was no relation between the number of regenerated fibers and the length of the ANS. There was no reduction in the number of blood vessels in the non-isolated ANSs. The results suggest that regeneration through an isolated acellular nerve segment exceeding 30 mm depends on cellular and humoral support from the near-nerve environment. Thus, the near-nerve environment is crucial for regeneration through long ANSs, and the importance of humoral, cellular and vascular support is discussed.

Gene transfer to Schwann cells after peripheral nerve injury: a delivery system for therapeutic agents.

We transferred a reporter gene to Schwann cells to test whether they might serve as an endoneurial delivery system for therapeutic proteins. A replication-defective adenoviral vector carrying the gene for beta-galactosidase (lacZ) was injected into the distal segment of intact or crushed sciatic nerves of adult rats, and the expression of lacZ was histochemically assessed. Less than 1% of the Schwann cells became reactive in intact nerves, but up to 18% of the proliferating Schwann cells of injured nerves expressed lacZ. Gene expression decayed with time but might persist for up to 2 months. It was enhanced by immunosuppression: daily cyclosporin A injections reduced both proliferation of Schwann cells and lymphocytic infiltration of the nerve, whereas tolerance induced by a single intrathymic injection of the vector 4 days after birth abolished the inflammatory response but not the proliferation of Schwann cells. The vector itself did not impede axonal regeneration. The results indicate that adenoviral gene transfer to Schwann cells in injured nerves is possible and suggest that induced production of neurotrophic factor may represent a therapeutic supplement to surgical nerve repair.

Effects of long-term phasic electrical stimulation on denervated soleus muscle: guinea-pig contrasted with rat.

Guinea-pig soleus muscles were denervated and electrically stimulated for periods of 43 to 66 days. Stimuli were in 1 s bursts of 40 Hz pulses, repeated every 5 min. Other guinea-pigs were denervated for 82 days without stimulation and, in a third group, the soleus muscle was necrotized and allowed to regenerate without reinnervation for 13-15 days. Isometric and isotonic recordings were made in vivo. Denervated guinea-pig muscles were embedded in epoxy resin for light and electron microscopy. Chronic stimulation of denervated guinea-pig soleus had no effects on the prolonged twitch or on reduced maximal shortening velocity, maximal rate of rise of tension and tetanic force. This contrasts with the slow-to-fast conversion produced by denervation and denervation-stimulation of rat soleus. Loss of force was much greater in rat than guinea-pig after denervation, and chronic stimulation increased force in rat to the same level as in guinea-pig after denervation (with or without stimulation). Eighty-day denervated guinea-pig soleus did not reveal those morphological signs of fibre breakdown and regeneration which are prominent in denervated rat soleus muscles. Those changes in rat resembled aneurally regenerated muscles in several aspects, especially the increased incidence of fibres with internal myo-nuclei which did not appear in guinea-pig soleus after denervation. Aneurally regenerated guinea-pig soleus became fast like aneurally regenerated rat muscle. Our data are compatible with the hypothesis that slow-to-fast transformation of denervated rat soleus is not directly brought about by chronic stimulation but by de-novo formation of fast-contracting regenerated fibres. The persistence of fibrillation in guinea-pig but not rat after denervation may account for the species difference.

Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

Fatal congenital myopathy with actin filament deposits.

We present the clinical and morphological findings in a case of progressive congenital myopathy. The symptoms present at birth included severe general muscular hypotonia, diffuse muscular atrophy, arthrogryposis, absence of spontaneous movements, and left ventricular hypertrophy. A biopsy specimen taken from the gastrocnemius muscle when the patient was 2 weeks old revealed deposits which consisted of actin filaments as shown by electron microscopy. The infant was occasionally respirator dependent but was mostly able to breathe unassisted. At the age of 5 months he died of respiratory failure. The actin filament deposits may explain the clinical findings.