Anal sphincter electromyography in patients with newly diagnosed idiopathic parkinsonism.

OBJECTIVES: The differential diagnosis of patients with idiopathic parkinsonism is difficult, especially early in the course of the disease. External anal sphincter electromyography (EAS-EMG) has been reported to be of value in the differential diagnosis between Parkinson's disease (PD) and multiple system atrophy (MSA). Patients with MSA are reported to have pathological EAS-EMG and patients with PD are reported to have significantly less pathological EAS-EMG results. Comparisons between patients with parkinsonian disorders have usually been made many years into the disease, and thus it is largely unknown if the results of EAS-EMG can be used to distinguish the different diagnoses in the early phase of the disease. MATERIALS AND METHODS: We investigated 148 newly diagnosed patients with idiopathic parkinsonism from a population-based incidence cohort (100 definite PD, 21 probable PD, 16 MSA, 11 progressive supranuclear palsy, and 40 controls) with EAS-EMG within 3 months of their first visit and, in the majority of patients, before start of treatment with dopaminergic drugs. The clinical diagnoses were made using established clinical diagnostic criteria after a median follow-up of 3 years. RESULTS: All patient groups had more pathological EAS-EMG results than controls. No EAS-EMG differences were found between the patient groups, especially not between PD and MSA. CONCLUSIONS: External anal sphincter electromyography examination cannot separate the different parkinsonian subgroups from each other in early course of the diseases.

Digital nerve somatosensory evoked potentials and MRI. Correlation analysis in patients with symptomatic cervical spine disorders.

OBJECTIVES: Analysis of the relationship between the symptoms, digital nerve somatosensory evoked potentials (D-SEP) and MRI, in patients with symptomatic cervical spine disorders (CSD). MATERIALS AND METHODS: MRI and D-SEP following electrical stimulation of digits I, III and V in 44 patients. RESULTS: Symptoms in the fingers correlated significantly with disk herniation at the corresponding cervical level and with spinal cord impingement at one or two adjacent rostral segments on MRI. D-SEP was abnormal in 52% of all patients. Among them, the groups with multiple and single level involved on MRI had 62% and 30% of abnormal somatosensory evoked potentials (SEP), respectively. Digit I-SEP abnormality was more often localized at the root level, while digit V-SEP at the spinal cord level above the dorsal nucleus. D-SEP correlated best with compression of the spinal cord at adjacent upper and especially the most rostral (C3-5) levels on MRI. CONCLUSIONS: Accurate correlation of D-SEP and symptoms with MRI is essential for correct localization of lesions in patients with CSD.

Expression of mRNA for plasminogen activators and protease nexin-1 in innervated and denervated mouse skeletal muscle.

Plasminogen activators (urokinase-type, u-PA and tissue-type, t-PA) are serine proteases that have been suggested to play important roles in synaptic remodeling. The enzymatic activity of u-PA in particular has previously been shown to increase dramatically after denervation of skeletal muscle. Using (32)P-labeled riboprobes and Northern blots the expression of mRNA for u-PA, t-PA and the inhibitor protease nexin-1 (PN-1) has been studied in innervated and 1-10-days denervated hind-limb muscle from mouse. Using RNA extracted from innervated and 6-days-denervated mouse hemidiaphragm muscles the expression of these mRNAs has also been investigated in synaptic and extrasynaptic muscle regions. For both u-PA and t-PA the observed autoradiographic signals were similar for RNA extracted from innervated and denervated leg muscles. The signals were also similar for RNA extracted from perisynaptic and extrasynaptic regions of hemidiaphragm muscle but u-PA signals were lower in denervated than in innervated hemidiaphragm. No such difference was observed for t-PA. PN-1 mRNA levels were also found to decrease after denervation in the hemidiaphragm but no substantial decrease was observed in denervated hind-limb muscles. No difference was observed between PN-1 expression in perisynaptic and extrasynaptic regions. The effect of denervation on PA enzymatic activity in skeletal muscle is therefore likely to be mediated at some post-transcriptional level.

Quantitative electromyography of the external anal sphincter in Parkinson's disease and multiple system atrophy.

The distinction of multiple system atrophy (MSA) from Parkinson's disease (PD) can be difficult, especially early in the disease. In MSA degeneration of sacral anterior horn cells (Onuf's nucleus) results in denervation-reinnervation of anal and urethral sphincter muscles, which can be recognized as neurogenic electromyographic (EMG) changes of motor unit potentials. Sphincter EMG has therefore been recommended as a test for distinguishing MSA from PD. Our results confirm the presence of marked neurogenic EMG changes of the external anal sphincter muscle in patients with probable MSA compared to healthy controls. However, in patients with probable PD, our quantitative EMG data show a scatter from normal to marked neurogenic changes and the degree of EMG abnormality is correlated to the duration of the disease. Thus an abnormal sphincter EMG cannot be taken as a strong indicator of MSA rather than PD in the individual patient, especially in long-standing cases.

Muscle morphology and mitochondrial investigations of a family with autosomal dominant cerebellar ataxia and retinal degeneration mapped to chromosome 3p12-p21.1.

The autosomal dominant cerebellar ataxias (ADCA) are a group of neurodegenerative disorders with ataxia and dysarthria as early and dominant signs. In ADCA type II, retinal degeneration causes severe visual impairment. ADCA type II has recently been mapped to chromosome 3p by three independent groups. In the family with ADCA type II studied here, the disease has been mapped to chromosome 3p12-p21.1. Histochemical examination of muscle biopsies in 5 cases showed slight neurogenic atrophy and irregular lobulated appearance or focal decreases of enzyme activity when staining for NADH dehydrogenase, succinic dehydrogenase and cytochrome oxidase. Ragged-red fibres were scarce. Electron microscopic examination showed uneven distribution of mitochondria with large fibre areas devoid of mitochondria and/or large subsarcolemmal accumulations of small rounded mitochondria, and frequent autophagic vacuoles. These vacuoles contained remnants of multiple small rounded organelles, possibly mitochondria, and had a remarkably consistent ultrastructural appearance. Biochemical investigation of mitochondrial function showed reduced activity of complex IV and slightly reduced activity of complex I in the respiratory chain in a severely affected child while no abnormalities were found in his affected uncle.

Endocytotic activity of mouse skeletal muscle fibres after long-term denervation.

The endocytotic activity of skeletal muscle fibres and its relation to the denervated endplate region has been studied using horseradish peroxidase (HRP) as marker for endocytosis. In muscles denervated for a short time period (10-20 days) HRP-uptake occurred in small segments of the muscle fibres near the centre of the muscle (endplate region). After long-term denervation (6-12 months) similar segments with high endocytotic activity were seen preferentially in more peripheral parts of the muscle fibres. Ultrastructural characteristics of segments with high endocytotic activity from long-term denervated muscle fibres include a proliferating transverse tubular system, HRP-containing bodies of different sizes with some very large vacuoles extending over several sarcomeres. These characteristics are similar to those described previously for HRP-uptake in the endplate region of short-term denervated muscle (Tågerud et al., J. Neurol. Sci., 75 (1986) 141) except that no recognizable endplate structures were observed in the present study. The results are discussed in relation to the fate of the denervated endplate and the receptive capacity for synapse formation in long-term denervated muscle.

Dextrans as markers for endocytosis in innervated and denervated skeletal muscle.

Fluorescence-labeled dextrans were evaluated as markers for endocytosis in skeletal muscle. Fluorescein isothiocyanate (FITC)-labeled dextrans (average molecular weight 3900 to 71200) showed a higher uptake in denervated than in innervated muscle both in vitro and in vivo. The in vitro uptake of FITC-dextran (35.600) increased linearly with time at 37 degrees C, and was almost completely inhibited by low temperature (4 degrees C). The uptake was not a pure bulk uptake, because a saturable component was evident from the concentration dependence and from competition experiments with unlabeled dextran. The uptake of FITC-labeled or rhodamine B isothiocyanate (RITC)-labeled dextrans in denervated muscle occurred mainly in small segments of the fibers centered around the denervated endplate region. However, not all denervated fibers showed such segments. Periodic acid Schiff's base staining for carbohydrates stained dextrans in denervated muscle fibers. Some staining, probably of lysosomes, was also observed in denervated muscle not exposed to dextran.

Trapezius muscle changes unrelated to static work load. Chemical and morphologic controlled studies of 22 women with and without neck pain.

From a cross-sectional study of 82 women who were engaged in assembly work that involved static muscle loading of the shoulder muscles, 11 cases with complaints of neck tension (all except 1 arising at work) and 11 individually matched, exposed control cases without neck pain were studied. In addition, 10 matched, unexposed control cases were studied. Upon histochemical examination and study of the trapezius muscle, morphologic changes of type ragged red fibers were found in 8/11 neck-pain cases, in 7/11 exposed controls, and in 4/10 unexposed controls. The pathologic and clinical importance of rare, ragged red fibers in the trapezius muscle thus seems uncertain.

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres.

Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.

High endocytotic and lysosomal activities in segments of rat myotubes differentiated in vitro.

Endocytosis and the lysosome system have been studied in rat myotubes differentiated in vitro. Horseradish peroxidase was used as marker for endocytosis and was found to accumulate unevenly in the myotubes. Small segments of myotubes display very high endocytotic activity. Similar segments contained numerous lysosomes, as seen by the accumulation of neutral red or histochemical staining for acid phosphatase. The segments also contained accumulations of acetylcholine receptors as determined by binding of tetramethyl rhodamine-labelled alpha-bungarotoxin. Unstained segments in living cultures could be recognized by phase-contrast microscopy since they often appeared somewhat dilated and were not as well spread on the culture surface as the main parts of the myotubes. Ultrastructurally, the segments contained an intensely proliferating tubular system in communication with the extracellular space, which therefore probably represents the developing transverse tubular system. The segments also contained endocytosed marker within large phagosomes. Contractile filaments occurred in the segments but were frequently less well-organized than in other parts of the myotubes. The described characteristics of the segments in rat myotubes differentiated in vitro bear resemblance to some of the characteristics of the denervated endplate region of adult muscle.

An ultrastructural study of the segmental uptake of horseradish peroxidase in the endplate region of denervated skeletal muscle fibres.

The segmental uptake of horseradish peroxidase (HRP) in the endplate region of denervated skeletal muscle fibres has been studied ultrastructurally using a method for selecting single muscle fibres with high segmental peroxidase staining from denervated mouse tibialis anterior muscle. Segments containing large peroxidase positive phagosomes could already be seen 10-15 min after i.v. injection of HRP. Such segments were still present 24 h after HRP injection. The localization of phagosomes, deep in the fibres rather than immediately under the sarcolemma, suggests that the uptake occurs from t-tubuli. Vivid proliferation of t-tubuli, consisting of vesiculation, enlargement and encircling of cytoplasmic components, was also observed. The HRP accumulates in phagosomes of varying size and shape. Similar membrane-limited bodies without or with very weak peroxidase staining were also observed. The peroxidase-positive phagosomes participate in autophagic processes as suggested by their content of undegraded cellular material. Golgi profiles, which occurred deep in the muscle fibres, and enlarged components of the sarcoplasmic reticulum were frequently encountered in the segments. Myofibrillar degeneration occurs in the segments and progresses with time after denervation. The described segments may be related to the increased membrane turnover in denervated muscle fibres and/or they may be related to processes aimed at establishing new synaptic contacts.

Effects of botulinum toxin induced muscle paralysis on endocytosis and lysosomal enzyme activities in mouse skeletal muscle.

The effects of botulinum toxin (type A) induced muscle paralysis on endocytosis and lysosomal enzyme activities in skeletal muscle were compared with the effects of surgical denervation. Muscle atrophy, measured as decrease in total muscle protein content, was as large or larger after botulinum toxin treatment as after denervation. Endocytic activity, measured as the in vitro uptake of horseradish peroxidase, and the specific activities of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase and cathepsin D were all increased six days after denervation. Only the specific activity of cathepsin D was increased six days after botulinum toxin poisoning. The uptake of horseradish peroxidase and the specific activity of N-acetyl-beta-D-glucosaminidase were also increased eleven days after poisoning. Transverse sections of eleven days botulinum poisoned muscles from animals injected with horseradish peroxidase showed fibres with dense peroxidase staining similar to those seen in denervated muscle although they seemed to occur less frequently. The results show that increases in endocytic activity and lysosomal enzyme activities may occur in skeletal muscle without the presence of degenerating axons. The differences in effects of surgical denervation and botulinum toxin induced paralysis are discussed in terms of what is known about the mechanism of action of botulinum toxin and the possible functional roles of the two lysosomal enzymes studied.

Biochemical and ultrastructural effects of chloroquine on horseradish peroxidase uptake and lysosomal enzyme activities in innervated and denervated mouse skeletal muscle.

The effects of chloroquine treatment on horseradish peroxidase (HRP) uptake and lysosomal enzyme activities in innervated and denervated mouse skeletal muscle have been studied using biochemical, histochemical and ultrastructural techniques. Chloroquine treatment caused a large (59-101%) increase in the activity of cathepsin D in both innervated and denervated muscle. The activity of N-acetyl-beta-D-glucosaminidase also increased slightly in denervated muscle. No effect was observed on acid phosphatase activity. The in vivo uptake of HRP in innervated and denervated muscle was unaffected by chloroquine treatment. The results show that the activities of certain lysosomal enzymes may increase in skeletal muscle without an increase in endocytic activity. This is discussed in comparison to what is seen in denervated and dystrophic muscle. Histochemical and ultrastructural studies showed the HRP uptake to occur segmentally in denervated muscle fibres from untreated as well as chloroquine-treated animals. Ultrastructurally the peroxidase-positive phagosomes occurring in these segments were found to contain increased levels of undegraded material after chloroquine treatment suggesting that these phagosomes are of a lysosomal nature and also participate in autophagic processes.

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle.

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.

Ultrastructural and biochemical changes in electrically stimulated dark cutting beef.

In dark cutting beef, with a high ultimate pH, ultrastructural and biochemical changes were followed during the very early post-mortem period in non-stimulated and electrically stimulated muscles. The consumption of ATP was extremely rapid in the electrically stimulated dark cutting muscle. In these samples pronounced changes in the ultrastructure were seen in the form of heavy contractions and complete disorganization of the tissue. The changes could have been caused by a combined effect of super-contractions and proteolytic activity. The changes seen in electrically stimulated samples were not reflected in improvements of the instrumentally evaluated tenderness.

Uptake of horseradish peroxidase in denervated skeletal muscle occurs primarily at the endplate region.

The spatial distribution of horseradish peroxidase (HRP) uptake has been studied by light- and electron microscopy in the denervated hemidiaphragm of the mouse. Segments with high HRP uptake were observed in a band centrally located in the denervated muscle. This distribution is similar to the well-known innervation pattern of the diaphragm. Ultrastructural studies demonstrated a high incidence of postsynaptic folds in close proximity of fibre areas with high intracellular content of HRP. 8-12 days after denervation a large number of fibres showed segments of high HRP uptake. 2-4 days after denervation very few such segments were observed. Biochemical studies also demonstrated an increase in HRP uptake after denervation occurring primarily in the endplate region. The activities of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase, acid phosphatase and cathepsin D all increased after denervation, most prominently in the endplate region. It is suggested that the observed segmental uptake of HRP and lysosomal activation reflects a process for rapid membrane turnover in denervated muscle.

Lysosomes in skeletal muscle following denervation. Time course of horseradish peroxidase uptake and increase of lysosomal enzymes.

The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2-14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase. It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.