Muscle-specific kinase antibodies: a novel cause of peripheral nerve hyperexcitability?

INTRODUCTION: Antibodies that target the postsynaptic neuromuscular junction (NMJ) protein, muscle-specific kinase (MuSK), have been associated with myasthenia gravis (MG), often with cramps and fasciculations, after administration of acetylcholinesterase inhibitors (AChE-I). METHODS: In this report, 2 patients are described with elevated MuSK antibodies and evidence of peripheral nerve hyperexcitability (PNH) unrelated to AChE-I medication. RESULTS: Patient 1 presented with facial neuromyotonia and fasciculations, without overt weakness. EMG studies demonstrated myokymic discharges in facial muscles, with bursts of discharges after voluntary activation, and widespread fasciculation potentials in limb muscles. Patient 2 presented with bulbar weakness and fasciculations in the tongue and limbs, initially diagnosed as bulbar-onset amyotrophic lateral sclerosis. Subsequent investigation identified the presence of MuSK antibodies. CONCLUSIONS: We hypothesize that MuSK antibodies may induce these phenotypes through disruptive actions at the NMJ, in particular the binding of acetylcholinesterase (AChE) to MuSK via its collagen Q (ColQ) tail, producing a reduction in synaptic AChE activity.

Nuevas estrategias terapéuticas para elevar el colesterol unido a lipoproteínas de alta densidad / New therapeutic strategies to increase high-density lipoprotein cholesterol

Los marcados efectos antiaterogénicos y antioxidantes de las partículas de lipoproteínas de alta densidad (HDL) justifican la búsqueda de nuevas estrategias que mejoren no tanto los valores de colesterol unido a HDL (cHDL), sino que también promuevan un transporte reverso de colesterol más eficiente. Este capítulo revisa fármacos que han sido testados en investigación animal y que se han mostrado capaces de aumentar el eflujo de colesterol (agonistas LXR), promover la captación hepática de cHDL (inactivadores parciales de SRB1) o de incrementar la capacidad antioxidante (antiinflamatoria) de las HDL (D4F, miméticos de apo A-I). Sólo este último, junto con la perfusión de apo A-I Milano y la administración de un PPAR-α/γ (aleglitazar) han sido evaluados en humanos, mostrando resultados prometedores, lo que justifica la inversión en nuevos proyectos de investigación en este campo (AU)

The search for new strategies to improve not only HDL-cholesterol levels but also to promote more efficient reverse cholesterol transport is justified by the marked antiatherogenic and antioxidant effects of high-density lipoprotein (HDL) particles. The present article reviews the drugs tested in animal research that have been shown to be capable of increasing cholesterol efflux (LXR agonists), promoting HDL-cholesterol uptake by the liver (partial inactivators of SRB1 ) or increasing the antioxidant (antiinflammatory) capacity of HDL (D4F, apo A-I mimetics). Only the latter, together with apo A-I Milano infusion and administration of a PPARα/γ (aleglitazar) have been evaluated in humans and have shown promising results, thus justifying investment in further research in this field (AU)

[Effect of intraoperative radiation therapy on enzyme histochemistry of expression muscle of guinea pigs after injury of facial nerve].

PURPOSE: To explore the mechanism of facial nerve recovery after radiotherapy by simulating surgical treatment of parotid gland carcinoma with reserving facial nerve and studying the ability of aerobic metabolism, transmission of neurotransmitter, variation of ultrastructure of motor end and mitochondrion after radiotherapy during operation. METHODS: Animal models of treatment group(15Gy) and lethal dose group(17Gy) were established. Succinic dehydrogenase(SDH) and acetylcholine lipase(AchE) of orbicularis oris were measured after radiotherapy. Student's t test was used for statistical analysis. RESULTS: The activity of SDH and AchE declined after radiotherapy. There was significant difference between treatment group and lethal dose group. One month later, there was a significant improvement in the activity of SDH and AchE in treatment group, while that of lethal dose group continued to decline. CONCLUSION: After radiotherapy during parotid gland surgery, the activity of SDH and AchE, transmission of neurotransmitter, and the ability of aerobic metabolism decreased. The ultrastructure of motor end and mitochondrion was destroyed. The variation returned to preoperative levels in treatment group, while did not in lethal dose group.

COX-2 expression in striated muscle under physiological conditions.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.

Morphological study of two human facial muscles: orbicularis oculi and corrugator supercilii.

Human facial muscles are unique in that they do not cross joints and they function either to open and close the apertures of the face or to tug the skin into intricate movements producing facial expressions. Compared to other skeletal muscles of the body, little is known about the microscopic architecture and organization of facial muscles. It was hypothesized that facial muscles with different roles would possess differences in their cellular organization and morphology that would reflect their unique function. The palpebral orbicularis oculi (oo) and the corrugator supercilii (cs) were studied because they are in close topographical proximity to one another and share the same nerve supply and embryonic origin. This study compared the two muscles which were procured as biopsies from cosmetic surgery procedures. Architectural and morphological features were elucidated using a combination of conventional histological stains, immunocytochemistry and histochemistry. Quantitative measures of fiber sizes, shapes, and fiber-type distributions were performed along with measures of capillary area per unit of contractile area (capillary index). Fiber-type profiles and motor end-plates were demonstrated by using antibodies to fast and slow myosins, as well as to neurofilament protein. The oo was shown to differ significantly from the cs on the basis of fiber shapes, sizes, and types. The oo muscle fibers were small, rounded, and 89% of them were of the fast-twitch (Type II) variety. The muscle fibers in the cs were larger, polygonal, and only 49% of them were of the fast-twitch variety. The capillary index of the cs was 2.4 times that of the oo.

Cleft lip: a histochemical and ultrastructural analysis of lip muscles.

AIMS: In order to evaluate the pathogenesis of cleft-lip in relation to both the anatomical and structural anomalies of the mesenchymal tissues, the authors concluded that the presence of structural anomalies in the examined tissues could not explain the malformation, but might be a consequence of it. Delayed muscular development, asymmetrical distribution of the muscular fibres and their anomalous insertion suggest that the anatomical/functional loss clinically detectable in the orbicular muscle could be the result of a perinatal dysmorphological process rather than of a simple mesenchymal hypoplasia. METHODS: Schendel et al. suggested that a metabolic defect in the mitochondrial function could cause a deficiency in cell migration and proliferation responsible for the malformation in question. To establish whether the pathogenesis of the cleft-lip is associated with an alteration in mitochondrial functionality, eight patients affected by unilateral cleft-lip were subjected to a biopsy of the orbicular muscle during the course of reparative surgery. RESULTS: The results obtained showed: 1) a great variation in the size of muscle fibres; 2) the absence of ragged red fibres; 3) a normal oxidative function in the muscle fibres examined; 4) the absence of typologically significant groupings positive for myofibral ATPases. Furthermore, the morphology of the mitochondria was preserved in all cases and neither inclusions nor morphological or volumetric changes were detected. CONCLUSIONS: This preliminary data did not confirm the constant presence of mitochondrial pathology responsible for the malformation in question. In our opinion, the growth deficiency of the maxillary segment could be ascribed to the cicatrization of the surgical repair of the cleft-lip.

Doxorubicin chemomyectomy is enhanced when performed two days following bupivacaine injections: the effect coincides with the peak of muscle satellite cell division.

PURPOSE: Doxorubicin is effective in permanently removing muscle after direct injection into the eyelid for treatment of blepharospasm and hemifacial spasm. However, patients often require two or more injection series before full abatement of their spasms is achieved. Local anesthetics cause muscle necrosis, followed by regeneration, a process that requires activation and division of muscle satellite cells. This study examined whether the muscle toxicity of doxorubicin could be amplified by injection of doxorubicin into the eyelid of rabbits 2 days after a local anesthetic injury, perhaps exploiting the toxic effects of doxoribicin on satellite cells at the peak time of their division after injury. METHODS: Rabbit eyelids received two series of injections of bupivacaine and hyaluronidase spaced 18 hours apart. Two days later, the eyelids were injected with either 0.5 or 1 mg doxorubicin. Animals were monitored daily for onset and duration of skin injury. After 1 month, the eyelids were assessed for muscle loss using histologic and morphometric techniques. RESULTS: Injection of doxorubicin during the peak of satellite cell activation and division 2 days after injury significantly increased muscle loss over doxorubicin alone. This treatment did not result in increased skin injury compared with doxorubicin alone. CONCLUSIONS: Permanent muscle loss was increased when doxorubicin was injected at the peak of satellite cell division 2 days after injury of the muscle with bupivacaine in rabbit eyelid, taking advantage of the antimitotic effects of doxorubicin on satellite cell division during the period of active regeneration. When local anesthetic injection immediately preceded the doxorubicin injection, increased myotoxicity was not seen. The injection of doxorubicin into muscle 2 days after a previous injury maximizes muscle loss. The increased muscle loss provided by this double treatment may decrease the number of injection visits required by blepharospasm and hemifacial spasm patients during their course of treatment, thus reducing the number of patients with side effects, which increases with repeated exposures of the eyelid to doxorubicin.

Expression of different isoforms of nitric oxide synthase in experimentally denervated and reinnervated skeletal muscle.

Denervated muscle fibers express enhanced levels of stress and apoptosis-associated proteins and undergo apoptosis. In experimentally denervated and reinnervated rat facial muscle, we now evaluate changes in the expression patterns of different isoforms of nitric oxide synthase (NOS)-generating nitric oxide (NO), which mediates oxidative stress and apoptosis. Physiological expression of NOS corresponds to a constant sarcolemmal staining pattern for neuronal NOS (nNOS) and a patchy sarcolemmal and weak sarcoplasmic labeling for the endothelial NOS-isoform, with no expression for inducible NOS (iNOS). Denervated muscle displayed distinct downregulation of nNOS with preserved expression of dystrophin. Also, denervated and immediately reinnervated muscle fibers showed decreased expression of nNOS. However, muscle fibers reinnervated for 10 weeks revealed a restored physiological expression of nNOS. There were no changes in the expression of endothelial and inducible NOS. As NO is known to induce growth arrest and collapse of neuronal growth cones, downregulation of NOS may contribute to promotion of axonal regeneration by aiding formation of new endplates. NO is upregulated in reinnervated muscle fibers and thus prevents polyneural hyperinnervation by extrajunctional synapses. Furthermore, downregulation of NOS during denervation is compatible with the finding that low levels of NO contribute to apoptosis instead of necrosis in disease states of oxidative stress.

Muscle-specific enzyme activity patterns of the capillary bed of human oro-facial, masticatory and limb muscles.

Enzyme-histochemical methods were used to analyse the activities of alkaline phosphatase (AP), dipeptidylpeptidase IV (DPP IV) and adenosine triphosphatase (ATPase) in capillaries of four different human oro-facial muscles, the major and minor zygomatic, the orbicularis oris and buccinator, one masticatory, the masseter and two limb muscles, the biceps brachii and first dorsal interosseus muscles. In all muscles, except for the orbicularis oris, the majority of the capillaries lacked enzyme activity. Therefore, none of these enzymes seems to be reliable as a general marker for human muscle capillaries. In general, the capillaries of the limb muscles and the major and minor zygomatic and the buccinator, were similar in their staining pattern for AP and ATPase, but differed in DPP IV staining. The orbicularis oris muscle differed from the other muscles by showing the largest proportion of capillaries with AP and ATPase activity. The masseter muscle had the largest proportion of capillaries stained for DPP IV. The muscle specific differences in enzyme activity of the capillaries are in agreement with our previous findings of specific differences between limb, oro-facial and masticatory muscles with respect to capillary supply and composition of fibre types and myosins. The results reflect functional specialization of the capillary bed of human muscles.

Gene regulation of cyclic AMP-dependent protein kinase subunits (C alpha, beta; RI alpha, beta and RII alpha, beta) in rat facial motoneurons after nerve transection.

In this study we investigated the changes in gene expressions of catalytic (C alpha, beta) and regulatory (RI alpha, beta and RII alpha, beta) subunits of cAMP-dependent protein kinase (PKA) in axotomized facial motoneurons of the rat. Nerve transection induced changes in the expression of C subunit messenger RNAs and RII subunit messenger RNAs. Control facial motoneurons had a high expression of both C alpha and C beta subunit messenger RNAs, but their expression declined after axotomy. The decrease was most pronounced at postoperative week 2 and returned to basal level within postoperative week 4. In contrast, the expression of both RII alpha and beta subunit messenger RNAs, which were low in control facial motoneurons, was increased after axotomy. Enhancement of RII subunits messenger RNAs was apparent during postoperative weeks 1 and 3, and then returned to the basal level. RI alpha, beta subunits messenger RNAs were strongly expressed in normal facial motoneurons, but were not clearly influenced by axotomy. These results indicate an attenuation of total PKA activity in axotomized facial motoneurons. Furthermore, such gene regulation may imply a change of the targets for PKA in facial motoneurons during the process of neurite regeneration.

Expression of NADPH-diaphorase activity in the facial motoneurons after compression of the facial nerve in the albino rat.

Compression of the rat facial nerve in its bony canal led to increased nitric oxide synthase activity in the facial motoneurons as revealed by NADPH-d histochemistry. The increase occurred steadily from 3 days to 6 weeks, peaking at 4 weeks after compression. Parallel to this was also a gradual return of facial function which reached its maximum at 4-5 weeks after compression.

Characterization of human oro-facial and masticatory muscles with respect to fibre types, myosins and capillaries. Morphological, enzyme-histochemical, immuno-histochemical and biochemical investigations.

This study provides a comparative characterization of four human oro-facial muscles, one masticatory muscle (the masseter) and two limb muscles, with respect to muscle fibre types, myosin isoforms and capillary supply. Enzyme-histochemical methods were used to evaluate the myofibrillar ATPase fibre type composition. Immuno-histochemical techniques were used to determine the expression of myosin heavy chain (MHC) isoforms in the different fibre types. The contents of MHCs and myosin light chains (MLC) in different muscles were analysed with electrophoretic methods. In addition, the capillary bed of the muscles was evaluated using both enzyme- and immuno-histochemical techniques. The fibre type compositions of the oro-facial and masseter muscles were found to be qualitatively and quantitatively different from each other and from those of limb muscles. In general, the oro-facial muscles contained a predominance of unusually high oxidative type II fibres, with a staining reaction for ATPase in between that of type IIA and type IIB fibres, termed type IIAB. In fact, one of the oro-facial muscles, the zygomatic minor, showed the highest type II fibre proportion ever reported in humans. This fibre type pattern is in contrast to that of the masseter muscle, which contains a majority of type I fibres, small diameter low oxidative type IIB fibres and a significant proportion of ATPase-intermediately stained fibres, termed IM, and IIC. Inter- and intra-muscular variability in fibre size and shape was considerable in both the oro-facial and masseter muscles. The oro-facial muscles were devoid of muscle spindles. The immuno-histochemical and biochemical analyses showed a characteristic myosin composition of each muscle. Notably, the results indicated the presence of a previously undetected fast MHC isoform in the oro-facial muscles, tentatively termed "fast F". The masseter contained unusual myosin isoforms, such as fetal and alpha-cardiac MHCs, and unique combinations of MHC isoforms which were not found in the limb or oro-facial muscles. The type IM and IIC fibres co-expressed slow and fast A MHCs in the oro-facial and limb muscles, but slow and a "fast B like" MHC in the masseter. Individual fibres in the oro-facial and limb muscles contained one or two MHC isoforms, whereas individual fibres in the masseter co-expressed up to four different MHC isoforms. On the basis of their pattern of expression of MHC isoforms, up to five fibre types could be distinguished in the oro-facial and limb muscles and eight in the masseter.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology of experimentally denervated and reinnervated rat facial muscle. I. Histochemical and histological findings.

The morphological changes in rat facial muscles were evaluated after permanent denervation and were compared with findings after immediate reinnervation. Thirty rats underwent transection of the left and right facial nerves immediately followed by hypoglossal-facial nerve anastomosis on the right side (muscular reinnervation) and removal of 8-10 mm of the facial plexus on the left side (permanent muscular denervation). Levator labii muscle samples of both sides were collected sequentially at 2, 6, 7, 10, 20, and 24 weeks after surgery and submitted to routine histological and enzyme histochemical staining procedures. In normal levator labii muscles a typical "chessboard" pattern was found, with type I fibers being smaller than type II fibers. These latter fibers also were more prevalent than the type I fibers. Among the type II fiber subtypes, the type IIB fibers were larger and more frequent. Two weeks after surgery, there were no differences between denervated facial muscles and those undergoing reinnervation. Both showed atrophic myofibers among normal-sized fibers and slight fibrosis. Those muscles denervated for more than 2 weeks displayed increasing fiber atrophy with frequent loss of typability, as well as proliferation of connective tissue and fat cells in perimysial and endomysial sites. After denervation for 20 weeks only a few atrophic fibers were found in wide areas of fibrosis and fat cells. Following nerve anastomosis the reinnervated levator labii muscle showed much less fiber atrophy. Regrowth to normal fiber diameters was found with only a few atrophic myofibers 10 weeks after anastomosis although a moderate fibrosis predominated at perimysial sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences in the orbicularis oculi muscle: conservation between species.

The orbicularis oculi muscle is a complex facial muscle involved in eyelid closure. The central parts of pretarsal and preseptal regions of the palpebral part of the orbicularis oculi muscle in rabbit and cynomolgus monkey lower eyelids were examined histologically and were analyzed for muscle fiber number, muscle fiber cross-sectional area and fiber type composition. Distinct regional differences were seen in the muscle fiber composition in these two regions of the muscle. The pretarsal portion of the muscle, that closest to the eyelid margin, was quite homogeneous and almost completely composed of type 2 fibers. These fibers were the smallest in cross-sectional area. Type 2 fibers also predominated in the preseptal portion of the muscle, but this region contained between 10 and 20% type 1 fibers. They appeared to be a gradient in muscle fiber size, whereby the fiber size increased as a function of the distance from the eyelid margin. The same pattern of regional differences were found in both rabbit and monkey orbicularis oculi. Thus, there is a clear conservation of these regional differences in these two species. While the developmental significance is unknown, the identification of this pattern may facilitate the evaluation of chemomyectomy agents for treatment of eyelid spasms in humans and allow a more accurate analysis of biopsy material from this muscle.

Histochemical mapping and fiber size analysis of mimic muscles.

Fourteen functionally relevant mimic muscles of nine human bodies were analyzed with respect to their muscle fiber sizes and their histochemical fiber type composition. In cryostat sections stained for actomyosin ATPase, type 1 and type 2 fibers were evaluated separately by means of computer-assisted image analysis. The fiber diameters varied between 20.24 and 41.45 microns. According to the proportions of the fiber types, the mimic muscles could be classified into three groups: (1) phasic muscles, with 14 to 15 percent type 1 fibers, (2) intermediate muscles, with 28 to 37 percent type 1 fibers, and (3) tonic muscles, containing 41 to 67 percent type 1 fibers. It is concluded that one has to consider this diversity of mimic muscles when planning the surgical reconstruction of facial paralysis.

Distribution of different fiber types in whole cross-sections of human platysma. A histological and morphometric study.

Analysis of cryostat cross-sections of the entire platysma muscle from human autopsies revealed enzyme histochemical and morphometric differences between normal human limb and facial muscles. The mean diameter of platysma fibers was about 50% of that of normal limb muscle fibers. Fiber type diameter increased from the medial to the lateral parts of the platysma. A variability coefficient of 356 indicated great variation in fiber caliber, with many fibers of 10 microns and less. Type I fibers showed an increase in density from the medial to the lateral parts of the muscle. The arrangement of histochemical fiber types was irregular with a tendency to form fiber type groupings.

Inactivation of type MM phosphoglycerate mutase by sulfhydryl group reagents during facial embryogenesis.

Type MM phosphoglycerate mutase from free dissected mandibular processes from embryonic rats was reversibly inactivated by tetrathionate, p-chloromercuribenzoate, and Hg2+. Titration with p-chloromercuribenzoate showed the existence of two sulfhydryl groups per enzyme subunit, the modification of which produced a progressive decline in enzyme activity. The apparent Km values for substrate and cofactor were not affected by tetrathionate treatment. Phosphoglycerate mutase inactivated by tetrathionate and by p-chloromercuribenzoate was unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P2. Glycerate-2,3-P2 protected against tetrathionate but failed to protect against Hg2+ and p-chloromercuribenzoate.

Histochemical characteristics of human mimic muscles.

Eight different mimic muscles of 13 human cadavers (7 male, 6 female) were studied by histochemical techniques. In sections stained for myosin ATPase the composition of fibre types was quantitatively evaluated by computer-assisted image analysis. The data were compared to those of 2 muscles in the lower limb of the same individuals. According to the percentage of the type I muscle fibres 3 groups of mimic muscles were distinguished: (1) the orbicularis oculi muscle (15%), (2) the major zygomatic, levator labii superioris, levator anguli oris, depressor anguli oris muscles and platysma (27-38%), and (3) the occipitofrontal and buccinator muscles (57-77%). For comparison, the gracilis and rectus femoris muscles were built up by 36% and 48% of type I fibres. The average diameters of fibres in mimic muscles were significantly less than in the 2 limb muscles. Differences in muscle fibre size between male and female specimens were not significant. The relevance of morphological characteristics of mimic muscles for facial expression and reconstructive surgery is discussed.