Antibodies against muscle-specific kinase impair both presynaptic and postsynaptic functions in a murine model of myasthenia gravis.

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

Age-related changes in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins, and subneural apparatuses.

We compared age-related changes in the intrinsic laryngeal muscles of aged and young adult rats by determining the number and diameter of muscle fibers, contractile muscle protein (myosin heavy chain isoforms, MHC) composition, and the morphology of the subneural apparatuses. In aged rats, both the numbers and the diameters of muscle fibers decreased in the cricothyroid (CT) muscle. The number of fibers, but not diameter, decreased in the thyroarytenoid (TA) muscle. In the posterior cricoarytenoid (PCA) muscle, neither the number nor the diameter of fibers changed significantly. Aging was associated with a decrease in type IIB and an increase in type IIA MHC isoform levels in CT muscle, but no such changes were observed in the TA or PCA muscles. Morphological examination of primary synaptic clefts of the subneural apparatus revealed that aging resulted in decreased labyrinthine and increased depression types in only the CT muscle. In the aged group, morphologically immature subneural apparatuses were found infrequently in the CT muscle, indicating continued tissue remodeling. We suggest, therefore, that age-related changes in the intrinsic laryngeal muscles primarily involve the CT muscle, whereas the structures of the TA and PCA muscles may better resist aging processes and therefore are less vulnerable to functional impairment. This may reflect differences in their roles; the CT muscle controls the tone of the vocal folds, while the TA and PCA muscles play an essential role in vital activities such as respiration and swallowing.

Fine structural study of the regeneration of muscle fibers in the rat soleus muscle during aging.

We examined the regeneration of muscle fibers in the soleus muscle of mature (12 months) and aged (24 and 27 months) rats by using electron microscopy. In both mature and aged muscles, regenerating muscle fibers were mainly formed within the scaffolds of basal laminae after necrosis. In the aged muscle, however, satellite cells within the scaffolds were occasionally destroyed, and immature muscle cells occurred in and around muscle bundles. These findings suggest that new muscle fibers formed in the interstitial spaces may contribute to the total number of regenerated muscle fibers. The origin of the immature muscle cells is briefly discussed.

Carrier cell-mediated cell lysis of squamous cell carcinoma cells by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus.

BACKGROUND: The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes: SCCA1 and SCCA2. SCCA1 gene is up-regulated in squamous cell carcinoma cells. We analyzed the proximal region of the SCCA1 promoter and the antitumor effect of oncolytic adenovirus driven by the SCCA1 promoter in squamous cell carcinoma cells. METHODS: The SCCA1 promoter was analyzed by dual luciferase assay and substituted with the E1A promoter to construct the oncolytic adenovirus to determine the squamous cell carcinoma-specific cell lysis. RESULTS: Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at -525 to -475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by four-fold. Oncolytic adenovirus driven by this SCCA1 enhancer-promoter complex specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of syngeneic squamous cell carcinoma cell tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. CONCLUSIONS: SCCA1 was up-regulated in squamous cell carcinoma cells and oncolytic adenovirus driven by SCCA1 promoter specifically killed these cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma.

A further observation of muscle spindles in the extensor digitorum longus muscle of the aged rat.

We observed three novel muscle spindles in the extensor digitorum longus muscle of the aged (20 months) rat. Two muscle spindles of the three contained thin muscle fibers lacking sensory innervation between the layers of the spindle capsule and within the periaxial space, respectively. The other one contained sensory-innervated thin muscle fibers with an indistinct equatorial nucleation between the layers of the spindle capsule. These findings suggest that the occurrence of thin muscle fibers may be intimately related to the degeneration and regeneration of extrafusal muscle fibers during aging and that these newly formed thin muscle fibers may often fail to receive sensory innervation.

Fine structural study of the innervation of muscle spindles in the internal oblique muscle of the abdominal wall in the adult mouse.

We examined by electron microscopy the innervation of muscle spindles in the internal oblique muscle of the mouse abdominal wall. In the equatorial region, in addition to the sensory innervation on individual intrafusal muscle fibers, sensory cross terminals were often observed between nuclear chain fibers. In the area from the juxtaequatorial region to the polar region, nuclear bag fibers were supplied by trail and plate-type motor endings, while nuclear chain fibers were innervated by sensory endings, being probably secondary sensory endings. From these findings, it is clear that the innervation patterns differ between two types of intrafusal muscle fibers.

A small lamellar corpuscle within a small nerve bundle outside the tendon of the rat soleus muscle.

A small nerve bundle outside the tendon of the adult rat soleus muscle contained a small lamellar corpuscle similar in structural organization to the ordinary paciniform corpuscle. A terminal axon composing this corpuscle was originated from a side branch of an afferent nerve fiber and surrounded by a number (approximately 15) of closely packed flattened lamellae of modified Schwann cells, while the stem nerve fiber freely terminated within the nerve bundle. These findings suggested that an afferent nerve fiber retracted after degeneration might extend a new branch within the nerve bundle and unexpectedly form a lamellar corpuscle within it.

Regeneration of muscle fibers in the extensor digitorum longus muscle of the aged rat.

Regeneration of muscle fibers was observed in the extensor digitorum longus (EDL) muscle of aged (24 and 27 months) Wistar rats. The aged muscles consisted almost exclusively of medium-sized muscle fibers. In addition to degenerating and/or atrophied muscle fibers, very small muscle fibers <10 mum in diameter were observed in some muscle bundles which sporadically distributed in the muscle. In the degenerating muscle fibers, satellite cells mostly appeared to be normal, possibly surviving within the scaffold of basal lamina to form new (regenerating) muscle fibers. However, some of the satellite cells were degenerated and destroyed, suggesting the decrease in number of muscle fibers. On the other hand, very small muscle fibers existed between small and/or medium-sized muscle fibers or in the wide interstitial spaces between them solitarily or in small groups. In addition, immature muscle cells having a centrally located nucleus and sporadically distributed myofilaments were observed among the small and/or medium-sized muscle fibers and partially lacked a layer of basal lamina. These immature muscle cells were often closely apposed to fibroblasts with some slender cytoplasmic processes and/or to each other without an interposing basal lamina. These findings suggest that in addition to satellite cells within the basal lamina tubes, some of the regenerating muscle fibers in the aged EDL muscle may be originated from mesenchymal cells such as fibroblasts in the interstitial spaces.

Scanning electron microscopic observation of the sensory region in the frog muscle spindle.

The equatorial sensory region of muscle spindles in the fourth toe extensor digitorum longus muscle of the adult frog was examined by scanning electron microscopy. Segments of this thin and long muscle after fixation were longitudinally cut with a razor blade and then treated with an HCl-hydrolysis method to remove connective tissues. Cells of the inner capsule extended thin and flattened cytoplasmic processes, showing a sieve-like appearance. Some specimens after a partial disruption of the inner capsule reevaluated at the fine structural level that numerous sensory terminals with varicose swellings longitudinally arranged along each intrafusal muscle fiber.

Novel muscle spindles containing muscle fibers devoid of sensory innervation in the extensor digitorum longus muscle of aged rats.

We examined the structural features of muscle spindles at the equatorial and juxtaequatorial regions in the extensor digitorum longus muscle of adult (12 months) and aged (25 months) rats. In aged muscle spindles, the lamellated layers of the spindle capsule were a little increased in number compared to those in the adult ones. Two novel muscle spindles were observed in the aged muscle. In one muscle spindle, the spindle capsule contained four thin intrafusal muscle fibers invested by the inner capsule and two muscle fibers between the layers of the spindle capsule. Serial semithin sections revealed that the latter lacked the investment of the spindle capsule at the polar region. The other muscle spindle contained four intrafusal muscle fibers: two thin sensory-innervated muscle fibers invested by the inner capsule and two thick muscle fibers similar in structural features to neighboring extrafusal muscle fibers and lacking sensory innervation within the wide periaxial space. These findings suggest that two muscle fibers between the layers of the spindle capsule may be invested by the newly formed capsular cells during aging, while two thick fibers within the periaxial space may fail to receive the sensory innervation during the early development and follow the course of extrafusal fiber differentiation.

A complex muscle fiber network in the cricothyroid muscle: a scanning electron microscopic study.

OBJECTIVES: To examine the three-dimensional ultrastructure of cricothyroid (CT) muscle fibers to elucidate their morphologic characteristics with regard to the specific functions of the muscle. STUDY DESIGN: An anatomic animal study. METHODS: The CT muscles of five adult rats were processed using the HCl-hydrolysis method to remove the peri- and intramuscular connective tissue components. The fine muscle fiber arrangements were observed by scanning electron microscopy. RESULTS: Complex muscle fiber interconnections (myomyous junctions) were identified in the muscles. Myomyous junctions were characterized by the connection of many villous processes that were 1 to 2 mum in diameter and 2 to 4 mum in length with a serrated appearance at the ends of a lateral branch or a bifurcating trunk of the muscle fibers. CONCLUSIONS: Myomyous junctions form a complex muscle fiber network, which is thought to synchronize the activity of the CT muscle fibers. This is the first three-dimensional demonstration of the muscle fiber network in the CT muscle; this network may play a major role in the functional specificity of the CT muscle.

Small clusters of granule-containing cells at the lateral side of the posterior cricoarytenoid muscle of the young adult rat.

Small clusters consisting of granule-containing cells, sustentacular cells and capillaries around them, similar in structure to the carotid body-like paraganglia, sometimes existed at the lateral side of the posterior cricoarytenoid (PCA) muscle of young adult (3 months) rats. Differing from the paraganglia, however, these cell clusters were discontinuously invested by slender cytoplasmic processes of fibroblasts. In individual granule-containing cells, granules varied in size and had a concentrically or eccentrically arranged, electron-dense material, resembling those of chromaffin cells of the adrenal medulla. A series of desmosome-like structures were frequently observed between adjacent granule-containing cells, but synapses between them were not necessarily clear. Nerve endings containing clear synaptic vesicles and occasional granulated vesicles, being possibly cholinergic in nature, sometimes formed synapses with the granule-containing cells, probably indicating that the granule-containing cells receive the efferent nerve innervation. On the other hand, the sustentacular cells lacked cytoplasmic granules and sent their cytoplasmic processes around the granule-containing cells. Capillaries in and around clustered cells were of the fenestrated type. From these findings, it is suggested that unlike the carotid body-like paraganglia, the noncapsulated cell clusters at the lateral side of the PCA muscle of the young adult rat may be identical to groups of extra-adrenal chromaffin tissues.

A further observation of the structural changes of microvessels in the extensor digitorum longus muscle of the aged rat.

We further examined the structural changes of microvessels in the extensor digitorum longus muscle of the aged (18 months) rat. Muscle bundles in this aged muscle constantly consisted of numerous large muscle fibers 50-60 mum in diameter and a few small muscle fibers <30 mum in diameter. Neuromuscular junctions (NMJs) in large muscle fibers often showed degenerative figures, thus degenerating muscle fibers. On the other hand, NMJs in small muscle fibers were mainly characterized by sparse and short junctional folds, being possibly in the course of regeneration. In some muscle bundles, the extracellular matrix was a little widened. Microvascular networks from arterioles to venules via capillaries seemed to vary in structural features between muscle bundles. In addition to the normal microvascular network consisting of microvessels with a round or oval vascular lumen during their course, two different types of microvascular networks were found. One type was characterized by the constriction of arterioles, capillaries and venules, probably representing a degenerative process of the microvascular network. In fact, uneven and compressed scaffolds of basal laminae of capillaries were often observed around these constricted microvessels. The other type consisted of arterioles and capillaries with an irregular slit-like vascular lumen and venules with a round or oval vascular lumen, and these capillaries had thick or two-layered basal laminae, being probably in the course of remodeling of the microvascular network. From these findings, it is suggested that the constriction and/or contraction of microvessels by smooth muscle cells and pericytes may be involved in the degeneration and remodeling of the microvascular network in the muscle bundles following degeneration and regeneration of the muscle fibers.

Temporal occurrence of immature skeletal muscle fibers in the sciatic nerve matrix regenerating within the silicone chamber after nerve transection in the normal rat.

We examined by light and electron microscopy the immature skeletal muscle fibers in the rat sciatic nerve regenerating within the silicone chamber 14 days after nerve transection. Small myelinated and nonmyelinated nerve fibers associated with Schwann cells from the proximal stump began to approach the midportion of the interstump zone. In the middle segment, fibroblasts or fibroblast-like mesenchymal cells and macrophages were observed everywhere in the newly formed matrix filled with exuded erythrocytes and fibrin clots. In addition to some fibroblast-like mesenchymal cells were closely apposed to each other. However, the proximal and distal segments contained immature muscle fibers with various amount of myofilaments and one or plural centrally located nuclei, thus indicating various phases of the early differentiation of skeletal muscle fibers similar to those observed during an early stage of developing muscle fibers. However, the precise origin of these skeletal muscle fibers remains to be determined.

Electron microscopic study of capillary network remodeling in the extensor digitorum longus muscle of normal adult rat.

Capillary networks demonstrate structural changes during maturation, aging, vascular disease, and cancer. Their morphological structure and function have an important influence on each other. Understanding the process of morphological vascular changes in the capillary network with advancing age may help overcome fatal vascular diseases. Aging-related structural changes of the capillary segments may accompany degeneration and regeneration of muscle fibers and serve to remodel the capillary network as a means of adapting to the changing environment. However, difficulty in obtaining human samples has hampered clarification of these microstructural changes. Herein, we examined serial ultrathin sections of capillary segments in the extensor digitorum longus muscle of normal mature (12 months old) rats in an attempt to analyze their structural changes. After bifurcation, a minimum of one capillary segment was filled with erythrocytes and was found to have fenestrations and plural endothelial disruptions, or pores, at the fenestrated portions. Some of the stagnated erythrocytes demonstrated extended protrusions, and their processes appeared to penetrate the basal lamina through the pores. These findings can also show that capillary segments are involved in partial remodeling of the capillary network. A better understanding of age-related structural changes of the capillary networks will help in fine-tuning novel vascular therapy for not only several fatal vascular diseases but also malignant tumors.

Scanning electron microscopic study of the muscle fiber arrangement in the rat cricopharyngeal muscle.

CONCLUSIONS: Myomyous junctions comprise a complex muscle fiber network, which is thought to synchronize the activity of the cricopharyngeal (CP) muscle fibers. Myomyous and myotendinous junctions explain the heterogeneity in muscle fiber length which contributes to the efficient behavior of the muscle. This scanning electron microscopy (SEM) study demonstrated the complex muscle fiber arrangement of the CP muscle and improved on the previous description of its morphological specificity. OBJECTIVE: To examine the 3D ultrastructure of the inferior pharyngeal constrictor muscle fibers to obtain further knowledge of their morphological characteristics with regard to the specific functions of the muscle in deglutition. MATERIAL AND METHODS: Six adult rats were used. Their CP and thyropharyngeal (TP) muscles were obtained and processed using the HCl hydrolysis method to remove i.m. connective tissue. The fine muscle fiber structure was observed by means of SEM. RESULTS: Multifaceted muscle fiber interconnections (myomyous junctions) were identified in the CP muscle. The myomyous junctions were characterized by the tight connection of many finger-like processes at the ends of a lateral branch or bifurcating trunk of the muscle fibers. In addition, muscle fibers occasionally tapered and ended within the muscle belly, forming myotendinous junctions. The TP muscle lacked these structures.

Alterations in erythrocyte membrane lipid and its fragility in a patient with familial lecithin:cholesterol acyltrasferase (LCAT) deficiency.

Lecithin:cholesterol acyltrasferase (LCAT) plays a key role in the cholesterol metabolism-mediated esterification of free cholesterol into the cholesterol ester in normal plasma. Familial LCAT deficiency is frequently associated with anemia. Using biochemical and physiological techniques, the erythrocytes of this patient were investigated to gain an insight into the relationship between the abnormalities of lipid metabolism and erythrocyte membrane fragility. Abnormal erythrocytes, so-called Target cells and/or Knizocytes, were observed at 20% in our patient's erythrocytes. Moreover, the mean corpuscular volume of the patient's cells was 7% greater than that of a normal individual. In the membrane lipids of the patient's erythrocytes, cholesterol and phosphatidylcholine increased, and phosphatidylethanolamine decreased. The electron spin resonance technique with a fatty acid spin probe showed that the membrane fluidity was more elevated than that of normal cells in spite of the increase in cholesterol content and the cholesterol/phospholipid ratio of the membrane of patient's erythrocytes. The patient's abnormally shaped erythrocytes were less deformed than those of the normal individual under high shear stress. The partial depletion of membrane cholesterol from the patient's erythrocytes was demonstrated by incubation with normal plasma with LCAT activity. The increment of transformed erythrocytes during the incubation could be prevented by cholesterol depletion from the patient's erythrocyte membrane. These findings indicate that normochromic anemia of the patient might be caused by erythrocyte fragility resulting from decreased deformity and/or abnormal shape of the cells due to abnormal lipid composition in the membrane.

Ganglion cells in the posterior cricoarytenoid muscle of the rat following experimental denervation.

OBJECTIVE: To investigate morphological changes of the i.m. ganglion cells in the posterior cricoarytenoid (PCA) muscle of the rat following denervation of the recurrent laryngeal nerve. MATERIAL AND METHODS: The recurrent laryngeal nerve on the left side of the rat was resected. Three weeks after transection, the PCA muscle was removed for morphological study using light and electron microscopy. RESULTS: No morphological changes were found in the i.m. ganglion cells in the PCA muscle, even though the myelinated nerve fibers were destroyed and had disappeared in ramified i.m. bundles. Around the cell body, numerous non-myelinated nerve fibers were found; these contained a large number of clear, spherical synaptic vesicles approximately 50 nm in diameter and several dense-cored vesicles approximately 100 nm in diameter. In contrast, neuromuscular junctions in most muscle fibers with partially disoriented and/or disintegrated myofibrils showed degenerative figures. In some instances, however, multiple nerve terminals were detected in contact with the postsynaptic membrane. Like the varicose swellings of non-myelinated nerve fibers around the ganglion cell body, these nerve terminals contained, in addition to clear synaptic vesicles (50 nm in diameter), several dense-cored vesicles (100 nm in diameter). CONCLUSION: We suggest that i.m. ganglion cells in the rat PCA muscle may supply postganglionic nerve fibers to the denervated neuromuscular junctions after transection of the nerve.

A novel nerve bundle containing thin muscle fibers in the posterior cricoarytenoid muscle of the marmoset.

We examined a novel nerve bundle in the posterior cricoarytenoid muscle of the marmoset. This intramuscular nerve bundle contained two thin muscle fibers about 10 microm in diameter, like intrafusal muscle fibers in the muscle spindle. These thin muscle fibers were individually surrounded by nerve bundles consisting of numerous nonmyelinated nerve fibers. Individual nerve axons contained clear synaptic vesicles and large granulated vesicles, being possibly cholinergic (parasympathetic) in nature. These nerve axons were often in contact with the muscle fiber with and without an interposing basal lamina. Two thin muscle fibers gradually terminated in the endoneural connective tissue around myelinated and nonmyelinated nerve fibers during their course. The innervation of thin muscle fibers in the novel nerve bundle is briefly discussed.

Carotid body-like tissues around the rat posterior cricoarytenoid muscle.

We examined the carotid body-like tissues around the posterior cricoarytenoid (PCA) muscle of the rat by light and electron microscopy. One branch after bifurcation of the inferior (recurrent) laryngeal nerve frequently formed a small ganglion at the lateral side of this muscle and sometimes contained paraganglion cells (granule-containing cells). In addition, encapsulated structures (paraganglia) enveloped by a few layer of capsular cells were often observed on and near the muscle. Moreover, granule-containing cells resembling the encapsulated paraganglion cells were found in clusters outside the small nerve. These clustered cells were incompletely surrounded by fibroblastic processes. In addition to synapses between adjacent cells, afferent and efferent synapses were distinguished between nerve endings and these cells, possibly receiving both afferent and efferent innervation. These findings suggest that the clustered granule-containing cells outside the small nerve in the proximity of the PCA muscle may function as chemoreceptor cells as well as the paraganglion cells within the nerve bundles and the encapsulated paraganglia.