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The authors investigated the process of denervation and reinnervation of the interarytenoid (IA) muscle in the guinea pig using transmission electron microscopy and glycogen depletion technique after unilateral transection of the recurrent laryngeal nerve (RLN) and superior laryngeal nerve to clarify the innervation pattern of the unpaired IA muscle. Anastomosis between the bilateral arytenoid branches was confirmed in the belly of the IA muscle. Five weeks after transection, all of the IA muscle fibers appeared to have been reinnervated by the contralateral RLN. As the arytenoid branch of the RLN runs together with that of the contralateral RLN in a single intramuscular nerve funiculus, it is possible that collateral sprouting branches grow and extend into the adjacent denervated Schwann's sheaths. The authors conclude that the unpaired IA muscle, as a whole, receives specific motor nerve supply from the bilateral RLNs, although each muscle fiber is innervated unilaterally. [ABSTRACT FROM AUTHOR]

Capillaries in the posterior cricoarytenoid and the arytenoid muscles of the guinea pig were examined by electron microscopy 3 days after transection of unilateral recurrent laryngeal nerve. The denervated posterior cricoarytenoid muscle had neuromuscular junctions (NMJs) lacking terminal axons, while in the unpaired arytenoid muscle, normal and denervated NMJs coexisted among muscle fibres being seemingly normal. Capillaries in both muscles were almost of the continuous type. However, 10% of capillaries around the denervated NMJs and among some muscle fibres in both muscles, often had a small number (<10) of fenestrae bridged by a single-layered diaphragm. The untreated normal muscles contained only continuous capillaries. These findings suggest that denervation may induce the fenestrated changes of intramuscular capillaries. [ABSTRACT FROM PUBLISHER]

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., (© The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

Although replication-competent viruses have been developed to treat cancers, their cytotoxic effects are insufficient, as infection is inhibited by the generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with replication-competent adenovirus were incubated with target cancer cells in a high titer of anti-adenovirus antibody. Carrier cells were injected into syngeneic subcutaneous tumors after immunization with adenovirus. Carrier cell-derived cell fragments containing viral particles were engulfed by proliferative target cancer cells. This engulfment-mediated transfer of adenovirus was not inhibited by the anti-adenovirus antibody and enabled repetitive infection. After the induction of anti-adenoviral cytotoxic T-lymphocyte (CTL) responses by immunization with adenovirus, administration of carrier cells infected with a replication-competent adenovirus induced complete tumor regression. Adenovirus-GM-CSF augmented the anti-tumor effect of carrier cells by increasing anti-adenoviral and anti-tumoral CTL responses and decreased the number of injections of carrier cells required to induce complete tumor regression. This novel carrier cell-mediated viral transfection system might prove useful in a variety of cancer therapies.

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.

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.

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.

Age-related remodeling of the hypopharyngeal constrictor muscle was studied by comparing the morphological features of the subneural apparatus (SNA) of the thyropharyngeal (TP) and cricopha ryngeal (CP) muscles in young and old rats. Scanning electron microscopy revealed that the TP and CP muscles had both gutter- and depression-type SNAs, although different proportions of the two types of apparatus were found in each muscle. In young-adult rats, the gutter-type SNA was predominant in the TP muscle, whereas in the CP muscle the depression type was predominant. By contrast, in old rats, the depression-type SNA was predominant in the TP muscle, while no such transformation of the dominant SNA was noted in the CP muscle. In addition, the number of type IIb and type I muscle fibers was lower and greater, respectively, in older animals compared with those of younger animals. Furthermore, there were more type IIc fibers in the TP muscle of old rats, but no difference in the CP muscle between young and old animals. These findings suggest that the transformation of SNAs and the muscle fibers of the hypopharyngeal constrictor muscle takes place during aging and that their remodeling processes differ in the TP and CP muscles.

We employed light and electron microscopy to examine the innervation of terminal arterioles in the rat posterior cricoarytenoid (PCA) muscle over a three-week period before and after unilateral denervation of the recurrent nerve. Observations showed an occasional single small-sized myelinated nerve fiber around the terminal arteriole in both normal and denervated PCA muscles. One axon after the last node of Ranvier of the small-sized myelinated nerve fiber, together with some nonmyelinated nerve fibers, was enwrapped by the Schwann cell and ran along the terminal arteriole, forming varicose swellings with numerous mitochondria along its course. In one instance, a small-sized myelinated nerve fiber in the denervated PCA muscle ramified into several branches containing numerous mitochondria. These nerve endings mainly existed in the adventitia, consisting of some layers of fibroblasts and collagen fibrils, and some of them were close to the vascular smooth muscle cells with only an intervening basal lamina. These findings suggest that the mitochondria-filled nerve endings may be sensory in nature, and function in response to the expansion and contraction of blood vessels caused by increased and/or decreased blood flow or intravascular pressure.