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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Structural changes of microvessels in the extensor digitorum longus muscle of the aged rat.

We examined the structural changes of microvessels (arterioles, capillaries, and venules) in the extensor digitorum longus muscle of the aged (27 months) rat. Muscle bundles in the aged muscle almost consisted of medium-sized muscle fibers which were peculiar in the aged EDL muscle. However, microvessels in and around these muscle bundles varied in shape. Degenerating capillaries and scaffolds of basal laminae remaining after necrosis of preexisting capillaries were frequently observed around these medium-sized muscle fibers. In addition, the vascular lumen was often very narrow or irregular slit-like. In terminal and precapillary arterioles, the endothelium and smooth muscle cells showed a constricted appearance and their vascular lumen was often irregular slit-like, probably playing an important role in intercepting the blood flow into the disrupted capillaries. Moreover, some venules had the slit-like vascular lumen, sawtooth-like endothelium and thick or multilayered basal laminae, and occasional erythrocytes were found outside the endothelium, probably indicating that these venules are in the course of regeneration. These findings suggest that in addition to the frequent destruction of capillaries, the structural changes of arterioles and venules may be involved in remodeling the microvasculature of the muscle bundles after maturation of regenerating muscle fibers.

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.

Carrier cell-mediated delivery of a replication-competent adenovirus for cancer gene therapy.

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.

The sensory region of muscle spindles in the posterior cricoarytenoid muscle of the marmoset.

We examined muscle spindles in the posterior cricoarytenoid (PCA) muscle of the marmoset. The spindle capsule contained only one intrafusal muscle fiber which did not form a typical aggregation of equatorial nuclei such as bags and chains. The intrafusal fiber was innervated by sensory endings which branched and arranged irregularly. These sensory endings lay on the surface of the fiber in shallow grooves or deeply penetrated into it. These findings suggest that the muscle spindles in the marmoset PCA muscle are strikingly different in the structure of intrafusal fibers in the arrangement of sensory endings from those in common mammalian skeletal muscles.