Mentalis nerve branches supplying the lower lip revisited: a study of human fetuses and donated elderly cadavers.

PURPOSE: Little information is known about the mentalis nerve course from the lower lip approximation margin (free margin) to the upper lip. Likewise, no difference in nerve distribution has been observed between the cutaneous and mucosal parts of the lip. Therefore, this study reexamined mentalis nerve morphology. METHODS: For macroscopic observations, three fresh cadavers were dissected (one male and two females; aged 78-93). We also evaluated histological sections obtained from five donated elderly cadavers (two males and three females, aged 82-96 years) and 15 human fetuses (11-40 weeks or crown-rump length 80-372 mm). Immunohistochemical analysis for S100 protein and tyrosine hydroxylase was performed. RESULTS: In both fetuses and adult cadavers, one to three nerve branches ran upward in the submucosal tissue from the mental foramen. Near the free margin of the lip, some branches passed through the orbicularis oris muscle layer toward the lip skin, whereas others followed a reversed J-shaped course along the free margin. Nerve twigs ran in parallel beneath the mucosa, whereas wavy nerve twigs attached to the basal lamina of the lip epidermis. The difference in nerve endings abruptly occurred at the skin-mucosal junction. Tyrosine hydroxylase-positive sympathetic nerve twigs surrounded arteries and formed a branch composed of S100-negative unmyelinated fibers. CONCLUSION: The lower lip skin was innervated by a perforating branch passing through the orbicularis oris muscle, that was different from the lip mucosa. A sudden change in the nerve ending configuration at the mucocutaneous junction seemed to develop postnatally.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Effects of Myostatin on Nuclear Morphology at the Myotendinous Junction.

Myostatin (Myo) is known to suppress skeletal muscle growth, and was recently reported to control tendon homeostasis. The purpose of the present study was to investigate the regulatory involvement of Myo in the myotendinous junction (MTJ) in vivo and in vitro. After Achilles tendon injury in mice, we identified unexpected cell accumulation on the tendon side of the MTJ. At postoperative day 7 (POD7), the nuclei had an egg-like profile, whereas at POD28 they were spindle-shaped. The aspect ratio of nuclei on the tendon side of the MTJ differed significantly between POD7 and POD28 (p = 4.67 × 10-34). We then investigated Myo expression in the injured Achilles tendon. At the MTJ, Myo expression was significantly increased at POD28 relative to POD7 (p = 0.0309). To investigate the action of Myo in vitro, we then prepared laminated sheets of myoblasts (C2C12) and fibroblasts (NIH3T3) (a pseudo MTJ model). Myo did not affect the expression of Pax7 and desmin (markers of muscle development), scleraxis and temonodulin (markers of tendon development), or Sox9 (a common marker of muscle and tendon development) in the cell sheets. However, Myo changed the nuclear morphology of scleraxis-positive cells arrayed at the boundary between the myoblast sheet and the fibroblast sheet (aspect ratio of the cell nuclei, myostatin(+) vs. myostatin(-): p = 0.000134). Myo may strengthen the connection at the MTJ in the initial stages of growth and wound healing.

Development and Regeneration of Muscle, Tendon, and Myotendinous Junctions in Striated Skeletal Muscle.

Owing to a rapid increase in aging population in recent years, the deterioration of motor function in older adults has become an important social problem, and several studies have aimed to investigate the mechanisms underlying muscle function decline. Furthermore, structural maintenance of the muscle-tendon-bone complexes in the muscle attachment sites is important for motor function, particularly for joints; however, the development and regeneration of these complexes have not been studied thoroughly and require further elucidation. Recent studies have provided insights into the roles of mesenchymal progenitors in the development and regeneration of muscles and myotendinous junctions. In particular, studies on muscles and myotendinous junctions have-through the use of the recently developed scRNA-seq-reported the presence of syncytia, thereby suggesting that fibroblasts may be transformed into myoblasts in a BMP-dependent manner. In addition, the high mobility group box 1-a DNA-binding protein found in nuclei-is reportedly involved in muscle regeneration. Furthermore, studies have identified several factors required for the formation of locomotor apparatuses, e.g., tenomodulin (Tnmd) and mohawk (Mkx), which are essential for tendon maturation.

Development and growth of median structures in the human tongue: A histological study using human fetuses and adult cadavers.

Glossectomy is a surgical procedure performed to remove all or part of the tongue in patients with cancer. The removal of a significant part of the tongue has a marked effect on speech and swallowing function, as patients may lose not only the tongue muscles but also the median lingual septum (MLS). Therefore, to achieve successful tongue regeneration, it is necessary to investigate the developmental processes of not only the tongue muscles but also the MLS. This study was conducted to clarify the mutual development of the tongue muscles and the MLS in human fetuses. Serial or semi-serial histological sections from 37 embryos and fetuses (aged 5-39 weeks) as well as nine adults were analyzed. The MLS appeared at Carnegie stage 15 (CS15), and until 12 weeks of gestation, abundant fibers of the intrinsic transverse muscle crossed the septum in the entire tongue. However, in near-term fetuses and adults, the contralaterally extending muscles were restricted to the deepest layer just above the genioglossus muscle. This finding indicates that the crossing transverse muscle showed the highest density at mid-term. A thorough understanding of both the MLS and the tongue muscles is necessary for successful tongue regeneration.

Regeneration process of myotendinous junction injury induced by collagenase injection between Achilles tendon and soleus muscle in mice.

Recently, it has become clear that peri-muscular tissues play a significant role in the deterioration of muscle function. Understanding the function and regeneration of muscle, as well as its surrounding tissues, is crucial to determining the causes of muscular illnesses. However, the regeneration process of the myotendinous junction (MTJ), the most closely related peri-muscular tissue, is still unknown. Therefore, we generated a mouse model of MTJ injury by collagenase injection and searched for the process of regeneration of the MTJ and its adjacent regions. The MTJ region was damaged by collagenase injection, which greatly increased the tendon cross sectional area. Collagenase injections increased the proportion of myofibers with a central nucleus, which is a characteristic of regenerating muscle. The collagenase injection group had myofibers with central nuclei and expressing MTJ markers. Additionally, we measured the length of MTJs using serial cross sections of the soleus muscle and discovered that MTJs at 2 weeks after collagenase injection were shorter compared to the control group, with a propensity to progressively recover their length over time. The results showed that MTJs undergo morphological regeneration even when severely damaged, and that this regeneration occurs in conjunction with muscle regeneration. We anticipate that these findings will be valuable in upcoming research on motor unit regeneration.

Analysis of Bone Mineral Density and Bone Quality of Cortical Bone in the Human Hyoid Body and Histological Observation of the Entheses.

The hyoid is the only bone in the human body that is completely independent, not forming a joint with any other bone; its position is maintained by the suprahyoid and infrahyoid muscles, as well as several ligaments. The purpose of this study was to ascertain the effect of the functional pressure arising from these muscles and ligaments on the hyoid body structure from its bone mineral density, bone quality, and histological observations. The area between the mesial-most part of each lesser horn and the center of the hyoid body was divided equally into four measurement regions. We conducted histological investigations at each measurement region and observed the entheses. To analyze bone mass and bone quality, we also measured bone mineral density (BMD) and analyzed biological apatite (BAp) crystallite orientation in the same regions. Histological observations identified periosteal insertions and fibrocartilaginous entheses. There was no significant difference in BMD between any of the measurement regions, but the preferential orientation of BAp crystallites was stronger in the infrahyoid muscles and ligaments, where fibrocartilaginous entheses are found, than in other places. This suggests that the functional pressure at these sites might exert a major effect not only on the morphological characteristics of the entheses but also on bone quality.

Downregulation of SOX9 expression in developing entheses adjacent to intramembranous bone.

Entheses are classified into three types: fibrocartilaginous, fibrous, and periosteal insertions. However, the mechanism behind the development of fibrous entheses and periosteal insertions remains unclear. Since both entheses are part of the temporomandibular joint (TMJ), this study analyzes the TMJ entheses. Here, we show that SOX9 expression is negatively regulated during TMJ enthesis development, unlike fibrocartilage entheses which are modularly formed by SCX and SOX9 positive progenitors. The TMJ entheses was adjacent to the intramembranous bone rather than cartilage. SOX9 expression was diminished during TMJ enthesis development. To clarify the functional role of Sox9 in the development of TMJ entheses, we examined these structures in TMJ using Wnt1Cre;Sox9flox/+ reporter mice. Wnt1Cre;Sox9flox/+ mice showed enthesial deformation at the TMJ. Next, we also observed a diminished SOX9 expression area at the enthesis in contact with the clavicle's membranous bone portion, similar to the TMJ entheses. Together, these findings reveal that the timing of SOX9 expression varies with the ossification development mode.

Histology of the optic nerve head with special reference to the layer-specific distribution of composite fibers at and near the lamina cribrosa: An immunohistochemical study using specimens from elderly donated cadavers.

BACKGROUND: This study aimed to demonstrate the composite fibers of the lamina cribrosa (LC) and their layer-specific distributions. The elastic fiber-rich septa, showing a cribriform arrangement in the optic nerve, may continue into the LC. METHODS: Orbital content, including the long course of the optic nerve, was obtained from 25 elderly cadavers. Sagittal and cross-sections were prepared from each specimen. In addition to elastica Masson staining, immunohistochemistry was performed for elastin, glial fibrillary acidic protein (GFAP), S100 protein (S100), and CD68 in microglia. RESULTS: The LC beam usually had fewer elastic fibers than the septa, but an elastic fiber-rich zone was observed along the scleral flange. GFAP-positive fibers were rich in the prelaminar area, whereas S100-positive fibers were rich in all layers of the LC. Double-positive (GFAP+/S100+) fibers were present in the prelaminar area. In contrast, S100-single positive fibers were evident in the LC and retrolaminar areas and were likely to insert into a sclera-choroid border area. The density of macrophages and microglia was not different between the septa and LC. Individual variations were observed in the distribution and density of the nerve-associated fibrous tissues. CONCLUSION: The LC beam was quite different from the septa in the composite fibers and architecture. Transverse fibers, dominant in the LC beam, corresponded to fibrous processes of astrocytes and other nerve-associated fibrous tissues. Many of these nerve elements suggest low mechanical properties of the LC.