Katsuya Kami

Effects of chronic treadmill running on neurogenesis in the dentate gyrus of the hippocampus of adult rat.

Proliferating astrocytes and proliferating neuroblasts have been observed in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus of adult rats under normal conditions. However, whether these proliferating cells are stimulated by running has not been determined. Using immunohistochemical techniques, we examined the effects of chronic treadmill running on proliferating astrocytes (PCNA+/GFAP+ cells), proliferating neuroblasts (PCNA+/DCX+ cells) and newly generated postmitotic neurons (DCX+/NeuN+ cells) in the DG of the hippocampus of adult rats and also characterized the morphological features of PCNA+/GFAP+ cells and PCNA+/DCX+ cells. PCNA+/GFAP+ cells with few processes and PCNA+/DCX+ cells without long processes were detected in the SGZ, and we determined that these are morphological features of the astrocytes and neuroblasts with proliferative ability. Chronic treadmill running (at a speed of 22 m/min, 30 min/days for 7 days) significantly increased the numbers of PCNA+/GFAP+ cells and DCX+/NeuN+ cells, and the number of PCNA+/DCX+ cells tended to increase by chronic treadmill running. These results indicate that chronic treadmill running stimulates the proliferation of astrocytes in the SGZ. Furthermore, the present study indicates that chronic treadmill running increases DCX+/NeuN+ cells that are detected in a transient stage during the neuronal maturation process. These events may be the cellular basis mediating both running-induced increases of new neurons in the DG of the hippocampus and running-induced improvement of learning and memory functions of adult rats.

In vivo activation of STAT3 signaling in satellite cells and myofibers in regenerating rat skeletal muscles

Although growth factors and cytokines play critical roles in skeletal muscle regeneration, intracellular signaling molecules that are activated by these factors in regenerating muscles have been not elucidated. Several lines of evidence suggest that leukemia inhibitory factor (LIF) is an important cytokine for the proliferation and survival of myoblasts in vitro and acceleration of skeletal muscle regeneration. To elucidate the role of LIF signaling in regenerative responses of skeletal muscles, we examined the spatial and temporal activation patterns of an LIF-associated signaling molecule, the signal transducer and activator transcription 3 (STAT3) proteins in regenerating rat skeletal muscles induced by crush injury. At the early stage of regeneration, activated STAT3 proteins were first detected in the nuclei of activated satellite cells and then continued to be activated in proliferating myoblasts expressing both PCNA and MyoD proteins. When muscle regeneration progressed, STAT3 signaling was no longer activated in differentiated myoblasts and myotubes. In addition, activation of STAT3 was also detected in myonuclei within intact sarcolemmas of surviving myofibers that did not show signs of necrosis. These findings suggest that activation of STAT3 signaling is an important molecular event that induces the successful regeneration of injured skeletal muscles.

Localization of leukemia inhibitory factor and interleukin-6 messenger ribonucleic acids in regenerating rat skeletal muscle

In the present study, we characterized both temporal and spatial expression patterns of leukemia inhibitory factor (LIF) and interleukin‐6 (IL‐6) messenger ribonucleic acids (mRNAs) in injured skeletal muscle using in situ hybridization. LIF and IL‐6 mRNAs were expressed in mononucleated cells and damaged muscle cells. Further, signals for LIF mRNA were also detected in Schwann cell‐like cells of intramuscular nerves. These results suggest that the earliest events involved in the repair of injured muscles and nerves may be triggered by these cytokines.

Gene Expression of Receptors for IL-6, LIF, and CNTF in Regenerating Skeletal Muscles

The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor α (IL-6R), LIF receptor β (LIFR), and CNTF receptor α (CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons.

LOCALIZATION OF MYOGENIN, C-FOS, C-JUN, AND MUSCLE-SPECIFIC GENE MRNAS IN REGENERATING RAT SKELETAL MUSCLE

It has been suggested that myogenin is an important factor for the differentiation of myoblasts and that its function in myogenesis is regulated by proto-on-cogenes in in vitro experiments. We have characterized the spatial and temporal expression patterns of myogenin, c-fos, c-jun, and muscle creatine kinase mRNAs during the skeletal muscle regeneration process using in situ hybridization histochemistry. Myogenin transcripts are first detected in the myonuclei/nuclei of satellite cells at 6 h after induction of regeneration. Myogenin mRNA is expressed in desmin-positive myoblasts, yet no muscle creatine kinase mRNA is detected in this cell type. Both the muscle creatine kinase and myogenin mRNAs are expressed in the newly formed myotubes, but not at earlier stages. Transcripts for c-fos and c-jun mRNAs are expressed first in the myonuclei/nuclei of satellite cells at 3 h post-trauma. c-jun mRNA is expressed in both myoblasts and myotubes, while c-fos mRNA was not detected in these cells. These results suggest that myogenin plays important roles in the regeneration of injured muscle and that c-jun and c-fos may have different roles in this process.

Alterations of M-cadherin, neural cell adhesion molecule and β-catenin expression in satellite cells during overload-induced skeletal muscle hypertrophy

Aim:  Neural cell adhesion molecule (NCAM) and M‐cadherin are cell adhesion molecules expressed on the surface of skeletal muscle satellite cell (SC). During myogenic morphogenesis, M‐cadherin participates in mediating terminal differentiation and fusion of myoblasts by forming a complex with β‐catenin and that NCAM contributes to myotube formation by fusion of myoblasts. Hypertrophy and hyperplasia of functionally overloaded skeletal muscle results from the fusion with SCs into the existing myofibres or new myofibre formation by SC–SC fusion. However, the alterations of NCAM, M‐cadherin and β‐catenin expressions in SCs in response to functional overload have not been investigated.

Galectin-1 is a novel factor that regulates myotube growth in regenerating skeletal muscles.

Adult skeletal muscles have a vigorous regenerative capacity in response to chemical, mechanical or physical injuries. Muscle satellite cells play a critical role in skeletal muscle regeneration. Activated satellite cells (myoblasts) proliferate and then differentiate. Differentiated myoblasts fuse with each other to form multinucleated myotubes, and the growth of myotubes is induced by both fusion with additional myoblasts and reinnervation of motor neurons. Cellular and molecular events underlying the regenerative processes are regulated by critical factors, which are produced by satellite cells, myoblasts, myotubes, extracellular matrix and inflammatory cells. Galectin-1 is abundantly synthesized in adult skeletal muscles, but its roles in muscle regeneration have not been fully elucidated. We reviewed previous studies on the function of galectin-1 regarding myogenesis in vivo and in vitro, and discussed the roles of this lectin in regenerating skeletal muscles based on our observations. In intact adult muscles, galectin-1 was associated with basement membranes of myofibers. After muscle injury, galectin-1 immunoreactivity was increased within the cytoplasm of activated satellite cells. Thereafter, differentiated myoblasts lost galectin-1 immunoreactivity, but galectin-1 expression associated with basement membranes was detected in myotubes. Administration of anti-galectin-1 antibody, which perturbs the function of galectin-1, decreased the size of myotubes. Furthermore, muscle injury induced abundant expression of galectin-1 in damaged intramuscular nerve axons. We conclude that galectin-1 is a novel factor that promotes both myoblast fusion and axonal growth following muscle injury, and consequently, regulates myotube growth in regenerating skeletal muscles.

Features and a possible role of Mash1-immunoreactive cells in the dentate gyrus of the hippocampus in the adult rat

Neurogenesis occurs throughout life in both the subventricular zone (SVZ) and subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus in the adult brain. In the SVZ, it has been demonstrated that transit-amplifying neural progenitor cells, which appear between neural stem/progenitor cells (NSPCs) and neuroblasts during the neuronal differentiation process, express mammalian achaete-scute homolog 1 (Mash1), which regulates differentiation during neurogenesis. Although Mash1-positive cells (Mash1+ cells) are observed in the SGZ, the importance of Mash1 in hippocampal neurogenesis is not sufficiently understood. In the present study, using immunohistochemical techniques, we examined whether Mash1+ cells in the SGZ act as transit-amplifying neural progenitor cells, and whether chronic treadmill running can induce alterations of the Mash1+ cells in the SGZ of the DG. The present results indicated that Mash1 immunoreactivity is detected in proliferative cells, and that astrocytes or NSPCs and neuroblasts express Mash1. A quantitative analysis of Mash1-positive astrocytes or NSPCs and Mash1-positive neuroblasts indicated that approximately 90% of Mash1+ cells did not belong to astrocytic and neuronal cells. Furthermore, chronic treadmill running induced an increase in the number of proliferating Mash1+ cells. The present study suggests that the majority of the Mash1+ cells in the SGZ may be transit-amplifying neural progenitor cells. In addition, the proliferation of Mash1-positive transit-amplifying neural progenitor cells may contribute to the exercise-induced neurogenesis that is associated with the improvement of learning and memory function.

1250 Localization of LIF and LIFR and mRNAs in regenerating rat skeletal muscle

SOICHIRO ITOH, HIROTAKE SAMEJIMA, TSUYOSHI OHTA, KIYOSHI MOCHIDA, KENICHI SHINOMIYA Into the Silastic tube of 15-mm length Type1 collagen gel (G-group) or fibers (F-group), collagen fibers coated with laminih (L-group) or a synthetic laminin peptide containing the YIGSR sequence (Y-group) were packed, which were grafted to the sciatic nerve of Wistar rats. As a control the empty Silastic tube was grafted, and animals were sacrificed 8 weeks after operation. Minifascicle formation was found among the space of the degenerated collagen fibers in the F-, Land Y-group. Immunocytochemistry using anti-S-loo, anti-GFAP and anti-laminin antibody revealed that the infiltrating cells with elliptic nucleus into the degenerated collagen fibers might be Schwann cells. The density of axons and the % axon area in the Lor Y-group were significantly higher than those in the F-group. These results suggest that the collagen fiber becomes scaffold of the growth cone or Schwann cells, and that YIGSR sequence of laminin promotes the nerve regeneration in vivo.