Calculated data showing relative changes in relative amounts of oxy/deoxy-Hb (values 8 min. after blood pooling started were set as standards).

<p>The data are expressed as mean ± S.E.M. The averages and significance levels were calculated based on the measurements of the 7 subjects. Statistical significance at <i>P</i><0.05 is denoted by * and <i>P</i><0.01 is denoted by **.</p

Intramuscular pH changes in the triceps surae muscle during transcutaneous application of CO₂ using ³¹P-MRS.

<p>The graph shows that pH decreases during the accumulation of CO₂. (n = 5) Graph data are expressed as means ± S.E.M.</p

Rationale of the experimental outline.

<p>(A) The application of our novel system for transcutaneous application of CO₂ (For the upper limb). (B) The application of our novel system for transcutaneous application of CO₂ (For the lower limb).</p

pH changes in CO₂-absorbing solution during transcutaneous CO₂ application through the rat skin with or without the CO₂ hydrogel.

<p>The graph shows pH changes during CO₂ application with or without the CO₂ hydrogel. (n = 6). Graph data are expressed as means ± S.E.M.</p

Novel system for transcutaneous CO₂ application in the forearm with NIRS probe.

<p>Novel system for transcutaneous CO₂ application in the forearm with NIRS probe.</p

Western blot analysis of myogenic differentiation-related proteins.

<p>WT: wild type, KO: knockout. The expression levels of <i>MyoD</i>, <i>myogenin</i>, and <i>Pax7</i> was increased 3 days after injury in WT mice, but not in p21KO mice.</p

Immunofluorescence analysis of muscle basement membrane and plasma membrane.

<p>The expression of DAPI, laminin, and dystrophin was examined in wild-type (WT; upper panel) and p21 knockout (KO; lower panel) mice. (A) Expression in control, (B) Expression in 3 days, (C) Expression in 14 days, and (D) Expression in 28 days post-operation. (Scale bar = 50μm). (E) Quantification of laminin expression, (F) Quantification of dystrophin expression. (A-F) The injured membrane structure was almost repaired 14 days after injury in WT group, but not in the p21 KO group (p < 0.05).</p

P21 Deficiency Delays Regeneration of Skeletal Muscular Tissue

<div><p>The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. <i>Cyclin D1</i> mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of <i>MyoD</i>, <i>myogenin</i>, and <i>Pax7</i> indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the <i>in vivo</i> healing process in muscular injury.</p></div

Measurement of oxygenated and deoxygenated Hb concentration during transcutaneous application of CO₂ using NIRS.

<p>(A) Continuous measurement of oxy and deoxy-Hb concentrations using NIRS with pooling blood by a pneumatic tourniquet. The bold lines demonstrate the CO₂ group data. All data show the changes in Hb concentrations from the starting point to the end point of measurement (n = 7). Data show the decrease in oxy-Hb and the increase in deoxy-Hb after pooling blood, followed by the greater decrease in oxy-Hb and greater increase in deoxy-Hb in CO₂ group after transcutaneous CO₂ application. (B) Relative changes in amounts of oxy/deoxy-Hb (the values 8 min. after blood pooling started were set as standards). Graph data are expressed as mean ± S.E.M. The averages and significance checks were calculated based on measurements of the 7 subjects. Statistical significance at P<0.05 is denoted by *, and P<0.01 is denoted by **. The graph shows a significant decrease in oxy-Hb and increase in deoxy-Hb in the CO₂ group.</p

Measuring device to validate CO₂ hydrogel <i>in vitro</i> using rat skin.

<p>Measuring device to validate CO₂ hydrogel <i>in vitro</i> using rat skin.</p