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

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

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

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

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

Transcutaneous Application of Carbon Dioxide (CO₂) Induces Mitochondrial Apoptosis in Human Malignant Fibrous Histiocytoma <em>In Vivo</em>

<div><p>Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells <em>in vitro</em>. We also demonstrated that transcutaneous application of carbon dioxide (CO₂) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO₂ exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO₂ induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO₂ treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO₂ treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO₂ may represent a novel therapeutic tool in the treatment of human MFH.</p> </div

Evaluation of mitochondrial induced apoptosis in CO₂ or control treated tumors.

<p>(A) DNA fragmentation analysis of tumor samples from CO₂ treated and control mice two weeks post-treatment by immunofluorescence. (<i>Blue</i>, nuclear; <i>Green</i>, apoptosis nuclear) (B) DNA fragmentation was assessed by flow cytometry in CO₂ treated tumors (<i>Blue dots</i>) and control tumors (<i>Red</i>) two weeks post-treatment. (C) Immunoblot analyses determined that increased expression of the cleavage products of caspase 3 and 9, and PARP occurred in the CO₂ treated tumors compared to the control tumors. Tubulin was used as an endogenous loading control. (D) Immunoblot analysis of cytochrome c and Bax in mitochondrial and cytoplasmic fractions of CO₂ treated and control tumors. Tubulin was used as an endogenous loading control. (C, D) Positive bands in immunoblot analyses were semiquantified using densitometrical analyses using the Image J program (NIH, USA, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>).</p

Effect of transcutaneous application of CO₂ on MFH cell growth <i>in vivo</i>.

<p>Mice were treated with CO₂ or control air three days after MFH cell implantation. Treatment was administered twice weekly for two weeks. (A) MFH tumors in CO₂ treated and control mice, two weeks post-implantation. (B) Tumor volume (mm³) in CO₂ treated or control mice was monitored for two weeks post-implantation. (C) Body weight (g) of CO₂ treated or control mice was monitored for two weeks post-implantation. Data represent the mean ± S.E of at least three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01).</p