The Relationship Between Breathing, Muscles, and the Skeletal System

Title: Topical cutaneous application of carbon dioxide via a hydrogel for improved fracture repair: results of phase I clinical safety trial

Authors: Niikura T, Iwakura T, Omori T, Lee SY, Sakai Y, Akisue T, Oe K, Fukui T, Matsushita T, Matsumoto T, Kuroda R.

Journal: BMC Musculoskelet Disord. 2019 Nov 25;20(1):563. doi: 10.1186/s12891-019-2911-7. PMID: 31766994; PMCID: PMC6878668.

Link to full text: Topical cutaneous application of carbon dioxide via a hydrogel for improved fracture repair: results of phase I clinical safety trial

Abstract: Background: Clinicians have very limited options to improve fracture repair. Therefore, it is critical to develop a new clinically available therapeutic option to assist fracture repair biologically. We previously reported that the topical cutaneous application of carbon dioxide (CO2) via a CO2 absorption-enhancing hydrogel accelerates fracture repair in rats by increasing blood flow and angiogenesis and promoting endochondral ossification. The aim of this study was to assess the safety and efficacy of CO2 therapy in patients with fractures.

Methods: Patients with fractures of the femur and tibia were prospectively enrolled into this study with ethical approval and informed consent. The CO2 absorption-enhancing hydrogel was applied to the fractured lower limbs of patients, and then 100% CO2 was administered daily into a sealed space for 20 min over 4 weeks postoperatively. Safety was assessed based on vital signs, blood parameters, adverse events, and arterial and expired gas analyses. As the efficacy outcome, blood flow at the level of the fracture site and at a site 5 cm from the fracture in the affected limb was measured using a laser Doppler blood flow meter.

Results: Nineteen patients were subjected to complete analysis. No adverse events were observed. Arterial and expired gas analyses revealed no adverse systemic effects including hypercapnia. The mean ratio of blood flow 20 min after CO2 therapy compared with the pre-treatment level increased by approximately 2-fold in a time-dependent manner.

Conclusions: The findings of the present study revealed that CO2 therapy is safe to apply to human patients and that it can enhance blood flow in the fractured limbs.

Title: Transcutaneous carbon dioxide application accelerates muscle injury repair in rat models

Authors: Akahane S, Sakai Y, Ueha T, Nishimoto H, Inoue M, Niikura T, Kuroda R.

Journal: Int Orthop. 2017 May;41(5):1007-1015. doi: 10.1007/s00264-017-3417-2. Epub 2017 Feb 16. PMID: 28210805.

Link to PubMed: Transcutaneous carbon dioxide application accelerates muscle injury repair in rat models

Abstract: Purpose: Skeletal muscle injuries are commonly observed in sports and traumatology medicine. Previously, we demonstrated that transcutaneous application of carbon dioxide (CO2) to lower limbs increased the number of muscle mitochondria and promoted muscle endurance. Therefore, we aimed to investigate whether transcutaneous CO2 application could enhance recovery from muscle injury.

Methods: Tibialis anterior muscle damage was induced in 27 Sprague Dawley rats via intramuscular injection of bupivacaine. After muscle injury, rats were randomly assigned to transcutaneous CO2-treated or -untreated groups. From each group, three rats were sacrificed at weeks one, two, four and six. At each time point, histology and immunofluorescence analyses were performed, and changes in muscle weight, muscle weight/body weight ratio, muscle fibre circumference, gene expression levels and capillary density were measured.

Results: Injured muscle fibres were completely repaired at week six in the CO2-treated group but only partially repaired in the untreated group. The repair of basement and plasma membranes did not differ significantly between groups. However, expression levels of genes and proteins related to muscle protein synthesis were significantly higher in the CO2-treated group and significantly more capillaries four weeks after injury.

Conclusion: Transcutaneous CO2 application can accelerate recovery after muscle injury in rats.

Title: Transcutaneous carbon dioxide application suppresses bone destruction caused by breast cancer metastasis

Authors: Takemori T, Kawamoto T, Ueha T, Toda M, Morishita M, Kamata E, Fukase N, Hara H, Fujiwara S, Niikura T, Kuroda R, Akisue T.

Journal: Oncology Reports, 40, 2079-2087. https://doi.org/10.3892/or.2018.6608

Link to full text: Transcutaneous carbon dioxide application suppresses bone destruction caused by breast cancer metastasis

Abstract: Hypoxia plays a significant role in cancer progression, including metastatic bone tumors. We previously reported that transcutaneous carbon dioxide (CO2) application could decrease tumor progression through the improvement of intratumor hypoxia. Therefore, we hypothesized that decreased hypoxia using transcutaneous CO2 could suppress progressive bone destruction in cancer metastasis. In the present study, we examined the effects of transcutaneous CO2 application on metastatic bone destruction using an animal model. The human breast cancer cell line MDA-MB-231 was cultured in vitro under three different oxygen conditions, and the effect of altered oxygen conditions on the expression of osteoclast-differentiation and osteolytic factors was assessed. An in vivo bone metastatic model of human breast cancer was created by intramedullary implantation of MDA-MB-231 cells into the tibia of nude mice, and treatment with 100% CO2 or a control was performed twice weekly for two weeks. Bone volume of the treated tibia was evaluated by micro-computed tomography (µCT), and following treatment, histological evaluation was performed by hematoxylin and eosin staining and immunohistochemical staining for hypoxia-inducible factor (HIF)-1α, osteoclast-differentiation and osteolytic factors, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclast activity. In vitro experiments revealed that the mRNA expression of RANKL, PTHrP and IL-8 was significantly increased under hypoxic conditions and was subsequently reduced by reoxygenation. In vivo results by µCT revealed that bone destruction was suppressed by transcutaneous CO2, and that the expression of osteoclast-differentiation and osteolytic factors, as well as HIF-1α, was decreased in CO2-treated tumor tissues. In addition, multinucleated TRAP-positive osteoclasts were significantly decreased in CO2-treated tumor tissues. Hypoxic conditions promoted bone destruction in breast cancer metastasis, and reversal of hypoxia by transcutaneous CO2 application significantly inhibited metastatic bone destruction along with decreased osteoclast activity. The findings in this study strongly indicated that transcutaneous CO2 application could be a novel therapeutic strategy for treating metastatic bone destruction.

Title: Effects of the duration of transcutaneous CO2 application on the facilitatory effect in rat fracture repair

Authors: Oda T, Iwakura T, Fukui T, Oe K, Mifune Y, Hayashi S, Matsumoto T, Matsushita T, Kawamoto T, Sakai Y, Akisue T, Kuroda R, Niikura T.

Journal: J Orthop Sci. 2020 Sep;25(5):886-891. doi: 10.1016/j.jos.2019.09.017. Epub 2019 Oct 18. PMID: 31635930.

Link to full text: Effects of the duration of transcutaneous CO2 application on the facilitatory effect in rat fracture repair

Abstract: Background: Carbon dioxide therapy has been reported to be effective in treating certain cardiac diseases and skin problems. Although a previous study suggested that transcutaneous carbon dioxide application accelerated fracture repair in association with promotion of angiogenesis, blood flow, and endochondral ossification, the influence of the duration of carbon dioxide application on fracture repair is unknown. The aim of this study was to investigate the effect of the duration of transcutaneous carbon dioxide application on rat fracture repair.

Methods: A closed femoral shaft fracture was created in each rat. Animals were randomly divided into four groups: the control group; 1w-CO2 group, postoperative carbon dioxide treatment for 1 week; 2w-CO2 group, postoperative carbon dioxide treatment for 2 weeks; 3w-CO2 group, postoperative carbon dioxide treatment for 3 weeks. Transcutaneous carbon dioxide application was performed five times a week in the carbon dioxide groups. Sham treatment, where the carbon dioxide was replaced with air, was performed for the control group. Radiographic, histological, and biomechanical assessments were performed at 3 weeks after fracture.

Results: The fracture union rate was significantly higher in the 3w-CO2 group than in the control group (p < 0.05). Histological assessment revealed promotion of endochondral ossification in the 3w-CO2 group than in the control group. In the biomechanical assessment, all evaluation items related to bone strength were significantly higher in the 3w-CO2 group than in the control group (p < 0.05).

Conclusions: The present study, conducted using an animal model, demonstrated that continuous carbon dioxide application throughout the process of fracture repair was effective in enhancing fracture healing.

Title: Topical cutaneous application of CO2 accelerates bone healing in a rat femoral defect model

Authors: Kuroiwa Y, Fukui T, Takahara S, Lee SY, Oe K, Arakura M, Kumabe Y, Oda T, Matsumoto T, Matsushita T, Akisue T, Sakai Y, Kuroda R, Niikura T.

Journal: BMC Musculoskelet Disord. 2019 May 22;20(1):237. doi: 10.1186/s12891-019-2601-5. PMID: 31113412; PMCID: PMC6530028.

Link to full text: Topical cutaneous application of CO2 accelerates bone healing in a rat femoral defect model

Abstract: Background: Bone defects may occur because of severe trauma, nonunion, infection, or tumor resection. However, treatments for bone defects are often difficult and have not been fully established yet. We previously designed an efficient system of topical cutaneous application of carbon dioxide (CO2) using a novel hydrogel, which facilitates CO2 absorption through the skin into the deep area within a limb. In this study, the effect of topical cutaneous application of CO2 on bone healing was investigated using a rat femoral defect model.

Methods: In this basic research study, an in vivo bone defect model, fixed with an external fixator, was created using a rat femur. The affected limb was shaved, and CO2 was applied for 20 min/day, 5 days/week. In the control animals, CO2 gas was replaced with air. Radiographic, histological, biomechanical, and genetic assessments were performed to evaluate bone healing.

Results: Radiographically, bone healing rate was significantly higher in the CO2 group than in the control group at 4 weeks (18.2% vs. 72.7%). The degree of bone healing scored using the histopathological Allen grading system was significantly higher in the CO2 group than in the control group at 2 weeks (1.389 ± 0.334 vs. 1.944 ± 0.375). The ultimate stress, extrinsic stiffness, and failure energy were significantly greater in the CO2 group than in the control group at 4 weeks (3.2 ± 0.8% vs. 38.1 ± 4.8%, 0.6 ± 0.3% vs. 41.5 ± 12.2%, 2.6 ± 0.8% vs. 24.7 ± 5.9%, respectively.). The volumetric bone mineral density of the callus in micro-computed tomography analysis was significantly higher in the CO2 group than in the control group at 4 weeks (180.9 ± 43.0 mg/cm3 vs. 247.9 ± 49.9 mg/cm3). Gene expression of vascular endothelial growth factor in the CO2 group was significantly greater than that in the control group at 3 weeks (0.617 ± 0.240 vs. 2.213 ± 0.387).

Conclusions: Topical cutaneous application of CO2 accelerated bone healing in a rat femoral defect model. CO2 application can be a novel and useful therapy for accelerating bone healing in bone defects; further research on its efficacy in humans is warranted.

Keywords: Bone defect; Bone healing; CO2.

Title: Transcutaneous carbon dioxide application with hydrogel prevents muscle atrophy in a rat sciatic nerve crush model

Authors: Nishimoto H, Inui A, Ueha T, Inoue M, Akahane S, Harada R, Mifune Y, Kokubu T, Nishida K, Kuroda R, Sakai Y.

Journal: J Orthop Res. 2018 Jun;36(6):1653-1658. doi: 10.1002/jor.23817. Epub 2017 Dec 19. PMID: 29193246.

Link to full text: Transcutaneous carbon dioxide application with hydrogel prevents muscle atrophy in a rat sciatic nerve crush model

Abstract: The acceleration of nerve regeneration remains a clinical challenge. We previously demonstrated that transcutaneous CO2 application using a novel hydrogel increases the oxygen concentration in local tissue via an "artificial Bohr effect" with the potential to prevent muscle atrophy. In this study, we investigated the effect of transcutaneous CO2 administration on limb function after peripheral nerve injury in a rat sciatic nerve injury model. In total, 73 Sprague-Dawley rats were divided into a sham group, a control group (crush injury to sciatic nerve and no treatment) or a CO2 group (crush injury with transcutaneous CO2 application). CO2 was administered percutaneously for 20 min five times per week. Scores for the sciatic function index and pinprick test were significantly higher in the CO2 group than control group. The muscle wet weight ratios of the tibialis anterior and soleus muscles were higher in the CO2 group than control group. Electrophysiological examination showed that the CO2 group had higher compound motor action potential amplitudes and shorter distal motor latency than the control group. Histological examination of the soleus muscle sections at postoperative week 2 showed shorter fiber diameter in the control group than in the CO2 group. The mRNA expression of Atrogin-1 and MuRF-1 was lower, mRNA expression of VEGF and myogenin and MyoD was higher in CO2 group at postoperative week 2 compared to the control group.

Clinical significance: Transcutaneous CO2 application has the therapeutic potential to accelerate the recovery of muscle atrophy in peripheral nerve injury. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1653-1658, 2018.

Keywords: Bohr effect; muscle atrophy; peripheral nerve injury; transcutaneous carbon dioxide application.

Title: Carbon Dioxide Water Bathing Enhances Myogenin but Not MyoD Protein Expression after Skeletal Muscle Injury

Authors: Nonaka K, Akiyama J, Tatsuta N, Une S, Ito K, Ogaya S, Kataoka M, Iwata A, Okuda K.

Journal: J Phys Ther Sci. 2013 Jun;25(6):709-11. doi: 10.1589/jpts.25.709. Epub 2013 Jul 23. PMID: 24259835; PMCID: PMC3805014.

Link to full text: Carbon Dioxide Water Bathing Enhances Myogenin but Not MyoD Protein Expression after Skeletal Muscle Injury

Abstract: [Purpose] We reported that carbon dioxide (CO2) water bathing accelerates skeletal muscle regeneration; however, the underlying mechanism was unclear. MyoD and myogenin play roles in muscle regeneration, and the purpose of this study was to determine changes in MyoD and myogenin caused by CO2 water bathing after injury. [Subjects] Sixteen female Wistar rats (n = 4 per group) were used. [Methods] The rats were divided into four groups: no-injury (NI), injury (IC), injury + tap water bathing (ITW), and injury + CO2 water bathing (ICO2). Muscle injury was induced by injection of bupivacaine hydrochloride into the left tibial anterior (TA) muscles. Tap water and CO2 (1,000 ppm) water bathing were performed at 37 °C for 30 minutes once a day. The left TA muscles were removed 4 days after injury, and the expressions of MyoD and myogenin were measured. [Results] MyoD and myogenin were increased in the IC, ITW, and ICO2 groups compared with the NI group. Although the MyoD level was similar in the IC, ITW, and ICO2 groups, myogenin increased more in the ICO2 group than in the IC and ITW groups. [Conclusion] CO2 water bathing after muscle injury appears to induce an increase in the expression of myogenin.

Keywords: CO2 water bathing; Myogenic regulatory factors; Skeletal muscle injury.

Title: Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury

Authors: Hirota J, Hasegawa T, Inui A, Takeda D, Amano-Iga R, Yatagai N, Saito I, Arimoto S, Akashi M.

Journal: Int Wound J. 2023 Apr;20(4):1151-1159. doi: 10.1111/iwj.13974. Epub 2022 Oct 17. PMID: 36250918; PMCID: PMC10031219.

Link to full text: Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury

Abstract: In postoperative patients with head and neck cancer, scar tissue formation may interfere with the healing process, resulting in incomplete functional recovery and a reduced quality of life. Percutaneous application of carbon dioxide (CO2 ) has been reported to improve hypoxia, stimulate angiogenesis, and promote fracture repair and muscle damage. However, gaseous CO2 cannot be applied to the head and neck regions. Previously, we developed a paste that holds non-gaseous CO2 in a carrier and can be administered transdermally. Here, we investigated whether this paste could prevent excessive scarring and promote muscle regeneration using a bupivacaine-induced rat model of muscle injury. Forty-eight Sprague Dawley rats were randomly assigned to either a control group or a CO2 group. Both groups underwent surgery to induce muscle injury, but the control group received no treatment, whereas the CO2 group received the CO2 paste daily after surgery. Then, samples of the experimental sites were taken on days 3, 7, 14, and 21 post-surgery to examine the following: (1) inflammatory (interleukin [IL]-1β, IL-6), and transforming growth factor (TGF)-β and myogenic (MyoD and myogenin) gene expression by polymerase chain reaction, (2) muscle regeneration with haematoxylin and eosin staining, and (3) MyoD and myogenin protein expression using immunohistochemical staining. Rats in the CO2 group showed higher MyoD and myogenin expression and lower IL-1β, IL-6, and TGF-β expression than the control rats. In addition, treated rats showed evidence of accelerated muscle regeneration. Our study demonstrated that the CO2 paste prevents excessive scarring and accelerates muscle regeneration. This action may be exerted through the induction of an artificial Bohr effect, which leads to the upregulation of MyoD and myogenin, and the downregulation of IL-1β, IL-6, and TGF-β. The paste is inexpensive and non-invasive. Thus, it may be the treatment of choice for patients with muscle damage.

Title: Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats

Authors: Koga T, Niikura T, Lee SY, Okumachi E, Ueha T, Iwakura T, Sakai Y, Miwa M, Kuroda R, Kurosaka M.

Journal: J Bone Joint Surg Am. 2014 Dec 17;96(24):2077-84. doi: 10.2106/JBJS.M.01498. PMID: 25520342.

Link to PubMed: Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats

Abstract: Background: We previously demonstrated that topical cutaneous application of CO2, by means of a hydrogel in which the CO2 readily dissolves, increases blood flow and oxygen dissociation from hemoglobin in the soft tissues surrounding bone. In the present study, we utilized a rat fracture model to test the hypothesis that application of this treatment to fractured limbs would accelerate fracture repair.

Methods: A closed femoral shaft fracture was created in each rat. Topical cutaneous application of CO2 by means of a hydrogel was performed five times a week for up to four weeks in the CO2/hydrogel group (n = 60). Sham treatments were performed in the control group (n = 60). Radiographic, histological, immunohistochemical, laser Doppler perfusion imaging, real-time polymerase chain reaction, and biomechanical assessments were performed.

Results: Radiographic fracture union was evident at week 3 in twelve (86%) of fourteen animals in the CO2/hydrogel group compared with five (36%) of fourteen in the control group (p < 0.05; 95% CI [confidence interval] for the difference in union rate, 2.26% to 99.64%). Histological assessment revealed promotion of endochondral ossification in the CO2/hydrogel group. Immunohistochemical assessment at week 2 showed significantly greater capillary density in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 161 to 258 per mm(2)). Laser Doppler perfusion imaging demonstrated that the blood flow in the fractured limb was significantly greater at weeks 2 and 3 in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 8.4% to 22.4% and 6.7% to 19.0%, respectively). Gene expression of chondrogenic, osteogenic, and angiogenic markers was significantly greater in the CO2/hydrogel group at several time points. Ultimate stress, extrinsic stiffness, and failure energy (relative to the contralateral limb) were significantly greater in the CO2/hydrogel group at week 3 (p < 0.05; 95% CI for the difference, 24.8% to 67.5%, 4.0 % to 22.7%, and 9.6% to 58.8%, respectively). There were no significant differences between the groups with respect to any outcome measure at week 4.

Conclusions: Topical cutaneous application of CO2 by means of a hydrogel accelerated fracture repair in association with the promotion of angiogenesis, blood flow, and endochondral ossification.

Title: Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats

Authors: Inoue M, Sakai Y, Oe K, Ueha T, Koga T, Nishimoto H, Akahane S, Harada R, Lee SY, Niikura T, Kuroda R.

Journal: J Orthop Sci. 2020 Mar;25(2):338-343. doi: 10.1016/j.jos.2019.03.024. Epub 2019 Apr 25. PMID: 31031109.

Link to PubMed: Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats

Abstract: Background: Muscle atrophy causes difficulty in resuming daily activities after a fracture. Because transcutaneous carbon dioxide (CO2) application has previously upregulated oxygen pressure in the local tissue, thereby demonstrating its potential in preventing muscle atrophy, here we investigated effects of CO2 application on muscle atrophy after femoral shaft fracture.

Methods: Thirty fracture model rats were produced and randomly divided into a no treatment (control group) and treatment (CO2 group) groups. After treatment, the soleus muscle was dissected at post-fracture days 0, 14, and 21. Evaluations were performed by measuring muscle weight and performing histological examination and gene expression analysis.

Results: Muscle weight was significantly higher in the CO2 group than in the control group. Histological analysis revealed that the muscle fiber cross-sectional area was reduced in both groups. Nevertheless, the extent of atrophy was lesser in the CO2 group. Muscle fibers in the control group tended to change into fast muscle fibers. Vascular staining revealed that more capillary vessels surrounded the muscle fibers in the CO2 group than in the control group. Messenger RNA (mRNA) analysis revealed that the CO2 group had a significantly enhanced expression of genes that were related to muscle synthesis.

Conclusion: Transcutaneous CO2 application may be a novel therapeutic strategy for preventing skeletal muscle atrophy after fracture.

Title: The antioxidant action of СО2-one of the universal mechanisms of carboxytherapy

Authors: Drogovoz, S. M., K. O. Kalko, L. B. Ivantsyk, А. L. Shtroblya, Yu V. Stoletov, K. V. Drogovoz, and V. F. Ostasko.

Journal:

Link to full text: The antioxidant action of СО2-one of the universal mechanisms of carboxytherapy

Abstract: Carboxytherapy (carbon dioxide treatment) is a promising area and an alternative method of treating many diseases. Carbon dioxide (CO2) is constantly present in the body, being the end product of cellular metabolism. It accumulates in tissues, diffuses into the blood, and is transferred to the lungs in three forms: dissolved in plasma, in bicarbonates, and as oxyhemoglobin in erythrocytes. CO2 is totally indispensable for the normal course of biochemical, humoral, and tissue processes in the body, as it is a pacemaker of these processes.
Materials and research methods. Scientific publications in foreign and domestic journals on relevant topics over the past 5 years, Internet resources.
Research results and their discussion. Analysis of the above changes in the body under the action of CO2 and states of biochemical markers (ADMA and d-ROM, ROS, NO, NOS) under oxidative stress indicate that CO2 has antioxidant properties, and carboxytherapy is a pathogenetic therapy for diseases which pathogenesis includes oxidative stress. The ROS-inhibitory effect of CO2 is universal for different cell types and, obviously, for different species of living organisms.
Conclusions. Thus, it has been established that CO2 is a universal inhibitor of the reactive oxygen species generation by various human and animal cells. These data allow explaining the physiological and pathophysiological (medical) effects of CO2 in the mechanism of carboxytherapy in a new way from the standpoint of the biochemical role of ROS.
Key words: antioxidant action, carbon dioxide, carboxytherapy.

Title: Carbon dioxide-rich water bathing enhances collateral blood flow in ischemic hindlimb via mobilization of endothelial progenitor cells and activation of NO-cGMP system

Authors: Irie H, Tatsumi T, Takamiya M, Zen K, Takahashi T, Azuma A, Tateishi K, Nomura T, Hayashi H, Nakajima N, Okigaki M, Matsubara H.

Journal: Circulation. 2005 Mar 29;111(12):1523-9. doi: 10.1161/01.CIR.0000159329.40098.66. PMID: 15795362.

Link to full text: Carbon dioxide-rich water bathing enhances collateral blood flow in ischemic hindlimb via mobilization of endothelial progenitor cells and activation of NO-cGMP system

Abstract: Background: Carbon dioxide-rich water bathing has the effect of vasodilatation, whereas it remains undetermined whether this therapy exerts an angiogenic action associated with new vessel formation.

Methods and results: Unilateral hindlimb ischemia was induced by resecting the femoral arteries of C57BL/J mice. Lower limbs were immersed in CO2-enriched water (CO2 concentration, 1000 to 1200 mg/L) or freshwater (control) at 37 degrees C for 10 minutes once a day. Laser Doppler imaging revealed increased blood perfusion in ischemic limbs of CO2 bathing (38% increase at day 28, P<0.001), whereas N(G)-nitro-L-arginine methyl ester treatment abolished this effect. Angiography or immunohistochemistry revealed that collateral vessel formation and capillary densities were increased (4.1-fold and 3.7-fold, P<0.001, respectively). Plasma vascular endothelial growth factor (VEGF) levels were elevated at day 14 (18%, P<0.05). VEGF mRNA levels, phosphorylation of NO synthase, and cGMP accumulation in the CO2-bathed hindlimb muscles were increased (2.7-fold, 2.4-fold, and 3.4-fold, respectively) but not in forelimb muscles. The number of circulating Lin-/Flk-1+/CD34- endothelial-lineage progenitor cells was markedly increased by CO2 bathing (24-fold at day 14, P<0.001). The Lin-/Flk-1+/CD34- cells express other endothelial antigens (endoglin and VE-cadherin) and incorporated acetylated LDL.

Conclusions: Our present study demonstrates that CO2 bathing of ischemic hindlimb causes the induction of local VEGF synthesis, resulting in an NO-dependent neocapillary formation associated with mobilization of endothelial progenitor cells.

Title: Evidence-based hydro- and balneotherapy in Hungary--a systematic review and meta-analysis

Authors: Bender T, Bálint G, Prohászka Z, Géher P, Tefner IK.

Journal: Int J Biometeorol. 2014 Apr;58(3):311-23. doi: 10.1007/s00484-013-0667-6. Epub 2013 May 16. PMID: 23677421; PMCID: PMC3955132.

Link to full text: Evidence-based hydro- and balneotherapy in Hungary--a systematic review and meta-analysis

Abstract: Balneotherapy is appreciated as a traditional treatment modality in medicine. Hungary is rich in thermal mineral waters. Balneotherapy has been in extensive use for centuries and its effects have been studied in detail. Here, we present a systematic review and meta-analysis of clinical trials conducted with Hungarian thermal mineral waters, the findings of which have been published by Hungarian authors in English. The 122 studies identified in different databases include 18 clinical trials. Five of these evaluated the effect of hydro- and balneotherapy on chronic low back pain, four on osteoarthritis of the knee, and two on osteoarthritis of the hand. One of the remaining seven trials evaluated balneotherapy in chronic inflammatory pelvic diseases, while six studies explored its effect on various laboratory parameters. Out of the 18 studies, 9 met the predefined criteria for meta-analysis. The results confirmed the beneficial effect of balneotherapy on pain with weight bearing and at rest in patients with degenerative joint and spinal diseases. A similar effect has been found in chronic pelvic inflammatory disease. The review also revealed that balneotherapy has some beneficial effects on antioxidant status, and on metabolic and inflammatory parameters. Based on the results, we conclude that balneotherapy with Hungarian thermal-mineral waters is an effective remedy for lower back pain, as well as for knee and hand osteoarthritis.

Title: The Effectiveness of Manual Therapy in the Cervical Spine and Diaphragm, in Combination with Breathing Reeducation Exercises, in Patients with Non-Specific Chronic Neck Pain: Protocol for Development of Outcome Measures and a Randomized Controlled Trial

Authors: Tatsios PI, Grammatopoulou E, Dimitriadis Z, Koumantakis GA.

Journal: Diagnostics (Basel). 2022 Nov 4;12(11):2690. doi: 10.3390/diagnostics12112690. PMID: 36359533; PMCID: PMC9689657.

Link to full text: The Effectiveness of Manual Therapy in the Cervical Spine and Diaphragm, in Combination with Breathing Reeducation Exercises, in Patients with Non-Specific Chronic Neck Pain: Protocol for Development of Outcome Measures and a Randomized Controlled Trial

Abstract: Until now, non-specific chronic neck pain has mainly been considered as a musculoskeletal system dysfunction, with associated psychological involvement due to its prolonged or recurrent nature. However, patients with non-specific chronic neck pain frequently additionally exhibit respiratory dysfunction. Emerging evidence suggests that addressing the respiratory dysfunction in these patients will provide additional therapeutic benefits in musculoskeletal and respiratory-related outcomes for several reasons (biomechanical, biochemical, and psychological). Motor control dysfunction of the muscles surrounding the spine (diaphragm included) negatively affects the mechanics and biochemistry of breathing (pH-homeostasis). An impaired and ineffective breathing pattern has been recognized as the primary source of many unexplained symptoms (anxiety, depression, confusion, chest pain, hypocapnia, and breathlessness) in patients with non-specific chronic neck pain. The proposed protocol's purpose is dual: to assess the relative effectiveness of manual therapy in the cervical spine and the diaphragm, in combination with breathing reeducation exercises, along with cervical spine manual therapy or usual physical therapy care on the underlying dysfunctions in patients with non-specific chronic neck pain via a randomized controlled clinical trial, and to validate part of the outcome measures. Several musculoskeletal and respiratory dysfunction outcomes will be employed to delimit the initial extent and level of dysfunction and its resolution with the treatments under study.
Keywords: breathing exercises; breathing reeducation; chronic neck pain; diaphragm; manual therapy; respiratory dysfunction.

Title: The effect of transcutaneous application of carbon dioxide (CO₂) on skeletal muscle

Authors: Oe K, Ueha T, Sakai Y, Niikura T, Lee SY, Koh A, Hasegawa T, Tanaka M, Miwa M, Kurosaka M.

Journal: Biochem Biophys Res Commun. 2011 Apr 1;407(1):148-52. doi: 10.1016/j.bbrc.2011.02.128. Epub 2011 Mar 1. PMID: 21371433.

Link to PubMed: The effect of transcutaneous application of carbon dioxide (CO₂) on skeletal muscle

Abstract: In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1α) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1α-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO(2) increased blood flow and a partial increase of O(2) pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO(2) to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO(2) application caused: (1) the gene expression of PGC-1α, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO(2) may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.