Author name: Steve Scott

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.

Abstract Background: Joint contractures are a major complication in patients with spinal cord injuries. Positioning, stretching, and physical therapy are advocated to prevent and treat contractures; however, many patients still develop them. Joint motion (exercise) is crucial to correct contractures. Transcutaneous carbon dioxide (CO2) therapy was developed recently, and its effect is similar to that of exercise. This therapy may be an alternative or complementary approach to exercise. Question/purposes: Using an established model of spinal cord injury in rats with knee flexion contractures, we sought to clarify whether transcutaneous CO2 altered (1) contracture, as measured by ROM; (2) muscular and articular factors contributing to the loss of ROM; (3) fibrosis and fibrosis-related gene expression in muscle; and (4) the morphology of and fibrosis-related protein expression in the joint capsule. Methods: Thirty-six Wistar rats were divided into three equal groups: caged control, those untreated after spinal cord injury, and those treated with CO2 after spinal cord injury. The rats were treated with CO2 from either the first day (prevention) or 15th day (treatment) after spinal cord injury for 2 or 4 weeks. The hindlimbs of rats in the treated group were exposed to CO2 gas for 20 minutes once daily. Knee extension ROM was measured with a goniometer and was measured again after myotomy. We calculated the muscular and articular factors responsible for contractures by subtracting the post-myotomy ROM from that before myotomy. We also quantified histologic muscle fibrosis and evaluated fibrosis-related genes (collagen Type 1, α1 and transforming growth factor beta) in the biceps femoris muscle with real-time polymerase chain reaction. The synovial intima’s length was measured, and the distribution of fibrosis-related proteins (Type I collagen and transforming growth factor beta) in the joint capsule was observed with immunohistochemistry. Knee flexion contractures developed in rats after spinal cord injuries at all timepoints. Results: CO2 therapy improved limited-extension ROM in the prevention group at 2 weeks (22° ± 2°) and 4 weeks (29° ± 1°) and in the treatment group at 2 weeks (31° ± 1°) compared with untreated rats after spinal cord injuries (35° ± 2°, mean difference, 13°; 39° ± 1°, mean difference, 9°; and 38° ± 1°, mean difference, 7°, respectively) (95% CI, 10.50-14.86, 8.10-10.19, and 4.73-9.01, respectively; all p < 0.001). Muscular factors decreased in treated rats in the prevention group at 2 weeks (8° ± 2°) and 4 weeks (14°± 1°) and in the treatment group at 2 weeks (14 ± 1°) compared with untreated rats (15° ± 1°, 4.85-9.42; 16° ± 1°, 1.24-3.86; and 17° ± 2°, 1.16-5.34, respectively; all p < 0.05). The therapy improved articular factors in the prevention group at 2 weeks (4° ± 1°) and 4 weeks (6° ± 1°) and in the treatment group at 2 weeks (8° ± 1°) compared with untreated rats (10° ± 1°, 4.05-7.05; 12° ± 1°, 5.18-8.02; and 11° ± 2°, 1.73-5.50, respectively; all p < 0.05). CO2 therapy decreased muscle fibrosis in the prevention group at 2 weeks (p < 0.001). The expression of collagen Type 1, α1 mRNA in the biceps femoris decreased in treated rats in the prevention group at 2 and 4 weeks compared with untreated rat (p = 0.002 and p = 0.008, respectively), although there was little difference in the expression of transforming growth factor beta (p > 0.05). CO2 therapy did not improve shortening of the synovial intima at all timepoints (all p > 0.05). CO2 therapy decreased transforming growth factor beta immunolabeling in joint capsules in the rats in the prevention group at 2 weeks. The staining intensity and Type I collagen pattern showed no differences among all groups at all timepoints. Conclusion: CO2 therapy may be useful for preventing and treating contractures after spinal cord injuries. CO2 therapy particularly appears to be more effective as a prevention and treatment strategy in early-stage contractures before irreversible degeneration occurs, as shown in a rat model. Clinical relevance: Our findings support the idea that CO2 therapy may be able to improve the loss of ROM after spinal cord injury.

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.

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 CO2 increased blood flow and a partial increase of O2 pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO2 to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO2 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 CO2 may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.

Abstract Carbon dioxide is a physiologic compound present in our body, mainly as a result of cellular metabolism. The frequency of carboxytherapy use by dermatologists and cosmetologists increased significantly in the second half of the 20th century due to the fact that it improves blood circulation within skin tissues. This article focuses on the use of carboxytherapy in case of various skin problems, such as stretch marks, scars, loss of elasticity, redundancy of fatty tissue, cellulite, morphea, and alopecia. The review of sparse studies that are available indicate increasing interest in this method.

A 60-year-old woman presented with a chief complaint of scarring along the sites of a submental incision and a right preauricular incision that had been made 5 years earlier during a rhytidectomy procedure. Examination revealed tethering, hypertrophy, and erythema of the submental incision (Figure 1, A) and widening of the preauricular incision (Figure 2, A). The patient did not wish to undergo any scar revision surgeries and instead opted for noninvasive treatment with injections of carbon dioxide (CO2) gas (carboxytherapy). She underwent 10 treatments, administered 1 to 2 weeks apart. She was satisfied with the results (Figures 1, B, and 2, B). CO2 is a nontoxic gas that is naturally produced by cells as they undergo the process of metabolism. CO2 therapy for cutaneous use was introduced in France in 2000.1 Carboxytherapy has been used to treat stretch marks, skin laxity, cellulite, dark circles under the eyes, and scars. It is believed that its mechanism of action is twofold. First, the mechanical injection of the CO causes the destruction of fat cells and the interruption of surrounding connective tissue. Second, as the CO2 accumulates in tissues, it induces dilation of capillaries and stimulates an inflammatory response that increases collagen deposition and reorganization and eventually improves skin texture and tone. The injection of CO2 has been shown to be relatively safe, with little to no toxicity.1-5 As such, carboxytherapy can be used as an adjunct to liposuction in order to remove any irregularities. Furthermore, its revascularization and collagen-remodeling effects make carboxytherapy useful for improving unsightly scars.1-3,5 Treatment with carboxytherapy is relatively painless. The injection is carried through a 30-gauge, 5/8-inch needle with the bevel directed toward the skin. Patients typically experience a light tingling sensation at the injection site that soon dissipates. The area might feel warm for 10 to 20 minutes, and there may be some ecchymosis at the injection site. The subcutaneous crepitus that ensues is usually resolved within 1 hour, and patients are asked to avoid hot baths, hot tubs, and saunas for about 4 hours. A typical treatment protocol consists of 10 to 20 weekly treatments. Results are usually noticed after the fifth treatment, and improvement is seen with subsequent treatment. Because of the relative absence of toxicity, ease of use, and predictable results, carboxytherapy is an excellent addition to the armamentarium of cosmetic surgeons.

Abstract For a successful conventional or superficial liposuction, it is necessary to consider the competence of the surgeon who is to administer the procedure necessary for this type of surgery as well as the physical and psychological evaluation of the determined patient. A poor result often is related to the persistence of adipose tissue irregularity in the form of fatty tissue accumulation. This complication, common to this type of surgery, has called for research to determine methods for its treatment. Carbon dioxide (CO2) therapy refers to the transcutaneous and subcutaneous administration of CO2 for therapeutic purposes. This treatment originated at the Royal Spas of France in 1932 with the treatment of patients affected by obliteration of arteriopathies. Recent studies have demonstrated the effect of subcutaneous CO2 therapy performed to improve local parameters of circulation (performed by Doppler, laser-Doppler, and trans-cutaneous partial pressure of oxygen determination), and to reduce localized adiposities (verified reporting variations in maximum circumference and performing histologic studies). With these results, the absence of toxicity, and the relevant side effects related to this treatment taken into consideration, the Plastic Surgery Unit of Siena has been committed to researching the role that CO2 therapy can play in the treatment of skin irregularity and as a complement to liposuction. The authors report their experience using Carbomed programmable automatic CO2 therapy apparatus and 30GA1/2 0,3X13 microlance needles for the treatment of patients with adipose tissue accumulations located on the thighs and knees. In their study, 42 patients were divided into three groups: A, B, and C. In Group A, only liposuction was performed. In group B 3 weeks after liposuction CO2 therapy was administered in two weekly subcutaneous applications of CO2 for 10 consecutive weeks. In group C, CO2 therapy alone was administered with the same contingencies used for group B (two weekly subcutaneous applications of CO2 for 10 consecutive weeks). The objective was to assess the effectiveness of CO2 therapy for skin irregularity and as a complement to liposuction for adipose tissue accumulation by reporting variations in circumference and skin elasticity monitored by the Cutometer SEM 474 in all treated areas. The data obtained were analyzed statistically. Values of p less than 0.05 were considered significant. The authors report their experience and the results achieved from the study.

Abstract Diabetic foot foot ulcer (DFU) is a serious complication of diabetes mellitus, and possibly the major morbidity of the diabetic foot. It is the most common foot injury in diabetic patients and can lead to lower-extremity amputation. Management of DFU requires a systematic knowledge of the major risk factors for amputation, frequent routine evaluation, scrupulous preventive maintenance, and correction of peripheral arterial insufficiency. Carboxytherapy refers to the subcutaneous injection of CO2 to improve the microcirculation and promote wound healing by stimulating the microcirculation. Since optimal ulcer healing requires adequate tissue perfusion, it is considered that carboxytherapy could be useful in the treatment of DFU. The present prospective clinical study included 40 patients with different sizes and types of chronic DFU. In addition to cleaning of the wound, antibiotics, and debridement as necessary, the treatment protocol included blood sugar control, medication, healthy habits, no weight-bearing, and carboxytherapy. The results showed that this treatment which included carboxytherapy promoted wound healing and prevented amputation. These positive effects should be confirmed through a complete study that includes different clinical and instrumental parameters.

Abstract Background: The inefficient healing of chronic wounds is a result of poor blood perfusion at the wound and surrounding tissues. Artificially applied carbon dioxide (CO2) has the potential to improve the perfusion and oxygenation of tissues, hence is useful for the healing of chronic wounds. Objective: The aim of the present study was to determine the effect of a transcutaneous application of physiological vasodilator gaseous CO2 on cutaneous blood flow. Methods: Laser Doppler (LD) flux in cutaneous microcirculation, skin temperature, electrocardiogram and arterial blood pressure were measured simultaneously in a group of 33 healthy men, aged 21-28 years, during rest and a 35-minute CO2 therapy. One lower limb of each subject represented the studied extremity, being exposed to gaseous CO2. The contralateral limb was the control, being exposed to air. Each limb was sealed in a plastic bag. Results: During CO2 therapy the LD flux in the studied extremity increased from 5.8 PU ± 3.9 PU to 30.3 PU ± 16.7 PU (mean ± standard deviation; paired t-test, p < 0.001), while that in the control extremity did not change significantly. Conclusions: Our results confirm a local vasodilatory effect of applied CO2 therapy. This finding indicates its potential clinical use. Keywords: CO2 therapy; Physiological measurements; dry CO2 bath; laser Doppler fluxmetry.