Heritage and Technology

Hisae Hayashi 1, Sumio Yamada, Yoshitaka Kumada, Hiroshi Matsuo, Takanobu Toriyama, Hirohisa Kawahara

ABSTRACT
Objective: We investigated the effect of immersion of feet in CO2-enriched water for preventing expansion and formation of ischemic ulcer in critical limb ischemia of diabetic patients after surgical revascularization.
Materials and methods: Eligible patients were allocated CO2 group (CO2 immersion plus standard care) or control group (standard care alone) and were followed up for 3 months after surgical revascularization. The end point is defined as an expansion of a target ulcer (more than 101% of original size) or the formation of new ulcers during the follow-up period.
Results: Fifty-nine patients out of originally enrolled 66 patients with type II diabetes were included in intention-to-treat population. The cumulative prevention rate for ischemic ulcer after 3 months was 97.1% in the CO2 group, while,- in the control group, it was 77.8%, i.e., significantly lower than the CO2 group (P = 0.012, log-rank test). The transcutaneous oxygen pressure increased significantly only in the CO2 group, from 56 ± 14 to 63 ± 15 mmHg (P < 0.01, Wilcoxon signed rank test), in 3 months.
Conclusion: These results suggest that addition of CO2 immersion to standard care of critical limb ischemia in diabetic patients improves early postoperative outcome after vascular surgery.

Paramjit S. Tappia, Brett Hiebert, Rohan Sanjanwala, Paul Komenda, Chris Sathianathan, Amarjit S. Arneja and Bram Ramjiawan

ABSTRACT
Ineffective healing and treatment of foot ulcers can lead to an infection and gangrene of the wound area that ultimately results in the loss of the limb. The incidence of foot ulcers is higher in patients with diabetes, peripheral vascular disease and kidney disease. Accordingly, this study was undertaken to assess the ability of foot bathing in CO2-enriched water to heal foot ulcers. The design was a double-blinded, randomized, placebo-controlled study. Patients with at least one foot ulcer were randomized to receive either a treatment with bath therapy at 37 ± 0.5 °C containing either 1000–1200 ppm CO2-enriched tap water (the intervention) or non-carbonated tap water at 37 ± 0.5 °C (the control group). Treatment was conducted three times/week for 15 min per session for up to 16 weeks for a total of 48 treatment sessions. Before and at the end of every treatment month, wound size, wound area oxygenation and the ankle brachial index were measured. In addition, the McGill pain questionnaire was conducted. Blood was also collected at these time points (for a total of five collections) for the measurement of different biomarkers. While no significant differences (p < 0.05) in the group/time interaction effect were observed, a clear separation within the wound area reduction/wound area/oxygenated Hb outcomes was seen between placebo (control) and treatment (CO2) group. This pilot study is suggestive that bathing in CO2-enriched water may accelerate the healing of foot ulcers.

Cesare Brandi 1, Luca Grimaldi, Giuseppe Nisi, Anna Brafa, Alessandra Campa, Massimiliano Calabrò, Matteo Campana, Carlo D’Aniello

ABSTRACT
A wound is defined as chronic when it does not heal according to the normal repair times and mechanisms. This particular condition may be principally due to local hypoxia. Carbon dioxide (CO(2)) therapy refers to the transcutaneous or subcutaneous administration of CO(2) for therapeutical effects on both microcirculation and tissue oxygenation. In this study, we report the clinical and instrumental results of the application of CO(2) in the therapy of chronic wounds. The study included 70 patients affected by chronic ulcers. The patients were selected by aetiology and wound extension and equally divided into two homogeneous groups. In group A, CO(2) therapy was used in addition to the routine methods of treatment for such lesions (surgical and/or chemical debridement, advanced dressings according to the features of each lesion). In group B, patients were treated using routine methods alone. Both groups underwent to instrumental (laser doppler flow, measurement of TcPO(2)), clinical and photographic evaluation. In the group that underwent subcutaneous treatment with CO(2) therapy, the results highlighted a significant increase in tissue oxygenation values, which was confirmed by greater progress of the lesions in terms of both healing and reduction of the injured area. Considering the safety, efficacy and reliability of this method, even if further studies are necessary, we believe that it is useful to include subcutaneous carbon dioxide therapy in the treatment of wounds involving hypoxia-related damage.

Helena Ban Frangež, Zoran Rodi, Jana Miklavčič and Igor Frangež

ABSTRACT
Aim: Diabetic symmetrical peripheral neuropathy is a common complication of diabetes mellitus. Patients treated with transcutaneous CO2 application for chronic wounds reported an improvement in peripheral sensations. This study aimed to evaluate the effect of transcutaneous application of gaseous CO2 on diabetic symmetrical peripheral neuropathy. Methods: A prospective randomized, double-blind study was performed at the University Medical Center Ljubljana between September 2019 and September 2020. Sixty consecutive patients with diabetes with a unilateral chronic wound were randomized into either a study group that received transcutaneous CO2 therapy or a control group that received placebo treatment with air. Results: Vibration, monofilament sensation, and temperature of the big toe improved significantly in the study group (p < 0.001, for vibration sensation, monofilament test and temperature of the big toe), but not in the control group (p = ns for all evaluated outcomes). Conclusion: According to our results, a transcutaneous application of gaseous CO2 shows promising results in treating diabetic symmetrical peripheral neuropathy. Considering the major consequences of sensory loss leading to foot ulceration and possibly amputation, we believe this treatment approach deserves future attention and investigation as a treatment modality of diabetic symmetrical peripheral neuropathy.

Igor Frangez, Jure Colnaric, Danijela Truden

ABSTRACT
In neuropathic foot ulcers, the most prominent finding is the loss of peripheral sensation and is typically seen in diabetic patients. In addition, vasculopathy may lead to foot ulcerations in diabetic patients. CO2 therapy was found to improve chronic wound healing in patients with vascular impairment. It refers to the transcutaneous and subcutaneous application of CO2 as well as CO2 water baths for therapeutic purpose. In the method used, gaseous CO2 is applied transcutaneously using the PVR system®. CO2 is applied by means of a single-use, low-density polyethylene bag which is wrapped around the leg being treated and secured with an elastic strap. The advantages of this method, compared to injecting CO2 into subcutaneous tissue, are non-invasiveness, the absence of pain and protection against infection. Compared to CO2 balneotherapy this approach enables the use of higher CO2 concentrations, application to chronic wound patients and, with appropriate precautionary measures, prevents the increase of CO2 in the surrounding air. Finzgar et al. observed that the transcutaneous application of gaseous CO2 caused a significant increase in the Laser Doppler (LD) flux in cutaneous microcirculation in vivo in humans. The favourable clinical and microcirculatory effects of gaseous CO2 have further been observed in studies of patients with intermittent claudication as well as patients with primary and secondary Raynaud’s phenomenon. The reviewed studies suggest that the increased delivery of CO2 to the ulcerated area will cause vasodilation and an increase in blood flow. The improved angiogenesis and oxygenation will result in healing of the chronic wound. This principle may be applied in the treatment of diabetic foot ulceration. Moreover, the effect on blood flow may also be important in preventive and curative treatment of patients with impaired mobility due to organic or functional causes. Further work is needed for the development of therapeutic strategies to optimize CO2 use in diabetic foot patients.

Miha Finžgar, Helena Ban Frangež, Ksenija Cankar, Igor Frangež

ABSTRACT
Introduction: Microvascular function is impaired in patients with diabetes mellitus (DM) and is involved in numerous DM complications. Several microvascular-supporting interventions have been proposed of which the transcutaneous application of gaseous CO2 (hereinafter CO2 therapy) is one of the most promising. The aim of present study was to determine the effect of repeated CO2 therapies on the cutaneous microvascular function in DM patients with diabetic foot ulcers.
Methodology: A total of 42 subjects with at least one chronic diabetic foot ulcer were enrolled in the study. They were divided into the experimental group (21 subjects aged 64.6 ± 11.6 years) that underwent 4-week-long treatment with transcutaneous application of gaseous CO2 (hereinafter CO2 therapies), and the placebo group (21 subjects aged 65.0 ± 10.7 years) that underwent 4-week-long placebo treatment with transcutaneous application of air. Before the first and after the last treatment in both groups, laser Doppler (LD) flux in foot cutaneous microcirculation, heart rate, and arterial blood pressure measurements were carried out during rest and local thermal hyperaemia (LTH) provocation test.
Results: In the experimental group the following statistically significant changes were observed after the completed treatment 1) increased mean relative powers of LD flux signals during rest in the frequency bands related to NO-independent endothelial (0.07 ± 0.055 vs. 0.048 ± 0.059, p = 0.0058), NO-mediated endothelial (0.154 ± 0.101 vs. 0.113 ± 0.108, p = 0.015), and neurogenic (0.17 ± 0.107 vs. 0.136 ± 0.098, p = 0.018) activity; 2) decreased resting LD flux (35 ± 29 PU vs. 52 ± 56 PU; p = 0.038); and 3) increased peak LD flux as a function of baseline during LTH (482 ± 474%BL vs. 287 ± 262%BL, p = 0.036); there were no statistically significant changes observed in the placebo group. No systemic effects were observed in none of the two groups by means of mean values of heart rate and arterial blood pressure.
Conclusions: Repeated CO2 therapies improves the microvasular function in DM patients without any systemic side effects.

Milos Macura, Helena Ban Frangez, Ksenija Cankar, Miha Finžgar, Igor Frangez

ABSTRACT
Chronic wounds in diabetics are difficult to treat, therefore, adjuvant therapies have been investigated. Bathing in CO2‐rich water (spa therapy) has been known in Europe for decades for its positive effect on peripheral vascular disorders. Recently, much effort has been invested in developing optimal application methods of CO2. Uses include subcutaneous injections of CO2, bathing in CO2‐enriched water, and transcutaneous application of CO2. To verify the effect of transcutaneous application of gaseous CO2 on the healing of chronic diabetic wounds, a randomized double‐blind clinical research was designed. The research included 30 and 27 wounds in the study and control groups, respectively. In addition to standard treatment, patients in the study group received 20 therapies with medical‐grade CO2 gas and the control group received the same treatment with air. Results showed significantly faster healing in the study group: 20 of the 30 wounds in the study group were healed compared with none in the control group. Mean wound surface and volume in the study group was reduced significantly (surface: 96%, P = .001, volume: 99%, P = .003) compared with a small reduction in the control group (surface: 25%, P = .383, volume: 27%, P = .178).
Considering our results, transcutaneous application of gaseous CO2 is an effective adjuvant therapy in diabetic chronic wound treatment.

Tomohiro Matsumoto 1, Masayuki Tanaka, Ryosuke Nakanish, Mihi Takuwa, Takumi Hirabayashi, Kohei Ono, Takuya Ikeji, Noriaki Maeshige, Yoshitada Sakai, Toshihiro Akisue, Hiroyo Kondo, Akihiko Ishihara, Hidem Fujino

ABSTRACT
Hyperglycemia impairs oxidative capacity in skeletal muscle. Muscle oxidative capacity is regulated by peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α). Transcutaneous carbon dioxide (CO2) enhances PGC-1α expression in skeletal muscle. Therefore, the aim of this study was to clarify the effects of CO2 therapy on muscle oxidative capacity impaired by streptozotocin (STZ)-induced hyperglycemia. Eight-week-old male Wistar rats were randomly divided into 4 groups: control, CO2 treatment, STZ-induced hyperglycemia, and STZ-induced hyperglycemia treated with CO2. STZ-induced hyperglycemia resulted in a decrease of muscle oxidative capacity and decreased PGC-1α and cytochrome c oxidase subunit 4 (COX-4) expression levels; while, application of transcutaneous CO2 attenuated this effect, and enhanced the expression levels of endothelial nitric oxide synthesis (eNOS). These results indicate that transcutaneous CO2 improves impaired muscle oxidative capacity via enhancement of eNOS and PGC-1α-related signaling in the skeletal muscle of rats with hyperglycemia.

Yan-Jun Xu, Vijayan Elimban, Sukhwinder K Bhullar, Naranjan S Dhalla

ABSTRACT
The effects of CO2 water-bath therapy on the hind limb of diabetic animals with or without peripheral ischemia were examined. Diabetes was induced in rats by administering streptozotocin (65 mg·kg-1), and the animals were then divided into 3 groups. After 4 weeks, peripheral ischemia was induced by ligation of the femoral artery for 2 weeks in 2 groups (diabetic ischemic) of diabetic rats, whereas the femoral artery was not occluded in the third group (diabetic). All these animals were subjected to water-bath therapy (with or without CO2 mixing; 20 min·day-1 for 5 days·week-1) for a period of 4 weeks. Both peak and mean flows, unlike minimal flow, in diabetic ischemic limbs were increased about a twofold by CO2 water-bath treatment. Morphological examination of hind limb tissue sections revealed about a twofold increase in the small artery count in diabetic ischemic animals upon CO2 water-bath treatment. These results indicate that CO2 water-bath therapy augments the blood flow and development of angiogenesis in the skeletal muscle of diabetic ischemic animals and thus may be of some benefit for the treatment of peripheral arterial disease in diabetes.

Vijayan Elimban, Yan-Jun Xu, Sukhwinder K. Bhullar, Naranjan S. Dhalla

ABSTRACT
Previously, it was shown that both blood flow and angiogenesis in the ischemic hind limb of diabetic rats were increased upon CO2 treatment for 4 weeks. In the present study, we have compared the effects of 6 weeks CO2 therapy in diabetic rats with or without peripheral ischemia. Diabetes was induced in rats by a tail vein injection of streptozotocin (65 mg/kg body weight), whereas peripheral ischemia was produced by occluding the femoral artery at 2 weeks of inducing diabetes. Both diabetic and diabetic-ischemic animals were treated with or without CO2 water-bath at 37 °C for 6 weeks (30 min/day; 5 days/week) starting at 2 weeks, after the induction of ischemia. CO2 treatment did not affect heart rate and R-R interval as well as plasma levels of creatine kinase, glucose, cholesterol, triglycerides and high density lipoproteins. Unlike the levels of plasma Ox-LDL, MDA and TNF-α, the levels of NO in diabetic group were increased by CO2 water-bath treatment. On the other hand, the levels of plasma Ox-LDL and MDA were decreased whereas that of NO was increased without any changes in TNF-α level in diabetic-ischemic animals upon CO2 therapy. Treatment of diabetic animals with CO2 increased peak, mean and minimal blood flow by 20, 49 and 43% whereas these values were increased by 53, 26 and 80% in the diabetic-ischemic group by CO2 therapy, respectively. Blood vessel count in diabetic and diabetic-ischemic skeletal muscles was increased by 73 and 136% by CO2 therapy, respectively. These data indicate that peripheral ischemia augmented the increase in blood flow and development of angiogenesis in diabetic skeletal muscle upon CO2 therapy. It is suggested that greater beneficial effects of CO2 therapy in diabetic-ischemic animals in comparison to diabetic group may be a consequence of difference of changes in the redox-sensitive signal transduction mechanisms.