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 CO2group, 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.
The current clinical consensus is that critical limb ischemia (CLI) with multi-level-disease requires adjunctive treatments after surgical revascularization.1) Adjunctive treatments should prevent postoperative problems such as amputation, ulcer exacerbation and graft occlusion. Although recent studies have focused on the effects of medication,2, 3) a number of alternatives including spinal cord stimulation,4) hyperbaric therapy,5) and an intra-muscular injection of autologous bone-marrow mononuclear cells,6) which may be also beneficial in revascularized limbs. However, the efficacy as adjunctive treatments for revascularized limbs remains unclear.
In 1997, Hartmann et al.7) reported that immersion in water enriched with carbon dioxide (CO2) had positive microcirculatory effects. In 2002, we demonstrated that CO2 immersion increased the blood flow of feet to the much higher extent than the plain water, and it improved the limb salvage rate in CLI patients without revascularization option.8)Using experimental animals, Irie et al.9) recently showed that CO2immersion induced local plasma vascular endothelial growth factor (VGEF) production, resulting in NO-dependent neocapillary formation associated with mobilization of endothelial progenitor cells. These results suggest that CO2 immersion could be an effective adjunctive treatment to prevent early postoperative amputation and ulcer exacerbation.
Based on these previous findings, the present study evaluated the hypothesis that immersion of feet in CO2-enriched water can prevent expansion or formation of ischemic ulcer after surgical revascularization of CLI in patients with advanced type II diabetes.
Materials and Methods
Patients and Setting
Study was conducted at Nagoya Kyoritsu Hospital between November 2004 and November 2007 for diabetic patients after lower limb revascularization. Two experienced surgeons diagnosed and performed the surgical revascularization. The patients were eligible for participation if they had CLI clinically defined by extremity pain at rest requiring use of analgesics for at least 2 weeks or the presence of ischemic ulcer before surgical revascularization, and had type C or D lesions10) in the infrainguinal artery that were detected by preoperative angiography. In addition they had hemodynamic failure in the below knee artery that were detected by postoperative ultrasonography. Patients were excluded from the study if they had ulcers that expose bone, tendon or fascia, because the immersion of these feet in water may increase the risk of severe infection. Patients were ineligible if they had clinically infected ulcers, severe heart failure (New York Heart Association Class III or IV), malnutrition (serum albumin < 2.5 g/dL), or a history of autoimmune disease affecting the vascular system. Patients were excluded if they enrolled in a clinical evaluation of another wound-care device or drug.
All patients provided written informed consent in order to participate in this study, which was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the School of Health Sciences, Nagoya University, Nagoya, Japan (approval number 5-512), and was approved by the Ethics Committee of Nagoya Kyoritsu Hospital, Nagoya, Japan.
Seventy limbs of 59 patients were enrolled in this study. The patients were randomly assigned to receive either topical treatment (CO2immersion plus standard care; CO2 group) or standard care alone (control group) using the concealed envelope method.
Both groups received medication of Ticlopidine (300−600 mg/day), Cilostazol (200 mg/day), or Sarpogrelate (200−300 mg/day) in addition to Aspirin (81−162 mg/day). Patients with ischemic ulcers received wound cleansing, debridement (as required at the discretion of the vascular surgeons) and dressing changes. All patients were required to wear treatment shoes that provide depressurization for ischemic ulcers.
Carbon Dioxide-enriched Water Bath (CO2 Immersion)
The CO2 group immersed their feet in CO2-enriched water (depth of 20–30 cm, 37–38°C, duration for 10 minutes) every day, during hospitalization. After discharge, they received CO2 immersion at least 3 times per week for the next 3 months at our outpatient clinic. The CO2-enriched water is made with an extra corporeal circulation system (MRE-SPA-MD, Mitsubishi Rayon Engineering, Tokyo, Japan).8, 9) It can instantly enrich 5 L/min of tap water (pH 6.8) with CO2 (free CO2concentration, 1,000 to 1,200 mg/l, pH 4.6). Patients recorded the immersion dates, which were confirmed by the investigators.
The primary endpoint was defined as the expansion of a target ulcer to 101% or more of the original size and/or formation of a new ulcer during the follow-up period. Patients were censored from the analysis at the time of their last study contact if they were lost to follow up before the assessment at 3 months after surgery (for discontinued therapy due to hospitalization, withdrew consent, or social problems). All time-dependent occurrences of endpoints were quantified within the study period in both groups. The patients were also evaluated with respect to hemodynamic parameters at the end of follow-up period and the values were compared with those obtained at immediately after surgery. All measurements were performed by four expert physical therapists.
Ischemic ulcers were photographed from the same direction every two weeks. Dimensions were determined from the longest edge-to-edge measurement (length) of the ulcer and the longest ulcer dimension perpendicular to the length (width).11) Ulcer size was defined as: length × width of ulcer (cm).
Transcutaneous oxygen pressure (tcPO2) and transcutaneous carbon dioxide pressure (tcPCO2) were measured using an oxymonitor (PO850, Sumitomo Electric System Solutions, Tokyo, Japan). Skin blood-flow (SBF) was measured using a laser Doppler flow-meter (ALF-21, Advance, Tokyo, Japan). Measurement sites were located on the dorsal surface of the affected feet of patients seated (probe 80–85 cm below heart level) in a standardized environment with an ambient temperature of 25 ± 2˚C, relative humidity 60%, and air movement at < 0.1 m/s. After swabbing the skin with alcohol, the tcPO2 probe was positioned 3.0 cm proximally to the first interdigital fold as the skin was heated to 43.5˚C. The laser Doppler probe was positioned 2.5 cm proximally to the fourth interdigital fold.12) When a stable steady-state was achieved (20 min after fixing the probes), data were continuously acquired for 5 min using an automated recorder (Power Lab, Bio Research Center, Tokyo, Japan), and the mean value during the final minute was adopted.
All patients rested for 10 min in the supine position, and then ankle pressure (AP), ankle brachial pressure index (ABI), toe pressure (TP), and toe brachial pressure index (TBI) were measured using an ABI-form (BP-203RPEII, Colin, Tokyo, Japan). The ABI and TBI were calculated as the ratio of ankle systolic pressure divided by brachial systolic pressure and as the ratio of hallucis systolic pressure divided by brachial systolic pressure, respectively.
Demographic and baseline variables were compared using Chi-square test and Mann-Whitney test. Prevention of ischemic ulcer expansion was estimated using the Kaplan-Meier method. The cumulative values of preventing expansion or formation of ischemic ulcer were analyzed using a log-rank test. The analysis was conducted based on the principle of the intention-to-treat.13) Changes in variables from baseline to after 3 months and differences between the groups were analyzed using Wilcoxon signed rank test and Mann-Whitney test based on per-protocol. P-values < 0.05 were considered to indicate statistical significance. All data were analyzed using Statistical Package for the Social Sciences, version 15.0 (SPSS Inc., Chicago, Illinois).
The patients were allocated CO2 group (34 limbs of 28 patients) and control group (36 limbs of 31 patients). The CO2 group and control group did not differ with respect to baseline characteristics (Table 1).
The distribution of conduit type, bypass procedure and values for immediately postoperative hemodynamic measurements were similar for both groups (Table 2). There were many cases who did not have a suitable autogenous vein for distal bypass graft. Namely, 16 out of 28 patients in CO2 group (57%) and 18 out of 31 patients in control group (58%) have undergone the coronary bypass operation using great saphenous veins. 5 patients in each group underwent infragenicular revascularization with autogenous vein. Before surgery, the enrolled 31 limbs (44%) showed AP < 50mmHg, and another 39 limbs (56%) showed either TP or tcPO2 in consistent with the CLI criteria.1)
Effects of CO2 immersion on Ischemic Ulcer Expansion after Surgical Revascularization
Fig. 1 shows Kaplan-Meier plots demonstrating the effect of CO2immersion on ischemic ulcer expansion or formation after surgical revascularization. In the control group, the rate of prevention of ischemic ulcer (Y axis) began to decrease after day 20 and reached 77.8% after 90 days. In contrast, in the CO2 group, the rate of prevention remained at 100% until 60 days and then slightly decreased to 97.1% at 90 days. In other words, ischemic ulcers were exacerbated in 1 limb in the CO2group, while, in control group, new ulcer developed in 2 limbs and pre-existing ulcers became exacerbated in 6 limbs. The difference in the cumulative values of the prevention of ischemic ulcer expansion after 3 months in the CO2 group and control group was statistically significant (P = 0.012). In exacerbated limbs, expansion of a target ulcer > 150% of the original size were commonly observed. Among them, major amputation was performed in two limbs in the control group, but in none in the CO2 group. Deep wound infection was observed 4 limbs in the control group only. The ulcer size (length × width, in cm) was significantly reduced in the CO2 group (2.9 ± 2.5 to 1.6 ± 1.9 in 13 limbs over 3 months; P < 0.001), but not in the control group (2.4 ± 1.6 into 2.4 ± 1.4 in 8 limbs, over 3 months; P = 0.06).
Among enrolled, 11 patients (6 in CO2 group and 5 in control group) were discontinued due to several reasons: 2 died (one in each group), 1 for exacerbation of congestive heart failure (in CO2 group), 2 due to the second surgery for graft failure (one in each group), and 6 were censored by consent withdrawn or social problems (three in each group).
Effect of CO2 Immersion on the Tissue Oxygen Pressure
The mean tcPO2 value increased in 3 months in the CO2 group, while that in the control group did not increase during this period. Accordingly, after 3 months, tcPO2 became significantly higher in the CO2 group than in the control group. However the mean tcPCO2 and SBF did not change in both groups (Table 3). Other parameters, AP, ABI, TP, TBI, also did not change significantly.
In the present study, we report that the expansion and formation of ischemic ulcers was successfully prevented for 3 months by immersing feet in CO2-enriched water after surgical revascularization for CLI in diabetic patients. Consistently, the tcPO2 values were remarkably improved in the CO2 group, but remained unchanged in the control group. These results strongly suggest that CO2 immersion is effective against ulcer exacerbation, presumably due to the elevation of tcPO2.
Previously, the rates of limb salvage as well as that of graft patency were followed for relatively long periods after surgical revascularization of CLI patients.14–17) These reports suggested that despite patent grafts of infrainguinal bypass, patients with end-stage renal diseases often developed extensive necrosis of extremities that resulted in amputation. In the early postoperative stage, ulcer exacerbation could often be overlooked; however, it leads to major complications. Therefore, early postoperative ulcer expansion and new ulcer formation should be evaluated as well as the graft patency. Ulcer healing after revascularization may be affected by many factors including nutrition (represented by serum albumin level), preoperative ulceration, and lesion severity.18) Amann et al.19) identified the importance of microcirculatory perfusion as the ultimate cause of ischemic tissue loss. Ubbink et al.20)suggested that ischemic ulcers of non-reconstructable CLI might heal without major intervention if local microcirculation is well preserved.
Herein, we showed that average of tcPO2 in the CO2 group increased more than 10% after 3 months, whereas that of the control group remained unchanged. Hartmann et al.7) demonstrated that immersion in CO2-enriched water increases tcPO2 by 10% and immediately increases laser Doppler output three-fold in patients with mild peripheral occlusive arterial disease. In this context, we previously showed that immersion in CO2-enriched water causes vasodilation, even in patients with non-reconstructable CLI and improved the limb salvage of CLI patients.8)This work led us to examine the efficacy of CO2 immersing as an adjunctive treatment following surgical revascularization. The increased supply of oxygen may be an underlining mechanism for the prevention against ulcer expansion and new ulcer formation. Using experimental animals, it has been shown that CO2 immersion results in a NO-dependent increase in collateral blood perfusion, induction of regional VEGF synthesis, and mobilization of endothelial-lineage progenitor cells into the circulation.9) These results suggest that CO2 immersion improves subcutaneous microcirculation.
The tcPO2 value has been used as an index to quantify local microcirculation in ischemic regions to determine the amputation level and predict therapeutic effects.21–23) Furthermore, the tcPO2 value has confirmed a substantial intra-class correlation coefficient and variability, and a smaller variance than that of the AP, TP, and ABI measured by trained observers.24) In the CO2 group, the tcPO2 improved by > 10% from baseline indicates clinically significant changes.25) The tcPO2 was elevated by CO2 immersion, without affecting the blood pressure at either the ankle or toe. These results suggest that the physiological mechanism for prevention of ulcer exacerbation by CO2 immersion is the improvement of subcutaneous microcirculation.
Serious complications, such as major amputation of limb and/or deep wound infection with target ulcer, occurred only in the control group. It is tempting to speculate that CO2 immersion prevents bacterial infection. The pH of CO2-enriched water is acidic (pH 4.6) and stabilizes hypochlorous acid (HOCl); thus, it exhibits a strong bactericidal action, since stabilized HOCl has potential pharmaceutical applications in the control of soft tissue infection.26) However, further investigation with a larger number of patients will be required to confirm this notion.
Finally, we must mention the study population. All of the enrolled patients suffer from type II diabetes and approximately 70% of them are receiving hemodialysis due to chronic renal failure. Also, they have been suffering from coronary artherosclerosis and often received coronary bypass operation using autogenous veins. This caused the shortage of the available vein and this was one of the reasons why the large number of revascularization was limited above knee vessels and only 10 limbs out of 70 underwent infragenicular revascularization with autogenous vein (Table 2). Though there were no significant difference in patients' characteristics between CO2 group and control group, the ratio of smokers and duration of the hemodialysis tend to be slightly higher and longer in control group than CO2 group. These factors might modify the results, so the further investigation will be needed to clarify this point. As CO2 immersion increases local oxygen supply by improving subcutaneous microcirculation, such a regimen could be used as an adjunctive treatment for the prevention of ischemic ulcer expansion at limb with CLI, not only in diabetic patient but also in general CLI. To prove this, a long-term follow-up study is on the way with a larger number of patients suffering from various diseases, with or without CO2immersion.
The authors thank all staff members of the Department of Rehabilitation at Nagoya Kyoritsu Hospital for their expert technical participation.
We also thank Dr. Shonen Yoshida and Mr. Hiroshi Takahashi (Division of Outcome Research, Nagoya Kyoritsu Hospital) and Dr. Hideki Ishii (Department of Cardiology, Nagoya University Graduate School of Medicine and Nagoya University Hospital) for their critical reading of the manuscript.
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