A Prospective, Randomized, Controlled Study of Hyperbaric Oxygen Therapy: Effects on Healing and Oxidative Stress of Ulcer Tissue in Patients with a Diabetic Foot Ulcer

Abstract

Although hyperbaric oxygen (HBO) therapy has been reported to help heal chronic foot ulcers in patients with diabetes mellitus (DM), production of HBO-related oxidative stress is a concern. To assess the therapeutic effect and oxidative stress of HBO, a 2-week, prospective, randomized, controlled clinical study was conducted from January 1, 2010 to January 1, 2012 among 36 consecutively admitted patients with diabetic foot ulcers (DFU).

Average patient age was 60.08 ± 5.97 years and average DM duration was 16.4 ± 11.3 years; 86.1% had type 2 DM, and 47.2% had Wagner grade-III foot ulcers. Patients randomized to the control group (n = 18) received standard care including offloading, wound debridement, and glucose control. HBO treatment group patients (n = 18) received standard care and twice-daily HBO sessions for 90 minutes at 2.5 atmospheres absolute (ATA) 5 days a week for 2 weeks. Transcutaneous oxygen pressure (TcPo₂) at the edge of the ulcer and wound size were measured at baseline and after 7 and 14 days of treatment. Ulcer tissues were harvested on days 7 and 14 to determine oxidative stress by measuring malondialdehyde (MDA) and antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]) levels. Compared to baseline, TcPo2 in the HBO group increased on day 7 (477.8 ± 118.2 mm Hg versus 37.06 ± 5.23 mm Hg, P <0.01) and day 14 (501.1 ± 137.7 mm Hg versus 35.61 ± 4.85 mm Hg, P <0.01). Ulcer size reduction in the HBO group was greater than that of the control group (42.4% ± 20.0% versus 18.1% ± 6.5%, P <0.05). MDA levels, SOD, and CAT were all significantly higher in the HBO than in the control group on day 14 (P <0.05). The results of this study suggest HBO treatment for 2 weeks initiates a healing response in chronic DFUs, but the observed oxidative stress in local ulcer tissue may offset this effect long-term. Until needed additional research has been conducted, prolonged and/or inappropriate HBO treatment should be avoided. {C}

 Potential Conflicts of Interest: This study was supported by research funding from the Subei People’s Hospital of Yangzhou University.

Introduction

  Chronic lower extremity ulcers constitute a severe complication of diabetes mellitus (DM). Approximately 15% of patients with DM will develop a lower-extremity ulcer during the course of their disease process,¹ accounting for 20% of the hospital admissions of patients with DM. In addition, 85% of patients with DM who ultimately undergo a lower-extremity amputation have a history of foot ulcers.²

  Hyperbaric oxygen (HBO) therapy involves inhalation of 100% oxygen inside a pressurized hyperbaric chamber.³ Results of multicenter, randomized, controlled trials by Abidia et al⁴ and Veves et al⁵ reported HBO could be a factor in healing chronic foot ulcers in persons with DM, most likely by restoring fibroblast growth and collagen production⁶ and improving the environment necessary for wound healing by diminishing tissue swelling and improving circulation.⁷ However, resultant HBO oxidative-related stress remains a concern.

  Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage.⁸ Low-level ROS has been shown in in vitro and in vivo studies to be an essential mediator of intracellular signaling⁹ and efficient wound angiogenesis,¹⁰ but excessive production of ROS or impaired detoxification of this aggressive molecule causes oxidative stress, which has been identified as an important feature in the pathogenesis of chronic, nonhealing wounds, as noted in a review of the literature.¹¹ HBO is reported in an in vivo study to enhance the production of ROS and oxidative stress in several tissues,¹² as well as to influence the enzymatic antioxidant defense system.^13,14

  The purpose of this prospective, randomized, controlled pilot study was to investigate the effect of HBO on diabetic foot ulcer (DFU) healing and oxidative stress — ie, whether it was a negative influence on DFU healing.

Methods

  Patients. Potential participants were inpatients consecutively admitted to the author’s unit of the hospital for treatment of chronic wounds from January 1, 2010 to January 1, 2012. Patients were eligible to participate if they met the following inclusion criteria: diagnosis of DM; at least one full-thickness wound below the ankle (Wagner grades III or less) for >3 months; history of receiving standard care for >2 months; normal palpation of arterial pulses at lower extremities; normal lower limb Doppler scan results; TcPo2 >30 mm Hg at the dorsum of the foot; and no abnormal x-ray findings that may be indicative of chronic bone infection. Excluded were patients with wounds classified as more severe than Wagner grade III; TcPo2 at the dorsum of the foot with ulcer <30 mm Hg (ie, severe arteriopathy) in room air; upper respiratory infection; emphysema; history of thoracic surgery; malignant disease; middle ear barotraumas; pregnancy; and smoking or abstention <1 month. The study was approved by the Human Investigation Committee of SuBei’s hospital of Yangzhou University. All participants provided written informed consent.

  Study protocol. Using a randomization table, participants were assigned to the HBO or control group. Patients in both groups were hospitalized for 2 weeks during the study and provided standard care, which was evaluated and performed in this study by a multidisciplinary team comprising the department of plastic surgery, endocrinology, and HBO. Standard care included offloading, footwear (featuring extra depth, arch support, and custom-molded inserts), and nonweight-bearing of the affected foot. All patients were prescribed oral antibiotics based on bacterial culture test results (including Gram-positive, Gram-negative, and anaerobes) and drug sensitivity test of ulcer tissue. Blood glucose levels were monitored and maintained at <8 mmol/L with subcutaneous insulin injections if necessary. Dressings were changed daily; silver-impregnated dressing was used in infected ulcers and absorptive cotton in uninfected ulcers. Ulcers were cleansed with saline solution, and daily wound curettage or debridement (provided by two specified surgeons) of necrotic tissue and surrounding callus was performed to get a well-bleeding granulating base. Ulcer tissue samples for biochemical assay were obtained during the curettage or debridement session. Patients randomized to the HBO group also received HBO therapy following the protocol specified by Kessler et al¹⁵ — ie, 100% oxygen, twice a day, administered for 90 minutes at 2.5 times atmospheres absolute (ATA), 5 days a week for 2 weeks (20 treatment sessions). HBO was provided via a multiperson hyperbaric chamber (K018YX-10-8, XinYing, Hang Zhou City, China). Each HBO session included 15 minutes of compression time, three 30-minute HBO periods with two 5-minute intervals in room air, and a 15-minute decompression period.

  TcPo2 and ulcer area measurement. TcPo2 levels were measured (PeriFlux 5000, Perimed, Sweden) on day 0 (baseline) as well as before and after the HBO therapy session on day 7 and day 14. Measurements were taken on proximal noninflamed skin 1 cm from the ulcer edge, regardless of the specific location of the ulcer on foot. Ulcer size was measured using standardized photographs taken on day 0, day 7, and day 14. Although photographs were computerized using the Image J program (version 1.42I, NIH, US), changes in the ulcer area were expressed using percentage of reduction of ulcer area compared to individual baseline of day 0. Day 7 and day 14 measurements were obtained by two physicians blinded to the patient group.

  Ulcer biopsy. After session 10 and session 20 of HBO therapy, biopsy samples of granulation tissue were taken from the ulcer bed or lateral wall, gently rinsed with physiological saline solution, and stored at -80˚ C for further biochemical analysis.

  Enzyme-linked immunosorbent assay (ELISA) for MDA. The tissue samples were lysed in a modified radio-immunoprecipation assay (RIPA) buffer. After centrifuge at 4˚ C, the supernatants were collected and protein concentration was determined. According to the manufacturer’s direction, the supernatant then was tested with commercially available ELISA kits (Cell Biolabs, Inc., San Diego, CA) to determine MDA.

  Western-blot for antioxidant enzymes. The protein of each tissue sample was extracted as above, and 40 µg of protein was resuspended in 40 µL of Laemmli sample buffer and 5% 2-mercaptoethanol and heated for 10 minutes. The samples were loaded in 12% sodium dodecyl sulfate (SDS) polyacrylamide gels. After separation by SDS-polyacrylamide gels (SDS-PAGE), proteins were transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes and blocked in blocking solution (0.05% Tween 20, 5% skimmed milk in Tris-buffered saline [TBS]). Then the membranes were incubated with primary antibodies at 4˚ C overnight. After washing three times in TBS buffer (Tris-Base, NaCl, Tween 20, pH 7.4) for 15 minutes, the immunoblots were immersed in the secondary antibodies for 1 hour with agitation. The specific complex was detected by an enhanced chemiluminescence detection system. The density levels of bands were quantified by using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).

  Real-time polymerase chain reaction (RT-PCR) for expression of antioxidant enzyme-relative genes, ribonucleic acid (RNA) extraction, and complementary deoxyribonucleic acid (cDNA) synthesis. Total RNA from ulcer tissues homogenate was isolated using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA). The isolation was performed according to the manufacturer’s instructions and quantified by measuring the absorbance at 260 nm; the RNA quality was determined by measuring the 260-280 ratio. The cDNA synthesis was performed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions: 1.5 µg of total RNA from each sample was added to a mixture of 2.0 µL of 10x reverse transcriptase buffer, 0.8 µl of 25x dNTP mix (l00 mM), 2.0 µL of 10x reverse transcriptase random primers, 1.0 µL of MultiScribe reverse transcriptase, and 3.2 µL of nuclease-free water. The final reaction mixture was kept at 25˚ C for 10 minutes, heated to 37˚ C for 120 minutes, heated for 85˚ C ° for 5 seconds, and then cooled to 4˚ C.

  Quantification of messenger RNA (mRNA) expression by RT-PCR. Quantitative analysis of mRNA expression of target genes was performed by real time-PCR. cDNA from the above preparation was subjected to PCR amplification using 96-well optical reaction plates in the ABI Prism 7500 System (Applied Biosystems, Life Technologies, Carlsbad, CA). The 25-µL reaction mixture contained 0.1 µL of 10 µM forward primer and 0.1 µL of 10 µM reverse primer (40 µM final concentration of each primer), 12.5 µL of SYBR Green Universal Mastermix, 11.05 µL of nuclease-free water, and 1.25 µL of cDNA sample. The RT-PCR data were analyzed using the relative gene expression method, as described in the Applied Biosystems User Bulletin No. 2.¹⁶ The data are presented as the fold change in gene expression normalized to the endogenous reference gene (-actin) and relative to a calibrator.

  The TcPo2 and ulcer area data collection, as well as the biopsy of ulcer tissue obtained on day 7 and 14, usually occurred following HBO session 10 and 20, and the biochemical tests were run as soon as possible in next several days. The investigators and the physicians in charge of biochemical testing did not intervene in daily routine clinical management of the patients.

  Statistical analysis. Statistical analyses were performed using the statistical package SPSS for Windows version 16.0 (SPSS, Inc., Chicago, IL). A significance level of  = 0.05 was used for all tests. Discrete variables were summarized as frequencies and continuous variables as mean ± SD if normally distributed and as medians and 25th and 75th percentiles if not normally distributed. An independent-samples t-test was used for continuous variables normally distributed. For the variables not normally distributed, Wilcoxon’s test was performed. To determine differences between the two groups in the frequency such as in DM type and oral antibiotic therapy, X² two-tailed Fisher’s exact test analysis was used.

Results

  During the enrollment period, a total of 81 eligible participants was registered: 43 (53.1%) were excluded because they did not meet the study inclusion criteria and two (2.5%) did not consent to participate. Thirty-six (36, 44.4%) patients were enrolled, randomized, and finished their protocol of 2 weeks/20 sessions. Patients in the HBO group received a total of 720 treatment sessions. No serious complications such as death or amputation or other adverse reactions such as barotraumatic otitis, dizziness, seizures, or pneumothorax occurred.

  At baseline, no significant differences in characteristics were noted between the HBO and control groups (see Table 1).

  The average TcPo2 before HBO treatment was 37.06 ± 5.23 mm Hg in room air and 477.8 ± 118.2 mm Hg on day 7 (P <0.01). On day 14 (session 20), the average was 35.61 ± 4.85 mm Hg before and 501.1 ± 137.7 mm Hg after treatment. This difference was statistically significant (P <0.01).

  None of the ulcers in either treatment group healed during the 2-week study. On day 7, the average percent reduction in ulcer size was 12.3% ± 1.9% in the control and 15.0% ± 5.7% in the HBO group. On day 14, the average percent reduction in ulcer area was significantly greater in the HBO (42.4% ± 20.0%) than in the control group (18.1% ± 6.5%, P <0.05) (see Figure 1).

  On day 7, the average level of MDA was not significantly different between the two treatment groups (29.9 ± 7.7 pmol/L in the HBO and group and 34.1 ± 10.5 pmol/L in the control group). On day 14, the difference in MDA between groups was significantly different (92.6 ± 18.8 in the HBO compared to 29.0 ± 4.3 pmol/mg in the control group, P <0.05).

  Western-blot analysis. Results of Western-blot analysis showed similar expression between HBO and control group on both day 7 and day 14. On day 7, protein levels of SOD-1 and CAT were also not significantly different between the control and the HBO group, but on day 14 they were significantly higher in the HBO (average 16.2 ± 8.2 versus 26.3 ± 5.4 to SOD and 11.9 ± 3.7 versus 12.2 ± 8.3 to CAT) than in the control group (average 43.6 ± 5.0 versus 9.8 ± 7.1 to SOD and 27.6 ± 10.1 versus 3.1 ± 1.7 to CAT, P <0.05) (see Figure 2).

  Expression of antioxidant enzymes-relative genes. On day 14, but not on day 7, the mRNA expression levels of SOD-1, CAT genes were significantly higher in the HBO group than in the control group (1.33 ± 0.28 versus 0.72 ± 0.11 to SOD and 2.18 ± 0.46 versus 1.27 ± 0.26 to CAT ([P <0.05]) No differences in the GPx-1 gene were observed. At baseline, when comparing the HBO group with the control group, the GPx-1 gene was 0.42 ± 0.08 versus 0.37 ± 0.19 at day 0, 0.71 ± 0.33 versus 0.66 ± 0.09 at day 7, and 0.98 ± 0.16 versus 1.14 ± 0.63 at day 14 (P＞>0.05) (see Figure 3).

Discussion

  Diabetic foot ulceration is a condition of multifactoral etiology that, according to the International Working Group on the Diabetic Foot,¹⁷ requires a multidisciplinary approach, including relief of pressure, restoration of skin perfusion, treatment of infection, metabolic control, and local wound care. When that standard of care fails, advanced treatment modalities may be considered.

  In this study, change in ulcer area after 2 weeks was significantly different between the HBO and control group. Results of a prospective multicenter trial by Sheehan et al¹⁸ have shown that percent change in DFU size at 4 weeks is a predictor of complete healing after 12 weeks. The response observed during this 2-week study suggests wounds in the HBO group were on a healing trajectory. However, it is not known how long this effect will last. According to a meta-analysis of nine randomized controlled trials (N = 471) by Kranke et al,¹⁹ HBO treatment could improve the healing rate of DFUs (wound size reduction) in the short term (especially at 6 weeks) but not in the long term. However, the strength of this conclusion was tempered by the design and reporting flaws of the studies analyzed.

  The results of biochemical tests regarding oxidative stress in the present study might help explain the reported short- and long-term outcomes. In this study, along with the treatment effect of HBO, oxidative stress increases significantly in HBO group — especially after 20 sessions — as evidenced by the increased expression of MDA and some antioxidant enzymes such as SOD-1 and CAT, as well as the upregulated genes SOD-1 and CAT. Oxidative stress is an unavoidable byproduct of aerobic metabolism and perhaps is dangerous to all life forms due to a large number of oxygen radicals and other activated oxygen species. Failure to inhibit oxidative stress would induce apoptosis or even necrosis, damaging cell protein, membrane lipid, and DNA.²⁰ In the present study, MDA as a degraded product of polyunsaturated lipids by ROS,²¹ and three important antioxidant enzymes — ie, SOD-1, CAT, and GPx-1, which are inducible under conditions of oxidative stress adaptation²⁰ — were used to evaluate the level of oxidative stress. MDA increased slightly at session 10 and significantly at session 20, together with the increased expression levels of proteins and genes SOD-1 and CAT after 14 days of treatment. This suggests that as the number of HBO sessions increases, ROS and oxidative stress accumulate. This, in turn, has been identified as an important feature in the pathogenesis of chronic, nonhealing wounds¹¹ and might temper the long-term treatment effectiveness of HBO.

Limitation

  The small sample size, although similar to sample sizes in other studies,^5,22,23 may limit the external validity of these results. In addition, the number of adverse effects that occur during HBO treatment is controversial.

  The short duration (2 weeks and 20 HBO sessions) resulted in a decrease in ulcer size but a potentially negative effect of oxidative stress, which probably affects long-term effectiveness.

  Another study limitation is that the control group received standard care only. The provision of hyperbaric air intervention in the control group could have provided more insight, especially with respect to the incidence of adverse effects. Also, pathology needs to confirm whether ill effects might be the result of HBO settings/provision, such as high gas pressure or high-concentration oxygen.

  The medical history of the participants in this study was not a consideration. Information regarding potentially influential diseases — eg, stroke, heart failure — and detailed ulcer treatment history were not collected.

  All of the above support a need for multicenter, large-sample, long-term, randomized, double-blind studies to provide more meaningful and persuasive results about the effects of HBO on ulcer outcomes as well as oxidative stress. The potential benefits of exogeneous antioxidant medications during HBO treatment also should be investigated.

Conclusion

  In this study, the percent reduction in ulcer size after 2 weeks of HBO treatment was significantly greater than in the control group, suggesting a positive effect of HBO on ulcer healing. At the same time, the results showed that HBO treatment could induce oxidative stress in local ulcer tissue that could accumulate and might offset long-term healing. Until more research has been conducted, it seems prudent to avoid prolonged, inappropriate, and/or unnecessary HBO treatments.

Acknowledgment

  The authors thank the participants and the colleagues of the departments of endocrinology and HBO in Subei People’s Hospital of Yangzhou University. They thank Ms. Sharon Longo and Mr. Jie Li of the Department of Neurosurgery, Upstate Medical University, Syracuse, NY, for their support on biochemical trials. This study was supported by research funding from the Subei People’s Hospital of Yangzhou University.

Drs. Ma, Li, Shi, Hou, and Chen are medical doctors and Dr. Du is a medical doctor and Associate Professor, Department of Plastic Surgery, Subei People’s Hospital, Yangzhou University, Yangzhou, Jiangsu 225001, China. Please address correspondence to: Jin Du, MD, Subei People’s Hospital, Yangzhou University, 98 Nantongxilu, Yangzhou, Jiangsu 225001, China; email: dujinhaha2004@yahoo.com.cn.

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