The Effect of Hyperbaric Oxygen Therapy on Oxidative Stress and Inflammation in Patients With Diabetic Foot Ulcers
Conditions: Diabetic Foot Ulcer Neuropathic
Sponsor: National Polytechnic Institute, Mexico

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NATIONAL POLYTECHNIC INSTITUTE 
SUPERIOR SCHOOL OF MEDICINE 
DIVISION OF POSTGRADUATE STUDIES 
 
                            
The effect of hyperbaric oxygen therapy on oxidative stress and 
inflammation in patients with diabetic foot ulcers 
 
Principal Investigator: 
Modesto Gómez López 
Molecular Biology of the Inflammatory Process, School of Medicine, National Polytechnic 
Institute. 
 
Research associates: 
Nadia Mabel Pérez Vielma 
Sección de Estudios de Posgrado e Investigación, Centro Interdisciplinario de Ciencias de 
la Salud, Unidad Santo-Tomas, Instituto Politécnico Nacional, 
María Magdalena Valencia Gutiérrez 
Sección de Estudios de Posgrado, Escuela Superior de Medicina, Instituto politécnico 
Nacional. 
Jennifer Viridiana Sánchez Camacho 
Sección de Estudios de Posgrado, Escuela Superior de Medicina, Instituto politécnico 
Nacional. 
José Enrique González Hernández 
Clínica Especializada en el manejo de diabetes, de la Secretaría de Salud CDMX  
, Ángel Miliar García 
Laboratorio de Biología Molecular, Escuela Superior de Medicina, Instituto Politécnico 
Nacional. 
César Ochoa 
College of Osteopathic Medicine of the Pacific, Pomona, CA, USA and Center for Clinical, 
Research at WUHS, Pomona, CA. 
 Jonathan Labovitz 
Clinical Education and Graduate Placement, Western University of Health Sciences, 
College of  Podiatric Medicine, 309 East 2nd Street, Pomona, CA 91766, USA 
Mexico, CDMX, December 28th, 2018 

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The effect of hyperbaric oxygen therapy on oxidative stress and 
inflammation in patients with diabetic foot ulcers 
 
Background   
 
Type 2 diabetes mellitus (T2DM) is one of the most significant public health 
conditions worldwide (1, 2). Patients present a metabolic disorder originated by the 
combination of a deficiency in insulin synthesis by pancreatic β-cells and insulin 
resistance in the specific tissues where they carry out their action, resulting in 
hyperglycemia (3, 4). 
 
The severity of insulin resistance depends on numerous factors, such as weight, 
age, heredity, oxidative stress, and endocrine history (4-8). As a net effect, glucose 
metabolism is modified, resulting in elevated fasting glucose levels or glucose 
intolerance and will manifest T2DM within 3 to 5 years (9). On the other hand, 
hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric 
Medical Society (UHMS) as treatment where a patient intermittently breathes 100% 
oxygen at a pressure above sea level pressure (a pressure greater than 1 
atmosphere absolute, ATA) (10, 11) and its benefits have been demonstrated in 
stroke, through the promotion of cerebral oxygenation, metabolic improvement, 
anti-inflammatory barrier, protection of cerebral blood flow, modulation of 
intracranial pressure, decrease of oxidative stress and apoptosis, increase of 
vascular and neural regeneration (12).  
   
HBOT induces an increase of 100% of oxygen in blood and tissues during the 
procedure helping to maintain the integrity and function of tissues and cells (13), 
stimulates the antioxidant response in plasma and regulates the processes of 
angiogenesis, vascular tone by increasing the levels of vascular endothelial growth 
factor (VEGF) and decreasing the levels of endothelin-1 (ET-1). Different factors 
such as age, oxidative stress, diabetes and chronic inflammation (14, 15), play an 
important factor in the development of diabetic foot or diabetic neuropathy (16, 17). 

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It has been described that the hyperbaric chamber increases the levels of 
circulating free fatty acids, so that chronic inflammation could be associated with 
an increase in the production of reactive oxygen species (ROS) and a decrease in 
antioxidant activity (18). 
 
HBOT consists of breathing 100% pure oxygen in a pressurized environment 
(hyperbaric chamber) to at least 1.4 atmospheres absolute (ATAs). The scientific 
basis for the use of oxygen at elevated pressures is based on the application of the 
Henry, Dalton and Boyle gas laws. There are other effects, the volumetric ones 
with an increase of 10 to 15 times the amount of oxygen in the plasma fraction, 
therefore, the diffusion distance from the vascular space to the tissues increases, 
and this is directly proportional to the pressure used (19, 20). The aim of this 
treatment is to evaluate the effect of HBOT on gene expression of superoxide 
dismutase 1 and 2 (SOD1 and SOD2), pro-inflammatory cytokines (TNFα, IL-1β, 
IL-12, IL-4, as well as oxidative (NLRP3) and antioxidant (GPX2) enzymes in 
patients with diabetic foot. 
 
Intermittent exposure to hyperbaric oxygen increases oxygen saturation by 100% 
in the blood (21, 22) and stimulates various biological processes that promote 
healing in numerous clinical conditions in which hypoxia is the dominant element 
(23-25). Hyperbaric oxygen therapy stimulates plasma antioxidant responses and 
regulates angiogenesis and vascular tone processes by increasing vascular 
endothelial growth factor levels and decreasing endothelin-1 levels. Different 
factors including age, oxidative stress (OS), diabetes and chronic inflammation 
play an important factor in the development of diabetic foot (26-27). It is assumed 
that the hyperbaric chamber increases the levels of circulating free fatty acids, and 
chronic inflammation may be associated with an increase in the production of 
reactive oxygen species (ROS) and a decrease in antioxidant activity (28). 
 
 
 

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Justification: 
 
Mexico is a country where there is a high population of patients with diabetic foot 
and little research in response to non-invasive treatments such as hyperbaric 
chamber. Therefore, studies are needed to determine the mechanisms by which 
complications are triggered in these patients and to determine possible molecular 
markers for their timely treatment. 
 
Research question: 
 
In Mexican patients diagnosed with diabetic foot, what are the gene expression 
changes in inflammation and oxidative stress in diabetic foot patients treated with 
hyperbaric oxygen? 
 
Hypothesis: 
 
Patients diagnosed with diabetic foot will have significant changes after hyperbaric 
treatment such as increased gene expression in the antioxidant response and 
decreased gene expression in the inflammatory response. 
 
General Objective:  
 
To evaluate gene expression changes in inflammation and oxidative stress in 
diabetic foot patients treated with hyperbaric oxygen. 
 
Specific objectives: 
 
 To evaluate the clinical and biochemical characteristics of the included patients 
according to sex. 

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 To evaluate the gene expression of SOD, CAT, GPX, GRd, NLRP3, IL-1b, Casp-
1, IL-4, IL-6, IL-10 and IL-13 in the basal state, at 12 and 30 sessions of treatment 
in the hyperbaric chamber.  
 
Methodology: 
 
Type of study: quasi-experimental, longitudinal, prolective, analytical study. 
 
Characteristics of the population: 
 
Women and men older than 18 years, with Diabetes type 2, residents of the 
CDMX, with diabetic foot with hyperbaric treatment, attended at the hospital of 
specialties in diabetes, secretary of health, accepted and signed the informed 
consent form.  
 
Selection criteria: 
 
Men and women with diabetic foot and hyperbaric treatment. 
Ages 30-60 years. 
Signed informed consent form. 
 
Exclusion: 
 
Patients with diabetic foot without presence of dermatologic lesions. 
 
Elimination: 
 
Patients who decided to abandon the protocol. 
Incomplete data for any variable. 
 
 

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Sample size: 
 
15 patients with diabetic foot. 
 
Operationalization of variables: 
 
Variable 
Type 
Measuring unit 
Age 
Quantitative 
Years 
Sex 
Qualitative 
Male/ female 
Systemic blood 
pressure 
Quantitative  Milimeters of mercury 
Waist  
Quantitative 
Centimeters 
Dislipidemias 
Qualitative 
Positive or negative 
Anaemia 
Qualitative 
Positive or negative 
Tabaquism 
Qualitative 
Positive or negative 
Charcot’s foot 
Qualitative 
Positive or negative 
Chronic kidney 
disease by CKD-
EPI I-III 
Qualitative 
Positive or negative 
Insuficiencia 
venosa periférica 
Qualitative 
Positive or negative 
Glucosa en sangre 
Quantitative 
milligrams/ deciliters 
Colesterol 
Quantitative 
milligrams/ deciliters 
Triglicéridos 
Quantitative 
milligrams/ deciliters 
Basal glycosylated 
hemoglobin basal 
Quantitative 
Percentage 
Creatinina  
Quantitative 
milligrams/ deciliters 
Tasa de filtrado 
glomerular 
Quantitative 
milliliters/ square meter of body surface area 
glycosylated 
Quantitative 
Percentage 

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hemoglobin at 3 
months 
Wagner's 
classification for 
diabetic feet 
Qualitative 
Wagner  
Grade 0: Absence of ulcers in a high-risk foot. 
Grade 1: Superficial ulcer involving the full 
thickness of the skin but no underlying 
tissues. 
Grade 2: Deep ulcer, penetrating to ligaments 
and muscles but not involving bone or 
abscess formation. 
Grade 3: Deep ulcer with cellulitis or abscess 
formation, almost always with osteomyelitis. 
Grade 4: Localized gangrene. 
Grade 5: Extensive gangrene involving the 
entire foot. 
Gene expression 
SOD, CAT, GPX, 
GRd, NLRP3, IL-
1b, Casp-1, IL-4, 
IL-6, IL-10 e IL-13. 
Qualitative 
International units  
 
Procedure: 
 
1. Patients attended at the diabetic foot clinic in the CDMX were invited to 
participate. 2.  
2. An informed consent process was carried out with the patient's signature to 
agree to participate in the study. 
3. Questions were asked about their personal pathological and non-pathological 
history.  
4. Three tubes of blood samples were taken for determination of blood biometry, 
urea, creatinine, cholesterol, triglycerides, glycosylated hemoglobin. A 4 ml tube 

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with EDTA was taken to measure the gene expression of SOD, CAT, GPX, 
GRd, NLRP3, NLRP3, IL-1b, Casp-1, IL-4, IL-6, IL-10 and IL-13. 
5. Complementary DNA (cDNA) synthesis was carried out following the 
instructions of the Transcriptor First Strand cDNA Synthesis kit (Roche 
Diagnostics), using a mixture of random hexamer primers, nitrogenous bases 
(A, T, G and C), reaction buffer, RNase inhibitor and Reveso Transcriptase 
enzyme. The retrotranscription reaction was carried out in a thermocycler 
(Eppendorf Mastercycler) programmed at an initial temperature of 25°C for 15 
minutes, 45°C for 60 minutes and 72°C for 15 minutes. 
6. The qRT-PCR technique to determine relative expression, a "master mix" (10 
μL) was prepared in microtubes for the LightCycler nano (Roche Diagnostics), 
the mix was composed of 100 nM forward oligonucleotides, 100 nM reverse 
oligonucleotides, 100 nM hydrolysis probe (Human Universal Probe Library, 
Roche Diagnostics), 2 µL TaqMan master (Taq-polymerase), 0. 5 U LightCycler 
Uracil-DNA glycosylase, and 2 μL of DNAc (500 ng). The mixture was 
subjected to different temperature changes in the LightCycler Nano Real-Time 
PCR System (Roche Diagnostics) under the following conditions: initial 
incubation (denaturation) at 95 °C for 10 minutes, followed by 45 cycles of: 
denaturation (95 °C for 10 seconds), allylation of oligos to cDNA (60 °C for 30 
seconds), and extension (copying) of the cDNA strand (72 °C for 1 minute). The 
specific sequences used to design the oligonucleotides used in qRT-PCR were 
designed 
using 
the 
online 
software 
ProbeFinder: 
https://lifescience.roche.com/shop/en/us/overviews/brand/universal-probe-
library. 
7. Relative RNAm expression of genes was normalized to the relative expression 
levels of the housekeeping gene (18s). Relative quantification (normalization) of 
gene expression was determined using the 2-ΔΔΔCT method. 
 
 
 
 

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Statistical analysis 
 
In the analysis, qualitative variables are presented in frequencies and percentages, 
compared with the Chi-square test; quantitative variables according to the Shapiro-
Wilk normality test, normally distributed variables are presented in means and 
standard deviation, compared with Student's t-test, those related with ANOVA test; 
quantitative variables of free distribution are presented in medians and interquartile 
ranges, compared with Mann Whitney U-test, those related with Kruskall-Wallis 
test. Data analysis was carried out with SPSS version 23, Graph Pad Prisma. A 
value p<0.05 was taken as statistical significance, 95% confidence interval 
(95%CI). 
 
Ethical aspects: 
 
The project was carried out in accordance with the General Health Law on Health 
Research published in the Official Journal of the Federation on February 7, 1984, 
published in the Official Journal of the Federation the General Health Law, 
regulating the third paragraph of Article 4 of the Political Constitution of the United 
Mexican States, which came into force on July 1 of the same year; as well as the 
guidelines of the Declaration of Helsinki of the World Medical Association. of the 
Political Constitution of the United Mexican States, which came into force on July 
1st of the same year; as well as the guidelines of the Declaration of Helsinki of the 
World Medical Association on Ethical Principles for Medical Research Involving 
Human Subjects according to the 64th General Assembly, Fortaleza, Brazil, 
October 2013. The study is of greater than minimal risk, adhering to Chapter 1, 
Article 17, Section III of the ethical aspects of research on human beings, the 
authorization of the participants will be requested through an Informed Consent 
Letter (ICF) where the objective of the study and its significance, the procedures, 
the potential benefits and the possible risks will be stated; the name of those 
responsible for the study will also be mentioned and the autonomy and freedom 
they have to participate will be clarified.   

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In addition, according to the Helsinki declaration, specifically paragraph 33 of the 
last amendment of 2013, we will mention to the patient the possible benefits, risks, 
costs and efficacy of any new intervention, being evaluated by comparison with the 
best proven interventions, in this study the patient will be told which intervention 
could be more effective. As part of the ethical considerations, it will be informed 
that 14 ml of blood (one tablespoonful) will be drawn and that this amount does not 
put the health of the participants at risk, producing minimal discomfort with the aim 
of studying the presence of biochemical alterations. 
 
Biosafety aspects.  
 
- The present study has safety implications when obtaining blood samples, which 
can be considered infectious-contagious material. In the present study, the authors 
and co-authors are aware of and adhere to the General Law of Ecological 
Equilibrium and Environmental Protection and the General Law for the Prevention 
and Integral Management of Waste, which are of public order and social interest, 
Official Mexican Standard NOM-CRP-001-ECOL/1993, which establishes the 
characteristics of hazardous waste, the list of such waste and the limits that make 
a waste hazardous due to its toxicity to the environment; Official Mexican Standard 
NOM-002-SCT/2003 List of the most commonly transported Hazardous 
Substances and Materials; Official Mexican Standard NOM-002-SCT/2003 List of 
the most commonly transported Hazardous Substances and Materials; Official 
Mexican Standard NOM-002-SCT/2003 List of the most commonly transported 
Hazardous Substances and Materials. 
 
As the information will be obtained through consultation with highly trained 
personnel with more than 10 years of experience, without endangering the health 
or physical integrity of health personnel or patients, the environment will not be 
affected since we have the training for the handling of RPBI, with full respect for 

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the rights of patients avoiding any unnecessary physical or mental suffering or 
damage as dictated by the Nuremberg Code 1947. 
 
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