## 2 Materials and Methods  
The research was designed as a prospective randomized, double-blind study performed at the University Medical Center Ljubljana between September 2019 and September 2020 (Clinicaltrial.gov ID: NCT04561609). The study included patients with diabetes with a unilateral chronic wound and symptoms of DSPN with scores above three on the Michigan neuropathy scoring instrument (MNSI) [23]. This study included only patients without previous amputations to fully evaluate the vibration sensation and monofilament test on all typical points. Patients with asymmetrical neuropathy due to other causes were excluded. Additionally, patients with comorbidities where CO2-induced vasodilation could theoretically worsen their condition (deep vein thrombosis, chronic kidney diseases grade III and IV, chronic heart diseases NYHA III and IV, patients with known malignant diseases, patients with progressive infection, signs of systemic infection with elevated inflammatory markers, or osteomyelitislis) and patients with alcohol abuse were also excluded. Sixty patients who met the defined period's inclusion criteria were randomized into a study or a control group. The detailed description of the transcutaneous CO2 therapy using a peripheral vascular rehabilitation system (PVR System; Derma Art, Brezice, Slovenia) is described in a previous publication [16]. Patients in the study group received transcutaneous CO2 therapy for 45 min per session every day (from Monday to Friday) for four weeks (20 sessions altogether). Instead of gaseous CO2, the air was used as a placebo treatment on patients in the control group. Before inclusion, patients were familiarized with the study protocol, received all the relevant information on the study from the medical staff, and afterward gave their written informed consent. Both patients and the doctor who evaluated their sensation status before and after the completing treatments were blind for group assignment (study or control). The nurse performing the transcutaneous CO2 therapy used a random number generator to randomize patients into one of the two groups. After completing the study, data were compared for the two groups, which were subsequently disclosed as study or control groups. The basic patient characteristics are presented in Table 1.  
The main outcome measure was an improvement of DSPN based on vibration sensation, monofilament test. Additionally, we measured the toe skin temperature. Vibration sensation (measured before the first and after the last CO\({}_{2}\) or placebo therapies) was performed using a 128 Hz tuning fork on five standard points on each foot (on the skin above the bony prominence of the first metatarasophalangeal joint (MPT), the base of the hallux, malleolus, diaphysis of the tibia, and tibia tuberosity). Results are presented as the number of points or scores (out of ten) with no vibration sensation [24]. Monofilament testing was performed before the first and after the last CO\({}_{2}\) or placebo therapies using Semmes-Weinstein monofilament (SWM) 10 g on eight standard points of each foot (on the plantar surface of the big toe, the third and fifth toes, on the plantar surface of the first, third and fifth metatararsal heads, on the plantar arch and the plantar surface of the calcaneus). Results are presented as the number of points or scores (out of 16) with no sensation [25]. An additional outcome measure was the temperature measured on the plantar aspect of the big toe using an infrared thermometer (Voltcraft IR 260-8S, Voltcraft, London, United Kingdom). Temperature measurement was standardized--before the first treatment and before the 20 th treatment (to exclude increased temperature during CO\({}_{2}\) therapy due to vasodilation). Patients were left to lie on the examination table barefoot for 20 min to adjust to the room temperature. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (approval ID: KME 84/02/16).