Objective, measurable assessment of the elasticity of the skin around the eyes following the carboxytherapy treatment

Carboxytherapy is one of the most popular procedures used in dermatology. The treatments are known to be minimally invasive and highly effective for various skin disorders. Therefore, the research carried out in the paper is purposeful and addresses an important area of aesthetic therapies.

tissue perfusion, as a result of higher partial CO 2 pressure (hypercapnia). Temporary hypoxia is a powerful stimulus to the initiation of angiogenesis. 1,2 In the skin, carbon dioxide reacts with water which leads to the formation of carbonic acid and subsequent lowering of tissue pH. The decrease in pH translates into weaker bonds between hemoglobin and oxygen (Bohr effect). In consequence, vasodilatation of microcirculation and increased blood flow are observed. 3 Few aesthetic treatments induce long-lasting effects related with the improvement of microcirculation which seems to be necessary for a proper functioning of skin cells, for example, fibroblasts. It has been demonstrated that carboxytherapy stimulates the degradation of old collagen and enhances the production of new collagen. 4 Moreover, it increases the amount of elastin fibers. 5 After series of treatments, new collagen and new capillary vessels became arranged in a pattern characteristic for the young skin. 4 Carboxytherapy has been reported to affect collagen production and induce overall improvement of skin condition, including dark circles reduction, however, its impact on skin elasticity (measured with the use of MPA system) has not been studied. Instrumental MPA methods designed to measure the mechanical properties of the skin are the most objective technique of the assessment of the effectiveness of aesthetic treatments. Moreover, the evaluation of skin quality, including its flexibility, represents the most objective and reliable criteria. 6 The evaluation of effects solely on the basis of the analysis of photographs is subjective, and therefore, it can provide limited data on the efficiency of tested treatments.

| Study group
The study involved 39 patients (35 women, four men), aged 25-55 (mean age: 38 years) with periorbital skin changes due to aging (wrinkles, loss of skin elasticity). All individuals signed the informed consent. None of participants performed any aesthetic treatments within the treated area before the inclusion into this study. Exclusion criteria included treatment with carbonic anhydrase inhibitors, history of cardiac and circulatory failure, anemia, stroke, tumors, severe respiratory failure, connective tissue diseases, infectious diseases, bleeding diathesis, treatment with anticoagulants, history of cerebrovascular events, pregnancy, and lactation. 1 Enrolled patients were not allowed to use any dermatological and cosmetic products to the treatment area around the eyes within 6 h before the treatment.

| Methods
Five sessions of carboxytherapy were performed at one-week intervals, with the use of disposable needles 32G. Prior to procedure, the periorbital area was disinfected. The procedure involved intradermal, controlled injection of purified carbon dioxide (CO 2 ). A 32-gauge needle was inserted at an angle of 30° to a depth of 0.75-1 mm at one point near the left and right eye (approximately 1.5 cm below the angle of the lateral eye).
Laparoscopic CO 2 which is approved for medical use was utilized for the procedure. The treatment of eye area lasted a few minutes.
The treatment was performed with the device equipped with heating system, adjustable CO 2 flow rate (from 10 cc/min), and controlled CO 2 dosing (from 0.5 cc). A total of 3 cc of gas was used per one side with the same flow rate for both eyes. Before each treatment procedure, patients were asked about skin reactions and the possible complications. The procedure itself started with the preparation of the eye area. Make-up was removed (if necessary), and the skin was thoroughly degreased and disinfected with cosmetic pads and ethanol. A disposable gas supply tube and a disposable sterile needle were used during the procedure. Each time, before the initiation of the procedure, the hose was vented.
It took a few minutes until the gas was "absorbed" and the pressure on the eyeball subsided.
The effectiveness of the treatment was assessed with the use of Cutometer device (Courage + Khazaka electronic GmbH, Germany) and based on photographic documentation. Cutometer MPA 580 was used to assess changes in the elasticity of upper skin layer before the start of the series of treatments and 2 weeks after the last treatment. This device uses negative pressure which deforms the skin mechanically and enables the measurement of the following parameters (tested in this study): • R2 -a measure of the overall skin elasticity, including viscous changes of the skin for both deformation and retraction; it measures the ratio of "skin final retraction" to "final deformation"; the increase in this parameter indicates an increase in skin elasticity; • R7 -the measure of immediate elastic recovery after complete deformation, which is related to elasticity; The higher are the values (approaching 100%), the better is skin elasticity.
The photographic documentation was made using the

| Statistical analysis
The mean and standard deviation (mean ± SD) and median and interquartile range median (25%; 75%) were used for the description of parameters. For data showing no departures from normality (according to Shapiro-Wilk's test) and equal variances, standard t-test was used to express the differences between groups. In other cases, Mann-Whitney U test was performed. In case of repeated measurements, a paired t test was performed and Wilcoxon signed-rank test was applied, respectively. Statistical assessment of categorical variables was carried out using chi-square tests (with Fisher's exact test when appropriate). p values of < 0.05 were considered as statistically significant.

| RE SULTS
In our study, statistically significant differences in R2 and R7 parameters between the baseline and after treatment state were observed ( Table 1).
The R2 and R7 parameters of the Cutometer are the most frequently used in the literature to assess skin elasticity. In our study, we observed statistically higher R2 parameter values after the carboxytherapy. In 29 out of 39 subjects, the carboxytherapy treatment significantly improved skin tension and elasticity. Such treatment also considerably improved skin flexibility (R7 parameter). Favorable changes occurred in 28 individuals (Tables 1 and 2). The comparison of therapy effects in age groups (25-40 and 41-55 years) did not reveal any statistically significant results, which means that carboxytherapy-related improvement of skin elasticity is independent of age.
Also, photographic documentation demonstrated visual reduction in the number and depth of wrinkles as well as skin smoothing ( Figure 1). Again, the achieved effects were independent of patients' age.

| DISCUSS ION
To the best of our knowledge, this is the first study describing the effects of carboxytherapy treatment on skin elasticity within periorbital area, measured with the use of Cutometer. Cutometer measurements were made before the start of the series and 2 weeks after the last treatment in accordance with suggestion to perform such analyses after 2 weeks from the completion of therapy. El-Domyati M et al. 6 showed the results of biopsy made after 2 weeks from the last session of carbotherapy showed that the newly formed finer  carboxytherapy were properly organized in a normal lattice pattern, while the interfibrillar spaces were became reduced in a histochemical evaluation. 6 The results of many studies indicate that tissue stretching during  tests may concern the comparison of cutometric measurements with a different objective and instrumental assessment of flexibility or texture, profilometry, or skin imaging.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest. The authors themselves are responsible for the content and writing of the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within this article. Derived data supporting the findings of this study are available from the corresponding author AK on request.