Methods

Study design and ethical considerations

The study protocol was approved by the Institutional Ethics Committee (approval number 6.308.240) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Random allocation was not performed due to the exploratory nature of this pilot study and logistical constraints related to surgical scheduling. This design may introduce allocation bias, which should be addressed in future randomized controlled trials. Both groups were followed using the same assessment schedule. Saliva samples and pain evaluations were performed at three predefined time points: T0 (1–6 h before surgery), T1 (48 h postoperatively), and T2 (7 days postoperatively). These assessments were conducted for both the PBM and control groups to allow longitudinal comparisons of inflammatory and oxidative stress biomarkers as well as postoperative pain outcomes. As a pilot study, no formal sample size calculation was performed. The sample size was determined based on feasibility considerations, participant availability, and the exploratory nature of the study. The primary objective was to generate preliminary data and estimate effect sizes to support the design of future randomized controlled trials.

The selection of assessment time points was based on key phases of the postoperative inflammatory and healing response. The baseline measurement (T0) was obtained 1–6 h before surgery to capture preoperative biomarker levels without surgical influence. The 48-hour postoperative assessment (T1) was chosen because the early inflammatory response and oxidative stress typically peak within the first two days following surgical trauma. The 7-day assessment (T2) corresponds to the transition between the inflammatory and early proliferative phases of wound healing, when initial tissue repair and symptom improvement are expected.

Participants and group allocation

Eligible participants were women aged 18 years or older scheduled for elective abdominoplasty. Exclusion criteria included acute inflammatory or infectious conditions, use of photosensitizing medications, HIV infection, pregnancy, lactation, and tattoos on the abdominal region.

Participants were allocated into two groups: the intervention group (PBM group), which received photobiomodulation after surgery, and the control group, which received standard postoperative care without PBM. All participants received the same postoperative physiotherapy instructions, including posture adjustments and wound care. The study was not prospectively registered, which limits transparency. Future studies will be prospectively registered to strengthen methodological rigor.

Photobiomodulation protocol

A single PBM session was applied to investigate the immediate postoperative biological response and to reflect the clinical feasibility of PBM use prior to hospital discharge. PBM was applied using an 808 nm infrared laser device (Elight IRL, DMC, São Carlos, Brazil) in cluster mode, with continuous emission and the applicator positioned perpendicular to the abdominal skin at a distance of 1 cm. Each of the four infrared laser diodes delivered an output power of 100 mW for 30 s, corresponding to 3 J per irradiation site. During each application, the four infrared diodes were activated simultaneously, resulting in a total cluster output power of 400 mW and an energy delivery of 12 J per cluster position. The energy density was calculated using a beam area of 2.26 cm² per irradiation site, resulting in 1.33 J/cm². This beam area was obtained from the optical specifications of the device and was used for all dosimetric calculations.

According to the manufacturer’s specifications, the wavelength tolerance was ± 10 nm and the output power variability was ± 20% of the nominal value. These values represent the operational tolerances of the device and do not indicate equipment malfunction. The device contains four red laser emitters and four infrared laser emitters. In the present study, only the four infrared emitters were activated, operating simultaneously at a nominal wavelength of 808 nm (± 10 nm).

A custom template based on the anatomical vascular zones described by Huger (1979) was developed to standardize the irradiation area. The template encompassed a 36 × 9 cm anatomical region and contained 144 predefined irradiation sites spaced 1 cm apart, with 72 sites per hemibody. The sites were aligned with anatomical landmarks, including the iliac crests and costal margins. A dermatographic pencil was used to transfer these reference points to the skin surface, ensuring reproducible positioning of the laser applicator throughout the procedure. The marked sites did not correspond to holes, perforations, or openings in the template; rather, they represented predefined reference locations used to standardize PBM application.

PBM was applied sequentially, with the cluster positioned for 30 s at each treatment location. During each application, the four infrared laser diodes emitted simultaneously, delivering 3 J to each of four predefined irradiation sites. Therefore, although 144 irradiation sites were marked on the skin, only 36 sequential cluster positions were required because four sites were treated simultaneously at each position. The applicator was repositioned according to a predefined sequence to ensure that all 144 marked sites were irradiated once, without repeating any marked irradiation site during successive applications. Consequently, the total irradiation time was approximately 18 min (36 positions × 30 s). The total energy delivered to the treated area was 432 J (144 irradiation sites × 3 J per site). The handheld laser device was manually operated throughout the procedure.

All safety protocols were followed, including the use of protective eyewear by both patients and operators and equipment disinfection before and after each application.

Standard postoperative care

All participants received the same standard postoperative care routinely provided after abdominoplasty. This included analgesic medication as prescribed by the surgical team, wound care and dressing management, recommendations for rest and gradual return to daily activities, and standardized physiotherapy instructions aimed at promoting mobility and preventing postoperative complications.

Importantly, these physiotherapy guidelines were part of the routine postoperative care protocol of the surgical service and were provided to both groups equally. Therefore, they were not considered an additional intervention within the context of this study.

Analgesic prescriptions followed the standard institutional postoperative protocol and were applied equally to both groups. Therefore, postoperative analgesic management was not expected to introduce systematic differences between groups.

Saliva collection and biomarker analysis

Saliva samples were collected at three time points: T0 (1–6 h before surgery, in-hospital collection), T1 (48 h postoperatively, home collection), and T2 (7 days postoperatively, home collection).

Unstimulated saliva samples were collected using Salivette® devices. Participants were instructed to collect samples in the morning while fasting and to avoid eating, drinking, or performing oral hygiene procedures for at least one hour prior to collection in order to minimize variability in salivary biomarker levels.

For T1 and T2, patients performed home collection using the Salivette® kits. Samples were temporarily stored in home freezers and subsequently transported to the laboratory. A video call was conducted at each time point to verify correct sample collection and to assess pain using the Visual Analog Scale (VAS).

Upon arrival at the Cellular and Molecular Immunology Laboratory (UFCSPA), samples were stored at − 80 °C until biochemical analysis.

Saliva deproteinization

To prepare samples for biochemical analysis, 500 µL of saliva were mixed with 500 µL of absolute ethanol at room temperature. The mixture was then centrifuged at 3,000 rpm for 10 min. Protein precipitation was achieved by centrifugation, and the resulting supernatant was collected and stored in microtubes at − 80 °C for subsequent assays.

Cytokines (TNF-α and IL-10) analysis

TNF-α and IL-10 levels were quantified using ELISA kits (PeproTech/Thermo Fisher Scientific) according to the manufacturer’s instructions. Detection ranges were 23–3000 pg/mL.

Nitrite quantification (Griess reaction)

Nitrite levels were measured as an indirect indicator of nitric oxide (NO) availability using the Griess reaction. For each well of a 96-well plate, 50 µL of deproteinized saliva were added along with 50 µL of Griess reagent, which consisted of 5% phosphoric acid (H₃PO₄), 2% sulfanilamide, 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride (NED), and ultrapure water. The plate was incubated for 1 h at 37 °C, and absorbance was read at 540 nm using a spectrophotometer. Concentrations were determined based on a standard curve.

Lipid peroxidation assay (MDA-TBARS test)

Lipid peroxidation was assessed using the MDA-TBARS method as described by Ohkawa et al., [21]. A total of 200 µL of deproteinized saliva were pipetted into separate test tubes. Each tube received 375 µL of acetic acid (2.5 M, pH 3.4), 375 µL of thiobarbituric acid (0.8%), and 500 µL of sodium dodecyl sulfate (8.1%). The reaction mixture was incubated in a water bath at 100 °C for 1 h, followed by centrifugation at 5,000 g for 15 min. The resulting supernatant was transferred to a 96-well plate and read at 532 nm using a microplate spectrophotometer. Final concentrations were calculated from absorbance values using a standard curve.

Statistical analysis

Statistical analyses were performed using Python (version 3.12) with appropriate statistical libraries, including SciPy and StatsModels. Data were tested for normality using the Shapiro-Wilk test. For continuous variables, non-parametric comparisons between groups were performed using the Mann-Whitney U test. Repeated measures over time were analyzed using the Friedman test. Correlations were assessed using Spearman’s rank correlation coefficient. Multiple linear regression was used to assess predictors of biomarker levels (age, BMI, physical activity, surgical history). A significance level of p < 0.05 was adopted. In addition to traditional significance testing, effect sizes were calculated to quantify the magnitude of differences between groups and across time points. Between-group differences (PBM vs. Control) were assessed using Cohen’s d, while within-group changes across time points were evaluated with Cohen’s dz for paired samples. Effect sizes were interpreted according to conventional thresholds (small = 0.2, medium = 0.5, large = 0.8). This approach provides complementary information to p-values, allowing a more nuanced interpretation of clinical relevance beyond statistical significance.