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. 2026 Jun 15;58(8):656–671. doi: 10.1002/lsm.70164

Evaluation of the Efficacy of Photobiomodulation Therapy, Therapeutic Ultrasound, and Boric Acid in the Prevention of Intra‐Abdominal Adhesions in an Experimental Rat Model: A Macroscopic, Histopathological, and Gene Expression Analysis

Sıtkıcan Okur 1,✉, Büşra Baykal 1, Latif Emrah Yanmaz 2, Ayşe Gölgeli Bedir 1, Damla Tuğçe Okur 3, Mümin Gökhan Şenocak 1, Ferda Turgut 4, Serkan Yildirim 5, İsmail Bolat 5, Betül Apaydin Yildirim 6, Özlem Özdemir Tozlu 7
PMCID: PMC13580430  PMID: 42296279

ABSTRACT

Objectives

This study aimed to evaluate the efficacy of photobiomodulation therapy (PBMT), therapeutic ultrasound (TUS), boric acid (BA), and their combinations in preventing experimentally induced intra‐abdominal adhesions in rats.

Materials and Methods

Ninety‐six male Wistar rats (14 weeks old) were randomly assigned to eight groups (Control, Laparotomy, Physiological Saline, BA, PBMT, TUS, BA + PBMT, and BA + TUS; n = 12/group). Adhesions were surgically induced. Treatments were applied once daily for 7 consecutive days as follows: BA (8 mg/kg, intraperitoneal), PBMT (904 nm, 3 J/cm2 applied to the incision site), and TUS (1 MHz, 0.5 W/cm2 applied to the incision site). On Day 8, rats were euthanized, and peritoneal fluid and adhesion tissue samples were collected for macroscopic scoring, histopathology, biochemical assays, and gene expression analyses.

Results

PBMT alone significantly reduced adhesion scores compared with the control (p < 0.05) and showed the most pronounced antifibrotic effect, while BA and TUS, alone or in combination, had limited impact. BA treatment increased malondialdehyde (MDA) levels (p < 0.05), indicating higher oxidative stress, whereas PBMT enhanced antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT) (p < 0.05). Gene expression analysis revealed that PBMT modulated cytokines linked to inflammation, particularly tumor necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β1).

Conclusion

PBMT demonstrated significant potential in reducing intra‐abdominal adhesions by alleviating inflammation, controlling oxidative stress, and promoting tissue repair. BA and TUS showed limited efficacy under the present experimental conditions, and BA in particular was associated with increased oxidative stress and inflammatory responses. Further research with optimized dosing, extended treatment durations, and combination strategies is warranted.

Keywords: boric acid, inflammation, intra‐abdominal adhesion, oxidative stress, photobiomodulation therapy, therapeutic ultrasound

1. Introduction

Postoperative intra‐abdominal adhesions are a frequent complication of abdominal surgery, occurring in up to 90%–95% of patients and often leading to serious clinical issues such as intestinal obstruction, chronic pain, infertility, and difficulties in repeat operations. Adhesion formation is primarily driven by fibrin deposition following peritoneal injury and an impaired ability to clear fibrin at the affected sites [1]. Current preventive measures focus on minimizing surgical trauma, controlling inflammation, and promoting fibrin absorption, yet no universally effective or safe method has been established. Thus, identifying an ideal strategy or agent to prevent intra‐abdominal adhesions remains an important unmet clinical need [2].

Boron is officially recognized as a trace element by the World Health Organization in 1980, and boric acid (BA) has been shown to possess antioxidant, antimicrobial, and anti‐inflammatory properties, as well as the ability to activate numerous enzymes [3]. Building on this evidence, BA has attracted growing interest in surgical medicine as a potential anti‐adhesive material, owing to its broad‐spectrum antiseptic activity, diverse therapeutic applications, rapid absorption with urinary excretion, and relatively short half‐life of about 1 day. Traditionally employed in wound care, vaginal and eye health, ear infections, foot odor control, and even swimming pool maintenance, BA is now being explored for its role in the development of surgical anti‐adhesive polymers [4]. The mechanism of action of borate is thought to involve the formation of complexes with sugar‐containing nucleotides (e.g., NAD+), thereby disrupting multiple enzyme activities.

Photobiomodulation therapy (PBMT) has been clinically applied to treat inflammatory diseases since 1981, showing benefits, such as reducing edema, enhancing tissue repair, and providing pain relief through mitochondrial chromophore activation. Although its anti‐inflammatory effects have sometimes been debated, recent studies demonstrate dose‐dependent benefits of PBMT in both in vitro and in vivo models, particularly in reducing cytokine‐mediated joint inflammation [5]. Therapeutic ultrasound (TUS) is a commonly used physical therapy technique that operates through high‐frequency sound waves. These vibrations promote cavitation, which enhances cell permeability and facilitates intercellular communication across articular surfaces and membranes, an effect that underlies its anti‐inflammatory properties [6]. In addition, stable cavitation can slow the conduction velocity of C fibers, thereby contributing to pain relief. When applied in continuous mode, ultrasound generates a rise in tissue temperature. The primary therapeutic advantages of this thermal effect include decreasing muscle spindle activity, which in turn helps alleviate muscle spasms and pain [7].

The aim of this study is to experimentally induce intra‐abdominal adhesions in a rat model and to evaluate the preventive efficacy of PBMT, TUS, and BA through macroscopic assessment, histopathological examination, and gene expression analyses. It is hypothesized that PBMT, TUS, and BA will significantly reduce the severity of intra‐abdominal adhesions compared to placebo, and that these effects will be supported by histopathological findings and gene expression profiles.

2. Materials and Methods

2.1. Animals and Ethics

Ninety‐six male Wistar rats (14 weeks old; 300–400 g) were randomized into eight groups (n = 12) using an online randomization tool (www.random.org.tr). To minimize potential confounders, cage positions were rotated weekly, and all treatments were administered at consistent times of day by the same operator. Sample size was calculated with G*Power 3.1.9.7 (α = 0.05, power = 0.95) based on Urkan and Güven [3], indicating a minimum of 12 rats per group (Table 1). Animals were acclimatized for at least 2 weeks, habituated to handling and device exposure (with the device off), and housed under a 12 h light/12 h dark cycle at 22°C ± 1°C and 50% humidity with ad libitum access to pellet feed and water. Animals were naive (no previous procedures) and confirmed healthy upon arrival by a veterinarian. All experimental procedures were approved by the Atatürk University Faculty of Veterinary Medicine Animal Ethics Committee (Approval No. 2022/4‐53; Date: March 29, 2022). Animals were housed in standard polycarbonate cages (five per cage) without additional environmental enrichment, in accordance with institutional guidelines.

Table 1.

Experimental groups and treatment procedures.

Group Group name Adhesion model Treatment
1 (n = 12) Control (C) + None
2 (n = 12) Laparotomy − Abdominal cavity opened and closed without any treatment procedure
3 (n = 12) Saline (S) + Intra‐abdominal irrigation with 2 mL saline for 7 consecutive days
4 (n = 12) Boric acid (BA) + Intra‐abdominal irrigation with 2 mL saline containing dissolved BA for 7 consecutive days
5 (n = 12) PBMT + Photobiomodulation therapy applied to the incision site for 7 consecutive days postoperatively
6 (n = 12) TUS + Therapeutic ultrasound applied to the incision site for 7 consecutive days postoperatively
7 (n = 12) BA + PBMT + Single intra‐abdominal irrigation with 2 mL saline containing dissolved BA + PBMT applied to the incision site for 7 consecutive days postoperatively
8 (n = 12) BA + TUS + Single intra‐abdominal irrigation with 2 mL saline containing dissolved BA + TUS applied to the incision site for 7 consecutive days postoperatively
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2.2. Experimental Design and Adhesion Induction

All rats received subcutaneous butorphanol (1 mg/kg) as premedication [8]. Adhesions were induced using the peritoneal button formation (PBF) model, chosen for its reproducibility [9]. Anesthesia was induced with intramuscular xylazine (10 mg/kg) and ketamine (100 mg/kg). After povidone‐iodine skin preparation, a 3 cm midline laparotomy was performed. Four peritoneal folds (1 cm2) were created with hemostats and ligated at the base using 6‐0 polypropylene sutures (0.2 cm spacing). Abdominal closure was achieved with continuous PDS 4‐0 (subcutis) and interrupted nylon 4‐0 (skin) [10]. No routine postoperative analgesia was administered in order to avoid potential confounding effects of analgesic agents on inflammatory, oxidative stress, and gene expression outcomes. However, all animals were closely monitored at regular intervals for clinical signs of pain or distress, including reduced mobility, abnormal posture, piloerection, decreased food intake, and vocalization. A predefined humane endpoint and rescue analgesia protocol were established, and animals showing significant pain or distress would have received appropriate analgesic treatment or been humanely euthanized. No animals required rescue analgesia during the study period. The laparotomy group served as a negative control, in which the abdominal cavity was opened and closed without inducing the adhesion model. The laparotomy group was not included in the comparative statistical analysis, as no adhesion induction procedure was performed in this group. Therefore, it was considered a procedural control rather than a treatment group and was used only for descriptive comparison to confirm the absence of spontaneous adhesion formation. This approach was adopted to avoid introducing bias into the comparative statistical analysis, as the laparotomy group did not undergo adhesion induction and therefore was not directly comparable to treatment groups in terms of pathophysiological conditions.

2.3. Interventions

2.3.1. Boric Acid

BA (Eti Boron) was sterilized with ethylene oxide and prepared as a 0.5% solution (pH 4.9). Rats received intraperitoneal injections of BA at a dose of 8 mg/kg (2.5 mL in sterile saline) once daily for 7 consecutive postoperative days. Care was taken to avoid direct instillation onto adhesion sites. The administered volume remained within the safety threshold of ≤ 10 mL/kg, and dosing was selected based on previously reported subtoxic and fibroblast‐compatible concentrations. A saline‐only group (2 mL) was included as a vehicle control [11, 12].

2.3.2. Photobiomodulation Therapy

PBMT (904 nm GaAs, 25 mW; Lasermed 2200, MLA1/25 probe) was applied cutaneously over the incision area. The treatment was delivered in contact mode, with a spot size of 0.5 cm2, at a dose of 3 J/cm2 for 60 s per session, beginning immediately postoperatively and repeated once daily for 7 consecutive days (total energy 1.5 J). The probe was positioned lateral to the incision and oriented at a 90° angle toward the peritoneum, without scanning. Output power was verified prior to each session using a calibrated power meter. PBMT was well tolerated without anesthesia, whereas sham animals underwent identical handling with the device turned off. All treatments were performed by the same operator at consistent times, and protective goggles were worn by both personnel and animals.

2.3.3. Therapeutic Ultrasound

TUS was applied with a portable device (Chattanooga, USA) at 1 MHz, 0.5 W/cm2, 1:4 duty cycle (20%). Sessions began immediately postoperatively and continued once daily for 7 days, lasting 3 min each. A gel‐coupled transducer was applied laterally to the incision at 90° to the peritoneum. Sham animals underwent identical handling with the device off. Parameters were within published safety and efficacy ranges.

2.4. Adhesion Assessment

2.4.1. Ultrasonographic Evaluation

Due to the high efficacy of the PBF model, adhesion formation was monitored non‐invasively by ultrasonography (Vetus 8, Mindray) to minimize unnecessary sacrifice. In control and laparotomy groups, ultrasonography was performed on postoperative Day 7 under sedation with medetomidine (100 µg/kg IM) and butorphanol (2 mg/kg IM). Following ventral shaving, rats were placed in dorsal recumbency and examined with a 5–13 MHz micro‐convex probe via a ventral approach. Atipamezole was administered at the end of the procedure to reverse medetomidine effects.

2.4.2. Macroscopic Evaluation

Rats were sacrificed on postoperative Day 8, corresponding to the peak adhesion formation period (7–10 days). Euthanasia was performed with pentobarbital sodium (100 mg/kgpoe, tail vein) [13]. A U‐shaped abdominal incision was made, and adhesions were graded using the Nair scoring system [14] by two blinded pathologists. Macroscopic adhesion score (Nair scoring system) was defined as the primary outcome measure and was used as the basis for sample size calculation. Treatment allocation was known to the operator performing the interventions (S.O.) but was concealed from the pathologists performing macroscopic and histopathological scoring, and from the personnel conducting biochemical and gene expression analyses.

2.4.3. Histological and Immunohistochemical Analyses

Tissues were fixed in 10% buffered formalin (48 h), paraffin‐embedded, and sectioned at 4 µm. Hematoxylin–eosin (HE) staining was used for inflammation and fibrosis scoring by two blinded pathologists according to established criteria. Masson's Trichrome staining (Facepath Anilin Blue kit, Barcode 8681065132824) was performed to evaluate collagen deposition.

2.4.4. Immunohistochemistry

Deparaffinized sections mounted on poly‐l‐lysine slides underwent 3% H2O2 block, citrate buffer antigen retrieval (pH 6.1), protein blocking, and incubation with primary antibodies against TNF‐α and NF‐κB. Detection used biotinylated secondary antibodies, streptavidin‐peroxidase, DAB chromogen, and Mayer's hematoxylin counterstain. Slides were examined under a Zeiss AXIO microscope.

2.4.5. Double Immunofluorescence

Sections were incubated with anti‐α‐SMA followed by FITC‐conjugated secondary antibody (ab6785, 1:1000), then anti‐Collagen I followed by Texas Red secondary (ab6719, 1:1000). DAPI mounting medium (D1306) was applied, and fluorescence was assessed under a Zeiss AXIO microscope.

2.5. Biochemical and Molecular Analyses

2.5.1. Cytokines and Oxidative Stress

On Day 8, plasma was stored at −80°C for enzyme‐linked immunosorbent assay (ELISA) assays of TNF‐α, IL‐6, IL‐1β, and TGF‐β1, as well as oxidative stress markers (MDA, GSH, SOD, CAT, and GPx). Adhesion tissues were rinsed, blotted, and stored at −80°C. ELISAs followed manufacturers' instructions (BioTek μQuant MQX200).

MDA: Thiobarbituric acid method (~535 nm absorbance). SOD: Inhibition of NBT reduction in xanthine/xanthine oxidase system (560 nm). CAT: Molybdate–H2O2 complex method (405 nm). Tissue MDA was quantified as nmol/g using a 1,1,3,3‐tetraethoxypropane standard curve.

2.5.2. Hypoxia and Angiogenesis Biomarkers

Hypoxia and angiogenesis‐related biomarkers, including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), and hypoxia‐inducible factor 1‐alpha (HIF‐1α), were measured in tissue homogenates. Quantification of these proteins was performed using commercially available ELISA kits, following the manufacturer's protocols in accordance with standard laboratory procedures. Briefly, tissue samples were homogenized under controlled conditions to preserve protein integrity, and supernatants were collected after centrifugation. The concentrations of VEGF, CTGF, and HIF‐1α were then determined spectrophotometrically by comparing absorbance values with standard curves generated from known concentrations of recombinant proteins. All assays were conducted in duplicate to ensure accuracy and reproducibility of the results.

2.5.3. Gene Expression (RT‐qPCR)

Primers for TNF‐α, TGF‐β1, and VEGF were designed with Primer‐BLAST. From ~50 mg tissue, total RNA was isolated using QIAzol, chloroform‐phase separation, isopropanol precipitation, and ethanol washing. RNA (2–5 µg) was DNase I‐treated and reverse‐transcribed (miScript kit, Applied Biosystems). qPCR was performed using SYBR Green Rox Master Mix (QIAGEN system), with GAPDH as the housekeeping gene. Reactions were run in triplicate, and results were analyzed by the 2−ΔΔCt method.

2.6. Western Blot Analysis

Proteins were extracted (Thermo 87790), quantified by BCA assay, separated on 10% SDS‐PAGE, and transferred to nitrocellulose membranes (Bio‐Rad Trans‐Blot Turbo). Membranes were blocked (5% milk/PBST), probed with primary antibodies, followed by HRP‐conjugated secondaries (1:3000), and visualized using ECL. Densitometric analysis was normalized to β‐actin.

2.7. Statistical Analysis

Statistical analyses were performed using SPSS v25. Normality was tested with the Shapiro–Wilk test. Parametric data were analyzed by one‐way ANOVA with Tukey's post hoc test, while non‐parametric data were compared using the Kruskal–Wallis test followed by Dunn's post hoc test. Results are presented as mean ± SD (and SE where relevant) or median (min–max). A p‐value < 0.05 was considered statistically significant.

3. Results

3.1. Study Flow and Group Disposition

During the study, one animal each from the Control (C), TUS, and TUS + BA groups and two animals each from the BA and PBMT + BA groups died. No mortality was observed to be directly related to the experimental interventions, and deaths were considered incidental based on clinical observations. Extra animals were included based on a priori power analysis and used as replacements when losses occurred. Replacement animals were re‐randomized according to the original randomization scheme, ensuring group balance.

3.2. Ultrasonographic Confirmation and Group Exclusion

After adhesion induction with the PBF model (Figure 1A), abdominal ultrasonography identified adhesion bridges in all groups except Laparotomy, in which no adhesions were detected (Figure 1B). The laparotomy group, in which no adhesions were detected, was retained as a negative control to confirm that adhesion formation was specifically related to the experimental model. Adhesions of varying severity were confirmed in the remaining groups (Figure 1C), after which animals were sacrificed for macroscopic and laboratory analyses. These findings further support the role of the laparotomy group as a valid negative control, confirming that adhesion formation was specifically associated with the experimental induction model rather than surgical exposure alone.

Figure 1.

Figure 1

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Experimental peritoneal adhesion model in rats showing (A) the surgical procedure, (B) representative abdominal ultrasonography images for adhesion detection, and (C) macroscopic views scored according to the Nair system (0–4) (n = 12 per group).

3.3. Macroscopic Adhesion Scores

Adhesion grading revealed a significant difference between Control and PBMT (PBMT showing improvement; p < 0.05), although median adhesion scores were similar across most groups. Statistical analysis based on rank distribution revealed a significant difference between the Control and PBMT groups (p < 0.05), while no consistent pairwise differences were observed among the remaining groups (Table 2).

Table 2.

Comparison of adhesion scores among the groups.

Groups
Control S BA PBMT TUS PBMT + BA PBMT + TUS
Score 3 (2–3)a 2 (1–3)ab 2 (1–3)ab 2 (1–3)b 2 (1–3)ab 2 (1–3)ab 2 (1–2)ab
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Note: The Kruskal–Wallis test was performed to determine differences among groups. Subsequently, a multiple comparison Dunn's test was conducted to identify the specific groups responsible for the differences. Letters indicate significant differences between groups (p < 0.05). Data are presented as median (range). Although median adhesion scores appear similar across groups, statistical differences are based on rank distribution and variability within groups as determined by the Kruskal–Wallis and Dunn's post hoc tests.

3.4. Plasma Oxidative Stress and Antioxidant Markers

MDA (nmol/L): BA (48.1 ± 15.8) was higher than all groups (Control 31.3 ± 6.7; PBMT 22.9 ± 2.1; PBMT + BA 26.4 ± 8.0; S 19.4 ± 0.9; TUS 31.9 ± 4.8; TUS + BA 24.3 ± 4.5; p < 0.05). No other between‐group differences (p > 0.05, Figure 2A). SOD (EU/mL protein): PBMT (26.9 ± 1.4) and S (28.8 ± 3.0) were higher than Control (22.7 ± 2.5; p < 0.05); other comparisons non‐significant (p > 0.05, Figure 2B). CAT (kU/L): PBMT (440 ± 53) and S (451 ± 42) were significantly higher than the Control group (304 ± 43; p < 0.001), indicating an enhanced antioxidant response in these groups. However, no significant differences were observed among the remaining groups (Figure 2C). GSH (mg/L): S (408 ± 43) was higher than all groups (p < 0.05). Control (246 ± 17.4) did not differ from non‐S groups (p > 0.05, Figure 3A). GPx (ng/L): PBMT (31.7 ± 3.7) and S (33.2 ± 4.9) were higher than Control (24 ± 1.9) and BA (22.1 ± 2.0; p < 0.05, Figure 3B).

Figure 2.

Figure 2

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Plasma oxidative stress and antioxidant parameters, including malondialdehyde (MDA) levels (A), superoxide dismutase (SOD) activity (B), and catalase (CAT) activity (C) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

Figure 3.

Figure 3

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Plasma antioxidant parameters, including reduced glutathione (GSH) levels (A) and glutathione peroxidase (GPx) activity (B) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

3.5. Plasma Cytokines and Growth Factors

CTGF (pg/mL): No differences versus Control (145 ± 5.7; p > 0.05). BA (148 ± 8.6) was higher than PBMT (125 ± 3.9), S (125 ± 8.5), and TUS + BA (125 ± 3.1; p < 0.05, Figure 4A). HIF‐1α (ng/L): Control (509 ± 117) was higher than S (372 ± 39.8; p < 0.05); other comparisons versus Control non‐significant. BA (631 ± 64) was higher than PBMT (417 ± 64), S, and TUS + BA (468 ± 58; p < 0.05, Figure 4B). VEGF (ng/L): No significant group differences (p > 0.05, Figure 4C) TGF‐β1 (pg/mL): Control (31.4 ± 6.5) differed from the other groups (p < 0.05). BA (34.8 ± 5.0) was higher than PBMT (28.4 ± 1.7) and S (26.5 ± 3.1; p < 0.05, Figure 5A). TNF‐α (ng/L): PBMT (119 ± 5.2) and S (118 ± 5.9) were lower than Control (144 ± 8.1; p < 0.05); other comparisons versus Control non‐significant. BA (155 ± 9.3) was higher than all groups except Control and TUS (148 ± 8.8; p < 0.05, Figure 5B). IL‐1 (ng/L): BA (332 ± 65) was higher than Control (252 ± 26) and higher than all other groups (p < 0.05, Figure 5C). IL‐6 (pg/L): No differences versus Control (78 ± 6.3; p > 0.05). S (67 ± 5.8) was lower than TUS (83.1 ± 4.8) and BA (83.2 ± 7.4; p < 0.05, Figure 5D).

Figure 4.

Figure 4

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Plasma levels of connective tissue growth factor (CTGF) (A), hypoxia‐inducible factor‐1α (HIF‐1α) (B), and vascular endothelial growth factor (VEGF) (C) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

Figure 5.

Figure 5

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Plasma levels of transforming growth factor‐β1 (TGF‐β1) (A), tumor necrosis factor‐α (TNF‐α) (B), interleukin‐1 (IL‐1) (C), and interleukin‐6 (IL‐6) (D) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

3.6. Tissue Oxidative Stress Markers

MDA (nmol/g): BA (29.2 ± 1.8) was higher than all groups (p < 0.05). Control (17.1 ± 3.3) did not differ from non‐BA groups (p > 0.05, Figure 6A) SOD (EU/g protein): No significant differences (p > 0.05, Figure 6B) CAT (U/g): No differences versus Control (342 ± 32.9; p > 0.05). BA (326 ± 73) was lower than S (394 ± 24; p < 0.05, Figure 6C).

Figure 6.

Figure 6

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Oxidative stress parameters in adhesion tissue, including malondialdehyde (MDA) levels (A), superoxide dismutase (SOD) activity (B), and catalase (CAT) activity (C) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

3.7. Histopathology and Masson Trichrome

Control: Severe adhesions, inflammation, angiogenesis, and hyperemia with edematous fold thickening (Figure 7‐1A); Masson: moderate connective tissue, mild muscle in adhesion areas (Figure 7‐2A). S: Mild adhesions/inflammation/angiogenesis; moderate hyperemia (Figure 7‐1B); Masson: severe connective tissue, moderate muscle (Figure 7‐2B). BA: Moderate adhesions/inflammation/angiogenesis/hyperemia (Figure 7‐1C); Masson: severe connective tissue, moderate muscle (Figure 7‐2C). PBMT: Reduced adhesion formation with moderate inflammatory infiltration, decreased angiogenesis, and less pronounced hyperemia compared to the control group (Figure 7‐1D). Masson: moderate connective tissue, mild muscle (Figure 7‐2D). TUS: Severe adhesions/inflammation/angiogenesis/hyperemia (Figure 7‐1E); Masson: severe connective tissue, mild muscle (Figure 7‐2E). PBMT + BA: Moderate adhesions/inflammation/angiogenesis; mild hyperemia (Figure 7‐1F); Masson: severe connective tissue, moderate muscle (Figure 7‐2F). TUS + BA: Moderate adhesions/inflammation/angiogenesis; mild hyperemia (Figure 7‐1G); Masson: severe connective tissue, moderate muscle (Figure 7‐2G).

Figure 7.

Figure 7

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Histological appearance of adhesion tissues stained with hematoxylin and eosin (HE) (upper panels) and Masson's trichrome (lower panels) in the experimental groups. (A) Control group showing severe adhesions, marked inflammatory cell infiltration, angiogenesis, and hyperemia with edematous thickening. (B) Saline (S) group showing mild adhesions, mild inflammation and angiogenesis, and moderate hyperemia. (C) Boric acid (BA) group showing moderate adhesions, inflammation, angiogenesis, and hyperemia. (D) PBMT group showing reduced adhesion formation, moderate inflammatory infiltration, decreased angiogenesis, and less pronounced hyperemia compared to control. (E) TUS group showing severe adhesions, inflammation, angiogenesis, and hyperemia similar to the control group. (F) PBMT + BA group showing moderate adhesions, inflammation, and angiogenesis with mild hyperemia. (G) TUS + BA group showing moderate adhesions, inflammation, and angiogenesis with mild hyperemia. Masson's trichrome staining demonstrates collagen deposition patterns, with increased connective tissue density particularly in BA and combination groups (n = 12 per group).

3.8. Immunohistochemistry and Double Immunofluorescence

Control: Severe TNF‐α (Figure 8‐1A) and NF‐κB (Figure 8‐2A); mild α‐SMA and moderate COL1 (Figure 9A). S: Mild TNF‐α/NF‐κB; severe α‐SMA and COL1 (Figures 8‐1B,2B and 9B). BA: Mild TNF‐α/NF‐κB; severe α‐SMA and COL1 (Figures 8‐1C,2C and 9C). PBMT/TUS: Severe TNF‐α/NF‐κB with mild α‐SMA and moderate COL1 (Figures 8‐1D,E, 2D,E and 9D,E). PBMT + BA/TUS + BA: Moderate TNF‐α/NF‐κB with mild α‐SMA and moderate COL1 (Figures 8‐1F,G, 2F,G and 9F,G). Quantitative scoring and statistics are provided in Figure 10.

Figure 8.

Figure 8

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Immunohistochemical staining of adhesion tissues demonstrating tumor necrosis factor‐α (TNF‐α) and nuclear factor kappa B (NF‐κB) expression across experimental groups. (A–G) TNF‐α expression: The control group shows strong immunoreactivity, indicating severe inflammation. The saline (S) and BA groups exhibit mild expression. The PBMT and TUS groups demonstrate persistent strong staining, while PBMT + BA and TUS + BA groups show moderate expression levels. (A–G) NF‐κB expression: A similar pattern is observed, with strong expression in the control group, mild staining in S and BA groups, and moderate to strong expression in treatment groups. Combination groups demonstrate moderate expression levels. Overall, PBMT‐treated groups show partial modulation of inflammatory markers, while BA‐related groups demonstrate variable expression patterns consistent with increased oxidative and inflammatory responses (n = 12 per group).

Figure 9.

Figure 9

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Peritoneal tissue from the Control (A), S (B), BA (C), PBMT (D), TUS (E), PBMT + BA (F), and TUS + BA (G) groups. Intracytoplasmic αSMA expression in muscle tissues (FUTC) and Col1 expression in connective tissue cells (Texas Red) were detected by immunofluorescence (IF). Scale bar: 200 µm (n = 12 per group).

Figure 10.

Figure 10

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Comparison of TNF‐α, NF‐κβ, αSMA, and COL1 expression levels across experimental groups. Data include immunohistochemical and immunofluorescence staining results along with statistical analyses. Values are presented as mean ± SEM. Each bar represents the mean gene expression level for the respective group, and the error bars indicate the standard error (SE). The p‐values shown above indicate the level of statistical significance for pairwise comparisons (n = 12 per group).

3.9. Gene Expression (RT‐qPCR)

TGF‐β1: Control and S were similar; increases were observed in PBMT, TUS, and BA. BA + PBMT and BA + TUS were above Control, but not statistically significant (p > 0.05). (Figure 11A). TNF‐α: S was lower than Control. PBMT and TUS were higher than Control, with a greater increase in BA. BA + PBMT showed a significant elevation (p < 0.05), whereas BA + TUS showed a significant reduction (p < 0.001, Figure 11B). VEGF: S, PBMT, TUS, and BA were similar to Control. BA + PBMT increased, BA + TUS was comparable to Control; no significant differences overall (p > 0.05, Figure 11C).

Figure 11.

Figure 11

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Relative mRNA expression levels of transforming growth factor‐β1 (TGF‐β1) (A), tumor necrosis factor‐α (TNF‐α) (B), and vascular endothelial growth factor (VEGF) (C) in the control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS groups, as determined by RT‐PCR; data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (n = 12 per group).

3.10. Western Blot

TNF‐α protein expression was significantly increased in BA + PBMT versus Control, with other groups comparable to Control. β‐Actin confirmed equal loading (Figure 12).

Figure 12.

Figure 12

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Evaluation of TNF‐α and actin protein levels by Western blot analysis. The upper panel shows TNF‐α protein levels, while the lower panel shows actin protein levels used as a loading control. Groups: Control, S, PBMT, TUS, BA, BA + PBMT, and BA + TUS. Actin bands confirm equal protein loading for each lane (n = 12 per group).

4. Discussion

In this study, the effects of BA, PBMT, and TUS on the prevention of experimentally induced intra‐abdominal adhesions were investigated, and the impact of their combinations was evaluated. Macroscopic assessment revealed that all groups exhibited a numerical reduction in adhesion scores. However, only the PBMT group demonstrated a statistically significant decrease in adhesion scores compared to the control group. This finding is consistent with the therapeutic benefits of PBMT reported in the literature for various inflammatory pathologies [5, 15, 16].

PBMT is known to accelerate tissue repair, reduce inflammation and edema, alleviate pain, and stimulate cellular functions. These effects are thought to be triggered by the absorption of laser photons by chromophores such as cytochrome c oxidase within mitochondria [17]. Activation of cytochrome c oxidase increases ATP production, thereby enhancing cellular function and promoting tissue recovery [18]. Our findings suggest that PBMT may have contributed to the reduction of adhesion scores through these biological mechanisms. However, a partial discrepancy was observed between macroscopic outcomes and histological/immunohistochemical findings. While PBMT significantly reduced macroscopic adhesion scores, its effects on inflammatory markers such as TNF‐α and NF‐κB, as well as histopathological parameters, were not consistently aligned across all analyses. This inconsistency may be attributed to differences in the sensitivity and timing of macroscopic versus microscopic assessments, as well as the multifactorial nature of adhesion formation. Therefore, the anti‐adhesive effect of PBMT observed at the macroscopic level may not be solely explained by classical inflammatory pathways. In particular, since inflammation is a critical factor in the initiation and progression of adhesion formation, the anti‐inflammatory effect of PBMT could play a central role in reducing peritoneal injury response. By attenuating inflammation, PBMT may prevent excessive fibrin deposition and fibroblast activation, thereby limiting adhesion development.

Interestingly, in the PBMT + BA combination group, the expected therapeutic effect was not observed. This outcome may be related to repetitive or traumatic intraperitoneal injections, which can cause peritoneal surface injury and subsequently increase adhesion formation. Similarly, Eroğlu and Uyanıkgil highlighted that repeated intraperitoneal interventions may enhance adhesion scores, as foreign body exposure, infections, or chemical irritants within the peritoneal cavity are well‐established triggers for adhesion development [19]. Furthermore, vascular or tissue injury during injection may lead to local bleeding, which in turn promotes inflammation and adhesion formation [20]. However, this explanation does not fully account for the lack of similar findings in the saline group, suggesting that the observed increase in oxidative stress markers in the BA group is more likely related to its biochemical effects rather than mechanical irritation alone. Furthermore, the absence of an enhanced therapeutic effect in the PBMT + BA group may be attributed to the opposing biological mechanisms of these interventions. While PBMT exerts well‐documented anti‐inflammatory and antioxidative effects, BA may induce pro‐inflammatory and oxidative responses, thereby potentially counteracting the beneficial effects of PBMT.

In the BA group, plasma MDA levels were significantly higher compared to the other groups. While low‐dose BA has been reported to exert minimal effects on MDA, higher doses have been shown to elevate oxidative stress markers [21, 22]. This phenomenon may be explained by two possible mechanisms: (i) repetitive BA administration could induce oxidative stress and lipid peroxidation, thereby elevating MDA levels; (ii) although a non‐toxic dose was selected, higher BA concentrations may suppress antioxidant defense systems. In line with previous studies [21], no significant differences were observed in SOD, CAT, and GSH levels between the BA and control groups. Additionally, the markedly higher IL‐1 levels in the BA group suggest that BA may enhance pro‐inflammatory cytokine production [23]. Taken together, these findings do not support a clearly beneficial effect of BA under the present experimental conditions. On the contrary, BA administration was associated with increased oxidative stress and a more pro‐inflammatory profile, suggesting that its proposed anti‐adhesive or protective role should be interpreted with caution. Conversely, PBMT treatment led to increased plasma SOD, CAT, and GPx levels compared to controls. These histopathological findings are consistent with the macroscopic adhesion scores. The observed increase in CAT activity further supports the role of PBMT in enhancing the antioxidant defense system, suggesting a protective effect against oxidative stress‐induced tissue damage. Previous reports have shown that PBMT enhances SOD activity, reduces oxidative damage, and promotes recovery in different tissues [24, 25]. The rise in CAT levels following photostimulation may reflect compensatory activation of antioxidant systems against free radical production [26]. By preventing reactive nitrogen species generation and modulating antioxidant enzyme activity, PBMT appears to protect cellular integrity [27, 28].

Moreover, PBMT reduced TNF‐α levels significantly compared to controls, in agreement with its reported ability to modulate inflammatory mediator expression [29]. Although the precise cellular mechanisms remain under debate, photobiological effects of PBMT are initiated by photon absorption by photoacceptors, leading to secondary messenger cascades that regulate inflammation and tissue repair [17, 30]. With respect to gene expression analyses, TGF‐β1 and TNF‐α expression showed modest but not statistically significant alterations across the treatment groups. While BA + PBMT and BA + TUS combinations elevated TGF‐β1 expression compared to controls, the lack of significance limits the biological interpretation. Given the well‐established role of TGF‐β1 in fibroblast activation, extracellular matrix deposition, and tissue remodeling processes across different tissues [31], future studies with optimized dosing and longer treatment durations are warranted. BA + PBMT significantly enhanced TNF‐α expression, suggesting a potential synergistic pro‐inflammatory effect, whereas BA + TUS decreased TNF‐α expression, highlighting the modulatory capacity of ultrasound on inflammation [5]. However, it should also be considered that, depending on the applied parameters, TUS may exert pro‐inflammatory effects, which could have influenced the observed outcomes.

In terms of VEGF expression, no statistically significant differences were detected among the groups, although the BA + PBMT group demonstrated a trend toward higher levels. This observation is consistent with previous reports indicating that the effects of PBMT on VEGF are highly dependent on treatment parameters such as wavelength, dose, and timing [32]. For example, Colombo et al. showed that PBMT at 660 nm promoted neovascularization in a rodent wound healing model, although the increase in VEGF expression was not consistently significant [33]. Such discrepancies suggest that the modulation of VEGF by PBMT may vary according to the experimental model and the phase of tissue repair being evaluated. Considering that excessive VEGF‐driven angiogenesis can also contribute to adhesion formation, these findings highlight the importance of precise regulation and optimal timing of VEGF modulation to achieve clinically relevant benefits [34].

Our findings are in line with previous reports where anti‐inflammatory agents such as meloxicam and methylprednisolone effectively reduced postoperative intra‐abdominal adhesions through suppression of inflammatory cell infiltration and fibrosis [35, 36]. Similarly, in our study, PBMT demonstrated a significant reduction in adhesion scores, which may be attributed to its anti‐inflammatory effects, including the modulation of cytokine expression (e.g., TNF‐α, TGF‐β1) and attenuation of oxidative stress. Collectively, these results reinforce the notion that targeting inflammatory pathways, whether through pharmacological agents or biophysical approaches such as PBMT, represents a promising strategy in adhesion prevention.

This study has several limitations. First, not all possible therapeutic combinations were tested; in particular, the PBMT + TUS and BA + PBMT + TUS groups were not included. This restricts the ability to draw conclusions regarding the synergistic or antagonistic effects of multi‐modal therapies. Second, animals that did not develop adhesions following laparotomy were excluded, which precluded assessment of the biochemical impact of laparotomy alone. However, the laparotomy group was retained as a procedural negative control to verify that adhesion formation was not a consequence of surgical exposure alone. Although it was not included in statistical comparisons due to the absence of adhesion induction, its inclusion provides important contextual validation of the experimental model. While this decision was taken to minimize unnecessary sacrifice and ensure animal welfare, it remains a limitation in interpreting the role of surgical trauma in adhesion development. In addition, the absence of routine postoperative analgesia may be considered a limitation; however, this approach was intentionally adopted to avoid interference with inflammation‐related outcome measures. Strict welfare monitoring, predefined humane endpoints, and a rescue analgesia protocol were implemented throughout the study period. Finally, dosing and treatment parameters may not fully reflect optimal clinical conditions, and longer term or repeated follow‐up studies are necessary to validate the findings. Despite these limitations, the study provides valuable comparative data and highlights promising avenues for future experimental and clinical investigations. Furthermore, the relatively small sample size per group and the single sacrifice time‐point (Day 8) limit the precision of the estimates and preclude conclusions about long‐term adhesion outcomes. Regarding generalizability, the PBF model in rats is a well‐established surrogate for human postoperative adhesion formation; however, differences in peritoneal physiology between rodents and humans should be considered when extrapolating these findings to clinical practice.

5. Conclusion

This study demonstrated that PBMT significantly reduced adhesion formation compared to the control group, whereas BA and TUS alone or in combination showed limited effects. The results also indicate that PBMT and BA may influence inflammatory and oxidative stress markers, suggesting potential roles in modulating cellular responses. However, limitations such as non‐optimized doses and treatment durations may have affected the efficacy of BA and TUS. Future investigations should explore different dosing strategies, treatment durations, and combinations to optimize these therapeutic approaches. Long‐term and clinical studies are warranted to validate the safety, durability, and translational potential of these interventions for preventing intra‐abdominal adhesions.

Author Contributions

Conceptualization: Sıtkıcan Okur and Latif Emrah Yanmaz. Methodology: Sıtkıcan Okur, Latif Emrah Yanmaz, and Ayşe Gölgeli Bedir. Formal analysis and investigation: Büşra Baykal, Damla Tuğçe Okur, Mümin Gökhan Şenocak, Ferda Turgut, Serkan Yıldırım, and İsmail Bolat. Writing – original draft preparation: Sıtkıcan Okur and Büşra Baykal. Writing – review and editing: Sıtkıcan Okur, Latif Emrah Yanmaz, Ayşe Gölgeli Bedir, Betül Apaydın Yıldırım, and Özlem Özdemir Tozlu. Supervision: Sıtkıcan Okur and Latif Emrah Yanmaz. Funding acquisition: Sıtkıcan Okur. Resources: Sıtkıcan Okur.

Ethics Statement

All procedures were performed in accordance with institutional and national guidelines for the care and use of laboratory animals. Approval was obtained from the Atatürk University Faculty of Veterinary Medicine Animal Ethics Committee (Decision No. 53; Meeting 2022/4, March 29, 2022).

Conflicts of Interest

The authors declare no conflicts of interest. This study was supported by TÜBİTAK (project no. 123O826, 3501 Program). The funder had no role in study design, data collection, analysis, interpretation, or manuscript preparation.

Protocol Registration

No study protocol was pre‐registered prior to the commencement of this study.

Acknowledgments

The authors thank the staff of the Atatürk University Experimental Research Center (ATADEM) for their assistance in animal care and surgical procedures, and the Pathology and Genetics Laboratory teams for their support during analyses. This study was supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) under project number 123O826 (3501 Program).

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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