It has been hypothesized that HBO can mediate postoperative adhesion formation in the intra-abdominal region. To explore the anti-adhesion activity of HBO therapy, this study performed an in vivo study involving the treatment of rats using various HBO protocols (Figure 1 and Table 1). Upon completion of HBO therapy, adhesion formation was measured using an adhesion severity scoring system as well as the intra-abdominal adhesion scoring method. Our results, summarized in Figure 2A,B, indicate that the formation of adhesions following intra-abdominal surgery decreased with HBO treatment in a dose-dependent manner (* p < 0.05 and ** p < 0.01 vs. control group).

This study employed Protocol II to explore the possible effects of pressure and oxygen on adhesion formations in rats, following intra-abdominal surgery. A comparison of results in Figure 3A,B indicate that the formation of adhesions in Group B (a pressure of 1 ATA with 100% O₂) was reduced to a greater extent than those in Group A (room air, 1 ATA). These results indicate that oxygen may play an important role in decreasing the formation of postoperative adhesions (** p < 0.05 vs. Group A). As shown in Figure 3, pressure induced a more significant decrease in Group C (a pressure of 2.5 ATA with 100% O₂) than in Group B (^# p < 0.05 vs. Group B). This indicates that pressure was an important regulator in preventing the formation of adhesions following intra-abdominal surgery. From these assays, we also observed that rats exposed to HBO exhibited an oxygen-dependent suppression of adhesion formation following intra-abdominal surgery (^& p < 0.05 vs. Group C). These observations imply that the reduction in adhesion formation in HBO treated rats depended on the dosage and pressure of the supplied oxygen. Taken together, these results suggest that HBO has a potent anti-adhesion effect following intra-abdominal surgery.

Adhesion formation is clinically significant and a leading cause of postoperative morbidity, including small bowel obstruction [14]. The cause of elevated peritoneal adhesion formation rates in some individuals remains unknown. Currently, three major approaches are taken to prevent or reduce post-surgical adhesion: minimizing surgical trauma, anti-fibrotic drugs, and the use of barriers [15]. A variety of materials, such as barrier membranes [16], gels [17] and degradable biocompatible polymers [18], have been applied to reduce adhesion formation by maintaining separation between serosal surfaces [19]. However, these materials are not widely used, due to problems associated with clinical application as well as other limiting factors [20]. Moreover, these anti-adhesive strategies can increase the risk of intestinal anastomotic failure [21].

HBO therapy may be useful in managing adhesive intestinal obstruction associated with abdominal surgery [22]. Our findings further suggest that HBO₂ can lead to complete healing or clinically significant improvements in two thirds of patients suffering from chronic radiation enteritis [23]. Knowledge of the various HBO protocols used to treat peritoneal adhesion formation is required before these methods will approach clinical applicability.

Rat experimental protocols in this study were conducted in accordance with the regulations stipulated by the Institutional Animal Care and Use Committee of the College of Medicine, National Cheng Kung University. Male Wistar Rats were purchased from the College of Medicine, National Cheng Kung University. The animals were individually housed in stainless steel cages at 22 °C ± 2 °C on a 12 h light/dark cycle.

The male Wistar rats, weighing 270 to 300 grams, were anaesthetized with 50 mg/kg of intraperitoneal sodium Zoletil 50 (Virbac, Carros, France). The abdominal area was then shaved and prepared for aseptic surgery. A 3-cm midline laparotomy was performed. The cecum was identified and exteriorized and an area measuring approximately 1 cm × 2 cm on the ventral surface was abraded using a 1 cm × 2 cm sterile electrocautery tip cleaner until petechial hemorrhages appeared over the entire area. The cecum was abraded under a microscope to prevent occult cecum perforation. The cecum was then reinserted into the abdomen and a 1 cm × 2 cm section of the peritoneum as well as a superficial layer of underlying muscle from the right abdominal wall were excised by 8-mm punch biopsy.

Protocol I: In this experiment, a total of 24 rats were randomly assigned to four groups. In the three HBO treatment groups (n = 12), HBO sessions lasted 90 minutes and oxygen pressure was 2.5 ATA. The control group (n = 12) received no HBO treatment. The experimental design was based on the novel protocol of HBO therapy illustrated in Figure 1.

Protocol II: In this experiment, a total of 15 rats were randomly assigned to five groups. HBO therapy was performed once daily for 7 days. The rats were treated in a small research chamber. To ensure that the rats were supplied 100% oxygen, the chamber was flushed with oxygen for 10 min prior to the initiation of compression to vent the ambient air. The HBO treatments lasted for 90 min, including 10 min of compression and 10 min of decompression. The experimental design was based on the novel protocol of HBO therapy listed in Table 1.

The rats received HBO treatments from day 0 to day 14 after surgery. Copies of the adhesion area were calculated using Image J. (National Institutes of Health, Bethesda, MA, USA). Results were expressed as a percentage of adhesion area in the control group, which was considered to be 100%.

Intra-abdominal adhesions were graded blindly by two surgeons according to the method of Nair et al. [24]. Observers blind to the group assignment of the animals separately assessed the number and severity of adhesions using a well-documented adhesion severity scoring system [25]. In this evaluation, Grade 0 indicates no adhesions; Grade 1 indicates a single adhesion; Grade 2 indicates mild adhesions; Grade 3 indicates moderate adhesions, and Grade 4 indicates severe adhesions on the abdominal wall.

All data were reported as the mean (±SEM) of at least three separate experiments. Statistical analysis was performed using a t-test or one-way ANOVA with post-hoc test and the level of significance was set at p < 0.05 or 0.01.