Acute respiratory distress syndrome (ARDS) is a condition characterized by acute-onset hypoxemia and bilateral pulmonary edema resulting from increased alveolocapillary membrane permeability, which cannot be fully explained by cardiac failure [1]. Currently, there is no specific drug or treatment available for ARDS. Supportive care focuses on mechanical ventilation to prevent further lung injury and managing refractory hypoxemia, which are cornerstone interventions for ARDS. Improving arterial oxygenation and minimizing potential tissue damage are essential goals of treatment. [2, 3]. In addition to these strategies, hyperbaric oxygen therapy (HBO) and medical ozone therapy are being explored. HBO therapy has physiological effects such as improving oxygenation, increasing antimicrobial activity, modulating inflammation and immune function, and promoting angiogenesis [4, 5]. Medical ozone therapy involves administering a specific mixture of ozone and oxygen gases into the body’s circulation or cavities. Ozone therapy is known to modulate phagocytic activity and the expression of antioxidant enzymes in the immune system. Both clinical and experimental studies have shown that ozone therapy is beneficial in inflammatory conditions such as infected wounds, chronic skin ulcers, burns, and advanced ischemic diseases [6].

Human studies on ARDS provide valuable information concerning the onset and progression of physiological and inflammatory changes in the lungs. However, the numerous variables present in critically ill patients make it challenging to evaluate alternative therapies in humans. This underscores the importance of animal models. When developing experimental animal models, it is crucial to choose models specifically suited to the factor under investigation, as these models can be influenced by various factors. There are numerous direct and indirect techniques available for inducing experimental ARDS in animal models. One direct approach involves the administration of a bacterial product, such as intratracheal bacteria or lipopolysaccharides (LPS), to cause lung injury, while another method includes the administration of an acid, such as hydrochloric acid (HCl), to induce aspiration [7].

In this study, we aimed to investigate the effects of HBO and medical ozone therapies on ARDS through experimental methods. Our hypothesis was that HBO and medical ozone therapies would improve recovery and oxygenation in a rat model of experimentally induced ARDS. In this context, the primary objective of our study was to induce direct lung injury by intratracheal administration of Escherichia coli in rats and compare the macroscopic and histopathological effects of HBO and ozone therapies on lung injury. The secondary objective was to measure and compare the levels of pro-inflammatory and anti-inflammatory cytokines, namely interleukin (IL)-1β, IL-6, vascular endothelial growth factor (VEGF), total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), partial pressure of oxygen (PO₂), partial pressure of carbon dioxide (PCO₂), in plasma samples from all groups of rats.

Thirty-two female Wistar albino rats were included in the study and divided into four groups, each containing eight rats. The rats were approximately 2.5 months old (2–3 months) with an average weight of 250 grams (200–300 grams). The animals were randomly divided into four groups, housed in cages containing eight rats each, and observed in the laboratory environment for one week before any procedures. During the study, the rats were provided ad libitum access to tap water and standard rodent feed. They were kept in a controlled environment with a temperature of 23–25°C, a 12-hour light/dark cycle, and 50–60% humidity.

On the day of the study, the rats were transported to the research laboratory and weighed. Anesthesia was induced using intraperitoneal injections of 10 mg/kg ketamine hydrochloride (Ketalar®, Eczacıbaşı İlaç Sanayi ve Ticaret A.Ş., Istanbul) and 90 mg/kg xylazine (Rompun®, Bayer Türk Kimya Sanayi, Istanbul), with additional injections planned as necessary. Absence of response to painful stimuli was considered indicative of sufficient anesthesia depth. All groups received live E. coli bacteria (10⁹ CFU/100 grams body weight) via intratracheal injection. All injections were performed simultaneously, considering biological rhythms. Clinical monitoring of all subjects included daily assessments of activity, appetite, and respiratory rate [8]. Treatments commenced 10 hours after the administration of live E. coli.

The experimental groups were defined as follows:

In Group S, intraperitoneal saline was administered for five days following intratracheal E. coli injection. In Group C, intraperitoneal cefepime HCL (50 mg/kg) was administered for five days [9]. In Group HBO, intraperitoneal cefepime HCl (50 mg/kg) was supplemented with HBO administered over five days, with three sessions per day at eight-hour intervals on the first day and two sessions per day at 12-hour intervals for the subsequent days. Each session lasted 90 minutes at a pressure of 2.5 atmospheres absolute (ATA), for a total of 11 HBO therapy sessions over five days [9]. Before therapy, the experimental pressure chamber was ventilated with 100% oxygen under normobaric conditions for 10 minutes. It was then compressed with 100% oxygen to 2.5 ATA (49 feet of seawater), and the rats inhaled 100% oxygen continuously at this depth for 60 minutes, followed by decompression with 100% oxygen over 15 minutes. In Group OT, intraperitoneal cefepime HCL (50 mg/kg) was combined with intraperitoneal oxygen/ozone gas mixture (0.7 mg/kg) administered once daily for five days [10].

For sample collection, the rats were anesthetized with 10 mg/kg ketamine hydrochloride and 90 mg/kg xylazine. Under sterile conditions, the abdominal cavity was opened, and the abdominal aorta was clamped. Blood samples were collected using heparinized syringes, and the rats were sacrificed under anesthesia. Blood samples were centrifuged at 4,000 rpm for 15 minutes, and the resulting heparinized plasma was transferred to Eppendorf tubes and stored at -80°C for cytokine measurement. Following sacrifice, the rats’ thoracic cavities were opened, and the lung tissues were excised. The left lung tissue was frozen at -80°C for pathological and biochemical analysis, while the right lung tissue was fixed in 10% formaldehyde for histological examination.

Cytokines such as IL-6, IL-1β, and VEGF were measured in plasma and tissue samples using enzyme-linked immunosorbent assay with a BIO-TEK ELX800 device. TAS and TOS levels were determined using colorimetric methods with a MINDRAY BS-400 device. Blood gas analyses were performed using a Radiometer ABL 800 FLEX blood gas analyzer (Radiometer, Copenhagen, Denmark). PCO₂ and PO₂ were measured based on the amperometric principle.

The right lung tissues were excised immediately before sacrifice, fixed in Bouin’s solution for 24 hours, and embedded in paraffin and labeled after standard tissue processing steps. Four-micron-thick tissue sections were obtained using a microtome, flattened in a water bath, and placed on slides. Hematoxylin and eosin (H&E) staining was used to examine lung tissue architecture, while Gomori trichrome staining was performed to identify collagen deposits. Gomori-stained sections were evaluated for the severity of interstitial fibrosis using the Ashcroft scale. Using this semi-quantitative grading system, pulmonary fibrosis was scored on a scale of 0–8, based on the following criteria: 0, normal lung; 1, minimal fibrous thickening of alveolar or bronchiolar walls; 2–3, moderate thickening without significant disruption of lung architecture; 4–5, marked fibrosis with fibrous bands or small fibrous masses disrupting the structure; 6–7, severe distortion with extensive fibrous areas; and 8, total fibrous obliteration. The average score for all sections represented the fibrosis score for that lung segment.

A total of 32 Wistar albino rats were used in the study. Two rats died shortly after the E. coli injection during the initial phase of the study and were excluded from further analysis. Blood and tissue samples were not collected from these rats, and they were excluded from statistical evaluations. The remaining 30 rats were divided into four groups: Group S, Group C, Group HBO, and Group OT.

Table 1 presents the comparisons of the IL-1β, IL-6, VEGF, TAS, TOS, OSI, PO₂, PCO₂, and lactate levels in serum samples obtained from the groups. The PO₂ level was significantly higher in Groups HBO and OT compared to the other groups (p < 0.001). The PCO₂ level was significantly lower, while the lactate level was significantly higher in Group HBO compared to the other groups (p < 0.001 and p = 0.005, respectively). TAS (p = 0.040) and TOS (p < 0.001) levels were significantly higher in Group S than in the other groups (p < 0.001). OSI values were also significantly higher in Group S (p < 0.001). When VEGF, a cytokine responsible for angiogenesis, was examined in serum, no significant increase was observed in Groups S, C, or HBO. However, VEGF levels were significantly elevated in Group OT (p = 0.003).

Upon examining VEGF levels in lung tissue, Group OT was found to have significantly higher levels than the other groups (p < 0.001). The histopathological Ashcroft score in Group HBO was significantly higher compared to the other groups (p < 0.001) (Table 2).

The relationships between the Ashcroft score of the groups and the serum levels of IL-1β, IL-6, VEGF, TAS, TOS, OSI, PO₂, PCO₂, and lactate are summarized in Table 3. A statistically significant, positive, and excellent correlation was found between the Ashcroft grade of Group OT and serum VEGF levels (r = 0.770, p = 0.025).

The relationships between the Ashcroft score of the groups and the lung levels of IL-1β, IL-6, VEGF, TAS, TOS, and OSI are presented in Table 4. A strong negative correlation was identified between the Ashcroft score of Group C and lung TAS levels (r = -0.733, p = 0.039).

Studies conducted on ARDS have shown that applying the significant hypotheses developed regarding the treatment methods required for repair mechanisms in humans is highly challenging. The variability of clinical conditions in humans and the experimental nature of these treatments make their application difficult [7, 11]. As a result, animal models play a crucial role. There are various models in which animal lungs are damaged directly, indirectly, or through a combination of these mechanisms [11]. The experimental models involve the intratracheal or intranasal administration of bacterial agents (e.g., live bacteria) and bacterial products (e.g., LPS) or the gastric aspiration of strong acids (e.g., HCl) to directly induce lung injury [11].

In a study published by Kim et al., the intratracheal administration of live E. coli was used as a method to induce ARDS. This approach demonstrated that acute lung injury closely resembled the human condition, characterized by alveolar hemorrhage, leukocyte infiltration, and increased alveolar wall thickness, achieving a 30% greater similarity to human ARDS [12]. In our study, to achieve results closest to human pathophysiology, live E. coli was administered to rats to induce lung injury. The significantly lower PO₂ levels observed in the blood gas analyses of rats in the untreated Group S, compared to those receiving HBO or ozone therapy, suggest successful lung injury, evidenced by disrupted alveolar structure, increased edema, and fibrosis tissue. The levels of oxidative and antioxidant stress markers, namely TAS, TOS, and OSI, were significantly elevated in Group S. In addition, although the levels of pro-inflammatory cytokines IL-1β and IL-6 were not statistically significant, they were numerically higher compared to other groups. These findings indicate the presence of sepsis-related inflammation and impaired oxygenation in Group S, while HBO and ozone therapy improved oxygenation. Ozone therapy demonstrated a higher anti-inflammatory efficacy compared to HBO, and most importantly, the VEGF-mediated angiogenesis induced by ozone therapy helped reduce tissue perfusion imbalance.

Perng et al. applied HBO therapy at 3 ATA in rats with ARDS induced by LPS and observed improved hypoxemia in the HBO group, while the untreated group exhibited persistent hypoxemia [13]. Bai et al. similarly reported improved PO₂ levels following HBO therapy for LPS-induced acute lung injury [14]. Furthermore, Peng et al. found that HBO administration in rats led to transient hyperoxia followed by a hypoxemia phase [10]. In our study, consistent with the literature, oxygenation improved in Group HBO. However, unlike the above-mentioned studies, we observed higher lactate levels in this group.

A meta-analysis by Smit et al., evaluating 60 animal experiments, reported that hyperoxia caused systemic vasoconstriction, more pronounced in the intestines and skin, and could lead to not only pulmonary side effects but also hemodynamic changes [15]. In the current study, despite the high PO₂ levels observed in the blood gas analyses of Group HBO, the elevated lactate levels suggest that hyperoxia-induced vasoconstriction may have disrupted tissue perfusion balance.

In a study by Yamanel et al., sepsis was induced in rats via E. coli, and HBO therapy at 2.8 ATA was applied twice daily. HBO therapy resulted in lower levels of hemorrhage, leukocyte infiltration, and alveolar septal thickening compared to other groups [9]. In another study, Yılmaz et al. applied HBO therapy at 2.5 ATA to rats with acute lung injury induced by intraperitoneal sepsis. Histopathological examination showed that hemorrhage, fibrosis, and lung collapse were most prominent in the HBO group [16]. In light of these findings, in our study, we applied HBO therapy at 2.5 ATA, which is well-tolerated by rats, and our histopathological findings corroborated the literature. We observed increased alveolar septal thickening, edema, and leukocyte infiltration in Group HBO, along with fibrosis prominently highlighted by different staining techniques. This underscores the importance of lung-protective ventilation strategies.

Although ozone is a potent oxidant, it does not exacerbate tissue inflammation. On the contrary, ozone acts as an effective modulator of biological oxidative stress [17]. In a study by Yamanel et al., histopathological examination revealed that ozone therapy in rats with ARDS resulted in reduced levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha and IL-1β, as well as significant reductions in lung injury scores [9]. Similarly, Yılmaz et al. found significantly higher levels of IL-10, an anti-inflammatory parameter, in ozone-treated rats with ARDS. Consistent with these findings, in our study, although statistical differences in IL-6 and IL-1β levels were not observed in the group treated with ozone therapy, the levels were numerically lower compared to the untreated control group. We can consider that this reduction in inflammation compared to the control group may be due to the anti-inflammatory effect of ozone therapy.

In a study conducted by Yılmaz et al., when examining blood gas parameters, the highest PO₂ and the lowest PCO₂ levels were found in the group treated with ozone therapy. Histopathologically, the least damage was observed in the ozone-treated group [16]. In our study, Group OT exhibited significantly higher PO₂ values compared to the other groups. Consequently, lactate levels were lower in Group OT than in Group HBO. This finding supports our hypothesis that ozone therapy reduces alveolar damage and, therefore, tissue perfusion imbalance. These results suggest that ozone therapy is more effective than other treatments in improving blood gas parameters and that alveolar damage is less pronounced in ozone-treated subjects. Furthermore, ozone increases the production of 2,3-DPG (2,3-diphosphoglycerate) in erythrocytes, shifting the oxyhemoglobin dissociation curve to the left and enhancing oxygen delivery to tissues [4]. The lower lactate levels in Group OT, compared to Group HBO, indicate better perfusion. This effect was demonstrated histopathologically, as Group OT showed less fibrosis, edema, and cellular density in the tissues compared to Group HBO.

Numerous clinical studies in the literature have demonstrated the antioxidant and anti-inflammatory effects of ozone therapy [18–20]. However, Yılmaz et al. did not find significant differences in antioxidant levels in ozone-treated rats with lung injury [10]. In contrast, Yamanel et al. reported significantly higher superoxide dismutase and glutathione peroxidase activities, which are antioxidant enzymes [9]. In our study, the lack of significant differences in TAS, TOS, and OSI levels in Group OT suggests that the ozone therapy applied did not result in pronounced antioxidant effects. This highlights the importance of the ozone dose used. Ozone doses below 10 µg/mL per mL of blood are considered to be biologically ineffective as they are almost completely neutralized by plasma antioxidants [6]. Our study indicates that higher concentrations of ozone may be required to achieve antioxidant effects.

Endothelial damage is a significant prognostic factor in ARDS, and VEGF is one of the markers of endothelial damage. Studies have shown that VEGF production in lung tissue decreases in ARDS [21, 22]. In the study using a bacterial-induced lung injury model in rats to investigate VEGF expression in the lungs, Maitre et al. found a decrease in both VEGF protein and VEGF mRNA levels in the ARDS-induced group. In addition, they observed reduced VEGF protein levels in the bronchoalveolar lavage fluid [22]. Similarly, Koyama et al. demonstrated reduced VEGF levels in bronchoalveolar lavage fluid in individuals with chronic lung damage, pulmonary sarcoidosis, or smoking-related lung injury [23]. In the current study, VEGF levels in lung tissue were found to be lower in the untreated Group S compared to the other groups. The reduction in VEGF levels in lung tissue is associated with decreased lung perfusion in ARDS, supporting findings from the literature [21, 22, 24]. Although there are no prior studies on the effects of ozone therapy on VEGF levels in animal models of lung injury, our study found a statistically significant increase in VEGF levels in Group OT compared to other groups. This suggests that tissue perfusion imbalance was less pronounced in Group OT compared to Group HBO. We also observed increased VEGF levels in both blood and lung tissue in Group OT. We hypothesize that ozone therapy in ARDS contributes to lung recovery by reducing lung injury via VEGF-mediated angiogenesis and its fibrosis-reducing effects.

Our review of the literature revealed no previous studies investigating the effects of HBO and ozone therapy on VEGF levels in lung injury. Therefore, our study represents a novel contribution to this field.

ARDS lacks a specific treatment method and is managed through supportive care. HBO and ozone therapies have the potential to mitigate lung injury in ARDS by reducing inflammation and oxidative stress. Ozone therapy appears particularly noteworthy for its ability to improve oxygenation and reduce lung injury by enhancing angiogenesis. The development and implementation of such innovative treatment methods offer promise for reducing ARDS-related mortality rates and improving patient outcomes. Future clinical studies will undoubtedly shed light on the applicability of these treatment methods and provide guidance on the feasibility of their implementation.