Protective effects of hyperbaric oxygen and iloprost on ischemia/reperfusion-induced lung injury in a rabbit model

Forty New Zealand white male rabbits, weighing 2.5 to 3 kg, were randomly allocated into one of five study groups. The experiment was continued until eight animals in each group survived during the entire procedure and during the postoperative 4 h. The HBO group received a single session of HBO treatment the IL group received 25 ng/kg/min infusion of IL; the HBO + IL group received both HBO and IL; the control group received only 0.9% saline; and the fifth group was the sham group. HBO treatment and IL infusion were administrated during 60 min of ischemia and 60 min of reperfusion in the treatment groups.

Initial anesthesia was achieved with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg) without endotracheal intubation; then followed by 25 mg/kg fractionally in order to allow the animals to have spontaneous respiration. All animals received a similar volume of maintenance fluids (0.9% sodium chloride, 20 ml/h) for the whole procedure. An arterial catheter (20-gauge) was placed in an ear artery to monitor blood pressure (Petas KMA 800, Ankara, Turkey). Each animal received 150 U/kg heparin intravenously for anticoagulation. Animals were placed in the supine position. The infrarenal abdominal aorta was exposed through a midline laparotomy incision and a transperitoneal approach, with the abdominal contents reflected to the right. The aorta was exposed from the left renal artery down to the aortic bifurcation. In all animals, except the sham controls (n = 8), the aorta was cross-clamped just proximal to the aortic bifurcation using atraumatic arterial bulldog clamps (Vascu-statt, Scanlan International, St. Paul, MN, USA). The clamp was removed after 60 min and restoration of blood flow was verified visually. Four hours after the intervention, all animals were humanely killed by a lethal cardiac injection of pentobarbital (100 mg/kg), immediately after which a median sternotomy was used to extract lung tissues for histopathological examination by light microscopy and biochemical investigations by MDA assay. The left lungs were removed, fixed with 10% tamponated formalin and stored for 24 hours. A sagittal section was obtained at the level of the hilum. These tissues were fixed again with 10% formalin for 2 days. Three animals died during the procedure, one in the HBO group during anesthetic induction, one in the IL group due to surgical intervention, and one in the sham group due to hemorrhage. They were excluded from the study.

Animals received HBO therapy in an animal monoplace chamber during 60 min of ischemia and 60 min of reperfusion period at 2.5 atmospheric pressure. Before pressurization, 100% medical oxygen was flushed through the chamber for 10 min to displace ambient air. The oxygen pressure was then increased slowly and reached 2.5 ATA in 5 min. The chamber was ventilated during HBO therapy to avoid carbon dioxide (CO₂) accumulation. Oxygen content was monitored continuously and maintained ≥98.5% (Nellcor:Draeger, Hemel Hempstead, Hertfordshire, UK). The concentration of CO₂ was not allowed to rise above 0.1%. An environmental control system maintained the inner temperature and relative humidity at 25 ± 1 °C and 50 ± 20%, respectively. After 120 min at 2.5 ATA, the chamber was decompressed to normal atmospheric pressure in 5 min.

Blood pH, pO₂ (mmHg), pCO₂ (mmHg), bicarbonate (HCO₃; mmol/L), sodium (Na⁺), potassium (K⁺), creatine kinase (CPK; IU/L), lactate dehydrogenase (LDH; IU/L) and TNF-α (pg/mL) values were determined 1 h after the onset of ischemia and 1 h after the onset of reperfusion. Blood pH, pO₂, pCO₂, HCO₃, Na⁺ and K⁺ values were determined using a blood gas analyzer (Ciba Corning Blood Gas Analyzer Model 860, Ciba Corning Diagnostics Corp., Irvine, CA, USA). Plasma CPK and LDH levels were measured in lithium heparinized plasma using automated enzyme reactions (automated analysis for Hitachi System 717, Boehringer Mannheim Diagnostica, Mannheim, Germany).

TNF-α assays were performed according to the method described by Pizarro et al. using the quantitative sandwich enzyme immunoassay technique (Quantikine M, R&D Systems, Minneapolis, MN, USA) [13]. Lipid peroxidation in the lung tissue was evaluated by measuring the level of MDA, which is the end product of lipid peroxidation. MDA was measured in plasma and the lung as an indicator of free radicals. Plasma and tissue (lung) MDA levels were determined with spectrophotometry using thiobarbituric acid-reactive substances [14].

After standard tissue preparation, 5 μm tissue sections were obtained. The specimens were stained with hematoxylin-eosin and examined with light microscopy (Eclipse E200; Nikon, Tokyo, Japan). All histopathological changes were detailed in each lung tissue, including inflammatory cell infiltration, alveolar edema, congestion and preservation of the alveolar septum. At least two different sections were explored in each specimen. The same pathologist, who was blinded to the study, assigned a score of 0 to 4 on the basis of congestion, interstitial edema, polymorphonuclear leukocyte infiltration and alveolar hemorrhage as follows: 0, normal histological appearance; 1, mild and focal changes; 2, moderate and multifocal changes; 3, marked and multifocal changes; 4, marked and diffuse changes.

Data were analyzed using the Statistical Package for Social Sciences (SPSS) software (version 10.0 for Windows). All differences associated with a chance probability of 0.05 or less were considered statistically significant. All data are presented as mean ± SD. Statistical analysis was performed using analysis of variance (ANOVA) test. One-way ANOVA test was followed by post-hoc Dunnett's test.