Experimental Animals
This experimental study was conducted at the Gazi University Laboratory Animal Breeding and Experimental Research Center (GÜDAM) in accordance with ARRIVE guidelines. The study protocol was approved by the local ethics committee of the Gazi University Animal Experiments (G.Ü.ET-20.042), Ankara, Turkey. All the animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
In the study, 28 male Wistar albino male rats weighing between 275 and 325 gr were used. The rats were kept at a temperature of 20–21°C, with 12 h of night and 12 h of daytime, and fed until 2 h before anesthesia.
Animal Groups and Study Design
Twenty eight rats were randomly assigned to four groups: control (Group C), boric acid (Group BA), cecal ligation and puncture (Group CLP), and boric acid administered one hour after CLP (Group CLP + BA) (Table 1).
Table 1
Animal groups and study design.
Group C | Sham (Laparotomy only) | n = 6 |
|---|
Group BA | Sham + BA (Laparotomy and boric acid administration) | n = 6 |
Group CLP | Cecal ligation and perforation | n = 8 |
Group CLP + BA | Cecal ligation and perforation + boric acid (1 h after the sepsis model) | n = 8 |
Anesthesia and Surgical Protocol
Anesthesia was induced with an intramuscular injection of 50 mg/kg of ketamine hy-drochloride (Ketalar®, Pfizer, Istanbul, Turkey) combined with 10 mg/kg of xylazine hy-drochloride (Alfazyne® 2% vial, Ege Vet, Kemalpaşa/İzmir, Turkey). All surgical interventions were conducted under aseptic conditions, with the rats undergoing aseptic skin preparation and being positioned supine under a heating lamp.
Group C
Anesthesia was administered, followed by a laparotomy. The cecum was isolated without any perforation, puncture, or ligation. The abdominal cavity was then surgically closed
Group BA
Following anesthesia, a laparotomy was performed. Similar to the control group, the cecum was isolated without perforation, puncture, or ligation. The abdomen was closed, and boric acid 500 mg/kg was administered via intraperitoneal injection
Group CLP
After anesthesia, a laparotomy was carried out, the cecum was distended with stool, ligated with 3/0 silk just below the ileocecal valve, and punctured twice on the anterior surface using an 18-gauge Intraket needle. The abdomen was subsequently surgically closed.
Group CLP + BA
Anesthesia was induced, and CLP was performed as previously described. The abdomen was surgically closed, followed by the administration of 500 mg/kg via intraperitoneal injection one hour later.
Euthanasia and Tissue Removal
All the rats were sacrificed 24 h after the operation; rats were anesthetized with ketamine (50 mg/kg) and xylazine (10 µg/kg) and sacrificed by collecting blood (5–10 ml) from the intracardiac. After heartbeat and respiration ceased, rats were monitored for a further 2 min to confirm death. Lung and renal samples were stored at -80˚C for biochemical analysis and immersed in 10% neutral buffered formalin for histopathological assessment.
Histopathological Evaluation of Kidney and Lung Tissues
Rat lung and kidney tissue samples were embedded in paraffin following routine tissue processing. Tissue sections of 5 µm thickness were cut from paraffin blocks using a microtome (Leica RM2245, Germany) and stained with hematoxylin and eosin (H&E) to analyze histopathological changes. H&E-stained tissue sections were examined under a computer-assisted light microscope (Leica DM 4000B, Germany) and micrographs were captured using the Leica LAS V4.9 software.
H&E-stained kidney sections were observed under 400× magnification and kidney injury were evaluated considering the pathological changes including interstitial edema, peritubular capillary dilatation, vacuolization, ablation of tubular epithelial cells from basement membrane, loss of brush border in proximal tubular epithelium, cellular swelling and nuclear defragmentation. Each criterion was scored between 0 and 3 (0 = none, 1 = mild, 2 = moderate and 3 = severe changes) in specimen from all animals [8].
H&E-stained lung specimens were examined under 200× and 400× magnification and lung injury were assessed in respect to pathological changes involving alveolar wall thickening, capillary congestion, intraalveolar hemorrhage, interstitial neutrophil infiltration and intraalveolar neutrophil infiltration. Each parameter was scored between 0 and 3 (0 = none, 1 = mild, 2 = moderate and 3 = severe changes) in lung specimen from each animal [9].
Biochemical Assessment
The lung and kidney tissue was rapidly frozen in liquid nitrogen and subsequently stored at -80°C until biochemical analyses were performed. The tissue was evaluated for Total Antioxidant Status (TAS), Total Oxidant Status (TOS), Oxidative Stress Index (OSI), and Paraoxonase-1 (PON-1) activity. To prevent thawing and preserve tissue integrity, all sample processing was conducted swiftly. Approximately 80–100 mg of lung and kidney tissue was dissected using sterile lancets, weighed, and pulverized in liquid nitrogen to create a fine powder. The powdered tissue was then transferred into homogenization tubes and diluted at a 1:10 (w/v) ratio with a 140 mM potassium chloride (KCl) solution. Homogenization was performed at 50 rpm for 2 minutes using a homogenizer, with the tubes placed in an ice-filled beaker to maintain low temperatures. The homogenates were centrifuged at 3000 rpm for 10 minutes, and the supernatants were carefully collected for analysis. Total Antioxidant Status (TAS) was measured using a commercially available kit (RelAssay Diagnostics). A 30 µL aliquot of the sample was mixed with 500 µL of measurement buffer, and the absorbance was initially recorded at 660 nm. Following the addition of a chromogenic reagent and a 5-minute incubation at 37°C, a second absorbance measurement was taken. TAS values were expressed in **Trolox equivalents. Total Oxidant Status (TOS) was also determined using a RelAssay Diagnostics kit. A 75 µL sample was combined with the assay buffer, and absorbance was first measured at 530 nm. After adding a pro-chromogenic solution and incubating for 5 minutes at 37°C, a second absorbance reading was recorded. TOS results were expressed in hydrogen peroxide (H₂O₂) equivalents. All measurements were conducted in triplicate, and the average values were reported. The oxidative stress index (OSI) was calculated using the following formula: OSI (arbitrary units, AU) = (TOS (µmolH2O2equivalent/L)) / (TAS(µmolH2O2equivalent/L)) × 100. The assay sensitivities were 0.02 mmol/L for TAS and 0.05 mmol/L for TOS. The kits were validated in prior studies, which have been cited to ensure transparency. The results were expressed in mmol/L.
PON enzyme activity was determined spectrophotometrically. The enzyme assay was based on the prediction of p-nitrophenol at 412 nm. An enzyme unit was defined as the amount of enzyme that catalyzes the hydrolysis of 1 µmol of substrate at 25°C. PON activity was calculated and expressed as U/L.
Statistical Analysis
Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) 20.0 for Windows was used for all statistical analyses. Each categorical variable was analyzed using the Kolmogorov–Smirnov test. Biochemical and histopathological parameters were performed using one‑way ANOVA followed by Tukey's post hoc test. Statistical significance was set at p < 0.05. All values are expressed as mean ± standard deviation (SD) or standard error of mean (SEM).