A total of 62 male Sprague-Dawley (SD) rats (weight,270–300 g; age, 10 weeks) were obtained from the Experimental Animal Center of Jeonbuk National University (Iksan, Republic of Korea). They were housed at a temperature of 23±2°C and humidity of 60±10% under a 12-h light/dark cycle. They were supplied with free access to food and water. All experimental protocols were approved based on ethical procedures and scientific care by the Institutional Animal Care and Use Committee of Jeonbuk National University (approval no. JBNU2020–084).

Experimental animals were stratified in three categories [a sham operation group, CA under normothermia, and CA and hypothermia treatment (HT)] as follows: i) group I, a sham group (n=5) was maintained under normothermia conditions without CA; ii) group II, a normothermia group without hypothermia (33°C) treatment following CA (n=17); and iii) group III (n=40), a group that underwent CA under normothermia and were treated with HT after CA for 2 h (n=17), 4 h (n=13) and 6 h (n=10) following ROSC, where all rats were reheated to normothermia.

CA and CPR were performed as previously described (24,25) with minor modifications (Fig. 1). Briefly, the rats were anesthetized with2–3% isoflurane and mechanically ventilated to maintain respiration using a rodent ventilator (Harvard Apparatus). To monitor peripheral oxygen saturation (SpO₂), a pulse oximetry oxygen saturation probe (Nonin Medical, Inc.) was attached to the left foot. Body temperature was maintained at 37±0.5°C during and following the CA surgery. To monitor electrocardiogram (ECG) changes, electrocardiographic probes (Cytiva) were placed on the limbs to provide three-lead data, and which were monitored continuously. The left femoral artery and right femoral vein were separately cannulated to monitor the mean arterial pressure (MAP) (MLT 1050/D; ADInstruments, Ltd.) and intravenous injection.

Following a 5-min stabilization period, vecuronium bromide (2 mg/kg; Gensia Sicor Pharmaceuticals, Inc.) was intravenously administered, anesthesia was stopped and mechanical ventilation was withdrawn. A MAP below 25 mm-Hg and subsequent pulseless electric activity were used to define CA (25,26). CA was confirmed at3–4 min following vecuronium bromide injection. At 5 min following CA, CPR was initiated by intravenously administering a bolus injection of epinephrine (0.005 mg/kg; Sigma-Aldrich; Merck KGaA) and sodium bicarbonate (1 mEq/kg; Sigma-Aldrich; Merck KGaA) followed by mechanical ventilation with 100% oxygen and manual chest compressions at a rate of 300/min until MAP reached 60 mm-Hg and electrocardiographic activity was observed. Once the animal was hemodynamically stable and spontaneously breathing (usually at 1 h following ROSC), the catheters were removed and the animal was extubated.

The body temperature of the normothermia group was maintained at 37±0.5°C during and following the CA surgery and maintained until the rats were sacrificed according to the time schedule. In the hypothermia group, CA was established at normal temperature; then, the body temperature was maintained with ice packs and fans at 33±0.5°C immediately following CPR for 2, 4 and 6 h, and they were rewarmed rapidly with the heating pad until the desired temperature (37±0.5°C) was achieved. The rats were then returned to their cages until they were sacrificed one day following CPR/ROSC. The body temperature was monitored using a rectal temperature sensor (27).

An intraperitoneal injection of 30 mg/kg pentobarbital sodium (JW Pharm Co., Ltd.) was used to anesthetize all animals. Blood was collected from the abdominal veins of each animal in each group. Serum was collected by blood centrifugation (2,774 × g, 15 min, 4°C) and was preserved at −80°C until analysis. The levels of blood urea nitrogen (BUN) and creatinine in the serum were determined according to methods outlined by the International Federation of Clinical Chemistry (28) using an automated chemical analyzer Hitachi 2070 (Hitachi, Ltd.). All assays were conducted in triplicate using fresh serum.

The rats were deeply anesthetized by an intraperitoneal injection of 200 mg/kg pentobarbital sodium (JW Pharm Co., Ltd.), and they were perfused transcardially with 0.1 M of phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in 0.1 M of phosphate buffer (PB; pH 7.4). Kidneys were isolated from each animal and fixed with 4% paraformaldehyde in 0.1 M PB (pH 7.4) at room temperature during the 1 week, then sliced sagittally, embedded in paraffin and sectioned (6 µm).

H&E staining was performed to examine pathological changes in the kidneys according to a previously described procedure (29). PAS staining was performed to examine changes in glomeruli according to previously described procedures (30–32). Masson's trichrome staining method was used for defining tubular injury, considering tubular dilatation, tubular atrophy, tubular cast formation, vacuolization, degeneration, interstitial fibrosis and sloughing of tubular epithelial cells, or thickening of the tubular basement membrane according to previously described procedures (33,34).

A total of 2 experienced pathologists evaluated histopathological changes in a double-blinded manner. Images of 10 stained sections/rat were captured at ×400 magnifications using a Leica DM 2500 light microscope (Leica Microsystems GmbH). A total of 10 fields were analyzed in each section. Histopathological analysis of renal lesions was performed according to previously described procedures (35,36). Briefly, lesions were categorized as no significant microscopic lesions (NSML), minimal, mild, moderate, or marked lesions, respectively, graded using the following scale with the blind test: normal, 0 points; <25% damage, 1 point;26–50% damage, 2 points;51–75% damage, 3 points; and76–100% damage, 4 points. Glomerular lesions were defined by loss of cellular elements, collapse of capillary lumen, amorphous hyaline material with or without adhesions to the Bowman's capsule (30–32) and scored by the following numeric scales: no damage, 0 points; very mild, 1 point; mild, 2 points; moderate, 3 points; and severe, 4 points. Tubular injury was scored by the following scoring system: no tubular injury, 0 points;1–9% of tubules injured, 1 point;10–25% of tubules injured, 2 points;26–50% of tubules injured, 3 points; 51–75% of tubules injured, 4 points; and at least 76% of tubules injured, 5 points (33,34).

MDA concentration in the renal cortex was evaluated according to a previously described protocol (23,37,38). In short, the homogenization and centrifugation of the renal tissues were performed at 8,832 × g for 10 min at 4°C, and the supernatant was collected and stored at −80°C for MDA analysis. MDA content was determined according to the instructions of TBARS assay kit (cat. no. 10009055; Cayman Chemical Company).

IHC was performed with SOD-1, SOD-2, CAT and GPX to study changes in antioxidant immunoreactivities in the kidney. IHC was carried out according to our previously described method (22). In brief, the sections (6 µm) were incubated with primary goat anti-SOD1 (1:500; cat. no. SAB2500976; Sigma-Aldrich; Merck KGaA), goat anti-SOD2 (1:1,000; cat. no. SAB2501676; Sigma-Aldrich; Merck KGaA), rabbit anti-CAT (1:1,000; cat. no. ab16731; Abcam) and rabbit anti-GPX (1:1,000; cat. no. ab22604; Abcam) overnight at 4°C, followed by the biotinylated-conjugated anti-rabbit (1:250; cat. no. BA-1000-1.5; Vector Laboratories, Inc.) and the biotinylated-conjugated anti-goat (1:250; cat. no. BA-5000-1.5; Vector Laboratories, Inc.) secondary antibodies for 2 h at 24°C and developed using Vectastain ABC (Vector Laboratories, Inc.). Then, they were visualized with 3,3′-diaminobenzidine solution (in 0.1 M Tris-HCl buffer).

Leica DM 2500 microscope was used to image the sections at a magnification of ×400. A total of 10 sections/rat were selected and 10 areas were captured. ImageJ threshold analysis software version 1.52a (National Institutes of Health) was used to measure the percent (%) of relative optical density (ROD).

All experiments were repeated in triplicate. Graph Pad Prism version 5.0 (GraphPad Software, Inc.) was used to analyze the data, which were expressed as the means ± standard error of the mean (SEM) values. Survival was analyzed using Kaplan-Meier statistics and the log-rank test. MAP and peripheral oxygen were compared using one- and two-way repeated-measures of analysis of variance to assess the effect of time. To determine the significance of differences, post hoc analyses were conducted using Tukey's test for all pairwise multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

There was no statistically significant difference among the groups regarding baseline characteristics, including body weight, MAP and SpO₂ (Table I and Fig. 2). The induction of CA occurred 3–4 min following the intravenous injection of vecuronium bromide (2 mg/kg). CA was confirmed with an isoelectric ECG, SpO₂ and MAP, and these changed as expected according to the experimental protocol (Fig. 2A-C). As revealed in Fig. 2D, the body temperature was different among all groups following ROSC.

As revealed in Fig. 3, the survival rate of each group was evaluated at one day post-CA. The rate of Group II was 42.9%. In group III, the rate following 2 h-HT was 42.9%, the rate following 4 h-HT was 57.1% and the rate following 6 h-HT was 71.4%. In this experiment, there was no difference between the rats in Group II and the rats with 2 h-HT in Group III.

As demonstrated in Fig. 4B, the serum BUN level in Group I was 13.8 mg/dl one day post-CA. In group II, the BUN level was significantly increased to 35.3 mg/dl. In Group III, the BUN level was 30.7 mg/dl following 2 h-HT, 25.0 mg/dl following 4 h-HT and 22.7 mg/dl following 6 h-HT. In addition, as revealed in Fig. 4C, the serum creatinine level in Group I was 0.23 mg/dl. In group II, the creatinine level was significantly increased to 0.43 mg/dl. In group III, the creatinine levels were lower than that observed in Group II as follows: 0.39 mg/dl following 2 h-HT, 0.37 mg/dl following 4 h-HT and 0.36 mg/dl following 6 h-HT.

In Group I (sham), intact histological structures were revealed by H&E, PAS and Masson's trichrome staining (Fig. 5A). The interstitial fibrosis was not detected in all groups, meanwhile in Group II, CA-induced renal histopathology was examined at one day following ROSC using H&E, PAS and Masson's trichrome staining. Severe CA-induced kidney injury was significantly increased in the proximal tubules and glomeruli; particularly, the brush borders of the renal tubular epithelial cells were seriously eroded (Fig. 5A and B). In addition, in this group, glomerular capillaries were dilated with inflammatory cells, and interstitial edema and acute renal tubular necrosis were serious as compared with those observed in Group I (Fig. 5A).

In Group III, kidney injury was attenuated at one day following ROSC (Fig. 5A and B). In particular, CA-induced injury in the proximal tubules was significantly decreased following 6 h-HT as compared with that observed in Group II. In addition, local expansion of the proximal tubules was decreased as compared with that revealed in Group II (Fig. 5A). For the glomeruli, 6 h-HT significantly attenuated glomerular injury as compared with that revealed in Group II (Fig. 5A and B).

As revealed in Fig. 4A, the level of MDA in the renal cortex was significantly increased at one day following CA in Group II compared with Group I. However, in Group III, the level of MDA was significantly decreased following 4 h- and 6 h-HT. It was also decreased in the 2 h-HT, but there was no statistically significant difference compared with Group II.

In Group I, normal SOD-1, SOD-2, GPX and CAT immunoreactivities were evaluated, revealing that they were principally located in the tubules (Fig. 6A). In Group II, SOD-1, SOD-2, GPX and CAT immunoreactivities were significantly reduced at one day following ROSC as compared with those revealed in Group I (Fig. 6A and B).

In Group III, SOD-1, SOD-2, GPX and CAT immunoreactivities following 2 h-HT were not significantly different from those observed in Group II (Fig. 6A and B). In the case of 4 h-HT, the four immunoreactivities were significantly higher than those revealed in Group II (Fig. 6A and B), demonstrating that, in particular, GPX immunoreactivity was significantly higher compared with the other immunoreactivities. In the case of 6 h-HT, all immunoreactivities were higher than those identified in the rats which received 4 h-HT, revealing that the ROD of each SOD-1, SOD-2, GPX and CAT immunoreactivity was 78.4, 67.4, 86.5 and 79.5%, respectively, as compared with Group I (Fig. 6A and B).