Oxidative stress and NF-KB/iNOS inflammatory pathway as innovative biomarkers for diagnosis of drowning and differentiating it from postmortem submersion in both fresh and saltwater in rats

Animals

Forty-five albino rats (adult male, 250–300 g and 12–16 weeks old) were acquired from the experimental animals growing centre at Minia University. The rats were acclimated for two weeks in a well-ventilated laboratory with regulated temperature (22 ± 2 ºC) and lighting (12-h light/dark cycle) in standard cages provided with ad libitum standard diet feeding and tap water drinking.

Ethical approval

The experiment was done in compliance with the ethical committee's recommendations and guidelines for animal use and care at Minia University, approval No. 814:6:2023.

Water sample collection

We collected the experiment's freshwater from the Nile River near Minia Governorate in Egypt (salinity of 0.5 per cent) [12] and the experiment's saltwater from the Red Sea near Hurghada Town (salinity of 40.6 per cent) [13].

Experimental design

We randomly allocated the rats into five groups, nine rats per group: control group (C); Freshwater-drowned group (FD); Freshwater postmortem submersion group (FPS); saltwater-drowned group (SD); saltwater postmortem submersion group (SPS).

Control group (C)

Rats were anaesthetised with 40 mg/kg body weight Na thiopental injected intraperitoneally, and then the sacrifice was made by cervical dislocation.

Freshwater-drowned group (FD) and saltwater-drowned group (SD)

To imitate actual drowning, rats were injected intraperitoneally with Na thiopental (40 mg/kg body weight), and then the cage was submerged for one minute in a basin of water (50 × 150 × 50 cm) filled with 50 L of the collected freshwater and saltwater, respectively, with breathing allowed for another minute, this process was repeated until death.

Freshwater postmortem submersion group (FPS) and saltwater postmortem submersion group (SPS)

Rats were anaesthetised with 40 mg/kg body weight Na thiopental injected intraperitoneal, euthanised by cervical dislocation, and then dipped in fresh and saltwater, respectively.

After the autopsy, the rats' lungs in each group were dissected; the left lungs were prepared for histological and immunohistochemical inspection, while the right lungs were prepared for other biochemical studies.

Biochemical analysisSample collection

Tissue homogenates: the tissues were chopped into small pieces and rinsed in ice-cold PBS (0.01 M, pH = 7.4) to completely remove blood. After weighing the tissue pieces, a glass homogenizer on ice was used to homogenize them in PBS (tissue weight (g): PBS (mL) volume = 1:9). Sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze–thaw cycles to break down the cells further. The homogenates were then centrifuged for 5 min at 5000 × g to obtain the supernatant.

Measurement of lung oxidative stress/antioxidant biomarkers

Biochemical estimation of lung lipid peroxides done with the use of thiobarbituric acid by reacting with malondialdehyde (MDA), resulting in a pink product whose absorbance can be determined at 534 nm [14].

The Griess reaction, in which nitrite reacts with a solution containing naphthyl ethylenediamine and sulfanilamide to produce a brilliant reddish-purple hue measured at 540 nm, was used to calculate the total nitrite/nitrate (NOx) content of the lung tissue [15].

The Nishikimi and Yogi [16] approach was utilised to determine the superoxide dismutase enzyme (SOD) concentration. The basis of this test is that SOD can inhibit nitroblue tetrazolium dye reduction by phenazine methosulfate, and the enzyme activity was determined using a spectrophotometer at 560 nm.

Reducing 5'-dithiobis-2-nitrobenzoic acid to a yellow hue using Beutler's [17] technique is a chemical approach for measuring the quantity of reduced glutathione (GSH) in lung tissue, the absorbance of a reduced chromogen at 405 nm shows a direct correlation with the concentration of GSH.

Assessment of VCAM-1, COX-2 levels in the lung using ELISA assay

VCAM-1 and COX-2 levels in the lung were tested using ELISA kits according to the producer's instructions. Catalog No; E-EL-R106196T and CSB-E13399r, respectively.

The quantitative sandwich enzyme immunoassay method was used in this assay. COX-2 and VCAM-1 specific antibodies have been pre-coated onto a microplate. Once the standards and samples were pipetted into the wells, any marker present was bound by the immobilized antibody. Any unbound substances were removed, and then a biotin-conjugated antibody specific to each marker was added to the wells. After washing, avidin-conjugated Horseradish Peroxidase (HRP) was added to the wells. The wash was repeated to remove any unbound avidin-enzyme reagent. After that, a substrate solution was added to the wells, and colour developed in proportion to the amount of each marker bound in the initial step. The intensity of the colour was measured after the colour development was stopped. The results were calculated by the professional software "Curve Expert" to make a standard curve.

Relative measurement of NF-kB and iNOS gene expression using (RT-PCR)

TRI REAGENT solution (Ambion, Warrington, UK, Cat no: TR 118) was used to extract total RNA from 100 μg of rat lungs. The following primers were employed in a thermal cycler (Applied Biosystems GeneAmp® 5700 fast, Cambridge LTD., UK) with 5 μg of total RNA according to the kit's instructions (Promega GoTaq® 1-Step RT-qPCR syber green with ROX Vial Cat no: 53711–5399). The Nanodrop was used to ascertain the RNA's concentration and purity.

After an initial denaturation at 95 °C for 10 min, PCR amplification was carried out using a cycling protocol consisting of 40 cycles (denaturation at 95 °C for 10 s, annealing and extension at 72 °C for 30 s). The levels of gene expression in each sample were compared to those in a control group. The comparative threshold cycle approach (Ct) was used to determine the relative expression levels of the NF-kB and iNOS genes [18]. The GADPH gene was utilised as the reference point for all values.

The primers used were:

NF-KB gene; F: 5′- GAG AAG AAC AAG AAA TCC TAC CCA C, 3′

R: 5′- TCC ATT TGT GAC CAA CTG AAC G -3′

iNOS gene; F: 5′-CAC CAC CCT CCT TGT TCA AC,3′

R: 5′-CAA TCC ACA ACT CGC TCC AA-3′.

GADPH gene; F: 5′- GTA TTG GGC GCC TGG TCA CC -3′,

R: 5′- CGC TCC TGG AAG ATG GTG ATG G -3.

Histopathological examinationLight microscopic study (H&E)

Left lung lobes tissue samples were processed into paraffin blocks straight after being fixed in a 10% buffered formalin solution. Hematoxylin and eosin (H&E) stains were used on serial slices of tissue (5–7 µm) [19]. Two blinded histologists evaluated the histological and immunohistochemical slides for the experimental groups.

To determine and score the intensity of the lung injury. Thickened interalveolar septum, vascular congestion, and infiltration of inflammatory cells were used to compare the histopathological alterations among the experimental groups. Damage severity was measured and categorised as follows: Scores of—were deemed normal, + mild, + + moderate, and + + + severe [20].

Immunohistochemistry

Anti-caspase 3 (CAT#ab32351) and Anti-tumour Necrosis Factor Alpha (TNF-α) (CAT#ab220210) were purchased from Abcam, Egypt. Anti-cyclooxygenase 2 (COX 2) (CAT#bs-10411R-TR; Bioss; Egypt), Anti-iNOS (CAT#GB11119; Servicebio, Egypt) and anti-nuclear factor kappa B (NF-κB) (CAT#bs-20159R-TR; Bioss; Egypt) antibodies were also used. All antibodies were utilized in immunohistochemistry experiments as directed by the respective manufacturers.

Sections were deparaffinised, rehydrated, and then subjected to a 20-min antigen retrieval in EDTA buffer in the microwave. Sections were immersed in 0.01 per cent hydrogen peroxide for 10–15 min to inhibit endogenous peroxidase activity. Anti-caspase 3 (1:200) and anti-TNF-α (1:100) polyclonal primary antibodies were then used to incubate sections at 4 °C overnight and, after that, incubation for 30 min with the secondary antibody. The sections were then cleaned and stained with DAB solution for 10 min after being incubated with horseradish peroxidase Envision kit (DAKO) for 20 min. Hematoxylin counterstaining, dehydration, clearing, and mounting were done after sections had been washed in PBS).

Light microscopic images were taken by BX51-microscope and linked to the computer programmed with software LC—micro-application. The image j analysis—software assessed caspase 3, TNF alpha, COX 2 and iNOS immunoreactivity percentages [21] in randomly chosen nine non-overlapping fields at magnification × 400 from each animal in each group. Also number of NF-κB positive nuclei was calculated in randomly chosen nine non-overlapping fields at magnification × 400 from each animal in each group.

Statistical analysis

Graph Pad Prism was used for the statistical analysis of the data (version 8.01 for Windows, Graph Pad Software, San Diego, California, USA, www.graphpad.com). We calculated the mean and the standard error of the mean. Values were expressed using the mean and the standard error of the mean. Multiple comparisons were made using the one-way ANOVA, then the Tukey-Kramar post hoc test, and we used the student t-test to see whether there were statistically significant differences between each two groups; P-values less than 0.05 are considered statistically significant.

Sample size calculation

A sample size of 9 rats in each group was determined to provide 80% power for a one-way ANOVA test at the level of 0.05 significance using G Power 3.1 9.2 software.