Genetic Ablation of Nrf2 Enhances Susceptibility to Acute Lung Injury After Traumatic Brain Injury in Mice

Mouse Model of TBI.

All procedures were approved by the Institutional Animal Care Committee and were in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Breeding pairs of Nrf2-deficient ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Homozygous wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were generated from inbred heterozygous Nrf2 (+/−) mice (17). Genotypes of Nrf2 (−/−) and Nrf2 (+/+) mice were confirmed by polymerase chain reaction (PCR) amplification of genomic DNA isolated from the blood. PCR amplification was carried out by using three different primers: 5′-TGGACGGGACTATTGAAGGCTG-3′ (sense for both genotypes), 5′-CGCCTTTTCAGTAGATGGAGG-3′ (antisense for wild-type) and 5′-GCGGATTGACCGTAATGGGATAGG-3′ (antisense for LacZ). Age- and weight-matched adult male mice (6–8 weeks, 28–32 g) were separated into four groups: I, sham + wild-type (Nrf2 +/+); II, TBI + wild-type (Nrf2 +/+); III, sham + deficient (Nrf2 −/−); IV, TBI + deficient (Nrf2 −/−).

The mouse model of TBI was employed as described (18) with recent minor modification (19). Mice were anesthetized by intraperitoneal injection with sodium pentobarbital (50 mg/kg). A round, flat and 6 mm diameter Teflon impounder was centered between the ears and eyes. TBI was induced by a 100 g weight dropped from a 12 cm height along a stainless steel string, which translated into 1200 g/cm. Brain injury-induced apnea was then treated for 3 mins with 100% oxygen administration and chest compression to stimulate the respiration (20). This model is generally associated with 20% of mortality within the first 5 mins post-injury and no delayed mortality was observed thereafter. Sham mice were subjected to identical treatment with the exception that no injury was performed. After operation procedures, the mice were returned to their cages. Heart rate, arterial blood pressure and rectal temperature were monitored, and the rectal temperature was kept at 37 ± 0.5°C (physical cooling if required) throughout the experimental and recovery periods.

At 24 h following sham or injury, mice were sacrificed for sample collection. For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, mice (n =6 per group) were transcardially perfused with cold saline (4°C) followed by 4% neutral-buffered formalin. The lungs were harvested, stored overnight in 4% neutral-buffered formalin, and embedded in paraffin. For wet/dry weight ratio assay, RNA reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and enzyme activity assay, mice (n =6 per group) were exsanguinated by cardiac puncture. The lungs were excised and the wet/dry weight ratio was immediately determined. Specimens of lung tissue were stored in liquid nitrogen.

Capillary Permeability in Lung Tissue.

PCP was determined by Evans blue (EB) extravasation method as previously described (21). Evans blue (50 mg/kg; Sigma Chemical Co., St. Louis, MO) in 250 μl of 0.9% saline was injected intravenously via the left internal vein at 30 mins before sacrifice. The mice were exsanguinated by cardiac puncture. The lung tissue was excised and weighed. To each sample, 1.5 ml formamide was added and incubated at 37°C for 24 h. After filtration with glass filter, the absorbance of the filtrate was measured at 620 nm using a Beckman spectrophotometer. The total amount of dye can be calculated by means of a standard calibration curve. The capillary permeability in lung tissue was shown as the micrograms of Evans blue in every milligram of tissue (n =6 per group).

Wet/Dry Weight Ratio in Lung Tissue.

Pulmonary edema was determined using the wet/dry method as previously described (22). The lung tissue samples were weighed before and after drying in a desic oven for 72 h at 80°C.

TUNEL Staining and Quantitation of Apoptotic Cells.

The formalin-fixed, paraffin-embedded sections (4 μm) were detected for apoptotic cells by the TUNEL method. The procedures were according to the kit instructions (ISCDD, Boehringer Mannheim, Germany). Microscopy of the stained tissue sections was performed by a pathologist blinded to the experimental condition. The extent of lung damage was evaluated by the apoptotic index (AI) which was the percent of average number of TUNEL-positive cells in each section counted in 10 microscopic fields.

RNA Extraction and RT-PCR.

The pulmonary mRNA expression levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and antioxidant/detoxifying enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase α1 (GST-α1), were determined by RT-PCR. Total RNA was extracted with TriPure Reagent (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the manufacturer’s instructions. The cDNA synthesis from the isolated RNA was performed using a reverse transcriptional system. Briefly, 5 μg of total lung RNA was reversely transcribed using 0.5 μg oligo(dT)15 U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (Promega, Madison, WI, USA). The cDNA was then amplified by PCR using various primer sets: TNF-α, 5′-ACGGCATGGATCTCAAAGAC-3′ and 5′-GGTCACTGTCCCAGCATCTT-3′; IL-1β, 5′-GAGTGTGGATCCCAAGCAAT-3′ and 5′-CTCAGTG-CAGGCTATGACCA-3′; IL-6, 5′-AGTTGCCTTCTTGG-GACTGA-3′ and 5′-GCCACTCCTTCTGTGACTCC-3′; NQO1, 5′-CATTCTGAAAGGCTGGTTTGA-3′ and 5′-CTAGCTTTGATCTGGTTGTCAG-3′; GST-α1, 5′-AAGCCAGGACTCTCACTA-3 ′ and 5 ′-CTGCTGATTCTGCTCTTG-3′; β-actin, 5′-AGTGTGACGTTGACATCCGTA-3′ and 5′-GCCAGAGCAGTAATCTCCTTCT-3′. PCR was started within 5 mins incubation at 94°C followed by a three-step temperature cycle: denaturation at 94°C for 30 s, annealing at 53–55°C for 50 s, and extension at 72°C for 1 min for 25 to ~30 cycles. A final extension step at 72°C for 8 mins was included after the final cycle to complete polymerization. PCR products were detected by agarose gel electrophoresis in 1.5% NuSieve agarose gels (FMC, USA) and visualized by ethidium bromide staining. The intensity of the bands was quantified using Glyko Bandscan software, and the ratios of each gene product to β-actin product were used as indices of inflammatory cytokines and antioxidant/detoxifying enzymes mRNA expression.

ELISA.

The pulmonary protein expression levels of inflammatory cytokines were quantified using ELISA kits specific for mouse cytokines according to the manufacturers’ instructions (TNF-α from Diaclone Research, France; IL-1β, IL-6 from Biosource Europe SA, Belgium). Values were expressed as pg/mg protein.

Enzyme Activity Assay for Lung Tissue.

Lung tissue was dissected and homogenized in ice-cold 10 mM Tris-HCl (pH 7.8). The homogenates were centrifuged at 12,000 × g for 15 mins at 4°C. Protein concentration of the resulting supernatant was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL). NQO1 enzyme activity was determined by calculating the dicumarol-sensitive fraction of 2,6-dichlorophenol-indo-phenol reduction (23). Reactions consisting of 30 μg protein, 25 mM Tris-HCl (pH 7.4), 0.7 mg/ml crystalline bovine serum albumin, 5 μM FAD, 0.2 mM NADH, and 0 or 20 μM dicoumarol were preincubated for 10 mins at 25°C (final concentrations in 200 μl of reaction volume). To initiate the reaction, 40 μM of 2,6-dichlorophenol-indo-phenol was added, and the initial velocity of the reduction of dichlorophenol-indo-phenol was measured spectrophotometrically at 540 nm. The extinction coefficient for 2,6-dichlorophenol-indo-phenol was 2.1 × 10⁴ M/cm. The total GST enzyme activity assay consisted of 100 μg protein, 1 mM 1-chloro-2,4-dinitrobenzene, and 1 mM glutathione at 37°C in 100 mM potassium phosphate buffer (pH 6.5) (final concentrations in 150 μl of reaction volume) (24). The reaction was monitored at 340 nm and the non-enzymatic slope was subtracted from the total observed slope. The extinction coefficient for 1-chloro-2, 4-dinitrobenzene was 9600 M/cm. All values were expressed as nmol/min/mg protein.

Statistical Analysis.

Software SPSS 13.0 was used for the statistical analysis. All data were expressed as mean ± standard error (SE). Student’s t test was used to analyze the differences between the sham and TBI groups within a single genotype as well as between genotypes. Statistical significance was accepted at P < 0.05.

Pulmonary Capillary Permeability.

As shown in Figure 1, the PCP level in both sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice was similar. An increased level of PCP was detected in both Nrf2 (+/+) and Nrf2 (−/−) mice at 24 h after TBI. However, the pathological change was more severe in Nrf2 (−/−) mice than in Nrf2 (+/+) mice.