Nitrosative and oxidative stresses contribute to post-ischemic liver injury following severe hemorrhagic shock: the role of hypoxemic resuscitation

Abstract

Purpose: Hemorrhagic shock and resuscitation is frequently associated with liver ischemia-reperfusion injury. The aim of the study was to investigate whether hypoxemic resuscitation attenuates liver injury.

Methods: Anesthetized, mechanically ventilated New Zealand white rabbits were exsanguinated to a mean arterial pressure of 30 mmHg for 60 minutes. Resuscitation under normoxemia (Normox-Res group, n = 16, PaO(2) = 95-105 mmHg) or hypoxemia (Hypox-Res group, n = 15, PaO(2) = 35-40 mmHg) followed, modifying the FiO(2). Animals not subjected to shock constituted the sham group (n = 11, PaO(2) = 95-105 mmHg). Indices of the inflammatory, oxidative and nitrosative response were measured and histopathological and immunohistochemical studies of the liver were performed.

Results: Normox-Res group animals exhibited increased serum alanine aminotransferase, tumor necrosis factor--alpha, interleukin (IL) -1β and IL-6 levels compared with Hypox-Res and sham groups. Reactive oxygen species generation, malondialdehyde formation and myeloperoxidase activity were all elevated in Normox-Res rabbits compared with Hypox-Res and sham groups. Similarly, endothelial NO synthase and inducible NO synthase mRNA expression was up-regulated and nitrotyrosine immunostaining increased in animals resuscitated normoxemically, indicating a more intense nitrosative stress. Hypox-Res animals demonstrated a less prominent histopathologic injury which was similar to sham animals.

Conclusions: Hypoxemic resuscitation prevents liver reperfusion injury through attenuation of the inflammatory response and oxidative and nitrosative stresses.

Figure 1. Serum levels of alanine aminotransferase (ALT) denoting the degree of hepatocellular injury.

Box plots show the median (lines), interquartile ranges (boxes) and 5 and 95 percentiles (whiskers) of the three groups during the experiment. ⁺ p<0.05 Sham vs. Normox-Res; *p<0.05 Normox-Res vs. Hypox-Res.

Figure 2. Indices of oxidative stress.

Hepatic tissue levels of malondialdehyde (MDA, panel A), glutathione (GSH, panel B), the percentile ratio of reduced to total GSH (R/T, panel C) and the geometric mean fluorescence intensities of neutrophils and monocytes of peripheral blood which reflect reactive oxygen species (ROS) production (GMFI, panel D). Box plots showing the median (lines), interquartile ranges (boxes), and the 5 and 95 percentiles (whiskers) of the three groups at the end of the experiment. ⁺ p<0.05 Sham vs. Normox-Res; *p<0.05 Normox-Res vs. Hypox-Res.

Figure 3. Serum cytokines.

Serum levels of Tumor Necrosis Factor (TNF) – α, Interleukin (IL) - 1β and IL-6. Box plots showing the median (lines), interquartile ranges (boxes) and 5 and 95 percentiles (whiskers) of the three groups at the end of the experiment. ⁺ p<0.05 Sham vs. Normox-Res; *p<0.05 Normox-Res vs. Hypox-Res; # p<0.05 Sham vs. Hypox-Res group.

Figure 4. Histopathological and immunohistochemical morphological changes.

Slices for histology (left column) were stained with hematoxylin/eosin, while for nitrotyrosine (NT) immunohistochemical detection (right column) were stained with the chromogene 3,3 diaminobenzidine tetrahydrochloride. As shown, the central vein originated from a Normox-Res animal is completely destructed and the adjacent hepatocytes have been intensively stained for NT. In contrast, in the case of hypoxemic animals the respective region is well preserved with limited staining for NT, an appearance similar to that of sham animals. These differences are shown in the box plot in where the median (lines) histological and NT scores, interquartile ranges (boxes), and 5 and 95 percentiles (whiskers) of the three groups at the end of the experiment are presented. ⁺ p<0.05 Sham vs. Normox-Res; *p<0.05 Normox-Res vs. Hypox-Res.

Figure 5. RT-PCR results for e-NOS and i-NOS.

Upper panel: Real Time amplification plots of a positive for endothelial NO synthase (e-NOS) and inducible NO synthase (i-NOS) genes sample. Lower panel: Agarose gel electrophoresis (2% agarose, 80Volts, 200 mA) of RT-PCR products A: agarose gel for e-NOS (180 bp). 1. 100 bp DNA ladder 2. Sample positive for the e-Nos gene 3. Sample positive for the e-Nos gene 4. Negative control (ddH₂O as template) B: agarose gel for i-NOS (119 bp). 1. Sample positive for the i-Nos 2. Sample negative for the i-Nos 3. Negative control (ddH₂O as template) 4. 100 bp DNA ladder C: agarose gel for GAPDH (209 bp). 1. Sample positive for the housekeeping gene (GAPDH) 2. Sample positive for the housekeeping gene (GAPDH) 3. Negative control (ddH₂O as template) 4. 100 bp DNA ladder.