Acute lung injury using oleic acid in the laboratory rat: establishment of a working model and evidence against free radicals in the acute phase

doi:10.1016/S0149-7944(02)00775-4

Current Surgery

Volume 60, Issue 4, July–August 2003, Pages 412-417

https://doi.org/10.1016/S0149-7944(02)00775-4 Get rights and content

Abstract

Objective

To determine the optimal model of acute respiratory distress syndrome (ARDS) using oleic acid in our laboratory and to measure the presence or absence of free radicals in this model.

Design

This protocol consisted of 2 phases. During the first phase, various conditions were tested, to include different doses (30 or 50 microliters) of oleic acid, different levels of support (with and without mechanical ventilation), and different injury time periods (sacrifice 4 or 8 hours after injection). During the second phase, animals were randomly assigned to experimental (injured) and control (noninjured) groups for the measurement of free radicals by nitrotyrosine Western blot and by the conversion of hydroethidine to ethidium bromide by superoxide.

Setting

Multidisciplinary laboratory and animal surgery suite.

Participants

Twenty-seven male Sprague–Dawley rats.

Results

During the first phase, several animal deaths occurred in the high-dose, ventilated groups, whereas there were no deaths in the nonventilated animals. On hematoxylin and eosin stain, injury was greatest in the animals that received the higher dose of oleic acid and that were sacrificed at 8 hours. In the protocol’s second phase, oxygen radical assays were negative for all experimental and control lungs.

Conclusions

During this study, we successfully established a working animal model of ARDS for our laboratory. Our findings to date suggest that free radicals do not contribute to oleic acid lung injury in the early stages.

Introduction

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) make up a spectrum of disease that is commonly defined as “acute non-cardiogenic edematous lung injury.”1 They are among the leading causes of death in intensive care units, with a mortality rate of 30% to 50%.2, 3 Acute lung injury and ARDS are caused by an inciting insult, such as systemic release of endotoxin, aspiration, or fat embolism, which then triggers a series of pathologic events involving cytokines, neutrophils, proteases, lipid mediators, and free radicals.1

The role of free radicals in ARDS is well established.4, 5, 6 Neutrophils recruited to the lungs release superoxide during the “oxidative burst.”7, 8 Stimulation by inflammatory mediators may also lead to the production of superoxide and nitric oxide by alveolar macrophages, and lung epithelial, endothelial, and interstitial cells.7 Nitric oxide and superoxide react to create peroxynitrite, a potent oxidant that damages alveolar epithelium and pulmonary surfactant.9, 10, 11 Peroxynitrite nitrates the phenolic rings of proteins, such as tyrosine, creating nitrotyrosine.12, 13 Peroxynitrite and nitrotyrosine are present in both animals and humans with ARDS.14, 15, 16 Nitrotyrosine is quite stable and can be measured in tissue homogenates as a marker of oxygen radicals. In our laboratory, we have previously successfully measured superoxide radicals in an intestinal ischemia-reperfusion model by injecting hydroethidine (HE) and measuring the conversion of HE to ethidium bromide by superoxide (presented in poster form at the Pacific Coast Surgical Association, February 2002). Others have used a similar method as well.17

There are many methods of inducing ARDS in animals. These include the administration of intratracheal lipopolysaccharide,18 intratracheal HCL,19 repeated saline lavage,20 endotoxin infusion,21 and intravenous injection of oleic acid.22, 23, 24 One-time intravenous injection of oleic acid has been shown to induce lung injury and ARDS within minutes to hours.22, 23, 24 Methods in the literature vary in terms of type of animal used, dosage of oleic acid, injury time period, and level of anesthesia/ventilatory support. In addition, the role of free radicals in oleic acid-induced lung injury is controversial.25, 26, 27, 28, 29 We embarked upon this study to determine the optimal model of ARDS using oleic acid in our laboratory. We were especially interested in determining whether mechanical ventilation was necessary and/or helpful in establishing a well-controlled model of oleic acid-induced injury. In addition, we set out to measure the presence or absence of free radicals in this model.

Section snippets

Materials/methods

This study was approved by the Institutional Animal Care and Use Committee and the Institutional Review Board at Madigan Army Medical Center. We used male Sprague–Dawley rats, 4 to 6 weeks old, weighing 250 to 350 g. The protocol was divided into 2 phases. (See Table 1.) During phase I, there were 8 groups, each containing 2 to 4 animals. The groups received 2 different doses (30 or 50 μL) of oleic acid, were either mechanically ventilated or kept in an oxygen chamber, and were sacrificed 4 or

Results

Nonventilated animals were observed closely in an oxygen chamber and did not exhibit any signs of pain or distress. There were no deaths prior to euthanasia in these groups. The ventilated animals were maintained under general anesthesia and thus were not distressed. There were no deaths prior to euthanasia in the low-dose (30 μL) ventilated animals. Three ventilated animals received the higher dose of oleic acid (50 μL). One of these animals died 1 hour after oleic acid injection, and 1 died 3

Discussion

The purpose of this study was to establish a working animal model of the acute respiratory distress syndrome in our laboratory, to determine the practicability and necessity of mechanically ventilating a rat with lung injury, and to determine the level of injury produced by different doses of oleic acid at 2 different time points after injection. In addition, we set out to determine whether free radicals contribute to the injury pattern caused by oleic acid.

The oleic acid model described in the

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^☆: The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the United States Government, the Department of Defense, or the Department of the Army.

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Gary P. Wratten symposiumAcute lung injury using oleic acid in the laboratory rat: establishment of a working model and evidence against free radicals in the acute phase☆

Abstract

Objective

Design

Setting

Participants

Results

Conclusions

Introduction

Section snippets

Materials/methods

Results

Discussion

Lancet

Chest

Chest Surg Clin N Am

Mayo Clin Proc

Clin Chest Med

J Biol Chem

J Pharmicol Toxicol Methods

Free Radic Biol Med

J Surg Res

Clin Chest Med

Clin Chest Med

Br J Anaesth

Oxidant stress and adult respiratory distress syndrome

Eur Respir J

Oxygen free radicals in ARDS, septic shock and organ dysfunction

Intensive Care Med

Superoxide in the pulmonary circulation

Pharmacol Ther

Superoxide release from neutrophils causes a reduction in nitric oxide gas

Am J Physiol Lung Cell Mol Physiol

Nytrotyrosine formation and its role in various pathological conditions

Free Radic Res

Mechanisms of peroxynitrite-induced injury to pulmonary surfactant

Am J Physiol

Nitric oxide, superoxide, and peroxynitritethe good, the bad, and the ugly

Am J Physiol

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury

J Clin Invest

Evidence for in vivo peroxynitrite production in human acute lung injury

Am J Respir Crit Care Med

Ni tric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome

Am J Respir Crit Care Med

Gary P. Wratten symposium
Acute lung injury using oleic acid in the laboratory rat: establishment of a working model and evidence against free radicals in the acute phase☆