Acute Lung Injury Caused by High Tidal Volume in a Rat Pneumonia Model

Research Article

Austin J Emergency & Crit Care Med. 2015;2(2): 1015.

Acute Lung Injury Caused by High Tidal Volume in a Rat Pneumonia Model

Julie Chi Chow1#, Wei-Lun Liu2#, Chih-Cheng Lai2, Khee-Siang Chan3, Chin-Ming Chen3,4,5*, Kuo-Chen Cheng6,7,8 and Willy Chou4,5

1Department of Pediatric, Chi Mei Medical Center, Taiwan

2Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Taiwan

3Department of Intensive Care Medicine, Chi Mei Medical Center, Taiwan

4Chang Jung Christian University, Taiwan

5Department of Recreation and Health-Care Management, Chia Nan University of Pharmacy & Science, Taiwan

6Section of Respiratory Care, Chi Mei Medical Center, Taiwan

7Department of Internal Medicine, Chi Mei Medical Center, Taiwan

8Department of Safety Health and Environment, Chung Hwa University of Medical Technology, Taiwan #Both had an equal contribution on this paper

*Corresponding author: Chin-Ming Chen, Department of Intensive Care Medicine, Chi-Mei Medical Center, 901 Chung Hwa Road, Yang Kang City, Tainan, 71044, Taiwan

Received: January 11, 2015; Accepted: February 20, 2015 Published: February 24, 2015


Background: To establish animal model of two-hit model for ventilator induced lung injury after pneumonia.

Methods: Male Sprague-Dawley rats (300 – 400g) were intratracheally challenged with lipopolysaccharide (LPS) as a first hit to induce lung inflammation. Rats were then randomized 24 hours later to receive mechanical ventilation as a second hit, with either an injurious strategy of high tidal volume (TV) of 22 mL/kg and zero positive end-expiratory pressure (PEEP) (high volume group, HV) or a protective strategy of low TV of 6 mL/kg with PEEP of 5 cm H2O (low volume group, LV), along with a fraction of inspired oxygen of 40 % during the experimental period. There were 4 groups (n = 8-10 rats/group): (1) HV + placebo; (2) HV + LPS; (3) LV + placebo; and (4) LV + LPS.

Results: After 4-hours of ventilator use, each group had a similar hemodynamic status (mean arterial pressures and heart rates) and arterial pH, PaCO2, and HCO3 values. However, as compared with the other groups, group 2 (HV + LPS) had lower arterial O2 and lung compliance, worse lung edema, higher total and neutrophil cell counts in lung lavage fluid, and increased lung elastance and some lung cytokines.

Conclusion: Inadequate ventilator settings may cause severe lung injury that is a complication after LPS induced pneumonia, as evidenced in our animal model by worse lung compliance, elastance, oxygenation, inflammatory cells, cytokines, and lung edema, which comply with evidence in the literature. Clinicians should be cautious regarding possible lung injury by inappropriate ventilator settings.

Keywords: Acute lung injury; Lipopolysaccharide; Mechanical ventilation; Pneumonia; Rat model


ALI: Acute Lung Injury; ARDS: Acute Respiratory Distress Syndrome; BAL: Bronchoalveolar Lavage; CXCL1: chemokine ligand 1; Fio2: Fraction of Inspired Oxygen; HV: High Volume; IL: Interleukin; LPS: Lipopolysaccharide; LV: Low Volume; MAP: Mean Arterial Pressure; MV: Mechanical Ventilation; PEEP: Positive Endexpiratory Pressure; TNF: Tumor Necrosis Factor; TV: Tidal Volume; VILI: Ventilator-Induced Lung Injury; W/D: Wet To Dry


Pneumonia that is complicated by acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), is the leading cause of death in critically ill patients, and often requires mechanical ventilation (MV) [1]. Although MV provides essential life support, and the weaning rate may be as high as 90% in selected patients with planned extubation [2], mechanical stresses produced by MV can lead to over-distension of lung units or shear forces that are generated during repetitive opening and closing of atelectatic lung units or biotrauma. This can induce or enhance an inflammatory response that can also worsen lung injury as a ventilator-induced lung injury (VILI), with characteristics similar to those caused by ARDS [3,4]. One large multicenter trial demonstrated the importance of VILI by using ventilation with a lower tidal volume (TV; 6 mL/ kg) versus traditional TV (12 mL/kg) in which the lower TV improved survival [5]. The cytokines response can also be reversed by reinstitution of lung protective mechanical ventilation after injurious ventilator strategy [6].

Inappropriate ventilator strategies preceded by hemorrhagic shock and followed by reperfusion or intratracheal lipopolysaccharide (LPS) administration can cause a so-called “two-hit injury”; this makes the lung more susceptible to mechanical ventilation injury [7,8]. The spectrum of injuries includes disrupting endothelial and epithelial cells, increasing endothelial and epithelial permeability, neutrophil infiltration, enhanced production of inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-a, and subsequent permeability pulmonary edema, hyaline membranes, and decreased lung compliance [7-9]. Clinically IL-8 levels, the equivalent of chemokine ligand 1 (CXCL1) in rats, are elevated in patients with ARDS and mechanical stress [10,11].

Thus, the aim of this study was to determine whether a large TV could augment the inflammatory response in rat lungs after they received an intra-tracheal instillation of LPS. LPS instillation to induce lung injury mimics the clinical syndromes of pneumonia and ARDS and can be used to explore any injury augmentation from large TV during MV in lungs with underlying injuries. We also investigated rats’ lung mechanics, blood gases, augmented inflammatory responses, and lung injuries.

Materials and Methods

Animal preparation

All experiments complied with protocols approved by the Institutional Animal Care and Use Committee of Chi-Mei Hospital. Briefly, LPS (Escherichia coli O55:B5, Sigma, St Louis, MO, USA) was administered intratracheally at 0.1 mg/kg into male Sprague- Dawley rats (300 – 400 g) under anesthesia (sodium pentobarbital, intra-peritoneal injection) as a first hit to induce lung inflammation. Animals were allowed to recover from anesthesia and returned to their cages with food and water provided ad libitum.

Experimental protocol

Rats were block randomized 24 hours after the intratracheal challenge with LPS and were anesthetized with sodium pentobarbital. For each rat, the right carotid artery was cannulated (Angiocath IV Catheter; 24-gauge) to monitor mean arterial pressure (MAP) and to collect samples and for blood gas measurements. A tracheostomy was performed, and a 14-gauge cannula (Angiocath IV Catheter; 2.1 x 48 mm) was inserted into the trachea. The rats were ventilated with a Servo 300 ventilator (Siemens, Sweden) using a TV of 6 ml/ kg, positive end-expiratory pressure (PEEP) of 5 cm H2O, and a respiratory rate of 50 breaths/min with a fraction of inspired oxygen (FiO2) of 40 %. A 24 gauge intravenous cannula was inserted and kept in the tail vein to infuse anesthesia or lactated Ringer’s solution to maintain adequate ventilator synchrony or blood pressure.

Thirty minutes later, rats were randomly assigned to receive mechanical ventilation at two different ventilator settings over a 4-h period. For a high volume (HV) group (injurious strategy), rats were ventilated with a TV of 22 ml/kg and zero PEEP. For a low volume (LV) group (protective strategy), rats were ventilated with a TV of 6 ml/kg with PEEP of 5cm H2O. The minute volume was maintained as constant during the entire study period by adjusting the respiratory rate to 16-18 breaths/min in the HV group. The fraction of inspired oxygen was maintained as noted above. MAP was maintained above 70 mmHg after ventilation randomization.

Rats were randomly assigned to 4 groups (10 rats/group) in a blinded manner: Group 1: HV + placebo; Group 2: HV + LPS; Group 3: LV + placebo; Group 4: LV + LPS.

Measurements of lung mechanics and blood gases and tissue sampling

Airway pressures (peak and plateau pressures) were recorded using a data acquisition system (National Instrument DAQ Card 700, Austin, TX) at a sampling rate of 200 Hz (ICU Lab, Kleis TEK Engineering, Bari, Italy). Lung elastance was calculated using the formula: (Plateau Pressure – PEEP)/TV. Blood gases were checked at the beginning of the study period and hourly until the end of the study period. Rats were humanely sacrificed at the conclusion of the experiment by sodium pentobarbital overdose. The lungs were excised via a midline sternotomy and inflated twice to an airway pressure of 30cm H2O to generate static pressure-volume curves by manually injecting 0.5 to 1cm3 aliquots of air in a stepwise manner starting at atmospheric pressure and continuing until achieving an airway pressure of 30cm H2O. This was followed by generating a deflation curve by withdrawing air using a similar step-wise approach. Volumes were maintained at each step for 6 seconds before the next air injection.

The left lungs were lavaged (bronchoalveolar lavage, BAL) for cell differentiation assays. The right upper lungs were used to measure wet to dry (W/D) lung weight ratios. The remaining part of the right lung was removed, homogenized in Triton X solution, and then centrifuged (5804R; Eppendorf, Brinkmann Instruments, Westbury, NY) at 1,000 x g at 4°C for 10 min. After centrifugation, the supernatant was collected to determine cytokine levels (IL-1, CXCL1, and TNF-a). Analysis of cytokines (IL-1, CXCL1, and TNF-a) in the plasma (n=5 for each group) and BAL fluid (n=8-10 for each group) was done in a blinded fashion by technicians using DuoSet ELISA Development kits (R & D Systems, Minneapolis, MN, USA).


Lung injury scores included alveolar collapse, alveolar hemorrhage, perivascular edema, alveolar polymorphonuclear leukocytes, membranes, and alveolar edema [7,12]. Scoring was done by a pathologist who was blinded to the experimental groups. Five regions for each specimen were examined. An injury score of 0–3 (0 = normal; 1 = mild; 2 = moderate; 3 = severe) was assigned to each region and used to calculate a total score for lung injury (n=5 for each group), which was evidenced by previous literatures [7,12].

Statistical analysis

Results are given as means ± SEM’s. Group comparisons used two-way analysis of variance (ANOVA) for repeated measures. One-way analysis ANOVA followed by a t-test was used, when appropriate. Post-hoc analysis used a Bonferroni test. A p-value of <0.05 was considered significant.


After 4-hours of ventilator use, the volume of fluid that had been infused was identical (≈ 2.5 ml lactated Ringer’s solution) in all rats during the mechanical ventilation period. Each group of rats had a similar hemodynamic status (mean arterial pressures and heart rates) at baseline and during mechanical ventilation (Figure 1).