INTRODUCTION:

Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS), remain the leading factor of morbidity and mortality in critically ill patients^1,2.

ALI affect more than 1,90,000 patients with an associated 74,500 deaths per year in the United States³. Whereas when we conducted an observational study in Medical Intensive Care Unit of tertiary care hospital, Mumbai for a period of June - October 2011, ARDS rate was found to be 33% amongst all admitted cases with a case fatality rate of 45%. Therefore, improved treatment and prevention strategies are warranted to minimize the mortality associated with this disease. There is a need to develop animal model which mimics ALI/ARDS in humans and to evaluate effective treatment modality.

ALI is characterized by an extensive neutrophil influx into the lung, interstitial edema, the expression of proinflammatory mediators, and damage of the lung epithelium and endothelium⁴, fibrin leakage into the alveoli, and increased pulmonary levels of cytokines, chemokines, and other proinflammatory mediators with chest x-rays showing bilateral pulmonary infiltrates⁵.

Current interest in ARDS, spurred by the lack of an effective therapy, has stimulated the development of a wide variety of animal models, using a range of insults in a range of species, including the rat, pig, dog, sheep and primate.

ALI/ARDS is a clinical syndrome defined by⁶ acute hypoxemic respiratory failure, bilateral pulmonary infiltrates consistent with edema and normal cardiac filling pressures. The etiology of ALI/ARDS is based on different underlying pathophysiological mechanisms⁷. Direct (e.g. concomitant existing pneumonia, aspiration of gastric content, inhalation injury, near drowning) and indirect (e.g. concomitant existing sepsis, multiple trauma, multiple blood transfusion, burns, acute pancreatitis, drug overdose) conditions associated with ARDS have to be distinguished.

This OA animal model of fat embolism-induced ARDS represents one of the various diseases that make up the syndrome of ARDS⁸.

Furthermore, OA has been implicated both in the pathogenesis of ARDS and as a prognostic factor for ARDS. The observed elevation of OA levels in plasma and BAL samples from patients with ARDS is particularly interesting considering that OA-induced lung injury is an extensively used experimental model of ARDS⁹. Thus, we propose that OA represents simple ARDS model and a prognostic factor for this syndrome also that OA itself is a mediator of ARDS. The effect of IV administration of oleic acid (OA) on the lungs of several animal species is well established as a model of acute diffuse lung injury resembling the initial phase of ARDS.

The experiments were performed to investigate the oleic acid induced changes in lung variables for the measure of ALI, inflammatory mediators and to test the hypothesis that intravenous administration of oleic acid in the rat may serve as a relevant model to study human ALI/ARDS so as to assess various formulations in the treatment of syndrome.

Subjects and Methods:

Animals:

Male Wistar rats (180–220 g) obtained from Haffkine Institute were kept at a regular 12-h light dark cycle, with a temperature of 22±3⁰C temperature. Food and water were given ad libitum. The research project was approved by the Animal Care Ethics Committee of the Bombay Veterinary College, Mumbai.

Chemicals:

Oleic acid, Bovine Serum Albumin and Phosphate buffer saline were obtained from Himedia Laboratories, India. The Interleukine (IL) - 1β, 6 and tumor necrosis factor (TNF)-α enzyme-linked immunoassay (ELISA) kits were purchased from Krishgen BioSystems, India.

Administration of Oleic acid:

Animals were anesthetized by intra peritoneal injection of Ketamine (75 mg/kg). Initially to investigate the time- and dose-dependent responses to OA-induced lung injury, OA was administered to animals at different doses – 150 and then 250 μl/kg of OA suspended in BSA. It was observed that intravenous administration of 250 μl/kg OA suspended in 250 μl of BSA produced a pronounced lung injury post-challenge.

Experimental Design:

Two groups each containing 48 animals were used in this study: (a) Control group, i.e., animals receiving 0.1% bovine serum albumin only; (b) animals receiving 300 μl of an OA suspension immediately after vortexing (50 μl of pure OA suspended in 250 μl of 0.1 percent BSA).

After set periods of time (4, 8, 24 and 30 h) group of 12 animals per time point were sacrificed to assess respiratory functions.

Assessment of Respiratory Function:

While developing an animal model, from a group of 12 animals, at each time interval, 2 were subjected to chest X-rays. Plethysmograph readings were taken for all the animals. Bronchoalveolar lavage fluid (BALF) collection was done in 6 animals. Left lung wet/dry weight ratio and histopathological examination was performed on right lungs for 6 animals after sacrificing them. Thus assessment of respiratory functions was carried out using following methods:

· Chest X-ray:

Chest x-rays were made of anaesthetized animals at exposure of 2.5 kV and 42 mA for a fraction of 0.4 seconds.

· Plethysmograph readings:

At each time interval, each animal was placed in a double chamber plethysmograph to measure respiratory rate and tidal volume.

· Bronchoalveolar lavage fluid (BALF) collection:

At each time interval, lungs were lavaged in situ by infusion of 15 ml (in 5 ml aliquots) sterile normal saline solution via a cannula ligated in the trachea. The BALF was collected into plastic tubes on ice.

BALF was centrifuged at 2000 rpm at 4⁰C for 10 min. After centrifugation of the volumes of BALF, 2 samples of 2 ml were taken from the supernatant and were stored at -20⁰ C for analysis of cytokines IL-1β, IL-6 and TNF-α. Total cell count and differential cell counts were done from cytocentrifuge smears. Total protein concentration in BALF was estimated according to the Biuret method.

· Cytokine testing:

Levels of IL-1β, IL-6 and TNF-α in the BALF were determined using enzyme-linked immunosorbent assay kits. Samples and standards were twice assayed spectrophotometrically using a microplate reader. Results were recorded as optical densities, plotted against the linear portion of the standard curve, and expressed as pictograms of cytokine/millimeter of BALF.

· Lung wet/dry weight ratio:

To determine lung wet/dry weight ratio animals were sacrificed and the thorax was opened. The trachea was separated from thymus and esophagus and cut just below the larynx. The lungs still connected to the heart were dissected. The left lung was excised and immediately weighed using a precision balance, then re-weighed after being dried for 24 hours in an incubator at 90⁰C. The wet/dry ratio was calculated by dividing the wet weight by the dry weight.

· Histopathological analysis of lungs:

Right lungs from animals in each experimental group were immersed in 10% formaldehyde fixative for 24 h and were then rinsed with tap water to remove the formaldehyde. For light microscopic observation, tissues were dehydrated with graded alcohol and then embedded in paraffin at 60⁰C. A series of microsections (5mm) was stained with haematoxylin–eosin using standard histological techniques and observations were performed under microscope at a magnification of ×100. Slides were examined by a veterinary pathologist for evidence of inflammatory changes.

RESULTS:

After induction of lung injury using intravenous Oleic acid in the dose of 250 µl/kg in rats, following results were obtained.

Rat Chest X-rays:

After intravenous injection of Oleic Acid in anaesthetized rats serial chest x-rays were taken at baseline, 8, 24 and 30 h for 2 rats from OA and control group.

Figure A demonstrates chest x-ray taken in normal rat - does not show any infiltrates.

Figure B 8 h after the administration of iv OA showed scattered, patchy bilateral infiltrates predominantly in the lower zones.

Figure: Rat chest x-rays after Oleic acid challenge

Figure C the x-ray taken 24 h after OA administration revealed diffuse bilateral pulmonary infiltrates in all quadrants.

Figure D the final x-ray taken after 30 h showed patchy infiltrates, less than the previous x-ray taken at 24 h.

Plethysmograph readings:

Time-course analysis of plethysmograph readings taken in Wistar rats after Oleic Acid injection showed a gradual increase in respiratory rate (RR) from a baseline of 60 – 110 breaths per minute to a peak of 134 breaths per minute at the end of 24 h which returned to normal level of 84 breadths per minute at the end of 30 h. All these values differed significantly from control values at 4, 8, 24 and 30 h after Oleic acid instillation. (ANOVA test, P < 0.05)

Similarly it was noted that the tidal volume (TV) decreased to a low of 0.97 ml at the end of 24 h of OA administration and gradually increased to 1.15 ml at the end of 30 h of OA administration. All these values differed significantly from control values at 24 and 30 h after Oleic acid instillation. (ANOVA test, P < 0.01)

Table: Assessment of respiratory function at various time intervals after OA challenge (N = 6, Mean ± SEM)

Parameter

At 4 h

At 8 h

At 24 h

At 30 h

OA group

Control group

OA group

Control group

OA group

Control group

OA group

Control group

RR (breaths/min)

106.08±

6.79

84.89±

5.47

123.29±

3.49

93.76±

6.90

134.37±

7.20

73.40±

4.93

85.5± 4.9

64±

3.72

TV (ml)

1.14±

0.13

1.21±

0.20

1.03±

0.11

1.56± 0.30

0.97±

0.10

2.00± 0.30

1.15± 0.23

2.07± 0.30

TLC (cells/mm³)

9358.33±

1566.87

3181.33±

610.25

10403.33±

1711.65

3030± 819.54

20566.67±

4912.21

2975±

455.29

8421.67± 527.45

3985±

618.14

PMN (%)

64.67±

3.30

34±

5.39

66.33±

2.48

43.83±

2.49

70.17±

2.65

49.33±

6.27

47.17±

3.79

34.67±

4.65

Total Protein

(g/dL)

0.42±

0.03

0.32±

0.03

0.58±

0.03

0.37±

0.05

0.62±

0.08

0.32±

0.03

0.3±

0.04

0.22±

0.03

IL-1β (pg/ml)

59.98±

7.2

29.98±

5.78

92.46±

16.59

30.33± 6.1

163.4± 35.61

37.04± 6.34

85.25± 8.39

33±

8.95

IL-6 (pg/ml)

1678.15±

356.69

34.7±

7.75

2697.47± 462.45

52.75± 8.53

3040.7± 434.71

51.55± 6.43

1179.23±222.69

46.51± 10.29

TNF-α (pg/ml)

226.92±

19.18

46.3±

12.11

337.75±

49.86

29.23± 6.17

444±

71.86

44.96± 2.16

245.35±26.87

37.19± 4.07

Lung Wet/Dry ratio

4.39±

0.28

3.00±

0.30

4.92±

0.46

3.25± 0.26

5.36±

0.22

3.54± 0.37

2.89± 0.27

2.57± 0.27

Bronchoalveolar lavage fluid analysis:

Following intravenous administration of OA, the maximum TLC was noted to be 20566 cells/mm³ at 24 h and it declined towards baseline at the end of 30 h as 8421 cells/mm³. All these values differed significantly from control values at all time intervals. (ANOVA test, p < 0.05)

Peak in PMN number of 70% was seen at 24 h. Similarly decreasing to baseline of 34% at the end of 30 h. the increase in PMN number was significant as compared to control group at 4, 8 and 24 h (ANOVA test, p < 0.01).s

The total proteins in BALF also increased from 0.42 g/dL at the end of 4 h to 0.58 g/dL at the end of 8 h and peaked to a level of 0.62 g/dL after 24 h and then declined to 0.3 g/dl after 30 h of OA administration. The increase in total protein concentration was significant at 8 and 24 h in comparison to control group (ANOVA test, p < 0.01). Epithelial cells were also present in BALF.

TLC in BALF after OA administration. Data are expressed as mean ± SD (N = 6 for all groups). * P < 0.05 compared with the control group.

Neutrophil number in BALF after OA administration. Data are expressed as mean ± SD (N = 6 for all groups). * P < 0.05 compared with the control group.

Total protein in BALF after OA administration. Data are expressed as mean ± SD (N = 6 for all groups). * P < 0.01 compared with the control group.

BALF Cytokine levels:

It was noted that all the cytokine levels rose significantly (ANOVA test, p < 0.001) after the induction of injury with Oleic Acid and reached a peak level at the end of 24 h before gradually declining at the end of 30 h.

IL-1β, IL-6 and TNF-α levels in BALF.Stands for p < 0.001 as compared to animal challenged with control. (N = 6)

Lung Wet/Dry weight Ratio:

Significant increase in lung oedema was seen from 4 to 24 h (ANOVA test, p < 0.01) with a maximum value of 5 and decreasing to 3.10 at the end of 30 h.

Lung wet/dry weight ratio after OA administration. Data are expressed as mean ± SD. * P < 0.01 compared with the control group.

Histopathological examination:

Figure A: 4 h after OA administration there was an influx of inflammatory cells is seen in the alveolar space. Note the patchy nature of the injury and vascular congestion.

Figure B: 8 h after instillation of OA, diffuse inflammatory reaction with large number of polymorphs, lymphocytes and exudative fluid in the alveolar spaces and septal thickening is seen.

Figure C: 24 h after administration of OA, intense inflammatory response with exudation of fluid, white blood cells and proteinaceous material found in the alveolar spaces. Areas of focal hemorrhage are also seen. There is loss of normal architecture of the alveolar spaces.

Figure D: Decrease in exudative fluid, inflammatory cells and areas of haemorrhage are noted 30 h after administration of Oleic acid. However loss of cytoarchitecture of alveolar bed and areas of patchy fibrosis also noted.

(A) 4 h after OA administration

(B) 8 h after OA administration

(C) 24 h after OA administration

(D) 30 h after OA administration

Figure 1: Histopathological examination of lung samples after OA challenge

DISCUSSION:

Oleic acid-induced lung injury, a well-described laboratory model for acute pulmonary injury in the rat and other species, causes morphologic and cellular changes similar to human adult respiratory distress syndrome (ARDS). Experiments were performed to study of course of events following administration of intravenous Oleic acid and so as to evaluate the effects of various formulations in the Oleic acid ALI/ARDS animal model.

Radiological changes after OA challenge included bilateral pulmonary infiltrates and patchy areas of consolidation. There are very few studies where radiological evidence of injury have been demonstrated. We demonstrated peak radiological changes at the end of 24 h post lung injury.

We observed tachypnea, respiratory distress and decreasing tidal volume using time-course analysis of plethysmograph readings taken in rats after Oleic Acid injection. A peak respiratory rate of 134 breaths per minute at the end of 24 h which returned to normal level of 84 breadths per minute at the end of 30 h was noted. Similarly it was noted that the tidal volume (TV) decreased to a low of 0.97 ml at the end of 24 h of OA administration and gradually increased to 1.15 ml at the end of 30 h of OA administration.

Dan Liu et al, too, observed a peak in RR of 130-160 breaths per minute after administration of Oleic acid in Sprague-Dowley rats and Kate G. Davidson reported a drop in tidal volume to 1.2 ml at the end of 4 h after Oleic acid infusion.

These observations were similar to our observations and confirmed the successful induction of ALI/ARDS.

Following intravenous administration of OA, the maximum TLC was noted to be 20566 cells/mm³ and Peak in PMN number of 70% was seen at 24 h.

Brett Volpe et al noted a TLC of 87 lakh whereas PMN count of 50%. Such a high number of cells could be attributed to different strain of Rat - Fischer rats used by them.

The total proteins in BALF increased to a maximum of 0.62 g/dL after 24 h and then declined to 0.3 g/dl after 30 h of OA administration.

Dan Liu et al¹⁰also reported a total protein count of 0.5 at the end of 12 hrs after OA infusion. Whereas Brett Volpe et al noted a total protein count of 0.3 in BALF at the end of 24 h. The possible reason could be the dose of Oleic acid used by them was 30 μL as against we conducted a study using 50 μL dose of Oleic acid.

Cytokine storm was noted at the end of 24 h of OA administration with a peak levels of 1163.4 pg/ml, 3040.7 pg/ml and 444 pg/ml for IL-1β, IL-6 and TNF-α respectively.

Muiris T Kennedy et al observed BALF IL-6 level of 2000 pg/ml at the end of 6 hrs post Oleic acid stimulation which was similar to our reading. Dan Liu et al¹⁰ noted levels of TNF-α ranging from 525-600 pg/ml and IL-1β from 25-35 pg/ml post injury in mice. These differences in values of IL-1β and TNF-α may be due to species variation.

Significant increase in lung edema was seen from 4 to 24 h with a maximum value of 5 and decreasing to 3.10 at the end of 30 h. Dan Liu et al¹⁰ also reported lung wet/dry weight ratio of 6.04 at the end of 12 h after Oleic acid infusion.

In summary, the data presented in this study demonstrated that OA animal model of fat embolism-induced ARDS represents one of the various diseases that make up the syndrome of ARDS.

REFERENCES:

1. Costa, E.L., et al., 2006. The lung in sepsis: guilty or innocent? Endocr. Metab. Immune Disord. Drug Targets 6, 213–216.

2. Frutos-Vivar, F., et al., 2006. Epidemiology of acute lung injury and acute respiratory distress syndrome. Semin. Respir. Crit. Care Med. 27, 327–336.

3. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD. Incidence and outcomes of acute lung injury. N Engl J Med. 2005;353:1685–1693.

4. Goodman, R. B., R. M. Strieter, D. P. Martin, K. P. Steinberg, J. A. Milberg, R. J. Maunder, S. L. Kunkel, A. Walz, L. D. Hudson, and T. R. Martin. 1996. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 154:602–611.

5. Matthay, M. A., G. A. Zimmerman, C. Esmon, J. Bhattacharya, B. Coller, C. M. Doerschuk, J. Floros, M. A. Gimbrone, Jr., E. Hoffman, R. D. Hubmayr, et al. Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group. Am. J. Respir. Crit. Care Med. 167: 1027–1035.

6. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;342: 1334–1349..

7. European Medicines Agency Guideline On Clinical Investigation Of Medicinal Products In The Treatment Of Patients With Acute Respiratory Distress Syndrome, 2007

8. Volpe et al

9. Oleic Acid Inhibits Alveolar Fluid Reabsorption A Role in Acute Respiratory Distress Syndrome? Istva´n Vada´sz, Rory E. Morty, Markus G. Kohstall, Andrea Olschewski, Friedrich Grimminger, Werner Seeger, and Hossein A. Ghofrani

Received on 25.03.2014 Modified on 02.04.2014

Research J. Pharm. and Tech. 7(8): August 2014 Page 882-888