Histology

Lungs were harvested and fixed in 4% formaldehyde. The tissues were then trimmed in a standard position and put in embedding cassettes. One cassette was prepared per animal. Paraffin blocks were sectioned at ~ 4 μm thickness, put on glass slides, and stained with hematoxylin and eosin (H&E). Pictures were taken using an Olympus microscope (BX60, serial NO. 7D04032) at objective magnification of × 4 and × 10 and microscope’s Camera (Olympus DP73, serial NO. OH05504).

A quantitative analysis for acute lung injury (ALI) was performed using a severity scoring scale of 0–2, based on the American Thoracic Society Documents, 2011 [16]. Analysis was performed by a certified veterinarian pathologist (Patho-logica Ltd., Ness Ziona, Israel) who was blinded to experimental treatment.

Neutrophils: Not visible within the field—a score of 0; 1–5 neutrophils—1; more than 5 neutrophils—2.

Fibrin: Not visible within the field—a score of 0; a single well-formed band of fibrin within the airspace—1; multiple eosinophilic membranes—2.

Thickened alveolar walls: Due to technical artifacts, only septal thickening that is equal or greater than twice normal was considered. Less than × 2—score 0; × 2–× 4—score 1; more than × 4—score 2.

The analysis was based on measurements of 20 fields, using objective magnification of × 4 and × 10 (HPF).

Neutrophil cell count was performed using MATLAB color-based, brightness-based, and morphological-based segmentation. The cells were counted from a rectangle of 88,892 μm².