Question

How can you histologically analyze the degree of pathology and inflammation in the lungs of mice following infection with streptococcus pneumonia? (with tissue was fixed, for what duration it was fixed, what staining was used to visualize the tissue and provide pictures identifying markers of inflammation inflammatory cell types, damages to tissue and final graphics)

Answer 1

The method is as follow:

Histopathology

Mice will be anesthetized and killed by cervical dislocation 48 h after infection with S. pneumoniae strain or depending on strain used for infection time may vary, respectively, after which their lungs were inflated with 4% formalin, fixed for at least 48 h in situ, removed, embedded in paraffin, cut into 5-mm-thick sections, and adhered to slides. Staining will then be performed with H&E . Sections will be obtained. H&E sections were evaluated based on the distribution and severity of pneumonia, vasculitis, and overall tissue damage. Scores ranging from 0 to 5 were assigned, whereby 0, no finding (i.e., pneumonia, vasculitis, or tissue damage); 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. A moderate score for the pneumonia classification was defined as affecting 25% of the lobe, and moderate vasculitis was defined as focal areas of dense cellular infiltration around veins or blood vessels. Moderate overall tissue damage was characterized by a large, acute area (∼200 by 4000 mm) of infiltrating cells, with some appearing necrotic in the perivascular and peribronchiolar space.

H&E is the combination of two histological stains: hematoxylin and eosin. The hematoxylin stains cell nuclei blue, and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors.

Infection with S. pneumoniae. (A-H) Lung histology of mice after transnasal infection with S. pneumoniae (5 x 10 6 CFU/ mouse) revealed widely expansive (A), suppurative to necrotizing bronchopneumonia (a-d), predominantly infiltrated by neutrophils within bronchial lumina (B) and alveoli (C) and large areas of necrosis and hemorrhage (D, arrowhead). Additional features included marked neutrophilic infiltration and edema of perivascular spaces (E), severe alveolar edema (F, asterisk) and necropurulent pleuritis (G, arrowhead) and steatitis (H).