Fig. 6

Cellular recovery and regeneration of injured lungs. Histologic analysis of airway and vascular recovery. a Large airway: Alcian blue 0 h with marginating PMNs (arrows) and 36 h with goblet cells (arrow), periodic acid-Schiff (PAS) stain demonstrating loss of epithelial integrity and airway cilia at 0 h and restoration of ciliated epithelial lining at 36 h (arrows). b Distal airway: Alcian blue and PAS stain 0 h with alveolar edema (star), pentachrome 0 h with alveolar infiltrates (arrow). c Vascular network: Alcian blue 0 h with marginating PMNs (arrows), PAS stain 0 h with perivascular edema (star), pentachrome 0 h with intimal thickening (star). d Neutrophil quantification. e Myeloperoxidase (MPO) activity. f Macrophage quantification by CD163 immunostaining. Student’s t-tests, ***p < 0.001, **p < 0.01, or p > 0.05, not significant (n.s.). g TUNEL staining of injured lungs at 0 h and 36 h. h Metabolic activity of injured and control lungs at 0 h and 36 h, Student’s t-test for Injured versus Control, not significant (n.s.). i Aquaporin 5 (Aqp 5) and (j) ZO-3 immunostaining of injured lungs at 0, 18, and 36 h. Recovery of surfactant in type II cells: (k) Loss of surfactant protein C (SPC) at 0 h, with recovery at 36 h. l Transmission electron micrograph (TEM) of type II cell with loss of lamellar bodies containing surfactant (white arrow) at 0 h, and TEM of type II cell with lamellar bodies containing surfactant (white arrow) with active surfactant secretion (black arrow) at 36 h. m Function of type II pneumocytes demonstrated by uptake of fluorescently labeled surfactant protein B (BODIPY-SBP) at 36 h. n ZO-1 immunostaining at 0, 18, 36 h. o Quantification of connexin 43⁺ cells at 0, 18, 36 h (Student’s t-tests, ***p < 0.001), with p connexin 43 immunostaining at 36 h. q CD31 immunostaining at 36 h. r TEM (pseudocolored) of pulmonary capillaries at 0 h showing an extravascular red blood cell (red) indicating alveolar hemorrhage (star), disruption of type I cell membrane (purple, arrow), and intra-capillary leukocyte (orange) adjacent to microvascular endothelial cell (blue), and 36 h demonstrating intact blood–gas barrier. Vascular viability and function at 36 h: uptake of s CSFE and t acetylated low-density lipoprotein (acLDL). All graphs represent data from n = 8 lungs. All values represent mean ± standard deviation