Lung injury after asphyxia and hemorrhagic shock in newborn piglets: Analysis of structural and inflammatory changes

Abstract

Objective: Asphyxia of newborns is a severe and frequent challenge of the peri- and postnatal period. The purpose of this study was to study early morphological, immunological and structural alterations in lung tissue after asphyxia and hemorrhage (AH).

Methods: 44 neonatal piglets (age 32 hrs) underwent asphyxia and hemorrhage (AH) and were treated according to the international liaison committee of resuscitation (ILCOR) guidelines. For this study, 15 piglets (blood transfusion (RBC) n = 9; NaCl n = 6, mean age 31 hrs) were randomly picked. 4 hours after ROSC (return of spontaneous circulation), lung tissue and blood samples were collected.

Results: An elevation of myeloperoxidase (MPO) activity was observed 4 hrs after AH accompanied by an increase of surfactant D after RBC treatment. After AH tight junction proteins Claudin 18 and junctional adhesion molecule 1 (JAM1) were down-regulated, whereas Occludin was increased. Furthermore, after AH and RBC treatment dephosphorylated active form of Connexin 43 was increased.

Conclusions: AH in neonatal pigs is associated with early lung injury, inflammation and alterations of tight junctions (Claudin, Occludin, JAM-1) and gap junctions (Connexin 43) in lung tissue, which contributes to the development of lung edema and impaired function.

Fig 1. Lung injury after AH.

A) Horovitz oxygenation index calculated as ratio of arterial oxygen partial pressure and the fractional inspired oxygen at baseline (BL), ROSC (return of spontaneous circulation) and 4 hrs after asphyxia. B) Lung injury score determined in H.E. stained lung tissue 4 hrs after asphyxia and hemorrhage (AH). C) Representative images of H. E. staining. D) Results of IHC staining of surfactant protein D in lung tissue. E) representative images of surfactant D staining. * p = 0.05, n.s. = not significant, graphical representation as mean ± SEM, NaCl n = 6, RBC n = 9, sham n = 6.

Fig 2. Local inflammation and activation of the complement system after AH.

A) Myeloperoxidase (MPO) activity obtained from lung tissue samples. B) local amount of C5a receptor 1 and C) complement factor 5a receptor 2 protein in lung tissue normalized to the total protein amount of the samples, as well as representative western blots and loading control bands (GAPDH). * p = 0.05; n.s. = not significant, graphical representation as mean ± SEM, NaCl n = 6, RBC n = 9, sham n = 6.

Fig 3. No elevation of apoptotic cells in lung tissue after AH.

A) Results from caspase 3 analysis. B) TUNEL positive nuclei in percentage of whole cell count by DAPI staining; n.s. = not significant; graphical representation as mean ± SEM, NaCl n = 6, RBC n = 9, sham n = 6.

Fig 4. Alterations in cell cell contacts after AH.

Results of IHC staining of A) Claudin 18, B) Occludin and C) JAM-1 of lung tissue after asphyxia. Alterations in gap junction protein amount of D) Connexin 43 and E) Protein amount of Connexin 40. * p = 0.05, n.s. = not significant, graphical representation as mean ± SEM, NaCl n = 6, RBC n = 9, sham n = 6.