Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 1;315(5):L816-L833.
doi: 10.1152/ajplung.00249.2018. Epub 2018 Sep 13.

Former-preterm lambs have persistent alveolar simplification at 2 and 5 months corrected postnatal age

Mar Janna Dahl  1 , Sydney Bowen  1 , Toshio Aoki  1 , Andrew Rebentisch  1 , Elaine Dawson  1 , Luke Pettet  1 , Haleigh Emerson  1 , Baifeng Yu  1 , Zhengming Wang  1 , Haixia Yang  1 , Chong Zhang  2 , Angela P Presson  2   3 , Lisa Joss-Moore  1 , Donald M Null  4 , Bradley A Yoder  1 , Kurt H Albertine  1
Affiliations

Former-preterm lambs have persistent alveolar simplification at 2 and 5 months corrected postnatal age

Mar Janna Dahl et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Preterm birth and mechanical ventilation (MV) frequently lead to bronchopulmonary dysplasia, the histopathological hallmark of which is alveolar simplification. How developmental immaturity and ongoing injury, repair, and remodeling impact completion of alveolar formation later in life is not known, in part because of lack of suitable animal models. We report a new model, using former-preterm lambs, to test the hypothesis that they will have persistent alveolar simplification later in life. Moderately preterm lambs (~85% gestation) were supported by MV for ~6 days before being transitioned from all respiratory support to become former-preterm lambs. Results are compared with term control lambs that were not ventilated, and between males (M) and females (F). Alveolar simplification was quantified morphometrically and stereologically at 2 mo (4 M, 4 F) or 5 mo (4 M, 6 F) corrected postnatal age (cPNA) compared with unventilated, age-matched term control lambs (4 M, 4 F per control group). These postnatal ages in sheep are equivalent to human postnatal ages of 1-2 yr and ~6 yr, respectively. Multivariable linear regression results showed that former-preterm lambs at 2 or 5 mo cPNA had significantly thicker distal airspace walls ( P < 0.001 and P < 0.009, respectively), lower volume density of secondary septa ( P < 0.007 and P < 0.001, respectively), and lower radial alveolar count ( P < 0.003 and P < 0.020, respectively) compared with term control lambs. Sex-specific differences were not detected. We conclude that moderate preterm birth and MV for ~6 days impedes completion of alveolarization in former-preterm lambs. This new model provides the opportunity to identify underlying pathogenic mechanisms that may reveal treatment approaches.

Keywords: alveolar capillary growth; alveolar formation; bronchopulmonary dysplasia; lung development; neonatal chronic lung disease.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Feeding protocol and timeline for term control (T) and former-preterm (FPT) lambs. T lambs received ewe’s colostrum for 24 h after vaginal delivery and then ewe’s replacement colostrum. FPT lambs were fed ewe’s replacement colostrum for the first 2 days of life and then ewe’s milk replacement formula. Solid foods were introduced at 30 days postnatal age (PNA) [corrected postnatal age (cPNA) for FPT lambs]. T and FPT lambs were weaned from milk at 2 mo PNA (cPNA for FPT lambs) and fed solid foods ad libitum. T lambs were born at term gestation (~150 days gestation); FPT lambs were born at ~128 days gestation. T and FPT lambs were kept in the laboratory for two epochs: 2 mo PNA and 2 mo cPNA, respectively (A), or 5 mo PNA and 5 mo cPNA, respectively (B).
Fig. 2.
Fig. 2.
Scatter plot of repeated measures of weight by month stratified by experimental group and sex. Weight trajectory for the former-preterm (FPT) lambs progressively fell below the weight trajectory for the term control (T) group. Weight at 5 mo was significantly less for the FPT group vs. the T group (*P < 0.05). Sample size of 4/group, except for 5-mo female FPT group (n = 6).
Fig. 3.
Fig. 3.
Predicted marginal means from the linear mixed effects regression model comparing experimental groups (A) and sex (B) over time for the 5-mo group of lambs. A: at birth, weight was not significantly different between former-preterm (FPT) lambs and term control (T) lambs. However, at 5 mo of postnatal age (corrected postnatal age for FPT lambs), weight of FPT lambs was significantly less than weight of T lambs (*P < 0.05). B: no differences in weight based on sex were detected. Sample size of 4/group, except for 5-mo female FPT group (n = 6).
Fig. 4.
Fig. 4.
Histology of lung tissue from term control (T) and former-preterm (FPT) lambs for the 2-mo and 5-mo groups. Each column of panels is the same magnification (refer to corresponding scale bars). Terminal respiratory units (TRU) have fewer and shorter alveolar secondary septa and thicker distal airspace walls among FPT lambs (D, H, L, P) than among T lambs (A, E, I, M). Higher magnification views of the regions in each TRU (*) are shown in the side columns. These views show that alveolar secondary septa appear shorter and thicker (arrowhead), and distal airspace walls appear thicker (arrow), among FPT lambs (C, G, K, O) than among the matched T lambs (B, F, J, N). Figures were adjusted automatically by the digital camera system for black correction and white balance. All figures were adjusted the same.
Fig. 5.
Fig. 5.
Morphometric indexes of alveolar structural development for term control (T; open symbols) and former-preterm (FPT; filled symbols) lambs for the 2-mo and 5-mo groups. Multivariable linear regression models were completed to compare T lambs vs. FPT (control for sex) and to compare males (squares) vs. females (circles; control for T/FPT). Distal airspace walls were significantly thicker (A and B), volume density of secondary septa was significantly lower (C and D), and radial alveolar count was significantly lower (E and F) among FPT lambs (*) vs. matched T lambs, after control for sex, for both the 2-mo and 5-mo groups (Table 4). Whiskers are means ± SD for sample size of 4/group, except for 5-mo female FPT group (n = 6).
Fig. 6.
Fig. 6.
Capillary localization in lung tissue sections from term control (T) and former-preterm (FPT) lambs for the 2-mo and 5-mo groups. Each image is the same magnification. Immunohistochemical localization of CD-31 protein highlights endothelial cells with brown immunostain (arrows) among the 2-mo group (A–D) and 5-mo group (E–H). Figures were adjusted automatically by the digital camera system for color correction and white balance. All figures were adjusted the same.
Fig. 7.
Fig. 7.
Stereological indexes of capillary development for term control (T; open symbols) and former-preterm (FPT; filled symbols) lambs for the 2-mo and 5-mo groups. Capillary surface density (A and B) is referenced to equal epithelial cell surface density C and D). Multivariable linear regression models were completed to compare T lambs vs. FPT (control for sex), and to compare male (squares) vs. females (circles; control for T/FPT). No differences were detected for capillary surface density or epithelial cell surface density for the 2-mo or 5-mo groups (Table 4). Whiskers are means ± SD for sample size of 4/group, except for 5-mo female FPT group (n = 5, because paraffin tissue sections from the lung of 1 of the 6 female FPT lambs would not immunostain for platelet endothelial cell adhesion molecule 1).
Fig. 8.
Fig. 8.
Normalized mRNA levels of caspase-3, p53, c-myc, transforming growth factor-β (TGFβ), vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2) in term control (T; open symbols) and former-preterm (FPT; filled symbols) lambs for the 2-mo and 5-mo groups. Multivariable linear regression models were completed to compare T lambs vs. FPT (control for sex), and to compare males (squares) vs. females (circles; control for T/FPT). For the 2-mo group, normalized mRNA level of TGFβ (D) and VEGF (E) was significantly lower among FPT lambs (*) vs. matched T lambs, after controll for sex (Table 4). No differences were detected for caspase-3 (A), p53 (B), c-myc (C), and VEGF-R2 (F) normalized mRNA level for the 2-mo group. For the 5-mo group, normalized mRNA level of TGFβ (D) was significantly higher, whereas VEGF (E) was significantly lower, among the FPT lambs (*) vs. matched T lambs, after control for sex (Table 4). No differences were detected for caspase-3 (A), p53 (B), c-myc (C), and VEGF-R2 (F) normalized mRNA level for the 5-mo group. Also for the 5-mo group, normalized mRNA level for caspase-3 (A) and p53 (B) was significantly lower among male lambs (†) vs. female lambs, after control for T/FPT (Table 4). Whiskers are means ± SD for sample size of 4/group, except for 5-mo male FPT group (n = 3, because of mRNA degradation among the frozen lung tissue samples for 1 of the 4 males) and 5-mo female FPT (n = 6).
Fig. 9.
Fig. 9.
Normalized protein abundance of cleaved caspase-3, proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor receptor 2 (VEGF-R2) in term control (T; open symbols) and former-preterm (FPT; filled symbols) lambs for the 2-mo and 5-mo groups. Multivariable linear regression models were completed to compare T lambs vs. FPT (control for sex) and to compare males (squares) vs. females (circles; control for T/FPT). For the 2-mo group, normalized protein abundance of PCNA (C) was significantly lower among the FPT lambs (*) vs. matched T lambs after control for sex (Table 4). Also, for the 2-mo group, normalized protein abundance of PCNA (C) was significantly lower among male lambs (squares) (†) vs. female lambs (circles) after control for T/FPT (Table 4). No differences were detected for cleaved caspase-3 (A) and VEGF-R2 (E) normalized protein abundance for the 2-mo group. For the 5-mo group, normalized protein abundance of cleaved caspase-3 (B) and VEGF-R2 (F) was significantly greater among the FPT lambs (*) vs. matched T lambs after control for sex (Table 4). No difference was detected for PCNA (D) protein abundance. Also, for the 5-mo group, normalized protein abundance of VEGF-R2 was significantly lower among male lambs (†) vs. female lambs after control for T/FPT (Table 4). Whiskers are means ± SD for sample size of 4/group, except for for the female FPT group (n = 6). Order of gel bands is aligned with graphic results for consistent presentation. Black lines between gel lanes separate groups of lambs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abdullah OM, Seidel T, Dahl M, Gomez AD, Yiep G, Cortino J, Sachse FB, Albertine KH, Hsu EW. Diffusion tensor imaging and histology of developing hearts. NMR Biomed 29: 1338–1349, 2016. doi:10.1002/nbm.3576. - DOI - PMC - PubMed
    1. Abman SH. Bronchopulmonary dysplasia: “a vascular hypothesis”. Am J Respir Crit Care Med 164: 1755–1756, 2001. doi:10.1164/ajrccm.164.10.2109111c. - DOI - PubMed
    1. Agostoni C, Buonocore G, Carnielli VP, De Curtis M, Darmaun D, Decsi T, Domellöf M, Embleton ND, Fusch C, Genzel-Boroviczeny O, Goulet O, Kalhan SC, Kolacek S, Koletzko B, Lapillonne A, Mihatsch W, Moreno L, Neu J, Poindexter B, Puntis J, Putet G, Rigo J, Riskin A, Salle B, Sauer P, Shamir R, Szajewska H, Thureen P, Turck D, van Goudoever JB, Ziegler EE; ESPGHAN Committee on Nutrition . Enteral nutrient supply for preterm infants: commentary from the European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition. J Pediatr Gastroenterol Nutr 50: 85–91, 2010. doi:10.1097/MPG.0b013e3181adaee0. - DOI - PubMed
    1. Albertine KH. Utility of large-animal models of BPD: chronically ventilated preterm lambs. Am J Physiol Lung Cell Mol Physiol 308: L983–L1001, 2015. doi:10.1152/ajplung.00178.2014. - DOI - PMC - PubMed
    1. Albertine KH, Dahl MJ, Gonzales LW, Wang ZM, Metcalfe D, Hyde DM, Plopper CG, Starcher BC, Carlton DP, Bland RD. Chronic lung disease in preterm lambs: effect of daily vitamin A treatment on alveolarization. Am J Physiol Lung Cell Mol Physiol 299: L59–L72, 2010. doi:10.1152/ajplung.00380.2009. - DOI - PMC - PubMed

Publication types

  NODES
INTERN 2
twitter 2