Medical Molecular Morphology

, Volume 51, Issue 3, pp 166–175 | Cite as

Morphological characterization of pulmonary microvascular disease in bronchopulmonary dysplasia caused by hyperoxia in newborn mice

  • Hidehiko NakanishiEmail author
  • Shunichi Morikawa
  • Shuji Kitahara
  • Asuka Yoshii
  • Atsushi Uchiyama
  • Satoshi Kusuda
  • Taichi Ezaki
Original Paper



Pulmonary microvascular injury is associated with the pathogenesis of bronchopulmonary dysplasia (BPD). To characterize the mechanisms of pulmonary vascular disease resulting from BPD, we studied the ultrastructural changes affecting pulmonary microvasculature.


Newborn ICR mice were exposed to 85% hyperoxia or normoxia for 14 days, and then normal air replacement conditions for the following 7 days. At postnatal day (P)14 and P21, lungs were harvested for ultrastructural examination and assessment of pulmonary hypertension.


The ultrastructure of pulmonary microvasculature in the hyperoxia-exposed lungs revealed a collapsed capillary lumen. This was due to the abnormal morphology of endothelial cells (ECs) characterized by heterogeneously thick cytoplasm. Compared to normal air controls, the specimens displayed also remarkably thick blood–air barriers (BABs), most of which were occupied by EC layer components. Structural changes were accompanied by increased pulmonary artery medial thickness and right ventricular hypertrophy (RVH). Moreover, abnormalities in ECs persisted even after exposure to 7 days of normal air replacement conditions. Results were confirmed by morphometric quantification.


Our results suggest that the abnormal morphology of capillary ECs and thick BABs correlates with pulmonary artery remodeling and RVH. These ultrastructural changes might represent possible mechanisms of secondary pulmonary hypertension in BPD.


Bronchopulmonary dysplasia Hyperoxia Newborn Pulmonary hypertension Pulmonary microvascular disease 



This study was supported by the Medical Research Institute, Tokyo Women’s Medical University. We would like to thank T. Matsuda, Tohoku University, K. Chou, Hokkaido University, and S. Shimizu, Tokyo Women’s Medical University, for their excellent support during lung structural analysis in this study. We would like to thank Editage ( for English language editing.


This study was supported by a Takako Satake Research Fellowship Grant (Grant Number 54) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI (Grant Number 16K10111).

Compliance with ethical standards

Conflict of interest

Teijin Pharma Ltd., Tokyo, Japan kindly provided the home oxygen condenser (TO-90-7H) without any compensation. They had no control over the interpretation, writing, or publication of this study. The authors have no financial relationships relevant to this article to disclose.

Supplementary material

795_2018_182_MOESM1_ESM.tif (2.5 mb)
Supplementary material 1 Chronic exposure to 85% oxygen for 14 days (O2-14d) in the developing lung caused abnormal alveolar development as indicated by: a increased mean chord length (Lm), b increased percentage of air space volume density (% AVD), and c decreased septal density. These parameters 30 did not improve under normal air replacement conditions after hyperoxia (O2-Air 21d). Data are expressed as means ± standard deviations (SDs); *P < 0.05 vs. Air-14d, P < 0.05 vs. Air-21d; n = 6 in each group (TIF 2542 KB)


  1. 1.
    Husain AN, Siddiqui NH, Stocker JT (1998) Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 29:710–717CrossRefPubMedGoogle Scholar
  2. 2.
    Jobe AH, Bancalari E (2001) Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163:1723–17239CrossRefPubMedGoogle Scholar
  3. 3.
    Abman SH (2001) Bronchopulmonary dysplasia: “a vascular hypothesis”. Am J Respir Crit Care Med 164:1755–1756CrossRefPubMedGoogle Scholar
  4. 4.
    Kunig AM, Balasubramaniam V, Markham NE et al (2005) Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats. Am J Physiol Lung Cell Mol Physiol 289:L529–L535CrossRefPubMedGoogle Scholar
  5. 5.
    Lin YJ, Markham NE, Balasubramaniam V et al (2005) Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats. Pediatr Res 58:22–29CrossRefPubMedGoogle Scholar
  6. 6.
    Wilson WL, Mullen M, Olley PM et al (1985) Hyperoxia-induced pulmonary vascular and lung abnormalities in young rats and potential for recovery. Pediatr Res 19:1059–1067CrossRefPubMedGoogle Scholar
  7. 7.
    de Visser YP, Walther FJ, Laghmani el H et al (2009) Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury. Respir Res 10:30CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bhat R, Salas AA, Foster C et al (2012) Prospective analysis of pulmonary hypertension in extremely low birth weight infants. Pediatrics 129:e682–e689CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Khemani E, McElhinney DB, Rhein L et al (2007) Pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era. Pediatrics 120:1260–1269CrossRefPubMedGoogle Scholar
  10. 10.
    Slaughter JL, Pakrashi T, Jones DE et al (2011) Echocardiographic detection of pulmonary hypertension in extremely low birth weight infants with bronchopulmonary dysplasia requiring prolonged positive pressure ventilation. J Perinatol 31:635–640CrossRefPubMedGoogle Scholar
  11. 11.
    Nakanishi H, Uchiyama A, Kusuda S (2016) Impact of pulmonary hypertension on neurodevelopmental outcome in preterm infants with bronchopulmonary dysplasia: a cohort study. J Perinatol 36:890–896CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bhatt AJ, Pryhuber GS, Huyck H et al. (2001) Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am J Respir Crit Care Med 15;164:1971–1980CrossRefGoogle Scholar
  13. 13.
    Nakanishi H, Sugiura T, Streisand JB et al (2007) TGF-β neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung. Am J Physiol Lung Cell Mol Physiol 293:L151–L161CrossRefPubMedGoogle Scholar
  14. 14.
    Roberts RJ, Weesner KM, Bucher JR (1983) Oxygen-induced alterations in lung vascular development in the newborn rat. Pediatr Res 17:368–375CrossRefPubMedGoogle Scholar
  15. 15.
    Mastin JP, Shelburne JD, Thet LA (1988) Subcellular changes in capillary endothelial cells during repair of hyperoxic lung injury. J Appl Physiol 64:689–696CrossRefPubMedGoogle Scholar
  16. 16.
    Kistler GS, Caldwell PR, Weibel ER (1967) Development of fine structural damage to alveolar and capillary lining cells in oxygen-poisoned rat lungs. J Cell Biol 32:605–628CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Durr RA, Dubaybo BA, Thet LA (1987) Repair of chronic hyperoxic lung injury: changes in lung ultrastructure and matrix. Exp Mol Pathol 47:219–240CrossRefPubMedGoogle Scholar
  18. 18.
    Cho SJ, George CL, Snyder JM et al (2005) Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol 33:622–628CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Berger J, Bhandari V (2014) Animal models of bronchopulmonary dysplasia. The term mouse models. Am J Physiol Lung Cell Mol Physiol 307:L936–L947CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Tschanz SA, Burri PH (2002) A new approach to detect structural differences in lung parenchyma using digital image analysis. Exp Lung Res 28:457–471CrossRefPubMedGoogle Scholar
  21. 21.
    Rasband WS. ImageJ, Bethesda, (2015) Maryland USA1997-2012 [cited 2015 August 12]. Available from:
  22. 22.
    Weibel ER (1979) Stereological methods. Academic Press, LondonGoogle Scholar
  23. 23.
    Hislop A, Reid L (1976) New findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. Br J Exp Pathol 57:542–554PubMedPubMedCentralGoogle Scholar
  24. 24.
    Pappas CT, Obara H, Bensch KG et al (1983) Effect of prolonged exposure to 80% oxygen on the lung of the newborn mouse. Lab Invest 48:735–748PubMedGoogle Scholar
  25. 25.
    Warner BB, Stuart LA, Papes RA et al (1998) Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol 275:L110–L117PubMedGoogle Scholar
  26. 26.
    Bucher JR, Roberts RJ (1981) The development of the newborn rat lung in hyperoxia: a dose-response study of lung growth, maturation, and changes in antioxidant enzyme activities. Pediatr Res 15:999–1008CrossRefPubMedGoogle Scholar
  27. 27.
    Randell SH, Mercer RR, Young SL (1990) Neonatal hyperoxia alters the pulmonary alveolar and capillary structure of 40-day-old rats. Am J Pathol 136:1259–1266PubMedPubMedCentralGoogle Scholar
  28. 28.
    Shaffer SG, O’Neill D, Bradt SK et al (1987) Chronic vascular pulmonary dysplasia associated with neonatal hyperoxia exposure in the rat. Pediatr Res 21:14–20CrossRefPubMedGoogle Scholar
  29. 29.
    Li C, Fu J, Liu H et al (2014) Hyperoxia arrests pulmonary development in newborn rats via disruption of endothelial tight junctions and downregulation of Cx40. Mol Med Rep 10:61–67CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hou A, Fu J, Yang H et al (2015) Hyperoxia stimulates the transdifferentiation of type II alveolar epithelial cells in newborn rats. Am J Physiol Lung Cell Mol Physiol 308:L861–L872CrossRefPubMedGoogle Scholar
  31. 31.
    Ahlfeld SK, Gao Y, Conway SJ et al (2015) Relationship of structural to functional impairment during alveolar-capillary membrane development. Am J Pathol 185:913–919CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Maniscalco WM, Watkins RH, Pryhuber GS et al (2002) Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons. Am J Physiol Lung Cell Mol Physiol 282:L811–L823CrossRefPubMedGoogle Scholar
  33. 33.
    Bachiller PR, Nakanishi H, Roberts JD Jr (2010) Transforming growth factor-beta modulates the expression of nitric oxide signaling enzymes in the injured developing lung and in vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 298:L324-L334. CrossRefGoogle Scholar

Copyright information

© The Japanese Society for Clinical Molecular Morphology 2018

Authors and Affiliations

  • Hidehiko Nakanishi
    • 1
    Email author
  • Shunichi Morikawa
    • 2
  • Shuji Kitahara
    • 2
    • 3
  • Asuka Yoshii
    • 2
    • 4
  • Atsushi Uchiyama
    • 1
  • Satoshi Kusuda
    • 1
  • Taichi Ezaki
    • 2
  1. 1.Department of Neonatology, Maternal and Perinatal Center Neonatal DivisionTokyo Women’s Medical UniversityTokyoJapan
  2. 2.Department of Anatomy and Developmental BiologyTokyo Women’s Medical University School of MedicineTokyoJapan
  3. 3.Department of Radiation Oncology, Harvard Medical SchoolMassachusetts General HospitalBostonUSA
  4. 4.Department of Pharmacology and Experimental TherapeuticsBoston University School of MedicineBostonUSA

Personalised recommendations