Long-term pulmonary and neurodevelopmental impairment in a fetal growth restriction rabbit model

Abstract

Fetal growth restriction (FGR) remains one of the main obstetrical problems worldwide, with consequences beyond perinatal life. Animal models with developmental and structural similarities to the human are essential to understand FGR long-term consequences and design novel therapeutic strategies aimed at preventing or ameliorating them. Herein, we described the long-term consequences of FGR in pulmonary function, structure, and gene expression, and characterized neurodevelopmental sequelae up to preadolescence in a rabbit model. FGR was induced at gestational day 25 by surgically reducing placental blood supply in one uterine horn, leaving the contralateral horn as internal control. Neonatal rabbits born near term were assigned to foster care in mixed groups until postnatal day (PND) 21. At that time, one group underwent pulmonary biomechanical testing followed by lung morphometry and gene expression analysis. A second group underwent longitudinal neurobehavioral assessment until PND 60 followed by brain harvesting for multiregional oligodendrocyte and microglia quantification. FGR was associated with impaired pulmonary function and lung development at PND 21. FGR rabbits had higher respiratory resistance and altered parenchymal biomechanical properties in the lungs. FGR lungs presented thicker alveolar septal walls and reduced alveolar space. Furthermore, the airway smooth muscle content was increased, and the tunica media of the intra-acinar pulmonary arteries was thicker. In addition, FGR was associated with anxiety-like behavior, impaired memory and attention, and lower oligodendrocyte proportion in the frontal cortex and white matter. In conclusion, we documented and characterized the detrimental pulmonary function and structural changes after FGR, independent of prematurity, and beyond the neonatal period for the first time in the rabbit model, and describe the oligodendrocyte alteration in pre-adolescent rabbit brains. This characterization will allow researchers to develop and test therapies to treat FGR and prevent its sequelae.

Figure 1

Postnatal survival and biometrics of FGR and control rabbits. (A) Postnatal survival of 155 rabbits from 30 litters. Controls without littermates were excluded from further analysis. (B) Birth weight of animals that survived until last assessment time point (n, FGR = 16; control = 41). (C) Weight evolution of animals that survived until last assessment time point. Data were analyzed using a linear mixed-effects model. Values as mean ± SD, **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Pulmonary functional tests at postnatal day 21. (A) Results from forced oscillation maneuvers, showing increased tissue damping and elastance in lung parenchyma from FGR rabbits. (B) Increased resistance in the respiratory system, and in the conducting airway in the FGR group. Data were analyzed using a linear mixed-effects model. Values as mean ± SD, **p < 0.01; ****p < 0.0001.

Figure 3

Histology in postnatal day 21 rabbit lungs (FGR = 6; controls = 10). (A) Alveolar morphometric data. (B) Representative images of histological H&E-stained slides, with arrows showing alveolar walls. (C) Airway smooth muscle content. (D) Representative α-SMA images of conducting airways, smooth muscle is dark brown. (E) Pulmonary arteries medial thickness. F. Representative α-SMA images of peripheric pulmonary arteries. Data were analyzed using a linear mixed-effects model. Values as mean ± SD, or median and IQR according to data characteristics. *p < 0.05; ***p < 0.001.

Figure 4

(A, C) Individual scores of neurobehavioral assessment at postnatal days 1 and 7, respectively. (B, D) Representative images of kittens at each gestational age. FGR n = 10; controls n = 22. Values as median and IQR, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Neuropathology in postnatal day 60 rabbit brains. (A) Percentage of oligodendrocytes in front and midbrain structures. (B) Representative images from frontal cortex, showing olig2 + cells (arrows) and neurons (arrowheads) from FGR and control brains. (C) Density of microglia in front and midbrain structures. (D) Representative images from the frontal cortex, showing Iba1 + cells (arrows) and neurons (arrowheads) from FGR and control brains. CA3: cornu ammonis 3. Data were analyzed using a linear mixed-effects model. Values as mean ± SD, **p < 0.01.