Limited effects of preterm birth and the first enteral nutrition on cerebellum morphology and gene expression in piglets

Abstract

Preterm pigs show many signs of immaturity that are characteristic of preterm infants. In preterm infants, the cerebellum grows particularly rapid and hypoplasia and cellular lesions are associated with motor dysfunction and cognitive deficits. We hypothesized that functional brain delays observed in preterm pigs would be paralleled by both structural and molecular differences in the cerebellum relative to term born piglets. Cerebella were collected from term (n = 56) and preterm (90% gestation, n = 112) pigs at 0, 5, and 26 days after birth for stereological volume estimations, large-scale qPCR gene expression analyses (selected neurodevelopmental genes) and western blot protein expression analysis (Sonic Hedgehog pathway). Memory and learning was tested using a T-maze, documenting that preterm pigs showed delayed learning. Preterm pigs also showed reduced volume of both white and gray matter at all three ages but the proportion of white matter increased postnatally, relative to term pigs. Early initiation of enteral nutrition had limited structural or molecular effects. The Sonic Hedgehog pathway was unaffected by preterm birth. Few differences in expression of the selected genes were found, except consistently higher mRNA levels of Midkine, p75, and Neurotrophic factor 3 in the preterm cerebellum postnatally, probably reflecting an adaptive response to preterm birth. Pig cerebellar development appears more affected by postconceptional age than by environmental factors at birth or postnatally. Compensatory mechanisms following preterm birth may include faster white matter growth and increased expression of selected genes for neurotrophic factors and regulation of angiogenesis. While the pig cerebellum is immature in 90% gestation preterm pigs, it appears relatively mature and resilient toward environmental factors.

Keywords: Enteral and parenteral nutrition; neonatal brain development; postconceptional age; prematurity.

Figure 1

Time lines for Experiments 1 and 2. The numbers on the preterm and term lines indicate postnatal days, whereas the numbers on the postconceptional age (PCA) line indicate postconceptional age, starting at day 106. The 12‐day difference in birth age between the groups is shown as “−12” on the line for term pigs. The T‐maze test was performed only for pigs in Experiment 1. Tissue sampling was done only for pigs from Experiment 2.

Figure 2

T‐maze experiment. T‐maze test using term (black symbols, n = 6) and preterm (white symbols, n = 17) pigs. Each symbol represents average performance (mean ± SEM) of all pigs and of all ten trials for each of six consecutive days (A1–A6), starting on postnatal day 15. The term pigs reached the learning criterion (80% correct choices) 3 days before the preterm pigs (*P < 0.05).

Figure 3

Postnatal comparison of cerebellar volumes. For both white matter (A) and gray matter (B), the cerebellar volumes were smaller for preterm (gray bars), relative to term (white bars) pigs at all measured ages (*P < 0.05, **P < 0.01, ***P < 0.001). The gray/white matter ratio (C) was higher in preterm pigs at birth (*P < 0.05).

Figure 4

Development of cerebellar gray and white matter volumes. Each symbol shows a volume estimate for a single pig (black circles = gray matter, open circles = white matter. Dotted lines display the linear fit of volume as a function of postconceptional age of PCA 106, 118, 123, and 144 corresponding to preterm postnatal age 0, and term postnatal ages 0, 5, and 26 days. The shaded gray area shows the prediction intervals for the linear fits. The black bars shows means ± confidence intervals for PCA 111 and 132, corresponding to preterm postnatal age 5, and 26 days. The observed means for PCA 111 and 132 were not significantly different from the expected values calculated from the growth curves (P > 0.05). The total relative growth from 90% gestation to 26 days in term pigs was 108 and 64% for white and gray matter, respectively.