Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction

Abstract

The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at −80 C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level.

Structural Brain Changes during the Neonatal Period in a Rabbit Model of Intrauterine Growth Restriction

“…This result goes in line with previous evidences in which positive correlation between O4-OL in the CC and birth weight were also found on postnatal day 7 in a moderate IUGR rat model [28]. Going in the same line, a recent article from our group has demonstrated impairments in the O4-OL differentiation after 5 days of incubation in a novel neurosphere culture obtained from our animal model [42]. Disruption in the oligodendrocyte maturation process dur-DOI: 10.1159/000512948 ing neurodevelopment is of relevance as it has been linked with neurodevelopmental impairments later on [15,20,29,38,72].…”

“…In comparison to in vivo studies, the in vitro experimental setting is even more controlled allowing to obtain significant differences even with a smaller number of animals. In fact, a recent study from our group working on the same IUGR rabbit model but performing histological analyses in in vitro conditions has demonstrated impairments in the O4-OL using 4 animals from each group [42]. Another limitation of our study is that we did not explore if the results reported here could be extrapolated to other brain regions and in different time points.…”

“…A total of 6 pregnant rabbits were included in the IUGR induction protocol for 25 days of gestation. The selection of 6 pregnant rabbits was done following previous experience from the group [31, 35, 42]. Considering fetal mortality related to the IUGR model, 12 IUGR models and 18 controls were theoretically obtained at the time of delivery.…”

“…WM Evaluation in CC: pre-OLs, Microglia, and Astrocytes Following previous studies with the same animal model, 4 brains per experimental group would be required for the assessment of oligodendrocytes [42]. However, due to differences in the methodology used (in vitro vs. ex vivo), 8 brains per experimental group were finally included.…”

Docosahexaenoic Acid and Lactoferrin Effects on the Brain and Placenta in a Rabbit Model of Intrauterine Growth Restriction 

“…At 25 days of gestation, a total of 19 pregnant rabbits were subjected to the IUGR induction protocol. The selection of the sample size was made taking into account the mortality related to the IUGR model following previous experience in the group 7,14,29,60 . This sample size would allow us to include the required number of animals for each analysis of the study following previous evidence (see each paragraph for detailed information).…”