Developmental susceptibility of neurons to transient tetrahydrobiopterin insufficiency and antenatal hypoxia–ischemia in fetal rabbits

Abstract

Tetrahydrobiopterin (BH 4 ) is important for normal brain development as congenital BH 4 deficiencies manifest movement disorders at different childhood ages. BH 4 transitions from very low levels in fetal brains to higher 'adult' levels postnatally, with the highest levels in the thalamus. Maternal supplementation with BH 4 -precursor, sepiapterin, reduces postnatal motor deficits and perinatal deaths following 40-min fetal hypoxia-ischemia (HI) at 70% gestation, suggesting brain BH 4 is important in improving function after HI. We tested the hypothesis that the intrinsically low concentrations of BH 4 made fetal neurons vulnerable to added insults. Brains were obtained from either naïve fetal rabbits or after 40-min HI, at 70% (E22) and 92% gestation (E29). Neuronal cultures were prepared from basal ganglia, cortex and thalamus, regions with different intrinsic levels of BH 4 . Cultures were grown with or without added BH 4 to 48 hours. Cell survival and mitochondrial function were determined by flow cytometry. At E22, thalamic cells had the lowest survival rate in a BH 4 -free milieu, in both control and HI groups, while BH 4 supplementation ex vivo increased neuronal survival in only HI cells. Neuronal survival was similar in all regions without BH 4 at E29. BH 4 supplementation increased cell survival and cells with intact mitochondrial membrane potential, from basal ganglia and cortex, but not thalamus. After E29 HI, however, the benefit of BH 4 was limited to cortical neurons. We conclude that BH 4 is important for fetal neuronal survival following HI especially in the premature thalamus. Supplementation of BH 4 has a greater benefit at an earlier gestational age.

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon

“…However, we were unable to support the survival of the neural cells in culture beyond 7 days. Our research is aligned with other studies showing that it is difficult to extend the culture period of fetal neurons in vitro [143132]. Despite the incorporation of novel reagents, such as RA in combination with other prosurvival factors such as bFGF, we still could not support survival long-term in the dish.…”

Neuronal vulnerability to fetal hypoxia-reoxygenation injury and motor deficit development relies on regional brain tetrahydrobiopterin levels

“…The present study also explains the discrepancy of ex vivo survival of neuronal cells isolated from different brain regions being unrelated to the differences in endogenous levels in our previous publication [9]. Based on tetrahydrobiopterin levels, the E22 thalamus neurons at 70% gestation were expected to show increased resistance to H–I injury compared to cortical neurons with a comparatively lower tetrahydrobiopterin content.…”

“…Previously we showed that cumulative increases of tetrahydrobiopterin in the thalamus, basal ganglia, and cortex occur in naïve fetal brains between 70%–92% gestation, suggesting a role in normal development [8]. In a study to examine if tetrahydrobiopterin influenced neuronal survival, neuronal cultures from different brain regions of basal ganglia, cortex, and thalamus at 70% and 92% gestation, we showed differential regional susceptibility to ex vivo tetrahydrobiopterin levels, especially following H–I, with the thalamus being most vulnerable in the preterm age [9]. Increasing tetrahydrobiopterin, however, improved neuronal survival after H–I especially in the premature thalamus [9].…”

Tetrahydrobiopterin in antenatal brain hypoxia-ischemia-induced motor impairments and cerebral palsy

“…Different survival rates are found in ex vivo primary neuronal cultures from the basal ganglia, cortex, and thalamus following in vivo HI. Thalamic cells from 70% gestation exhibit the lowest survival rate in a BH 4 -free milieu, in control and HI groups [71] . HI but not control cells are amenable to rescue with supplemented ex vivo BH 4 , suggesting a causal relationship between HI injury and BH 4 -dependent pathways.…”

“…The fetal rabbit model indicates that BH 4 pathway is a target of HI ( Fig. 4 ), and a two-hit model likely explain susceptibility to injury [71] . The first condition necessary is low developmental, like in prematurity, BH 4 levels.…”