Does growth restriction increase the vulnerability to acute ventilation-induced brain injury in newborn lambs? Implications for future health and disease.

Fetal growth restriction (FGR) and preterm birth are frequent co-morbidities, both are independent risks for brain injury. However, few studies have examined the mechanisms by which preterm FGR increases the risk of adverse neurological outcomes. We aimed to determine the effects of prematurity and mechanical ventilation (VENT) on the brain of FGR and appropriately grown (AG, control) lambs. We hypothesized that FGR preterm lambs are more vulnerable to ventilation-induced acute brain injury. FGR was surgically induced in fetal sheep (0.7 gestation) by ligation of a single umbilical artery. After 4 weeks, preterm lambs were euthanized at delivery or delivered and ventilated for 2 h before euthanasia. Brains and cerebrospinal fluid (CSF) were collected for analysis of molecular and structural indices of early brain injury. FGRVENT lambs had increased oxidative cell damage and brain injury marker S100B levels compared with all other groups. Mechanical ventilation increased inflammatory marker IL-8 within the brain of FGRVENT and AGVENT lambs. Abnormalities in the neurovascular unit and increased blood-brain barrier permeability were observed in FGRVENT lambs, as well as an altered density of vascular tight junctions markers. FGR and AG preterm lambs have different responses to acute injurious mechanical ventilation, changes which appear to have been developmentally programmed in utero.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses.

OBJECTIVE: To study the consequences of glucocorticoid treatment in fetal growth restriction (FGR) on cardiac function. SETTING: Laboratory. SAMPLE: Sheep. METHODS: Growth restriction was induced in sheep fetuses using single umbilical artery ligation (SUAL) on days 105-110 of gestation (term 147). Control fetuses were not ligated. Betamethasone (BM) (11.4 mg intramuscularly) or saline was administered to ewes on days 5 and 6 after surgery. Ewes were anaesthetised on day 7, the fetuses were removed, and their hearts were mounted on a Langendorff apparatus. Balloon catheters were inserted into the right and left ventricles. OUTCOME MEASURES: Ventricular contractile function and infarct area following ischaemia/reperfusion. RESULTS: The SUAL resulted in FGR (body weight 77% of control). The FGR was associated with increases in basal left ventricular pressure development and rates of contraction and relaxation. Right ventricular contraction was unaffected. Following brief ischaemia/reperfusion, the infarct area in FGR hearts was increased four-fold compared with controls. Antenatal BM resulted in a proportional increase in heart size and coronary flow, especially in FGR fetuses, and left ventricular pressure and heart rate responses to ß-adrenoceptor activation were increased. CONCLUSIONS: Fetal hearts rapidly adapt to FGR to maintain substrate delivery to the brain and heart. The FGR greatly enhanced the area of ischaemia, with implications for susceptibility in postnatal life. Antenatal BM treatment does not interfere with these cardiac changes but appears to increase left ventricle ß-adrenoceptor responsiveness, which may render the offspring vulnerable to subsequent cardiac dysfunction.

Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain.

The neurosteroid allopregnanolone (AP) is a GABAergic agonist that suppresses central nervous system (CNS) activity in the adult brain, and by reducing excitotoxicity is considered to be neuroprotective. A role for neurosteroids in the developing brain, particularly in late gestation, is still debated. The aim of this study was to investigate effects on proliferation and cell death in the brain of late gestation fetal sheep after inhibition of AP synthesis using finasteride, a 5alpha-reductase type 2 (5alpha-R2) inhibitor. Catheters were implanted in fetal sheep at approximately 125 days of gestation. At 3-4 days postsurgery, fetuses received infusions of either finasteride (20 mg/kg/h; n=5), the AP analogue alfaxalone (5 mg/kg/h; n=5), or finasteride and alfaxalone together (n=5). Brains were obtained at 24 h after infusion to determine cell death (apoptotic or necrotic) and cell proliferation in the hippocampus and cerebellum, areas known to be susceptible to excitotoxic damage. Finasteride treatment significantly increased apoptosis (activated caspase-3 expression) in hippocampal CA3 and CA1, and cerebellar molecular and granular layers, an effect abolished by co-infusion of alfaxalone and finasteride. Double-label immunohistochemistry showed that both neurons and astrocytes were caspase-3 positive. Finasteride treatment also increased the number of dead (pyknotic) cells in the hippocampus and cerebellum (Purkinje cells), but not when finasteride+alfaxalone was infused. Cell proliferation (Ki-67-immunoreactivity) increased after finasteride treatment; double-labeling showed the majority of Ki-67-positive cells were astrocytes. Thus, steroids such as AP appear to influence the constitutive rate of apoptosis and proliferation in the hippocampus and cerebellum of the fetal brain, and suggest an important role for neurosteroids in the development of the brain.

Inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in the late gestation fetal sheep.

Allopregnanolone (AP) is a potent GABAergic agonist that suppresses CNS activity, seizure threshold, and excitotoxicity in the adult brain. AP is present in the fetal sheep brain and increases rapidly after asphyxial insult due to increased 5alpha-reductase type-2 (5alphaR-2) expression. The aim of this study was to use finasteride to suppress fetal neurosteroid synthesis, and then determine the effect on brain injury, particularly in the hippocampus, of asphyxia induced in utero by brief occlusion of the umbilical cord. Catheters and an inflatable umbilical cord cuff were implanted in fetal sheep at approximately 125 days gestation. Five days later the fetuses received either finasteride (20 mg/kg/h) or vehicle (40% hydroxypropyl-beta-cyclodextrin) for 2 h. The umbilical cord was occluded (UCO) for 5 min at 30 min after starting the infusion. The fetal brain was obtained 24 h later for examination of activated caspase-3 expression as an index of apoptosis, and to measure AP content. Finasteride treatment alone significantly reduced AP content and increased the number of caspase-3 positive cells in the hippocampus, cerebellum, and the subcallosal bundle, indicating that AP modulates the normal rate of apoptosis in the developing brain. UCO in vehicle and finasteride-treated fetuses produced a similar, marked decrease in O2 saturation (5.8+/-0.6%), but after finasteride treatment UCO caused a significantly greater increase in the number of caspase-3 positive cells in the hippocampal cornu ammonis 3 (CA3) (57.3+/-1.6%) compared with the vehicle-treated fetuses. Thus, 5alpha-reduced steroids such as AP may be protective in reducing cell death following acute fetal asphyxia. Perturbation of normal fetal neurosteroid levels in late gestation (e.g. due to preterm birth, or maternal synthetic steroid treatment to induce fetal lung maturation) could adversely affect brain development and increase its vulnerability to injury.