Duration of fetoscopic spina bifida repair does not affect the central nervous system in fetal lambs

Abstract

Prenatal spina bifida aperta (SBA) repair improves neurologic outcomes, yet comes with a significant risk of prematurity and uterine scar-related complications. To reduce those, different fetoscopic techniques, e.g. with varying number of ports, are being explored. This has an impact on the duration of the procedure, potentially affecting central nervous system (CNS) development. Both the condition and anesthesia can impact the CNS, particularly the hippocampus, a region vital for prospective and episodic memory. Previous animal studies have shown the potential influence of anesthesia, premature delivery, and maternal surgery during pregnancy on this area. To compare the effects of 2- versus 3-port fetoscopic SBA repair in the fetal lamb model using neuron count of the hippocampus as primary outcome. Based on the hippocampal neuron count from previous lamb experiments, we calculated that we required five animals per group to achieve a statistical power of ≥ 80%. A SBA defect was created in fetal lambs at 75 days of gestation (term: 145 days). At 100 days, fetuses underwent either a 2-port or 3-port fetoscopic repair. At 143 days, all surviving fetuses were delivered by cesarean section, anesthetized and transcardially perfused with a mixture of formaldehyde and gadolinium. Next, they underwent neonatal brain and spine MRI following which these organs were harvested for histology. Hippocampus, frontal cortex, caudate nucleus, and cerebellum samples were immunostained to identify neurons, astrocytes, microglia, and markers associated with cell proliferation, myelination, and synapses. The degree of hindbrain herniation and the ventricular diameter were measured on MRI images and volumes of relevant brain and medulla areas were segmented. Both treatment groups included five fetuses and nine unoperated littermates served as normal controls. The durations for both skin-to-skin (341 ± 31 vs 287 ± 40 min; P=0.04) and fetal surgery (183 ± 30 vs 128 ± 22; P=0.01) were longer for the 2-port than for the 3-port approach. There were no significant differences in neuron density in the hippocampus, frontal cortex and cerebellum. In the caudate nucleus, the neuron count was higher in the 2-port group (965 ± 156 vs. 767 ± 92 neurons/mm2, P=0.04). There were neither differences in proliferation, astrogliosis, synaptophysin, or myelin. The tip of the cerebellar vermis was closer to the foramen magnum in 2-port animals compared to 3-port animals (-0.72 ± 0.67 mm vs. -2.47 ± 0.91 mm, P=0.009). There were no significant differences in the ratio of hippocampus, caudate nucleus, or cerebellar volume to body weight. For the spine, no differences were noted in spine volume/body weight ratio for the lower (L1/L2), mid (L3/L4), and higher (L5/6) levels. Compared to controls, in repaired animals the cerebellar vermis tip lays closer to the foramen magnum, parietal ventricles were enlarged and medulla volumes were reduced. In the experimental spina bifida fetal lamb model, a 2-port repair took 40% longer than a 3-port repair. There was however no indication of any relevant morphologic differences in the fetal brain.