Early neonatal loss of inhibitory synaptic input to the spinal motor neurons confers spina bifida-like leg dysfunction in a chicken model

Abstract

Spina bifida aperta (SBA), one of the most common congenital malformations, causes lifelong neurological complications, particularly in terms of motor dysfunction. Fetuses with SBA exhibit voluntary leg movements in utero and during early neonatal life, but these disappear within the first few weeks after birth. However, the pathophysiological sequence underlying such motor dysfunction remains unclear. Additionally, because important insights have yet to be obtained from human cases, an appropriate animal model is essential. Here, we investigated the neuropathological mechanisms of progression of SBA-like motor dysfunctions in a neural tube surgery-induced chicken model of SBA at different pathogenesis points ranging from embryonic to posthatch ages. We found that chicks with SBA-like features lose voluntary leg movements and subsequently exhibit lower-limb paralysis within the first 2 weeks after hatching, coinciding with the synaptic change-induced disruption of spinal motor networks at the site of the SBA lesion in the lumbosacral region. Such synaptic changes reduced the ratio of inhibitory-to-excitatory inputs to motor neurons and were associated with a drastic loss of γ-aminobutyric acid (GABA)ergic inputs and upregulation of the cholinergic activities of motor neurons. Furthermore, most of the neurons in ventral horns, which appeared to be suffering from excitotoxicity during the early postnatal days, underwent apoptosis. However, the triggers of cellular abnormalization and neurodegenerative signaling were evident in the middle- to late-gestational stages, probably attributable to the amniotic fluid-induced in ovo milieu. In conclusion, we found that early neonatal loss of neurons in the ventral horn of exposed spinal cord affords novel insights into the pathophysiology of SBA-like leg dysfunction.

Suppression of GABAergic transmission in the spinal dorsal horn induces pain-related behavior in a chicken model of spina bifida

“…1 and Suppl. Video 1) and motor dysfunction [21], consistent with reports of human neonates with SBA [6, 8, 10]. Therefore, pathological characterization of spinal motor networks of SBA chicks would provide insight into the progression of SBA-like neurological complications.…”

“…To produce SBA chicks, surgical manipulation of the neural plate was carried out as described previously [19, 21]. Briefly, the eggshell and amnion were opened and placed under a stereomicroscope to determine the developmental stage of the embryo.…”

“…Spinal cord sections from the open defect area (lumbosacral region) from SBA chicks, and from a similar location in the normal chicks, were collected on embryonic day (ED)-14, ED-18, PD-2, PD-4, and PD-10. The incisions of the roof plates of the neural tubes of SBA embryos were confirmed by observing open defects on the backs of the chicks, and the induction of SBA in the chicks post-hatching was confirmed by observing open defects on the backs of the chicks with leg dysfunction ([21]; Fig. 1a; Suppl.…”

“…Immunofluorescence staining was performed as described previously [21]. For double immunofluorescence staining, the sections were incubated for 60 h at 4°C in a solution containing rabbit polyclonal anti-GABA (1:3,000; Sigma) plus mouse monoclonal anti-CB or mouse monoclonal anti-CR (1:1000; SWANT) antibodies; and rabbit polyclonal anti-caspase 3 (1:1000; Bioss) plus mouse monoclonal anti-CB (1:1000; SWANT) antibodies.…”

“…These facts led us to postulate the existence of cross-talk between SBA-related pain complications and alteration in GABAergic transmission in the spinal cord dorsal horn. Accordingly, we explored the kinetics of GABAergic transmission in the spinal cord dorsal horn from gestation to post-hatching in a chicken model of SBA to obtain pathophysiological data on the sequence of events associated with SBA-like neurological disorders [19–21].…”