Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Abstract

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Premature Neural Progenitor Cell Differentiation Into Astrocytes in Retinoic Acid-Induced Spina Bifida Rat Model

“…Astrocytes respond to stress or tissue damage within the CNS by proliferating, activating, and interacting with other cell types through signaling molecules (Alonso, 2005). Therefore, it is possible that these cells are responding to the tissue damage induced by the enzymatic action of the amniotic fluid insult (Botto et al, 1999;Oria et al, 2019); however, the mechanism behind this reaction remains unclear and should be a focus of future pre-clinical studies.…”

Comparison of inherited neural tube defects in companion animals and livestock

“…Anencephaly involves the absence and degeneration of all, or part of the brain as a result of the failure of the cranial neural tube to close (Cantile & Youssef, 2016; Wallingford et al, 2013; Wood & Smith, 1984). Cranioschisis (lack of closure of the skull bones) is often associated with exencephaly, a term describing the exposure of the brain tissue, which typically results in full or partial anencephaly over time (Oria et al, 2019; Wood & Smith, 1984).…”

“…Many rodent models have been developed and are typically involved in identifying candidate genes for human studies and elucidating pathways involved in NTD formation (Copp et al, 2003; Copp & Greene, 2010; Harris & Juriloff, 2010; Oria et al, 2019). As noted above, laboratory rodents contributed strongly to the understanding of NTDs.…”

“…As noted above, laboratory rodents contributed strongly to the understanding of NTDs. In the case of meningomyelocele, studies of spinal cord damage after exposure to amniotic fluid and neuronal tissue differentiation in affected curly‐tail mice encouraged the investigation of surgical repair of lesions in humans (D. S. Heffez, Aryanpur, Hutchins, & Freeman, 1990; Oria et al, 2019; Selçuki, Manning, & Bernfield, 2001). Furthermore, identification of the role of inositol in the developing rat embryo (9‐day‐old) has led to clinical trials studying the possibility of inositol supplementation to prevent folate‐resistant defects of NTDs (Cockroft, 1988; Greene et al, 2016).…”