Pathogenesis of cerebral malformations in human fetuses with meningomyelocele

Abstract

Background: Fetal spina bifida aperta (SBA) is characterized by a spinal meningomyelocele (MMC) and associated with cerebral pathology, such as hydrocephalus and Chiari II malformation. In various animal models, it has been suggested that a loss of ventricular lining (neuroepithelial/ependymal denudation) may trigger cerebral pathology. In fetuses with MMC, little is known about neuroepithelial/ependymal denudation and the initiating pathological events.The objective of this study was to investigate whether neuroepithelial/ependymal denudation occurs in human fetuses and neonates with MMC, and if so, whether it is associated with the onset of hydrocephalus.

Methods: Seven fetuses and 1 neonate (16-40 week gestational age, GA) with MMC and 6 fetuses with normal cerebral development (22-41 week GA) were included in the study. Identification of fetal MMC and clinical surveillance of fetal head circumference and ventricular width was performed by ultrasound (US). After birth, MMC was confirmed by histology. We characterized hydrocephalus by increased head circumference in association with ventriculomegaly. The median time interval between fetal cerebral ultrasound and fixing tissue for histology was four days.

Results: At 16 weeks GA, we observed neuroepithelial/ependymal denudation in the aqueduct and telencephalon together with sub-cortical heterotopias in absence of hydrocephalus and/or Chiari II malformation. At 21-34 weeks GA, we observed concurrence of aqueductal neuroepithelial/ependymal denudation and progenitor cell loss with the Chiari II malformation, whereas hydrocephalus was absent. At 37-40 weeks GA, neuroepithelial/ependymal denudation coincided with Chiari II malformation and hydrocephalus. Sub-arachnoidal fibrosis at the convexity was absent in all fetuses but present in the neonate.

Conclusion: In fetal SBA, neuroepithelial/ependymal denudation in the telencephalon and the aqueduct can occur before Chiari II malformation and/or hydrocephalus. Since denuded areas cannot re-establish cell function, neuro-developmental consequences could induce permanent cerebral pathology.

Figure 1

Ependymal denudation and secondary astroglial reaction at the aqueduct of a fetus with MMC (39 weeks GA). A. Transverse section through the aqueduct with immunostaining for caveolin. The ependymal cells are strongly reactive. Arrows indicate an area devoid of ependyma. SA = aqueduct of Sylvius. Scale bar = 100 μm. Insert: Detailed magnification of immature ependyma immunoreacting with anti-caveolin. Scale bar = 20 μm. B. Section adjacent to that of previous figure, stained with haematoxylin-eosin. Arrows indicate an area devoid of ependyma. Scale bar = 50 μm. C. Section adjacent to that of previous figure immunostained with anti-GFAP. Immunoreactive cell processes and cells (astrocytes) are confined to the denuded area (white star). Subependymal neuropil of adjacent areas lined by ependymal cells has only few astrocytes (small arrow) and virtually no astrocyte processes (black stars). The large arrow points to the border of the denuded area. Scale bar = 50 μm. Top insert: Detailed magnification of the non-reactive ependymal and subependymal neuropil. Scale bar = 30 μm. Bottom insert: numerous astrocytes (arrow) in the vicinity of the denuded area. Scale bar = 35 μm.

Figure 2

Haematoxylin-eosin staining of the aqueduct throughout gestation. A. Transverse section through a wide-open aqueduct of a control fetus (40 weeks GA). Scale bar = 500 μm. B. Transverse section through the aqueduct of a fetus with MMC (22 weeks GA). The lumen appears narrow or slit like. Scale bar = 100 μm. C. Transverse section through the aqueduct of a fetus with MMC (21 weeks GA). Several infoldings are present (i.e. forking). Scale bar = 100 μm. D. Secondary damage at the aqueduct of a fetus with MMC (21 weeks GA). Arrowheads indicate haemosiderophages (I), gliosis (II), and recent bleeding (III). Scale bar = 100 μm. E. Transverse section through the aqueduct of a fetus with MMC (37 weeks GA). The figure shows sub-ventricular rosette formation (R). Scale bar = 50 μm.

Figure 3

Ependymal denudation and subcortical heterotopias. A. Nestin staining of the aqueduct of a SBA fetus of 21 weeks GA. Arrowheads indicate intact (I) and denuded (II) ependymal lining. At the denuded area, the figure shows reduction of nestin-positive cells (brown; DAB) indicative of progenitor cell loss. Scale bar = 100 μm. B. Haematoxylin-eosin staining of the telencephalon of a fetus with MMC (16 weeks GA). Arrowheads indicate subcortical heterotopias associated with ependymal denudation of the lateral ventricle. Scale bar = 500 μm.

Figure 4

Histological and ultrasound data in pre- and postnatal SBA according to gestational age. Histological and US findings are illustrated in chronological order, according to gestational (GA). The time axis is in the middle part of the figure. Histological data of aqueduct and convexity are indicated at the upper part of the figure. US data of ventricular size (Chiari II malformation, ventriculomegaly and macrocephaly) are indicated at the lower part of the figure. During the first half of gestation, neuroepithelial/ependymal denudation is observed before the onset of Chiari II malformation and hydrocephalus.