Chlorambucil-induced postclosure exencephaly and axial skeletal abnormalities in rat fetuses

Abstract

Open neural tube defects (NTD) are reported to arise from failure of the embryonic neural folds to close or rupture of a previously closed neural tube. The critical period for dysmorphogenesis of the neural tube by either mechanism is of clinical importance. We had previously reported that single doses of cyclophosphamide (CPA), administered to pregnant rats resulted in reopening of the closed neural tube. The objective of the present study was to determine if other antineoplastic drugs also had similar ability to cause rupture of the closed neural tube. Therefore, single doses of chlorambucil dissolved in bicarbonate buffer were administered to Wistar rats on one of gestation days (GD) 11 through 14 (i.e., well after closure of the neural tube) and fetuses were examined on GD 20. It was observed that the window of susceptibility extended from GD 11 through 14 and that a single dose of 10 mg/kg of the drug was most effective. The affected fetuses had the prosencephalon and mesencephalon parts of the brain protruding through a wide opening of the cranial roof. The exposed brain tissue was hemorrhagic and covered by a thin, transparent and often porous membrane. The cranial vault was missing. Numerous malformations of the axial skeleton and of several nonneural organs were found to accompany the brain defect. Electron microscopic and light microscopic studies revealed extensive cell death, fragmentation, and phagocytosis of dead cells and vacuolization of the neuroepithelium (NE) within 12 h of the drug treatment. Cell death per se was not pronounced in the cranial mesenchyme (ME). The vacuoles in the NE coalesced into small cavities. The ME failed to proliferate adequately and organize itself into the cranial vault primordium. Hemorrhage and disorganization of the NE was progressive. The fluid that escaped into the interstitium seemed to extend into the cysts that developed in the ME externally and into the ventricular system internally. The edema of the ME thus might have contributed to disruption of precursor mesenchymal tissue and consequently to malformation of the cranial bones. Large hematomas and cysts also developed on the basal aspect of the crumbling brain vesicles and appeared to lift the neural tissue out of the shallow cranial cavity. As previously reported for CPA, these data provide evidence for the ability of chlorambucil to cause postclosure exencephaly in rat fetuses for a susceptible period that is considerably long.