Our previous collaborational study has shown that ETB−/− rats can be rescued from premature death when colostomy is performed at neonatal period (Stamp et al., 2015). This operation enables us to keep ETB−/− rats survive up to four to six weeks when they are sacrificed. The present study confirms that ETB−/− rats have similar weight gain as their ETB+/+ and ETB+/− littermates that receive the same colostomy surgery. This indicates that the effects on cell death in the brain are unlikely related to malnutrition. However, rats with colostomy have significantly lower body weight than sham-operated ETB+/+ rats, which suggests that colostomy per se compromises body growth. The mechanisms are related to decreased fluid and electrolyte absorption and accelerates gastric emptying as in human with colostomy. Therefore, the comparison of brain development has to be between different genotypes with colostomy. There was no difference in the pain behaviors as well as the general body size of the rats.
The effect of null mutation of EDNRB was further examined on cell survival in the cerebellum, the cerebral cortex, the dentate gyrus, and the hippocampus by comparing ETB−/−, ETB+/−, and ETB+/+ littermates that received colostomy. The deficiency of EDNRB substantially increased cell death in the cerebellum, hippocampus, dentate gyrus of adult ETB−/− rats, compared with ETB+/+ littermates. However, no such changes were observed in the cerebral cortex.
EDNRB receptor expression is differentially regulated during early and later brain development. In embryonic embryos, EDNRB is abundantly expressed in cells lining the ventricles, but its expression is substantially shown decreased in the cortex and subventricular zones at postnatal day 14 (Leonard et al., 2015). In contrast, the expression of EDNRB in the cerebellum and hippocampus persist in juvenile rats (Tsaur et al., 1997). EDNRB is generally related to the development of neural crest linage. Growing evidence shows its regulatory effects on a number of regions of the brain, including the cerebellum, hippocampus, and early cerebral cortex. This study further shows increased pathological cell death persists in juvenile ETB−/− rats in the cerebellum and the hippocampus where EDNRB is normally expressed at this age (Chen et al., 2021). It is unlikely that the adverse effects are related to the nutritional status. We have not observed a significant increase in cell death in the cerebral cortex, which is consistent with significant decrease in the EDNRB expression within the first two weeks following birth in the cerebral cortex (Puppala et al., 2015). Taken together, the mutated EDNRB effects in different regions of the brain further support a receptor mediated events during development. The signaling pathways are followed and the research will be reported in our further paper.
HSCR is associated with a variety of congenital abnormalities in the CNS, including microcephaly, agenesis of the corpus callosum, asymmetry of lateral ventricles, central hypoventilation, sensorineural deafness, seizures, mental retardation and autonomic nervous abnormalities (Croaker et al., 1998; Mowat et al., 1998; Staiano et al., 1999). Although HSCR in humans is polygenic, the EDNRB mutation causes a substantial proportion of sporadic and familial cases (Amiel et al., 2008; Puffenberger, 2003). The current report on brain structural changes has less been studied in human brain with HSCR. Our results in ETB−/− rats may allow us to extrapolate that the major effects on human HSCR with congenital EDNRB mutation are associated to the early development and increased cell death, especially in the cerebellum and hippocampus. In addition, cerebral cortical cell protection may be critically important to the juvenile survival in the disease, which are explored further by our group.