Brain size reductions associated with endothelin B receptor mutation, a cause of Hirschsprung’s disease

Background: ET_B has been reported to regulate neurogenesis and vasoregulation in foetal development. Its dysfunction was known to cause HSCR, an aganglionic colonic disorder with syndromic forms reported to associate with both small heads and developmental delay. We therefore asked, "is CNS maldevelopment a more general feature of ET_B mutation?" To investigate, we reviewed the micro-CT scans of an ET_B^−/− model animal, sl/sl rat, and quantitatively evaluated the structural changes of its brain constituents. Methods: Eleven neonatal rats generated from ET_B^+/− cross breeding were sacrificed. Micro-CT scans were completed following 1.5% iodine-staining protocols. All scans were reviewed for morphological changes. Selected organs were segmented semi-automatically post-NLM filtering: TBr, T-CC, T-CP, OB, Med, Cer, Pit, and S&I Col. Volumetric measurements were made using Drishti rendering software. Rat genotyping was completed following analysis. Statistical comparisons on organ volume, organ growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control groups based on autosomal recessive inheritance. One-way ANOVA was also performed to evaluate potential dose-dependent effect. Results: sl/sl rat has 16.32% lower body weight with 3.53% lower growth rate than the control group. Gross intracranial morphology was preserved in sl/sl rats. However, significant volumetric reduction of 20.33% was detected in TBr; similar reductions were extended to the measurements of T-CC, T-CP, OB, Med, and Pit. Consistently, lower brain and selected constituent growth rates were detected in sl/sl rat, ranging from 6.21% to 11.51% reduction. Lower organ volume/bodyweight ratio was detected in sl/sl rats, reflecting disproportional neural changes with respect to body size. No consistent linear relationships exist between ET_B copies and intracranial organ size or growth rates. Conclusion: Although ET_B^−/− mutant has a normal CNS morphology, significant size reductions in brain and constituents were detected. These structural changes likely arise from a combination of factors secondary to dysfunctional ET-1/ET-3/ET_B signalling, including global growth impairment from HSCR-induced malnutrition and dysregulations in the neurogenesis, angiogenesis, and cerebral vascular control. These changes have important clinical implications, such as autonomic dysfunction or intellectual delay. Although further human study is warranted, our study suggested comprehensive managements are required for HSCR patients, at least in ET_B^−/− subtype.