Using Micro-CT scanning to identify structural changes in Craniofacial, Neural, and Cardiovascular systems of spotting-lethal rat, a model of Hirschsprung disease

Abstract

HSCR has long been regarded as an enteric disease for its well-known colonic aganglionosis. However, the fact of it being a polygenic neurocristopathy begs the question: is structural growth retardation a more general feature in HSCR individuals? To clarify, we examine the multi-organ changes within the sl/sl rat, which is an autosomal recessive HSCR model with high genetic penetrance that arises from ETB knockout mutation.

This thesis contributes to improving our understanding in modified soft tissue imaging workflow and multi-systemic changes associated with HSCR using an animal model.

A. Imaging methodology: 1. Micro-CT scanning can be a useful structural-preserving visualization tool for studying external morphology, neuroanatomy, and cardiovascular anatomy in small animals. 2. Successful micro-CT scanning of soft tissue requires contrast staining, which can be achieved with iodine or PTA. Iodine is a more effective and safer choice among the two. 3. Ex vivo micro-CT scans can offer structural details comparable to those of 4x H&E light micrographs. 4. Image degradation of in vivo micro-CT scan can be improved by raising the voltage and/or current of X-ray source. 5. Post-acquisition NLM filtering is an effective enhancement technique for in vivo micro-CT scans.

B. Global structural changes associated with HSCR model: 1. The HSCR model, sl/sl rat, is associated with subtle body growth impairment during early life; this impairment may progressively worsen with age. 2. The craniofacial changes in sl/sl rat share some resemblances to those seen in domestication syndrome, including a flatter cranium, a smaller maxilla, a larger mandible, a disproportionally shorter but wider nose, a shrunken molar bed and reduced superior incisor dimensions. It may therefore be prudent to suspect that HSCR patients may have similar dysmorphology and thus increased risks for obstructive sleep apnoea and dental malocclusion. 3. sl/sl rat has a grossly normal neural morphology with intact constituents; however, significant structural reductions of 20.33% in brain volume and 7.35% in brain growth rate are detected. Similar reductions are seen in cerebral cortex, caudate putamen, olfactory bulb, medulla, and pituitary gland. These findings suggest that HSCR patients may exhibit varying degrees of brain reductions and subsequent neurological dysfunction, either subtle or overt. 4. Despite having an impaired endothelin signalling, congenital conotruncal cardiac malformations are not detected in sl/sl rat. 5. Cardiac morphology is grossly normal in sl/sl rat; however, marked reductions are detected in heart volume (40%), growth rate (20%), and organ volume/bodyweight index (25%). Measurements of LA, LV, RA, and RV share similar reducing trends. Our findings are not only consistent with prior notions that HSCR patients have elevated risks for congenital heart diseases, but also suggest that structural cardiac anomaly may be a more general feature. Thus, long-term follow-up and systematic assessments may be recommended clinically.

The above findings support the concept that HSCR, at least with the ETB-/- subtype, is likely a multi-organ condition, with significant changes to craniofacial, neurological, and cardiovascular systems.