Immunohistochemical confirmation of the presence of smooth muscle in the normal neonatal anorectum and in neonates with anorectal malformations

To evaluate the presence of smooth muscle in relation to the anorectum in neonates that are normal and in neonates with anorectal malformations using immunohistochemical staining of antibodies specific to smooth muscle actin (SMA). Research ethics committee approval was obtained to study historical neonatal tissue sections. 7-15-mu thickness, sagittal sections of both normal anorectums (NA) and neonates with anorectal malformations (ARM) were available to study. The fragile tissue predates the development of charged glass microscope slides that are inherent to contemporary immunohistochemistry (IHC). Normal appendix sections on non-charged slides were used to establish and optimise a novel, steam generated heat, antigen retrieval protocol. This technique of "gentle" antigen retrieval allows preservation of tissue architecture whilst maintaining adequate staining of tissue on non-charged glass microscope slides. NA and ARM tissue was stained and compared to control NA and ARM sections. SMA staining was observed in sagittal sections of both normal neonatal anorectums and neonates with anorectal malformations, confirming the presence of smooth muscle. This preliminary work confirms the presence of smooth muscle in the anorectum of neonates that are normal and in neonates with anorectal malformations. It is likely that the smooth muscle complex demonstrated represents the internal anal sphincter in the normal anorectum. It is postulated that the smooth muscle complex seen in anorectal malformations may represent a similar internal anal sphincter complex which maybe of functional significance. Further IHC of tissue sections will allow reconstruction of the exact three-dimensional smooth muscle structure of the anorectum in neonates that are normal and neonates with anorectal malformations.