Proceedings of The Physiological Society

Future Physiology (Leeds, UK) (2017) Proc Physiol Soc 39, PC17

Poster Communications

Can archived human gastrointestinal specimens be utilised to study interstitial cell biology?

H. El-Kuwaila1

1. university of leeds, Leeds, United Kingdom.

Interstitial cells (ICs) are attracting increasing research attention due to their crucial role in regulating bowel motility. The biology of IC is important in health and in a range of diseases including gastrointestinal, urinary tract and vascular diseases. However, interstitial cells are not identifiable using conventional histopathological techniques such as haematoxylin and eosin and also because of the need to use fresh tissue samples and specific antigen markers. Therefore, novel approaches are needed to obtain robust histological data using archived samples. Developing reliable methods that can be used to visualise ICs in archived tissues would increase the number of available specimens and limit the need for a fresh tissue. Moreover, normative data is also difficult to obtain in humans, since true control tissues are rarely removed from healthy humans. Therefore, this study aimed to compare staining for IC markers in fresh and archived specimens; optimise the examination of archived formalin-fixed paraffin-embedded (FFPE) tissue; explore the potential to identify cKit mRNA in archived tissue using in situ hybridisation. Immunohistochemistry was performed on archived specimens (n=15) and fresh specimens fixed in three different fixatives from patients with Hirschsprung's disease (n=8) and patients undergoing colostomy closure (n=9). Antigen retrieval on archived tissue using citrate based solutions was compared to borohydrate and EDTA solutions. In situ hybridisation for cKit was performed on FFPE tissue specimens using RNAscope®. The results showed that without antigen retrieval, stronger positive staining was obtained for cKit antigen in fresh samples compared to archived samples. Examination of antigen retrieval solutions revealed that citrate-based procedures produced superior staining compared to using borohydrate and EDTA. Preliminary RNAscope® results showed positive detection of cKit mRNA around the myenteric plexus. The ability to use archived tissue to study IC is a valuable resource for future research in this field. We showed that while the archived tissue was ideal for examining IC, the use of retrieval methods enhanced the identification of ICs, with citrate-based solution showing the optimal results. We also showed that In situ hybridisation can be used to examine the distribution of mRNA relevant to IC biology. Key words: Interstitial cells, Hirschsprung's disease, archived FFPE.

Where applicable, experiments conform with Society ethical requirements