immunostaining-to-visualize-murine-enteric-nervous-system-development

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

University of Wisconsin School of Medicine and Public Health

Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed.

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers.

Parenteral nutrition (PN) is a necessary therapy used to feed patients with gastrointestinal dysfunction. Unfortunately, PN results in intestinal atrophy and changes to host immune function. PN may also induce additional effects on gut motility that we hypothesized would result from changes in the enteric nervous system.

Colonic enteric nervous system analysis during parenteral nutrition.

Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model.

Article	Year
Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed. BMC research notes\| PubMed ID: 21134271	2010
Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers. The Journal of comparative neurology\| PubMed ID: 21858821	2012
Colonic enteric nervous system analysis during parenteral nutrition. The Journal of surgical research\| PubMed ID: 23601532	2013
Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease. Journal of pediatric surgery\| PubMed ID: 25092084	2014

Christopher S. Erickson

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