Laser capture microdissected mucosa versus whole tissue specimens for assessment of radiation-induced dynamic molecular and pathway changes in the small intestine.
BACKGROUND
The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis.


RESULTS
RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples.


CONCLUSIONS
LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.
Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America. jzheng@uams.edu
Junying
J
Zheng
Sarita
S
Garg
Junru
J
Wang
David S
DS
Loose
Martin
M
Hauer-Jensen
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D053456:Caspase 14 / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects; D016207:Cytokines / Q000378:metabolism; D004260:DNA Repair / Q000528:radiation effects; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D061346:Laser Capture Microdissection / Q000379:methods*; D008297:Male; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D015398:Signal Transduction / Q000528:radiation effects; D013997:Time Factors; D059467:Transcriptome / Q000528:radiation effects
D016207:Cytokines; D053456:Caspase 14