Original Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Apr 14, 2009; 15(14): 1708-1718
Published online Apr 14, 2009. doi: 10.3748/wjg.15.1708
Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells
Stephan Gehring, Edmond Sabo, Maryann E San Martin, Elizabeth M Dickson, Chao-Wen Cheng, Stephen H Gregory
Stephan Gehring, Maryann E San Martin, Elizabeth M Dickson, Chao-Wen Cheng, Stephen H Gregory, Department of Medicine, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI 02903, United States
Edmond Sabo, Department of Pathology, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI 02903, United States
Stephan Gehring, Zentrum für Kinder- und Jugendmedizin der Universitätsmedizin der Johannes Gutenberg-Universität, 55101 Mainz, Germany
Author contributions: Gehring S, San Martin ME developed the LCM isolation method of Kupffer cells; Gehring S, San Martin ME, Dickson EM, Cheng CW performed the experiments; Sabo E analyzed the microarray data; Gregory SH designed and supervised the study; Gehring S and Gregory SH wrote the manuscript.
Correspondence to: Dr. Stephen H Gregory, Department of Medicine, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, 432 Pierre M. Galletti Building, 55 Claverick Street, Providence, RI 02903, United States. sgregory@lifespan.org
Telephone: +1-401-4447369
Fax: +1-401-4447524
Received: October 22, 2008
Revised: March 7, 2009
Accepted: March 14, 2009
Published online: April 14, 2009
Abstract

AIM: To develop a method of labeling and micro-dissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM).

METHODS: Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model.

RESULTS: mRNA extracted from the microdissected cell population displayed marked increases in colony-stimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury.

CONCLUSION: The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

Keywords: Kupffer cells, India ink, Laser capture microdissection, Bile duct ligation, DNA microarray