title: Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.
abstract: OBJECTIVE
  This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis.
  
  
  METHODS
  Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R).
  
  
  RESULTS
  The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis.
  
  
  CONCLUSIONS
  The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.
authors: Department of Urology, Kobe City Medical Center General Hospital, 2-1-1, Minatojima-Minamimachi, Chuo-ku, Kobe, Japan, suginoy@kcho.jp.
authors: Yoshio
authors: Y
authors: Sugino
authors: Katherine J
authors: KJ
authors: O'Malley
authors: Zhou
authors: Z
authors: Wang
authors: Pradeep
authors: P
authors: Tyagi
authors: Lori A
authors: LA
authors: Birder
authors: Osamu
authors: O
authors: Ogawa
authors: Naoki
authors: N
authors: Yoshimura
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D003520:Cyclophosphamide / Q000633:toxicity; D003556:Cystitis / Q000235:genetics* / Q000473:pathology; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation; D061346:Laser Capture Microdissection / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis / Q000235:genetics; D043587:Receptor, Muscarinic M3 / Q000096:biosynthesis / Q000235:genetics; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000235:genetics*; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology*; D019459:Urothelium / Q000378:metabolism / Q000473:pathology
chemical_list: D000199:Actins; D003520:Cyclophosphamide; D012333:RNA, Messenger; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C498862:Acta2 protein, rat