title: Three-dimensional imaging of tumor angiogenesis.
abstract: OBJECTIVE
  To three-dimensionally visualize the microvessel environment of tumor angiogenesis by confocal laser scanning microscopy (CLSM).
  
  
  METHODS
  To reveal underlying mechanisms of tumor angiogenesis, a 7, 12-dimethylbenz(a) anthracene-induced rat cancer model was used. For demonstrating tumor vasculature, fluorescence injection method (FITC-conjugated gelatin solution) was employed. FITC gelatin was injected into the left ventricle of the rat heart. After complete perfusion, the mammary glands were resected, fixed under ice cold conditions and subjected to immunohistochemistry (IHC) for tumor cells. The LSM-410 (Carl Zeiss, Jena, Germany) was employed on thick sections (300-2,000 microns) to elucidate detailed microvessel networks (MVN) and tumor cells.
  
  
  RESULTS
  Tumor vasculature on thick sections was clearly detected by CLSM at the maximum focus depth of 2,000 microns. Three-dimensional (3-D), reconstructed images of normal mammary glands showed regular and linear MVN. In DMBA-induced mammary cancer, vascular density of MVN was markedly increased and showed an anastomosing, irregular MVN pattern. Furthermore, focal segmentation and tortuous, branching patterns of microvessels were also seen.
  
  
  CONCLUSIONS
  Application of the fluorescence injection method and IHC using CLSM was very useful for studying the 3-D relationship between tumor angiogenesis and neoplastic epithelial changes. These results suggest that application of this technique is ideal for studying 3-D imaging of tumor angiogenesis.
authors.affiliation: Laboratories for Structure and Function Research, Tokai University School of Medicine, Isehara, Japan. itohj@is.icc.u-tokai.ac.jp
authors.forename: J
authors.initials: J
authors.lastname: Itoh
authors.forename: K
authors.initials: K
authors.lastname: Yasumura
authors.forename: T
authors.initials: T
authors.lastname: Takeshita
authors.forename: H
authors.initials: H
authors.lastname: Ishikawa
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: K
authors.initials: K
authors.lastname: Ogawa
authors.forename: K
authors.initials: K
authors.lastname: Kawai
authors.forename: A
authors.initials: A
authors.lastname: Serizawa
authors.forename: R Y
authors.initials: RY
authors.lastname: Osamura
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D004195:Disease Models, Animal; D005260:Female; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007150:Immunohistochemistry; D007633:Keratins / Q000276:immunology; D015674:Mammary Neoplasms, Animal / Q000098:blood supply* / Q000139:chemically induced / Q000473:pathology*; D008833:Microcirculation; D018613:Microscopy, Confocal / Q000379:methods; D009389:Neovascularization, Pathologic / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000906:Antibodies; D015127:9,10-Dimethyl-1,2-benzanthracene; D007633:Keratins