Abstract
This study describes a new technique for the visualisation and quantitation of glandular epithelial cell proliferation in gastrointestinal mucosa using computerised three-dimensional reconstruction. The tissue used in this study was colorectal biopsy tissue infiltrated in vitro with bromodeoxyuridine (BrdU), although the method could be applied to any gastrointestinal site labelled with any specific marker for cell proliferation. The method is as follows. Five-micron-thick serial sections (> 100) were cut from colorectal biopsies infiltrated in vitro with BrdU. After labelling all the sections for BrdU-positive cells using standard immunohistochemistry, colorectal glands were identified which were completely sectioned within the series. Each microscopic image of the sectioned gland was orientated, digitised and stored using a Kontron image analyser. On each of the stored images, the crypt profile, the positive cells and the negative cells were interactively marked and digitally stored. Using three-dimensional (3-D) reconstruction software, the outer surface of the crypt, the total positive and the total negative fractions could be viewed in three dimensions. The total BrdU-positive cell number could be automatically calculated for the complete crypt or, alternatively, compartmental analysis of the labelling pattern within the crypt could be obtained. This represents a powerful technique: it does not require orientation, it can be carried out on complex glandular structures and is not affected by the biases involved in measuring labelling indices from single tissue sections.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Agard D. A. Optical sectioning microscopy: cellular architecture in three dimensions. Annu Rev Biophys Bioeng. 1984;13:191–219. doi: 10.1146/annurev.bb.13.060184.001203. [DOI] [PubMed] [Google Scholar]
- Braendgaard H., Gundersen H. J. The impact of recent stereological advances on quantitative studies of the nervous system. J Neurosci Methods. 1986 Oct;18(1-2):39–78. doi: 10.1016/0165-0270(86)90112-3. [DOI] [PubMed] [Google Scholar]
- Brakenhoff G. J., van der Voort H. T., van Spronsen E. A., Nanninga N. 3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy. Scanning Microsc. 1988 Mar;2(1):33–40. [PubMed] [Google Scholar]
- Clarke R. M. Mucosal architecture and epithelial cell production rate in the small intestine of the albino rat. J Anat. 1970 Nov;107(Pt 3):519–529. [PMC free article] [PubMed] [Google Scholar]
- Ferguson A., Sutherland A., MacDonald T. T., Allan F. Technique for microdissection and measurement in biopsies of human small intestine. J Clin Pathol. 1977 Nov;30(11):1068–1073. doi: 10.1136/jcp.30.11.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goodlad R. A., Levi S., Lee C. Y., Mandir N., Hodgson H., Wright N. A. Morphometry and cell proliferation in endoscopic biopsies: evaluation of a technique. Gastroenterology. 1991 Nov;101(5):1235–1241. doi: 10.1016/0016-5085(91)90072-s. [DOI] [PubMed] [Google Scholar]
- Goodlad R. A., Madgwick A. J., Moffatt M. R., Levin S., Allen J. L., Wright N. A. Prostaglandins and the gastric epithelium: effects of misoprostol on gastric epithelial cell proliferation in the dog. Gut. 1989 Mar;30(3):316–321. doi: 10.1136/gut.30.3.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kett P., Geiger B., Ehemann V., Komitowski D. Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy. J Microsc. 1992 Aug;167(Pt 2):169–179. doi: 10.1111/j.1365-2818.1992.tb03227.x. [DOI] [PubMed] [Google Scholar]
- Takahashi T., Iwama N. Architectural pattern of gastric adenocarcinoma--a 3-dimensional reconstruction study. Virchows Arch A Pathol Anat Histopathol. 1984;403(2):127–134. doi: 10.1007/BF00695229. [DOI] [PubMed] [Google Scholar]
- Takahashi T., Iwama N. Atypical glands in gastric adenoma. Three-dimensional architecture compared with carcinomatous and metaplastic glands. Virchows Arch A Pathol Anat Histopathol. 1984;403(2):135–148. doi: 10.1007/BF00695230. [DOI] [PubMed] [Google Scholar]
- Wright N. A., Carter J., Irwin M. The measurement of villus cell population size in the mouse small intestine in normal and abnormal states: a comparison of absolute measurements with morphometric estimators in sectioned immersion-fixed material. Cell Tissue Kinet. 1989 Nov;22(6):425–450. doi: 10.1111/j.1365-2184.1989.tb00227.x. [DOI] [PubMed] [Google Scholar]