Intestinal Preparation Techniques for Histological Analysis in the Mouse

Jonathan M. Williams1, Carrie A. Duckworth2, Kate Vowell3, Michael D. Burkitt2, D. Mark Pritchard2

1 Pathology and Pathogen Biology, Royal Veterinary College, Hatfield, United Kingdom, 2 Gastroenterology Research Unit, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom, 3 Mary Lyon Centre, Medical Research Council Harwell, Oxford, United Kingdom
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.2
Online Posting Date:  June, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The murine intestinal tract represents a difficult organ system to study due to its long convoluted tubular structure, narrow diameter, and delicate mucosa which undergoes rapid changes after sampling prior to fixation. These features do not make for easy histological analysis as rapid fixation in situ, or after simple removal without careful dissection, results in poor postfixation tissue handling and limited options for high quality histological sections. Collecting meaningful quantitative data by analysis of this tissue is further complicated by the anatomical changes in structure along its length. This article describes two methods of intestinal sampling at necropsy that allow systematic histological analysis of the entire intestinal tract, either through examination of cross sections (circumferences) by the gut bundling technique or longitudinal sections by the adapted Swiss roll technique, together with basic methods for data collection. © 2016 by John Wiley & Sons, Inc.

Keywords: crypt; histology; intestine; mouse; villus

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Histology
  • Strategic Planning
  • Basic Protocol 1: Preparation of Intestines for Cross Sectional Histological Analysis (Gut Bundling Technique)
  • Alternate Protocol 1: Preparation of Intestines for Longitudinal Histological Analysis (Adapted Swiss Roll Technique)
  • Support Protocol 1: Quantification of Epithelial Cell Features (Cell Scoring)
  • Support Protocol 2: Measurement of Villus/Crypt Lengths Using ImageJ
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Intestines for Cross Sectional Histological Analysis (Gut Bundling Technique)

  Materials
  • Mouse
  • PBS, ice cold (Sigma, cat. no. P4417)
  • Formalin, prepared fresh (3.75% [w/v] formaldehyde in PBS or 10% neutral buffered formalin solution [Sigma, cat. no. HT501128])
  • Paraffin wax
  • Forceps, 12‐cm dressing and 12.5‐cm curved
  • Scissors, 10‐cm or 12‐cm sharp‐tipped
  • 4‐Metric sofsilk sutures (e.g., Tyco Healthcare)
  • Dissection mat or board
  • Petri dish (plastic or glass)
  • 20‐ml syringe with 23‐G, 2.5‐cm needle
  • 5‐ml syringe with 26‐G needle
  • White card, ∼7 cm × 7.5 cm (to fit into a 30‐ml universal container; see Fig.  )
  • 30‐ml universal container
  • Fume hood or downdraft table
  • Scalpel with no. 22 blade
  • Ruler
  • 3M Micropore surgical tape (2.5 cm width)
  • Solvent resistant pencil or pen
  • Tissue cassettes (e.g., Sigma)
  • Embedding Mold
  • Additional reagents and equipment for serial dehydration (see Duckworth et al., )

Alternate Protocol 1: Preparation of Intestines for Longitudinal Histological Analysis (Adapted Swiss Roll Technique)

  Materials
  • Mouse
  • Formalin, prepared fresh (3.75% [w/v] formaldehyde in PBS or 10% neutral buffered formalin solution [Sigma, cat. no. HT501128])
  • Forceps, 12‐cm dressing, 12.5‐cm curved, and 11.5‐cm self‐closing
  • Scissors, 10‐cm or 12‐cm sharp
  • Dissection mat or board
  • 5‐ml syringe equipped with 26‐G needle
  • Spring scissors, 10‐cm with a 4‐mm cutting blade (curved or straight as per personal preference)
  • 30‐G needle
  • Labeled containers, for storing samples
  • Double‐deep tissue cassette (e.g., IHC World)

Support Protocol 1: Quantification of Epithelial Cell Features (Cell Scoring)

  Materials
  • High‐quality H&E stained sections or immunohistochemically stained slides from treatment and control animals (see Duckworth et al., )
  • Standard light microscope
  • Multiple unit counter with totalizer (e.g., Thomas Scientific) or computer with appropriate software (e.g., Microsoft Excel; Microsoft Notepad)

Support Protocol 2: Measurement of Villus/Crypt Lengths Using ImageJ

  Materials
  • H&E stained cross sections of small intestine (prepared as per gut bundling technique)
  • Microscope (e.g., Olympus BX43 upright microscope with Olympus SC‐30 digital camera)
  • Appropriate image acquisition software (e.g., Olympus cellSens)
  • Hemocytometer grid or other scale of known size (for calibration)
  • Appropriate image analysis software (e.g., ImageJ available at http://rsb.info.nih.gov/ij/download.html)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003‐1007. doi: 10.1038/nature06196.
  Booth, C. and Potten, C.S. 2002. The Intestine as a Model for Studying Stem‐Cell Behavior. In Tumor Models in Cancer Research, Cancer Drug Discovery and Development (B.A. Teicher, ed.) pp. 337‐357. Humana Press, Totowa, N.J.
  Burkitt, M.D., Hanedi, A.F., Duckworth, C.A., Williams, J.M., Tang, J.M., O'Reilly, L.A., Putoczki, T.L., Gerondakis, S., Dimaline, R., Caamano, J.H., and Pritchard, D.M. 2015. NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice. J. Pathol. 236:326‐336. doi: 10.1002/path.4527.
  Duckworth, C.A. and Pritchard, D.M. 2009. Suppression of apoptosis, crypt hyperplasia, and altered differentiation in the colonic epithelia of bak‐null mice. Gastroenterology 136:943‐952. doi: 10.1053/j.gastro.2008.11.036.
  Duckworth, C.A., Burkitt, M.D., Williams, J.M., Parsons, B.N., Tang, J.M.F., and Pritchard, D.M. 2015. Murine models of helicobacter (pylori or felis)‐associated gastric cancer. Curr. Protoc. Pharmacol. 69:14.34.1‐14.34.35. doi: 10.1002/0471141755.ph1434s69.
  Duckworth, C.A., Clyde, D., Worthley, D.L., Wang, T.C., Varro, A., and Pritchard, D.M., 2013. Progastrin‐induced secretion of insulin‐like growth factor 2 from colonic myofibroblasts stimulates colonic epithelial proliferation in mice. Gastroenterology 145:197‐208. doi: 10.1053/j.gastro.2013.03.012.
  FELASA Working Group on Revision of Guidelines for Health Monitoring of Rodents and Rabbits, Mahler Convenor, M., Berard, M., Feinstein, R., Gallagher, A., Illgen‐Wilcke, B., Pritchett‐Corning, K., and Raspa, M. 2014. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab. Anim. 48:178‐192. doi: 10.1177/0023677213516312.
  Hummel, K.P., Richardson, F.L., and Fekete, E. 1968. Anatomy. In Biology of the Laboratory Mouse, 2nd ed. (E.L. Green, ed.). Dover Publications, New York.
  Kilkenny, C., Browne, W., Cuthill, I.C., Emerson, M., and Altman, D.G. 2010. Animal research: Reporting in vivo experiments: The ARRIVE guidelines. Br. J. Pharmacol. 160:1577‐1579. doi: 10.1111/j.1476‐5381.2010.00872.x.
  Leblond, C.P. and Stevens, C.E., 1948. The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100:357‐377. doi: 10.1002/ar.1091000306.
  Lefrançois, L. and Lycke, N. 2001. Isolation of mouse small intestinal intraepithelial lymphocytes, peyer's patch, and lamina propria cells. Curr. Protoc. Immunol. 17:3.19.1–3.19.16. doi: 10.1002/0471142735.im0319s17.
  Matsuo, K., Ota, H., Akamatsu, T., Sugiyama, A., and Katsuyama, T., 1997. Histochemistry of the surface mucous gel layer of the human colon. Gut 40:782‐789. doi: 10.1136/gut.40.6.782.
  Moolenbeek, C. and Ruitenberg, E.J., 1981. The “Swiss roll”: A simple technique for histological studies of the rodent intestine. Lab. Anim. 15:57‐59. doi: 10.1258/002367781780958577.
  Moore, R., Carlson, S., and Madara, J.L., 1989. Villus contraction aids repair of intestinal epithelium after injury. Am. J. Physiol. 257:G274‐G283.
  Potten, C.S. 1990. A comprehensive study of the radiobiological response of the murine (BDF1) small intestine. Int. J. Radiat. Biol. 58:925‐973. doi: 10.1080/09553009014552281.
  Potten, C.S. and Grant, H.K., 1998. The relationship between ionizing radiation‐induced apoptosis and stem cells in the small and large intestine. Br. J. Cancer 78:993‐1003. doi: 10.1038/bjc.1998.618.
  Potten, C.S., Owen, G., and Roberts, S.A., 1990. The temporal and spatial changes in cell proliferation within the irradiated crypts of the murine small intestine. Int. J. Radiat. Biol. 57:185‐199. doi: 10.1080/09553009014550431.
  Ritsma, L., Ellenbroek, S.I.J., Zomer, A., Snippert, H.J., de Sauvage, F.J., Simons, B.D., Clevers, H., and van Rheenen, J. 2014. Intestinal crypt homeostasis revealed at single‐stem‐cell level by in vivo live imaging. Nature 507:362‐365. doi: 10.1038/nature12972.
  Schneider, C.A., Rasband, W.S., and Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9:671‐675. doi: 10.1038/nmeth.2089.
  Scudamore, C.L. 2014. A Practical Guide to the Histology of the Mouse. Wiley Blackwell, Hoboken, N.J.
  Snipes, R.L., 1981. Anatomy of the cecum of the laboratory mouse and rat. Anat. Embryol. 162:455‐474. doi: 10.1007/BF00301871.
  Treuting, P.M. and Dintzis, S.M., 2012. Lower gastrointestinal tract. In Comparative Anatomy and Histology (P.M. Treuting, S. Dintzis, D. Liggitt, and C.W. Frevert, eds.) pp. 177‐192. Academic Press, San Diego.
  Washington, M.K., Powell, A.E., Sullivan, R., Sundberg, J.P., Wright, N., Coffey, R.J., and Dove, W.F., 2013. Pathology of rodent models of intestinal cancer: Progress report and recommendations. Gastroenterology 144:705‐717. doi: 10.1053/j.gastro.2013.01.067.
  Watson, A.J.M. and Pritchard, D.M., 2000. Lessons from genetically engineered animal models: VII. Apoptosis in intestinal epithelium: lessons from transgenic and knockout mice. Am. J. Physiol. Gastrointest. Liver Physiol. 278:G1‐G5.
  Williams, J.M., Duckworth, C.A., Watson, A.J.M., Frey, M.R., Miguel, J.C., Burkitt, M.D., Sutton, R., Hughes, K.R., Hall, L.J., Caamaño, J.H., Campbell, B.J., and Pritchard, D.M., 2013. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Dis. Model. Mech. 6:1388‐1399. doi: 10.1242/dmm.013284.
  Young, B., Lowe, J.S., Stevens, A., and Heath, J.W. 2006. Wheater's Functional Histology: A Text and Colour Atlas, 5th ed. Elsevier, New York.
Internet Resources
  http://rsb.info.nih.gov/ij/download.html
  ImageJ download site
  http://www.informatics.jax.org/greenbook/frames/frame13.shtml
  Biology of the Laboratory Mouse eBook. Adapted for the Web by Mouse Genome Informatics, The Jackson Laboratory.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library