Pathology Evaluation of Developmental Phenotypes in Neonatal and Juvenile Mice

Brad Bolon1, Susan Newbigging2, Kelli L. Boyd3

1 GEMpath, Inc., Longmont, 2 Toronto Centre for Phenogenomics, Toronto, Ontario, 3 Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.31
Online Posting Date:  September, 2017
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Abstract

Necropsy (or autopsy) is the post mortem dissection of an animal to examine and collect organs and tissues in order to understand the effects and causes of disease. The systematic harvesting of samples at necropsy is an essential step in defining the reason for an unexpected death and in characterizing the features (i.e., phenotype) of a newly discovered condition. Phenotypic evaluation of young (neonatal and juvenile) mice emphasizes morphologic (macroscopic and microscopic) techniques and biochemical (clinical chemistry, hematologic, and molecular) analyses. This paper describes the most common procedures utilized to gather phenotypic data from neonatal and juvenile mice, with advanced alternatives for preparing special specimens (e.g., blood smears, electron microscopic samples). These techniques are applicable to young mice of all strains and are effective regardless of the fundamental cause, including genetically engineered or spontaneous mutations and exposure to pathogens or xenobiotic agents (i.e., foreign chemicals). © 2017 by John Wiley & Sons, Inc.

Keywords: developmental pathology; anatomic pathology; clinical pathology; genetically engineered mouse; neonatal; phenotyping; teratogen

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Conventional Mouse Necropsy with Immersion Fixation
  • Support Protocol 1: Lung Inflation with Fixative
  • Support Protocol 2: Blood Collection
  • Support Protocol 3: Blood Smear Preparation
  • Support Protocol 4: Tissue Touch Preparation
  • Alternate Protocol 1: Necropsy with Perfusion Fixation
  • Alternate Protocol 2: Whole‐Body Sampling of Neonatal Mice
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Conventional Mouse Necropsy with Immersion Fixation

  Materials
  • Mouse
  • 70% or 95% (v/v) ethanol in a spray bottle
  • 10% (v/v) neutral buffered formalin (NBF; e.g., Sigma‐Aldrich, cat. no. HT501128)
  • Individual animal necropsy record (IANR; optional): paper or electronic (see Supplemental Form 1 in Supporting Materials)
  • Euthanasia apparatus: e.g., carbon dioxide chamber (all ages) or ice‐cold (4°C) platform covered with paper towel (up to PND 7) (see Strategic Planning)
  • Digital balance, calibrated (preferred range: 0.001 g)
  • Weigh boats (optional; for weighing organs)
  • Digital calipers or metric ruler (preferred length: 10 cm, with millimeter markings)
  • Absorbent bench protector, e.g., laboratory “diaper” or shelf liner (VWR, cat. no. 56617‐006 or 54110‐106)
  • Biohazard trash liners, 2‐gallon (Fisher Scientific, cat. no. 03‐411‐700)
  • Dissection board (cardboard, cork, plastic, or wax)
  • Dissection pins, hypodermic needles, or rubber bands (for securing animals to dissection board)
  • Blotting paper (e.g., paper towels)
  • Cotton‐tipped applicators (e.g., Q‐tip; optional)
  • Specimen container
  • Permanent black marker (for labeling specimen container)
  • Tissue cassettes:
  • regular depth (e.g., Fisher Scientific, cat. no. 22‐272416)
  • one‐ or six‐chamber mesh‐top (e.g., Fisher Scientific, cat. no. 15‐182‐706H or 15‐182‐705C)
  • Solvent‐resistant marking pen (e.g., Ted Pella, cat. no. 22315) or no. 2 pencil (for labeling tissue cassettes)
  • Surgical tools:
  • Scissors, blunt‐sharp
  • Scissors, sharp‐sharp (two pairs)
  • Forceps, rat‐toothed
  • Forceps, serrated (two pairs if no rat‐toothed forceps are available)
  • Razor blade, single‐sided carbon steel edge
  • Petri dish (glass or plastic; optional)
  • Card stock (i.e., a 3 × 5–in. index card; optional)
  • Stereomicroscope (i.e., dissecting microscope; optional)
  • Microscope‐mounted digital camera (optional; for photodocumentation)
  • Additional reagents and equipment for organ inflation with fixative, blood collection, blood smears, and tissue touch preparations (see Support Protocols 1–4)

Support Protocol 1: Lung Inflation with Fixative

  Additional Materials (also see protocol 1Basic Protocol)
  • 1‐ml syringe
  • 23‐G hypodermic needle or 24‐G polypropylene catheter (e.g., SurFlash; Terumo, cat. no. SR*FNP2419)
  • Non‐sterile suture (e.g., Fisher Scientific, cat. no. 14‐516‐130) or regular sewing thread
  • Hemostat (Halstead mosquito forceps; Fisher Scientific, cat. no. 13‐812‐10)

Support Protocol 2: Blood Collection

  Additional Materials (also see protocol 1Basic Protocol)
  • For collection of pooled blood (select one):
  • Capillary tubes and sealing putty (for hematocrit measurements)
  • 1‐ml syringe
  • Blood collection tube (e.g., BD Microtainer tubes, cat. no. 365973, lavender top)
  • Physiological saline (optional) (pH 7.4, ideally near normal body temperature of ∼38°C; for extending volume of small samples)
  • Syringe and needle for intracardiac blood collection:
  • For mice up to 14 days of age: 500‐μl insulin syringe with 27‐G hypodermic needle (Terumo, cat. no. SS05M2713)
  • For mice 15 days and older: 1‐ml tuberculin syringe with 23‐G needle

Support Protocol 3: Blood Smear Preparation

  Materials
  • Blood sample (see protocol 3)
  • Plain glass slides

Support Protocol 4: Tissue Touch Preparation

  Additional Materials (also see protocol 1Basic Protocol)
  • Plain glass slides

Alternate Protocol 1: Necropsy with Perfusion Fixation

  Additional Materials (also see protocol 1Basic Protocol)
  • Fixative (select one):
  • 10% (v/v) neutral buffered formalin (NBF; e.g., Sigma‐Aldrich, cat. no. HT501128)
  • Methanol‐free 4% formaldehyde (often called 4% paraformaldehyde as it is prepared from paraformaldehyde powder, e.g., Sigma‐Aldrich, cat. no. 158127)
  • Modified Karnovsky's solution: methanol‐free 2% formaldehyde (from paraformaldehyde powder) and medical‐grade 2.5% glutaraldehyde (e.g., Sigma‐Aldrich, cat. no. G5882)
  • Anesthetic agent (select one):
  • Isoflurane (Forane) for inhalation
  • Pentobarbital, ketamine/xylazine, ketamine/medetomidine, or 2,2,2‐tribromoethanol for injection
  • Phosphate‐buffered saline (PBS), pH 7.4
  • Anesthetic chamber or 1‐ml syringe and 23‐G hypodermic needle
  • Perfusion apparatus and appropriate port (select one):
  • Variable‐flow peristaltic perfusion pump (e.g., Fisherbrand, cat. no. 13‐876‐1) with 24‐G polypropylene straight catheter (e.g., SurFlash; Terumo, cat. no. SR*FNP2419)
  • 3‐ml syringe with 23‐ or 25‐G butterfly catheter (e.g., Vacuette, Greiner Bio‐One, cat. no. 450090 or 450092)
NOTE: Ports of higher gauges may be better for neonates (up to 7 days), but with care any gauge from 23 to 25 will suffice for young individuals at all ages. The advantage of using catheters is that they connect to the perfusion apparatus by long, flexible tubing, so pressure‐induced oscillations of the port tip associated with movement of the perfusion apparatus are less likely to damage the heart tissue relative to a rigid hypodermic needle of the same gauge for the butterfly catheter.

Alternate Protocol 2: Whole‐Body Sampling of Neonatal Mice

  Additional Materials (also see protocol 1Basic Protocol)
  • Fixative (select one):
  • Bouin's solution (e.g., Sigma‐Aldrich, cat. no. HT10132)
  • Modified Davidson's solution (e.g., Fisher Scientific, cat. no. NC0557391)
  • Specimen container (minimum size: 50‐ml conical centrifuge tube)
  • Tissue cassettes, extra deep (e.g., Tissue‐Tek Mega‐Cassettes, Sakura Finetek, cat. no. 4173)
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Figures

Videos

Literature Cited

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  Brayton, C., & Treuting, P. M. (2012). Phenotyping. In P. M. Treuting & S. M. Dintzis (Eds.), Comparative anatomy and histology: A mouse and human atlas (pp. 7–14). San Diego: Academic Press (Elsevier).
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  Newbigging, S., Ward, J. M., & Bolon, B. (2015). Necropsy sampling and data collection for studying the anatomy, histology, and pathology of mouse development. In B. Bolon (Ed.), Pathology of the developing mouse: A systematic approach (pp. 133–173). Boca Raton, FL: CRC Press (Taylor & Francis).
  Office of Animal Care and Use (OACU) & Animal Research Advisory Committee (ARAC) (2013). Guidelines for the euthanasia of rodent fetuses and neonates. Retrieved from http://oacu.od.nih.gov/ARAC/documents/rodent_euthanasia_pup.pdf [Retrieved May 1, 2014, from U.S. National Institutes of Health]
  Papaioannou, V. E., & Behringer, R. R. (2005). Mouse phenotypes: A handbook of mutation analysis. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
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  Richey, L. J., & Li, N. (2014). How to fix and prepare tissue for histology submission. Retrieved from http://sites.tufts.edu/histopath/files/2014/06/how‐to‐prepare‐tissues‐for‐histology‐submission.pdf [Retrieved May 9, 2017]
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Key References
  Bolon, 2015b. See above.
  These books detail essential concepts of the anatomy, physiology, and pathology of developing mice.
  Kaufman, 1992. See above.
  Kaufman & Bard, 1999. See above.
  Newbigging et al., 2015. See above.
  Papaioannou & Behringer, 2005. See above.
  Rugh, 1990. See above.
  Theiler, 1972. See above.
Internet Resources
  Gage, G. J., Kipke, D. R., & Shain, W. (2012). Whole animal perfusion fixation for rodents. Journal of Visualized Experiments, 65, 3564. doi: 10.3791/3564. https://www.jove.com/video/3564/whole‐animal‐perfusion‐fixation‐for‐rodents
  Videos and protocols for perfusion fixation and necropsy of adult rodents using techniques that can be adapted for use in neonatal and juvenile mice.
  Parkinson, C. M., O'Brien, A., Albers, T. M., Simon, M. A., Clifford, C. B., & Pritchett‐Corning, K. R. (2011). Diagnostic necropsy and selected tissue and sample collection in rats and mice. Journal of Visualized Experiments, 54, 2966. doi: 10.3791/2966. https://www.jove.com/video/2966/diagnostic‐necropsy‐selected‐tissue‐sample‐collection‐rats
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