Histopathological Evaluation of Skeletal Muscle with Specific Reference to Mouse Models of Muscular Dystrophy

Rebecca L. Terry1, Dominic J. Wells2

1 Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire, 2 Neuromuscular Group, Department of Comparative Biomedical Sciences, Royal Veterinary College, London
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.19
Online Posting Date:  December, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The muscular dystrophies are a diverse group of degenerative diseases for which many mouse models are available. These models are frequently used to assess potential therapeutic interventions and histological evaluation of multiple muscles is an important part of this assessment. Histological evaluation is especially useful when combined with tests of muscle function. This unit describes a protocol for necropsy, processing, cryosectioning, and histopathological evaluation of murine skeletal muscles, which is applicable to both models of muscular dystrophy and other neuromuscular conditions. Key histopathological features of dystrophic muscle are discussed using the mdx mouse (a model of Duchenne muscular dystrophy) as an example. Optimal handling during dissection, processing and sectioning is vital to avoid artifacts that can confound or prevent future analyses. Muscles carefully processed using this protocol are suitable for further evaluation using immunohistochemistry, immunofluorescence, special histochemical stains, and immuoblotting. © 2016 by John Wiley & Sons, Inc.

Keywords: cryosectioning; dystrophy; histopathology; mdx; muscle; necropsy

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Skeletal Muscle Removal, Freezing, and Cryosectioning
  • Basic Protocol 2: H&E Staining
  • Basic Protocol 3: Analysis of H&E Histology
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Skeletal Muscle Removal, Freezing, and Cryosectioning

  Materials
  • Mice: test strain and control mice of the same genetic background (e.g., mdx and C57Bl/10)
  • Isopentane (2‐methylbutane, C 5H 12)
  • Liquid nitrogen (LN2) in a Dewar
  • Sodium pentobarbital (overdose given intraperiotneally in mice), optional
  • 70% (v/v) ethanol
  • Optimal Cutting Temperature (OCT) Embedding compound (e.g., Bright Cryo‐m‐Bed, Bright Instruments)
  • Surgical instruments (Fine Science Tools, Interfocus; “Student‐quality” instruments are adequate for dissection) including:
    • Curved forceps (Dumont no.7; cat. no. 91197‐00)
    • Fine scissors (cat. no. 91460‐11)
    • Surgical scissors (cat. no. 91402‐12)
  • 22‐mm diameter cork discs (label in advance), packs of 100 (Bright Instruments)
  • Aluminum foil (label in advance)
  • Fine permanent marker
  • Animal transport boxes
  • Metal container for isopentane
  • Dissecting microscope (optional)
  • 22‐G needles
  • Long forceps (∼30‐cm; Fine Science Tools, cat. no. 11000‐30 or similar)
  • Tissue paper
  • Single‐edged razor blade
  • Storage box
  • −80°C freezer
  • Cryostat
  • Superfrost Plus glass slides (VWR International)
  • Slide racks
  • Paintbrushes, mixture of firm and soft bristles, 0.5‐cm width heads or similar
  • Microcentrifuge tubes for collecting intermediate sections

Basic Protocol 2: H&E Staining

  Materials
  • Slides of frozen muscle sections, defrosted and air‐dried ( protocol 1)
  • Harris’ Hematoxylin (Sigma, cat. no. HHS16l): diluted 1:1 with tap water and filtered
  • Eosin (Gurr Certistain): diluted 1% (w/v) in tap water and filtered before use (shelf life 1 to 2 months)
  • Acid alcohol [70% industrial methylated spirit (IMS)/1% HCl made in fume hood]
  • IMS diluted to 70%, 80%, and 90% solutions with deionized water
  • Xylene
  • DPX mounting medium
  • Pencil
  • Coplin jars/slide racks and glass staining trays
  • Coverslips
  • Glass rods
  • Forceps
  • Tissue paper
  • Slide boxes
  • Brightfield microscope ± camera
NOTE: IMS is used as a cheaper alternative to ethanol.

Basic Protocol 3: Analysis of H&E Histology

  Additional Materials (also see protocol 2)
  • Slide boxes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Arechavala‐Gomeza, V., Kinali, M., Feng, L., Brown, S.C., Sewry, C., Morgan, J.E., and Muntoni, F. 2010. Immunohistological intensity measurements as a tool to assess sarcolemma‐associated protein expression. Neuropathol Appl Neurobiol 36(4):265‐274. doi: 10.1111/j.1365‐2990.2009.01056.x.
  Barton, E., Lynch, G., and Khurana, T. 2015. Measuring isometric force of isolated mouse muscles in vitro [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.1.2.002.pdf [accessed 1st July].
  Briguet, A., Courdier‐Fruh, I., Foster, M., Meier, T., and Magyar, J.P. 2004. Histological parameters for the quantitative assessment of muscular dystrophy in the mdx‐mouse. Neuromuscul Disord 14(10):675‐682. doi: 10.1016/j.nmd.2004.06.008.
  Bulfield, G., Siller, W.G., Wight, P.A., and Moore, K.J. 1984. 'X chromosome‐linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A 81(4):1189‐1192. doi: 10.1073/pnas.81.4.1189.
  De Luca, A. 2014. Use of treadmill and wheel exercise for impact on mdx mice phenotype [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.001.pdf [accessed 1st July].
  Dubach‐Powell, J. 2014. Quantitative determination of muscle fiber diameter (minimal Feret's diameter) and percentage of cental nuclei [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.1.2.001.pdf [accessed 2nd July].
  Godfrey, C., Muses, S., McClorey, G., Wells, K.E., Coursindel, T., Terry, R.L., Betts, C., Hammond, S., O'Donovan, L., Hildyard, J., El Andaloussi, S., Gait, M.J., Wood, M.J., and Wells, D.J. 2015. How much dystrophin is enough: The physiological consequences of different levels of dystrophin in the mdx mouse. Hum. Mol. Genet. 1;24(15):4225‐4237 doi: 10.1093/hmg/ddv155.
  Grange, R. 2015. Use of treadmill and wheel exercise to assess dystrophic state [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.003.pdf [accessed 1st July].
  Greaves, P., Chouinard, L., Ernst, H., Mecklenburg, L., Pruimboom‐Brees, I.M., Rinke, M., Rittinghausen, S., Thibault, S., Von Erichsen, J., and Yoshida, T. 2013. Proliferative and non‐proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium. J. Toxicol. Pathol. 26(3 Suppl):1S‐26S. doi: 10.1293/tox.26.1S.
  Gregorevic, P., Meznarich, N.A., Blankinship, M.J., Crawford, R.W., and Chamberlain, J.S. 2008. Fluorophore‐labeled myosin‐specific antibodies simplify muscle‐fiber phenotyping. Muscle. Nerve 37(1):104‐106. doi: 10.1002/mus.20877.
  Grounds, M. 2014. Quantification of histopathology in haematoxylin and eosin stained muscle sections. [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.1.2.007.pdf [accessed 1st July].
  Grounds, M.D., Radley, H.G., Lynch, G.S., Nagaraju, K., and De Luca, A. 2008. Towards developing standard operating procedures for pre‐clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol. Dis. 31(1):1‐19. doi: 10.1016/j.nbd.2008.03.008.
  Grounds, M.D., Terrill, J.R., Radley‐Crabb, H.G., Robertson, T., Papadimitriou, J., Spuler, S., and Shavlakadze, T. 2014. Lipid accumulation in dysferlin‐deficient muscles. Am. J. Pathol. 184(6):1668‐1676. doi: 10.1016/j.ajpath.2014.02.005.
  Gutpell, K.M., Hrinivich, W.T., and Hoffman, L.M. 2015. Skeletal muscle fibrosis in the mdx/utrn± mouse validates its suitability as a murine model of Duchenne muscular dystrophy. PLoS One. 10(1):e0117306. doi: 10.1371/journal.pone.0117306.
  Kalmar, B. and Greensmith, L. 2012. In Vivo Assessment of Mouse Hindlimb Muscle Force, Contractile, and Fatigue Characteristics, and Motor Unit Number. Curr. Protoc. Mouse Biol. 2(1):89‐101. doi: 10.1002/9780470942390.mo110155.
  Kalmar, B., Blanco, G., and Greensmith, L. 2012. Determination of Muscle Fiber Type in Rodents. Curr. Protoc. Mouse. Biol. 2(3):231‐243. doi:10.1002/9780470942390.mo110229.
  Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M., and Altman, D.G. 2010. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol. 8(6):e1000412. doi: 10.1371/journal.pbio.1000412.
  Kornegay, J.N., Spurney, C.F., Nghiem, P.P., Brinkmeyer‐Langford, C.L., Hoffman, E.P., and Nagaraju, K. 2014. Pharmacologic management of Duchenne muscular dystrophy: Target identification and preclinical trials. ILAR J. 55(1):119‐149. doi: 10.1093/ilar/ilu011.
  Lynch, G. 2014. Measuring isometric force of isolated mouse skeletal muscles in situ [online], available: http://www.treat‐nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.2.005.pdf [accessed 1st July].
  Mojumdar, K., Liang, F., Giordano, C., Lemaire, C., Danialou, G., Okazaki, T., Bourdon, J., Rafei, M., Galipeau, J., Divangahi, M., and Petrof, B.J. 2014. Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2. EMBO Mol. Med. 6(11):1476‐1492. doi: 10.15252/emmm.201403967.
  Moorwood, C., Liu, M., Tian, Z., and Barton, E.R. 2013. Isometric and eccentric force generation assessment of skeletal muscles isolated from murine models of muscular dystrophies. J. Vis. Exp. (71):e50036. doi: 10.3791/50036.
  Nagaraju, K. and Gordish, H. 2014. Serum creatine kinase analysis in mouse models of muscular dystrophy [online], available: http://www.treat‐nmd.eu/downloads/file/sops/md/MD_M.2.2.001.pdf [accessed 10th March].
  Qiu, K., Falk, D.J., Reier, P.J., Byrne, B.J., and Fuller, D.D. 2012. Spinal delivery of AAV vector restores enzyme activity and increases ventilation in Pompe mice. Mol. Ther. 20(1):21‐27. doi: 10.1038/mt.2011.214.
  Ruegg, M. and Meinen, S. 2014. Histopathology in Masson Trichrome stained muscle sections [online], available: http://www.treat‐nmd.eu/downloads/file/sops/cmd/MDC1A_M.1.2.003.pdf [accessed 1st July].
  Spurney, C.F., Gordish‐Dressman, H., Guerron, A.D., Sali, A., Pandey, G.S., Rawat, R., Van Der Meulen, J.H., Cha, H.J., Pistilli, E.E., Partridge, T.A., Hoffman, E.P., and Nagaraju, K. 2009. Preclinical drug trials in the mdx mouse: Assessment of reliable and sensitive outcome measures. Muscle Nerve. 39(5):591‐602. doi: 10.1002/mus.21211.
  Stapleton, D.I., Lau, X., Flores, M., Trieu, J., Gehrig, S.M., Chee, A., Naim, T., Lynch, G.S., and Koopman, R. 2014. Dysfunctional muscle and liver glycogen metabolism in mdx dystrophic mice. PLoS One. 9(3):e91514. doi: 10.1371/journal.pone.0091514.
  Terry, R.L., Kaneb, H.M., and Wells, D.J. 2014. Poloxomer 188 has a deleterious effect on dystrophic skeletal muscle function. PLoS One. 9(3):e91221. doi: 10.1371/journal.pone.0091221.
  van Putten, M., Hulsker, M., Young, C., Nadarajah, V.D., Heemskerk, H., van der Weerd, L., 't Hoen, P.A., van Ommen, G.J., and Aartsma‐Rus, A.M. 2013. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double‐knockout mice. FASEB J. 27(6):2484‐2495. doi: 10.1096/fj.12‐224170.
  Wells, K.E., McMahon, J., Foster, H., Ferrer, A., and Wells, D.J. 2008. Gene delivery to dystrophic muscle. Methods Mol. Biol. 423:421‐431. doi: 10.1007/978‐1‐59745‐194‐9_33.
  Whitmore, C. and Morgan, J. 2014. What do mouse models of muscular dystrophy tell us about the DAPC and its components?. Int. J. Exp. Pathol. 95(6):365‐377. doi: 10.1111/iep.12095.
  Wooddell, C.I., Zhang, G., Griffin, J.B., Hegge, J.O., Huss, T., and Wolff, J.A. 2010. Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice. Muscle Nerve. 41(4):487‐499. doi: 10.1002/mus.21527.
Internet Resources
  http://www.treat‐nmd.eu/research/preclinical/dmd‐sops
  Standard operating procedures from the above site/are listed in the Literature Cited.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library