Myofiber Damage Evaluation by Evans Blue Dye Injection

Christine I. Wooddell1, Hannah G. Radley‐Crabb2, Jacob B. Griffin1, Guofeng Zhang2

1 Roche Madison Inc., Madison, Wisconsin, 2 School of Anatomy and Human Biology, The University of Western Australia, Crawley, Australia
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110141
Online Posting Date:  December, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Evans blue dye (EBD) can be used in live mice to study muscle pathology or injury, including exercise‐induced muscle damage. EBD is excluded from intact cell membranes but leaks into cells, including muscle fibers, when the cell membrane is ruptured. EBD can be visualized by its autofluorescence under a fluorescence microscope. EBD‐stained myofibers can be quantified from microscope images of muscle cross‐sections. These myofibers are often in clusters that lend themselves to morphometric analysis. When the damaged myofibers are interspersed among intact myofibers, however, a more suitable approach is to count individual myofibers in the field of view. A much faster approach to measure EBD in muscles from different strains of mice or between treatment groups is to extract the EBD from muscle samples and quantitate it using a spectrophotometric microplate reader. The advantages and disadvantages of using each of these approaches are discussed here. Curr. Protoc. Mouse Biol. 1:463‐488 © 2011 by John Wiley & Sons, Inc.

Keywords: Evans blue dye; skeletal muscle; mouse model; mdx mouse

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Delivery of EBD
  • Basic Protocol 2: Morphometric Quantitation of Areas within the Muscle
  • Basic Protocol 3: Determining Percentagesl OF EBD‐Positive Myofibers
  • Support Protocol 1: Counting Percentage of EBD+ Myofibers
  • Support Protocol 2: Production of Mouse Polyclonal Antibodies to Dystrophin
  • Basic Protocol 4: Spectrophotometric Quantitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Delivery of EBD

  • Mice
  • Evans blue dye (Sigma‐Aldrich, cat. no. E2129) prepared at 5 mg/ml with sterile physiological saline (0.90% NaCl) (see recipe)
  • 1% to 2% isoflurane
  • Phosphate buffered saline (PBS)
  • Heat lamp or 50‐ml conical tube containing warm water (50°C)
  • 1‐ml syringe with 30‐G needle for i.v. injection or 500‐µl or 1‐ml syringe with 27‐G needle for i.p. injection
  • Anesthesia machine with animal chamber or an animal holder to restrain the mouse during injection

Basic Protocol 2: Morphometric Quantitation of Areas within the Muscle

  • Tragacanth gum (Sigma Aldrich, cat. no. G1128)
  • Isopentane
  • Liquid nitrogen
  • Isoflurane
  • Acetone
  • Xylene
  • DPX mountant glue (BDH, cat. no. 36029.4H)
  • Cork (∼1 cm3 per sample)
  • Cryostat
  • Glass histology slides (VWR Superfrost Plus Micro slides, cat. no. 48311‐703)
  • Glass cover slips
  • Fluorescence microscope (band pass: excitation 515 to 560 nm; low pass: emission 590 nm) with camera and image capture software (e.g., Leica DM RBE microscope, a personal computer, a Hitachi HVC2OM digital camera, Image Pro Plus 6.2 software, and Vexta stage movement software)

Basic Protocol 3: Determining Percentagesl OF EBD‐Positive Myofibers

  • Tissue‐Tek O.C.T. compound (Sakura Finetek, cat. no. 4583)
  • Mice
  • 3% to 5% isoflurane
  • Liquid nitrogen
  • Styrofoam container for holding liquid nitrogen and Styrofoam float for holding embedding molds while freezing in liquid nitrogen (Fig. )
  • Peel‐A‐Way disposable embedding molds, truncated (8 × 8 × 20–mm; PolySciences, cat. no. 18985)

Support Protocol 1: Counting Percentage of EBD+ Myofibers

  • Frozen tissue (see protocol 3)
  • 2% to 4% formalin in PBS
  • PBS
  • Mouse anti‐dystrophin polyclonal antibodies diluted as needed in PBS (see protocol 5)
  • FITC‐conjugated goat anti‐mouse IgG (FAB‐specific; Sigma‐Aldrich, cat. no. F8771) diluted 1:400 in PBS
  • Cryostat
  • Glass histology slides (VWR Superfrost Plus Micro slides, cat. no. 48311‐703)
  • Paraffin pen
  • Microscope, excitation filters (FITC and CY3), camera

Support Protocol 2: Production of Mouse Polyclonal Antibodies to Dystrophin

  • pDNA that produces human or mouse dystrophin (prepare or order endotoxin‐free plasmid DNA)
  • Sterile, physiological saline solution
  • C57Bl/10ScSn‐Dmdmdx/J (mdx‐10ScSn) mice (Jackson Laboratories)
  • 1% to 2% isoflurane anesthesia
  • Ketoprofen
  • Serum tubes with gel and clot activator
  • 50‐ml polypropylene tubes
  • Heating pad at 37°C
  • Scalpel
  • Latex tourniquet
  • 4‐0 absorbable sutures
  • 1‐ml syringes
  • Additional reagents and equipment for muscle harvesting, processing, and immunostaining (see protocol 3 and protocol 4) and HLV injection (Hagstrom et al., )

Basic Protocol 4: Spectrophotometric Quantitation

  • EBD (see recipe)
  • N,N‐dimethyl formamide (DMF)
  • Liquid nitrogen
  • Muscle samples
  • Spreadsheet (e.g., Microsoft Excel)
  • Mortar and pestle (mortar must be cooled with liquid N 2)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Rotator (e.g., Rugged rotator, Glas‐Col) or equivalent
  • Microcentrifuge
  • 96‐well UV‐transparent microplates (BD Falcon, cat. no. 353261)
  • Microplate reader capable of reading absorbance at 630 nm (SpectraMAX Plus, Molecular Devices)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Archer, J.D., Vargas, C.C., and Anderson, J.E. 2006. Persistent and improved functional gain in mdx dystrophic mice after treatment with L‐arginine and deflazacort. Faseb J. 20:738‐740.
   Bates, M.K., Zhang, G., Sebestyen, M.G., Neal, Z.C., Wolff, J.A., and Herweijer, H. 2006. Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA. Biotechniques 40:199‐208.
   Bostick, B., Yue, Y., Long, C., Marschalk, N., Fine, D.M., Chen, J., and Duan, D. 2009. Cardiac expression of a mini‐dystrophin that normalizes skeletal muscle force only partially restores heart function in aged mdx mice. Mol. Ther. 17:253‐261.
   Brussee, V., Tardif, F., and Tremblay, J.P. 1997. Muscle fibers of mdx mice are more vulnerable to exercise than those of normal mice. Neuromuscul. Disord. 7:487‐492.
   Chakkalakal, J.V., Harrison, M.A., Carbonetto, S., Chin, E., Michel, R.N., and Jasmin, B.J. 2004. Stimulation of calcineurin signaling attenuates the dystrophic pathology in mdx mice. Hum. Mol. Genet. 13:379‐388.
   Cifuentes‐Diaz, C., Frugier, T., Tiziano, F.D., Lacene, E., Roblot, N., Joshi, V., Moreau, M.H., and Melki, J. 2001. Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J. Cell Biol. 152:1107‐1114.
   Coulton, G.R., Morgan, J.E., Partridge, T.A., and Sloper, J.C. 1988. The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation. Neuropathol. Appl. Neurobiol. 14:53‐70.
   Doherty, K.R. and McNally, E.M. 2003. Repairing the tears: Dysferlin in muscle membrane repair. Trends Mol. Med. 9:327‐330.
   Durbeej, M., Sawatzki, S.M., Barresi, R., Schmainda, K.M., Allamand, V., Michele, D.E., and Campbell, K.P. 2003. Gene transfer establishes primacy of striated vs. smooth muscle sarcoglycan complex in limb‐girdle muscular dystrophy. Proc. Natl. Acad. Sci. U.S.A. 100:8910‐8915.
   Fougerousse, F., Bartoli, M., Poupiot, J., Arandel, L., Durand, M., Guerchet, N., Gicquel, E., Danos, O., and Richard, I. 2007. Phenotypic correction of alpha‐sarcoglycan deficiency by intra‐arterial injection of a muscle‐specific serotype 1 rAAV vector. Mol. Ther. 15:53‐61.
   Goyenvalle, A., Vulin, A., Fougerousse, F., Leturcq, F., Kaplan, J.C., Garcia, L., and Danos, O. 2004. Rescue of dystrophic muscle through U7 snRNA‐mediated exon skipping. Science 306:1796‐1799.
   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. 31:1‐19.
   Hagstrom, J.E., Hegge, J., Zhang, G., Noble, M., Budker, V., Lewis, D.L., Herweijer, H., and Wolff, J.A. 2004. A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol. Ther. 10:386‐398.
   Hamer, P.W., McGeachie, J.M., Davies, M.J., and Grounds, M.D. 2002. Evans blue dye as an in vivo marker of myofibre damage: Optimizing parameters for detecting initial myofibre membrane permeability. J. Anat. 200:69‐79.
   Kobinger, G.P., Louboutin, J.P., Barton, E.R., Sweeney, H.L., and Wilson, J.M. 2003. Correction of the dystrophic phenotype by in vivo targeting of muscle progenitor cells. Hum. Gene Ther. 14:1441‐1449.
   Lefaucheur, J.P., Pastoret, C., and Sebille, A. 1995. Phenotype of dystrophinopathy in old mdx mice. Anat. Rec. 242:70‐76.
   Li, D., Yue, Y., and Duan, D. 2008. Preservation of muscle force in Mdx3cv mice correlates with low‐level expression of a near full‐length dystrophin protein. Am. J. Pathol. 172:1332‐1341.
   Marcaletti, S., Thomas, C., and Feige, J.N. 2011. Exercise performance tests in mice. Curr. Protoc. Mouse Biol. 1:141‐154.
   Matsuda, R., Nishikawa, A., and Tanaka, H. 1995. Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: Evidence of apoptosis in dystrophin‐deficient muscle. J. Biochem. 118:959‐964.
   McGeachie, J.K., Grounds, M.D., Partridge, T.A., and Morgan, J.E. 1993. Age‐related changes in replication of myogenic cells in mdx mice: Quantitative autoradiographic studies. J. Neurol. Sci. 119:169‐179.
   McNeil, P.L. and Kirchhausen, T. 2005. An emergency response team for membrane repair. Nat. Rev. Mol. Cell Biol. 6:499‐505.
   Minetti, G.C., Colussi, C., Adami, R., Serra, C., Mozzetta, C., Parente, V., Fortuni, S., Straino, S., Sampaolesi, M., Di Padova, M., Illi, B., Gallinari, P., Steinkuhler, C., Capogrossi, M.C., Sartorelli, V., Bottinelli, R., Gaetano, C., and Puri, P.L. 2006. Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors. Nat. Med. 12:1147‐1150.
   Miura, P., Chakkalakal, J.V., Boudreault, L., Belanger, G., Hebert, R.L., Renaud, J.M., and Jasmin, B.J. 2009. Pharmacological activation of PPARbeta/delta stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice. Hum. Mol. Genet. 18:4640‐4649.
   Piers, A.T., Lavin, T., Radley‐Crabb, H.G., Bakker, A.J., Grounds, M.D., and Pinniger, G.J. 2011. Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice. Neuromuscul. Disord. 21:132‐141.
   Reeve, E.B. 1957. The contribution of I 131‐labeled proteins to measurements of blood volume. Ann. N.Y. Acad. Sci. 70:137‐147.
   Richard, I., Roudaut, C., Marchand, S., Baghdiguian, S., Herasse, M., Stockholm, D., Ono, Y., Suel, L., Bourg, N., Sorimachi, H., Lefranc, G., Fardeau, M., Sebille, A., and Beckmann, J.S. 2000. Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis‐associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice. J. Cell Biol. 151:1583‐1590.
   Shavlakadze, T., White, J., Hoh, J.F. Rosenthal, N., and Grounds, M.D. 2004. Targeted expression of insulin‐like growth factor‐I reduces early myofiber necrosis in dystrophic mdx mice. Mol. Ther. 10:829‐843.
   Sher, R.B., Aoyama, C., Huebsch, K.A., Ji, S., Kerner, J., Yang, Y., Frankel, W.N., Hoppel, C.L., Wood, P.A., Vance, D.E., and Cox, G.A. 2006. A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis. J. Biol. Chem. 281:4938‐4948.
   Sokolow, S., Manto, M., Gailly, P., Molgo, J., Vandebrouck, C., Vanderwinden, J.M., Herchuelz, A., and Schurmans, S. 2004. Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3. J. Clin. Invest. 113:265‐273.
   Straub, V., Rafael, J.A., Chamberlain, J.S., and Campbell, K.P. 1997. Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J. Cell Biol. 139:375‐385.
   Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J.M., and Davies, K. 1998. Expression of full‐length utrophin prevents muscular dystrophy in mdx mice. Nature Med. 4:1441‐1444.
   van Putten, M., de Winter, C., van Roon‐Mom, W., van Ommen, G.J., 't Hoen, P.A., and Aartsma‐Rus, A. 2010. A 3 months mild functional test regime does not affect disease parameters in young mdx mice. Neuromuscul. Disord. 20:273‐280.
   Vilquin, J.T., Brussee, V., Asselin, I., Kinoshita, I., Gingras, M., and Tremblay, J.P. 1998. Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise. Muscle Nerve 21:567‐576.
   Voisin, V., Sebrie, C., Matecki, S., Yu, H., Gillet, B., Ramonatxo, M., Israel, M., and De la Porte, S. 2005. l‐Arginine improves dystrophic phenotype in mdx mice. Neurobiol. Dis. 20:123‐130.
   Wang, B., Li, J., Qiao, C., Chen, C., Hu, P., Zhu, X., Zhou, L., Bogan, J., Kornegay, J., and Xiao, X. 2008. A canine minidystrophin is functional and therapeutic in mdx mice. Gene Ther. 15:1099‐1106.
   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. 41:487‐499.
   Zhang, G., Wooddell, C.I., Hegge, J.O., Griffin, J.B., Huss, T., Braun, S., and Wolff, J.A. 2010. Functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection. Hum. Gene Ther. 21:221‐237.
PDF or HTML at Wiley Online Library