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Skeletal Muscle Regeneration in the Mouse pp 101–127Cite as

Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1460))

Abstract

The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoDCre × R26mTmG reporter mouse that elegantly distinguishes muscle (MyoDCre-driven GFP+) from the non-myogenic constituents (Tomato+); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.

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References

  1. Demer JL (2007) Mechanics of the orbita. Dev Ophthalmol 40:132–157

    Article  PubMed  PubMed Central  Google Scholar 

  2. Kirillova I, Gussoni E, Goldhamer DJ, Yablonka-Reuveni Z (2007) Myogenic reprogramming of retina-derived cells following their spontaneous fusion with myotubes. Dev Biol 311:449–463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Stuelsatz P, Shearer A, Li Y, Muir LA, Ieronimakis N, Shen QW, Kirillova I, Yablonka-Reuveni Z (2015) Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency. Dev Biol 397:31–44

    Article  CAS  PubMed  Google Scholar 

  4. Stuelsatz P, Shearer A, Yablonka-Reuveni Z (2014) Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin. Dev Biol 385:366–379

    Article  CAS  PubMed  Google Scholar 

  5. Fischer MD, Gorospe JR, Felder E, Bogdanovich S, Pedrosa-Domellof F, Ahima RS, Rubinstein NA, Hoffman EP, Khurana TS (2002) Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol Genomics 9:71–84

    Article  CAS  PubMed  Google Scholar 

  6. Lucas CA, Hoh JF (1997) Extraocular fast myosin heavy chain expression in the levator palpebrae and retractor bulbi muscles. Invest Ophthalmol Vis Sci 38:2817–2825

    CAS  PubMed  Google Scholar 

  7. Porter JD, Khanna S, Kaminski HJ, Rao JS, Merriam AP, Richmonds CR, Leahy P, Li J, Andrade FH (2001) Extraocular muscle is defined by a fundamentally distinct gene expression profile. Proc Natl Acad Sci U S A 98:12062–12067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Spencer RF, Porter JD (2006) Biological organization of the extraocular muscles. Prog Brain Res 151:43–80

    Article  PubMed  Google Scholar 

  9. Couly GF, Coltey PM, Le Douarin NM (1992) The developmental fate of the cephalic mesoderm in quail-chick chimeras. Development 114:1–15

    CAS  PubMed  Google Scholar 

  10. Noden DM, Francis-West P (2006) The differentiation and morphogenesis of craniofacial muscles. Dev Dyn 235:1194–1218

    Article  CAS  PubMed  Google Scholar 

  11. Valdez G, Tapia JC, Lichtman JW, Fox MA, Sanes JR (2012) Shared resistance to aging and ALS in neuromuscular junctions of specific muscles. PLoS One 7:e34640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yu Wai Man CY, Chinnery PF, Griffiths PG (2005) Extraocular muscles have fundamentally distinct properties that make them selectively vulnerable to certain disorders. Neuromuscul Disord 15:17–23

    Article  CAS  PubMed  Google Scholar 

  13. Schoser BG, Pongratz D (2006) Extraocular mitochondrial myopathies and their differential diagnoses. Strabismus 14:107–113

    Article  PubMed  Google Scholar 

  14. Kaminski HJ, Richmonds CR, Kusner LL, Mitsumoto H (2002) Differential susceptibility of the ocular motor system to disease. Ann N Y Acad Sci 956:42–54

    Article  PubMed  Google Scholar 

  15. Ahmadi M, Liu JX, Brannstrom T, Andersen PM, Stal P, Pedrosa-Domellof F (2010) Human extraocular muscles in ALS. Invest Ophthalmol Vis Sci 51:3494–3501

    Article  PubMed  Google Scholar 

  16. Kaminski HJ, al-Hakim M, Leigh RJ, Katirji MB, Ruff RL (1992) Extraocular muscles are spared in advanced Duchenne dystrophy. Ann Neurol 32:586–588

    Article  CAS  PubMed  Google Scholar 

  17. Khurana TS, Prendergast RA, Alameddine HS, Tome FM, Fardeau M, Arahata K, Sugita H, Kunkel LM (1995) Absence of extraocular muscle pathology in Duchenne’s muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing. J Exp Med 182:467–475

    Article  CAS  PubMed  Google Scholar 

  18. Porter JD, Karathanasis P (1998) Extraocular muscle in merosin-deficient muscular dystrophy: cation homeostasis is maintained but is not mechanistic in muscle sparing. Cell Tissue Res 292:495–501

    Article  CAS  PubMed  Google Scholar 

  19. Porter JD, Merriam AP, Hack AA, Andrade FH, McNally EM (2001) Extraocular muscle is spared despite the absence of an intact sarcoglycan complex in gamma- or delta-sarcoglycan-deficient mice. Neuromuscul Disord 11:197–207

    Article  CAS  PubMed  Google Scholar 

  20. Porter JD (1998) Commentary: extraocular muscle sparing in muscular dystrophy: a critical evaluation of potential protective mechanisms. Neuromuscul Disord 8:198–203

    Article  CAS  PubMed  Google Scholar 

  21. Andrade FH, Porter JD, Kaminski HJ (2000) Eye muscle sparing by the muscular dystrophies: lessons to be learned? Microsc Res Tech 48:192–203

    Article  CAS  PubMed  Google Scholar 

  22. Porter JD, Merriam AP, Khanna S, Andrade FH, Richmonds CR, Leahy P, Cheng G, Karathanasis P, Zhou X, Kusner LL, Adams ME, Willem M, Mayer U, Kaminski HJ (2003) Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice. FASEB J 17:893–895

    CAS  PubMed  Google Scholar 

  23. Karpati G, Carpenter S (1986) Small-caliber skeletal muscle fibers do not suffer deleterious consequences of dystrophic gene expression. Am J Med Genet 25:653–658

    Article  CAS  PubMed  Google Scholar 

  24. Karpati G, Carpenter S, Prescott S (1988) Small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy. Muscle Nerve 11:795–803

    Article  CAS  PubMed  Google Scholar 

  25. Turner PR, Westwood T, Regen CM, Steinhardt RA (1988) Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature 335:735–738

    Article  CAS  PubMed  Google Scholar 

  26. Zeiger U, Mitchell CH, Khurana TS (2010) Superior calcium homeostasis of extraocular muscles. Exp Eye Res 91:613–622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ragusa RJ, Chow CK, St Clair DK, Porter JD (1996) Extraocular, limb and diaphragm muscle group-specific antioxidant enzyme activity patterns in control and mdx mice. J Neurol Sci 139:180–186

    Article  CAS  PubMed  Google Scholar 

  28. Matsumura K, Ervasti JM, Ohlendieck K, Kahl SD, Campbell KP (1992) Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 360:588–591

    Article  CAS  PubMed  Google Scholar 

  29. Porter JD, Rafael JA, Ragusa RJ, Brueckner JK, Trickett JI, Davies KE (1998) The sparing of extraocular muscle in dystrophinopathy is lost in mice lacking utrophin and dystrophin. J Cell Sci 111(Pt 13):1801–1811

    CAS  PubMed  Google Scholar 

  30. McDonald AA, Hebert SL, McLoon LK (2015) Sparing of the extraocular muscles in mdx mice with absent or reduced utrophin expression: a life span analysis. Neuromuscul Disord 25:873–887

    Article  PubMed  Google Scholar 

  31. Kallestad KM, Hebert SL, McDonald AA, Daniel ML, Cu SR, McLoon LK (2011) Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis. Exp Cell Res 317:873–885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Porter JD, Israel S, Gong B, Merriam AP, Feuerman J, Khanna S, Kaminski HJ (2006) Distinctive morphological and gene/protein expression signatures during myogenesis in novel cell lines from extraocular and hindlimb muscle. Physiol Genomics 24:264–275

    Article  CAS  PubMed  Google Scholar 

  33. Pacheco-Pinedo EC, Budak MT, Zeiger U, Jorgensen LH, Bogdanovich S, Schroder HD, Rubinstein NA, Khurana TS (2009) Transcriptional and functional differences in stem cell populations isolated from extraocular and limb muscles. Physiol Genomics 37:35–42

    Article  CAS  PubMed  Google Scholar 

  34. Hughes MO (2007) A pictural anatomy of the human eye/anophthalmic socket: a review for ocularists. J Ophthal Prosthet 12:51–63

    Google Scholar 

  35. Lee SH, Wong M, Yap S (2015) Anatomy—muscles of the eye. http://rodsncones.blogspot.com/2014/05/anatomy-muscles-of-eye.html. Accessed 8 Dec 2015

  36. LifeMap (2015) Extraocular muscles anatomy. http://discovery.lifemapsc.com/library/images/extraocular-skeletal-muscle-anatomy. Accessed 8 Dec 2015

  37. Montgomery TM (2015) The extraocular muscles. http://www.tedmontgomery.com/the_eye/eom.html. Accessed 8 Dec 2015

  38. Carry MR, O'Keefe K, Ringel SP (1982) Histochemistry of mouse extraocular muscle. Anat Embryol (Berl) 164:403–412

    Article  CAS  Google Scholar 

  39. Demer JL, Oh SY, Poukens V (2000) Evidence for active control of rectus extraocular muscle pulleys. Invest Ophthalmol Vis Sci 41:1280–1290

    CAS  PubMed  Google Scholar 

  40. Demer JL (2003) Evidence for a pulley of the inferior oblique muscle. Invest Ophthalmol Vis Sci 44:3856–3865

    Article  PubMed  Google Scholar 

  41. Ruskell GL, Kjellevold Haugen IB, Bruenech JR, van der Werf F (2005) Double insertions of extraocular rectus muscles in humans and the pulley theory. J Anat 206:295–306

    Article  PubMed  PubMed Central  Google Scholar 

  42. Kono R, Poukens V, Demer JL (2005) Superior oblique muscle layers in monkeys and humans. Invest Ophthalmol Vis Sci 46:2790–2799

    Article  PubMed  Google Scholar 

  43. Zhou Y, Liu D, Kaminski HJ (2010) Myosin heavy chain expression in mouse extraocular muscle: more complex than expected. Invest Ophthalmol Vis Sci 51:6355–6363

    Article  PubMed  PubMed Central  Google Scholar 

  44. Rossi AC, Mammucari C, Argentini C, Reggiani C, Schiaffino S (2010) Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles. J Physiol 588:353–364

    Article  CAS  PubMed  Google Scholar 

  45. Rubinstein NA, Porter JD, Hoh JF (2004) The development of longitudinal variation of Myosin isoforms in the orbital fibers of extraocular muscles of rats. Invest Ophthalmol Vis Sci 45:3067–3072

    Article  PubMed  Google Scholar 

  46. Zhu Q, Hillmann DJ, Henk WG (2000) Observations on the muscles of the eye of the bowhead whale, Balaena mysticetus. Anat Rec 259:189–204

    Article  CAS  PubMed  Google Scholar 

  47. Zhou JB, Ge S, Gu P, Peng D, Chen GF, Pan MZ, Qu J (2011) Microdissection of guinea pig extraocular muscles. Exp Ther Med 2:1183–1185

    PubMed  PubMed Central  Google Scholar 

  48. Clarkson C, Brown A, Ekenstedt K, Fletcher TF (2015) Orbit, eyeball & related structures. http://vanat.cvm.umn.edu/carnLabs/Lab24/Lab24.html. Accessed 12 Nov 2015

  49. Marques MJ, Pertille A, Carvalho CL, Santo Neto H (2007) Acetylcholine receptor organization at the dystrophic extraocular muscle neuromuscular junction. Anat Rec 290:846–854

    Article  CAS  Google Scholar 

  50. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102:777–786

    Article  CAS  PubMed  Google Scholar 

  51. Yablonka-Reuveni Z (2011) The skeletal muscle satellite cell: still young and fascinating at 50. J Histochem Cytochem 59:1041–1059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kanisicak O, Mendez JJ, Yamamoto S, Yamamoto M, Goldhamer DJ (2009) Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD. Dev Biol 332:131–141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45:593–605

    Article  CAS  PubMed  Google Scholar 

  54. Chapman VM, Miller DR, Armstrong D, Caskey CT (1989) Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice. Proc Natl Acad Sci U S A 86:1292–1296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS (1996) Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet 5:1149–1153

    Article  CAS  PubMed  Google Scholar 

  56. Banks GB, Combs AC, Chamberlain JS (2010) Sequencing protocols to genotype mdx, mdx(4cv), and mdx(5cv) mice. Muscle Nerve 42:268–270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lu QL, Morris GE, Wilton SD, Ly T, Artem'yeva OV, Strong P, Partridge TA (2000) Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion. J Cell Biol 148:985–996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Belart.com (2015) mortar w/bowl & housing, liquid nitrogen-cooled (Bel-Art# 372600000) https://www.belart.com/bel-art-h37260-0000-liquid-nitrogen-cooled-stainless-steel-ladle-and-reservoir-6-diameter-4-height.html. Accessed 7 Dec 2015

  59. Stuelsatz P, Keire P, Almuly R, Yablonka-Reuveni Z (2012) A contemporary atlas of the mouse diaphragm: myogenicity, vascularity, and the Pax3 connection. J Histochem Cytochem 60:638–657

    Article  PubMed  PubMed Central  Google Scholar 

  60. Yablonka-Reuveni Z, Danoviz ME, Phelps M, Stuelsatz P (2015) Myogenic-specific ablation of Fgfr1 impairs FGF2-mediated proliferation of satellite cells at the myofiber niche but does not abolish the capacity for muscle regeneration. Front Aging Neurosci 7:85

    Article  PubMed  PubMed Central  Google Scholar 

  61. Day K, Shefer G, Richardson JB, Enikolopov G, Yablonka-Reuveni Z (2007) Nestin-GFP reporter expression defines the quiescent state of skeletal muscle satellite cells. Dev Biol 304:246–259

    Article  CAS  PubMed  Google Scholar 

  62. Yablonka-Reuveni Z, Christ B, Benson JM (1998) Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program. Anat Embryol (Berl) 197:421–437

    Article  CAS  Google Scholar 

  63. IHC-World (2015) Tris-EDTA buffer antigen retrieval protocol. http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm. Accessed 7 Dec 2015

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Acknowledgements

We are grateful to Lindsey Muir for her helpful comments on the manuscript. We also express gratitude to our former lab members, Irina Kirillova and Andrew Shearer for their important contributions during the initial phase of our studies leading to the ocular/periocular isolation protocol described here. Our study of mouse extraocular muscles and their satellite cells is currently supported by grants to Z.Y.R. from the National Institute of health (AG035377 and NS088804) and by an AFM-telethon fellowship to P.S. (#18574). Z.Y.R. acknowledges additional support during the preparation of this chapter from the National Institutes of Health (NS090051).

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  1. Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA, USA

    Pascal Stuelsatz & Zipora Yablonka-Reuveni Ph.D.

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  1. Pascal Stuelsatz
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  2. Zipora Yablonka-Reuveni Ph.D.
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Correspondence to Zipora Yablonka-Reuveni Ph.D. .

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  1. Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Michael Kyba

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Stuelsatz, P., Yablonka-Reuveni, Z. (2016). Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells. In: Kyba, M. (eds) Skeletal Muscle Regeneration in the Mouse. Methods in Molecular Biology, vol 1460. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3810-0_9

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  • DOI: https://doi.org/10.1007/978-1-4939-3810-0_9

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