Electron microscopy in the investigation and diagnosis of muscle disease

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1 Electron microscopy in the investigation and diagnosis of muscle disease Roy Weller Clinical Neurosciences University of Southampton School of Medicine

2 Normal Muscle

3 Normal Muscle

4 The Sarcomere The names for the different parts are derived from German

5 The Sarcomere H-zone (from the German heller", brighter) Inside the H-zone is a thin M-line (from the German Mittelscheibe, the disc in the middle of the sarcomere) formed of cross-connecting elements of the cytoskeleton I-band = Isotropic in polarised light as the actin filaments are thin and do not interfere with the light A-band = Anisotropic in polarised light as the myosin filaments are thicker and do interfere with the light Z-line = Zwischenscheibe the disc in between the I bands

6 TS through an A-band

7

8

9 Sarcomeres

10 T-tubes and Lateral Sacs

11 Myofibils Nucleus EM of muscle fiber Sarcolemma Basal lamina or Basement membrane Pathology and Genetics of Muscle Disease Book to be published 2013 Satellite cell Macrophages Perivascular Lymphocytes Regenerating myoblast

12 Neurogenic muscle disease

13 Neurogenic Muscle Disease ATPase stain Normal Chequer-board Pattern Denervation small group atrophy Reinnervation and fibre type grouping

14 Sarcolemmal disorders including Duchenne musclar dystrophy

15 Muscular Dystrophies associated with the lack of Sarcolemmal proteins Basement Membrane

16 Duchenne Muscular Dystrophy Dystrophin Immunocytochemistry- Normal

17 Normal Duchenne Muscular dystrophy: separation of basement membrane from the muscle fibre

18 Duchenne Muscular dystrophy: separation of basement membrane from the muscle fibre

19 Ion Channel disorders

20 Nerve and muscle end plate Cholinesterase in the Basement Membrane Sarcolemma Genetic Ca Disorders of Sarcoplasmic reticulum 2+ Ion Channels Mitochondria Glycogen and lipid ATP Ca 2+ T-tube Release and uptake of Ca2+ Muscle fibril contraction and relaxation

21 T-tubules and Lateral Sacs

22 D Myopathy with Tubular Aggregates

23 Tubular Aggregates

24 Tubular Aggregates

25 Tubular Aggregates

26 Mitochondrial myopathies

27 Mitochondrial Cytopathy (Myopathy)

28 Mitochondrial myopathy 1 μm resin section

29 Mitochondrial Cytopathy (Myopathy) (megaconial)

30 Inclusions in Mitochondrial Cytopathy (Myopathy)

31 Inclusions in Mitochondrial Cytopathy (Myopathy)

32 Cytoplasmic Inclusions

33 Cytoplasmic Inclusions

34 Cytoplasmic Inclusions some myofibrillar myopathies

35 Cytoplasmic Inclusion

36 Sarcoglycan & Dystroglycan Complexes Laminin 2 Caveolin Basement Membrane Sarcolemma Dystrophin F-Actin Z-band A-Band Plectin Desmin etc Actin Myosin Z-bands Desmin & intermediate filamentassociated proteins αb-crystallin Desmin etc Myofibrillar myopathies Emerin Lamin A&C Nucleus

37 Reducing body reduces menadione nitro-blue-tetrazolium (NBT) mutation in the FHL1 gene and other disorders

38 Z-band disorders rod bodies

39 Nemaline myopathy defects in thin filament proteins eg actin

40 Toxic myopathy due to Senna

41 Muscle fibre destruction and regeneration For example inflammatory myopathies Polymyositis, dermatomyositis, Inclusion Body Myositis, parasitic infections But also Dystrophies

42 Inflammatory cells in the endomysium surrounding atrophic muscle fibres

43 Hunkin Muscle fibre destruction influenza-a myopathy macrophages and regenerating myoblasts

44 Normal Necrosis 24hrs Regeneration 10-14days 30days 30days Focal necrosis and regeneration of skeletal muscle

45 Satellite Cell source of regenerating myoblasts

46 Normal basement membrane (Laminin) Trypsin destroys the basement membrane Barium chloride destroys only the muscle fibres Is an intact basement membrane tube essential for successful muscle fibre regeneration? Role of the basement membrane in the regeneration of skeletal muscle. Caldwell CJ, Mattey DL, Weller RO Neuropathol Appl Neurobiol Jun;16:

47 Scanning Electron microscopy Barium chloride destroys only the muscle fibres Basement membrane tubes are preserved

48 Scanning Electron microscopy: Macrophages on basement membrane tubes: -- Barium chloride treated muscle

49 * Scanning Electron microscopy: Macrophages within basement membrane tube: - - Barium chloride treated muscle

50 Macrophages and regenerating myoblast within basement membrane tube: -- Barium chloride treated muscle

51 Basement membrane closely invests a regenerating myoblast in Trypsin treated muscle in which the muscle fibre and the basement membrane were destroyed

52 5 days recovery: which is the Trypsin-treated muscle?

53 Inclusion Body Myositis

54 Inclusion Body Myositis

55 Inclusion Body Myositis

56 Inclusion Body Myositis

57 Inclusion Body Myositis

58 Inclusion Body Myositis

59 Inclusion Body Myositis

60 Electron microscopy in the evaluation of individual cases Dr Ingrid Mazanti Dr Mark Walker

61

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