MUSCLE FIBER NECROSIS

General features   Necrosis & Rhabdomyolysis   Stages     Early Features       Metabolic damage       Sarcolemmal membrane damage       C5b-9 deposition       Macrophage activation       Fixed muscle, Paraffin sections     Later features       Phagocytosis & Regeneration       Myofiber collapse       Eosinophils   Distribution of Necrotic fibers     Clustered       Immune (Vascular)         Regional Ischemic Immune         NXP-2       Hereditary (Stretch-induced)         Myopathic grouping     Perifascicular: IMPP     Scattered   Necrosis: Types & Features     Muscle Fiber Patterns       Membrane damage         Delta lesions       Metabolic failure, Diffuse       Peripheral, Histiocyte-related       Dark   Etiologies     Differential Diagnosis       Necrosis       Necrosis, Immune     Immune       Antibody-related         HMGCR         Mi-2         NXP-2         SRP         BCIM       LHIM       Macrophage activation       RIIM     Phosphorylase deficiency     Toxicity       Alcohol       Statin

Segmental Necrosis of Muscle fibers: Varied stages

Necrosis: General features & Stages ¹

Cell & Metabolic Conparative features Morphology   Dark   Metabolic failure   Membrane damage   Large Histiocyte   Phagocytosis	Necrosis, Early Stages: Muscle Fiber Patterns of Pathology
	Myofiber Necrosis   Metabolic     Muscle Fiber cytoplasm       Pale stain: Diffuse     Myonuclei: Lose staining	Myofiber Necrosis   Sarcolemmal Membrane Δ     Muscle Fiber Cytoplasm       Pale stain: "C" or Δ Lesion       Dark stain: Around C-region     Myonuclei: Lose staining	Myofiber Necrosis   Dark Necrosis     Muscle Fiber Cytoplasm       Dark stain: Diffuse     Myonuclei: Lose staining	Myofiber Necrosis   Histiocyte-Associated     Neighboring histiocytes       Around edge of fiber     Muscle Fiber Cytoplasm       Stain: Present
	Later Stages: Histiocyte Invasion & Phagocytosis of Muscle fibers			Later stage: Fiber Degeneration
	Myofiber Necrosis   Cell invasion     Stage: Intermediate       Muscle Fiber         Macrophage invasion	Myofiber Necrosis   Phagocytic   Stage: Late     Muscle Fiber       No cytoplasm remaining       Replaced by histiocytes	Myofiber Necrosis   Collapse   Stage: Very Late     Muscle Fiber       Space: Collapsed     Histiocytes       Few remaining       Many have migrated away	Myofiber Necrosis   Primary Fiber Degeneration     Stage: Late     Muscle fiber       Cytoplasm: Fragmented; Lost     Histiocytes: Few or None

• Necrosis: Cellular & metabolic changes
  • Cytoplasmic swelling (oncosis)
  • Rupture of plasma membrane
  • Cytoplasmic organelles: Swelling; Loss
  • Chromatin: Condensation, moderate
  • Absent: Apoptotic & Autophagic markers
  • Necrosis in muscle
    • Associated with either
      • Metabolic failure: Ca⁺⁺ leak into cytoplasm
      • Break in, or loss of, muscle surface fiber membrane
    • Results in: Irreversible damage to whole, or segment of, muscle fiber
    • All organelles involved
  • Associated metabolic alterations
    • No common biochemical denominator
    • Mitochondrial
      • Uncoupling
      • Production of reactive oxygen species (ROS)
      • Mitochondrial membrane permeabilization: Often controlled by cyclophilin D
      • Ca⁺⁺ overload
    • Lysosomal
      • ROS production by Fenton reactions
      • Membrane permeabilization
    • Nuclear
    • Hyperactivation of PARP-1
    • Hydrolysis of NAD⁺
    • Lipid degradation
      • Activation of phospholipases, lipoxygenases & sphingomyelinases
    • Ca⁺⁺
      • Activation of non-caspase proteases: Calpains; Cathepsins
• Necrotic muscle fiber morphology: Patterns during necrosis
  • Necrosis due to: Metabolic failure
    • Myofiber changes: Sarcolemma & Cytoplasm
      • H&E & Gomori Trichrome
        • Cytoplasm staining: Pale
        • Intermyofibrillar network: Absent
        • Myonuclei: Absent or reduced staining
      • Necrotic muscle fibers have reduced or absent staining with
        • NADH
        • Mitochondrial (SDH)
        • Myosin ATPase
      • Necrotic muscle fibers: Molecular & cellular markers
        • Cytoplasm of necrotic muscle fibers: Stains for
          • C_5b-9 Complement (Membrane attack complex (MAC))
          • IgM
          • Ca⁺⁺: Increased in necrotic muscle fibers
        • Around the rim of necrotic muscle fibers: Alkaline phosphatase
        • Muscle fiber phagocytosis
          • Histiocytic (Phagocytic) cells stain for: Acid phosphatase; Esterase; HAM56; CD68; MHC Class I
      • Ultrastructure
        • Loss of plasma membrane
        • Myofibrils: Lysed or Lost
        • Mitochondria: Abnormal
        • Late: Granular or filamentous debris in fibers
      • Rare in intrafusil muscle fibers
  • Necrosis due to: Membrane damage
    • Early damage pattern
      • Focal susarcolemmal area: Delta (Focal, subsarcolemmal) lesion: Pale stain
      • Other remaining cytoplasm: Dark stain
    • Disorders
      • Hereditary: Dystrophinopathy & Other sarcolemmal protein disorders
      • Toxic: Statin; Lysolecithin
  • Necrosis due to: Ischemia
    • Early fiber damage pattern: "Dark" necrosis
      • Cytoplasm of involved muscle fibers: Diffusely dark on H&E & Gomori trichrome stains
    • Later fiber damage: 2 patterns
      • Pale fragmented, or No, cytoplasm staining + No, or few, associated histiocytes
      • Invasion, Phagocytosis & replacement of fibers by histiocytes
    • Disorders with "Dark" necrosis
      • RIIM
      • Rhabdomyolysis with Phosphorylase deficiency
      • BACM
  • Necrosis due to: Large Histiocyte-mediation
  • Necrosis due to: Necroptosis (Regulated)
    • Programmed cell death
    • Stimuli
      • TNF-α : Pro-necrotic in muscle fibers
      • Death domain receptors: TNFR1 , Fas/CD95 , TRAIL-R
      • Toll-like receptors: TLR3 , TLR4
    • Pathway: Kinase RIP1 (RIPK1)
      • Molecules upregulated: RIPK1 , RIPK3 , MLKL
        
      • Execution pathway
        • RIP1–RIP3 necroptotic complex
        • Production of mitochondrial reactive oxygen species (ROS)
        • Depletion of cell energy under apoptotic-deficient conditions
      • Caspase-independent
    • Histologic marker: phospho-MLKL
• Necrosis in muscle: Stages
  • Early necrosis: Patterns
    • Cytoplasmic pallor: "Metabolic" pattern
    • Sectoral lesion & Hypercontraction
    • Subsarcolemmal lesion
  • Phagocytosis (Clean-up) of necrotic muscle fibers
    • Early: Infiltration of muscle fibers by histiocytic cells
      • Proliferation or Migration of Phagocytic cells
      • Attraction of Phagocytic cells to necrotic muscle fiber
      • Phagocytosis of necrotic muscle fibers
      • Associated macrophages: M1 type
        • Classically activated inflammatory macrophages
        • Infiltrate at early time points after injury
        • Generate inflammatory cytokines
        • Sustain activation & proliferation of muscle stem cells
    • Late: "Collapse"
      • Migration of macrophages away from necrotic muscle fiber
      • "Collapse" of region with necrotic muscle fiber
  • Cell remnant disposal
    • Related enzyme: MAP kinase phosphatase-1 (MKP-1; DUSP1)
  • Cell proliferation
    • Muscle precursors
      • Function: Formation of regenerating muscle fibers
      • Origins
        • Satellite (Stem) cells
        • Vessel-associated muscle stem cells (Mesoangioblasts)
          • Myogenic precursors
          • Express pericyte markers
          • Cross vessel walls
    • Connective tissue & Fat: Fibro/adipogenic progenitors (FAPs)
      • Sustain satellite cell-driven myogenesis
      • May differentiate into myoblasts, adipocytes or fibroblasts
    • M2 macrophages
    • Presence: Associated with tissue "Inflammation"
    • Late: Muscle healing
      • FAPs & Stem cells undergo apoptosis

Muscle Fiber Necrosis (Rhabdomyolysis episode): Morphology

H & E Stain	← Normal muscle fiber
	← Necrotic muscle fiber: Pale; Little phagocytosis
	← Necrotic muscle fiber: Phagocytosis by macrophages
	← Hypercontracted necrotic muscle fiber
	← Perimysium

H & E Stain

H&E stain
Necrotic muscle fibers: Pale; Little phagocytosis (Arrows)
Gomori trichrome stain	H&E Necrotic muscle fiber, Early Fiber is pale & enlarged. Internal nuclei may reflect invasion by macrophages.

Gomori trichrome stain

Necrotic muscle fibers: Internal architecture has lost NADH staining (Arrow) NADH stain

Sudan black stain

H&E stain

Membrane damage at several regions of individual fibers: Associated with clear regions in cytoplasm H&E stain

H&E stain

H&E stain

NADH stain

NADH stain

Congo red stain

H&E stain

Necrotic muscle fibers: Phagocytosis (Arrows)

Gomori trichrome stain

H&E stain

NADH stain

Esterase stain

Acid phosphatase stain Necrosis: Varied Stages   Early: Pale stained muscle fibers   Later: Muscle fibers progressively replaced by acid phosphatase + cells (Right) Acid phosphatasae stain

Esterase stain Necrotic muscle fiber, Late Muscle fiber is invaded and   replaced by phagocytes.

Esterase stain

H&E stain

Acid phosphatasae stain

H&E stain

Acid phosphatase stain

MHC Class I stain Very late stage necrosis: MHC Class I   Stains remaining cells phagocytsing collapsed muscle fibers   Stains capillaries (Normal)   No staining of most muscle fibers (Normal)

Alkaline phosphatase stain Necrosis: Alkaline phosphatase stains around the rim of muscle fibers

Toluidine blue stain Necrotic muscle fiber (Longitudinal section)   Pale   Invaded by cells
Toluidine blue stain Necrotic muscle fiber (Cross-section)   Pale   Invaded by cells	Toluidine blue stain Necrotic muscle fiber   Pale   Invaded by cells

Toluidine blue stain Cells in necrotic fiber

Toluidine blue stain Non-necrotic, abnormal muscle fiber (Left): Dilated SR

Necrotic muscle fibers: Complement C5b-9 (MAC) deposition

Stain: C5b-9 components of complement (Membrane attack complex (MAC)) Necrosis: C5b-9 deposition diffusely within muscle fiber cytoplasm

Necrosis: Ongoing

Necrosis: Early stage   Pale muscle fiber cytoplasm   Lipid droplets   Two other muscle fibers have large lipid droplets but are not pale & necrotic H&E stain

H&E stain

H&E stain

Necrosis associated with Eosinophils

H&E stain

H&E stain

Gomori trichrome stain

H&E stain

H&E stain

H&E stain

Necrosis: Scattered Muscle fibers

H&E stain

H&E stain

C5b-9 stain

H&E stain

H&E stain

H&E stain

H&E stain