Axon loss
  Large vs Small
  Large & Small
  Differential fascicular loss
  Schwann cell Δ
    Bungner bands
    Collagen pockets
Wallerian degeneration

Gomori trichrome stain
Mild loss of myelinated
axons (red) in a fascicle

Oppenheim 1894

Myelinated axons: Loss

Gomori trichrome stain
Myelinated Axon loss: Moderate

Gomori trichrome stain

VvG stain

Myelinated Axon loss: Severe

Myelinated Axon loss: Large > Small

Toluidine blue stain
Myelinated Axon loss: Small > Large

VvG stain

Axons, Large & Small: Comparative changes

Moderate loss of large axons (Arrow); Small axons are relatively preserved

Neurofilament stain

Axon loss, severe: Large & Small axons are both markedly reduced

Neurofilament stain
Myelinated axons: Severe loss

VvG stain

Wallerian Degeneration 5

    Axon degeneration & loss
    Myelin fragmentation
    Myelin degradation
  After WD
    Axon regeneration
    Bungner band
    Collagen pockets
Alternate Degeneration Process
  Trophic-withdrawal Degeneration


Wallerian Degeneration: Principles & Features

  • Wallerian degeneration: Definition
  • Wallerian Degeneration: Morphological & other changes in nerve constituents
    • Stimulus for Wallerian degeneration
      • Distal axon loses connection with proximal axon
    • Axon changes 7
      • Early
        • Minutes: Changes in axon segments near transsection
          • Short-distance (200 μM): Acute axon degeneration (AAD)
          • Mediated by
            • Extracellular Ca++ influx
              • Anterogradely conduced wave in distal axon
            • Activation of calpain
              • Ca++-dependent serine-threonine protease
              • Intracellular enzyme
          • Inhibited by: Ca++ channel blockers
        • Hours to 2 days
          • Regions near injury: Accumulation of organelles & mitochondria
            • Dystrophic bulbs
            • Occur at both transected ends
            • Mechanism: Anterograde & retrograde axon transport
          • Proximal & Distal axon: Retraction from injury site
          • Distal axon
            • Remains morphologically intact & electrically excitable
            • Axon transport (anterograde & retrograde) continues
          • Proximal stump: Produces sprouts within hours after axotomy
      • Later (≥ 3 days): Changes in distal axon
        • Endoplasmic reticulum: Loss of structure
        • Cytoplasm: Neurofilament & Cytoskeleton Degradation
          • Associated with influx of Ca++
          • Activation of calpain
          • Autophagy-related
        • Mitochondria: Swelling
        • Distal axon morphology: Granular degeneration
          • Becomes fragmented
          • Phagocytosis
          • Direction after focal injury: Proximal to Distal; Rate of up to 24 mm/hr
      • Timing
        • Degeneration begins several days (4 to 10) after axonal transection
        • Before degeneration the distal axon segment may remain electrically excitable
        • Delayed by
          • Temperature: Reduced
          • Extracellular Ca++: Lowered
          • Ca++ channel (L-type) blockers
          • Mutations or Loss
            • NMNAT1: WldS
            • NOS knockout
            • DR6 (TNFRSF21) 9
            • SARM1 8
              • Required for early injury induced axon degeneration
              • Loss: Slows axon degeneration
              • Toll/Interleukin-1 receptor (TIR) domain
                • Possesses intrinsic NAD+ cleavage (NADase) activity
                • Promotes axon degeneration
            • DLK (MAP3K12) : Loss of function
            • jnk (MAPK8) : Signaling requires DR6
          • Inhibition: JNK kinase ; GSK3; IKKB (IKBKB)
        • More rapid
          • Galectin-3 loss
            • Increased pro-inflammatory cytokines
              • IL-1β, TNF-α, Toll-like receptor (TLR)-2 & -4
            • Increased phagocytic capacity of Schwann cells & macrophages
        • No effect: NGF
      • Molecular events 1
        • Early in axons
          • Loss of m-Calpain
          • Ca++ entry
        • Associated cytokines
          • Early: TNFα & IL-1α
          • After delay: IL-1β
        • Inhibitory molecule: OX2 (CD200) inhibits macrophage lineage cells
        • Not related to bcl-2 or caspase activation
      • Molecules upregulated in neurons after axotomy
        • STAT-3 protein : Associated with CNTF stimulation
        • Activating transcription factor 3 : Acts as heterodimer with jun proteins
        • Nna1 (ATP/GTP-Binding protein 1; AGTPBP1) : Motor neurons
          • Putative zinc carboxypeptidase
          • Presumed nuclear localization
          • Adenosine triphosphate/guanosine triphosphate binding motif
          • Mutations: Purkinje cell degeneration (pcd) mouse
        • Nerve injury associated kinase: Sensory neurons
      • Molecules upregulated in nerve distal to transection
        • Early activation of erbB2 4
          • Related to: Schwann cell demyelination after axotomy
          • Time course
            • Early activation: Occurs 10 to 180 minutes after nerve damage
            • erbB2 also increased late (days) after nerve transection
          • Anatomy
            • Originates in microvilli of Schwann cells, in direct contact with axon
            • Localized to nodal region of myelinating Schwann cells
            • Activation occurs near & distal to nerve transection site
          • Related features
            • MAPK is also activated early after nerve transection
            • ATP mimetic PKI166 (Blocks erbB2 activation)
              • Reduces ovoid accumulation in Schwann cell cytoplasm
            • Neuregulin coreceptor erbB3 participates in the rapid activation
            • Neuregulin in vitro
              • Induces demyelination
              • Mimics early response of Schwann cells to nerve damage
        • Ninjurin1
          • Adhesion molecule
          • Induced in injured DRG neurons & Schwann cells
        • Ninjurin2
          • Adhesion protein
          • Expressed constitutively by mature sensory neurons
          • Induced in Schwann cells in distal segment of lesioned nerve
        • Glial cell line-derived neurotrophic factor (GDNF)
        • GDNF family receptor α1 (GFRα1)
        • Disintegrin CRII-7/rMDC15
          • ADAM (a disintegrin and metalloprotease) gene family
        • FGF-2
        • IL-6: Pain-inducing cytokine
        • TNF-α : Macrophage recruitment from the periphery
        • SDF-1γ (Stromal cell-derived factor (SDF)-1 isoform)
      • Molecules reduced in nerve distal to transection
        • SCG10 (Stathmin-like 2; STMN2)
          • Reduced in distal stump before morphologic changes
          • Promotes regeneration in proximal stump
      • Mice with slow Axon degeneration
        • C57BL/Wlds
          • Genetics
            • Mutation: 85 kb tandem triplication on distal mouse chromosome 4
            • Mutated region contains 2 associated genes
              • Nicotinamide mononucleotide adenylyltransferase (NMNAT1; D4Cole1e)
              • 5' end of ubiquitination factor E4B (Ube4b)
          • Proteins
            • NMNAT1
              • Subcellular location: Nuclear; May act in cytoplasm
              • Expressed in: Skeletal muscle, Heart, Liver, Kidney & Brain
              • Function: NAD biosynthesis
              • Probably the component responsible for axonal protection
                • NMNAT1 enzyme activity required for axon protection
                • Sirt1 (NAD-dependent deacetylase)
                  • Downstream of Nmnat
                  • Contributes to axonal protection
            • E4B
              • Subcellular location: Cytoplasmic
              • Expressed in: Skeletal muscle, Ovary, Testis & Heart
              • Functions
                • Binds to ubiquitin moieties of conjugates
                • Catalyzes ubiquitin chain assembly
            • WldS mutation
              • Chimeric gene product: 1st 70 AA Of Ube4b + NMNAT1 full sequence
              • May augment Nmnat2
                • Maintains Nmnat enzyme activity in distal axons after injury
              • Loss of NMNAT2 causes axon loss in vitro
              • Site of action may be axonal ER/Golgi or mitochondria
          • Mutation effects on proteins: Increased expression
          • Mouse effects
            • Wallerian degeneration delayed by 3 to 4 weeks
            • Axons less susceptible to vincristine toxicity
            • pmn mouse: Slower progression of disease
            • SOD1/ALS (SOD1-G93A) mouse 3
              • Slightly longer survival
              • Delayed denervation at NMJ
        • Neuronal nitric oxide synthase knockout 2
          • Slow Wallerian degeneration
          • Delayed regeneration
          • Incomplete pruning of axon sprouts: Enhanced number of axons
    • Myelin
      • Early
        • Widening of Schmidt-Lantermann incisura
        • Ovoid formations begin at these loci
      • Paranodal myelin retraction
      • Myelin "collapse" & fragmentation
      • Degenerative changes of myelin occur
        • Within Schwann cells
        • Distal to proximal direction
        • Small axons before large axons
        • May signal phagocytic macrophages
    • Schwann cells
      • Proliferate: Especially non-myelinating Schwann cells
      • Auto-autophagy: Myelinating Schwann cells 6
        • Contain & Degrade: Myelin debris (Myelinophagy); MPZ & MBP proteins
        • Autophagy markers increased: LC3-II; Wipi2
        • Most prominent in 1st week after nerve injury
        • Less autophagy by CNS oligodendroglia after axon damage
      • Form Bands of Bungner
        • Definition: Arrays of Schwann cells & processes within basement membrane
        • Provide substrate for axonal regeneration
        • Schwannn cells atrophy and disappear if axonal regeneration does not occur
    • Phagocytes (Macrophages): Degradation to lipid debris
      • Origin
        • Mostly hematogenous incoming
        • Invasion of nerve 3 to 4 days after axon transection
      • Phagocytosis of sudanophilic (lipid) debris: Appear as foamy cells
      • Complement is necessary for phagocytosis
      • Clear axonal & myelin debris
      • Course: Cells may persist for 3 to 7 months
    • Fibroblasts
      • Proliferate during 1st week
      • Migrate adjacent to degenerating fibers
      • Produce some collagen
    • Blood-nerve barrier
      • Loses integrity during early degeneration & regeneration
      • Re-established over months
  • Alternate axon degeneration pathway: Trophic withdrawal induced
    • Stimulus: Loss of trophic factors (NGF)
    • Site of degeneration: distal axon
    • Pathway components
      • Membrane related: p75; DR6
      • Bax
      • Caspases: 9; 6; 3


Oppenheim 1894

Wallerian Degeneration: Pathology

Hypoglossal nerve: 5 days after nerve section


Axons: Degeneration, Ongoing


Neurofilament stain
Reduced, or absent, staining of large axons within pale areas that normally contain axons & myelin
  Clusters of small, unmyelinated, axons are normally stained
Compare to: Control nerve

Neurofilament stain

Myelin is still present

VvG stain

Loss of Large Axons
  Absent, or Reduced, Axons (Yellow; Neurofilament stain) inside Myelin basic protein stained myelin
    Areas where axons are lost are black
  Myelin basic protein stain (Red) is variable and irregular
  Small axons (Green) are preserved
  See: Control nerve for comparison

Neurofilament (Green) & Myelin basic protein (Red) stain

Non-myelinating Schwann cells
  Normally present in clusters
  Chronic large axon loss produces a more diffuse distribution

NCAM stain

Axon degeneration: Ongoing
  Fixed nerve
Axon & Myelin Degeneration: Many circular compact myelin profiles with
  Irregular, pale or dark, central regions
  No phagocytes or fragmentation
  Irregular morphology

Axon Degeneration: Patterns & Morphology

Aggregated organelles
  Clustered in region of axon within compact myelin
  Myelin often has abnormal structure
Aggregated organelles
  Remnants of axon within remaining compact myelin
  No associated phagocytic cells

From: Robert Schmidt MD

From: Robert Schmidt MD

Axoplasm Pallor
  Axoplasm is pale
  Organelle aggregates are present
  Structure of compact myelin is disrupted

Axoplasm: Pale
  Outer myelin layers have abnormal structure

Axoplasm Aggregates

Axoplasm: Dark & Homogeneous

Myelin Breakdown: Fragmentation & Degeneration in Schwann cells

Gomori trichrome stain
Wallerian degeneration: Early; Myelin fragmentation
  Myelin in phagocytic, post-myelinating cells: Clustered red (GT) or Black (VvG) endoneurial stain

VvG stain

Acid phosphatase stain
Wallerian degeneration: Myelin fragmentation stage
  Large endoneurial cells (red) contain prominent lysosomal activity

Acid phosphatase stain

Large, Post-myelinating Schwann cells contain
  Fragmented myelin: In various shapes
  Lipid droplets: Small, Rounded & Clear

Toluidine blue stain

Myelin fragmentation in Post-myelinating Schwann cells

Electron micrograph: From Robert E Schmidt MD
Early phase: Schwann cell
  Myelin, compact: Fragmentation inside Schwann Cell
  Schwann cell characteristic: Surrounded by Basal lamina (Below; Arrow)
  Axon is lost

Electron micrograph: From Robert E Schmidt MD

Myelin Ovoids

Myelin ovoids: Longitudinal section of nerve

Toluidine blue stain

Toluidine blue stain

Myelin Ovoids: Teased axons
Myelin Remnants
  Irregular laminated structure
  Within Schwann cell
  No associated axon

Robert E Schmidt MD

Myelin Degradation
  Schwann Cells contain
    Lipid debris
    Myelin fragments
  Axons: Lost

Electron micrograph: From Robert E Schmidt MD

Electron micrograph: From Robert E Schmidt MD

Myelin Degradation to Lipids

Acid phosphatase stain
Phagocytic cells, Endoneurial

Toluidine blue stain

CD68 stain

Phagocytes (vacuolated) in endoneurium
Large cells: Contain myelin debris or round, clear lipid droplets.
Normal axons with thin & thick myelin sheaths may also be present.

Lipid droplets in Schwann Cells & Macrophages
  Schwann cells (Dark arrows) &
  Macrophages (Light arrows)
    Contain lipid droplets &
      Myelin debris

Macrophages, Perivascular
  • Contents
    • Lipid droplets
    • Myelin debris, small
  • Location
    • In & around vessel wall

From R E Schmidt MD

Axons, Regenerating
  Surrounded by Schwann cell cytoplasm
  Thinly myelinated axon on far right

From R E Schmidt MD

From R E Schmidt MD

Denervated Schwann cell bands
  (Bands of Bungner)

  • Due to: Myelinated axon loss
  • Basement membrane: Irregular
  • Size: > 3 μM

From R E Schmidt MD

Collagen Pockets

RE Schmidt MD

Collagen pockets
  Collagen fibrils surrounded
    by cell processes
  More common with
    Loss of small axons
    Increased age
  No axon regeneration

Electron micrograph: From Robert E Schmidt MD

Electron micrograph: From Robert E Schmidt MD

Loss of Unmyelinated & Myelinated Axons

Neurofilament stain (Green) + NCAM stain (Red)
Unmyelinated axons
  Normal: Yellow (Green axon + Red NCAM stained Schwann cell)
  Lost: Red (NCAM stained Schwann cell without axons) (arrow)
Myelinated axons
  Reduced numbers of larger green (neurofilament) profiles
    Remaining large axons often have central yellow region of stain
  See: Control (Below)

Control nerve

Neurofilaments (Green); NCAM (Red)

Skin: Normal & Axon loss

Skin: Normal innervation

PGP 9.5 stain: Glenn Lopate


Skin: Pathologic innervation

Beaded axons

PGP 9.5 stain: Glenn Lopate
Axon loss


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