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

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

Oppenheim 1894

Nerve Injury


Electrophysiology: Changes after nerve transection

Wallerian degeneration

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
        Neurofilament loss
      Irregular structure
        To lipids
    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
    • Wallerian degeneration: 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
        • Before degeneration
          • Distal axon segment may remain electrically excitable
          • Sensory responses persist 2 to 3 days longer than motor
          • Conduction failure may precede axon degeneration
          • Time to loss of excitability
            • Facial nerve: 4 to 7 days
            • Arm nerves, Motor: 5 to 7 days
            • Arm nerves, Sensory: 7 to 10 days
        • Degeneration
          • Begins several days (4 to 10) after axonal transection
          • Progression
            • Possible length dependence
            • May be from proximal to distal axon or diffuse
          • More rapid with shorter distal stump
        • 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
          • Genetics
            • Disorder: CONDCA
            • Mutations (Animal): 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
    • Wallerian degeneration: Myelin changes 10
      • Early
        • Anatomical: Widening of Schmidt-Lantermann incisura
          • Ovoid formations begin at these loci
        • Molecular
          • Expression of Phospholipase A2 (Lipolytic enzyme)
          • Activation of neuregulin-ErbB2 signaling (Demyelinating mechanisms)
          • Actin polymerization
          • E-cadherin recycling
      • Paranodal myelin retraction
      • Myelin "collapse" & fragmentation
        • Myelin Degeneration: Causes
        • Degenerative changes of myelin: Patterns
          • Initial
            • Within "Demyelinating" (Post-myelinating) Schwann cells
            • Subcelllular: Begins along Schmidt-Lanterman clefts
            • Associated with: c-Jun activation; Increased autophagic activity
          • Anatomic along nerve: Distal to proximal direction
          • Axon types: Small axons before Large axons
          • Molecular markers of activity
            • MLKL 11
              • Increased in Schwann cells & Macrophages
              • Binds to sulfatide
              • Myelin degradation associated with phosphorylation of MLKL serine 441
              • MLKL knockout: Reduces or delays myelin breakdown & axon regeneration
            • Myelin basic protein (MBP) still present in myelin fragments
          • Consequence: Often signal, or attract, phagocytic macrophages
          • Ovoid formation
        • Primary ovoids: Early change (1 to 2 days)
          • Intracellular (Schwann cell) myelin: Fragmentation
          • Abaxonal Schwann cell cytoplasm
            • Dilated
            • Contains rough endoplasmic reticulum (RER) & vesicles
        • Secondary ovoids (Myelinosomes): Later change (3 to 7 days)
          • Pinched off from primary ovoid
          • Compact myelin structures in Schwann cell cytoplasm
          • Exocytosed from Schwann cell cytoplasm
            • Into abaxonal extracellular space
            • Myelin debris: May be further degraded by macrophages
        • Macrophages
          • ? Attracted by cytokines
          • Phagocytosis & degradation of myelin debris
    • 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 Büngner
        • Definition: Arrays of Schwann cells & processes within basement membrane
        • Molecular: Büngner band Schwann cells express both NCAM & P0 protein
        • Provide substrate for axonal regeneration
        • Long-term: 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

    Damage & Loss
    Relationship to myelin
    Structure changes
    Fragmentation in Schwann cells
    Lipid & Myelin debris
End Stage
  Collagen pockets
  Bungner bands

Waller illustration: 5 days after nerve section (Hypoglossal nerve)


Axons: Degeneration, Ongoing, Early


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
  Axons (Yellow & Green; Neurofilament stain)
    Absent, or Reduced large axons: Axons inside Myelin basic protein stained myelin (Schwann cells)
    Areas where axons are lost are black
    Small axons are relatively preserved
  Myelin basic protein stain (Red)
    Shapes: Variable and irregular
    No associated axons
    Central dark areas where axons are lost
  See: Control nerve for comparison

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

Schwann cells, Non-myelinating
  Normal: Present in clusters
  Chronic large axon loss produces a more diffuse distribution

NCAM stain
Schwann cells, Myelinating
  Normal: Express NCAM mainly in adaxonal cytoplasm
  Early axon degeneration: Increased expression diffusely in Schwann cell cytoplasm (Below)_

NCAM stain

Axon Degeneration: Patterns & Morphology

Aggregated organelles
  Clustered regionally in axon: Within compact myelin; Near abnormal myelin structure
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 & Axon degeneration: Ongoing, Early

  Fixed nerve

Toluidine blue stain
Axon & Myelin Degeneration: Many circular compact myelin profiles with
  Irregular, pale or dark, central regions
  No phagocytes or fragmentation
  Irregular morphology

Toluidine blue stain
Axon degeneration: Early
  Irregular myelin figures
  Some remaining axons have dark axoplasm
  No histiocytes with lipid droplets in cytoplasm

Toluidine blue stain

Myelin Breakdown: Fragmentation & Degeneration in Schwann cells

Gomori trichrome stain
Wallerian degeneration: Early; Myelin fragmentation
  Myelin in phagocytic, post-myelinating ("Demyelinating") 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 ("Demyelinating") Schwann cells
    Fragmented myelin (Light arrow): In various shapes & stages of degeneration
    Lipid droplets (Dark arrow): Small, Round & Clear

Toluidine blue stain

Myelin fragmentation in Post-myelinating ("Demyelinating") Schwann cells

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

"Demyelinating", Autophagic Schwann cell
  Contains: Myelin fragments & Lipid droplets

Robert E Schmidt MD

Myelin Degradation: Later phase
  "Demyelinating" Schwann Cells contain
    Lipid debris
    Myelin fragments, small
  Axons: Lost

Electron micrograph: From Robert E Schmidt MD

Electron micrograph: From Robert E Schmidt MD

Axon Degeneration: Ongoing, Weeks; Myelin Degradation to Lipids

Acid phosphatase stain
Phagocytic cells, Endoneurial

Toluidine blue stain

CD68 stain

Toluidine blue stain
Wallerian degeneration: Ongoing
  Irregular, large myelin figures
  Many histiocytes with lipid droplets in cytoplasm
  Axons are degraded & lost

Toluidine blue stain

Toluidine blue stain
Wallerian degeneration: Ongoing myelin degradation
  Irregular, large myelin figures
  Many histiocytes with lipid droplets in cytoplasm
  Axons are degraded & lost

Toluidine blue stain

Toluidine blue stain

Toluidine blue 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.

Myelin Degradation: Comparative stains

Ultrastructure: Myelin debris & Lipid droplets in Schwann cells

Toluidine blue stained plastic sections
Myelin profiles & Lipid droplets in phagocytic (Probably Schwann) cells

Toluidine blue stained plastic sections

Neurofilament stain = Green; Myelin basic protein stain = Red
Wallerian Degeneration, Weeks
  Regions of Myelin basic protein (Red) often have no associated axons
  Loss of small unmyelinated axons (Green)
Control Nerve
  Myelin basic protein (Red) surrounds large axons (Yellow)
  Normal clusters of small unmyelinated axons (Green)

Neurofilament stain = Green; Myelin basic protein stain = Red

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

Myelin Ovoids & Remnants

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

Axons, Regenerating

  Surrounded by Schwann cell cytoplasm
  Unmyelinated (Below)
  Thinly myelinated axon (Far right)

From R E Schmidt MD

From R E Schmidt MD

Denervated Schwann cell bands
  (Bands of Büngner)

  • 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: Mild loss
  Normal: Yellow (Green axon + Red NCAM stained Schwann cell)
  Lost: Non-myelinating Schwann cells (NCAM stained, Red) without associated axons (Arrow)
Myelinated axons
  Reduced numbers of larger green (neurofilament) profiles
    Remaining large axons often have central yellow region of stain
    More severe axon loss
    Control (Below)

Control nerve

Neurofilaments (Green); NCAM (Red)

Skin: Normal & Axon loss

  Technical features

Skin Biopsy: General

Skin: Normal innervation

PGP 9.5 stain: Glenn Lopate


Skin: Pathologic innervation

Beaded axons

PGP 9.5 stain: Glenn Lopate
Axon loss


Myelin Artefact

Vesiculated myelin

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