Neuromuscular

AXON LOSS

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

Gomori trichrome stain
Myelinated Axons (Red)
Loss: Moderatly servere

Oppenheim 1894


Nerve Injury

Degrees

Electrophysiology: Changes after nerve transection

Wallerian degeneration


Myelinated axons: Loss


Myelinated Axon loss: Moderate



VvG stain

Gomori trichrome stain

Myelinated Axon loss: Severe

Axons, Large & Small: Comparative changes

Myelinated Axon loss: Large > Small

Toluidine blue stain
Myelinated Axon loss: Small > Large

VvG stain

Large axons: Moderately severe loss
Small Axons: Relatively preserved:
  Many Small axons, but Few large, myelinated axons
  Small axons are diffusely distributed, not clustered, in endoneurium

Neurofilament stain
Remaining Large Myelinated Axon (Arrow)

Neurofilament stain

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

Neurofilament stain
Myelinated axons: Severe loss

VvG stain

Wallerian Degeneration 5

Pathology
  Stages
    Axon degeneration & loss
      Early
        Neurofilament loss
        Myelin
        Ultrastructure
    Myelin
      Irregular structure
      Degradation
        Fragmentation
        Intermediate
        Ovoids
        To lipids
      Remnants
      Also: Eosinophilic vasculitis
  Post-WD
    Axon regeneration
    Bungner band
    Collagen pockets
Molecules
Principles
Alternate Degeneration Process
  Trophic-Withdrawal

Waller


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
        • WD Delayed by: Molecules & Factors
          • Temperature: Reduced
          • Extracellular Ca++: Lowered
          • Ca++ channel (L-type) blockers
          • Mutations or Loss
            • NMNAT1: WldS
            • NOS knockout
            • DR6 (TNFRSF21) 9
            • SARM1 inhibition 8
              • SARM1 structures
                • Forms octameric ring: via SAM domain
              • SARM1 Loss
                • Axon degeneration: Slowed
                • Types of axon degeneration affected
                  • Transsection
                  • Dying back (Chemotherapy)
              • SARM1 activity
                • Required for early injury induced axon degeneration
                • SARM1 activated by
                  • TNF-α (Neuro-Inflammatory signal)
                  • MLKL (Necroptosis-like signal): Via inducing loss of
                    • NMNAT2 ; STMN2
                  • Axotomy
                    • Blocks delivery of labile axon survival factors
                    • Factors normally inhibit SARM1
                • Mechanism of SARM1 action: Stimulates NAD+ cleavage & loss
                  • Active SARM1 protein domain
                    • Toll/Interleukin-1 receptor (TIR)
                    • Possesses intrinsic NAD+ cleavage (NADase) activity
                  • NADase activity increased
                    • NAD+ converted to ADPR (ADP Ribose) products
                    • Leads to Ca++ influx & Axon degeneration
              • SARM1 required for: Vincristine & Bortezomib induced axon degeneration 12
                • Vincristine
                  • Stimulates axon autonomous degeneration via MAPK pathway
                  • Mediated by MAP3K12 & MAP3K13
                • Bortezomib
                  • Induces axon degeneration via neuron cell body
                  • Mediated by
                    • Activated caspases (Caspase-3 cleavage) in axon
                    • Transcriptional regulation
                  • Apoptosis-like
                  • Similar mechanism to NGF withdrawal
            • DLK (MAP3K12) : Loss of function
            • jnk (MAPK8) : Signaling requires DR6
          • Inhibition: JNK kinase ; GSK3; IKKB (IKBKB)
        • WD 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
      • WD: 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
        • 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
            • Mixed lineage kinase domain-like protein (MLKL) 11
              • Increased in Schwann cells & Macrophages
              • Binds to sulfatide
              • Final executioner of canonical necroptosis
              • Induces loss of axon survival factors NMNAT2 & STMN2 to activate SARM1
              • 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

Remak

Oppenheim 1894

Wallerian Degeneration: Pathology

Axons
  Early
    Damage & Loss
    Relationship to myelin
  Regeneration
Myelin
  Early
    Structure changes
  Degeneration
    Early
    Fragmented in Schwann cells
  Degradation
    Lipid & Myelin debris
  Ovoids
End Stage
  Collagen pockets
  Bungner bands

Waller illustration

  5 days after nerve section (Hypoglossal nerve)

 

Axon Degeneration: Patterns & Morphology


Axons: Degeneration, Ongoing, Early

Histology
Morphology & Ultrastructure
Myelin changes

Axon Loss: Early

Neurofilament stain of Axons: Reduced or Absent

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

Loss of Myelinated Axons: Early


Myelinated Axon Degeneration: Early


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

Axon Loss, Early: Myelin changes

Myelin is still present

VvG stain

Neurofilament-stained axons: Lost within regions of myelin (MBP stain)
Axons: Neurofilament stain
  Reduced or Absent staining for Large axons (Yellow)
  Staining for Small axons (Green) remains
  Mechanism of axon loss: SARM1
Myelin-Basic Protein (MBP) (Red): Present in remaining myelin
  Regions with MBP stain are abnormal: Costain with NCAM

Neurofilament stain (Green or Yellow); Myelin Basic Protein stain (Red)

Wallerian Degeneration, Days to Few Weeks
  Regions of Myelin basic protein (Red) often have no associated axons
  Loss of small unmyelinated axons (Green)

Neurofilament stain = Green; Myelin basic protein stain = Red

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
Also see
  Control nerve
  Moderately later pathology
  Large Axon loss: Chronic

Loss of Large Axons: Moderately later than above
  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)
    Abundant
    Shapes: Variable and irregular
    No associated axons
    Central dark areas where axons are lost

Regions of Myelin Basic Protein (Red) have no associated Neurofilament-stained (Yellow) axons

Neurofilament (Green) & Myelin basic protein (Red) stain
Also See
  Control nerve for comparison
  Axon loss: Earlier

Wallerian Degeneration: Schwann cells, Myelinating

NCAM stain
  Axon Degeneration, Early (Above): Increased NCAM expression in Schwann cell cytoplasm
  Normal (Below): Express NCAM mainly in adaxonal cytoplasm

NCAM stain
Normal nerve: Schwann cells, Non-myelinating
  Normal (NCAM+) Non-myelinating Schwann cells
    Present in clusters between myelinated axons
    Not present in most regions of myelin sheath
  Also see: Large axon loss, Chronic

Abnormal Co-localization of NCAM & MBP in myelin remnants (Yellow)

NCAM (Green) + Myelin basic Protein (MBP) (Red) stain
Also see: Control nerve

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

Wallerian Degeneration: Intermediate stages

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
Also see: Normal control nerve

VvG stain


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

Acid phosphatase stain

Large, Post-myelinating (Autophagic) Schwann cells
  Autophagic
  Contain
    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 (Autophagic) Schwann cells


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

Autophagic Schwann cell
  Contains: Myelin fragments & Lipid droplets

Robert E Schmidt MD


Myelin Degradation: Later phase
  Autophagic 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
Macrophages


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

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

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







Phagocytic cells: Contain Myelin & Lipid 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

Myelin Ovoids: Teased axons
  Top: Myelinated axon, control (Node of Ranvier at Arrow)
  2nd row: Schwann cell sheath with no remaining myelin fragments or axon
  Below: Myelin ovoids & remnants along paths of previously degenerated myelinated axon

Myelin Remnants
Myelin Remnants: Features
  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 Myelinated, & Some Unmyelinated, Axons

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: Severe loss
  Markedly reduced numbers of larger green (neurofilament) axons
    Few remaining large axons are small
  See
    More severe small axon loss
    Control (Below)

Neurofilament stain (Green) + NCAM stain (Red)

Large Axons: Nearly complete loss (Chronic)
  Large axons: None stained
  Small axons: Many preserved
  Myelin basic protein: None present

Neurofilaments (Green); MBP (Red)

Control nerve

Neurofilaments (Green); NCAM (Red)
Myelinated Axons (Large, Green)
  2 sizes: Large & Intermediate
  No associated NCAM positive (red or yellow) cells
Unmyelinated axons (Small, Yellow)
  Occur in clusters
  Co-stain (Yellow) for neurofilaments & surrounding non-myelinating Schwann cells

Also see
  Large axon loss
  Large & Small axon loss

Skin: Normal & Axon loss

Biopsy
  General
  Images
  Technical features

Skin Innervation & 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


Go to Normal nerve
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8/6/2020