Axon loss

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

Neuropraxia
- Axon: Anatomically intact: No Wallerian degeneration
- Anatomy
  - Especially affects: Large myelinated axons
  - Pathology: Focal demyelination or dysfuntion
- Physiology: Nerve conduction block
- Clinical pattern: Motor > Sensory
Axonotmesis
- Axons: Discontinuous Wallerian degeneration
- Epineurium: Continuous
Neurotmesis
- Complete nerve disconnection

Electrophysiology: Changes after nerve transection

CMAP
- Normal for 2 to 3 days
- Reduced amplitude: Reaches nadir at 7 to 10 days
SNAP
- Amplitude: Reaches nadir at day 10 or 11
EMG
- Recruitment reduced: Immediately after transection
- Fibrillation potentials & Positive waves: Onset
  - Typical: 14 days
  - May be shorter with lesion close to muscle

Wallerian degeneration

Motor axons: Onset 3 to 5 days
Sensory axons: Onset 6 to 10 days

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
Myelin
Irregular structure
Degradation
Fragmentation
Intermediate
Ovoids
To lipids
Remnants
Also: Eosinophilic vasculitis
Macrophages
Schwann cells
Ultrastructure
Early
Lipid debris
Ovoids
Schwann cells, Autophagic
Post-WD
Axon regeneration
Bungner bands
Collagen pockets
Molecules
Principles
Alternate Degeneration Process
Trophic-Withdrawal

Axon Loss & Wallerian Degeneration
Event Timing in Nerve

Minutes
- Ca⁺⁺ wave
- MAPK: Signal activation
- Mitochondria: Transport ↓
Hours
- 2nd Ca⁺⁺ wave
- NMNAT2: Degradation
- SARM1: Activation
- NAD⁺: Depletion
- NMN: Accumulation
- ATP: Loss
- Mitochondria: Fragment
- Axon: Autophagy ↑
Days to Weeks
- Neurofilament: Loss
- Axon: Disintegration
- Myelin: Damage & Loss
  - Schwann cells
  - Histiocytes
- Macrophages
  - Entry & Activation
- Debris
  - Clearance
  - Remnants
Weeks to Months
- Büngner bands

Myelin Ovoids

Waller

Remak

Wallerian Degeneration: Principles & Features

Wallerian degeneration: Definition

Sequence of Axon & Myelin degeneration
Location: Segment of nerve distal to a site of transection
Stages
- Axons: Loss of Structure & Degeneration
  - NMNAT2: Protects axon from degeneration
  - SARM1: Facilitates axon degeneration
- Myelin & Myelin proteins
  - Structural fragmentation in Schwann cells
  - Degradation to lipid in Schwann cells & Macrophages
  - Associated with cells: Histiocyte-predominant
- Schwann cells without axons
  - Form Büngner bands
  - Facilitate axon regeneration
Electrodiagnostic
? Similar process: Dying back axon degeneration

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 (≥ 1 to 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
        
        NMNAT2 ⁸
        
        General
        
        Chaperone
        Aids in refolding of misfolded proteins
        
        NAD+ biosynthesis
        
        Catalyzes
        
        Nicotinamide mononucleotide (NMN) →
        Nicotinamide adenine dinucleotide (NAD+)
        
        Controls SARM1 activation
        
        NAD+: Inhibits SARM1 (Inactive TIR domains)
        
        NMN: Associated with SARM1 activation (Active TIR domains)
        
        Present in axon cytoplasm
        
        Carried down axon by anterograde axon transport
        Short half-life: Rapidly lost after axon transection
        2 pools: Vesicular & Non-vesicular
        
        Axons
        
        Protects from Wallerian degeneration
        Required for: Axon growth & maintenance
        NMNAT2 loss: Prevents axon regeneration
        
        Disease: Polyneuropathy & Erythromelalgia
        
        NOS knockout
        DR6 (TNFRSF21) ⁹
        SARM1 ↓ Activity (Inhibition) ⁸
        
        Toll-like receptor adapter protein
        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
        
        Causes damage by: Reducing NAD+ in axon cytoplasm
        
        SARM1 activated by
        
        High NMN/NAD+ ratio
        TNF-α (Neuro-Inflammatory signal)
        MLKL (Necroptosis-like signal): Via inducing loss of
        
        NMNAT2 ; STMN2
        
        Axotomy
        
        Blocks delivery of labile axon survival factors (NMNAT2)
        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
        
        Disease association: ALS & SPG ¹³
        
        Gain of function polymorphisms
        
        Frequency: 0.12% vs Not seen in controls
        
        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: MOX2 (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) ¹⁴
      - Reduced in distal stump before morphologic changes
      - Promotes regeneration in proximal stump
      - Heterozygous knockout: Motor axon damage
      - Reduced in spinal cord in ALS
  - Mice with slow Axon degeneration
    - C57BL/Wld^s
      - 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 NNMNAT
        
        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
        
        Causes ectopic localization of NMNAT1 (NAD+ biosynthesis enzyme) to axons
        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
    - Anatomic
      - Schmidt-Lantermann incisura: Widened
      - Myelin layers: Irregular
      - 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
      - Wallerian degeneration
      - Primary demyelinating disorders
        
        Immune (Segmental demyelination)
        
        Toxic
        
        Lysophosphatidylcholine
        Tellurium
        
        Diphtheria
        
        Calcium ionophore
    - 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 Schwann cell & Macrophage activity
        
        Mixed lineage kinase domain-like protein (MLKL) ¹¹
        
        Location: 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): 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
  - Changes
    - Proliferation: Especially non-myelinating Schwann cells
    - De-differentiation: 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 of myelin breakdown products to lipid debris
  - Origin
    - Mostly hematogenous incoming
    - Recruitment regulated by: Raf�MEK�ERK mitogen-activated protein kinase
    - 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
Schwann cells
Autophagic
Ultrastructure

Waller illustration

5 days after nerve section (Hypoglossal nerve)

Axon Degeneration: Patterns & Morphology

Axons: Degeneration, Ongoing, Early

Axon Loss
Histology
Morphology/Ultrastructure
Very Early
Axoplasm loss
Organelle aggregation

Myelin Pathology
Histology
Periphery
C_5b-9 deposition
MxA+ cells
Myelin Ovoids/Remnants
Schwann cells
Autophagic
Ultrastructure
Inner layers
Compact
Outer layers
Later stages
Ovoids
Autophagic Schwann cells
Cells with Lipid debris

Degeneration of Myelinated Axons: Ultrastructure

Axon Loss: Early

Neurofilament stain of Axons: Reduced or Absent
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
Also see: Control Nerve

Neurofilament stain (Green or Yellow); 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

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

Myelinated Axon Degeneration: Very Early

Axon Pathology

From: R Schmidt

WD Very Early: Axoplasm Aggregates

WD Very Early: Axoplasm: Dark & Homogeneous

WD Very Early: Axoplasm
Pale
Few organelles

WD Very Early: Axoplasm, Ultrastructure
Aggregated organelles
Clustered regionally in axon: Near mildly abnormal myelin structure

From: R Schmidt

WD Very Early: Axoplasm, Ultrastructure
Volume: Reduced
Structure
Irregular or Pale
May contain vesicles or organelles
Myelin sheath: May appear very thick compared to axon size

From: R Schmidt

From: R Schmidt

WD Very Early: Axoplasm, Ultrastructure
Volume: Reduced
Structure
Irregular or Pale
May contain vesicles or organelles

WD Moderately Early: Axoplasm loss & Pathology

From: R Schmidt

Axoplasm
Volume: Reduced
Structure: May be irregular
Myelin sheath: Appears very thick compared to axon size

From: R Schmidt

From: R Schmidt

Axoplasm
Volume: Reduced
Structure: May be irregular
Myelin sheath: Appears thick compared to axon size

From: R Schmidt

Axoplasm
Blebs: May be present between damaged axon & myelin (Below)

Myelin ± Axon Pathology: Early

From: R Schmidt

Axoplasm
Clustered organelles
Myelin: Early WD Changes
Internal Pathology: Bleb, from internal layers of myelin sheath, indents axon
Sheath: Thick compared to axon size

WD Very Early
Myelin
Abnormal structure of internal layers
Axon
Aggregated organelles
Clustered regionally in axon: Near abnormal myelin structure

WD Very Early
Axon
Aggregated Organelles
Remnants of axon within remaining compact myelin
Myelin: Irregular internal areas
No associated phagocytic cells

From: Robert Schmidt MD

From: Robert Schmidt MD

WD Early
Myelin: Blebs from inner myelin layers indent axons

Compact Myelin: Abnormal Structure

Axoplasm Pallor
Axoplasm is pale
Organelle aggregates are present
Myelin Early WD Changes
Structure of compact myelin is disrupted

Outer Myelin Layers: Abnormal Structure

Axoplasm
May be: Pale or Aggregated
Myelin Early WD Changes
Myelin outer layers: Abnormal structure
Remaining myelin: Thick compared to axon size

WD, Early: Ab-Axonal Schwann cell Cytoplasm
Pale background
Irregular aggregates

Wallerian Degeneration

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

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

Axon Loss: Myelin Pathology

H&E stain

Myelin
Stain: Reduced or lost
Structure: Lost or clumped

Gomori trichrome stain

VvG stain

H&E stain

Myelin
Stain: Reduced or lost
Structure: Lost or clumped

Gomori trichrome stain

Gomori trichrome stain

Myelin is still present

VvG stain

Many Myelin sheaths are paler stained

VvG stain

Wallerian Degeneration: Schwann cells, Myelinating: Molecular pathology
NCAM expressed in myelin sheath

NCAM stain

Axon Degeneration, Early/Myelin Pathology (Above): Increased NCAM expression in Schwann cell cytoplasm/Myelin sheath
Normal (Below): NCAM expressed 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

Autophagic Schwann cells

Acid phosphatase stain

Acid phosphatase stains
Fragmented myelin
Scattered endoneurial histiocytes

Acid phosphatase stain

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: Schwann cell pathology

C_5b-9 stain

C_5b-9: Stains Schwann cell processes around myelin sheaths

C_5b-9 stain

MxA stain

MxA: Stains Schwann cell processes around myelin sheaths

MxA stain

MxA 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

Myelin & Axon Degeneration: Ongoing, Weeks; Myelin Degradation to Lipids

Ultrastructure

Macrophages & Phagoocytic cells ¹⁵

Sources
- Intrinsic
- Recruitment from circulation
- Schwann cell differentiation
Recruitment factors
- Interleukin-17B (IL-17B) : Binds to IL-17 receptor B
- Chemokine, CC motif, ligand 2 (CCL2)

Acid phosphatase stain
Phagocytic cells, Endoneurial

Toluidine blue stain

CD68 stain
Macrophages

Toluidine blue stain

Wallerian degeneration: Ongoing
Irregular, large myelin figures
Many histiocytes: Contain small round 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

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: Features

Irregular laminated structure
Within Schwann cell
No associated axon

Histiocytes: Debris- & Lipid-containing

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.

Toluidine blue stained plastic sections

Myelin profiles & Lipid droplets in phagocytic (Probably Schwann) cells

Toluidine blue stained plastic sections

Early WD: Histiocytes
Myelin
Myelin outer layers: Irregular structure
Schwann cell, Ab-Axonal cytoplasm
Contains myelin fragments
Histiocytes
Present in areas around axon

Histiocytes with Myelin fragments around a cell with a large, partially degraded Myelin sheath

Myelin Degradation: Late stage with Lipid droplets & some Myelin fragments

Ultrastructure: Myelin debris & Lipid droplets in Schwann cells & Histiocytes
Some cells have mostly myelin debris (Upper right)
Some cells have many clear, round lipid droplets

Histiocyte: Endoneurial
Contains many clear, round Lipid droplets
Surface: Shows several processes extending into endoneurium

Lipid droplets in Schwann Cells & Macrophages

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

Phagocytic cells: Contain Myelin & Lipid debris

Wallerian Degeneration, Later stage: Histiocytes with Lipid debris around Endoneurial Microvessels

From R E Schmidt MD

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
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

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

RE Schmidt MD

Electron micrograph: From Robert E Schmidt MD

Collagen Pockets
Collagen fibrils surrounded by Schwann cell processes

Electron micrograph: From Robert E Schmidt MD

Schwann cell processes within & around collagen fibrils

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: Co-stains (Yellow) on many smaller axons

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

Utility: Reliable test to document loss of small axons in skin
Measurement: Intraepidermal nerve fiber density (IENFD)
Patterns of axon loss & damage
- Length dependent
  - Definition: More loss of axons in distal than proximal leg
  - Symptoms: Distal; Symmetric
  - Associations
    - Disorders that damage axons
    - Diabetes: More common than in Non-length dependent small fiber neuropathies
- Non-length dependent
  - Definition: Similar, or more, loss of axons at proximal compared to distal locations
  - Association: Small fiber ganglionopathy
  - Symptoms
    - Involvement of face, mouth, trunk, upper limbs, or muscle
  - Disease associations
    - IgM antibodies vs TS-HDS
    - IgG antibodies vs FGFR3
    - Acute onset small fiber neuropathies
    - Idiopathic
    - Fibromyalgia-like syndromes
    - Systemic immune disorders: Sjögren syndrome
- Axon beading: May be early sign of axon damage
- Painful neuropathies
  - May have reduced miR-146a & miR-155 expression in painful regions
- Normal numbers of small axons
  - May occur in: Hereditary pain syndromes
Autonomic C-fibers
- Vasomotor: Blood vessel walls
- Sudomotor: Sweat glands
- Pilomotor: Arrector pilorum smooth muscle