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PERIPHERAL NERVE: Normal features

Age changes   Pi granules Blood-Nerve barrier Elzholz body Endoneurium Epineurium Perineurium Morphology   Sural nerve     Adults       Axons         Unmyelinated         Myelinated           Schmidt-Lanterman Cleft             Morphology           Node of Raniver             Ultrastructure       Epineurium       Perineurium       Schwann cells       Vessels         Endoneurial         Epineurial     Children   Infraorbital nerve   Sciatic nerve (Adult)   Myelin     Sciatic     Sural Mouse

NCAM stain Non-Myelinating (Remak) Schwann cells in clusters (NCAM stain) Mild staining is also present in some myelin sheaths

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Epineurium

• Connective tissue & vessels surrounding nerve fascicles
• Collagen
  • Bundles: Oriented longitudinally
  • More dense near fascicles
  • Fibril width: 60 to 110 nM diameter
  • Type I > III
• Vessels (Vasa nervorum)
  • Arteries
  • Veins
  • Lymphatics
• Nerves
  • Axons: Small, unmyelinated; In vessel walls, Artery >> Vein
  • Non-myelinating Schwann cells: NCAM+; Surround unmyelinated axons
• Pathology: Vasculitis & Inflammatory vasculopathies

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Perineurium Structure Lamellated structure surrounding all nerve fascicles Width Thicker Larger fascicles More proximal nerve: Up to 15 layers At nerve branches Thin: Near distal nerve terminals Continues in CNS: Pia-arachnoid PNS Capsule of some sensory receptors Not: Muscle basal lamina Vessels: Trans-perineurial Smooth muscle layer Continuous in arterioles Incomplete in veins Arteriole size: 10–25 μM May have associated axons Separated from endoneurium by: Subperineurial space Embryology Development period: 12 to 14 weeks Origin: Mesenchymal cells (Fibroblasts) Neurofibromin Associated with: Arrangement & formation of perineurium Perineurial cells Shape: Flattened cells in concentric layers Surrounded by: Basement membrane Thicker than Schwann cell basement membrane May surround one, or several, neighboring perineurial cells Cytoplasm Rarified except in perinuclear region Contains filaments similar to smooth muscle cells Many pinocytotic vescicles Cytoplasm: Linoleic acid high Other molecules Surface complement regulators & inhibitors C1IHN, C4BP, CD59, Clusterin Tight junctions Link adjacent perineurial cells Blood-Nerve barrier Origin ? Fibroblasts Differentiation maintained by desert hedgehog from Schwann cells Neurofibromin Molecular & Ultrastructure Collagen fibrils Width: 40 to 64 nM Contain: Collagen III > I "Raccoon tail" (Long-spaced collagen, Luse body) Non-branching filaments (10 to 12 nM): Oxytalan Laminin isoforms: A, B2 & S chains Stains Epithelial membrane antigen (EMA) C5b-9 complement PAS: Stains proteoglycans CALD1 SLC16A1 Junction proteins in perineurium ZO-1 (TJP1) Tricellulin Occludin: Tight junction Claudin-1 Claudin-3 Claudin-19: Tight junction JAM-C: Adherens junction Cadherin-5: Adherens junction Functions Modulates external stretching forces Regulates endoneurial pressure & milieu Part of blood-nerve barrier: With endoneurial blood vessels Pathology

Early Illustration of Perineurium: Ranvier

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Endoneurium

• Anatomy
  • Region inside perineurium
  • Contiguous with sub-arachnoid space
• Contents
  • Axons
  • Schwann cells & Myelin
    • 80% to 90% of endoneurial nuclei are Schwann cells
    • Collagen IV: Component of Schwann cell basement membrane
  • Endoneurial microvessels: Pathology
  • Disease: HIEM
  • Features
    • Orientation: Longitudinal with many oblique & transverse anastomoses
    • Size: 9 μM mean diameter; Range 5 to 22 μM
    • Density: 60–100 per mm² in human sural nerve: Lower with age
  • May stain for
    • Alkaline phosphatase
    • UEAI lectin: Endothelial cells
    • Scaffolding proteins: Link endothelial cell membranes to cytoplasmic components
      • ZO-1 (TJP1)
      • CLDN5
  • Endoneurial microvessels: Endothelial cells
    • Anatomy: Single layer next to vessel lumen
    • Linked by tight junctions
      • Claudins
      • Tight junction proteins
    • Molecules: Mitogen receptors
      • Glial-derived neurotrophic factor (GFRα1)
      • Vascular endothelial growth factor (VEGFR2; KDR)
      • Basic fibroblast growth factor (FGFR1)
      • Transforming growth factor-β (TGFRI/II)
      • Glucocorticoid (GR; NR3C1)
    • May contain Weibel–Palade bodies: ? Produce factor VIII-related antigen
    • Surrounded by collagen
    • No fenestrae or associated glial cells
    • Form part of blood-nerve barrier
    • Tight junction proteins in endoneurial capillaries
      • Claudin-5: Endothelial; Schmidt-Lanterman incisures
      • Occludin
      • ZO-1
  • Collagen (50 to 65 nM)
    • Collagen III > I
    • More tightly packed near Schwann cell basement membrane
  • Interstitial space
  • Other cells
    • Fibroblasts: Few (10%); More frequent near endoneurial vessels; No basement membrane
    • Macrophages: Resident
• Under pressure relative to epineurium

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Blood-Nerve Barrier ¹

• Anatomy
  • Locations
    • Perineurium: Very impermeable; High pinocytotic activity
    • Endoneurial microvessels: More permeable than perineurium
  • Tight junctions between
    • Endothelial cells in microvessels
    • Innermost perineurial epithelioid myofibroblast cells
• Molecules
  • Barrier proteins
    • ZO-1 (TJP1)
      • Intracellular
      • Connects tight junction proteins to actin cytoskeleton
    • Claudin-1 (CLDN1)
      • Locations: Perineurium, Mesaxon, Schmidt-Lanterman incisures; Paranodal loops of myelinated Schwann cells
      • Barrier function
      • Disease: Ichthyosis, Leukocyte vacuoles, Alopecia, Sclerosing cholangitis (ILVASC)
    • Claudin-3 (CLDN3)
      • Locations: Schmidt-Lanterman incisures; Mesaxon
    • Claudin-4 (CLDN4) : BNB tight junctions
    • Cadherin-5 (CDH5)
      • Endothelial
      • Adherens junctions
      • Locations: Endoneurial vessel endothelium; Schmidt-Lanterman incisures; Mesaxon
      • CLDN5 knock-out: Increased permeability for molecules < 800 Da
    • Occludin (OCLN)
      • Tight junction membrane protein
      • Present in most BBB regions: Perineurium; Endothelial microvessel endothelium; Schmidt-Lanterman incisures
      • Disease: Pseudo-TORCH syndrome 1
    • Tricellulin (MARVELD2)
      • PN locations: Mesaxon, Schmidt-Lanterman incisures, Paranodal loops
      • Tricellular tight junctions
      • Epithelial barriers
      • Regulates passage of macromolecules through tight junctions
      • Disease: Non-syndromic hearing loss (DFNB49)
    • α1 catenin (CTNNA1) : F-actin cytoskeleton linking to adherens junctions
  • Transporters: Influx & Efflux
    • Alkaline phosphatase
    • Glucose transporter-1 (SLC2A1)
    • Monocarboxylate transporter-1 (SLC16A1)
    • Creatine transporter (SLC6A8)
    • Cationic amino acid transporter-1 (SLC7A5)
    • γ-glutamyl transpeptidase
    • p-glycoprotein (ABCB1; PGY1)
    • SLC1A1
• Functions
  • Endoneurial ionic microenvironment: Regulation
  • Transendothelial electrical resistance (TEER): High
  • Permeability to solutes & macromolecules: Low
  • Transendothelial water flux (hydraulic conductivity): Low
• Possible Disorders
  • AIDP
  • CIDP
  • HIV sensory neuropathy
  • HIEM

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Adults: Sural nerve

Structure Fascicles

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Nerve Structure

H&E stain Normal Endoneurium, Perineurium, Epineurium & Vessels

Nerve fascicles Contain normal numbers of myelinated axons in endoneurium VvG stain

Gomori trichrome stain

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VvG stain

H&E stain

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Nerve (Sural): Axons Normal
Large & Small axons in endoneurium
Large axons: Surrounded by clear myelin halos
Small axons: Clustered among large myelinated axons

Neurofilament stain

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Toluidine blue stain

Axon morphology (Adult): 3 size populations
• Large: Thick myelin sheath
• Intermediate: Thin myelin sheath
• Small: Unmyelinated axons
• Compare to: Infant

Toluidine Blue stained plastic section

Electron micrograph (Robert E Schmidt MD)

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Small, Unmyelinated Axons

From Robert E Schmidt MD

Small axons in Clusters
  Several are often surrounded by processes from single Schwann cell

Electron micrographs   (Robert E Schmidt MD) Unmyelinated axons   Surrounded by     Schwann cells

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Nerve (Sural): Schwann cells, Non-myelinating

NCAM1 stain

Neural Cell Adhesion Molecule (NCAM1)
  NCAM is abundant in non-myelinating (Remak) Schwann cells that surround Unmyelinated axons
  NCAM is also present in
    Adaxonal Schwann cell cytoplasm around myelinated axons
    Myelin sheaths of some larger axons
Myelinated axons: Are generally smaller than in the more proximal sciatic nerve

NCAM stain

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NCAM in Non-myelinating Schwann cells
  Surrounds most small unmyelinated axons (Yellow)
  Not present around large, myelinated axons (Green)
  May occur in the center of larger axons (Yellow)
See: Loss of small axons

Neurofilament stain (Green) NCAM stain (Red) Overlap (Yellow)

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NCAM & P0
  Normal: Mostly present in different endoneurial cells
  NCAM
    Non-myelinating Schwann cells (Green)
    Small amounts in non-compacted areas of myelinating Schwann cell cytoplasm (Yellow)
  P0: Myelin & Myelinating Schwann cell cytoplasm (Red)
  Schwann Cell Pathology
    Büngner bands: NCAM & P0 present in same Schwann cells
    Onion bulbs

NCAM stain (Green) p0 stain (Red)

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NCAM & MBP
  Normal: Mostly present in different endoneurial cells
  NCAM
    Non-myelinating Schwann cells (Green)
    Small amounts in non-compacted areas of myelinating Schwann cell cytoplasm (Yellow)
  MBP: In Myelin surrounding larger axons (Red)

NCAM stain (Green) MBP stain (Red) Epineurial vessels & Supporting cells in endoneurium ATPase pH 4.3 stain Epineurial Vessels VvG stain Epineurial vessels: Normal Artery (Black arrow): Linear fibrils near lumen; Thick wall Vein (White arrow): Interlaced, thin fibrils around outside of vessel; Thin wall Toluidine blue stain Gomori trichrome stain Myelinated axons: Normal numbers Epineurial vessels   Intermediate-size & Small   Alkaline phosphatase: Stains endothelium in some epineurial and endoneurial vessels Alkaline phosphatase stain Congo red stain Nodes of Ranvier Non-myelinated axon segment   between 2 myelin internodes Anatomy Axon at node Diameter: Reduced Organelle density Increased Length: ~ 1 μm Schwann cells Loops around axon No myelin Electron micrographs   (Robert E Schmidt MD) Schmidt-Lanterman Cleft (Incisure) From: R Schmidt Schmidt-Lanterman Cleft: Extends through myelin from inside to outside From: R Schmidt Pi (Reich) Granules Electron micrograph (Robert E Schmidt MD) Pi Granules Location   Myelinating Schwann cells   Subcellular     Often adjacent to nucleus     Some near paranode Staining   Metachromatic: Toluidine blue, Hirsch–Peiffer & Methylene blue stains   Frozen sections: Acid phosphatase, Sudan black & PAS Size: 1 μM Structure: Lamellar; 5 nm periodicity Age   More common in older patients   Not present < 8 years of age Disease associations   Non-specific   Not present in recently remyelinated axons Pi granules: Few or many in cytoplasm of myelinating Schwann cells From R Schmidt Elzholz body Location: Schwann cell cytoplasm Structure: Round; Myelin-like periodicity Coomposition: Lipid Cause: ? Myelin remodeling Mast cell Myelin & Schwann cells: Sural nerve P0 (Red)   Prominent in myelin around most (large & small) myelinated axons   Smallest axons (Green), often in clusters, have no myelin or associated P0 staining Neurofilament stain (Green) P0 stain (Red) Myelin basic protein (MBP) in Myelin (Red)   Most prominent in myelin around largest myelinated axons   In myelin around fewer axons than P0 (Above) Neurofilament stain (Green) Myelin basic protein stain (Red) Non-myelinating Schwann cells Nerve: Sciatic Neurofilament stain Large & small axons: Non-uniform distribution within a fascicle Neurofilament stain Large & small axons: Non-uniform distribution among fascicles Neurofilament stain   Regions with varied numbers of large & small axons Neurofilament stain Neurofilament stain Neurofilament stain Neurofilament stain Non-myelinating Schwann cells NCAM1 stain   Regions have varied numbers and distributions of NCAM+ Schwann cells NCAM1 stain NCAM1 stain   Regions with varied numbers of NCAM+ Schwann cells NCAM1 stain Myelin Components Neurofilament stain (Green) P0 stain (Red) P0 (Red)   Prominent in myelin around most (large & small) myelinated axons   Smallest axons (Green), often in clusters, have no myelin or associated P0 staining Neurofilament stain (Green) P0 stain (Red) Myelin basic protein (MBP) (Red)   Most prominent in myelin around largest myelinated axons Neurofilament stain (Green) Myelin basic protein stain (Red) P0 vs Myelin Basic Protein (MBP) in Myelin P0 stain (Green) Myelin basic protein stain (Red) P0 protein   Associated with large & small axons   More around periphery of myelin sheath Myelin basic protein   Mainly associated with largest axons   More toward inside of myelin sheath P0 stain (Green) Myelin basic protein stain (Red) Nerve: Infraorbital (Branch of Trigeminal) H&E stain Myelinated Axons: Abundant all through endoneurium Gomori trichrome stain VvG stain Myelinated Axons: Abundant all through endoneurium H&E stain Gomori trichrome stain Acid phosphatase stain Infraorbital Nerve   Acid phosphatase positive cells scattered in endoneurium (Above)   Alkaline phosphatase positive vessels: Few in endoneurium (Below) Alkaline phosphatase stain ATPase activity: Abundant in endoneurial cells ATPase pH 4.3 stain Infraorbital Nerve   Larger myelinated axons: Many   Small unmyelinated axons: Fewer than in sural nerve Neurofilament stain NCAM stain Infraorbital Nerve   Non-myelinating (Remak) Schwann cells : Fewer than in sural nerve NCAM stain NCAM: Present in Schwann cells   Non-myelinating (Remak) Schwann cells   Myelinating Schwann cells: Adaxonal & Abaxonal cytoplasm NCAM stain Infraorbital Nerve   Larger myelinated axons (Green): No associated NCAM; Many   Small unmyelinated axons (Yellow): Fewer than in sural nerve Neurofilament stain (Green) NCAM stain (Red) Infraorbital Nerve: P0 (Red)   Prominent in myelin (Red) around most (large & small) myelinated axons   Smallest axons (Green) have no myelin or associated P0 staining Neurofilament stain (Green) P0 stain (Red) Infraorbital Nerve   Myelin basic protein (MBP) is markedly reduced in myelin around axons compared to sural nerve Neurofilament stain (Green) Myelin basic protein stain (Red) P0 stain (Green) Myelin basic protein stain (Red) VvG stain Nerve: Infant Toluidine blue stained plastic sections Nerves from Infants   Axons are mildly small   Myelin sheaths around large axons are moderately thin   Compare to: Adult Toluidine blue stained plastic sections Nerve from neonate Few axons are myelinated. Fibroblasts are prominent. Normal nerve: Mouse Axon populations: 3   Myelinated, Large   Myelinated, Small   Unmyelinated From: RE Schmidt Return to Biopsy illustrations Return to Neuromuscular Home Page Return to Nerve biopsy References 1. Exp Neurol 2020 Mar 3;328:113272 4/28/2024