Residency · Residency · Pathology

Peripheral Nerve and Muscle Biopsy Interpretation

Overview

Nerve and muscle biopsies require specialized processing beyond routine formalin fixation. Interpretation demands familiarity with enzyme histochemistry, semi-thin resin sections, and electron microscopy. While genetic and serologic testing increasingly replace biopsy for many conditions, tissue diagnosis remains essential for vasculitis, amyloidosis, and diagnostically challenging myopathies.

Peripheral Nerve Biopsy

Indications

The most common current indications for nerve biopsy include vasculitis, amyloid neuropathy, chronic inflammatory demyelinating polyneuropathy (CIDP) when clinical and electrophysiologic data are equivocal, leprosy, sarcoidosis, lymphoma or other neoplastic infiltration, and hereditary neuropathies when genetic testing is inconclusive.

Processing Requirements

The sural nerve (a sensory nerve at the lateral ankle) is the most common biopsy site. The fascicle must be split into portions for three processing methods: formalin-fixed paraffin-embedded (FFPE) tissue for H&E, trichrome, and Congo red staining; glutaraldehyde-fixed tissue for resin (Epon) embedding yielding toluidine blue semi-thin (1-micron) sections and electron microscopy; and fresh-frozen tissue for immunofluorescence if vasculitis or immune-mediated disease is suspected.

Normal Nerve Anatomy

The epineurium is the outer connective tissue sheath. The perineurium surrounds individual fascicles and forms the blood-nerve barrier. The endoneurium surrounds individual nerve fibers within fascicles. Myelinated fibers include large fibers (motor, proprioception) and small fibers (touch, temperature), showing a bimodal distribution on fiber density histograms. Unmyelinated fibers are small fibers (pain, autonomic) visible only on electron microscopy.

Patterns of Nerve Injury

Axonal Degeneration (Wallerian Degeneration)

Axonal degeneration results in loss of axons and their myelin sheaths. Myelin ovoids are degenerating myelin fragments forming ovoid structures. Clusters of regenerating small myelinated fibers (regenerative clusters) indicate attempted repair. Causes include diabetes, toxic neuropathy, vasculitis, and nutritional deficiency.

Demyelination

Demyelination produces loss of myelin with preservation of axons. Segmental demyelination is loss of myelin at individual internodes. Remyelination produces thin myelin sheaths disproportionate to axon diameter, best appreciated on semi-thin sections. Onion bulb formation consists of concentric layers of Schwann cell processes around demyelinated/remyelinated fibers, indicating chronic demyelination-remyelination cycles (seen in CIDP, CMT1, and Refsum disease). Causes include CIDP, Guillain-Barre syndrome (acute), hereditary neuropathies (CMT1A, CMT1B), and anti-MAG neuropathy.

Specific Entities

Vasculitic Neuropathy

Vasculitic neuropathy shows necrotizing vasculitis of epineurial arterioles (small vessels) with fibrinoid necrosis of the vessel wall and transmural inflammatory infiltrate. Asymmetric or patchy axonal loss reflects ischemic damage, and hemosiderin deposits indicate prior hemorrhage. It may be isolated to the nerve (nonsystemic vasculitic neuropathy) or part of systemic vasculitis (PAN, GPA, eosinophilic GPA). Combined nerve-muscle biopsy increases diagnostic sensitivity.

Amyloid Neuropathy

Amyloid neuropathy shows Congo red-positive amyloid deposits in the endoneurium and vessel walls with apple-green birefringence under polarized light. Types include AL (light chain) and ATTR (transthyretin, either hereditary or wild-type). Mass spectrometry-based amyloid typing on FFPE tissue is the gold standard for classification. Nerve biopsy may be the presenting manifestation of systemic amyloidosis.

CIDP

CIDP shows endoneurial and epineurial inflammation (lymphocytes, macrophages), active demyelination with macrophage-mediated myelin stripping (visible on electron microscopy), onion bulb formation in chronic cases, and subperineurial edema. It responds to immunosuppressive therapy (IVIG, steroids, plasmapheresis).

Hereditary Neuropathies (CMT)

CMT1A (PMP22 duplication) shows a demyelinating pattern with prominent onion bulb formation and uniformly slow nerve conduction velocities. CMT2 (various genes) shows an axonal degeneration pattern with normal or mildly reduced conduction velocities. Genetic testing is now first-line, and biopsy is less frequently performed.

Muscle Biopsy

Indications

Muscle biopsy is indicated for inflammatory myopathy (dermatomyositis, polymyositis, IBM), muscular dystrophy when genetic testing is inconclusive, metabolic myopathy (glycogen storage, mitochondrial), congenital myopathy, and vasculitis.

Processing Requirements

Open biopsy is preferred, with vastus lateralis or deltoid being the most common sites. Fresh-frozen sections are essential (not FFPE for primary analysis). Enzyme histochemistry requires fresh-frozen tissue and includes H&E, modified Gomori trichrome (for ragged red fibers), NADH-TR, ATPase (for fiber typing), cytochrome c oxidase (COX), succinate dehydrogenase (SDH), PAS (glycogen), oil red O (lipid), acid phosphatase, and nonspecific esterase. Formalin-fixed tissue is used for IHC (MHC class I, complement, dystrophin, sarcoglycans). Glutaraldehyde-fixed tissue provides material for electron microscopy (mitochondrial myopathy, inclusion body myositis).

Normal Muscle Histology

Normal muscle shows polygonal fibers of uniform size with peripheral nuclei. Type 1 (slow twitch, oxidative) and Type 2 (fast twitch, glycolytic) fibers alternate in a checkerboard pattern on ATPase staining. Endomysial connective tissue is minimal, and internal nuclei are rare (less than 3 percent of fibers).

Patterns of Muscle Injury

Neurogenic Atrophy

Neurogenic atrophy produces small angulated fibers (denervated fibers) and target fibers, which show a three-zone pattern on NADH-TR (central pale zone, dark ring, normal periphery) and are pathognomonic of denervation. Fiber type grouping represents loss of the normal checkerboard pattern with groups of same-type fibers from collateral reinnervation sprouting. Group atrophy shows clusters of small fibers of the same type from failed reinnervation.

Myopathic Changes

Myopathic changes include fiber size variation (mix of hypertrophic and atrophic fibers, rounded rather than angulated), internal nuclei (greater than 3 percent of fibers), split fibers, necrosis and regeneration (pale fragmented necrotic fibers and basophilic regenerating fibers with large nuclei), and increased endomysial connective tissue with fatty replacement.

Inflammatory Myopathies

Dermatomyositis (DM)

The pathognomonic finding is perifascicular atrophy, consisting of 2 to 4 layers of atrophic fibers at the fascicle periphery. Additional features include perimysial and perivascular inflammation (CD4-positive T-cells, B cells, plasmacytoid dendritic cells), complement (C5b-9/MAC) deposition on capillaries (microangiopathy), capillary dropout, MHC class I upregulation in perifascicular fibers, and MxA protein expression in perifascicular fibers (interferon-mediated). Dermatomyositis-specific autoantibodies include anti-Mi-2, anti-NXP2, anti-TIF1-gamma, and anti-MDA5.

Polymyositis (PM)

PM shows endomysial CD8-positive T-cell infiltrate surrounding and invading non-necrotic muscle fibers with diffuse MHC class I upregulation and no perifascicular atrophy. It is a diagnosis of exclusion, and many cases previously classified as PM are now reclassified as IBM, anti-synthetase syndrome, or necrotizing autoimmune myopathy. True PM is rare.

Inclusion Body Myositis (IBM)

IBM is the most common inflammatory myopathy in patients over 50 years and combines inflammatory and degenerative features. It shows endomysial CD8-positive T-cell inflammation with invasion of non-necrotic fibers (like PM) plus rimmed vacuoles (basophilic-rimmed vacuoles on H&E and trichrome), eosinophilic cytoplasmic inclusions, congophilic (amyloid) deposits within fibers, ragged red fibers and COX-negative fibers (mitochondrial dysfunction), and p62-positive cytoplasmic aggregates. Unlike PM and DM, IBM is resistant to immunosuppressive therapy.

Immune-Mediated Necrotizing Myopathy (IMNM)

IMNM shows prominent necrosis and regeneration with minimal inflammation. It is associated with anti-SRP or anti-HMGCR antibodies (statin-associated). Additional features include complement deposition on the sarcolemma, macrophage-predominant infiltrate in necrotic fibers, and MHC class I upregulation. It responds to aggressive immunosuppression.

Muscular Dystrophies

Dystrophin immunostaining is absent in Duchenne muscular dystrophy (DMD) and reduced or patchy in Becker muscular dystrophy (BMD). Sarcoglycan complex IHC evaluates limb-girdle muscular dystrophies. Merosin (laminin alpha-2) is assessed for congenital muscular dystrophy. Emerin and lamin A/C are evaluated for Emery-Dreifuss muscular dystrophy. The dystrophic pattern shows necrosis/regeneration, fiber size variation, internal nuclei, fibrosis, and fatty replacement.

<image>A medical illustration of peripheral nerve biopsy findings. Panel A (Normal nerve, semi-thin section): Toluidine blue-stained cross-section showing a nerve fascicle with evenly distributed large and small myelinated fibers with dark myelin rings. Panel B (Axonal neuropathy): Loss of myelinated fibers with scattered myelin ovoids (degenerating myelin) and clusters of thinly myelinated small regenerating fibers. Panel C (Demyelinating neuropathy with onion bulbs): Semi-thin section showing concentric layers of Schwann cell processes forming onion bulb formations around demyelinated axons, with thinly remyelinated fibers in the center. Panel D (Vasculitic neuropathy): Epineurial arteriole showing fibrinoid necrosis of the vessel wall with transmural inflammatory infiltrate, adjacent hemosiderin deposits, and asymmetric loss of myelinated fibers.</image>

<image>A medical illustration of inflammatory myopathies. Panel A (Dermatomyositis): Perifascicular atrophy with 2-4 layers of small, pale, atrophic fibers at the fascicle periphery, perimysial inflammation with CD4+ T-cells and B cells. Panel B (Inclusion body myositis): Endomysial inflammation with CD8+ T-cells invading a non-necrotic fiber, rimmed vacuoles (basophilic-rimmed on modified Gomori trichrome stain) within muscle fibers, and a COX-negative fiber nearby. Panel C (Immune-mediated necrotizing myopathy): Multiple necrotic fibers (pale, fragmented) and basophilic regenerating fibers with minimal inflammatory infiltrate, complement C5b-9 deposition on sarcolemma shown in IHC inset. Panel D (Normal vs. dystrophy): Dystrophin IHC showing normal continuous sarcolemmal staining (left) versus complete absence in Duchenne muscular dystrophy (right).</image>

<image>A medical illustration of neurogenic and metabolic muscle pathology. Panel A (Neurogenic atrophy): Small angulated fibers scattered among normal-sized fibers, a target fiber with three-zone pattern on NADH-TR stain (central pale zone, dark intermediate ring, normal periphery). Panel B (Fiber type grouping): ATPase stain showing loss of normal checkerboard pattern with groups of same-type fibers from collateral reinnervation. Panel C (Ragged red fibers): Modified Gomori trichrome stain showing a fiber with subsarcolemmal red granular accumulation (abnormal mitochondria), with corresponding SDH stain showing a strongly reactive blue fiber (SDH-hyperintense) and COX stain showing the same fiber as COX-negative. Panel D (Glycogen storage myopathy): PAS stain showing excessive glycogen accumulation within muscle fibers, acid phosphatase stain showing vacuoles with lysosomal activity in Pompe disease.</image>

Clinical Pearls

Sural nerve biopsy must be split for three processing methods (formalin, glutaraldehyde for resin, and fresh-frozen). If the entire specimen is placed in formalin, semi-thin sections and electron microscopy cannot be performed, and diagnostic yield drops dramatically. Vasculitic neuropathy is now the most common indication for nerve biopsy; the key findings are fibrinoid necrosis of epineurial vessels with asymmetric fascicular axon loss, and a combined nerve-muscle biopsy increases diagnostic sensitivity from approximately 50 percent to 75 percent.

Perifascicular atrophy is pathognomonic for dermatomyositis and can be present even without skin findings. Its presence should prompt evaluation for underlying malignancy (especially in adults with anti-TIF1-gamma antibodies). Rimmed vacuoles on muscle biopsy strongly suggest inclusion body myositis, and unlike PM and DM, IBM does not respond to immunosuppressive therapy, making accurate distinction critical for management.

Muscle biopsy for suspected myopathy must be processed fresh-frozen, not in formalin. Enzyme histochemistry (ATPase, NADH-TR, COX, SDH) requires fresh tissue and is essential for diagnosis of mitochondrial myopathies, denervation, and fiber typing. Dystrophin IHC (using antibodies to N-terminus, C-terminus, and rod domain) should be performed on any biopsy showing a dystrophic pattern in a young male to distinguish DMD (absent staining) from BMD (reduced/patchy staining).

References

• Dubowitz V, Sewry CA, Oldfors A. Muscle Biopsy: A Practical Approach. 5th ed. Elsevier; 2021.
• Dyck PJ, Thomas PK. Peripheral Neuropathy. 4th ed. Elsevier Saunders; 2005.
• Mammen AL. Autoimmune myopathies: autoantibodies, phenotypes, and pathogenesis. Nat Rev Neurol. 2011;7(6):343-354.
• Collins MP, et al. Peripheral Nerve Society guideline on the classification, diagnosis, investigation, and immunosuppressive therapy of non-systemic vasculitic neuropathy. J Peripher Nerv Syst. 2010;15(3):176-184.
• Greenberg SA. Inclusion body myositis: clinical features and pathogenesis. Nat Rev Rheumatol. 2019;15(5):257-272.

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