How to Work Up a Nerve Biopsy

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Introduction  Tissue processing  Evaluation  Teased fiber preparation  Anatomy

Introduction: Head

Objectives: Pathologic study of nerve tissue is especially useful for the recognition of interstitial events such as inflammation or vascular alterations that cannot be inferred from clinical or electrophysiologic findings and may provide insight into an underlying mechanism or cause.

Three major types of pathology

Tissue processing: Head

The fresh specimen should be separated into two half, one-half fix in formalin and the other half fix in gluteraldehyde- formaldehyde. However, everything can be done with formalin fixed tissue.

The segment for paraffin sectioning should be infiltrated in toto, sectioned after infiltration. Excessive loose connective tissue attached to the nerve bundle should be removed and embedded in a separate block. In this way, it will be easier to obtain good transverse section of the nerve bundle.

Good transverse paraffin sections should be obtained. Initially, HE, trichrome, and Congo red stained sections plus 5 unstained (immunostaining ready) sections should be obtained. Elastic stain, PTAH stain, and extra levels should be ordered if the suspicion of vasculitis is high. (PTAH in fact works better than trichrome if only the assessment of fiber lost is the only reason for trichrome stain. In addition, it has the advantage of detecting minute amount of fibrin deposition).

Semithin sections stained with toulidine blue or acid-fushin/ toulidine blue should be obtained from both ends of the gluteraldehyde fixed segment.

The rest of the specimen should be submitted for teased fiber preparation in toto.

Evaluation:  Head

Paraffin sections:

Semithin Sections

Teased fiber changes:  Head

Normal fibers

Wallerian degeneration: these fibers demonstrate fragmented myelin that forms ovoids and myelin balls over several internodes.

Paranodal swelling: defined as a paranodal expansion of the axonal diameter exceeding 150% of the average internodal diaeter. It may be present on one or both sides of the nodal gap.

Paranodal demyelination: a nodal gap distance, devoid of any myelin, that exceeds the internodal mean diameter of the same fiber.

Excessive myelin wrinkling: irregular infoldings and superimposed myelin folds, involving one third or more of the internodal length in such a way that the greatest diameter is equal toor exceeds 150% of the smallest diameter. Very difficult to be separated from artifacts.

Intercalated internode: a fiber shows one remyelinated internode surrounded on both sides by normally myelinated internodes. This is regarded as a product of the repair process that follows paradnodal demyelination.

Segmental demyelination: active demyelination with myelin fragmentation and ovoids within one internode that is surrounded by normally myelinated internodes is an extremely rare change.

Segmental remyelination: a fiber showing two or more remyelinated intercalated internodes surrounded by normally myelinated internodes.

Regeneration: a sequence of short, thinly myelinated internodes less than 50% of a normal internode length of the same fiber diameter.

Anatomy:  Head

Blood-nerve-barrier: this barrier is formed by the perineurial cells and the endothelial cells of the blood vessels. This barrier is not present in the dorsal root ganglia or in autonomic ganglia. These sites in the PNS are vulnerable to certain toxins such as mercury.

Blood vessels: Occasional thin rims of lymphocytes around small epineurial vessels can occur in normal nerve or non-inflammatory neuropathies.

Classification of myelinated fibers according to their caliber:

Diameter of fibers: Myelinated fibers exhibit a bimodal distribution of fiber diameter in the normal nerve with peaks of distribution at 5 and 13 micron and a range of 2 to 20 micron. Most axons above 3 micron are myelinated. Unmyelinated fibers range from 0.2 to 3 micron and with peaks of distribution at 1.5 micron.

Endoneurium: The endoneurium has positive pressure in reference to the perineurium. This expansile tendency and the elastic properties of perineurium create the uniformly circular shape of each fascicle. Except at the branching point, normal fascicles that are free of artifacts should appear circular.

Giant fibers: Giant fibers can be seen in toxic neuropathies and giant axonal neuropathy (an autosomal recessive disorder).

Length of nodes: the distance between each node along a myelinated fiber is approximately proportional to the thickness of the myelin sheath with a range of 200 to 1500 micron. The Schwann cell nucleus is usually sited around the middle of the internode.

Number of fibers: in a transverse section of a human sural nerve, there are approximately 8,000 myelinated fibers per mm2, whereas the unmyelinated axons are more numerous at 30,000 per mm2. Unmyelinated fibers are more numerous than myelinated fibers in mixed peripheral nerves by a factor of 3 or 4:1.

Perineurium: consists of concentric layers of flatten perineurial cells [EMA(+), S100(-), Leu7(-), Laminin (variable+), collagen 4 (variable+)] separated by layers of collagen. In the sural nerve, there are 8-12 layers of perineurial cells. The number of layers decreases progressively. The perineurium blends with the pia-arachnoid. EM: Perineurial cells are joined by tight junction but arachnoidal cells have intermediate filaments and are interconnected by well-formed desmosomes. Perineurial cells also have a discontinsuous pericellular basement membrane and pinocytic vesicles, features that are not seen in arachnoidal cells.

Remak cells: All axonal satellite cells are generally included under the term "Schwann cells", but some writers distinguish those that surround unmyelinated fibers as Remak cells.

Renaut bodies: Renaut bodies are present in 2-7.5% of sural nerve biopsies. They are 30-200 mm oval to round bodies, usually subperineurially located, with their long axis runing parallel to the long axis of the nerve. They tend to collapse on formalin fixed sections but the shape is maintained in gluteraldehyde or B5 fixed tissue. They may also have a circumferential disposition around the entire fascicle and produces the so called “subpeirneuraial edema”.On cross sections, they are whorled fibrillary structures that may contain few vessels and mast cells but not axons. In glutyaraldehyde or B5 fixed tissue, they are lightly eosinophilic, and lightly stained with touluidine blue and Alcian blue but not with PAS or Congo red. They should not be mistaken as amyloid or immunoglobulin deposits.

Type of cells: about 90% of the nuclei in cross sections belong to Schwann cells, 5% to fibroblasts, and 5% to other cells such as mast cells and macrophages. Mast cells are rare but are normal constituents of the endoneurium and are also seen in sensory ganglia and in the epineurial sheath of peripheral nerve. Macrophages make up about 2-5% of endoneurial cells.

Unmyelinated fiber: they are better visualized by silver technique than with immunostaining against neurofilament. Schwann cells associated with unmyelinated fibers are more likely to be GFAP positive.