Introduction

From a practical point of view, the pathology of peripheral nerves falls into two main categories: (a) peripheral neuropathies, which are diagnosed and treated by physicians and for which an elective nerve or muscle biopsy may be performed as a diagnostic procedure rather than as a therapeutic exercise, and (b) tumors and traumatic lesions, which are removed surgically mainly as a therapeutic measure to alleviate symptoms.

For the diagnosis of peripheral neuropathies, a detailed knowledge of the structure, immunohistochemistry and ultrastructure of peripheral nerves, and clinicopathological correlations is essential. The diagnosis of tumors and traumatic lesions, conversely, relies more on identifying the cellular components within the lesion and their interrelationships. This chapter, therefore, concentrates first on how to identify different cellular components in normal peripheral nerves and, second, on how knowledge of the normal structure of peripheral nerves can be used to identify and assess pathological lesions.

Development of the Peripheral Nervous System

The first anatomical evidence of nervous system differentiation is the neural plate, which develops as a thickened specialized area in the middorsal ectoderm of the late gastrula stage of the developing embryo. This zone later becomes depressed along the axial midline to form a neural groove that folds inward to form the neural tube. Before fusion is completed, groups of cells become detached from the lateral folds of the neural plate to form the neural crests. Anteriorly, neural crests are located at the level of the presumptive diencephalon and extend backward along the whole neural tube.

The neural crest yields pluripotent cells endowed with migratory properties. In the peripheral nervous system, the neural crest is the source of neurons and satellite cells in the autonomic and sensory ganglia; ectodermal placodes may also give rise to ganglion cells in the cranial region. Schwann cells are also derived from the neural crest. Migrating pluripotent neural crest cells and their subsequent development is determined and progressively limited, perhaps by the inductive effect of neuregulins and their receptors erbB2 and erbB3, by environmental factors, and by relations with other cell types . The transcription factor Sox-10, that is initially expressed in the earliest migrating neural crest cells, appears to be intimately involved in the development of Schwann cells from the neural crest. Interestingly, the major myelin protein, P0, is also a transcriptional target for Sox-10 .

Many of the events that occur during the later stages of development of peripheral nerves are recapitulated during the regeneration that follows nerve damage in postnatal life. Developing neuroblasts of the dorsal root ganglia (posterior sensory root ganglia) extend neurites both centrally into the neural tube and toward the periphery. Developing motor neurons in the anterior lateral parts of the neural tube extend their neurites toward the periphery. Schwann cells derived from the neural crest become associated with the developing peripheral nerves and eventually form myelin around many of the axons. The proximal portions of the anterior horn cell axons and the central axons of the sensory ganglion cells are myelinated within the neural tube by oligodendrocytes.

Growth of Axons

One of the major questions that has been raised is how neuronal processes grow over long distances and arrive at specific terminal regions. Genetic determinants, growth factors, and the extracellular matrix appear to play important roles in the appropriate guidance of neuronal processes. In 1909, Santiago proposed the concept of neurotrophic substances to explain the directionality and specificity of axonal growth in the developing nervous system, but it was not until the 1960s that nerve growth factor (NGF) was discovered by Rita Levi-Montalcini and Stanley Cohen, as a target-derived neurotrophic factor that supports the survival and differentiation of sensory and autonomic ganglia in the peripheral nervous system (6).

Nerve growth factor is a protein composed of three subunits alpha (α), beta (β), and gamma (γ) but only the β-NGF has nerve growth promoting activity. Beta-NGF in humans is a 14.5 KDa polypeptide, γ-NGF is an arginyl esterase, whereas the function of the α subunit is not known. Other substances that participate in axon growth are members of the NGF family [such as brain-derived neurotrophic factor (BDNF)]; neurotrophins 3 (NT-3), 4/5 (NT-4/5), and 6 (NT-6); semaphoring-3A, neuropilin-1, and ephrin . The tips of growing axons possess multiple surface receptors for soluble and bound molecules that provide information for the axons' growth course . Nerve growth factor interacts with the NGF receptor on the surface of the axon and promotes motility of the growing tip of the axon by interaction with the cytoskeleton of the cell. Mitochondria, neurotubules, neurofilaments, actin filaments, and some cisternae of smooth endoplasmic reticulum are incorporated into the axonal growth cone by axoplasmic flow. In addition to its growth promoting properties, NGF also promotes the early synthesis of neurotransmitters.

Schwann cells in the developing nerve produce NGF and possess NGF receptors on their surface membranes, but expression of these receptors diminishes markedly as the peripheral nerve matures. As NGF binds to Schwann cell receptors and becomes concentrated on the surface of the primitive Schwann cell, it provides a chemotactic stimulus for growing axons . Failure of trophic interactions between the target organ and its innervation may result in nerve dysfunction. Indeed, cases of human neuropathies have been attributed to deficiency of neurotrophic factors; important data that provides a rational basis for the clinical use of neurotrophic agents in peripheral neuropathies.

The extracellular matrix also plays an important role in axonal growth and guidance. The tip of the growing axon has receptors for adhesion to extracellular substances such as collagen, fibronectin, laminin, and entactin; binding of extracellular components to these receptors promotes elongation of axons and stimulates cytoskeletal protein synthesis and therefore cell movement and axon growth. Some of these extracellular components are found within or near basement membranes surrounding Schwann cells .

General Pathology of Peripheral Nerves

The general pathological reactions of peripheral nerves are, for most practical purposes, limited to (a) axonal degeneration and regeneration and (b) segmental demyelination and remyelination. Hypertrophic changes with onion-bulb formation occur most commonly as a result of recurrent segmental demyelination and are most often seen in hereditary neuropathies.

Axonal Degeneration and Regeneration

If a neuron in the anterior horn of the gray matter of the spinal cord or in a dorsal root ganglion dies, its axon degenerates and no regeneration occurs. Such neuronal destruction is seen in poliomyelitis, motor neuron disease (amyotrophic lateral sclerosis), spinal muscular atrophy, and infarction of the spinal cord. Dorsal root ganglion cells may be lost in viral infections such as varicella zoster or in a variety of hereditary sensory neuropathies. If an axon in a peripheral nerve is injured, for example, by trauma, entrapment, or ischemia, the distal end of the axon degenerates and subsequently regeneration occurs from the proximal stump of the damaged axon. The success of the regeneration depends on the distance of the site of damage from the nerve end organ (either motor endplate or sensory nerve ending) and the amount of scarring or other obstruction laid in the path of the regenerating axons.

Axonal degeneration was described by Waller in 1850 in London and the eponym wallerian degeneration is still used. Much of the fundamental work on nerve degeneration and regeneration, however, was performed by Cajal in the early part of the twentieth century. Twenty-four hours after nerve injury, most myelinated and nonmyelinated axons start to show degenerative changes. There is retraction of myelin from the nodes of Ranvier and dilatation of Schmidt-Lanterman incisures in the proximal as well as in the distal stump. By 48 hours, myelin and axon changes become more obvious as the axon disintegrates and myelin sheaths become disrupted and form globules in which axon fragments are enclosed. Disintegration of the myelin appears to start with dilatation of the Schmidt-Lanterman incisures during the first day or two after injury. Myelin debris is initially birefringent and has a lamellated ultrastructure, as does normal myelin. But, as proteins break down and lysosomal enzymes become active around the myelin debris , cholesterol within the myelin is esterified to cholesterol esters, and lipid debris loses its birefringence in polarized light and its lamellated ultrastructure to become amorphous globular lipid, which now stains strongly with Sudan dyes and oil red O. The Marchi technique also differentiates between normal myelin and degenerating myelin .

During the second week after nerve injury, much of the myelin debris is removed from the distal part of the nerve, and regenerative features become more prominent. Axon fragments and myelin debris are broken down by both Schwann cells and macrophages. Schwann cells directly attract macrophages by secretion of different proteins, probably regulated by autocrine circuits involving the neuropoietic cytokines, IL-6, and leukemia inhibitory factor . Macrophages, in addition to their phagocytic function may help to promote nerve repair through the elaboration of Schwann cell mitogens and may also affect neurons and axonal growth directly through the release of neurotrophins.

Although Schwann cell mitoses are seen as early as 24 hours after nerve injury, the peak of proliferation is between 3 and 15 days after nerve damage. As Schwann cells proliferate, they form columns surrounded by basement membrane; often redundant, old Schwann cell basement membrane is associated with these bands. Regenerating axons grow along the bands; and, if regeneration fails, the bands shrink and Schwann cells may disappear and become replaced by fibrous tissue.

Several easily detectable histological changes occur during axonal regeneration The neuron cell bodies in the anterior horns of the spinal cord or in the dorsal root ganglia show changes of chromatolysis during the first three weeks after axonal injury. The nerve cell perikaryon swells by some 20%, and the nucleus becomes eccentric, as does the nucleolus. Nissl substance (a mixture of rough endoplasmic reticulum and polyribosomes) is dispersed so that the cytoplasm becomes pale when stained by H&E or by the Nissl stain. During this stage of chromatolysis, there is a marked increase in polyribosomal ribonucleic acid (RNA) with an upregulation of a number of regeneration-associated genes, including those encoding growth-associated protein-43 (GAP 43), cytoskeleton protein-23, and α-tubulin, and in peptides such as galanin and vasoactive intestinal polipeptide (VIP), reflecting the metabolic events involved in axon regeneration. Regenerative changes in axons are seen within the first few hours after nerve damage but are most easily detected 5 to 20 days after injury. Using immunocytochemistry, GAP 43 can be identified in regenerating axons 4 to 21 days after injury . The proximal stump of the axon swells to create a balloonlike structure, often 50 μm in diameter and 100 μm in length. The balloons are filled with organelles and fibrils, which can be detected by electron microscopy; they can be visualized by light microscopy using immunocytochemical stains for neurofilament protein, GAP 43, or silver stains such as were used by Cajal when he first described them. Myelin sheaths become stretched around the swollen axon balloons.

Starting around the fourth day after injury, multiple nerve sprouts, or neurites, extend from the axon balloon (growth cone) and grow distally at 1 to 2.5 mm/day. As the neurites enter the bands, they become invaginated into the surface of the Schwann cell and, if growth continues, they become myelinated. Unmyelinated fibers regenerate in a similar way, but they are smaller and no myelin sheaths form around them. Regenerating neurites can be detected in the classical way by silver staining; but, in cross sections of peripheral nerve, they are best demonstrated in 0.5- to 1-μm resin sections or by electron microscopy. Characteristically, regenerating axons form clusters encircled by a single basement membrane. In the light microscope, these clusters are recognized by the close association of small, thinly myelinated axons within the nerve; myelinated nerve fibers in a normal nerve are well-separated from each other by endoneurial collagen.

Axon growth and regeneration are stimulated by nerve growth factor that is synthesized by Schwann cells, fibroblasts, and macrophages and transported back along the axon by retrograde axoplasmic transport to stimulate nerve cell protein synthesis. In addition to growth factors, there appears to be topographical affinity between regenerating axons and certain pathways; for example, it appears that regenerating tibial nerve axons grow toward the distal tibial nerve rather than toward the distal peroneal nerves. Connective tissue elements may also play a role in guiding regenerating axons . Neurite outgrowth promoting factors on cell surfaces (cell adhesion molecules) or in the extracellular matrix promote extension of the axon by providing an appropriate adhesivness in the substrate . Both neurotrophic and neurite outgrowth promoting factors are essential for axonal growth after injury .

The success of regeneration, with axons reaching effective end organs, may be influenced by several factors. If the injury is far proximal from the end organ, few regenerating axons may make effective reconnections. But, regeneration over short distances may be very effective in the peripheral nervous system. The presence of scar tissue or discontinuity of anatomical pathways may also inhibit regeneration. A number of grafting techniques are employed to overcome this problem. If regeneration to the distal stump of a nerve is blocked by scar tissue, axons may grow outside the original course of the nerve and even back alongside the proximal stump (terminals of Perroncito); thus, small bundles of regenerating neurites, often surrounded by perineurial cells, form amputation neuromas. Microscopically, there are interlacing bundles containing axons surrounded by myelin sheaths and with fine perineurial coverings. Immunocytochemistry for neurofilament proteins (axons), EMA, and Glut-1 (perineurial cells), and S-100 (Schwann cells) may be very useful in establishing the structure and identity of the nerve bundles in an amputation neuroma. Immunohistochemical and radioimmunoassay data have shown a focal accumulation of sodium channels within the tips of injured axons that may be responsible, in part, for the ectopic axonal excitability and the resulting abnormal sensory phenomena (pain and paresthesia) which frequently complicate peripheral nerve injury . Macropaghes migrate into the neuroma within the first two weeks after the injury, and later they are seen with numerous large cytoplasmic vacuoles filled with myelin fragments. This suggests that macrophages may also participate in the genesis of chronic pain after the neuroma has formed possibly by: (a) creating demyelinating axonal regions susceptible to external stimuli; (b) by releasing substances that influence regeneration of axons; or (c) by direct action on the denuded remodelling membranes .

Axon degeneration, often with regeneration, is a feature of numerous peripheral neuropathies, including those associated with diabetes, amyloidosis, infections (such as leprosy), sarcoidosis, paraneoplastic syndromes, vascular disease, and metabolic diseases. Most toxic neuropathies result in chronic axonal degeneration at the extreme distal ends of sensory and motor nerves (distal axonopathies). The distal ends of long tracts in the spinal cord (dorsal columns and corticospinal tracts) are often affected as well as the peripheral nerves. Timely withdrawal of the toxin may allow effective regeneration to occur, but only in the peripheral nerves, not in the spinal cord. Many peripheral neuropathies induced by the diseases itemized above are slowly progressive, so that nerve biopsies in these conditions do not usually reveal the early stages of axonal degeneration and regeneration. More frequently, the histological picture is characterized by loss of large myelinated axons and, to a lesser extent, loss of small myelinated and unmyelinated axons. Nerve root or peripheral nerve compression and trauma to peripheral nerve trunks result in axonal degeneration. Regeneration may be recognized in transverse sections of peripheral nerve by the presence of clusters. In teased fiber preparations, short internodes in the distal part of the nerve indicate that axonal degeneration and regeneration have occurred in the past .

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     外周神经系统

    引言

    从实践观点出发，外周神经病理分为两大块，（A）外周神经疾病，主要由内科医生诊治，因此选择性的神经或肌肉活检通常做为诊断手段而不是治疗手段。（B）肿瘤及创伤性病变，这要由外科医生来处理，目的是减轻症状。

     要诊断外周神经疾病，对外周神经的结构、免疫组织化学、亚显微结构，以及临床病理相关性等方面知识的掌握是非常重要的。而对于肿瘤及创伤性病变的诊断，更多的依赖于病变内部细胞成分及它们之间相互关系的识别。因此，在这一章节，我们首先讲解怎么辨识正常外周神经内的不同细胞，其次是讲解怎样用正常外周神经结构的知识去解释病变。

Schwann Cells and Myelination

Schwann cells move freely between and around developing peripheral nerve axons, forming primitive sheaths around the neurites and growing in parallel with them. Contact with axons stimulates Schwann cell division in vitro. In vivo Schwann cell multiplication virtually ceases in the normal adult animal, but mitotic activity is induced by peripheral nerve damage. It is thought that exposure of the axon to the Schwann cell following loss of myelin sheaths (demyelination) or during axonal regeneration following axonal degeneration (wallerian degeneration) promotes Schwann cell division and that the relationship between Schwann cells and axons in the normal nerve induces some sort of contact inhibition in the Schwann cells. If axon regeneration does not occur following axon damage, Schwann cells gradually decrease in number, suggesting that Schwann cell growth and survival depend on contact with axons. Experimental evidence also suggests that continued axon regeneration depends on the presence of Schwann cells.

By the ninth week of gestation, fascicles of the human sural nerve are identifiable and contain large axon bundles surrounded by Schwann cell processes. Between weeks 10 and 15, Schwann cells extend several long flattened processes that wrap around large clusters of fine axons. At this stage, two to four Schwann cells are located within a common basement membrane and form Schwann families.

Myelination of peripheral nerves in humans commences between the twelfth and eighteenth week of gestation. Initiation of myelination depends on the diameter of the axon and its association with Schwann cells. By the time that axons have increased in diameter to between 1.0 and 3.2 nm, they are in a 1:1 relationship with Schwann cells and have either formed mesaxons or membrane spirals with compact sheaths of 3 to 15 layers. The reason why some nerves become myelinated and others do not is not clear. Schwann cells around myelinated fibers and around unmyelinated fibers are both able to produce myelin, but the factors that determine whether myelination occurs are unknown. Certain transcription factors, such as Krox-20 and Oct-6, are known to be involved in the myelination program. In Oct-6 null mice, for instance, myelination is severely delayed, while in Krox-20 null mice myelination fails completely. Schwann cells in developing and regenerating peripheral nerves also express high levels of the neurotrophin receptor p75NTR. Neurotrophins are a family of proteins that play a variety of functions in the development and maintenance of the peripheral nervous system. Certain glycoproteins, such as myelin-associated glycoproteins, are believed to participate in establishing specific Schwann cell axon interactions in the developing peripheral nervous system.

Experimental studies have shown that axons may induce the formation of myelin if the unmyelinated sympathetic chain is grafted onto a myelinated nerve such as the saphenous nerve. Schwann cells that had not previously formed myelin will do so if they come into contact with large, regenerating axons that were previously myelinated. It appears also that Schwann cells may influence the caliber of axons since axonal diameter may be decreased markedly in some hereditary demyelinating neuropathies in which there is a genetic defect in Schwann cells and in myelination. It has been demonstrated that myelinating Schwann cells control the number and phosphorylation state of neurofilaments in the axon, leading to enlargement of the axon itself. Conversely, absence of myelin results in fewer neurofilaments, reduced phosphorylation levels, and therefore smaller axon diameters. Myelin-associated glycoprotein (MAG) acts as a myelin signal that modulates the caliber of myelinated axons. Maintenance of an axon therefore appears to depend not only on influences from the neuron cell body but also on interactions of the axon with the accompanying Schwann cells.

Some 70% of axons within a mixed sensory nerve, such as the sural nerve, are very small and will become segregated into groups of 8 to 15 axons lying in longitudinal grooves within one Schwann cell; these will form the unmyelinated fibers within the peripheral nerve. Thus, all axons in the peripheral nervous system are invaginated into the surfaces of Schwann cells, but myelin sheaths only form around the larger axons, which represent only a small proportion of peripheral nerve fibers.

Anatomy of Peripheral Nerves

An understanding of the anatomy of peripheral nerves is essential for the interpretation of clinical signs and symptoms and for planning an autopsy to investigate a patient with a peripheral neuropathy.

Major nerves, such as the sciatic and median nerves, contain motor, sensory, and autonomic nerve fibers; they are thus compound nerve trunks. It was Sir Charles Bell, the Scottish physician, who first demonstrated that motor function lay in the anterior roots; Magendie, the French physiologist, showed that the sensory function lay in the posterior roots. This (anterior-motor; posterior-sensory) is known as the Bell-Magendie law. Motor nerves are derived from anterior horn cells in the spinal cord or from defined nuclei in the brainstem. The initial segment of the axon lies within the central nervous system and is ensheathed by myelin formed by oligodendrocytes. As the axons pass out of the brainstem or spinal cord they become myelinated by Schwann cells. Anterior spinal roots join the posterior roots as they pass through the intervertebral foramina to form peripheral nerve trunks. Cranial nerves leave the skull through a number of different foramina. The junction point between oligodendrocytes and the Schwann sheath of the cranial nerves, known as Obersteiner-Redlich zone (O-Rz), has some clinical significance. For example, the pulsatile compression of the O-Rz by a vessel in some exit foramina may be responsible for the clinical symptoms of trigeminal and glossopharyngeal neuralgia, hemifacial spasm, torticollis spasmodicus, or even symptoms of essential hypertension when a vascular cross-compression of the left vagus nerve occurs

Motor nerves end peripherally at muscle endplates and many of the sensory nerves are associated with peripheral sensory endings. The cell bodies of sensory nerves lie outside the central nervous system in the dorsal root ganglia or in cranial nerve ganglia. Each ganglion contains numerous, almost spherical neurons (ganglion cells) with their surrounding satellite cells. Such satellite cells are derived from the neural crest and have an origin similar to that of Schwann cells. Satellite cells have been referred to in the past by a large variety of names such as amphicyte, capsular cells, perisomatic gliocyte, or perineuronal satellite Schwann cells.

Dorsal root ganglion cells were first described by the Swiss anatomist Albert von Kolliker in 1844. They are examples of pseudounipolar cells, which means that a single, highly coiled axon, or stem process, arises from each perikaryon; but, at varying distances from the neuron, there is a T- or Y-shaped bifurcation, always at a node of Ranvier, with the formation of central and peripheral axons. Thus, the initial segment of axon gives the impression that the cell is a unipolar neuron when it actually has two axons. The central axon passes into the spinal cord, either to synapse in the posterior sensory horn of gray matter or to pass directly into the dorsal columns. Peripheral axons pass into the peripheral nerves.

Autonomic nerves are either parasympathetic or sympathetic. Preganglionic parasympathetic fibers pass out of the brainstem in the cranial nerves III, VII, IX, and X and from the sacral cord in the second and third sacral nerves. Postganglionic neurons are situated near or within the structures being innervated. Sympathetic preganglionic fibers arise from neurons in the intermediolateral cell columns of gray matter in the thoracic spinal cord and pass out in thoracic anterior roots. These preganglionic fibers are myelinated and reach the sympathetic trunk through the corresponding anterior spinal roots; they synapse with the sympathetic ganglion cells in paravertebral or prevertebral locations. The autonomic nervous system innervates viscera, blood vessels, and smooth muscle of the eye and skin.

Histology, Immunocytochemistry, and Ultrastructure of Peripheral Nerves

Components of the Nerve Sheath

Macroscopic inspection of a normal peripheral nerve reveals glistening white bundles of fascicles bound together by connective tissue. The intraneural arrangement of fascicles is variable and changes continuously throughout the length of every nerve. Damaged peripheral nerves are often gray and shrunken due to the loss of myelin. Microscopically, transverse sections of a peripheral nerve show how endoneurial compartments containing axons and Schwann cells are surrounded by perineurium to form individual fascicles embedded in epineurial fibrous tissue.

Epineurium

The epineurium consists of moderately dense connective tissue binding nerve fascicles together. It merges with the adipose tissue that surrounds peripheral nerves, particularly in the subcutaneous tissue. In addition to fibroblasts, the epineurium contains mast cells. Although mostly composed of collagen, there are elastic fibers in the epineurium so that, when a specimen of unfixed nerve is removed from the body, there is some elastic recoil of the epineurium. The amount of epineurial tissue varies and is more abundant in nerves adjacent to joints. As nerve branches become smaller to consist of only one fascicle, epineurium is no longer present. In nerves that consist of several fascicles, one or more arteries, veins, and lymphatics run longitudinally in the epineurium parallel to the nerve fascicles (the vasa nervorum). Inflammation and occlusion of such arteries is an important cause of nerve damage in vasculitic diseases. The overgrowth of epineurial adipose tissue produces the so-called lipofibromatous hamartoma, which classically affects the hands and is associated with enlargement of the affected digit.

Perineurium

Originally described by Friedrich G.J. Henle in the nineteenth century, the perineurium has, in the past, been known by a variety of different terms, such as mesothelium, perilemma, neurothelium, perineurothelium, and, more recently, perineurial epithelium.

Based on the pioneer work of the 1995 Nobel Prize winners Christiane Nüsslein-Volhard and Wieschaus, an intercellular signaling molecule secreted by Schwann cells known as Desert Hedgehog, was described, that functions as an important molecule in the formation of the perineurium. Apparently this molecule signals to the surrounding connective tissue cells to organize the perineurium.

The perineurium consists of concentric layers of flattened cells separated by layers of collagen. The number of cell layers varies from nerve to nerve and depends on the size of the nerve fascicle. In the sural nerve, for example, there are 8 to 12 layers of perineurial cells, but the number of layers decreases progressively so that a single layer of perineurial cells surrounds fine distal nerve branches. Perineurial cells eventually fuse to form the outer-core of the terminal sensory endings in pacinian corpuscles and muscle spindles. In motor nerves, the perineurial cells form an open funnel as the nerve ends at the motor endplate. Paraganglia of the vagus nerve may lie just underneath the perineurium.

By electron microscopy, perineurial cells are seen as thin sheets of cytoplasm containing small amounts of endoplasmic reticulum, filaments, and numerous pinocytotic vesicles that open on to the external and internal surfaces of the cell. Basement membrane is usually seen on both sides of each perineurial lamina. Numerous cell junctions, including well-formed tight junctions (zonulae occludentes), are present between adjacent perineurial cells and appear to be critical for the formation of the blood-nerve barrier. Claudins are integral membrane proteins that play a major role in tight junctions and are present in normal and neoplastic perineurium. Claudins comprise a group of approximately 20 different proteins that are exclusively localized in tight junctions . In peripheral nerves, claudin-1 expression is largely limited to perineurial cells but is also present in paranodal regions and in the outer mesaxon along internodes. When tracer substances such as ferritin and horseradish peroxidase are injected into the blood, they do not enter peripheral nerves. Their entry is prevented by tight junctions in endoneurial capillaries and by the tight junctions in the inner layers of the perineurium. Thus, there is a blood-nerve barrier analogous to the blood-brain barrier. The blood-nerve barrier is present soon after birth and may prevent the entry of drugs and other substances into nerves that may otherwise interfere or block nerve conduction. No such blood-nerve barrier exists in the dorsal root ganglia or in autonomic ganglia; these sites in the peripheral nervous system are vulnerable to certain toxins, such as mercury.

If the perineurium is injured, there is breakdown of the blood-nerve barrier and perineurial cells migrate into the endoneurium to surround small fascicles of nerve fibres. This is classically seen in amputation neuromas but is also observed in focal compressive lesions of nerve. The swelling of the nerve and the concentric arrangement of the perineurial cells in the compressive lesions spawned the term localized hypertrophic neuropathy, but it is quite different from hypertrophic neuropathy, in which Schwann cells form whorls around individual axons in response to recurrent segmental demyelination (see below).

Whereas the epineurial sheath of the nerve is continuous with the dura mater at the junction of spinal nerves and spinal nerve roots, the perineurium blends with the pia-arachnoid. There are some morphological similarities between perineurium and arachnoid cells, although arachnoid cells are not usually coated by basement membrane. Immunocytochemically, perineurial cells and pia-arachnoid cells are positive for epithelial membrane antigen (EMA) and vimentin but are negative for S-100 protein and CD57. Perineurial cells also express insulin-dependent glucose transporter protein I (Glut-1).

Epithelial membrane antigen belongs to a heterogenous family of highly glycosylated transmembrane proteins found originally on the surface of mammary epithelial cells  but which are also present in the cells of virtually all epithelial tumors. However, EMA is not restricted to epithelial structures and has been identified on plasma cells and on cells in certain lymphomas and soft tissue tumors. Perineurial cells, arachnoid, and pia share certain ultrastructural characteristics and express EMA and vimentin in their cytoplasm. Immunohistochemistry has demonstrated that perineurial cells proliferate in some conditions, such as traumatic neuroma, Morton's neuroma, neurofibroma, solitary circumscribed neuroma, neurothekeoma, pacinian neuroma, and in the mucosal neuromas associated with multiple endocrine neoplasia (vide infra).

Some tumor cells break through the perineurial sheath to grow along the perineurial space; perineurial invasion has been correlated with decreased survival times in some cancers. The problem for the histopathologist, however, is that sometimes perineurial invasion cannot be unequivocally determined on hematoxylin and eosin (H&E)stained sections. Immunocytochemistry for Glut-1, EMA, and claudin-1 may be used to rapidly and accurately assess the presence of perineurial invasion. Care must be taken, however, when examining cases of vasitis nodosa, in which benign proliferating ductules may be found within the perineurium and endoneurium. Nerve involvement has also been reported in fibrocystic disease of the breast, normal and hyperplastic prostate, and normal pancreas.

Endoneurium

The endoneurium is the compartment that contains axons and their surrounding Schwann cells, collagen fibers, fibroblasts, capillaries, and a few mast cells.

In cross sections of peripheral nerves, some 90% of the nuclei belong to Schwann cells, 5% to fibroblasts, and 5% to other cells (such as mast cells and capillary endothelial cells). Within the endoneurium, CD34+ bipolar cells with delicate dendritic processes have been identified and are distinct from Schwann cells. Similar cells have been identified in peripheral nerve sheath tumors in various proportions.

Some investigators have observed endoneurial dendritic cells, distinct from Schwann cells and conventional fibroblasts, that may function as phagocytes under certain conditions. In this regard, it has been described within the human endoneurium, an intrinsic population of immunocompetent and potentially phagocytic cells (endoneurial macrophages), that share several lineage-related and functional markers with macrophages and may represent the peripheral counterpart of del-Rio-Hortega cells (microglia) of the CNS.

Nerve fibers may be myelinated or unmyelinated but not all nerves have the same nerve fiber composition. Most biopsies of peripheral nerves in humans are taken from the sural nerve at the ankle, and it is the composition of this nerve that has been most closely studied  . Fibroblasts are ultrastructurally identical to fibroblasts elsewhere in the body. Mast cells are a normal constituent of the endoneurium and are also seen in sensory ganglia and in the epineurial sheath of peripheral nerves. There is an increase in the number of mast cells in some pathological conditions such as axonal (wallerian) degeneration and in some neoplastic entities such as von Recklinghausen's disease (neurofibromatosis). A characteristically high number of mast cells is seen in neurofibromas, but they are only present in the Antoni B areas of schwannomas. Mast cells are thought to influence growth of neurofibromas because some of their mediators may also act as growth factors. Apparently the inciting factor for mast cell migration into nerve sheath tumors is Kit ligand. Mast cell stabilizers are claimed to reduce proliferation and itching of neurofibromas. Following nerve injury, there is breakdown of the blood-nerve barrier as endoneurial vessels become permeable to fluid and protein; this increase in permeability may be related to the release of biogenic amines from mast cells within the endoneurium. Proteases released from mast cells have a high myelinolytic activity and may play a role in the breakdown of myelin in certain demyelinating diseases.

Collagen within the endoneurial compartment is highly organized and forms two distinct sheaths around myelinated and unmyelinated nerve fibers and their Schwann cells. The outer endoneurial sheath (of Key and Retzius) is composed of longitudinally oriented large diameter collagen fibers; the inner endoneurial sheath (of Plenk and Laidlaw) is composed of fine collagen fibers oriented obliquely or circumferentially to the nerve fibers. The term neurilemma has been applied to the combined sheath formed by the basement membrane of the Schwann cell and the adjacent inner endoneurial sheath. Thus the term neurilemmoma is inappropriate when used to describe tumors of Schwann cell origin (schwannomas). The longitudinal orientation of collagen fibers in the outer endoneurial sheath, together with the Schwann cell basement membrane tubes, may play an important role in guiding axons as they regenerate following peripheral nerve damage.

Renaut bodies  are seen not infrequently in the endoneurium of human peripheral nerves. Described in the nineteenth century by the French physician Joseph Louis Renaut, they are cylindrical (circular in cross section), hyalin bodies attached to the inner aspect of the perineurium. Composed of randomly oriented collagen fibers, spidery fibroblasts, and perineurial cells, Renaut bodies stain positively with Alcian blue because of the presence of acid glycosaminoglycans. The rest of the endoneurium also contains Alcian blue positive mucoproteins. Renaut bodies express vimentin and EMA and produce extracellular matrix highly enriched in elastic fiber components. In longitudinal section, they may extend for some distance along the nerve and end in a blunt and abrupt fashion. These bodies are more prominent in horses and donkeys than in humans. Their precise function is not known, but Renaut himself thought that they may act as protective cushions within the nerve. They increase in number in compressive neuropathies and in a number of other neuropathies, including hypothyroid neuropathy, and may be a reaction to trauma.

Blood Supply of Peripheral Nerves

Vasa nervorum supplying peripheral nerves are derived from a series of branches from associated regional arteries. Branches from those arteries enter the epineurium to form an intercommunicating or anastomosing plexus. From that plexus, vessels penetrate the perineurium obliquely and enter the endoneurium as capillaries often surrounded by pericytes. Tight junctions between the endothelial cells of the endoneurial capillaries constitute the blood-nerve barrier.

Complete infarction of peripheral nerves is very uncommon, probably due to the rich anastomotic connections of epineurial arteries. However, inflammation and thrombotic occlusion of epineurial arteries is seen in vasculitides, and occlusion by emboli occurs in patients with atherosclerotic peripheral vascular disease; both these disorders result in ischemic damage to peripheral nerves with axonal degeneration and consequent peripheral neuropathy.

Nerve Fibers

Most peripheral nerves contain a mixture of myelinated and unmyelinated nerve fibers. As the axons are oriented longitudinally along the nerve, quantitative estimates of the number of fibers in the nerve and their diameters are only adequately assessed in exact transverse sections. Longitudinal sections of peripheral nerve are less valuable than transverse sections, but teased nerve fibers  are very valuable for detecting segmental demyelination and remyelination and for assessing past axonal degeneration and regeneration.

In a transverse section of a human sural nerve, there are approximately 8,000 myelinated fibers/mm², whereas the unmyelinated axons are more numerous at 30,000 myelinated fibers/mm². Peripheral nerve fibers are classified as class A, class B, and class C fibers, according to their size, function, and the speed at which they conduct nerve impulses. Class A fibers are myelinated and are further subdivided into six groups covering three size ranges. The largest are 10 to 20 nm diameter myelinated fibers that conduct at 50 to 100 m/sec; myelinated fibers 5 to 15 nm in diameter conduct at 20 to 90 m/sec, and 1 to 7-nm diameter myelinated fibers conduct at 12 to 30 m/sec. Class B fibers are myelinated preganglionic autonomic fibers some 3 nm in diameter and conducting at 3 to 15 m/sec. Unmyelinated fibers are small (0.2 to 1.5 nm in diameter), conduct impulses at 0.3 to 1.6 m/sec, and include postganglionic autonomic and afferent sensory fibers, including pain fibers.

Correlation of Normal Histology with the Pathology of Peripheral Nerves

Handling and Preparation of Peripheral Nerve Biopsy and Autopsy Specimens

The sural nerve is the nerve that is most commonly biopsied in the investigation of peripheral neuropathies. It is a sensory nerve so that in some motor neuropathies it may be totally normal, in which case examination of small branches of motor nerves within a muscle biopsy may be more fruitful. At autopsy, a wider range of motor and sensory nerves may be sampled, depending on the clinical picture. Whether taken at biopsy or autopsy, peripheral nerves are very easily damaged. The myelin sheaths are semiliquid and may be crushed by indelicate handling. The specimen should be gripped at only one end and then gently dissected free before laying it, very gently stretched, on a piece of dry card and placing it in fixative or in liquid nitrogen for snap freezing. Fresh, frozen nerve should be used for enzyme and lipid histochemical studies whereas formalin-fixed nerve can be embedded in paraffin for the application of routine stains and immunocytochemistry. Although formalin-fixed material can be used for the preparation of 0.5- to 1-μm resin-embedded sections and for electron microscopy, ideally the tissue should be fixed in glutaraldehyde and postfixed in osmium for ultrastructural studies. Teased fibers can be prepared from either glutaraldehyde- or formalin-fixed material.

The method of preparation really depends on the information sought. Frozen sections are ideal for detecting abnormal lipids, such as sulfatide in metachromatic leukodystrophy, and for detecting the cholesterol ester droplets of degenerating myelin by staining for Sudan red or oil red O. Increased lysosomal enzyme activity as in Krabbe's leukodystrophy or in human and experimental neuropathies in which axonal degeneration or segmental demyelination is suspected can be detected in frozen sections stained histochemically for acid phosphatase. Brief formalin or glutaraldehyde fixation can be used in some cases for electron microscopic enzyme histochemistry. Frozen sections can also be used for immunofluorescence for the detection of immunoglobulin binding to myelin sheaths in paraproteinemias. Transverse frozen sections of nerve are ideal for these purposes although they are often more difficult to prepare than longitudinal sections.

There is a variety of methods of preparing and examining fixed specimens of peripheral nerve, and each method reveals different information. Ideally, exact transverse sections should be cut from the peripheral nerve; occasionally, longitudinal sections are also useful, particularly for detacting regenerating axons by immunocytochemistry. Paraffin-embedded sections can be stained for a variety of histological stains and for immunocytochemistry to reveal nerve components. Blood vessels and inflammatory exudates are ideally studied in paraffin sections, but quantitation of nerve fibers, the detection of axon degeneration and regeneration, and the assessment of segmental demyelination and remyelination are more satisfactory in 0.5- to 1-μm toluidine blue stained resin sections or by electron microscopy. The presence of amyloid in the endoneurium or giant axons in some hereditary neuropathies and in some toxic neuropathies can be detected both in paraffin- and in resin-embedded sections. Teased preparations are most useful for detecting segmental demyelination and remyelination and for assessing whether axonal degeneration and regeneration have occurred within the nerve in the past.

Peripheral Neuropathies

The pathological diagnosis of a peripheral neuropathy usually requires close clinicopathologic correlation and knowledge of the electrophysiologic data, such as nerve conduction velocities and electromyography. Moderate slowing of nerve conduction velocities usually indicates loss of large myelinated fibers, whereas excessive slowing of conduction velocity suggests that segmental demyelination has occurred. Although there are a number of specific histopathological features that aid in the diagnosis of peripheral neuropathy [e.g., amyloid, the presence of M. leprae bacilli, abnormal lipids such as sulfatide within the nerve, giant axons, and vasculitis], for the most part, assessment of peripheral nerve pathology depends on detection and quantitation of general pathological features and good clinicopathological correlation.