Skin Nerve Anatomy: Neuropeptide Distribution and Its Relationship to Itch

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A.



Metze



Skin Nerve Anatomy



The integument of the body is innervated by large, cutaneous branches of

musculocutaneous nerves that arise segmentally from the spinal nerves. In the

face, branches of the trigeminal nerve are responsible for cutaneous innervation. The main nerve trunks enter the subcutaneous fat tissues, divide, and

form a branching network at the dermal–subcutaneous junction. This deep

nervous plexus supplies the deep vasculature, adnexal structures, and sensory

receptors. Subsequently, the nerve fibers reorganize into small nerve bundles,

which ascend along with the blood vessels and lymphatic vessels to form a

network of interlacing nerves beneath the epidermis (i.e., the superficial nerve

plexus of the papillary dermis) (2).

The cutaneous nerves contain only sensory or autonomic nerve fibers.

The sensory nerves conduct afferent impulses from the periphery to their cell

body in the dorsal root ganglia, or, for the face, to the trigeminal ganglion.

Cutaneous sensory neurons are unipolar; one branch of a single axon extends

from the cell body toward the periphery, and the second one extends toward

the central nervous system. As many as 1000 afferent nerve fibers may innervate 1 cm2 of the skin. The sensory innervation follows well-defined segments

(i.e., dermatomes); however, an overlapping innervation may occur. Autonomic postganglionic fibers are codistributed with the sensory nerves until

they arborize into the terminal autonomic plexus, which supplies skin glands,

blood vessels, and arrector pili muscles.

As the peripheral nerves approach the skin and branch, the number of

myelinated fibers decreases and the epineural connective tissue sheaths that

surround the larger nerve trunks disintegrate (Fig. 1). In the dermis, perineural layers and the endoneurium envelop the primary neural functional unit

(i.e., the Schwann cell–axon complex). The multilayered perineurium comprises flattened cells and collagen fibers, and serves mechanical as well as

barrier functions. The perineural cells are surrounded by a basement membrane, possess intercellular tight junctions of the zonula occludens type, and

show high pinocytotic activity. The endoneurium is composed of fine connective tissue fibers, fibroblasts, capillaries, and, occasionally, a few histiocytes and mast cells. The endoneural tissue is separated from the Schwann

cells by a basement membrane and subserves nutritive functions for the

Schwann cells (3).

The Schwann cell–axon complex consists of the cytoplasmic processes

of the neurons that propulse the action potentials and the enveloping

Schwann cells. The peripheral axon may be myelinated or unmyelinated. In

myelinated nerve fibers, the Schwann cell membranes wrap themselves

around the axon repeatedly, thus forming the regular concentric layers of

the myelin sheath. In nonmyelinated nerves, several axons are found in the



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Figure 1 Larger subcutaneous nerve trunk with disintegration of epineural connective tissue sheaths (E). Perineural layers (P) and the endoneurium envelop the

primary neural functional unit (i.e., the Schwann cell–axon complex). Routine histology, H and E staining.



cytoplasm of Schwann cells, forming characteristic polyaxonal units (Fig. 2).

However, these axons are not enclosed beyond the initial stage of enfolding

and therefore are invested with only a single or a few layers of Schwannian

plasma membranes without formation of thick lipoprotein sheaths (4). This

intimate relationship implies a crucial role of Schwann cells for the development, mechanical protection, and function of the nerves. In addition, the

Schwann cells serve as a tube to guide regenerating nerve fibers. The axons are

long and thin cytoplasmic extensions of the neurons that may reach a length

of more than 100 cm. Ultrastructurally, the cytoplasm of the axons contains

neurofilaments belonging to the intermediate filament family, mitochondria,

longitudinally orientated endoplasmic reticulum, neurotubuli, and small

vesicles and granules that represent packets of neurotransmitters and neuropeptides (3).

In general, unmyelinated-type C-fibers constitute autonomic and sensory fibers whereas myelinated-type A-fibers correspond to a subgroup of

sensory neurons. The myelinization of the axons allows for a high conduction

velocity of 4–70 m/sec compared to a lower speed of 0.5–2 m/sec in the

unmyelinated fibers. The sensory myelinated fibers are further divided based

on their diameter and conduction speed into rapidly conducting A-subcate-



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Figure 2 In nonmyelinated nerves, several axons are found in the cytoplasm of a

Schwann cell forming the polyaxonal unit. The axons contain secretory granules. A

basement membrane (arrow) separates the Schwann cell–axon complex and endoneural tissue (E). Electron microscopy.



gories and slowly conducting A-subcategories. Because the conduction

velocity of the action potentials of individual axons remains constant and

myelinated and unmyelinated fibers show no overlap, this feature is a useful

tool in the classification of sensory nerve fibers. Several neurophysiological

experiments have shown that the A-fibers conduct tactile sensitivity, whereas

A-fibers and C-fibers transmit temperature, noxious sensations, and itch (5).

The detection of the fine nerve fibers can be achieved by impregnation

with silver salts, vital and in vitro methylene blue staining, and histochemical

reaction for acetylcholinesterase (6). Immunohistochemistry allows for visualization of constitutional and structural proteins in the peripheral nervous

system. Protein gene product 9.5 (PGP 9.5) is a pan neuronal cytoplasmic

protein that is invariably expressed over the entire length of the axons (7).

Other less sensitive neuronal markers are neurofilament proteins (NFs) (Fig.

3; see color insert), neuron-specific enolase (NSE), nerve growth receptor

(NGF), synaptophysin (membrane protein of neural vesicles), nerve cellspecific clathrin (LCb subunit, neuronal-coated vesicles involved in receptormediated endocytosis), and calcium-binding S-100 protein—the latter being

also expressed in Schwann cells. Myelinated nerve fibers stain for myelin basic

protein (a component of the myelin sheath), N-CAM (CD56), leu 7 (CD57, a

marker for a subset of natural killer lymphocytes that crossreacts with a

myelin-related glycoprotein) (8).



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Figure 3 Dermal nerve fibers as stained for neurofilaments (arrows) in close

proximity to blood vessels and inflammatory cells (star). Immunoperoxidase staining.

(See color insert.)



In the upper dermis, small myelinated nerve fibers are only surrounded

by a monolayer of perineural cells and a small endoneurium whereas in thin

peripheral branches of unmyelinated nerve fibers, perineural sheaths are

absent (9). After losing their myelin sheaths, cutaneous nerves terminate

either as free nerve endings or in association with receptors, such as Merkel

cells or special nerve end organs.

Beneath the epidermis, nerve fibers obtain a coiled configuration and

multiple varicosities. According to the concept of Cauna (10), the terminal

part of cutaneous nerve fibers ramifies and forms brushlike ‘‘penicillate’’ free

nerve endings. Skin nerves terminate not only in the subepidermal connective

tissues or close to skin appendages, but also intraepidermally (Fig. 4; see color

insert).

Visualization of intraepidermal nerves is not possible on light microscopy. However, silver impregnation techniques and immunohistochemistry

have clearly identified nerve fibers in all layers of the epidermis. PGP 9.5

proved to be the most sensitive immunomarker for intraepidermal nerve

fibers. Other proteins such as NF, NSE, N-CAM, clathrin, nerve growth

factor, pituitary adenylate cyclase-activating polypeptide (PACAP), and gMSH show a more variable expression (11–14). Some of the nerve fibers have

been shown to go straight up to the superficial layers; others follow a more

tortuous pattern or show some branching (Fig. 5; see color insert).



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Figure 4 Small sensory nerve fiber (arrow) as visualized by the expression of CGRP

in the papillary dermis. Positive immunofluorescence staining for CGRP depicted in

red pseudocolors. Confocal laser scanning microscopy. (See color insert.)



Figure 5 Intraepidermal nerve fiber as immunostained for PGP 9.5 (arrows). The

tortuous course can be best demonstrated by optical sectioning using confocal laser

scanning microscopy (optical sections a–c). Keratinocytes (K), junctional zone of

epidermis, and dermis (stars). The positive immunofluorescence staining is depicted in

red pseudocolors. (See color insert.)



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Intraepidermal nerves may occur at every site of the hairy and glabrous

skin. The density of intraepidermal nerves, as evaluated by immunoreactivity

for PGP 9.5, is within the range of 2.9–9.6 per 1000 basal keratinocytes (14).

The highest number of intraepidermal nerves has been found in facial skin

(15). According to some authors, the number of nerves seems to decrease from

the trunk to the distal parts of the limbs (13), whereas others found the

opposite. Apart from using detection systems with different sensitivities, sun

exposure, age, and other factors may account for the conflicting results. For

example, intraepidermal nerve fibers may not be distributed evenly in normal

human skin but may be present focally so that the epidermis may lack fibers in

small biopsy specimens. In general, immunostaining of the distal parts of

nerves for neuropeptides is difficult because retrograde axonal transport

results in low peripheral concentrations (16).

In vivo pretreatment of the skin with the neuropharmacological agent,

capsaicin, induces loss of most, but not all, of the epidermal fibers staining,

suggesting that these are sensory fibers of the unmyelinated C-type (7).

However, in addition to sensory functions, intraepidermal nerve fibers fulfill

neurotrophic functions on keratinocytes and enable the neuroimmunological

modulation of Langerhans cell functions (17,18).

B.



Sensory Receptors



The sensory receptors of the skin can be divided into free and corpuscular

nerve endings. Corpuscular endings contain both neural and nonneural

components and are of two main types: nonencapsulated Merkel’s ‘‘touch

spot’’ and encapsulated receptors (19,20). In the past, many of the free and

corpuscular nerve endings in humans and animals have been associated with

specific sensory functions according to their distribution and complex architecture. Because it is difficult to identify specific sensory modalities within

individual terminal axons by means of neurophysiological techniques, many

of the assumptions remain speculative (21,22).

The free nerve endings of nonmyelinated C-fibers and, to some extent,

of small myelinated A-fibers are widely distributed throughout the body and

form the majority of the sensory receptors. In humans, the ‘‘free’’ nerve

endings do not represent naked axons but remain covered by small cytoplasmic extensions of Schwann cells and a basement membrane that may show

continuity with that of the epidermis (23). The terminal of the axon may be

beaded and, besides mitochondria, harbors vesicles and granules filled with

sensory neuropeptides.

The free nerve endings of ‘‘polymodal’’ C-fibers are chemosensitive,

mechanosensitive, and thermosensitive, and mediate multiple sensory modalities such as touch, temperature, pain, and itch. Alterations of sensory nerves,



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such as axonal deposition of polyvinylpyrrolidone (PVP) (24) and hydroxyethyl starch (25,26), will induce a highly characteristic burning pruritus of

noninflamed skin. Micrographic recordings have clearly shown that the

sensation of itch is transmitted by a subpopulation of unmyelinated Cpolymodal nociceptive neurons (27).

The terminals of nociceptive neurons are free nerve endings in the

dermis and epidermis (Figs. 4 and 5; see color insert). Interestingly, after

removing the epidermis, itch is not inducible, whereas pain still can be

provoked. It can be speculated that rubbing, scratching, and pressing will

temporarily relieve the sensation of itch by impairment of superficial intraepidermal nociceptors responsible for the generation of itch. In the epidermis,

‘‘innervation patches’’ show up, which can be identified in confocal microscopy as one morphological terminal field coming from the same dermal nerve

bundle (13). Physiologically, two-point discrimination of itch may be attributed to this distribution mode of intraepidermal nerve fibers (14).

The sensory neurons express specific receptors for histamine, serotonin, prostaglandin, and bradykinin. It can be hypothesized that pruritogenic

agents specifically bind to itch receptors on the surface of chemosensitive

nerve endings and thereby cause firing of the axons. The existence of specific

binding sites on chemosensitive neurons may account for the observation

that experimental pruritus induced by intradermal histamine injection, but

not of pruritus induced by mucunain, is blocked by systemically administered H1 antagonists (28). Others, such as vanilloid receptors, bind exogenous capsaicin, are heat-sensitive, and can be sensitized by protons (see

also Chapter 28). Only recently, a proteinase-activated receptor 2 (PAR2)—

which is activated by trypsin and related proteases, including mast cell

tryptase—has been localized to sensory nerve fibers (29). The activation of

these receptors leads to depolarization of nerve fibers and release of

neuropeptides.

C.



Sensory Neuropeptides



Skin nerves express a battery of biologically active peptides that are synthesized in the neural cell body of the dorsal root ganglions, subjected to

posttranslational modification to the active form, packed in storage granules,

and transferred to the nerve terminals in the skin. Finally, depolarization of

peripheral nociceptive nerve endings will release neuropeptides from the

neurosecretory granules (30).

In dermal nerve fibers, staining has been demonstrated for the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene–related

peptide (CGRP), vasoactive intestinal peptide (VIP), galanin (Gal), g-mela-



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nocyte-stimulating hormone (g-MSH), atrial natriuretic peptide (ANP),

peptide histidine methionine (PHM), neurotensin (NT), and dynorphin

(Dyn) (18,30,31). Epidermal nerves have shown immunoreactivity for SP,

NKA, CGRP, and, more variably, for neuropeptide Y (NPY), VIP, somatostatin (Som), and h-endorphin (11,14). Some of the immunohistochemical

findings are still contradictory, but the list of neuropeptides is ever-increasing.

A differential expression pattern has been described for sensory and

autonomic nerve fibers (Fig. 6; see color insert). Although SP, NKA, CGRP,

VIP, Som, and Gal have been demonstrated in sensory nerves, VIP, PHM,

and NPY seem to occur in autonomic nerves. More than one sensory

neuropeptide may be colocalized in a single nerve fiber whereas neuropeptides

in the autonomic system occur together with classical neurotransmitters.

However, many of these neuropeptides are not only expressed in skin nerves

but may be derived from keratinocytes, Merkel cells, endothelial cells,

fibroblasts, and immune cells (18).

Neuropeptides diffuse to specific receptors on blood vessels, skin glands,

epidermis, connective tissue cells, and immune cells where they mediate

biological responses. In contrast to neurotransmitters, reuptake is not the

major mechanism of inactivation. The action of neuropeptides is limited by



Figure 6 Expression of sensory neuropeptides within axons of different nerve fibers

(a–c) suggesting a variable codistribution of autonomic and sensory fibers. Positive

immunofluorescence staining for CGRP depicted in red pseudocolors. Confocal laser

scanning microscopy. (See color insert.)



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hydrolytic enzymes including tryptase, neutral endopeptidase, and angiotensin-converting enzyme (30).

D.



Neurogenic Inflammation and Neuroimmunology



The function of sensory nerves is not only to conduct nociceptive information

to the central nervous system for further processing; sensory fibers also have

the capacity to respond directly to noxious stimuli by initiating a local

inflammatory reaction. Noxious stimulation of polymodal C-fibers produces

action potentials that travel centrally to the spinal cord and, in a retrograde

fashion, along the ramifying network of axonal processes. The antidromic

impulses that start from the branching points cause secretion of neuropeptides stored along the peripheral nerves. A consequence of their effects

on vessels and resident inflammatory cells in close proximity is arteriolar

vasodilation (flare reaction) and increased permeability of the postcapillary

venules (whealing). Neurogenic inflammation can be elicited by antidromic

electrical stimulation of sensory nerves and administration of histamine or

various neuropeptides, and can be abolished by denervation, capsaicin, and

local anesthetics (32).

Overall, the nature of the flare and wheal reaction is far more complex

than previously thought. Apart from direct initiation of vasodilation, and

leakage of plasma and inflammatory cells, neuropeptides may exert their

effects via the activation of mast cells (33). Ultrastructural and more recent

immunohistochemical findings suggest a close proximity of mast cells to

neuropeptide-containing nerves (34,35). Hence, neuropeptide release from

nerves has been suggested to induce mast cell degranulation. However, even

potent mast cell-activating neuropeptides induce histamine release in concentrations that seem not to be present in vivo (see also Chapter 15). Other

experiments and stimulation of nerves in mast cell-deficient mice support the

notion that mast cells are not essential for neurogenic inflammation (32). The

observation of histamine-immunoreactive nerves in the skin of Sprague–

Dawley rats even suggests a more direct route of cutaneous histamine effects,

mediated exclusively by the peripheral nervous system (36).

Recent studies strongly suggest an interaction between the nervous

system and the immune system far beyond that described for the classical

model of axon flare reaction (37). The close anatomical association of

cutaneous nerves with inflammatory and immunocompetent cells and the

well-recognized immunomodulatory effect of many neuropeptides indicate

the existence of a neuroimmunological network. Nerves have been described

in the Peyer’s patches and the spleen. In the skin, lymphocytes are regularly

found in close proximity to small nerve fibers (Metze, personal observation).

This anatomical association is consistent with the capacity of SP to influence



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T-cell proliferation and homing (38,39). By the release of CGRP and

substance P, some cutaneous nerve fibers may activate polymorphonuclear

cells (40) and stimulate macrophages (41). Secretory neuropeptides further

stimulate endothelial cells to transport preformed adhesion molecules, such

as P-selectin and E-selectin, from intracellular Weibel–Palade bodies to the

endothelial surface and thereby enhance chemotactic functions (42). Moreover, substance P stimulates the production of proinflammatory as well as

immunomodulating cytokines and, conversely, cytokines such as interleukin

(IL)-1 enhance the production of SP in neurons (43,44).

Nerve fibers are also intimately associated with monocytoid cells,

including Langerhans cells. Immunohistochemical results strongly suggest

that intraepidermal nerve fibers are capable of depositing SP, CGRP, and

MSH at or near Langerhans cells. Via receptor binding, neuropeptides inhibit

the function of immunocompetent cells and induce tolerance to potent

contact allergens (17,45). In addition, subepidermal and epidermal nerve

fibers may recruit Langerhans cells and, vice versa, Langerhans cells induce

nerve differentiation via interleukin-6, nerve growth factor, and fibroblast

growth factor (FGF). These findings strongly support the concept of an

interaction between the immune system and the neuroendocrine system in the

skin.

The complex innervation of the skin with sensory nerve fibers and the

potential release of a variety of neuropeptides imply a participation of

neuroimmunological mechanisms in many skin diseases and related symptoms including itch.



E.



Itchy Skin Diseases



1.



Atopic Dermatitis



Pruritus is a cardinal symptom of atopic dermatitis; still, its mechanism and

association with the cutaneous nervous system have not been fully understood. Increased numbers of neurofilament-positive, CGRP-positive, and SPpositive nerve fibers were observed in the papillary dermis, at the dermal–

epidermal junction, in the epidermis, and around sweat glands (46–49).

Different densities of PGP 9.5-positive peripheral nerves were found in acute,

lichenified, and prurigo lesions in comparison to noninvolved skin of patients

with atopic dermatitis (50).

Electron microscopical investigation of lesional skin of atopic patients

revealed hyperplastic nerve fibers with enlarged axons (49,50) possibly due to

an increased release of nerve growth factor and NT-4 by basal keratinocytes

(51). In addition, axons focally lost their surrounding cytoplasm of Schwann

cells and thus communicated directly with dermal cells. These axons con-



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tained many mitochondria and neurofilaments, with abundant neurovesicles

confirming a high content of neuropeptides.

In general, an increase of sensory—but a decrease of adrenergic—

autonomic nerve fibers was observed, indicating a differential role of primary

afferent and autonomic nerve fibers for the pathophysiology of pruritus in

atopic dermatitis (47).

Apart from alterations in the neuropeptide profile of nerve fibers, their

receptors as well as neuropeptide-degrading enzymes may play a crucial role

in the pathophysiology of pruritus in atopic dermatitis. Because PAR-2

immunoreactivity is enhanced in atopic dermatitis patients (M. Steinhoff,

unpublished observation), it can be hypothesized that tryptase may activate

PAR-2 in inflammatory conditions when there is mast cell infiltration and

degranulation. Thus, PAR-2 may be involved in the pruritus of patients with

atopic dermatitis, explaining why atopic patients show a rather weak response

following treatment with antihistamines. Moreover, IL-2 and other cytokines,

as released from various cutaneous and immune cells during inflammation,

have been demonstrated to induce pruritus and activate neuropeptide release

from sensory nerves in the skin of patients with atopic dermatitis (52,53).

2.



Dry Skin (Xerosis)



Itch in the dry skin of older patients, or in atopic patients, is a common

symptom that may be attributed to a high density of nerve C-fibers within the

epidermis. Only recently, animal studies showed that dry skin induces the

expression of nerve growth factor in keratinocytes, leading to the elongation

and penetration of sensory nerve fibers into the epidermis (Takamori et al.,

International Workshop for Study of Itch, Singapore, 2001). In addition, dry

skin reflects an increased transepidermal water loss due to an incomplete

arrangement of intercellular lipid lamellae in the stratum corneum (54,55).

This impaired barrier function in the skin supports the entrance of irritants

and itchy agents (56). Additionally, pH changes within the skin can be

assumed to activate itch receptors (57).



F.



Prurigo Nodularis



Prurigo nodularis is a distressing condition characterized by intensely pruritic, lichenified, or excoriated papules and nodules first described by Hyde in

1909. It represents a cutaneous reaction pattern to repeated rubbing or

scratching caused by pruritus of a different origin, rather than a disease per

se (28). In fact, prurigo nodularis is common in patients with atopic dermatitis

and other itchy dermatoses such as scabies, dry skin, and bullous pemphigoid.

In addition, prurigo nodularis often signals systemic diseases.