C-Fiber Nociceptors

CMHs are commonly encountered cutaneous afferents, and activity of sufficient magnitude in these fibers is thought to evoke a burning pain sensation. The size of the receptive field appears to scale with the size of the animal. Typical values for monkey are between 15 and 20 mm² (LaMotte and Campbell 1978), and for human they are near 100 mm² (Schmidt et al 1997). There are often discrete areas of mechanical sensitivity (hot spots) within the receptive field, but in many fibers the areas of mechanical responsiveness tend to fuse over the region of the receptive field. Most CMHs respond to chemical stimuli (though not as well as A-fiber nociceptors; Davis et al 1993b) and can therefore be considered polymodal.

Responses to heat stimuli have been studied in considerable detail. The response of a typical CMH to a random sequence of heat stimuli ranging from 41–49°C is shown in Figure 1-1A. It can be seen that the response increases monotonically with stimulus intensity over this temperature range, which encompasses the pain threshold in humans. One ion channel involved in the transduction of heat at nerve terminals is thought to be the neuronal transient receptor potential ion channel V1 (TRPV1); activity in this channel increases with increasing temperature (Caterina et al 1997). A detailed description of the neuronal ion channels involved in stimulus transduction is presented in Chapter 2 (for review see Dubin and Papapoutian 2010). Signal transduction molecules on keratinocytes may also play a role in heat transduction by inducing the release of adenosine triphosphate (ATP), which activates purinergic receptors (P2X₃ and P2Y₂) on the free nerve endings (see Fig. 1-4).

Two types of heat response are observed following a stepped heat stimulus. Quick C (QC) fibers exhibit their peak discharge during the rising phase of the heat stimulus, whereas slow C (SC) fibers exhibit their peak discharge during the plateau phase (Fig. 1-2B). The heat thresholds (Fig. 1-2C) and mechanical thresholds of QC fibers are significantly lower than those of SC fibers, thus suggesting that they may be located more superficially in the epidermis. QC fibers respond more vigorously to pruritic stimuli than do SC fibers, which suggests that they may be important in itch sensations (Johanek et al 2008).

Thermal modeling studies combined with electrophysiological analysis have indicated that (1) the heat threshold of CMHs depends on the temperature at the depth of the receptor and not the rate of increase in temperature, (2) transduction of heat stimuli (conversion of heat energy to action potentials) occurs at different skin depths for different CMHs (Tillman et al 1995b), and (3) suprathreshold responses of CMHs vary directly with the rate of increase in temperature (Tillman et al 1995a, 1995b; Yarnitsky et al 1992). The depth of the heat-responsive terminals of CMHs varies quite widely (ranging from 20–570 μm; Tillman et al 1995b). When a stepped temperature stimulus is applied to the skin, the temperature increases in the subsurface levels more slowly because of thermal inertia. The disparity in the surface temperature and the temperature at the level of the receptor varies directly with depth and indirectly with time. Given that the depth of CMH terminals varies widely, true heat thresholds are obtained when the rate of increase in temperature is very gradual or when the duration of the stimulus is very long. Although the literature reflects a wide range of heat thresholds for CMHs, when tested with these types of heat stimuli, the heat threshold of the majority of CMHs is in a remarkably narrow range of 39–41°C (Tillman et al 1995b).

The response of CMHs is also strongly influenced by the stimulus history. Both fatigue and sensitization are observed. One example of fatigue is the observation that the response to the second of two identical heat stimuli is substantially less than the response to the first stimulus. This fatigue is dependent on the time between stimuli, with full recovery taking longer than 10 minutes. A similar reduction in the intensity of pain after repeated heat stimuli is observed in human subjects (LaMotte and Campbell 1978). Fatigue is also apparent in Figure 1-1A, where the response to a given stimulus varied inversely with the intensity of the preceding stimulus. A decrease in the response to heat is also observed following mechanical stimuli applied to the receptive field or electrical stimuli applied to the nerve trunk (Peng et al 2003). This suggests that fatigue in response to a given stimulus modality can be induced by heterologous stimulation, that is, by excitation with a stimulus of a different modality. Interestingly, recovery from cross-modal or heterologous fatigue is faster than recovery from fatigue induced by a stimulus of the same modality. Presumably, this is because these heterologous stimuli do not activate and therefore do not fatigue the stimulus transduction apparatus in the same way. Alternatively, fatigue may arise from independent effects on spike initiation (from antidromic stimulation) and transduction (from natural stimulation at the receptive field). Fatigue in response to heat stimuli is also seen in vitro when small (and presumably nociceptive) dorsal root ganglion (DRG) cells are repetitively tested with heat stimuli (Greffrath et al 2002). The enhanced response, or sensitization, that may occur in CMHs after tissue injury is described below in the section on hyperalgesia.

Responses to mechanical stimuli are covered in more detail later. Suffice it here to indicate that CMHs usually display a slowly adapting response to mechanical stimuli of a given force. As noted later, MSA CMHs have a graded response to punctate stimuli, but their stimulus–response functions become saturated at levels substantially below the threshold for pain.

C-fiber MIAs are heterogeneous with regard to responses to chemical and heat stimuli, and some respond only to mechanical stimuli (but of course with a very high mechanical threshold). The sensitivity to mechanical stimuli has no obvious correlation to the heat threshold (Davis et al 1993b). In contrast to CMH afferents, some C-fiber MIAs in humans are vigorously excited when challenged with histamine or capsaicin. In addition, the activity observed in these C-fiber MIAs parallels the duration of the perception of itch (histamine) or burning pain (capsaicin) (Schmelz et al 1997, 2000b). C-fiber MIAs may therefore act as chemosensors. In addition to pronounced chemosensitivity, these fibers have some other interesting properties that could account for pain in response to tonic pressure stimuli or the neurogenic flare response (see below).

Low-threshold C-fiber mechanoreceptors that do not respond to heat have been described in the cat (Bessou and Perl 1969) and rabbit (Shea and Perl 1985). In primates, including humans, these fibers have been found in proximal areas of the body (Kumazawa and Perl 1977, Nordin 1990) and the hairy skin on the forearm (Vallbo et al 1999). These afferents are strongly activated by innocuous mechanical stimuli moved slowly across the receptive field, but they also respond to pinprick stimuli. The neuronal activity in these fibers is not critical for the perception of touch and, according to one imaging study, leads to the activation of the insular but not the sensory cortex (Olausson et al 2003). Low-threshold C-fiber mechanoreceptors are thought to mediate the sensation of “pleasant” touch and may therefore play an important role in “affiliative” behavior (Vallbo et al 1999, Wessberg et al 2003, Löken et al 2009).

Some mechano-insensitive C fibers are reported to be activated by non-noxious and noxious cold and hot stimuli. It has been hypothesized that activity in these afferents may mediate the “hot–burning” sensations caused by such stimuli. These afferents may also be involved in mediating psychophysical phenomena such as “paradoxical heat” or the thermal grill illusion (Campero et al 2009).

C-fiber afferents differ not only in their receptive features but also in their conductive properties. In fact, their conductive and receptive properties appear to correlate. When unmyelinated C-fiber afferents are activated repetitively by electrical stimuli, their conduction latency increases gradually (i.e., the conduction velocity of the afferent decreases). In addition, with increasing stimulation frequency, the amount of this activity-dependent slowing increases. Slowing in C-fiber MIAs is greater than in C-fiber MSAs (Weidner et al 1999), and mechanosensitive nociceptive afferents show more pronounced slowing than do cold-sensitive C fibers, low-threshold C fibers, or sympathetic efferent C fibers (Gee et al 1996, Serra et al 1999, Obreja et al 2010, Ringkamp et al 2010). This difference in slowing properties indicates that the ion channels responsible for conduction may be different and suggests that the ion channels responsible for spike initiation at the receptive terminal may also differ between C-fiber classes.

A-Fiber Nociceptors

A-fiber nociceptors are thought to evoke pricking pain, sharpness, and perhaps aching pain. As a general rule, A-fiber nociceptors do what C-fiber nociceptors do, but do it more robustly. They respond at higher discharge frequencies, and the discriminable information supplied to the CNS is greater (e.g., Slugg et al 2000).

Two types of A-fiber nociceptors are apparent (Dubner et al 1977, Treede et al 1998). A summary of their properties is presented in Table 1-1. Type I fibers are typically responsive to heat, mechanical, and chemical stimuli and may therefore be referred to as AMHs or polymodal nociceptors. Because the heat thresholds are high with short-duration stimuli (typically >53°C), the responsiveness of these fibers to heat has in some studies been overlooked. Consequently, these fibers have been called high-threshold mechanoreceptors (HTMs) by many investigators (e.g., Burgess and Perl 1967). Heat sensitivity in type I fibers is most likely mediated by the vanilloid receptor–like protein 1 (VRL1, renamed TRPV2) since it has a similar high threshold for activation by heat and is expressed in neurons with small myelinated axons (Caterina et al 1999). When heat thresholds are determined with long-duration temperature stimuli, however, thresholds are in the mid-40–50°C range (Treede et al 1998). Type I AMHs are seen in hairy and glabrous skin (Campbell et al 1979) and have also been described in the cat and rabbit (Fitzgerald and Lynn 1977, Roberts and Elardo 1985). The mean conduction velocity of type I AMHs in the monkey is 25 m/sec and extends as high as 55 m/sec. Thus, by conduction velocity criteria, type I AMHs fall into a category between that of Aδ and Aβ fibers. Nearly all type I AMHs are MSAs. Their receptive field size is similar to that of CMHs, but the presence of “hot spots” in response to mechanical stimuli is much more obvious.

Type II A-fiber nociceptors were encountered only infrequently in early studies. It turns out that this is because the thresholds to mechanical stimuli place the majority of these fibers in the MIA category. Many have no demonstrable response to mechanical stimuli. When an unbiased electrical search stimulus is used, however, the prevalence of type I and type II A-fiber nociceptors in the hairy skin of the primate is similar. They do not occur in the glabrous skin of the hand (where type I AMHs are prevalent). Their mean conduction velocity, 15 m/sec, is also lower than that of type I AMHs. Their responses to heat resemble those observed in CMHs, and they may also be mediated by the vanilloid receptor 1 (VR1 or TRPV1). Responses to endogenous inflammatory/algesic mediators resemble those seen with type I A-fiber nociceptors (Davis et al 1993b).

Examples of the differing responses of the two types of A-fiber nociceptors to a heat stimulus are shown in Figure 1-3. Type I fibers exhibit a distinctive, gradually increasing response to heat. They sensitize to burn and chemical injury and probably play a role in the development of hyperalgesia. Type II fibers respond to heat in similar fashion to CMHs: early peak frequency and a slowly adapting response (Treede et al 1995). As noted later, type II A-fiber nociceptors are thought to signal first pain sensation in response to heat and may also contribute to pain caused by the application of capsaicin to the skin (Ringkamp et al 2001).

The conduction velocity of small myelinated Aδ fibers is, by definition, faster than that of unmyelinated C fibers. However, the terminal cutaneous branches of nociceptive Aδ fibers may conduct at a velocity characteristic of unmyelinated fibers (i.e., <2 m/sec) (Peng et al 1999). In addition, these unmyelinated terminals may branch off the main axon several centimeters proximal to their cutaneous receptive field.

Nociceptors Can Be Classified by Molecular Markers

The anatomical and biochemical features of nociceptive afferents have been studied extensively to correlate these features with their receptive properties. A wide range of cell markers have been used to classify nociceptive afferents and to study their peripheral and central projections. These markers include molecules expressed on the cell surface (e.g., receptors, glycoconjugates), molecules stored and released from nociceptive afferents (e.g., peptides), and enzymes. Expression of receptors for neurotrophic factors is of interest since these factors may regulate the sensitivity of nociceptive afferents in physiological and pathological states such as inflammation and neuropathy. The size of neuronal populations expressing or co-expressing different markers varies between species (Zwick et al 2002) and changes with the developmental stage (Molliver et al 1997, Guo et al 2001). Inflammation of the innervated tissue or a peripheral nerve lesion can cause substantial changes in the expression of these molecules. With the ongoing discovery of new marker molecules and the refinement of histological techniques, classification of nociceptive afferents is undergoing constant change and revision. Despite these “challenges,” however, classification of nociceptive afferents based on the expression of biochemical markers is instructive inasmuch as certain different neuronal populations are distinguishable across species. Sophisticated genetic manipulations have allowed the peripheral and central projections of defined neuronal populations to be studied in great detail. In addition, ablation experiments have been used to study the role of defined neuronal populations in animal behavior.

The cell bodies of nociceptive somatic and visceral afferents are located in DRGs. Slowly conducting Aδ and C fibers, including nociceptors, have small cell bodies (Lawson and Waddell 1991). Some of these are labeled with an antibody directed against a neurofilament protein (NF200) and are therefore thought to correspond to the somata of small myelinated Aδ afferents.

Small DRG cells are subdivided into peptidergic neurons (i.e., neurons containing peptides such as substance P [SP], calcitonin gene–related peptide [CGRP], and somatostatin [SST]) and “non-peptidergic” neurons. In the rat, about 40% of DRG cells, 50% of C fibers, and 20% of Aδ fibers are classified as peptidergic (McCarthy and Lawson 1989, Lawson et al 1996). Non-peptidergic, nociceptive neurons contain fluoride-resistant acid phosphatase (FRAP) (Silverman and Kruger 1988a), and their somata and axons bind the plant isolectin B4 (IB4) from Griffonia simplicifolia (Silverman and Kruger 1988b). It is common practice to classify neurons as “peptidergic” or “non-peptidergic” based on their binding of IB4. However, considerable co-localization of SP or CGRP and IB4 or FRAP has been reported in rats but less so in mice (Carr et al 1990, Wang et al 1994, Bergman et al 1999, Price and Flores 2007). In vivo intracellular recordings combined with immunohistochemistry have shown that cells containing SP or CGRP or cells binding IB4 are nociceptive and that non-nociceptive cells do not label with these markers (Lawson et al 1997, 2002; Gerke and Plenderleith 2001).

A group of mas-related genes (Mrgs) have been discovered that are selectively expressed in small DRG neurons and encode G protein–coupled receptors (GPCRs) (Dong et al 2001). Independently, sensory neuron–specific GPCRs (so-called sensory neuron–specific receptors [SNSRs]) in which the encoding genes were identical to some of the previously described Mrgs were identified shortly thereafter (Lembo et al 2002). For some Mrgs (MrgA–C) identified in mice, no ortholog genes exist in human or non-human primates, but closely related Mrgs (so-called MrgXs) have been identified. For other Mrgs (MrgD–G), however, ortholog genes exist in humans. Mrgs are expressed mainly in non-peptidergic, IB4-positive neurons, with some Mrgs being expressed in distinct IB4 subpopulations. In in vitro recordings, MrgD⁺ DRG cells showed characteristics typical of nociceptors (e.g., broad action potentials, expression of tetrodotoxin [TTX]-resistant sodium channels) (Drussor et al 2008). Receptors encoded by Mrgs respond to a variety of ligands, including β-alanine, cortistatin, peptides derived from different opioid precursors, and different RFamide peptides (Dong et al 2001, Han et al 2002, Lembo et al 2002, Robas et al 2003, Shinohara et al 2004), and they probably modulate excitability and sensitivity in this class of nociceptive afferents.

Expression of some markers appears to be related to the peripheral target tissue innervated by the neuron. Thus, almost all visceral afferents are peptidergic, but only about half the afferents projecting to the skin are (e.g., Perry and Lawson 1998) and only a small percentage of afferents projecting to muscle are labeled with IB4 (Plenderleith and Snow 1993, Ambalavanar et al 2003). MrgD-positive fibers exclusively innervate the skin, and they terminate in more superficial skin layers than do their peptidergic counterparts (Fig. 1-4) (Zylka et al 2005). Peptidergic and non-peptidergic afferents project to distinct dorsal horn laminae, with peptidergic fibers projecting mainly to lamina I and lamina II outer and IB4-binding afferents projecting preferentially to lamina II inner (e.g., Hunt and Rossi 1985, Silverman and Kruger 1988b; but see also Woodbury et al 2000).

Although all nociceptive neurons depend on nerve growth factor (NGF) during early development, in the adult only peptidergic neurons express its receptor TrkA (tropomyosin-related kinase A) (Averill et al 1995). In contrast, most IB4-positive DRG cells do not express TrkA (Molliver et al 1995, but see also Kashiba et al 2001) but express one of the glial-derived neurotrophic factor (GDNF) family receptors (GDNFRα1–4) together with receptor tyrosine kinase Ret (Bennett et al 1998, Orozco et al 2001).

Peptidergic and non-peptidergic neurons express different receptors involved in signal transduction, and they may therefore display different sensitivity to a given stimulus. Thus the P2X₃ receptor, which mediates nociceptor excitation by ATP, is primarily expressed in IB4-positive neurons (Vulchanova et al 1998). In contrast, TRPV1, which mediates responses to heat, capsaicin, and protons, is expressed in only a minority of IB4-positive cells in mice (Zwick et al 2002). In rats, however, this segregation is less obvious since about half of both IB4-positive and -negative cells express TRPV1 (Caterina et al 1997; Michael and Priestley 1999; Guo et al 1999, 2001). Species differences also exist in the co-expression of different Mrgs and their co-expression with other markers of nociceptive neurons (Zylka et al 2003).

Coupling between C-Fiber Nociceptors

Activation of one fiber by action potential activity in another is referred to as coupling. Coupling of action potential activity occurs between C fibers in the normal peripheral nerve of the monkey (Meyer et al 1985a). Coupling frequently involves conventional CMHs. Coupling is eliminated by injecting small amounts of local anesthetic at the receptive field of the CMH, thus indicating that the site of coupling is near the receptor. Collision studies indicate that the coupling is bidirectional. Sympathetic fibers do not appear to be involved in this coupling as demonstrated by experiments in which the sympathetic chain is stimulated or ablated (Meyer and Campbell 1987). The role of coupling is unknown but it may relate to the flare response or other efferent functions of nociceptors (see below). Coupling between peripheral nerve fibers is also one of the pathological changes associated with nerve injury (e.g., Blumberg and Jänig 1982, Meyer et al 1985b). In this case, coupling occurs at the site of axotomy.

Anatomical Studies of Cutaneous Nociceptors

Immunostaining for protein gene product (PGP) 9.5, a carboxy-terminal ubiquitin hydrolase, has proved particularly sensitive in identifying small-diameter afferents in the skin (Hsieh et al 1996). Vertical sections reveal that epidermal axons emerge from the superficial dermal nerve plexuses running beneath the epidermis. Schwann cells encase the axons at the dermal level, but as the axons rise into the epidermis between keratinocytes, the Schwann cell encasements are lost (Kruger et al 1981). Both clear round and large dense-core vesicles are noted at the epidermal penetration site. The vesicles are similar morphologically to vesicles present in other cells involved in hormone and neurotransmitter secretion. It is presumed that these vesicles secrete their contents into tissues on activation (see the efferent role of nociceptors below). Some of these fibers appear to innervate Langerhans cells. In small-fiber neuropathies in which patients have pain and deficits in cutaneous pain sensibility, these axonal terminals stained by PGP 9.5 are markedly decreased (Holland et al 1998).

As illustrated in Figure 1-4, free nerve endings can be traced far into the epidermal layer. These free nerve endings are probably sensory and serve the sensations of pain, temperature, and itch. The parent axons of these unmyelinated terminals are probably both myelinated and unmyelinated. Some of these free nerve endings are peptidergic and contain SP or CGRP (Gibbons et al 1987). Others are non-peptidergic and reach into the superficial layers of the epidermis.

Nociceptors and Pain in Response to Heat Stimuli

Nociceptors and Pain in Response to Controlled Mechanical Stimuli

Nociceptors and Cold Pain Sensation

Cold pain differs from heat pain in a number of important factors: (1) the cold pain threshold (≈14°C on hairy skin; Harrison and Davis 1999) is much farther from resting skin temperature (33°C) than the heat pain threshold (about 45°C), (2) the slope of the stimulus–response function is much steeper for heat pain than for cold pain (Morin and Bushnell 1998), and (3) the lag in response between stimulus onset and pain report suggests that cold pain is subserved by deeper receptors whereas heat pain seems to be subserved by superficial receptors. Klement and Arndt (1992) demonstrated that cold pain could be evoked by cold stimuli applied within the veins of human subjects. A local anesthetic applied within the vein, but not in the overlying skin, abolished cold pain sensibility. It is therefore possible that cold pain is served, at least in part, by vascular receptors.

Just as the sensation of warmth is served by a specific set of primary afferents (predominantly C fibers), the sense of cooling is served by a specific set of primary afferents (i.e., cold fibers). Cold fibers are predominantly of the A type. They exhibit ongoing activity at room temperature, and their response increases markedly with gentle cooling. Stimuli that induce cold pain are not encoded well by these cold fibers. Although the majority of nociceptors have some response to ice stimuli applied to the skin, Simone and Kajander (1997) showed that all A-fiber nociceptors respond to cold stimuli below 0°C. C-fiber nociceptors may play a role in signaling cold pain sensation as well (LaMotte and Thalhammer 1982). A non-selective cation channel has been identified (called ANKTM1 or transient receptor potential ankyrin 1 [TRPA1]) that has an activation threshold (17.5°C) comparable to the cold pain threshold (Story et al 2003). This channel is found in a subset of nociceptive sensory neurons that are responsive to intense heat and capsaicin. However, the role of TRPA1 in mediating noxious cold is still debated.

Nociceptors and Chemically Evoked Sensations

Many chemical agents produce pain when applied to the skin. In many cases the pain from these agents probably results from tissue injury and is therefore indirect. (Chemical mediators associated with inflammation are described later.) One exception that has received a lot of attention is capsaicin. Intradermal injection of capsaicin produces intense burning pain that lasts for several minutes. When capsaicin is injected into the receptive field of C-fiber MSAs, the response is weak (relative to the heat response) and of short duration (Baumann et al 1991). In contrast, A-fiber and C-fiber MIAs exhibit a long-lasting, vigorous response to capsaicin (Schmelz et al 2000b, Ringkamp et al 2001), thus suggesting that these fibers are responsible for the pain induced by capsaicin. The pungent effects of capsaicin appear to be mediated by the TRPV1 receptor expressed on nociceptive fibers. This receptor appears to be activated by heat and protons (acid) as well.

Another chemical of interest is histamine, which produces a long-lasting itch when applied to the skin. Injection of histamine into the receptive field of C-fiber MSAs leads to a lasting response (Johanek et al 2008). Iontophoresis of histamine into the receptive field of a subpopulation of C-fiber MIAs also produces a vigorous, long-lasting response (Schmelz et al 1997), which suggests that both CMHs and C-fiber MIAs may play a role in histamine-induced itch. Histamine probably activates nociceptors via the H₁ receptor located on peripheral terminals.

Because cowhage spicules produce an intense itch that is not blocked by topical antihistamines (Johanek et al 2008), and they provide a useful tool to investigate the chronic itch in patients that is resistant to antihistamine treatment. In about half of normal subjects, cowhage-induced itch is greatly attenuated during selective blockade of myelinated fibers. Although C-fiber MIAs do not respond to cowhage, QC fibers and A-fiber nociceptors respond vigorously to cowhage (Ringkamp et al 2011). The active ingredient in cowhage is the cysteine protease mucunain, which activates nociceptive terminals via protease-activated receptor 2 (PAR-2) and PAR-4 (Reddy et al 2008).

Hyperalgesia to Heat Stimuli

We first consider the situation in which a burn injury is applied to the skin and the test stimulus is heat applied to the location of the burn injury. When a burn is applied to the glabrous skin of the hand, marked hyperalgesia to heat develops as shown in Figure 1-9A (Meyer and Campbell 1981a). The hyperalgesia is manifested as a leftward shift of the stimulus–response function that relates the magnitude of pain to stimulus intensity. For example, the 41°C stimulus was not painful before the burn but after the injury was as painful as the 49°C stimulus before the injury.

Peripheral Sensitization as a Mechanism for Primary Hyperalgesia to Heat Stimuli

Substantial evidence favors the concept that the primary hyperalgesia to heat stimuli that develops at the site of a burn injury is mediated by sensitization of nociceptors (Meyer and Campbell 1981a, LaMotte et al 1982). Sensitization is defined as a leftward shift of the stimulus–response function that relates the magnitude of the neural response to stimulus intensity. Sensitization is characterized by a decrease in threshold, an augmented response to suprathreshold stimuli, and ongoing spontaneous activity. These properties correspond to the properties of hyperalgesia (Table 1-2).

To explain the hyperalgesia that occurs with a burn on the glabrous skin of the hand, a correlative analysis of subjective ratings of pain in humans with responses of nociceptors (CMHs and type I AMHs) in anesthetized monkeys was performed (Meyer and Campbell 1981a). Test heat stimuli were applied to the glabrous skin of the hand before and after a 53°C, 30-second burn. The burn led to prominent hyperalgesia in the human subjects (Fig. 1-9A). The CMHs showed a decreased response following the burn (Fig. 1-9C), whereas the type I AMHs were markedly sensitized (Fig. 1-9B). Thus, it is likely that for thermal injuries on the glabrous skin of the hand, AMHs, not CMHs, code for the heat hyperalgesia.

Sensitization is not a uniform property of nociceptors. Tissue type and the nature of the injury are important variables. For example, CMHs that innervate hairy skin become sensitized, whereas as described above, CMHs that innervate the glabrous skin of the hand do not become sensitized to a burn injury (Campbell and Meyer 1983). Thus, CMHs appear to play a role in accounting for hyperalgesia to heat stimuli on hairy skin (LaMotte et al 1983). These data support the conclusion that the hyperalgesia to heat stimuli that occurs at the site of an injury is due to sensitization of primary afferent nociceptors.

Hyperalgesia to Mechanical Stimuli

Distinguishing hyperalgesia to mechanical stimuli in the primary and secondary zones may be incorrect in some respects since the mechanism for hyperalgesia in the two zones may have some common elements. The mechanisms discussed in this section, however, will be limited to those applicable to the primary zone.

Different forms of mechanical hyperalgesia have been characterized. One form is evident when the skin is gently stroked with a cotton swab and is referred to as “stroking hyperalgesia,” “dynamic hyperalgesia,” or “allodynia.” The second form of hyperalgesia is evident when punctate stimuli, such as von Frey probes, are applied and, accordingly, has been termed “punctate hyperalgesia.” Hyperalgesia to tonic stimulation with a blunt probe, called “pressure hyperalgesia,” and impact hyperalgesia to shooting small bullets against the skin at a controlled velocity have also been described in the primary hyperalgesic zone (Kilo et al 1994). As discussed in the later section on secondary hyperalgesia, the mechanism for these different forms of mechanical hyperalgesia is probably different. Stroking hyperalgesia is thought to be signaled by low-threshold mechanoreceptors, whereas punctate hyperalgesia is mediated at least in part by nociceptors. Pressure hyperalgesia and impact hyperalgesia are probably mediated by sensitized C fibers. Another form of mechanical hyperalgesia termed “progressive tactile hypersensitivity,” which may contribute to the allodynia associated with inflammation, has been described (Ma and Woolf 1997).

Nociceptor Sensitization as a Mechanism for Mechanical Hyperalgesia in the Primary Zone

Primary hyperalgesia to mechanical stimuli appears to be due, at least in part, to sensitization of primary afferent nociceptors to mechanical stimuli. This sensitization is manifested in several ways.

Loss of Central Inhibition as a Mechanism of Mechanical Hyperalgesia in the Primary Zone

Under usual circumstances, production of pain from activation of nociceptors with mechanical stimuli is inhibited in the CNS by the concurrent activation of low-threshold mechanoreceptors (e.g., Bini et al 1984). There is evidence that injury decreases the responsiveness of low-threshold mechanoreceptors. Hyperalgesia to mechanical stimuli in the primary zone could therefore be due to injury to low-threshold mechanoreceptors, which would lead to central disinhibition of nociceptor input and thus result in enhanced pain (i.e., hyperalgesia).

Arachidonic Acid Metabolites

The prostaglandins, thromboxanes, and leukotrienes are a large family of arachidonic acid metabolites collectively known as eicosanoids. The eicosanoids are generally considered to not activate nociceptors directly but rather enhance the sensation of pain in response to natural stimuli and other endogenous chemicals by increasing the frequency of action potential firing (for reviews see Schaible et al 2002, Cunha and Ferreira 2003, Momin and McNaughton 2009). A sensitizing and direct excitatory effect of PGE₂ and PGI₂, however, has been demonstrated in afferents innervating joints. Prostaglandins are synthesized by the constitutive enzyme cyclooxygenase-1 (Cox-1) and by Cox-2, an enzyme induced in peripheral tissues by inflammation (Ballou et al 2000). Several prostaglandins, PGI₂, PGE₁, PGE₂, and PGD₂, are considered to play a role in inflammatory pain and hyperalgesia. Prostaglandins reduce the threshold for initiation of action potentials and increase the excitability of sensory neurons by decreasing the threshold for activation of a nociceptor-specific voltage-activated Na current, Na_v1.8, and increasing intracellular cyclic adenosine monophosphate (cAMP) levels (England et al 1996, Gold et al 1996). The prostaglandin-induced increase in firing frequency may also result from an increase in the hyperpolarization-activated current (Ih), which leads to faster depolarization toward the action potential threshold, the consequence of which is a decrease in the time interval between successive action potentials (Momin and McNaughton 2009). Of the leukotrienes (metabolites of the lipoxygenase pathway), LTD₄ and LTB₄ have been suggested to play a role in hyperalgesia (Levine et al 1984) and in sensitization to mechanical stimuli (Martin et al 1987).

Bradykinin

Several lines of evidence suggest that bradykinin may also play a critical role in inflammatory pain and hyperalgesia (see Couture et al 2001, Meini and Maggi 2008 for reviews). Bradykinin is released on tissue injury (e.g., from plasma), is present in inflammatory exudates, and excites and sensitizes unmyelinated and myelinated nociceptors to natural stimuli (Beck and Handwerker 1974, Khan et al 1992). Administration of exogenous bradykinin produces pain and transient hyperalgesia to heat in humans (Manning et al 1991). Bradykinin acts on B₁ and B₂ receptors to induce nociceptor sensitization by activation of phospholipase C (PLC) and protein kinase C (PKC), production of arachidonic acids, and modulation of the TRPV1 channel (see the section on the vanilloid receptor below) (Reeh and Sauer 1997, Banik et al 2001).

Protons

The low pH levels found in inflamed tissue have led to the hypothesis that local acidosis may contribute to the pain and hyperalgesia associated with inflammation. Continuous administration of low-pH solutions in humans causes pain and hyperalgesia to mechanical stimuli (Steen and Reeh 1993). This correlates with the observation that protons selectively activate nociceptors and produce sensitization of nociceptors to mechanical stimuli. Excitation of nociceptors by protons does not undergo tachyphylaxis or adaptation, and a synergistic excitatory effect of protons and a combination of inflammatory mediators has been reported (Steen et al 1996).

A class of acid-sensing ion channels (ASICs), a subgroup of the degenerin/epithelial sodium channel (DEG/ENaC) family of proteins, has emerged as sensors of low pH (see Holzer 2009, Sluka et al 2009 for review). ASICs signal moderate decreases in extracellular pH, in contrast to TRPV1, which is activated by severe acidosis (pH values below 6). ASIC1A and ASIC3 have been identified in DRG neurons, and their expression is increased by inflammation, nerve injury, and bone cancer, thus suggesting that ASICs may play a role in mediating or modulating pain in these conditions. The observation that a non-selective ASIC inhibitor, amiloride, reduces cutaneous acid-evoked pain in humans suggests that ASICs may be a potential therapeutic target for inflammatory pain (Ugawa et al 2002).

Serotonin

Mast cells, on degranulation, release platelet-activating factor, which in turn leads to the release of serotonin (5-hydroxytryptamine [5-HT]) from platelets. Serotonin causes pain when applied to a human blister base (Richardson and Engel 1986) and can activate nociceptors (Lang et al 1990). Serotonin can also potentiate the pain induced by bradykinin and enhance the response of nociceptors to bradykinin. Additional evidence for a role of 5-HT in nociception stems from observations that 40% of lumbar DRG neurons, mostly small to medium-sized cells, are immunoreactive for the 5-HT_2A receptor and many of these cells also express the TRPV1 receptor (Van Steenwinckel et al 2009).

Histamine

Release of SP from nociceptor terminals can cause the release of histamine from mast cells. Histamine can lead to a variety of responses, including vasodilatation and edema. The role of histamine in pain sensation is less clear since application of exogenous histamine to the skin produces itch and not pain sensations (Simone et al 1991a). Histamine excites polymodal visceral nociceptors, especially when applied in high concentrations (Koda et al 1996), and potentiates the responses of nociceptors to bradykinin and heat (Mizumura et al 1995). Mechanosensitive cutaneous nociceptors in rats and humans respond only weakly to histamine (Lang et al 1990), but a subpopulation of mechano-insensitive C fibers was vigorously excited by histamine (Schmelz et al 1997). Activation of histamine H₃ receptors, a ligand-gated ion channel that modulates the influx of Na⁺, however, leads to decreased release of inflammatory peptides and reduced pain and inflammation (Cannon et al 2007).

Purines

During inflammation and tissue injury, purines such as adenosine and its mono- or polyphosphate derivatives (AMP, ADP, ATP) may be released or leak into the extracellular space and activate nociceptors (for review see Burnstock 2009). Platelets are a rich source of ATP, and aggregation of platelets or lysis of cells can lead to release of ATP. Adenosine and its phosphates have been reported to induce pain in a human blister base. Intra-arterial or intradermal injection of adenosine also causes pain, and intravenous/intracoronary infusion of adenosine induces angina-like symptoms (Sylvén et al 1986). In animals, adenosine enhances the response to formalin, presumably via the A₂ receptor. Animals lacking the adenosine A_2a receptor are hypoalgesic to heat stimuli (Ledent et al 1997).

A number of lines of evidence support the potential role of ATP as a peripheral mediator of pain. ATP is found at increased levels at sites of inflammation and can activate nociceptors. Psychophysical studies in humans indicate that iontophoresis of ATP into normal skin results in dose-related pain. ATP-induced pain is dependent on capsaicin-sensitive neurons; repeated topical application of capsaicin reduces the ATP-induced pain to 25% of normal. In addition, the ATP-induced pain is increased two- to three-fold when iontophoresed into skin made hyperalgesic by acute capsaicin treatment or by ultraviolet inflammation. Thus, in inflammatory conditions ATP may activate nociceptors and serve as an endogenous mediator of pain (Hamilton et al 2000). In human microneurographic studies, injection of ATP activated 60% of mechano-responsive and mechano-insensitive C-nociceptive fibers without sensitizing these fibers to mechanical or heat stimuli (Hilliges et al 2002).

Receptors for ATP have been found on primary sensory neurons both in the DRG and in the periphery. Multiple purinergic (P2) receptors have been suggested to be involved in pain signaling and modulation. ATP presumably activates nociceptive neurons in normal skin via the P2X₃ receptor and the heteromeric P2X₂/P2X₃ receptor (Chen et al 1995, Lewis et al 1995, Cook et al 1997). Messenger RNA for most of the P2X receptors (1–6) has been found in DRG neurons. In particular, both mRNA for the P2X₃ receptor and the receptor protein itself have been found in small-diameter neurons in the DRG. Local intradermal injection of agents activating P2X receptors results in dose-related pain behavior in rodents that is mediated by capsaicin-sensitive neurons (Bland-Ward and Humphrey 1997) and enhanced pain behavior in response to formalin (Sawynok and Reid, 1997). The proportion of C-fiber nociceptors responding and the magnitude of their response are increased by P2X agonists in inflamed skin. Activation of homomeric P2X₃ receptors is thought to contribute to acute nociception and inflammatory pain, whereas activation of heteromeric P2X_2/3 receptors appears to modulate the longer-lasting nociceptive sensitivity associated with nerve injury or chronic inflammation (Burnstock 2009).

Recently, it has been suggested that peripheral adenosine receptors may also be involved in the modulation of inflammatory pain. A₁ adenosine receptors are expressed in DRG cells, and peripheral activation of these receptors results in a reduction in inflammatory hyperalgesia via interactions with the nitric oxide/cyclic guanosine monophosphate/protein kinase G intracellular signaling pathways (Lima et al 2010).

Cytokines

During inflammation, cytokines (e.g., interleukin-1β [IL-1β], tumor necrosis factor α [TNF-α], IL-6) are released by a variety of cells (e.g., macrophages, Schwann cells) and regulate the inflammatory response (see Miller et al 2009, Schaible 2010). Clinical studies have shown that TNF-α levels in synovial fluid are increased in painful joints (Shafer et al 1994). Treatment with antibodies against TNF-α has been reported to improve the symptoms accompanying rheumatoid arthritis, including pain (Elliott et al 1994). Studies in animals have demonstrated mechanical and thermal hyperalgesia after systemic or local injection of IL-1, IL-6, and TNF-α. Additionally, treatment with antiserum against TNF-α is able to inhibit or delay the onset of hyperalgesia in experimental models of inflammation (Woolf et al 1997).

Cytokines may excite nociceptors either by rapid alterations in the properties of ion channels expressed in sensory neurons; indirectly by stimulating the release of other mediators such as prostaglandins, neurotrophins, and ATP; and by longer-term changes resulting from new gene transcription. Direct excitation and sensitization of nociceptive afferent fibers to thermal and mechanical stimuli have been shown for IL-1β and TNF-α (Fukuoka et al 1994). When applied along a peripheral nerve, TNF-α induces ectopic activity in nociceptive afferent fibers (Sorkin et al 1997).

IL-6 in combination with its soluble IL-6 receptor can sensitize nociceptors to heat as evidenced by increased heat-evoked intradermal release of CGRP (Obreja et al 2002). Other cytokines, IL-1β and TNF-α, also produce transient sensitization of heat-evoked release of CGRP from nociceptors in rat skin (Oprée and Kress 2000). IL-6–deficient mice show reduced mechanical and thermal hyperalgesia following inflammation (Xu et al 1997). These studies provide evidence for a role of cytokines in inflammation-associated hyperalgesia. The sensitization of nociceptors by cytokines may be mediated by p38-induced phosphorylation of TTX-resistant sodium channels, as well as by up-regulation of TRPV1 expression and function (Jin and Gereau 2006; for review see Ma and Quirion 2007).

Excitatory Amino Acids

A number of excitatory amino acids (EAAs) and peptide receptors are present at post-synaptic sites in the dorsal horn. These receptors have been found on DRG cells and the presynaptic terminals of primary afferents and are considered to play a role in the modulation of nociceptive impulses (see Carlton 2001, Goudet et al 2009). The most studied EAA, glutamate, can act either through ligand-gated ion channels (ionotropic glutamate receptors [iGluRs]) or through G protein–coupled metabotropic receptors (mGluRs). Based on sequence homology and physiological and pharmacological properties, the mGluRs have been further divided into three groups—group I (mGluR 1 and 5), group II (mgluR 2 and 3), and group III (mGluR 4, 6, 7, and 8). iGluR, mGluR1, and mGluR5 receptors have been identified on unmyelinated axons in the skin (Bhave et al 2001, Zhou et al 2001). About 40% of lumbar DRG cells contain mGluR2/3 immunoreactivity, and a majority of these cells are IB4⁺ small cells.

Several lines of evidence indicate a role of peripheral mGluRs in nociception and inflammatory pain. Peripheral application of glutamate activates nociceptors, and peripheral administration of ligands binding to glutamate receptors induces pain behavior in animals. Involvement of peripheral iGluR, mGluR1, and mGluR5 in formalin-induced pain behavior and glutamate-induced thermal hyperalgesia has been demonstrated (Davidson et al 1997). Intraplantar, but not intrathecal or intracerebroventricular administration of an mGluR5 antagonist reduced inflammatory hyperalgesia. Neurons in the DRG can be double-labeled with antisera for mGluR5 and VR1, thus suggesting that mGluR5 is expressed on the peripheral terminals of nociceptive neurons and contributes to inflammatory hyperalgesia (Walker et al 2001). In particular, mGluR1 activates PLC, which leads to release of Ca²⁺ from intracellular stores and activation of PKC.

Endogenous sources of glutamate in the periphery include plasma, macrophages, epithelial and dendritic cells in the epidermis and dermis, and Schwann cells. In addition, peripheral processes of the primary afferents contain glutamate, and nociceptor stimulation can cause peripheral release of glutamate from the terminals of these afferents.

Peripheral mGluRs are also considered to have antinociceptive effects. Peripheral administration of group II mGluR agonists blocks PGE₂-induced thermal hyperalgesia, and activation of these receptors results in depression of the responses of nociceptors sensitized by exposure to formalin or inflammatory soup (Yang and Gereau 2002, Du 2008). These observations suggest that selective group II agonists may be a therapeutic target for inflammatory pain states.

Nerve Growth Factor

NGF may contribute to inflammatory pain via direct and indirect mechanisms (for review see Pezet and McMahon 2006, Watson et al 2008). Pro-inflammatory cytokines stimulate the release of NGF from various sources, including fibroblasts, keratinocytes, Schwann cells, and inflammatory cells (lymphocytes, macrophages, and mast cells). NGF stimulates mast cells to release histamine and serotonin. NGF can also induce heat hyperalgesia by acting directly on the peripheral terminals of primary afferent fibers (Chuang et al 2001). Transgenic animals modified to overexpress NGF show hyperalgesic pain behavior (Davis et al 1993a). NGF sensitizes nociceptors and may alter the distribution of Aδ fibers such that a greater proportion of fibers have nociceptor properties (Stucky et al 1999). NGF has been implicated in the inflammation-induced changes in nociceptor response properties, such as an increase in the incidence of ongoing activity, increase in the maximum fiber following frequency, and changes in the configuration of the action potential of DRG neurons (Djourhi et al 2001). The inflammation-induced changes in nociceptive neurons are prevented by sequestration of NGF (Koltzenburg et al 1999). Cultured DRG neurons from inflamed animals exhibit spontaneous activity, and cultured DRG neurons from non-inflamed animals exhibit spontaneous activity when cultivated for 1 day with NGF (Kasai and Mizumura 2001). These studies suggest that in inflamed rats NGF may play a role in inducing spontaneous activity in DRG neurons.

NGF modulates the activity of ligand- and voltage-gated ion channels involved in nociception, such as TRPV1, P2X₃, ASIC3, and Na_v1.8. NGF potentiates responses of the TRPV1 receptor (see the section on the vanilloid receptors), and NGF-induced hyperalgesia is absent in TRPV1 knockout mice (Chuang et al 2001). NGF-induced hyperalgesia may be mediated via its actions on the TTX-resistant sodium channel Na_v1.8. NGF-induced thermal hyperalgesia failed to develop in mice with a mutation in the Na_v1.8 gene (Kerr at al 2001). Binding of NGF to TrkA stimulates the mitogen-activated protein kinase (MAPK), phosphatidyl-3′-kinase (PI3K), and PLC-γ intracellular signal transduction pathways (for details see Cheng and Ji 2008). Potential clinical therapeutic approaches being explored include humanized monoclonal antibodies to NGF or its tyrosine kinase receptor TrkA and sequestration of NGF via soluble receptor protein that binds NGF.

Other Receptors

A number of other receptor systems have been reported to play a role in the peripheral modulation of nociceptor responsiveness.

Peripheral Modulators of Nociceptor Activity

GPCRs, present on the plasma membrane and terminals of nociceptive neurons, play an important role in the modulation of pain signaling. GPCRs involved in antinociceptive mechanisms include opioid, cannabinoid, SST, muscarinic acetylcholine, γ-aminobutyric acid (GABA_B), mGlu, and α₂-adrenergic receptors (Fig. 1-13, for review see Pan et al 2008). Most GPCR agonists that have antinociceptive action are coupled to G_i/o proteins, which modulate voltage-gated Ca²⁺ channels and result in a decrease in presynaptic Ca²⁺ entry and inhibition of neurotransmitter release. GPCRs also modulate an inwardly rectifying K⁺ channel, the GIRK channel, which plays a critical role in maintaining resting membrane potential and excitability.

The GPCRs on peripheral nociceptors are attractive potential therapeutic targets for the development of new drugs that may have some benefit, in contrast to the more traditional analgesics, which work at the level of the CNS. Drugs acting at the periphery and inhibiting the generation and signaling of nociceptive input toward the spinal cord and the brain may prevent central plastic changes such as wind-up and central sensitization. In addition, these drugs may provide analgesia without the undesirable adverse effects, such as sedation, dizziness, and cognitive dysfunction, associated with drugs acting on the CNS system (see Stein et al 2009).

Secondary Hyperalgesia to Mechanical but Not Heat Stimuli

Primary hyperalgesia is characterized by the presence of enhanced pain in response to heat and mechanical stimuli, whereas secondary hyperalgesia is characterized by enhanced pain in response to only mechanical stimuli (e.g., Ali et al 1996). In one study in which the sensory changes that occur in the zones of primary and secondary hyperalgesia were compared (Raja et al 1984), burn injuries were induced in two locations on the glabrous skin of the hand in human subjects (Fig. 1-14). Within minutes of the injury, lightly touching the skin at the site of the two burns, as well as in a large area surrounding the burns, caused pain. The decrease in the pain threshold to von Frey hairs in the primary (injured) zone was similar to that in the area of secondary hyperalgesia (Fig. 1-14B). Marked hyperalgesia to heat was observed in the area of primary hyperalgesia (site A, the injury site, Fig. 1-14C). In the uninjured region between the two burns, however, the painfulness of the heat stimuli actually decreased (Fig. 1-14D). Notably, the area between the burns was hypo-algesic to heat while being hyperalgesic to mechanical stimuli.

Spreading Sensitization of Nociceptors Does Not Occur

Activation of nociceptors leads to a flare response (discussed in more detail below). This response is neurogenic in the sense that it depends on intact innervation of the skin by nociceptors. The flare response extends well outside the area of initial injury. One explanation for the flare response is that it involves spreading activation of nociceptors. Activation of one nociceptor leads to the release of chemicals that activate neighboring nociceptors, which leads to further release of chemicals and activation of additional nociceptors. Lewis (1942) believed that a similar mechanism, which he termed spreading sensitization, accounted for secondary hyperalgesia. Activation and sensitization of one nociceptor lead to spread of this sensitization to another nociceptor, possibly because of the effects of a sensitizing substance released from the nociceptor initially activated. Another theoretical possibility is that coupling between nociceptors increases after injury.

Several lines of evidence indicate that spreading sensitization does not occur:

Other differences exist between flare and secondary hyperalgesia (LaMotte et al 1991):

Central Mechanisms of Secondary Hyperalgesia

If peripheral sensitization does not account for secondary hyperalgesia, the mechanisms noted in Figure 1-10C–F should be examined in the CNS. Indeed, it has been relatively easy to demonstrate enhanced responsiveness of CNS neurons to mechanical stimuli after cutaneous injury (e.g., Simone et al 1991b). Substantial evidence favors the following important tenet: the peripheral signal for pain does not reside exclusively with nociceptors. Under pathological circumstances, other receptor types, which are normally associated with the sensation of touch, acquire the capacity to evoke pain. This principle applies not only to secondary hyperalgesia but also to neuropathic pain states in general. This condition arises in part through augmentation of the responsiveness of central pain-signaling neurons to input from low-threshold mechanoreceptors, a phenomenon often termed central sensitization.

Many of the insights acquired about secondary hyperalgesia have been gained from studies with capsaicin. Investigators have been drawn to the use of capsaicin as the “injury” stimulus for several reasons:

LaMotte and colleagues performed a number of pivotal experiments to determine the relative importance of peripheral and central sensitization in secondary hyperalgesia (LaMotte et al 1991). To test whether peripheral nerve fibers are sensitized, capsaicin was administered under conditions of a proximal nerve block, and the magnitude of hyperalgesia was determined after the effects of the anesthetic dissipated. When the relevant nerve is blocked proximal to the capsaicin injection site, the CNS is spared the nociceptive input generated at the time of injection. The effects of capsaicin on the peripheral nervous system are not affected (e.g., a flare develops) since the nerve block is proximal to the area of capsaicin application. Figure 1-15 shows the results of this experiment in one subject. No hyperalgesia was present after the block had worn off. Thus, when the CNS is spared the input of nociceptors at the time of the acute insult, hyperalgesia does not develop (LaMotte et al 1991, Pedersen et al 1996).

Additional evidence that central sensitization, not peripheral sensitization, plays a major role in secondary hyperalgesia includes the following:

Different Mechanisms for Stroking and Punctate Hyperalgesia

Two distinct forms of mechanical hyperalgesia are observed in the zone of secondary hyperalgesia: punctate hyperalgesia and stroking hyperalgesia. Hyperalgesia to blunt pressure is not observed in the secondary zone (Koltzenburg et al 1992). We will first consider stroking hyperalgesia (also called allodynia). Stroking hyperalgesia appears to be mediated by activity in low-threshold mechanoreceptors. When a pressure cuff was used to selectively block myelinated fibers, the pain in response to stroking disappeared at a time when touch sensation was lost but heat and cold sensations were still present (LaMotte et al 1991, Koltzenburg et al 1992). This is also true in patients with stroking hyperalgesia from neuropathic pain (Campbell et al 1988b). In another series of experiments, Torebjörk and colleagues (1992) performed intraneural microstimulation in awake human subjects. As shown in Figure 1-16, stimulation of primary afferent fibers normally concerned with tactile sensibility evoked pain when (but not before) secondary hyperalgesia was produced.

Punctate hyperalgesia is manifested by heightened pain associated with the application of small, stiff, or sharp probes to the skin (e.g., von Frey monofilaments). Several lines of evidence indicate that punctate hyperalgesia has a different neural mechanism than stroking hyperalgesia does and is mediated by central sensitization to activity in nociceptors:

The pain in response to a controlled punctate stimulus does not vary significantly across the zone of secondary hyperalgesia but decreases precipitously at the border (Huang et al 2000). This suggests that the sensitization responsible for secondary hyperalgesia is an all-or-nothing phenomenon. In addition, subjects were able to grade the magnitude of pain from stimuli of different intensity. Interestingly, although the threshold for pain in response to punctate stimuli decreases in the zone of secondary hyperalgesia (Magerl et al 1998), the threshold for touch detection increases (Magerl and Treede 2004).

Model for Stroking Hyperalgesia

From the above we know that secondary hyperalgesia to stroking stimuli appears to be due to sensitization of central pain-signaling neurons to the input from low-threshold mechanoreceptors (Fig. 1-17). In normal skin, activity in low-threshold mechanoreceptors signals touch sensation (Fig. 1-17A). As a result of the barrage of activity in nociceptors, sensitization occurs in the CNS such that input from low-threshold mechanoreceptors gains access to the pain system (Fig. 1-17B). Now, light touching of the skin is painful. Plasticity in the response of second-order neurons in the dorsal horn appears to be a major factor that accounts for this central sensitization (see Chapter 6 for detailed discussion). However, another possibility that involves plasticity in primary afferents is that mechanoreceptors gain access to nociceptive neurons by means of a presynaptic link (Cervero et al 2003; see discussion below on primary afferent depolarization).

Model for Punctate Hyperalgesia

Punctate hyperalgesia appears to be mediated by central sensitization to nociceptor input. However, most nociceptors respond to heat stimuli. Why is there not hyperalgesia to heat stimuli in the secondary hyperalgesic zone? One possibility is that this central sensitization involves a mechano-specific channel. In this model, punctate hyperalgesia is mediated by mechano-specific nociceptive afferents that project via sensitized mechano-specific interneurons to central pain-signaling neurons. Support for this hypothesis comes from experiments in which the skin was pretreated with topical capsaicin to eliminate epidermal nerve fibers that are sensitive to heat (Nolano et al 1999). Such treatment led to a lack of pain in response to heat stimuli; however, secondary hyperalgesia to punctate stimuli developed after the injection of capsaicin into nearby untreated skin (Fig. 1-18; Fuchs et al 2000). Additional experiments with selective nerve fiber blocks revealed that the punctate hyperalgesia disappeared when Aδ fibers were blocked (Magerl et al 2001). Thus, punctate hyperalgesia appears to be signaled by Aδ-fiber afferents that are insensitive to capsaicin and heat.

One well-studied form of central sensitization, termed wind-up, is characterized by a slowly increasing response of central neurons to repeated C-fiber stimulation at rates greater than 0.3 Hz (e.g., Mendell and Wall 1965). The perceptual correlate of wind-up is temporal summation (Price et al 1977). The finding that temporal summation does not change in the zone of secondary hyperalgesia argues against wind-up as a mechanism for secondary hyperalgesia (Magerl et al 1998).

Ectopic Sensitivity Develops in Injured Fibers

When a nerve is severed, the nociceptors are also severed. The injured (transected) nociceptors could in principle function abnormally at the site of nerve transection (the neuroma). Indeed, abnormal spontaneous activity has been observed in A and C fibers originating from a neuroma (see Chapter 64). Given that a substantial proportion of C-fiber afferents are nociceptors, it is likely that this spontaneous activity is in fact occurring in nociceptive afferents. In patients with a painful neuroma and hyperalgesia, locally anesthetizing the neuroma may eliminate the pain and hyperalgesia (Gracely et al 1992). Thus, ongoing activity arising from nociceptive fibers in the neuroma contributes to the ongoing pain and hyperalgesia after nerve injury and may contribute to phantom limb pain (see Chapter 67).

Ectopic mechanical sensitivity also develops in experimental neuromas. Tapping at the site of a neuroma leads to a neural response. This may account for the observation that tapping on a neuroma is quite often found to be painful (Tinel’s sign). Superficial neuromas, which are more prone to accidental mechanical stimulation, or neuromas that are in locations associated with high mechanical stress are more likely to be painful. One strategy to alleviate neuroma pain is to resect the neuroma and move the nerve to a deep location. Since neuromas form when a nerve is cut, removing a neuroma in essence is a neuroma relocation operation. Neuroma relocation effectively relieves pain enduringly in at least some cases (Burchiel et al 1993).

The Role of the Intact Nociceptor

Several lines of evidence suggest that uninjured, intact nociceptors that share the nerve of the injured fibers play a role in neuropathic pain. Much of the evidence comes from animal models of neuropathic pain in which the L5 spinal nerve is cut and ligated (Kim and Chung 1992). This injury leads to behavioral signs of hyperalgesia to mechanical and heat stimuli applied to the ipsilateral foot. Using this model, we have learned the following: (1) Dorsal rhizotomy of the lesioned L5 root does not reverse the hyperalgesia regardless of whether this is done pre-emptively (before the L5 spinal nerve is severed) or after the lesion. Thus, interruption of input from the injured L5 spinal nerve fails to reverse the hyperalgesia in the foot, which indicates that ectopic activity from the injured nerve is not essential for the development of neuropathic pain (Li et al 2000). (2) Electrophysiological recordings from the uninjured L4 spinal nerve (the root that most overlaps the innervation territory of the L5 root) reveal abnormal spontaneous activity in C-fiber nociceptors. The spontaneous activity appears to emanate at least in part from the skin (Wu et al 2001). (3) Molecules related to pain (e.g., CGRP, brain-derived neurotrophic factor [BDNF], VR1) are up-regulated in L4 DRGs (Fukuoka et al 2000). (4) Expression of the TTX-resistant sodium channel Na_v1.8 increases in the sciatic nerve (Gold et al 2003).

Additional evidence for a contribution of non-axotomized nociceptors comes from clinical studies demonstrating that distal therapies are effective in neuropathic pain states. Capsaicin causes degeneration of the cutaneous terminals of nociceptors that express TRPV1 (Nolano et al 1999). Capsaicin applied to the skin can alleviate the pain associated with nerve injuries (e.g., Robbins et al 1998). This clinical effect can be understood only by invoking a role of cutaneous nociceptors that survive the injury. Moreover, since the toxicity of capsaicin appears to be restricted to the skin, it is the cutaneous terminal of the nociceptor that must be generating pain.

Wallerian Degeneration and Neuropathic Pain

When the L5 spinal nerve is cut, the axons distal to the cut undergo wallerian degeneration. In the peripheral nerve, axons from intact nerve roots are in close proximity to degenerating axons and thus are exposed to diffusible factors released into the endoneurial space or at the nerve terminals. These factors could be derived from Schwann cells or macrophages and could affect nociceptive terminals directly or indirectly by an alteration in the cell bodies of nociceptors. As illustrated in Figure 1-20, wallerian degeneration may play a role in neuropathic pain by producing sensitization of primary afferent nociceptors and/or by leading to the development of central sensitization.

Sympathetically Maintained Pain Is a Receptor Disorder

Clinical studies support the concept that catechol sensitivity may develop in nociceptors after partial nerve injury. For example, intraoperative stimulation of the sympathetic chain induces pain in patients with causalgia (Walker and Nulson 1948, White and Sweet 1969). Also, physiological activation of sympathetic vasoconstrictor neurons leads to enhanced spontaneous pain and hyperalgesia in patients with SMP (Baron et al 2002). Pain is increased in the majority of patients with complex regional pain syndrome when sympathetic nervous system activity is evoked by a loud startling noise or by cooling their forehead (Drummond et al 2001). Injection of NE around stump neuromas or in the skin of patients with post-herpetic neuralgia induces an increase in spontaneous pain (Chabal et al 1992, Choi and Rowbotham 1997, Lin et al 2006). In SMP, anesthetic blockade of the sympathetic nervous system relieves the pain and hyperalgesia; intradermal injection of NE into the previously hyperalgesic area induces pain (Ali et al 2000). NE injected into normal subjects evokes little or no pain. This suggests that SMP does not arise from too much NE, but rather from the presence of adrenergic receptors in the skin that are coupled to nociceptors. Therefore, in SMP, the NE that is normally released from sympathetic terminals acquires the capacity to evoke pain.

Nerve Injury Induces Catechol Sensitization in Nociceptors

In addition to spontaneous activity, adrenergic sensitivity develops in nociceptors after nerve injury. In a primate model of an L6 spinal nerve lesion (Ali et al 1999), intact nociceptors innervating the foot exhibited spontaneous activity and a response to the α₁-adrenergic agonist phenylephrine applied to the receptive field. In monkeys in which no spinal nerve lesion was applied, little or no catechol sensitivity and spontaneous activity were present. Using a somewhat different injury model, studies in rabbits have also demonstrated catechol sensitization of intact nociceptors after injury to companion nerve fibers (Sato and Perl 1991). C fibers ending in a neuroma also display adrenergic sensitivity (e.g., Häbler et al 1987). Thus adrenergic mechanisms may play a role in activating injured nociceptors as well.

α₁-Adrenergic Agonists Activate Nociceptors Leading to Central Sensitization

Clinical studies support the postulate that SMP arises from expression of α-adrenergic receptors on the terminals of nociceptors. Systemic phentolamine, an α-adrenergic antagonist, relieves pain when given to patients with SMP (Raja et al 1991). Topical application of clonidine, an α₂-adrenergic agonist, to the painful skin of patients with SMP relieved hyperalgesia in the painful area (Davis et al 1991). Activation of α₂-adrenoceptors located on sympathetic terminals by clonidine blocks the release of NE. When phenylephrine, a selective α₁-adrenergic agonist, was applied to the clonidine-treated area, pain was rekindled in patients with SMP (Davis et al 1991). Thus, clinical data, as well as primate physiological data, suggest that in SMP, release of NE from the sympathetic terminals activates nociceptors that express α₁-adrenoceptors. The spontaneous activity and excitation of nociceptors by NE lead to central sensitization. Whether a change in phenotype or some other molecular change explains this nociceptor chemical sensitization is unanswered. Of some interest is the finding that the density of α₁-adrenoceptors in the epidermis of hyperalgesic skin of patients with complex regional pain syndrome is increased as measured with quantitative autoradiographic techniques (Drummond et al 1996). For SMP, procedures that reduce or eliminate excitation of α₁-adrenergic receptors lessen nociceptor activity and therefore lessen the hyperalgesia. Other potential central mechanisms that modulate nociception and emotional responses by adrenergic facilitation of nociceptive transmission in the dorsal horn or thalamus and/or by depletion of bulbospinal opioids have also been postulated (Drummond 2010).

Acknowledgment

We appreciate the technical assistance of T. V. Hartke and Ian Suk for contributing original artwork. This work was supported by National Institute of Health grants P01 NS 47399, NS-14447, and NS-26363.

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