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The transition from acute to chronic pain states may be the most important challenge in research to improve clinical treatment of debilitating pain. We describe a recently identified mechanism of neuronal plasticity in primary afferent nociceptive nerve fibers (nociceptors) by which an acute inflammatory insult or environmental stressor can trigger long-lasting hypersensitivity of nociceptors to inflammatory cytokines. This phenomenon, “hyperalgesic priming,” depends on the epsilon isoform of protein kinase C (PKCε) and a switch in intracellular signaling pathways that mediate cytokine-induced nociceptor hyperexcitability. We discuss the impact of this discovery on our understanding of, and ultimately our ability to treat, a variety of enigmatic and debilitating pain conditions, including those associated with repetitive-injury, and generalized pain conditions like fibromyalgia.
The notion that “chronic” pain is a phenomenon distinct from “acute” pain is widely accepted as common sense among both physicians and the lay public . Yet, there is no definition of chronic pain that distinguishes it mechanistically from acute pain. Current working definitions of chronic pain, for the most part resort to fixed temporal cut-offs after which point acute pain switches in name to chronic pain. The arbitrariness of this approach is amply emphasized by the range of calendar-based periods that are used, for example: 1 month , 3 months , 6 months , or 1 year .
Clinical experience, however, suggests that the difference between acute and chronic pain is not arbitrary, and that there can be a functional transition of pain states from acute to chronic [5-8]. This transition seems to be associated with a time-dependent disconnection of the generation of pain from the initial tissue injury, and decrease of responsiveness to some therapies that successfully treat acute pain . Alternatively acute pain may persist for long periods without ever undergoing a “chronicization”  of its underlying mechanism.
Understanding the cellular mechanisms underlying chronic pain states will be a critical step in the development of new therapies to specifically target the distinct mechanisms of chronic pain. Numerous studies have implicated plastic changes in central nervous system circuitry, driven by increased activity in nociceptive primary afferent nerve fibers . However, until recently, very little was known concerning the cellular changes underlying these very long-lasting alterations in excitability of primary afferents associated with chronic pain states.
In this review, we summarize a growing body of evidence that a transient insult can trigger long-lasting changes in primary afferent nociceptors, which prime them to become hyperresponsive to future mild insults that would normally not evoke pain in the un-primed state. The epsilon isoform of protein kinase C (PKCε) in the primary afferent nociceptor plays multiple crucial roles in this phenomenon , known as “hyperalgesic priming.” Emerging evidence suggests that hyperalgesic priming may be not only a basic pathophysiological mechanism of chronic re-injury pain, but may also be key to understanding some of the most perplexing chronic pain conditions, including pain syndromes that are stress-related (e.g., fibromyalgia, irritable bowel syndrome, or post-traumatic stress disorder), and neuropathic (e.g., associated with diabetes or chemotherapy for cancer or AIDS). Finally, we suggest that hyperalgesic priming is related to a broader range of other physiological phenomena in which excitable cells undergo PKCε-dependent priming in response to diverse insults.
Numerous animal models of long-lasting pain have been developed as a means to study mechanisms of chronic pain. However, most of these models are complicated by the presence of ongoing tissue injury (notably, a variety of models employ chronic inflammation induced by complete Freund’s adjuvant or carrageenan), preventing the distinction between mechanisms of ongoing acute pain and neuroplastic changes that specifically underlie chronic pain. Even commonly employed models of neuropathic pain involving mechanical damage to a peripheral nerve can involve ongoing post-operative irritation and inflammation of the nerve or spinal root .
Separating the mechanisms of acute and chronic pain was a key concern in initial investigations that led to the discovery of the phenomenon of hyperalgesic priming. The clinical observation that some chronic pain conditions can be initiated by one or more transient episodes of acute pain (e.g., complex regional pain syndrome type I, occupational repetitive stress disorders), a protocol was developed to temporally isolate mechanisms that maintain chronic pain from those that produce an antecedent acute pain  (Figure 1). A short-lived inflammation was induced in rat hind paw by intradermal injection of a very low dose of the inflammogen, carrageenan. The resultant inflammation (localized redness with minimal swelling) is associated with acute hyperalgesia, detected as a decrease in threshold for the withdrawal response to a mechanical pressure stimulus applied to the inflamed paw. Both the acute inflammation and associated hyperalgesia resolve within 4 days, leaving the animal with no signs of ongoing inflammation or hyperalgesia (indeed, carrageenan injection is often used as a model of a simple acute inflammatory pain). However, when the paw is challenged with a new inflammatory stimulus, even weeks later, a dramatically enhanced hyperalgesic response is apparent. Thus, injection of a low dose of an inflammatory cytokine, e.g., prostaglandin E2 (PGE2), which in a naïve rat paw would cause only a brief hyperalgesia lasting less than 4 hours, now evokes hyperalgesia lasting at least 24 hours. In addition to this prolongation, hyperalgesic priming also causes an increase in the magnitude of hyperalgesia: the dose-response relationship for PGE2-induced hyperalgesia is shifted to the left by more than an order of magnitude . Hyperalgesia induced by other endogenous inflammatory mediators, including serotonin and adenosine are similarly enhanced. This latent hyper-susceptibility to inflammatory hyperalgesia that is still present, undiminished, weeks later, is termed “hyperalgesic priming.” The potential clinical significance of this hypersensitivity to pro-inflammatory cytokines is emphasized by the fact that PGE2 is a target of the most commonly used class of drugs for treating pain, the non-steroidal anti-inflammatory analgesics (NSAIDs).
Initial investigations into the mechanism underlying hyperalgesic priming revealed that, in the primed state, PGE2-induced hyperalgesia is not only enhanced and prolonged, but it also involves a switch in intracellular signaling pathways. Mechanical hyperalgesia induced by PGE2 is normally mediated largely by activation of the adenylyl cyclase — cyclic AMP — protein kinase A (PKA) second messenger signaling cascade in nociceptive primary afferents , and thus is strongly attenuated by injection of PKA antagonists into the peripheral receptive fields of those nerve fibers. Following induction of hyperalgesic priming, however, the new, prolonged component of the response to PGE2 is not sensitive to PKA antagonism, but is instead greatly reduced by a selective inhibitor of the epsilon isoform of PKC (PKCε). Of note, the PKCε-mediated prolonged response to PGE2 in the primed state does not replace the normal PKA-mediated transient response, but is added to it.
In addition to playing a key role in the expression of priming-enhanced mechanical hyperalgesia, PKCε activity is also required to maintain the primed state (even when the state is latent, i.e., there is no inflammatory mediator inducing hyperalgesia). To demonstrate this role of PKCε, after carrageenan-induced hyperalgesic priming was established, antisense oligodeoxynucleotide was injected intrathecally to produce a transient (approximately 3- to 5-day) decrease in PKCε expression in sensory neurons  (Figure 2). Priming-induced enhancement of PGE2 hyperalgesia was not only blocked during this transient reduction in PKCε level, but also failed to reappear after the antisense treatment was discontinued and PKCε recovered . Thus, ongoing PKCε activity is essential to maintain, as well as initiate, the primed state.
The observation that PKCε is necessary to maintain hyperalgesic priming, suggested the possibility that stimulating PKCε activity could be sufficient to initiate priming. Indeed, hyperalgesic priming can be induced by a highly selective agonist of PKCε (pseudo-receptor octapeptide for activated PKCε, ψεRACK). The observation that hyperalgesic priming could be induced by doses of ψεRACK too low to produce any detectable hyperalgesia [12,17] suggests that a chronic state of hyperalgesic priming can be induced without an antecedent acute pain state.
Consistent with its having a peripheral mechanism, hyperalgesic priming is induced by a localized stimulus and is topographically limited to that region, requires PKCε activity in peripheral tissue, and is mimicked by selective stimulation of PKCε activity in the skin. More specifically, this peripheral PKCε-mediated mechanism must occur within the primary afferent nerve fiber itself because, among all the cells in the skin, only primary afferents are exposed to the spinally administered antisense oligodeoxynucleotides that reverse hyperalgesic priming.
Although the cellular mechanisms of hyperalgesic priming occur within the peripheral terminals of primary afferent nociceptors, the resultant abnormal afferent activity can trigger plastic changes in the central nervous system (“central sensitization”) that add an additional dimension of nervous system dysfunction to the chronic pain state. That initiation and maintenance of central sensitization is a consequence of primary afferent activity (whether evoked or spontaneous) is suggested by the ability of transient local anesthesia to terminate hyperalgesia both in animal models of central sensitization  as well as in pain patients whose conditions are thought to involve central sensitization, including chemogenic pain , surgical pain  and generalized pain syndromes (e.g., irritable bowel  and fibromyalgia ). Therefore, from a therapeutic perspective, it may be useful to note that elimination of hyperalgesic priming in the primary afferent would also be expected to ameliorate central sensitization; however, treatments directed at central sensitization are unlikely to provide lasting relief if priming of primary afferent nociceptor hyperexcitability is not addressed.
A variety of poorly-understood and intractable chronic pain conditions can be characterized as an injury-induced state of hyper-responsiveness to subsequent insults (Figure 3). Pain may subside by avoidance of re-injury, but the latent state of hyper-responsiveness persists. In this regard there is a basic resemblance of such conditions to the phenomenon of hyperalgesic priming, and the comparison may yield important insights to advance our understanding of the underlying pathophysiological mechanisms of chronic pain.
For example, repetitive strain injury (also known as cumulative trauma disorder or occupational overuse syndrome) is a condition produced by repetition of movements resulting in chronic hyper-susceptibility to pain from future repetitions of these normally innocuous movements. Temporary pain relief can be achieved with injection of local anesthetic , suggesting the importance of abnormal primary afferent activity in generating the pain.
Some forms of headache may also be interpreted as chronic re-injury pain conditions that are consistent with the mechanism of hyperalgesic priming. For example, the most common headache disorder, tension-type headache, can transition from an episodic form to a much more intractable chronic form . A recent longitudinal study has shown that this transition is associated with a decrease in the threshold for pain evoked by pressure stimuli . This observation seems consistent with the induction of hyperalgesic priming by muscle tension, leading to subsequent hyper-susceptibility to future pain in those muscles. Tonic decrease in pressure pain threshold could result from hyperresponsiveness of primed primary afferent nociceptors to ambient levels of cytokines. Central sensitization, which is often suggested as a mechanism of chronic tension-type headache could be a consequence of such hyperalgesic priming of muscle afferents.
While the ability of stressful stimuli to cause analgesia is well-known, it is also clear that repeated environmental stressors can induce a persistent hyperalgesic state . The hypothesis that PKCε-dependent hyperalgesic priming underlies long-lasting stress-induced enhancement of hyperalgesia was recently tested in animal experiments . Rats exposed to non-habituating sound stress exhibited marked increases in mechanical hyperalgesia evoked by local injections of prostaglandin E2 or epinephrine. This enhancement, which lasted for weeks, required concerted action of glucocorticoids and catecholamines at receptors located in the periphery on sensory afferents. The altered response to pronociceptive mediators involves a switch in G-protein coupling of their receptors from Gs to Gi [28,28], and for prostaglandin, emergence of novel dependence on PKCε and loss of dependence on protein kinase A  (Figure 3). Thus, stress induces a sustained condition of increased sensitivity to the hyperalgesic effects of pro-inflammatory cytokines that is indistinguishable from the phenomenon of hyperalgesic priming that is induced by inflammation. Because ongoing stress can cause a lasting elevation of circulating pro-inflammatory cytokines , hyperalgesic priming of primary afferents in stressed individuals might produce tonic pain.
Generalized pain syndromes (including fibromyalgia, irritable bowel and chronic fatigue) are very common clinical pain syndromes (see Table 1) characterized by widespread chronic musculoskeletal pain without overt clinical signs of underlying pathophysiology. Because of our lack of understanding of the underlying etiology, currently accepted therapies are symptomatic (e.g. antidepressants, muscle relaxants, anticonvulsants, exercise, cognitive therapy), and are able to relieve pain in only about half of patients .
We propose that a key mechanism underlying generalized pain syndromes is stress-induced hyperalgesic priming of primary afferent nerve fibers. Prominent clinical characteristics of generalized pain conditions like fibromyalgia are consistent with this idea. For example, symptoms are characterized by waxing and waning episodes of pain, between which patients remain in a latent state of hypersensitivty to future insults . Also consistent with a priming-like latent hyperexcitability of primary afferents, pain in fibromyalgia patients may be relieved by injection of local anesthetics at tender points  or even at symptom-free trigger points . Finally, stress can dramatically exacerbate symptoms in generalized pain syndromes—even the earliest descriptions of these conditions noted their obvious link to both stress and inflammatory conditions . Approximately half of all fibromyalgia patients report that initial onset of their chronic pain followed a traumatic event , and it has been suggested that fibromyalgia should be considered primarily as a stress disorder . Furthermore, it has also been suggested that due to the close association of fibromyalgia with conditions like rheumatoid arthritis, the concept of “primary” fibromyalgia (versus secondary fibromyalgia) may not be justified .
The hypothesized role of stress-induced hyperalgesic priming may help illuminate a peculiar clinical observation in fibromyalgia; although systemic steroids are generally effective in the treatment of acute inflammatory musculoskeletal pain , fibromyalgia patients treated with glucocorticoids have been reported to show worsened outcomes . This result is consistent with expected exacerbation of hyperalgesic priming by steroid drugs. Beyond its theoretical interest, this insight has practical importance, as one study reported 40% of elderly fibromyalgia sufferers received corticosteroids on the mistaken assumption that they had polymyalgia rheumatica or an inflammatory joint disease .
Hyperalgesic priming increases the response of nociceptive nerve fibers to pro-inflammatory cytokines, and therefore, our hypothesis predicts that these cytokines play a role in the pathogenesis of generalized pain syndromes like fibromyalgia. In fact, the observation of a much higher incidence of fibromyalgia in patients with inflammatory diseases (e.g., approximately half of patients with systemic lupus erythematosus  and one sixth of rheumatoid arthritis patients ) or infectious diseases (e.g., approximately one third of patients infected with human T cell lymphotropic virus type I ) suggests that proinflammatory cytokines may be an important factor [45,46]. Furthermore, many patients with fibromyalgia exhibit increased levels of proinflammatory cytokines (including prostaglandin E2, interleukins and tumor necrosis factor α) in plasma and tissues [47,48], and cytokine levels correlate with the severity of their pain . The observation that effective treatment of associated inflammatory disease (e.g., rheumatoid arthritis) may improve fibromyalgia pain , suggests that normalizing cytokine levels may correct the underlying pathology of fibromyalgia. Thus, cytokine antagonists might prove to be a useful novel tool in fibromyalgia therapy; in fact, intravenous immunoglobulin therapy (IVIg), one effect of which is reduction of cytokine levels , has yielded promising results in a small trial in fibromyalgia patients .
Finally, it is clear that different individuals are differentially susceptible to developing generalized pain syndromes during their lifetimes . The observation that exposure of humans to stress early in life, or even before birth, can induce life-long chronic susceptibility to develop pain syndromes, for example , migraine headaches , raises the intriguing possibility that early stress could trigger permanent changes in pain pathways in the nervous system. Consistent with this idea, recent studies found that maternal separation of neonatal rats can cause the animals in adulthood to exhibit pain symptoms resembling irritable bowel syndrome . Whether stress-induced hyperalgesic priming could underlie such long-term changes is not known since, at present, the persistence of hyperalgesic priming has only been studied up to 3 weeks.
Although not ordinarily classified as generalized pain syndromes, other chronic conditions could also involve a component of stress-induced hyperalgesic priming. For example, stress-induced exacerbation of pain in rheumatoid arthritis37 and colitis38 is well recognized, and this effect might arise from stress-induced priming of nociceptors making them more susceptible to the hyperalgesic effect of the elevated levels of inflammatory cytokines present in these inflammatory diseases [56,57]. In addition, stress itself increases cytokine levels , which would further exacerbate pain.
A number of other stress-related psychological disorders that are not ordinarily thought of as primarily pain conditions are, however, associated with generalized pain. For example, generalized pain may be the major debilitating symptom in post-traumatic stress disorder (PTSD) [58,59], chronic fatigue syndrome  and depression . The tight linkage between such stress-related psychological disorders and pain (e.g., the “depression-pain dyad”) may reflect that stress not only induces psychological symptoms, but may also directly activate the neuropathological mechanism of hyperalgesic priming.
Other chronic pain disorders, classified as “neuropathic” because they are caused by damage or dysfunction in the nervous system, are notoriously resistant to treatment even with potent narcotic analgesics . Much research into the stress-induced exacerbation of neuropathic pain has focused on direct interaction of stress-activated sympathetic nerve fibers with sensory nerve fibers at sites of nerve injury [63,64], and consistent with this idea, destruction of sympathetic nerve fibers (sympathectomy) relieves symptoms in some of these patients. However, sympathectomy is often ineffective , and recent experimental evidence suggests that the dominant effect of stress in some neuropathic pain conditions may be the induction of hyperalgesic priming -- painful peripheral neuropathy induced by alcohol consumption in rats is abolished when stress axes are ablated .
Our proposal that hyperalgesic priming is a principal mechanism in an array of chronic pain syndromes might appear to result in an incongruous grouping of pain conditions that have traditionally been considered to be separate entities. To the contrary, however, this proposal conforms with a growing awareness that the attempt to create specific diagnostic categories has under-emphasized commonalities among these conditions. Thus, for example, in view of the difficulty in separating them diagnostically, and that their coexistence within patients is so common, it has also been suggested that fibromyalgia and irritable bowel syndrome should be considered different expressions of a single pathogenetic process , and it has been questioned whether posttraumatic stress disorder, tenderness, and fibromyalgia syndrome are in fact different entities . Even repetitive stress disorders, which seem more localized and linked to more specific injuries, are now recognized to share with generalized pain syndromes a very strong pathophysiological component of psychosocial stress .
This review is primarily concerned with the role of PKCε-dependent changes in neuronal excitability in the transition from acute to chronic pain, which has been termed “hyperalgesic priming.” However, it seems unlikely that this cellular mechanism has evolved specifically to modulate pain sensitivity, and thus it may be informative to view it in the wider context of excitable cells in general. We speculate that “hyperalgesic” priming constitutes one aspect of a more basic phenomenon in which PKCε in excitable cells responds to stressful events by altering the cells’ reactivity to future insults. This idea converges with the emerging concept of PKCε signaling as a “stress-sensing” cellular mechanism .
Probably the most intensively investigated function of PKCε is its role in cardiac preconditioning , a phenomenon that conforms well with the concept of PKCε as a stress-sensing trigger of altered excitability. In cardiac preconditioning a brief episode of ischemia followed by reperfusion induces changes in cardiac myocytes, lasting for weeks, that enable them to survive a future, more severe, ischemic event. Activation of PKCε is both required and sufficient to trigger the cascade of signaling pathways that ultimately alter the response of calcium handling mechanisms to ischemic stress.
In a similar phenomenon, termed neuronal preconditioning, a variety of sublethal cellular stressors can prepare neurons to respond to a subsequent episode of lethal ischemia via PKCε-dependent mechanisms . Perhaps more closely related to the hyperexcitability which is the hallmark of hyperalgesic priming, ischemic stress can also induce long-term hyperexcitability of hippocampal neurons that involves the activation of L-type calcium channels and NMDA receptors . It has been suggested that PKCε activity may be crucial to this hyperexcitability, and as such is a potential target of anti-epileptic therapy .
Finally, anxiety may be viewed as another type of chronic hyper-reactive state in which repeated stressors induce exaggerated fear responses to future stress. From this perspective, it is intriguing to note that knockdown of PKCε in the amygdala can reduce anxiety-like behavior in mice (induced by exposure to open field or elevated maze) . Perhaps affective disorders characterized by excessive anxiety, like post-traumatic stress disorder, represent a central nervous system correlate of PKCε-dependent hyperalgesic priming of primary afferent nerve fibers.
In summary, an understanding of PKCε-dependent hyperalgesic priming may provide fundamental insights into the cellular mechanisms that underlie a diverse range of pain conditions, including what may be the most important single issue for improving the clinical treatment of pain: cellular mechanisms that cause the transition from acute to chronic pain states. PKCε presents a promising target for the development of novel pharmacological strategies for the treatment of pain: expression of PKCε is preferentially expressed in nociceptive neurons, and it plays a crucial role in chronic hyperexcitability but its inhibition causes little disruption in normal sensory function.
In addition to the possibility of defining a key mechanism of nociceptor hyperexcitability in chronic pain, research into hyperalgesic priming might help illuminate other puzzling issues in nociceptor physiology. For example, opioid-induced hyperalgesia is a paradoxical increase in pain associated with the development of tolerance to opiate analgesic drugs, which complicates opiate withdrawal and is thought to underlie analgesic rebound headache. That opioid-induced hyperalgesia has been found to be PKC-dependent and to involve a switch in G-protein-coupling (of the μ-opioid receptor)  suggests a possible mechanistic relationship to hyperalgesic priming.
Finally, recognizing parallels between hyperalgesic priming and a broad range of other stress-induced states of altered cellular reactivity, may not only encourage new lines of investigation in these disparate fields of investigation, but perhaps lead to a more unified concept of the physiological role of PKCε.
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