In general, cytokines can be grouped into three families: interleukins, interferons, and tumor necrosis factors. All three families of cytokines are extremely potent, small soluble proteins that regulate the amplitude and duration of immune and inflammatory states through autocrine or paracrine actions in an effort to maintain tissue homeostasis. Originally classified as lymphokines and monokines to indicate their cellular source, it is now understood that all nucleated cells are capable of synthesizing and responding to these inflammatory mediators, including cells within the central nervous system (CNS). For the scope of this chapter, cytokines will be classified in terms of their regulation of the inflammatory cascade and are thus termed either proinflammatory or antiinflammatory. This chapter will focus on tumor necro- sis factor α (TNF), Interleukin (IL)-1β, and IL-6 as proinflammatory cytokines, and IL-10 in the context of being an antiinflammatory cytokine.
In general, peripheral cytokines are undetectable in the absence of tissue insult or injury. In contrast, within the CNS, cytokines are constituitively expressed at low levels with beneficial physiological functions.1,2 This difference in basal expression suggests that cytokines expressed in the CNS may have very different actions from those expressed peripherally. Within the CNS, many cytokines are known to function as neuromodulators and as such can be beneficial and necessary or detrimental, depending on their relative concentrations. For example, in the hippocampus, IL-1β at low levels (fM) augments long-term potentiation, while at higher levels (nM-pM) IL-1β inhibits long-term potentiation.3 Additional support for the neuromodulatory role of cytokines in the CNS come from studies which have found that IL-1β and TNF are important mediators of sleep and sickness responses.3,4 Conversely, proinflammatory cytokines such as TNF and IL-1β have been postulated to exert detrimental effects in central nervous system disease states such as multiple sclerosis, ischemia, Alzheimer’s disease, and HIV-associated dementia.5–9
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6.2.1 CYTOKINESIN PAIN
Early work with lipopolysaccharide (LPS)-induced illness reported marked hyper- algesia (increased response to a noxious stimuli) as a hallmark of illness behaviors.
LPS-induced hyperalgesia was found to correlate with increases in peripheral and central proinflammatory cytokines such as TNF, IL-1β, and IL-6.10–12 This early work in LPS-induced hyperalgesia led to the initial hypothesis that TNF, IL-1β, and IL-6 possess nociceptive actions in both the peripheral and central nervous systems in the absence of injury. In the periphery, both intraperitoneal and intraplantar IL-1β or TNF have been reported to produce short-lived dose-dependent hyperalge- sia.10,13–19 Furthermore, direct application of TNF on the sural nerve has been shown to produce C-fiber nociceptor sensitization,20,21 while administration to the sciatic nerve resulted in mechanical allodynia (response to a normally non-noxious stimuli) and thermal hyperalgesia.22 In an analogous manner, intracerebroventricular admin- istration of TNF, IL-1β, or IL-6 has been found to elicit hyperalgesia23–25 while intrathecal IL-6 had been reported to mediate allodynia in the absence of injury.26 Taken together, these data implicate three nociceptive processing regions at which TNF, IL-1β, and IL-6 may modulate pain responses following injury: in the periphery at the site of injury, in the spinal cord at the initial site of signal integration and processing, or in the brain at the site of supraspinal processing.
A potential role for peripheral actions of IL-1β and TNF in the genesis or maintenance of pain states following injury has been supported by observations of their upregulation in various peripheral inflammatory models that result in hyperal- gesia and allodynia.13,15,16 Pharmacological blockade of proinflammatory cytokines with IL-1 receptor antagonist (IL-1ra), anti-TNF serum, thalidomide, or antiinflam- matory IL-10 or IL-4 has been found to delay the onset and attenuate hyperalgesia in models of peripheral inflammatory pain.13,16,27–30 TNF has also been implicated in neuropathic pain models, such as chronic constriction injury (CCI), through the use of TNF receptor antibodies, thalidomide, and antiinflammatory IL-10.31–33 These pharmacological interventions have been shown to decrease TNF expression endo- neurially and this decrease paralleled the attenuation of mechanical allodynia or thermal hyperalgesia. These data highlight the vast literature that supports a potential role for peripheral cytokines in the modulation of pain behaviors in both inflamma- tory and neuropathic pain models.
In addition to peripheral expression of proinflammatory cytokines following injury, our laboratory has shown that several neuropathic models induce increased TNF, IL-1β, and IL-6 mRNA and protein in the spinal cord in a manner that parallels the development of allodynia.26,34–38 A similar upregulation of IL-1β expression in the spinal cord has been found in both formalin and zymosan peripheral inflammatory models as well as in the L5 spinal nerve transection mononeuropathy model.39 Further support of central proinflammatory cytokines in nociception have come from the observations that intrathecal application of IL-6 evokes hyperalgesia in a sciatic cryoneurolysis model that normally does not produce hyperalgesia.26 From studies utilizing pharmacological antagonism as well as studies in both genetically modified transgenic and knockout mice, numerous other laboratories have also implicated an important role of central cytokines in nociception. Pharmacological antagonism with
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intrathecal administration of IL-1ra or antibodies to the IL-1 receptor has produced attenuation in pain behaviors in both the formalin40 and CCI41 models, lending further support for an important role of central cytokines in the modulation of neuropathic pain. Similarly, transgenic mice overexpressing TNF on the glial fibrillary acidic protein (GFAP) promoter exhibited increased allodynia following L5 spinal nerve transection as compared to wild-type counterparts.42 Conversely, in response to nerve injury, IL-6 knockout mice have been shown to exhibit delayed mechanical allodynia44 without developing heat or pressure hypersensitivity.43 These studies highlight the importance of central TNF, IL-1β, and IL-6 in nociception following nerve injury.
Unfortunately, trying to decipher the role of cytokines in the genesis of pain states is very complex. This complexity can be further highlighted by the observa- tions that exogenous application of IL-1β can be both pro- and antinociceptive.45–47 A recent study demonstrated that high-dose intrathecal application of IL-1β was antinociceptive in a carrageenan model of inflammation, but application of IL-1β neutralizing antibody, without the addition of exogenous IL-1β, did not alter carra- geenan hyperalgesia.46 These findings coupled with previous research in the formalin model40 suggest that the increase in endogenous IL-1β following inflammatory injury is not sufficient to activate antinociceptive pathways and may even be pronociceptive.
These observations of dose-dependent effects of IL-1β in the CNS are not unique to pain models. In ischemia models, TNF and IL-1β have been found to be both neurotoxic and neuroprotective depending on their concentration, the concentration of their endogenous antagonists, as well as the time at which they are expressed following injury.3,5,7 Extrapolation from the ischemia literature would suggest that the study of central cytokine contributions to nociception must not only include the study of TNF, IL-1β, and IL-6 expression but also the temporal pattern of this expression as well as the balance of this expression with the endogenous antagonists.
6.2.2 ROLEOF CYTOKINESIN CENTRAL SENSITIZATION
Proinflammatory TNF, IL-1β, and IL-6 are postulated to contribute to central sen- sitization.1,2 Central sensitization produces lower thresholds and spontaneous ectopic neuronal firing48 which are manifested as hyperalgesia and allodynia at the behavioral level. The expression of cytokine receptors on neurons provides a mechanism by which TNF, IL-1β, and IL-6 may directly sensitize neurons; for example, IL-1β has been found to act directly on neurons to increase axonal transport and release of substance P, a potent nociceptive substance.49–51 Similarly, exogenous TNF or IL-6 has been shown to induce substance P synthesis and release in sympathetic gan- glia.52,53 In addition to a direct action on neurons, proinflammatory cytokines may indirectly affect neurons via interactions with the surrounding glial population. IL- 1β and substance P have been found to synergistically induce the release of IL-6 and prostaglandins from human spinal cord astrocytes.54 Furthermore, TNF and IL- 1β are known to be inducers of both microglial and astrocytic activation in vitro as well as in vivo.7,55
Activation of spinal glial (microglia and astrocytes) cells is important to the development of both allodynia and hyperalgesia. Administration of glial metabolic
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inhibitors in zymosan and formalin models has been shown to attenuate hyperalge- sia.40,59 In addition, our laboratory has repeatedly shown spinal glial activation in a number of rodent neuropathy models.35,36,56,57 Activated glial cells synthesize and secrete proinflammatory mediators such as cytokines (TNF, IL-1β, IL-6), nitric oxide (NO, through the induction of iNOS), and prostaglandins (through the induction of COX-2),7,58,59 all of which are potent nociceptive substances. Further neuronal sen- sitization is possible when TNF interacts with astrocytes, thereby increasing intra- cellular Ca2+ and inducing depolarization. Depolarization results in decreased glutamate uptake, leaving excessive glutamate in the synaptic cleft which produces aberrant or ongoing neuronal firing.60,61 Together this suggests that glial-derived cytokines or cytokine-induced glial activation are involved in the genesis and main- tenance of persistent neuropathic pain states.
6.2.3 RODENT MODELSOF PERSISTENT PAIN
There are numerous, well-established animal models of neuropathic pain.56,62–67 Our laboratory has developed two peripheral neuropathy models in the rat using freeze lesions.56,57,65 Although the freeze lesion is unique because it produces a regenerative permissive injury, the extent of the nerve lesion precludes early onset testing of neuropathic behaviors. In addition, it is technically challenging to selectively freeze the L5 spinal nerve with available cryoprobes. For these reasons, we adopted a modified Chung model and exclusively use this model for our neuropathic pain studies. From multitudinous studies, we have determined that a simplified L5 spinal nerve transection vs. L5 and L6 spinal nerve ligation (Chung model) produces robust mechanical allodynia and thermal hyperalgesia with quick onset and long duration in the nerve injury group as well as eliciting minimal inflammatory sham surgery behaviors. The results in this chapter utilize this L5 spinal nerve transection model for the neuropathic pain studies. As seen in Fig. 6.1, mechanical allodynia increases with time following L5 spinal nerve transection.
Our laboratory also has experience with models of low back pain associated with lumbar radiculopathy (i.e., lumbar dorsal and ventral root injury proximal to the dorsal root ganglion).38 The mechanisms that give rise to low back pain associated with or without lumbar radiculopathy remain obscure. During the past decade, there has been increasing interest in the mechanisms of low back pain/radiculopathy. Several useful animal models of lumbar nerve root injury68–73 established two specific mechanisms at the injury site level, specifically mechanical deformation of the nerve roots, and biologic or biochemical activity of the disc tissue. However, the exact contribution of each component and its full elucidation have thwarted the scientific community.
To this end, our laboratory further characterized the original Kawakami/Weinstein rat model of radiculopathy in which we investigated the type of suture material and extent of the lumbar root injury.38 In order to better understand radicular pain mechanisms, we examined spinal glial activation and IL-1β expression following lumbar nerve root injuries (loose ligation of only L5 dorsal and ventral roots with chromic gut or silk, or tight ligation with silk). We concluded that root injury via a chemically induced and/or mechanically induced factor evokes pain behaviors and activates glial cells and IL-1β expression.
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