Marucia Chacur

Spinal Glia and Proinflammatory Cytokines Mediate Mirror-Image Neuropathic Pain in Rats

Mirror-image allodynia is a mysterious phenomenon that occurs in association with many clinical pain syndromes. Allodynia refers to pain in response to light touch/pressure stimuli, which normally are perceived as innocuous. Mirror-image allodynia arises from the healthy body region contralateral to the actual site of trauma/inflammation. Virtually nothing is known about the mechanisms underlying such pain. A recently developed animal model of inflammatory neuropathy reliably produces mirror-image allodynia, thus allowing this pain phenomenon to be analyzed. In this sciatic inflammatory neuropathy (SIN) model, decreased response threshold to tactile stimuli (mechanical allodynia) develops in rats after microinjection of immune activators around one healthy sciatic nerve at mid-thigh level. Low level immune activation produces unilateral allodynia ipsilateral to the site of sciatic inflammation; more intense immune activation produces bilateral (ipsilateral + mirror image) allodynia. The present studies demonstrate that both ipsilateral and mirror-image SIN-induced allodynias are (1) reversed by intrathecal (peri-spinal) delivery of fluorocitrate, a glial metabolic inhibitor; (2) prevented and reversed by intrathecal CNI-1493, an inhibitor of p38 mitogen-activated kinases implicated in proinflammatory cytokine production and signaling; and (3) prevented or reversed by intrathecal proinflammatory cytokine antagonists specific for interleukin-1, tumor necrosis factor, or interleukin-6. Reversal of ipsilateral and mirror-image allodynias was rapid and complete even when SIN was maintained constantly for 2 weeks before proinflammatory cytokine antagonist administration. These results provide the first evidence that ipsilateral and mirror-image inflammatory neuropathy pain are created both acutely and chronically through glial and proinflammatory cytokine actions.

A new model of sciatic inflammatory neuritis (SIN): induction of unilateral and bilateral mechanical allodynia following acute unilateral peri-sciatic immune activation in rats

&NA; Immune activation near healthy peripheral nerves may have a greater role in creating pathological pain than previously recognized. We have developed a new model of sciatic inflammatory neuritis to assess how such immune activation may influence somatosensory processing. The present series of experiments reveal that zymosan (yeast cell walls) acutely injected around the sciatic nerve of awake unrestrained rats rapidly (within 3 h) produces low threshold mechanical allodynia in the absence of thermal hyperalgesia. Low (4 &mgr;g) doses of zymosan produce both territorial and extra‐territorial allodynia restricted to the ipsilateral hindpaw. Higher (40–400 &mgr;g) doses of zymosan again produce both territorial and extra‐territorial allodynia. However, allodynia is now expressed both in the ipsilateral as well as contralateral hindpaws. Several lines of evidence are provided that the appearance of this contralateral (‘mirror’) allodynia reflects local actions of zymosan on the sciatic nerve rather than spread of this immune activator to the general circulation. Since many clinical neuropathies result from inflammation/infection of peripheral nerves rather than frank physical trauma, understanding how immune activation alters pain processing may suggest novel approaches to pain control.

Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats

Recent evidence suggests that spinal cord glia can contribute to enhanced nociceptive responses. However, the signals that cause glial activation are unknown. Fractalkine (CX3C ligand‐1; CX3CL1) is a unique chemokine expressed on the extracellular surface of spinal neurons and spinal sensory afferents. In the dorsal spinal cord, fractalkine receptors are primarily expressed by microglia. As fractalkine can be released from neurons upon strong activation, it has previously been suggested to be a neuron‐to‐glial signal that induces glial activation. The present series of experiments provide an initial investigation of the spinal pain modulatory effects of fractalkine. Intrathecal fractalkine produced dose‐dependent mechanical allodynia and thermal hyperalgesia. In addition, a single injection of fractalkine receptor antagonist (neutralizing antibody against rat CX3C receptor‐1; CX3CR1) delayed the development of mechanical allodynia and/or thermal hyperalgesia in two neuropathic pain models: chronic constriction injury (CCI) and sciatic inflammatory neuropathy. Intriguingly, anti‐CX3CR1 reduced nociceptive responses when administered 5–7 days after CCI, suggesting that prolonged release of fractalkine may contribute to the maintenance of neuropathic pain. Taken together, these initial investigations of spinal fractalkine effects suggest that exogenous and endogenous fractalkine are involved in spinal sensitization, including that induced by peripheral neuropathy.

Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10

Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine.

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine‐induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin‐1 (IL1), interleukin‐6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine‐induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti‐CX3CR1 also blocked fractalkine‐induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine‐induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine‐induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine‐induced pain facilitation. Lastly, NO appears to be importantly involved, as l‐NAME, a broad‐spectrum NO synthase inhibitor, also blocked fractalkine‐induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron‐to‐glia signal triggering pain facilitation.

Peri-sciatic proinflammatory cytokines, reactive oxygen species, and complement induce mirror-image neuropathic pain in rats

&NA; In inflammatory neuropathy, immune activation near intact peripheral nerves induces mechanical allodynia. The identity of the peripheral immune product(s) that lead to these changes in pain behavior is unknown. The present series of studies utilized the sciatic inflammatory neuropathy (SIN) model to examine this question. Here, inflammatory neuropathy is created by injecting an immune activator (zymosan) around one sciatic nerve via an indwelling catheter. Our prior studies demonstrated that peri‐sciatic zymosan activated macrophages and neutrophils to release proinflammatory cytokines and reactive oxygen species (ROS). In addition, zymosan is a classical activator of the complement cascade. Thus the present series of experiments examined whether any of these inflammatory mediators are involved in the initial induction of SIN‐induced ipsilateral or bilateral allodynias. Peri‐sciatic injection of selective inhibitors/antagonists revealed that a number of immune products are early mediators of the resultant allodynias, including proinflammatory cytokines (tumor necrosis factor, interleukin‐1, and interleukin‐6), ROS, and complement. Thus these immune‐derived substances can markedly alter sensory nerve function at mid‐axon.

Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants.

The ability of Lys49 and Asp49 phospholipases A(2) (PLA(2)), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20 micro g/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA(2) with p-bromophenacyl bromide (p-BPB), which abrogates its PLA(2) activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA(2), caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA(2) did not show any nociceptive effect. Hyperalgesia induced by both PLA(2)s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B(2) receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFalpha) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA(2)-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA(2). The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by N(g)-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA(2)s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA(2)s exert their algogenic actions through different molecular mechanisms.

Pharmacological modulation of hyperalgesia induced by Bothrops asper (terciopelo) snake venom

The ability of Bothrops asper snake venom to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of the venom (5-15 microg/paw) caused a dose and time-related hyperalgesia, which peaked 2h after venom injection. Bothrops asper venom-induced hyperalgesia was blocked by the bradykinin B(2) receptor antagonist HOE 140 and attenuated by dexamethasone, an inhibitor of phospholipase A(2). Inhibition of the lipoxygenase pathway by NDGA abrogated the algogenic phenomenon. The hyperalgesic response was not modified by pretreatment with indomethacin, an inhibitor of the cyclo-oxygenase pathway, by meloxicam, an inhibitor of the type 2 cyclo-oxygenase pathway, by the PAF receptor antagonist BN52021 or by anti-TNF-alpha or anti-interleukin 1 antibodies. Intraplantar injection of the venom also caused an oedematogenic response which was not modified by any of these pharmacological treatments. These results suggest that hyperalgesia induced by Bothrops asper venom is, at least partially, mediated by bradykinin, phospholipase A(2) activity and leukotrienes. Distinct mechanisms are involved in the development of hyperalgesia and oedema induced by the venom.

Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia‚ proinflammatory cytokines and nitric oxide

&NA; Snakebites constitute a serious public health problem in Central and South America, where species of the lancehead pit vipers (genus Bothrops) cause the majority of accidents. Bothrops envenomations are very painful, and this effect is not neutralized by antivenom treatment. Two variants of secretory phospholipases A2 (sPLA2), corresponding to Asp49 and Lys49 PLA2s, have been isolated from Bothrops asper venom. These sPLA2s induce hyperalgesia in rats following subcutaneous injection. However, venom in natural Bothrops bites is frequently delivered intramuscularly, thereby potentially reaching peripheral nerve bundles. Thus, the present series of experiments tested whether these sPLA2s could exert pain‐enhancing effects following administration around healthy sciatic nerve. Both were found to produce mechanical allodynia ipsilateral to the injection site; no thermal hyperalgesia was observed. As no prior study has examined potential spinal mechanisms underlying sPLA2 actions, a series of anatomical and pharmacological studies were performed. These demonstrated that both sPLA2s produce activation of dorsal horn astrocytes and microglia that is more prominent ipsilateral to the site of injection. As proinflammatory cytokines and nitric oxide have each been previously implicated in spinally mediated pain facilitation, the effect of pharmacological blockade of these substances was tested. The results demonstrate that mechanical allodynia induced by both sPLA2s is blocked by interleukin‐1 receptor antagonist, anti‐rat interleukin‐6 neutralizing antibody, the anti‐inflammatory cytokine interleukin‐10, and a nitric oxide synthesis inhibitor (l‐NAME). As a variety of immune cells also produce and release sPLA2s during inflammatory states, the data may have general implications for the understanding of inflammatory pain.

Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators.

&NA; Snakebites are a relevant public health problem in Central and South America. Snake bite envenomations cause intense pain, not relieved by anti‐venom. The fangs of many species are short, causing subcutaneous injection. Fangs of larger species inflict subcutaneous or intramuscular envenomation. To understand pain induced by subcutaneous venom, this study examined spinal mechanisms involved in pain‐enhancing effects of subcutaneous Lys49 and Asp49 secretory phospholipase‐A2 (sPLA2), two components of Bothrops asper snake venom showing highly different enzymatic activities. Unilateral intraplantar sPLA2‐Lys49 (catalytically inactive) or sPLA2‐Asp49 (catalytically active) into rat hindpaws each induced mechanical hyperalgesia (Randall–Selitto test), whereas only catalytically active sPLA2‐Asp49 caused mechanical allodynia (von Frey test). Effects induced by both sPLA2s were inhibited by intrathecal fluorocitrate, a reversible glial metabolic inhibitor. In support, immunohistochemical analysis revealed activation of dorsal horn astrocytes and microglia after intraplantar injection of either sPLA2. Spinal proinflammatory cytokines, nitric oxide, and prostanoids each appear to be involved in the pain‐enhancing effects of these sPLA2s. Blockade of interleukin‐1 (IL1) inhibited hyperalgesia induced by both sPLA2s, while leaving allodynia unaffected. Blockade of tumor necrosis factor reduced responses to sPLA2‐Asp49. An inhibitor of neuronal nitric oxide synthase, 7‐nitroindazole (7‐NI), inhibited hyperalgesia induced by both sPLA2s, without interfering with allodynia induced by sPLA2‐Asp49. On the other hand, l‐N6‐(1‐iminoethyl)lysine (l‐NI), an inhibitor of the inducible nitric oxide synthase, did not alter any sPLA2‐induced effect. Lastly, celecoxib, an inhibitor of cyclooxygenase‐2, attenuated sPLA2 actions. These data provide the first evidence of spinal mediators involved in pain facilitation induced by subcutaneous venoms.