Linda Sorkin - US grants

Recent observations suggest that antibody-antigen reactions along sensory axons, such as occurs following recognition of the anti-GD2 antibody for it membrane constituent antigen, may lead to complement fixation and an associated local release of tumor necrosis factor-a (TNF) from mast cells and Schwann cells. TNF forms membrane na+ pores providing a novel mechanism for initiating ectopic depolarization of axons at non terminal sites. This alteration in sensory processing is independent of peripheral receptors or structural changes of the axon. Systemic administration of antiGD2, as an immunotherapeutic agent, leads to severe pain and allodynia in man. Thia allodynia can be modeled in rats: administration of anti-GD2 antibody results in ongoing activity in afferent fibers as well as nociceptor sensitization. Endogenous TNF is released within nerves in many pain states, including local inflammation and injury. focal administration of TNF to the nerve trunk also elicits discharge in nociceptive afferents at frequencies consistent with the development of central sensitization. This proposal will investigate the hypothesis that anitGD2 antibody alterations of nociception are induced by an immune response initiated case along the peripheral nociceptive axons. Behavioral responses of rats treated with anti-GD2 to a range of cutaneous stimuli will be measured. Mechano-allodynia and thermal hyperalgesia will be tested. Dependence of the pan state on complement fixation, prostanoids and cytokine release will be explored. Ongoing activity and sensory thresholds will be recorded for identified primary afferent fibers, concentrating on A and C fibers thought to transmit pain information. Manipulations of the same variables used to examine generation of behavioral pain states will be used as pretreatments before recording excitability of nerve fibers. In this way, parallel changes between pain behavior and electrophysiology will be assessed. TNF, prostanoids and local increases in acidity will be administered directly to the nerve trunk to ascertain if these substances are sufficient to trigger the cascade. Results will begin to delineate the pharmacological sequence of events by which administration of GD2 antibody produces pain. This work will be the first step in determining if mid axonal generation of increased afferent throughput, mediated by TNF, is a common element in pathological pain states associated with tissue and nerve injury. This series of events along the axon represents a fundamentally different way for explaining the generation of many anomalous pain states and may thus initiate development of novel approaches to treatment.

Recent observations suggest that antibody-antigen reactions along sensory axons, such as occurs following recognition of the anti-GD2 antibody for it membrane constituent antigen, may lead to complement fixation and an associated local release of tumor necrosis factor-a (TNF) from mast cells and Schwann cells. TNF forms membrane na+ pores providing a novel mechanism for initiating ectopic depolarization of axons at non terminal sites. This alteration in sensory processing is independent of peripheral receptors or structural changes of the axon. Systemic administration of antiGD2, as an immunotherapeutic agent, leads to severe pain and allodynia in man. Thia allodynia can be modeled in rats: administration of anti-GD2 antibody results in ongoing activity in afferent fibers as well as nociceptor sensitization. Endogenous TNF is released within nerves in many pain states, including local inflammation and injury. focal administration of TNF to the nerve trunk also elicits discharge in nociceptive afferents at frequencies consistent with the development of central sensitization. This proposal will investigate the hypothesis that anitGD2 antibody alterations of nociception are induced by an immune response initiated case along the peripheral nociceptive axons. Behavioral responses of rats treated with anti-GD2 to a range of cutaneous stimuli will be measured. Mechano-allodynia and thermal hyperalgesia will be tested. Dependence of the pan state on complement fixation, prostanoids and cytokine release will be explored. Ongoing activity and sensory thresholds will be recorded for identified primary afferent fibers, concentrating on A and C fibers thought to transmit pain information. Manipulations of the same variables used to examine generation of behavioral pain states will be used as pretreatments before recording excitability of nerve fibers. In this way, parallel changes between pain behavior and electrophysiology will be assessed. TNF, prostanoids and local increases in acidity will be administered directly to the nerve trunk to ascertain if these substances are sufficient to trigger the cascade. Results will begin to delineate the pharmacological sequence of events by which administration of GD2 antibody produces pain. This work will be the first step in determining if mid axonal generation of increased afferent throughput, mediated by TNF, is a common element in pathological pain states associated with tissue and nerve injury. This series of events along the axon represents a fundamentally different way for explaining the generation of many anomalous pain states and may thus initiate development of novel approaches to treatment.

DESCRIPTION (provided by applicant): NMDA receptor (NMDAr) activation allows Ca2+ flux across the membrane that induces spinal sensitization, activation of signal transduction cascades and hyperalgesia. However, activation of Ca2+ permeable non-NMDA receptors (Ca-permeable non-NMDAr), without any help from NMDAr, can also produce synaptic strengthening and activation of signal transduction cascades. Recent work using NMDAr antagonist-insensitive models of pain shows that intrathecal administration of a Ca2+ permeable non-NMDA receptor antagonist, blocks or reverses secondary mechanical allodynia. The same agent is without effect in some NMDA-sensitive (dependent) models of secondary hyperalgesia. This leads to the hypothesis: Ca2+ permeable AMPA receptor activation can induce spinal sensitization and hyperalgesia via an initiating mechanism distinct from NMDA receptor activation. A 3rd group of pain models is partially dependent on NMDAr activation. Thermal and mechanical hyperalgesia following intraplantar carrageenan are blocked equally well by either NMDA or Ca-permeable non-NMDAr antagonists. After capsaicin or C-fiber stimulation, ERK (member of the mitogen-activated protein kinase signaling cascade) and transcriptional factor CREB (cAMP responsive binding protein) become phosphorylated. However, less than half of the phosphorylation is blocked by NMDAr antagonist pretreatment. The remainder is elicited by agents acting on receptors other than NMDAr, perhaps including Ca-permeable non-NMDAr. If Ca-permeable non-NMDAr are responsible, this implies a convergence between NMDAr- and Ca-permeable non-NMDAr-induced pathways prior to ERK activation. As models of burn and post-surgical pain, in which Ca-permeable non-NMDAr presumably appears to play a role, resemble clinical conditions, this novel ionotropic pathway probably plays a significant part in generation of at least some clinical pain states. We propose to investigate the effects of Ca-permeable non-NMDAr activation on ERK and CREB phosphorylation (using Western blots and immunohistochemistry), expression of c-fos and development of hyperalgesia.

DESCRIPTION (provided by applicant): Following nerve injury, pro-inflammatory cytokines increase within dorsal root ganglia (DRG) and spinal cord where they activate mitogen-activated protein kinases (MAPK), including p38 kinase (p38). Activation of p38 alters multiple proteins in primary afferent fibers and dorsal horn cells, and its inhibition reduces pain behavior following nerve injury. This application proposes to dissect the molecular pathway upstream and downstream of p38 activation in response to spinal nerve ligation, a model of neuropathic pain. Aim 1 will concentrate on nerve injury-induced changes in p38 and its isoforms. Aim 2 will look at upstream regulators and aim 3 at potential downstream effectors. We will systematically examine co-variance of pain behavior (mechanical and thermal sensitivity) with changes in activation and/or expression of cytokines (TNF and IL- 1), MKK3, MKK6, p38 isoforms and MAPKAP-2, a p38 substrate kinase. Changes in protein expression (Western blots) will be followed, in some instances, by a kinase assays to verify activity, and immunohistochemistry to ascertain cellular localization. Spinal cord and the DRG will be examined separately to determine if the cascade differs between the two locations. Inhibition or loss of individual elements in the cascade will allow us to determine what elements "downstream" of the blockade are reduced. Blockade of individual elements will occur using antisense for individual p38 isoforms, knock out mice and receptor specific pharmacological antagonists. Parallel experiments examining pain behavior will allow us to extend our findings past co-variance and to determine if blockade of any substance, within the DRG or spinal cord, is either necessary or sufficient to reduce pain behavior. Delineation of these patterns and mechanistic cause/effect relationships will increase our ability to methodologically develop rational treatments for neuropathic pain by providing us with more selective targets. This approach has the potential for avoiding some of the problems that occur with complete inhibition of p38 function.

DESCRIPTION (provided by applicant): Following nerve injury, pro-inflammatory cytokines increase within dorsal root ganglia (DRG) and spinal cord where they activate mitogen-activated protein kinases (MAPK), including p38 kinase (p38). Activation of p38 alters multiple proteins in primary afferent fibers and dorsal horn cells, and its inhibition reduces pain behavior following nerve injury. This application proposes to dissect the molecular pathway upstream and downstream of p38 activation in response to spinal nerve ligation, a model of neuropathic pain. Aim 1 will concentrate on nerve injury-induced changes in p38 and its isoforms. Aim 2 will look at upstream regulators and aim 3 at potential downstream effectors. We will systematically examine co-variance of pain behavior (mechanical and thermal sensitivity) with changes in activation and/or expression of cytokines (TNF and IL- 1), MKK3, MKK6, p38 isoforms and MAPKAP-2, a p38 substrate kinase. Changes in protein expression (Western blots) will be followed, in some instances, by a kinase assays to verify activity, and immunohistochemistry to ascertain cellular localization. Spinal cord and the DRG will be examined separately to determine if the cascade differs between the two locations. Inhibition or loss of individual elements in the cascade will allow us to determine what elements "downstream" of the blockade are reduced. Blockade of individual elements will occur using antisense for individual p38 isoforms, knock out mice and receptor specific pharmacological antagonists. Parallel experiments examining pain behavior will allow us to extend our findings past co-variance and to determine if blockade of any substance, within the DRG or spinal cord, is either necessary or sufficient to reduce pain behavior. Delineation of these patterns and mechanistic cause/effect relationships will increase our ability to methodologically develop rational treatments for neuropathic pain by providing us with more selective targets. This approach has the potential for avoiding some of the problems that occur with complete inhibition of p38 function.

[unreadable] DESCRIPTION (provided by applicant): Injury and inflammation leads to debilitating and persistent hyperalgesia. This hyperalgesia reflects sensitization of peripheral nerve terminals evoked by a rapid expression of proinflammatory factors as well as a facilitation of spinal nociceptive processing. Studies of peripheral systems have now begun to suggest that tissue injury triggers not only inflammation, but also a well-orchestrated series of events that lead to reversal of the inflammatory state. In this regard, lipoxins represent a unique class of lipid mediators that can function as "braking signals" in inflammation. Lipoxin A4 (LXA4) is generated via the lipoxygenase pathway during cell-cell interactions. LXA4 binds to a G protein-coupled receptor (ALXR), reducing chemokine and cytokine production and stimulating monocytes and macrophages to nonphlogistic activity. The resolving and anti-inflammatory role of peripheral lipoxin raises the provocative hypothesis that similar systems may also down regulate injury induced spinal facilitation. In preliminary work, intrathecal LXA4 and LXA4 analogues indeed had potent antihyperalgesic effects in inflammation-evoked hyperalgesia. In a subsequent pilot study, ALXR expression was detected throughout na[unreadable]ve rat spinal parenchyma and was unexpectedly found to be co-localized with astrocytes. Current work suggests that spinal astrocytes sustain inflammatory and neuropathic pain states though an enhancement of neuronal excitability. This linkage opens the possibility that lipoxins regulate spinal nociceptive processing though their effects upon astrocytic activation. This proposed work will: i) characterize the effects of intrathecally delivered structurally-related lipoxin molecules in a model of inflammatory pain; ii) determine the spinal distribution and regulation of the enzymes necessary for lipoxin synthesis, iii) determine if ALXR expression varies with progression of inflammation induced painful conditions and iv) start examining the possible molecular mechanism by which ALXR activation may control astrocyte activation. Targeting mechanisms that counter-regulate the spinal consequences of inflammation suggests a novel endogenous mechanism by which persistent pain can be controlled. Moreover, these studies will additionally inform us regarding the expanding role of spinal non-neuronal cells in persistent pain states. Current medication for chronic pain relief is surprisingly limited. Lipoxins represent a novel class of anti- inflammatory agents that may offer a new therapeutic strategy by which pain can be reduced. The aim of this proposal is to investigate the role of lipoxins in conditions associated with persistent inflammatory pain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dynamic changes in AMPA receptor (AMPAr) density and phosphorylation as well as number of Ca2+ permeable AMPAr in plasma membrane contribute to long term potentiation and synaptic strengthening. Tumor necrosis factor (TNF), a glial product, has been shown to elicit insertion of Ca2+ permeable AMPAr in plasma membrane of hippocampal neurons. In spinal cord, TNF contributes to chronic pain following a variety of injuries. The mechanism by which TNF enhances dorsal horn neuronal activity is unknown. I propose that following peripheral inflammation, glial TNF, working through a PI3K/Akt pathway, phosphorylates GluR1 subunits at 2 distinct sites, causing the AMPAr containing them to be inserted into plasma membranes. The increased AMPAr contain a disproportionate number that are Ca2+permeable, further contributing to synaptic strengthening. I will measure P-GluR1, insertion of AMPAr into the membrane and number of dorsal horn cells with functional Ca2+permeable AMPAr as a result of paw inflammation. I will use a combination of Western blots, subcellular fractionation, kainite induced cobalt loading and confocal microscopy. Spinal administration of specific inhibitors to every step in the proposed pathway will demonstrate that these elements are necessary for pain behavior and GluR1 insertion into neuronal plasma membranes as well as the sequence of events. Finally we will use 2 strains of knock in mice that do not phosphorylated GluR1 at individual serine residues. Use of these mice will further delineate the pathway and specify the necessary point of action of multi-purpose kinases. These experiments will further our understanding of fundamental spinal cord mechanisms. Ultimately, it will allow us to more logically and successfully design selective agents to treat chronic pain. PUBLIC HEALTH RELEVANCE: Tissue inflammation induces spinal cord release of TNF, sensitization of spinal neurons, and pain behavior. The mechanism by which TNF sensitizes pain transmission neurons is unknown. The proposal explores the possibility that TNF, via several identified steps, causes insertion of more and more active glutamate receptors into neuronal membranes to make them more excitable.