Inflammation-induced GluA1 trafficking and membrane insertion of Ca2+ permeable AMPA receptors in dorsal horn neurons is dependent on spinal tumor necrosis factor, PI3 kinase and protein kinase A.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.

Selective stimulation of either tumor necrosis factor receptor differentially induces pain behavior in vivo and ectopic activity in sensory neurons in vitro.

Recent studies suggest that tumor necrosis factor-alpha (TNF) sensitizes primary afferent neurons, and thus facilitates neuropathic pain. Here, we separately examined the roles of tumor necrosis factor receptor (TNFR) 1 and 2 by parallel in vivo and in vitro paradigms using proteins that selectively activate TNFR1 or TNFR2 (R1 and R2). In vivo, intrathecally injected R1, but not R2 slightly reduced mechanical and thermal withdrawal thresholds in rats, whereas co-injection resulted in robust, at least additive pain-associated behavior. In vitro, the electrophysiological responses of dorsal root ganglia (DRG) from rats with spinal nerve ligation were measured utilizing single-fiber recordings of teased dorsal root filaments. In naïve DRG, only R1 (10-1000 pg/ml) induced firing in Ass- and Adelta-fibers, whereas R2 had no effect. In injured DRG, both R1 and R2 at significantly lower concentrations (1 pg/ml) increased discharge rates of Adelta-fibers. Most interesting, in adjacent uninjured DRG, R2 and not R1, increased ectopic activity in both Ass- and Adelta-fibers. We conclude that TNFR1 may be predominantly involved in the excitation of sensory neurons and induction of pain behavior in the absence of nerve injury, TNFR2 may contribute in the presence of TNFR1 activation. Importantly, the effects of individually applied R1 and R2 on injured and adjacent uninjured fibers imply that the role of TNFR2 in the excitation of sensory neurons increases after injury.

[Spinal glutamate receptor antagonists differentiate primary and secondary mechanical hyperalgesia caused by incision]. / Spinale Glutamatrezeptorantagonisten. Differenzierung von primärer und sekundärer mechanischer Hyperalgesie nach operativer Schnittinzision im Tierexperiment.

Secondary mechanical hyperalgesia has been demonstrated in postoperative patients indicating that central sensitization occurs after surgery. However, the underlying mechanisms are unknown. Here, we studied the role of spinal AMPA/kainate receptors for pain behaviors indicating secondary hyperalgesia caused by gastrocnemius incision in the rat. These were reduced by NBQX, a selective antagonist of AMPA/kainate receptors. However, administration of NMDA receptor antagonists caused no or only a modest decrease in behaviors for secondary hyperalgesia but produced associated motor deficits and supraspinal side effects. We further determined that only secondary mechanical hyperalgesia was reversed by JSTX, a selective antagonist of calcium-permeable AMPA receptor; primary mechanical hyperalgesia and guarding behavior were unchanged. These findings indicate that JSTX influenced a spinal amplification process that leads to secondary hyperalgesia but does not contribute to primary hyperalgesia and guarding after incision. This amplification process likely requires Ca(2) influx through spinal AMPA/KA (but not NMDA) receptors. Behaviors for secondary mechanical hyperalgesia after incision can be inhibited without affecting primary mechanical hyperalgesia and guarding. Mechanisms for central sensitization causing secondary hyperalgesia in postoperative patients may therefore be separated from spontaneous pain and hyperalgesia that arises adjacent to the area of the incision.

Spinal nerve ligation induces transient upregulation of tumor necrosis factor receptors 1 and 2 in injured and adjacent uninjured dorsal root ganglia in the rat.

Evidence indicates a role for tumor necrosis factor-alpha (TNF) in neuropathic pain. We correlated pain behavior in response to mechanical stimulation with immunoreactivity for TNF receptor (TNFR) 1 and 2 at 6, 24, 76 and 120 h following L5 and L6 spinal nerve ligation (SNL). Allodynia began in both L4 and L5 dermatomes within 6 h following SNL, peaking by 24 h. In L5 (injured) dorsal root ganglia (DRG), TNFR1 and TNFR2 levels displayed a bimodal increase, peaking at 6 and 120 h after SNL. In L4 (uninjured) DRG, TNFR1 and TNFR2 immunoreactivity peaked at 24 h returning to basal levels by 120 h. TNFR upregulation in injured and adjacent uninjured DRG neurons may be essential for mediating enhanced TNF effects and thus contribute to the development of pain-related behavior.

Antibody directed against GD(2) produces mechanical allodynia, but not thermal hyperalgesia when administered systemically or intrathecally despite its dependence on capsaicin sensitive afferents.

Anti-GD(2) antibodies have been shown to be effective for immunotherapy of neuroblastoma and other GD(2) enriched malignancies. Infusion of anti-GD(2) antibodies frequently causes spontaneous pain and allodynia for the duration of the immunotherapy and occasionally longer lasting neuropathic pain. Bolus intravenous injection of anti-GD(2) in rats initiates mechanical allodynia as measured by withdrawal threshold of the hindpaws. In this study, thermal thresholds were measured prior to and for up to 6 h following systemic anti-GD(2) administration in adult rats. In addition, both thermal and mechanical thresholds were tested following intrathecal administration of anti-GD(2) and IgG(2a). Murine anti-GD(2) elicited mechanical allodynia when administered into either the vasculature or the intrathecal space. Effective systemic doses were 1--3 mg/kg as previously shown. Intrathecally, optimal doses ranged from 0.01 to 0.1 ng; a higher dose was ineffective. Thermal hyperalgesia was not observed via either route of administration. Intrathecal pretreatment 48--72 h prior to the experiment with capsaicin at doses sufficient to cause a 50% depletion of dorsal horn CGRP, caused a total blockade of the mechanical allodynia indicating an involvement of peptidergic fine afferent fibers. It is likely that the antibody reacts with an antigen on peripheral nerve and/or myelin to initiate its effect. The lack of observed thermal hyperalgesia is surprising especially in light of the capsaicin-associated blockade, however, it is consistent with several other immune system related models of pain.

Epineurial application of TNF elicits an acute mechanical hyperalgesia in the awake rat.

Tumor necrosis factor alpha (TNF) injected into the sciatic nerve and neutralizing antibodies to its receptor injected around the nerve are respectively associated with inducing and blocking pain behavior beginning 1 to 3 days post-injection. This study examined the acute effects of TNF applied around the nerve trunk on the mechanical threshold (determined with von Frey hairs) and withdrawal latency to radiant heat. TNF (0.9 and 7.7 ng in 90 microL) injected onto the nerve via an indwelling catheter elicited a decrease in mechanical threshold. Following the low dose of TNF, no change in thermal latency was observed; after the 7.7 ng dose, thermal thresholds decreased and returned to baseline multiple times within the 3-hour observation period. Identical doses of TNF injected near, but not on the nerve, 90 ng of TNF injected on the nerve, and vehicle were without effect on either modality. These data indicate that effects of acutely administered TNF to the nerve trunk are capable of producing modality specific pain behavior. These changes may represent a first step in TNF-induced neuropathic pain.

Isoprostanes, novel eicosanoids that produce nociception and sensitize rat sensory neurons.

Isoprostanes are a novel class of eicosanoids primarily formed by peroxidation of arachidonic acid. Because of their potential as inflammatory and/or hyperalgesic agents whose formation is largely independent of cyclooxygenases, we examined whether 8-iso prostaglandin E(2) (8-iso PGE(2)) or 8-iso prostaglandin F(2alpha) (8-iso PGF(2alpha)) reduces mechanical and thermal withdrawal threshold in rats, and whether they sensitize rat sensory neurons. Injection of 1 microg of 8-iso PGE(2) (in 2.5 microl) into the hindpaw of rats significantly reduced mechanical and thermal withdrawal thresholds, whereas 1 microg of 8-iso PGF(2alpha) elicited a transient decrease in only the mechanical withdrawal threshold. Both isoprostanes enhanced the firing of C-nociceptors in a concentration-dependent manner when injected into peripheral receptive fields. Exposing sensory neurons grown in culture to 1 microM 8-iso PGE(2) or 8-iso PGF(2alpha) augmented the number of action potentials elicited by a ramp of depolarizing current. In contrast, 8-iso PGE(2) but not 8-iso PGF(2alpha) enhanced the release of substance P- and calcitonin gene-related peptide-like immunoreactivity from isolated sensory neurons. Ten micromolar 8-iso PGE(2) stimulated peptide release directly, whereas treatment with 1 microM 8-iso PGE(2) augmented the release evoked by either bradykinin or capsaicin. Pretreating neuronal cultures with the nonsteroidal anti-inflammatory drug ketorolac did not alter the sensitizing action of 8-iso PGE(2) on peptide release, suggesting that this action of the isoprostane was not secondary to the production of prostaglandins via the cyclooxygenase pathway. These data support the notion that isoprostanes are an important class of inflammatory mediators that augment nociception.

Nociceptive and inflammatory effects of subcutaneous TNFalpha.

Tumor necrosis factor alpha (TNF) is a potent pro-inflammatory cytokine that produces pain and hyperalgesia following injection. Its algesic effects are due to sensitizing actions on nociceptive primary afferents and to the upregulation of other pro-inflammatory and algesic proteins. In anesthetized rats, we investigated the effect of subcutaneously injected TNF on background activity and mechanical sensitivity of C nociceptors of the sural nerve, as well as its effects on cutaneous plasma extravasation. TNF sensitized C nociceptors dose-dependently; the optimal dose (5 ng) lowered threshold in 66.7% of the tested fibers. This sensitization occurred within 30 min and could last for 2 or more hours. Injected TNF had no effect on Abeta mechanoreceptive fibers. In addition, TNF evoked ongoing activity in 14% of C nociceptors and caused significant and dose-related increases in vascular permeability in glabrous skin. Our data suggest that TNF released during disease or after tissue injury participates in the generation of hyperalgesia and inflammation.

Antibody activation and immune reactions: potential linkage to pain and neuropathy.

Immune responses are an input source of modulation/modification for the peripheral nervous system that can result in pain and/or peripheral neuropathy. The resulting pain can be a significant debilitating component of many diseases as well as an untoward side effect of treatment. This paper briefly describes three sources of peripheral neuropathy generated in the presence of, or associated with, an immune response. Two are classified as autoimmune diseases. The body, in an attempt to rid itself of a tumor or an invading bacterial infection or virus, attacks its nervous system due to molecular mimicry; this results in, respectively, paraneoplastic neuropathy or inflammatory polyneuropathy. The third neuropathic pain syndrome is iatrogenic and occurs after administration of an antibody to GD2 ganglioside as an immunotherapy for neuroblastoma. This paper will attempt to point out some common elements in their neuropathologies and mechanisms.

Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Tumour necrosis factor-alpha induces ectopic activity in nociceptive primary afferent fibres.

Tumour necrosis factor-alpha, a pro-inflammatory cytokine, is expressed endoneurially following a variety of local and systemic pathophysiological insults which give rise to pain. We administered tumour necrosis factor-alpha to pentobarbital-anaesthetized rats, either topically along a restricted portion of the sciatic nerve or injected subcutaneously within the distribution of the sural nerve. Single nociceptive primary afferent fibres were assessed for ectopic discharge and receptor sensitization. Low concentrations (0.001-0.01 ng/ml) of tumour necrosis factor-alpha applied along the nerve elicited a dose-dependent, rapid onset (1-3 min) increase in discharge; higher concentrations led to reduced firing rates. C-fibres developed higher mean firing frequencies than A delta-fibres. Bursting frequency in both fibre types reached several (6) Hz. No change in mechanical threshold was observed. Intradermal injection (50 pg in 50 microliters) led to ectopic discharge and a decrease in mechanical threshold; these effects developed at different rates, suggesting multiple actions of the cytokine. Our data suggest that acute application of tumour necrosis factor-alpha to the axon can lead to aberrant electrophysiologic activity independent of peripheral receptor involvement. This low level of ectopic firing of nociceptive axons may produce wind-up in dorsal horn neurons or may, by itself, be interpreted as pain.

An animal model of pain produced by systemic administration of an immunotherapeutic anti-ganglioside antibody.

For the management of pediatric neuroblastoma, a promising experimental treatment includes slow systemic infusion of a human/ mouse chimeric monoclonal antibody against the GD2 ganglioside. Beneficial actions are however, accompanied by severe pain and altered cardiovascular tone. The pain is conventionally controllable with moderate to relatively high doses of intravenous morphine. An animal model was established to examine the change in nociceptive threshold produced by anti-GD2-antibody. Rats given bolus injections of antibody through an in-dwelling jugular catheter developed a quantifiable mechanical allodynia. At higher doses, allodynia and touch evoked agitation began within the first 15-min test interval, was maximal within the first hour, and for some doses was still present, although greatly reduced at 24 and 48 h. Rapid administration of antibody led to an increase in mean resting blood pressure of 12 mmHg +/- 1.8 (P < or = 0.02) and the development of a prolonged cardiovascular response to an otherwise innocuous stimulus. These observations demonstrate that the pain associated with monoclonal antibody treatment can be modeled in animals. This approach has potential for defining the pharmacology of the allodynia and ways in which the pain state may be ameliorated.

Electrophysiological characteristics of primary afferent fibers after systemic administration of anti-GD2 ganglioside antibody.

An animal model showing mechanical allodynia following systemic bolus injection of a human/mouse chimeric monoclonal antibody to the GD2 ganglioside (ch14.18) has been established (e.g. pain behavior generated by a light tactile stimulus). This is of clinical relevance since ch14.18 is a promising experimental treatment for pediatric neuroblastoma. The present study examined the hypothesis that allodynic effects of the anti-GD2 antibody are mediated by actions on cutaneous nerve fibers. After determining the basal magnitude of the mechanical stimulus required to produce withdrawal, ch14.18, a murine form of the anti-GD2 antibody of IgG2a isotype (14G2a), a control murine anti-melanoma antibody of IgG2a isotype (9.2.27) or saline was injected through a previously implanted jugular cannula. The experimenter was blinded to the syringe contents. Withdrawal threshold was tested at 15 min intervals for 1 h. After administration of either ch14.18 or 14G2a mechanical allodynia typically started within the first 15 min and persisted throughout the hour of behavioral testing. In the control antibody group, a modest change in tactile withdrawal threshold was observed at the 60 min time point only. Rats were then anesthetized with pentobarbital and prepared for single fiber recordings from the sural nerve. Fibers were classified based on conduction velocity, as A beta (> 25 m/s), A delta (2-25 m/s) or C (< 2 m/s). Background activity (BA) was observed in a significant number of A delta (12/61) and C (32/42) fibers in both anti-GD2 treated groups compared to the anti-melanoma antibody (1/17 A delta and 2/10 C fibers) and saline (0/26 A delta and 0/19 C fibers) treated groups. Mean mechanical threshold for A delta fibers in all three antibody treated groups was significantly reduced compared to the saline control; this was not observed for C-fibers in any group. Intravenous bolus injection (15 mg/kg) and infusion of lidocaine (plasma level 0.3-2.2 micrograms/ml) both reduced anti-GD2 associated BA. These data demonstrate that mechanical-allodynia could be produced by action(s) of the anti-GD2 antibody (direct or indirect) on peripheral nerves and suggest intravenous lidocaine as part of the analgesic regimen accompanying anti-GD2 antibody treatment.

The effects of A- and C-fiber stimulation on patterns of neuropeptide immunostaining in the rat superficial dorsal horn.

The present study determines the effects of sciatic nerve stimulation at intensities that activate A-fibers alone or both A- and C-fibers on immunostaining for substance P (SP), cholecystokinin-octapeptide (CCK-8), galanin (GAL), dynorphin (DYN) and vasoactive intestinal polypeptide (VIP) in the superficial dorsal horn of the rat spinal cord. The goal of this study is to provide a more precise spatial localization of the sites of release or accumulation of these compounds in relation to specific types of stimuli. Following A-fiber stimulation, there was no significant change in immunostaining for any of these compounds. However, A- and C-fiber stimulation resulted in major changes. For SP, CCK-8, GAL and DYN there was a large and significant loss of immunostaining in medial regions of the dorsal horn. This is the area where sciatic nerve primary afferent fibers terminate and the depletion is probably correlated with activity in these fibers. By contrast, VIP immunostaining is increased in the lateral part of the superficial cord, which is outside of the central sciatic afferent fiber terminations. This indicates that the increase is not in the fine sciatic sensory axons that are directly stimulated. As a final point, the fact that C-fiber but not A-fiber stimulation causes marked changes in the immunocytochemical distribution of all these compounds is further evidence, albeit indirect, that they are involved in nociceptive information processing.

Neural changes in acute arthritis in monkeys. IV. Time-course of amino acid release into the lumbar dorsal horn.

Extracellular levels of amino acids were measured during the development of experimental arthritis in anesthetized monkeys. Levels of glutamate, aspartate, glycine, serine, glutamine, taurine, cysteic acid and asparagine were each measured in consecutive 30 min samples before, during and for several hours after injection of kaolin and carrageenan into the articular capsule of one knee. Samples were obtained via a microdialysis probe placed in the lumbar dorsal horn ipsilateral to the injected knee and assayed using HPLC with fluorescence detection. Glutamate, aspartate, glycine and serine increased transiently following intra-articular injection of inflammatory agents. During this period glutamine levels decreased. A second phase of release then occurred which included more prolonged changes in amino acid levels that were sometimes of greater magnitude than those immediately following the injection. In animals which were later observed to have depletion of SP in the dorsal horn of the inflamed side, taurine levels increased starting after the Glu, Asp and Gly had plateaued at near baseline concentrations. Thus during the first stages of joint inflammation EAAs are released into the dorsal horn, followed by increased levels of IAAs, possibly representing activation of the descending endogenous analgesia system. This phase is followed by a semiacute response consisting in part of increased extracellular levels of SP and Tau. While SP is presumably part of an ascending nociceptive transmission system, Tau could be part of a second system aimed at reducing excessive neural activity including neural transmission resulting in intense maintained pain.