Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

BACKGROUND: Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. METHOD: Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. RESULTS: An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. CONCLUSIONS: Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Origins of antidromic activity in sensory afferent fibers and neurogenic inflammation.

Neurogenic inflammation results from the release of biologically active agents from the peripheral primary afferent terminal. This release reflects the presence of releasable pools of active product and depolarization-exocytotic coupling mechanisms in the distal afferent terminal and serves to alter the physiologic function of innervated organ systems ranging from the skin and meninges to muscle, bone, and viscera. Aside from direct stimulation, this biologically important release from the peripheral afferent terminal can be initiated by antidromic activity arising from five anatomically distinct points of origin: (i) afferent collaterals at the peripheral-target organ level, (ii) afferent collaterals arising proximal to the target organ, (iii) from mid-axon where afferents lacking myelin sheaths (C fibers and others following demyelinating injuries) may display crosstalk and respond to local irritation, (iv) the dorsal root ganglion itself, and (v) the central terminals of the afferent in the dorsal horn where local circuits and bulbospinal projections can initiate the so-called dorsal root reflexes, i.e., antidromic traffic in the sensory afferent.

Effects of intraplantar botulinum toxin-B on carrageenan-induced changes in nociception and spinal phosphorylation of GluA1 and Akt.

Increasing evidence suggests that botulinum neurotoxins (BoNTs) delivered into the skin and muscle in certain human and animal pain states may exert antinociceptive efficacy though their uptake and transport to central afferent terminals. Cleavage of soluble N-methylaleimide-sensitive attachment protein receptor by BoNTs can impede vesicular mediated neurotransmitter release as well as transport/insertion of channel/receptor subunits into plasma membranes, an effect that can reduce activity-evoked facilitation. Here, we explored the effects of intraplantar botulinum toxin- B (BoNT-B) on peripheral inflammation and spinal nociceptive processing in an inflammatory model of pain. C57BL/6 mice (male) received unilateral intraplantar BoNT (1 U, 30 µL) or saline prior to intraplantar carrageenan (20 µL, 2%) or intrathecal N-methyl-D-aspartate (NMDA), substance P or saline (5 µL). Intraplantar carrageenan resulted in edema and mechanical allodynia in the injected paw and increased phosphorylation of a glutamate subunit (pGluA1ser845) and a serine/threonine-specific protein kinase (pAktser473) in spinal dorsal horn along with an increased incidence of spinal c-Fos positive cells. Pre-treatment with intraplantar BoNT-B reduced carrageenan evoked: (i) allodynia, but not edema; (ii) pGluA1 and pAkt and (iii) c-Fos expression. Further, intrathecal NMDA and substance P each increased dorsal horn levels of pGluA1 and pAkt. Intraplantar BoNT-B inhibited NMDA, but not substance P evoked phosphorylation of GluA1 and Akt. These results suggest that intraplantar toxin is transported centrally to block spinal activation and prevent phosphorylation of a glutamate receptor subunit and a kinase, which otherwise contribute to facilitated states.

Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -ß (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-ß antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

The search for novel analgesics: targets and mechanisms.

The management of the pain state is of great therapeutic relevance to virtually every medical specialty. Failure to manage its expression has deleterious consequence to the well-being of the organism. An understanding of the complex biology of the mechanisms underlying the processing of nociceptive information provides an important pathway towards development of novel and robust therapeutics. Importantly, preclinical models have been of considerable use in determining the linkage between mechanism and the associated behaviorally defined pain state. This review seeks to provide an overview of current thinking targeting pain biology, the use of preclinical models and the development of novel pain therapeutics. Issues pertinent to the strengths and weaknesses of current development strategies for analgesics are considered.

Modulation of peripheral inflammation by the spinal cord.

The central nervous system, and the spinal cord in particular, is involved in multiple mechanisms that influence peripheral inflammation. Both pro- and anti-inflammatory feedback loops can involve just the peripheral nerves and spinal cord or can also include more complex, supraspinal structures such as the vagal nuclei and the hypothalamic-pituitary axis. Analysis is complicated by the fact that inflammation encompasses a constellation of end points from simple edema to changes in immune cell infiltration and pathology. Whether or not any of these individual elements is altered by any potential mechanism is determined by a complex algorithm including, but not limited to, chronicity of the inflammation, tissue type, instigating stimulus, and state/tone of the immune system. Accordingly, the pharmacology and anatomical substrate of spinal cord modulation of peripheral inflammation are discussed with regard to peripheral tissue type, inflammatory insult (initiating stimulus), and duration of the inflammation.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: potential roles in acute inflammatory pain.

PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The α-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3Kß isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The δ-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the γ-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3Kß antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the γ-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

Chimeric antibody c.8B6 to O-acetyl-GD2 mediates the same efficient anti-neuroblastoma effects as therapeutic ch14.18 antibody to GD2 without antibody induced allodynia.

BACKGROUND: Anti-GD2 antibody is a proven therapy for GD2-positive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients. METHODOLOGY/PRINCIPAL FINDINGS: We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and in vivo in an animal model. CONCLUSION/SIGNIFICANCE: The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6-which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects-provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma.

TNF-α triggers rapid membrane insertion of Ca(2+) permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury.

Excitotoxicity (caused by over-activation of glutamate receptors) and inflammation both contribute to motor neuron (MN) damage in amyotrophic lateral sclerosis (ALS) and other diseases of the spinal cord. Microglial and astrocytic activation in these conditions results in release of inflammatory mediators, including the cytokine, tumor necrosis factor-alpha (TNF-α). TNF-α has complex effects on neurons, one of which is to trigger rapid membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, and in some cases, specific insertion of GluA2 lacking, Ca(2+) permeable AMPA receptors (Ca-perm AMPAr). In the present study, we use a histochemical stain based upon kainate stimulated uptake of cobalt ions ("Co(2+) labeling") to provide the first direct demonstration of the presence of substantial numbers of Ca-perm AMPAr in ventral horn MNs of adult rats under basal conditions. We further find that TNF-α exposure causes a rapid increase in the numbers of these receptors, via a phosphatidylinositol 3 kinase (PI3K) and protein kinase A (PKA) dependent mechanism. Finally, to assess the relevance of TNF-α to slow excitotoxic MN injury, we made use of organotypic spinal cord slice cultures. Co(2+) labeling revealed that MNs in these cultures possess Ca-perm AMPAr. Addition of either a low level of TNF-α, or of the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to the cultures for 48 h resulted in little MN injury. However, when combined, TNF-α+PDC caused considerable MN degeneration, which was blocked by the AMPA/kainate receptor blocker, 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), or the Ca-perm AMPAr selective blocker, 1-naphthyl acetylspermine (NASPM). Thus, these data support the idea that prolonged TNF-α elevation, as may be induced by glial activation, acts in part by increasing the numbers of Ca-perm AMPAr on MNs to enhance injurious excitotoxic effects of deficient astrocytic glutamate transport.

Carrageenan induced phosphorylation of Akt is dependent on neurokinin-1 expressing neurons in the superficial dorsal horn.

BACKGROUND: Paw carrageenan induces activation of phosphatidylinositol 3-kinase (PI-3K) and Akt in dorsal horn neurons in addition to induction of pain behavior. Spinal PI-3K activation is also thought to be required for inflammation-induced trafficking of GluA1, AMPA receptor subunits, into plasma membranes from cytosol. Phosphorylation of Akt has a unique time course. It occurs first in the superficial dorsal horn (0.75 h), then soon dissipates and is followed an hour later by Akt phosphorylation in deeper dorsal horn laminae, primarily lamina V. Initially, we wished to determine if Akt phosphorylation in the deeper laminae were dependent on the presence of lamina I, neurokinin receptor bearing projection neurons. As the study progressed, our aims grew to include the question, whether carrageenan-induced GluA1 subunit trafficking was downstream of Akt phosphorylation. RESULTS: Rats pretreated with spinal saporin conjugated to a stabilized form of substance P had substantial loss of neurons with neurokinin 1 receptors throughout their superficial, but not deep dorsal horns. Animals pre-treated with substance P-saporin exhibited no change in locomotor ability and a small, but significant decrease in carrageenan-induced mechanical allodynia when compared to animals pre-treated with spinal saporin alone. Importantly, carrageenan-induced phosphorylation of Akt was blocked, in the substance P-saporin treated group, throughout the spinal cord grey matter. In marked contrast, carrageenan induced-trafficking of the GluA1 receptor subunit increased equivalently in both treatment groups. CONCLUSIONS: We infer from these data that 1) phosphorylation of Akt in the deep dorsal horn is dependent on prior activation of NK1 receptor bearing cells in superficial dorsal horn, and 2) there are parallel spinal intracellular cascades initiated by the carrageenan injection downstream of PI-3K activation, including one containing Akt and another involving GluA1 trafficking into neuronal plasma membranes that separately lead to enhanced pain behavior. These results imply that the two pathways downstream of PI-3K can be activated separately and therefore should be able to be inhibited independently.

A pilot study of the effects of cannabis on appetite hormones in HIV-infected adult men.

RATIONALE: The endocannabinoid system is under active investigation as a pharmacological target for obesity management due to its role in appetite regulation and metabolism. Exogenous cannabinoids such as tetrahydrocannabinol (THC) stimulate appetite and food intake. However, there are no controlled observations directly linking THC to changes of most of the appetite hormones. OBJECTIVES: We took the opportunity afforded by a placebo-controlled trial of smoked medicinal cannabis for HIV-associated neuropathic pain to evaluate the effects of THC on the appetite hormones ghrelin, leptin and PYY, as well as on insulin. METHODS: In this double-blind cross-over study, each subject was exposed to both active cannabis (THC) and placebo. RESULTS: Compared to placebo, cannabis administration was associated with significant increases in plasma levels of ghrelin and leptin, and decreases in PYY, but did not significantly influence insulin levels. CONCLUSION: These findings are consistent with modulation of appetite hormones mediated through endogenous cannabinoid receptors, independent of glucose metabolism.

Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior.

In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca(2+) permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100microg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5microg) and LY294002; 50 and 100microg) and Akt (Akt inhibitor IV; 3microg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and alpha-motor neurons (peak 45min) and later (peak 2h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.

Monoclonal antibodies against GD(2) ganglioside, such as ch14.18, the human-mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody's ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1mg/kg). Injection of ch14.18 (1mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies.

Behavioral models of pain states evoked by physical injury to the peripheral nerve.

Physical injury or compression of the root, dorsal root ganglion, or peripheral sensory axon leads to well-defined changes in biology and function. Behaviorally, humans report ongoing painful dysesthesias and aberrations in function, such that an otherwise innocuous stimulus will yield a pain report. These behavioral reports are believed to reflect the underlying changes in nerve function after injury, wherein increased spontaneous activity arises from the neuroma and dorsal root ganglion and spinal changes increase the response of spinal projection neurons. These pain states are distinct from those associated with tissue injury and pose particular problems in management. To provide for developing an understanding of the underlying mechanisms of these pain states and to promote development of therapeutic agents, preclinical models involving section, compression, and constriction of the peripheral nerve or compression of the dorsal root ganglion have been developed. These models give rise to behaviors, which parallel those observed in the human after nerve injury. The present review considers these models and their application.

Secondary hyperalgesia in the rat first degree burn model is independent of spinal cyclooxygenase and nitric oxide synthase.

Various animal models of pain are dependent on activation of different glutamate receptor subtypes. First degree burn of the paw elicits a secondary hyperalgesia that is dependent on Ca2+ permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not N-methyl-D-aspartate (NMDA) receptors. The present study takes advantage of that specificity by examining the effects of spinal pretreatments of agents on this secondary hyperalgesia. Rats with indwelling intrathecal catheters were pretreated with agents prior to paw injury. Mechanical withdrawal thresholds were measured before, and for three h after the injury. Spinal pretreatment with cyclooxygenase (10 and 30 microg (S)-(+)-ibuprofen; and 3 and 30 microg ketorolac) and nitric oxide synthase (33 and 100 microg N(G) Nitro-L-arginine methyl ester hydrochloride (L-NAME) and 10 microg thiocitrulline) inhibitors resulted in no specific anti-allodynia. In contrast, ziconotide (0.3, 1.0 and 3 microg), the N-type voltage gated calcium channel antagonist was very effective in blocking burn-induced sensitivity at all doses used. l-type (Diltiazam 230 microg) and P-type (Agatoxin IVA 0.3 microg) calcium channel blockers produced intermediate effects. Thus, cyclooxygenase and nitric oxide synthase are assumed not to be downstream of Ca2+ permeable AMPA receptors. Voltage gated calcium channels blockers could exert their effects either pre- or post-synaptically.

Secondary hyperalgesia in the postoperative pain model is dependent on spinal calcium/calmodulin-dependent protein kinase II alpha activation.

BACKGROUND: Spinally administered non-N-methyl-D-aspartate (NMDA), but not NMDA, receptor antagonists block primary (1 degree) and secondary (2 degrees) mechanical hyperalgesia and spontaneous pain after plantar incision. Hyperalgesia after thermal stimulation is also mediated by non-NMDA, but not NMDA, receptors. Although previous pain behavior studies in the thermal stimulus model demonstrated distinct protein kinase involvement downstream from spinal non-NMDA receptor activation, protein kinase signaling mechanisms have not been examined in the postoperative pain model. In the present study, we investigated whether spinal calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) mediates 1 degree and/or 2 degrees hyperalgesia and spontaneous pain behavior after plantar incision. METHODS: Catheterized rats received a 1 cm incision in the hindpaw and were tested over 2 days for responses to mechanical stimulation adjacent to or 1 cm away from the incision site. Some rats received intrathecal (IT) pretreatment with a CaMKIIalpha inhibitor (14, 34, or 104 nmol KN-93) or vehicle (5% dimethyl sulfoxide in sterile saline). Separate groups received IT 34 nmol or 104 nmol KN-93 and were tested for hindpaw weight bearing. Lumbar spinal cords were extracted 1 h after incision or sham treatment to measure phosphorylated CaMKIIalpha and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid GLUR1-831 in Western immunoblots. RESULTS: Incision increased spinal CaMKIIalpha and GLUR1-831 phosphorylation. Although pretreatment with all doses of IT KN-93 reduced the development of 2 degrees hyperalgesia, only 34 nmol KN-93 appeared to have an effect on 1 degrees hyperalgesia. IT KN-93 did not affect nonevoked pain. CONCLUSION: Spinal sensitization underlying incision-evoked hyperalgesia involves spinal CaMKIIalpha activation and enhanced spinal alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor (AMPA) function.

Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization.

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.

Covariance among age, spinal p38 MAP kinase activation and allodynia.

UNLABELLED: This study examined effects of age (young rats, approximately 35 days, vs mature rats, approximately 75-110 days) on spinal nerve ligation (SNL)-induced tactile allodynia and phosphorylation of p38 (as measured by phospho-p38 MAP kinase [P-p38]) in dorsal root ganglia and spinal cord. Effects of SNL combined with spinal nerve transection also were assessed. Mature rats displayed milder SNL-induced allodynia than young rats. Addition of spinal nerve transection distal to the ligation in older animals resulted in an allodynia comparable to that seen in young animals. In DRG, both groups displayed early (5 h) and late (10 days) peaks in P-p38 following surgery as compared to naïve rats. Tight nerve ligation plus transection had no additional effect on P-p38 levels in DRG. In spinal cord, young rats had increased levels of P-p38 from 5 h to 3 days after SNL. Phosphorylated p38 levels then decreased, with a second peak at 10 days. In contrast, spinal cord from mature rats showed less early p38 phosphorylation, although they also displayed a late 10-day peak. Addition of a transection to the ligation produced restoration of the early peak along with intensification of allodynia. Alterations of spinal P-p38 at early time points thus seem to covary with intensity of tactile allodynia. PERSPECTIVE: Age and modifications to spinal nerve ligation, a common model of neuropathic pain, influence spinal p38 phosphorylation and allodynia. Early levels of spinal P-p38 seem to covary with allodynia intensity. This may mean that small variations of an injury could affect the therapeutic window of a p38 antagonist.