Judith A. Strong

Neuropathic pain: Early spontaneous afferent activity is the trigger

&NA; Intractable neuropathic pain often results from nerve injury. One immediate event in damaged nerve is a sustained increase in spontaneous afferent activity, which has a well‐established role in ongoing pain. Using two rat models of neuropathic pain, the CCI and SNI models, we show that local, temporary nerve blockade of this afferent activity permanently inhibits the subsequent development of both thermal hyperalgesia and mechanical allodynia. Timing is critical—the nerve blockade must last at least 3–5 days and is effective if started immediately after nerve injury, but not if started at 10 days after injury when neuropathic pain is already established. Effective nerve blockade also prevents subsequent development of spontaneous afferent activity measured electrophysiologically. Similar results were obtained in both pain models, and with two blockade methods (200 mg of a depot form bupivacaine at the injury site, or perfusion of the injured nerve just proximal to the injury site with TTX). These results indicate that early spontaneous afferent fiber activity is the key trigger for the development of pain behaviors, and suggest that spontaneous activity may be required for many of the later changes in the sensory neurons, spinal cord, and brain observed in neuropathic pain models. Many pre‐clinical and clinical studies of pre‐emptive analgesia have used much shorter duration of blockade, or have not started immediately after the injury. Our results suggest that effective pre‐emptive analgesia can be achieved only when nerve block is administered early after injury and lasts several days.

Robust Increase of Cutaneous Sensitivity, Cytokine Production and Sympathetic Sprouting in Rats with Localized Inflammatory Irritation of the Spinal Ganglia

We investigated the role and mechanisms of inflammatory responses within the dorsal root ganglion (DRG) in the development of chemogenic pathological pain. DRG inflammation was induced by a single deposit of the immune activator zymosan in incomplete Freunds adjuvant in the epidural space near the L5 DRG via a small hole drilled through the transverse process. After a single zymosan injection, rats developed bilateral mechanical hyperalgesia and allodynia which began by day 1 after surgery, peaked at days 3-7, and lasted up to 28 days. The number of macrophages in ipsilateral and contralateral DRGs increased significantly, lasting over 14 days. Robust glial activation was observed in inflamed ganglia. Cytokine profile analysis using a multiplexing protein array system showed that, in normal DRG, all but interleukin (IL)-5, IL-10 and granulocyte-macrophage colony stimulating factor (GM-CSF) were detectable with concentrations of up to 180 pg/mg protein. Local inflammatory irritation selectively increased IL-1beta, IL-6, IL-18, monocyte chemoattractant protein-1 (MCP-1), and growth-related oncogene (GRO/KC) up to 17-fold, and decreased IL-2 and IL-12 (p70) up to threefold. Inflaming the DRG also remarkably increased the incidence of spontaneous activity of A- and C-fibers recorded in the dorsal root. Many of the spontaneously active A-fibers exhibited a short-bursting discharge pattern. Changes in cytokines and spontaneous activity correlated with the time course of pain behaviors, especially light stroke-evoked tactile allodynia. Finally, local inflammation induced extensive sprouting of sympathetic fibers, extending from vascular processes within the inflamed DRG. These results demonstrate the feasibility of inducing chronic localized inflammatory responses in the DRG in the absence of traumatic nerve damage, and highlight the possible contribution of several inflammatory cytokines/chemokines to the generation of spontaneous activity and development and persistence of chemogenic pathologic pain.

Early Blockade of Injured Primary Sensory Afferents Reduces Glial Cell Activation in Two Rat Neuropathic Pain Models

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Systemic antiinflammatory corticosteroid reduces mechanical pain behavior, sympathetic sprouting, and elevation of proinflammatory cytokines in a rat model of neuropathic pain.

Background:Chronic pain models are commonly defined as either nerve-injury or inflammation models, but recent work suggests inflammatory processes are important in nerve injury–induced pain. Methods:In the rat spinal nerve ligation model, the authors examined effects of systemic corticosteroid triamcinolone acetonide (TA) on the cytokine protein profile and sympathetic sprouting in the axotomized sensory ganglia, excitability of sensory neurons, and mechanical sensitivity. Results:By postoperative day 3, marked increases (5- to 16-fold) in monocyte chemoattractant protein-1, growth-related oncogene (GRO/KC or CXCL1), and interleukin (IL)-6 were observed, whereas IL-4 and IL-2 levels fell more than fourfold. The increased cytokines and number of sympathetic basket formations in the sensory ganglia were reduced toward normal values by TA given starting at the time of injury. Interleukin-4 and IL-2 levels were not restored by TA. Systemic TA also reduced the firing rate and incidence of bursting activity, but not the overall incidence of spontaneous activity, in large- and medium-sized neurons. Mechanical hypersensitivity on postoperative day 3 was reduced by TA, and some effect could still be observed 4 days after cessation of TA. However, starting TA at day 7 was ineffective. Conclusions:Several components of the spinal nerve injury model are responsive to corticosteroid, suggesting inflammatory processes are important in the development of neuropathic pain. The observation that TA was effective when given starting at the time of injury suggests that steroid treatment might alter the development of chronic pain after surgical procedures that involve nerve injury, such as amputation or hernia repair.

The Chemokine CXCL1/Growth Related Oncogene Increases Sodium Currents and Neuronal Excitability in Small Diameter Sensory Neurons

BackgroundAltered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons.ResultsWe report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 – 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS.ConclusionMany studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.

Endomorphins fully activate a cloned human mu opioid receptor.

Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin‐1 and endomorphin‐2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin‐stimulated increase of cAMP in a dose‐dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein‐activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose‐dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [d‐Ala2,N‐Me‐Phe4,Gly‐ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G‐protein‐activated inwardly rectifying potassium (GIRK) channels.

Genotype and smoking history affect risk of levodopa‐induced dyskinesias in Parkinson's disease

Parkinsons disease (PD) patients vary widely in their response to levodopa treatment, and this variation may be partially genetic in origin. We determined whether particular dopamine and opioid receptor polymorphisms were associated with risk of earlier onset of dyskinesia side effects during levodopa therapy. Smoking status was also examined. The 92 subjects were recruited from the movement disorders clinic of a neurology practice associated with a medical school. All were adult‐onset PD patients who had been taking levodopa at least 5 years and/or had developed levodopa‐induced dyskinesia. Carrying the G‐allele of the A118G single nucleotide coding region polymorphism of the mu opioid receptor, as well as a history of never smoking, were independently associated with increased risk of earlier onset of dyskinesia (P = 0.05 and 0.02, respectively). One genotype of the D2 dopamine receptor intronic dinucleotide repeat polymorphism (14 repeats/15 repeats, with frequency of 6%) was also associated with earlier dyskinesia (P = 0.003). History of smoking has previously been associated with reduced risk of developing PD. Our results suggest that smoking history may also influence the response to levodopa, with contribution comparable to those of individual genes including the mu opioid receptor and D2 dopamine receptor.

Local inflammation in rat dorsal root ganglion alters excitability and ion currents in small-diameter sensory neurons.

Background: Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity. Methods: The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3–5 days) of L4 and L5 ganglia altered voltage-gated K+ and Na+ currents. Results: Tetrodotoxin-sensitive Na+ currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na+ currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K+ currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold. Conclusions: Neuronal inflammation per se, in the absence of nerve injury, causes large increases in Na channel density and enhanced excitability. The unusual finding of increased K+ current may reflect regulation of excitability in the face of such large increases in Na+ current.

Knockdown of sodium channel NaV1.6 blocks mechanical pain and abnormal bursting activity of afferent neurons in inflamed sensory ganglia.

&NA; Knocking down expression of the sodium channel isoform NaV1.6 in lumbar sensory ganglia blocked development of pain behaviors and neuronal hyperexcitability induced by local inflammation of the ganglion. &NA; Inflammatory processes in the sensory ganglia contribute to many forms of chronic pain. We previously showed that local inflammation of the lumbar sensory ganglia rapidly leads to prolonged mechanical pain behaviors and high levels of spontaneous bursting activity in myelinated cells. Abnormal spontaneous activity of sensory neurons occurs early in many preclinical pain models and initiates many other pathological changes, but its molecular basis is not well understood. The sodium channel isoform NaV1.6 can underlie repetitive firing and excitatory persistent and resurgent currents. We used in vivo knockdown of this channel via local injection of siRNA to examine its role in chronic pain after local inflammation of the rat lumbar sensory ganglia. In normal dorsal root ganglion (DRG), quantitative polymerase chain reaction showed that cells capable of firing repetitively had significantly higher relative expression of NaV1.6. In inflamed DRG, spontaneously active bursting cells expressed high levels of NaV1.6 immunoreactivity. In vivo knockdown of NaV1.6 locally in the lumbar DRG at the time of DRG inflammation completely blocked development of pain behaviors and abnormal spontaneous activity, while having only minor effects on unmyelinated C cells. Current research on isoform‐specific sodium channel blockers for chronic pain is largely focused on NaV1.8 because it is present primarily in unmyelinated C fiber nociceptors, or on NaV1.7 because lack of this channel causes congenital indifference to pain. However, the results suggest that NaV1.6 may be a useful therapeutic target for chronic pain and that some pain conditions may be mediated primarily by myelinated A fiber sensory neurons.

Function of γ-Aminobutyric Acid Receptor/Channel ρ1Subunits In Spinal Cord

γ-Aminobutyric acid (GABA) receptor/channel ρ1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel ρ1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel ρ1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel ρ1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel ρ1 subunit mutant mice (rho1-/-). GABA receptor/channel ρ1 subunit expression in the rho1-/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho1-/- mice. A decreased threshold for mechanical pain in the rho1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel ρ1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.