Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors

<p>Abstract</p> <p>Background</p> <p>Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.</p> <p>Results</p> <p>Focal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP-10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons derived from LPC-injured animals responded with increased [Ca²⁺]_ifollowing exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-<b>[R]</b>) or its inactive enantiomer (CCR2 RA-<b>[S]</b>) by intraperitoneal (i.p.) injection. CCR2 RA-[<b>R</b>] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia.</p> <p>Conclusion</p> <p>These results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain.</p

Chemokines and pain mechanisms

The development of new therapeutic approaches to the treatment of painful neuropathies requires a better understanding of the mechanisms that underlie the development of these chronic pain syndromes. It is now well established that astrocytic and microglial cells modulate the neuronal mechanisms of chronic pain in spinal cord and possibly in the brain. In animal models of neuropathic pain following peripheral nerve injury, several changes occur at the level of the first pain synapse between the central terminals of sensory neurons and second order neurons. These neuronal mechanisms can be modulated by pronociceptive mediators released by non neuronal cells such as microglia and astrocytes which become activated in the spinal cord following PNS injury. However, the signals that mediate the spread of nociceptive signaling from neurons to glial cells in the dorsal horn remain to be established. Herein we provide evidence for two emerging signaling pathways between injured sensory neurons and spinal microglia: chemotactic cytokine ligand 2 (CCL2)/CCR2 and cathepsin S/CX3CL1 (fractalkine)/CX3CR1. We discuss the plasticity of these two chemokine systems at the level of the dorsal root ganglia and spinal cord demonstrating that modulation of chemokines using selective antagonists decrease nociceptive behavior in rodent chronic pain models. Since up-regulation of chemokines and their receptors may be a mechanism that directly and/or indirectly contributes to the development and maintenance of chronic pain, these molecular molecules may represent novel targets for therapeutic intervention in sustained pain states

Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy

<p>Abstract</p> <p>Painful distal sensory polyneuropathy (DSP) is the most common neurological complication of HIV1 infection. Although infection with the virus itself is associated with an incidence of DSP, patients are more likely to become symptomatic following initiation of nucleoside reverse transcriptase inhibitor (NRTI) treatment. The chemokines monocyte chemoattractant protein-1 (MCP1/CCL2) and stromal derived factor-1 (SDF1/CXCL12) and their respective receptors, CCR2 and CXCR4, have been implicated in HIV1 related neuropathic pain mechanisms including NRTI treatment in rodents. Utilizing a rodent model that incorporates the viral coat protein, gp120, and the NRTI, 2'3'-dideoxycytidine (ddC), we examined the degree to which chemokine receptor signaling via CCR2 and CXCR4 potentially influences the resultant chronic hypernociceptive behavior. We observed that following unilateral gp120 sciatic nerve administration, rats developed profound tactile hypernociception in the hindpaw ipsilateral to gp120 treatment. Behavioral changes were also present in the hindpaw contralateral to the injury, albeit delayed and less robust. Using immunohistochemical studies, we demonstrated that MCP1 and CCR2 were upregulated by primary sensory neurons in lumbar ganglia by post-operative day (POD) 14. The functional nature of these observations was confirmed using calcium imaging in acutely dissociated lumbar dorsal root ganglion (DRG) derived from gp120 injured rats at POD 14. Tactile hypernociception in gp120 treated animals was reversed following treatment with a CCR2 receptor antagonist at POD 14. Some groups of animals were subjected to gp120 sciatic nerve injury in combination with an injection of ddC at POD 14. This injury paradigm produced pronounced bilateral tactile hypernociception from POD 14–48. More importantly, functional MCP1/CCR2 and SDF1/CXCR4 signaling was present in sensory neurons. In contrast to gp120 treatment alone, the hypernociceptive behavior associated with the injury plus drug combination was only effectively reversed using the CXCR4 antagonist AMD3100. These studies indicate that the functional upregulation of CCR2 and CXCR4 signaling systems following a combination of gp120 and an NRTI are likely to be of central importance to associated DSP and may serve as potential therapeutic targets for treatment of this syndrome.</p

CCR2 receptor antagonist (CCR2 RA-) administration reversed existing nociceptive behavior

<p><b>Copyright information:</b></p><p>Taken from "Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors"</p><p>http://www.molecularpain.com/content/3/1/38</p><p>Molecular Pain 2007;3():38-38.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2228278.</p><p></p> Animals were subjected to a nerve demyelination injury on day 0 and nociceptive behavior was assessed for 28 days. On days 14 and 28 post-surgery, animals received 5 mg/kg CCR2 RA-[] or 5 mg/kg of its inactive enantiomer, (CCR2 RA-[], or saline by intraperitoneal injection, and behavioral responses were tested 1 h later. Administration of the CCR2 RA-[] to focal nerve demyelination injured rats resulted in a significant bilateral increase of mN force required to elicit a paw withdrawal compared with vehicle-treated controls and animals subjected to CCR2 RA-[]. Nociceptive behavior in vehicle-treated controls and animals subjected to CCR2 RA-[] differed significantly from day 0 pre-injury baseline responses (*p < 0.01). Data represent means ± SE

Colocalization of IP-10 immunoreactivity (-ir) and isolectin B4 (IB)-binding neurons in the lumbar DRG of naïve rat and rats subjected to LPC-induced nerve injury

<p><b>Copyright information:</b></p><p>Taken from "Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors"</p><p>http://www.molecularpain.com/content/3/1/38</p><p>Molecular Pain 2007;3():38-38.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2228278.</p><p></p> IB-binding in rat DRG neurons distinguishes a population of C-fiber nociceptors. ) The majority of IP-10-ir cells were limited to medium diameter neurons in the lumbar DRG from vehicle-treated rats (red arrows). ) IB-binding small diameter presumptive nociceptors (green arrows) did not colocalize with IP-10-ir lumbar DRG neurons from vehicle-treated rodents at POD7 (, merged images). ) Lumbar DRG ipsilateral to focal nerve demyelination exhibited numerous medium and small diameter IP-10-ir neurons at POD7. Limited numbers of neurons were positive for both IP-10-ir (, red arrows) and IB4-binding (, green arrows) on POD7 (, merged images). ) Many IP-10-ir neurons (red arrows) colocalized with IB-binding neurons (, green arrows) at POD14 (, merged images). Yellow arrows indicate colocalized cells. Scale bar is 100 μm

Mean threshold force required for paw withdrawal to Von Frey stimulation at 1, 3, 7, 14, 21, 28, 35 and 42 days following LPC-induced focal nerve demyelination

<p><b>Copyright information:</b></p><p>Taken from "Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors"</p><p>http://www.molecularpain.com/content/3/1/38</p><p>Molecular Pain 2007;3():38-38.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2228278.</p><p></p> Each data point is the mean threshold (± SE) force on the hindpaw ipsilateral (black circle) or contralateral (white circle) to the focal nerve injury site eliciting a withdrawal response (n = 10). Reduced behavioral thresholds for the hindpaw ipsilateral to the nerve lesion were significantly different from pre-operative baseline on postoperative days 1–28. The threshold force for the hindpaw contralateral to the nerve lesion did not reach significance until postoperative day 3, and significant differences were observed until postoperative day 28. The time course of sham injury (n = 6) is also represented but did not differ from the uninjured animals. Analysis was performed using two-way ANOVA followed by the Bonferroni post-hoc pair-wise comparisons (*p < 0.01).LPC-induced focal nerve demyelination did not produce changes in thermal responses as assessed by the Hargreaves test. Each bar is the mean withdrawal latency (± SE) of the hindpaw ipsilateral (white bar) or contralateral (black bar) to the focal nerve demyelination injury at postoperative day 7 and 14 (n = 10)

Expression of CCR2 mRNA and protein immunoreactivity in rat lumbar DRG ipsilateral to focal nerve demyelination

<p><b>Copyright information:</b></p><p>Taken from "Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors"</p><p>http://www.molecularpain.com/content/3/1/38</p><p>Molecular Pain 2007;3():38-38.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2228278.</p><p></p> ) Lumbar DRG removed from vehicle-treated animals at POD7 did not exhibit CCR2 mRNA expression (n = 5). ) Many lumbar DRG neurons in vehicle-treated rats sensory neurons were positive for isolectin IB, a neuronal phenotype that distinguishes some C-fiber nociceptors (green cells). There was no evidence of CCR2 protein expression in sham animals (n = 5). ) Lumbar DRG neurons from nerve-injured rats on POD7 exhibited CCR2 mRNA transcripts in some small and medium diameter neurons (black arrows). Open black arrowhead indicates a neuron without CCR2 mRNA transcripts (n = 4). ) Lumbar DRG neurons from a rat subjected to focal nerve demyelination exhibited few CCR2 immunopositive (white arrows) sensory neurons (n = 4). ) Many lumbar DRG neurons on POD14 exhibited CCR2 mRNA transcripts (black arrows). Open arrowhead indicates non-labeled neuron. ) CCR2 immunoreactivity was present in an increased number of neurons at POD14 (white arrows; n = 5). Scale bar is; 30 μm (, ), 50 μm (, , ), and 100 μm ()

Chemokines increased Ca2+i levels in acutely isolated rat DRG cells following focal demyelination injury

<p><b>Copyright information:</b></p><p>Taken from "Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors"</p><p>http://www.molecularpain.com/content/3/1/38</p><p>Molecular Pain 2007;3():38-38.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2228278.</p><p></p> The figure shows examples of responses of cells acutely isolated from rat DRGs ipsilateral to the nerve injury at various days after a focal demyelination injury. Under normal conditions, cells rarely respond to any chemokine but did respond to other stimuli such as high K or ATP (). However, there was an increased responsiveness of the cells, the majority of which could be characterized as neurons, between post-operative days 14–28 (). The frequency of the responses to chemokines returned to approximately the same level as control animals by post-operative day 35 (). For all experiments, MCP-1 (), IP-10 (), RANTES (), SDF1 () were applied at a concentration of 100 nM. Capsaicin (), high K () and ATP () were applied at concentrations of 100 nM, 50 mM and 100 uM, respectively