A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain

Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f);Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.     

Sensory neuron targeting by self-complementary AAV8 via lumbar puncture for chronic pain

Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin gene-related peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-beta-endorphin (ppbetaEP) led to significant (P < 0.0001) reversal of mechanical allodynia for >/=3 months. The antiallodynic effect could be reversed by the mu-opioid antagonist naloxone 4 months after gene transfer (P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with ppbetaEP was equally effective (P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.     

Targeting voltage-gated sodium channels for treatment for chronic visceral pain

Voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability, and their abnormal activity is related to several pathological processes, including cardiac arrhythmias, epilepsy, neurodegenerative diseases, spasticity and chronic pain. In particular, chronic visceral pain, the central symptom of functional gastrointestinal disorders such as irritable bowel syndrome, is a serious clinical problem that affects a high percentage of the world population. In spite of intense research efforts and after the dedicated decade of pain control and research, there are not many options to treat chronic pain conditions. However, there is a wealth of evidence emerging to give hope that a more refined approach may be achievable. By using electronic databases, available data on structural and functional properties of VGSCs in chronic pain, particularly functional gastrointestinal hypersensitivity, were reviewed. We summarize the involvement and molecular bases of action of VGSCs in the pathophysiology of several organic and functional gastrointestinal disorders. We also describe the efficacy of VGSC blockers in the treatment of these neurological diseases, and outline future developments that may extend the therapeutic use of compounds that target VGSCs. Overall, clinical and experimental data indicate that isoform-specific blockers of these channels or targeting of their modulators may provide effective and novel approaches for visceral pain therapy.     

Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells

The present study evaluated the role of N-methyl-d-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E₂ (PGE₂), which was significantly attenuated by injection of the NMDAR antagonist d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE₂-induced hyperalgesia in thepaw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE₂-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE₂-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.The involvement of excitatory amino acids in the transmission of the nociceptive information from the primary afferent neurons to the spinal cord is, at present, supported by an immense number of studies (1–3). In fact, a role of the amino acid glutamate (GLU) as a synaptic mediator has been demonstrated by electrophysiological experiments that showed its release by stimulation and consequent increase in the probability that the target cell will fire an action potential (3, 4). In addition, glutamatergic receptors, especially the N-methyl-d-aspartate receptor (NMDAR), detected throughout the entire nervous system (5, 6), have been associated with the development and maintenance of spinal cord neuron sensitization (7, 8). Stimulation of spinal neurons by GLU through NMDARs was also associated to inflammatory processes (3, 9). For instance, sensitization of spinal nerves has been frequently related to the wind-up phenomenon, an increase in the electrical activity of spinal cord neurons and pain sensation independent of primary nociceptor input (10).The detection of glutamatergic receptors in the presynaptic membrane of afferent fibers associated with nociception and hyperalgesia raised the hypothesis that GLU released into the synaptic cleft could also activate receptors expressed in the central terminals of primary afferent neurons (11, 12). Moreover, the possibility that GLU is involved in nociceptor sensitization was strengthened by reports of the expression of NMDARs in nociceptors (13) and their role in acute and persistent inflammatory mechanical hyperalgesia in the rat paw (13, 14). In addition, the glutamatergic role in the sensitization of the primary sensory nociceptive neuron is indirectly supported by GLU-induced neuronal depolarization (4).Previous studies from our group have stressed the importance of GLU in the sensitization of the primary sensory neuron with the introduction of the concept of glutamate retrograde sensitization (15–17), focusing especially on the role of NMDARs in this process. In those studies, we demonstrated that the intrathecal (i.t.) injection of GLU, NMDA, or AMPA (another ionotropic glutamatergic receptor agonist) induced mechanical hyperalgesia in the rat hind paws in a dose-dependent manner. However, because the intraplantar (i.pl.) injection of either morphine or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) [previously demonstrated, along with dipyrone and diclofenac, to cause local analgesia (18–20)], ipsilaterally antagonized hyperalgesia induced by i.t. NMDA, but not AMPA, we have proposed that the primary nociceptive neuron is the main site of action of the i.t.-injected NMDA. Moreover, i.t. administration of the NMDA antagonists d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) or MK801, but not of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited the hyperalgesia induced by i.pl. injection of prostaglandin E₂ (PGE₂) or carrageenan. These results suggested that the maintenance of nociceptor sensitization by inflammatory stimuli depends on a continuous spinal release of GLU that acts on presynaptic NMDA-type receptors (15, 17). Furthermore, the proposal of a GLU-dependent retrograde sensitization of the primary sensory neuron was supported by the additional demonstration that the selective knockdown of NaV1.8 (SNS/PN3) sodium channels [described to be expressed only in nociceptors and associated with inflammatory sensitization (21)] in the dorsal root ganglion (DRG) by i.t. treatment with antisense oligodeoxynucleotides (AS-ODNs) abolished the mechanical hyperalgesia induced by i.t. administration of NMDA (16).Those data, and the fact that the cerebrospinal fluid, in addition to being in contact with the spinal cord, also bathes part of the DRG (22, 23), led to the suggestion that i.t.-injected drugs could directly reach the DRGs of the primary sensory neurons (24). Thus, considering the methodology used to demonstrate the retrograde sensitization (15, 17), we raised the possibility that i.t-administered drugs actually targeted receptors located at the DRG and not at the presynaptic membrane in those experiments.In the present study, we investigated whether NMDARs expressed at the DRG participate in the modulation of the excitability of primary afferent neurons during inflammatory nociceptive sensitization. We tested whether the intraganglionar (i.gl.) injection of NMDA, into the fifth lumbar (L5)-DRG, had a direct hyperalgesic effect and whether i.gl. injection of the NMDAR antagonist d-AP-5 could inhibit the nociceptor sensitization induced by i.t. NMDA or i.pl. PGE₂ administration. The specificity of ganglionar and spinal GLU receptors was tested by comparing the effects of selective antagonists for NMDA and AMPA receptors. We also evaluated the functional expression of NMDARs in DRG primary cultures in experiments using a fluorescent calcium indicator. Finally, we investigated the individual relevance of each subunit of NMDAR for the development of i.pl. PGE₂-induced hyperalgesia and in the activation of NMDARs in DRG cultures.     

Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons

Purinergic ionotropic P2X7 receptors (P2X7Rs) are closely associated with excitotoxicity and nociception. Inhibition of P2X7R activation has been considered as a potentially useful strategy to improve recovery from spinal cord injury and reduce inflammatory damage to trauma. The physiological functions of P2X7Rs, however, are poorly understood, even though such information is essential for making the P2X7R an effective therapeutic target. We show here that P2X7Rs in satellite cells of dorsal root ganglia tonically inhibit the expression of P2X3Rs in neurons. Reducing P2X7R expression using siRNA or blocking P2X7R activity by antagonists elicits P2X3R up-regulation, increases the activity of sensory neurons responding to painful stimuli, and evokes abnormal nociceptive behaviors in rats. Thus, contrary to the notion that P2X7R activation is cytotoxic, P2X7Rs in satellite cells play a crucial role in maintaining proper P2X3R expression in dorsal root ganglia. Studying the mechanism underlying the P2X7R-P2X3R control, we demonstrate that activation of P2X7Rs evokes ATP release from satellite cells. ATP in turn stimulates P2Y1 receptors in neurons. P2Y1 receptor activation appears to be necessary and sufficient for the inhibitory control of P2X3R expression. We further determine the roles of the P2X7R-P2Y1-P2X3R inhibitory control under injurious conditions. Activation of the inhibitory control effectively prevents the development of allodynia and increases the potency of systemically administered P2X7R agonists in inflamed rats. Thus, direct blocking P2X7Rs, as proposed before, may not be the best strategy for reducing pain or lessening neuronal degeneration because it also disrupts the protective function of P2X7Rs.     

A possible role for saliva as a diagnostic fluid in patients with chronic pain

Objectives:

The focus of this review was on proteins and peptides found in saliva. Of greatest interest were those neuropeptides relevant to nociception and to the pathogenesis of chronic pain syndromes. An additional goal was to develop a standardized protocol to collect saliva for laboratory assessment.

Methods:

Data were obtained through discussion with experts at the medical schools in San Antonio and Heidelberg and a Medline literature search involving all relevant studies from 1966 to 1997. The literature search was based on the following key terms: saliva, serotonin, neuropeptide, substance P (SP), calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF).

Results:

The mean concentration of SP in the saliva of healthy normal controls ranged from 9.6 to 220 pg/mL. Generally, the concentration of SP was approximately three times higher in saliva than in plasma. In a number of painful conditions, particularly tension headache, substantial elevations of salivary SP were found. Mean values for salivary CGRP in healthy controls were approximately 22 pmol/L and were significantly elevated in patients with migraine attacks or cluster headache. There were no data to indicate prior quantitative determination of NGF in human saliva.

Conclusions:

After sampling and processing techniques have been standardized,measurement of neuropeptides in human saliva could provide a valuable tool for the study of patients with chronic painful disorders such as rheumatoid arthritis, osteoarthritis, and even fibromyalgia syndrome.     

A model of neural cross-talk and irritation in the pelvis: implications for the overlap of chronic pelvic pain disorders

BACKGROUND & AIMS: Irritable bowel syndrome, interstitial cystitis, and other chronic pelvic pain (CPP) disorders often occur concomitantly. Neural cross-talk may play a role in the overlap of CPP disorders via the convergence of pelvic afferents. We investigated the hypothesis that afferent irritation of one pelvic organ may adversely influence and sensitize another via neural interactions. METHODS: We measured pelvic organ smooth muscle and striated muscle reflexes during micturition and colorectal distention (CRD) in urethane-anesthetized rats. The effects of acute cystitis on distal colonic sensory thresholds to CRD and the effects of acute colonic irritation on micturition parameters were assessed. RESULTS: External urethral sphincter (EUS) electromyography (EMG) was typical for the rat, with phasic firing during micturition. External anal sphincter EMG also showed phasic firing during micturition in synchrony with EUS activity but, in addition, showed both tonic bursts and phasic firing independent of EUS activity. Before bladder irritation, graded CRDs to 40 cm H2O produced no notable changes in abdominal wall EMG activity. Following acute bladder irritation, dramatic increases in abdominal wall EMG activity in response to CRD were observed at much lower distention pressures, indicating colonic afferent sensitization. Analogously, following acute colonic irritation, bladder contraction frequency increased 66%, suggesting sensitization of lower urinary tract afferents. CONCLUSIONS: We report compelling evidence of bidirectional cross-sensitization of the colon and lower urinary tract in a novel experimental model. This cross-sensitization may account for the substantial overlap of CPP disorders; however, further studies are needed to fully characterize these pathways.     

Trypsin mediates nociception via the proteinase-activated receptor 2: a potentially novel role in pancreatic pain

BACKGROUND & AIMS: The pathogenesis of pain in pancreatitis remains poorly understood. We hypothesized that trypsin, a key inflammatory mediator in this condition, can also activate nociceptive neurons via the proteinase-activated receptor 2. METHODS: Double immunohistochemical staining of T8 to T12 dorsal root ganglia sections was performed with antibodies against proteinase-activated receptor 2 and vanilloid receptor 1, a marker for primary nociceptive neurons. In vivo nociceptive activity was measured by FOS immunoreactivity in thoracic spinal dorsal horn segments after intrapancreatic administration of proteinase-activated receptor 2 agonists. Pain behavior was assessed by visceromotor reflex activity in response to noxious stimulation of the pancreas with proteinase-activated receptor 2 agonists. RESULTS: Proteinase-activated receptor 2 was expressed by virtually all nociceptive neurons in thoracic dorsal root ganglia. Intraductal trypsin, in subinflammatory concentrations, activated spinal dorsal horn neurons in a dose-dependent manner, as measured by FOS expression. Both trypsin and a proteinase-activated receptor 2-specific peptide agonist induced a behavioral pain response when infused into the pancreatic duct of awake rats. Preinfusion of the pancreatic duct with proteinase-activated receptor 2-specific activating peptide desensitized the response to trypsin. CONCLUSIONS: Our findings suggest a novel proteinase-activated receptor 2-mediated role for trypsin in the pathogenesis of pancreatic pain and one that is independent of its inflammatory effect.     

The role of the p75 neurotrophin receptor in the morphology of dorsal root ganglion cells in streptozotocin diabetic mice: effects of sciatic nerve crush

Aims/hypothesis Neuronal dorsal root ganglion (DRG) cells seem to be vulnerable in diabetes. The aim of this study was to determine whether the p75 neurotrophin receptor stimulates perikaryal shrinkage and neuronal death, and further accelerates neuronal DRG cell loss after axotomy in a mouse model of diabetes.Methods Nine non-diabetic BALB/c p75^+/+ mice, seven diabetic BALB/c p75^+/+ mice, nine non-diabetic p75^–/– mice and nine diabetic p75^–/– mice received a unilateral sciatic nerve crush 1 to 2 days after streptozotocin treatment. Tissues were fixed 28 days later by vascular perfusion, and the volume and number of the fifth lumbar DRG neurons were obtained using assumption-free stereological techniques.Results In diabetic p75^+/+ mice there was a 9% reduction in the perikaryal volume of the DRG A cells (p<0.05) and a 10% reduction in the perikaryal volume of the DRG B cells (p<0.05) on the non-crushed side compared with in non-diabetic p75^+/+ mice. However, neuronal cell number was not reduced. Conversely, no perikaryal shrinkage of A cells or B cells occurred on the non-crushed side in diabetic p75^–/– mice, and no neuronal cell loss was observed. Following nerve crush, there was a loss of B cells in non-diabetic p75^+/+ mice (37±6%) and in diabetic p75^+/+ mice (36±4%). In non-diabetic p75^–/– mice, no neuronal cell loss occurred after crush, whereas in diabetic p75^–/– mice the loss of B cells (14±4%) was small but significant (p<0.02).Conclusions/interpretation In experimental diabetes the p75 neurotrophin receptor is involved in neuronal DRG cell body shrinkage without loss of neuronal DRG cells. Following sciatic nerve crush, DRG cell loss is not accelerated in diabetic p75^+/+ mice.     

The role of chronic pain and current substance use in predicting negative social support among disadvantaged persons living with HIV/AIDS

Chronic pain and substance use can strain the supportive relationships of persons with serious chronic illness, which may increase the likelihood of receiving negative, rather than positive, social support from informal caregivers and social network members. To our knowledge, this is the first study to longitudinally examine the effects of chronic pain and substance use on negative social support. The sample (N?=?383) comprised disadvantaged, primarily African-American, persons living with HIV/AIDS with a history of injection drug use, 32.4% of whom reported frequent or constant pain in the prior 6 months. Using factor analysis and structural equation modeling, current substance use and greater levels of chronic pain positively predicted negative social support 12 months later, after controlling for baseline negative support, viral load, age and sex. We also found a significant interaction effect such that among those not using substances, there was a significant positive association between pain and negative support, but no such association among those currently using substances. The findings emphasize the importance of treatment of chronic pain and substance use in the supportive functioning of social networks of a disadvantaged population with serious chronic conditions and persistent health disparities.