Junhui Du

Peripheral and central sensitization in remote spinal cord regions contribute to central neuropathic pain after spinal cord injury

ABSTRACT Central neuropathic pain (CNP) developing after spinal cord injury (SCI) is described by the region affected: above‐level, at‐level and below‐level pain occurs in dermatomes rostral, at/near, or below the SCI level, respectively. People with SCI and rodent models of SCI develop above‐level pain characterized by mechanical allodynia and thermal hyperalgesia. Mechanisms underlying this pain are unknown and the goals of this study were to elucidate components contributing to the generation of above‐level CNP. Following a thoracic (T10) contusion, forelimb nociceptors had enhanced spontaneous activity and were sensitized to mechanical and thermal stimulation of the forepaws 35 days post‐injury. Cervical dorsal horn neurons showed enhanced responses to non‐noxious and noxious mechanical stimulation as well as thermal stimulation of receptive fields. Immunostaining dorsal root ganglion (DRG) cells and cord segments with activating transcription factor 3 (ATF3, a marker for neuronal injury) ruled out neuronal damage as a cause for above‐level sensitization since few C8 DRG cells expressed AFT3 and cervical cord segments had few to no ATF3‐labeled cells. Finally, activated microglia and astrocytes were present in thoracic and cervical cord at 35 days post‐SCI, indicating a rostral spread of glial activation from the injury site. Based on these data, we conclude that peripheral and central sensitization as well as reactive glia in the uninjured cervical cord contribute to CNP. We hypothesize that reactive glia in the cervical cord release pro‐inflammatory substances which drive chronic CNP. Thus a complex cascade of events spanning many cord segments underlies above‐level CNP.

N-methyl-D-aspartate-induced excitation and sensitization of normal and inflamed nociceptors.

The present study investigates the contribution of peripheral N-methyl-D-aspartate (NMDA) receptors to acute nociception and persistent inflammatory pain in the rat. Immunohistochemical localization of the NMDA receptor one (NMDAR1) subunit demonstrates that 47% of unmyelinated axons in the normal digital nerve are positively labeled. In concert with the overall progression of inflammation following injection of complete Freunds adjuvant (CFA) in the hind paw, a significant increase in the proportion of NMDAR1-labeled unmyelinated digital axons occurs at 2 and 7, but not 14 days following hind-paw inflammation. In behavioral studies, we confirm an increased mechanical sensitivity in CFA-injected hind paws. Furthermore, activation of NMDA receptors following intraplantar NMDA (1.0 mM) in normal animals results in a mechanical sensitivity similar to that observed in inflamed animals. Conversely, a low concentration of NMDA (0.5 mM) that has little affect on mechanical thresholds in normal animals produces a significant increase in mechanical sensitivity in the inflamed state. CFA-induced mechanical sensitivity involves NMDA-receptor activation demonstrated by the observation that injection of MK-801 alone into the inflamed hind paw returns mechanical sensitivity to normal (pre-inflammation) levels. In single-unit studies, there is a dose-dependent increase in NMDA-induced nociceptor activity in both normal and inflamed skin, but the amount of NMDA required to induce activation is reduced in inflamed skin. In addition, NMDA-induced discharge rates and percentage of NMDA-activated nociceptors are significantly increased in inflamed compared with normal skin, and this activation can be blocked by co-administration of MK-801. Exposure of nociceptors in normal skin to 1 mM NMDA sensitizes the units to reapplication of NMDA and to heat. Nociceptors that demonstrate sensitization to heat in persistent inflammation show an enhanced sensitization when exposed to exogenous NMDA. Thus, peripheral NMDA receptors not only play an important role in modulating the responses of nociceptors in normal skin, but their upregulation and activation on peripheral nociceptors contributes significantly to the mechanical sensitivity and heat sensitization that accompanies persistent inflammation.

Glutamate-induced excitation and sensitization of nociceptors in rat glabrous skin

&NA; Anatomical studies demonstrate the presence of glutamate receptors on unmyelinated axons in peripheral cutaneous nerves. Pharmacological studies show that intraplantar injection of glutamate or glutamate agonists in the glabrous skin results in nociceptive behaviors. The present study describes a novel in vitro skin‐nerve preparation using the glabrous skin from the rat hindpaw. In the first series of experiments, recordings were obtained from 141 fibers that responded to a strong mechanical search stimulus. Based on their conduction velocity they were classified as C (27%), A&dgr; (28%) and A&bgr; (45%) fibers. The C and A&dgr; fibers typically exhibited sustained firing during suprathreshold mechanical stimuli whereas both rapidly (66%) and slowly (34%) adapting responses were obtained from A&bgr; fibers. Noxious heat excited 46% of the C fibers but only 12% of the A&dgr; units. In another series of experiments application of an ascending series of glutamate concentrations (10, 100, 300, and 1000 &mgr;M) to A&dgr; (n=14) and C (n=19) nociceptors resulted in a significant excitation of 43% (6/14) A&dgr; fibers and 68% (13/19) C fibers. At these concentrations, there was no excitation of A&bgr; units (n=13). Superfusion of the receptive fields of either mechanoheat‐sensitive A (AMH, n=10) or C fibers (CMH, n=12) for 2 min with 300 &mgr;M glutamate resulted in sensitization of 90% (9/10) AMH and 92% (11/12) CMH fibers to subsequent thermal stimulation. This was evidenced by a significant (1) decrease in thermal threshold for activation, (2) increase in discharge rate, and (3) increase in peak instantaneous frequencies during the second heat trial. Glutamate‐induced sensitization to heat occurred in the absence of either a glutamate‐induced excitation or an initial heat response. Exposure of A&dgr; or C fibers to glutamate did not result in a decrease in von Frey thresholds. These data provide a physiological basis for the nociceptive behaviors that arise following intraplantar injection of glutamate or glutamate agonists. Furthermore, demonstration of glutamate‐induced excitation and heat sensitization of nociceptors indicates that local or topical administration of glutamate receptor antagonists may have therapeutic potential for the treatment of pain.

Somatostatin receptors on peripheral primary afferent terminals: Inhibition of sensitized nociceptors

&NA; Somatostatin (SST) is in primary afferent neurons and reduces vascular and nociceptive components of inflammation. SST receptor (SSTR) agonists provide analgesia following intrathecal or epidural administration in humans, but neurotoxicity in the central nervous system (CNS) has been reported in experimental animals. With the rationale that targeting peripheral SSTRs would provide effective analgesia while avoiding CNS side effects, the goals of the present study are to investigate the presence of SSTRs on peripheral primary afferent fibers and determine the behavioral and physiological effects of the SST agonist octreotide (OCT) on formalin‐induced nociception and bradykinin‐induced primary afferent excitation and sensitization in the rat. The results demonstrate that: (1) SSTR2as are present on 11% of peripheral primary afferent sensory fibers in rat glabrous skin; (2) intraplantar injection of OCT reduces formalin‐induced nociceptive behaviors; (3) OCT reduces, in a dose‐dependent fashion, responses to thermal stimulation in C‐mechanoheat sensitive fibers; and (4) OCT reduces the responses of C‐mechanoheat fibers to bradykinin‐induced excitation and sensitization to heat. Each of these actions can be reversed following co‐injection of OCT with the SSTR antagonist cyclo‐somatostatin (c‐SOM). Thus, activation of peripheral SSTRs reduces both inflammatory pain and the activity of sensitized nociceptors, avoids deleterious CNS side effects and may be clinically useful in the treatment of pain of peripheral origin.

Metabotropic glutamate 1α receptors on peripheral primary afferent fibers : their role in nociception

Several lines of evidence indicate that Group I metabotropic glutamate (mGlu) 1alpha receptors are involved in the processing of nociceptive information in the spinal cord. The goals of the present study are to document the role of mGlu1alpha receptors in peripheral nociception. To accomplish this we investigate the presence of mGlu1alpha receptors on peripheral primary afferent fibers and determine the behavioral effects of (S)-3,5-dihydroxyphenylglycine (S-DHPG), which is an mGlu1/5 receptor agonist and (RS)-1-aminoindan-1, 5-dicarboxylic acid (AIDA), a selective mGluR1alpha antagonist, on mechanical and thermal sensitivity and formalin-induced nociceptive behaviors. The anatomical studies at the electron microscopic level demonstrate that 32.4+/-2.9% of the unmyelinated axons and 21.6+/-4.7% of the myelinated axons are positively immunostained for mGlu1alpha receptors. Intraplantar injection of 0.1 or 1 mM S-DHPG results in a significant increase in mechanical sensitivity that persists for more than 60 min and this effect is blocked by co-injection of S-DHPG with 1 mM AIDA. Intraplantar injection of 40 microM AIDA+2% formalin significantly attenuates phase 2 lifting/licking and flinching behavior and this AIDA-induced effect is blocked with co-injection of 1 microM S-DHPG. In behavioral tests, intraplantar S-DHPG (0.1, 1.0, 10 mM) does not change tail flick latencies or paw withdrawal latencies to heat stimulation. These data indicate that mGlu1alpha receptors are present on peripheral cutaneous axons and activation of peripheral mGlu1alpha receptors contributes to mechanical allodynia and inflammatory pain but not thermal hyperalgesia.

Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel

&NA; Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that ∼60% of SSTR2a‐labeled cells are positive for TRPV1. Conversely, ∼33% of TRPV1‐labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 &mgr;M octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) ‐induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co‐application of the SSTR antagonist cyclo‐somatostatin (c‐SOM). In vitro studies using a skin‐nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 &mgr;M OCT significantly reduces CAP‐induced activity and can prevent CAP‐induced desensitization. Furthermore, blockade of peripheral SSTRs with c‐SOM dramatically enhances CAP‐induced behaviors and nociceptor activity, demonstrating SSTR‐induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP‐induced excitation and does not effect OCT‐induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors.

Chronic Spontaneous Activity Generated in the Somata of Primary Nociceptors Is Associated with Pain-Related Behavior after Spinal Cord Injury

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptors in vivo are unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neurons in vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Kainate-induced excitation and sensitization of nociceptors in normal and inflamed rat glabrous skin

This study investigates contributions of peripheral kainate receptors to acute nociception and persistent inflammatory pain in rat. Immunohistochemical analysis of kainate receptor expression using antibodies recognizing glutamate receptor subunits 5, 6, and 7 demonstrates that 28% of unmyelinated axons in normal digital nerve are positively labeled. Following intraplantar injection of complete Freunds adjuvant, a significant increase in glutamate receptor subunits 5, 6, and 7-labeled axons occurs at 2 days (40%), but not 7 (31%) or 14 days (28%) post-complete Freunds adjuvant. In behavioral studies, we confirm an increased mechanical sensitivity in complete Freunds adjuvant-injected hind paws. Furthermore, activation of kainate receptors following intraplantar injection of 1.0 mM kainate in normal animals results in a mechanical sensitivity similar to that observed in inflamed animals. A 1.0 mM kainate injection into inflamed hind paws further enhances the mechanical sensitivity. Injection of the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (0.1 mM) reverses complete Freunds adjuvant-induced mechanical sensitivity through a local effect. In single unit recordings from nociceptors in a glabrous skin-nerve preparation, mechanical sensitization is present in inflamed skin evidenced by a decrease in mechanical threshold and an increase in discharge rate during a suprathreshold, constant force stimulus. Thermal sensitization is also present evidenced by a decrease in heat threshold. There is a dose-dependent increase in kainate-induced nociceptor activity in both normal and inflamed skin but the kainate required to induce activation is reduced in inflamed skin. Although proportions of kainate-activated nociceptors are the same in normal and inflamed skin, the kainate-induced mean discharge rate is significantly enhanced in inflamed skin. Exposure of normal and inflamed nociceptors to 0.3 mM kainate sensitizes fibers to re-application of kainate and heat. This sensitization is blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione or the glutamate receptor subunit 5 selective antagonist 3S,4aR,6S,8aR-6-[4-carboxy-phenyl] methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid. The data indicate that peripheral kainate receptors not only play an important role in normal nociception but also contribute to mechanical sensitivity and heat sensitization accompanying inflammatory pain.

Group II metabotropic glutamate receptor activation on peripheral nociceptors modulates TRPV1 function

Transient receptor potential vanilloid 1 (TRPV1) receptors are critical to nociceptive processing. Understanding how these receptors are modulated gives insight to potential therapies for pain. We demonstrate using double labeling immunohistochemistry that Group II metabotropic glutamate receptors (mGluRs) are co-expressed with TRPV1 on rat dorsal root ganglion (DRG) cells. In behavioral studies, intraplantar 0.1 microM APDC, a group II agonist, significantly attenuates capsaicin-induced nociceptive behaviors through a local effect. The APDC-induced inhibition of capsaicin responses is blocked by 1 microM LY341495, a group II antagonist. At the single fiber level, nociceptor responses to capsaicin are significantly decreased following exposure to APDC and this effect is blocked by LY341495. Finally, activation of peripheral group II mGluRs inhibits forskolin-induced thermal hyperalgesia and nociceptor heat sensitization, suggesting group II receptors are negatively coupled to the cAMP/PKA pathway. The data indicate that group II mGluRs and TRPV1 receptors are co-expressed on peripheral nociceptors and activation of mGluRs can inhibit painful sensory transmission following TRPV1 activation. The data are consistent with group II and TRPV1 receptors being linked intracellularly by the cAMP/PKA pathway. Peripheral group II mGluRs are important targets for drug discovery in controlling TRPV1-induced nociception.

Pro-nociceptive role of peripheral galanin in inflammatory pain

&NA; We investigated the peripheral function of galanin (GAL) in capsaicin (CAP)‐induced inflammatory pain. Intraplantar GAL (0.1 ng/&mgr;l) alone does not produce nociceptive behaviors. However, ipsilateral but not contralateral GAL at low doses (0.1 ng/&mgr;l) significantly increases CAP‐evoked nociceptive behaviors approximately twofold. This effect is attributed to activation of peripheral GAL receptor 2 (GalR2) because a selective GalR2 agonist (AR‐M1896) mimics the pro‐nociceptive actions of GAL. Recording from nociceptors confirms that GAL does not modify activity of nociceptors but markedly enhances CAP‐induced excitation of these fibers. CAP produces a discharge rate of 0.15±0.05 impulses/s which increases to 0.54±0.17 impulses/s following CAP+GAL. Immunohistochemical studies indicate GalR2 are highly expressed (65.8%) in L5 dorsal root ganglion (DRG) cells. Also, 44.5% GalR2‐positive DRG neurons label for the capsaicin receptor (vanilloid receptor 1, VR1) while 61.7% of VR1‐positive DRG neurons label for GalR2; 28.1% of total DRG neurons are double‐labeled supporting the hypothesis that GAL‐induced effects are mediated by GalR2 on capsaicin‐sensitive primary afferents. Furthermore, 68.0% unmyelinated and 23.1% myelinated digital nerve axons label for GalR2, indicating the receptor is transported out to the periphery. Immunostaining for GAL peptide in digital nerves labels 46.4% unmyelinated and 27.1% myelinated axons, suggesting that afferents are a major source of ligand for peripheral GalR2. These results suggest that peripheral GAL has an excitatory role in inflammatory pain, likely mediated by peripheral GalR2 and that GAL can modulate VR1 function.