Shengtai Zhou

Peripheral administration of NMDA, AMPA or KA results in pain behaviors in rats

The present study investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor subtypes in peripheral pain transmission. Activation of NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-pro- pionic acid (AMPA) and kainate acid (KA) receptors in glabrous skin of the rat hindpaw resulted in mechanical allodynia and mechanical hyperalgesia. These agonist- induced pain behaviors were attenuated following peripheral injection of appropriate antagonists (MK-801 and CNQX). Thus, activation of NMDA, AMPA or KA receptors at the level of the peripheral nerve terminal can produce nociceptive behavior. These data suggest that topical application of glutamate receptor antagonists may be useful in treating pain disorders. Since all three receptor subtypes are involved in peripheral pain transmission, however, it will be necessary to antagonize multiple glutamate receptor subtypes to achieve effective pain relief.

Opioid receptors on peripheral sensory axons

Opioid receptors have been demonstrated by light microscopic techniques in fine cutaneous nerves in naive animals. The present study extends these findings by showing that 29 and 38% of unmyelinated cutaneous sensory axons can be immunostained for mu- or delta-opioid receptors respectively. Local cutaneous injection of DAMGO, a mu-opioid ligand, ameliorates the nociceptive behaviors caused by local cutaneous injection of glutamate, a purely nociceptive chemical stimulus showing that the mu-receptors are functional. By contrast the delta-opioid ligand [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE) had no effect on these behaviors. These findings indicate a wider function for opioid receptors in naive animals than previously envisioned.

Peripheral glutamate release in the hindpaw following low and high intensity sciatic stimulation

The present study demonstrates that following A and/or C fiber stimulation of the sciatic nerve, glutamate levels increase significantly in the hindpaw extracellular space. In hindpaw dialysate, electrical stimulation (5 min) of the sciatic nerve at 2×, 20×, 50× or 200× threshold current required to produce a muscle twitch resulted in peak glutamate increases of 120.8 ± 9%, 134.3 ± 5%, 153.9 ± 10% and 150.5 ± 5% of basal levels, respectively. Application of 1% capsaicin to the sciatic nerve (10 min) to selectively activate C fibers resulted in a peak glutamate increase of 130.8 ± 8% of basal levels. Aspartate levels did not change significantly in either paradigm. These data indicate that low and high intensity stimulation can result in peripheral release of glutamate, providing a major source of ligand for the glutamate receptors localized on peripheral primary afferents.

Localization and activation of substance P receptors in unmyelinated axons of rat glabrous skin.

Immunohistochemical staining for the substance P (neurokinin INK1) receptor labels 32% of the unmyelinated axons in the glabrous skin of the rat hindpaw. This is the first demonstration of substance P (SP) receptors associated with the membranes of primary afferent fibers. Injection of SP into the subcutaneous tissue of the third hind toe results in behavioral changes interpreted as mechanical hyperalgesia and mechanical allodynia. These nocifensive behaviors can be blocked by the NK1 antagonist CP99,994-1. The presence of peripheral axons immunolabelled for NK1 receptors and the demonstration that exogenous peripheral SP causes nocifensive behaviors would seem to indicate that SP can have a direct effect on sensory afferents with activation of these receptors resulting in fine afferent firing and thus the pain-related behaviors. Additionally, the presence of round, clear vesicles in some of the SP receptor-labelled axons suggests the presence of autoreceptors since it is known that many primary sensory afferents contain SP.

Evidence for the interaction of glutamate and NK1 receptors in the periphery

It is known that Substance P (SP) enhances glutamate- and N-methyl-D-aspartate (NMDA)-induced activity in spinal cord dorsal horn neurons and that this enhancement is important in the generation of wind-up and central sensitization. It is now known that SP and glutamate receptors are present on sensory axons in rat glabrous skin. This raises the issue as to whether SP and glutamate interact in the periphery. Using the tail skin in rats, the present study demonstrates 1) that unmyelinated axons at the dermal-epidermal junction immunostain for antibodies directed against NMDA, non-NMDA or SP (NK1) receptors; 2) that glutamate injected into the tail skin results in dose-dependent nociceptive behaviors interpreted as mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia, which are blocked following co-injection with glutamate antagonists; 3) that peripheral injection of SP potentiates glutamate-induced nociceptive behaviors in that the co-injection of SP+glutamate results in a significantly longer duration of behavioral responses compared to the responses seen following injection of either substance alone. These data provide support for the hypothesis that primary afferent neurons might well be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons.

Attenuation of formalin-induced nociceptive behaviors following local peripheral injection of gabapentin

&NA; Gabapentin (GP) has been shown to have antihyperalgesic properties and the site of drug action is reported to be the central nervous system. The goal of the present study was to determine whether GP also has a peripheral site of action. Rats received intraplantar 20‐&mgr;l injections of 6, 60 or 600 &mgr;g GP+2% formalin, 300 or 600 &mgr;g S‐(+)‐3‐isobutylgaba+2% formalin, 600 &mgr;g R‐(−)‐3‐isobutylgaba+2% formalin or formalin alone. The two lower doses of GP significantly reduced flinching and lifting/licking behavior during phase 2; however, phase 1 behaviors were unaffected, 600 &mgr;g GP significantly reduced these nociceptive behaviors during both phases. 600 &mgr;g S‐(+)‐3‐isobutylgaba also reduced formalin‐induced nociceptive behaviors; however, 600 &mgr;g of the isomer R‐(−)‐3‐isobutylgaba had no effect. The antihyperalgesic effect of GP (1) was not due to a systemic effect since animals injected with 600 &mgr;g GP in one hindpaw and 2% formalin into the contralateral hindpaw developed nociceptive behaviors which were no different than those seen in animals injected with formalin alone; (2) was not due to a local anesthetic effect since needle sticks within the drug‐injected region evoked paw withdrawal behavior which was not different from pre‐drug levels; (3) was blocked by 20 &mgr;l d‐serine but not by l‐serine. Although the mechanism of action of GP has yet to be elucidated, these results indicate that GP has a peripheral site of action and thus may offer a novel therapeutic agent for topical or local treatment of pain of peripheral origin.

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.

Peripheral GABAA receptors: evidence for peripheral primary afferent depolarization

We propose that the primary afferent depolarization that follows GABA(A) receptor activation in the spinal cord also occurs in the periphery. As evidence, the present study localizes beta2/beta3 and alpha1 subunits of the GABA(A) receptor on 10-14% of the unmyelinated primary afferents axons in the glabrous skin of the cat paw. Behavioral studies demonstrate that local peripheral injection of the GABA(A) agonist muscimol at a low concentration (2.0 microM) attenuates, and at a high concentration (1 mM) enhances, formalin-induced nociceptive behaviors. Intraplantar injection of muscimol alone at a high dose evokes thermal hyperalgesia. Bicuculline, a GABA(A) antagonist, prevents these muscimol-induced changes in behavior. The muscimol-induced effects are due to local rather than systemic or central activation of GABA(A) receptors, as such effects are not observed in the contralateral paw. We interpret these findings to indicate that activation of GABA(A) receptors by low concentrations of muscimol depolarizes peripheral primary afferent terminals, a phenomenon we call peripheral primary afferent depolarization, in turn reducing the size of the peripheral action potentials and concomitantly reducing the amount of algogenic substances released from the peripheral terminals of these fibers. This sequence of events presumably results in a reduction in nociceptor activation. Higher concentrations of muscimol further depolarize GABA(A) receptor-containing terminals, which then initiates action potentials in nociceptors analogous to the appearance of dorsal root reflexes that arise following activation of GABA(A) receptors on central primary afferent terminals. These latter events reverse the analgesic effects of GABA(A) ligands and lead to potentiation of nociceptive input. Thus, the present study provides anatomical and behavioral evidence supporting a bimodal role for GABA(A) receptors in the modulation of peripheral nociceptive transmission.

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.