Activation of Peripheral NMDA–Nitric Oxide Cascade in Formalin Test

Experiments were conducted according to a protocol approved by the Sapporo Medical University Animal Care and Use Committee. The animals used in this study were male Sprague-Dawley rats (weighing 300–350 g; Japan SLC, Hamamatsu, Japan), which were housed individually in a temperature-controlled room (at 21 ± 1°C) with a 12-h light–dark cycle and given free access to food and water.

Animals were anesthetized with 50 mg/kg sodium pentobarbital intraperitoneally, and additional sodium pentobarbital was administered throughout the experiment to maintain areflexia. A microdialysis probe (A-I-4-03; Eicom, Kyoto, Japan) was inserted subcutaneously into the bilateral glabrous skin of the hind paw, as described previously. 8The probe was perfused with artificial cerebrospinal fluid (ACSF) (140 mm NaCl, 4.0 mm KCl, 1.26 mm CaCl², 1.15 mm MgCl², 2.0 mm Na²HPO⁴, and 0.5 mm NaH²PO⁴; pH 7.4), at a constant flow rate of 3 μl/min for 120 min to establish a diffusion equilibrium, followed by sample collection to obtain basal release levels of NO²⁻and NO³⁻. In another series, during general anesthesia (isoflurane in oxygen), the right sciatic nerve was exposed in the upper thigh. A transection of the nerve was made just distal to the greater trochanter, with a 2-cm distal stump excision. Seven days after surgery, a microdialysis probe was inserted subcutaneously according to the method described previously.

The drugs and chemicals used in the experiment were N  ^G-monomethyl-l-arginine acetate (l-NMMA; Research Biochemicals, Natrick, MA), D ,l-2-amino-5-phosphonovaleric acid (AP-5; Sigma Chemical, St Louis, MO) and NMDA (Sigma Chemical). l-NMMA, AP-5, and NMDA were dissolved in physiologic saline. The pH of each solution was adjusted to 7.4.

After sample collection to obtain the basal level, the probe was perfused with ACSF, l-NMMA (40 mm), or AP-5 (10 mm) at a constant flow rate of 3 μl/min. Thirty minutes after perfusion, the sample was collected to obtain the basal level of NO metabolites (total amount of NO²⁻and NO³⁻[NO²⁻–NO³⁻]) and a 50μl volume of 5% formalin was injected into the plantar surface of the right hind paw using a 29-gauge needle. Perfusate collection was started with a 4-min delay because of the dead space of the outflow catheter to sample subcutaneous perfusate from the time of formalin injection. In another series of rats, NMDA (5 mm) was perfused through a microdialysis probe for 20 min. Samples were collected in polypropylene tubes for 5 min at each sampling time point, and the samples were immediately analyzed for NO²⁻–NO³⁻. After the microdialysis study, the animals were killed with a overdose of sodium pentobarbital.

The concentration of NO²⁻–NO³⁻in the dialysate was analyzed using an automated NO-detecting high-performance liquid chromatography system (ENO-10; Eicom). NO²⁻–NO³⁻in the dialysate was separated by a reverse-phase separation column packed with polystyrene polymer (4.6 × 50 mm; NO-PAK; Eicom), and NO³⁻was reduced to NO²⁻in a reduction column packed with copper-plated cadmium filling (NO-RED; Eicom). NO²⁻was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The separation and reduction columns and the reaction coil were placed in a column oven that was set at 35°C. The absorbance of the color of the product dye at 540 nm was measured using a flow-through spectrophotometer (NOD-10; Eicom). The mobile phase, which was delivered by a pump at a rate of 0.33 ml/min, was 10% methanol containing 0.15 m NaCl–NH⁴Cl and 0.5 g/l 4Na-EDTA. The Griess reagent, which was 1.25% HCl that contained 5 g/l sulfanilamide with 0.25 g/l N-naphthylethylenediamine, was delivered at a rate of 0.1 ml/min. The contamination of NO²⁻–NO³⁻in ACSF and the reliability of the reduction column were evaluated in each experiment. To determine the in vitro  recovery of NO²⁻and NO³⁻across the dialysis probe, a microdialysis probe was put into aliquots at a 10-mm concentration of NaNO²and NaNO³and perfused with Ringer’s lactate solution at a constant flow rate of 3 μl/min at room temperature. The in vitro  recovery was estimated based on the levels of NO²⁻and NO³⁻in the 5-min dialysate sample.

The NO²⁻–NO³⁻concentration was represented as the mean ± SD of percentage basal values. The statistical significance of differences between groups was assessed by a two-way analysis of variance for repeated measures, followed by the Scheffé F test. Changes in NO²⁻–NO³⁻during NMDA perfusion were compared with baseline values using one-way analysis of variance, followed by the Scheffé F test. P < 0.05 was considered to be statistically significant.

A preliminary study showed that dialysis equilibrium was obtained within 120 min after the start of ACSF perfusion at a constant flow rate of 3 μl/min, and basal values were stable for at least 240 min (data not shown). The basal value of NO²⁻–NO³⁻was 13.19 ± 1.42 pmol/10 μl. The in vitro  recovery of NO²⁻–NO³⁻was estimated to be 68.97 ± 0.02% (n = 6) across the dialysis probe.

Subcutaneous injection of formalin significantly (P < 0.05) increased the concentrations of NO²⁻–NO³⁻on the side ipsilateral to the injection, but not on the contralateral side (fig. 1). Ipsilateral NO²⁻–NO³⁻concentration significantly increased immediately after formalin injection (P < 0.05), and the increases were observed for 100 min. The increase in the NO²⁻–NO³⁻concentration after formalin injection was suppressed significantly (P < 0.05) during l-NMMA perfusion (fig. 2). l-NMMA alone did not affect the basal value of NO²⁻–NO³⁻concentration (data not shown). In the animals with denervated sensory nerves in the hind paws, the concentrations of NO²⁻–NO³⁻did not show significant changes after formalin injection (fig. 3).

The perfusion of AP-5 through the microdialysis probe suppressed significantly (P < 0.05) the formalin-induced increase in NO²⁻–NO³⁻concentration, as shown in figure 4. Figure 5shows the effects of the perfusion of NMDA through the microdialysis probe on the concentration of the NO²⁻–NO³⁻. NMDA perfusion increased the concentrations of NO²⁻–NO³⁻significantly (P < 0.05).

In the current study, injection of formalin into the plantar surface of the rat hind paw significantly increased the subcutaneous concentration of NO²⁻–NO³⁻on the side ipsilateral, but not the side contralateral, to the injection. This increase was suppressed by the perfusion of NOS inhibitor l-NMMA. The perfusion of competitive NMDA antagonist AP-5 also suppressed the formalin-induced increase in the concentration of NO²⁻–NO³⁻. In addition, the subcutaneous perfusion of NMDA alone increased the concentration of NO²⁻–NO³⁻. In the animals that had undergone sciatic nerve sectioning, the increase in the concentration of NO²⁻–NO³⁻was not observed. These data show that subcutaneous formalin induced the peripheral release of NO through activation of an NMDA–NO cascade in the peripheral nervous system.

Several lines of observations suggest that peripheral NO acts as a pronociceptive mediator. 9–11Human and animal studies support a role of NO in mediating peripheral nociceptive transmission. 12–15Intracutaneous injection of NO precursors in humans evoke pain in a dose-dependent manner, 12,14suggesting that NO may activate cutaneous nociceptors directly or indirectly. In the mouse, the NOS inhibitor N  ^G-nitro-l-arginine methylester hydrochloride (l-NAME), if coadministered with intraplantar formalin, exhibited antinociceptive activity in a dose-dependent manner. 13Intraarticular injection of l-NAME resulted in a complete reversal of heat hyperalgesia in arthritic model of rats. 15It is suggested that NO is released during the inflammation and that it acts to increase vascular permeability to different chemical substances activating specific receptors involved in pain signaling. 16It is conceivable that endogenous NO is released at the site of acute inflammation. 17In the formalin test, it has been hypothesized that the first phase of formalin-induced behavior reflects formalin-induced activation of C-fiber primary afferent nociceptors, and that the second phase is an associated phase of inflammation in which various chemical mediators are able to alter the functions of peripheral afferent fibers. 18–20As shown in the current study, formalin induces the elevation of the subcutaneous concentration of NO²⁻–NO³⁻on the ipsilateral side of the hind paw; however, the increase does not show biphasic change. Peripheral NO was released in acute nociception and the inflammatory stage.

Nitric oxide is synthesized from the terminal guanidino nitrogen atoms of the amino acid l-arginine by an enzyme, NOS. 21Because NO has a short half-life, it does not diffuse for a long distance. Therefore, NO must be synthesized locally in the formalin-injected site. Different types of NOS exist in different cells. 2Neuronal NOS is expressed constitutively in neurons. Inducible NOS (iNOS) originally was described in macrophages and hepatocytes but also is found in central nervous system glial cells. Endothelial NOS is expressed constitutively in endothelial cells. Possible peripheral sources of NO include the peripheral afferent fibers, neutrophils, infiltrating leukocytes, and the endothelial cells lining the capillaries. In the current study, l-NMMA perfusion suppressed the increase in concentration of NO²⁻–NO³⁻after formalin injection. Because l-NMMA is a nonselective NOS inhibitor, it is unclear which type of NOS would be associated with production of NO. Peripheral NO may be generated through neuronal NOS within nociceptors because neuronal NOS-like immunoreactivity is expressed in small- and medium-diameter dorsal root ganglion neurons 22,23; neuronal NOS-like immunoreactivity is colocalized in some dorsal root ganglia neurons with substance P–like and calcitonin gene-related-peptide–like immunoreactivity 22,24; and NOS-like immunoreactivity is coexpressed in some dorsal root ganglia neurons with substance P–like or calcitonin gene-related-peptide–like immunoreactivity and is reduced greatly in neonatal rats treated with capsaicin. 22,25Selective neuronal NOS inhibitor 7-nitro-indazole (7-NINA) exhibits antinociceptive activity if injected intraperitoneally into mice, by producing dose-related inhibition of the second phase of hind paw licking after subplantar injection of formalin. 26Salvemini et al.  27reported that NO produced by constitutive NOS (neuronal or endothelial NOS) was involved in the development of inflammation at early time points (< 4 h) after subcutaneous administration of carrageenan, and NO produced by inducible NOS was involved in the maintenance of the inflammatory response at later time points (> 5 h). It is possible that the NO released after formalin injection may be generated by the neuronal NOS. The increase in NO²⁻–NO³⁻concentration after formalin injection was suppressed by NMDA receptor antagonist AP-5, and NMDA perfusion caused the release of NO²⁻–NO³⁻. These facts suggest that NO is produced by neuronal NOS because NMDA receptors exist in the peripheral nervous system. 28The current results, in which the concentration of NO²⁻–NO³⁻did not increase in the rats with denervated sensory nerves, imply that an important source of NO is peripheral afferent fibers.

Several studies have shown that peripheral glutamate has nociceptive and inflammatory roles. Application of glutamate to skin evokes pain-related behaviors, 28,29and peripherally administered glutamate antagonists can prevent the nociception produced by inflammation. 29,30Thus, glutamate receptor activation should play an important role in nociceptive signaling at the level of the peripheral nerve terminal in the skin. In our previous report, the increase in glutamate concentration in the plantar surface of the rat hind paw was observed on the side ipsilateral, but not the side contralateral, to the subcutaneous injection of formalin. 8We demonstrated that perfusion with the NMDA antagonist AP-5 suppressed the increase in NO after formalin injection, and that NMDA perfusion induced the increase in NO concentration. These facts suggest that formalin evokes the release of glutamate, which may induce the activation of a peripheral NMDA–NO cascade. This cascade was activated throughout the current experiment; the concentration of NO²⁻–NO³⁻did not show the biphasic changes seen in the behavioral study. A possible explanation for this discrepancy is that the central inhibitory systems (descending inhibitory system and intraspinal inhibitory system) are activated in the period between first and second phases in the formalin test. 31–33We previously reported that intrathecal pretreatment with yohimbine and methysergide produced greater increases in the number of flinches in phase 1, the interphase period, and phase 2 after formalin injection than without pretreatment. 31Formalin injection induced increases in noradrenaline, 4-hydroxy-3-methoxyphenylglycol (MHPG), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in both the ipsi- and contralateral dorsal halves. 31Henry et al.  32compared the nociceptive behaviors produced by a single hind paw injection of formalin with those produced by two formalin injections administered 20 min apart. They showed that there was a second decrease in nociceptive responses after the second injection, suggesting that the interphase period in the formalin test results from active inhibition. Matthies and Franklin 33reported that decerebrate rats (those having undergone transection of the brain at the rostral metencephalon or mesencephalon) did not show the abatement in the interphase period in the formalin test, which suggests that the cessation of the nociceptive behavior at the interphase period may be caused by a central inhibitory modulation of nociceptive behaviors. In summary, subcutaneous formalin injection induces peripheral release of NO, the production of which is mediated by activation of NMDA receptors in the peripheral nervous system. This peripheral NMDA–NO cascade could contribute to formalin-induced nociception.