Peripheral nitric oxide in carrageenan-induced inflammation

Brain Research 912 (2001) 171–175 www.elsevier.com / locate / bres

Research report

Peripheral nitric oxide in carrageenan-induced inflammation Keiichi Omote*, Koji Hazama, Tomoyuki Kawamata, Mikito Kawamata, Yoshito Nakayaka, Masaki Toriyabe, Akiyoshi Namiki Department of Anesthesiology, Sapporo Medical University School of Medicine, South-1, West-16, Chuoku, Sapporo 060 -8543, Japan Accepted 22 June 2001

Abstract Recent studies have suggested that nitric oxide (NO) peripherally produced by different nitric oxide synthase (NOS) isoforms contributes to edema formation and development of hyperalgesia. The present study was designed to examine the effects of NOS isoforms on NO release in carrageenan-induced inflammation at various time points. A microdialysis probe was implanted subcutaneously into the glabrous skin of hindpaws of Sprague–Dawley rats under pentobarbital anesthesia. After sample collection to obtain the basal level of the 2 total amount of nitrite and nitrate (NO 2 2 / NO 3 ), modified Ringer solution, a non-selective NOS inhibitor, NG monomethyl-L-arginine acetate ( L-NMMA), or an iNOS inhibitor, aminoguanidine hemisulfate (AG) was perfused through the microdialysis probe. 2 mg of carrageenan was injected into the plantar surface of the probe-implanted hindpaw. Carrageenan was also injected in rats that had 2 undergone sciatic nerve sectioning. Carrageenan significantly increased the dialysate concentrations of NO 2 2 / NO 3 for more than 8 h. 2 2 L-NMMA suppressed the carrageenan-induced increase in NO 2 / NO 3 concentration. Although AG did not suppress the increase in NO 22 / NO 32 for the first 2 h after carrageenan injection, significant suppression of the increase in NO 22 / NO 32 was observed from 2.5 h after carrageenan injection. In the rats in which the sciatic nerves had been denervated, the increases in concentrations of NO 22 / NO 32 were completely suppressed up to 3 h and partially suppressed 4.5–8 h after carrageenan injection. The results of the current study show that carrageenan induces peripheral release of NO, the production of which is mediated by nNOS in the early phase and by both nNOS and iNOS in the late phase of carrageenan-induced inflammation.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Carrageenan; Inflammation; Nitric oxide; Nitric oxide synthase

1. Introduction The intraplantar injection of carrageenan elicits an inflammatory response that is characterized by a timedependent increase in paw edema, neutrophil infiltration, and increased levels of various mediators in the paw exudates and by development of hyperalgesia to thermal and mechanical stimuli. Edema formation in the rat hindpaw following injection of carrageenan has been described as a biphasic event consisting of a relatively rapid early phase followed by a more sustained late phase [4]. Recent studies have indicated that nitric oxide (NO) *Corresponding author. Tel.: 181-11-6112-111 ext. 3568; fax: 18111-6319-683. E-mail address: [email protected] (K. Omote).

plays a role in edema formation and development of hyperalgesia in tissue injury and inflammation. Subcutaneous formalin injection induces peripheral release of NO [15]. NO dilates microvascular blood vessels [2] and promotes microvascular permeability, resulting in edema formation [3,6]. NO also increases the synthesis / release of pro-inflammatory mediators such as cytokines and reactive oxygen species [12] and prostanoids [18], resulting in promotion of inflammatory reaction. In addition, peripheral administration of the nitric oxide synthase (NOS) inhibitor NG monomethyl-L-arginine acetate ( L-NAME) has been found to reduce both mechanical and thermal hyperalgesia induced by hindpaw injection of carrageenan, indicating that NO plays a role in the development of hyperalgesia [11,14]. Thus, peripherally released NO contributes to the development of edema and hyperalgesia in tissue injury and inflammation.

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02733-0

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NO is not normally present in its free radical form in the mammalian body and must be synthesized in a process involving NOS. NOS exists in one of three isoforms, two constitutive isoforms that are always present within the body (neuronal and endothelial NOS; nNOS and eNOS, respectively) and one isoform that is not normally present and must be synthesized de novo (inducible NOS; iNOS). The nNOS and eNOS are primarily, but not exclusively, found within the nervous system and endothelial tissue, respectively, while iNOS is commonly found in a variety of cell types, including macrophages, chondrocytes and neutrophils, but is also found in central nervous system glial cells [9]. We previously suggested that NO was produced and released after formalin-induced injury in the peripheral nervous system [15]. Although the location and identity of the NOS isoform responsible for NO synthesis at the site of inflammation have remained unclear, recent evidence suggests that in carrageenan-induced inflammation, different NOS isoforms may play roles at various points during the time course of an injury [4]. The purpose of the present study was to investigate which isoform of NOS is responsible for the production and release of NO in the early and late phases of carrageenan-induced inflammation.

2. Materials and methods 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 (21618C) room with a 12-h light–dark cycle and given free access to food and water.

2.1. Animal preparation Animals were anesthetized with intraperitoneal sodium pentobarbital (50 mg / kg), and additional sodium pentobarbital was administered throughout the experiment to maintain areflexia. A microdialysis probe (A-I-4-03, Eicom, Japan) was subcutaneously inserted into the right glabrous skin of the hindpaw, as described previously [15,16]. The probe was perfused with modified Ringer solution (140 mM NaCl, 4.0 mM KCl, 1.26 mM CaCl 2 , 1.15 mM MgCl 2 , 2.0 mM Na 2 HPO 4 , and 0.5 mM NaH 2 PO 4 , pH 7.4) at a constant flow rate of 3 ml / min for 120 min to establish a diffusion equilibrium, followed by sample collection to obtain basal release levels of NO metabolites (total amount of NO 22 and NO 32 , NO 22 / NO 32 ). In another series, under 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 the surgery, a microdialysis probe was

subcutaneously inserted into the right hindpaw according to the method described above.

2.2. Drugs The drugs and chemicals used in the present experiment were L-NMMA (a non-selective NOS inhibitor; Research Biochemicals, Natick, MA, USA), aminoguanidine hemisulfate (AG, an iNOS inhibitor; Sigma, St. Louis, MO, USA) and Lambda carrageenan (Sigma). L-NMMA, AG and carrageenan were dissolved in modified Ringer solution. The pH of all solutions was adjusted to 7.4.

2.3. Microdialysis study Following sample collection to obtain the basal level, the probe was perfused with modified Ringer solution, L-NMMA (40 mM) or AG (40 mM) at a constant flow rate of 3 ml / min. Thirty minutes after the perfusion, a sample 2 was collected to obtain the control value of NO 2 2 / NO 3 as pre-carrageenan injection value, and 2 mg of carrageenan in a volume of 50 ml was injected into the plantar surface of the right hindpaw with a 27 gauge needle. Perfusate collection was started with a 4-min delay because of the dead space of the out-flow catheter in order to sample subcutaneous perfusate from the time of carrageenan injection. Samples were collected in polypropylene tubes for 5 min at each sampling time point, and the samples for 2 determination of NO 2 2 / NO 3 were immediately analyzed. After the microdialysis study, the animals were killed with an overdose of sodium pentobarbital. 2 2.4. Analysis of NO 2 2 /NO 3 2 NO 2 in the dialysate was analyzed using an 2 / NO 3 automated NO detector high-performance liquid chormatography (HPLC) system (ENO-10, Eicom). NO 2 2 and NO 32 in the dialysate were separated by a reversed-phase separation column packed with polystyrene polymer (NO2 PAK, 5034.6 mm, Eicom), and NO 2 3 was reduced to NO 2 in a reduction column packed with copper-plated cadmium filling (NO-RED, Eicom). NO 2 2 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 358C. The absorbance of the color of the product dye at 540 nm was measured by 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–NH 4 Cl and 0.5 g / l 4Na-EDTA. The Griess reagent, which was 1.25% HCl containing 5 g / l sulfanilamide with 0.25 g / l N-naphthylethylenediamine, was delivered at a rate of 0.1 ml / min. The contamination of 2 NO 2 2 / NO 3 in modified Ringer solution and the reliability of the reduction column were examined in each experiment.

K. Omote et al. / Brain Research 912 (2001) 171 – 175 2 To determine the in vitro recovery of NO 2 2 and NO 3 across the dialysis probe, a microdialysis probe was put into aliquots at a 10 mM concentration of NaNO 2 and NaNO 3 and perfused with modified Ringer’s solution at a constant flow rate of 3 ml / min at room temperature. The in vitro recovery was estimated on the basis of the levels of 2 NO 2 2 and NO 3 in a 5-min dialysate sample.

2.5. Statistical analysis The NO 22 / NO 32 concentration is expressed as mean6S.E. of percentage of the control value. The statistical significance of differences between groups was assessed by two-way analysis of variance (ANOVA) for repeated measures followed by Scheffe’s F-test. A P value ,0.05 was considered to be statistically significant.

3. Results A preliminary study showed that dialysis equilibrium was obtained within 120 min after starting of modified Ringer solution perfusion at a constant flow rate of 3 ml / min and basal values were stable for more than 8 h 2 (data not shown). The basal value of NO 2 2 / NO 3 was 14.1561.27 pmol / 10 ml. The in vitro recovery of NO 2 2 / NO 2 3 was estimated to be 71.2160.03% (n56), across the dialysis probe. L-NMMA and AG alone did not affect basal values of 2 NO 2 2 / NO 3 concentration (data not shown). The pre-carrageenan value of NO 22 / NO 32 concentration during the

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perfusion with modified Ringer, L-NMMA or AG was defined as control value. Injection of carrageenan significantly (P,0.01) in2 creased the concentration of NO 2 2 / NO 3 (Fig. 1). The 2 2 increase in NO 2 / NO 3 concentration peaked at 2.5 h after the injection of carrageenan and remained elevated for more than 8 h. The increase in NO 22 / NO 32 concentration after carrageenan injection was significantly (P,0.01) suppressed by L-NMMA perfusion during the experiment (Fig. 1). Although AG perfusion did not suppress the increase in 2 NO 2 2 / NO 3 for the first 2 h after injection of carrageenan, a significant (P,0.01) suppression of the increase in 2 NO 2 2 / NO 3 was observed from 2.5 h after carrageenan injection (Fig. 1). 2 In denervated animals, the concentrations of NO 2 2 / NO 3 did not show significant changes for 3 h after carrageenan 2 injection. Significant (P,0.01) increases in NO 2 2 / NO 3 concentration were observed at 3.5 h after carrageenan injection and the concentration remained elevated thereafter, but the concentration was significantly (P,0.01) lower than that the group of Ringer solution at 4.5 h after carrageenan injection and remained lower thereafter (Fig. 1).

4. Discussion This study has demonstrated that carrageenan causes the production and release of NO at the injured site. Perfusion of a nonselective NOS inhibitor, L-NMMA, suppressed the release of NO following carrageenan injection in this

2 Fig. 1. Time courses of NO 2 2 / NO 3 concentrations after carrageenan injection and the effects of L-NMMA, AG and neurectomy. Data are represented as the mean6S.E. from the six rats in each group. *P,0.05, showing statistical significance of the difference from Ringer.

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study. Perfusion of an inducible NOS inhibitor, AG, suppressed the release of NO 2.5–8 h after carrageenan injection. Neurectomy completely suppressed NO release up to 3 h and partially suppressed NO release 4.5–8 h after carrageenan injection. These findings indicate that nNOS contributes to the NO production in both the early and late phase, as well as iNOS which only contributes to the late phase. The roles of peripheral NOS isoforms in edema formation and development of hyperalgesia have been investigated in some behavioral studies. Regarding paw edema formation, Salvemini et al. [17] reported that the nonselective NOS inhibitors L-NMMA and L-NAME given intravenously 1 h before or after carrageenan administration inhibited paw edema formation at all time points. The selective iNOS inhibitors N-iminoethyl-L-lysine ( L-NIL) and AG failed to inhibit carrageenan-induced paw edema formation for the first 4 h following carrageenan administration but inhibited paw edema formation at subsequent time points (from 5–10 h). Although pretreatment with intraperitoneal administration of an nNOS inhibitor, 7nitro-indazole (7-NI), inhibited both early-phase and latephase hindpaw edema formation, 7-NI proved to be a more effective inhibitor of early- (but not late-) phase hindpaw edema response to carrageenan [4]. The results described above support our findings that NO would be produced by nNOS in the early phase and by both nNOS and iNOS in the late phase of carrageenan-induced inflammation. Since the nNOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM) or 7-NI has been shown to attenuate edema formation in the rat by stimulation of the sensory saphenous nerve (i.e., neurogenic inflammation), it is believed that NO produced by nNOS induces the release of neuropeptides (e.g., substance P and calcitonin-gene related peptide) from sensory nerve terminals, resulting in the development of neurogenic edema [8,21]. Regarding the development of hyperalgesia, the nNOS inhibitors 7-NI and TRIM administered intraperitoneally 2.5 h after carrageenan injection inhibited both mechanical and thermal hyperalgesia in the rat [5]. Intraarticular administration of 7-NI, 4.5 h after onset of carrageenan-induced joint inflammation, attenuated thermal hyperalgesia [11]. These findings suggest that NO produced by nNOS contributes to the development of hyperalgesia in the late phase. Salvemini et al. [17] detected the presence of iNOS mRNA in the rat hindpaw and iNOS protein in soft tissue of the rat hindpaw at 3 and 6 h after carrageenan administration, respectively. This supports our results showing the suppression of NO release by an iNOS inhibitor from 2.5 h after carrageenan injection. Salvemini et al. also suggested that infiltrating neutrophils were not the source of iNOS since pretreatment with colchicines suppressed neutrophil infiltration but did not inhibit the iNOS mRNA expression or the elevated levels of NO metabolites in the paw exudates. However, the precise cellular location of iNOS in the carrageenan-injected

hindpaw has not been elucidated. It seems likely that iNOS generation occurs in macrophages present at the inflammatory site, although other cell types, including bone osteoblasts [7] and glia [19], in which cytokine-induced iNOS formation has been noted, cannot be excluded. This study has shown that neurectomy and iNOS inhibitor completely suppressed NO release in early and late phases, respectively. Though it appears evident that nNOS and iNOS contribute to NO production, a contribution from eNOS cannot be ruled out. To clarify the role of eNOS in carrageenan-induced NO production, a further study is needed. At present, highly selective inhibitors of nNOS, iNOS and eNOS are not available. Although AG has been used as iNOS inhibitor, this agent also possesses a weak pharmacological character of cNOS inhibitor [13]. 7-NI has been known as nNOS inhibitor, but this agent has been reported to be a relatively potent inhibitor of iNOS enzyme activity [1]. Furthermore, 7-NI cannot be dissolved in water and can only be dissolved in arachis oil after warming and sonication. Therefore, the perfusion with 7-NI through a microdialysis probe is not appropriate to microdialysis study. For these reasons, we performed an experiment using rats with denervation of sciatic nerve in order to determine the role of nNOS after carrageenan in the present study. It has been reported that dorsal rhizotomy [20] or peripheral denervation [10] reduces the severity of inflammation in arthritic rats. These results might reflect the decreasing in NO production due to nNOS reduction following denervation. In summary, carrageenan induces peripheral release of NO, the production of which is mediated by nNOS in the early phase of inflammation and by both nNOS and iNOS in the late phase of inflammation. The production and release of NO by these NOSs are thought to contribute to tissue injury- and inflammation-induced edema and hyperalgesia.

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