Articular Incapacitation Research Articles

Neutrophils-derived peroxynitrite contributes to acute hyperalgesia and cell influx in zymosan arthritis

We investigated the contribution of neutrophils to joint hyperalgesia and peroxynitrite formation in zymosan arthritis. Rats received 1 mg zymosan intra-articular, and joint hyperalgesia was measured using the rat knee-joint articular incapacitation test. After 6 h, joint exudates were collected by aspiration for the assessment of cell influx, myeloperoxidase activity, and nitrite (as an index of nitric oxide formation) levels. Nitrotyrosine content, used as an index of peroxynitrite formation, was measured in joint exudates, using enzyme-linked immunosorbent assay. A group of rats was rendered neutropenic through the administration of a rabbit anti-rat neutrophil antibody (2 ml kg(-1), i.p.) 30 min before injection of 1 mg zymosan intra-articular. Other groups received uric acid (100 or 250 mg kg(-1), i.p.), the peroxynitrite scavenger, 30 min before 1 mg zymosan intra-articular. Controls received the vehicle. The significant inhibition of joint hyperalgesia in neutropenic animals was associated to significantly decreased cell influx, myeloperoxidase activity, nitric oxide, and nitrotyrosine levels in the joint exudates, as compared to naive rats. Uric acid administration inhibited both hyperalgesia and cell influx, as compared to controls. Neutrophils are involved in both nitric oxide and peroxynitrite formation in zymosan arthritis, thereby contributing to acute joint hyperalgesia. Scavenging of reactive nitrogen species (e.g. peroxynitrite) inhibits neutrophil migration and joint hyperalgesia in the acute phase of zymosan arthritis in rats.

Contribution of TNFα, IL-1β and CINC-1 for articular incapacitation, edema and cell migration in a model of LPS-induced reactive arthritis

The protective effect of anti-CINC-1, -TNFα and -IL-1β antisera on articular inflammatory incapacitation, articular diameter and synovial fluid cell content, which are correlated to nociception, edema and cell migration, respectively, were evaluated in a rat model of LPS-induced reactive arthritis. In this model, Escherichia coli lipopolysaccharide (LPS; 30 ng) was injected in a knee-joint previously primed with carrageenan (300 μg). Articular incapacitation was evaluated hourly by the automated registering of the knee-joint function during animal walking, and the knee-joint edema was evaluated by measuring the articular diameter increase. After 6 h, the animals were euthanized for collecting synovial fluid for the evaluation of cell migration. LPS produced dose-dependent incapacitation and edema. Anti-TNFα, -IL-1β, and -CINC-1 antisera (20 and 40 μl) were used as pretreatment into knee-joint before LPS injection. At higher dose, Anti-TNFα and anti-CINC-1 were able to inhibit incapacitation, articular edema and mononuclear (MON) migration. Anti-IL1β did not affect incapacitation at any dose, although inhibited edema and cell migration. Surprisingly, the higher dose of anti-IL1β antisera did not inhibit cell migration, although inhibited articular edema. These findings corroborate the role TNFα has in different forms of arthritis, but points out the idea that CINC-1 (the homologue for human IL-8) may constitute a promising target for reactive arthritis management. Indeed, the potent antiedematogenic effect, and principally the anti-migration effect of anti-CINC-1, raises the possibility of a better control of disease progression than with anti-IL-1β therapies.

ANTI-INFLAMMATORY AND ANTI-NOCICEPTIVE ACTIVITY OF RISEDRONATE IN EXPERIMENTAL PAIN MODELS IN RATS AND MICE

1. The antinociceptive effect of risedronate in experimental pain models in rats and mice was investigated in the present study. 2. Rats received zymosan intra-articularly (i.art.) into the right knee joint and the nociceptive response was assessed using the articular incapacitation test. Joint washouts were used for determining cell influx, tumour necrosis factor (TNF)-alpha and leukotriene (LT) B4 levels. 3. Mice received either zymosan (1 mg) or acetic acid (0.6%) i.p. and the nociceptive response was measured as the number of writhings between 0 and 30 min after the stimuli. Control animals received i.p. injections of saline. 4. Groups were pretreated with risedronate (5-500 microg/kg, s.c.) and compared with vehicle (saline)-treated (NT) animals. One group of rats was cotreated with the micro-opioid receptor antagonist naloxone (2 mg/kg, s.c.) prior to risedronate, followed by 1 mg zymosan i.art. 5. Risedronate, at 100 and 500 microg/kg, significantly and dose-dependently inhibited the nociceptive response in the writhings test (P < 0.05), inhibiting responses to acetic acid by 65.4 and 49.2%, respectively, and to zymosan by 72.9 and 71.9%, respectively. 6. Pretreatment with risedronate also significantly (P < 0.05) and dose-dependently inhibited the articular incapacitation in zymosan-arthritis. 7. Risedronate, at 50 microg/kg, significantly inhibited TNF-alpha release as compared with the NT group (39.4 +/- 9.8 vs 145.6 +/- 43.3 pg/mL TNF-alpha, respectively). 8. Risedronate, at 50 and 100 microg/kg, significantly inhibited LTB4 release into the joints compared with the NT group (2883.1 +/- 73.2, 1911.5 +/- 205.3 and 4709.9 +/- 237.2 pg/mL, respectively). These effects of risedronate were associated with a significant reduction in the inflammatory cell infiltration. 9. Cotreatment with risedronate and naloxone did not reverse the antinociceptive effects of risedronate in zymosan-arthritis. 10. This is the first demonstration that risedronate displays intrinsic antihypernociceptive activity. This effect is associated with reduced cell infiltration and inhibition of TNF-alpha and LTB4 release and is not linked to an endogenous opioid-release mechanism.

Anti-nociceptive effect of thalidomide on zymosan-induced experimental articular incapacitation

The anti-nociceptive effect of thalidomide on zymosan-induced articular knee joint incapacitation in rats was investigated. Thalidomide (5–45 mg/kg), given 30 min before but not 2 h after the intra-articular injection of zymosan, inhibited the nociceptive response in a dose-dependent manner. Furthermore, thalidomide pretreatment significantly reduced the concentration of tumor necrosis factor-alpha (TNF-α, − 68.4%) in the exudate of zymosan-injected joints, but not those of interleukin-1β, interleukin-6, CINC-1 or interleukin-10. The expression of TNF-α, determined by immunohistochemical staining, in synovial tissues obtained from articular joints injected with zymosan was also inhibited by thalidomide pretreatment. The anti-nociceptive effect of thalidomide was not reversed by the co-administration of an opioid receptor antagonist, naloxone, suggesting that endogenous opioids do not mediate the anti-nociceptive effect of thalidomide in this model. In conclusion, the anti-nociceptive activity of thalidomide in zymosan-induced articular incapacitation is associated with the inhibition of TNF-α by resident synovial cells.

Blockade of leukotriene B 4 prevents articular incapacitation in rat zymosan-induced arthritis

We investigated whether leukotrienes mediate cell influx and articular incapacitation in zymosan-induced arthritis. Rats received 1 mg zymosan intra-articularly (i.a.). The hyperalgesia was measured using the rat articular incapacitation test. Cell influx, leukotriene B 4 and prostaglandin E 2 levels were assessed in the joint exudate, at 6 h. Groups received either the leukotriene B 4 synthesis inhibitor MK 886 (3-[1-(4-chlorobenzyl)-3- t-butyl-thio-5-isopropylindol-2-yl)]-2,2-dimethylpropanoic acid 30 min before or 2 h after the zymosan; 0.3–3 mg kg −1 i.p.), the leukotrienes synthesis inhibitor BWA 4C ( N-(3-phenoxycinnamyl)-acetohydroxamic acid—2 h after the zymosan; 10 μg i.a.) or the peptido-leukotrienes antagonist sodium montelukast (30 min before and 2 h after the zymosan; 10 mg kg −1 per os). MK 886 inhibited the articular incapacitation and cell influx, while reducing leukotriene B 4, but not prostaglandin E 2 levels. BWA 4C inhibited the articular incapacitation. Sodium montelukast did not affect either of the parameters. The data suggest that leukotriene B 4 is involved in cell influx and articular incapacitation in zymosan arthritis.

Efeito de inibidores da sintase de óxido nítrico na dor inflamatória articular e influxo celular da artrite induzida por zymosan em ratos

OBJETIVO: Investigar o papel do oxido nitrico (NO) no influxo celular (IC) e a incapacitacao articular (IA) na artrite por zymosan (AZy). METODOS: Ratos Wistar receberam injecao de Zy (1mg) no joelho direito. Grupos-controles receberam salina. A IA foi avaliada pelo Teste de incapacitacao articular para ratos. Apos sacrificio, o IC e a liberacao de PGE2 foram avaliados no lavado articular. Avaliou-se o edema pela diferenca peso umido/peso seco do tecido articular. Grupos foram tratados i.p. com L-NAME - inibidor de sintases de NO (NOS) (LN=30-300mg/kg) ou aminoguanidina (AG= 30-300mg/kg) - inibidor seletivo de iNOS 30 min antes ou 2 h apos o Zy. RESULTADOS: O pre-tratamento com L-NAME ou AG inibiu de forma dose-dependente a IA e o IC, mas nao o edema ou a liberacao de PGE2, sendo maximo para LN ou AG (100mg/kg). L-arginina (1g/kg) co-injetada com LN (100mg/kg) reverteu a inibicao da IA e do IC. D-NAME (100mg/kg) nao teve efeito. Co-injecao de naloxona (antagonista opioide) reverteu o efeito de LN e AG sobre a IA. A pressao arterial media elevou-se (p<0,01) apos LN (100mg/kg), mas nao com AG (100mg/kg). LN e AG (100mg/kg) dados 2 h apos Zy nao tiveram efeito. Azul de metileno (inibidor de GMPc), e nao hemoglobina (scavenger de NO), reduziu IA e IC (p<0,01). Nitroprussiato de sodio (doador de NO) dado 2 h apos Zy, reduziu a IA, mas nao o IC (p<0,01). CONCLUSOES: A antinocicepcao na AZy por inibicao da iNOS so ocorre como pre-tratamento, por efeito antiinflamatorio via GMPc, independentemente de NO exogeno ou de PGE2, associada a liberacao de opioides endogenos. Um doador de NO produz analgesia na AZy. Enfim, o trabalho demonstra efeito analgesico de um doador de NO em um modelo de periartrite, abrindo a perspectiva do uso dessas substâncias em humanos.

Nitric oxide and cyclooxygenase may participate in the analgesic and anti-inflammatory effect of the cucurbitacins fraction from Wilbrandia ebracteata

Wilbrandia ebracteata is a medicinal plant from South America used in folk medicine for the treatment of chronic rheumatic diseases. We have shown that the high performance liquid chromatography-characterized (HPLC) dichloromethane fraction isolated from Wilbrandia ebracteata (WEDC) inhibits the parameters observed in experimental models of inflammation in vivo and in vitro. In the present study, we extend our previous observations on the analgesic effects of WEDC by investigating its actions using the hot plate test and zymosan-induced writhing test in mice, as well as zymosan-induced arthritis in rats evaluating articular inflammatory pain, cell migration and determination of NO release into the joint exudate. The effect of WEDC on the activity of COX-1 and COX-2 in vitro and its ulcerogenic capacity in vivo were also investigated. The oral treatment of the animals with WEDC (1–10 mg/kg) produced a significant, dose-dependent reduction of articular incapacitation and abdominal contortions in the writhing test. The same effect was not observed in the hot plate and rota-rod tests. WEDC also reduced nitrite release into the zymosan-inflamed joints. In the evaluation of COX activity, we observed that WEDC was able to selectively inhibit COX-2 but not COX-1 activity in COS-7 cells. Moreover, WEDC treatment did not show gastrointestinal toxicity. Our data confirm the anti-nociceptive activities of the WEDC and indicate that this effect could be associated with inhibition of cyclooxygenase-2 (COX-2) and nitric oxide release. The effects could be attributed to cucurbitacins since several of these were isolated from the WEDC.

Antinociceptive effects of interleukin-4, -10, and -13 on the writhing response in mice and zymosan-induced knee joint incapacitation in rats.

The antinociceptive effects of interleukin (IL)-4, -10, and -13 were investigated in two different experimental pain models. Our results showed that pretreatment (30 min) with IL-4 (1-5 ng/animal), IL-10 (0.4-10 ng/animal), or IL-13 (0.4-2.5 ng/animal) inhibited the writhing response induced by the i.p. administration of acetic acid (53-89%) or zymosan (63-74%) in mice, and the knee joint incapacitation induced by i.a. injection of zymosan (49-66%) in rats. Neither of the cytokines affected the pain elicited in mice using the hot-plate test. This analgesic effect of IL-4, -10, and -13 was not reversed by the combined pretreatment with the opioid receptor antagonist naloxone. IL-4, -10, or -13 significantly inhibited the release of both tumor necrosis factor (TNF)-alpha (60, 53, and 100%, respectively) and IL-1beta (80, 100, and 100%, respectively) by mice peritoneal macrophages obtained after local (i.p.) injection of zymosan. Antisera against IL-4, -10, and -13 potentiated both the zymosan-induced writhing response and the articular incapacitation. Our results demonstrate that IL-4, -10, and -13 display analgesic activity that is probably not due to endogenous opioid release. This analgesic effect could be related to a peripheral mechanism, probably via the inhibition of the release of the pro-inflammatory cytokines TNF-alpha and IL-1beta by resident peritoneal macrophages.

Effects of endothelin-1 on inflammatory incapacitation of the rat knee joint.

This study assessed the possible local nociceptive and hyperalgesic properties of endothelin-1 (ET-1) in the rat knee-joint incapacitation test, in which animals are placed for 1 min/h on a revolving (3 rpm) metal cylinder and nociception is measured as the time the hindpaw of the injected limb was off the cylinder (i.e., paw elevation time, PET). Carrageenan (Cg; 150 micrograms/joint), E. coli LPS (1 microgram/joint), and ET-1 (120 or 240 pmol/joint) each increased PET persistently, unlike sarafotoxin S6c (120-240 pmol/joint) or PBS. ET-1 (15 and 30 pmol/joint, 30 min before) did not cause incapacitation per se but potentiated PET induced by Cg, increasing the area under the curve (AUC in arbitrary units, 0-6 h) from 105 +/- 9 to 165 +/- 10 and 169 +/- 25, respectively. Prior Cg injection (300 micrograms/joint, 72 h before) sensitized the joint to incapacitation triggered by restimulation with either Cg (300 micrograms/joint), LPS (1 microgram/joint), or ET-1 (30 pmol/joint). Treatment with bosentan (10 mg/kg i.v., 15 min before joint stimulation) did not affect PET values in naive animals to Cg or LPS, but significantly reduced the upregulated response evoked by restimulation with LPS (but not Cg), from 465 +/- 24 to 290 +/- 49 (AUC 0-12 h). Therefore, ET-1 triggers nociception and hyperalgesia in the naive knee joint of the rat, perhaps via ETA receptors. Although local endogenous ETs may not have a role in inflammatory nociception in the naive joint, they may participate in articular incapacitation induced by restimulation with LPS. This latter finding could be relevant to the etiology of pain associated with chronic arthritic diseases.

Analgesic and antiinflammatory activities ofAgeratum conyzoidesin rats

The water soluble fraction (WSF) obtained from a hydroalcohol extract of A. conyzoides, a medicinal plant used in Brazilian folk medicine, was evaluated for possible analgesic and antiinflammatory activities. It was demonstrated that WSF (20–50 mg/kg; i.p.) treatment reduced the articular incapacitation induced by carrageenin (300 μg) in rats. In this model, naloxone (2 mg/kg) blocked the analgesic action of morphine (2 mg/kg) but did not change the WSF antinociceptive effect. It suggests that endogenous opioids are not involved in the WSF antinociceptive effect. The neutrophil migration induced by carrageenin (300 μg) injection into rat peritoneal cavities and into 6-day-old subcutaneous air-pouches was significantly inhibited (p <0.05) by WSF pre-treatment (30 and 50 mg/kg; s.c.). At the same dose WSF also inhibited (p <0.05) the carrageenin (400 μg/paw)-induced oedema, but failed to modify the oedema induced by dextran (100 μg/paw). Furthermore, the increase in the cutaneous vascular permeability induced by the potent leukocyte chemotactic agent LTB4 (39 ng co-injected with 500 ng iloprost, i.d.) was significantly blocked by WSF (30 mg/kg; i.p.). However, in the same dose WSF caused a 2-fold increase in the vascular permeability induced by histamine (10 μg), a direct vasoactive mediator. These results suggest that WSF can inhibit the inflammatory reactions induced by neutrophil mobilizing stimuli. © 1997 John Wiley & Sons, Ltd.

Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics

A new behavioral test is described in which quantitation is independent of the observer and is sensitive to all classes of analgesics. A computer-assisted device measures the period during which a rat hind paw fails to touch the surface of a rotating cylinder for 1 min (paw elevation time). Intra-articular injection of carrageenin induces a progressive and dose-dependent incapacitation of the limb. The maximum paw elevation time is attained 3–4 h after carrageenin challenge. The model showed dose-dependent sensitivity to 1. (a) a central acting opiate (morphine, ID 50 = 1.5 mg/kg, i.p.), 2. (b) cyclooxygenase inhibitors (indomethacin, ID 50 = 0.8 mg/kg, i.p.; diclofenac, ID 50 = 0.22 mg/kg, i.p.), and 3. (c) peripheral analgesics which directly antagonize nociceptor hypersensitivity: dipyrone (ID 50 = 21 mg/kg, i.p.), N-methyl-nalorphine (ID 50 = 14 mg/kg, i.p.) and BW443C (ID 50 = 17.5 mg/kg, i.p.). The knee-joint carrageenin incapacitation was also blocked by the sympatholytics, propranolol and guanethidine. After the blockade by either indomethacin or guanethidine, intra-articular injections of prostaglandin E 2 or dopamine, respectively, reversed carrageenin-induced incapacitation. These results suggest that during inflammatory articular incapacitation cyclooxygenase and sympathomimetic mediators are involved, as has been suggested for the rat paw carrageenin hyperalgesia test and formalin test.

On the analgesic action of peripheral acting opiates

A new behavioral test is described in which quantitation is independent of the observer and is sensitive to all classes of analgesics. A computer-assisted device measures the period during which a rat hind paw fails to touch the surface of a rotating cylinder for 1 min (paw elevation time). Intra-articular injection of carrageenin induces a progressive and dose-dependent incapacitation of the limb. The maximum paw elevation time is attained 3–4 h after carrageenin challenge. The model showed dose-dependent sensitivity to 1.(a) a central acting opiate (morphine, ID50 = 1.5 mg/kg, i.p.),2.(b) cyclooxygenase inhibitors (indomethacin, ID50 = 0.8 mg/kg, i.p.; diclofenac, ID50 = 0.22 mg/kg, i.p.), and3.(c) peripheral analgesics which directly antagonize nociceptor hypersensitivity: dipyrone (ID50 = 21 mg/kg, i.p.), N-methyl-nalorphine (ID50 = 14 mg/kg, i.p.) and BW443C (ID50 = 17.5 mg/kg, i.p.). The knee-joint carrageenin incapacitation was also blocked by the sympatholytics, propranolol and guanethidine. After the blockade by either indomethacin or guanethidine, intra-articular injections of prostaglandin E2 or dopamine, respectively, reversed carrageenin-induced incapacitation. These results suggest that during inflammatory articular incapacitation cyclooxygenase and sympathomimetic mediators are involved, as has been suggested for the rat paw carrageenin hyperalgesia test and formalin test.