Maria S. de Abreu Castro

Role of tachykinin NK receptors on the local and remote injuries following ischaemia and reperfusion of the superior mesenteric artery in the rat.

Neuropeptides acting on tachykinin NK receptors play an important role in the amplification of inflammatory responses. We have assessed the effects of tachykinin NK receptor blockade on the injuries following intestinal ischaemia and reperfusion (I/R) in rats. The tachykinin NK1 receptor antagonist SR140333 dose‐dependently (0.05 to 0.5 mg kg−1) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. A structurally‐distinct NK1 receptor antagonist, CP99,994, but not tachykinin NK2 or NK3 receptor antagonists also suppressed mild I/R injury. Neonatal pretreatment with capsaicin effectively depleted sensory neurons and abrogated the injuries following mild I/R. Treatment with SR140333 (0.5 mg kg−1) significantly reversed severe reperfusion‐induced local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and blood neutropaenia, but did not prevent the lethality associated with severe I/R. Post‐ischaemic treatment with SR140333 significantly inhibited the elevations of TNF‐α in the intestine and lung, but not serum, following severe I/R. The increase in the concentrations of IL‐10 in the lung and serum were also suppressed. Post‐ischaemic blockade of tachykinin NK1 receptors markedly inhibited the local and remote injuries, but not lethality, following reperfusion of the SMA in rats. Neuropeptides, possibly substance P, released from sensory nerves appear to account for the activation of these tachykinin NK1 receptors. Antagonists of the tachykinin NK1 receptor may be useful adjuncts in the treatment of the injuries which occur following reperfusion of an ischaemic vascular territory.

Opioidergic orofacial antinociception induced by electroacupuncture at acupoint St36

The participation of opioids in the antinociceptive effect of electroacupuncture was evaluated in terms of nociception produced by thermal stimuli applied to the face of male Wistar rats, weighing 180-230 g. Electrical stimulation (bipolar and asymmetric square wave with 0.5 mA intensity for 20 min) of acupoint St36, located in the anterior tibial muscle 10 mm distal to the knee joint, induced antinociception in the present model, which was maintained for 150 min. Acupoint LI4, located in the junction of the first and second metacarpal bones, did not achieve antinociception at any frequency studied (5 Hz: 1.7 +/- 0.1; 30 Hz: 1.8 +/- 0.1; 100 Hz: 1.7 +/- 0.1 vs 1.4 +/- 0.2). The antinociception obtained by stimulation of acupoint St36 was only achieved when high frequency 100 Hz (3.0 +/- 0.2 vs 1.0 +/- 0.1) was used, and not with 5 or 30 Hz (1.2 +/- 0.2 and 0.7 +/- 0.1 vs 1.0 +/- 0.1). The antinociceptive effect of acupuncture occurred by opioid pathway activation, since naloxone (1 and 2 mg/kg, subcutaneously) antagonized it (1.8 +/- 0.2 and 1.7 +/- 0.2 vs 3.0 +/- 0.1).

Participation of ATP-sensitive K+ channels in the peripheral antinociceptive effect of fentanyl in rats

We examined the effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and cesium on the ability of fentanyl, a clinically used selective micro-opioid receptor agonist, to promote peripheral antinociception. Antinociception was measured by the paw pressure test in male Wistar rats weighing 180-250 g (N = 5 animals per group). Carrageenan (250 microg/paw) decreased the threshold of responsiveness to noxious pressure (delta = 188.1 +/- 5.3 g). This mechanical hyperalgesia was reduced by fentanyl (0.5, 1.5 and 3 microg/paw) in a peripherally mediated and dose-dependent fashion (17.3, 45.3 and 62.6%, respectively). The selective blockers of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 microg/paw) and tolbutamide (80, 160 and 240 microg/paw) dose dependently antagonized the antinociception induced by fentanyl (1.5 microg/paw). In contrast, the effect of fentanyl was unaffected by the large conductance Ca2+-activated K+ channel blocker ChTX (2 microg/paw), the small conductance Ca2+-activated K+ channel blocker apamin (10 microg/paw), or the non-specific K+ channel blocker TEA (150 microg/paw), 4-AP (50 microg/paw), and cesium (250 microg/paw). These results extend previously reported data on the peripheral analgesic effect of morphine and fentanyl, suggesting for the first time that the peripheral micro-opioid receptor-mediated antinociceptive effect of fentanyl depends on activation of ATP-sensitive, but not other, K+ channels.

Mechanisms underlying the inhibitory effects of tachykinin receptor antagonists on eosinophil recruitment in an allergic pleurisy model in mice

The activation of tachykinin NK receptors by neuropeptides may induce the recruitment of eosinophils in vivo. The aim of the present study was to investigate the effects and underlying mechanism(s) of the action of tachykinin receptor antagonists on eosinophil recruitment in a model of allergic pleurisy in mice. Pretreatment of immunized mice with capsaicin partially prevented the recruitment of eosinophils after antigen challenge, suggesting the potential contribution of sensory nerves for the recruitment of eosinophils Local (10–50 nmol per pleural cavity) or systemic (100–300 nmol per animal) pretreatment with the tachykinin NK1 receptor antagonist SR140333 prevented the recruitment of eosinophils induced by antigen challenge of immunized mice. Neither tachykinin NK2 nor NK3 receptor antagonists suppressed eosinophil recruitment. Pretreatment with SR140333 failed to prevent the antigen‐induced increase of interleukin‐5 concentrations in the pleural cavity. Similarly, SR140333 failed to affect the bone marrow eosinophilia observed at 48 h after antigen challenge of immunized mice. SR140333 induced a significant increase in the concentrations of antigen‐induced eotaxin at 6 h after challenge. Antigen challenge of immunized mice induced a significant increase of Leucotriene B4 (LTB4) concentrations at 6 h after challenge. Pretreatment with SR140333 prevented the antigen‐induced increase of LTB4 concentrations. Our data suggest an important role for NK1 receptor activation with consequent LTB4 release and eosinophil recruitment in a model of allergic pleurisy in the mouse. Tachykinins appear to be released mainly from peripheral endings of capsaicin‐sensitive sensory neurons and may act on mast cells to facilitate antigen‐driven release of LTB4.

Pharmacological Characterization of Sephadex-lnduced Oedema in Rat Paws: Predominant Role of Serotonin and Platelet-Activating Factor

An intravenous injection of Sephadex beads has been used to induce lung inflammation and bronchial hyperreactivity in small animals. In the present study, we injected Sephadex beads (0.3-5.5 mg/paw) into rat paws and followed the resulting inflammation plethysmometrically. Our results show that Sephadex beads induced a significant and dose-dependent increase in the hindpaw volume at 5 min; it was maximal at 30-60 min and declined at 4 h. However, the paw volume remained significantly increased for up to 21 days. The initial 4-hour-oedema was confirmed by histopathology of the paw tissues, but the persistent increase in paw volume was related to a chronic inflammatory (granulomatous) response. The Sephadex-induced oedema was predominantly due to serotonin (5-HT) release since specific antagonists such as methysergide (1 mg/kg) and pizotifen (0.1-2 mg/kg) administered both systemically and locally were able to inhibit the oedema (10-100 microgram/paw) as could pretreatment with compound 48/80. In addition, platelet-activating factor (PAF) was also shown to be involved, since systemic pretreatment using the specific PAF antagonist BN 52021 (1 mg/kg) was able to inhibit the increase in paw volume induced by Sephadex. Effective doses of indomethacin (2 mg/kg), L-NAME (1 mg/kg), pyrilamine (1-2 mg/kg), ondansetron (1 mg/kg) and HOE 140 (1 mg/kg) did not affect the Sephadex-induced oedema, thus ruling out the participation of prostaglandins, nitric oxide, histamine, 5-HT3 receptors and bradykinin in its development. Since the late increases in paw volume induced by Sephadex were reduced by pretreatment of the animals with the immunosuppressive drugs rapamycin and dexamethasone but not cyclosporin, our results also suggested that distinct immunological pathways may be involved in the modulation of the chronic phase of inflammation induced by Sephadex beads in rat paws.

Evidence for a role of capsaicin-sensitive sensory nerves in the lung oedema induced by Tityus serrulatus venom in rats.

In the most severe cases of human envenoming by Tityus serrulatus, pulmonary oedema is a frequent finding and can be the cause of death. We have previously demonstrated a role for neuropeptides acting on tachykinin NK(1) receptors in the development of lung oedema following i.v. injection of T. serrulatus venom (TsV) in experimental animals. The present work was designed to investigate whether capsaicin-sensitive primary afferent neurons were a potential source of NK(1)-acting neuropeptides. To this end, sensory nerves were depleted of neuropeptides by neonatal treatment of rats with capsaicin. The effectiveness of this strategy at depleting sensory nerves was demonstrated by the inhibition of the neuropeptide-dependent response to intraplantar injection of formalin. Pulmonary oedema, as assessed by the levels of extravasation of Evans blue dye in the bronchoalveolar lavage and in the left lung, was markedly inhibited in capsaicin-treated animals. In contrast, capsaicin treatment failed to alter the increase in arterial blood pressure or the lethality following i.v. injection of TsV. Our results demonstrate an important role for capsaicin-sensitive sensory nerves in the cascade of events leading to lung injury following the i.v. administration of TsV.

The endogenous cytokine profile and nerve fibre density in mouse ear Leishmania major-induced lesions related to nociceptive thresholds

Several reports have shown that cutaneous leishmaniasis lesions are painless, suggesting that Leishmania infection interferes with pain perception. Comparisons of inflammation-induced hyperalgesia between BALB/c and C57BL/6 mice have been little explored in the literature, and comparative data regarding nociception in leishmaniasis are non-existent. In susceptible BALB/c mice and resistant C57BL/6 mice that were intradermally inoculated with a low dose of Leishmania major in the ear, we investigated the variation in nociception over a 12-wk period post-infection and this variations association with the structure of nerve fibres and the presence of endogenous cytokines that are classically considered hyper- or hypo-nociceptive. Infected BALB/c mice presented susceptibility and severe lesions. Infected C57BL/6 mice exhibited resistance and healing lesions. The immune response involved pro- and anti-inflammatory cytokine secretion, respectively. The infection-induced hypoalgesia in BALB/c mice after wks 9 was accompanied by decreased levels of IL-6 and IL-10 in ear tissue with intact nerves. C57BL/6 mice showed short-lived hyperalgesia in wks 2, which was related to increased local levels of IL-6, KC/CXCL-1, TNF-α and IL-10 and a decrease in nerve density. The increase in pro-inflammatory cytokine IL-6, KC/CXCL-1 and TNF-α levels during hyperalgesia suggested a role for these mediators in afferent nerve sensitisation, which was secondary to the inflammatory damage of nerve fibres stained by PGP 9.5. In contrast, the mechanisms of hypoalgesia may include the downregulation of cytokines, the preservation of the structure of nerve endings, and as yet uninvestigated unidentified differences in neurotransmitter release or a direct role of the parasites in the context of the progressive and permissive inflammatory response of BALB/c mice.