Shinobu Sakurada

The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord

A capsaicin test involving peripheral nociception, which produces behaviour similar to that elicited by formalin, is described in mice. Capsaicin was injected subcutaneously (s.c.) into the dorsal surface of a hindpaw and the time the animals spent licking the paw was recorded. Doses of capsaicin of 6.25-1600 ng induced nociception, during a period of 5 min, starting immediately after injection and disappearing completely at 10 min. Intrathecally (i.t.) administered [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), a tachykinin antagonist and [D-Phe7,D-His9]substance P (6-11), a selective antagonist of substance P (SP), inhibited the capsaicin-induced behaviour, in a dose-dependent manner. This licking behaviour was also inhibited by intrathecal administration of SP antiserum but not by somatostatin (SOM) antiserum. Intrathecal pretreatment with capsaicin resulted in a marked reduction of the licking response, following subcutaneous injection of capsaicin into the paw. Capsaicin-induced licking was not affected by intrathecal administration of cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-(OBz)-Thr], a SOM antagonist and by intrathecal pretreatment with cysteamine, a SOM depletor. This nociceptive test may allow discrimination between SP- and SOM-mediated responses in the spinal cord of the mouse.

Role of histamine H1 receptor in pain perception: a study of the receptor gene knockout mice

To study the participation of histamine H(1) receptors in pain perception, histamine H(1) receptor knockout mice were examined for pain threshold by means of three different kinds of nociceptive tasks. These included assays for thermal nociception (hot-plate, tail-flick, paw-withdrawal), mechanical nociception (tail-pressure), and chemical nociception (abdominal constriction, formalin test, capsaicin test) which evoked pain by the activation in nociceptive Adelta and C fibers. The mutant mice lacking histamine H(1) receptors showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in histamine H(1) receptor knockout mice significantly decreased when compared to wild-type mice. This data indicates that histamine plays an important role in both somatic and visceral pain perceptions through histamine H(1) receptors. The difference in the effect of histamine H(1) receptors antagonist, the active (D-) and inactive (L-) isomers of chlorpheniramine on ICR mice further substantiates the evidence of the role of histamine H(1) receptors on pain threshold.

Characterization of the hyperalgesic effect induced by intrathecal injection of substance P

Substance P (SP) was administered to awake rats by injection into the lumbar subarachnoid space via an indwelling cannula. Intrathecal (i.t.) injection of substance P produced a dose-related hyperalgesic response in the tail-pressure assay. This hyperalgesic effect peaked at 1 min and returned to control level within 15 min. Tachyphylaxis to the action of substance P was not observed by successive intrathecal injections. The hyperalgesic effect induced by substance P was increased by pretreatment with naloxone and blocked by a large dose of morphine. A synthetic analogue (D-Pro2, D-Trp7,9)-substance P, was not found to block the action of substance P on mechanical responses. These results suggest that substance P apparently produces a direct action on spinal substance P receptors and the antagonistic effect of morphine on the hyperalgesia induced by substance P may be mediated through a postsynaptic mechanism in the spinal cord.

Antinociception induced by CP 96,345, a non-peptide NK-1 receptor antagonist, in the mouse formalin and capsaicin tests

The non-peptide NK-1 receptor antagonist, CP 96,345, has been evaluated for antinociceptive activity in two chemical pain models in the mouse. CP 96,345, injected intrathecally (i.t.) 5 min prior to 2.0% formalin, produced significant antinociception in both the early and late phases of the formalin-induced paw licking procedure. Antinociception could also be observed during the late phase by treatment with CP 96,345 after formalin. In the capsaicin (CAP) test, i.t. injection of CP 96,345 produced a dose-dependent reduction of the paw-licking response at doses much less than antinociceptive doses in the formalin test. Naloxone did not affect antinociception in either test. CP 96,345 evoked a reversible deficit in motor performance as assayed by the rotarod test. The results indicate that i.t. CP 96,345 is antinociceptive in the capsaicin test at doses showing no overt behavioural effects but there is an overlap in doses producing antinociceptive and motor effects in the formalin test.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

INVOLVEMENT OF NITRIC OXIDE IN SPINALLY MEDIATED CAPSAICIN- AND GLUTAMATE-INDUCED BEHAVIOURAL RESPONSES IN THE MOUSE *

The intrathecal (i.t.) injection of capsaicin (0.1 nmol/mouse) through a lumbar puncture elicited scratching, biting and licking responses. Pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (320 nmol), by i.t. injection, resulted in a significant inhibition of the behavioural response produced by i.t. capsaicin (0.1 nmol/mouse). Similar behavioural responses were induced by i.t. injections of NMDA (0.4 nmol), kainate (0.05 nmol) or AMPA (0.05 nmol), which were all inhibited by co-administration of L-NAME (20-80 nmol). L-Arginine (600 mg/kg, i.p.) but not D-arginine (600 mg/kg, i.p.) reversed the inhibitory effect of L-NAME on capsaicin-, NMDA-, kainate- and AMPA-induced behavioural response. Scratching, biting and licking responses induced by tachykinin receptor agonists, substance P, [Sar9,Met(O2)11]substance P, neurokinin A and neurokinin B were not affected by co-administration of L-NAME (40 and 80 nmol). These results suggest that spinal nitric oxide may play a significant role in mechanisms of the behavioural response to capsaicin, probably through the release of glutamate, but not tachykinins.

Enhanced antinociception by intracerebroventricularly and intrathecally-administered orexin A and B (hypocretin-1 and -2) in mice

Orexins are neuropeptides located exclusively in neurons of the lateral hypothalamic area, which send projections to most monoaminergic nuclei, such as noradrenergic locus coeruleus, dopaminergic ventral tegmental areas, and histaminergic tuberomammillary nuclei. The present work was carried out to examine the role of orexins in nociception in mice. C57BL/6 mice were administered with orexin A and B intracerebroventricularly (i.c.v.), intrathecally (i.t.) and subcutaneously (s.c.) to reveal the sites of action of these peptides and to examine the pain thresholds using four kinds of nociceptive tasks. Orexins showed antinociceptive effects in all four types of assays for thermal (hot-plate, tail-flick, paw-withdrawal), mechanical (tail-pressure), chemical (formalin, capsaicin and abdominal stretch) nociceptions and nociceptin-induced behavioral responses, when administered i.c.v. or i.t., whereas the s.c. administration was ineffective. The antinociceptive effects of orexin A were more remarkable than those of orexin B. The i.c.v. administration of orexin A was as effective as, or more potent than the i.t. administration. The effects of orexin A were completely blocked by adenosine type 1 receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and theophylline, but not by naloxone, suggesting a possible involvement of the adenosine-containing neurons and/or the adenosine pathway in these orexin actions. The i.c.v. administration of nociceptin had no significant effects on orexin expression in the brain and spinal cord. The present findings suggest that orexins have an antinociceptive role in at least four different types of pains, probably acting on both the brain and spinal cord.

Differential effects of intraplantar capsazepine and ruthenium red on capsaicin-induced desensitization in mice

Intraplantar injection of capsaicin (1.6 microg/paw) into the mouse hindpaw produced an acute paw-licking/biting response. This study was designed (1) to investigate the antinociceptive effects of intraplantar administration of capsazepine, a competitive vanilloid receptor antagonist, and ruthenium red, a noncompetitive antagonist, in the nociceptive licking/biting response induced by intraplantar injection of capsaicin, and (2) to determine whether these compounds were able to prevent capsaicin-induced desensitization in mice. Both capsazepine and ruthenium red produced a dose-dependent reduction in the capsaicin-induced nociceptive response. In licking/biting response to intraplantar capsaicin, ruthenium red was more potent than capsazepine in producing antinociceptive activity as assayed by the capsaicin test. The first injection of capsaicin induced a profound desensitization to the second and third injections of capsaicin at the interval of 15 or 30 min. The capsaicin-induced desensitization was prevented dose-dependently by antinociceptive doses of capsazepine, whereas ruthenium red in doses exhibiting antinociceptive activity was without effect on capsaicin-induced desensitization. The present results suggest that both capsazepine and ruthenium red can produce a local peripheral antinociceptive action, which may be mediated by inhibiting the membrane ion channel activated by capsaicin. In addition, these data suggest that capsazepine may act in the mechanism clearly different from ruthenium red in the capsaicin-induced nociceptive desensitization.

The effects of substance P analogues on the scratching, biting and licking response induced by intrathecal injection of N‐methyl‐d‐aspartate in mice

1 Intrathecal (i.t.) administration of N‐methyl‐d‐aspartate (NMDA) elicited a dose‐dependent behavioural response consisting of licking, biting and scratching in mice. 2 Repeated i.t. injections of 0.4 nmol NMDA, at 5 min intervals, resulted in the rapid development of desensitization to this NMDA‐induced behavioural phenomenon. 3 The NMDA‐induced response was dose‐dependently inhibited by the simultaneous injection of a selective NMDA‐receptor antagonist, d‐2‐amino‐5‐phosphonovaleric acid. 4 The substance P (SP) analogues [d‐Pro2, d‐Trp7,9] SP and [d‐Arg1, d‐Trp7,9, Leu11] SP (spantide) inhibited NMDA‐induced behavioural responses in a dose‐dependent manner. However, [d‐Phe7, d‐His9] SP (6–11), a SP analogue selective for neurokinin1 (NK1) receptors, failed to inhibit NMDA‐induced responses even at a dose of 4.0 nmol. 5 These results indicate that NMDA‐induced behavioural responses are mainly mediated through NMDA receptors without affecting NK1 receptors in the spinal cord.

Behavioural characterization of substance P-induced nociceptive response in mice

Intrathecal injection of substance P produced a behavioural syndrome, consisting of reciproacal hindlimb scratching and biting or fore- and hind-licking. Pretreatment with either an analogue of substance P, (D-Pro2, D-Trp7,9)-substance P (DPDT-SP) or (D-Arg1, D-Pro2,4, D-Trp7,9, Leu11)-substance P, given intrathecally, reduced the response to substance P in a dose-dependent manner. The behaviour induced by substance P was also inhibited by intrathecal, intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) injection of morphine. Intrathecal or subcutaneous injection of naloxone showed a biphasic effect on substance P response; the substance P-induced nociceptive response was increased by a small dose of naloxone, while it was inversely decreased by a large dose of naloxone. The results with analogues of substance P support the hypothesis that substance P, injected intrathecally, acts directly on substance P receptors in the spinal cord. The nociceptive response induced by substance P appears to be controlled by endogenous opioids in the spinal cord.