Tail Withdrawal Test Research Articles

Blockade of CCK-B receptors by L-365,260 induces analgesia in the squirrel monkey

The potential antinociceptive effects of the selective cholecystokinin-B (CCK-B) antagonist L-365,260 were examined in the squirrel monkey tail withdrawal test. Pain threshold was measured in 6 male monkeys by recording the latency to remove the tail from a warm (55 °C) water bath. L-365,260 at doses of 100 ng/kg to 100 μg/kg significantly elevated tail withdrawal latencies throughout a 2 h test period. These data provide the first evidence that blockade of CCK-B receptors induces analgesia in primates.

Region-specific noradrenaline depletion by neonatal DSP-4: functional consequences and effect on a coexisting neurotransmitter

Noradrenaline (NA) depletion following neonatal treatment of rats with DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine) was not accompanied by any changes in neuropeptide tyrosine (NPY) levels. After 85–95% NA depletion in the spinal cord of DSP-4-treated rats, the reaction time in the tail withdrawal test was shorter than in the controls but the endogenous activation of the sympathetic nervous system in the baroreceptor reflex remained unchanged. These results suggest that the NA neurons in the spinal cord have an inhibitory function the processing of incoming sensory information.

Effect of subtype-selective opioid receptor blockers on nitrous oxide antinociception in rats

Nitrous oxide antinociception in rats was evaluated by the warm water tail withdrawal test following central pretreatment with blockers of various opioid receptor subtypes. The analgesic dose, 50% (AD 50) value for nitrous oxide antinociception was significantly elevated by MR-2266 (which is relatively selective for κ-opioid receptors) and increased to a lesser degree by ICI-174,864 (which is selective for δ-opioid receptors). However, pretreatment with β-funaltrexamine (which is selective for μ-opioid receptors), even at extremely high doses, was ineffective in altering the AD 50 for nitrous oxide antinociception. According to these findings, nitrous oxide antinociception, as evaluated in this paradigm, appears to be mediated by κ- and possibly δ- but not μ-opioid receptors.

Comparative pharmacological and biochemical studies between butorphanol and morphine

A number of in vivo and in vitro studies were undertaken to compare the pharmacological and biochemical effects of the partial agonist, butorphanol, with that of morphine. Both compounds were equipotent antinociceptive agents in the rat tail withdrawal test. In the acetic acid writhing test butorphanol had approximately 3.5 times the antiwrithing activity on a molar basis than morphine. In a study of the effects of these compounds on body temperature, butorphanol as well as morphine produced hyperthermia after acute dosing. Additionally, butorphanol produced a profound diuresis and decrease in urine osmolality after acute administration. In contrast, morphine produced an antidiuresis throughout most of the study period with no significant changes in urine osmolality from control. Butorphanol administration had no effect on respiratory rate, while morphine markedly decreased respiratory rate. In in vitro radioligand displacement studies, butorphanol was a potent competitor against 3H-DAGO, 3H-DPDPE, and 3H(−)-EKC binding, exhibiting 3, 10, and 30 times more activity, respectively, than morphine. Both compounds were weak inhibitors of 3H-(+)-SKF 10047 binding, yielding IC 50 values of in excess of 1 μM. The results indicate that butorphanol has multiple actions on the opioid receptor system, and shares similarities as well as differences in its mechanism(s) of actions with morphine.

The response to analgesia testing is affected by gonadal steroids in the rat

The effect of gonadal steroids on the response to analgesia testing was determined in castrated male and female rats and castrated male and female rats treated with testosterone proprionate (TP) and estradiol benzoate (EB), respectively. The time to respond to a noxious somatic stimulus in the form of heat was assessed using the tail withdrawal test (tail withdrawal from hot water) and hot plate test (the time to paw lick or jump). In male rats, castration resulted in a significant reduction of the reaction time for tail withdrawal. This effect was reversed by treatment with TP. The time to paw lick or jump in male rats was also diminished by castration. Treatment with TP resulted in a partial reversal of the effect of castration on this response. In castrated female rats, the time required for tail withdrawal was decreased by castration and increased by treatment with EB. The reaction time to the hot plate in female rats was diminished by castration and further reduced by EB administration. These data indicate that gonadal steroids influence the response to a noxious heat stimulus in male and female rats and that the effect may vary according to sex and the way in which the stimulus is applied.

Opioid δ-receptor involvement in supraspinal and spinal antinociception in mice

The possibility that the opioid δ-receptor mediates antinociception in tests where heat is the noxious stimulus was investigated using highly selective μ- and δ-agonists and -antagonists. Antinociceptive dose-response curves were constructed for μ ([ d-Ala 2, NMePhe 4, Gly-ol]enkephalin, DAGO; morphine) and δ ([ d-Pen 2, d-Pen 5]enkephalin, DPDPE)-agonist in the absence, and in the presence of the μ non-surmountable antagonist, β-funaltrexamine (β-FNA) or the δ-antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu- OH, where Aib is α-amino-isobutyric acid). Agonists and ICI 174,864 were given alone or in the same intracerebroventicular (i.c.v.) or intrathecal (i.th.) injection to mice 20 min prior to testing in the warm-water (55 °C) tail-withdrawal test (+10 min for i.th. DPDPE); β-FNA was given as a single i.c.v. or i.th. pretreatment dose (20 and 0.01 nM, respectively) 4 hr with β-FNA resulted in a rightward displacement of the DAGO and morphine antinociceptive dose-response lines, but failed to displace the i.c.v. DPDPE curve. Similarly, i.th. pretreatment with β-FNA displaced the i.th. morphine dose-response curve to the right without affecting the i.th. DPDPE antinociceptive dose-response line. ICI 174,864 (1 and 3 μg) produced a dose-related antagonism of i.c.v. or i.th. DPDPE, but did not alter the antinociceptive effects of DAGO or morphine given by the same routes. Co-administration of ICI 174 864 (μg) i.c.v. morphine in β-FNA pretreated (but not control) mkce resulted in a further rightward displacement of the morphine dose-response line. The effective antagonism of DPDPE but not morphine or DAGO antinociception by ICI 174,864, together with the effectiveness of β-FNA against morphine and DAGO but not DPDPE antinociception, provide strong and direct evidence for the involvement of cerebral and spinal δ-receptors in the mediation of antinociception in tests where heat is employed as the noxious stimulus. Additionally, the effectiveness of ICI 174,864 against morphine in β-FNA pretreated (but not control) mice demonstrates a δ-effect of morphine, in vivo.

Stereospecific potentiation of opiate analgesia by cocaine: predominant role of noradrenaline

Cocaine hydrochloride (50 mg) pellets implanted subcutaneously in male Wistar rats potentiated the analgesia of morphine, levorphanol, methadone and buprenorphine as measured by the tail-withdrawal test. Potentiated opiate analgesia was abolished by naloxone and further enhanced by desipramine and phenoxybenzamine. Yohimbine, α-methyl p-tyrosine, haioperidol, zimelidine, methysergide, p-chlorophenylalanine produced no significant effect on potentiated opiate analgesia. Pseudococaine (dextro-cocaine), which is several-fold less potent than cocaine as an inhibitor of noradrenaline and dopamine reuptake in the CNS, had no significant effect on opiate analgesia. Analgesia produced by low doses of baclofen, a GABA agonist, was also not potentiated by cocaine. This study suggests a predominant role for noradrenaline in the Stereospecific potentiation of opiate analgesia by cocaine.

Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: Studies with receptor selective agonists

The apparent affinity of naloxone at cerebral and spinal sites was estimated using selective mu [D-Ala 2, Gly-ol 5]-enkephalin (DAGO) and delta [D-Pen 2, D-Pen 5]enkephalin] (DPDPE) opioid agonists in the mouse warm water tail-withdrawal test in vivo; the mu agonist morphine was employed as a reference compound. The approach was to determine the naloxone pA 2 using a time-dependent method with both agonist and antagonist given intracerebroventricularly (i.c.v.) or intrathecally (i.th.); naloxone was always given 5 min before the agonist. Complete time-response curves were determined for each agonist at each site in the absence, and in the presence, of a single, fixed i.c.v. or i.th. dose of naloxone. From these i.c.v. or i.th. pairs of time-response curves, pairs of dose-response lines were constructed at various times; these lines showed decreasing displacement with time, indicative of the disappearance of naloxone. The graph of log (dose ratio - 1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this plot, the apparent pA 2 and naloxone half-life was calculated at each site and against each agonist. The affinity of naloxone was not significantly different when compared between agonists after i.c.v. administration. A small difference was seen between the affinity of i.th. naloxone against DPDPE and DAGO; the i.th. naloxone pA 2 against morphine, however, was not different than that for DPDPE and DAGO. The naloxone half-life varied between 6.6 and 16.9 min, values close to those previously reported for this compound. These results suggest that the agonists studied may produce their i.c.v. analgesic effects at the same receptor type or that alternatively, the naloxone pA 2 may be fortuitously similar for mu and delta receptors in vivo. Additionally, while the affinity of naloxone appears different for the receptors activated by i.th. DAGO and DPDPE, further work may be necessary before firm conclusions regarding the nature of the spinal analgesic receptor(s) can be drawn.

ChemInform Abstract: Racemic and Optically Active 1,3,3‐Trimethyl‐4‐phenyl‐4‐(propionyloxy)piperidine.

AbstractDas Piperidin (II) sowie (+)‐ und (‐)‐(II) werden dargestellt, von denen (:‐)‐(II) keine antinociceptive Wirkung besitzt.

Racemic and optically active 1,3,3-trimethyl-4-phenyl-4-(propionyloxy)piperidine.

The preparation and resolution of 1,3,3-trimethyl-4-phenyl-4-(propionyloxy)piperidine (5,3-methylprodine) are described, and the results of the antinociceptive activities of the products by hot-plate (mice) and tail-withdrawal (rats) tests are shown to support proposals made from a recent analysis of the stereochemical structure-activity relationships of C-methyl derivatives of the reversed ester of meperidine. Data of absolute configuration were obtained by X-ray crystallography of a hydrobromide salt.

Electrolytic raphe magnus lesions block analgesia induced by a stress-morphine interaction but not analgesia induced by morphine alone.

Morphine analgesia in the tail withdrawal test was examined in rats which had had lesions of the nucleus raphe magnus (NRM) or sham operations. Half of the rats were tested while restrained, whereas the other half were not restrained. In the sham-operated rats, restraint potentiated the analgesic response to morphine. Lesions of the NRM had no effect on unrestrained rats but reduced the morphine analgesia exhibited by restrained rats so that it was indistinguishable from that of unrestrained rats. The NRM seems to be involved in analgesia induced by an interaction of morphine and restraint but not in analgesia induced by morphine alone.

Evidence that stress augments morphine analgesia by increasing brain tryptophan

Morphine analgesia measured by the tail withdrawal test was examined in rats that were either restrained or left free during testing. It was found that restraint potentiated morphine analgesia and decreased the latency of the peak analgesic effect. Methysergide, a serotonin antagonist, and valine, which prevents the increase in brain tryptophan induced by restraint, blocked the effect of restraint on morphine analgesia. Valine did not alter analgesia in unrestrained rats. An increase in brain tryptophan uptake induced by stress is suggested as a possible mechanism by which stress can interact with pain modulation systems.

Binding characteristics and analgesic activity of D-Ala 2-Leu 5-enkephalin chloromethyl ketone

The chloromethyl ketone derivative of D-Ala 2-Leu 5-enkephalin (DALECK) was synthesized and its potency was tested in competing for 3H-naloxone binding sites and inducing analgesia. It was established that the compound is a potent affinity reagent at alkaline pH, blocking selectively and irreversibly the high-affinity (K D<1 nM) binding site. Intracisternally given DALECK showed a long-lasting, dose-dependent antinociceptive effect in the rat tail-withdrawal test. This could be completely antagonized by naloxone administration showing the reversible nature of DALECK in this in vivo assay. It is suggested that DALECK binds reversibly to the morphine receptor which mediates analgesia but irreversibly to the enkephalin receptor, the function of which remains to be elucidated.

Capsaicin and nociception in the rat and mouse. Possible role of substance P.

Newborn or adult rats and mice were treated with capsaicin. The effect of systemic or intrathecal treatment on thermonociception, chemonociception, content and release of immunoreactive substance P (I-SP) was investigated. Treatment of two day old rats caused a small, but life-long elevation of the hot plate or tail withdrawal latency. Treatment of adult rats led to a large increase in the reaction time on the hot plate for 4--10 days but the tail withdrawal latency was only slightly elevated for not more than 1--2 days. Mice treated on the 2nd day of life had normal reaction times on the hot plate and a small and inconsistent prolongation of the tail withdrawal latency. In contrast, mice treated on day 7, 10 or as adults had greatly prolonged latencies in both tests for at least 3 months. The changes in latencies were not affected by naloxone or methysergide. Responses to noxious chemical stimuli were moderately inhibited in mice treated on the 2nd day of life, but almost abolished in mice treated on day 7, 10 or as adults. Neonatal capsaicin treatment of rats resulted in a depletion of I-SP in spinal cord and sciatic nerve for 20 months. Capsaicin-evoked release of I-SP from rat spinal cord was reduced by 93% after neonatal treatment, but only by 69% 2 weeks after adult treatment. Treatment of mice on day 2 caused a similar decrease of the I-SP content in spinal cord and of the capsaicin-evoked I-SP release (88%) as treatment on day 4 or 7 although behavioral changes were different. After treatment of adult mice release of I-SP was reduced by 93%. Capsaicin administered intrathecally to rats or mice depleted I-SP in the spinal cord but not in the sciatic nerve. The animals were almost insensitive to noxious heat (tail withdrawal test) and to local application of mustard oil or capsaicin to the hindpaw. Chemosensitivity of the eye, however, remained unchanged. The experiments indicate that systemic or intrathecal capsaicin treatment of rats or mice affects thermo- and chemonociception but species differences were found. It appears, furthermore, that changes in substance P alone cannot explain all the observed behavioral effects after capsaicin treatment.

Effect of capsaicin on morphine analgesia--possible involvement of hypothalamic structures.

1. Rats were treated with 50 mg/kg capsaicin on the second day of life or at the age of 2--3 months. The effect of morphine on the nociceptive threshold, as determined by the reaction time in tail withdrawal test, was measured 3--4 and 1--2 months after capsaicin pretreatment, respectively. 2. The analgesic effect of morphine was markedly attenuated in rats treated with capsaicin in the adult age, while neonatal capsaicin treatment did not affect morphine analgesia. 3. Pretreatment of adult rats with capsaicin results in the impairment of certain hypothalamic preoptic neurones, while neonatal capsaicin treatment induces selective degeneration of chemosensitive primary sensory neurones without affecting hypothalamic neurones. Therefore, it is suggested that in the analgesic effect of morphine the capsaicin-sensitive neurones of the preoptic area are involved, and the contribution of spinal mechanisms might be of minor importance. Thus, the preoptic region may be an important link in endogenous pain controlling systems.

Synthetic analgesics. Synthesis and pharmacology of the diastereoisomers of N-(3-methyl-1-(2-phenylethyl)-4-piperidyl)-N-phenylpropanamide and N-(3-methyl-1-(1-methyl-2-phenylethyl)-4-piperidyl)-N-phenylpropanamide.

The synthesis of the respective diastereoisomers and enantiomers of N-[3-methyl-1-(2-phenylethyl)-4-piperidylj-Nphenylpropanamide and N-[3-methyl-l-(l-methyl-2-phenylethyl)-4-piperidyl]-N-phenylpropanamide is reported. Analgesic activity is evaluated in the tail withdrawal test in rats. cis-(+)-N-[3-Methyl-l-(2-phenylethyl)-4-piperidyll-N-phenylpropanamide (23) is found to be an extremely potent analgesic, up to 6684 times morphine. Compound 23 has a fast onset of action, a shorter duration of action than morphine, and an unusually high safety margin. As part of a continuing effort to develop novel analgesic agents a series of methyl-substituted derivatives of fentanyl (1) was prepared. Fentanyl, a well-known analgesic characterized by high potency, a rapid onset, and short duration of action,lg2 belongs to a series N-[l-(2-phenylethyl)-4-piperidyl]-N-phenylpropanamide~.~ At the time of the peak effect (Table IV) 1 is about 300 times more potent than morphine in the tail withdrawal test in rats..4 It is known that methyl substitution in the side chain (Y to the basic nitrogen of 1 (compound 2) enhances the analgesic a~tivity.~ On the other hand, the activity-enhancing effects of 3-methyl substitution in the piperidine ring of 4-phenylpiperidine analgesics are well documented.”* These considerations have led to synthesis of the different diastereoisomers and enantiomers of N-[3-methyl-l(2-phenylethyl)-4-piperidyl]-N-phenylpropanamide (3, R = H) and N-[3-methyl-l-(l-methyl-2-phenylethyl)-4-piperidyll-N-phenylpropanamide (3, R = Me). The recent publication of Riley, et UL.,~ has prompted us to report our results.