Morphine withdrawal modifies antinociceptive effects of acute morphine in rats

Repeated opioid use is known to cause tolerance of antinociceptive effects. Whether opioid abstinence modifies antinociceptive effects is unknown. Here we reported that morphine withdrawal for 18 h and 4 days after repeated morphine treatment largely reduced tail-flick latencies compared with control, while the rats showed severe withdrawal syndromes. However, the latencies and withdrawal syndromes were restored to control level at 20 days withdrawal. Similarly, antinociceptive effects of acute morphine were decreased at 18 h and further decreased at 4 days but restored to control level at 20 days withdrawal. Behavioral stress that was given to the rats at 18 h withdrawal further reduced tail-flick latencies and antinociceptive effects. Conversely, the glucocorticoid receptor antagonist RU38486 increased tail-flick latencies and antinociceptive effects at 4 days withdrawal. These results suggest that morphine withdrawal could evoke behavioral stress to modify antinociceptive effects, implicating a significant influence of opioid abstinence on chronic pain treatment.     

Nociceptive reactions during stimulation of immunity in rats with various individual resistance to stress

Various behavioural nociceptive reactions and individual resistance against stress were studied under conditions of stimulation of the immune processes by various techniques. The research problems included a study of influence of the immune stimulation with preparation "Imunofan" upon pain responses depending on individual resistance of animals to a stress, and the obtained results were compared with similar data in natural model of immune activation. To reveal central immune regulation of nociceptive reactions, imunofan was injected into brain ventricles. The work was carried out in 43 "Wistar" adult male rats. Free "open field" behaviour of animals was recorded to define a stress-resistance. Following nociceptive reactions, tail-flick to thermal stimuli; start, escape, jumping and vocalization to electrical skin stimulation, were studied. It was shown that intramuscular injection of imunofan (0.01 ml, 0.005% solution) depressed an active behaviour of animals in open field and reduced pain thresholds. This hyperalgesia was much higher in non-resistant rats in comparison to the resistant ones. Similar results were obtained in natural activation of immunity caused by operative procedure necessary for injection of imunofan into ventricles. Intracerebroventricular injections were accompanied by stronger and more complex changes of pain sensitivity.     

Role of giant cell nuclei of the reticular formation in mechanisms of analgesia during auricular electroacupuncture and action of morphine

The activity of antinociceptive mechanisms induced by auricular electroacupuncture (AEA) and intraperitoneal injection of morphine (M) was studied on rats subjected to bilateral lesion of gigantocellular nuclei (GCN). It was shown that under AEA hot-plate (HP) and tail-flick (TF) latencies (L) significantly increased as compared to the baseline level. A comparison of L shows that HP and TF in the experimental group were significantly shorter than in the control group. After M injection the rats of both the experimental and control groups showed a significant increase of HP L and TF L as compared to the baseline level, but there was no difference in L between the groups. It is concluded that neurochemical systems of GCN are involved in the mechanism of antinociception elicited in AEA while the mechanisms of antinociceptive effect of M do not involve GCN.     

Relationship between the Plasma Testosterone Level and Pain Reaction Times in Male Rats

Male prepubertal (about 4 weeks old) Wistar rats were used to estimate the pain reaction times using the tail-flick and hot-plate models; the testosterone concentration in the blood plasma was measured in all the animals before the tests. The same sets of animals were kept for the next 4 weeks under standard conditions; the experiment was repeated, and pain reaction times were also evaluated in the 8-week-old rats with blood samples collected to determine the plasma testosterone level. The results showed significant (P < 0.01) increases in the pain reaction times in both pain models in pubertal animals observed in a parallel manner with a corresponding significant (P < 0.01) increase in the plasma testosterone level. Therefore, age and sex are important factors in the choice of animals in pain experiments.     

Tail-flick latency and self-mutilation following unilateral deafferentation in rats

Unilateral deafferentation induced by transection of the C(4)-C(8) dorsal roots of spinal cord, followed by a complex of abnormal self-mutilating behavior, is interpreted as an animal model of chronic nociception. The objective of our study was to test the differences in tail-flick latency between intact control and unilaterally deafferented animals and to assess the changes in their acute nociceptive sensation. The initial hypothesis was that deafferentation-induced painful sensation might cause stress-induced analgesia that should be manifested as prolonged tail-flick latency. The experiment was carried out on 11 male and 10 female adult Wistar rats. The tail-flick latency was repeatedly measured over a period of 10 consecutive weeks both in the preoperative baseline period and following multiple cervical dorsal rhizotomy. Contrary to our hypothesis, unilateral deafferentation was followed by a significant shortening of the tail-flick latency both in males and females. In deafferented animals, compared to the controls, variations of tail-flick latency were reduced. In individual animals after deafferentation, concurrent dynamic changes were observed in self-mutilating behavior, in a loss and regaining of body weight, and in tail-flick latency. Our data suggest that changes in tail-flick latency may be interpreted in terms of central sensitization and that tail-flick latency might be considered as a useful marker of chronic nociception.     

Analgesic efficacy of orally administered buprenorphine in rats

The analgesic effect of orally administered buprenorphine was compared with that induced by a standard therapeutic injected dose (0.05 mg/kg of body weight, s.c.) in male Long-Evans rats. Analgesia was assessed by measuring pain threshold, using the hot-water tail-flick assay before and after administration of buprenorphine. The results suggest that a commonly used formula for oral buprenorphine in flavored gelatin, at a dose of 0.5 mg/kg, does not increase pain threshold in rats. Instead, oral buprenorphine doses of 5 and 10 mg/kg were necessary to induce significant increases in pain threshold. However, these doses had to be administered by orogastric infusion because the rats would not voluntarily eat flavored gelatin containing this much buprenorphine. The depth of analgesia induced by these infused doses was comparable to that induced by the clinically effective s.c. treatment (0.05 mg/kg).     

Features of active control used in pharmacological studies

The effects of 5 injections of salt solution and unfractionary heparin in dose 0.36 microgram/kg (Serva, Germany 10 kDa, activity 180 U/mg) have been studied in Wistar rats. It was found that two injections of salt solution were enough to form a stable defensive state in all rats which was manifested as an expectation of pain in tail-flick testing. The defensive motivation provoked by the injections negatively influenced the learning process as saline-induced rats refused to solve a food-getting task in a problem situation. Explorative and locomotor activities were depressed in these rats and were accompanied by numerous stressful and neurotic-like manifestations. Unlike saline-rats, practically all heparinized-rats instead of 45% of intact rats were able to solve a cognitive task despite the injections. Anxiety was decreased, but sensitivity to different external factors was increased in the heparin-induced rats. Formed habit in these rats was characterized by a high organization and stability. However, the majority heparin effects in tail-flick test were discovered when comparing the heparin-induced rats with intact ones and were not observed in comparison with the saline-rats. It is suggested that the saline-control should be considered as specific control having defensive features which are necessary to take into account in interpretation of effects of other pharmacological preparations.     

Effect of 5,7-dihydroxytryptamine on pain sensitivity and analgesic activity of morphine

Electron microscopy of rats ultrathin sections from dorsal and central raphe nucleus and spinal cord after 5,7-dihydroxytryptamine intracisternal microinjection (200 micrograms) has revealed neurones and axonal terminals distruction, which associated with tail-flick hypoalgesia and blood pressure nociceptive reactions diminished. In this condition the morphine (2 mg/kg) analgesia and drug depressive effect on pain hemodynamic manifestations increase significantly.     

Effect of naloxone on hypoalgesia in rats exposed to prolonged immobilization stress

Significant hypoalgesia (tail-flick reflex at 60 degrees C) was observed in rats during the whole period of 24-hour immobilization in cramped cages, as compared to food-and-water deprived and control animals. This hypoalgesia was not antagonized by naloxone (1 mg/kg), however, the animals periodically receiving the drug during immobilization revealed aggressiveness and significant hypoalgesia after immobilization was discontinued (30-120 min observation).     

Intrathecal pertussis toxin attenuates the morphine withdrawal syndrome in normal but not in arthritic rats

The effect of intrathecal pertussis toxin on morphine dependence was studied in rats suffering from chronic pain (Freund's adjuvant-induced arthritis). Animals were rendered tolerant-dependent by subcutaneous implantation of 3 pellets of 75 mg morphine base each. In both, normal and arthritic animals, 1 microgram pertussis toxin reduced the analgesia induced by morphine in the tail-flick test. Naloxone (1 mg/kg, s.c.) precipitated a withdrawal syndrome in arthritic animals that was milder in respect to the one produced in normal rats. Pretreatment with pertussis toxin significantly diminished the incidence of withdrawal signs such as jumps, squeak on touch, chattering, ptosis, body shakes and diarrhoea in tolerant-dependent normal rats, while this effect could not be observed in animals suffering from chronic pain. This differential activity of the toxin could be due to the altered tonus of certain neurotransmitter systems that accompanies the chronic situation of pain.     

Site specific 1:1 opioid:albumin conjugate with in vitro activity and long in vivo duration

A site-specific 1:1 dynorphin A-(1-13)-NH(2) derivative conjugated specifically to Cys 34 on human serum albumin (CCI-1035) was shown to be an opioid receptor agonist in vitro and to be a long lasting antinociceptive agent when administered intravenously to mice as assessed by an acetic acid writhing assay. When 10 micromol/kg of CCI-1035 was administered to mice, rapid antinociception was observed within 5 min following intravenous bolus injection and was sustained beyond 8 h. Antinociceptive activity was absent in a heat induced pain model using a mouse tail-flick assay. This finding represents the first report of a 1:1 albumin opioid conjugate retaining potent in vivo activity equal to or greater than dynorphin A, accompanied by a dramatic extension in duration of action. This novel site-specific bioconjugation technology produces an agent that may be useful for peripheral pain therapy.     

Antinociceptive Effects in Normal and Diabetic Rats Exposed to 50?Hz Magnetic Field

The effects of repeated applications of alternating sinusoidal 50 Hz magnetic field, MF (B = 5.0 mT, 165-minlong sessions everyday, each including four 30-min-long exposures separated by 15-min-long intervals, carried out for 30 days), on thermonociception (estimated using the tail-flick test) were examined in intact rats, diabetic rats (induction by streptozotocin), and diabetic rats treated with insulin. Exposures to MF led to mild (several percent) increases in the tail-flick latency, TFL, immediately after each session and a sustained rather significant rise in this parameter (increment up to 40 %) developing with some delay, from 2 to 4 days. The latter effect was limited in time (lasting 2 days long); then, the TFLs tended to rapidly return to initial (or nearly initial) values. This intense hypoalgesic effect induced by MF exposures was observed on days 3 and 4 in normal rats, on days 4 and 5 in diabetic rats, and on days 5 and 6 in diabetic rats treated with insulin. Significant increases in the mean arterial blood pressure were observed in diabetic rats; exposures to MF exerted no significant influence on this parameter in both normal and diabetic animals. Mechanisms of the development of diabetic neuropathy and those of the hypoalgesic actions of MF are discussed. The MF-induced antinociception seems to be, in future, an attractive choice for the relief of acute and chronic symptoms in diabetic neuropathy, but further detailed studies are necessary to find optimum MF parameters, modes of application, and “time windows.”     

Different alpha-receptor subtypes are involved in clonidine-produced analgesia in different pain tests

Dose-response curves for clonidine-produced analgesia in rats were constructed using the tail-flick and formalin tests. Subsequently, the relative role of alpha 1 and alpha 2 receptors in clonidine analgesia in each of these tests was determined using systemic administration of vehicle controls, tolazoline, yohimbine and prazosin prior to injection of an ED50 dose of clonidine. Clonidine was found to be significantly more potent in the formalin test than in the tail-flick test. Furthermore, clonidine analgesia in the tail-flick test was completely antagonized by tolazoline and yohimbine, but not by prazosin, whereas clonidine was antagonized by tolazoline and prazosin, but not by yohimbine in the formalin test. The implications of these findings with regard to the contributions of different alpha-receptor subtypes to clonidine-produced analgesia in different pain tests are discussed.     

LVV-hemorphin 7 and angiotensin IV in correlation with antinociception and anti-thermal hyperalgesia in rats

Hemorphins, a family of atypical endogenous opioid peptides, are produced by the cleavage of hemoglobin β-chain. Hemorphins were proved to bind to the μ-opioid receptors (agonist) and angiotensin IV receptors (insulin-regulated aminopeptidase; IRAP) (inhibitor). Among the hemorphins, LVV-hemorphin-7 (LVV-H7) was found to be abundant and with a longer half life in the CNS. Using intrathecal and intracerebroventricular injections, LVV-H7 and angiotensin IV were given to the rats, which were then subjected to the plantar test and the tail-flick test. Our results showed that LVV-H7 attenuated carrageenan-induced hyperalgesia at the spinal level, which could not be reversed by the co-administration of naloxone. At the supraspinal level, LVV-H7 also produced a significant anti-hyperalgesia effect but with a lower extent. Angiotensin IV showed a similar anti-hyperalgesia effect at the spinal level, but had no effect at the supraspinal level. In the tail-flick test and paw edema test, both peptides showed no effect. These results suggest that LVV-H7 mainly exert the anti-hyperalgesia effect at the spinal level, possibly through IRAP but not μ-opioid receptors. In addition, we observed the expression of IRAP in the CNS of animals with/without carrageenan-induced hyperalgesia. Our results showed a significant expression of IRAP in the spinal cord of rats. However, there was no significant quantitative change of IRAP after the development of hyperalgesia. The serum level of LVV-H7 was also found to be with no change caused by hyperalgesia. These results indicated that the endogenous LVV-H7 and IRAP may not regulate the severity of hyperalgesia through a quantitative change.     

Central antinociceptive effects of lysine-vasopressin and an analogue

Vasopressin (VP) neurons project to extrahypothalamic sites involved in pain perception, including the substantia gelatinosa of the spinal cord as well as the trigeminal and vagus nerves. Previous studies have reported antinociceptive activity following intracerebroventricular (ICV) or subcutaneous (SC) VP injections (16–100 μg) on the tail-flick test while hyperalgesia has been observed in rats either genetically deficient in VP or treated with antisera to VP. The present study investigated whether nanogram (ng) doses of lysine-vasopressin (LVP) and a VP analogue with prolonged activity increased tail-flick latencies and flinch-jump thresholds following ICV or SC injections. LVP (150 and 500 ng, ICV) significantly increased tail-flick latencies while the analogue 1-deamino-(8-Lys-N^?-(Gly-Gly-Gly))-VP (500 ng, ICV) produced more powerful and prolonged analgesia. In contrast, latencies were not increased by SC injections of LVP (150–1500 ng). Further, flinch-jump thresholds were affected minimally by either ICV or SC LVP injections. These data suggest a role for VP in pain modulation and a central site of this action.     

Inflammation and pain sensitivity: effects of leukotrienes D4, B4 and prostaglandin E1 in the rat paw

Leukotrienes (LT's) and prostaglandins (PG's) have been proposed as mediators of vascular permeability changes in inflammatory reactions. Also, prostaglandins, especially of the E-type, have been shown to enhance pain responses. In the present studies in rats, the effects of LTB4 and LTD4 on edema and pain thresholds were examined in combination with PGE1 and/or brewer's yeast. Subplantar injections of LTD4 or LTB4 induced small increases in paw thickness which were potentiated by the co-administration of PGE1. LTD4 alone had no significant effect on the development of the yeast paw edema. LTB4 was found to reduce significantly the yeast edema and this reduction could be reversed by administration PGE1. A small but significant decrease in pain threshold was caused by PGE1 and this was significantly enhanced in the presence of LTD4. LTB4, like PGE1, was found to cause slight hyperalgesia but no synergy between the two agents was observed. LTD4 was found to have no effect on the initial hypoalgesia or subsequent development of hyperalgesia caused by brewer's yeast. Both LTB4 and PGE1, however, prevented the initial hypoalgesia and significantly reduced the latency for development of yeast induced hyperalgesia. These effects of LTB4 are discussed in terms of possible release of cyclooxygenase products.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

Antinociceptive effects of intrathecal endomorphin-1 and -2 in rats

Endomorphin-1 and endomorphin-2 were recently postulated to be endogenous mu-opioid receptor agonists. We have investigated the antinociceptive and antihyperalgesic effects of intrathecally administered endomorphins in cumulative doses (0.1-100 microg) on acute and inflammatory pain sensations in awake rats. In the tail-flick test, both peptides caused a dose-dependent short-lasting antinociception, except at the highest dose, which caused motor impairment also. The dose-response curves revealed the development of acute tolerance (tachyphylaxis) to endomorphin. Similarly in the carrageenan-injected paw, the endomorphins (10 microg) exerted transient antinociceptive effects. These are the first data to demonstrate decreased responsivity in models of both acute and inflammatory pain after intrathecal administration of endomorphin-1 and -2 in awake rats.     

Paradoxical correlations of the effectiveness of the intranasal and intraperitoneal administration of dermorphins in rats

The analgetic activity of dermorphin and its analogue A-2 was assessed by the tail-flick test. Following intraperitoneal administration at doses 5 mg/kg and above the peptides showed significant effects. After intranasal application of the peptides a significant effect was observed in the range of low doses 0.001-0.1 mg/kg. After intranasal application of high dermorphin doses (1 or 5 mg/kg) the analgetic activity decreased. The effect of analogue A-2 lasted longer after intranasal, than after intraperitoneal administration. It is assumed that the neurophysiological mechanisms of the analgetic activity of dermorphins depend on the route of their administration.     

Antinociceptive and behavioral activation responses elicited by d-Pro(2)-endomorphin-2 in the ventrolateral periaqueductal gray are sensitive to sex and gonadectomy differences in rats

Sex differences have been observed in antinociception after morphine administered into either the lateral ventricles, rostral ventromedial medulla, or ventrolateral periaqueductal gray such that male rats exhibit significantly greater antinociception than female rats. Adult gonadectomy produced small, but significant changes in morphine antinociception relative to same-sex sham-operated controls. The present study examined whether sex and adult gonadectomy differences were observed in antinociceptive responses after D-Pro(2)-Endomorphin-2 (1-50 microg) elicited from the ventrolateral periaqueductal gray (vlPAG) on the tail-flick and jump tests in rats, and compared these effects with morphine antinociception. D-Pro(2)-Endomorphin-2 antinociception in the vlPAG was significantly greater in estrous-phase, sham-operated and ovariectomized female rats relative to sham-operated and castrated male rats on the tail-flick, but not jump test that differed markedly from the greater magnitude of morphine antinociception noted for male rats on both tests. In testing whether D-Pro(2)-Endomorphin-2's antinociceptive sex differences were secondary to alterations in activity, similar decreases in the pattern of total activity were observed after D-Pro(2)-Endomorphin-2 in the vlPAG in male and female rats. In evaluating whether male and female rats differed in their behavioral activation responses after D-Pro(2)-Endomorphin-2 in the vlPAG, significantly more excessive grooming, seizures, barrel rolls and explosive running behaviors were observed after D-Pro(2)-Endomorphin-2 in male, but not female rats during the precise periods of time when they were failing to display robust antinociceptive responses on the tail-flick test. Thus, the different patterns of sex differences after D-Pro(2)-Endomorphin-2 in the vlPAG appear to be attributable to sex-dependent alterations in behavioral activation rather than nociceptive processing per se.