Potentiation of morphine analgesia with co-administration of Nimodipine in Rat, Tail-Flick test study: Implication in the treatment of chronic pain.

Background: Opioids like morphine produce side effects ranging from nausea and vomiting, pruritus, over sedation, dizziness and urinary retention to respiratory depression. Particularly, on chronic administration, it leads to development of tolerance. Combining opioids with certain other drugs (adjuvant analgesics) like ketamine, which is an N-methyl-D-aspartate (NMDA) receptor antagonist, not only increases the analgesia, but also reduces the dose of opioids. Previous research done in our laboratory and outside suggests that nimodipine, an L-type calcium channel blocker (L-CCBs), could be one such adjuvant drug. Aims & Objective: To study of morphine-induced analgesia and the development of morphine tolerance & effect of nimodipine on morphine-induced analgesia and tolerance. Materials and Methods: The experimental work was divided into 2 parts: (i) Part I – Study of morphine induced analgesia and the development of morphine tolerance; and (ii) Part II – Study the effect of nimodipine on morphine-induced analgesia and tolerance. Adult Wistar rats (n=24) received either normal saline, L-CCB (Nimodipine), Morphine or both drugs (Morphine + Nimodipe). Tail-Flick test was done after 40 minutes of injection. To compare the control with treated groups, statistical analysis of the values of Tail-flick latency was done by Kruskal Wallis one way ANOVA, followed by "Tukey's Multiple Comparison Test” (multiple range 't' test) (p<0.05 was taken to be significant). Results: The values of tail-flick latency were almost equal to baseline values for group I, throughout the experiment, while for group II, values of tail-flick latency were almost equal to the cut off time (9.15 ± 1.762), at day 1, but gradually the values decreases over the time period of experiment and at the end of experiment, tail-flick values reaches to base line value. Tail-flick latency for nimodipine was the same as for saline. Values of tail-flick latency for group IV were higher in comparison with group II. Conclusion: The present study indicates that antagonist of L-VGCCs, particularly nimodipine, may enhance the analgesic potency of opioids like morphine and also delayed the development of opioid tolerance.

Chronic spinal infusion of loperamide alleviates postsurgical pain in rats.

Plantar incision in rat generates spontaneous pain behaviour. The opioid drug, morphine used to treat postsurgical pain produces tolerance after long-term administration. Loperamide, a potent mu-opioid agonist, has documented analgesic action in various pain conditions. However, loperamide analgesia and associated tolerance following continuous spinal administration in postsurgical pain has not been reported. Chronic spinal infusion of drugs was achieved using intrathecal catheters connected to osmotic minipump. Coinciding with the onset of spinal infusion of loperamide or morphine, rats were subjected to plantar incision. Pain-related behaviour was assessed by Hargreaves apparatus (thermal hyperalgesia) and von Frey filaments (mechanical allodynia). Morphine and loperamide (0.5, 1 and 2 µL/h) induced analgesia was observed until 7th day post-plantar incision in Sprague-Dawley rats. Morphine and loperamide produced dose-dependent analgesia. Loperamide, in the highest dose, produced analgesia till 7th day. However, the highest dose of morphine produced inhibition of thermal hyperalgesia till 5th day and mechanical allodynia only till 3rd day post-plantar incision. Morphine and loperamide produced analgesia in postsurgical pain, which may be mediated through different mechanisms. Longer duration of analgesia with loperamide could probably be due sustained blockade of calcium channels.

Nimodipine down-regulates CGRP expression in the rat trigeminal nucleus caudalis.

L-type calcium channel blockers like verapamil are used in the prophylaxis of migraine. However, their effect on the expression of CGRP in the trigeminal nucleus caudalis (TNC) is unknown. It is important because an earlier study had shown that olcegepant, a CGRP receptor antagonist, acts at the level of the trigeminal spinal nucleus rather than the trigeminal ganglia. Nimodipine was used in the present study as it crosses the blood-brain barrier. The objective of the study was to determine the pattern of expression of calcitonin gene-related peptide (CGRP) in the TNC after administration of nimodipine and/or morphine. Wistar rats were injected with saline, morphine, nimodipine or morphine + nimodipine for 14 days. Subsequently, the lowest part of the medulla oblongata containing the spinal nucleus was removed and processed for immunohistochemical localization of CGRP. The density of expression was quantified using Image J software. The results were statistically analyzed. CGRP expression was noted over the superficial part of the TNC, which decreased significantly after nimodipine administration. Conversely, morphine produced an up-regulation. The expression was unchanged with reference to saline in the morphine + nimodipine treated group. Decreased expression of CGRP in the trigeminal nucleus caudalis after nimodipine is being reported for the first time. Also, whether CGRP expression can be used as a marker for predicting the therapeutic efficacy of an anti-migraine drug is currently being investigated.

Small-sized neurons of trigeminal ganglia express multiple voltage-sensitive calcium channels: A qualitative immunohistochemical study.

The cell bodies of pseudounipolar neurons of the trigeminal ganglia have been presumed to play a supportive role to neurites, which transmit various sensations like pain from the periphery to the brain stem. However, several studies have recently shown that these neuronal cell bodies could modulate the afferent stimuli by up-regulating various ion channels and also by increasing the synthesis of neuropeptides like calcitonin gene-related peptide (CGRP). Since voltage-sensitive calcium ion channels (VSCCs) determine neuropeptides/ neurotransmitters released by neurons, the aim of the present study was to localize the various VSCCs (N-, P/Q-, L-, T- and R-types) in the trigeminal ganglia neurons by immunohistochemistry. The results showed that all the VSCCs are expressed by the cell bodies of neurons though the small-sized neurons showed higher expression of these channels. The small-sized neurons were identified by immunohistochemical localization of CGRP, the most common neuropeptide for pain transmission in the trigeminal ganglia neurons. Some of these channels (N, P/Q and T types) were also expressed on the cell surface though previous electrophysiological studies have shown the expression of all the channels on the cell surface. It is suggested that the cell bodies could play a more active role than hereto ascribed to these, in the modulation of sensory stimuli.

Nimodipine is more effective than nifedipine in attenuating morphine tolerance on chronic co-administration in the rat tail-flick test.

Opioids, when co-administered with L-type calcium channel blockers (L-CCBs) show morphine like higher antinociceptive effect. This antinociceptive effect has been further investigated using a different experimental paradigm. The effect of two different L-CCBs (nifedipine and nimodipine) on morphine-induced antinociception was studied by the tail-flick test (40 min after morphine administration) in adult Wistar rats. A fixed-dose of nimodipine or nifedipine (2 mg/kg, once daily) was combined with a fixed dose of morphine (10 mg/kg, twice daily) for 10 days. Co-administration of L-CCBs significantly increased the antinociceptive effect of morphine, even 12 hr after administration. Also, nimodipine was more effective than nifedipine. Nimodipine was further studied using a higher and escalating doses of morphine (20-30 mg/kg twice daily for 14 days). Nimodipine increased the antinociceptive effect of morphine in the latter part of the study (days nine to fourteen) though significant difference was observed on 11th evening and 12th morning. No obvious adverse effects were observed in the present study. The results show for the first time that nimodipine is more effective than nifedipine and that these L-CCBs continue to be effective, even 12 hr after administration in the tail-flick test.

Enhanced analgesic effect of morphine-nimodipine combination after intraspinal administration as compared to systemic administration in mice.

Calcium plays an important role in the pathophysiology of pain. A number of studies have investigated the effect of L-type calcium channel blockers on the analgesic response of morphine. However, the results are conflicting. In the present study, the antinociceptive effect of morphine (2.5 microg) and nimodipine (1 microg) co-administered intraspinally in mice was observed using the tail flick test. It was compared to the analgesic effect of these drugs (morphine - 250 microg subcutaneously; nimodipine - 100 microg intraperitoneally) after systemic administration. Nimodipine is highly lipophilic and readily crosses the blood brain barrier. Addition of nimodipine to morphine potentiated the analgesic response of the latter when administered through the intraspinal route but not when administered through systemic route. It may be due to direct inhibitory effect of morphine and nimodipine on neurons of superficial laminae of the spinal cord after binding to mu -opioid receptors and L-type calcium channels respectively.

Acute analgesic effect of loperamide as compared to morphine after intrathecal administration in rat.

Loperamide, a mu opioid receptor agonist, which is commonly used as an antidiarrhoeal agent has been reported to possess analgesic activity after intrathecal administration. However, the exact analgesic profile, i.e., onset, duration and intensity of analgesia in relation to morphine is not fully known. In the present study, the acute analgesic effect of loperamide (5 microg) was compared with that of morphine (5 microg) and morphine + loperamide (5 microg of each) using the tail flick method after intrathecal administration. Naloxone (5 mg/kg) reversibility of the analgesic effect was also studied. The analgesic response of loperamide was significantly higher than morphine. Even after 22 hr, maximum possible effect was greater than 49%. Naloxone partially antagonized the analgesic effect of loperamide. This suggested that loperamide may be acting through blockade of Ca2+ channels besides activating mu opioid receptors. Loperamide may prove to be a better substitute for morphine as spinal analgesic.

Expression of mu-opioid receptors in developing rat spinal cord: an autoradiographic study.

The expression of mu-opioid receptors in the developing rat spinal cord (Postnatal days 7, 14, 30) was studied by autoradiography using [3H]DAMGO. When compared to camera lucida drawings, the receptor was noted over the entire gray matter and dorsal root ganglia at postnatal days 7 and 14. At postnatal day 30, the receptor expression decreased over the gray matter except the superficial laminae (laminae I and II). At all age groups studied, a higher expression of the receptor was noted over the superficial laminae. The study shows that micro-opioid receptors appears early in postnatal development and attains mature receptor distribution relatively late in ontogeny, suggesting a possible role in the normal development of nervous system. This is affirmed by an impairment of psychomotor development in babies born to mothers, addicted to opiates.

Up-regulation of micro-opioid receptors in the spinal cord of morphine-tolerant rats.

Though morphine remains the most powerful drug for treating pain, its effectiveness is limited by the development of tolerance and dependence. The mechanism underlying development of tolerance to morphine is still poorly understood. One of the factors could be an alteration in the number of micro-receptors within specific parts of the nervous system. However, reports on changes in the micro-opioid receptor density in the spinal cord after chronic morphine administration are conflicting. Most of the studies have used subcutaneously implanted morphine pellets to produce tolerance. However, it does not simulate clinical conditions, where it is more common to administer morphine at intervals, either by injections or orally. In the present study, rats were made tolerant to morphine by injecting increasing doses of morphine (10-50 mg/kg, subcutaneously) for five days. In vitro tissue autoradiography for localization of micro-receptor in the spinal cord was done using [3H]-DAMGO. As compared to the spinal cord of control rats, the spinal cord of tolerant rats showed an 18.8% increase or up-regulation in the density of micro-receptors in the superficial layers of the dorsal horn. This up-regulation of micro-receptors after morphine tolerance suggests that a fraction of the receptors have been rendered desensitized, which in turn could lead to tolerance