Antagonism of stimulation-produced analgesia by naloxone and N-methyl-D-aspartate: role of opioid and N-methyl-D-aspartate receptors

Abstract

The present study aims to investigate the influence of electrical stimulation of periaqueductal gray (PAG) following peripheral nerve injury and its modulation by naloxone and N-methyl-D-aspartate (NMDA). Chronic neuropathic pain was induced by chronic constriction injury of the sciatic nerve, and subsequently a cannula was implanted in the PAG area for the purpose of electrical stimulation and intra-PAG drug administration. Intra-PAG administration of morphine, ketamine, and their combination were found to elicit antinociceptive response on hot-plate test. Electrical stimulation of PAG was also observed to demonstrate decreased pain response on hot-plate test, and this effect was reversed by the administration of naloxone, NMDA, and their combination, when injected into the PAG area. These findings suggest that apart from the opioid receptors, probably NMDA receptors also have a role to play in stimulation-produced analgesia.

Keywords

periaqueductal gray NMDA naloxone chronic neuropathic pain hot-plate test

Introduction

A large number of investigations have demonstrated that electrical stimulation of periaqueductal gray (PAG) produces analgesia in experimental animals.^1–5 There have been reports suggesting the μ-opioid receptors located in the PAG to be involved in the mechanism of stimulation-produced analgesia (SPA).^2,6,7 Furthermore, naloxone was found to antagonize the SPA partially, suggesting the role of other mechanisms such as the presence of endogenous morphine-like substance to produce analgesia.² Few studies have also shown that inactivation of N-methyl-D-aspartate (NMDA) receptors produced antinociception of supraspinal origin, and this effect was partially mediated by brain opioids.⁷ Moreover, it has been proposed in various studies that descending pain modulation system from PAG to spinal cord plays an important role in opiate-mediated analgesic processes.^8–10

Earlier studies have indicated that in conditions of chronic neuropathic pain, there is increased thermal and mechanical hyperalgesia probably due to upregulation of NMDA receptors at the site of injury and also due to NMDA-receptor-mediated central changes in synaptic excitability.^11,12 The chronic constriction injury (CCI) results in the Wallerian degeneration of a substantial number of, but not all, axons distal to the loose ligatures, with greater than 80% loss of myelinated fibers and 60–80% loss of unmyelinated fibers.¹³ Proximal to the loose ligatures, the proximal stumps of degenerating axons intermingle with spared axons,^13,14 leading to injured and uninjured neurons residing in L4 and L5 dorsal root ganglion. Moreover, recent evidence indicates that CCI also affects the excitability properties of second-order sensory neurons within the spinal cord entry zone, which contributes to the hyperalgesia and allodynia following CCI.¹⁵

In view of the involvement of the opioid system along with other systems, it can be anticipated that peripheral nerve injury may influence the manifestation of SPA. Therefore, in the present study, we examined the influence of SPA from the PAG area following peripheral nerve injury and its modulation by naloxone and NMDA.

Materials and methods

Animals

Healthy male Wistar rats weighing 180–250 g were used for the study. The animals were procured from the Central Animal House, University College of Medical Sciences, Delhi. Animals were housed in groups of 4–5 per cage with free access to pellet diet and water in a temperature-controlled facility (22 ± 2°C). All the experiments were performed at daytime between 0930 and 1530 hours. The study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985) and with the recommendations and approval of the Institutional Animal Ethics Committee, University College of Medical Sciences, Delhi.

Drugs and dosing schedule

Morphine sulfate was obtained from Government Opium and Alkaloid Works, India. Ketamine hydrochloride, naloxone hydrochloride, and NMDA were obtained from Sigma, USA. Morphine, ketamine, naloxone, and NMDA were used in doses of 5, 100, 1, and 10 µg/rat, respectively. All the drugs were injected in the PAG area, using a microinjection syringe (Hamilton) attached to a 30-gauge stainless steel injector. The injected volume was 0.5 µl delivered over a period of 2 min. Groups involving administration of more than one drug were scheduled such that the peak effect of all the administered drugs is attained at the same time.

Hot-plate test

The response to thermal stimuli was assessed using the hot-plate test.¹⁶ Rats were placed on a hot-plate, whose temperature was set at 55°C. The latency response to either a hind paw lick or a jump was recorded. A cutoff time of 60 s was kept to avoid any tissue damage.

CCI of sciatic nerve

The CCI of sciatic nerve was used as a model for the induction of neuropathic pain. Animals were anaesthetized with chloral hydrate (350 mg/kg). Thereafter, the right sciatic nerve was exposed at the level of mid-thigh and two ligatures were tied proximal to the trifurcation of sciatic nerve according to the modified method of Bennett and Xie.¹⁷ The ligation was of sufficient strength to produce a slight indentation of the nerve but not to induce muscle twitch. The wound was closed and neosporin powder was sprayed. A sham surgery was performed on the left side, where the nerve was exposed, but no ligature was made. Animals were treated with an antibiotic (gentamicin, intraperitoneal [i.p.]) and allowed to recover for 14 days following which they were subjected to the hot-plate test (as described below) and the latency to lick/jump were recorded.

Electrical stimulation of PAG

The rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.) and placed in a stereotaxic apparatus. A midline incision was given on the muscles over the skull bones and the muscles retracted. A burr hole was drilled and the holes made on the skull surface. The guide cannula (26 gauge) was implanted stereotaxically into the PAG area using the coordinates (–5.5 mm) posterior to bregma, 0.5 mm lateral, and 7 mm below the skull surface according to the atlas of Paxinos and Watson.¹⁸ Each cannula was fitted with an indwelling occlusion stylet to prevent blockage. Following surgery, rats were injected with gentamicin for 3 days and housed individually. The animals were allowed postoperative recovery for 7 days. The PAG area was electrically stimulated (50 Hz, 0.2 ms, 10 V for 30 s) through a stimulator (Medicare, India), using an insulated bipolar electrode (diameter of each wire was 130 µm), via the same guide cannula.¹⁹

Histology

For histological verification of electrode placement, rats were deeply anesthetized and anodal electrolytic lesions (0.25 mA, 10 s) were made through the stimulating electrode. The animals were perfused transcardially with a 10% formalin solution containing 5% potassium ferrocyanide to produce a Prussian blue reaction to mark the iron deposited from the stimulating electrode tip. The brains were removed, sectioned (50 μm), stained with neutral red dye, and observed under the microscope.

Statistical analysis

Results were expressed as percentage of maximal possible effect (% MPE) and was calculated from the formula: % MPE = 100 × (test latency – control latency)/(cutoff time – control latency), where test latency = latency to lick hind paw after drug administration following induction of neuropathic pain; control latency = latency to lick hind paw after induction of neuropathic pain without the administration of drug. The data showing % MPE were expressed as mean ± standard error of mean (SEM) and the results were analysed for significant differences between groups and within groups by analysis of variance (ANOVA) followed by post hoc Tukey’s test. P values less than 0.05 were considered significant.

Results

Effect of intra-PAG administration of morphine, ketamine, and their combination on hot-plate test latency

The effect of administration of morphine, ketamine, and their combination on hot-plate latency (HPL) was observed following peripheral nerve injury. Morphine-treated rats demonstrated significant increase in hind-paw licking latency, and thereby increase in % MPE (p < 0.001) as compared to the control group (saline treated). Similarly, intra-PAG ketamine also demonstrated significant increase in HPL (p < 0.05) as compared to control (Table 1 ). Combination of morphine and ketamine showed further increase in % MPE (p < 0.001; Table 1).

Table 1.

Effect of intra-PAG electrical stimulation (STIM) and administration of morphine, ketamine, and their combination on hot-plate latency (HPL) expressed in seconds, following chronic constriction injury (CCI) in rats (n = 8)

Treatment (µg/rat)	HPL before CCI (on Day 0)	HPL after CCI (at Day 14; A)	HPL after drug treatment/STIM (B)	% MPE (A and B)
Control (saline)	8.18 ± 0.37	3.95 ± 0.23^a	3.17 ± 0.23	−1.32 ± 0.09
Morphine (5)	8.97 ± 0.41	4.75 ± 0.20^b	14.53 ± 0.45	17.69 ± 0.98^c
Ketamine (100)	7.11 ± 0.89	3.10 ± 0.45^d	6.06 ± 0.56	5.2 ± 0.46^e
Ketamine (100) + morphine (5)	8.46 ± 0.40	4.02 ± 0.32^f	20.37 ± 1.85	29.24 ± 3.18^c,g,h
STIM only	9.25 ± 0.38	3.82 ± 0.29ⁱ	7.85 ± 0.42	7.36 ± 0.63^j,g,k

^a p < 0.0001 versus control (HPL before CCI).

^b p < 0.0001 versus morphine (HPL before CCI).

^c p < 0.001 versus control.

^d p < 0.001 versus ketamine (HPL before CCI).

^e p < 0.05 versus control.

^f p < 0.0001 versus ketamine + morphine (HPL before CCI).

^g p < 0.001 versus morphine.

^h p < 0.001 versus ketamine.

ⁱ p < 0.0001 versus STIM only (HPL before CCI).

^j p < 0.01 versus control.

^k p < 0.001 versus ketamine + morphine.

Effect of electrical stimulation of the PAG area

Electrical stimulation of the PAG (PAG-Stim) followed by the saline treatment showed significant increase in HPL as compared to the control group (saline-treated following CCI). The PAG-Stim + saline group showed significantly increased HPL compared to the HPL of the same group at 14 days following CCI.

Effect of intra-PAG administration of NMDA, naloxone, and their combination following electrical stimulation of PAG area

Administration of naloxone after PAG-Stim significantly increased the pain response compared to the PAG-Stim + saline group, thus reversing the effect of SPA (Table 2 ). Similarly, NMDA treatment after PAG-Stim antagonized the effect of SPA. Administration of NMDA + naloxone after PAG-Stim further increased the pain response significantly as compared to the other groups (Table 2).

Table 2.

Effect of electrical stimulation (STIM) of periaqueductal gray on hot-plate latency (HPL) expressed in seconds, following chronic constriction injury (CCI), and its modulation by NMDA and naloxone in rats (n = 8)

Treatment (µg/rat)	HPL before CCI (on Day 0)	HPL after CCI (at Day 14)	HPL after STIM (A)	HPL after drug treatment (B)	% MPE (A and B)
STIM only	9.25 ± 0.38	3.82 ± 0.29^a	7.85 ± 0.42^b	–	–
STIM + saline (control)	9.24 ± 0.88	3.92 ± 0.26^c	7.95 ± 0.45^b	7.72 ± 0.44	−0.40 ± 0.01
STIM + naloxone (1)	9.51 ± 0.64	5.11 ± 0.46^d	9.4 ± 0.60^e	4.6 ± 0.58	−9.51 ± 0.67^f
STIM + NMDA (10)	9.6 ± 0.71	3.31 ± 0.20^g	7.7 ± 0.89^h	4.38 ± 0.58	−6.42 ± 0.87^f
STIM + NMDA (10) + naloxone (1)	8.62 ± 0.66	3.03 ± 0.21ⁱ	8.76 ± 0.56^j	2.3 ± 0.34	−12.68 ± 1.10^f,k,l

^a p < 0.0001 versus STIM only (HPL before CCI).

^b p < 0.0001 versus control (HPL after CCI).

^c p < 0.0001 versus STIM + saline (HPL before CCI).

^d p < 0.0001 versus STIM + naloxone (HPL before CCI).

^e p < 0.0001 versus STIM + naloxone (HPL after CCI).

^f p < 0.001 versus control (% MPE).

^g p < 0.0001 versus STIM + NMDA (HPL before CCI).

^h p < 0.001 versus STIM + NMDA (HPL after CCI).

ⁱ p < 0.0001 versus STIM + NMDA + naloxone (HPL before CCI).

^j p < 0.0001 versus STIM + NMDA + naloxone (HPL after CCI).

^k p < 0.05 versus STIM + naloxone (% MPE).

^l p < 0.01 versus STIM + NMDA (% MPE).

Discussion

A large number of investigations have demonstrated that electrical stimulation of PAG produces analgesia in experimental animals.^1–5 In the present study, we have attempted to explore the role of NMDA receptors along with the opioid receptors in SPA in animals with peripheral nerve injury. In the preliminary experiments on neuropathic pain, we found reduced HPL, 2 weeks following CCI of sciatic nerve. This could be due to the upregulation of glutamate receptors following nerve injury, and NMDA-receptor channel activation due to loss of inhibitory γ-aminobutyric acid (GABA) control, ectopic impulse generation, and alteration in phenotype of damaged nerve.^20,21

Despite the fact that several studies have demonstrated the efficacy of opioids in certain types of neuropathic pain, it still remains unclear whether they are useful in allodynia caused due to CCI of sciatic nerve.^22,23 In recent reports, we have demonstrated that uncompetitive NMDA-receptor antagonists, such as ketamine, potentiate the analgesic action of morphine in inflammatory pain and also prevent the development of tolerance, when administered intraperitoneally.^24,25 Moreover, microinjections of opiates into the PAG have been known to attenuate neuropathic pain symptoms in rats.²⁶ However, Smith et al.²⁷ have demonstrated the inefficacy of NMDA antagonist ketamine (at doses 0.1–100 µg) when administered directly into the PAG to elicit antinociceptive response. In the present study, we have administered a high dose of ketamine (100 µg) in the PAG area following CCI of sciatic nerve and have observed decreased pain response to hot-plate test. Moreover, coadministration of morphine with ketamine into the PAG area enhanced the analgesic response in the animals. These findings further insinuate the role of NMDA receptors along with opioid receptors present in the PAG area in evoking analgesic response in neuropathic pain. This effect can be attributed to the NMDA-receptor-mediated central changes in synaptic plasticity and also increased sensitivity of these receptors in the dorsal horn of the spinal cord after peripheral nerve injury.^11,12,28 Various studies have already suggested the role of opioid receptors in SPA,^1–5 but the involvement of other mechanisms cannot be ruled out. There have been reports suggesting that inhibition of nociceptive dorsal horn neurons by PAG Stim was mediated in part by the release of serotonin, norepinephrine, and inhibitory amino acids in the spinal cord. Direct or indirect role of NMDA receptors have not been much investigated.

In this study, we have observed reversal of SPA after administration of naloxone (1 µg). Moreover, administration of NMDA (10 µg) also attenuated the effect of SPA. The doses of NMDA and naloxone were selected on the basis of earlier studies and pilot experiments performed in our laboratory.^29,30 However, in the pilot studies, administration of higher doses of NMDA (25 µg) resulted in seizures in all the animals. Hence, the dose was reduced gradually and 10 µg was selected, since it did not cause seizures. When both NMDA and naloxone were coadministered following PAG-Stim, there was enhanced attenuation of SPA. These observations point toward the involvement of NMDA receptors in addition to the opioid receptors in PAG area to induce antinociceptive response in chronic neuropathic pain. There could be a possible interaction between these receptors at this level, which is reflected by increased attenuation of SPA from the PAG, when both NMDA and naloxone were coadministered.

In conclusion, administration of intra-PAG morphine, ketamine, and their combination were found to elicit antinociceptive response on hot-plate test. Electrical stimulation of PAG was also observed to demonstrate decreased pain response on hot-plate test, though the analgesic effect was significantly less than that of morphine, when administered in the PAG area. This analgesic effect produced by the electrical stimulation was reversed by the administration of naloxone, NMDA, and their combination, when injected into the PAG area. The result of injecting both NMDA and naloxone following electrical stimulation was not observed to be additive, thus indicating a modulation of the receptors at the level of PAG. Hence, the present study shows that SPA is probably mediated partly through opioid and NMDA receptors and this mechanism can be made use of in clinical settings to relieve pain due to chronic nerve injury. Electrical stimulation can serve as a useful aid to enhance pain relief in patients, but the intensity and duration of the current and voltage to be applied needs to be standardized in future experiments before they can be tested on patients.

Footnotes

Acknowledgements

The authors would like to thank Dr Prem Suman for her invaluable guidance on the project.

The work was supported by Indian Council of Medical Research [grant number 45/12/2008/PHA/BMS], New Delhi.

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