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Antiallodynic Effects of Endomorphin-1 and Endomorphin-2 in the Spared Nerve Injury Model of Neuropathic Pain in Mice

Wang, Chang-lin PhD; Yang, Dai-jun MSc; Yuan, Bi-yu MSc; Qiu, Ting-ting MSc

doi: 10.1213/ANE.0000000000002318
Pain and Analgesic Mechanisms: Original Laboratory Research Report
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BACKGROUND: The spared nerve injury (SNI) model is a new animal model that can mimic several characteristics of clinical neuropathic pain. Opioids are recommended as treatment of neuropathic pain. Therefore, the present study was conducted to investigate the antinociceptive effects of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given centrally and peripherally in the SNI model of neuropathic pain in mice.

METHODS: The SNI model was made in mice by sparing the sural nerve intact, when the other 2 of 3 terminal branches of the sciatic nerve (common peroneal and tibial nerves) were tightly ligated and cut. Von Frey monofilaments were used to measure the SNI-induced mechanical allodynia-like behavior. The antiallodynic effects of EM-1 and EM-2 were determined after central and peripheral administration in the SNI model of neuropathic pain. Also, the specific opioid receptor antagonists were used to determine the opioid mechanisms of EMs involved in neuropathic pain. Values were expressed as the mean ± standard deviation.

RESULTS: Our results showed that the SNI mice developed prolonged mechanical allodynia-like behavior in ipsilateral paw after surgery, with the withdrawal threshold value being 0.061 ± 0.02 g after 14 days. EM-1 and EM-2 produced significant antiallodynic effects in ipsilateral paw after intracerebroventricular (i.c.v.) administration, more effective than that of morphine. The peak withdrawal thresholds of 10 nmol EM-1 and EM-2 determined at 5 minutes after injection were 0.92 ± 0.36 and 0.87 ± 0.33 g, respectively, higher than that of morphine (0.46 ± 0.20 g). Moreover, both EMs (10 nmol, i.c.v.) exerted significant antiallodynic effects in the contralateral paw, whereas no significant antinociceptive activity was seen after i.c.v. administration of morphine with equimolar dose. It was noteworthy that EM-1 and EM-2 produced antinociception through distinct μ1- and μ2-opioid receptor subtypes, and the EM-2-induced antiallodynia contained an additional component that was mediated by the release of endogenous dynorphin A, acting on κ-opioid receptor. In addition, the antiallodynic activities of peripheral administration of EM-1, EM-2, and morphine were also investigated. Intraplantar, but not subcutaneous administration of EM-1 and EM-2 also exhibited potent antinociception, establishing the peripheral and local effects. Both μ1- and μ2-opioid receptor subtypes, but not the δ- or κ-opioid receptors were involved in the peripheral antiallodynia of EMs.

CONCLUSIONS: The present investigation demonstrated that both EM-1 and EM-2 given centrally and peripherally produced potent antiallodynic activities in SNI mice, and differential opioid mechanisms were involved.

Published ahead of print August 4, 2017.

From the School of Life Science and Technology, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China.

Published ahead of print August 4, 2017.

Accepted for publication May 17, 2017.

Funding: Supported by grants from the National Natural Science Foundation of China (No. 31100562), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. ES201512), State Key Laboratory of Medicinal Chemical Biology, NanKai University (No. 2017024), Special fund of Harbin innovative talent in science and technology research (2014RFQXJ178), Program for Interdisciplinary Basic Research of Science-Engineering-Medicine in Harbin Institute of Technology (IBRSEM).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Chang-lin Wang, PhD, School of Life Science and Technology, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China. Address e-mail to wangcl@hit.edu.cn.

Chronic pain has been estimated to impair a patient’s quality of life and affects one-sixth of the population.1,2 Neuropathic pain is a common chronic pain state that is caused by nerve injury or diseases affecting the somatosensory system, either centrally or peripherally.3 The clinical features of neuropathic pain are characterized by spontaneous pain (nonstimulus evoked symptoms), allodynia (perception of pain elicited by normally nonnoxious stimuli), and hyperalgesia (increased pain responses or decreased thresholds to normally noxious stimuli).4,5 Changes of neural plasticity including structure, function, and organization of neurons are responsible for the altered sensitivity characteristics of neuropathic pain.

An animal model is of great use for research because of its specific characteristics that resemble a human disorder or disease. Over the last decades, numerous animal models of peripheral nerve injury and the subsequent neuropathic pain have been developed and characterized. These animal models can simulate human neuropathic pain signs, including spontaneous pain, hyperalgesia, and allodynia-like behavior, which make them valuable tools for laboratory experiments.6 Previously, the most prominent models are the chronic constriction injury (CCI) model,6 the partial sciatic nerve ligation model,7 and the spinal nerve ligation model.8 However, the CCI and partial sciatic nerve ligation models lead to considerable variability in the number of peripheral axon responders, and abnormal pain sensation and behavior. In addition, the surgical procedure of the spinal nerve ligation model is relatively invasive and difficult to perform. In 2000, a novel model of peripheral nerve damage, termed the spared nerve injury (SNI) model, was firstly developed by Decosterd and Woolf.9 This model consists of sparing the sural nerve intact, when the other 2 of 3 terminal branches of the sciatic nerve (common peroneal and tibial nerves) are tightly ligated and cut.9 The surgical procedure of SNI is easy to perform and reproducible, and long-lasting mechanical allodynia-like response is observed in the spared sural nerve skin territory in both rats and mice.9–11 Thus, the SNI model has been found most valuable in studies on neuropathic pain and its treatment, and allowed for clinical screening for novel analgesic compounds.

Opioids, like morphine, remain the most effective broad-spectrum analgesics available for the treatment of moderate-to-severe acute pain, but serious side effects limit their therapeutic use. These include constipation, respiratory depression, development of tolerance, and physical dependence.12 In addition, morphine exhibits low efficacy against neuropathic pain. Therefore, the development of novel opioid analgesics has been a longstanding research goal.13 Many naturally occurring peptides are potential neuropharmaceuticals, and the study of endogenous neuropeptides, like opioid peptides, provides a rational and potentially powerful approach in the design of peptide therapeutics.14 Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are endogenous neuropeptide ligands that displayed high affinity and selectivity for μ-opioid receptor, firstly isolated from bovine brain in 1997 by Zadina et al.15 Both EMs produced potent antinociceptive activity in rodent models of acute16,17 and neuropathic18 pain with reduced side effects than opioid alkaloids. After IV administration, EMs were found to induce cardiorespiratory depression only at high dose.19 Particularly, centrally administered EMs elicited no significant reward behavior20 or locomotor activity.21 Also, it was reported that both EMs produced potent antidepressant-like activity in mice,22 while depression is 1 of the side effects associated with opioid use due to opioid-induced hormonal changes. Even opioids which exert potency in nociceptive pain reportedly lack potent analgesic effects against neuropathic pain, or show efficacy only at high doses.23 Alternatively, EMs are more effective than the majority of the opioids against neuropathic pain even at low doses, indicating that EMs have the potential to be pharmaceutical agents for the treatment of neuropathic pain.18 Since their discovery, the analgesic effects of EMs against neuropathic pain have been studied centrally and peripherally using sciatic nerve crushing or a CCI model.18,24,25 Also, numerous EMs analogs administered systemically showed antineuropathic effects in CCI-rat model.26–28 However, to date, no study investigated the effects of EMs on neuropathic pain induced by the SNI model, which produced prolonged and substantial changes in mechanical sensitivity.9–11 Therefore, in the present study, we characterized the antinociceptive effects of EM-1 and EM-2 given centrally and peripherally in the SNI model of neuropathic pain in mice. Also, the specific opioid receptor antagonists were used to determine the opioid mechanisms of EMs involved in neuropathic pain.

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METHODS

Animals and Drugs

Male Kunming strain mice weighing 20 to 23 g were used. Animals were housed in an animal room that was maintained at 22°C ± 2°C with a 12-hour light: 12-hour dark cycle, and given free access to food and water. All animals were cared for and experiments were performed in accordance with the European Community guidelines for the use of experimental animals (2010/63/EU). All the protocols in this study were approved by the Ethics Committee of Harbin Institute of Technology, China.

EM-1 and EM-2 were synthesized by liquid-phase method and characterized by reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry, and their bioactivity was recorded by a model BL-420F system (Taimeng Technology and Market Corporation of Chengdu, China) as described in our previous study.29,30 Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory, Shenyang, China. Naloxone hydrochloride, naloxone methiodide, β-funaltrexamine hydrochloride (β-FNA), naloxonazine dihydrochloride, naltrindole isothiocyanate hydrochloride (NTI), and nor-binaltorphimine hydrochloride (nor-BNI) were purchased from Sigma-Aldrich (St. Louis, MO). The antibodies against dynorphin A(1–17) (anti-Dyn A antibody), [Met5]enkephalin, [Leu5]enkephalin were obtained from Abcam (Cambridge, United Kingdom). Briefly, these antibodies did not show cross-reactivity with EM-1 and EM-2. All compounds were dissolved in sterilized saline and stored at −20°C. For the antagonistic study, EM-1 and EM-2 were dissolved together with the specific opioid receptor antagonists in saline. The aliquots were thawed and used on the day of the experiment. During an experiment, the drug solutions were kept on crushed ice. The antinociceptive effects of EM-1, EM-2, and morphine were determined by intracerebroventricular (i.c.v.), intraplantar (i.pl.), and subcutaneous (s.c.) administration 14 days after SNI.

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Surgery of SNI Model

Surgical procedures were performed under general anesthesia with 60 mg/kg pentobarbital sodium in the way of intraperitoneal injection. The surgery was made as described by Decosterd and Woolf.9 SNI was made with a 10.0 silk tight ligation of the 2 branches of the left sciatic nerve, the common peroneal, and the tibial nerves followed by transaction and removal of a 2- to 4-mm nerve portion. The third branch, the sural nerve, remained intact and any contact or stretch to this nerve was carefully avoided.9 The animals were allowed to survive for at least 14 days after surgery.

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Implantation of Cannula Into the Ventricle for i.c.v. Administration

The surgical implantation of cannula was conducted in an aseptic environment. Male Kunming mice weighing 18 to 22 g were anesthetized with 60 mg/kg pentobarbital sodium by intraperitoneal injection and placed in a stereotaxic apparatus. The incision area of the scalp was shaved, and a sagittal incision was made in the midline, exposing the surface of the skull. A single hole was drilled through the skull at 2.5 mm posterior and 1 mm lateral from the bregma. A stainless steel guide cannula was implanted 3 mm ventral from the surface of the skull for i.c.v. administration, and fixed to the skull using dental cement. To prevent occlusion, a dummy cannula was inserted into the guide cannula. The dummy cannula protruded 0.5 mm from the guide cannula. After surgery, the animals were allowed to recover for at least 4 days. During this time, mice were gently handled daily to minimize the stress associated with manipulation of the mice throughout the experiments.

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Drug Administration

For i.c.v. administration, drugs were injected in a volume of 4 μL at a constant rate of 10 μL/min, followed by 1 μL of saline to flush in the drug using a 25-μL microsyringe. Vehicle control animals received appropriate saline. After completion of the behavioral test, the proper injection site was verified in pilot experiments by administration and localization of methylene blue dye. Only the data from those animals with dispersion of the dye throughout the ventricles were used in this study. For peripheral administration, mice received a s.c. or i.pl. injection at a volume of 100 or 20 μL drugs or saline, respectively.

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Behavior Test

Mechanical allodynia was examined 2 to 14 days after SNI. Animals were placed in transparent plastic domes on a metal mesh floor with a hole size 2 × 3 mm. After 30-minute habituation, the threshold for paw withdrawal (both ipsi- and contralateral sides) was measured by grade-strength von Frey monofilaments (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g).9–11 Monofilaments were perpendicularly applied to the lateral plantar surface of the hind paw with sufficient force to cause filament bending. Testing started with filament 0.008 g and positive response was determined by paw withdrawal response to 1 of 5 repetitive stimuli. In the case of negative response, the next stiffer monofilament was applied. The monofilament that first evoked a positive response was designated as the threshold in grams.9–11

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Statistical Analysis

Values were expressed as mean ± standard deviation. The extent and duration of analgesia were estimated using area under the curve (AUC). The AUC values depicting withdrawal threshold versus time were computed by trapezoidal approximation over the period 0 to 40 minutes. Responses were analyzed with a 1-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for comparison of multiple groups. P <.05 was used as the criterion for statistical significance. A 2-way ANOVA was used to analyze the effects of different groups (SNI ipsilateral, SNI contralateral, sham ipsilateral, and sham contralateral) and time period, including the interaction between group and time period. Also, the time-courses for effects of i.c.v. or i.pl. administration of different doses of drugs, and the dose and time interactions were analyzed using the 2-way ANOVA. P ≤ .05 was used as the criterion for statistical significance. As a primary aim to choose sample size for a further experiment, GraphPad StatMate 2.0 was used to analyze statistical power and sample size. For a pilot study of the effects of drugs on the ipsilateral withdrawal threshold, the estimated standard deviation of drug-treated group (1 nmol EM-1, i.c.v.) and control group (saline, i.c.v.) was 0.349 and the alpha level was defined as 0.05 (2 tailed) for sample size justification. The results from StatMate showed the tradeoff among sample size, the difference you can detect, and the power. The preliminary experiment showed that the difference between means of drug-treated group and control group at 5 minutes after administration was 0.495, then the sample size was justified to obtain a power more than 80%. By viewing the report of StatMate, the sample size in each group should be more than 9. Therefore, based on sample size justification and our previous studies, we chose sample size “n” being 9 to 13 in this study.

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RESULTS

SNI-Induced Mechanical Allodynia-Like Behavior Measured by von Frey Monofilaments

Figure 1.

Figure 1.

Figure 2.

Figure 2.

The SNI surgical procedure is shown in Figure 1. After SNI surgery, mice developed mechanical allodynia-like behavior to von Frey hair stimulation as shown by the decrease in paw withdrawal threshold ipsilateral to the nerve injury (Figure 2). The mechanical allodynia was clearly present ipsilaterally 2 days after SNI surgery, compared with the sham group in ipsilateral paw (F(3,44) = 36.72; P <.001). The SNI mice continued to exhibit pronounced mechanical allodynia for the study period. Fourteen days after surgery, the withdrawal threshold value of the ipsilateral paw was 0.061 ± 0.02 g, significant lower than the sham-operated withdrawal threshold of 1.2 ± 0.29 g (F(3,44) = 43.96; P <.001). Also, mechanical withdrawal threshold of response decreased significantly, albeit less pronounced, in the contralateral paw compared to the sham group (F(3,44) = 43.96; P <.001, 14 days after surgery). The 2-way ANOVA of the present data revealed significant effects for group and time period, F(3,396) = 292.5, P <.001 and F(8,396) = 23.43, P <.001, respectively, as well as, a statistically significant group and time period interaction, F(24,396) = 12.42, P <.001. There was no significant change of either ipsilateral or contralateral paw in the sham group from the presurgery withdrawal threshold baseline (P >.05).

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Antiallodynic Effects of i.c.v. Administration of EM-1, EM-2, and Morphine in SNI Mice

Table 1.

Table 1.

Figure 3.

Figure 3.

Figure 3 shows the dose- and time-related antinociceptive effects of EM-1, EM-2, and morphine on neuropathic pain induced by mice SNI model after i.c.v. administration. Both EM-1 and EM-2 (i.c.v., 1–10 nmol) dose-dependently increased the ipsilateral withdrawal threshold in mice (F(3,42) = 20.46, P <.001 for EM-1 and F(3,41) = 24.09, P <.001 for EM-2 at 5 minutes, respectively, Figure 3A, B). The peak analgesic effects of EM-1 and EM-2 were observed at 5 minutes after i.c.v. injection, then returned to the preinjection level 30 minutes after injection. In contrast, morphine, a kind of opioid alkaloid, also produced significant antiallodynic effects ipsilaterally, reached its peak 20 minutes after injection, and later gradually declined with a duration of action up to 60 minutes (F(3,38) = 15.4, P <.001 at 20 minutes, Figure 3C). However, the antinociceptive potency of morphine was lower than EM-1 and EM-2. At the dose of 10 nmol, the peak withdrawal threshold values of EM-1 and EM-2 determined at 5 minutes after injection were 0.92 ± 0.36 and 0.87 ± 0.33 g, respectively, higher than that of morphine with the value being 0.46 ± 0.20 g determined at 20 minutes after injection. The 2-way ANOVA of data of EM-1 revealed significant effects for both dose and time, F(3,252) = 47.7, P <.001 and F(5,252) = 81.66, P <.001, respectively, as well as a statistically significant dose and time interaction, F(15,252) =11.43, P <.001. The 2-way ANOVA of data of EM-2 and morphine also indicated that the dose and time interactions were statistically significant (F(15,246) = 13.94, P <.001 and F(18,266) = 4.77, P <.001, respectively). The dose–response curves of paw withdrawal threshold induced by EMs and morphine are shown in Figure 3E. Also, the extent and duration of antiallodynia were estimated by AUC values (0–40 minutes) illustrated in Table 1. The potency order of these drugs was EM-1 > EM-2 > morphine. Moreover, we found that both EMs exerted significant antiallodynic effects in the contralateral paw (P <.05 and P <.01), whereas no significant antinociceptive activity of morphine was seen at a dose of 10 nmol after i.c.v. administration (P >.05; Figure 3D).

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Opioid Mechanisms of Antiallodynic Effects Elicited by i.c.v. Administration of EM-1 and EM-2

To investigate the opioid mechanisms of i.c.v. administration of EM-1 and EM-2 on the antinociception in ipsilateral paw of SNI mice, the antagonistic effects of the specific opioid receptor antagonists and antibodies against endogenous opioid peptides were determined. Figure 4A, B shows the effects of i.c.v. coadministration of various opioid receptor antagonists on the antinociception of EM-1 and EM-2. Naloxone (40 nmol, i.c.v.), the broad-spectrum opioid receptor antagonist, almost fully attenuated the antiallodynic activities of both EM-1 and EM-2 (10 nmol, i.c.v.), which indicated that central opioid receptors were involved (F(5,61) = 60.98, P <.001 for EM-1; F(5,60) = 112.7, P <.001 for EM-2). The specific μ-opioid receptor antagonist, β-FNA, could also completely antagonizes the antiallodynic effects produced by EM-1 and EM-2 (P <.001). It is noteworthy that naloxonazine, a selective μ1-opioid receptor antagonist, did not significantly block the antiallodynia of EM-1 (P >.05), but partially reversed the effects of EM-2 (F(5,60) = 112.7, P <.001). In addition, the antinociception of both EMs were not significantly modified by NTI, a selective δ-opioid receptor antagonist (P >.05), indicating that δ-opioid receptor was not involved. Interestingly, nor-BNI, the κ-opioid receptor antagonist, largely reversed the effects of EM-2 against neuropathic pain (F(5,60) = 112.7, P <.001), but not EM-1 (P >.05). Thus, differential opioid mechanisms were involved in the central antinociceptive activities of EM-1 and EM-2.

Figure 4.

Figure 4.

Because EMs exhibit high affinity and selectivity for μ-opioid receptor, but not δ- or κ-opioid receptor, it is unlikely that the EM-2-induced antinociception is mediated by a direct stimulation of κ-opioid receptor. Dynorphin A has been proposed to be the endogenous opioid ligand for κ-opioid receptor. Therefore, in the present study, the effects of i.c.v. pretreatment with antibodies against dynorphin A(1–17) and δ-endogenous opioid peptides [Met5]enkephalin, [Leu5]enkephalin on the antiallodynia of EM-2 were examined. As shown in Figure 4C, the antinociception of EM-2 were not affected by i.c.v. pretreatment with either [Met5]enkephalin or [Leu5]enkephalin antibodies (P >.05), indicating δ-endogenous ligands were not involved. However, anti-Dyn A antibody significantly blocked the EM-2-elicited effects against neuropathic pain (F(3,35) = 4.761, P <.01), which indicated that central administration of EM-2 may induce the release of endogenous dynorphin A(1–17), acting on κ-opioid receptor to produce antinociception in the brain.

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Antiallodynic Effects of Peripheral Administration of EM-1, EM-2, and Morphine in SNI Mice

Opioid receptors that expressed in the peripheral nervous system, such as afferent axons may also contribute to the antinociception of opioids against neuropathic pain. Therefore, the antiallodynic activities of peripheral administration of EM-1, EM-2, and morphine were investigated to explore whether the peripheral opioid receptors were involved. In this study, we examined the effects of EM-1, EM-2, and morphine in neuropathic mice after i.pl. directly injected to the injured paw (ipsilateral paw), and after systemic s.c. administration. In Figure 5A–C, locally (i.pl.) administered EM-1, EM-2, and morphine (10–100 nmol) significantly increased the ipsilateral withdrawal threshold in a dose-dependent manner (F(3,39) = 20.19, P <.001 for EM-1, F(3,39) = 24.33, P <.001 for EM-2 at 5 minutes and F(3,40) = 23.83, P <.001 for morphine at 10 minutes, respectively). Both EMs reached their maximal antiallodynic response at 5 minutes after i.pl. administration, which was similar to central administration of them. Nevertheless, the peak antiallodynic effects produced by morphine were observed at 10 minutes after i.pl. injection, different from i.c.v. injection, but still with a long duration of action being about 60 minutes. Surely, morphine also displayed less potent effects on neuropathic pain compared to EM-1 and EM-2 after i.pl. administration. The 2-way ANOVA of data of EM-1, EM-2, and morphine indicated significant interactions for both dose and time period (F(15,240) = 10.45, P <.001 for EM-1, F(15,234) = 8.78, P <.001 for EM-2 and F(18,280) = 8.15, P <.001 for morphine, respectively). The dose–response curves of paw withdrawal threshold induced by EM-1, EM-2, and morphine are shown in Figure 5D, and the AUC values (0–40 minutes) of EMs and morphine (100 nmol, i.pl.) are illustrated in Table 2. The order expressing the potency of antiallodynia was EM-2 > EM-1 > morphine.

Table 2.

Table 2.

Figure 5.

Figure 5.

To compare the peripheral and local effects of EM-1, EM-2, and morphine, the antinociception of s.c. administration of these compounds was also investigated. Our results showed that the antiallodynic effects of both EM-1 and EM-2 at a dose of 30 mg/kg were not statistically significant versus saline control, whereas morphine (2 mg/kg, s.c.) produced significant and prolonged antiallodynic effects measured in the ipsilateral paw (F(3,41) = 40.3, P <.001 at 20 minutes, Figure 5E). The maximal withdrawal threshold value was 0.36 ± 0.15 g measured at 20 minutes after systemic injection.

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Effects of i.pl. Coadministration of Various Opioid Receptor Antagonists on the Antiallodynic Effects Elicited by EM-1 and EM-2

Figure 6.

Figure 6.

To determine the role of opioid receptors on the antiallodynic effects of i.pl. administered EM-1 and EM-2, the effects of coadministration of the specific opioid receptor antagonists were studied. As illustrated in Figure 6, naloxone and naloxone methiodide (400 nmol, i.pl.), which has limited access to the brain, significantly antagonized the antinociception of EM-1 and EM-2 (100 nmol, i.pl.) (F(6,64) = 94.77, P <.001 for EM-1; F(6,67) = 170.2, P <.001 for EM-2), validating the role of local peripheral opioid receptors in neuropathic pain. Furthermore, the antiallodynic effects elicited by both EM-1 and EM-2 was fully blocked by β-FNA, a specific μ-opioid receptor antagonist (P <.001), and partially reversed by naloxonazine, a selective μ1-opioid receptor antagonist (P <.001), indicating the observed effects were mediated by both μ1- and μ2-opioid receptor subtypes. On the contrary, i.pl. coadministered NTI or nor-BNI did not alter the antinociceptive effects of EM-1 and EM-2, excluding the role of δ- and κ-opioid receptors (P >.05).

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DISCUSSION

Neuropathic pain is a devastating consequence of injury or diseases affecting the somatosensory component of the peripheral or central nervous system at any level.3 In recent years, several mechanisms underlying the initiation and maintenance of neuropathic pain have been elucidated. These included peripheral sensitization of nociceptors, ectopic excitability of afferent neurons, primary sensory degeneration, central sensitization comprising pronociceptive facilitation, disinhibition of nociception, and abnormal descending modulatory pathways. However, pharmacological management of neuropathic pain remains a great clinical challenge.31,32 Numerous therapeutic trials and treatment options are available for relieving neuropathic pain. Opioids are recommended as effective analgesic agents for the treatment of neuropathic pain.33

Over the past decades, numerous animal models of neuropathic pain have been developed and characterized.6–11 These neuropathic pain models showed great value in the development of novel analgesic drugs. The SNI model is a new animal model that can mimic several characteristics of clinical neuropathic pain closely, and the surgical procedure is easy to perform, and reproduce.6–11 Therefore, the present study was conducted to investigate the antinociceptive effects of EMs in the mice SNI model of neuropathic pain after central and peripheral administration.

In this study, we observed that the SNI mice developed prolonged mechanical allodynia-like behavior in ipsilateral paw after surgery, which were consistent with previous studies.9–11,34 Notably, in contrast to the report of Bourquin et al,10 our present data indicated that the injured mice also displayed mechanical allodynia in the contralateral paw. There is no reasonable explanation for this discrepancy. Perhaps, diverse surgical procedure, behavior assessment, and strain of mice may account for these differences. Nevertheless, our results were consistent with other animal models of neuropathic pain,7,8 where contralateral effects have been observed. Similar results were also reported in the SNI model of rats.34 It may be explained that the contralateral changes were mediated by commissural interneurons, which altered gene expression in the contralateral sensory neurons, and ultimately change behavioral response to contralateral stimulation.35,36

It is widely accepted that opioids have an important role in the treatment of neuropathic pain.37,38 Our present results demonstrated that EM-1, EM-2, and morphine produced significant antiallodynic effects in ipsilateral paw of SNI mice after i.c.v. administration. Unlike the antinociception of opioid alkaloid, the alleviation of allodynia of i.c.v. administered EMs appeared shorter than that of morphine (30 minutes in contrast to 60 minutes after injections). The possible reason is that most opioid peptides, like EMs, can be easily degraded by various peptidases and proteases,39 while morphine is more resistant to enzymatic degradation. The prolonged antiallodynic effects of morphine may also be due to persistent activation of down-stream kinases.40 Interestingly, at the same dose, morphine displayed less potent antinociceptive effects in ipsilateral paw of SNI mice compared with EM-1 and EM-2. Furthermore, both EMs exerted significant antiallodynic effects in the contralateral paw, whereas no significant antinociceptive activity was seen after i.c.v. administration of morphine with equimolar dose. These data indicated that EMs were more effective than morphine against neuropathic pain, which were consistent with previous reports.18,24,25 The exact reason for the different analgesic potencies of EMs and morphine is not clear. One possible explanation is the differential opioid receptor responsiveness induced by peptide agonists and morphine. It has been reported that both EM-1 and EM-2 induced internalization of the μ-opioid receptor.41 However, morphine, the opioid alkaloid, did not cause the receptor internalization, which might easily induce tolerance development and subsequently loose its effectiveness in neuropathic pain.42 Another explanation is different μ-opioid receptor subtypes in the brain may mediate the differential antiallodynic activities of opioids agonists.

Additionally, the opioid mechanisms of antiallodynic effects induced by central administration of EM-1 and EM-2 were investigated. i.c.v. coadministration of nonselective opioid receptor antagonist naloxone fully attenuated the antinociception of both EM-1 and EM-2 in neuropathic pain, indicating a central opioid mechanism. However, the antinociception of EM-2, but not EM-1, was partially reversed by naloxonazine, a selective μ1-opioid receptor antagonist, which demonstrated that EM-1 and EM-2 may produce antinociception through distinct μ1- and μ2-opioid receptor subtypes. Furthermore, the antiallodynic effects elicited by EM-2 contained an additional component that was mediated by the release of endogenous dynorphin A, acting on κ-opioid receptor. These results were consistent with previous investigations which reported that EM-1 and EM-2 produced central antinociception in acute pain through different actions at μ1- and μ2-opioid receptor subtypes,43 and the release of the endogenous κ-opioid ligand dynorphin A was involved in the antinociception of EM-2.17,44–47

Besides the central nervous system, all 3 opioid receptors (μ, δ, and κ) have been found on cell bodies of sensory neurons in the dorsal root ganglia, and on the peripheral terminals of primary afferent neurons.48 Neuropathic pain resulting from peripheral nerve injury is a pathological state which may change the opioid receptors expression in peripheral sensory neurons. Several studies have reported that μ-, δ-, and κ-opioid receptors expression was upregulated in neuropathic pain.49–51 It should be mentioned that the overexpression of peripheral opioid receptors and increased binding affinity in neuropathic pain may make them more accessible for both endogenous and exogenous opioids. Therefore, the antiallodynic activities of peripheral administration of EM-1, EM-2, and morphine were also investigated to explore the role of peripheral opioid receptors. In the present study, we found locally (i.pl.) administered EM-1 and EM-2 dose-dependently increased the ipsilateral withdrawal threshold, in contrast to noneffective systemic (s.c.) administration. These observations were in agreement with previous report,24 establishing the peripheral and local effects of EMs.

Furthermore, the present results showed that the antinociception of EM-1 and EM-2 was antagonized by both brain-permeable naloxone and naloxone methiodide, a nonselective peripherally restricted opioid receptor antagonist, validating the local and peripheral nature of opioid receptors in neuropathic pain. It is noteworthy that the antiallodynic effects elicited by both EM-1 and EM-2 were fully blocked by β-FNA and partially reversed by naloxonazine, indicating the observed effects were mediated by both μ1- and μ2-opioid receptor subtypes. By contrast, the antinociceptive effects of EM-1 and EM-2 were not altered by i.pl. coadministration of NTI or nor-BNI, excluding the role of δ- and κ-opioid receptors. These results were inconsistent with the above-mentioned opioid mechanisms induced by central administration of EM-1 and EM-2. The exact reason for this discrepancy is unclear. There is biochemical and pharmacological evidence supporting the existence of μ-opioid receptor subtypes. At least 2 subtypes, μ1- and μ2-opioid receptor, have been proposed.52 Autoradiographic studies showed that μ1- and μ2-opioid receptor subtypes are localized in the spinal cord and brain, exerting modulation of nociception.53 The supraspinal and spinal antinociception was mainly mediated by μ1- and μ2-opioid receptors, respectively. Notably, previous investigation indicated that multiple μ1-opioid receptors, μ1-opioid receptor subtype-1 and μ1-opioid receptor subtype-2, were involved in the antinociception of EM-2 given spinally44 and supraspinally.45 The additional component of EM-2-elicited antinociception that was mediated by the release of endogenous κ-opioid agonist dynorphin A may be produced by stimulation of the μ1-opioid receptor subtype-1, which is extremely sensitive to β-FNA and naloxonazine.44,45 Based on the present data, we propose that the μ1-opioid receptor subtype-1 is likely to be exclusively distributed centrally, but not peripherally, to induce the release of endogenous κ-opioid agonist, because the κ-opioid receptor was not involved in the antinociception of peripheral administration of EM-2. In addition, both EM-1 and EM-2 might bind with μ1-opioid receptor subtype-2 and μ2-opioid receptor subtype in the peripheral level to produce antinociception. Further studies should be addressed to confirm our hypothesis. Thus, the differential antiallodynic effects of EM-1 and EM-2 given centrally and peripherally may be due to activation of different subtypes of μ-opioid receptors.

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CONCLUSIONS

In summary, our present study indicated that the SNI mice developed prolonged mechanical allodynia-like behavior in ipsilateral paws. Both EM-1 and EM-2 given centrally produced significant antiallodynic effects, more effective than morphine. Additionally, we demonstrated that EM-1 and EM-2 may produce antinociception through distinct μ1- and μ2-opioid receptor subtypes, and the antiallodynic effects elicited by EM-2 contained an additional component that was mediated by the release of endogenous κ-opioid agonist. Moreover, i.pl.-administered EM-1 and EM-2 also exhibited potent antinociceptive effects, establishing the peripheral and local effects. Both μ1- and μ2-opioid receptor subtypes, but not the δ- or κ-opioid receptors, were involved in the peripheral antiallodynia of EMs. Taken together, both EM-1 and EM-2 given centrally and peripherally induced potent antiallodynic activities in SNI mice by differential opioid mechanisms. This investigation is of great value in the development of novel opioid therapeutics against neuropathic pain.

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DISCLOSURES

Name: Chang-lin Wang, PhD.

Contribution: This author helped design the whole study and analyze the data, and wrote the manuscript.

Name: Dai-jun Yang, MSc.

Contribution: This author helped conduct the study and analyze the data.

Name: Bi-yu Yuan, MSc.

Contribution: This author helped conduct the study and analyze the data.

Name: Ting-ting Qiu, MSc.

Contribution: This author helped conduct the study and analyze the data.

This manuscript was handled by: Jianren Mao, MD, PhD.

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