Antinociceptive Effects and Interaction Mechanisms of Intrathecal Pentazocine and Neostigmine in Two Different Pain Models in Rats

Huang, Huiying; Bai, Xiaohui; Zhang, Kun; Guo, Jin; Wu, Shaoyong; Ouyang, Handong

doi:https://doi.org/10.1155/2022/4819910

Pain Research and Management

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Abstract Introduction Materials and Methods Results Discussion Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 4819910 | https://doi.org/10.1155/2022/4819910

Antinociceptive Effects and Interaction Mechanisms of Intrathecal Pentazocine and Neostigmine in Two Different Pain Models in Rats

Huiying Huang,¹Xiaohui Bai,^2,3Kun Zhang,²Jin Guo,²Shaoyong Wu,²and Handong Ouyang²

Academic Editor: Vahid Rakhshan

Received01 Sept 2021

Revised10 Mar 2022

Accepted20 Apr 2022

Published18 May 2022

Abstract

Background. Pentazocine produces a wide variety of actions in the treatment of perioperative analgesia. Neostigmine is a cholinesterase inhibitor used to antagonize the residual effects of muscle relaxants and also produces an analgesic effect. Objectives. To investigate the analgesic effects of intrathecally injected pentazocine and neostigmine and their interaction. Methods. Sprague–Dawley rats were used to test the analgesic effect of pentazocine and neostigmine using the paw formalin pain model and the incision mechanical allodynia model. Pentazocine (3, 10, 30, and 100 μg), neostigmine (0.3, 1, 3, and 10 μg) or a pentazocine-neostigmine mixture were separately injected to evaluate their antinociceptive effects alone on the treatment groups. The corresponding control group received an intrathecal injection containing the same volume of saline. The formalin pain test, or the plantar incision pain behavior test were performed 30 minutes later. Isobolographic analysis was used to evaluate the interaction between pentazocine and neostigmine. Intrathecally administered selective mu-opioid receptor antagonist CTAP, selective kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), nonselective opioid receptor antagonist naloxone, and muscarinic acetylcholine receptor antagonist atropine were also used to test the possible interaction mechanism. These antagonists were used 30 minutes before the pentazocine and neostigmine mixtures which were intrathecally injected. Results. Intrathecally administered pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone had a marked dose-related impact on suppressing the biphasic responses in the formalin test. Pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone attenuated the mechanical allodynia in a plantar incision model in a dose-dependent manner. Isobolographic analysis revealed that the mixture of intrathecal pentazocine and neostigmine synergistically decreased both phase I and II activity in the formalin test and mechanical allodynia in the plantar incision model. Pretreatment of intrathecally administered nor-BNI, naloxone, atropine, but not CTAP, antagonized the analgesic effect of the pentazocine-neostigmine mixture. Conclusions. All of these results suggest that the combined application of pentazocine and neostigmine is an effective way to relieve pain from formalin and acute incision mechanical allodynia. The synergistic effect between pentazocine and neostigmine is mostly attributed to the kappa-opioid receptor and the cholinergic receptor in the spinal cord.

1. Introduction

Pentazocine was previously classified as a kappa-opioid receptor agonist and a mu-opioid receptor antagonist, but researchers later determined it as a mixed kappa-opioid receptor agonist and a partial mu-opioid receptor agonist [1–3]. It shares lots of the side effects of other opioids such as constipation and nausea, but it produces less central nervous system depression. While it seldom affects the mood after short-term use, it sometimes causes hallucinations, nightmares, and delusions [4]. Intravenously administered pentazocine can reduce both the incidence and severity of itching in women treated with subarachnoid opioids during cesarean section [5]. Pentazocine is also effective in alleviating postoperative pain and is commonly used as an analgesic in the perioperative period [6]. Three major opioid receptor systems, mu-opioid, delta-opioid, and kappa-opioid have been characterized with respect to the signal transduction pathway leading to pain modulation. Activation of the kappa-opioid system within the nucleus accumbens may circumvent pain-induced affective disorders [7], and µ-heterodimerization may be a potential target in a spinal nerve injury neuropathic pain model [8]. Spinal cord cholinergic receptors and acetylcholine (Ach) participate in the transmission and regulation of pain, and acetylcholine receptors have been identified as a target for pain control for decades [9, 10]. Ach is released in response to pain stimuli at the spinal cord and brain stem level [11]. Cholinesterase inhibitor physostigmine has been shown to relieve clinical postoperative pain [12]. And neostigmine, a clinical cholinesterase inhibitor, is widely used as an anesthetic to reverse nondepolarizing neuromuscular blockers. It is also regularly combined with atropine as atropine can block the muscarinic acetylcholine receptor [13]. Neostigmine produces a dose-dependent analgesic after spinal and peripheral administration in preclinical and clinical trials [14, 15]. It inhibits cholinesterase and results in much more ACh at sites of cholinergic transmission. Direct activation of cholinergic receptors or the pharmacological blocking of acetylcholinesterase to amplify endogenous acetylcholine action has been proven to alleviate pain in rodents and humans [9]. A reduction in cholinergic modulation may also be integral to mPFC deactivation for neuropathic pain, as well as underscore mPFC related cognitive shortfalls related to such pain [16]. In the process of anesthetic resuscitation, pentazocine is sometimes used as an analgesic, and neostigmine as a muscle relaxant antagonist. However, there is no research on the combined effects of pentazocine and neostigmine for postoperative pain treatment. In this experiment, the actual effects of pentazocine and neostigmine were assessed using a formalin-induced pain model and the plantar incisional pain model. To explore its possible mechanism of action, we intrathecally administered the selective kappa-opioid receptor antagonist nor-Binaltorphimine, the selective mu-opioid receptor antagonist CTAP, the nonselective opioid receptor antagonist naloxone, and the muscarinic acetylcholine receptor antagonist atropine.

2. Materials and Methods

2.1. Animals and Drug Administration

This research was approved by the Animal Care and Use Committee of Sun Yat-Sen University (No. L10202020000X, Guangzhou, China). Three hundred and eighty male Sprague–Dawley (SD) rats weighing 200 to 250 g were used. Rats were kept in separate cages with 50 to 60% humidity at 24°C with free access to food and water. All surgical procedures were performed with the rats under isoflurane (1–3%) inhalation. Nor-Binaltorphimine 10 μg (nor-BNI, Abcam ab120078), CTAP 10 μg (R&D, 1560/1), naloxone 10 μg (Merck, 465-65-6), and atropine 10 μg (Merck, 51-55-8) were administered 30 minutes before the pentazocine (Merck, 359-83-1)-neostigmine (Merck, 114-80-7) mixture.

2.2. Drugs

The drugs nor-Binaltorphimine 10 μg, CTAP 10 μg, naloxone 10 μg, atropine 10 μg, pentazocine (3–100 μg), neostigmine (0.3–10 μg), and pentazocine-neostigmine were respectively dissolved in 10 μl of normal saline and were administered intrathecally [17–20]. The antagonists were intrathecally administered as described elsewhere [21].

2.3. Treatment Schedule and Experimental Design

In this study, we designed four independent experiments, and our study established n = 10 rats for each experimental group. All the rats were randomly divided into different groups.

Experiment I: Male SD rats (n = 130) were divided into 13 groups and intrathecally administered (i.t.) 30 minutes before the formalin test with different doses of pentazocine (3 μg, 10 μg, 30 μg, and 100 μg, i.t., n = 10), neostigmine (0.3 μg, 1 μg, 3 μg, and 10 μg, i.t., n = 10), saline alone (10 μl, i.t., n = 10), or the pentazocine-neostigmine mixture (1/2ED₅₀, 1/4ED₅₀, 1/8ED₅₀, and 1/16ED₅₀, i.t., n = 10) (Figure 1(a)).

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(c)

Experiment II: Male SD rats (n = 130) were divided into 13 groups and intrathecally administered (i.t.) 4 hours after a plantar incision model with different doses of pentazocine (3 μg, 10 μg, 30 μg, and 100 μg, i.t., n = 10), neostigmine (0.3 μg, 1 μg, 3 μg, and 10 μg, i.t., n = 10), saline alone (10 μl, i.t., n = 10), or the pentazocine-neostigmine mixture (1/2ED₅₀, 1/4ED₅₀, 1/8ED₅₀, and 1/16ED₅₀, i.t., n = 10) (Figure 1(b)).

Experiment III: Male SD rats (n = 120) were divided into 12 groups and several antagonists were intrathecally administered (i.t.) 30 minutes before the administration of the pentazocine (30 μg)-neostigmine (3.0 μg) mixture. Saline alone (10 μl), nor-Binaltorphimine (10 μg), CTAP (10 μg), naloxone (10 μg), and atropine (10 μg), were intrathecally administered 30 minutes before the administration of the pentazocine (30 μg)-neostigmine (3.0 μg) mixture in the formalin test and incision pain model (Figure 1(c)).

2.4. Intrathecal Injection

Under isoflurane (1–3%) inhalation anesthesia, a sterile needle attached to a 25 μl micro-injector was inserted into the intervertebral space between L5 and L6 in rats. A sudden and slight flick of the tail indicated that the needle entered the subarachnoid space, where 10 μl of the specific therapeutic drug or vehicle was delivered for more than 30 seconds [21]. The needle was held in the same position for an additional 15 seconds to ensure diffusion prior to removal. Thirty minutes after intrathecal injection the behavioral tests were performed.

2.5. The Formalin Test

A 30-gauge needle was used to inject 50 μl of 5% formalin subcutaneously into the plantar surface of the left hind paw [17]. Next, the rats were placed in a transparent organic glass cylinder (20 cm × 30 cm) for observation. A mirror was placed under the cylinder at a 45° angle. Immediately after injection, the rat exhibited the behavioral plantar pain phenomenon exhibiting spontaneous flinching, withdrawing, and licking of the injected paw. Pain behavior was quantified by recording the number of paw flinches for 1-minute periods from 1 to 2 minutes and 5 to 6 minutes and then at 5-minute intervals between 10 minutes and 60 minutes after the formalin injection. We observed two phases of paw flinching behavior. The first stage in the pain model (the interval between 0 and 6 minutes after the formalin injection) is the initial acute pain response, followed by the second stage, persistent pain (starting about 10 minutes after the formalin injection).

2.6. The Plantar Incision Model

In order to simulate acute postoperative pain, we used the rat plantar incision model [22]. The rats were anesthetized with 2–3% isoflurane. Make a 1 cm longitudinal incision at the plantar surface of the right hind foot 0.5 cm from the heel end. The skin, fascia, and underlying flexor muscles were cut, and the wound was sutured with 5–0 nylon sutures after sufficient hemostasis. The sham control group rats were anesthetized without incision. Four hours after the pain model was established, the rat behavior test was performed.

2.7. Mechanical Allodynia

Mechanical allodynia (other pain) is a painful sensation triggered by an innocuous stimulus, such as a light touch [23]. Rats were placed on a mesh floor individually covered with a clear plastic cage and allowed to acclimate for 30 minutes. Paw withdrawal response to mechanical stimulation was detected by the calibrated von Frey hairs method. Mechanical sensitivity was assessed using the von Frey hairs (0.4 g, 0.6 g, 1 g, 2 g, 4 g, 6 g, 8 g, 10 g, and 15 g) up-down method as previously described [24].

2.8. Isobolographic Analysis

To test the interaction between pentazocine and neostigmine, we performed an isobolographic analysis. First, each ED₅₀ value (effective dose producing a 50% maximal possible effect) was identified by the dose-response curves for each of the two drugs. In the formalin test, the time response data are presented as the number of paw flinches in the 1-minute time frames of 1 to 2 minutes and 5 to 6 minutes in the first phase and then at 5-minute intervals during the period from 10 minutes to 60 minutes in the second phase. In order to get the ED₅₀, the flinches were converted into a percentage maximal possible effect (%MPE). We defined separately the value of 50%MPE as ED₅₀ calculated by the following formula in the two stages of formalin test:

In the plantar incision model, the 50% MPE (ED₅₀) was calculated using the following formula [25]:

Next, the respective ED₅₀ values (1/2, 1/4, 1/8, and 1/16) for each drug were coadministered. The experimental ED₅₀ for the mixture was calculated by the dose-response curves of the mixture. The expected additive ED₅₀ values for pentazocine and neostigmine were determined by an isobologram. The x and y axes in the isobologram represent the ED₅₀ values of each drug, respectively. The lines connecting the ED₅₀ points are the theoretical additive lines and the theoretical additive points for the drug combinations. The experimental values below the lines of additivity indicate a synergistic interaction [25].

2.9. Statistical Analysis

All data were expressed as the means ± SEM. The Shapiro–Wilk test was used to detect the normality of the data distribution. To compare the differences between each dose of pentazocine, neostigmine, and the mixtures of pentazocine and neostigmine in two stages of the formalin test and the von Frey test, we used one-way analysis of variance (ANOVA) or two-way ANOVA followed by the Bonferroni post hoc test. The differences between the experimental ED₅₀ for pentazocine, neostigmine, and the mixtures, and the expected additive ED₅₀ values for pentazocine, neostigmine, and the mixtures were evaluated by the one-way ANOVA in the formalin test and the von Frey test. The criterion for statistical significance was a . Statistical tests were performed with SPSS 21.0 software (SPSS, USA).

3. Results

3.1. The Effects of Pentazocine and Neostigmine on Formalin-Induced Pain

The number of flinches for each minute is plotted versus the time after the formalin injection into the hind paw. Two phases of paw flinching behavior were separately quantified. Intrathecal administration of pentazocine (Pen) and neostigmine (Neo) decreased the number of paw flinches. Pentazocine (3–100 μg) relieved the formalin-induced phase I pain (one-way ANOVA, F_4,45 = 9.990, ) (Figure 2(b)) and phase II pain (one-way ANOVA, F_4,45 = 20.675, ) (Figure 2(c)). Neostigmine (0.3–10 μg) also relieved the formalin induced phase I pain (one-way ANOVA, F_4,45 = 10.649, ) (Figure 2(e)) and phase II pain (one-way ANOVA, F_4,45 = 16.748, ) (Figure 2(f)). In phases I and II, pentazocine’s calculated ED₅₀ values were 91.1 ± 5.4 μg and 101.2 ± 6.2 μg, respectively. Neostigmine's ED₅₀ values in phases I and II were 8.93 ± 0.59 μg and 9.2 ± 0.55 μg, respectively (Figures 2(i) and 2(j), Table 1).

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Figure 2

(a-f). Intrathecal injection of pentazocine (3–100 μg) and neostigmine (0.3–10 μg) induced a dose-dependent inhibition against the formalin-induced pain responses in both phases. (g-h). Injecting pentazocine or neostigmine attenuated the plantar incision-induced mechanical allodynia. (i-k). Dose-response curves of intrathecal pentazocine and neostigmine for flinching during phase I (i) and phase II (j) in the formalin test (k). Data are expressed as the maximal possible effect (% MPE). Each point on the graph represents the mean ± SEM. Compared with the saline group, , , , n = 10 rats in each group).

3.2. The Effects of Pentazocine and Neostigmine on Plantar Incision-Induced Mechanical Allodynia

Behavior was tested 30 minutes after the intrathecal injection. Intrathecal injection of pentazocine (3–100 μg) produced a dose-dependent inhibition against the plantar incision-induced mechanical allodynia (repeated measure, two-way ANOVA, F_4,36 = 29.178, ) (Figure 2(g)). Intrathecal injection of neostigmine (0.3–10 μg) produced a dose-dependent inhibition against the plantar incision-induced mechanical allodynia (repeat measure, two-way ANOVA, F_4,36 = 23.751, ) (Figure 2(h)). The calculated ED₅₀ values of pentazocine and neostigmine were 98.0 ± 5.5 μg and 10.2 ± 0.55 μg, respectively (Figure 2(k), Table 1).

3.3. Isobolographic Analyses

We used the ED₅₀ of pentazocine and neostigmine in phase I to evaluate their interaction in the formalin pain model. The ED₅₀ of pentazocine and neostigmine was 91.1 μg and 8.93 μg in phase I, respectively. Intrathecal administration of the pentazocine-neostigmine mixtures (1/2 ED₅₀, 1/4 ED₅₀, 1/8 ED₅₀, and 1/16 ED₅₀) decreased the number of paw flinches (Figure 3(a)). In phase I, the pentazocine-neostigmine mixtures relieved the pain induced by formalin (one-way ANOVA, F_4,45 = 34.687, ) (Figure 3(b)). In phase II, the pain was relieved by the pentazocine-neostigmine mixtures (one-way ANOVA, F_4,45 = 99.844, ) (Figure 3(c)). In the plantar incision pain model, pentazocine ED₅₀ was 98 μg and neostigmine ED₅₀ was 10.2 μg. Intrathecal administration of the pentazocine-neostigmine mixtures (1/2 ED₅₀, 1/4 ED₅₀, 1/8 ED₅₀, and 1/16 ED₅₀) significantly attenuated the mechanical allodynia (repeat measure, two-way ANOVA, F_4,36 = 50.640, ) (Figure 3(d)).

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The experimental ED₅₀ for pentazocine and neostigmine were 30.3 ± 2.5 μg and 2.95 ± 0.3 μg in phase I, respectively (Figure 4(a)). The experimental ED₅₀ for pentazocine and neostigmine were 34.4 ± 3.0 μg and 3.1 ± 0.24 μg in phase II, respectively (Figure 4(b)). The expected additive ED₅₀ values for pentazocine and neostigmine were 45.9 ± 3.9 μg and 4.47 ± 0.43 μg in phase I, respectively (Figure 4(a)). The expected additive ED₅₀ values for pentazocine and neostigmine were 50.8 ± 3.5 μg and 4.58 ± 0.42 μg in phase II, respectively (Figure 4(b)). The experimental values for the pentazocine-neostigmine mixtures decreased significantly () below the lines of additivity indicating a synergistic effect (Figures 4(a) and 4(b) and Table 1). The experimental ED₅₀ for pentazocine and neostigmine were 34 ± 2.2 μg and 3.3 ± 0.27 μg, respectively (Figure 4(c)). The expected additive ED₅₀ values for pentazocine and neostigmine were 51.0 ± 3.0 μg and 4.85 ± 0.4 μg, respectively (Figure 4(c)). The experimental values for the pentazocine-neostigmine mixtures decreased significantly () below the lines of additivity, indicating a synergistic effect (Figure 4(c) and Table 1).

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Figure 4

Shows the interaction between intrathecal pentazocine and neostigmine in phases I (a) and II (b) of the formalin tests and the plantar incisional pain model (c) which were analyzed using an isobologram. The x- and y-axes represent the ED₅₀ dose of pentazocine and neostigmine, respectively. The lines connecting the ED₅₀ points are the theoretical additive lines and the theoretical additive points for the drug combinations. The theoretical additive value was significantly higher than the experimental ED₅₀ value of the combination of the two drugs. The experimental ED₅₀ values were significantly below the lines of additivity, indicating a synergistic interaction.

3.4. Intrathecal Antagonist Test

We used the selective κ-opioid receptor antagonist nor-Binaltorphimine (10 μg, nor-BNI), the selective µ-opioid receptor antagonist CTAP (10 μg), the nonselective opioid receptor antagonist naloxone (10 μg), and the muscarinic acetylcholine receptor antagonist atropine (10 μg) to evaluate the possible synergistic effect mechanisms between pentazocine and neostigmine. Nor-BNI, naloxone, and atropine, but not CTAP, attenuated the analgesic effect of the pentazocine-neostigmine mixture against formalin-induced pain (Figures 5(a) and 5(b)) and the plantar incision-induced mechanical allodynia (Figure 5(c)).

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Figure 5

To evaluate the possible interaction mechanism between pentazocine and neostigmine, nor-Binaltorphimine (nor-BNI, 10 μg), CTAP (10 μg), naloxone (NAL10 μg), and atropine (ATR 10 μg), were intrathecally administered 30 minutes before the administration of the pentazocine (30 μg)-neostigmine (3.0 μg) mixture. (a) Nor-BNI, naloxone, and atropine significantly antagonized the antinociception of the pen-neo mixture in the first phase of formalin pain (One-way ANOVA, F_5,54 = 36.437, ). (b) Nor-BNI, naloxone, and atropine significantly antagonized the antinociception of the pen-neo mixture in the second phase of formalin pain (One-way ANOVA, F_5,54 = 62.32, ). (c) Nor-BNI, naloxone, and atropine significantly antagonized the antinociception of the pen-neo mixture in the plantar incision pain model (One-way ANOVA, F_5,54 = 16.88, ). , , vs. Pen + Neo group, n = 10 rats in each group. Each bar represents the sum of flinches in (a) phase (I), (b) phase II, and (c) the mechanical withdrawal threshold (mean ± SEM) from 10 rats.

4. Discussion

The results show that pentazocine and neostigmine produce analgesic effects against formalin-induced pain and incision-induced mechanical allodynia. There was a synergistic effect between pentazocine and neostigmine in both pain models. The formalin test was chosen as it is a valuable method for studying nociception in detecting drug analgesic effects [26]. The test showed a biphasic pain response. The early phase (0–6 minutes, Phase I) was mainly caused by the activation of C-fiber due to the peripheral stimulus, while the late phase (10–60 minutes, Phase II) was caused by an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord [26]. In order to evaluate the analgesic effects of pentazocine and neostigmine in the postoperative period, we used the plantar incisional pain model [22].

As a mixed opioid agonist/antagonist, pentazocine’s analgesic mechanism is not entirely understood. The spinal µ- and κ-opioid receptors are considered the most important pathways mediating the analgesic effects of pentazocine [27]. Different administration methods and dosages are also important factors that affect its analgesic actions. The analgesic mechanism of pentazocine varies with different doses and administrations [27]. Its analgesic action shows a biphasic bell-shaped dose-response curve. Intravenous injection of pentazocine at a modest dose (30 mg/kg) exhibits a peak antinociceptive effect via the µ- and κ-opioid receptors [27]. When an intravenous injection of pentazocine reaches a dose of 100 mg/kg, its analgesic effect is mainly through the µ-opioid receptor, not the κ-opioid receptor, as this analgesic effect can be partly antagonized by the κ-opioid receptor agonist [27]. α-adrenergic receptors might be other analgesic pathways of pentazocine as phentolamine alone is effective in reducing pentazocine’s analgesic effects [28]. Nor-BNI is a highly selective kappa-opioid receptor antagonist that can partially antagonize the action of morphine and fentanyl [29]. In our results, the synergistic effects between pentazocine and neostigmine can be antagonized by the nonselective opioid receptor antagonist naloxone and the κ-opioid receptor antagonist nor-BNI, but not antagonized by the µ-opioid receptor antagonist CTAP. CTAP is a highly selective antagonist for µ-opioid receptors over δ- and κ-opioid receptors [30]. This shows that the combined medication of this dose is mostly via the κ-opioid receptor, but not the µ-opioid receptor, when intrathecally administered.

Systemic and spinal administration of acetylcholinesterase inhibitors and muscarinic receptor agonists can produce an analgesic effect [9, 12, 15]. Intrathecal injection of neostigmine and physostigmine produces dose-dependent antinociception effects and relieves allodynia in a dose-related manner [9, 12, 15]. The analgesic effect caused by neostigmine is mainly related to the release of acetylcholine and the activation of the muscarinic-acetylcholine receptor, as atropine blocks the muscarinic-acetylcholine receptor and antagonizes the analgesic effect [9, 12, 15]. However, when neostigmine is used as a nondepolarizing muscle relaxant antagonist, it is always used in combination with atropine as it antagonizes the muscarinic-acetylcholine receptor activity [13]. Normally, it inhibits acetylcholinesterase (AchE) and causes more ACh at sites of cholinergic transmission. ACh is released when physiological stimuli (pain) modulate the processing of pain at the spinal cord or brain stem level [11].

The administration of muscarinic receptor agonists and acetylcholinesterase inhibitors in the spinal cord can also result in antinociception [31]. The perioperative administration of physostigmine can reduce opioid consumption and peri-incisional mechanical allodynia [12]. Intrathecal neostigmine alone, or combined with clonidine, or opioids, has been successfully used for postoperative analgesic effects and pain relief [32], as it produces a longer effect with greater cardiovascular system reliability and fewer side effects. Epidural administration of neostigmine can prolong ropivacaine analgesia and reduce hourly ropivacaine consumption [33]. Intra-articular administration with a 500 μg dose of neostigmine is as effective as a postoperative analgesic and is not likely to significantly increase the adverse effects [11]. The sustained analgesic effects of neostigmine after surgery are also interpreted as a decrease in the activation of the descending pathway of pain-induced acetylcholine release [34]. In our results, the synergistic effects between pentazocine and neostigmine were antagonized by the muscarinic acetylcholine receptor antagonist atropine, indicating that the muscarinic acetylcholine receptor might also be an important pathway for pentazocine-neostigmine’s synergistic analgesic effect. The analgesic effect of intrathecal pentazocine and neostigmine may involve the pain descending inhibitory system. Pentazocine through the opioid and σ-receptor-independent pathway inhibits the norepinephrine transporter function and regulates the descending noradrenergic inhibitory system [35]. Previous works have identified that the spinal nicotinic acetylcholine receptors also affect pain regulation via the descending noradrenergic pathway [36]. We speculate that the descending inhibitory system might be another important pathway for the synergistic effect of pentazocine and neostigmine in formalin-induced pain and incision pain. Different doses of opioid receptor antagonists have different effects [37], and different types of opioid receptors show different effects in relieving thermal allodynia and mechanical pain [27]. This is the main research limitation of our work as we only used one antagonist dose and only two animal models.

We used isobolographic analysis to demonstrate the synergistic interaction between intrathecal pentazocine and neostigmine in both phases of the formalin test and the plantar incision model. There are several possibilities for this synergistic effect. Synergistic effect occurs when drugs have different effects at critical points along a common pathway [38]. The cholinesterase inhibitor modulates the transmission and processing of nociception according to the pre- and postsynaptic mechanisms, so simultaneous engagement of pre- and postsynaptic mechanisms may enhance the antinociception induced by either drug acting at one site independently [9, 16]. Moreover, two different receptors can simultaneously activate a common second messenger pathway in a single neuron and promote an effector mechanism [38, 39]. In this present study, the combined therapy of pentazocine and neostigmine produced a dose-dependent analgesic against formalin-induced pain and incisional mechanical allodynia. The combined use of pentazocine and neostigmine has a synergistic effect which may be related to the cholinergic system and the κ-opioid receptor at the spinal cord level.

Data Availability

All the original data can be obtained from the corresponding author of the article by e-mail (ouyhd@sysucc.org.cn).

Disclosure

Huiying Huang, Xiaohui Bai, Kun Zhang are the co-first authors.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

Huiying Huang, Xiaohui Bai, Kun Zhang contributed equally to this work. Shaoyong Wu and Handong Ouyang are co-corresponding authors. H. H., X. B., and H. O conceptualized the data. X. B., K. Z., S. W., and J. G. curated the data. H. O. did the supervision. H. H. and X. B. performed the data analysis. X. B. and H. O. wrote the original draft of the article.

Acknowledgments

The authors would also like to thank Mr. Christopher Lavender of Sun Yat-sen University Cancer Center's in-house Editorial Officer for the language editing of this article. This work was supported by the National Natural Science Foundation of China (82071237 and 81771192) and H. O.’s Clinical Medical Scientists Project of Sun Yat-sen University Cancer Center (PT09090101).

References

P. J. Fudala and R. E. Johnson, “Development of opioid formulations with limited diversion and abuse potential,” Drug and Alcohol Dependence, vol. 83, no. Suppl 1, pp. S40–S47, 2006.
View at: Publisher Site | Google Scholar
S. J. Mahapatra, S. Jain, S. Bopanna et al., “Pentazocine, a kappa-opioid agonist, is better than diclofenac for analgesia in acute pancreatitis: a randomized controlled trial,” American Journal of Gastroenterology, vol. 114, no. 5, pp. 813–821, 2019.
View at: Publisher Site | Google Scholar
T. Mori, J. Ohya, T. Itoh, Y. Ise, M. Shibasaki, and T. Suzuki, “Effects of (+)-pentazocine on the antinociceptive effects of (-)-pentazocine in mice,” Synapse, vol. 69, no. 3, pp. 166–171, 2015.
View at: Publisher Site | Google Scholar
J. L. Riley, B. A. Hastie, T. L. Glover, R. B. Fillingim, R. Staud, and C. M. Campbell, “Cognitive-affective and somatic side effects of morphine and pentazocine: side-effect profiles in healthy adults,” Pain Medicine, vol. 11, no. 2, pp. 195–206, 2010.
View at: Publisher Site | Google Scholar
M. Hirabayashi, K. Doi, N. Imamachi, T. Kishimoto, and Y. Saito, “Prophylactic pentazocine reduces the incidence of pruritus after cesarean delivery under spinal anesthesia with opioids: a prospective randomized clinical trial,” Anesthesia & Analgesia, vol. 124, no. 6, pp. 1930–1934, 2017.
View at: Publisher Site | Google Scholar
D. L. Robinson Jr., S. Nag, and S. S. Mokha, “Estrogen facilitates and the kappa and mu opioid receptors mediate antinociception produced by intrathecal (-)-pentazocine in female rats,” Behavioural Brain Research, vol. 312, pp. 163–168, 2016.
View at: Publisher Site | Google Scholar
N. Massaly, B. A. Copits, A. R. Wilson-Poe et al., “Pain-induced negative affect is mediated via recruitment of the nucleus accumbens kappa opioid system,” Neuron, vol. 102, no. 3, pp. 564–573 e6, 2019.
View at: Publisher Site | Google Scholar
V. Tiwari, S. Q. He, Q. Huang et al., “Activation of µ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents,” Pain, vol. 161, no. 4, pp. 842–855, 2020.
View at: Publisher Site | Google Scholar
P. V. Naser and R. Kuner, “Molecular, cellular and circuit basis of cholinergic modulation of pain,” Neuroscience, vol. 387, pp. 135–148, 2018.
View at: Publisher Site | Google Scholar
Z. Y. Wang, Q. Q. Han, M. Y. Deng et al., “Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors,” Biochemical and Biophysical Research Communications, vol. 525, no. 4, pp. 1087–1094, 2020.
View at: Publisher Site | Google Scholar
A. S. Habib and T. J. Gan, “Use of neostigmine in the management of acute postoperative pain and labour pain: a review,” CNS Drugs, vol. 20, no. 10, pp. 821–839, 2006.
View at: Publisher Site | Google Scholar
C. Klivinyi, G. Rumpold-Seitlinger, C. Dorn et al., “Perioperative use of physostigmine to reduce opioid consumption and peri-incisional hyperalgesia: a randomised controlled trial,” British Journal of Anaesthesia, vol. 126, no. 3, pp. 700–705, 2021.
View at: Publisher Site | Google Scholar
A. Sen, B. Erdivanli, Y. Tomak, and A. Pergel, “Reversal of neuromuscular blockade with sugammadex or neostigmine/atropine: effect on postoperative gastrointestinal motility,” Journal of Clinical Anesthesia, vol. 32, pp. 208–213, 2016.
View at: Publisher Site | Google Scholar
T. K. Oh, E. Ji, and H. S. Na, “The effect of neuromuscular reversal agent on postoperative pain after laparoscopic gastric cancer surgery: comparison between the neostigmine and sugammadex,” Medicine, vol. 98, no. 26, p. e16142, 2019.
View at: Publisher Site | Google Scholar
J. Eldufani and G. Blaise, “The role of acetylcholinesterase inhibitors such as neostigmine and rivastigmine on chronic pain and cognitive function in aging: a review of recent clinical applications,” Alzheimer's and Dementia: Translational Research & Clinical Interventions, vol. 5, no. 1, pp. 175–183, 2019.
View at: Publisher Site | Google Scholar
D. Radzicki, S. L. Pollema-Mays, A. Sanz-Clemente, and M. Martina, “Loss of M1 receptor dependent cholinergic excitation contributes to mPFC deactivation in neuropathic pain,” Journal of Neuroscience, vol. 37, no. 9, pp. 2292–2304, 2017.
View at: Publisher Site | Google Scholar
T. Yamamoto, O. Saito, K. Shono, and S. Tanabe, “Anti-hyperalgesic effects of intrathecally administered neuropeptide W-23, and neuropeptide B, in tests of inflammatory pain in rats,” Brain Research, vol. 1045, no. 1-2, pp. 97–106, 2005.
View at: Publisher Site | Google Scholar
K. A. Ahmad, R. M. Shoaib, M. Z. Ahsan et al., “Microglial IL-10 and beta-endorphin expression mediates gabapentinoids antineuropathic pain,” Brain, Behavior, and Immunity, vol. 95, pp. 344–361, 2021.
View at: Publisher Site | Google Scholar
H. Higuchi, S. Yamamoto, S. Ushio, T. Kawashiri, and N. Egashira, “Goshajinkigan reduces bortezomib-induced mechanical allodynia in rats: possible involvement of kappa opioid receptor,” Journal of Pharmacological Sciences, vol. 129, no. 3, pp. 196–199, 2015.
View at: Publisher Site | Google Scholar
I. J. Kim, C. H. Park, S. H. Lee, and M. H. Yoon, “The role of spinal adrenergic receptors on the antinociception of ginsenosides in a rat postoperative pain model,” Korean J Anesthesiol, vol. 65, no. 1, pp. 55–60, 2013.
View at: Publisher Site | Google Scholar
C. Mestre, T. Pelissier, J. Fialip, G. Wilcox, and A. Eschalier, “A method to perform direct transcutaneous intrathecal injection in rats,” Journal of Pharmacological and Toxicological Methods, vol. 32, no. 4, pp. 197–200, 1994.
View at: Publisher Site | Google Scholar
T. J. Brennan, E. P. Vandermeulen, and G. F. Gebhart, “Characterization of a rat model of incisional pain,” Pain, vol. 64, no. 3, pp. 493–502, 1996.
View at: Publisher Site | Google Scholar
S. Lolignier, N. Eijkelkamp, and J. N. Wood, “Mechanical allodynia,” Pfluegers Archiv European Journal of Physiology, vol. 467, no. 1, pp. 133–139, 2015.
View at: Publisher Site | Google Scholar
B. Nie, S. Zhang, Z. Huang et al., “Synergistic interaction between dexmedetomidine and ulinastatin against vincristine-induced neuropathic pain in rats,” The Journal of Pain, vol. 18, no. 11, pp. 1354–1364, 2017.
View at: Publisher Site | Google Scholar
H. Ouyang, P. Wang, W. Huang, Q. Li, B. Nie, and W. Zeng, “Spinal antinociceptive action of amiloride and its interaction with tizanidine in the rat formalin test,” Pain Research and Management, vol. 20, no. 6, pp. 321–326, 2015.
View at: Publisher Site | Google Scholar
M. Fischer, G. Carli, P. Raboisson, and P. Reeh, “The interphase of the formalin test,” Pain, vol. 155, no. 3, pp. 511–521, 2014.
View at: Publisher Site | Google Scholar
H. Shu, M. Hayashida, H. Arita et al., “Pentazocine-induced antinociception is mediated mainly by mu-opioid receptors and compromised by kappa-opioid receptors in mice,” Journal of Pharmacology and Experimental Therapeutics, vol. 338, no. 2, pp. 579–587, 2011.
View at: Publisher Site | Google Scholar
F. W. Foong and M. Satoh, “Neurotransmitter-blocking agents influence antinociceptive effects of carbamazepine, baclofen, pentazocine and morphine on bradykinin-induced trigeminal pain,” Neuropharmacology, vol. 23, no. 6, pp. 633–636, 1984.
View at: Publisher Site | Google Scholar
J. d. M. Turnes, E. I. Araya, A. R. Barroso et al., “Blockade of kappa opioid receptors reduces mechanical hyperalgesia and anxiety-like behavior in a rat model of trigeminal neuropathic pain,” Behavioural Brain Research, vol. 417, Article ID 113595, 2022.
View at: Publisher Site | Google Scholar
M. T. Calderwood, A. Tseng, and B. Glenn Stanley, “Lateral septum mu opioid receptors in stimulation of feeding,” Brain Research, vol. 1734, Article ID 146648, 2020.
View at: Publisher Site | Google Scholar
D. Dhanasobhon, M. C. Medrano, L. J. Becker et al., “Enhanced analgesic cholinergic tone in the spinal cord in a mouse model of neuropathic pain,” Neurobiology of Disease, vol. 155, Article ID 105363, 2021.
View at: Publisher Site | Google Scholar
N. Zhang and M. J. Xu, “Effects of epidural neostigmine and clonidine in labor analgesia: a systematic review and meta-analysis,” Journal of Obstetrics and Gynaecology Research, vol. 41, no. 2, pp. 214–221, 2015.
View at: Publisher Site | Google Scholar
M. Van de Velde, N. Berends, A. Kumar et al., “Effects of epidural clonidine and neostigmine following intrathecal labour analgesia: a randomised, double-blind, placebo-controlled trial,” International Journal of Obstetric Anesthesia, vol. 18, no. 3, pp. 207–214, 2009.
View at: Publisher Site | Google Scholar
H. R. Noori, S. Fliegel, I. Brand, and R. Spanagel, “The impact of acetylcholinesterase inhibitors on the extracellular acetylcholine concentrations in the adult rat brain: a meta-analysis,” Synapse, vol. 66, no. 10, pp. 893–901, 2012.
View at: Publisher Site | Google Scholar
G. Obara, Y. Toyohira, H. Inagaki et al., “Pentazocine inhibits norepinephrine transporter function by reducing its surface expression in bovine adrenal medullary cells,” Journal of Pharmacological Sciences, vol. 121, no. 2, pp. 138–147, 2013.
View at: Publisher Site | Google Scholar
D. Bagdas, G. Sevdar, Z. Gul et al., “(E)-3-furan-2-yl-N-phenylacrylamide (PAM-4) decreases nociception and emotional manifestations of neuropathic pain in mice by α7 nicotinic acetylcholine receptor potentiation,” Neurological Research, vol. 43, no. 12, pp. 1056–1068, 2021.
View at: Publisher Site | Google Scholar
C. Stein, “Opioid receptors,” Annual Review of Medicine, vol. 67, no. 1, pp. 433–451, 2016.
View at: Publisher Site | Google Scholar
A. Alcantara Montero, S. Balsalobre Gongora, D. M. Narganes Pineda, and B. Blanco Polanco, “Multimodal analgesia and pharmacological synergy in pain management,” SEMERGEN-Medicina de Familia, vol. 46, no. 4, pp. 284-285, 2020.
View at: Publisher Site | Google Scholar
N. P. Kazantzis, S. L. Casey, P. W. Seow, V. A. Mitchell, and C. W. Vaughan, “Opioid and cannabinoid synergy in a mouse neuropathic pain model,” British Journal of Pharmacology, vol. 173, no. 16, pp. 2521–2531, 2016.
View at: Publisher Site | Google Scholar

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