Introduction

Neuropathic pain is defined as ‘pain caused by a lesion or disease of the somatosensory nervous system’. It is a complicated syndrome underlain by multiple mechanisms, and the current therapies for it are not completely satisfactory (Jensen et al. 2011; Colloca et al. 2017). Some neuropathic pain patients suffer from various comorbid symptoms, such as sleep disturbances and psychiatric disorders (e.g. anxiety and depression). Both chronic pain and these comorbid symptoms lead to a vicious cycle that seriously affects the quality of life (Gustorff et al. 2008; Attal et al. 2011; Radat et al. 2013; Schaefer et al. 2014; Failde et al. 2018). For the management of neuropathic pain, several sets of international and regional guidelines recommend gabapentin and pregabalin as the first-line therapy (O’Connor and Dworkin 2009; Attal et al. 2010; Cruccu and Truini 2017). Gabapentin and pregabalin are selective ligands for the α2δ subunit of voltage-gated calcium channels (Gee et al. 1996; Li et al. 2011) and exert various pharmacological effects, including anticonvulsant, anxiolytic and antidepressant activities, as well as analgesia (Dooley et al. 2007; Stahl et al. 2013).

Mirogabalin {[(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid} is a newly synthesised potent and selective α2δ ligand. It has been licenced for the treatment of peripheral neuropathic pain including painful diabetic peripheral neuropathy and postherpetic neuralgia in Japan (Deeks 2019). We previously reported that mirogabalin has unique binding characteristics to α2δ subunits (Domon et al. 2018a) and shows potent and long-lasting analgesic effects in neuropathic pain models (Domon et al. 2018a, b) and fibromyalgia models (Saeki et al. 2019). Here, to provide further information on the pharmacological profile of mirogabalin and its utility for chronic pain therapy, we investigated its anxiolytic effects in a rat model of chronic constriction injury (CCI) as an experimental animal model for neuropathic pain.

Materials and methods

Animals

Male Crl:CD(SD) rats at 6 weeks of age were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). The animals were housed under standard laboratory conditions of temperature (21–27 °C), relative humidity (35–70%) and a 12-h light/dark cycle (lights on 06:00–18:00 h), with a commercial diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water available ad libitum. The animals were randomly allocated to the study groups using a computed randomisation procedure (IBUKI; Nihon Bioresearch Inc., Gifu, Japan) based on pain response and body weight.

All experiments were performed in accordance with the Guidelines for the Management and Welfare of Experimental Animals (Hashima Laboratory, Nihon Bioresearch Inc.; April 2, 2007, modified on August 27, 2010), and the Guidelines of the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd.

Chemicals

Mirogabalin besylate (code number DS-5565) was synthesised by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). The drug was dissolved in distilled water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) and administered orally at a volume of 10 mL/kg. Control groups received an equal amount of distilled water. The dose levels (expressed as free form) and pretreatment time of mirogabalin were determined based on our previous reports (Domon et al. 2018a, b; Saeki et al. 2019) and preliminary study (Supplementary material). The chemical structure of mirogabalin besylate is shown in Fig. 1.

Fig. 1
figure 1

Chemical structure of mirogabalin besylate (code number DS-5565)

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Preparation of CCI model

The CCI model was prepared in accordance with a previous report (Bennett and Xie 1988) with minor modifications. Under isoflurane anaesthesia (Isoflurane Inhalation Solution [Pfizer]; Pfizer Japan Inc., Tokyo, Japan), the left femoral skin was incised and the sciatic nerve was exposed. The sciatic nerve was loosely ligated at mid-thigh level with two surgical 4–0 silk threads at an interval of 1 mm. The surgical area was sutured with surgical threads and the animals received subcutaneous injection of ampicillin sodium (50 mg/0.25 mL/body, Viccillin® 1 g for injection; Meiji Seika Pharma Co., Ltd., Tokyo, Japan).

Assessment of pain response (von Frey test)

The plantar surface of the left hind paw was stimulated with von Frey filaments (bending force: 0.4, 0.6, 1, 2, 4, 6, 8 and 15 g; North Coast Medical Inc., Gilroy, CA, USA), and the 50% paw withdrawal threshold was determined by the up-down method (Dixon 1980; Chaplan et al. 1994). The von Frey test was conducted before and 7, 14 and 21 days after model preparation. CCI model rats with 50% paw withdrawal threshold of 6 g or lower on Day 21 were selected and assigned to the study groups. The actual success rate of CCI model was 90%.

Assessment of anxiety-related behaviour (elevated plus maze test)

The elevated plus maze test was conducted using a maze elevated 60 cm above the floor level, composed of two open arms and two closed ones (42 cm in length, 15 cm in width, wall height 30 cm) that crossed in the middle perpendicularly, and a central platform. The rats were placed individually in the central platform facing an open arm and allowed to explore for 5 min. The behaviour was analysed with a video tracking system (SMART; Panlab Inc., Holliston, MA, USA) and the following parameters were measured: (1) time spent in each arm and platform, (2) number of entries into each arm and platform, and (3) distance travelled in each arm and platform.

Assessment of depression-related behaviour (forced swimming test)

The forced swimming test, originally reported by Porsolt et al. (1977), was conducted using an automated analysis system (Shimamura et al. 2007) with minor modifications. Briefly, a small magnet (3 mm in width, 2 mm in depth, 3 mm in height) was fixed on each forelimb using surgical tape. Then, the rats were placed individually in a vertical acrylic cylinder (20 cm in diameter, 50 cm in height) containing water up to 25 cm at 24±2 °C. The duration of immobility was detected and analysed using an automated behaviour analytical system (MicroAct™; Neuroscience, Inc., Tokyo, Japan). Visual observation using video recordings was also conducted as a supplement. There was no distinction between the automated analysis and supplemental visual observation. The forced swimming test was conducted on two consecutive days (first trial for 15 min and second trial for 5 min).

Experimental schedule

This study consisted of a series of two experiments, namely, confirmation of anxiety- and depression-related behaviours (experiment 1), followed by the evaluation of drug effects (experiment 2). In experiment 1, the elevated plus maze test was conducted at 4 or 5 weeks after model preparation, and the forced swimming test was conducted at 6 or 8 weeks after model preparation. Based on the results of experiment 1, experiment 2 focused on the effects of mirogabalin on the anxiety-related behaviours observed at 5 weeks after model preparation. The drug was administered 2 h before undergoing the elevated plus maze test at 5 weeks after model preparation. The von Frey test was conducted after the elevated plus maze test (i.e., 4 h after drug administration). The study design and experimental schedule are summarised in Fig. 2.

Fig. 2
figure 2

Study design and experimental schedule for experiment 1 (top) and experiment 2 (bottom)

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Statistical analysis

All data are expressed as the mean ± standard error. Statistical analysis was performed using the F-test followed by Student’s or Aspin–Welch’s t test for two-group comparisons, and Bartlett’s test followed by Dunnett’s or Steel’s test for multiple-group comparisons. In the von Frey test, Wilcoxon’s rank sum test was used for two-group comparisons. Differences were considered significant when p<0.05. The SAS System (SAS Software Release 9.1.3; SAS Institute Japan Ltd., Tokyo, Japan) was used for these analyses.

Results

Experiment 1

In the von Frey test, CCI model rats showed sustained decreases in the 50% paw withdrawal threshold compared with sham rats (Fig. 3). In the elevated plus maze test, there were no statistically significant differences in any anxiety parameters between CCI model rats and sham rats at 4 weeks after model preparation (Fig. 4, top, middle and bottom). At 5 weeks after model preparation, CCI model rats exhibited significant decreases in time spent in the open arms and platform and significant increases in time spent in the closed arms (Fig. 4, top). The number of entries into the open arms significantly decreased in CCI model rats (Fig. 4, middle). The distance travelled in the open arms and platform significantly decreased in CCI model rats (Fig. 4, bottom).

Fig. 3
figure 3

Changes in 50% paw withdrawal thresholds in CCI model rats. Each value represents the mean ± standard error (n=10). **p<0.01: Significantly different from the sham control group with Wilcoxon’s rank sum test

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Fig. 4
figure 4

Anxiety parameters in elevated plus maze test in CCI model rats. Top: time spent in platform, open arms and closed arms. Middle: numbers of entries into platform, open arms and closed arms. Bottom: distance travelled in platform, open arms and closed arms. Each value represents the mean ± standard error (n=10). *p<0.05, **p<0.01: Significantly different from the sham control group with Student’s t test or the Aspin–Welch t test.

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In the forced swimming test, there were no statistically significant differences in the immobility time between CCI model rats and sham rats at 6 or 8 weeks after model preparation (Fig. 5).

Fig. 5
figure 5

Immobility times in forced swimming test in CCI model rats. Left: first trial for 15 min. Right: second trial for 5 min. Each value represents the mean ± standard error (n=10). *p<0.05: No significant differences are observed between the sham control group and the CCI group by Student’s t test

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Experiment 2

As in experiment 1, CCI model rats showed sustained decreases in the 50% paw withdrawal threshold (data not shown). At 5 weeks after model preparation, a single oral administration of mirogabalin (3 or 10 mg/kg) dose-dependently increased the 50% paw withdrawal threshold and the effect of 10 mg/kg was statistically significant upon comparison with the CCI control group (Fig. 6).

Fig. 6
figure 6

Analgesic effects of mirogabalin in CCI model rats. Mirogabalin besylate was administered orally (10 mL/kg), while the control group received distilled water. The dose levels are expressed as the levels of the free form of the drug. Each value represents the mean ± standard error (n=10). **p<0.01: Significantly different from the sham control group with Wilcoxon’s rank sum test. #p<0.05: Significantly different from the CCI control group with Steel’s test.

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In the elevated plus maze test, anxiety-related behaviours were confirmed in a similar fashion to that in experiment 1. Namely, CCI model rats exhibited significant decreases in time spent in the open arms, significant increases in time spent in the closed arms (Fig. 7, top), significant decreases in the number of entries into each arm and distance travelled in each arm (Fig. 7, middle and bottom). The decreased number of entries into each arm reflected that CCI model rats stayed in the closed arms for a long time and rarely moved to the other areas. A single oral administration of mirogabalin (3 or 10 mg/kg) alleviated these anxiety-related behaviours in a dose-dependent manner (Fig. 7, top, middle and bottom). In particular, mirogabalin at 10 mg/kg significantly recovered the decreased time spent in the open arms and the increased time spent in the closed arms, compared with those in the CCI control group (Fig. 7, top). Mirogabalin also recovered the decreased number of entries into each arm and the decreased distance travelled in each arm (Fig. 7, middle and bottom).

Fig. 7
figure 7

Anxiolytic effects of mirogabalin in elevated plus maze test in CCI model rats. Top: time spent in platform, open arms and closed arms. Middle: numbers of entries into platform, open arms and closed arms. Bottom: distance travelled in platform, open arms and closed arms. Mirogabalin besylate was administered orally (10 mL/kg), while the control group received distilled water. The dose levels are expressed as the levels of the free form of the drug. Each value represents the mean ± standard error (n=10). *p<0.05, **p<0.01: Significantly different from the sham control group with Student’s t test. #p<0.05, ##p<0.01: Significantly different from the CCI control group with Dunnett’s test

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Discussion

The present study indicated neuropathic pain-induced anxiety- or depression-related behaviours in a time-dependent manner. Indeed, CCI model rats developed sustained tactile allodynia within 2 weeks after model preparation, whereas anxiety-related behaviours were observed in the elevated plus maze test at 5 weeks but not at 4 weeks after model preparation. Furthermore, a prolonged immobility time as one of the depression-related behaviours was not observed in the forced swimming test even 6 or 8 weeks after model preparation. Similar phenomena have been reported in sciatic nerve cuffing model mice (Benbouzid et al. 2008) and partial sciatic nerve ligation model rats (Wang et al. 2015), in which anxiety-related behaviours but not depression-related behaviours developed under conditions of chronic neuropathic pain. On the other hand, it has been reported that depression-related behaviours tend to develop later than anxiety-related behaviours in sciatic nerve cuffing model mice (Yalcin et al. 2011). Therefore, it remains possible that depression-related behaviours might be observed more than 8 weeks after model preparation under our experimental conditions. In addition to the above findings, some studies have demonstrated the time-dependent development of anxiety- or depression-related behaviours in neuropathic pain models (Narita et al. 2006; Suzuki et al. 2007; Sawada et al. 2014; Yalcin et al. 2014; Alba-Delgado et al. 2016) and chronic inflammatory pain models (Narita et al. 2006; Wu et al. 2017), indicating that time might be one of the critical points in chronic pain-induced psychiatric disorders.

In the pharmacological evaluation, mirogabalin alleviated tactile allodynia in CCI rats as in the other experimental neuropathic pain models, such as streptozotocin-induced diabetes, partial sciatic nerve ligation and spinal cord injury rats (Domon et al. 2018a, b). Furthermore, mirogabalin alleviated anxiety-related behaviours in CCI rats in the elevated plus maze test. In addition, an interesting finding in this study was the decreased locomotor activity in CCI rats (i.e., decreases in the number of entries into each arm and distance travelled in each arm) and the effect of mirogabalin on that. In spite of sustained neuropathic pain state from 2 weeks to 5 weeks after model preparation, the decreased locomotor activity was observed at week 5 but not at week 4. Furthermore, the decreased locomotor activity was only observed in the open arms and platform but not in the closed arms even at week 5 in experiment 1. These results suggest that the decreased locomotor activity in CCI rats in the elevated plus maze test is one of anxiety-related behaviours, not pain-related behaviours, and that the recovery effect of mirogabalin on decreased locomotor activity depends on its anxiolytic activity rather than analgesic activity.

The standard α2δ ligands, gabapentin and pregabalin, have been reported to have anxiolytic effects in normal animals (Field et al. 2001; Belliotti et al. 2005; Lotarski et al. 2011) and experimental neuropathic pain models, such as CCI (Roeska et al. 2008; Grégoire et al. 2012; Harte et al. 2016), spinal nerve transection (Hasnie et al. 2007), partial sciatic nerve ligation (La Porta et al. 2016), spinal cord injury (Baastrup et al. 2011) and anti-retroviral-associated neuropathy (Wallace et al. 2008). In fact, pregabalin has been licenced and used for the treatment of generalised anxiety disorder in the EU (Montgomery 2006; Frampton 2014). In our preliminary study (Supplementary material), mirogabalin showed an anxiolytic effect in normal rats in the elevated plus maze test, and the effect of mirogabalin 10 mg/kg was comparable to that of diazepam 3 mg/kg. Interestingly, the anxiolytic effect of mirogabalin was relatively short-lasting compared with its sustained analgesic effects. The anxiolytic effect of mirogabalin 10 mg/kg peaked at 2 h after administration and returned to the control level after 4 h (Supplementary Fig. 1, top). By contrast, the analgesic effects of mirogabalin peaked at 4 h after administration and remained until 8 h or more after administration (Domon et al. 2018a, b). Therefore, time-course changes of the anxiolytic effects of mirogabalin were different from those of the analgesic effects. Similar findings were obtained for pregabalin. The analgesic effects of pregabalin peaked at 4 h after administration (Domon et al. 2018a, b), whereas the maximum anxiolytic effect of pregabalin 30 mg/kg was observed 1 h after administration and diminished after 2 h (Supplementary Fig. 1, bottom). Taken together, the discrepancy in time-course changes between anxiolytic and analgesic effects suggests that the anxiolytic effects of α2δ ligands are not dependent on their analgesic effects. It is not clear yet whether the anxiolytic effects of mirogabalin in CCI model rats depend on the primary anxiolytic activity or secondary effects via the analgesic activity. However, at least in part, the primary anxiolytic activity of mirogabalin is considered to contribute to the alleviation of anxiety-related behaviours in CCI rats. Anxiety disorders are highly prevalent in neuropathic pain patients and negatively influence the quality of life (Gustorff et al. 2008; Attal et al. 2011; Radat et al. 2013; Schaefer et al. 2014; Failde et al. 2018). The analgesics with anxiolytic effects might offer a significant benefit for neuropathic pain therapy.

In conclusion, CCI model rats showed anxiety-related behaviours in a time-dependent manner in the elevated plus maze test. Mirogabalin alleviated both the anxiety-related behaviours and tactile allodynia in CCI model rats. Mirogabalin may thus provide effective anxiety relief as well as pain relief in patients with neuropathic pain.