Effects of Mirogabalin on Hyperalgesia and Chronic Ocular Pain in Tear-Deficient Dry-Eye Rats

Abstract

Purpose: Patients with dry eye disease (DED) sometimes complain of ocular pain. DED-related ocular pain has many similarities with neuropathic pain. Mirogabalin, a novel ligand for the α₂δ subunit of voltage-gated calcium channels, is approved for treating neuropathic pain in Japan. This study aimed to investigate the effect of mirogabalin on hyperalgesia and chronic ocular pain in a rat DED model.

Methods: DED was induced in female Sprague Dawley rats by unilaterally excising the external lacrimal gland (ELG) and Harderian gland (HG). After 4 weeks of ELG and HG removal, tear production (pH threads) and corneal epithelial damage (fluorescein staining) were evaluated. Corneal hyperalgesia and chronic pain were analyzed, respectively, by measuring capsaicin-induced eye-wiping behavior and c-Fos expression in the trigeminal nucleus. Mirogabalin (10 or 3 mg/kg) was evaluated for effects on DED-induced hyperalgesia and chronic ocular pain.

Results: Tear production was significantly lower in DED-induced eyes than in control eyes. Corneal damage was significantly higher in DED eyes than in control eyes. Hyperalgesia and chronic ocular pain were detected 4 weeks after ELG and HG removal. Five days of mirogabalin administration significantly suppressed capsaicin-induced eye-wiping behavior, which indicated the suppression of ocular hyperalgesia. Administration of 10 mg/kg mirogabalin significantly reduced c-Fos expression in the trigeminal nucleus, which indicated the amelioration of chronic ocular pain.

Conclusions: Mirogabalin suppressed DED-induced hyperalgesia and chronic ocular pain in a rat DED model. Our findings suggested that mirogabalin might effectively alleviate chronic ocular pain in patients with DED.

Dry eye disease (DED) encompasses a variety of clinical manifestations, including ocular surface disorders, such as dryness, discomfort, pain, reduced tear secretion, and increased tear fluid evaporation.¹ DED is a very common condition; recent reports have indicated that approximately 11% of the world's population shows some signs of DED.² Some patients with DED have few signs on the ocular surface, but severe subjective symptoms, including a burning sensation, pain, hyperalgesia, and allodynia.^3,4 Our understanding of DED is growing, and, currently, it is thought that patients diagnosed with DED experience features of neuropathic pain.⁵ 

Pregabalin and gabapentin are recommended as first-line treatments for neuropathic pain.^6,7 Pregabalin and gabapentin inhibit the release of excitatory neurotransmitters by binding to the α2δ subunit of voltage-gated calcium channels in the central nervous system. It has been reported that gabapentin is effective on DED-related ocular pain.⁸ Moreover, pregabalin suppressed hypersensitivity and hyperalgesia in a rat model of DED.⁹ Mirogabalin is a novel gabapentinoid ligand for the α2δ subunit of voltage-gated calcium channels,¹⁰ which is administered orally.^11,12 In 2019, mirogabalin was approved for peripheral neuropathic pain, such as diabetic peripheral neuropathy and postherpetic neuralgia, in Japan. On the other hand, to our knowledge, no study has tested mirogabalin for ocular pain, including DED. 

The present study aimed to evaluate the efficacy of mirogabalin in a tear-deficient DED rat model. This model was created by removing the external lacrimal gland (ELG) and the Harderian gland (HG). 

Materials and Methods

Animals

Eight-week-old female Sprague Dawley (SD) rats were purchased from CLEA Japan, Inc. (Tokyo, Japan). The rodents were housed in the animal facility at Hokkaido University. Standard feed and water were provided ad libitum. All animal experiments were conducted according to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Additionally, the study was approved by the Ethics Review Committee for Animal Experimentation of Hokkaido University (#21-0058). 

Surgical Procedures

Surgery was conducted on the right side, where the ELG and HG were excised. Briefly, rats were anesthetized with intraperitoneal (IP) injections of ketamine (90 mg/kg) and xylazine (10 mg/kg). Prior to surgery, a drop of lacrimal gel (Scopisol solution for eyes; Senju Pharmaceutical Co., Ltd., Osaka, Japan) was administered to both eyes to protect the corneas from drying out. The right temporal skin was incised and the ELG was exposed and removed. Then, conjunctival tissue near the external eye angle was dissected, and the HG was removed. The skin incision was sutured with 5-0 nylon sutures (Blue nylon 5-0, Akiyama medical MFG., Co., Ltd., Tokyo, Japan) and antibiotic ointment (Tarivid ophthalmic ointment; Santen Pharmaceutical Co., Ltd., Osaka, Japan) was applied to prevent infection. Untreated rats were used as controls because the removal of the main lacrimal gland on one side may affect the contralateral side.¹³ 

Phenotype Analysis in the Dry-Eye Rat Model

Tear Production

Four weeks after ELG and HG removal, tear fluid volume was measured (DED n = 11 and control n = 11) with a cotton thread treated with phenol red (Zone-Quick, Tokyo, Japan). The top of the thread was placed on the lateral canthus of the eye for 30 seconds to allow the tear fluid to travel up the thread (by capillary action). The pH of the tear fluid changed the thread color, due to the phenol red. After 30 seconds, a ruler was used to measure the distance that tear fluids traveled up the thread. 

Fluorescein Staining

Corneal fluorescein staining was performed to assess the degree of corneal damage at 4 weeks after ELG and HG excision (DED n = 5 and control n = 11). After anesthetizing rats with ketamine and xylazine, Flores 0.7 mg ophthalmic test paper (AYUMI Pharmaceutical Corporation, Tokyo, Japan) was applied to the conjunctival sac. Then, the surface of the eye was examined with a slit-lamp and cobalt blue light. The degree of fluorescein staining was evaluated in the upper, middle, and lower trichome segments of the cornea. The results were rated on a 3-point scale (0 = no damage, 1 = partially damaged, 2 = more than half damaged, and 3 = totally damaged). The scores were summed to obtain the total score for each eye. 

Eye Wiping Behavior

To examine whether the ELG and HG excisions induced hyperalgesia in the cornea, capsaicin-induced eye wiping behavior was measured at 4 weeks after ELG and HG excision. A 10 mM capsaicin ethanol solution was prepared by dissolving 30.5 mg capsaicin in 90% ethanol. At the time of use, the solution was diluted with distilled water to obtain a 100 µM capsaicin solution. Rats were evaluated by placing 30 µL capsaicin solution into the eye, and counting the number of times eye wiping occurred within 2 minutes. 

Drug Administration

Mirogabalin besylate was provided from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Four weeks after ELG and HG removal, the rats received oral administrations of 10 mg/kg (n = 11) or 3 mg/kg (n = 10) of free-form mirogabalin through a stainless-steel tube under isoflurane anesthesia. Oral mirogabalin was administered for 5 consecutive days because when mirogabalin is administered to a healthy adult, mirogabalin reached the steady state by day 3.¹⁴ The vehicle group (n = 11) received the same volume of deionized water. 

Immunohistology

Tissue Preparation

To investigate whether chronic pain was induced in the dry eye model, we performed immunostaining to evaluate c-Fos expression, a marker of neuronal activity, in the trigeminal nucleus. Briefly, rats received a ketamine/xylazine mixture via an IP injection. Then, an incision was made through the skin and abdominal wall under the rib cage. The diaphragm was then incised to expose the pleural cavity. To avoid damaging the lungs, scissors were used to make an incision on one side of the ribs up to the clavicle. A similar incision was made on the contralateral side. Then, the sternum was lifted above the head and secured. A small incision was made at the posterior end of the left ventricle, and a plastic tube, connected to a syringe filled with 4% paraformaldehyde (PFA), was inserted. The right atrium was incised and manually refluxed with PFA. 

After perfusing, the brain was carefully removed and immersed in 4% PFA for at least 24 hours. Then, it was immersed in 30% sucrose in 1× phosphate buffered saline (PBS) until the brain sank. Next, the brain was embedded in a plastic mold (Tissue-Tek Cryomold; Sakura Finetek Japan Co., Ltd., Tokyo, Japan) with Optimal Cutting Temperature Compound (Sakura Finetek Japan Co., Ltd.), and the specimen block was frozen on dry ice. The specimen blocks were stored at −80°C. Frozen tissues were cut into sections (15–20 µm) with a Leica Microsystems cryostat. The sections were collected in 6-well inserts filled with 1× PBS. 

Immunohistochemistry

Immunofluorescence was performed with the free-floating technique. In short, prior to immunofluorescence, the tissue sections were washed 3 times in 1× PBS. Next, the sections were transferred into a 10-mL tube filled with10% goat serum (Thermo Fisher Scientific) and incubated for 30 minutes. The sections were then incubated at 4°C overnight with a primary rabbit monoclonal antibody that targeted phospho-c-Fos (c-Fos; 1:500, #5348; Cell Signaling Technology, Danvers, MA, USA). For fluorescence detection, the secondary antibody was Alexa Fluor 540 goat anti-rabbit IgG (1:500; Thermo Fisher Scientific). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the specimens were viewed with a fluorescence microscope (BZ-9000, Keyence). Cells located in the trigeminal nucleus that were stained with c-Fos and DAPI were counted to evaluate neuronal activity. 

Statistical Analysis

The Student's t-test and paired t-test were performed for statistical comparisons between groups. The one-way analysis of variance (ANOVA) and Tukey's HSD test were used to compare the means of three independent data sets. All results are expressed as the mean ± standard error and the number (n), as indicated. Differences between means were considered statistically significant when P values were < 0.05. 

Results

Effects of ELG and HG Excision on Tear Production and Corneal Surface

We initially examined how the ELG and HG excisions affected tear production. Four weeks after ELG and HG removal, tear fluid volume declined in DE eyes (5.5 ± 0.5 mm, n = 11), compared to control eyes (16.0 ± 1.4 mm, n = 11, P < 0.01; Fig. 1A). Fluorescein scores were significantly higher in DE eyes (2.2 ± 0.6, n = 5) than in control eyes (1.0 ± 0.2, n = 11, P < 0.05; Fig. 1B). 

Figure 1.

 

Tear production and corneal damage after ELG and HG excision. Dry eye disease (DED) was induced in the right eyes of rats by excising the external lacrimal gland (ELG) and Harderian gland (HG). The right eye of untreated rats was used as a control. (A) Tear fluid volume was significantly lower in DE eyes than in control eyes; n = 11 each; P < 0.01. (B) Fluorescein scores were significantly higher in DE eyes (n = 5) compared to control eyes (n = 11); P < 0.05.

ELG and HG Excision Induced Corneal Hyperalgesia

We evaluated corneal hyperalgesia with eye-wiping behavior induced by administering eye drops that contained capsaicin, an agonist of the transient receptor potential vanilloid 1 (TRPV1). Capsaicin administration induced eye wiping behavior significantly more frequently in DE eyes (38.9 ± 2.6 times/2 minutes, n = 11) than in control eyes (13.7 ± 2.6 times/2 minutes, n = 11, P < 0.01; Fig. 2A). This result suggested that ELG and HG excision induced corneal hyperalgesia. 

Figure 2.

 

Eye wiping behavior induced by capsaicin eye drops. (A) DE eyes were wiped significantly more frequently than control eyes; n = 11 each; **P < 0.01. (B) Administration of 10 mg/kg mirogabalin significantly reduced the number of eye wipes within 2 minutes in DE eyes compared to untreated DE eyes; n = 11 each; *P < 0.05. (C) Eye wiping behavior was also ameliorated with administration of 3 mg/kg mirogabalin; n = 10 each; *P < 0.05. (D) Vehicle administration did not significantly reduce the number of eye wipes in DE eyes; n = 11 each.

Mirogabalin Attenuated Hyperalgesia in the Cornea

We next examined whether corneal hyperalgesia could be attenuated with mirogabalin. The animals received either 10 mg/kg or 3 mg/kg mirogabalin for 5 days. At 3 hours after the last administration, we measured the frequency of eye-wiping behavior induced by capsaicin eye drops. Administration of 10 mg/kg mirogabalin reduced the number of eye wipes in DE eyes from 38.9 ± 2.6 (n = 11) to 31.0 ± 2.8 times/2 minutes (n = 11, P < 0.05; Fig. 2B). Corneal hyperalgesia was also alleviated with 3 mg/kg mirogabalin. Administration of 3 mg/kg mirogabalin significantly reduced eye-wiping behavior in dry eyes from 36.0 ± 4.0 (n = 10) to 25.4 ± 1.9 times/2 minutes (n = 10, P < 0.05; Fig. 2C). On the other hand, administration of vehicle did not suppress hyperalgesia in dry eyes (DED group = 39.0 ± 1.8 times/2 minutes, n = 11; DED + vehicle group = 37.0 ± 2.8 times/2 minutes, n = 11; Fig. 2D). 

Mirogabalin Reduced the Number of c-Fos-Positive Cells in the Trigeminal Nucleus After ELG and HG Removal

To investigate the induction of chronic corneal pain after removing the ELG and HG and the effects of mirogabalin, we counted the number of c-Fos-positive cells in the trigeminal nucleus (Fig. 3A). We evaluated four rats each from the control, the DE eye, and the mirogabalin-treated DE eye groups. The c-FOS-positive cell counts were averaged by evaluating several sections per rat. We found that the number of c-Fos-positive cells in the trigeminal nucleus was significantly higher in DE eyes (21.0 ± 3.3), compared to control eyes (10.9 ± 0.9, P < 0.05). The number of c-Fos-positive cells was also significantly reduced with the administration of 10 mg/kg mirogabalin (12.1 ± 1.6, P < 0.05; Fig. 3B). 

Figure 3.

 

The c-Fos-positive cells in the trigeminal nucleus. (A) Representative fluorescence photograph of a trigeminal nucleus section immunostained with anti-c-Fos antibody (red) and DAPI nuclear stain (blue). (B) Comparison of control eyes to DE eyes, before and after treatment with mirogabalin (10 mg/kg). The number of c-Fos-positive cells significantly increased in the trigeminal nuclei of DE eyes. Mirogabalin significantly reduced the number of c-Fos-positive cells in the trigeminal nuclei of DE eyes; n = 4 each. **P < 0.05 (Tukey's HSD test).

Discussion

In this study, we showed that hyperalgesia and chronic pain were induced at 4 weeks after removing the ELG and HG. We also found that hyperalgesia and chronic pain were suppressed by the oral administration of mirogabalin, based on the capsaicin-induced eye-wiping frequency and the c-Fos activity in the trigeminal nuclei of the medulla oblongata. 

Rats have two lacrimal fluid-producing tissues, the external and internal lacrimal glands. The HG is a lipid-secreting gland that occupies most of the orbit.¹⁵ The HG is well developed in rodents, such as rats and mice, and lagomorphs, such as rabbits. However, the HG was lost in primates during evolution. The functions of the lipids secreted by the HG in rodents are to lubricate and protect the cornea.¹⁶ Patients with DED have abnormal or impaired lipid secretion, based on lipid analyses in tear fluids.¹⁷ Thus, our DED rats may well mimic patients with DED in terms of reduced tear volume and abnormal lipid composition. 

Mirogabalin has shown good analgesic efficacy and tolerability in Asian patients with postherpetic neuralgia or diabetic peripheral neuropathic pain.^11,12 In addition, mirogabalin was reported to attenuate pain-related behaviors in a mouse model of post-traumatic trigeminal neuropathy.¹⁸ Accordingly, we hypothesized that neuropathic pain would occur in a rat DED model after excising the ELG and HG, and that mirogabalin might suppress the ocular symptoms. 

Patients with DED have been reported to show corneal hyperalgesia,^19,20 a characteristic symptom of neuropathic pain.²¹ It was recently suggested that patients with DED present with neuropathic ocular pain.^22,23 TRPV1, a representative pain receptor, is a polymodal neuronal ion channel receptor that is activated by multiple stimuli, including capsaicin, heat above 43°C, and acidic conditions.^24,25 Therefore, we used capsaicin eye drops to activate TRPV1, and the frequency of eye-wiping behavior was taken as an indicator of hyperalgesia. Our experiments showed that corneal hyperalgesia was induced in rats after the ELG and HG excisions. Moreover, corneal hyperalgesia was significantly suppressed with 5 days of oral mirogabalin (at both 3 mg/kg and 10 mg/kg). Therefore, we concluded that increased eye wiping behavior (or hyperalgesia), was a symptom of dry eye-induced neuropathic ocular pain, and it could be suppressed with mirogabalin, a drug for treating neuropathic pain. 

Pain sensed in the cornea is transmitted to the nasal subnucleus of the trigeminal spinal tract nucleus (Vi/Vc), and then, to the thalamus and primary somatosensory cortex.²⁴ In the present study, c-Fos expression in the Vi/Vc was significantly increased in ELG- and HG-excised rats. Because c-Fos positive cells in the somatic sensory neurons suggest persistent painful input, DE rats are presumed to have chronic eye pain. This chronic ocular pain was significantly suppressed by treating them with mirogabalin. These findings suggested that mirogabalin may be a new treatment option for patients with chronic ocular pain. 

Ocular pain in patients with DED is often intractable and does not improve with artificial tears alone.²⁶ The efficacy of gabapentin and pregabalin has been reported for ocular pain.^8,9 Mirogabalin has a higher affinity for the α2δ-1 subunit of the dorsal root ganglion and is slower to dissociate, resulting in a more sustained analgesic effect. These gabapentinoids also bind to the α2δ-2 subunit of the cerebellum and thus have central nervous system-specific adverse drug reactions, such as dizziness and somnolence.^27,28 In contrast, mirogabalin has a lower affinity for the α2δ-2 subunit of the cerebellum and dissociates more rapidly, thus causing less of this side effect.²⁹ Therefore, mirogabalin may be a safety and useful treatment option for ocular pain in patients with DED. 

Conclusion

Treatment with mirogabalin attenuated hyperalgesia and chronic pain induced in rats with ELG and HG resections. Our findings suggested that mirogabalin could be effective for treating corneal hyperalgesia and chronic ocular pain associated with DED. 

Acknowledgments

The authors thank Ikuyo Hirose and Shiho Yoshida (Hokkaido University) for their skillful technical assistance. 

This research was conducted with support from a research fund from Daiichi Sankyo Co., Ltd. 

Declaration of Interests: Yoshiaki Tagawa received a research grant from Daiichi Sankyo Co., Ltd. 

Disclosure: K. Kikuchi, None; Y. Tagawa, None; M. Murata, None; S. Ishida, None