Rilmenidine protects against joint damage in MIA-induced model of osteoarthritis in rats

doi:10.21203/rs.3.rs-2249355/v1

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Rilmenidine protects against joint damage in MIA-induced model of osteoarthritis in rats

https://doi.org/10.21203/rs.3.rs-2249355/v1

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Rilmenidine protects against joint damage in MIA-induced model of osteoarthritis in rats

Purpose: Osteoarthritis is a common problem, and its incidence significantly increases with age. Patients suffer from excruciating pain while moving, and currently, major treatment options consist of surgery. Rilmenidine is a potent antihypertensive agent with a high affinity for imidazoline and alpha₂ adrenergic receptors. Based on the knowledge that these receptors are also related to bone turnover and pain, we aimed to reveal the effect of rilmenidine on the osteoarthritis model in rats.

Methods: Monosodium iodoacetate(MIA) was used to induce osteoarthritis. Animals were treated with rilmenidine(0.5, 2 mg/kg) for 14 days. Hot plate test was performed to assess pain response before and end of the drug treatments, in addition to the walking track analysis. Twenty-four hours after the last drug treatment, serum levels of receptor activator of nuclear factor kappa-Β ligand(RANKL) and osteoprotegerin(OPG) were measured. Hematoxylin&eosin and safranin-O staining were used to evaluate MIA and rilmenidine induced changes in the hindlimb joints.

Results: Our results demonstrated that rilmenidine(2 mg/kg) prevented MIA-induced thermal hyperalgesia with improved walking behavior in the walking track test. Additionally, rilmenidine(2 mg/kg) also prevented MIA-induced increase in the RANKL and OPG levels in the serum. Histopathological analysis showed that rilmenidine was protective on joint capsule and matrix.

Conclusion: Our results suggest that rilmenidine showed the antinociceptive effect on MIA-induced OA via improving bone turnover.

Osteoarthritis

rilmenidine

MIA

RANKL

OPG

rat

Osteoarthritis (OA) is a degenerative joint condition that results in articular cartilage destruction. It is characterized by chronic pain, inflammation, and movement limitation (1). OA causes unbearable pain and decreases the quality of patients. However, several treatment modalities are present in the clinics their alleviative effects are limited (2). Excruciating pain due to the OA pathology arises from both peripheral input and central sensitization. However, central sensitization was shown to decrease after knee replacement surgery, and the remaining pain also suggests a central driving mechanism (3). Due to the adverse effects of potent analgesic drugs and opioid abuse, identifying alternative treatment options with a safer profile is still of utmost importance.

Pain in OA is associated with subchondral bone marrow lesions and the presence of synovitis (4). An increase in subchondral bone resorption and a decrease in subchondral plate thickness results in subchondral sclerosis and osteophyte formation (5). Because this pathology appears more in the older ages, experimental data about its progression and occurrence is hard to investigate. Therefore, experimental animal models provide helpful information to understand pathophysiology and treatment options deeper. When injected into rodents, MIA is a glycolytic inhibitor that causes behavioral, histological, and biochemical alterations in human OA (5). So, mimicking bone turnover and subchondral sclerosis with accompanying pain, the MIA-induced OA model helps understand new treatment modalities.

Increased turnovers precede massive changes in cartilage and cause an increase in inflammation and pain-related molecules in the joint (6). Particularly, osteoclasts and osteoblast activity are increased, followed by osteoprotegerin (OPG) and receptor activator of NF-kB ligand (RANKL) system interaction (7). Activation is associated with reduced extracellular pH and extracellular matrix proteases and cytokines release. This osteoclast activation also decreases osteochondral integrity and may reveal subchondral nerves to algesic factors present in the joint (8). It has already been demonstrated that agents acting at alpha₂ adrenergic or imidazoline receptors have antinociceptive and analgesic effects on both rodents and humans (9). One of these agents, rilmenidine, is a selective imidazoline receptor agonist and shown to enhance analgesic effects of ibuprofen inflammation-related pain. Clonidine, another alpha-2 adrenergic receptor analog, has been shown to increase bone resorption and has been shown to have potent analgesic effects, but consequences of this resorptive effect on bone tissue is still controversial (10, 11). Nevertheless, its effects on the opioidergic system still limit clonidine use.

The main aim of this study is to investigate the possible analgesic and protective effects of rilmenidine on the MIA-induced model of osteoarthritis. Considering the substantial antihypertensive effects of rilmenidine and the ages of patients who suffer from osteoarthritis, revealing its possible effects will provide vital information for investigating novel treatment options.

2.1. Animals

Thirty-two male Wistar Albino rats (150–300) were used in this study. Animals were procured from Ondokuz Mayıs University Vivarium after obtaining ethical approval from Ondokuz Mayıs University Ethical Committee of Experimental Animals (HAYDEK_2021/10). All animals were maintained in sterile cages, no more than five animals for each cage under the following conditions, 22 ± 0.5°C temperature, 55% humidity, 12/12-night cycle with access to food and water ad libitum. All efforts were made to reduce animal suffering. Experiments were conducted and reported according to the ARRIVE guidelines (12). After power analysis, the number of animals was determined to observe the effects with 95% power by G*power free software.

2.2. Chemicals and experiment design

Monosodium iodoacetate, indomethacin, ketamine, xylazine were purchased from Sigma Co (Bethesda, US). Rilmenidine was obtained from a local pharmacy (Hyperium®, Servier, France). Monosodium iodoacetate was freshly dissolved in phosphate-buffered saline and rilmenidine were dissolved in saline just before the treatments. Animals were daily weighed and observed for any movement abnormalities, which showed deficiencies in limb usage extracted from the study. Animals were anesthetized with ketamine: xylazine (80:10 mg/kg, i.p.). The osteoarthritis model was created according to the previous reports (13). 50 µl MIA was administered (3 mg/kg) through the patellar ligament of right foots intra-articularly for MIA, rilmenidine low-dose (0.5 mg/kg, n = 9) and rilmenidine (2 mg/kg, n = 9) high dose groups. Control rats (n = 5) were administered an equivalent volume of PBS. Rilmenidine low dose (0.5 mg/kg) and high dose (2 mg/kg) was administered orally for 14 days. The MIA group was administered orally with an equivalent amount of PBS as a rilmenidine vehicle for 14 days. Twenty-four hours after the last drug treatments, animals subjected to the hot plate and walking track tests and peripheral blood samples were obtained via tail veins and sacrificed by cervical dislocation. The animals' general health and synovial joints' changes were tracked till the end of the experiments. Treatment doses of rilmenidine were mainly selected according to the previous reports that rilmenidine treatment without significant effect on the locomotor activity due to its effect on blood pressure (14, 15).

2.3. Hot-plate test

The nociceptive response of the animals was evaluated by a hot-plate test (16). All animals were subjected to the hot-plate test before and after the all-drug treatments. As previously described, thermal hyperalgesia was evaluated with the hot-plate device (Ugo Basile, Italy). Animals were placed on a hot apparatus (55°C), and their response time (jumping, one hindlimb standing, licking continuously) was recorded.

2.4. Walking track analysis

Animals were evaluated for spatiotemporal gait analysis with walking track analysis (17). Although noticeable differences between quadrupedal and bipedal gait sequences are present, previous studies reported that rodent osteoarthritis models also reflect similar compensatory behavior to protect injured limbs. Rats were placed on an open white area after staining hind limbs with different colors of ink and allowed to explore freely. Following one minute, the test was ended, and inks densities on the white area were visualized.

2.5. Biochemical tests

Peripheral blood samples were taken after the end of the behavioral tests. Animals were placed on the plexiglass restrainer, and tail veins were visualized with a hot lamp. Serum was separated by centrifugation (1200xg, 5 min). Total amounts of RANKL and OPG were determined by commercially available ELISA kits, strictly following the manufacturer's instructions.

2.6. Histopathological analysis

Gross and histological changes were assessed to evaluate the induction of arthritis and the effects of drug treatments. Knee joints were carefully isolated and fixed in 10% formalin solution following 72-hour 10% formic acid decalcification. De-calcified samples were embedded to the paraffin blocks, and the frontal section of the medial aspect of the joints was sectioned to 250 µm sections. Inflammatory infiltrates and surrounding tissue damage was assessed with hematoxylin-eosin and safranin-O fast green staining (18).

2.7. Statistical analysis

All experimental data were imported to the GraphPad Prism software (v9.0, US). All data expressed as mean ± standard error of the mean (SEM). Data normalization was investigated by Shapiro-Wilk's test. Differences between experimental groups were evaluated with one-way ANOVA, two-way ANOVA, or Kruskal Wallis, Mann Whitney U tests. Multiple comparisons were performed with Tukey's or Duncan's tests. P values less than 0.05 are considered significant.

3.1. Animal weight and hot plate test

Weights of animals were determined 0th and 14th days of experiments. Because MIA injection could deteriorate the movement capabilities of animals, we expected a possible increase in the animal weight. However, there was no significant difference between weights of the animals, and two-way ANOVA results demonstrated that all animals got weight which is statistically significant (F(3, 56) = 5,93, p = .001, Fig. 1A). A hot plate test was used to assess mechanical hyperalgesia in all animals. Our results demonstrated that both MIA (3,15 ± 1,20) caused a significant decrease in the hot plate latency compared to control (8,80 ± 1,83, p < 0,001). In contrast both rilmenidine 0.5 (9,44 ± 1,79) and 2 (10,07 ± 2,29) mg/kg doses significantly improved MİA-induced decrease in hot plate latency (p < 0,001, Fig. 1B).

3.2. Walking track analysis

Rats were allowed to walk on the clean white papers to see possible movement preference. Our results showed that MIA changed animal hindlimb touch preference (Fig. 2). However, we did not see any change in the rilmenidine 0.5 mg/kg group, rilmenidine 2 mg/kg nearly reversed impaired hindlimb touch preference (Fig. 2).

3.3. Rilmenidine prevented MIA induced increase in the RANKL and OPG levels

Biochemical analysis was performed to determine RANKL and OPG levels in serum samples. MIA caused significant increase in the RANKL (256 ± 85,6) and OPG (89,4 ± 20,4) levels compared to the control (50,4 ± 20,4, p < 0,001 and p < 001, 29,2 ± 22,2, Fig. 3). Rilmenidine, at the dose of 0.5 mg/kg, failed to show any significant effect on MIA-induced increase on both RANKL (189 ± 43,2) and OPG (113 ± 23,3) levels compared to the MIA group (p = 0,058, p = 0,093, respectively, Fig. 3). In contrast, rilmenidine at the dose of 2 mg/kg significantly suppressed MIA-induced increase of RANKL (171 ± 51,3) and OPG (50,3 ± 17,3) levels compared to MIA (p = 0,013, p = 0,002, respectively, Fig. 3).

3.4. Rilmenidine prevented MIA-induced damage in the joint

H&E and safranin-O staining were performed to investigate changes in the joint tissue. Our H&E and safranin-O staining results demonstrated no control group joint surface abnormalities. Nevertheless, MIA caused significant damage on the joint surfaces, as seen with the decreased joint capsule volume and matrix tissue (Fig. 4). However, there was no significant difference between rilmenidine 0,5 mg/kg compared to MIA, rilmenidine at the dose of 2 mg/kg significantly improved joint capsule and attenuated MIA-induced damage (Fig. 4).

In this study, we demonstrated that rilmenidine at 2 mg/kg dose significantly suppressed MIA-induced thermal hyperalgesia. Additionally, rilmenidine also inhibited MIA-induced increase in the bone turnover markers, RANKL and OPG. Our results indicated that rilmenidine protected bone matrix and joint capsule with alleviating bone turnover, resulting in decreased pain response.

The increasing incidence of osteoarthritis in the elderly population anticipates a dramatic rise in the number of suffering people in the following decades (19). Because pain is the first reason that causes people to seek treatment options, the challenge to find alternative treatment options for pain management and OA pathology is still ongoing. Therefore, animal models such as MIA-induced osteoarthritis, which we used in our study, provide nearly all aspects for osteoarthritis pathology to investigate drugs that intervene with symptoms or progressive pathology in joints (20). However, the role of imidazoline receptors in pain has been well described in the last decades (21). Knowledge about the possible effect of rilmenidine, which is another imidazoline receptor-I and alpha₂ receptor agonist, is still limited. The analgesic and antinociceptive effect of clonidine, a structural analog of rilmenidine, is well-described (22). However, the complex interactions of clonidine with the opioidergic system and NMDA receptors restrict its use in pain management (23, 24). Therefore, we sought to delineate the effects of rilmenidine, which has a safer profile than clonidine. Rilmenidine is currently used for the treatment of hypertension. Considering hypertension is also related to aging, drugs like rilmenidine, which show both antihypertensive effects and beneficially effect joint damage seen in the OA, step forward as alternative treatment option. As far as we know, the only paper about the analgesic/antinociceptive effect of rilmenidine in its synergetic action with ibuprofen on formalin-induced pain (25). Our results demonstrated that rilmenidine prevented MIA-induced thermal hyperalgesia, in line with Soukupova et al. We also performed walking track analysis to understand animal preference, which shows pain-bearing and animal choice to avoid injury leg. However, our qualitative, rilmenidine treatment improved avoiding behavior that MIA caused.

RANKL and OPG are considered crucial bone remodeling regulators, which regulate cellular changes. RANKL enhances osteoclastogenesis while OPG inhibits it (26). Therefore, the imbalance between RANKL and OPG in OA indicates impaired bone turnover. An increase in RANKL and OPG levels in the serum was already demonstrated by several groups in OA patients (27). Additionally, increased expression of RANKL and OPG mRNA levels in patients with intervertebral disc degeneration also proves the importance of these proteins in bone turnover (28). Therefore, we investigated possible changes in the RANKL and OPG serum levels with safranin-O staining in joint tissue. Our results align with the previous studies that RANKL and OPG significantly increased serum levels after MIA injection, and rilmenidine prevented that increase. Additionally, MIA caused massive destruction in the joint capsule and bone matrix. Decreased chondrocyte size with matrix shows MIA-induced OA pathology, whereas rilmenidine, at the dose of 2 mg/kg, showed significant improvement in chondrocyte cells and matrix.

Therefore, our results suggest that rilmenidine improved MIA-induced damage in the bone, which might be related to its effect on bone turnover markers in the serum. Unfortunately, our study suffers from one major limitation. However, we showed the ameliorative effect of rilmenidine on MIA-induced increase in thermal hyperalgesia, OA also strongly correlated with mechanical hyperalgesia. However, we lack the laboratory equipment and proper test to show this. Thus, subsequent studies should investigate the complete antinociceptive/analgesic effects of rilmenidine.

Acknowledgement

We are grateful for the help of Soner Tiryaki and Elif Türkdönmez during our experiments.

Ethical Approval: On 18/03/2021, after deciding hypothesis, Dr. Osman Kukula explained the experimental design and provided ethical committee approval for animal experiments from Ondokuz Mayıs University Ethical Committee of Experimental Animals (HAYDEK). Relevant documentary added as a pdf folder.

Competing Interests: Nothing to declare

Authors’ Contributions: Caner Günaydın found the hypothesis, Mustafa Nusret Çiçekli contributed majority of the experiments, Seda Kırmızıkan and Selenay Sevinç Şarklıoğlu refined data, Caner Günaydın and Selenay Sevinç Şarklıoğlu wrote the manuscript. When completed these experiments Osman Kukula's laboratory and equipment was used.

Funding: This research was not funded.

Availability of Data and Materials: Not applicable.

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No competing interests reported.

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Rilmenidine protects against joint damage in MIA-induced model of osteoarthritis in rats

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Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Animals

2.2. Chemicals and experiment design

2.3. Hot-plate test

2.4. Walking track analysis

2.5. Biochemical tests

2.6. Histopathological analysis

2.7. Statistical analysis

3. Results

3.1. Animal weight and hot plate test

3.2. Walking track analysis

3.3. Rilmenidine prevented MIA induced increase in the RANKL and OPG levels

3.4. Rilmenidine prevented MIA-induced damage in the joint

4. Discussion

Declarations

References

Additional Declarations

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