Role of ERK in gender difference of fibromyalgia pain

Abstract

This study investigated the ERK pathway of the peripheral nervous system and discovered a gender-specific pattern of ERK activation in the dorsal root ganglion of an acid-induced chronic widespread muscular pain model. We employed a twice acid-induced chronic musculoskeletal pain model in rats to evaluate mechanical pain behavior in both male and female groups. We further conducted protein analysis of dissected dorsal root ganglions from both genders. Both male and female rats exhibited a similar pain behavior trend, with females demonstrating a lower pain threshold. Protein analysis of the dorsal root ganglion (DRG) showed a significant increase in phosphorylated ERK after the second acid injection in all groups. However, phosphorylation of ERK was observed in the dorsal root ganglion, with higher levels in the male ipsilateral group compared to the female group. Moreover, there was a no difference between the left and right sides in males, whereas the significant difference was observed in females. In conclusions, the administration of acid injections induced painful behavior in rats, and concurrent with this, a significant upregulation of pERK was observed in the dorsal root ganglia, with a greater magnitude of increase in males than females, and in the contralateral side compared to the ipsilateral side. Our findings shed light on the peripheral mechanisms underlying chronic pain disorders and offer potential avenues for therapeutic intervention.

Introduction

Fibromyalgia syndrome is a chronic disorder of the musculoskeletal system that is characterized by widespread pain and tenderness at specific tender points. It exhibits a higher prevalence in females, with a female-to-male ratio ranging from 80% to 96%. Females also tend to exhibit a greater sensitivity to pain.^1,2 Patients with fibromyalgia syndrome experience chronic pain that is challenging to manage with a monotherapy approach. Therefore, a combination of pharmacological and non-pharmacological therapies is typically recommended. ³ The pharmacological approach usually involves the use of antidepressants, anticonvulsants, muscle relaxants, and analgesics. The non-pharmacological approach includes exercise, cognitive-behavioral therapy, and complementary therapies such as acupuncture or massage. It is essential to understand that fibromyalgia is a complex condition, and treatment plans must be tailored to each patient’s unique symptoms and requirements. A multidisciplinary approach involving a team of healthcare professionals, such as primary care physicians, pain specialists, and mental health providers, may be beneficial in managing fibromyalgia symptoms. Some patients are misusing their prescription opioids by either not taking the medication appropriately or by taking additional opioids that are not prescribed. ⁴ Opioid abuse, particularly with morphine-based drugs, can lead to numerous adverse effects and even death, as well as significant financial burdens on healthcare systems. Although non-steroidal anti-inflammatory drugs (NSAIDs) do not carry the risks of respiratory depression, constipation, or addiction associated with opioids, they do carry the risks of gastrointestinal bleeding, ulcers, and renal impairment. ⁵ Chronic pain has negative impacts on physical and mental function and significantly affects quality of life. It is challenging to treat and imposes substantial costs on patients and the healthcare system. The mechanisms underlying chronic pain are still not fully understood. However, developing clinically feasible approaches to reduce drug abuse and its complications is critical, particularly through interventions in the peripheral nervous system.

The dorsal root ganglion (DRG) is anatomically accessible and a suitable target for treatment. Although the DRG is more peripheral in terms of neurological status, studies have shown that it may have better results than treating the spinal cord in the pain management. However, clinical experience has demonstrated that direct treatment of the nerve, such as excision, may cause more severe subsequent pain. Therefore, a more specific understanding of the role of the dorsal root ganglion in the pain process is necessary to seek definitive treatments. The dorsal root ganglion receives stimulus signals from the periphery and transmits them to the central nervous system, playing an irreplaceable role in the process of pain formation. However, an increasing number of studies have found that the dorsal root ganglion not only plays a role in transmitting messages but may also be involved in the mechanism of pain modulation.

Extracellular signal-regulated kinases (ERK) regulate cell proliferation and differentiation, with their activity controlled by phosphorylation. It is worth noting that ERK phosphorylation is increased in chronic pain. ⁶ Some research suggests that the binding of neurotrophins to their specific tyrosine kinase receptors or neuronal activity leading to glutamate release and binding to its ionotropic and metabotropic receptors can contribute to this increase. ⁷ Therefore, inhibiting ERK activation would be a promising therapeutic target for the treatment of chronic pain. ⁸ However, it is important to consider the effect of gender on acid-induced pain in rat models and the phosphorylation of ERK in female and male rat models to better understand the role of the dorsal root ganglion in chronic widespread musculoskeletal pain and whether it influences pain processing differently in different genders through ERK.

Materials and methods

Animal

Female and male Sprague-Dawley rats (2 months old) with an average weight of approximately 200 g were purchased from BioLASCO (Taipei, Taiwan) and housed in groups of three in a temperature-controlled room (22°C ± 1°C) with a 12-h light-dark cycle. Rats had ad libitum access to food and water throughout the experiment. All experimental procedures were approved by the Animal Care and Use Committee at China Medical University in Taichung, Taiwan and were conducted in accordance with the guidelines set forth by the National Institutes of Health (NIH).

Repeated acid solution injection (AI)-induced chronic widespread pain (CWP) rat model

The current study utilized a chronic widespread pain (CWP) animal model that has been previously described and slightly modified for the current experiment. Both female and male rats were divided into three groups: Naïve, Saline, and acid-treated (acid/MES) groups (N = 6/group), respectively. Acidic saline was freshly prepared using 10 mM 2-[N-morpholino] ethanesulfonic acid and adjusted to pH 4.0 with 1 N NaOH. The rats received two injections of 100 μL of acid 10 mM MES into the left gastrocnemius muscle on day 0 (D0) and day 5 (D5) (Figure 1). Pain behavior was measured up to 19 days, and the rats were sacrificed at 11 days for further analysis.

Figure 1.

Diagram of the experimental design. The study utilized female and male rats which were divided into three groups: Naïve, Saline, and acid treatment (AI/MES). Chronic pain was induced by administering acid treatment twice, on day 0 and day 5. Pain behavior was observed for up to 19 days, and the rats were sacrificed at day 11 for further analysis.

Nociceptive behavioral tests

Nociceptive behavioral tests were conducted to evaluate mechanical sensitivity using von Frey filaments (Stoelting, Wood Dale, IL). Rats were individually placed in a chamber (10 cm × 10 cm × 20 cm) of a Plexiglas cage on an elevated iron mesh floor. The von Frey filaments were applied to the plantar surface through the mesh openings using the up-down method. A series of von Frey filaments with logarithmically incremental stiffness (0.4, 1.0, 2.0, 4.0, 6.0, 10.0, 15.0, and 26.0 g) were presented perpendicularly to the plantar surface for 5–6 s for each filament. The mechanical threshold value was determined by calculating the 50% withdrawal threshold using the up-down method. Both hind paws were tested daily starting 2 days before the first acid injection, between two injections, and at time points after the second injection.

Western blotting

Bilateral dorsal root ganglia at L4-5 segments were dissected and homogenized to prepare tissue lysates and following the manufacturer’s protocol (Biochain Institute, Inc., Hayward, CA). 20 μL of protein lysate was separated on a 10% polyacrylamide-SDS gel in a glycine-Tris buffer and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then probed with rabbit anti-ERK (1:1000, #4695 Cell Signaling Technology, Danvers, MA) and anti-phospho-ERK (1:1000, #9101 Cell Signaling Technology, Danvers, MA) antibodies at 4°C overnight. After washing, the membrane was incubated with an HRP-conjugated anti-rabbit IgG secondary antibody (1:5000, 111-035-144 Jackson ImmunoResearch, West Grove, PA). The chemiluminescent signal was developed using HRP-reactive chemiluminescence reagents (WBKLS0500, Millipore Corporation, Billerica, MA) and detected with an ImageQuant LAS 4000 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Band intensity was quantified and normalized to that of α-tubulin. The results are presented as fold-change compared to control for statistical analysis.

Immunofluorescence staining

The rats were deeply anesthetized and then perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline. The lumbar dorsal root ganglia were dissected and cut into 15-μm-thick transverse sections using a cryostat (LEICA CM3050S, Nussloch, Germany). After washing with phosphate-buffered saline (PBS), the sections were treated with 0.3% hydrogen peroxide in 10% methanol/PBS for 30 min at room temperature (RT). The sections were then blocked with 3% normal goat serum in PBS for 1 h and incubated overnight at 4°C with rabbit anti-p-ERK antibody (1:400; #9101 Cell Signaling Technology, Danvers, MA). Next, the sections were incubated with a Cy3-conjugated secondary antibody (1:400, 111-165-003 Jackson ImmunoResearch, West Grove, PA) for 90 min at RT. We mixed anti-p-ERK (1:400) and anti-GFAP (astrocyte marker, 1:4000; Chemicon) antibodies for double staining, subsequent air-dried, dehydrated, and coverslips. All section images were captured using a CCD camera (ZEISS Axiocam 503 color, Jena, Germany) attached to a Zeiss Axio Imager A2 microscope (Göttingen, Germany), and the fluorescence intensity of spinal sections was analyzed using ZEISS software (ZEN; Zeiss, Jena, Germany) at a magnification of 40x for analysis.

Statistical analysis

One-way ANOVA was used to estimate the differences between three drugs. ANOVA with Scheffe’s post hoc comparisons was used. A paired t test was used to estimate the differences between each time and base time in acid group. A p value of less than 0.05 represented a statistically significant difference between compared data sets. All analyses were performed with the SAS statistical package 9.4 (SAS, Cary, NC).

Result

Pain behavior could be decreased by acid injection in vivo

Mechanosensitivity measurement is a crucial method for studying pain in animal models. To evaluate pain sensitivity in male and female rats, acid treatment was administered twice on day 0 and day 5 (Figure 1), followed by the use of Von Frey filaments to measure mechanosensitivity in the pain animal models. Our findings demonstrated that the pain threshold was reduced in both ipsilateral and contralateral sides of female and male rats after twice acid injection (Figure 2). We could observe bilateral chronic pain reaction lasting for 2 weeks after second acid injection. Saline and Naïve groups did not exhibit any effects, confirming the success of this model in both female and male rats. Therefore, twice acid treatment could be used to establish a generalized chronic pain model.

Figure 2.

Mechanosensory response by Von Frey filaments after acid treatment. Mechanosensitivity was evaluated for up to 19 days, revealing a decrease in pain threshold after the twice acid injections (indicated by the red arrow) in both ipsilateral and contralateral sides of female and male rats. No changes were observed in the saline and Naïve groups. These results are presented in panels (a) and (b) for the female ipsilateral and contralateral sides, and panels (c) and (d) for the male ipsilateral and contralateral sides. #p < .05; ##p < .01; ###p < .001 versus BL. *p < .05; **p < .01; ***p < .001 versus acid groups. Rat number = 6-12 for each group at individual time points.

Activate ERK phosphorylation in DRG after acid treatment

The objective of this study was to explore the potential involvement of ERK activation in pain behavior. Immunofluorescence analysis demonstrated that acid treatment induced significant phosphorylation of ERK in the dorsal root ganglia (DRG) of male and female rats on day 11, as depicted in Figure 3. Moreover, subsequent analysis revealed that ERK was primarily expressed in satellite glia, rather than neurons, in both male and female sections (Figure 4). These results suggest a putative role for ERK activation in pain behavior, specifically through its activation in satellite glia in the DRG.

Figure 3.

ERK involved in acid injection rat model. Phosphorylated ERK activation was visualized by immunofluorescence following 11 days of acid injection in bilateral L4-5 dorsal root ganglia. The study groups included Naïve, Saline, and acid-treated groups. The male groups are represented in (A-C), while the female groups are shown in (D-F). Red indicated phosphorylated ERK.

Figure 4.

Phosphorylation of ERK expressed in satellite glia. Immunofluorescence was utilized to visualize the activation of phosphorylated ERK in satellite glia (indicated by an arrow) of bilateral L4-5 dorsal root ganglia after 11 days of acid injection. Red fluorescence represented phosphorylated ERK (A and D); Green fluorescence represented GFAP (B and E); The merged images were presented in C and F. The male groups were shown in (A-C), while the female groups were represented in (D-F).

Additionally, western blot analysis showed that acid treatment significantly increased ERK1/2 protein phosphorylation in both the ipsilateral and contralateral regions of male and female rats (Figure 5). Figure 6 revealed that ERK1/2 phosphorylation was significantly increased in the ipsilateral region of male rats compared to that of female rats. Although we did not see a statistically significant differences of contralateral region between male and females, there was a trend of more prominent ERK activation of male groups. Finally, we compared the state of ERK phosphorylation in the right and left sides of female rats and found that ERK phosphorylation was significantly higher in the right side than in the left side. Even without significant differences of males while comparing both sides, we could also notice the similar trend of rightside p-ERK dominant as female rats. (Figure 7). These findings suggest the differential expression of p-ERK on DRG between genders in the acid injection pain model.

Figure 5.

Expression of phosphorylated ERK in different group. Western blot analysis showed the levels of phosphorylated ERK (pERK1 and 2) in the ipsilateral and contralateral L4-5 dorsal root ganglia following 11 days of acid injection in female and male rats. The analyzed proteins included Naïve, saline, and acid treatment groups. The quantitative data presented the protein levels of pERK1 and 2 in the dorsal root ganglia of both female and male rats. (a) Female; (b) Male. **, p < .01; ***, <0.001.

Figure 6.

Effects of acid treatment on ERK phosphorylation in the ipsilateral and contralateral dorsal root ganglia. Western blot analysis revealed a significant increase in phosphorylated ERK in both ipsilateral (a) and contralateral (b) sides of the dorsal root ganglia. The quantitative data show the levels of phosphorylated ERK1 and 2 normalized to α-tubulin. *, p < .05; **, p < .01.

Figure 7.

Effects of acid treatment on ERK phosphorylation in left and right side of female and male rat. The western blot analysis showed a statistically significant increase in phosphorylated ERK1 and 2 in the right side of female rats compared to the left side (A). In male rats, ERK phosphorylation was elevated on both sides, but no significant difference was observed between the left and right sides. ***, p < .001.

Discussion

Clinically, it has been observed that females have a higher prevalence of chronic pain. Our previous studies have indicated that the expression of phosphorylated ERK protein in animal models of ovariectomy is lower than in the control group. The activation of ERK protein in the spinal cord by hormones may affect the expression of pain threshold, which could be a contributing factor to the higher prevalence of chronic pain in females. However, this pathway belongs to the central nervous system signaling, and peripheral nerves may also participate in pain regulation mechanisms. Therefore, we applied an animal model of chronic widespread muscle pain by repeatedly injecting acid to investigate the potential involvement of peripheral nerves in pain mechanisms. Our results showed that acid injections reduced the pain threshold in female and male rats and activated ERK protein in the dorsal root ganglia on the seventh day. The levels of phosphorylated ERK protein in both ipsilateral and contralateral sides of female and male rats increased after acid injection. Moreover, the level of phosphorylated ERK protein in the contralateral side was significantly higher than ipsilateral side for female rats. The same trend was observed in male rats, with the level of phosphorylated ERK protein in the contralateral side being higher than that in female rats.

In our previous research, we found that acid injection could activate ERK protein in the spinal cord, which in turn affects pain behavior expression, with hormone making the pain more prominent. ⁹ This phenomenon like the condition of clinical chronic muscle pain disease, where female rats have lower pain thresholds and stronger responses to pain. However, the activation of ERK in the spinal cord is more prominent on the ipsilateral side after acid injection, while in the dorsal root ganglion, it is more prominent on the contralateral side, even in male rat with more significant activation of the protein. We compared the difference in ERK activation in the dorsal root ganglion of male and female rat after acid injection, the results showed that male rat had more pronounced activation expression than female rat. Regardless of gender, there was also a higher activation level in the contralateral group after acid injection. This phenomenon may be related to mirror-image pain which is the mechanical hypersensitivity on the uninjured mirror-image side.^10–12

The satellite glia was activated in the contralateral DRG, TNF-a was also increased, which stimulates the production of nerve growth factor (NGF) in the unilateral spinal nerve ligation (SNL) study.^13–15 This phenomenon occurs in the DRG rather than the spinal cord, consistent with our results that peripheral nerves may participate in the pain formation process. Previous studies have found that satellite glia in the DRG may play a major role in the pain mechanism. Immunofluorescence staining revealed predominant ERK activation in the peripheral regions of DRG neurons following acid injection, which coincides with the location of satellite glia. These findings suggest that satellite glia may play a role in the development of acid-induced pain. We chose the DRG on day 7 after the second acid injection to observe ERK expression, the time point with the obvious decrease in pain threshold. However, this is a model of stable chronic pain induced by twice acid injections, and analyzing DRG at earlier or later time points may yield different results. Clinically, chronic musculoskeletal pain such as fibromyalgia is popular in women, while male patients have more comorbidities. ¹⁶ The reasons for this difference are unknown, but we hope that there will be increasing evidence to show that pain mechanisms in different genders are different.

The signal transmission and nerve fiber connections in DRG are very delicate, with communication occurring in various directions. This may be why contralateral activation is more prominent. Some signals may first travel to the contralateral side before returning to the ipsilateral side, resulting in an observable change in contralateral response in the earlier stages but a return to ipsilateral response over time. Therefore, examining ERK expression at different time points is necessary. Our data showed that double staining for Satellite glia and ERK were performed to examine the involvement of satellite glia in the ERK signal in DRG. We cannot exclude the possibility of involvement from other cells such as neurons or tissues. In the future work, we will plant to investigate whether inhibit ERK signal could decrease pain through ERK inhibitor.