G protein‐coupled estrogen receptor in the rostral ventromedial medulla contributes to the chronification of postoperative pain

Abstract Aims Chronification of postoperative pain is a common clinical phenomenon following surgical operation, and it perplexes a great number of patients. Estrogen and its membrane receptor (G protein‐coupled estrogen receptor, GPER) play a crucial role in pain regulation. Here, we explored the role of GPER in the rostral ventromedial medulla (RVM) during chronic postoperative pain and search for the possible mechanism. Methods and Results Postoperative pain was induced in mice or rats via a plantar incision surgery. Behavioral tests were conducted to detect both thermal and mechanical pain, showing a small part (16.2%) of mice developed into pain persisting state with consistent low pain threshold on 14 days after incision surgery compared with the pain recovery mice. Immunofluorescent staining assay revealed that the GPER‐positive neurons in the RVM were significantly activated in pain persisting rats. In addition, RT‐PCR and immunoblot analyses showed that the levels of GPER and phosphorylated μ‐type opioid receptor (p‐MOR) in the RVM of pain persisting mice were apparently increased on 14 days after incision surgery. Furthermore, chemogenetic activation of GPER‐positive neurons in the RVM of Gper‐Cre mice could reverse the pain threshold of pain recovery mice. Conversely, chemogenetic inhibition of GPER‐positive neurons in the RVM could prevent mice from being in the pain persistent state. Conclusion Our findings demonstrated that the GPER in the RVM was responsible for the chronification of postoperative pain and the downstream pathway might be involved in MOR phosphorylation.


| INTRODUC TI ON
Postoperative pain is a common puzzle that bothering a large number of perioperative patients. 1,2 To make matters worse, approximately 10% -50% patients will suffer the long-term postoperative pain, which transfers from acute pain and represents a huge potential of chronic pain. 3,4 This unexplained, persistent, and recurring chronic pain is not sensitive to the existing analgesic measures, which is a hard part of clinical treatment. [4][5][6] Thus, it is of great clinical significance to study the mechanisms underlying the occurrence and development of postoperative pain and find out a new therapeutic target.
Rostral ventromedial medulla (RVM), as the last functional nucleus of descending pain modulation system in the brain, has been regarded as a gateway that controls the pain regulation signaling transmitted from periaqueductal gray (PAG) to the dorsal horn of the spinal cord, which has a bidirectional role in pain facilitation or inhibition. 7 It has been proved that the descending modulation of RVM determined whether acute pain will develop into chronic pain to a large extent. 8,9 Moreover, RVM is also the major site for analgesic effects of endogenous or exogenous opioids, [10][11][12][13] since silencing the dual GABA/enkephalinergic RVM neurons can substantially increase behavioral sensitivity to heat and mechanical stimuli in adult mice. 14 G protein-coupled estrogen receptor (GPER), or recognized as G protein-coupled receptor 30 (GPR30), has been introduced as the plasma membrane to evoke rapid non-genomic estrogenic signaling, 15 which is widely distributed in different organs with the nervous system included. 16 Some studies have shown that estrogen and its nuclear receptors (ERα and ERβ) play a critical role in pain regulation. [17][18][19][20] However, few reports have directly investigated estrogenic effects on the descending pain modulation system, es-

| Plantar incision-induced pain model
The plantar incision surgery was conducted following the protocol from Pogatzki, E. M, et al. 22 Under anesthesia with 2% isoflurane, the right hind paw of mouse (or rat) was prepared with iodophor, then a 5 mm (10 mm for rat) longitudinal incision was made through the skin and fascia 2 mm (5 mm for rat) away from the edge of the heel with a sterile No. 11 scalpel. To leave the origin and insert intact, the muscle was elevated using a curved tweezer. The incision was sutured with 8-0 nylon and then dressed with antibiotic ointment. A homoeothermic blanket was used to maintain the temperature at 37℃ during the surgery.

| Behavioral tests
Mechanical and thermal pain thresholds were both tested. Mice were habituated to the test environment for 30 min every day from 3 days before measurement. Mice in quiet state or less autonomous activities were deemed as well acclimatized.

| Mechanical allodynia test
Von Frey filaments (0.008-2 g for mouse, 0.5-12 g for rat) were vertically applied to the hind paw adjacent to the incision for 1 s.
The same filament was stimulated 5 times with an interval of more than 1 min. Three or more times obvious painful behavioral response such as foot licking or foot withdrawal was considered as effective pain reaction, and the gram was recorded as the mechanical pain threshold.

| Hot plate test
Similarly, after the mice were habituated, they were gently placed on the hot plate at 52.5°C (IITC Life Science Inc.). The response latency to either lick, retraction, or jump was recorded as the nociceptive endpoint. In the absence of a response, mice were removed from the hot plate at 30 s to avoid tissue injury. Each mouse was measured 3 times, and the mean value was regarded as the thermal pain threshold.

| RVM stereotaxic injection
The Gper-Cre mice were anesthetized with sodium pentobarbital (0.06 mg g −1 , i.p.) and secured in a stereotactic frame (RWD Instruments). A midline scalp incision was made and a hole was drilled on the skull to allow passage of glass pipette filled with the virus.

| Drug administration
Clozapine N-oxide (CNO, 5 mg, Sigma) was initially dissolved with 1 ml saline (0.9%) and stored at 4℃ according to the method previously described. 23 CNO (3 mg kg −1 , i.p.) was administrated intraperitoneally per day from 10 to 14 days after incision surgery. An equal volume of 0.9% saline was given to control mice.

| Western blot analysis
RVM samples of different groups of mice were extracted and transferred from 10% SDS-PAGE to nitrocellulose membranes.
Following inhibition with 5% skimmed milk for 1 h, they were

| RT-PCR
The total RNA of RVM were extracted by Vazyme kit and transcribed to cDNA with superscript II and random hexamer primers. PCR amplification was done using the follow-
Finally, the fluorescence images were recorded with a laser-scanning confocal microscope (FV3000, Olympus Corp.). C-Fos staining was chosen to reflect the activated neurons. All images were analyzed by Image J to calculate the number of c-Fos positive neurons. Data were obtained from three or four animals in each group, and eight slices or ten slices were chosen from each animal (eight slices for RVM, ten slices for spinal cord) to sum up in total.

| Statistical analysis
Statistical tests were performed by GraphPad Prism 7.0 (GraphPad Software Inc.). All data are shown as mean ± SEM. Data normality was verified by Shapiro-Wilk test. The results of behavioral tests were analyzed with two-way repeated measures ANOVA followed by the Bonferroni post hoc test. One-way ANOVA analysis followed by the Tukey post hoc test was employed for multiple group comparisons.
The level of statistical significance was expressed as p-value <0.05.

| Chronification of acute pain induced by plantar incision surgery
Among perioperative patients with postoperative pain caused by surgeries, a certain proportion will develop into chronic pain. 25 To better study the mechanisms of chronification of acute postoperative pain, plantar incision was applied to simulate surgical trauma. The behavioral tests with von Frey and hot plate were conducted to detect both thermal and mechanical pain thresholds before the incision surgery (baseline) and 1, 3, 5, 7, 10, 14 days after the incision surgery.
Behavioral results showed that there were no obvious changes both in von Frey test (p > 0.05, Figure 1A) and hot plate (p > 0.05, Figure 1B Figure S1). The recovery progress of plantar incision ( Figure 1C) was relatively similar between the pain persisting mice and pain recovery mice at different time points. The plantar appearance of naive mice without incision surgery showed almost no change at different time points (p > 0.05, Figure   S2). Furthermore, HE examinations ( Figure 1D) showed no obvious increase of fibrosis and inflammation in the pain persisting mice on 14 days after incision surgery compared with naive mice and pain recovery mice. In this way, we established a model of chronification of acute postoperative pain induced by plantar incision surgery.

| The expression of GPER in RVM is significantly increased in the pain persisting mice
Previous research has shown that GPER is abundantly distributed in the central nervous system (CNS) and participates in pain modulation. 26,27 To best of our knowledge, RVM serves as one of the important functional nuclei for the descending pain modulation in the CNS and is associated with the occurrence and progression of chronic pain. 28,29 Here, we clarified the distribution of GPER in the RVM by immunofluorescence staining (Figure 2A).  Figure 3C) or GPER + neurons (Pain persisting vs. Naive, p < 0.01; Pain persisting vs. Pain recovery, p < 0.01; Figure 3D) was markedly higher compared with naive rats or pain recovery rats, respectively, suggesting that the activated GPER neurons in RVM can play a vital role in the chronification of postoperative pain. Furthermore, the type of activated cells in RVM are neurons rather than neuroglial cells, such as astrocytes and microglias ( Figure S3).

| Phosphorylation of MOR in the RVM is increased in the pain persisting mice
Among the pain-related nuclei in the CNS, RVM is a main area where endogenous and exogenous opioids exert analgesic functions 13 with high expression of MOR. 30,31 Previous study has reported that there is a subset of neurons in RVM-expressed MOR for maintaining thermal hyperalgesia in neuropathic pain rats. 32 In addition, activation of MOR in RVM can produce antinociception. 33 To further assess the effect of MOR on the chronification of postoperative pain, the phosphorylation of MOR in the RVM was detected by immunoblotting ( Figure 4A). The results demonstrated that the ratio of p-MOR to MOR was increased significantly in the pain persisting mice compared with naive mice or pain recovery mice (Pain persisting vs. Naive, p < 0.001; Pain persisting vs. Pain recovery, p < 0.01;  Figure 4D). Collectively, these results indicate that the increased level of p-MOR in RVM is associated with the chronification of postoperative pain.

| Chemogenetic activation or inhibition of GPER-positive neurons in the RVM affects the chronification of postoperative pain
To further verify the role of GPER in the RVM during pain chronification, we used chemogenetic method to specifically activate GPER-positive RVM neurons of Gper-Cre mice. Gper-Cre mice were constructed by CRISPR/Cas9 technique and identified by PCR combined with agarose gel electrophoresis ( Figure S4). The schematic diagram for animal preparation and behavioral tests were illustrated in Figure 5A With daily intraperitoneal administration of CNO or saline into pain recovery mice from 10 to 14 days after incision surgery, behavioral tests were conducted at 30 min after CNO/saline administration and on the next day before CNO/saline administration again, respectively. We observed that both PWT and PWL showed a significant decline 30 min after CNO administration on 10 and 11 days after incision surgery, whereas both PWT and PWL showed almost no changes after saline administration ( Figure S5A,B). Furthermore, the significant difference between before CNO administration and 30 min after CNO administration disappeared from 12 days after incision surgery because the daily baseline of pain thresholds has decreased in CNO-treated mice before CNO administration ( Figure   S5A,B). More importantly, we found that the daily mechanical and thermal pain thresholds tested before CNO or saline administration were both gradually decreased in CNO-treated pain recovery mice from 11 to 15 days after incision surgery compared with the saline- Pain recovery + Saline, p < 0.05; Figure 5J). These results imply that GPER-positive neurons in the RVM can facilitate the phosphorylation of MOR, which is an underlying mechanism for the recurrence of postoperative pain in pain recovery mice.
The rostral ventromedial medulla (RVM) has been regarded as an essential brainstem structure that exhibits descending pain modulation via projections to the spinal dorsal horn. Therefore, we explored the effects of chemogenetic activation of GPER-positive neurons on the activation of dorsal horn neurons. The results showed that the amounts of c-Fos + neurons in the dorsal horn of lumbar spinal cord in pain persisting and pain recovery mice treated by CNO were both higher than that in saline-treated mice (Pain persisting vs. Pain recovery + Saline, p < 0.001; Pain recovery + CNO vs. Pain recovery + Saline, p < 0.01; Figure 6A,B).
Additionally, we also explored the effects of chemogenetic activation of RVM GPER-positive neurons on pain persisting mice. We found that both PWT and PWL showed a significant decline 30 min after CNO administration only on the first day of CNO administration and subsequently passed off ( Figure S6A,B). Furthermore, the behavioral results showed that the daily PWT and PWL tested before CNO or saline administration were both gradually decreased in CNO-treated mice from 13 to 15 days after incision surgery and

| DISCUSS ION
Acute pain is almost ubiquitous after surgery, and it can be con- In this study, we used the plantar incision pain model to simulate the acute pain and the transition from acute pain to chronic pain (TAPCP) following common surgeries, such as breast and thoracic surgery, orthopedic surgery, limb surgery, and arterial bypass surgery. Currently, the thorough mechanisms of TAPCP after surgery are complex and poorly understood. Previous research has shown that peripheral and central sensitization are crucial in TAPCP as well as the persistent postoperative pain. 5,38 In general, continuous or intense peripheral nociceptive stimulation may induce central sensitization. The RVM, composed of the adjacent reticular formation and nucleus raphe magnus, has been regarded as a key player in the descending pain modulation systems. 28 It receives information from different central nuclei, including PAG, parabrachial complex (PB), and hypothalamus. 8 The descending PAG-RVM pathway, which modulates ascending nociceptive transmission at the spinal cord dorsal horn, is involved in the occurrence of chronic pain. 39 Many evidences suggest that an imbalance between descending facilitation and descending inhibition, particularly heightened descending facilitation from RVM, may underlie central sensitization in various pathological pain conditions. 4 0- 45 Our results showed that the number of activated GPER + neurons of RVM in chronic postoperative pain rats was remarkably higher compared with pain recovery rats or naive rats, suggesting that GPER + neurons in the RVM can regulate the chronification of postoperative pain.
Perioperative patients often trend to feel anxious or depressed generally summarized as the perioperative negative emotions. 46,47 Accumulating evidence suggests that negative emotions may promote the susceptibility of chronic postoperative pain. 48 Besides, the secreted estrogen in the CNS is closely related to negative emotions and exerts functions when combined with GPER. 49,50 In our previous study, the regulation of estrogen synthase aromatase on visceral pain has been investigated, 51 also indicating to further study the roles of GPER in the occurrence and progression of chronic postoperative pain. Over recent years, there has been a growing attention on the effects of GPER regulating pain. As reported by Luo et al., GPER may accelerate the development of bone cancer pain through suppressing inhibitory transmission and inducing excitatory transmission in the spinal cord. 52 In addition, GPER in the basolateral amygdala is closely associated with pain-related anxiety. 49 Furthermore, GPER antagonist G15 could efficiently suppress formalin-induced spontaneous biphasic nociceptive responses. 53 However, the role of GPER in the RVM during pain regulation has not been studied.
Based on these, we investigated that the expression of GPER in the RVM under different postoperative pain states and observed that GPER was highly expressed in mice with chronic postoperative pain.
Moreover, we demonstrated that GPER-positive neurons in the RVM could regulate the chronification of postoperative pain by chemogenetic techniques.
Many studies have shown that the dysfunction of the endogenous opioid analgesic system is responsible for chronic pain. [54][55][56] The activation of MOR by opioids, such as morphine, tends to occur at the RVM where it exerts analgesic effects. 31,57-59 Maione et al. have founded that the interaction between MOR and TRPV1 in the RVM can induce analgesia and activate glutamate transmission. 60 Furthermore, physical activity can activate MOR in RVM to prevent the increase in paw withdrawal frequency through modulation of serotonin transporter (SERT). 61 Besides, it has been proved that estrogen may negatively affect MOR-mediated signaling in the dentate gyrus, the hypothalamus, and the medial preoptic nucleus, probably via increasing phosphorylation of MOR. [62][63][64][65] Our study revealed that the ratio of p-MOR to MOR was markedly elevated in the RVM of chronic postoperative pain mice compared with pain recovery mice and that the ratio of p-MOR to MOR was restored in pain recovery mice by chemogenetic activation of GPER-positive neurons in the RVM. These results suggest that increased phosphorylation of MOR is a potential mechanism of chronification of postoperative pain.
Additionally, many recent studies have indicated that certain cytokines and chemokines as well as inflammation pathways are involved in the molecular regulation of neuropathic pain, 66-68 which provided a speculation that the regulation of GPER in chronic postoperative pain may also involve inflammatory signaling mechanisms. We will explore this speculation in future study.
In conclusion, our findings provided strong evidence that GPER expression and the number of activated GPER + neurons were both upregulated in the RVM of chronic postoperative pain animals.
Chemogenetic activation of GPER-positive neurons makes the pain recovery mice present the phenotype of chronic postoperative pain, and the underlying mechanism may be to promote the phosphorylation of MOR. These findings indicate that GPER can serve as a new target for preventing TAPCP after surgery and treating chronic postoperative pain.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.