The Role of Toll-Like Receptor 4 Mediates Microglial Activation during Remifentanil-Induced Hyperalgesia in Rats

Background: Opioids can induce a state of nociceptive sensitization, also known as opioid-induced hyperalgesia. Nevertheless, the exact mechanism is still unclear. The following study investigates the role of Toll-like receptor 4 (TLR4) in the microglia activation during remifentanil—induced hyperalgesia in rats’ model of incisional pain. Methods: Mechanical allodynia induced by remifentanil was established in adult male Sprague–Dawley rats with incisional pain. Paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWTL) were performed to evaluate mechanical and thermal hyperalgesia. The 32-G catheter intrathecal placement was used to deliver a specific TLR4 antagonist (LPS-RS).Western blot analysis was performed to measure the expression of the TLR4 and Iba-1, while Immunoﬂuorescence staining was used to investigate the cell type and cell activation. ResultsIncisionalpain-remifentanil decreased the PWT and PWTL, upregulated the expression of TLR4 and microglia activation in the spinal cord. On the contrary, the intrathecal delivery of LPS-RS at the dose of 25 μg significantly decreased mechanical allodynia and prevented the upregulation of TLR4 induced by incisional pain-remifentanil Conclusion: These findings suggest that TLR4 signaling pathway has an important role in incisional pain-remifentanil hyperalgesia, and that it could serve as the therapeutic target for persistent postsurgical pain In the present study we demonstrated the involvement of TLR4 signaling pathway in spinal cord in the mechanical allodynia induced by incisional pain-remifentanil. We found that intraoperative remifentanil infusion induced the upsurge of TLR4 and microglia activation in spinal cord, while the inhibition of TLR4 signaling using LPS-RS reduced mechanical allodynia and prevented the upregulation of TLR4 and microglia activation induced by incisional pain-remifentanil. Our findings revealed that TLR4 signaling pathway has an important role in incisional pain-remifentanil hyperalgesia, and that it could serve as the therapeutic target for persistent postsurgical pain.

pain and morphine demand 9 10 . Also, remifentanil-induced hyperalgesia caused by its faster and more frequent characteristic than that induced by other opioids, has become a focus problem 11 12 13 .
Thus far, it has been proposed numerous opioid-receptor-dependent neuronal mechanisms of OIH 14 . In addition to activating classical opioid receptors, previous studies have shown that morphine activates toll-like receptor-4 (TLR4) on glia, triggering proinflammatory mediator release, which in turn activates a serious of cascade events that enhance nociception 15 . While neuronal morphine actions are analgesic, concurrent production of neuroexcitatory substances by glial cells

Animals
Adult male Sprague-Dawley rats weighing 240 to 260 g were taken from Central Animal center, Anhui Medical University, China. Every four animals are raised in groups and adapted to the housing environment for one week. The ambient temperature is 22 ± 1 ºC, the relative humidity is 50 ± 10%, and the light and dark period is 12 / 12 hr. The protocol had been approved by the Ethical Committee of Anhui Medical University. In addition, all animal studies (including the rat euthanasia procedure) were done in compliance with the regulations and guidelines of Anhui Medical University institutional 4 animal care and conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the Institutional Animal Care and Use Committee (IACUC) guidelines.

Surgery procedure
The plantar incision was performed according the method described by Brennan et al 27 . Rats were anesthetized by nasal mask with sevoflurane (induction, 3%; surgery, 1%). The plantar surface of the right hind paw was disinfected with 5 % povidone-iodine solution and the animal foot through a hole in a sterile drape. A 1cm longitudinal incision through the skin and fascia starts at 0.5 cm from the margin of the heel, extending to the toes of the right hind paw. Using tweezers to raise the plantar muscle and cut it longitudinally to keep the muscle source and insertion intact. After stopping bleeding with gentle pressure, seal the skin with two 5 -0 nylon mattress sutures. The wound was covered with erythromycin ointment. The incision was checked daily to exclude animals with signs of infection or dehiscence from the study. To evaluate mechanical hyperalgesia, paw withdrawal threshold (PWT) was assessed by Von Frey filaments (Cat.38450, Ugo Basile, Varese, Italy). Each animal was placed alone in a cage (20 cm×20 cm ×20 cm) with a wire mesh grid floor in a quiet room. To avoid disruption of the wound, Von Frey filaments were inserted vertically to the hind paw plantar surface adjacent to the incision. Each rat 5 was tested three times at intervals of 5 minutes.
Paw withdrawal thermal latency (PWTL) measured with test equipment (BME410C, Institute of Biological Medicine, Academy of Medical Science, China) to evaluate thermal hyperalgesia. A transparent plastic chamber (22 cm × 12 cm × 12 cm) with a glass floor (2 mm thick) was used to place rats. The plantar surface adjacent to the wound of right hind paw was focused by a radiant heat source under the glass floor. The time from onset of radiant heat to withdrawal of the rat hind paw was defined as the withdrawal latency to the heat stimulation. In order to prevent tissue damage, a cut off time of 25s was established. Each rat was tested three times at intervals of 5 minutes. Thermal latency was defined as the mean of three responses.

Experimental Protocol and Grouping
Rats were randomly divided into five groups (num=8/group): group N, rats underwent a sham operation and were administered with same volume of saline ; group I, rats underwent a incision operation and received saline; group R, received a subcutaneous injection of remifentanil but did not undergo surgery; group R+I, rats underwent a incision operation and received subcutaneous injection of remifentanil; group L, received an intrathecal injection of LPS-RS 30 min before plantar incision in remifentanil-treated rats. Incision surgery and drug injection were performed simultaneously.
The same investigator performed all the experiments. For von Frey and plantar tests, baseline responses were obtained one day before surgery after animals became familiar with the special assessment conditions without nociceptive stimulation. According to the above protocol, the experiments were performed 1 day later. PWT and PWTL tests were conducted at 2, 6, 24 and 48 hours postoperative. After the behavioral testing (48h after operation), immunofluorescence staining and Western blot analysis specimens were collected.

Immunofluorescence
Rats transcardially perfusion with saline, followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH = 7.4) While under deep anesthesia (5% sevoflurane). The lumbar segments (L4-6) of the spinal cord were then dissected and fixed in the same fixative for 3 h and then replaced with 30% sucrose overnight. Cryostat sections(10 mm) were cut and incubated with 20% 6 normal goat serum at room temperature for 30 minutes and then diluted with primary antibody anti-Iba-1(microglia marker 1:500; Abcam, Cambridge, UK, ab5076)) for 24 h at 4°C.
After incubation at 4 °C for one night, the sections were incubated in Cy3-conjugated and FITCconjugated secondary antibodies for 1h at room temperature. A fluorescence microscope (Leica, Frankfurt, Germany) was used to examine the section and images were captured using a Leica DFC350 FX camera. Each group included four rats for immunofluorescence quantification, and tissue sections of each animal were randomly selected for analysis.

Western Blotting
The lumbar segments (L4-6) of the spinal cord were removed rapidly and snap frozen in liquid nitrogen while under deep anesthesia (5%sevoflurane). Tissue samples were homogenized in lysisbuffer solution.The supernatant was obtained by centrifugation for 15 min, at 4°C for 13000 rpm.
Bradford method, a detergent-compatible protein assay with a bovine serum albumin as standard is used to determine the protein concentration. Samples (80μg) were separated on SDS-PAGE (10%) and transferred to PVDF membrane. The filter membranes were blocked with 5% nonfat milk for 1h at room temperature and incubated with rabbit antibody against TLR4 (1:500; Abcam, Cambridge, UK, ab13556), iba1(1:2000; Abcam, Cambridge, UK, ab5076) at 4°C overnight. TBST buffer was used to wash the membrane and then the membrane was incubated for 1h at room temperature with secondary antibody conjugated with horseradish peroxidase, observed in ECL solution for 1 minute, and then exposed for 1-10 minutes. The membrane was reprobed with antibody against beta actin

Immunofluorescence staining of microglia activation in spinal cord
Immunofluorescence staining was performed to localize and assess microglia activation in the spinal cord during the maintenance of hyperalgesia induced by intraoperative remifentanil infusion. The phosphorylation Iba-1 was located in the spinal cord was showed by typical photomicrographs ( Figure   3A). The mean optical density of Iba-1 in the spinal cord was summarized in Figure 3B. The microglia activation in the spinal cord was weak in rats receiving sevoflurane and saline without surgery, However there was a significant increase in the group I, R, R+I, and L compared to group N (all P < 0.01). Moreover, Intraoperative Remifentanil infusion significantly enhanced microglia activation in the spinal cord (P < 0.01). Conversely, pretreatment with LPS-RS could reduce the microglia activation in spinal cord caused by intraoperative infusion of remifentanil (P <0.01).

Western Blot Analysis
Western blot was used to quantify the expression of Iba-1 and TLR4 in the spinal cord during the Accordingly, the time points observed in this study were 2, 6, 12, 24 and 48h postoperative, and the time of taking materials was 48h after operation with the strongest hyperalgesia. The major shortcoming of this study is that it did not include TLR4 Immunofluorescence staining to further verify the TLR4 expression on the microglia. 9

Conclusion
In the present study we demonstrated the involvement of TLR4 signaling pathway in spinal cord in the mechanical allodynia induced by incisional pain-remifentanil. We found that intraoperative remifentanil infusion induced the upsurge of TLR4 and microglia activation in spinal cord, while the inhibition of TLR4 signaling using LPS-RS reduced mechanical allodynia and prevented the upregulation of TLR4 and microglia activation induced by incisional pain-remifentanil. Our findings revealed that TLR4 signaling pathway has an important role in incisional pain-remifentanil hyperalgesia, and that it could serve as the therapeutic target for persistent postsurgical pain.

Declarations
Ethics approval and consent to participate