Animals
In all experiments we used Adult male Sprague-Dawley rats (240–260 g, provided by the Laboratory Animal Center of the Military Medical Science Academy of the PLA ). The animals were fed confirming to the protocols approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. All experimental operations were approved by the committee of experimental animals of Tianjin Medical University.
Drugs
Remifentanil hydrochloride purchased from RenFu Co. (Guangzhou, China) was dissolved in saline (NaCl 0.9%) and infused intravenously at a rate of 1.2 µg·kg− 1·min− 1 for 60 min, based on a previously reported dose[16]. Controls received the same volume of saline under identical conditions. Sevoflurane was purchased from Maruishi Pharmaceutical Co. (Osaka, Japan). IL-1ra was purchased from Abcam (Cambridge, England). A438079 was purchased from Tocris Bioscience (Bristol, England). Sterile saline was the vehicle for all drugs, except acYVAD-cmk [3% (vol/vol) DMSO]. (+)-Naloxone, ac-YVAD-cmk and DMSO were purchased from Sigma Aldrich (St. Louis, MO, USA). For intrathecal drug delivery, the catheters were preloaded with drugs at the distal end in a total volume and delivered over 20–30 s once the catheter was in position. Intrathecal doses were as follows: IL-1ra: 100 µg in 10 µL; (+)-naloxone: 1200 µg in 10 µL; A438079: 600 ng in 10 µL; and ac-YVADcmk: 20 µg in 10 µL, based on a previously reported dose[17]. Respective equivolume vehicles were used as controls.
Intrathecal Catheter Implantation
The intrathecal catheter implantation was on the basis of previously described methods[17, 18]. Briefly, an intrathecal polyethylene (PE-10) catheter was inserted into the subarachnoid space at the level of the spinal cord lumbar enlargement segments through an incision at the atlanto-occipital membrane. After a 1-week recovery, rats that showed any neurologic impairment according to locomotor function testing were discarded.
Anesthesia And Surgical Procedure
Animals were anesthetized with sevoflurane (induction, 3.0% v/v; surgery, 1.0% v/v) via a nose mask. We used the postoperative pain model described by Brennan et al[19]. We made a 1-cm longitudinal incision with a number 11 blade through the skin and fascia of the plantar aspect of the left hind paw. The incision started 0.5 cm from the proximal edge of the heel and extended towards the toes. Then, we elevated the plantaris muscle and incised longitudinally. The muscle origin and insertion remained intact. After haemostasis was achieved, we closed the skin with two 4 − 0 silk sutures. We covered the wound with erythromycin ointment for prevention of infection.
Experimental Protocol
Experiment One: The changes in mechanical and thermal hyperalgesia, IL-1β expression, GLT-1, Phospho-NR1 and the indicators of NLRP3 inflammasome activation in RIH (TLR4, P2 × 7R, NLRP3 and caspase-1).
The animals were randomly divided into 4 groups (n = 8) based on a randomization list provided by the Department of Anaesthesiology, Tianjin Medical University General Hospital according to relevant Standard Operating Procedure(computer-generated random number system): Sham + NS group: animals underwent a sham operation with NS infusion; Sham + Remifentanil group: animals underwent a sham operation with a remifentanil infusion; Incision + NS group: animals underwent a surgical incision with an NS infusion; and Incision + Remifentanil group: animals underwent a surgical incision with a remifentanil infusion. Remifentanil hydrochloride was dissolved in saline (NaCl 0.9%) and infused intravenously at a rate of 1.2 µg·kg− 1·min− 1 for 60 min. The animals in the Sham + NS and Incision + NS groups received the same volume of saline under identical conditions. The withdrawal threshold and withdrawal latency to mechanical and thermal stimulation, respectively, were evaluated at baseline (24 h before remifentanil infusion) and 2 h, 6 h, 24 h and 48 h after remifentanil infusion. The L4-L6 segments of the spinal cord were collected after the last behavioural test to measure the expression of IL-1β as well as GLT-1, Phospho-NR1 and the indicators of NLRP3 inflammasome activation (TLR4, P2 × 7R, NLRP3 and caspase-1).
Experiment Two: The effects of an IL-1β inhibitor, IL-1ra, on mechanical and thermal hyperalgesia, GLT-1, and Phospho-NR1 in RIH.
The animals were randomly divided into 3 groups (n = 8): Sham + NS + vehicle group: animals underwent a sham operation with an NS and vehicle infusion; Incision + Remifentanil + vehicle group: animals underwent a surgical incision with a remifentanil and vehicle infusion; and Incision + Remifentanil + IL-1ra: animals underwent a surgical incision with a remifentanil infusion and IL-1ra. Remifentanil was administered as in the above experiment. IL-1ra (100 µg in 10 µL) was intrathecally administered immediately before remifentanil infusion. The animals in the Sham + Remifentanil + vehicle group and Incision + Remifentanil + vehicle group received the same volume of the vehicle under identical conditions. The withdrawal threshold and withdrawal latency to mechanical and thermal stimulation, respectively, were performed at baseline (24 h before remifentanil infusion) and 2 h, 6 h, 24 h and 48 h after remifentanil infusion. The L4-L6 segments of the spinal cord were collected after the last behavioural test to measure the levels of GLT-1 and Phospho-NR1.
Experiment Three: The effects of (+)-naloxone (a TLR4 inhibitor), A438079 (a P2 × 7R inhibitor) and ac-YVADcmk (a caspase-1 inhibitor) on mechanical and thermal hyperalgesia, IL-1β, GLT-1, and Phospho-NR1 in RIH.
The animals were randomly divided into 5 groups (n = 8): Sham + NS + vehicle group: animals underwent a sham operation with an NS and vehicle infusion; Incision + Remifentanil + vehicle group: animals underwent a surgical incision with remifentanil and vehicle infusion; Incision + Remifentanil+(+)-naloxone group: animals underwent a surgical incision with a remifentanil infusion and (+)-naloxone; Incision + Remifentanil + A438079 group: animals underwent a surgical incision with a remifentanil infusion and A438079; and Incision + Remifentanil + ac-YVAD-cmk group: animals underwent a surgical incision with a remifentanil infusion and ac-YVAD-cmk. Remifentanil was administered as in the above experiment. (+)-Naloxone (1200 µg in 10 µL), A438079 (600 ng in 10 µL), and ac-YVAD-cmk (20 µg in 10 µL) were intrathecally administered immediately before remifentanil infusion. The animals in the Sham + NS + vehicle group and Incision + Remifentanil + vehicle group received the same volume of the vehicle under identical conditions. The withdrawal threshold and withdrawal latency to mechanical and thermal stimulation, respectively, were performed at baseline (24 h before remifentanil infusion) and 2 h, 6 h, 24 h and 48 h after remifentanil infusion. The L4–L6 segments of the spinal cord were collected after the last behavioural test to measure the levels of IL-1β, GLT-1 and Phospho-NR1.
Behavioural Tests
Mechanical Hyperalgesia
We used an electronic Von Frey filament (BSEVF3, Harvard Apparatus, USA) to measure the paw withdrawal threshold (PWT). Animals were placed in wire chambers (20 cm × 20 cm × 20 cm) with a grid bottom. The animals were allowed to acclimatize for 1 h before testing. The filament was applied vertically to the area adjacent to the wound of the left hind paw with increasing pressure. the PWT was defined as the pressure (g) at which the rat flinched, shook, or licked its paw. The test was repeated three times at 5-minute intervals. The mean of the 3 trials was regarded as the PWT. We used a cut-off pressure of 60 g to avoid tissue damage.
Thermal Hyperalgesia
We used an intellective hot plate equipment (YLS-6B, Zheng Hua Biological Instrumentation Co., China) to measure the paw withdrawal latency (PWL). Animals were allowed to acclimatize to the environment for 1 h before testing. They were placed on a hot plate (50℃). The PWL was defined as the time (s) when the rat appeared positive response (a clear paw withdrawal). The test was repeated three times at 10-minute intervals. The mean of the 3 trials was regarded as the PWL. We used a cut-off time of 30 s to avoid tissue damage.
Tissue Preparation
We used the method of overdose anesthesia for euthanasia. Animals were anesthetized with pentobarbital (130 mg/kg) for intraperitoneal injection. The L4 − 6 spinal cord was rapidly removed and frozen in liquid nitrogen.
Quantification of mRNA via Real-time PCR
The levels of IL-1β mRNA in the spinal cord were determined after the last behavioural test. The L4 − 6 spinal cord was removed and frozen in liquid nitrogen. Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA). Then, total RNA was transcribed into cDNA with a cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The expression of IL-1β mRNA was determined by real-time PCR using SYBR Green PCR Master Mix (Roche, Mannheim, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Each test was run in triplicate. The primers of IL-1β and GAPDH were designed and synthesized by SBS Genetech Co., Ltd. (IL-1β Forward 5’-GAGGCTCCATCTCCAAGGAC-3’, Reverse 3’-ACTGTGTGTGACAGGTTGGA-5’; GAPDH Forward 5’-TGATGGGTGTGAACCACGAG-3’, Reverse 3’-ATCACGCCACAGCTTTCCAG-5’). IL-1β gene expression was calculated with the delta-delta-Ct method[20].
Protein Analysis via Western Blot
The tissues from the L4 − 6 spinal cord were removed after the last behavioural test and frozen in liquid nitrogen. Tissues were homogenized in an SDS sample buffer containing proteinase inhibitors. The homogenate was centrifuged at 4 ℃(12,000 rpm, 10 minutes), and the he supernatant liquor was pipette as the total protein. The protein content was determined using the bicinchoninic acid (BCA) assay method. Before the proteins were transferred to PVDF membranes, they were separated on an SDS-PAGE gel. The membranes were blocked with 5% non-fat milk for 1 h on the shaker
and subsequently incubated with primary antibody rabbit anti-phospho-NR1 (1:500, ab75680, Abcam), rabbit anti-GLT-1 (1:1000, ab106289, Abcam), rabbit anti-TLR4 (1:1200, ab13867, Abcam), rabbit anti-P2 × 7R (1:1000, ab48871, Abcam), rabbit anti-NLRP3 (1:1000, ab214185, Abcam) and rabbit anti-caspase-1 antibodies (1:1000, ab62698, Abcam) overnight at 4 ℃. The membranes were washed with TBST buffer for 30 min and incubated with secondary antibody (goat anti-rabbit IgG, HRP, 1:5000, ab7090, Abcam) for 1 h at room temperature. The proteins were visualizedby enhanced
chemiluminescence detection (Millipore, Billerica, MA) using a Bio-Rad GS-700 imaging system with software (Bio-Rad, Hercules, CA,USA). β-Actin was used as a loading control. The results are expressed as the percentage of β-actin immunoreactivity.
Enzyme-linked Immunosorbent Assay (ELISA)
The level of IL-1β in the rat L4 − 6 ipsilateral dorsal spinal cord was detected by ELISA kits (Abcam) according to the attached instructions of the kits.
Statistical Analysis
GraphPad Prism 5 was used to generate the graphics and perform statistical analyses. All data were expressed as the mean ± standard deviation (SD). In nociceptive behavioural tests, a repeated measures analysis of variance (ANOVA) was performed to determine the overall differences in the PWT and PWL at each time point. To analyze the results of real-time PCR, western blot and ELISA, a one-way ANOVA was used. A P value less than 0.05 was considered statistically significant.