Materials and Methods
Animals
The male Sprague-Dawley rats (200−250g) were purchased from the Animals Experiment Center of Zhengzhou University (Zhengzhou, Henan, China). All rats were individually housed in clear plastic cages (water and food ad libitium,21 ± 1°C, humidity 45%−55%, 12−12h light/dark cycle). All experimental procedures were approved and reviewed by the Experimental Animals Care and Use Committee of Zhengzhou University, and consistent with the guidelines provided by the National Institutes of Health for the Care and Use of Laboratory Animals.
Intrathecal Catheterization
Intrathecal catheters were conducted following a previous study[17]. In brief, rats were anesthetized with phenobarbital (60 mg/kg,ip.), and then a sterile PE−10 catheter (inner diameter 0.3 mm,outer diameter 0.5 mm,Anilab Software & Instruments, Ningbo, Zhejiang,China) was insert into the subarachnoid cavity between L4 and L5 vertebral level, the catheter was fixed and tunneled to the back of neck. To confirm the placement of catheter, 10µl of 2% lidocaine was injected into subarachnoid through the catheter on the third day. Showing an immediate temporary motor block of both hind limbs considered to have proper placement. Then, rats were allowed to recover 7 days for the behavioral experiments.
Drug Administration
The morphine hydrochloride (2 µg/µl, Shenyang First Pharmaceutical Factory, Shenyang,China) was diluted in saline. The GLX351322 (Med Chem Express, MCE, China) was dissolved in 10% (v/v) DMSO (Solarbio® Life Sciences, Beijing, China) to 100 µM and stored at−80°C until use. The 4-PBA (Selleck Chemical, Houston, USA) was diluted in 30% (v/v) DMSO and stored at−80°C until use. GLX351322(20 µM,15 µl)[18], 4-PBA(100 µg)[19] or vehicle were administrated through the PE−10 catheter 30 min before morphine injection and followed by 10 µl saline to flush the PE−10 catheter.
Morphine Tolerance and Behavioral Assessment
Morphine (10 µg, twice daily) was intrathecally injected for 9 consecutive days to induce morphine tolerance. An equal volume of saline was received in control group at the same time points. The antinociceptive effect of morphine was assessed 30 min after morphine intrathecally injected by the hot water tail-flick assay from day 1 to day 9, and the tail-flick test was performed in the morning. Briefly, the animals were placed in a plastic box, and the distal one-third of the tail was immersed into the water at 50 ± 0.2°C. The latency response was defined as the rapid removal of the tail remaining in the hot water. A 15 s cutoff time was used to minimize the tail injury. The analgesic effect of morphine was used to evaluate by the percentage of maximal possible antinociceptive effect (%MPE), %MPE=[(test latency-baseline latency)]/(15s - baseline latency).
Western Blots
Western Blots were conducted by a previous research[20]. Briefly, after 9 days of behavioral tests, rats were anesthetized with pentobarbital sodium (i.p. 60mg/kg), the segments of L3-L5 spinal cord were rapidly removed and homogenized using radio immunoprecipitation assay lysis buffer combined with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor. Proteins concentration of the supernatants were gauged by using bicinchonininc acid (BCA) protein quantitation kit. Twenty-five µg proteins were separated and loaded on 10% or 12% SDS-PAGE, and transferred onto polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked by 5% bovine serum albumin (BSA) for 1.5h at room temperature and incubated overnight at 4°C with the primary antibodies (Table 1). Then, the membrane was washed and incubated with HRP-conjugated second antibodies. Finally, the membrane was detected with ECL reagent (K22030, Abbkine, Wuhan, China), and the results were measured and normalized to GAPDH using the Image Lab software (Bio-Rad,USA).
Table 1
The primary and secondary antibodies for WB and IF.
Antibody | Provider | Host | Catalog number | Dilution for WB | Dilution for IF |
NOX4 | Proteintech | Rabbit | 11587-AP | 1:1000 | 1: 40 |
BIP | Proteintech | Rabbit | 14347-AP | 1:1000 | 1: 35 |
PERK | Affinity | Rabbit | AF5034 | 1:1000 | |
P-PERK | Affinity | Rabbit | DF7576 | 1:1000 | 1: 35 |
IRE1 | Affinity | Rabbit | DF7709 | 1:1000 | |
P-IRE1 | Affinity | Rabbit | AF7150 | 1:1000 | 1: 40 |
ATF6 | Affinity | Rabbit | DF6009 | 1:1000 | 1: 35 |
LC3B | Abmart | Rabbit | T55992 | 1:1000 | 1: 35 |
P62 | Abmart | Rabbit | T55546 | 1:1000 | |
GAPDH | Proteintech | Rabbit | 10494−1-AP | 1:5000 | |
NeuN | Proteintech | Mouse | 66836−1-Ig | | 1: 100 |
GFAP | CST | Mouse | #3670 | | 1: 200 |
Iba−1 | Abcam | Goat | Ab5076 | | 1: 100 |
GAD67 | Abmart | Mouse | MG510328 | | 1: 40 |
Anti-Rabbit IgG HRP | Aspen | Goat | AS1107 | 1:5000 | |
CoraLite594 – conjugated Goat Anti-Rabbit IgG(H + L) | Proteintech | Goat | SA00013−4 | | 1:100 |
CoraLite488-conjugated Goat Anti-Mouse IgG(H + L) | Proteintech | Goat | SA00013−1 | | 1:100 |
CoraLite594 – conjugated Donkey Anti-Rabbit IgG(H + L) | Proteintech | Donkey | SA00013−8 | | 1:100 |
Fluorescein (FITC)–conjugated Affinipure Donkey Anti-Goat IgG(H + L) | Proteintech | Donkey | SA00003−3 | | 1:100 |
Immunofluorescence Staining
Rats were anesthetized with pentobarbital 60 mg/kg,i.p, and perfused with ice-cold 0.01M PBS and followed by 4%(v/v) paraformaldehyde (PFA) in 0.01M PBS. The L3-L5 spinal cord lumbar were immediately dissected and fixed in 4%(m/v) PFA at 4°C overnight, dehydrated in 20% and 30% sucrose solution for 48h at 4°C, and embedded in paraffin. The spinal tissue was sliced in 3 µm by a cryostat (RM2245, Leica, Germany). Next, the section was dried at 65°C for 1h, and rinsed with PBS, Tris-EDTA buffer (PH9.0) was used to perform antigen repair. The sections were blocked with 10% donkey serum for 40 min at 37°C, and then incubated with the primary antibody (Table 1) overnight at 4°C, washed with 0.01M PBS and incubated with the followed secondary antibody (Table 1). The images were captured by a fluorescence microscope (ZIESS, Germany), and were analyzed by Image-Pro Plus 4 software(Media Cybernetics, Maryland, USA).
For double immunofluorescence, the sections were incubated with each primary antibody (Table 1) and NeuN/GFAP/Iba−1 overnight at 4°C, and then washed with 0.01M PBS and mixed with the secondary antibody (Table 1) for 2 h at room temperature. The images were captured by a fluorescence microscope (ZIESS, Germany), and were analyzed by Image-Pro Plus 4 software (Media Cybernetics, Maryland, USA).
Transmission Electron Microscopic Examination
Rats were anesthetized with pentobarbital 60 mg/kg, i.p, and transcardially perfused with ice-cold 0.01 M PBS and 4%(m/v) PFA. The L3-L5 spinal cord were quickly removed and fixed in 2.5% (v/v) glutaraldehyde for 4h at 4°C, post-fixed with 1% osmium tetroxide in 0.01 M PBS for 2 h at 20°C, dehydrated and embedded in epoxy resin, the spinal segments were sliced in 60 nm thickness ultramicrotome and stained with 2%(v/v) uranyl acetate and lead citrate. All images were captured by a Hitachi 7700 TEM system.
Statistical Analyses
All the data were presented as mean ± SEM and analyzed using Graphpad Prism 9 (Graphpad Software,San Diego,USA). The data of behavioral tests were analyzed by two-way ANOVA followed by Bonferroni Post hoc tests to assess the changes between each group after drug injection each time. The results of western blot were analyzed by one-way ANOVA followed by Post hoc Tukey's tests. All the statistical significance were considered at P < 0.05.