1. Animals
Male C57BL/6J mice (The Laboratory Animal Center of the Military Medical Science Academy, Beijing, China) at 6–8 weeks of age weighing 20–25 g were utilized in the following experiments. Animals were housed in a controlled environment (12-h light/dark cycle, room temperature 20–25°C, 55–65% relative humidity) with free access to food and water. The mice used in the experiment experienced the least amount of discomfort possible. Protocols were approved by the Scientific and Ethics Committee of Tianjin Medical University and adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.
2. Surgical procedures
The following steps were taken in accordance with Bennett’s method[18]. Mice were anaesthetized with 2% isoflurane. Loose ligation of the sciatic nerve was carried out under aseptic conditions. Briefly, the right sciatic nerve was exposed at the level of the proximal thigh and freed of connective tissue. Four loose ligations of the sciatic nerve were performed without any constriction of the nerve. Ligatures were tied loosely around the nerve with 5 − 0 silicon-treated silk sutures at a spacing of 1 mm to preserve epineural circulation. Control animals underwent a sham operation in which the sciatic nerve was exposed but not ligated. The incision was closed in layers with sutures. Each group of mice had nerve injury for the observation of thermal latencies and mechanical thresholds up to postoperative day (POD) 14.
3. Reagents
GW7647 (PPARα agonist, Tocris Bioscience) was prepared at a concentration of 0.5 mg/mL in dimethyl sulfoxide (DMSO). Mice underwent a chronic constriction injury (CCI) procedure or sham surgery and were administered 5 mg/kg GW7647 1 h before CCI via intraperitoneal injection[19]. GW6471 (PPARα antagonist, Tocris Bioscience) was prepared at a concentration of 2 mg/mL in DMSO. Mice were intraperitoneally injected with 20 mg/kg GW6471 1 h before CCI[20]. Subsequent injections were administered every other day until the mice were sacrificed on POD 14.
4. Paw withdrawal mechanical threshold
Mechanical sensitivity was measured by the von Frey test, following previously reported methods[14]. Mice were kept in a cage with an elevated metal mesh floor in a quiet area for 30 minutes before the experiment. Several bending fibres of a von Frey calibrated monofilament (Stoelting Co., Wood Dale, IL, United States) were manually applied to the midplantar region of the hind paw. Positive reactions, which were characterized as quick paw withdrawal or licking, shaking or flicking of the paw in response to the applied stimulus, were counted. The threshold force of the reaction (in grams) was defined as the first filament that caused at least 2 positive reactions in 5 applications. To establish a baseline, the paw withdrawal threshold (PWT) was first measured 1 day prior to injury, and measurements were repeated on days 1, 3, 5, 7, 10, and 14 after injury.
5. Paw withdrawal thermal latency
As described by Heremanszm[21], using a glass cylinder, a mouse was confined to the heated surface of a hot plate that was maintained at a temperature of 52°C. The latency period before the mouse jumped or restrained/withdrew/licked the paw was recorded, with a cut-off time of 30 seconds to avoid damaging the skin of the mouse's hind paw. Each mouse was tested three times, with each test at least fifteen minutes apart to ensure experimental accuracy.
6. H&E staining
Sevoflurane was utilized as a deep anaesthetic before transcardial administration of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4; 4°C) and 37°C normal saline. The sciatic nerve was then promptly removed, preserved for 48 h in 10% buffered formalin, and prepared for standard paraffin histology. Haematoxylin and eosin (H&E) was used to stain 5-µm-thick paraffin-embedded slices from mice of the various groups[22].
7. Immunofluorescence
At the end of the experiment, mice were deeply anaesthetized with sevoflurane and perfused intracardially with PBS followed by fresh 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for tissue fixation. Following perfusion, the L4-6 spinal cord segments were removed from all mice. A corresponding segment of the L4-6 spinal cord was also harvested from a normal mouse. All tissues were postfixed in 4% paraformaldehyde for 7 h and 10%, 20%, and 30% sucrose solutions for 24 h. All spinal cord tissue was embedded in O.C.T., frozen, and cut into 5-µm-thick sections. After permeabilization and blocking, the sections were incubated with anti-PPARα (1:200, ab61182, Abcam, UK), anti-Iba-1 (1:200, Cat#17198, Cell Signaling Technology, Beverly, MA, USA), anti-GFAP (1:400, Cat#3670, Cell Signaling Technology, Beverly, MA, USA) and anti-NeuN (1:200, ab177487, Abcam, UK) primary antibodies at 4°C overnight. The sections were washed three times with PBS before being exposed for one hour to a suitable secondary antibody. Finally, DAPI was used to stain the nuclei in the sections. Fluorescence microscopy was used to observe the outcomes (Olympus, Tokyo, Japan).
8. Sample harvesting and processing
Two weeks following the nerve injury (POD 14) and after observation of NP behaviour was complete, mice were anaesthetized with inhalation of sevoflurane. Blood samples were taken from the tip of the heart. After standing at room temperature for 1 h, samples were centrifuged, and the supernatant was collected. Then, the mice were sacrificed, the colon was removed and cut open, and stool samples were taken carefully under the same aseptic conditions. Liquid nitrogen was used to store all collected specimens.
9. Microbial DNA extraction and sequencing
Using the CTAB/SDS technique, whole genomic DNA was extracted from the samples. Using 1% agarose gels, DNA concentration and purity were evaluated. Using barcode-specific primers, polymerase chain reaction (PCR) amplification of the 16S V3-V4 region was carried out (primer: 16S V3-V4: 341F-806R). The PCR products were then separated on a 2% agarose gel and purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Then, using the NEB Next® Ultra TM DNA Illumina Library Preparation Kit (NEB, USA), a sequencing library was created with an index code inserted in accordance with the manufacturer's instructions. The quality of the library was evaluated using an Agilent Bioanalyzer 2100 system and a Qubit@ 2.0 fluorometer from Thermo Scientific. Finally, paired-end reads of 250 bp/300 bp were generated after the library was sequenced on the Illumina MiSeq platform.
10. Analysis of 16S rDNA sequencing data
Paired-end reads from the initial DNA fragments were combined using FLASH. Due to the unique barcodes, paired-end reads were matched to each sample. The UPARSE software package's algorithms UPARSE-OUT ref and UPARSE-OTU were used to analyse the sequences. Internal Perl scripts were utilized to examine alpha (within-sample) and beta (among-sample) diversities. Sequences with a similarity of 97% were grouped under the same operational taxonomic unit (OTU). For each OTU, a representative sequence was chosen to gather classification data using the RDP classifier. To determine alpha diversity, we rarefied the OTU database and computed the Chao1, Simpson, and Shannon indices. Beta diversity was calculated using the unweighted UniFrac distance for principal coordinate analysis (PCoA) using the QIIME program.
11. LC‒MS/MS untargeted metabolomics analysis
After sample pretreatment, metabolites were extracted, and ultra-high-performance liquid chromatography (UHPLC) (1290 Infinity LC, Agilent Technologies) with quadrupole time-of-flight was used to analyse the results (AB Sciex TripleTOF 6600). The instrument was operated under the following conditions: chromatographic conditions: chromatographic column, ACQUITY UPLC BEH (2.1 mm ×100 mm, 1.7 µm); column temperature, 25°C; mobile phase, ammonium acetate and hydroxide mixed at 5 and 25 mM, respectively, in water (A) and acetonitrile (B); injection volume, 2 µL; and flow rate, 0.5 mL/min; mass spectrum conditions: ion source, ESI; Ion Source Gas1 (Gas1), 60; Ion Source Gas2 (Gas2), 60; curtain gas (CUR), 30; source temperature, 600°C, and IonSpray Voltage Floating (ISVF) ± 5500 V. Positive and negative ion scanning techniques were used for sample mass spectrum signal capture. Using XCMS software, we preprocessed the raw data and performed qualitative and quantitative analyses.
12. Statistical analysis
GraphPad Prism software (version 8.0) and SPSS statistical software (version 21.0) were used for the statistical analysis. The log rank test was used for analysis. All values are reported as the mean ± standard deviation (SD), and differences among means were determined using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. P < 0.05 was regarded as statistically significant for all tests.
Student's t test (SPSS 23.0) for ANOVA was used for statistical analysis of the data. The differences in microbial communities were investigated using analysis of similarities (ANOSIM), multiple response permutation procedures (MRPPs), and Bray‒Curtis differential distance matrix analysis. Linear discriminant analysis effect size (LEfSe) was used for quantitative biomarker analysis in different groups. P < 0.05 indicated statistical significance, and the significance test was a two-tailed test.