Animals
All experiments were approved and performed accordance to the Animal Care and Use Committee of Hubei University of Medicine (Hubei, China). Male C57BL6/J mice were provided by the Institute of Laboratory Animal Science, Hubei University of Medicine. Mice were randomly divided into four groups that are sham, BCP, BCP + Con-lv, and BCP + RNAi-lv. Nontargeting control lentivirus (Con-lv) or β2m RNA interference lentivirus (RNAi-lv) were accurately injected into the hippocampus according to the coodinates of the mice received carcinoma cells, respectively. Animals were kept under an autocontrolled environment of 12 h light/dark cycle with food and water provided ad libitum.
Construction of Recombined MHC-I RNAi-Lentivirus
We selected β2m as the siRNA target to specifically knockdown MHC-I expression according to the previous published reports (Elmer et al. 2013; Fu et al. 2016). The vectors GV248 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) and β2m gene (GenBank accession number NM_009735) were performed by GeneChem Co. Ltd (Shanghai, China). The control lentivirus was similar to β2m RNAi-lentivirus except for expressing shRNAs. The viral titre of lentivirus we used was 3×108 TU/ml.
Preparation of the Lewis Lung Carcinoma (LLC) Ccells
Murine LLC line LL/2 (LLC1) (CRL-1642) cells were cultured in Dulbecco’s minimum essential medium-high glucose (DMEM-high glucose, Gibco, Thermo Fisher Scientific) added with 10% fetal bovine serum (Gibco) and then placed in 5% CO2/95% O2 at 37 °C. Cells were harvested by trypsinization with 0.05% trypsin-EDTA (Gibco) and settled after centrifugation at 1000 rpm for 5 min. Then the precipitated tumor cells were resuspended with phosphate buffered saline (PBS) and diluted to a final concentration (1×107 cells/ml) for the BCP model established.
BCP Model Establishment
BCP model was established as the previous published reports (Honore and Mantyh 2000; Wang et al. 2020). Briefly, under the isoflurane (3% induction and 2% maintenance) anesthesia, the right leg was shaved and performed with 0.5% iodophor, and then a superficial incision was made on the skin overlying the patella. The tibia was discreetly exposed and drilled by a 23-gauge needle, then tumor cells (5 μl, 2×105 cells/ml) was injected into the intramedullary canal of the bone by a 10 μl microinjector. For the sham group, the boiled carcinoma cells were prepared in the same final concentration for injection.
Microinjection of Lentivirus in the Hippocampus
Animals were anesthetized by intraperitoneal injection of 2% phenobarbital sodium and placed on a stereotaxic apparatus (DW-2000, Chengdu Techman Software Co.,Ltd, China). An ophthalmic ointment application could be to keep the corneal lubricated. The scalp was shaved, 0.5% iodophor applied, and a midline incision made to expose the cranium. Then the cranium was drilled bilaterally with a dental drill to create burr holes following the coordinates for hippocampus: AP ± 2.0 mm, ML ± 1.6 mm, DV -2. 6 mm. Virus was loaded with standard glass capillaries (Sutter Instrument) and infused at a rate of 300 nl/min by a microsyringe pump (Longer Pump, TJ-1A). The injection needle was left for an additional 5-10 min and then slowly withdrawn. The incision was stitched and animals were allowed to recover over a heating pad.
Nociceptive Tests
Mechanical Hyperalgesia
Mechanical allodynia of all animals were evaluated with a dynamic plantar esthesiometer (Ugo Basile, Comerio, Italy) to record paw withdrawal threshold (PWT). Measurements were performed on day 0, 7, 14, and 21 after surgery. Mice were placed individual in perspex box with a wire mesh at the bottom in a quiet environment for 30 min. A perpendicular metal filament with 0.5 mm diameter was raised to the plantar surface of the ipsilateral hind paw of mice, and the force was continuous enhanced until the hindlimb was withdrawn. The final mechanical force threshold on the esthesiometer was recorded as the PWT of the mouse. Each mouse was tested three times at 10-min intervals, and the mean of three values was used to statistically analyze PWT of the mice.
Hot/Cold Plate Test
Mice were placed on a hot/cold-plate (Ugo Basile, Comerio, Italy) for 50 °C and 4 °C, respectively. The latency to first lift the right hind paw during 90 s in hot plate test and 120 s in cold plate test period were recorded. Values were averaged across two separate trials for each mouse at an interval of 30 min.
Assessment of Depressive-Like Behaviors
Forced Swim Test
Forced swim test is widely used for assessment depressive-like behavior in rodents. Each mouse was forced to swim for 6 min in a cylindrical plastic container (45 cm in height and 20 cm in diameter) filled with 30 cm in depth of water (25 ± 1 °C). The last 5 min of immobile time were recorded as a reflection of despair and hopeless behavior of the mouse.
Tail Suspension Test
The mouse’s tail at 1 cm from the tip was taped to a table about 50 cm above the surface and tested for 6 min. The total duration of immobility was measured in the last 5 min. Mice were considered immobile when they passive swaying and completely motionless.
Immunohistochemistry
Under 2% pentobarbital sodium anesthesia, mice were transcardially perfused with PBS followed by cold 4% paraformaldehyde (PFA, Sigma). The brain was quickly removed, postfixed within the same fixative at 4 °C overnight, and then disposed with 30% sucrose in PBS for 48 h at 4 °C. Brain was embedded with frozen section medium (O.C.T compound, SAKURA) and sectioned on a freezing microtome (CM1950, Leica, Germany) at 30 μm in coronal planes. Slices were blocked with 5% donkey serum in PBS for 10 min at room tempreture after PBS washing and incubated with rabbit/mouse anti MHC-I antibody (1:50, Proteintech, 15240-1-AP, 66013-1-Ig), rabbit anti Iba-1 antibody (1:500, Wako, 019-19741), rabbit anti DAP12 antibody (1:200, LSBio, LS-B9453-50), and sheep anti TREM2 antibody (1:50, R&D, AF1729) overnight at 4 °C. After washing with PBS three times the next day, slices were incubated with Alexa fluorescent conjugated secondary antibodies (711-605-152, 706-545-148, Jackson ImmunoResearch, US and ab150177, abcam, UK) in darkness for 40 min at 37 °C. Slices were captured with a laser scanning confocal microscope (Leica TCS SP8, Wetzlar, Germany). For the microglia cell count in hippocampus, images were examined from three independent experiments of three mice per group under the same conditions and then performing statistics and analysis.
RT-PCR
Mice were euthanized after the behavioral tests, and then hippocampus were quickly removed. Total RNA was extracted from the hippocampus followed by Trizol reagent (Invitrogen, 15596026) and cDNA was synthesized using reverse transcribed kit according to the manufacturer’s instructions (Takara, Japan). The targeted genes expression was measured by quantitative real time PCR on a SYBR Green qPCR Master Mix reagent system (Takara, Japan). The primer sequences of forward (F) and reverse (R) are synthesized by Sangon Biotech (Shanghai, China) and shown as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH):
F: 5´-GTGAAGGTCGGTGTGAAC-3´,
R: 5´-TGAGTGGAGTCATACTGGAA-3´;
MHC-I: F: 5´-GAACTGCTACGTAACACAGTTC-3´,
R: 5´-GTATGTATCAGTCTCAGTGGGG-3´;
TREM2: F: 5´-GGAACCGTCACCATCACTCT-3´,
R: 5´-ATGCTGGCTGCAAGAAACTT-3´;
DAP12: F: 5´-GATTGCCCTGGCTGTGTACT-3´,
R: 5´-CTGGTCTCTGACCCTGAAGC-3´.
Western Blot
Animals were euthanized after the behavioral tests, and then hippocampus were separated and put in ice-chilled radio-immunoprecipitation assay (RIPA) lysis buffer. The supernatant was extracted from hippocampal tissues after homogenization and disintegration. Protein concentration was detected by the BCA protein assay kit (P0012, Beyotime Biotechnology, Shanghai, China). The denatured protein samples (30 μg) were separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P PVDF membranes (IPVH00010, Millipore), and then membranes were blocked with 5% nonfat dry milk diluted in Tris-buffered saline with Tween 20 (TBST) for 90 min at room temperature (RT). Membranes were incubated with mouse anti-α-tubulin (1:5000, Sigma, T5168), rabbit anti MHC-I (1:1000, Proteintech, 15240-1-AP), rabbit anti DAP12 (1:1000, LSBio, LS-B9453-50), and sheep anti TREM2 (1:2000, R&D, AF1729) overnight at 4 °C. Then membranes were washed with TBST, and probed with anti-mouse, anti-rabbit, and anti-sheep secondary antibody conjugated with horseradish peroxidase (1:5000, Cell Signaling Technology, 7076; 1:5000, Cell Signaling Technology, 7074; 1:5000, Sigma, A3415) at RT for 90 min. The protein bands were detected by enhanced chemiluminescence (WBKLS0500, Millipore) after TBST washed and analyzed with Image Lab software (Bio-Rad).
Statistical Analyses
GraphPad Prism 8.0 software (La Jolla, USA) was applied to perform statistical analysis. All data were shown as mean ± S.E.M. T-tests and one-way analysis of variance (ANOVA) followed by post hoc Bonferroni’s test were performed to compare the differences between two groups and among multiple groups. P < 0.05 was identified as statistically significant. Sample size for each experiment is presented in the figure legends.