Construction of lentiviral vectors expressing PKCγ shRNA
The virus was packaged using the shRNA interference sequence TGAATGTGCACCGACGCTG plasmid pLVTHM (Shanghai Jikai Gene Co., Ltd,Shanghai,China ) and the lentiviral packaging plasmid constructed with the highly efficient and specific rat PKCγ gene. The shRNAs were cloned into lentiviral vector pLVTHM-GFP (Shanghai Jikai Gene Co., Ltd,Shanghai,China). To generate the lentivirus, the recombinant vector and packaged plasmids were cotransduced into 293T cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Lentivirus with the human U6 promoter carrying shPKCγ were generated as previously described[8]. He final titer of recombinant virus was 1.25 × 109TU/ml.
Preparation of carcinoma cell
the MADB-106 cells were maintained in Dulbecco's Modified Eagle's Medium(DMEM),supplemented with 10% Fetal bovine serum,100 unit/ml penicillin,and 100 μg/ml streptomycin;and cultured at 37℃ in the humidified atmosphere of 5% CO2;and then passaged weekly according to ATCC guidelines. For adminstration,cells were detached by scraping and then centrifuged at 900 rpm for 3 min. The pellet was suspended in Hank's balanced salt solution. Cells in logarithmic growth phase were selected for experiments and then used for intra-tibial injection.
Animals and grouping
40 female adult SD rats, weighing 180-200 g, were provided by the Experimental Animal Center of Shandong University (Jinan, China). All animal procedures were carried out in accordance with the recommendation of the Principles of Laboratory[9] and the ethics committee of the International Association for the Study of Pain[10]. The study was approved by the ethics committee for Animal Care and Use Committees of the Experimental Animal Center of the Second Hospital of Shandong University (Jinan, China) prior to the onset of the experiments (Permit number: KYLL-2017(LW)017). The number of animals was used as little as possible and their suffering was minimized to the lowest degree according to IASP guidelines[10]. All rats were maintained in the following identical conditions: A controlled temperature of 22of˚C, a 12 h light/dark cycle and ad libitum access to food and water. 1 week later, the rats were randomly and equally divided into five groups: normal control group (Naive group), sham operation group (Sham group), Bone cancer pain model group (BCP group), empty lentiviral vector group(Vehicle group)and PKCγ/shRNA recombinant lentiviral vector group (PKCγ group). Normal control group (Naive group): The experimenters were blinded to the pharmacological treatment while processing data and making exclusion decisions. Naïve group healthy rats without any treatment. Sham operation group (Sham group):unilateral intra-tibial injection of Normal saline. Bone cancer pain model group (BCP group): unilateral intra-tibial injection with 10 μl MADB-106 cells (cell density 4.8×109/ml) (from Cancer Institute of Concord Medical University of Chinese Academy of Medical Sciences). The initial treatment of Vehicle group and PKCγ group were the same as that of BCP group.
Recombinant lentivirus administration into rACC
Rats were implanted with stainless steel cannula for intra-rACC drug infusions. For microinjection studies, rats were anesthetized with intraperitoneal chloral hydrate (40 mg/kg), and were firmly fastened into a brain stereotactic apparatus with the lambda and bregma at horizontal level. A 30-gauge stainless steel cannula with a 33-gauge stainless steel stylet plug was bilaterally implanted 0.5 mm above the rACC injection site [2.6 mm anterior to bregma, 0.6 mm lateral from the midline,2.5 mm beneath the surface of the skull] or the prefrontal cortex(PFC) [2.6 mm anterior to bregma, 0.6 mm lateral from the mid-line, 3.7 mm beneath the surface of the skull] in-line with the atlas of Paxinos and Watson[11]. A 1μL Hamilton syringe with PE-10 tubing was linked to the cannula that extended 0.5 mm over the tip of the guide cannula. The cannula was fixed with denture cement, and all surgical procedures were performed under sterile conditions. Animals were allowed to recover for one week before the next experimental procedure. Before and At the end of the experiment, brains were sectioned for cresyl violet staining to verify cannula position and injection site. A week after operation, rACC were injected per hemisphere both with PKCγ/shRNA recombinant lentivirus 6.2×10^6 particles(1μL) in PKCγ/shRNA group and empty vector recombinant lentivirus 6.2×10^6 particles(1μL) in Vehicle group over a 5 min period.
Establishment of rat BCP model
Rat Bone Cancer Pain Model was established as previously described[4]. The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (300m/kg). Superficial incisions were made in the skin overlying the patella to expose the tibia head with minimal damage. A 23-gauge needle was inserted at the site of intercondylar eminence and pierced 5-10mm below the knee joint into the medullary cavity of tibia. In PKCγ/shRNA group and BCP group the Needle was then removed and 10 μL of mammary carcinoma cell suspension or 10 μL of heat-killed mammary carcinoma cell suspension into a 50 μL micropipette. The needle was slowly inserted into the tibia cavity and injected in the carcinoma cells. The injection site was closed with bone wax quickly after the syringe was removed to prevent the cell suspension from leaking out. The wound was sutured to avoid leaving a dead space and was disinfected with iodophors to prevent infection.
Pain thresholds analysis
Before baseline testing, the rats were habituated to the testing environment for 5 days. Baseline data were tested both before and after using the von Frey hair stimulation. 3 rats that showed obviously different data between these two tests were discarded. For the remaining animals internalized in the subsequent studies, the average of these two baseline tests was recorded as a baseline data. The experimental rats were placed in a plastic cage (10x10x15cm) with a Plantar Von Frey TM Dynamic Plantar Stimulator at the bottom, and the cage was placed on a wire mesh plate for experimental operation and observation. After 15 min accommodation, mechanical allodynia was measured as the hind paw withdrawal response to von Frey hair stimulation according to the up-down method. An ascending series of von Frey hair with logarithmically incremental stiffness (1.0, 2.0, 4.0, 6.0, 8.0,15.0 and 20.0g) were applied perpendicularly to the mid-plantar surface (avoiding the less sensitive tori) of each hind paw. The stimulus lasted for 10 seconds, and the interval between each measurement was 10 minutes. The minimum stimulus that caused rat paw withdrawal was defined as the mechanical withdrawal threshold.
Rats were placed under a cage on a glass plate that was elevated to allow maneuvering of a radiant heat source from below. Controlled radiant heat stimuli were applied to the plantar surface of the hind-paw (BME-410A bolometer, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences). The time from onset of radiant heat application to withdrawal of the rat’s hind paw was defined as the thermal withdrawal latency. The glass plate was kept dry and clean during the measurement. Both hind paws were tested independently with a 5 min interval between trials,so that pain can be restored to normal. A cut-off time of 20 s was imposed on the stimulus duration to prevent tissue damage. Each rat was tested on the paw three times and the average value was taken.
Western blot and immunofluorescence
A total of 14 days after operation, each group of rats were anaesthetized with pentobarbital prior to the perfusion of 100 ml NS through the ascending aorta and then rapidly sacrificed by decapitation. The rACC tissues were immediately removed five rats randomly selected at each time point for measurement) and snap frozen in liquid nitrogen until all the samples were collected for immunohistochemical DAB staining to detect the expression of PKCγ in rACC tissues. On the 7th day after operation, brain tissue neurons were taken from each of the five groups of rats, and the expression level of PKCγ subunit was measured by Western blot and immunofluorescence. On the 14th day after operation, the same operation as on the 7th day after surgery was performed again.
Fluorescent microscope test
After the perfusion, the rACC tissues were immediately removed, post-fixed in the same fresh fixative for 4 h at 4 ℃, and placed in 30% sucrose in 0.1 M phosphate buffer for 24 h at 4℃. The rACC tissues were cut into 20-μm-thick frontal sections with a cryostat. The sections were washed with PBS, mounted onto clean glass slides, dried at 4 ℃(protected from light), and cover-slipped with PBS containing 50% glycerin. The sections were observed with a fluorescent microscope (Leica, Wetzlar, Germany).
Statistical analysis
All results from the data analysis are shown as mean ± standard deviation (SD). An ANOVA test, followed by Student-Newman-Keuls (SNK) was used to compare the quantitative data between groups. A p value of less than 0.05 (two-tailed) was considered to indicate a statistically significant difference. Graph displays were performed using GraphPad Prism Software version 7.0.