Animals and grouping
Healthy adult female 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 line with the recommendation of the Principles of Laboratory [11]. The number of animals used was kept as small as possible, and animal suffering was minimized to the lowest degree according to the ethics committee of the International Association for the Study of Pain (IASP) [12]. 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) before the start of the experiments (Permit number: KYLL-2017 (LW) 017). All rats were maintained in the following identical conditions: a controlled temperature of 22 °C, a 12-hour light/dark cycle and ad libitum access to food and water. One week later, the rats were randomly divided into five groups (n=10/group): the blank control group (naive group), sham operation group (sham group), bone cancer pain model group (BCP group), BCP plus empty lentiviral vector group (vehicle group) and BCP plus PKCγ/shRNA recombinant lentiviral vector group (PKCγ group). Naive group: healthy rats without any treatment. Sham group: unilateral intra-tibial injection of normal saline. BCP model -treated groups (BCP group, vehicle group and PKCγ group): unilateral 10 μl intra-tibial injection of MADB-106 rat mammary carcinoma cells (cell density 4.6×108 cell/ml) (from Cancer Institute of Concord Medical University of Chinese Academy of Medical Sciences).
Preparation of MADB-106 rat mammary carcinoma cells
MADB-106 rat mammary carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM); supplemented with 10% foetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin; and cultured at 37 °C in a humidified atmosphere of 5% CO2. The cells were then passaged hebdomadally in terms of ATCC guidelines. For treatment, the cells were disengaged by scouring and then centrifuged at 900 rpm for 3 minutes. The pill was suspended in Hank's balanced salt solution. Cells in the logarithmic growth phase were selected for experiments and then used for intra-tibial injection.
Establishment of the rat BCP model
The bone cancer pain model of rats was established as previously described [4]. The rats were anaesthetized with an intraperitoneal injection of 10% chloral hydrate (300 mg/kg). Superficial incisions were made in the skin overlying the patella to expose the tibial head with minimal damage. A 23-gauge needle was inserted into the medullary cavity of the tibia, and 10 μl MADB-106 rat mammary carcinoma cell suspension (4.6×108 cells/ml) was slowly injected into the tibial cavity through the needle. The injection site was closed with bone wax immediately 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. The initial treatment of the vehicle group and PKCγ group was the same as that of the BCP group. In the sham group, unilateral intra-tibial injection of normal saline was used. No experimental procedures were performed in the naive group.
Construction of lentiviral vectors expressing PKCγ/shRNA
The lentiviral vectors expressing PKCγ/shRNA (LV-PKCγ/shRNA recombinant lentivirus) were packaged using the PKCγ interference sequence TGAATGTGCACCGACGCTG, the plasmid pLVTHM (Shanghai Gene Chem Gene Co., Ltd, Shanghai, China) and the lentiviral packaging plasmid. The PKCγ interference sequence was cloned into the lentiviral vector pLVTHM-GFP (Shanghai Gene Chem Gene Co., Ltd, Shanghai, China). Moreover, the lentiviral vector pLVTHM-GFP and packaged plasmids were co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The final titre of PKCγ/shRNA recombinant lentivirus was 1.25×109 TU/ml.
PKCγ/shRNA recombinant lentivirus administration into the rACC
7 days After treated with intra-tibial injection, rats were implanted with stainless steel cannulas for intra-rACC drug infusions. For the microinjection studies, rats were anaesthetized with intraperitoneal chloral hydrate (300 mg/kg) and were firmly fastened into a brain stereotactic apparatus with the lambda and bregma at the 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, and 2.5 mm beneath the surface of the skull] in-line with the atlas of Paxinos and Watson [13]. A 10 μl 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. Before and at the end of the experiment, the brains were sectioned for cresyl violet staining to verify the cannula position and injection site. The rats were monitored daily after surgery for signs of motor deficiency or infection. In the PKCγ group, 10 μl shRNA/PKCγ recombinant lentivirus (1.25×109 TU/ml) was injected into the bilateral rACC over 5 minutes. In the vehicle group, the same dose of empty recombinant lentivirus was injected. No experimental procedures were performed in the naive, sham and BCP groups.
Assessments of Pain-related behaviours
Before the baseline trial, The rats had a natural appearance and level of activity and ate regularly and were acclimated to the testing environment. The experimental rats were placed in a plastic cage (10×10×15 cm) with a Plantar von-Frey TM Dynamic Plantar Stimulator (Stoelting, USA) at the bottom, and the cage was placed on a wire mesh plate for the experimental operation and observation. After 15 minutes of acclimation, 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 hairs with logarithmically incremental stiffness (1.0, 2.0, 4.0, 6.0, 8.0, 15.0 and 20.0 g) were applied perpendicularly to the mid-plantar surface (avoiding the less sensitive tori) of each hind paw. The stimulus lasted for ten seconds, and the interval between each measurement was 10 minutes. The minimum stimulus that caused rat paw withdrawal was defined as the MWT.
Rats were placed under a cage on a glass plate that was elevated to allow manoeuvring 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 the onset of radiant heat application to the withdrawal of the hind paw was defined as the TWL. The glass plate was kept dry and clean during the measurement. Both hind paws were tested independently with a 5 minutes interval between trials so that pain could be restored to normal. A blocking time of 20 seconds was imposed on the stimulus duration to prevent tissue damage. The paw of each rat was tested three times, and the average value was taken. Both MWT and TWL were commonly used as the index to assess mechanical allodynia and thermal hyperalgesia and were measured during 3 weeks: pre-operative day 0 (baseline) and on days 3, 7, 14 and 21 following the intra-tibial injection.
Western blotting analysis
After the behavioural tests, rats in all groups were anaesthetized with an overdose of chloral hydrate before the perfusion of 100 ml of phosphate-buffered saline (PBS) through the ascending aorta and then rapidly sacrificed by decapitation. On days 7 or 21 after intra-tibial injection, the rACC tissues were immediately removed and frozen in liquid nitrogen, washed with cold PBS containing 2 mM EDTA and lysed with denaturing SDS-PAGE sample buffer using standard methods. Protein lysates were separated and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were blocked and then incubated with rabbit polyclonal anti-PKCγ antibody (dilution at 1:300; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. After the membranes were washed, they were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (dilution at 1:5,000; Santa Cruz) at room temperature for 2 hours. Western blotting was performed to detect the expression of PKCγ in rACC tissues.
Immunohistochemical analysis
To further verify the expression level of PKCγ in neurons after lentiviral vector injection, immunohistochemical analysis was performed. On the postoperative day 21, rats were deeply anaesthetized with an overdose of chloral hydrate and perfused transcardially with 100 ml of PBS, followed by 250 ml of ice-cold 4% paraformaldehyde. The rACC sections were removed and fixed at 4 °C for 5 hours and then transferred to 30% sucrose/PBS for 24 hours. rACC sections (20 mm) were incubated for 2 hours at room temperature in a blocking solution (3% normal goat serum) and then incubated for 48 hours at 4 ℃ with rabbit polyclonal anti-PKCγ antibody (dilution of 1:500; Santa Cruz). Following incubation, the tissue sections were washed and incubated for 3 hours at room temperature in the secondary antibody solution HRP-conjugated goat anti-rabbit IgG antibody (dilution of 1:2,000; Santa Cruz). The rACC sections were analysed using an LSM confocal imaging system (Carl Zeiss Japan, Tokyo, Japan).
Immunofluorescence analysis
On the postoperative day 21, rats were deeply anaesthetized with an overdose of chloral hydrate and perfused transcardially with 100 ml of PBS, followed by 250 ml of ice-cold 4% paraformaldehyde. The rACC tissues were subsequently cut into7 μm sections on a cryostat, and all sections were prefixed with acetone, blocked with goat serum at 37 ℃ and incubated overnight at 4 ℃. Following this, the tissue sections were washed and incubated for 2 hours in a dark room. The stained sections were scanned and images were subsequently captured using an inverted fluorescent microscope (Nikon Corporation, Tokyo, Japan).
Statistical analysis
Data are shown as the mean ± standard deviation (SD) and were analysed using SPSS 23.0 software (IBM SPSS, Armonk, NY, USA). Data from the pain-related behavioural assessments were analysed using a two-way repeated ANOVA (Analysis of Variance) to detect the difference among the groups; whereas One-way ANOVA followed by Student-Newman-Keuls (SNK) post hoc test was used to compare MWT and TWL at different time points and the differences in the numbers of PKCγ-immune-positive cells and protein expression levels of PKCγ among the groups. A P value of less than 0.05 (two-tailed) was considered to indicate a statistically significant difference.