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[8] and the ethics committee of the International Association for the Study of Pain[9]. 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[9].. 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 divided into four groups: normal control group (Naive group), sham operation group (Sham group), Bone cancer pain model group (BCP 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. 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 3 μl MADB-106 cells (cell density 5×106/ml) (from Cancer Institute of Concord Medical University of Chinese Academy of Medical Sciences). The initial treatment of PKCγ group was the same as that of BCP group,and then rACC was injected bilaterally with shRNA/NR2B recombinant lentivirus 6.2×106 particles on the 7th day after operation.
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. 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.
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. Animals 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 FreyTM 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 paw withdrawal latency (PWL). 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.
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 (4 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 and 14th day after operation, 4 rats were selected to take rACC brain tissue and the expression level of PKCγ subunit of neurons was assessed by Western blot.
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 5.0.