Up-regulation of PKCγ subunits of rACC neurons contributes to the development of pain sensitivity in bone cancer rats

Abstract Background: To explore the role of PKCγ subunits in rostral anterior cingulate cortex (rACC) neurons in bone cancer pain (BCP) development in rats. Methods: Healthy female Sprague-Dawley rats were randomly divided into five groups: the blank control (naive), sham operation (sham), BCP, BCP plus empty lentiviral vectors (vehicle) and BCP plus PKCγ/shRNA recombinant lentiviral vectors (LV-PKCγ/shRNA) (PKCγ) groups. The BCP, vehicle and PKCγ groups were intra-tibially injected with an MADB-106 rat mammary carcinoma cell suspension (10 μl, 4.6×106 cells/ml), whereas the sham group was intra-tibially injected with saline (10 μl). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were evaluated on preoperative day 0 (baseline) and days 3, 7, 14 and 21 after intra-tibial implantation. To downregulate PKCγ subunits in rACC neurons, the PKCγ group received a 10 µl bilateral rACC microinjection of shRNA/PKCγ recombinant lentivirus (1.25×109 TU/ml) on day 10 after intra-tibial implantation. The GFP expression of LV-shRNA/PKCγ was examined by microscopy. The vehicle group was injected with an equal dose of empty lentiviral vectors. Western blot, and immunohistochemical approaches were performed to assess the differential expression of PKCγ subunits in rACC neurons among these groups on postoperative days 7 or 21. Results: The baseline MWT and TWL values did not significantly differ among these five groups (P > 0.05). However, compared with the naive and sham groups, the tumour-bearing rats (BCP , vehicle and PKCγ groups) demonstrated marked mechanical allodynia and thermal hyperalgesia starting on postoperative day 7 following intra-tibial implantation of carcinoma cells (P < 0.05). Western blot on postoperative day 7 confirmed a significant increase in PKCγ expression in rACC neurons in the tumour-bearing rats (P < 0.05). However, from postoperative days 14-21, the LV-shRNA/PKCγ microinjection alleviated mechanical allodynia and thermal hyperalgesia in the PKCγ group (P < 0.05). Furthermore, western blot, and histological examination on postoperative day 21 indicated that PKCγ expression in bilateral rACC neurons was significantly lower in the PKCγ group than in the BCP and vehicle groups (P < 0.05). Conclusion: Upregulation of PKCγ subunits in rACC neurons of rats with bone cancer contributes to BCP development.