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

To explore the role of PKCγ subunits of rostral anterior cingulate cortex (rACC) neurons in the development of bone cancer pain in rats. Healthy female Sprague-Dawley rats were randomly divided into five groups: blank control group (naive group), sham operation group (sham group), bone cancer pain group (BCP group), BCP plus empty lentiviral vector group (vehicle group) and BCP plus PKCγ/shRNA recombinant lentiviral vector group (PKCγ group). The BCP group, vehicle group and PKCγ group received a 10 µl intra-tibial injection of MADB-106 rat mammary carcinoma cell suspension (4.6×10 8 cell/ml). In comparison, the sham group received a 10 µl intra-tibial injection of saline. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed on pre-operation day 0 (baseline) and days 3, 7, 14 and 21 after intra-tibial injection, respectively. To downregulate the PKCγ subunits of rACC neurons, the PKCγ group received a 10 µl bilateral rACC injection of shRNA/PKCγ recombinant lentivirus (1.25×10 9 TU/ml) on the day 7 after intra-tibial injection, whereas the vehicle group received an injection of the same dose of empty lentiviral vector. Western blotting, immunohistochemical and immunofluorescence analysis were performed to detect the different expression of PKCγ subunits in rACC neurons among these groups on postoperative days 7 or 21. No significant difference in the baseline of MWT and TWL was found among these five groups ( P > 0.05). However, compared with the naive group and sham group, the rats with bone cancer (BCP group, vehicle group and PKCγ group) demonstrated marked mechanical allodynia and thermal hyperalgesia that was evoked starting on postoperative day 7 following intra-tibial injection of carcinoma cells ( P < 0.05). Meanwhile, the western blotting analysis also confirmed that the expression of PKCγ

alleviated mechanical allodynia and thermal hyperalgesia ( P < 0.05).The present study indicates that up-regulation of PKCγ subunits of rACC neurons in bone cancer pain rats contributes to the development of bone cancer pain.

Background
The pathogenesis of bone cancer pain remains unknown, and there has been no effective treatment [1,2]. The anterior cingulate cortex (ACC) is an essential part of the cerebral cortex; in particular, the rostral ACC (rACC) is associated with pain perception and regulation [3][4][5]. Under the persistent action of noxious stimulation, neurons or synapses in the rACC, in terms of their structure and function, undergo long-term changes, collectively known as neuroplasticity. As a critical signalling molecule in cells, PKCγ plays a vital role in neuronal proliferation, differentiation, synapse formation, transmitter release, and long-term potentiation (LTP) of neuron excitability [6,7]. Previous studies have suggested that PKCγ is involved in the processing of peripheral pain signals and plays an essential role in the treatment of noxious stimulation in the dorsal horn of the spinal cord [6,[8][9][10]

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

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% CO 2 . The 5 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 (4.6×10 8 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.

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 (1.25×10 9 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 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-8 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 9 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.

Rats with bone cancer exhibit increased mechanical allodynia and thermal hyperalgesia
Comparison of the baseline in MWT and TWL, no significant differences in pain-related behavioural tests were demonstrated among these groups (P > 0.05). Furthermore, No significant differences in behavioural tests were measured among the naive and sham groups in the period of time examined (P > 0.05). However, following the intra-tibial injection, rats with bone cancer (the groups BCP, vehicle and PKCγ) demonstrated significantly decreased MWT and TWL on the postoperative day 7 compared with the naive and sham groups (P < 0.05). Moreover, the reduction of MWT and TWL in groups BCP and vehicle was not suspended until postoperative day 21 (Figure 1, 2). This suggests that the rats with the intra-tibial injection of mammary carcinoma cells consistently develop mechanical allodynia and thermal hyperalgesia.

Intra-tibial injection of mammary carcinoma cells specifically increases PKCγ protein expression levels in the rACC
Rats were sacrificed and then the rACC tissues were removed. The PKCγ protein expression in rACC neurons following the development of mechanical pain and thermal hyperalgesia was assessed on the day 7 after intra-tibial injection. As outlined in graphs, the expressions of PKCγ protein among the groups were examined by western blotting.  (Figure1, 2). This phenomenon indicates that mechanical allodynia and thermal hyperalgesia of rats in PKCγ group alleviate.

Bilateral intra-rACC injection of LV-PKCγ/shRNA decreases PKCγ protein expression levels in the rACC
The expression level of PKCγ protein in rACC neurons was evaluated following bilateral intra-rACC injection of LV-shRNA/PKCγ. Hence, rats were killed and then rACC tissues were removed on day 21 after intra-tibial injection. As shown in the graphs, the expressions of Bone cancer pain is one of the symptoms in terminal cancer patients, which has been described as a deep, burning-like chronic pain and has intense inflammatory and neuropathic components [1]. So far, although there are treatments such as opioid, diphosphonate, radiotherapy, chemotherapy and surgery for relieving cancer pain, it has been reported that many cancer patients have inadequate and undermanaged pain control [14,15]. Therefore, in order to solve this problem, the pathophysiological causes of bone cancer pain need to be further concerned. A large number of cancer pain animal models have been performed to examine the mechanisms that underlie tumour-evoked pain and 13 hyperalgesia. Using models in which mammary carcinoma cells are implanted into the tibial bone, researchers have begun to clarify the pathophysiological processes by which cancer produces pain [16]. In the present study, we discovered that the hind paw mechanical withdrawal threshold and thermal withdrawal latency gradually declined by the infusions of mammary carcinoma cells in bone from postoperative days 7-21. This suggests that bone cancer caused both induction and maintenance of the cancer-induced persistent nociception, which was pathologically and physiologically meet to the intended clinical situation.
The present study showed that intra-rACC injection of LV-PKCγ/shRNA alleviates mechanical allodynia and heat hyperalgesia in bone cancer rats. This suggests that rACC neurons play an essential role in the development of bone cancer pain. Some studies have also shown that the excitability of neurons in the supraspinal cord, such as the ACC, and the enhancement of synaptic transmission play essential roles in the development of chronic pain [17]. The ACC is a considerably large structure of the limbic system that reflects affective and motivational aspects of pain. ACC, especially the rACC, transmits and regulates the nociceptive information [18]. Imaging studies have also reported an increased ACC activity under noxious stimulation and chronic pain conditions [19]. Furthermore, we already have known that the efferent nerves from the ACC area innervate the grey matter around the midbrain aqueduct and the involvement of the rostral loop [20]. Studies have also demonstrated that spinal nociception is regulated by descending modulation from supraspinal structures, including neurons in the ACC and insular cortex [21]. These findings suggested that neuronal activity in the ACC may affect spinal nociception through descending modulatory systems. All of these results further demonstrate that enhanced nerve excitability of rACC region play a vital role in both the induction and maintenance of the bone cancer-induced mechanical and heat hyperalgesia.
According to the structural and functional characteristics of different subtypes, PKCs can be divided into conventional (α, β Ⅰ/Ⅱ, γ), novel (δ, ε, η, θ) and atypical (ζ, λ, ι, μ) forms [22]. The PKC family has a wide range of functions. When the cell membrane receptor coupled to phospholipase C is activated, DAG is produced, followed by activation of PKC, thus exerting a biological effect. Previous studies demonstrated that PKCs might be necessary in the processing of nociceptive information in chronic hyperalgesia. PKCγ activates the protein kinase system in neurons, thereby changes the phosphorylation state of the substrate and considers to be a central molecular integrator of nociceptive signalling. In particular, increasing evidence suggests that PKCγ is highly involved in central sensitization [23] as well as synaptic remodelling of neurons and long-term potentiation. Many studies have also indicated that some enhanced processes of reactivities, such as hyperalgesia, may be related to central sensitization. The molecular mechanisms underlying PKCγ-mediated pain hypersensitivity have been examined in recent studies. PKCγ in the trigeminal nucleus caudalis participated in the pathogenesis of chronic migraine [24]. Spinal protein kinase C was also involved in the induction and maintenance of both persistent spontaneous inching reflex and contralateral heat hyperalgesia in rats [25]. Huang also found that CCR5/PKCγ signalling pathway may contribute to the maintenance of BCP in rats [8]. Nevertheless, findings in rACC regions related to PKCγ-mediated pain have not been well established. In this present study, The PKCγ expression in the rACC was measured and found that BCP surgery markedly upregulated the expression of this protein, suggesting that increased PKCγ expression participated in inflammatory and neuropathic pain formation. Subsequently, we investigated whether PKCγ in the rACC played a critical role in the development of BCP in rats. The immunohistochemical staining, immunofluorescence staining, as well as western blotting also showed that the number of PKCγ immunoreactive neurons in rACC was significantly decreased following rats suffered from the injection of LV-PKCγ/shRNA. Therefore, these results suggest that BCP activated PKCγ in rACC neurons. We also found that the inhibition of PKCγ by LV-PKCγ/shRNA injection increased the hind paw TWL and MWT. However, the activation of PKCγ by BCP establishment reduced the paw TWL and MWT. It indicates that when PKCγ is silenced, or its function is inhibited, the hyperexcitability of rACC neurons can be no longer induced. Malmberg found that acute pain in PKCγ knockout mice was not affected significantly, while chronic pain was weakened, which is consistent with the present study [10]. The antiallodynic effects of PKCγ antagonists have also been reported in other animal models of chronic pain [8].
As universally used gene delivery systems, recombinant lentiviral vectors is capable of infecting the intermitotic cells and mitotic cells. Once a virus binds to a cell, its genes can be incorporated into the genomes of cells as a stable component of cytogenetics that can be passed on to its offspring during cell division. Meanwhile, the pathogenic genes of lentivirus have been deleted, so recombinant lentiviral vectors are used to express small interfering RNA (short interference RNA, siRNA) [26]. One way to deliver siRNA in vivo is to clone siRNA sequences into plasmid vectors as shRNA (short hairpin RNA). Viral delivery of shRNA expression cassettes allows efficient transduction in brain tissues. In our previous study, We successfully transfected LV-GluN2B/shRNA in rACC neurons and relieved the debilitating pain of bone cancer by selectively decreasing GluN2B expression levels in the rACC [27]. To further explore whether the PKC subunit of rACC neurons play a significant role in bone cancer pain, we injected PKCγ/shRNA recombinant lentiviral vectors into the bilateral rACC to silence the PKCγ subunits of rACC neurons after establish the BCP model.

Not applicable
Availability of data and material The datasets generated and analyzed during the current study are not publicly available due to copyright issues, but are available from the corresponding author on reasonable request.     Significance was defined as *P < 0.05, compared with naive or sham group; #P < 0.05, compared with BCP or PKCγ group. Data are presented as mean±SD for 3 rats per group.

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