Molecular Mechanism About The NR2B Subunit In The Rostral Anterior Cingulate Cortex Neurons Contributing To The Formation Of Pain Associated With Bone Cancer

BackgroundAccording to the statistics of WHO, there are about 10 million new cancer cases increased each year. With the continuous improvement of medical technology, the 5-year survival rate of cancer patients has also increased significantly, from the past 30% to 64%. It makes the problem of increase in patients with bone cancer-associated pain, a kind of chronic pain caused by primary or metastatic bone tumor. According to statistics,70% of cases with bone tumors are associated with varying degrees of pain, but there is no effective clinical treatment for them because the pathogenesis of cancer pain is still not clear and the traditional treatment methods such as the three step therapy get limited effectiveness and serious side effect. Quite a lot of clinical terminal bone cancer-associated pain patients are suffering and leading poor quality of life. There is clinical importance to treat bone cancer-associated pain patients effectively and to improve their quality of life. Therefore, to explore the pathogenesis of bone cancer-associated pain and to find effective treatment methods is an important subject to face in the treatment of cancer cases.Spinal dorsal horn is the primary central nervous system for pain formation, and many studies have shown that central sensitization of spinal dorsal horn plays an important role in formation and process of bone cancer-associated pain. The peripheral nociceptive information is transmitted to spinal dorsal horn, where it is gathered and integrated and transmitted to upper nerve centers, and then pain formed. However, some studies have indicated that the transmission of nociceptive information in the spinal dorsal horn is regulated by the upper nerve centers such as the medulla and the anterior cingulate cortex (ACC). There are two kinds of regulation, downward inhibition and facilitation, which play an important role in the formation of pain. To study how the upper nerve centers to regulate the spinal cord activity has become a hot spot in the field of pain research.The ACC is an important structure of the limbic system, whose afferent fibers can be derived from thalamus, such as the anterior medial thalamic nucleus, the inferior central nucleus, etc. Most of these thalamic nuclei can receive the nociceptive information from the spinal cord. Thus, the nociceptive information from the spinal cord can be passed to the ACC via the thalamus. The recording electrode was placed in the ACC, where the EPSPs can be generated by the truncated lower limb. ACC not only received the inputted information, also output signal to adjust the functions of other nuclear groups. For example, if the stimulated contralateral foot was cut, the response of stimulation will be strengthened on the normal foot. This suggests that the plasticity of rostral anterior cingulated cortex (rACC) neuron plays an important role in pain. Change in neuronal plasticity mediated by N-methyl-D-aspartic acid (NMDA) receptors mainly occurs in the form of NMDA receptor 2B (NR2B) subunit. These studies suggest that NR2B in neuron of rACC may play an important role in the formation of bone cancer-associated pain. This subject is to study the molecular mechanism about the NR2B subunit in rACC neuron involved in the formation of bone cancer-associated pain with methods of pain behavior, molecular biology and electrophysiology.Part I Expression of NR2B subunit in the rostral anterior cingulate cortex neuron in rat model of pain associated with bone cancerObjectiveTo observe the expression levels of the NR2B subunit in rACC in rats with bone cancer-associated pain.MethodsSprague Dawley adult rats, weighing 200-250g, were randomly divided with a random number table into blank control group (Naive group), sham operation group (Sham group) and bone cancer pain group (Tumor-bearing group). The Tumor-bearing group was inoculated in the proximal tibia bone marrow cavity with the MADB-106 rat mammary cancer cells suspension 10μl(cell density about 5×106/ml), while the Sham group was injected with the same volume of saline. At every 3 days the mechanical allodynia paw withdrawal threshold and the thermal hyperalgeisa paw withdrawal latency were measured to observe the changes of pain behavior before operation and within 21 days after operation. The expression level of NR2B subunit in the rACC neurons was detected by immunohistochemical technique and Western blot technique in 4 selected rats that had significant changes of pain behavior.ResultsThe mechanical allodynia paw withdrawal threshold and the thermal hyperalgeisa latency in the three groups are not significantly different in statistics (P> 0.05), but compared with the Naive group and the Sham group, they decreased significantly in the Tumor-bearing group from the 3rd day after operation, and the differences are statistically significant (P<0.05). In the Tumor-bearing group, they decreased the most at the 14th day after operation and are still significantly low at the 21st day after operation compared with the Naive group. This suggests that the hyperalgesia rat model is successfully reproduced. Western Blot analysis of rat rACC tissue before operation and at the 14th day after operation showed that the expression level of NR2B in rACC neuron in Tumor-bearing group was significantly higher at the 14th day after operation than that before operation, and also than that in control group (P<0.05).ConclusionRat model of bone cancer-associated pain was successfully reproduced by tibial intramedullary injection of MADB-106 rat mammary cancer cells after 14 days, and accompanied by high expression of.NR2B subunit in rACC neurons.Part Ⅱ The effect of intra-rACC administration of Ifenprodil, aNR2B antagonist, on mechanical allodynia threshold and thermal hyperalgesia latency in rat model of bone cancer-associated painObjectiveResults in the first part of the study show high level expression of NR2B subunit in rACC neuron induced by bone cancer-associated pain, then if NR2B antagonist administered in rACC can reduce the pain of bone cancer? This part is to observe the effect of Ifenprodil microinjection in rACC on rat mechanical allodynia threshold and thermal hyperalgesia latency with bone cancer-associated pain.MethodsA catheter was buried at the location of rACC, where was mapped based on the Paxinos and Watson brain mapping (1.7mm before the anterior fontanelle, 0.6mm side and 2.5mm deep), and after 7 days of observation, the model of bone cancer-associated pain was reproduced by tibial intramedullary injection of MADB-106 rat mammary cancer cell in the normal rats. The rats with bone cancer pain were randomly divided into 5 groups:Naive group, BCP group, BCP+NS group, BCP+Ifenprodil group and BCP+CNQX group. Ifenprodil 0.6μl (0.2g/l) was administered to the left and right rACC in the BCP+Ifenprodil group, while the same dose of normal saline or CNQX was given in the control group. The mechanical allodynia paw withdrawal threshold and the thermal hyperalgesia paw withdrawal latency were recorded by Frey Von method, and the expression of C-fos neurons at the spinal dorsal horn was detected by immunohistochemistry after the test of pain behavior.ResultsThere were no statistically significant defferences (P>0.05) in the mechanical allodynia paw withdrawal threshold and the thermal hyperalgesia paw withdrawal latency before drug administration between the 5 groups, but the differences of which between the control group and the BCD+Ifenprodil group were statistically significant after injection of Ifenprodil (P<0.05). The expression of C-fos at the spinal dorsal horn in BCD+Ifenprodil group was tested lower than that in control group and saline group by immunohistochemistry (P<0.05).ConclusionIfenprodil, the antagonist of NR2B, administered in rACC may ameliorate hyperalgesia of the rats with bone cancer-associated pain and can decrease the expression of C-fos neurons of the spinal dorsal horn.Part Ⅲ The construction of recombinant shRNA/NR2B (LV-GluN2B) and the detection of NR2B inhibition efficiencyObjectiveIfenprodil administered in rACC can reduce bone cancer-associated pain, but it is a short-acting agent with side effects, so it is necessary to find long-acting and safe analgesic measures for clinic. It has become a mature method to selectively silence the expression of target gene with small RNA, and the expression of NR2B in rACC neurons can be selectively silenced to achieve long-term analgesic effect. This study is to construct the siRNA/NR2B recombinant lentiviral vector for long term analgesia.MethodsComplied with the design principle of mRNA sequence and shRNA sequence of rat NR2B subunit in the gene bank, three 19nt target sequences were designed, and the DNA of the complementary strand was further synthesized. The DNA was inserted into the downstream of the pFU-GW-iRNA vector plasmid H1 promoter and then three recombinant plasmids pFU-GW-iRNA/NR2B containing shRNA/NR2B were obtained. A negative control plasmid was constructed at the same time.293T Cell was transfected with 3 recombinant plasmids and NR2B expression plasmid. The expression of target protein NR2B was detected with Western blot technique to determine the most effectively silencing recombinant plasmid to transfect into 293T cells, to package shRNA/NR2B recombinant lentiviral LV-GluN2B.ResultsElectrophoretic analysis of PCR products showed that the constructed recombinant plasmid containing 341bp band is NR2B-RNAi. Combined with sequencing, the target recombinant plasmid was successfully constructed. Western Blotting results showed that the knockout efficiency of Plv-NR2B3 was the highest to the target gene NR2B. By transfecting 293T cells, the recombinant lentivirus LV-GluN2B, which can effectively silence the target gene NR2B, was synthesized.ConclusionRecombinant NR2B-RNAi lentivirus LV-GluN2B was successfully constructed.Part Ⅳ Effect of selectively down-regulating expression of NR2B gene in rACC neuron on bone cancer-associated pain of ratObjectiveTo observe the therapeutic effect of down-regulating the expression of NR2B subunit through brain stereotaxic technique guided intra-rACC injection of shRNA/NR2B on rat model with bone cancer-associated pain.MethodsTibial cancer pain rats with buried catheters in rACC were randomly divided into three groups according to the random number table:normal saline group, LV-NC group and LV-GluN2B group,8 in each group. The mechanical pain threshold and the thermal pain latency were measured before medication and every 3 days after medication in rACC. At the most significant time of pain behavioral effect, tissue of rat rACC was taken, and the NR2B gene knockdown efficiency of LV-GluN2B was detected through immunohistochemistry, PCR and Western blot technique.ResultsBefore medication, there were no statistically significant differences between the three groups in mechanical allodynia paw withdrawal threshold and thermal hyperalgesia paw withdrawal latency(P> 0.05), and after intra-rACC injection of LV-GluN2B, the mechanical allodynia paw withdrawal threshold and thermal hyperalgesia paw withdrawal latency were significantly increased, and also significantly increased between the LV-GluN2B group and the normal saline group or LV-NC group (P<0.05). There was no significant difference before and after medication in the normal saline group and the LV-NC group (P> 0.05). Western Blot and PCR tests showed that the expression of NR2B protein and NR2B mRNA in LV-GluN2B group was lower than that in control group (P<0.05).ConclusionIntra-rACC administration of recombinant lentiviral LV-GluN2B may ameliorate the bone cancer pain through down-regulating the expression of NR2B.SignificanceThe first methods of pain behavior and molecular biology were used in this study and we observed that bone cancer-associated pain can lead to upregulating expression of NR2B in rACC neuron. Further method of injecting NR2B antagonist Ifenprodil reduced the bone cancer-associated pain. It is suggested that NR2B in rACC neuron plays an important role in bone cancer-associated pain. On this basis, in order to achieve long-term, effective and safe analgesia, the NR2B-RNAi recombinant lentivirus (LV-GluN2B) was constructed and highly selectively down-regulated the expression of NR2B in rACC neuron, and ameliorated the bone cancer-associated pain, providing a powerful experimental basis for the clinical treatment of bone cancer-associated pain.