Silencing Of NR2B Gene In Rat Anterior Cingulate Cortex (ACC)Neurons Controlling The Formalin-induced Pain-related Affect

BackgroundAs well-known, A line of investigation indicated the patients with chronic pain in clinic suffer from much more the emotional disturbance, such as anxiety, fear, pessimism, than pain itself. However during the past forty years,the date on the pain-related affective aspect are quite limited, due to the lack of a behavioral index or available animal model relating to the affective component. The current clinical management such as drug and block treatment are mostly for reliefing the feeling of acute pain or other types of pain,but it is very limited for the role of cancer pain associated with affective aspect.Therefor,finding a safe and effective method which not only reliefs the feeling of pain but also affects aspect assiociaten with pain,has become an important direction for the study of pain.Accumulated evidence implicates the anterior cingulate cortex (ACC) in pain processing. A number of studies have shown the involvement of excitatory amino acids, especially glutamate in synaptic transmission and signal processing in the ACC. Lei et al. demonstrated that blockade of NMD A receptors but not of AMPA/KA receptors in the rostral ACC (rACC) significantly inhibited formalin-induced conditioned place avoidance (F-CPA), which reflects the pain-related negative affective state and aversion learning produced by the nociceptive stimulation, and attenuated F-CPA retrieval-induced Fos expression in the ACC. Furthermore, Johansen and Fields demonstrated that glutamatergic activation in the ACC is necessary and sufficient for pain like aversion. It is therefore suggested that activation of glutamate NMDA receptors in the ACC is required for the induction of pain related negative affect.Functional NMDA receptors contain heteromeric combinations of the NR1subunit plus one or more NR2A-D, of which the NR1, NR2A and NR2B subunits are highly expressed in forebrain areas.Among NMDA receptor subtypes, the NR2B subunit containing receptors appear particularly important for nociception, thus leading to the possibility that NR2B selective antagonists may be useful in the treatment of chronic pain. However, several NR2B selective agents seem to block human ethera-go-go-related gene (HERG)-mediated K+currents and may thereby produce severe side effects by increasing the cardiac Q/T interval.RNA interference (RNAi) has become the method of choice for specifically suppressing the expression of any desired gene.and the lentiviral vector-mediated gene delivery system has several advantages over other viral or nonviral gene delivery systems, such as the high efficiency of gene transfer into a wide variety of cells including both dividing and non-dividing cells,long-term infection due to gene integration into the chromosome of host cells,and the absence of toxicity or immune response. Thus, we eventually selected lentivirus-mediated RNAi system targeting NR2B gene,which could be used to efficiently downregulate the expression of NR2B in ACC neurons.METHODS and RESULTS1. Silencing of NR2B gene in rat primary culture anterior cingulate cortex (ACC) neurons by lentiviral delivered shRNAMethods:Primary ACC cultures were prepared from the1-to2-day-old Sprague-Dawley rats, and were identified by the anti-MAP2monoclonal antibody and MTT. Primary Culture ACC Neurons which were right were divided into three groups:CONTROL group、NC-GFP-LV group and NR2B-RNAi-LV group. And Lentiviral (NR2B-RNAi-LV)transduct in primary cultured ACC neurons, the empty virals)transduct in NC-GFP-LV group and the same volume of salin transduct in CONTROL group.To determine the NR2B-RNAi-LV transduction efficiency in ACC neurons, GFP expression was examined by microscopy at different MOIs on days3-4after infection. The expressive level of NR2B Mrna protein in ACC were detected using RT-PCE and western bloting.Results:The efficiency of lentiviral vector transduction in ACC Neurons was98%under fluorescence microscopy in NC-GFP-LV group and NR2B-RNAi-LV group. But in CONTROL group there was no fluorescence observed.,NR2B mRNA expression decreased significantly in cells transfected with NR2B-RNAi-LV,as compared to that in untransfected cells(vs control group,q=6.0742,P<0.05)or negative transfection(vs NC-GFP-LV group,q=4.0379, P<0.05).However,NR2B expression level was similar between untransduced ACC Neuron and ACC Neurons infected by NC-GFP-LV. Protein levels of NR2B in the untransduced groups and NC-GFP-LV groups were similar,but reduced significantly in NR2B-RNAi-LV groups(q=9.3679, P<0.01;q=8.2702, P<0.01).As expected,there was no difference in the reduction of NR2B protein expression between the untransduced groups and NC-GFP-LV groups(q=1.0977, P>0.05).2. Experimental Studies on NR2B/RNAi recombinant lentivirus injected in ACC effect of formalin-induced pain-related affectMethods:Thirty eight rats were divided into three groups randomly, including the NR2B-RNAi-LV group, NC-GFP-LV group and Control group. Rat were placed in a stereotactic frame and injected NR2B/RNAi recombinant lentivirus in ACC(NR2B-RNAi-LV group-recombinant lenti-virus NR2B, NC-GFP-LV group-empty lentivirus containing no inserted sequence and Control group-injected the same volume saline).Brains were then sectioned coronally on a freezing microtome at thickness of35μM after injected7days to evaluate expression of EGFP,and the sections were observed directly under a fluorescent microscope. The ACC were removed for RNA and protein extraction, and the expressive level of NR2B Mrna protein in ACC were detected using RT-PCE and western bloting. Results:Intra-rACC administration of NR2B-RNAi-LV has no effect on formalin-induced biphasic nociceptive responses. Formalin-induced CPA can be established in CON group and NC-GFP-LV group,but was failure in NR2B-RNAi-LV group.And S-CPA can successfully established in, CON group、NC-GFP-LV group andNR2B-RNAi-LV group. Fluorescence images taken randomly show that there was a large number of fluorescence in NR2B-RNAi-LV group and NC-GFP-LVgroup,but there was no in CON group. NR2B mRNA expression is decreased7days after injection of NR2B-RNAi-LV compared with NC-GFP-LV group and CON group injected rat. But there was no difference between NC-GFP-LV group and CON group. Protein levels of NR2B in CON groups and NC-GFP-LV groups were similar,but reduced significantly in NR2B-RNAi-LV groups(q=6.5326,p<0.01;q=7.6534,p<0.01).As expected,there was no difference in the reduction of NR2B protein expression between CON groups and NC-GFP-LV groups(q=1.6523,p>0.05).3. Statistical AnalysisAll of the analyses were performed by SPSS12.0software package. Measurement data were presented as means±SD. The differences between multiple individual means were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between multiple individual rates were analyzed by T test. P<0.05was considered statistically significant in all tests.CONCLUSIONSThe NR2B/RNAi recombinant lentivirus shows high efficiency on infecting Primary Culture ACC Neurons and significantly inhibits the expression of NR2B in cells. Moreover, F-CPA and S-CPA shows microinjections of NR2B/RNAi recombinant lentivirus can control effectively formalin-induced pain-related affect,not affecting the acute nociceptive responses and cognitive function.