| BackgroundThe components of pain include pain sensation and pain-related emotion.The former is primarily a judgement of the character,intensity and location of the stimuli,and the latter is mainly the feeling of unpleasantness and pain related aversion.Neuropathic pain is one of the most common chronic pain,which is often characterized by hyperalgesia,allodynia and spontaneous pain.Neuropathic pain has an obvious influence on the quality of life,which often affects patients' sleep,work and life skills.It also increases the incidence of affective disorders such as depression and anxiety.These impacts not only bring physical and psychological harm to the patient,but long-term treatment also imposes a severe economic burden on their family.The rostral anterior cingulate cortex(rACC)neurons are important brain regions mediating the formation of chronic pain-related negative emotions.The activity of rACC neurons in the brain is increased after nerve injury,inhibition of activation of the neurons or surgical removal of them can significantly relieve chronic pain-related unpleasant reactions.Brain-derived neurotrophic factor(BDNF)is widely distributed in the central nervous system and plays an important role in learning,memory,and mediating synaptic transmission.As its highly specific receptor,TrkB is involved in many neuronal activities.Extracellular signal-regulated kinase(ERK)is one of the most important molecules in the MAPK family.Activation of ERK is essential in synaptic plasticity and long-term potentiation of neurons.Recent studies have shown that injection of TrkB receptor antagonists in rACC of rats can significantly inhibit the activation of ERK protein,and thus relieve pain related aversion emotional responses in rats with inflammatory pain.However,it is still unclear whether this pathway is involved in the formation of neuropathic pain-related negative emotional experiences.Therefore,in this study,a rat model of neuropathic pain was established by spared nerve injury(SNI),combined with a mechanical stimulus-induced conditioned place avoidance(CPA)behavioral experiment to comprehensively investigate whether BDNF signalling pathways in rACC neurons can induce the formation of neuropathic pain-related aversion and the exact molecular mechanism of this process.Chapter 1 Establishment of Neuropathic Pain Model in RatsObject This section aims to establish a rat model of neuropathic pain by spared nerve injury and explore whether the neuropathic pain is associated with the formation of pain related aversion based on the model.Methods SD rats were randomly divided into surgery group,sham operation group and blank control group.Rats in the surgery group(SNI group)underwent spared nerve injury;rats in the sham operation group(Sham group)underwent sham surgery;rats in the blank control group(Naive group)did not undergo any treatment.One day before surgery and 1,3,7,10,and 14 days after surgery,the mechanical pain threshold of the hind paw was measured by a Von Frey meter and expressed by PWT.On the 7th day after operation,rats in each group underwent mechanical stimulus-induced conditioned place avoidance training(MS-CPA)to detect the formation of pain related aversion.Results Compared with the control group,the paw withdrawal thresholds(PWT)began to decrease on the first postoperative day in the surgery group,and reached a plateau 7 days after surgery.In addition,the training results of conditioned place avoidance(CPA)showed that compared with the control group,only the surgery group produced significant CPA under mechanical stimulation,which reflected the formation of pain related aversion emotions.Conclusion The neuropathic pain model was successfully established by spared nerve injury(SNI),through which it was found that rats with neuropathic pain are often accompanied by pain related aversion emotional response.Chapter 2 Molecular Mechanism of BDNF in rACC Neurons of Rats Participating in the Formation of CPAObject This section aims to investigate how the BDNF protein in rACC of rats participates in the formation of conditioned place avoidance(CPA).Methods SD rats were randomly grouped.SNI group:surgery group,rats underwent spared nerve injury;Sham group:sham operation group,rats underwent sham surgery;Naive group:blank control group,rats did not undergo any treatment;CTX-B-R group:local injection of TrkB receptor antagonist CTX-B into rACC of rats in surgery group;CTX-B-P group:local injection of TrkB receptor antagonist CTX-B into PFC of rats in surgery group;BDNF-R group:local injection of exogenous BDNF into rACC of normal rats;BDNF-D group:local injection of DMSO into rACC of normal rats as control;BDNF-C group:local injection of CTX-B and exogenous BDNF into rACC of normal rats;local injection of CTX-B into rACC of normal rats;BDNF-P group:local injection of exogenous BDNF into PFC of normal rats.On the 7th day after operation,rACC and PFC brain tissues were taken respectively,and the expression of BDNF and its specific receptor TrkB was detected by ELISA,RT-PCR and Western blot techniques.All rats underwent conditioned place avoidance training(CPA)to detect the development of pain related aversion emotions.Results Compared with the control group,the expression of BDNF and TrkB in rACC neurons increased significantly only in the SNI rats,and they could produce obvious conditioned place avoidance(CPA).However,the early injection of TrkB receptor antagonist CTX-B in rACC(but not PFC)can block the formation of CPA.In addition,we also found that the injection of exogenous BDNF into rACC(but not PFC)of normal rats also induced the development of CPA,which could be blocked by the TrkB receptor antagonist CTX-B.Conclusion BDNF in rACC of rats participates in the formation of CPA by binding with its specific receptor TrkB.Chapter 3 BDNF/TrkB signaling pathway participates in the formation of neuropathic pain-related aversion by activating ERKObject This section aims to investigate whether and how the downstream molecule ERK participates in the formation of neuropathic pain related aversion emotional response.Methods SD rats were randomly grouped.SNI group:surgery group,rats underwent spared nerve injury;Sham group:sham operation group,rats underwent sham surgery;Naive group:blank control group,rats did not undergo any treatment;U0126-SNI group:local injection of highly selective MAPK inhibitor U0126(which can inhibit the activation of downstream ERK)into rACC of rats in surgery group;CTX-B group:local injection of TrkB receptor antagonist CTX-B into rACC of rats in surgery group;BDNF group:local injection of exogenous BDNF into rACC of normal rats;U0126 group:local injection of U0126 into rACC of normal rats;U0126-BDNF group:local injection of U0126 and exogenous BDNF into rACC of normal rats.On the 7th day after operation,rACC brain tissues were taken,and the expression of p-ERK was detected by immunohistochemistry,Western blot and immunofluorescence,etc.In addition,all rats underwent conditioned place avoidance(CPA)to detect the development of pain related aversion emotions.Results Compared with the control group,the expression of p-ERK in rACC neurons increased significantly only in the SNI rats,but the early injection of MAPK inhibitor U0126 in rACC could obviously block the activation of ERK.It was found that high expression of p-ERK can be significantly inhibited,when we injected TrkB receptor antagonist CTX-B in rACC neurons in advance.In addition,we also found that the injection of exogenous BDNF into rACC of normal rats can induce the activation of ERK and the formation of CPA,and this process can be blocked by p-ERK specific receptor antagonist U0126.High expression of p-ERK can be significantly inhibited after injecting TrkB receptor antagonist CTX-B in rACC neurons in advance.Conclusion The BDNF/TrkB signaling pathway in rACC of rats participates in the formation of neuropathic pain-related aversion by activating downstream ERK. |
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