| Objective: Chronic pain frequently exhibits a significant level of coexisting conditions with depression,and concurrently alleviating pain and depression symptoms poses a challenge.This study aims to investigate the potential of electroacupuncture(EA)to relieve pain and depression in rats by modulating hippocampal neuronal apoptosis and synaptic plasticity through the BDNF/TrKB/CREB signaling pathway.The findings aim to provide an experimental basis for the clinical use of EA in treating comorbid chronic pain and depression.Methods: A group of healthy male Sprague-Dawley(SD)rats,aged 8weeks and weighing approximately 250g(with a range of 220-280g),were carefully selected.Following a week of adaptive feeding,the rats were divided randomly into four groups: sham,model,medication,and electroacupuncture(EA)groups,with each group consisting of 15 rats.Inhalation anesthesia using 2% isoflurane was administered prior to injecting100 u L of complete Freund’s adjuvant(CFA)into the left hind paw’s plantar surface using a precise microinjector.On the 14 th day of the experiment,an additional 50 u L of CFA was injected to reproduce the chronic inflammatory pain and depression model.In contrast,the sham group received a similar volume of normal saline injected into the left plantar surface.Both the sham group and the model group were fed a standard diet without any further intervention.The medication group received oral administration of Duloxetine suspension at a dosage of 10mg/kg.The EA group underwent bilateral Hegu(LI 4)and Taichong(LR 3)EA treatment for 20 minutes,once a day,for a total of 14 sessions.Following the completion of all behavioral tests and 24 hours after the intervention,the rats were anesthetized and underwent laparotomy.The hearts were then perfused with 0.9% normal saline,and once the blood was drained,the rats were decapitated,and the hippocampus was carefully separated on ice.Key outcome measures included:(1)The pain behavior of the rats was assessed using the mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)tests.Additionally,the depression-like behavior was evaluated through the open field test(OFT),sucrose preference test(SPT),and forced swimming test(FST).(2)Pathological morphology of the hippocampal CA3 region and the number of Nissl bodies in neurons were observed using hematoxylin-eosin staining(HE)and Nissl staining.(3)The synaptic ultrastructure of hippocampal neurons was examined using a transmission electron microscope.(4)Td T-mediated d UTP Nick-End Labeling(TUNEL)staining was employed to detect the apoptosis of hippocampal neurons.(5)Western blot(WB)analysis was conducted to identify the proteins related to the BDNF/TrKB/CREB signaling pathway,as well as the apoptosis and synapse-related proteins mediated by this pathway in the rat hippocampus.The relative expression levels of BDNF,TrKB,CREB,Bcl-2,Bax,Caspase9,PSD-95,and Syn were determined.(6)Immunofluorescence(IF)detection of PSD-95 and Syn positive expression in the rat hippocampus was performed.(7)The content of the neurotransmitter glutamate(Glu)in the hippocampus was measured using ultraviolet colorimetry.(8)Immunohistochemistry(IHC)was utilized to assess the expression of BDNF and TrKB in the hippocampus.(9)Real-time polymerase chain reaction(RT-PCR)was employed to detect the expression of BDNF,TrKB,and CREB m RNA in the hippocampus.Results: 1.Behavioral changes in pain and depression in each rat group:(1)The results of the MWT indicated that on the 7th and 14 th days,the model group,medication group,and EA group showed significantly lower thresholds compared to the sham group(P < 0.05);on the 21 st and 28 th days,the medication group and EA group exhibited significantly higher thresholds than the model group(P < 0.05).(2)The results of the TWL showed that on the 7th and 14 th days,the model group,medication group,and EA group had significantly lower latent periods compared to the sham group(P < 0.05);on the 21 st and 28 th days,the medication group and EA group demonstrated significantly longer latent periods than the model group(P < 0.05).(3)The OFT results revealed that on the 14 th day,the total movement distance and central area stay time of the model group,medication group,and EA group were significantly lower than those of the sham group(P < 0.05).On the 28 th day,the total movement distance and central area stay time of the medication group and EA group were significantly higher than those of the model group(P < 0.05).(4)The SPT showed that on the 14 th day,the sugar preference index of the model group,medication group,and EA group was significantly lower than that of the sham group(P < 0.05);On the 28 th day,the preference rate for sugar water in the medication group and EA group was significantly higher than that in the model group(P < 0.05).(5)The results of the FST indicated that on the 14 th day,the model group,medication group,and EA group exhibited significantly longer immobility times compared to the sham group(P < 0.05);on the 28 th day,the immobility time of the medication group and EA group was significantly lower than that of the model group(P < 0.05).2.Pathological changes in the hippocampus of rats in each group:(1)The HE staining results showed that the hippocampal structure of the sham group appeared normal,with neatly arranged nerve cells,clear nucleoli,uniform staining,and no signs of apoptosis or necrosis.The model group displayed significant damage to the hippocampal tissue,with irregular arrangement of nerve cells,enlarged intercellular spaces,and observable dense and excessively stained neuron.A small number of cells were undergoing dissolution and disappearance.However,compared to the model group,the medication group and EA group exhibited alleviated damage,with neat arrangement of nerve cells and clear nucleoli.Only a few cells still showed nuclear pyknosis and deep staining.(2)The results of Nissl staining revealed no significant abnormalities in the morphology of neurons in the hippocampus of the sham group.Neuronal structure appeared clear,orderly,and abundant in Nissl bodies.In contrast,the model group exhibited loose arrangement of neurons,damaged and lost neurons,and fewer Nissl bodies in the hippocampus.The medication group and EA group showed less neuronal loss in the hippocampus,with tidy cell arrangement,clear nucleoli,and significantly increased Nissl bodies compared to the model group.3.Apoptosis of hippocampal neurons in each rat group:(1)TUNEL staining results demonstrated a significant increase in the number of apoptotic cells in the hippocampus of the model group rats compared to the sham group(P < 0.05).However,both the medication group and EA group exhibited a significant reduction in the number of apoptotic cells compared to the model group(P < 0.05).(2)The WB test results showed that in comparison to the sham group,the relative expression level of Bcl-2 protein in the hippocampus of the model group rats was significantly reduced(P < 0.05).Conversely,the relative expression of Bcl-2 protein significantly increased in the hippocampus of rats in the medication group and EA group(P < 0.05).Additionally,compared to the sham group,the relative expression levels of Bax and cleaved-Caspase9 proteins in the hippocampus of the model group rats were significantly elevated(P < 0.05).However,in the medication group and EA group,the relative expression levels of Bax and cleaved-Caspase9 proteins were significantly reduced relative to the model group(P < 0.05).4.Alterations in Synaptic Plasticity of Hippocampal Neurons in Each Group:(1)WB results demonstrated a significant decrease in the relative expression levels of PSD-95 and Syn protein in the hippocampus of the model group compared to the sham group(P < 0.05).In contrast,both the medication group and the EA group exhibited a substantial increase in the relative expressions of PSD-95 and Syn protein in comparison to the model group(P < 0.05).(2)IF staining revealed a notable reduction in the average optical density of PSD-95 and Syn protein in the hippocampus of the model group when compared to the sham group(P < 0.05).Conversely,both the medication group and the EA group displayed a significant increase in the average optical density of PSD-95 and Syn protein in comparison to the model group(P <0.05).(3)Transmission electron microscopy findings illustrated that the synaptic vesicle distribution in hippocampal neurons of the sham group was uniform,with no significant changes in the postsynaptic density area and synaptic cleft.In contrast,the model group exhibited decreased numbers of synaptic vesicles,reduced postsynaptic density area,and significantly increased synaptic cleft,indicating inhibited neural signal transduction.However,the medication group and the EA group displayed increased numbers of synaptic vesicles,thickened postsynaptic density area,and a decreased synaptic cleft in comparison to the model group.(4)UV colorimetry analysis revealed that the Glu content in the model group was significantly increased compared to the sham group(P < 0.05).Conversely,both the medication group and the EA group exhibited a substantial decrease in Glu content in comparison to the model group(P <0.05).5.Expressions of BDNF/TrKB/CREB signaling pathway-related proteins in the hippocampus of each group were as follows:(1)WB results indicated a significant decrease in the relative expressions of BDNF,TrKB,p-Trk B,and p-CREB proteins in the hippocampus of the model group when compared to the sham group(P <0.05).In contrast,both the medication group and the EA group demonstrated a substantial increase in the expressions of these proteins in comparison to the model group(P < 0.05).(2)IHC staining revealed a significant decrease in the positive expressions of BDNF and TrKB in the hippocampus of the model group compared to the sham group(P < 0.05).In contrast,both the medication group and the EA group exhibited a remarkable increase in the positive expressions of these proteins in comparison to the model group(P < 0.05).(3)RT-PCR results showed a significantly lower positive expression of BDNF,TrKB,and CREB m RNA in the hippocampus of the model group compared to the sham group(P < 0.05).Conversely,both the medication group and the EA group displayed a substantial increase in the expressions of these m RNAs in comparison to the model group(P < 0.05).Conclusion(s): The administration of CFA plantar injection in rats has been shown to lower the pain threshold and elicit behaviors indicative of depression.This is accompanied by observable damage to hippocampal tissue,characterized by irregularly arranged nerve cells,increased intercellular space,dissolution and disappearance of cells,and a decrease in Nissl bodies.Furthermore,there is an increase in neuronal apoptosis,impairment of synaptic structure and function,and a significant decrease in the expression of proteins related to the BDNF/TrKB/CREB signaling pathway.These findings suggest a close relationship between chronic pain-induced depression,neuronal apoptosis,and synaptic plasticity.The EA of acupoints "Hegu" and "Taichong" has been shown to influence neuronal apoptosis and synaptic plasticity through the up-regulation of proteins in the BDNF/TrKB/CREB signaling pathway.Additionally,this treatment has been found to preserve the morphological integrity of the hippocampus and neurons,as well as ameliorate behavioral symptoms associated with chronic pain and depression in rat models. |
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