Study On The Effect Of The Pathway From Ventral Covered Region To Nucleus Accumbens On Postoperative Pain

BachgroundPostoperative pain is acute pain that occurs after surgery,including somatic pain and visceral pain,mainly caused by a series of inflammatory mediators(including histamine,bradykinin,nerve growth factor,etc.)released by inflammatory cells and nerve endings when the tissue is injured To.Postoperative pain can cause symptoms such as respiratory function suppression,weakened immune function,and psychological disorders.If it is not effectively treated in time,it may develop into chronic pain and neuropathic pain,which will greatly affect the patient’s future life.Recent studies have shown that the ventral tegmental area(VTA)and nucleus accumbens(NAc)in the reward loop are involved in the regulation of analgesia.The nerve fibers of the VTA cell body project to the NAc,By activating Dopamine(DA)neurons in the ventral margin of the midbrain,it plays a role in the regulation of pain,indicating that there is a certain correlation between the reward system and the mechanism of analgesia.Data show that electrical stimulation of dopaminergic neurons in the striatum,nucleus accumbens,and ventral covered area can attenuate the pain response,and selective damage to dopaminergic neurons can cause hyperalgesia in animals.It shows that dopamine plays an important role in the reward system regulating pain.Injecting morphine and substance P analogs in the ventral tegmental area can reduce nociception,while injecting dopamine receptor antagonists into the nucleus accumbens reduces the analgesic effect of injecting morphine or substance P analogs in the ventral tegmental area.In addition,ankylosing harmful stimuli can lead to increased release of dopamine in the nucleus accumbens.It indicates that the analgesic effect of the ventral tegmental area may be through the projection of nerve fibers to the nucleus accumbens,but the molecular mechanism of postoperative pain has not been reported in the literature.ObjectiveIn this study,we used optogenetic technology and D2 receptor antagonists to investigate the role of dopaminergic neurons in the ventral tegmental area on the regulation of nucleus accumbens neurons in the development of pain by preparing a mouse model of incision pain.Methods1.Experiment animals: 6-8 weeks-old DAT-cre(Dopamine transporter,DAT)mice.Purchased by Shanghai Nanfang Biotechnology Development Co.,Ltd.purchased DAT-cre transgenic animals with the same background as C57B1/6,and the strain name was B6.SJL-Slc6a3tm1.1(cre)Bkmn/J.2.Groups and treat:Groups:Twelve C57BL/6 male mice were randomly divided into a control group(C group)and a model group(P group),n=6.DAT-cre mice were randomly divided into ChR2 control + model group(PC group),ChR2+ model group(P+ChR2 group),ChR2+DMSO group(ChR2+D),ChR2+ piperidone group(D2 receptor antagonist)Group ChR2+D2),n=6.Treat:Model group(P group): surgical treatment of left hind foot incision,n=6;Control group(Group C): the sham operation group,except that the left hind foot incision surgery is not performed,the rest of the treatment is the same as the model group,n=6;ChR2 control + model group(PC group): injection of ChR2 control virus,two weeks after expression,hind foot incision treatment,photostimulation operation 5 days after surgery,n=6;ChR2+ model group(P+ChR2 group): injection of ChR2 virus,two weeks after expression,a hind foot incision treatment,and photostimulation operation 5 days after surgery,n=6;ChR2+DMSO group(ChR2+D): after injection of ChR2 virus,two weeks after expression,a hindfoot incision treatment was performed.After DMSO injection for 5 minutes 5 days after surgery,a photostimulation operation was performed,n=6;ChR2+ piperidone group(D2 receptor antagonist group ChR2+D2): after injection of ChR2 virus,two weeks after expression,a hindfoot incision was made.After injection of piperidone 5 minutes 5 days after surgery,a light stimulation operation was performed,n= 6.3.Transfection of photosensitive virus: After anesthetizing the mice with isoflurane,according to the mouse map VTA(AP,-3.5 mm,LP,0.5 mm,DP,-3.8 mm)localization injection of the viral vector carrying the photosensitive gene ChR2(AAV2/9-EF1α-DIO-ChR2(H134R)-m Cherry),and its control(AAV2/9-EF1α-DIO-m Cherry),respectively named ChR2 group and ChR2 control group4.Viral expression detection: Two weeks after stereotactic injection of the virus,frozen sections were taken,and the expression level of ChR2 virus in the ventral tegmental area was detected under a fluorescent microscope.5.Tracer detection: stereotactic injection of Fluoro Gold(FG)retrograde tracer in the NAc area,brain perfusion is taken 3-5 days later,frozen sections are taken,and the fluorescent gold in the ventral tegmental area is observed under a fluorescent microscope Distribution.6.Preparation of postoperative pain model: a toe incision pain model was prepared according to the method of Brennan TJ [5].After isoflurane anesthesia,the model group mice made a 5 mm incision in the left hind heel Achilles tendon;the sham operation group The treatment is the same except that no hindfoot incision is made.7.Determination of mechanical pain threshold: using von frey filament measurement,light stimulation during the test,observe the changes in the nociceptive threshold of mechanical stimulation 1d before surgery,1 day,3 days,5 days,7 days,10 days after surgery.8.Immunofluorescence detection: After the Von-frey mechanical pain threshold was measured,the heart was quickly perfused with 4% paraformaldehyde,brain tissue was collected,frozen sections were taken,and immunofluorescence was used to detect the expression changes of D1 and D2 receptors in the nucleus accumbens.9.Western bloting detection: immediately after the Von-frey mechanical pain threshold was measured,the spinal cord L3-L5 was taken and protein expression was observed to observe the expression of p-PKA in the spinal cord.10.Statistical analysis: SPSS 21.0 was used for statistical analysis.The data results were expressed as mean ± standard error(Mean ± SEM).One-way analysis of variance or independent sample t test was used.P <0.05 was considered statistically significant.Results1)In the results of the mechanical pain threshold test,from the 1st day after surgery,the nociceptive threshold of the mechanical stimulus on the affected foot of the mice in the post-operative pain model group was significantly lower than that before the operation.There is a significant difference(***P<0.001),and this significant difference lasts until 5 days after surgery,and returns to the baseline value on the 7th day.In the ChR2+ model group,the nociceptive threshold of mechanical stimulation of the left foot was significantly higher than that of the control virus + model group at 5 days after surgery(***P<0.001).From the 5th day after operation,the nociceptive threshold of mechanical stimulation of the left foot of the ChR2+D2 antagonist group was significantly lower than that of the ChR2+DMSO group,and the difference was statistically significant(***P<0.001).2)The results of fluorescent gold expression indicate that there is fiber projection from the ventral tegmental area to the nucleus accumbens.3)Immunofluorescence results showed that after activation of dopaminergic neurons in the ventral tegmental area,the expression of D1、D2 receptors in the nucleus accumbens was significantly increased compared with the control group(**P<0.01).4)Western blot results showed that the expression of phosphorylated PKA(48.27±4.35)in the spinal cord of the ChR2+ model group that activated ventral tegmental dopaminergic neurons was significantly lower than that of the ChR2 control+model group(122.3±13.11)(*** P<0.001),the expression of phosphorylated PKA(103±5.74)in the D2 receptor antagonist group injected with piperidone in the nucleus accumbens was significantly higher than that in the ChR2+ model group(59.21±5.32)and ChR2+DMSO(58.56±7.15).(**P<0.01).Conclusion1.Exciting VTA dopaminergic neurons can relieve postoperative pain,activate NAc D1,D2 receptors,down-regulate the expression of phosphorylated PKA in the spinal cord,and antagonize NAc D2 receptors can reverse the relieving effect of excited VTA dopaminergic neurons on postoperative pain.2.The inhibitory effect of VTA dopaminergic neurons on postoperative pain may be related to activating the VTA-NAc dopamine pathway,activating the NAc DA receptor and reducing the phosphorylation of PKA in the spinal cord.