The Mechanism Of NMDA Receptor In Caudal Anterior Cingulate Cortex In Pain Emotion Regulation Of DPI Rats

Objective:Toothache is a common type of oral and maxillofacial pain with a high incidence, including acute and chronic pain,which can affect patients'eating and sleeping mood in severe cases.Due to the special nature of nerve distribution in the pulp tissue,pain often cannot be accurately located.Maxillofacial pain and body pain have different brain nerve conduction mechanisms,and the location and metabolic distribution of nociceptive stimuli in the cerebral cortex are still controversial.According to reference survey results,it is found that the anterior cingulate cortex?ACC?acts as a pain management area and is involved in the neural conduction mechanism of acute and chronic pain.ACC is divided into rostral anterior cingulate cortex?rACC?and caudal anterior cingulate cortex?cACC?.rACC plays a key role in pain and mood by regulating the levels of NMDA receptor subunits NR1,NR2B and NR2A,and NMDA receptor antagonist APV can reverse the phosphorylation level of subunits.Noxious stimuli can lead to activation of NMDA receptors in the cACC brain region.However,the specific mechanism by which NMDA receptor subunits NR1,NR2B,and NR2A in the cACC brain region are involved in pain is unclear.Some studies have confirmed that the cACC brain area is a key area involved in trigeminal nerve pain.Previous research of the research group showed that the Cg1 area of the cACC brain area was completely activated 3d after dental pulp injury?DPI?through micro-PET imaging.May be involved in the transmission of pulp-derived pain.Therefore,based on the previous research,this study selected 3 days after DPI as the research time point,and determined whether the APV can reverse the rat pulp-induced pain through behavioral evaluation of the rat face rubbing and forced swimming test.And determine whether APV can reverse the phosphorylation level of NMDA receptor subunits by Western Blot and laser confocal immunofluorescence technology;finally,using micro-PET imaging technology to determine whether APV can inhibit glucose metabolism in cACC brain area in DPI 3d.To further explore the nerve conduction mechanism of NMDA receptors in rat cACC region in dental pulp injury.Methods:1.cACC brain region implanted drug delivery tubeWith the assistance of the brain stereotactic instrument,the cACC brain area was embedded with a drug delivery tube according to the coordinate points.One week after the postoperative anti-infection recovery,a small amount of ink was injected into the cACC brain area to determine whether the position of the delivery tube was accurate.2.Construction of DPI rat modelForty-eight male SD rats were randomly selected.After intraperitoneal anesthesia,the high-speed turbine and a 1/2-round drill were used to intermittently remove the hard tissues of the maxillary first molar from the mesial fossa to expose the medullary cavity to form irreversible dental pulp origin.pain.Randomly divided into four groups:?1?SHAM group?n=12?:do nothing;?2?DPI group?n=12?:simply build DPI model;?3?DPI+NS group?n=12?:cACC brain area was injected with saline;?4?DPI+APV?n=12?:cACC brain area was injected with APV.3.Autoradiography Screening for APV ConcentrationAbout one week after the implantation tube was recovered,the cACC area was injected with different concentrations of APV:2nmol/?l,8nmol/?l,10nmol/?l,15nmol/?l,and subjected to[¹⁸F]-FDG autoradiography.4.Behavioral assessmentThe DPI rat model was constructed for two consecutive days before surgery and three consecutive days after surgery,and the body weight,diet,and drinking water of the rats were measured.In a quiet environment,a self-made face-shaping video recording device was used to record the total time for rats to wipe their faces within 20minutes;the water level in the swimming device was based on the rat's tail not touching the bottom,and the time for the rats to stand still within 5 minutes was recorded.The behavioral assessment method of cACC after 1 hour of APV was the same as before.5.Immunofluorescence detection of c-Fos and NMDA receptor subunit expression in cACC brainRats were anesthetized intraperitoneally,and brain tissue was obtained by infusion of paraformaldehyde.Laser confocal immunofluorescence technology was used to detect the expression of c-Fos,NR1,NR2A,NR2B in frozen brain tissue of DPI1-3d cACC.6.Western Blot to detect phosphorylation of NMDA receptor subunits in cACC brain regionIntraperitoneal anesthesia,fresh brain tissue was taken,cACC brain tissue protein was extracted,and Western Blot technique was used to detect the phosphorylation expression of NR2A subunit in cACC brain region at 1d,2d,3d,7d,and 14d after DPI.Isoflurane was lightly anesthetized,and rats were sacrificed after 1 hour of microinjection of APV.Fresh cACC brain tissue was taken and protein was extracted.Western Blot technology was used to detect phosphorylation levels of NR2A and NR2B subunits and c-Fos expression in DPI3d cACC brain regions.7.Micro-PET imaging technology to observe glucose metabolism in cACC brain areaSix rats were randomly selected from each group.After 20 minutes of cACC microinjection of APV,intraperitoneal injection of[¹⁸F]-FDG was performed.After absorption for 40 minutes,isoflurane was lightly anesthetized and a micro-PET scanner was placed for 10 minutes for static image scanning.Reconstruction,PMOD software analyzes glucose metabolism rate.Results:1.Ink experiment accurately locates cACC brain region drug delivery tube locationWith the help of a brain stereotactic instrument and a micro-injection pump,rats were sacrificed 10 minutes after the ink was injected into the cACC area.The brain tissue was removed,and the coronal plane of the brain tissue was cut along the injection part.Buried accurately.2.DPI model construction and evaluationFrom 2 days before DPI to 3 days after surgery,general behavioral results showed that the body weight,diet,and drinking water of DPI3d rats were significantly reduced,and the differences were statistically significant?P<0.05?.The behavioral results of rat rubbing showed that the total time of rubbing after DPI was increased?P<0.05?,and the result of forced swimming showed that the time of immobility was significantly prolonged?P<0.05?.The above results indicate that the DPI rat model was successfully constructed.DPI3d rats have pain and depression behavior,which is consistent with the previous results.The results of laser confocal immunofluorescence showed that the c-Fos was highly expressed in cACC brain regions of DPI 1-3d.3.NMDA receptor subunits NR1/NR2A/NR2B participate in cACC expression in DPI3d ratsLaser confocal immunofluorescence results showed that DPI3d cACC brain regions NR1,NR2A,and NR2B were highly expressed,indicating that NMDA receptors are involved in the conduction of cACC brain regions in DPI rats with acute pain.Western Blot detected DPI1d,2d,3d,7d,14d cACC region NMDA receptor subunit NR2A phosphorylation expression level,the results showed that DPI3d cACC region NR2A phosphorylation level expression was the most,the difference was statistically significant?P<0.05?,indicating acute Pulmonary-induced pain can up-regulate NMDA receptor NR2A phosphorylation.4.Autoradiography determines the optimal concentration of APV to be 10nmol/?l[¹⁸F]-FDG autoradiography showed that compared with APV concentrations of 2nmol/?l,8 nmol/?l,and 15 nmol/?l,10 nmol/?l APV injected into the cACC brain area could inhibit glucose metabolism in the cACC brain area.Therefore,in this study,10 nmol/?l APV was selected as the concentration for subsequent experiments.5.APV can reverse pain and emotion-related behaviorsDPI rats were examined 3 days after operation and 1 hour after cACC injection of APV.The results of rat surface scrubbing were compared with those in the SHAM,DPI,and DPI+NS groups?P<0.05?.This shows that APV may act on NMDA receptors in the cACC brain region,and then alleviate pain behavior in rats.DPI rats were subjected to forced swimming test 3 days after cACC injection of APV for 1 hour.The results showed that the rats in DPI+APV group had a longer time to stand still when swimming than those in DPI and DPI+NS groups?P<0.05?,indicating that Acting on the cACC brain region may inhibit depression in rats.6.APV can inhibit cACC glucose metabolism levelsMicro-PET imaging technique was used to detect the[¹⁸F]-FDG metabolic changes after 1 hour of injection of APV in the cACC area of DPI3d.The results of PMOD analysis showed that compared with the SHAM,DPI,DPI+NS group,cACC glucose metabolism in the DPI+APV group was inhibited statistically?P<0.05?.This indicates that APV injection in the DPI3d cACC brain area may inhibit NMDA receptors and thus inhibit glucose metabolism.7.APV down-regulates phosphorylation of NMDA receptor subunits NR2A/NR2B in cACC brain region and down-regulates c-Fos expressionWestern Blot results show that compared with DPI and DPI+NS groups,the phosphorylation level of NR2A and NR2B decreased in DPI+APV group,and the results were statistically different?P<0.001?.However,there was no significant difference in the expression of NMDA subunit NR1?P<0.001?.In addition,we also found that cACC brain area can reduce the expression of pain marker molecule c-Fos after injection of APV?P<0.001?.The results of this experiment indicate that APV can down-regulate the phosphorylation levels of NR2A and NR2B in cACC brain regions and down-regulate the expression of c-Fos.Conclusion:In this study,the DPI rat model was successfully constructed,and it was confirmed that NMDA receptors may be involved in the neural conduction mechanism of cACC brain region in acute pulp-induced pain.NMDA receptor antagonist APV may reverse pain and depression in rats.APV may act on cACC NMDA receptors and inhibit glucose metabolism.It may be involved in pain by reversing the NMDA receptor NR2A and NR2B phosphorylation levels.This study provides clinical guidance for the neural conduction mechanism of toothache.However,how the remaining subunit phosphorylation expression is worth further study...