| Background:Brachial plexus nerve injury is a common peripheral nerve injury.Brachial plexus avulsion injury（BPAI）is the most serious type of injury.It usually leads to not only sensorimotor dysfunction of the upper extremity,but also a high incidence rate of neuropathic pain.Approximately 30–80%of BPAI patients developed neuropathic pain,which is an intolerable neuropathic pain.It could greatly influence patients’life and work,and brings heavy burden to family and society.A painful limb is often more intolerable than a non-functioning extremity.It is also much more difficult to deal with.Acupuncture is an important method of traditional Chinese medicine.It has been used to treat various pain for more than 2,000 years.Electroacupuncture has also been reported by domestic scholars in the treatment of neuropathic pain induced by BPAI.One of the potential mechanisms of its therapeutic effect is modulation of the central nervous system.But the exact way of its action has not been fully addressed yet.In the present experiment,we established a neuropathic pain model induced by BPAI.The treatment of electroacupuncture was compared with sham acupuncture.Behavioral studies were applied in the evaluation of efficiency.Histomorphological study and small animal PET/CT were combined to investigate the long-term dynamic effects of electroacupuncture on the functional metabolism and microstructure of the brain.The results may demonstrate the underlying central plastic mechanism of electroacupuncture in the treatment of neuropathic pain following BPAI.Finally,we aimed to provide theoretical basis for the solution of treating neuropathic pain.Part Ⅰ.Behavioral Study in Rats with Neuropathic Pain Induced by Brachial Plexus Avulsion and Treated by ElectroacupunctureObjective:The therapeutic effect of electroacupuncture on neuropathic pain following brachial plexus avulsion injury were evaluated by the detection of mechanical and thermal pain thresholds in the intact forepawMethods:Thirty-two female SD rats,weighing 180-200 g,were randomly divided into4 groups:normal group（Group N）,model group（Group M）,sham acupuncture group（Group S）and electroacupuncture group（Group A）,with 8 rats in each group.In Groups M,S and A,the model of total brachial plexus avulsion was established through posterior cervical laminectomy.In Group A,electroacupuncture was given to the left side of the acupoints EX-C5,EX-C6 and EX-C7.In Group S,the acupoints were 3 mm lateral to the corresponding acupoints in Group A.There was no particular therapeutic intervention in Groups N and M.The mechanical withdrawal threshold（MWT）and thermal withdrawal latency（TWL）of the left forepaw were detected at the baseline and after surgery（3 days,7 days,2 weeks,3 weeks,1 month,2 months,3 months,and 4months,respectively）.the effect of electroacupuncture were evaluated.Results:（1）MWT of the left forepaws:There was no significant difference among the4 groups at the baseline（P>0.05）.On the 3rd day and 2nd week after model establishment,the MWT of the left forepaws was significantly lower in Group M,compared with Group N（P<0.05）.There was no significant difference between Groups S and A（P>0.05）.At the end of 2nd week,3rd week,1st month,2nd month,and 3rd month after model establishment（i.e.,at the end of the 1st,2nd,3rd,7th,and 11th week after the start of real/sham electroacupuncture application）.Compared with that in Group N,the MWT of the left forepaws was significantly lower in Group M（P<0.05）.Compared with that in Group S,The MWT of the left forepaws was significantly higher in Group A（P<0.05）.At the end of 4th month after model establishment（i.e.,at the end of 4th week after the stop of real/sham electroacupuncture application）,the MWT of the left forepaws was significantly lower in Group M compared with that in Group N（P<0.05）.Compared with that in Group S,the MWT of the left forepaws was significantly higher in Group A（P<0.05）.（2）TWL of the left forepaws:There was no significant difference among the 4 groups at the baseline（P>0.05）.On the 3rd day and at the end of the 1st and 2nd week after model establishment,the TWL of the left forepaws was significantly lower in Group M compared with that in Group N（P<0.05）.There was no significant difference between Group S and Group A（P>0.05）.At the end of the 3 week,1st month,2nd month and 3rd month after model establishment（i.e.,at the end of the 2nd,3rd,7th,and 11th week after the start of real/sham electroacupuncture application）,the TWL of the left forepaws was significantly lower in Group M compared with that in Group N（P<0.05）.The TWL of the left forepaws was significantly higher in Group A,compared with Group S（P<0.05）.At the end of the 4th month after model establishment（i.e.,at the end of the 4th week after the stop of real/sham electroacupuncture application）,the TWL of the left forepaws was significantly lower in Group M,compared with that in Group N（P<0.05）.The TWL of the left forepaws was significantly higher in Group A,compared with that in Group S（P<0.05）.Conclusion:The mechanical and thermal pain thresholds of the intact forepaws significantly decreased following brachial plexus avulsion injury.Compared with sham acupuncture,electroacupuncture on the left acupoints of EX-C5,EX-C6 and EX-C7could significantly relieve central sensitization of neuropathic pain.Pain threshold of the intact forepaws following brachial plexus avulsion was significantly elevated and the neuropathic pain was extenuated.The effect was maintained even at 1 month after the stop of treatment.Part Ⅱ.Brain Metabolic Mechanism Study in Rats with Neuropathic Pain Induced by Brachial Plexus Avulsion Injury and Treated by ElectroacupunctureObjective:The long-term changes of brain functional metabolism in rats of neuropathic pain induced by brachial plexus avulsion injury and treated by electroacupuncture were studied by small animal PET/CT.The central mechanism of electroacupuncture in treating neuropathic pain after brachial plexus root avulsion injury was also explored.Methods:A total of 32 female SD rats,weighing 180-200g,were randomly divided into4 groups:normal group（Group N）,model group（Group M）,sham acupuncture group（Group S）and electroacupuncture group（Group A）,8 rats in each group.In Groups M,S and A,the model of total BPAI was established through a posterior approach by hemilaminectomy.The acupoints of EX-C5,EX-C6 and EX-C7 on the left side of the neck were chosen for treatment in Group A.In Group S,the acupoints for treatment were located 3 mm laterally to corresponding acupoints in Group A without electrical outputs.There was no particular intervention in the Groups N and M.To investigate the glucose metabolism alteration,[fluorine-18]-fluoro-2-deoxy-D-glucose(18F-FDG)was applied to theses rats by tail intravenous injection and their brains were scanned by positron emission tomography/computed tomography（PET/CT）.The timepoints for examination were as follows:（1）at the baseline;（2）at the end of the 1st month following global BPAI（i.e.,the 3rd day after the start of electroacupuncture/sham acupuncture application）;（3）at the end of the 3rd month following global BPAI（i.e.,the end of the 11th week after the start of electroacupuncture/sham acupuncture application）;（4）at the end of the 4rd month following global BPAI（i.e.,the end of the 4th week after the stop of electroacupuncture/sham acupuncture application）.Results:（1）Activation of the neural circuits of pain:In the motor-sensor related regions of the brain,the metabolism of left piriform cortex,left mesencephalic region,and the right caudate putamen in group M were higher than that in group N.The metabolism of the left caudate putamen,the left piriform cortex,and left somatosensory cortex in Group M had lower metabolism than that in Group N.In the pain-related areas,the metabolism of right raphe nuclei and left periaqueductal grey matter in Group M were higher than that in Group N.In the emotion-cognition related regions,the metabolic level of left septum,right medial hypothalamus,right postural dorsal hippocampus,and left ventral tegmental area in Group M were higher than those in Group M.The posterolateral part of the right hippocampus,the anterior lateral part of the left hippocampus,the left nucleus of the left nucleus,the lateral part of the right hypothalamus,and the left septum in the model group were lower in metabolism than those in Group N.（2）Acupoint-specific neurological regulation:In the sensorimotor areas,the right somatosensory cortex,right motor cortex in Group A were higher metabolism than those in Group S.Metabolism in bilateral caudate putamen and the right dorsal lateral thalamic in Group A was lower than those in Group S.In the pain-related areas,left raphe nuclei in Group A was lower in metabolism than that in Group M;the metabolism of bilateral periaqueductal grey matter in Group A was lower than that in Group S.In the emotion-cognition areas,the metabolism of right medial prefrontal cortex,left retrosplenial cortex and right orbitofrontal cortex in Group A was higher than that in Group S.The ventral hippocampus,right amygdala,left anterolateral hippocampus,and the medial part of left hippocampus in Group A was lower in metabolism than that in Group S.（3）Central mechanism of the immediate analgesic effect of electroacupuncture:In the sensorimotor area,the metabolism of right motor cortex,right somatosensory cortex,right globus pallidus,right dorsolateral thalamus,left motor cortex,and right insular cortex in Group A was higher than those in Group M.Compared with Group M,the metabolism of left dorsolateral thalamus,left ventromedial nucleus,and the dorsal nucleus of the thalamus in Group A was lower.In the emotion-cognition related areas,the metabolism of right amygdala,bilateral medial prefrontal cortex,and the right ventral hippocampus in Group A was higher than those in the Group M.The metabolism of right septum,left ventral hippocampus,and right retrosplenial cortex in Group A was lower than those in Group M.（4）central mechanism of electroacupuncture analgesia post-effect:In the sensorimotor areas,metabolism in the right caudate putamen,left somatosensory cortex,left dorsolateral thalamus,right motor cortex,and left medial geniculate in Group A was higher than those in Group M.Metabolism in left somatosensory cortex,left motor cortex,right ventral pallidum,left parietal cortex,right somatosensory cortex,left lateral midline thalamus in Group A was lower than those in Group M.In the emotion-cognition related areas,metabolism in the left lateral hypothalamus and the right frontal cortex in Group A was higher than those in Group M.Metabolism in the left medial prefrontal cortex,left entorhinal cortex,and left amygdala in Group A was lower.（5）Two-way regulation mechanism of electroacupuncture analgesia:Compared with the sham electroacupuncture group,the electroacupuncture group significantly increased the metabolic link of the bilateral sensory-motor cortex one month after brachial plexus injury.The metabolic junction of the sensory motor cortex in the healthy limbs decreased three months after injury,and the post-effect of electroacupuncture treatment showed an increase in the metabolic connectivity associated with the sensorimotor cortex of the affected limb four months after injury.The changes in the metabolic connection of the sham electroacupuncture group were limited.Only 3 months after injury,the non-specific healthy dorsolateral thalamic brain area centered on the metabolic connection was enhanced,and the rest of the time was similar to the model group.Conclusion:After global BPAI,the function of the sensorimotor area in the brain significantly decreased.The excitability of the analgesic area decreased such as the raphe nuclei and the left periaqueductal grey matter,and the metabolism of emotion-cognition related areas also decreased.The central effect of sham electroacupuncture is more diffuse and non-specific.Electroacupuncture induces remodeling of the sensorimotor cortex and promotes the recovery of cognitive dysfunction in rats with neuropathic pain.Part Ⅲ.Brain Histomorphological Study of Electroacupuncture in the Treatment of Neuropathic Pain Induced by Brachial Plexus Avulsion Injury in RatsObjective:The present study investigated the changes of brain microstructures such as astrocytes,microglia,dendrites/dendritic spine in pain-related brain regions（e.g.,thalamus,primary somatosensory cortex）.The aim was exploring the mechanism of electroacupuncture in the treatment of neuropathic pain after brachial plexus avulsion.Methods:A total of 75 female SD rats,weighing 180-220g,were randomly divided into4 groups:normal group（Group N）,model group（Group M）,sham acupuncture group（Group S）and electroacupuncture group（Group A）.There were 15 rats in Group N and 20 rats in Groups M,S and A,respectively.In Groups M,S and A,the model of global BPAI was established through a posterior approach by hemilaminectomy.The acupoints of EX-C5,EX-C6 and EX-C7 on the left side of the neck were chosen for treatment in Group A.In Group S,the acupoints for treatment were located 3 mm laterally to corresponding acupoints in Group A without electrical outputs.Immunofluorescence study and Golgi staining were applied at 4 time points:（1）at the baseline;（2）on the 3rd day following global BPAI（i.e.,the 3rd day after the start of electroacupuncture/sham acupuncture application）;（3）at the end of the 1st month following global BPAI（i.e.,the end of the 3rd week after the start of electroacupuncture/sham acupuncture application）;（4）at the end of the 4rd month following global BPAI（i.e.,the end of the 4th week after the stop of electroacupuncture/sham acupuncture application）.At each timepoint,2 rats in each group were selected for the detection of immunofluorescence study and 3 rats for the Golgi staining（in Group N,the data at the second timepoint was the same as that at baseline）.The immediate and after effects of electroacupuncture or sham acupuncture were investigated.At each timepoint,the rats were sacrificed and the brain tissues were harvested for evaluating the effect of electroacupuncture in the microstructure of pain related areas in the brain in neuropathic pain rats following global BPAI.Results:（1）Immunofluorescence study:（1）S1 area:At baseline,there were no significant differences in the expression of GFAP and IBA-1 in bilateral S1 areas among the 4 groups.On the 3rd day after model establishment,the expression of GFAP and IBA-1 in bilaterally S1 significantly increased in Groups M,compared with that in Group N（P<0.05）.At the end of the 1st month following global BPAI,the expression of GFAP and IBA-1 in bilateral S1 areas in the Group M were higher than that in Group N（P<0.05）.The expression of GFAP and IBA-1 in the bilateral S1 in Group A was lower than that in Group S（P<0.05）.At the end of the 4rd month following global BPAI,the expression of GFAP and IBA-1 in the left S1 in the Group M was higher than that in Group N（P<0.05）,and IBA-1 in the right S1 in the Group M was higher than that in Group N（P<0.05）.The expression of GFAP and IBA-1 in the right S1 area in Group A was lower than that in Group S（P<0.05）.（2）Thalamus:At baseline,there was no significant difference in the expression of GFAP and IBA-1 in the bilateral thalamus among the 4 groups.On the 3rd day after model establishment,the expression of GFAP and IBA-1 in bilateral thalamus in Groups M was significantly higher compared with Group N（P<0.05）.At the end of the 1st month following global BPAI,the expression of GFAP and IBA-1 in bilateral thalamus was significantly higher in Group M than that in Group N（P<0.05）.The expression of GFAP and IBA-1 in the bilateral thalamus in Group A were significantly lower than that in Group S（P<0.05）.At the end of the 4rd month following global BPAI,the expression of GFAP in the bilateral thalamus area in Group M was higher than that in Group N（P<0.05）.The expression of GFAP and IBA-1 in the bilateral thalamus area in Group A was lower than that in Group S（P<0.05）.（2）Golgi staining:（1）S1 area:At baseline,there was no significant difference in the total number of dendritic intersections and dendritic spine density in the bilateral S1 areas.On the 3rd day after model establishment,the total number of intersection points and dendritic spine density in Groups M significantly decreased compared with that in Group N（P<0.05）.At the end of the 1st month following global BPAI,the total number of dendritic intersections and dendritic spine density of S1 in Group M was lower than that in Group N（P<0.05）.The total number of dendritic intersections and dendritic spine density of S1 in Group A increased compared with that in Group S（P<0.05）.At the end of the 4rd month following global BPAI,the total dendrites intersections and dendritic spine density of bilateral S1 in Group M was lower than that in Group N（P<0.05）.The total number of dendritic intersections and dendritic spine density of S1 in Group A was higher than that in the Group S（P<0.05）.（2）Thalamus:At baseline,there was no significant difference in the total number of dendritic junctions and dendritic spine density in the bilateral thalamus before surgery among the 4 groups.On the 3rd day after model establishment,the total number of intersection points and dendritic spine density decreased significantly in Groups M,compared with that in Group M（P<0.05）.At the end of the 1st month following global BPAI,the total number of dendritic intersections and dendritic spine density of the bilateral thalamus in Group M was lower than that in Group N（P<0.05）.The total number of dendritic intersections and dendritic spine density in the bilateral thalamus in Group A was higher than that in Group S（P<0.05）.At the end of the 4rd month following global BPAI,dendritic intersections and the total dendritic spine density of bilateral thalamus in Group M was lower than that in Group N（P<0.05）.The total number of dendritic intersections and dendritic spine density of bilateral thalamus of in Group A was higher than that in Group S（P<0.05）.Conclusion:The underlying mechanism of electroacupuncture in the treatment of neuropathic pain following global BPAI might be inhibition of the activation of astrocytes and microglia in bilateral S1 and thalamus.Plasticity changes of pyramidal cells in bilateral S1 and thalamus were also facilitated. |
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