Experimental Study Of Cholinergic Mechanisms Are Involved In The Pain Relieving Effect Of SCS

Neuropathic pain presents a therapeutic challenge since such pain is oftenresistant to conventional analgesic drugs including opioids. SCS has developedinto an indispensable tool in chronic pain management and about 60%-70% ofwell-selected patients suffering from neuropathic pain obtain satisfactory painrelief. However, despite research in recent years the mechanisms behind thebeneficial effects of SCS are poorly understood. Above all, cholinergicmechanisms in the pain relieving effect of SCS are still unclear. Purpose: In the present study, we created the neuropathic pain model in rats,observed the changes of acetylcholine level in dorsal horn induced by SCS indifferent groups of experimental animals classified by their behavior and theirreactivity to SCS, and clarified the acetylcholine receptor subtypes activated bySCS and their changes in order to investigate the relationship between thecholinergic system and SCS and provide the theoretical basis for the clinictreatments. Methods: PNI animal model was used in this study by ligating the sciaticnerve partially in SD rats. According to the different reactive threshold ofwithdraw, the experimental animals were categorized into tactile allodynia andnon-tactile allodynia groups and based on the reactivity to SCS, the animals wereclassified into responders to SCS and non-responders to SCS further. Throughmicrodialysis and HPLC, the changes of acetylcholine in dorsal horn wereinvestigated in the different groups of experimental animals. The acetylcholinereceptor subtypes activated by SCS were clarified by intrathecal administration ofthe different antagonists and the different receptor expressed levels as well as thechanges of the related neurotransmitter 5-HT were investigated by immuno-histochemistry.Results: The effect of SCS to the release of acetylcholine in the dorsal hornof spinal cord. The concentration of extracellular acetylcholine in spinal dorsalhorn 30min before SCS was regarded as the basal line. In the animal model ofallodynia, basal line of acetylcholine concentration in the group of responders toSCS was 2.9±0.3nmol/L (mean±SD). Compared to the basal line concentration,SCS obviously increased the extracellular acetylcholine concentration of spinaldorsal horn (7.8±0.5 nmol/L, P<0.05). 30min after SCS, the concentration was6.4±0.4 nmol/L (P<0.05) and 60min after SCS, the concentration was 4.1±0.7nmol/L (P>0.05). During the period of SCS, threshold of withdrawl in the injuriedside significantly increased (P<0.01) and before recovering to the previous basallevel, the increased effect lasted for 30min after SCS (P<0.05). Ach basalconcentration in the group of non-responders to SCS was 3.1±0.4nmol/L andSCS only induced slightly increase of Ach concentration (3.6±0.5nmol/L,P>0.05). Meanwhile, SCS couldn't induce the changes of threshold of withdrawlin the non-responders.When we admistrated nicotanic receptor antagonist (MCM) intrathecally tothe expermental animals, there was no obvious changes in the threshold ofwithdrawl compared to simply SCS treatment (P>0.05). However, when wetreated the animal with non-selective muscarinic acetylcholine receptor (mAchR)antagonist (ATR), anti-mechanical allodynia effect of SCS was blocked obviouslycompared to the simply SCS treatment (P<0.05). Anti-mechanical allodynia effectof SCS was blocked completely when compared ATR and MCM together(P<0.05). M1, M2 and M4 acetylcholine receptor antagonists could block SCSanti-allodynia effecs remarkably (P<0.05, n=7), among which the effect of M4antagonist (MT3) were strongest. However, Ach receptor antagonist had nosignificant influence to SCS-induced anti-mechanical allodynia effects.In the spinal cord of non-mechanical allodynia rats, M4 receptor distrabutedin the dorsal and ventral horns of the spinal cord wildly. Under microscope withlow magnification, high concentation of M4 receptor were found in the layer Ⅱ-Ⅳ of the dorsal horn, especially in the layerⅡ, in the layerⅠonly a few M4receptor distribution. In addition, there was no obvious difference of M4 receptorcontributions between the sciatic nerve injured side and un-injured side. Undermicroscope with high agnification, M4 receptors were punctuate structure anddistributed on the surface of most neurons. In the ventral horn of non-allodyniarats, M4 receptors were also observed in the surface of some neurons, includingmotor neurons. Moreover, there was the synaptic connection between motorneurons and M4 receptor positive neurons.In the non-responders to SCS of allodynic rats, M4 receptor only distributedin the superficial layer of spinal dorsal horn (layer Ⅰ and Ⅱ) and there was noexpression in the other region of spinal gray substance. There was no differenceof the M4 receptor expression between the sciatic nerve injured ipsilateral andcontralateral dorsal horns. Compared the non-allodynic rats, M4 receptor in thespinal gray substance of the responders to SCS decreased significantly andmainly contributed in the superficial layer of the dorsal horn. But in the rats ofnon-responders to SCS, M4 receptors still increased obviously in the greysubstance of spinal cord. Again, there was no difference of the M4 expressionbetween the sciatic nerve injured ipsilateral and contralateral dorsal horns.There was the high expression of 5-HT in the spinal dorsal horn ofof SCS was blocked completely when compared ATR and MCM together(P<0.05). M1, M2 and M4 acetylcholine receptor antagonists could block SCSanti-allodynia effecs remarkably (P<0.05, n=7), among which the effect of M4antagonist (MT3) were strongest. However, Ach receptor antagonist had nosignificant influence to SCS-induced anti-mechanical allodynia effects.In the spinal cord of non-mechanical allodynia rats, M4 receptor distrabutedin the dorsal and ventral horns of the spinal cord wildly. Under microscope withlow magnification, high concentation of M4 receptor were found in the layer Ⅱ-Ⅳ of the dorsal horn, especially in the layerⅡ, in the layerⅠonly a few M4receptor distribution. In addition, there was no obvious difference of M4 receptorcontributions between the sciatic nerve injured side and un-injured side. Undermicroscope with high agnification, M4 receptors were punctuate structure anddistributed on the surface of most neurons. In the ventral horn of non-allodyniarats, M4 receptors were also observed in the surface of some neurons, includingmotor neurons. Moreover, there was the synaptic connection between motorneurons and M4 receptor positive neurons.In the non-responders to SCS of allodynic rats, M4 receptor only distributedin the superficial layer of spinal dorsal horn (layer Ⅰ and Ⅱ) and there was noexpression in the other region of spinal gray substance. There was no differenceof the M4 receptor expression between the sciatic nerve injured ipsilateral andcontralateral dorsal horns. Compared the non-allodynic rats, M4 receptor in thespinal gray substance of the responders to SCS decreased significantly andmainly contributed in the superficial layer of the dorsal horn. But in the rats ofnon-responders to SCS, M4 receptors still increased obviously in the greysubstance of spinal cord. Again, there was no difference of the M4 expressionbetween the sciatic nerve injured ipsilateral and contralateral dorsal horns.There was the high expression of 5-HT in the spinal dorsal horn ofnon-allodynic rats and lest in the non-responders to SCS, which indicated therewas the relationship between the serotoninergic system and cholinergic system inthe spinal cord.