The Mechanisms Underlying Different Analgesic Effect Of Deep And Superficial Needling At Acupoint Housanli On Chronic Inflammatory Pain In The Rats

BackgroundNeedle insertion depth is an important factor in acupuncture treatment.Previous clinical studies have shown that there is difference of the curative effect between deep and superficial needling in treating diseases,yet the mechanism is still unknown.ObjectiveIn this study,we explored the mechanism of the different effect between deep and superficial needling by using a model of inflammatory pain induced by complete Freund’s adjuvant(CFA)in rats.Acupoint Housanli(ST 36,corresponding to Zusanli in human body)was chosen and high-sensitive neural tracing,immunofluorescence,behavior and molecular biology techniques were used to investigate the difference of innervation and chemical characteristics between superficial and deep tissue in the ST 36 region,analgesic effect between superficial and deep needling and its relationship with the changes of cell and chemical substances in local inflammatory region,dorsal root ganglia(DRG),spinal cord and acupoint region.The mechanism underlying anti-inflammatory and analgesic effect of the deep and superficial needling on chronic inflammatory model rats will be revealed systematically.Method1 The distribution of nerve fibers and immune cells in the ST 36 regionLocal tissues of ST 36 from three male Sprague Dawley(SD)rats were dissected out following perfusion with 4%paraformaldehyde,cut into sections,and stained with immunofluorescence method.The distribution of nerve fiber,mast cell,macrophage,dendritic cell and 5-heyroxytryptamine(5-HT)positive cell in local tissues of ST 36 region was determined.2 The differences of innervation and chemical characteristics of the deep and superficial layer in the ST 36 regionFour μl 0.1%neural tracer cholera toxin subunit B(CTB)was injected into the muscle or subcutaneous tissue beneath ST 36 in fourteen rats,respectively.In order to prevent leakage of CTB,the needle was kept in place for 1 min after injection and then pulled out slowly.Three days following injection,the distribution of motor and sensory neurons and transganglionic axonal terminals labelled with CTB was examined with immunofluorescence in the DRG,spinal cord and brainstem.Use the sections achieved from the above experiment to label the chemical characteristics of neurons related to skin and muscle tissues of ST 36 region.The antibodies labelling CTB and other chemical substance were from different animals.In this study,substance P(SP)and calcitonin gene-related peptide(CGRP)were labelled.3 The differences of the analgesic effect on inflammatory model rats exerted by the deep and superficial needling at ST 36Sixty-four SD rats were divided into control group,model group,deep needling group and superficial needling group at random.Control group:Under general anesthesia with isoflurane,50 μL 0.9%saline was injected into subcutaneous tissue of hind foot plantar with microinjector.Model group:Under general anesthesia with isoflurane,50 μL CFA was injected into subcutaneous tissue of hind foot plantar with microinjector.Deep needling group:After modeling,the needling was pierced perpendicularly into muscle tissue beneath ST 36.Connect Han’s Electro-Acupuncture Instrument with stimulus parameter 2/100 Hz,1-2-3 mA,for 10 min at each density level and 30 min in total.Superficial needling group:After modeling,the needling was pierced at 15° angles into the subcutaneous tissue beneath ST 36.The stimulus parameter is the same as Deep needling group.The mechanical pain threshold and thermal pain threshold of each group of rats were tested before modeling,48 h after modeling and 30 min after electropuncture(EA).After the pain threshold was tested,deep and superficial EA was given at ST 36.After EA and the pain threshold were tested,local inflammatory tissues from 8 rats were dissected out following perfusion with 4%paraformaldehyde,cut into sections,and stained with immunofluorescence method.The changes of mast cell,macrophage,neutrophil,langerhans cell and CGRP and SP positive nerve fiber were observed.The content of tryptase in fresh inflammatory region was tested by western blotting(WB)method in the other 8 rats of each group.4 The change of cell and chemical substance expression in ST 36 region exerted by the deep and superficial needling at ST 36The grouping,test of pain threshold and EA treatment were the same as the experiment 3.Each group had 16 rats.After EA and the pain threshold were tested,local tissues of ST 36 from 8 rats were dissected out following perfusion with 4%paraformaldehyde,cut into sections,and stained with immunofluorescence method.The changes of mast cell,macrophage,neutrophil,5-HT and CGRP and SP positive nerve fiber were observed.The content of tryptase in fresh ST 36 region was tested by WB method in the other 8 rats of each group.5 The change of cell and chemical substance expression in relevant DRG and spinal cord exerted by the deep and superficial needling at ST 36The grouping,test of pain threshold and EA treatment were the same as the above experiment 3.Each group had 16 rats.After EA and the pain threshold were tested,rat’s L3-L4 DRG and lumbar spinal cord from 8 rats were dissected out following perfusion with 4%paraformaldehyde,cut into sections,and stained with immunofluorescence method.The changes of SP,CGRP,vanilloid receptor 1(VR1),microglia,parvalbumin(PV),calbindin-D28k(CB),neuronal calcium-binding proteins 1(NECAB1),NECAB2 and neuronal nitric oxide synthase(nNOS)were observed.The content of PV,CB,NECAB1,NECAB2 and SP in fresh L1-L6 DRG and lumbar spinal cord was tested by WB and ELISA methods in the other 8 rats of each group.Results1 The distribution of nerve fibers and immune cells in the ST 36 regionThere were a lot of nerve fibers in the skin of the ST 36 region,which formed free nerve ending near epidermis.Part nerve fibers expressed CGRP,SP and tyrosine hydroxylase(TH).In muscles,most nerve existed as nerve tracts.Part of sensory nerve fibers were observed winding around the surface of intraspindle muscle and forming spiral nerve ending,and part forming flowering branch ending after repeated splitting.CGRP and SP positive nerve fibers distributed in skin and muscles of the ST 36 region,which was more intensive in skin.Besides,CGRP positive nerve fiber from spinal ventral horn formed a garland structure at neuromuscular junction,controlling muscle contraction.Mast cell,dendritic cell,macrophage and 5-HT positive cell mainly distributed in dermis and subcutaneous tissue,and there were lots of 5-HT positive cells around CGRP positive nerve fibers.2 The differences of innervation and chemical characteristics of the deep and superficial layer in the ST 36 regionAfter CTB was respectively injected into subcutaneous tissue and muscle tissue in ST 36 region,all labelled neuron and transganglionic axonal terminals appeared in DRG,spinal cord and nuclei gracilis on the ipsilateral side of the injection.Sensory neuron associated with superficial and deep layer of ST 36 region distributed at Li-L5 DRG,intensively in L4 DRG,and the number of sensory neurons associated with superficial tissue was about 1.9 times that of deep tissue.Sensory neuron can be divided into large(>50μm),medium(30-50μm)and small(<30 μm)categories according to its diameter.The proportion of large,medium and small sensory neuron associated with superficial tissue was 10.4%,34.4%and 55.2%,however,the proportion associated with deep tissue was 14.5%,37%and 48.5%.The proportion of small sensory neuron associated with superficial tissue was much higher than deep tissue.Part of sensory neuron associated with the superficial and deep tissues in the ST 36 region was CGRP and SP positive.Among them,the proportion of CGRP and SP positive sensory neuron in sensory neuron associated with superficial tissue was 28.7%and 38.4%.However,the proportion of CGRP and SP positive sensory neuron in sensory neuron associated with deep tissue was 39.2%and 19.4%.3 The anti-inflammatory and analgesic effect of deep and superficial EA at ST 36 on chronic inflammatory model ratsBefore modeling,the pain threshold of each group of rats was not different.After modeling,mechanical pain threshold of each model group declined outstandingly compared with the control group(P<0.001).After deep and superficial EA at ST 36,deep needling group and superficial needling group obviously increased mechanical pain threshold compared with the model group(P<0.001,P<0.01).While the mechanical pain threshold of superficial needling group was still lower than the control group(P<0.01).Before modeling,the thermal pain threshold of each group of rats was not different.After modeling,thermal pain threshold of each model group declined outstandingly compared with the control group(P<0.001).After deep and superficial EA at ST 36,deep needling group and superficial needling group obviously increased thermal pain threshold compared with the model group(P<0.05,P<0.001).While the thermal pain threshold of deep needling group was still lower than the control group(P<0.05).After modeling,mast cells in inflammatory tissue greatly increased and were activated.After the deep and superficial EA at ST 36,the number of mast cells reduced.After modeling,massive macrophage and neutrophil appeared in inflammatory tissue especially around vein,and the number of 5-HT positive cells increased.After the deep and superficial EA at ST 36,the number of macrophage and neutrophil declined obviously,release of 5-HT in inflammatory tissue was greatly reduced,and the number of 5-HT positive cells decreased compared with the model group(P<0.05).After modeling,the number of langerhans cells in inflammatory tissue decreased,and after the deep and superficial needling the number of positive cells increased.After modeling,CGRP and SP positive nerve fibers in inflammatory tissue increased.After the deep and superficial needling,the number of positive nerve fibers decreased.The WB results showed that compared with the control group,protein content of tryptase in inflammatory region increased in the model group(P<0.001)and decreased in the deep needling group compared with the model group(P<0.01).4 The influence on nerve fibers and immune cells in the ST 36 region exerted by the deep and superficial needling at ST 36After modeling,mast cell in the ST 36 regional was activated,and after the deep and superficial EA at ST 36 region,the number of mast cells was not reduced and most mast cells still kept be activated.The WB results showed that protein content of tryptase in the ST 36 region increased in the deep needling group compared with the control group(P<0.001).After modeling,the number of dendritic cells and 5-HT positive cells in the ST 36 region was not obviously changed,and the number of macrophage in ST 36 regional tissue was slightly increased,while after the deep and superficial EA at ST 36,the number of dendritic cells and 5-HT positive cells was obviously increased.5 The influence of deep and superficial EA at ST 36 on sensory neurons and chemical substance expression in relevant DRGAfter modeling,the number of CGRP,SP,VR1 and nNOS positive sensory neurons in DRG was increased and after the deep and superficial EA at ST 36,the number of these positive neurons was decreased.Most SP was co-expressed with CGRP.The ELISA results showed that SP protein content in DRG tended to increase after modeling and to decrease after the deep and superficial needling,but the difference in each group was not statistically significant.After modeling,the number of PV and CB positive neurons decreased in DRG,after the deep and superficial EA at ST 36,the number of positive neurons was increased.The WB results showed that after modeling,PV and CB protein content decreased in DRG(P<0.05),after the deep and superficial EA at ST 36 region,the protein content increased compared with the model group(P<0.05).6 The influence of deep and superficial EA at ST 36 on cells and chemical substance expression in relevant spinal cordCGRP and SP positive nerve fibers was projected on superficial spinal dorsal horn.After modeling,CGRP and SP positive axonal terminals and their receptor positive neurons obviously increased.The deep and superficial needling significantly decreased the density of CGRP positive axonal terminals and the number of CGRP receptor(P<0.001,P<0.01).However,only deep needling group decreased the density of SP positive axonal terminals.The ELISA results showed that SP protein content increased in the model group(P<0.05)and decreased in the deep needling group(P<0.05).After the modeling,the density of the VR1 positive fiber increased,and the fiber density decreased after the deep and superficial EA,and the reduction in superficial needling group was more obvious.Compared with the control group,the GAD density of the other three groups increased in spinal dorsal horns,and the increase of the deep needling group was more obvious.After modeling,microglia in the spinal dorsal horn were activated,and the number of nNOS positive neurons increased(P<0.01).The deep and superficial needling significantly inhibited the activation of microglia,and decreased nNOS-positive neurons(P<0.01,P<0.05).After modeling,the density of PV positive nerve fiber in substantia gelatinosa of spinal dorsal horn was declined,and the number of CB immune positive neurons in superficial spinal dorsal horn was decreased too,while the number of NECAB1 and NECAB2 positive neurons in spinal dorsal horn was increased.The deep and superficial EA increased the density of PV positive nerve fiber,the number of CB immune positive neurons,and decreased the number of NECAB1 and NECAB2 positive neurons.The WB results showed that both the deep and superficial needling increased PV protein content in spinal dorsal horn(P<0.05),while only the deep needling group increased CB protein content(P<0.05),and decreased NECAB1 and NECAB2 protein content(P<0.05).Conclusion1 Behavioral results show that both deep and superficial needling at ST36 increase the mechanical pain threshold and thermal pain threshold of chronic inflammatory model rats.Deep needling is better in improving the mechanical pain threshold,while superficial needling is more effective in improving the thermal pain threshold.2 The neuronal trace results show that the differences of innervation and chemical characteristics of deep and superficial local tissue in the ST 36 region may be the neuroanatomical basis for different effects of deep and superficial needling.3 The increase of immune cells in ST 36 region after deep and superficial needling may participate in the sensing and amplification of the acupuncture signal,so it may be the initiating factor of the effect of deep and superficial needling.Deep and superficial needling produce anti-inflammatory and analgesic effects by regulating the number and activity of immune cells in the inflammatory region.4 The decrease of the number of receptors in nociceptive neuron and the increase of content of buffered calcium-binding protein(CaBPs)in DRG can block the nociceptive impulse from the nociceptive neurons to the dorsal horm of the spinal cord,thereby playing an analgesic effect,which may be main mechanism the analgesic effect of superficial needling.5 Reduction of release of neurotransmitters and the number of CGRP receptors,inhibition of activation of microglia,increase of content of buffered CaBPs,and decrease in content of NECAB1/2 in the dorsal horn of the spinal cord lead to analgesic effect,which may be the main mechanism for deep needling.In summary,the peripheral immune cells in the acupoints and inflammatory region,the release of neurotransmitters,the changes in the number of receptors,the status of the microglia and intracellular CaBPs in the primary and secondary neurons of the DRG and the dorsal horn of the spinal cord may mediate the initial effects of deep and superficial needling and analgesic effects in rats with chronic inflammatory pain.