The Bilateral Neural Innervation Of Governor Meridian

BackgroundGovernor meridian is one important extra meridian,called the Sea of Yang Meridians,among meridian systems in the theory of the Traditional Chinese Medicine,which is mainly located on the dorsal midline of the back,following the path of the spinal cord up from the tail bone,across the top of the head,ending inside the mouth,on the upper gum.The meridians correspond to trajectories of peripheral nerves and blood vessels.The Governor meridian has 28 acupoints,covered most of the spinal segments and can have viscera regulation preference,especially gastric diseases.The effect of acupoints is related to its neurophysiological and anatomical features,and it has been reported that most high-frequency used acupoints located on or adjacent to peripheral nerve trunks,branches,or nerve endings,as well as blood vessels,and mast cells.Spinal nerve contains anterior ramus and posterior ramus.The anterior ramus includes intercostal/subcostal nerves suppling the thorax and abdomen midline.The posterior ramus includes peripherally posterior medial branch and posterior lateral branch.The dorsal midline is principally dominated by the posterior medial branch.Therefore,Governor meridian is primarily located on or adjacent to the posterior medial branch.Interestingly,almost all the dermatome maps proposed by Henry Head,Foerster and Lee etc.,show clear midline boundaries,which is contradictory to the concept of the receptive field of peripheral innervations.The size of the receptive field is largely a function of the branching characteristics of the afferent within the skin.Accordingly,receptive field of the dorsal midline of Governor meridian belongs to bilateral posterior medial branches.In this way,some anatomists and physicians acknowledged the variability of peripheral nerve distribution,as the midline overlap of cutaneous nerves,giving more evidence to question the dermatome maps existed.Therefore,we hypothesize that the Governor meridian distributed on the dorsal midline is of bilateral peripheral nerve innervations,which supports its efficacy on visceral functions in acupuncture therapy.Comparatively,the Bladder meridians have two main divisions,which are parallel with and lateral to the Governor meridian,for the first line～3 cm and for the second line～6cm in human,on the back bilaterally.Twenty-five acupoints scattered on the first lateral line and fourteen on the second line,covered all spinal segment innervation except T8 or T13.The same to the Governor meridian,the Bladder meridian have frequently been used to treat viscera diseases in acupuncture clinic,as cardiopulmonary and gastrointestinal dysfunction,but bilateral acupoints were selected simultaneously.Therefore,it is interesting to know whether ipsilateral acupoint in Bladder meridian is comparative to that of the Governor meridian in the same spinal segment.1 ObjectiveThis study aims to demonstrate the overlapped innervation of the Governor meridian from bilateral posterior ramus,and illustrate the superiority efficacy of acupuncture at Governor meridian on regulation of gastric motility,compared to those of the Bladder meridian throughout herpes zoster analysis,Evans blue extravasation,double fluorescent neural tracing retrogradely and anterogradely,and multichannel electrophysiological recording in the spinal cord dorsal horn,and gastric motility recordings.2 Methods2.1 Literature searching of herpes zosterThis study searched images of herpes zoster patients in Bing website.100 images which would accord with inclusion or exclusion criteria would be selected after screening.And they were divided into three parts:head/face,ventral side(thorax/abdomen),and dorsal side(back).The proportions of herpes region across the midline in different parts were calculated.2.2 The bilateral innervation of Governor meridian2.2.1 Evans blue extravasationWe examined the bilateral innervation of the Governor meridian by using plasma Evans blue(EB)extravasation with electric stimulation,performed on dorsal root or periphery nerve,to observe whether ipsilateral stimulation could produce a midline crossing of EB staining.A customized pair of hooked Ag/AgCl electrodes were used to hang one of these exposed nerves.The electrical current for nerve stimulation was generated by a STG stimulator.During the electrical stimulation,anesthetized rats were slowly injected with EB into the caudal vein.Fluorescence on the skin of each rat were collected and analyzed with in vivo imaging system after depilation.The proportion of EB dots crossing over the posterior or anterior midline produced by stimulation was accessed separately.2.2.2 Acupoints retrograde tracingDouble fluorescent neural retrograde tracing technique with the B subunit of cholera toxin Alexa Fluor 488 and 594(AF488-CTB,AF594-CTB)was applied on the Governor meridian and the 2nd line of Bladder meridian respectively.AF488-CTB,AF594-CTB was subcutaneously injected with 0.1%20μl into GV8 or BL47/BL48 by a microinjector.Three days later,the rats were transcardially perfused with 1×PBS followed by 4%paraformaldehyde(PFA).Then two sides of T7-10 dorsal root ganglia(DRGs)were harvested,post-fixed for 2 h,and cryoprotected in 25%sucrose overnight.The tissues were frozen and sectioned at 40μm with freezing microtome.Ultimately all the slices were obtained for each DRG.The number of AF488-CTB and AF594-CTB labeled neurons in bilateral DRGs(T7-T10)was calculated separately.2.2.3 Dorsal root ganglia anterograde tracingIn addition,AAV2/9-hSyn-EYFP or AAV2/9-hSyn-tdTomato was applied in bilateral DRGs(L4)to anterogradely label skins of Governor meridian.Male C57BL/6 mice was anesthetized with Tribromoethanol.The bilateral DRGs of L4 were exposed completely through separating the fascia of the intervertebral foramina bluntly.AAV2/9-hSyn-EYFP or AAV2/9-hSyn-tdTomato with 500nl was injected into bilateral DRGs(L4)respectively by a microinjector.The injection-rate was 200nl/min performed by a microinjection pump.After anterograde tracers were injected into DRGs for 6 weeks,the skin of Governor meridian(1cm×1cm)was harvested after transcardial perfusion with 1×PBS and 4%PFA.And the tissues were transparent by clearing solution.All the clearing steps were conducted at room temperature on the shaker.Samples were incubated with CUBIC solution 1 and solution 2 for 2 days respectively.Furthermore,primary antibodies anti-GFP and anti-RFP were used to incubate the samples.And then incubated with the Alexor Fluor 488 or 594 secondary antibodies.Finally,the tissues were incubated with CUBIC solution 3 until turning transparent.The final transparent tissues of Governor meridian were viewed and analyzed under a confocal imaging microscope.Three-dimensional reconstruction of the cutaneous innervation structure was performed by LAS X software.2.2.4 In vivo multichannel electrophysiological recordingIn the left dorsal horn of spinal cord,input projections from right lateral skin areas near to the midline Governor meridian was detected with in vivo multichannel electrophysiological recordings.Rats were performed laminectomy to expose spinal cord under the T10 to L1 vertebrae.Thirty-two array channels electrode were inserted into the left dorsal horn of spinal cord.The inserted angle was 77.5° and the depth was 800-1200μm.The records of spinal dorsal horn neurons were continuously monitored using a Cerebus5.0 acquisition system.The skin receptive area(right lateral skin areas near to the midline Governor meridian)was identified with multichannel electrophysiological recordings by gentle brushing.Left dorsal horn neurons activated by brushing near right to the midline were analyzed with NeuroExplorer 5.0 and Spike 2 software package.2.3 The acupoints of Governor meridian and Bladder meridian regulating gastric motilityFor functional verification,the effect of electroacupuncture on the gastric motility was compared by EA on GV6 of the Governor meridian with that on BL21 of the 1st lateral line and on BL50 of the 2nd lateral line of Bladder meridian.Three stainless-steel needles,0.3mm in diameter,were inserted into the back,one in GV6,the others in BL50 and BL21.And other three needles were inserted into the points 10mm near to these three acupoints.The paired stimulation electrodes were applied at GV6 and the point 10mm near to GV6.And other paired electrodes were applied at BL50,BL21 and their adjacent points.Needles were inserted 5mm deep into the skin.A Han’s Acupoint Nerve Stimulator was applied and the stimulus parameter was 1-2mA,0.6ms,10Hz,1min.Gastric pressure was recorded using the balloon pressure recording method.A pressure transducer was attached to the Quad Bridge Amplifier and it’s recorded by the Powerlab 8/30 data acquisation system.Then the balloon was filled with about 0.5-1mL pure water.Basic pressure was maintained at about 60cmH2O.The ratio of area under the curve(AUC)and wave peak were used for analysis.The effect of manual acupuncture(MA)on the gastric motility was compared among GV6,BL21and BL50.Three stainless-steel needles were inserted into the back respectively.The stimulus parameter was 2Hz,1min.Gastric pressure was recorded using the balloon pressure recording method as above.3 Result3.1 The bilateral innervation of Governor meridian3.1.1 Herpes zoster crossing the midlineImages of 100 herpes zoster patients according with to the inclusion or exclusion criteria were searched and selected from the Bing website randomly.35 images were of the head/face,30 images were of the ventral side,and 35 images were of the dorsal side.The proportions of herpes zoster crossing the midline were 45.7%,16.7%or 68.6%respectively.And the highest proportion is the shingles crossing the dorsal midline.3.1.2 EB dots crossing the dorsal midline produced by neurogenic inflammationDorsal root or intercostal nerves belonging to T8,T10-12 and L2-3 was stimulated electrically.And dorsal root,posterior ramus or posterior medial branch of spinal nerve involving T13-L4 was of stimulation.Ipsilateral electric stimulation of dorsal root or periphery nerve produced extravasation blue staining of EB dots across the midline of the back,accounting for 83.3%.Whereas,when the ipsilateral dorsal root or the intercostal nerves was stimulated,there was none crossing the anterior midline.3.1.3 The afferent neurons of the Governor meridian registered in bilateral DRGsWith retrograde tracing,the afferent neurons of the Governor meridian distributed in bilateral DRGs,whereas the inputs from the second line of the Bladder meridian registered to ipsilateral DRG mainly.The proportion of the bilateral DRG neurons by retrograde labeled in GV8 was 54.97%and 45.03%respectively.And proportion of ipsilateral and contralateral DRG neurons labeled by the left BL48/49 was 89.03%and 10.97%.3.1.4 Governor meridian distributed by bilateral afferent fibersThe abundant primary sensory fibers of Governor meridian were labeled by anterogradely neuronal tracers.AAV2/9-hSyn-EYFP labeled sensory nerve fibers from the left DRG could cross the posterior midline to the right side.AAV2/9-hSyn-tdTomato labeled sensory nerve fibers from the right DRG crossed the posterior midline to the left side.The distance of ipsilateral sensory nerve fibers across the posterior midline was 1.0600 ± 0.3905mm.The Governor meridian is distributed by sensory fibers from bilateral DRGs.3.1.5 Dorsal horn neurons of the spinal cord receiving neural inputs from the contralateral receptive field nearby Governor meridian.By in vivo multichannel electrophysiological recordings,it was observed that the left dorsal horn neurons of the spinal cord could receive neural inputs from the contralateral receptive field nearby the midline.And the contralateral respective field is about aside 0.5-1cm from Governor meridian.3.2 The acupoints of Governor meridian and Bladder meridian regulating gastric motility3.2.1 Different acupoints of Governor meridian and Bladder meridian regulating gastric motility by EAFor functional regulation of gastric motility such as the ratio of AUC and the ratio of wave peak,all the GV6,BL21 and BL50 could significantly inhibit gastric motility by MA(P<0.001).For the ratio of AUC,MA on GV6 produced more significant suppression than that on BL21(P<0.01)and BL50(P<0.01).Furthermore,compared with MA on BL21(P<0.05)and BL50(P<0.05),that on GV6 had significant differences in inhibiting gastric motility for wave peak ratio.3.2.2 Different acupoints of Governor meridian and Bladder meridian regulating gastric motility by MAFor the ratio of AUC and wave peak,all the GV6,BL21 and BL50 could significantly inhibit gastric motility by EA(P<0.001).EA on GV6 produced more significant suppression than that on BL21(P<0.05)and BL50(P<0.001)for AUC ratio.And there’s significant difference between EA on BL21 and BL50 on AUC ratio(P<0.01).In addition,compared with EA on BL21(P<0.01)and BL50(P<0.01),that on GV6 had significant differences in inhibiting gastric motility for wave peak ratio.EA on BL21 had significant differences than that on BL50(P<0.01)for wave peak ratio.4 Conclusions4.1 From the perspective of neuroanatomy,the present study demonstrated Governor meridian in the midline of the back with the bilateral innervations.4.2 Acupuncture on the Governor meridian has a better adjustment of gastric motility than that on the Bladder meridian laterally to the midline with ipsilateral innervations only.