Visualizing The Innervation Associated With Kidney And Its Yuan-,Luo-,Shu-,and Mu-Point

BackgroundThe correlation between viscera and somato is the basis and core of acupuncture theory.The meridian connects internal viscera with external acupoints,making them an integral whole.This kind of connection is bidirectional.The effect of viscera on the somato is mainly manifested in certain symptoms or physical signs when there is occurrences of pathological changes in the viscera,which may appear,such as hyperalgesia,tenderness,cord or nodular,etc at corresponding meridians or acupoints.Some researches have also shown that local physical properties of acupoints,such as temperature and electrical resistance of body surface are affected by related viscera lesions to a certain extent.The acupoints affected by different viscera diseases have site characteristics,which often occur on the specific acupoints related to the internal organs.The effect of body surface on viscera is that stimulating meridians or acupoints can regulate the function of corresponding viscera,which is also the most direct manifestation of somato-viscera correlation.In clinical acupoints selection,Yuan-,Luo-,Shu-,and Mu-points are used with high frequency,so it is also commonly chose to study the internal mechanism of correlation between viscera and body surface.The relationship between viscera and somato has some similarities with referred pain in modern medicine.Referred pain,also named as reflective pain,is pain perceived at a location other than the site of the primary lesion.This can also be regarded as the appearance of somato-viscera correlation in pathological state.There are three main proposed mechanisms for referred pain.The first one is peripheral nerve-branching theory,also regarded as pre-spinal convergence,holds that there are branches in the peripheral processes of sensory neurons in dorsal root ganglion,distributed in the viscera,and the body surface respectively.Sensations of internal organs and external sites are involved at the level of the spinal ganglion.The second is convergence-projection theory,which hypothesizes that the involved pain is caused by the convergence of the somato and visceral senses when sensory signals aretransmitted to central nervous system.This convergence can.occur at all levels of the spinal cord,thalamus,and cerebral cortex.Third,convergence-facilitation theory,is the hypothesis that when the visceral lesions occur,its afferent nerve endings release some neuroactive substances.These substances can reduce the threshold of pain at the end of the somatic nerve adjacent to the same segment,causing pain.All three hypothesis have certain basis,there is no definitive consensus regarding which is correct currently.But these studies also provide some morphological basis for the correlation of somato and viscera for acupuncture and moxibustion research.In addition to the sensory conduction pathway mechanism of referred pain,it also needs to clarify how stimulation of peripheral acupoints can regulate the function of viscera.The function of the viscera is controlled by autonomic nerve.The physiological activities of the autonomic nerve are not only dominated by the central nervous system,but also caused by the reflex of peripheral receptors.Stimullating signals from the somato can be transmitted to the autonomic nervous system via afferent nerve,which leads to the change of visceral function.A number of studies have shown that acupuncture at peripheral acupoints plays a good role in regulating autonomic nervous activity such as blood pressure,gland secretion,improving insomnia.Additionally,electrophysiological studies have recorded discharges of sympathetic nerve caused by acupuncture.Therefore,it is speculated that acupuncture may also take effect by acting on autonomic nervous activities.Corresponding morphological studies may provide further evidence to verify this mechanism.From the perspective of the meridian of foot-shaoyin,it connects to yuan-(Taixi,KI 3)and luo-points(Dazhong,KI 4)at limbs,shu-(Shenshu,BL 23)and mu-points(Jingmen,GB 25)at trunk.Related internal organ of the meridian is kidney,also contains the functional structures associated with the kidney.Adrenal gland is located above the medial side of the kidney and is encapsulated in the renal capsule together with it.Adrenal gland plays an important role in the metabolism of human body by secreting adrenal cortex and medullary hormones.At present,a large.number of domestic studies have confirmed that the promotion and warmth function of kidney-yang is closely related to hypothalamus-pituitary-adrenal axis.The extensive application and favourable curative effect of these specific acupoints in the treatment of urinary,reproductive,and endocrine diseases can be considered that peripheral acupoints have certain interaction with adrenal gland.ObjectiveTo observe regular innervation,morphological and chemical characteristics of afferent and efferent projected neurons associated with yuan-,luo-,shu-,mu-points area and kidney,adrenal gland in the rat,provide the neuroanatomical evidences that the multiple correlation of the kidney and its related specific acupoints with the sensory,motor and autonomic nervous systems at cellular level.Materials and methodsPart oneA total of 36 male Sprague Dawley rats(220±20 g)were randomly divided into Taixi,Dazhong,Shenshu,Jingmen,adrenal and kidney groups(n=6 in each group).Under respiratory anesthesia,rats received 4μL of AF488-CTB subcutaneous injections to one side of acupoint KI 3,KI 4,BL 23 and,GB 25 areas.Following longitudinal incision of skin,adrenal gland and kidney was exposed and 2μL of AF594-CTB was injected into them.Injections were performed using 10μL microsyringes.Two or three days after the application of tracers,bilateral sympathetic chains,thoracic(T)5 to lumbar(L)1 dorsal root ganglions(DRG)and spinal cords were dissected out from the rats that underwent transcardiac perfusion with 4%paraformaldehyde in 0.01 mol/L phosphate buffer.After storing in 0.01 mol/L phosphate-buffered saline containing 20%sucrose for 24 hours at 4℃,the materials were sliced into sections(40μm in thickness)and observed with fluorescent microscope.Part two12 male Sprague Dawley rats(220±20 g)were randomly divided into shenshu-adrenal and shenshu-kidney groups(n=6 in each group).Under respiratory anesthesia,rats received 4μL of AF488-CTB subcutaneous injections to one side of"Shenshu"(BL 23)area,at the same time,ipsilateral adrenal gland and kidney was injected into 2μL of AF594-CTB.Two or three days after the application of tracers,bilateral related nervous tissue were dissected out from the rats that underwent transcardiac perfusion.The materials were sliced into sections and observed with fluorescent microscope.Part ThreeThis part of study was conducted on 5 male Sprague-Dawley rats.After the rats underwent transcardiac perfusion,the local skin tissue of Shenshu(BL 23),adrenal gland and renal capsular were dissected out and stored in 0.01 mol/L phosphate-buffered saline containing 20%sucrose for 24 hours at 4℃.The tissues were stained using fluorescent immunohistochemical and histochemical techniques with TH(Tyrosine hydroxylase)and CGRP(Calcitonin gene related peptide),and then observed and recorded under fluorescent microscope and laser confocal microscope.ResultsPart OneNeurons labeled with AF488-CTB and AF594-CTB appeared at the ipsilateral side of the injection.The sensory neurons related to acupoint KI 3,KI 4,BL 23,GB 25,adrenal gland and kidney distributed mainly at L4-6,L3-6,T10-L1,T10-13,T10-L1 and T11-L1 DRGs respectively.Motor neurons associated with acupoint KI 3,KI 4,BL 23,GB 25 areas distributed mainly at anterior horn of spinal cord of L4-6,L3-6,T11-L1 and T11-13 segment respectively,anterior horn of cervical cord also existed some motor neurons related to BL 23 and GB 25 areas.For the sympathetic innervation,postganglionic neurons correlated with acupoints,adrenal gland and kidney located at the sympathetic chain of the corresponding segment respectively.Part TwoThe sensory neurons associated with both acupoint BL 23 and adrenal gland distributed from T10-L2 DRG with high concentration in T12-13 and T11-12 respectively,in which some of them were simultaneously labeled with both AF 488/594-CTB and located in T12-L1 DRG.For the sympathetic innervation,the postganglionic neurons correlated with BL 23 and adrenal gland were labeled with AF 488/594-CTB separately in the sympathetic chain at the lumbar segments,while the labeled preganglionic neurons were only observed at the lateral horn of T11-3 spinal segments in the cases of adrenal gland.In addition,the labeled motor neurons were mainly detected at the spinal ventral horn of cervical(C)7-8 and T11-L1 segments.In the cases of BL 23 and kidney double labeling,sensory neurons simultaneously labeled with both AF 488/594-CTB located at T12-13 DRG.The postganglionic neurons correlated with BL 23 and kidney were labeled with AF488/594-CTB separately in the sympathetic chain at lower thoracic sympathetic ganglion.No preganglionic neurons were observed at spinal cord.Part ThreeTH positive sympathetic nerve fibers and CGRP positive sensory nerve fibers were observed at local skin tissue of BL 23,adrenal gland and renal capsular.The distribution density of TH positive sympathetic nerve fibers was higher than CGRP positive sensory nerve fibers.Some of CGRP positive sensory nerve fibers were paralleled to TH positive sympathetic nerve fibers.Conclusions1.There exists a certain segmental distribution of the neurons related to the acupoints and viscera.Sensory neurons associated with BL 23 area and kidney(adrenal gland)converge and overlap at DRG,which indicates that a same sensory neurons in DRG may have peripheral branches innervated to visceral and somatic area respectively,suggesting that the signals from the area of the BL 23 and kidney(adrenal gland)may directly interact on each other through the same sensory neurons.2.The postganglionic sympathetic neurons associated with BL 23 area and kidney(adrenal gland)exist at a same sympathetic ganglion,indicating that peripheral acupoint may interact at viscera through sympathetic route.There is no direct relation with the preganglionic sympathetic neurons.3.The motor neurons labeled at the anterior horn of the spinal cord are only related to BL 23 area,suggesting that BL 23 does not affect the kidney(adrenal)through motor conduction pathway.