Morphological Observation And Tracing By HRP Of Acupoint-like Structures On The Body Surface Of The Cryprinus Carpiod

Acupoint is a clinical-needle-inserting position of the body in which used to be thought as the convergent part of "QI" and "blood" in Traditional Chinese Medicine(TCM).It is the reflex and treated point of the disease.Meridians are consisting of vessels and collaterals.It is the tunnel of QI-blood circulation in the body and the fundamental theory of TCM especially for acupuncture.The theory of acupoints and meridians used to elucidate the mechanism of function regulation of human body which is quite different from western medical and biological theory. They are the fundamental factors of the acupuncture and moxibustion discipline.And they are also the significant parts of TCM theory which strongly needed to be studied in the process of TCM modernization. Mechanism of acupoint can be illuminated clearly through deeply studying of the theory of acupoint and meridians,which can help us to know the function well and improve curative effect.How to explain acupoint and theory of meridians scientifically has significant meaning in communication TCM and modern medicine.With the development of science especially life science in recent half century,scientists have widely and penetratingly studied the theory of meridians,gained large amount of achievement and proved the objectivity of meridian phenomenon.But the various explanation and hypothesis about the essence of acupoint cannot make a perfect conclusion and clarify the mechanism.Researches on the correlation of acupoint and nerve have revealed that nerve and receptor have close relationship with meridian and acupoint.These researches focus on one point and its local morphological character,or the relationship with nerve segment and network and can not look the acupoint and meridian theory as a whole. Acupoint is a vital phenomenon in most of the living body.Based on the theory of evolution from low animal to high animal,we suppose that there be acupoint or acupoint-like structure in low animal bodies.Our previous researches have proved that body surface receptor of fish have an acupoint-like and meridian-like distribution.This study began with body surface receptor of fish and select Cryprinus carpiod as experimental animal to observe the distribution pattern of body surface receptor, meanwhile use the HRP tracer study to reveal the relationship between the receptor with the acupoint and meridian,in order to find out the biological evidence of acupoint by means of the comparative biological method.Experiment 1:morphological observation of acupoint-like structure on Cryprinus carpiod body surface1 Animals12 Cryprinus carpiods,6 Cryprinus carpiod in body length(4.0±0.42) cm,6 Cryprinus carpiods in body length(8.1±0.64) cm were used in the study,female and male without limited,buy from market,and was fed in fish bowl for 2 weeks.2 MethordsCryprinus carpiod(body length＜4.0±0.42＞cm) was put into 30μM fluorescent dye DiA water solution for 1 hour and then immersed in water for 30 minutes.After anesthetized with 2%diethyl ether in water solution, cryprinus carpiod was observed behind fluorescence microscope with B-2A colour-filter and photographed or put the picture together by image analysis system.This method was mainly used in the observation of the whole distribution of acupoint-like structure on the body surface.Put cryprinus carpiod(body length＜8.1±0.64＞cm ) into 30μM fluorescent dye DiA water solution for 7 days and then immerse them in water for 1 hour.After anesthetized with 2%diethyl ether in water solution,sometimes we pull out scales of fish backward if necessary(avoid to damage the skin between the scales) in order to clearly observe the nerve fiber direction,then observe behind fluorescence microscope with B-2A colour-filter and photograph.Draw pattern picture based on microscope photograph.This method mainly used in observing micro frame work of the acupoint-like structure and the nerve fiber direction and connection.3 Results3.1 We can conclude that there are acupoint-like structures on the cryprinus carpiod body surface through observation of the cryprinus carpiod as a whole.Its arrangement has obvious regular pattern:Ⅰ.Fluorescent labellings cluster on the body surface and the amount of every cluster is inequality.Ⅱ.Labelings around the eye distribute in radio-shape,linearity distribution along the front and back edge of gill cover.Ⅲ.Labelings on the soma part are obviously in linearity distribution and consist of 14 lines.Ⅳ.Tubiform structure in the soma part is lateral line canal,which fluorescent labelings in it too.3.2 We can find out the composition of the acupoint-like structure by observing cryprinus carpiod(body length＜8.1±0.64＞cm) body surface.The receptors in the labeling cluster have nerve fiber connection and join to one line towards proboscis side.Receptors on body surface and in lateral line canal are different in morphology.3.3 The direction and connection of the nerve fiber to the receptor can be clearly visualized in microscope of the cryprinus carpiod(body length＜8.1±0.64＞cm) dye for 7 days. Experiment 2:HRP tracer study of acupoint-like strcture on Cryprinus carpiod body surface1 Animals30 Cryprinus carpiods,length(12.2±0.62)cm,female and male without limited,buy from market,and feeding in fish bowl for 2 weeks.2 MethodsAfter anesthetize with 2%diethyl ether in water solution,30%HRP was injected into 11 points which is selected typically on the body surface by microsyringe.Before injection,the nerve fiber was broke down by needle.One position injection in each fish only,5-6μl in each point. Cryprinus carpiod survived for 36-48 hour after injection,and then was anesthetized with 2%diethyl ether in water solution,fixed in the devices designed by ourselves,exposed the heart and intubated through arterial cone.After perfusion and fixation,we cut frozen section next day,slice thickness is 30μm,then take TMB colorable reaction.Serial sections were gradient evaporated by alcohol,dimethyl benzene cleared,resin mounted, and observed.3 ResultsWe find that the positive cell totally emerged in the facial nucleus through our observation to all of the results,and most of them appeared in the same side of the facial nucleus of the injection position,only one specimen' spositive cell emerged in both sides of the facial nucleus.DiscussionWe have systematically observed the distribution of body surface receptor of cryprinus carpiod in vivo with fluorescence dye firstly.And find out its linearity arrangement and cluster distribution which similar to the character of acupoint and meridian distribution.The result of HRP tracer study show that the body surface receptor of cryprinus carpiod mainly projected to the facial nucleus.The information can converge and integrate in the facial nucleus and contact and interact with other nucleus in nerve central system,then influence the body function through efferent nerves.This entire net of sensory nerves on body surface conducts longitudinally first,and then enters the brain.It is quite different from the classical nerve theory which conducts horizontally according to nerve segment first and then conducts longitudinally.The former transmission way is more similar to the meridian theory of TCM certainly.So we think this study about the fish receptor on the body surface is meaningful for the meridian research in TCM.