Preliminary Study Of Meridian-like Structures In Transparent Fish By Injecting Alcian Blue

Meridian theory is one of the core contents of basic theory of traditional Chinese medicine.It has guided clinical practice for more than two thousand years effectively.However,the exploration of the essence of meridians just started in 1950-60’s.Scholars both at home and abroad did mounts of work around meridians by various kinds of modern science and technology.These works involved multi-field and multi-angle,including biophysics、anatomy、histology、biochemistry and so on,expecting to reveal the essence of meridians and to achieve the objective visualization of meridians.The school of body fluid represented by Zhang Weibo found the main content of meridians are interstitial tissues which was in favorable to the flow of interstitial fluid.After series of experiments,Professor Zhang proved meridians and collaterals were interstitial fluid channels characterized by low hydraulic resistance based on biological fluid mechanics principle.From the perspective of running interstitial fluid,meridians and collaterals could be visualized by injecting a kind of fluid which could be traced.And the anatomic location of meridians and collaterals could also be studied.Classical meridian theory indicates meridians and collaterals play the role of"running qi and blood、nourishing yin and yang、deciding live and death、administrating multiple diseases".In other words,both humans and animals’ life activities are regulated and controled by the meridians and collaterals system.Besides,many studies of acupuncture and moxibustion science and biophysics have proved meridians and collaterals system is biquitous in humans and animals’ bodies.For a long time,research on acupuncture and moxibustion and meridians mainly chose rat、mouse or rabbit as model organisms.They all belong to mammals while fish was barely used which was distantly related to human.Meridians or meridian-like structures should also exist in fish body.Observation revealed some species of fish,such as Gephyrocharax melanocheir fish and Pterophyllum scalarevar fish were translucent and oblate which was in favor of transmission of light and observation.Therefore,we conducted this preliminary study around the proposition whether meridian-like structures exist in transparent fish,trying to use Gephyrocharax melanocheir fish and Pterophyllum scalarevar fish as experimental animals in which alcian blue were injected.Experiment One Observation of side view and cross section after Gephyrocharax melanocheir fish was injected by alcian blue(AB)Objective:The objective of this experiment is to investigate the distribution of AB after it was injected in Gephyrocharax melanocheir fish and explore whether meridian-like structures were exist in transparent fish;Methods:Fourty-five Gephyrocharax melanocheir fish were anesthetized by tricaine solution respectively.25μl 0.1%AB was injected slowly into each fish at a single point near the vertebral column or dorsal fin by a micro-injection pump.After AB stopped moving,the fish was anesthetized to death.Pictures of the whole fish body were taken with a digital camera and a stereomicroscope to observe the distribution of AB from a side view.Then the fish was wrapped with embedding medium and was frozen.Then it was cut every other millimeter along the transverse plane which was perpendicular to the head-tail axis of the fish.Pictures of cross sections were taken using the digital camera to observe the distribution of AB;Results:Seven obvious blue longitudinal threadlike structures were found after observing the side views of fish.According to their locations,they were named back-middl line、abdomen-middle line、back-lateral line、lateral-middle line and three abdomen-lateral lines respectively.Besides,some short transverse branches also appeared.Pictures of cross sections showed these threadlike structures mainly distributed in dorsal median septum、ventral median septum、horizontal septum and intermuscular septa which all belonged to loose connective tissues.The positions of these threadlike structures were similar to“interspaces of muscles”which was described as meridians’ locations on human body in classical meridian theory.Conclusions:Meridian-like structures exist in Gephyrocharax melanocheir fish.The use of transparent fish may provide more convenient animal model for meridians’ tracing study.Experiment Two The study of dynamic migration of AB after injected in Gephyrocharax melanocheir fishObjective:The objective of this experiment is to investigate and summarize the dynamic migration of AB when it was injected into Gephyrocharax melanocheir fish;Methods:Thirty-six Gephyrocharax melanocheir fish were respectively anesthetized and injected according to the methods of experiment one.A digital camera was fixed right above the fish to record the process of injection and the dynamic migration of AB.Then the the length and speed of the migration and in the upper back and middle lateral of the fish body were calculated according to the videos;Results:The migration of AB appeared longitudinal both when it was in the upper back and in the middle lateral of the fish body.Statistic analysis found in the upper back the length was 11.30±6.63mm toward head and 7.23± 4.12mm toward tail which were significantly different(P<0.01).The movement had rates of 2.85 ±1.62mm/s toward head and 1.80±0.97mm/s toward tail which were also significantly different(P<0.01).Among the migration in the middle lateral of the fish body,the unidirectional migration from injection points toward tail account for 68.7%,the unidirectional migration from injection points toward head account for 9.4%,the bidirectional migration from injection points toward head and tail account for 21.9%;Conclusions:Intrinsic directional interstitial fluid flow may exist in the upper back and in the middle lateral of the fish body.The directional flow was similar to the trait of meridians running qi and blood.Experiment Three The study of the migration of AB after injected in different hydraulic resistance points in Pterophyllum scalarevar fishObjective:The objective of this experiment is to investigate and compare the migration of AB after it was injected in low hydraulic resistance points or high hydraulic resistance points in Pterophyllum scalarevar fish;Methods:Ten Pterophyllum scalarevar fish were randomly divided into low hydraulic resistance point group and high hydraulic resistance point group,five in each group.After they were anesthetized respectively,20μl 0.1%AB was slowly injected by micro-injection pump in a low hydraulic resistance point or a high hydraulic resistance point which was measured by measuring system of hydraulic resistance.After AB stopped moving,the fish was anesthetized to death.Pictures of the whole fish body were taken with a digital camera to observe the distribution of AB from a side view;Results:AB in four fish appeared long distance migration after it was injected in Pterophyllum scalarevar fish of low hydraulic resistance point group.AB tended to diffuse near injection points and one fish appeared long distance migration after it was injected in Pterophyllum scalarevar fish of high hydraulic resistance point group;Conclusion:The pattern that AB migrating for long distance after injected in low hydraulic resistance point preliminarily demonstrated the property of fluid constraint in low hydraulic resistance channel.