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Longitudinal directional movement of Alcian blue in Gephyrocharax Melanocheir fish: Revealing interstitial flow and related structure
World Journal of Acupuncture – Moxibustion 29 (2019) 127–132
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Experimental Research
Longitudinal directional movement of Alcian blue in Gephyrocharax Melanocheir fish: Revealing interstitial flow and related structure✰ Wei-bo ZHANG () a,∗, Xiao-jing SONG () a, Ze WANG () b, Guang-jun WANG () a, Shu-yong JIA () a, Yu-ying TIAN () a, Hong-yan LI () a a Department of biomedical engineering, Institute of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China ( , , 100086, ) b Department of Endocrinology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China (, , 100053, )
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Article history: Available online 30 May 2019 Keywords: Interstitial fluid Interstitial flow Meridians and collaterals Alcian blue Gephyrocharax Melanocheir fish
a b s t r a c t Background: Observing interstitial fluid (ISF) is very difficult because interstitial structure collapses and ISF disappears after tissue fixation. Additionally, ISF is colorless, and interstitial flow is weak in vivo. In order to view the interstitial flow, special dye and animal model was chosen to explore the movement characteristic of interstitial flow and related structure. Methods: The Gephyrocharax Melanocheir (GM) fish, a special animal with translucent body, were placed into 0.03 g/L tricain solution for anesthesia. 20–25 μL Alcian blue (AB) solution which can stain acid mucopolysaccharide immobilized by the collagen net in connective tissue was injected into each fish at a single point with a rate of 2 μL/min via a micro-injection pump. The process of infusion and the movement of the AB in fish were record by a digital camera. The (fresh) frozen sections of AB tracks tissue were performed to observe the morphological feature. Results: Several blue tracks were observed which were formed by longitudinal directional movements of AB solution. For back lateral track, the velocity and length of the movements were significantly fast and longer on the direction toward head than that toward tail (P < 0.01). For lateral middle track, the result was opposite, namely toward tail (P < 0.01). This phenomenon indicated an inherent ISF flow according to Darcy’s law. Morphological study showed these tracks were just in septa composed by connective tissue. The stained blue septa formed various shapes as interstitial space for ISF flow and connected with one another like a net. The finding can help us to understand the essence of meridian in traditional Chinese medicine (TCM). Conclusions: The dynamic asymmetry of the AB tracks revealed interstitial flow in the GM fish body. It implied an inherent interstitial flow along particular pathway formed by septa which may be a key to understand the nature and value of meridians and collaterals in health care. © 2019 Published by Elsevier B.V. on behalf of World Journal of Acupuncture Moxibustion House.
Introduction The cardiovascular system is well-known in the life sciences, and it plays the role of transporting body fluids. The fluid is blood when it is in blood vessels or lymph when it is in lymph vessels. The fluid that stays in neither blood vessels nor lymph vessels but in interstitial space is interstitial fluid (ISF). ISF had been thought to be in a unique gel phase and almost immobile, according to Guyton et al. in 1960s [1], which was the mainstream view for a long time. The water, ion and molecules exchange between blood
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Supported by the National Natural Science Foundation of China: 81173206. Corresponding author. E-mail address: [email protected] (W.-b. ZHANG).
vessels and interstitial space mainly through diffusion according to this model. In 1993, a two-phase model of ISF, gel and free fluid, was proposed by Aukland and Reed after elucidating a large amount of experimental data [2]. The other related work was the discovery of tissue channels by Casley–Smith in 1978, which were 50–100 nm in diameter and could be the structural basis of ISF flow [3]. In 2007, Swartz and Fleury published a review in which a 10 μm bead of interstitial fluid embedded in the fiber architecture of an in vitro fibrin gel was shown to be present by confocal reflectance microscopy [4]. The most recent finding was by Benias et al., who found ISF embedded in reticular collagen bundles when using probe-based Confocal Laser Endomicroscopy (pCLE), which is similar to Swartz’s finding but in vivo [5]. What does the ISF act like in tissue? Is it immobile or flowing? As noted by Aukland and
https://doi.org/10.1016/j.wjam.2019.05.008 1003-5257/© 2019 Published by Elsevier B.V. on behalf of World Journal of Acupuncture Moxibustion House.
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Reed, the fluid phase of ISF should be flowing all of the time via a “preferential channel”, which connects blood flow and lymph and is the important mechanism to prevent edema, but direct evidence for the existence of the fluid and the channel is scarce [2]. ISF flow is also called interstitial flow, and its functions were reviewed by Wiig and Swartz in 2012 [6]. However, neither pathway nor the speed of interstitial flow is known because of the lack of a technique for observing ISF in vivo. To view the interstitial flow, a special animal, Gephyrocharax Melanocheir (GM) fish, whose body is translucent, and Alcian blue dye (AB), which can stain acid mucopolysaccharide immobilized by a collagen net [7], were used. AB was injected into the fish, and several blue tracks were found [8]. The morphology of fish torso tissue revealed that the tracks were situated to various septa in fish composed of connective tissue [9], which is consistent with the new finding from Benias et al. Thus, the blue AB tracks can reflect the interstitial flow. Considering the two-phase model for ISF, the movement of AB includes diffusion and convection. Here, a more detailed dynamic process and morphological result were shown to deduce the inherent interstitial flow and related structures.
15 min until its gills stopped moving, which indicated that the fish had died. Sample processing and observation Specimens that were at the middle of the torso, including the epaxial muscle, spine and part of the hypaxial muscle, were cut instantly. Then, the specimens were embedded in OCT freezing medium and were rapidly frozen using an instant freezing platform (−40 °C) in a cryostat microtome (Thermo ScientificTM ), to ensure minimal water crystal formation. The specimens were embedded in either the sagittal or cross-section orientation. The frozen sections were prepared at 20 μm thickness, with a cross-sectional plane to the fish torso, or a sagittal plane. The specimens were mounted on glass slides. Then, they were air-dried and fixed with 10% formalin, washed with deionized H2 O, routine dehydrated, dipped in xylene for 3–4 min, and mounted under permount TM mounting medium. The structural features of the AB tracks were observed under an optical microscope. Results
Materials and methods Animals Totally 92 GM fish were carried, among which 83 fish were successfully experimented and others were dead during the anesthesia. The fish with the (3.9±0.19) cm in length, were purchased from Beijing Chaolaichun flower market and were used as experimental animals. They were kept in a fish bowl in water at a temperature of 24 °C and given full access to food and oxygen. AB (8 GX, Sigma Co., USA) was diluted to 0.1% (W/V) in distilled water and filtered through 0.22-μm pore-sized filter paper as the AB solution. Tricain powder was diluted to 0.03 g/L in water to keep the fish under anesthesia during the experiment. The experiment was approved by the institutional Animal Care and Use Committee (License number: AE20150118-001) of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Science. Anesthesia management The GM fish were treated by following a procedure described in document [9]. They were placed into 0.03 g/L tricain solution for anesthesia. When the fish body became inclined, it was taken out and fixed on a piece of sponge in a dish and submerged in tricain solution. AB injection and record To accomplish an easy comparison, AB was injected into each fish at a single point, which was close to the vertebral column (54 fish) or close to the dorsal fin (29 fish). The exact positions of the two injected points were described in previous paper [8]. The AB was injected using an insulin syringe needle (0.1 mm in diameter) connected to a micro-injector at a 45-degree angle to the fish body at a depth of approximately 1 mm. 20–25 μL AB solution was infused with a rate of 2 μL/min via a micro-injection pump (KDS310-PLUS; KD Scientific, USA). During the infusion, the narcosis of the fish was adjusted on the basis of whether the fish wagged its tail through a bottle of tricain solution, which was connected to a bottle of water via a three-way valve. The needle was pulled out after the AB stopped moving, and the residual dye on the surface of the fish body was washed away with water. A digital camera (Nikon D50 0 0, Japan) was fixed directly above the fish to record the process of infusion and the movement of the AB. After video recording, the fish was placed in tricain solution again for about
The positions of the blue tracks that appeared on the fish body and their names After injecting AB into the fish body, eight longitudinal threadlike blue tracks were observed from side views, which were repeatable and stable in position. These eight blue tracks were named according to their anatomic positions which were the back middle track (BMT), abdomen middle track (AMT), lateral middle track (LMT), back-lateral track 1 (BLT1), back-lateral track 2 (BLT2), abdomen-lateral track 1 (ALT1), abdomen-lateral track 2 (ALT2) and abdomen-lateral track 3 (ALT3). This part of the results has been published elsewhere in detail [9], and a brief introduction was given here. The BMT is the track in the middle of the back, which had four branches (BMT-1, BMT-2, BMT-3 and BMT-4) at different layers. The track that can be easily seen outside is BMT-1, which is along the edge of the dorsal fin. The AMT is located in the middle of the abdomen which is composed of three segments: the posterior margin of the abdomen (AMT-1), the superior margin of the anal fin (AMT-2) and the lower part of the spinal column (AMT-3). AMT-2 did not appear frequently and AMT-3 is difficult to be seen. The LMT is the track at the lateral middle, which stays in the horizontal septum, as revealed by the cross sections. The superficial part of the LMT (LMT-1) is exposed on the skin surface, which can be seen clearly and can extend to deep tissue (LMT2). Around the back of the fish between the BMT and LMT, there were two tracks, called the BLT1 and BLT2, which permeated each other to form a thick bond called BLT-B. Although the BLT-B did not belong to the longitudinal threadlike blue tracks. On the abdomen between LMT and AMT, three tracks could be recognized, which were called ALT1, ALT2 and ALT3, all of which started from the edge of the abdomen and extended to the tail. The positions of the above mentioned tracks are shown in Fig. 1. The movement of the back lateral track (BLT-B, BLT1 and BLT2) Within several minutes after injecting AB into a fish, diffusion of AB appeared first and turned into a track when it reached a position. Several moving tracks could be observed clearly from the side view. The first clear movement is BLT-B together with BLT1 and BLT2. BLT-B is a certain wide blue strip with two denser thin lines (BLT1, BLT2) at the two sides of the stripe. The movement appeared unequal in the two directions (toward the head and toward the tail), when injecting AB at the dorsal fin or at the middle of the vertebral column. Then, the average velocity and length of the migrations on the videos were calculated. The lengths of the
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The movement of AB along several routes simultaneously The video (Movie 4) showed that AB migrated along BLT-B, ALT1 and AMT simultaneously in different directions. Each track moved almost completely in one direction.
Fig. 1. The positions of longitudinal AB tracks on GM fish. Notes: BMT-1 is the surface branch of the back middle track along the dorsal fin. LMT-1 is the superficial part of the lateral middle track (LMT). BLT1 and BLT2 are the two tracks between LMT1 and BMT1. AMT-1 and AMT-2 are the two segments of the abdomen middle track (AMT). ALT1, ALT2 and ALT3 are three separated tracks between LMT-1 and AMT-1 [9].
movement toward the head and tail were significantly different (P < 0.01). The rate toward the head and tail were also significantly different (P < 0.01). The two groups of data were in accordance with a normal distribution, and the variance was similar (Fig. 2). In one video (Movie 1) in the online version of this paper, the movement of AB along BLT1 and BLT2 was faster than that along BLT-B. BLT1 developed only along a strip on the body surface of the fish, which could be seen by eye. The BLT-B turned down along the edge of the head when it reached the head.
The movement of the lateral middle track (LMT-1) Unlike BLT, which is mainly a wide strip, LMT-1 is a thin line on the surface and extends to deep tissue along the horizontal septum (LMT-2). In 54 fishes that were injected at the middle of the vertebral column, 32 fish showed movement of the lateral middle track, which was a repeatable phenomenon. The movements of AB along LMT-1 were unidirectional or bidirectional. Among the movements of AB along LMT-1, 22 appeared to be unidirectional migration from injection points toward the tail, which accounts for 68.7% of the total, and 3 appeared to have unidirectional migration from the injection points toward the head, which accounts for 9.4% of the total, while 7 showed bidirectional migration from the injection points toward the head and tail, which accounts for 21.9% of the total. In one video (Movie 2), a bidirectional migration was shown with a preferential direction to tail. The length and velocity toward tail were both significantly larger than that toward head (Fig. 3).
The movement of AB along AMT and a case of convection rather than diffusion In the 54 fishes that were injected with AB at the middle of the vertebral column, 32 fishes appeared to have movement along AMT, with almost a one-way movement down to the low edge of the fish first and then turning to the tail along the posterior margin, among which 15 extended to the middle of the abdomen along the posterior margin of the abdomen, which accounts for 46.9%, while 17 extended to the superior margin of the anal fin, which accounts for 53.1%. During the movement of a track, the head of the track usually appeared from dense to light, which indicates that a diffusion process occurred first. Nevertheless, in one video (Movie 3), we observed clearly a marching head of the track that was almost even, which indicates a convection rather than a diffusion at the moment.
Transverse and longitudinal movements The movements of AB were complicated and depended on the position of the injection point. Transverse and longitudinal movements could occur in sequence. In one case (Movie 5), AB moved first in many directions from the injection point. Then, in some directions, AB could move transversely along the linear marks between the muscles. When AB reached the route of BLT-B, the dye turned left and moved longitudinally along the route of BLT-B to the head and a moment later to the tail. Morphology of the AB track in GM fish Specimen sections showed that AB stained blue linear tracks were distributed among the muscles in the GM fish torso. They were septa composed of connective tissue. The septa that surrounded the back muscles integrated into a cavity that was in the center of the mediastinum, above the spine (Fig. 4(A), (B), and (F)). These septa integrated a cavity around the spine, also (Fig. 4(C) and (D)). In living fish, the cavity was filled with interstitial fluid. At one point in the cavity wall, two sides of the septa were integrated and formed a long branch. Some branches, AB tracks, which emanated from the cavity wall, went through the muscle tissue. The muscle phrenic divided the muscles tissue into individual sarcomeres (Fig. 4(A)—(D), and (J)). These connective tissue septa connected with one another, similar to in trees. After observing the sagittal plane section, it was found that there was a gap between the AB staining skin and the muscle in the back. The gap was the interstitial space filled with interstitial fluid for those cavities (Fig. 4(E)). The dynamic appearance of the AB tracks along BMT-1, BMT-4 in fish body arose from the fact that the AB fluid flowed in the cavity that was composed of connective tissues. The AB fluid in the cavity leaked into the branches of the septa, to stain the branches among the muscles on the side of the fish body. Discussion Observing ISF is very difficult because tissue fixation absorbs most of the water and destroys the structure that supports the ISF. More difficult is the observation of interstitial flow because it is weak and has no color compared with the blood flow. It is necessary to mark ISF in order to observe the ISF flow, while the marker can diffuse in the ISF, which could cover up the convection of the ISF itself. To solve this problem, Chery and Jain measured the interstitial convection and diffusion simultaneously using fluorescence recovery after applying the photobleaching (FRAP) technique. They observed the ear of a rabbit, which is translucent and thin. The movement of the fluorescence was considered to be only in two dimensions (x, y), to simplify the calculation of the Gaussian function [10]. In our study, the fish body is much thinner (2–3 mm) with respect to the length (37–43 mm), which is similar to the ear in the rabbit. Although the AB dye could move in three dimensions, two-dimensional movement along the length and width is the main stream for the dye, and it could be observed and recorded by a camera above the fish that was positioned perpendicular to fish body (side view). When the AB solution was injected, the local concentration of AB and the fluid pressure would immediately increase at the injection point. A diffusion and convection in all directions would occur, and the concentration represented by the denseness of the blue color decreased with the distance from the injection point. We have seen such a distribution of blue dye in most situations, usually with a large amount of blue color around the injection point at
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Fig. 2. Comparison between the movement of BLT in two directions. Note: TH represents the movement toward the head, while TT represents the movement toward the tail. The student’s two-sided paired t-test was used to analyze, n = 28.
Fig. 3. Comparison between the movement of LMT-1 in two directions. Note: TH represents the movement toward the head, while TT represents the movement toward the tail. The velocity and length opposite the moving direction are zero in the unidirectional ones. Wilcoxon rank-sum test was used to analyzed, n = 32.
Fig. 4. Structural features of AB tracks in GM fish (A—E, × 4; F, × 10). Notes: (A, B) Different depth sections of the same specimen. Dense net tissue was in the muscles. AB tracks were the connective tissue septa stained by AB. “a” is the side next to the abdomen, and “b” is the side next to the back. The red arrow indicates the spine. A cavity that surrounded the spine was formed with connective tissue septa stained by AB. There was some blank space around the spine in the cavity. Scale bar, 100 μm. (C, D) Different depth cross-sectional plane sections of the back muscle. “∗ ” indicates a cavity in the center of the mediastinum that was formed with the septa surrounded by the back muscles. Other AB tracks were connected with the cavity. The blank cavity should be filled with interstitial fluid. (E) Sagittal plane section of the back muscles. The AB track was the connective tissue of the back skin stained by AB. “∗ ” indicates the interstitial fluid space between the skin and muscles. The black arrow indicates the AB fluid. (F) Cavity stained by AB above the spine. The red arrow indicates the spine bone. (G) The red box indicates the resected specimen. (H) Distribution of BMT-1, BMT-2, BMT-3, and BMT-4. (I) The fresh frozen sections of fish were prepared at a 1 mm thickness. The red arrow indicates the AB fluid filled in the tissue channels among the muscle tissues. (J) The cavity surrounded by the spine. The green arrow indicates two sides of the cavity septa integrated into a branch.
the beginning of the injection. However, after a period of time, almost even AB appeared within a narrow area to form a blue track. By observing the cross sections, the denser AB was mainly located in subcutaneous tissue and intermuscular septa, which were connective tissue with a higher diffusion coefficient and lower hydraulic resistance than the cellular tissue. Two dynamic processes can cause the tracks: one process is the diffusion of solute of AB following the concentration gradient (∇ Xi ) and diffusion coefficient
(Di ), and the other process is the convection of solvent ( Nj ) according to Fick’s diffusion law (Eq. (1))
Ni = − DiC ∇ Xi +Xi
Nj
(1)
In our case, a solution of AB in water was injected continuously into the tissue, which could cause a pressure increase in the tissue to induce a convective flow that follows Darcy’s law, which
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Fig. 5. An illustration of exchange of materials between cells and blood via a capillary through diffusion and convection by interstitial flow. Notes: A, B, C, D represent cells at different places. a, b, c, d indicate the local milieu interne of the cells. The flow around cell A, if blocked, leads to marked changes in the homeostasis. The local micro-environment will depart from homeostasis and high concentration gradients of waste products, and nutrients will develop in spite of the fact that the blood flow could be in full operation. Enhancement of the interstitial flow within long-distance extracellular pathways with outflow in capillaries B, C and D and in lymph vessels can then play an important role in restoring the homeostasis in the tissue. This movement involves diffusion not only between cells and capillaries but also between cells and extracellular pathways that are very close to the cells. The ISF, which does not enter the lymph vessel, will continue to flow along but outside the lymph vessel in the extracellular pathways. This component of ISF can finally enter into the lymph vessels through increases in the pressure gradients through smooth muscle cell contractions and increased permeability of the cells of the lymph vessels.
describes the flow in porous media (Eq. (2)).
dQ ∇P =KA dt L
(2)
dQ/dt is the flux. K is the hydraulic conductivity, which relates to the flowing liquid. K = k/µ, k is a special hydraulic conductivity that is determined only by the property of tissue, and µ is the dynamic viscosity coefficient of the liquid. A and L are the area and length of the tissue. P is the pressure gradient. L/(K∗ A) is called the hydraulic resistance (Rh ) [7], which is the resistance of the tissue to fluid flow. Normally, an increase in the pressure at the injection point will drive the colored water to flow in all directions, which is indeed the situation at the beginning of the injection. However, a denser blue track appeared over a period of time after the injection, which indicates a preferential pathway with low hydraulic resistance (Rh ) and a higher diffusion coefficient (Di ). Benias et al. found ISF in many tissues, but the tissues under the dermis and between the muscles are difficult to observe because the laser cannot penetrate very deeply. Zhang et al. developed an instrument that can measure Rh in subcutaneous tissue [11], and they found a low hydraulic resistance channel along the meridians (LHRCM) on humans and pigs [12]. Fish are ancestors of mammals according to the theory of evolution. The LHRCM that exists in mammals could also exist in fish. We have compared the tracks that appeared on the fish with the acupuncture meridians on that human body and found a high level of accordance [9]. Thus, the finding of these tracks could illustrate the existence of LHRCM in fish. If the movement of AB is induced by only the convection of the injected water, then AB will move in two opposite directions when it reaches a channel of low hydraulic resistance. The two-way movements were actually observed, but in most cases, the movements of AB were one-way or asymmetric two-way along BLT, LMT and AMT. The situations were different in the three tracks. For BLT, most fish had two-way movement but a preferential tendency to the head with significant differences in the velocity and length between the two directions. For AMT, all of the fish had movement from the injecting site to the bottom along the middle of the abdominal wall and then turned to the tail along the superior margin of the anal fin, and most LMT were one-way movement toward the
tail. The preferential movement indicates inherent interstitial flow along these places. Meridians are a concept in traditional Chinese medicine (TCM). They are special longitudinal lines on the human body on which acupoints are located for acupuncture. There are more than ten meridians and hundreds of collaterals that branch from the meridians to form a net in which qi, a type of vital substance, runs in the net, according to TCM. Scientists in China and abroad have made a large amount of effort to search for qi and the meridians. In 1963, Bong-Han Kim in North Korea claimed to have found the meridian to be a tubal structure and node. He named the structure Bonghan duct and Bonghan node [13], but it could not be validated in other labs until 20 0 0 when Kwang-sup Soh found a novel threadlike structure similar to Bonghan duct and Bonghan node. The author renamed it primo vessel [14]. Zhang et al. elucidate that qi is free ISF, and the meridians are interstitial spaces for ISF flow, according to the traditional description that qi irrigates tissue in a way that is similar to fog and dew irrigating a crop [15]. However, directly observing the ISF and related structures is very difficult because the structure collapses during the fixations. Recently, the microstructure of ISF was observed by Benias et al., in their observations, ISF filled in a polygonal space and could become relatively isolated, to form water drops [5] similar to fog and dew. Additionally, the widespread existence of ISF is coincident with the qi transporting through the meridians and collaterals everywhere in the body, as described in TCM. Morphology can reveal the ISF structure, while it is difficult to know the flowing state. The separated interstitial spaces could seemingly interconnect with one another to form a channel, which helps the ISF flow and exhibits low hydraulic resistance, according to Darcy’s law. The finding of LHRCM illustrated the distribution of ISF coincidental with morphological findings that the meridians are located within connective tissue by Xie et al. [16] and Langevin and Yandow [17]. The AB dye application has a great advantage in that the acid mucopolysaccharide is embedded into collagen, for which the enveloped ISF could be stained during ISF movement. The 1-mmthick fresh frozen slice maintains the ISF and the related structures simultaneously (Fig. 4(I)), while only the related structures were
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left in the thin slice (Fig. 4(A)—(F)) because the fluid, including the AB dye that filled in the interstitial space, disappeared after the tissue section was air-dried and dehydrated. Comparing the two slices revealed the space for the ISF flow and related structures. Because a 1-mm slice is very thick and translucent, the shape and texture of the tissue around the ISF could be recognized in light conditions without staining, which could not be found by traditional histological methods. It was found that AB linear tracks were distributed among the muscles in the GM fish torso. They were septa composed of connective tissue, which were the muscle phrenic, to divide the muscle tissue into individual sarcomeres. In some parts of the fish body, the septa formed a cavity. The dynamic appearance of the AB tracks along BMT-1, BMT-4 in the fish body was that the AB fluid flowed in the cavity. At one point in the cavity wall, two sides of the septa integrated and formed a long branch. This structure composed by double layered connective tissues septa was similar to the structure mentioned by Sharma et al. [18]. Those branches, AB tracks, went through the muscle tissue. When the fluid pressure was sufficiently high, the AB fluid in the cavity diffused or leaked into the branches septa to stain the branches among the muscles on the side of the fish body. Those cavities and branches connected with each other, similar to a net. The net was composed of connective tissues in the fish body and provided the main structure for the interstitial flow, and it could be the structure of the meridians in TCM. The new finding by Benias et al. deduced the possibility that a large new organ could exist, while the functions of the “organ” remain to be studied. Meridians in TCM on the other hand play important roles in health and the formation of a disease. The reason that acupuncture can treat diseases is the opening of the meridians and the regulation of the yin and yang balance, which is similar to the idea of maintaining homeostasis in physiology, which could be done in reality by the flow of ISF. Blood is at the basis of homeostasis and must be maintained. However, most cells are not in blood but are surrounded by ISF, which exchanges materials with the blood and with the cells (Fig. 5). As the cells in the extracellular fluid consume nutrients and release metabolites constantly, the composition of the ISF changes. Additionally, the milieu interne would find it difficult to maintain homeostasis if the waste products were not cleaned out in time. The exchange between the cells and blood by diffusion through concentration gradients to drive the molecules is a way to maintain homeostasis, while interstitial flow provides a much more effective exchange compared with molecular diffusion. It can also drain large molecules into lymph, which cannot enter into the blood vessels. In this way, a successful homeostasis of the milieu interne is obtained through interstitial flow. Conclusion Longitudinal directional movement of AB fluid was shown in GM fish, which revealed the interstitial flow. Morphological study found the septa stained by AB formed various shapes around the
interstitial flow. It implies that the meridian structure of human body could be a channel formed by septa in which ISF flow. Acknowledgments Thanks Prof. Kjell Fuxe for his kindly help on revising part of the manuscript and supporting the works. Thank Xiaojing SONG and Ze WANG performing the experiment and writing a part of the manuscript. Thanks Shuyong JIA for preparing device of video observation and cross section. Thanks Yuying TIAN for performing the anaesthesia of MG fish. Thanks Guangjun WANG and Hongyan LI for participating in the manuscript drafting. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.wjam.2019.05.008. References [1] Guyton AC, Scheel K, Murphree D. Interstitial fluid pressure. III. Its effect on resistance to tissue fluid mobility. Circ Res 1966;19(2):412–19. [2] Aukland K, Reed RK. Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol Rev 1993;73(1):1–78. [3] Casley-Smith JR, Vincent AH. The quantative morphology of interstitial tissue channels in some tissues of the rat and rabbit. Tissue Cell 1978;10(3):571–84. [4] Swartz MA, Fleury ME. Interstitial flow and its effects in soft tissues. Annu Rev Biomed Eng 2007;9:229–56. [5] Benias PC, Wells RG, Sackey-Aboagye B, Klavan H, Reidy J, Buonocore D, et al. Structure and distribution of an unrecognized interstitium in human tissues. Sci Rep 2018;8:4947. doi:10.1038/s41598- 018- 23062- 6. [6] Wiig H, Swartz MA. Interstitial fluid and lymph formation and transport: physiological regulation and roles in inflammation and cancer. Physiol Rev 2012;92(3):1005–60. [7] Levick JR. Flow through interstitium and other fibrous matrices. Quart J Exp Physiol 1987;72(4):409–38. [8] Wang Z, Zhang WB, Jia SY, Tian YY, Wang GJ, Li HY. Finding blue tracks in GM fish similar to the locations of acupuncture meridians after injecting Alcian blue. J Acup Meridian Stud 2015;8(6):307–13. [9] Zhang WB, Wang Z, Jia SY, Tian YY, Wang GJ, Li HY, et al. Is there volume transmission along extracellular fluid pathways corresponding to the acupuncture meridians. J Acup Meridian Stud 2017;10(1):5–19. [10] Chary SR, Jain RK. Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc Natl Acad Sci USA 1989;86(14):5385–9. [11] Zhang WB, Tian YY, Li H, Zeng YJ, Zhuang FY. A method to measure hydraulic resistance of subcuitis continuously and the study of low hydraulic resistance points. ACTA Biophys Sin 1998;14(2):373–9. [12] Zhang WB, Tian YY, Li H, Tian JH, Luo MF, Xu FL, et al. A discovery of low hydraulic resistance channel along meridians. J Acup Meridian Stud 2008;1(1):20–8. [13] Kim BH. On the Kyungrak system. J Acad Med Sci 1963;90(5):1–41. [14] Lee BC, Yoo JS, Baik KY, Kim KW, Soh KS. Novel threadlike structures (Bonghan ducts) inside lymphatic vessels of rabbits visualized with a Janus Green B staining method. Anatom Rec 2005;286(1):1–7. [15] Zhang WB, Jia DX, Li HY, Wei YL, Huang Y, Zhao PN, et al. Understanding Qi running in the meridians as interstitial fluid flowing via interstitial space of low hydraulic resistance. Chin J Integr Med 2018;24(4):304–7. [16] Xie HR, Li FC, Zhang WB. Observation and analysis on the meridian collateral running track-related anatomical structure on the human body. Acup Res 2009;34(3):202–6. [17] Langevin HM, Yandow JA. Relationship of acupuncture points and meridians to connective tissue planes. Anat Rec 2002;269(6):257–65. [18] Sharma M, Rai P, Rameshbabu CS, Senadhipan B. Imaging of peritoneal ligaments by endoscopic ultrasound. Endosc Ultrasound 2015;4(1):15–27.
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Experimental Research
Longitudinal directional movement of Alcian blue in Gephyrocharax Melanocheir fish: Revealing interstitial flow and related structure✰ Wei-bo ZHANG () a,∗, Xiao-jing SONG () a, Ze WANG () b, Guang-jun WANG () a, Shu-yong JIA () a, Yu-ying TIAN () a, Hong-yan LI () a a Department of biomedical engineering, Institute of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China ( , , 100086, ) b Department of Endocrinology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China (, , 100053, )
a r t i c l e
i n f o
Article history: Available online 30 May 2019 Keywords: Interstitial fluid Interstitial flow Meridians and collaterals Alcian blue Gephyrocharax Melanocheir fish
a b s t r a c t Background: Observing interstitial fluid (ISF) is very difficult because interstitial structure collapses and ISF disappears after tissue fixation. Additionally, ISF is colorless, and interstitial flow is weak in vivo. In order to view the interstitial flow, special dye and animal model was chosen to explore the movement characteristic of interstitial flow and related structure. Methods: The Gephyrocharax Melanocheir (GM) fish, a special animal with translucent body, were placed into 0.03 g/L tricain solution for anesthesia. 20–25 μL Alcian blue (AB) solution which can stain acid mucopolysaccharide immobilized by the collagen net in connective tissue was injected into each fish at a single point with a rate of 2 μL/min via a micro-injection pump. The process of infusion and the movement of the AB in fish were record by a digital camera. The (fresh) frozen sections of AB tracks tissue were performed to observe the morphological feature. Results: Several blue tracks were observed which were formed by longitudinal directional movements of AB solution. For back lateral track, the velocity and length of the movements were significantly fast and longer on the direction toward head than that toward tail (P < 0.01). For lateral middle track, the result was opposite, namely toward tail (P < 0.01). This phenomenon indicated an inherent ISF flow according to Darcy’s law. Morphological study showed these tracks were just in septa composed by connective tissue. The stained blue septa formed various shapes as interstitial space for ISF flow and connected with one another like a net. The finding can help us to understand the essence of meridian in traditional Chinese medicine (TCM). Conclusions: The dynamic asymmetry of the AB tracks revealed interstitial flow in the GM fish body. It implied an inherent interstitial flow along particular pathway formed by septa which may be a key to understand the nature and value of meridians and collaterals in health care. © 2019 Published by Elsevier B.V. on behalf of World Journal of Acupuncture Moxibustion House.
Introduction The cardiovascular system is well-known in the life sciences, and it plays the role of transporting body fluids. The fluid is blood when it is in blood vessels or lymph when it is in lymph vessels. The fluid that stays in neither blood vessels nor lymph vessels but in interstitial space is interstitial fluid (ISF). ISF had been thought to be in a unique gel phase and almost immobile, according to Guyton et al. in 1960s [1], which was the mainstream view for a long time. The water, ion and molecules exchange between blood
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Supported by the National Natural Science Foundation of China: 81173206. Corresponding author. E-mail address: [email protected] (W.-b. ZHANG).
vessels and interstitial space mainly through diffusion according to this model. In 1993, a two-phase model of ISF, gel and free fluid, was proposed by Aukland and Reed after elucidating a large amount of experimental data [2]. The other related work was the discovery of tissue channels by Casley–Smith in 1978, which were 50–100 nm in diameter and could be the structural basis of ISF flow [3]. In 2007, Swartz and Fleury published a review in which a 10 μm bead of interstitial fluid embedded in the fiber architecture of an in vitro fibrin gel was shown to be present by confocal reflectance microscopy [4]. The most recent finding was by Benias et al., who found ISF embedded in reticular collagen bundles when using probe-based Confocal Laser Endomicroscopy (pCLE), which is similar to Swartz’s finding but in vivo [5]. What does the ISF act like in tissue? Is it immobile or flowing? As noted by Aukland and
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Reed, the fluid phase of ISF should be flowing all of the time via a “preferential channel”, which connects blood flow and lymph and is the important mechanism to prevent edema, but direct evidence for the existence of the fluid and the channel is scarce [2]. ISF flow is also called interstitial flow, and its functions were reviewed by Wiig and Swartz in 2012 [6]. However, neither pathway nor the speed of interstitial flow is known because of the lack of a technique for observing ISF in vivo. To view the interstitial flow, a special animal, Gephyrocharax Melanocheir (GM) fish, whose body is translucent, and Alcian blue dye (AB), which can stain acid mucopolysaccharide immobilized by a collagen net [7], were used. AB was injected into the fish, and several blue tracks were found [8]. The morphology of fish torso tissue revealed that the tracks were situated to various septa in fish composed of connective tissue [9], which is consistent with the new finding from Benias et al. Thus, the blue AB tracks can reflect the interstitial flow. Considering the two-phase model for ISF, the movement of AB includes diffusion and convection. Here, a more detailed dynamic process and morphological result were shown to deduce the inherent interstitial flow and related structures.
15 min until its gills stopped moving, which indicated that the fish had died. Sample processing and observation Specimens that were at the middle of the torso, including the epaxial muscle, spine and part of the hypaxial muscle, were cut instantly. Then, the specimens were embedded in OCT freezing medium and were rapidly frozen using an instant freezing platform (−40 °C) in a cryostat microtome (Thermo ScientificTM ), to ensure minimal water crystal formation. The specimens were embedded in either the sagittal or cross-section orientation. The frozen sections were prepared at 20 μm thickness, with a cross-sectional plane to the fish torso, or a sagittal plane. The specimens were mounted on glass slides. Then, they were air-dried and fixed with 10% formalin, washed with deionized H2 O, routine dehydrated, dipped in xylene for 3–4 min, and mounted under permount TM mounting medium. The structural features of the AB tracks were observed under an optical microscope. Results
Materials and methods Animals Totally 92 GM fish were carried, among which 83 fish were successfully experimented and others were dead during the anesthesia. The fish with the (3.9±0.19) cm in length, were purchased from Beijing Chaolaichun flower market and were used as experimental animals. They were kept in a fish bowl in water at a temperature of 24 °C and given full access to food and oxygen. AB (8 GX, Sigma Co., USA) was diluted to 0.1% (W/V) in distilled water and filtered through 0.22-μm pore-sized filter paper as the AB solution. Tricain powder was diluted to 0.03 g/L in water to keep the fish under anesthesia during the experiment. The experiment was approved by the institutional Animal Care and Use Committee (License number: AE20150118-001) of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Science. Anesthesia management The GM fish were treated by following a procedure described in document [9]. They were placed into 0.03 g/L tricain solution for anesthesia. When the fish body became inclined, it was taken out and fixed on a piece of sponge in a dish and submerged in tricain solution. AB injection and record To accomplish an easy comparison, AB was injected into each fish at a single point, which was close to the vertebral column (54 fish) or close to the dorsal fin (29 fish). The exact positions of the two injected points were described in previous paper [8]. The AB was injected using an insulin syringe needle (0.1 mm in diameter) connected to a micro-injector at a 45-degree angle to the fish body at a depth of approximately 1 mm. 20–25 μL AB solution was infused with a rate of 2 μL/min via a micro-injection pump (KDS310-PLUS; KD Scientific, USA). During the infusion, the narcosis of the fish was adjusted on the basis of whether the fish wagged its tail through a bottle of tricain solution, which was connected to a bottle of water via a three-way valve. The needle was pulled out after the AB stopped moving, and the residual dye on the surface of the fish body was washed away with water. A digital camera (Nikon D50 0 0, Japan) was fixed directly above the fish to record the process of infusion and the movement of the AB. After video recording, the fish was placed in tricain solution again for about
The positions of the blue tracks that appeared on the fish body and their names After injecting AB into the fish body, eight longitudinal threadlike blue tracks were observed from side views, which were repeatable and stable in position. These eight blue tracks were named according to their anatomic positions which were the back middle track (BMT), abdomen middle track (AMT), lateral middle track (LMT), back-lateral track 1 (BLT1), back-lateral track 2 (BLT2), abdomen-lateral track 1 (ALT1), abdomen-lateral track 2 (ALT2) and abdomen-lateral track 3 (ALT3). This part of the results has been published elsewhere in detail [9], and a brief introduction was given here. The BMT is the track in the middle of the back, which had four branches (BMT-1, BMT-2, BMT-3 and BMT-4) at different layers. The track that can be easily seen outside is BMT-1, which is along the edge of the dorsal fin. The AMT is located in the middle of the abdomen which is composed of three segments: the posterior margin of the abdomen (AMT-1), the superior margin of the anal fin (AMT-2) and the lower part of the spinal column (AMT-3). AMT-2 did not appear frequently and AMT-3 is difficult to be seen. The LMT is the track at the lateral middle, which stays in the horizontal septum, as revealed by the cross sections. The superficial part of the LMT (LMT-1) is exposed on the skin surface, which can be seen clearly and can extend to deep tissue (LMT2). Around the back of the fish between the BMT and LMT, there were two tracks, called the BLT1 and BLT2, which permeated each other to form a thick bond called BLT-B. Although the BLT-B did not belong to the longitudinal threadlike blue tracks. On the abdomen between LMT and AMT, three tracks could be recognized, which were called ALT1, ALT2 and ALT3, all of which started from the edge of the abdomen and extended to the tail. The positions of the above mentioned tracks are shown in Fig. 1. The movement of the back lateral track (BLT-B, BLT1 and BLT2) Within several minutes after injecting AB into a fish, diffusion of AB appeared first and turned into a track when it reached a position. Several moving tracks could be observed clearly from the side view. The first clear movement is BLT-B together with BLT1 and BLT2. BLT-B is a certain wide blue strip with two denser thin lines (BLT1, BLT2) at the two sides of the stripe. The movement appeared unequal in the two directions (toward the head and toward the tail), when injecting AB at the dorsal fin or at the middle of the vertebral column. Then, the average velocity and length of the migrations on the videos were calculated. The lengths of the
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The movement of AB along several routes simultaneously The video (Movie 4) showed that AB migrated along BLT-B, ALT1 and AMT simultaneously in different directions. Each track moved almost completely in one direction.
Fig. 1. The positions of longitudinal AB tracks on GM fish. Notes: BMT-1 is the surface branch of the back middle track along the dorsal fin. LMT-1 is the superficial part of the lateral middle track (LMT). BLT1 and BLT2 are the two tracks between LMT1 and BMT1. AMT-1 and AMT-2 are the two segments of the abdomen middle track (AMT). ALT1, ALT2 and ALT3 are three separated tracks between LMT-1 and AMT-1 [9].
movement toward the head and tail were significantly different (P < 0.01). The rate toward the head and tail were also significantly different (P < 0.01). The two groups of data were in accordance with a normal distribution, and the variance was similar (Fig. 2). In one video (Movie 1) in the online version of this paper, the movement of AB along BLT1 and BLT2 was faster than that along BLT-B. BLT1 developed only along a strip on the body surface of the fish, which could be seen by eye. The BLT-B turned down along the edge of the head when it reached the head.
The movement of the lateral middle track (LMT-1) Unlike BLT, which is mainly a wide strip, LMT-1 is a thin line on the surface and extends to deep tissue along the horizontal septum (LMT-2). In 54 fishes that were injected at the middle of the vertebral column, 32 fish showed movement of the lateral middle track, which was a repeatable phenomenon. The movements of AB along LMT-1 were unidirectional or bidirectional. Among the movements of AB along LMT-1, 22 appeared to be unidirectional migration from injection points toward the tail, which accounts for 68.7% of the total, and 3 appeared to have unidirectional migration from the injection points toward the head, which accounts for 9.4% of the total, while 7 showed bidirectional migration from the injection points toward the head and tail, which accounts for 21.9% of the total. In one video (Movie 2), a bidirectional migration was shown with a preferential direction to tail. The length and velocity toward tail were both significantly larger than that toward head (Fig. 3).
The movement of AB along AMT and a case of convection rather than diffusion In the 54 fishes that were injected with AB at the middle of the vertebral column, 32 fishes appeared to have movement along AMT, with almost a one-way movement down to the low edge of the fish first and then turning to the tail along the posterior margin, among which 15 extended to the middle of the abdomen along the posterior margin of the abdomen, which accounts for 46.9%, while 17 extended to the superior margin of the anal fin, which accounts for 53.1%. During the movement of a track, the head of the track usually appeared from dense to light, which indicates that a diffusion process occurred first. Nevertheless, in one video (Movie 3), we observed clearly a marching head of the track that was almost even, which indicates a convection rather than a diffusion at the moment.
Transverse and longitudinal movements The movements of AB were complicated and depended on the position of the injection point. Transverse and longitudinal movements could occur in sequence. In one case (Movie 5), AB moved first in many directions from the injection point. Then, in some directions, AB could move transversely along the linear marks between the muscles. When AB reached the route of BLT-B, the dye turned left and moved longitudinally along the route of BLT-B to the head and a moment later to the tail. Morphology of the AB track in GM fish Specimen sections showed that AB stained blue linear tracks were distributed among the muscles in the GM fish torso. They were septa composed of connective tissue. The septa that surrounded the back muscles integrated into a cavity that was in the center of the mediastinum, above the spine (Fig. 4(A), (B), and (F)). These septa integrated a cavity around the spine, also (Fig. 4(C) and (D)). In living fish, the cavity was filled with interstitial fluid. At one point in the cavity wall, two sides of the septa were integrated and formed a long branch. Some branches, AB tracks, which emanated from the cavity wall, went through the muscle tissue. The muscle phrenic divided the muscles tissue into individual sarcomeres (Fig. 4(A)—(D), and (J)). These connective tissue septa connected with one another, similar to in trees. After observing the sagittal plane section, it was found that there was a gap between the AB staining skin and the muscle in the back. The gap was the interstitial space filled with interstitial fluid for those cavities (Fig. 4(E)). The dynamic appearance of the AB tracks along BMT-1, BMT-4 in fish body arose from the fact that the AB fluid flowed in the cavity that was composed of connective tissues. The AB fluid in the cavity leaked into the branches of the septa, to stain the branches among the muscles on the side of the fish body. Discussion Observing ISF is very difficult because tissue fixation absorbs most of the water and destroys the structure that supports the ISF. More difficult is the observation of interstitial flow because it is weak and has no color compared with the blood flow. It is necessary to mark ISF in order to observe the ISF flow, while the marker can diffuse in the ISF, which could cover up the convection of the ISF itself. To solve this problem, Chery and Jain measured the interstitial convection and diffusion simultaneously using fluorescence recovery after applying the photobleaching (FRAP) technique. They observed the ear of a rabbit, which is translucent and thin. The movement of the fluorescence was considered to be only in two dimensions (x, y), to simplify the calculation of the Gaussian function [10]. In our study, the fish body is much thinner (2–3 mm) with respect to the length (37–43 mm), which is similar to the ear in the rabbit. Although the AB dye could move in three dimensions, two-dimensional movement along the length and width is the main stream for the dye, and it could be observed and recorded by a camera above the fish that was positioned perpendicular to fish body (side view). When the AB solution was injected, the local concentration of AB and the fluid pressure would immediately increase at the injection point. A diffusion and convection in all directions would occur, and the concentration represented by the denseness of the blue color decreased with the distance from the injection point. We have seen such a distribution of blue dye in most situations, usually with a large amount of blue color around the injection point at
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Fig. 2. Comparison between the movement of BLT in two directions. Note: TH represents the movement toward the head, while TT represents the movement toward the tail. The student’s two-sided paired t-test was used to analyze, n = 28.
Fig. 3. Comparison between the movement of LMT-1 in two directions. Note: TH represents the movement toward the head, while TT represents the movement toward the tail. The velocity and length opposite the moving direction are zero in the unidirectional ones. Wilcoxon rank-sum test was used to analyzed, n = 32.
Fig. 4. Structural features of AB tracks in GM fish (A—E, × 4; F, × 10). Notes: (A, B) Different depth sections of the same specimen. Dense net tissue was in the muscles. AB tracks were the connective tissue septa stained by AB. “a” is the side next to the abdomen, and “b” is the side next to the back. The red arrow indicates the spine. A cavity that surrounded the spine was formed with connective tissue septa stained by AB. There was some blank space around the spine in the cavity. Scale bar, 100 μm. (C, D) Different depth cross-sectional plane sections of the back muscle. “∗ ” indicates a cavity in the center of the mediastinum that was formed with the septa surrounded by the back muscles. Other AB tracks were connected with the cavity. The blank cavity should be filled with interstitial fluid. (E) Sagittal plane section of the back muscles. The AB track was the connective tissue of the back skin stained by AB. “∗ ” indicates the interstitial fluid space between the skin and muscles. The black arrow indicates the AB fluid. (F) Cavity stained by AB above the spine. The red arrow indicates the spine bone. (G) The red box indicates the resected specimen. (H) Distribution of BMT-1, BMT-2, BMT-3, and BMT-4. (I) The fresh frozen sections of fish were prepared at a 1 mm thickness. The red arrow indicates the AB fluid filled in the tissue channels among the muscle tissues. (J) The cavity surrounded by the spine. The green arrow indicates two sides of the cavity septa integrated into a branch.
the beginning of the injection. However, after a period of time, almost even AB appeared within a narrow area to form a blue track. By observing the cross sections, the denser AB was mainly located in subcutaneous tissue and intermuscular septa, which were connective tissue with a higher diffusion coefficient and lower hydraulic resistance than the cellular tissue. Two dynamic processes can cause the tracks: one process is the diffusion of solute of AB following the concentration gradient (∇ Xi ) and diffusion coefficient
(Di ), and the other process is the convection of solvent ( Nj ) according to Fick’s diffusion law (Eq. (1))
Ni = − DiC ∇ Xi +Xi
Nj
(1)
In our case, a solution of AB in water was injected continuously into the tissue, which could cause a pressure increase in the tissue to induce a convective flow that follows Darcy’s law, which
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Fig. 5. An illustration of exchange of materials between cells and blood via a capillary through diffusion and convection by interstitial flow. Notes: A, B, C, D represent cells at different places. a, b, c, d indicate the local milieu interne of the cells. The flow around cell A, if blocked, leads to marked changes in the homeostasis. The local micro-environment will depart from homeostasis and high concentration gradients of waste products, and nutrients will develop in spite of the fact that the blood flow could be in full operation. Enhancement of the interstitial flow within long-distance extracellular pathways with outflow in capillaries B, C and D and in lymph vessels can then play an important role in restoring the homeostasis in the tissue. This movement involves diffusion not only between cells and capillaries but also between cells and extracellular pathways that are very close to the cells. The ISF, which does not enter the lymph vessel, will continue to flow along but outside the lymph vessel in the extracellular pathways. This component of ISF can finally enter into the lymph vessels through increases in the pressure gradients through smooth muscle cell contractions and increased permeability of the cells of the lymph vessels.
describes the flow in porous media (Eq. (2)).
dQ ∇P =KA dt L
(2)
dQ/dt is the flux. K is the hydraulic conductivity, which relates to the flowing liquid. K = k/µ, k is a special hydraulic conductivity that is determined only by the property of tissue, and µ is the dynamic viscosity coefficient of the liquid. A and L are the area and length of the tissue. P is the pressure gradient. L/(K∗ A) is called the hydraulic resistance (Rh ) [7], which is the resistance of the tissue to fluid flow. Normally, an increase in the pressure at the injection point will drive the colored water to flow in all directions, which is indeed the situation at the beginning of the injection. However, a denser blue track appeared over a period of time after the injection, which indicates a preferential pathway with low hydraulic resistance (Rh ) and a higher diffusion coefficient (Di ). Benias et al. found ISF in many tissues, but the tissues under the dermis and between the muscles are difficult to observe because the laser cannot penetrate very deeply. Zhang et al. developed an instrument that can measure Rh in subcutaneous tissue [11], and they found a low hydraulic resistance channel along the meridians (LHRCM) on humans and pigs [12]. Fish are ancestors of mammals according to the theory of evolution. The LHRCM that exists in mammals could also exist in fish. We have compared the tracks that appeared on the fish with the acupuncture meridians on that human body and found a high level of accordance [9]. Thus, the finding of these tracks could illustrate the existence of LHRCM in fish. If the movement of AB is induced by only the convection of the injected water, then AB will move in two opposite directions when it reaches a channel of low hydraulic resistance. The two-way movements were actually observed, but in most cases, the movements of AB were one-way or asymmetric two-way along BLT, LMT and AMT. The situations were different in the three tracks. For BLT, most fish had two-way movement but a preferential tendency to the head with significant differences in the velocity and length between the two directions. For AMT, all of the fish had movement from the injecting site to the bottom along the middle of the abdominal wall and then turned to the tail along the superior margin of the anal fin, and most LMT were one-way movement toward the
tail. The preferential movement indicates inherent interstitial flow along these places. Meridians are a concept in traditional Chinese medicine (TCM). They are special longitudinal lines on the human body on which acupoints are located for acupuncture. There are more than ten meridians and hundreds of collaterals that branch from the meridians to form a net in which qi, a type of vital substance, runs in the net, according to TCM. Scientists in China and abroad have made a large amount of effort to search for qi and the meridians. In 1963, Bong-Han Kim in North Korea claimed to have found the meridian to be a tubal structure and node. He named the structure Bonghan duct and Bonghan node [13], but it could not be validated in other labs until 20 0 0 when Kwang-sup Soh found a novel threadlike structure similar to Bonghan duct and Bonghan node. The author renamed it primo vessel [14]. Zhang et al. elucidate that qi is free ISF, and the meridians are interstitial spaces for ISF flow, according to the traditional description that qi irrigates tissue in a way that is similar to fog and dew irrigating a crop [15]. However, directly observing the ISF and related structures is very difficult because the structure collapses during the fixations. Recently, the microstructure of ISF was observed by Benias et al., in their observations, ISF filled in a polygonal space and could become relatively isolated, to form water drops [5] similar to fog and dew. Additionally, the widespread existence of ISF is coincident with the qi transporting through the meridians and collaterals everywhere in the body, as described in TCM. Morphology can reveal the ISF structure, while it is difficult to know the flowing state. The separated interstitial spaces could seemingly interconnect with one another to form a channel, which helps the ISF flow and exhibits low hydraulic resistance, according to Darcy’s law. The finding of LHRCM illustrated the distribution of ISF coincidental with morphological findings that the meridians are located within connective tissue by Xie et al. [16] and Langevin and Yandow [17]. The AB dye application has a great advantage in that the acid mucopolysaccharide is embedded into collagen, for which the enveloped ISF could be stained during ISF movement. The 1-mmthick fresh frozen slice maintains the ISF and the related structures simultaneously (Fig. 4(I)), while only the related structures were
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left in the thin slice (Fig. 4(A)—(F)) because the fluid, including the AB dye that filled in the interstitial space, disappeared after the tissue section was air-dried and dehydrated. Comparing the two slices revealed the space for the ISF flow and related structures. Because a 1-mm slice is very thick and translucent, the shape and texture of the tissue around the ISF could be recognized in light conditions without staining, which could not be found by traditional histological methods. It was found that AB linear tracks were distributed among the muscles in the GM fish torso. They were septa composed of connective tissue, which were the muscle phrenic, to divide the muscle tissue into individual sarcomeres. In some parts of the fish body, the septa formed a cavity. The dynamic appearance of the AB tracks along BMT-1, BMT-4 in the fish body was that the AB fluid flowed in the cavity. At one point in the cavity wall, two sides of the septa integrated and formed a long branch. This structure composed by double layered connective tissues septa was similar to the structure mentioned by Sharma et al. [18]. Those branches, AB tracks, went through the muscle tissue. When the fluid pressure was sufficiently high, the AB fluid in the cavity diffused or leaked into the branches septa to stain the branches among the muscles on the side of the fish body. Those cavities and branches connected with each other, similar to a net. The net was composed of connective tissues in the fish body and provided the main structure for the interstitial flow, and it could be the structure of the meridians in TCM. The new finding by Benias et al. deduced the possibility that a large new organ could exist, while the functions of the “organ” remain to be studied. Meridians in TCM on the other hand play important roles in health and the formation of a disease. The reason that acupuncture can treat diseases is the opening of the meridians and the regulation of the yin and yang balance, which is similar to the idea of maintaining homeostasis in physiology, which could be done in reality by the flow of ISF. Blood is at the basis of homeostasis and must be maintained. However, most cells are not in blood but are surrounded by ISF, which exchanges materials with the blood and with the cells (Fig. 5). As the cells in the extracellular fluid consume nutrients and release metabolites constantly, the composition of the ISF changes. Additionally, the milieu interne would find it difficult to maintain homeostasis if the waste products were not cleaned out in time. The exchange between the cells and blood by diffusion through concentration gradients to drive the molecules is a way to maintain homeostasis, while interstitial flow provides a much more effective exchange compared with molecular diffusion. It can also drain large molecules into lymph, which cannot enter into the blood vessels. In this way, a successful homeostasis of the milieu interne is obtained through interstitial flow. Conclusion Longitudinal directional movement of AB fluid was shown in GM fish, which revealed the interstitial flow. Morphological study found the septa stained by AB formed various shapes around the
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