Journal of Acupuncture and Meridian Studies
Volume 2, Issue 3 , Pages 248-252, September 2009

Development of a Laparoscopic System for In Vivo Observation of the Bonghan Structure

Biomedical Physics Laboratory for Korean Medicine, School of Physics, Seoul National University, Seoul, Korea

Received 29 June 2009; accepted 15 July 2009.

Article Outline

Abstract 

Bonghan structures composed of corpuscles and ducts have been observed in numerous animal experiments. The structure has been suggested to be the physical substrate for acupuncture points and meridians used in traditional Chinese medicine. The corpuscle-duct structure on the organ surface has been extensively studied. However, it was only observed after the abdomen of a rat was fully exposed. It may be desirable to observe these structures under minimally invasive conditions. Our laparoscope is specifically designed for in situ, in vivo observation of the Bonghan structure on the surface of internal organs in rats. We made a laparoscope consisting of a borescope combined with an illumination module and an imaging data acquisition part. The Bonghan structure is transparent and is hard to see, even with the borescope. Spraying a specific dye, trypan blue and washing with saline is a necessary process to visualize the structure. For this reason, we devised a staining and washing system attached in parallel with the borescope in the laparoscopic system. The system operated successfully to reveal a Bonghan structure without surgery. This tool opens a host of possible applications such as observation of flow of stained liquid or drug in the Bonghan duct flowing from the skin to internal organs, and long-term observation of the physiological changes in Bonghan corpuscles.

Key Words:  Bonghan system , endoscope , in vivo observation , laparoscopy

 

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1. Introduction 

Considering the increasing acceptance of the therapeutic effects of acupuncture, a study on the existence of the physical substrate corresponding to classical acupuncture points and meridians may be of potential importance to modern biology. The Bonghan (BH) system was researched as a new circulatory system in the body after the first study was published in the early 1960s [1]. Based on the observations of animal experiments, the BH system has been shown to have a corpuscle and ductule structure [2]. The BH duct, especially, has quite a unique structure in which several sub-ducts compose a bundle. The diameter of each sub-duct is in the range of 5-30 μm [3]. This bundle structure, in which a main single duct is formed, usually contains no sub-ducts, and is clearly distinct from the blood vasculature and lymphatic system. A bundle of sub-ducts was also suggested as a pathway for the liquid and the ‘Sanal’ to complete the circulatory system with regeneration related physiological functions [3]. Through numerous animal experiments the corpuscle-duct structure has been extensively studied, and the structure is thought to be the physical substrate for acupuncture points and meridians used in traditional Chinese medicine 4, 5, 6, 7, 8.

Previous studies have shown that the BH structure may be divided into two types, depending on their location in the body: superficial and profound [2]. Superficial BH structures reside in the skin, and the profound (or deep) structures are in the surfaces of various internal organs. The appearance of the profound BH ducts is semi-transparent, or milky-colored, threadlike structures connected with profound BH corpuscles. Until now, surgery using full incision of the abdomen of an anesthetized animal was necessary for observation of the profound BH structure on organ surfaces. This surgi cal operation exposing the BH seriously affects the sample observations and alters or even damages the samples. Performing experiments with animals using minimal incisions is more appropriate. A less invasive surgical technique for searching organ surface BH structures should be introduced to keep physiological and morphological animal conditions intact.

One of the techniques for specifically searching the BH structures in the abdomen that may be implemented is laparoscopy. The laparoscope is an instrument through which structures within the abdomen and pelvis can be seen. After a small surgical incision in the abdominal wall, the laparoscope is inserted into the abdomen or pelvis. Several tubes can be pushed through the same incision or other small incisions permitting the introduction of probes and other instruments. In this way, a number of surgical procedures can be performed without the need for a large surgical incision. This kind of endoscopic system has been used widely in hospitals for medical diagnosis and treatments 9, 10, 11 and is still developing with the advance of related modern technologies 12, 13, 14, 15, 16, 17, 18.

This paper describes our developing laparoscopic system for searching the BH structure in the abdomen of small animals. The BH corpuscle-duct structure on the organ surface was studied only after abdominal surgery. It is now possible, with our imaging tool, to perform in vivo, in situ observation of the structure as intact as possible and to investigate its physiological function in the circulatory system. A schematic illustration of the structure of the lapar oscopic system is presented, and results of experiments with rats are reported. Some discus sions for future development are also presented.

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2. Materials and Methods 

2.1. The laparoscopic system 

The laparoscopic system optimized in this study is composed of three subsystems; the illumination part, the telescopic optical part, and the image data acquisition part. This system is developed for the investigation of the BH structures of small animals like rats. Additional manipulators and injection systems for staining the structure with some dyes such as trypan blue are also included. In general, the whole system could be divided into several parts; (1) image delivery, (2) manipulators, (3) illumination, (4) telescopic optical system and (5) image acquisition device. First, the image delivering part may be one of two types; fixed and flexible. In general, the fixed type is made of a rigid bore, and the flexible type is made possible by using flexible optical fibers. In our case, the fixed type is more suitable because of easy control on the position and direction of the bore tip. The size of the image delivering part depends on the animal size. The available distance of the bore for a rat should be in the range of 100-200 mm. The bore diameter is 2 mm for our current rat experiments. Several manipulators for adding dye and washing are also attached to the bore. The illumination part is composed of a light source, a collimator, filters, and a beam splitter. These components are optically arranged for selective uniform illuminations on the sample. The light source is a 100 watt halogen lamp and the light intensity is adjustable. Good optical design for the telescopic system is important because it determines the specifications of the whole laparoscopic system such as the field of view and magnifying power. Finally the image acquisition device is essential for taking high quality images. Currently, the subsystems for the laparoscope are combined and fixed in an optimal configuration, and only the distance between the internal organs of an animal and the tip of the bore is adjustable. For the construction of the laparoscopic system, we purchased two main components: a rigid type bore-scope kit (Super slim type in 2.4 mm diameter, Lee Technology Co., Korea) and a video microscopic system (Model: ICS-305B, Sometech Co., Korea) with a charged coupled device. They were used as the backbone for building the whole system. The actual setup for the system is shown in Figure 1.

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  • Figure 1. 

    Schematic diagram of the laparoscope to search for the Bonghan structure. The whole system is set on an optical table to minimize any external movement during the operation.

2.2. Animal preparation 

Eight-week-old Sprague-Dawley rats purchased from the Central Laboratory Animal, Inc. (Seoul, Korea) were housed in a 23°C temperature-controlled environment with proper relative humidity, a 12 hour light-dark cycle, and were fed food and water ad libitum. Procedures involving the animals and their care conformed to institutional guidelines in full compliance with current laws and policies [19]. Rats were anesthetized with urethane (1.5 g/kg) intraperitoneally, and the operations were performed under general anesthesia.

2.3. Operation process of searching BH structures 

The anesthetized rat was put on a movable stage so that search position could be adjusted. After a small incision of the skin at the center of the abdomen was made, the tip of the laparoscope was inserted. One of the manipulators was used for lifting the skin around the abdomen to clear the way for the tip and allow easy viewing with the system. For clear viewing inside the abdomen, an additional manipulator was used for lifting the skin of the abdomen around the incision area. Two small rubber hoses (2 mm diameter) were attached and inserted along the bore; one for dropping staining dye (trypan blue, 10%) and the other for dropping saline solution for washing. Drops of solutions were added by two syringes connected through hoses. Washing was performed immediately after each pouring of dye at a position. The time interval for a staining/washing process was completed within 10 seconds, and careful searches for the BH structure were made around the region.

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3. Results 

The focal length of our laparoscopic system was in the range between 0.7 mm and 9 mm, and the field of view varied from 0.8 mm to 30 mm. Ten times magnification was our maximum. The connection between the borescope and the CCD module is shown in more detail in Figure 2A. Figure 2B illustrates the borescope at the tip of which a syringe was attached, through a rubber hose, for dropping staining dye or wash solution. The areas we searched covered mostly the small and large intestine of rats. Sometimes the liver, stomach, spleen and bladder were reachable. Figure 2C shows one of the pictures from the rat experiments using the laparoscopic system with a small incision. The drastic effect of the staining dye is shown in Figure 3. Before staining in trypan blue, the structure in the field of view was transparent. After finishing the dye spraying and washing with saline solution, the stained structure was clearly visible as blue and threadlike. The moving images were recorded in 30 frames per second with a resolution of 640 × 480 pixels. These images are available as a supplement on the journal homepage (http://www.journalams.com). It was clearly demonstrated that only the BH structures were stained with trypan blue, whereas other structures such as blood vessels, internal organs, and the abdominal wall were not stained at all.

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  • Figure 2. 

    Illustrations for the connection between the borescope and the CCD (A). The staining dye is dropped through the rubber hose by using a syringe (B). Photograph of rat experiments with the laparoscope (C). The tip of the bore was inserted into the incised skin of the abdomen, and a manipulator was used to lift the skin.

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  • Figure 3. 

    Images from the laparoscopic system. The visualizing process is shown for the use of staining dye, trypan blue, in rat experiments. The process follows the direction of the arrows.

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4. Discussion and Conclusion 

Based on many clinical studies, acupuncture is thought to be a therapeutic treatment to regulate the function of internal organs by stimulating singular points on the skin. One of our recent experimental studies investigated tracing a path from acupoints to corresponding organs using subcutaneous injection of Alcian blue at certain acupoints such as BL23 and GB25 in rats [20]. After 2-7 hours following administration, Alcian blue-stained corpuscles (BH corpuscles) and spots along flow paths were observed from the injection points toward the cranial direction in the dorsal skin. These kinds of studies are a starting point showing the BH system as a flow path related to physiological functions. More refined studies may be implemented by using a less invasive instrument such as the laparoscopic system. This work is the first step towards keeping animals as intact as possible during experiments.

Through rat experiments, we found that the current system should be improved for finer image acquisitions. The major blurring problem during image acquisition is caused by animal movement during breathing and heart beats. The field of depth should be large enough to cover the longitudinal movements along the axis of view. For a clear moving image, the resolution of CCD should be greater than one million pixels. Illumination should be adjusted for more uniform reflection over the sample in the field of view.

Based on these rat experiments, we started to design another laparoscopic system to search for BH structures in larger animals, such as rabbits and dogs. The final aim of this development is the application to a surgical system for humans. Here, a laparoscopic system for searching the BH structure on the organ surface of small animals was developed. A trypan blue stained corpuscle-duct structure was found on the surface of internal organs of rats. This laparoscopic system is suitable for long-term and real-time observation of the BH structure in physiologically more intact conditions. This will enable more sophisticated experiments to be conducted, for example, in situ observation of liquid flow in the BH structure, and measurement of flow speed.

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Acknowledgments 

This work was supported by the Ministry for Health, Welfare and Family Affairs, and in part by the Mobase Co. Ltd.

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References 

  1. Soh KS . Bonghan circulatory system as an extension of acupuncture meridians . J Acupunct Meridian Stud . 2009;2:124–129
  2. Kim BH . On the acupuncture meridian system . J Jo Sun Med . 1963;90:6–35 [In Korean]
  3. Kim BH . The Kyungrak system . J Jo Sun Med . 1965;108:1–38 [In Korean]
  4. Lee C , Seol SK , Lee BC , Hong YK , Je JH , Soh KS . Alcian blue staining method to visualize Bonghan threads inside large caliber lymphatic vessels and X-ray microtomography to reveal their microchannels . Lymphat Res Biol . 2006;4:181–190
  5. Lee BC , Yoo JS , Ogay V , Kim KW , Dobberstein H , Soh KS , et al.   Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs . Microsc Res Tech . 2007;70:34–43
  6. Yoo JS , Kim MS , Sung B , Lee BC , Soh KS , Lee SH , et al.   Cribriform structure with channels in the acupuncture meridian-like system on the organ surfaces of rabbits . Acup Electrother Res . 2007;32:130–132
  7. Sung B , Kim MS , Lee BC , Yoo JS , Lee SH , Kim YJ , et al.   Measurement of flow speed in the channels of novel thread-like structures on the surfaces of mammalian organs . Naturwissenschaften . 2008;95:117–124
  8. Ogay V , Bae KH , Kim KW , Soh KS . Comparison of the characteristic features of Bonghan ducts, blood and lymphatic capillaries . J Acupunct Meridian Stud . 2009;2:107–117
  9. Narisawa T , Wong CQ , Weisburger JH . Evaluation of endoscopic examination of colon tumors in rats . Dig Dis . 1975;20:928–934
  10. Lee H , Ernst E . Acupuncture for GI endoscopy: a systematic review . Gastrointest Endosc . 2004;60:784–789
  11. Ravnic DJ , Konderding MA , Huss HT , Wolloscheck T , Pratt JP , Mentzer SJ . Murine microvideo endoscopy of the colonic microcirculation . J Surg Res . 2007;142:97–103
  12. Taylor P , Armstrong D , Linsell J , Power S , Mason R . Sequential endoscopy and biopsy of intact rat stomach—A new simple technique . Dig Dis Sci . 1988;33:321–323
  13. Pan YT, Wang ZG, Wu ZL, Jain A, Xie HK. Diagnosis of biological tissue morphology and function with endoscopic optical coherence tomography. Proceedings of the 2005 IEEE Engineering in Medicine and Biology 27th Annual Conference, September 1–4, 2005, Shanghai, China, 2005:7217–20.
  14. Fu L , Jain A , Xie H , Cranfield C , Gu M . Nonlinear optical endoscopy based on a double-clad photonic crystal fiber and a MEMS mirror . Optics Express . 2006;14:1027–1032
  15. Yelin D , Rizvi I , White WM , Motz JT , Hasan T , Bouma BE , et al.   Three-dimensional miniature endoscopy . Nature . 2006;443:765
  16. Bolenz C , Wenzel M , Cao Y , Trojan L , Hacker A , Arancibia MF , et al.   Newly developed mini-endoscope for diagnosis and follow-up of orthotopic bladder transitional-cell carcinoma in vivo . J Endourol . 2007;21:789–794
  17. Harada K , Miwa M , Fukuyo T , Watanabe S , Enosawa S , Chiba T . ICG fluorescence endoscope for visualization of the placental vascular network . Minim Invasive Ther Allied Technol . 2009;18:3–7
  18. Li B , Meng M . Computer-aided detection of bleeding regions for capsule endoscopy images . IEEE Transactions on Biomedical Engineering . 2009;56:1032–1039
  19. Guide for the Care and Use of Laboratory Animals . Washington DC: National Academy Press; 1996;
  20. Han HJ , Sung B , Ogay V , Soh KS . In vivo subcutaneous injection of Alcian blue at acupuncture points to trace a circulatory path from skin to organs . FASEB J . 2008;22:1198.6

PII: S2005-2901(09)60062-5

doi:10.1016/S2005-2901(09)60062-5

Journal of Acupuncture and Meridian Studies
Volume 2, Issue 3 , Pages 248-252, September 2009

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