림프 내 프리모시스템 관찰을 위한 프로토콜 개발

학위논문 (석사)-- 서울대학교 융합과학기술대학원 : 나노융합학과, 2013. 8. 송윤규.본 연구에서는 쥐의 림프 내 프리모시스템 (PVS) 가시화를 위하여 알시안블루 염료를 이용한 프로토콜을 확립 하였으며, 림프 내 프리모관과 림프관 내벽의 표면 특성을 원자힘 현미경(AFM, Atomic Force Microscope)을 이용하여 프리모관 표면에 있는 조직들과 림프관 내벽의 표면의 차이점이 무엇인지를 조사 분석하였다. 이 결과들을 바탕으로 프리모관의 x-ray microscopy 영상과 비교하였다. 토끼와 쥐 복대정맥 옆의 요추림프절에 알시안블루를 주입하여 림프관 안쪽에 떠있는 PVS 관찰 기술을 정립하였다. 이 프로토콜에 따라 림프관내 프리모관을 채취하여 형태적 특징을 DAPI 와 Phalloidin 염색 후 공초점현미경으로 확인하였다. 기존 연구에서 토끼와 쥐의 복부에 위치한 림프관에서만 관찰되었던 프리모시스템을, 본 연구에서는 흉관에서도 관찰하는 기법으로 확장하였다. 프리모시스템에 대한 H&E 및 면역염색으로 조직학적 분석을 하였다. DiI의 염색을 통해서 프리모관의 외막을 확인할 수 있었으며, H&E는 프리모관을 구성하는 세포의 세포질과 핵의 분포를 관찰할 수 있었다. EMP-3 면역 염색은 PVS의 외막이 상피세포로, vWF는 내부 세포가 내피 세포로 이루어져있음을 확인하였다. 프리모관은 EMP-3와 vWF에는 염색이 되지만 림프관의 중요한 마커로 사용되는 CD31이나 LYVE-1에는 염색이 되지 않는 특징을 가지고 있다. 이를 통해 림프관내 프리모관이 림프관과 구별되는 조직학적 특징을 가지고 있음을 확인하였다. 원자힘현미경을 이용하여 림프관의 내벽과 프리모관의 표면형상 분석을 시행하여 이들의 차이점을 규명하였다. 림프관의 내피세포는 탄성섬유로 이루어진 필라멘트가 interstitial collagen에 의해 부착되어있고, 그에 반해 PV의 표면은 상대적으로 매끈하고, 표면에 위치한 외공(hole)들을 관찰 할 수 있었다. PV의 외공은 PV의 표면과 내부의 연결 통로로서 selective channel 역할을 할 것으로 짐작된다. 결론적으로 림프관 내 프리모시스템의 해부학적 관찰 및 조직학적 분석 프로토콜을 개발했다. 원자힘현미경을 써서 프리모관의 표면특성을 조사하여 림프관 내벽과의 차이점을 규명했으며, x-ray microscopy 결과와 일치하는 프리모관의 외공구조를 자세히 밝혔다.The lymphatic primo vascular system (PVS) is the sub-system of the PVS that forms a network throughout an animal body as a third circulatory system in addition to the blood and lymph systems. In this study, the detailed protocol to observe the PVS in the lymph vessels near the caudal vena cava of a rat using the Alcian blue staining method was established. We investigated and analyzed the characteristics of the primo vessels and the inner wall surface of a lymph duct by atomic force microscopy. These data were compared with phase contrast x-ray microscope images of a primo vessel. The PVS floating inside lymphatic ducts was observed by injecting the Alcian blue into the lumbar lymph nodes near the caudal vena cava in rabbits and rats. . In previous studies, PVS was only observed within the abdominal lymph system but techniques were extended to the thoracic duct area in this thesis. After extracting the primo vessel, the morphological features were examined by staining them with Phalloidin and DAPI with the confocal laser scanning microscopy. For histological study of the PVS DiI, H&E and immune-histochemistry were performed. The DiI staining showed the outer membrane of the primo vessel and the H&E showed the distribution of cells and nuclei. The immuno staining with EMP-3 and vWF revealed that PVS had the EMP-3-positive epithelial layer and an inner vWF- positive endothelial layer, but not for CD31 or LYVE-1. These histological data provided the distinguishing features between lymph vessel and primo vessel. The ultra-structures of the primo vessel and the surface of lymph vessels inner wall were investigated using Atomic Force Microscope to find the differences between these two. Lymphatic endothelium layer were attached to filaments that were formed of many elastic fibers by interstitial collagen. On the other hand, the surface of a primo vessel (PV) was relatively smooth and had characteristic holes. These holes are considered to be size selective channels connecting outside and inside. As a conclusion, the detailed protocol was developed for the anatomical observations and histological analysis of the PVS. We investigated the primo vessel and the inner wall of lymph vessel using an atomic force microscope and found that they are distinctively different. We observed the holes on the surface of primo vessels which was consistent with the phase contrast x-ray microscopy.Table of Contents Abstract………………………………………………………… i Table of Contents…………….…….………………………… iii List of Figures and Tables ......................................... vii Chapter 1. Introduction 1.1 The Primo Vascular System.......……… 1 1.1.1 Morphology of Primo Vascular System…………… 3 1.1.2 Function of Primo Vascular System………………….6 1.2 Previous researches on Primo Vascular System in the Lymphatic system……………………………………….………7 References……………………………………………………… 10 Chapter 2. Microscopic Devices 2.1 Principle of Atomic Force Microscope………………… 12 2.1.1 Basic Principle………………………………………... 12 2.1.2 AFM Probe Deflection……………………………….. 12 2.1.3 Measuring Forces…………………………………….. 13 2.1.4 AFM Imaging Mode………………………………….. 14 2.2 Fluorescent Staining……………………………………… 17 2.2.1 Dapi………………………………………..…………. 17 2.2.2 Phalloidin………………………...…………………… 18 2.3 Fluorescence Stereo Microscope………………………19 2.3.1 Sample Preparation…………………………………… 20 2.3.2 Imaging Method……………………………………… 20 2.4 Confocal Laser Scanning Microscope…………………22 2.4.1 Imaging Mode………………………………………... 22 References……………………………………………………… 25 Primo Vascular System in the Lymphatic System Chapter 3. Protocol for the Observation of the Primo Vascular System in the Lymph Vessels of Rabbits 3.1 Introduction……………………………………………….…28 3.2 Materials…………………………………………………... 30 3.2.1 Reagents…………………………………………………. 30 3.2.2 Equipments……………………………………………… 31 3.2.3 Reagent setup……………………………………………. 32 3.3 Procedure…………………………………………..……… 33 3.3.1 Animal preparation……………………………………… 33 3.3.2 Surgical procedures and observations……………… 34 3.3.3 Preparation of isolated PVS tissue……………………39 3.3.4 Phalloidin and DAPI staining……………………………40 3.3.5 Time distribution during the entire procedure……….41 3.4 Anticipated results………………………………………….41 References…………………………………………………. 43 Chapter 4. Method for Observation of the Primo Vascular System in the Thoracic Duct of a Rat 4.1 Introduction………………………………………………….45 4.2 Materials and Methods……………………………………46 4.2.1 Animals………………………………………………….. 46 4.2.2 Surgery and Alcian blue injection………………………46 4.2.3 Staining and Microscopy……………………………….47 4.3 Anticipated results………………………………………… 48 4.4 Discussion…………………………………………………. 52 References…………………………………………… 54 Chapter 5. Observation of Primo Vascular System in Lymph Ducts of Rats 5.1 Introduction…………………………………………………. 55 5.2 Materials……………………………………………………. 57 5.2.1 Reagents………………………………………………... 57 5.2.2 Equipments……………………………………………... 58 5.3 Reagent setup……………………………………………… 59 5.3.1 Animals………………………………………………… 59 5.3.2 PBS……………………………………….......................59 5.3.3 Alcian blue staining dye………………………………...60 5.3.4 DAPI .………………………………………………….. 60 5.3.5 Phalloidin………………………………………………. 60 5.3.6 DiI……………………………………………………… 61 5.3.7 Harris H&E…………………………………………….. 61 5.3.8 Gordon & Sweets silver staining………………………62 5.3.9 EMP-3………………………………………………….. 64 5.3.10 VWF…………………………………………………... 64 5.3.11 IHC blocking solution………………………………… 64 5.4 Equipment setup…………………………………………… 66 5.5 Procedure…………………………………………………… 67 5.5.1 Animal preparation……………………………………... 67 5.5.2 Surgical procedures and observation……………… 68 5.5.2.1 Laparectomy…………………………………….. 68 5.5.2.2 Locating lumbar nodes………………………….. 69 5.5.2.3 Injection of AB and visualization of the PVS in lymph ducts……………………………………... 70 5.5.2.4 Observation of PVS in abdominal and thoracic lymph ducts………… 72 5.6 PVS Tissue Harvest and Fixation……………………..74 5.7 Analysis and Confirmation of the PVS………………… 75 5.7.1 Morphological analysis with DAPI and phalloidin staining……………………………………………………..… 75 5.7.2 Histology and Immunostaining…………………………77 5.8 Anticipated results………………………………………… 88 5.8.1 Anatomy………………………………………………... 88 5.8.2 Identification…………………………………………… 88 5.8.3 Histological analysis…………………………………… 89 5.9 Further Research…………………………………………..89 References…………………………………………………………91 Study on the Characteristics of Primo vessels with Atomic Force Microscope Chapter 6. Atomic Force Microscopy Study of Rat Primo Vessels of Rats 6.1 Introdution……………………………………………………94 6.2 Materials and Methods…………………………………… 97 6.2.1 Preparation of Rats and Surgical Procedure………… 97 6.3 Results…………………………………………………………98 6.3.1 Outer surface of Lymphatic vessel……………………98 6.3.2 Primo vessel surface…………………………………… 99 6.4 Discussion………………………………………………… 100 References……………………………………………………… 103 국문 초록………………………………………………………… 104 감사의 글………………………………………………………… 106Maste

Ultrastructure of a Mobile Threadlike Tissue Floating in a Lymph Vessel

Observations of the primo vascular system (PVS) floating in lymph ducts were reported by various groups. There have been, however, no studies on the ultrastructure of the entire cross section of a primo vessel (PV) inside a lymph vessel with a transmission electron microscope (TEM). In the current study we took the TEM images of a cross section of the PV inside a lymph vessel. We used the Alcian blue staining method for the finding of the target PV in a lymphatic vessel by injecting the dye into the inguinal lymph nodes. The stained PV was harvested together with the lymph vessel and some parts of the specimens were used for studying with optical microscopes. Some other parts were treated according to a standard protocol for TEM. As the results the TEM study revealed the loosely distributed collagen fibers with plenty of empty spaces and the lumens with the endothelial nuclei. It turned out to be very similar to the ultrastructure of the PVs observed on the surfaces of internal organs. It also showed how compactly the PV is surrounded with lymphocytes. In conclusion, the detailed morphological features like the distribution of fibers in the PV were revealed and shown to be similar to another kind of the PV on the surfaces of internal organs

Comparison of Alcian Blue, Trypan Blue, and Toluidine Blue for Visualization of the Primo Vascular System Floating in Lymph Ducts

The primo vascular system (PVS), floating in lymph ducts, was too transparent to be observed by using a stereomicroscope. It was only detectable with the aid of staining dyes, for instance, Alcian blue, which was injected into the lymph nodes. Some dyes were absorbed preferentially by the PVS than the lymph wall. It remains a standing problem to know what dyes are absorbed better by the PVS than the lymph walls. Such information would be useful to unravel the biochemical properties of the PVS that are badly in need for obtaining large amount of PVS specimens. In the current work we tried two other familiar dyes which were used in PVS research before. We found that Trypan blue and toluidine blue did not visualize the PVS. Trypan blue was cleared by the natural washing. Toluidine blue did not stain the PVS, but it did leave stained spots in the lymph wall and its surrounding tissues, and it leaked out of the lymph wall to stain surrounding connective tissues. These completely different behaviors of the three dyes were found for the first time in the current work and provide valuable information to elucidate the mechanism through which some special dyes stained the PVS preferentially compared to the lymphatic wall

Protocol for Detecting the Primo Vascular System in the Lymph Ducts of Mice

AbstractThe primo vascular system (PVS), which is the proposed conduit for the acupuncture Qi, is a complex network distributed throughout an animal's body. However, even with a microscope, it is not easily detectable because of its transparency. Thus, its existence is largely unknown in current anatomy. A convincing demonstration of its existence is needed. The lymph-primo vessel (PV), which is a subsystem of the PVS, is a very effective visual demonstration of the PVS. The lymph-PVS is a mobile threadlike structure floating in lymph ducts that has been observed in rabbits, rats, and mice by several independent teams. The involved techniques are novel and rather complicated; therefore, we have already provided detailed protocols for the surgery; for the injection of the staining dye; and for the detection, extraction, and identification of the PVS in rabbits and rats. However, the mouse is one of the most important laboratory animals used for various biomedical research purposes. For the convenience of researchers who wish to initiate the PVS experiments in mice, we provide a shortened version of the protocol, despite many similarities with previously published protocols. Thus, researcher can easily obtain the samples of the lymph-PVS of mice

Homing of the Stem Cells from the Acupoint ST-36 to the Site of a Spinal Cord Injury: A Preliminary Study

Homing of stem cells (SCs) to desired targets such as injured tissues remains a lingering problem in cell-based therapeutics. Studies on the biodistribution of intravenously administered SCs have shown the inefficacy of blood vessels as the homing path because most of the injected SCs are captured in the capillary beds of the lungs. We considered an alternative administration method using the acupuncture meridians or the primo vascular system. We injected SCs at the acupoint Zusanli (ST-36) below the knee of a nude mouse with a spinal cord injured at the thoracic T9-10 vertebrae. The SCs migrated from the ST-36, along the sciatic nerve, the lumbar 4-5, and then the spinal cord to the injury point T9-10. The SCs were not randomly scattered but were rather well aligned like marathon race runners, along the primo vascular system route toward the injury point. We observed the SCs at 1, 3, 6, 9, 12, and 15 hours after injection. The fast runners among the injected SCs took about 6 hours to reach the sciatic nerve, about 9 hours to reach the lumbar 4-5, and about 15 hours to reach the injury point T9-10. Keywords: primo vascular system (PVS), Zusanli (ST-36), stem cells, spinal cord injury (SCI), homin

A Method for the Observation of the Primo Vascular System in the Thoracic Duct of a Rat

Even though the primo vascular system (PVS) has been observed in large caliber lymph vessels by several independent teams, the presence of the PVS in the thoracic duct has been reported by only one team, probably because reproducing the experiment is technically difficult. This brief report presents a new, relatively straightforward method, which is a simple modification of the previous method of dye injection into the lumbar node, to observe the PVS in a thoracic duct of a rat by injecting Alcian blue into the renal node. When this new method was applied to a rat, the branching of the primo vessel in the thoracic duct was clearly displayed. Thus, this new method is expected to extend the network of the PVS from abdominal lymph ducts to thoracic ones

Distribution of Mast Cells and Locations, Depths, and Sizes of the Putative Acupoints CV 8 and KI 16

The anatomical locations and sizes of acupuncture points (APs) are identified in traditional Chinese medicine by using the cun measurement method. More precise knowledge of those locations and sizes to submillimeter precision, along with their cytological characterizations, would provide significant contributions both to scientific investigations and to precise control of the practice of acupuncture. Over recent decades, researchers have come to realize that APs in the skin of rats and humans have more mast cells (MCs) than neighboring nonacupoints. In this work, the distribution of MCs in the ventral skin of mice was studied so that it could be used to infer the locations, depths from the epidermis, and sizes of three putative APs. The umbilicus was taken as the reference point, and a transversal cross section through it was studied. The harvested skins from 8-week-old mice were stained with toluidine blue, and the MCs were recognized by their red-purple stains and their metachromatic granules. The three putative APs, CV 8 and the left and the right KI 16 APs, were identified based on their high densities of MCs. These findings also imply that acupuncture may stimulate, through MCs, an immune response to allergic inflammation

Distribution of Mast Cells and Locations, Depths, and Sizes of the Putative Acupoints CV 8 and KI 16

The anatomical locations and sizes of acupuncture points (APs) are identified in traditional Chinese medicine by using the cun measurement method. More precise knowledge of those locations and sizes to submillimeter precision, along with their cytological characterizations, would provide significant contributions both to scientific investigations and to precise control of the practice of acupuncture. Over recent decades, researchers have come to realize that APs in the skin of rats and humans have more mast cells (MCs) than neighboring nonacupoints. In this work, the distribution of MCs in the ventral skin of mice was studied so that it could be used to infer the locations, depths from the epidermis, and sizes of three putative APs. The umbilicus was taken as the reference point, and a transversal cross section through it was studied. The harvested skins from 8-week-old mice were stained with toluidine blue, and the MCs were recognized by their red-purple stains and their metachromatic granules. The three putative APs, CV 8 and the left and the right KI 16 APs, were identified based on their high densities of MCs. These findings also imply that acupuncture may stimulate, through MCs, an immune response to allergic inflammation