Volume 3, Issue 2, Pages 75-80 (June 2010)

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ABSTRACT

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Characteristic Features of a Nerve Primo-vessel Suspended in Rabbit Brain Ventricle and Central Canal

Received 6 July 2009; accepted 30 March 2010.

Abstract

Bonghan theory was proposed by Bonghan Kim to illustrate the anatomy and physiology of the acupuncture meridian system. One of his astonishing claims was the physical presence of the nerve primo-vessel, which can be involved with a regenerating system of nerves. Our previous work has shown that there is a nerve primo-vessel in brain ventricles and the central canal of the spine of a rabbit. In this study, confocal laser scanning microscopy, transmission electron microscopy, and high voltage electron microscopy demonstrated that a nerve primo-vessel comprised DNA particles, other microparticles, and rod-shaped nuclei encircled by helix-shaped actins. The nerve primo-vessel had acridine orange-stained DNA particles that varied in size and were in parallel. These characteristics of the nerve primo-vessel are crucial for a comprehensive understanding of their function in the central nervous system, which may be associated with nerve regeneration.

Key Words: brain ventricle , central canal , DNA , microparticles , nerve primo-vessel , nerve regeneration

Article Outline

• Abstract

• 1.. Introduction

• 2.. Materials and Methods

• 2.1.. Microscopy

• 2.1.1.. CLSM

• 2.1.2.. TEM and HVEM

• 3.. Results

• 4.. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

The meridian system has been regarded as one of the most fundamental concepts in traditional Chinese medicine. The Bonghan theory, proposed in the 1960s, insisted that the acupuncture meridian system was physically present as an anatomical structure different from the nerve or the cardiovascular networks in living organisms [1]. Bonghan theory enabled the Chinese meridian concept to be evaluated with diverse scientific methods [2]. Unfortunately, Bonghan theory has been neglected for over 40 years as it has been highly challenging to repeat the experiment as reported by Bonghan Kim. Only the Japanese anatomist, Fujiwara and Yu [3], partially succeeded in finding the same structures, including primo-vessels (Bonghan ducts) and primo-nodes (Bonghan corpuscles), on the internal organs and inside the blood vessels of rabbits.

Since early 2000, we have investigated the anatomical structures described in the Bonghan theory, i.e., Bonghan ducts and Bonghan corpuscles. Studies have been conducted in blood vessels [4, 5, 6, 7], lymph vessels [8, 9, 10, 11], on organ surfaces [12, 13, 14, 15, 16, 17], in rabbit brain ventricles, and in the central canal of the rabbit spinal cord [18]. Among these anatomical structures, Bonghan ducts and Bonghan corpuscles in blood vessels and on internal organs were confirmed by other research groups [19, 20]. We also investigated their characteristics by transmission electron microscopy (TEM), immunohistochemistry and proteomics [21, 22, 23]. Against the common view that ependymal cells in the ventricle of the brain can proliferate, but nerve tissue cannot, Kim [1] insisted that the nerve primo-vessel (NPV) had a regenerating function for the healing of damaged nerve tissue, including the brain and spine.

Our previous work has revealed the existence of an NPV [18]. To address its structural details, we employed confocal laser scanning microscopy (CLSM), TEM, and high voltage electron microscopy (HVEM) to observe DNA particles, other microparticles, and helix-shaped actin wound around the NPV nuclei. Based on these morphological characteristics of the NPV, we discuss potential functions and significance of the NPV in association with nerve regeneration.

2. Materials and Methods

Ten New Zealand white rabbits (female, 12-week-old) were obtained from the Jung Ang Laboratory Animal Company of Korea. The animals were raised at 23°C with 60% relative humidity under a 12 hour light/dark cycle. All animals had ad libitum access to food and water. The procedures involving the animals and their care were in full compliance with institutional regulations and current international laws and policies [24]. The rabbits were anesthetized by using an intraperitoneal injection of urethane (1.5 g/kg). Under deep anesthesia, the animals were decapitated without perfusion. After 1 hour freezing at −70°C, the brains were immediately isolated from the skulls. To maintain the original shape of the brain, an ice pack was placed beneath the isolated brain during dissection. The fourth ventricle was exposed and mildly cooled on an aluminum-foil-covered ice pack, followed by careful dissection under a stereomicroscope. Hematoxylin filtered through a 0.2 μm pore-sized membrane filter paper was poured into the exposed fourth ventricle of the brain, drop by drop, and left there for 1 to 2 minutes. After hematoxylin in situ staining of the exposed fourth ventricle, we applied 0.1 M phosphate buffered saline (pH 7.4) drop by drop into the fourth ventricle. The pink-colored choroid plexus in the fourth ventricle, which was readily identified by its many capillaries, was carefully removed. After staining and washing, a thin threadlike structure emerged along the midline of the fourth ventricle. The threadlike structure led to the third ventricle via the aqueduct. The in situ features were recorded using a CCD camera (DP70; Olympus Co., Tokyo, Japan) coupled to a stereomicroscope (SZX12; Olympus Co.).

We cut the threadlike structure, an NPV, in the middle of the mesencephalic aqueduct and fixed it in neutral phosphate-buffered formalin (pH 7.4) for CLSM. For TEM and HVEM, we fixed the NPV in a modified Karnovsky's fixative.

2.1. Microscopy

2.1.1. CLSM

The NPV was stained using a DNA staining dye, acridine orange (Sigma, St. Louis, MO, USA). CLSM was used to examine optical sections of the threadlike structure. For the nucleus and F-actin staining of the NPV, we used 15 μM propidium iodide (Sigma) and 330 nM Alexa Fluor 488 phalloidin dye (Molecular Probes, Eugene, OR, USA), respectively. After characterization of the nuclei of the threadlike structure, 10 μM of a phospholipid staining dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes), was applied to visualize the microparticles inside the NPV.

2.1.2. TEM and HVEM

Specimens were collected as described above for CLSM. They were fixed with a modified Karnovsky's fixative, consisting of 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2), by exposing the tissue three times, each for 10 minutes. Specimens were post-fixed with 1% (w/v) osmium tetroxide in the same buffer at 4°C for 2 hours and were washed briefly with distilled water twice. The post-fixed specimens were stained en bloc with 0.5% (w/v) uranyl acetate at 4°C overnight, and dehydrated in a graded ethanol series: 30%, 50%, 70%, 80%, 95%, and 100% three times, each for 10 minutes. The specimens were further treated with propylene oxide twice, each for 10 minutes, and embedded in Spurr's resin.

Ultrathin sections (approximately 60 nm in thickness) were cut with a diamond knife using an ultramicrotome (MT-X; RMC, Tucson, AZ, USA). The sections were mounted on copper grids and stained with 2% uranyl acetate and Reynolds' lead citrate, each for 7 minutes. They were examined with a transmission electron microscope (LIBRA 120; Zeiss, Oberkochen, Germany) operated at an accelerating voltage of 120 kV In addition, semithin sections (1 μm thick) were made and examined by HVEM (JEM-ARM 1300S; JEOL, Tokyo, Japan) operated at an accelerating voltage of 1,250 kV.

3. Results

As shown in Figure 1, which was published in our previous work [18], we were able to visualize the NPV in brain ventricles of a rabbit using hematoxylin. We intentionally held the NPV on the tip of an acupuncture needle to show that the NPV was suspended in the cerebrospinal fluid of the brain ventricle. The inset image of the NPV in Figure 1 shows how strongly the NPV was stained by hematoxylin. Also, the NPV is transparent, as shown by an overlapped region of the NPV (indicated by the arrow). The inset is presented in this study for the first time.

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Figure 1. Representative pictures of a nerve primo-vessel (NPV; Bonghan duct) in the brain ventricle and central canal of the spinal cord in rabbit. (A) The NPV, indicated by four arrows, lies in the mesencephalic aqueduct and fourth ventricle of rabbit brain, suspended by an acupuncture needle indicated by a dotted arrow. Scale bar, 2 mm. (B) Opened central canal of rabbit spinal cord before staining. There is no structure visualized inside the opened central canal. Scale bar, 50 μm. (C) After hematoxylin staining, NPV (arrows) emerges in the central canal of the rabbit spinal cord. Scale bar, 100 μm.

Figure 2A shows many microparticles stained by DiI that are in parallel. One of the microparticles may also correspond to TEM images of the micro-particles in the NPV (Figure 2B). Figure 3 demonstrates that the NPV has acridine orange-stained DNA particles that vary in size, and are aligned in parallel. For comparison between the CLSM and the HVEM images, we marked a dotted square, which corresponds to Figure 4. Figure 4 shows a round structure with high electron density, enveloped by two layers of membranes. As shown in Figure 5, the NPV has a long rod-shaped nucleus (stained with propidium iodide), which was encircled by helix-shaped F-actins (phalloidin staining).

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Figure 2. (A) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) stained nerve primo-vessel (NPV; Bonghan duct). DiI staining in NPV appears as aligned parallel dots. Several DiI stained dots are shown using rectangles. Scale bar, 10 μm. (B) Transmission electron microscopic image of NPV corresponding to a rectangle seen in (A). Dots stained by DiI are microparticles. Scale bar, 500 nm. (C) Magnified image of dotted rectangle in (B) shows empty vesicles and material-containing vesicles. Scale bar, 200 nm.

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Figure 3. Confocal laser scanning microscopic image of a nerve primo-vessel (Bonghan duct) stained by acridine orange, which binds DNA. DNA stained primo-vessel was optically sectioned from (A) to (D). Bright green signal indicates DNA which varies according to size and distribution. (B,C,D) Triangles indicate a cluster of DNA similar to that in the nucleus. Arrows indicate DNA particles in parallel. One tiny DNA green signal in (C) is intentionally marked by a dotted square for comparison with a high electron density structure taken using high voltage electron microscopy, as in Figure 4. All scale bars, 10 μm.

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Figure 4. High voltage electron microscopic image of a high electron density structure in a nerve primo-vessel (Bonghan duct). This round structure consists of a double outermost membrane which envelops the high electron density materials.

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Figure 5. Confocal laser scanning microscopic images of nucleus (red), stained by propidium iodide, enveloped by helix-shaped actin (green), stained by phalloidin. Clear images of helix-shaped actin are indicated by arrows. The primo-vessel sample was optically sectioned using 2.4 μm steps. Scale bar, 10 μm.

4. Discussion

According to the Bonghan theory, the NPV has the unique function of maintaining the vitality of the nervous system by supplying essential nutrients to the nerves [1]. Here, we investigated the morphological characteristics of the NPV and revealed DNA particles and microparticles aligned in parallel and long rod-shaped nuclei encircled by helix-shaped actin. Cell-free DNA, or fragmented DNA particles, has emerged as an important etiological parameter in cancer biology [25]. A recent issue in neuroscience has been the occurrence and role of microparticles, and their possible therapeutic implications for protection against and treatment of stroke [26, 27]. Therefore, we propose that the fragmented DNA and microparticles found in the NPV are related to the physiological function of the NPV in the brain and spine. Helix-shaped actin may be involved in the transportation of these particles in parallel inside the NPV. Interestingly, the helix-shaped actin observed in NPV has mainly been found in plants [28]. In one of our previous studies on the Bonghan duct, we observed that the Bonghan duct and a plant stem have similar morphological characteristics [29].

By definition, microparticles are vesicle-type byproducts that bud off from cells but do not contain nuclei [30], implying that microparticles share the same original membrane as their parent cells. Recently, microparticles and their original cells were found to have the same surface markers [31, 32], indicating that the specific surface markers on the microparticles in the NPV could be used to determine their origin.

An important function of the NPV may be related to the many observed fragmented DNA particles. From a medical point of view, fragmented DNA in living organisms has been considered to be a byproduct of cell death with no specific function. However, Kim claimed that the fragmented DNA, so called “primo-microcells” (Sanals), or “living eggs,” could form cells in damaged tissues during regeneration [33]. According to the Bonghan theory, the many fragmented DNA particles in the NPV are transported as Sanals to damaged nerve tissue and form cells for regeneration. The Bonghan system may, therefore, play a significant role in future regeneration medicine.

Acknowledgments

This work was supported by the Systems Biology Infrastructure Establishment Grant provided by the Gwangju Institute of Science and Technology in 2010.

References

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a Biomedical Physics Laboratory for Korean Medicine, Department of Physics and Astronomy, Seoul National University, Seoul, Korea

b Pharmacopuncture Medical Research Center, Korean Pharmacopuncture Institute, Seoul, Korea

c National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea

Corresponding author. Biomedical Physics Laboratory for Korean Medicine, Department of Physics and Astronomy, Seoul National University, 599 Gwanak, Gwanak-gu, Seoul 151-747, Korea

PII: S2005-2901(10)60015-5

doi:10.1016/S2005-2901(10)60015-5

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