Temporal Change of Alcian Blue-Stained Primo Vascular System in Lymph Vessels of Rats.

This study aims to investigate the temporal change of a vascular system now known as the primo vascular system (PVS). We used Alcian blue (AB) dye for imaging the distribution of the PVS in lymphatic vessels. The target lymph vessels were chosen as they are easily accessible from the skin, and long-term observation is possible with intact physiological conditions due to a minimal surgical procedure. AB solution was injected into the inguinal lymph node and the target lymph vessels were located along the superficial epigastric vessels. The imaging system allowed processing for extraction of images showing changes in the AB intensity of the visualized PVS components. This newly developed procedure can be used for further study on various dynamic processes of PVS in lymph vessels.

Electromechanical method coupling non-invasive skin impedance probing and in vivo subcutaneous liquid microinjection: controlling the diffusion pattern of nanoparticles within living soft tissues.

Transdermal drug delivery is the way to transport drug carriers, such as nanoparticles, across the skin barrier to the dermal and/or subcutaneous layer. In order to control the transdermal drug delivery process, based on the heterogeneous and nonlinear structures of the skin tissues, we developed a novel electromechanical method combining in vivo local skin impedance probing, subcutaneous micro-injection of colloidal nanoparticles, and transcutaneous electrical stimulation. Experiments on the nude mice using in vivo fluorescence imaging exhibited significantly different apparent diffusion patterns of the nanoparticles depending on the skin impedance: Anisotropic and isotropic patterns were observed upon injection into low and high impedance points, respectively. This result implies that the physical complexity in living tissues may cause anisotropic diffusion of drug carriers, and can be used as a parameter for controlling drug delivery process. This method also can be combined with microneedle-based drug release systems, micro-fabricated needle-electrodes, and/or advanced in vivo targeting/imaging technologies using nanoparticles.

Discovery of a novel fibrous tissue in the spinal pia mater by polarized light microscopy.

Abstract It is very well known that spinal meninges are composed of three layers, dura, arachnoid and pia mater, and that the main components of pia mater are collagen and reticular fibers. However, the distribution of those fibers has not been extensively investigated but just described as a mesh of fibers. In this study, we detected novel structures, which are composed of unidirectionally arranged fibers, in a rat spinal pia mater by using a polarized light microscope. They were seen as three parallel lines, one of which ran along a posterior spinal vein and the rest two of which ran along a pair of posterior spinal arteries. Histological analysis including Masson's trichrome, picrosirius-red staining, Gordon & Sweet's staining and immunohistochemistry with anti-collagen type 1 and 3 antibodies uncovered that they are mainly composed of collagen fibers and some reticular fibers. In addition, a putative primo vessel was detected in the novel fibrous tissue, which was proven out to be different from a blood vessel. In conclusion, we report a newly detected fibrous structure in the spinal pia mater, which may contribute to provide tensile force to the spinal meninges and to harbor the primo vascular system inside.

Novel threadlike structures on the surfaces of mammalian abdominal organs are loose bundles of fibrous stroma with microchannels embedded with fibroblasts and inflammatory cells.

Novel threadlike structures (NTSs) on the surfaces of mammalian abdominal organs have recently attracted interests regarding their ability to transport fluid, enable cell migration, and possibly facilitate cancer metastasis. Nevertheless, histological studies of NTSs have been sporadic and often have inconsistent interpretations of the NTS internal structure. In this article, we provide a synthetic and consistent view of the NTS internal structure: the NTS is a loose bundle of fibrous stroma that forms interstitial channels and microsinusoids infiltrated with inflammatory cells. The fibroblasts are embedded in the stroma and mostly aligned along the major axis of the NTS. The sinusoids, which are in inconsecutive cross sections, have boundaries more or less delineated by extracellular fibers, partly surrounded by endothelial-like cells, or both. We compare these morphological features to other well-known connective tissues (i.e., trabecular meshwork and lymphatic capillary) and discuss the biomechanical and biological functions of NTSs based on their structural characteristics.

Primo vascular system and its potential role in cancer metastasis.

The primo vascular system (PVS) is a newly found organ, which is distributed throughout the entire body. The system is composed of nodes storing many small cells and thin vessels branching out from the nodes. Inside the vessel there are multiple subvessels. The PVS is found in and on most organs, including the brain, and interestingly inside some lymph and blood vessels. The PVS is normally difficult to visualize due to its semitransparent optical property and its small size, which may be the main reason why it was not discovered until recently. The diameter of primo vessels (PVs) is in the range of 20-50 µm and the size of a primo node (PN), 100-1,000 µm. The outermost layer of the PVS is more porous than that of blood or lymph capillary vessels, and the nuclei of the PVS endothelial cells are rod shaped. Important PVS properties reported are: in the fluid inside the PVS, there are cells presenting stem cell markers CD133, Oct4, and Nanog, which may imply that this system has a role in regeneration. Another very important finding is its potential relevance to cancer. According to results from an animal study using xenografts of various cancer types (lung, ovarian, skin, gastric cancer, and leukemia), as the tumor grows, the PVS is formed in a high density in the vicinity of the tumor. In addition, it was shown that PVs connect the primary and secondary tumors and that cancer cells were transported via the PVs in an active manner. In this report, we illustrated the formation of the PVS in breast cancer, and using the green fluorescent protein-expressing gastric cancer cell lines, we observed the cancer cell movement from the primary to the secondary sites during the cancer progression.

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field.

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.

Ultra-weak photon emission during wrist curl and cycling exercises in trained healthy men.

Human ultra-weak photon emission (UPE) is related to the activity of respiratory chain and oxygen consumption. Investigations on UPE and its response to exercise are almost non existent. Since human UPE is an indicator of reactive oxygen species (ROS) levels, we used exercises as a model to study UPE. To continue the research on the relationship between human UPE and exercise, it was decided to carry out measurements of UPE in response to different exercise modes with 20 healthy male subjects. The performed exercises were wrist curls with a dumbbell and indoor cycling. Regarding wrist curl exercises, 70% of the subjects for the first exercise and 65% for the second exercise did not show any significant changes in UPE. Also, the statistical analysis did not show significant changes of the UPE levels. In terms of cycling exercise, 85% of subjects did not show any significant increase of UPE. The gathered data showed that a majority of the subjects didn't show an increase of the UPE during both types of exercises. Our results imply that the UPE is not only affected by oxygen consumption, but also by the intensity, the type of exercise, and the physiology of the subject.

Fine structure of extracellular fibers in primo-nodes and vessels.

To investigate the extracellular matrices in the primo-nodes (PNs) and vessels (PVs) (Bonghan corpuscles and ducts), fine structure of the fibers comprising the extracellular matrices was studied in relation to the diameter and the periodicity to identify the types of the fibers. Electron micrographs of the fibers from the previously published works on the PNs and PVs were analyzed by using MATLAB programming and ImageJ software. The extracellular fibrous components of the PNs and PVs could be classified generally into two groups--thin collagen fibrils and thick non-collagenous wavy bent fibers. The thick bent fibers were fibrin-like. The diameters of the thick bent fibers were 30-200 nm and they were highly bent. This group included organ surface PNs and lymphatic PNs/PVs. The diameters of the collagen fibrils were 30-70 nm. Organ surface, heart, and hypodermis PVs were categorized in this group. The periodicity of bent fibers in an organ surface PN was 16.1 +/- 0.2 (SE) nm, and the periodicity of collagen fibrils in the hypodermis PVs was 37.4 +/- 0.5 (SE) nm. Further investigations are required to reveal the physiological implications of the two types of fibers in the PNs and PVs.

Network of endocardial vessels.

BACKGROUND: Although there have been reports on threadlike structures inside the heart, they have received little attention. We aimed to develop a method for observing such structures and to reveal their ultrastructures. METHODS: An in situ staining method, which uses a series of procedures of 0.2-0.4% trypan blue spraying and washing, was applied to observe threadlike structures on the surfaces of endocardia. The threadlike structures were isolated and observed by using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). RESULTS: Networks of endocardial vessels (20 µm in thickness) with expansions (40-100 µm in diameter) were visualized; they were movable on the endocardium of the bovine atrium and ventricle. CLSM showed that (1) rod-shaped nuclei were aligned along the longitudinal direction of the endocardial vessel and (2) there were many cells inside the expansion. TEM on the endocardial vessel revealed that (1) there existed multiple lumens (1-7 µm in diameter) and (2) the extracellular matrices mostly consisted of collagen fibers, which were aligned along the longitudinal direction of the endocardial vessel or were locally organized in reticular structures. CONCLUSION: We investigated the endocardial circulatory system in bovine cardiac chambers and its ultrastructures, such as nucleic distributions, microlumens, and collagenous extracellular matrices.

Electrical characterization of proposed transpositional acupoints on the urinary bladder meridian in a rat model.

Specific electrical characteristicsof acupointswere investigated on the urinary bladder (BL) meridian in 14 rats. BL acupointsand non-acupoints on the back were selected and their electrical voltages were measured by using aSPACsystem.The mean voltages of each point or each line were statistically analyzed by using the ANOVA test.The BL meridian showed voltages higher than those of the reference line (P < .05). Bilateral 1st BL lines presented higher voltages than bilateral 2nd BL lines (P < .05). Most BL acupoints had voltageshigher than those for the corresponding reference points (P < .05). In particular, theright BL16 exhibited the biggest difference from the reference point, followed by the left extra BL point-2, the right BL27, the left BL17, and theleft BL45. Additionally, the distributions of neurofilamentsfor several points were investigated by using immunohistochemistry. There was a trend for the BL acupoints to have larger numbers of neurofilaments than the reference points, and that trend seemed to be directly proportional to the difference in voltage between the points.In conclusion, BL acupoints on the back in ratsexhibited specific electric and histologic characteristics. Therefore, those acupointsmay be utilized to investigate the efficacy of acupuncturewith laboratory animals.

In situ microextraction method to determine the viscosity of biofluid in threadlike structures on the surfaces of mammalian organs.

We report a method to measure the viscosity of microL volumes of biofluid obtained from threadlike structures (NTSs) on the surfaces of mammalian (rabbit) internal organs. The fluid was mechanically microextracted in situ from NTSs on the organ surfaces by a glass capillary connected to an extractor. From the Brownian motion of the 0.8+/-0.1microm diameter granules in the extracted fluid, the fluid viscosity was determined to be 1.4+/-0.1mPa s at room temperature. This viscosity is comparable to the viscosity of rabbit blood plasma.

Effects of Lac operon activation, deletion of the Yhha gene, and the removal of oxygen on the ultra-weak photon emission of Escherichia coli.

We observed a relation between gene activity and ultra-weak photon emission (UPE). By comparing the UPEs of E. coli with the LacI gene present and deleted we found that more gene activity produced higher UPE. This relation was further confirmed by studying the UPE of the E. coli with and without the Yhha gene. We interpreted that a higher aminoacyl t-RNA synthetase activity, which used ATP from the respiratory chain, could increase the emission. Satisfying the increased need of ATP by the E. coli through an increase of respiratory chain activity, which has reactive oxygen species (ROS) as a byproduct, results in a higher rate of photon emission. To ensure that oxygen is at the origin of this emission, we replaced the air by pure nitrogen. After 30 min, it was observed that the emission levels equaled the emission levels of the sterile medium. We could therefore conclude that the source of the photon emission would be affected by genetic activity and is oxygen related.

Enhancement of ultraweak photon emission with 3 MHz ultrasonic irradiation on transplanted tumor tissues of mice.

We investigated photon emissions of various bio-samples which were induced by ultrasonic stimulation. It has been reported that ultrasonic stimulations induced the thermal excitation of the bio-tissues. After ultrasonic stimulation, any measurement of photon radiation in the visible spectral range has not been carried out yet. The instruments consisted of electronic devices for an ultrasonic generator of the frequency 3 MHz and a photomultiplier tube (PMT) system counting photons from bio-tissues. The transplanted tumor tissues of mice were prepared for the experiments and their liver and spleen tissues were also used for the controls. It was found that the continuous ultrasonic stimulations with the electrical power 2300 mW induced ultraweak photon emissions from the tumor tissues. The number of induced photon was dependent of the type of the tissues and the stimulation time intervals. The level of photon emission was increased from the mouse tumor exposed to the ultrasonic stimulations, and the changes were discriminated from those of the spleens and livers.

In vivo visualization of bonghan ducts inside blood vessels of mice by using an Alcian blue staining method.

An in vivo method using Alcian blue (AB) was developed for visualizing floating threadlike tissues inside blood vessels of mice. These novel structures called intravascular Bonghan ducts (IBHDs) are considered as extension of acupuncture meridians. For in vivo imaging of IBHDs, AB solution (pH 7.4) that stains mucopolysaccharides like hyaluronic acid was used. After injecting AB solution into the femoral vein of a mouse, the threadlike structures, stained deep blue, inside the inferior vena cava. The histological results, such as hematoxylin, eosin, and AB staining, show the compositions of the cells and the extracellular matrix in the IBHD. Further studies are needed to investigate their physiological functions, especially in relation with those of other circulatory systems.

Development of a fluorescence stereomicroscope and observation of Bong-Han corpuscles inside blood vessels.

A fluorescence stereomicroscope system was developed in order to observe in situ the distribution of nuclei in intravascular Bong-Han ducts and corpuscle tissues by injecting acridine orange, which stained specifically nuclei. Intravascular Bong-Han corpuscles, connected with Bong-Han ducts could be detected in the aortas of rats, mice, and rabbits.

4 Hz magnetic field decreases oxidative stress in mouse brain: a chemiluminescence study.

We investigated the effects of delta and theta brain wave frequency magnetic fields (3, 4, and 5) on mouse brain by detecting photonic oxidative stress makers; spontaneous photon emission (SPE) and lucigenin and tert-butyl hydroperoxide (TBHP) induced chemiluminescences (CL). For this purpose, Balb/C mice were exposed to 3, 4, and 5 Hz magnetic fields (MF) at 0.7 mT for 3 h, respectively. After that we monitored SPE and lucigenin and TBHP-induced CL of the homogenates of mice brains. There was a significant decrease in SPE in the 4 Hz MF-exposed group. Lucigenin-induced CL was also significantly decreased only in the 4 Hz MF-exposed group. TBHP-induced CL was also distinctively decreased by all frequencies, 3, 4, and 5 Hz MF exposures. These results showed that oxidative stress in a mouse brain was decreased by 4 Hz MF. We suggest that the application of 4 Hz MF will contribute to magnetic field therapy.

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.

Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs.

The ultrastructures of novel threadlike structures (NTSs) and corpuscles on the surfaces of internal organs of rats were investigated using electron microscopy. The samples were studied in situ by using a stereomicroscope and were taken for further morphological analysis. Scanning electron microscope (SEM) images revealed a bundle structure of threadlike tissue, which was composed of several 10-micro m-thick subducts. The surfaces of the corpuscles were rather coarse and fenestrated. The corpuscles had cucumber-like shapes with an average length of about 2 mm and a thickness of about 400 micro m. Transmission electron microscope (TEM) images disclosed disordered collagen fibers, which formed the extracellular matrix of the threadlike tissue, and immune-function cells, like macrophages, mast cells, and eosinophils. Sinuses of various diameters, which were thought to be cross-sections of the lumens of the subducts, were observed in the TEM, cryo-SEM and focused-ion-beam SEM images. These SEM images were obtained for the first time to reveal the detailed structure of the NTSs that were only recently discovered.

Immunohistochemical and electron microscopic study of the meridian-like system on the surface of internal organs of rats.

Threadlike structures on the surfaces of mammalian internal organs were recently observed, and they can be the anatomical basis of the meridian-like network of internal organs as detected by the Bi-Digital O-Ring test imaging method. Morphological and histological studies were performed to reveal their novel features by using confocal and electron microscopy, and there is evidence that reveals that they have a ductal structure in which some liquid flows. They form a part of the network of acupuncture meridians that is thought to be a circulatory system distributed throughout an animal body. In order to confirm further the circulatory function it was necessary to provide evidences for the existence of endothelial cells at the inner boundaries of the microchannels that form the threadlike ducts. In this work we performed immunohistochemical studies for the identification of endothelial cells. In addition, we identified the extracellular matrices between the microchannels in the threadlike structure by using antifibronectin, elastin, laminin, and collagen type IV. Since immunohistochemical data are not sufficient for definite proof we supplemented the analysis with ultra structural information by using transmission electron microscopy (TEM). Yet TEM images show only ultrathin sections (50 nm) of a specimen, we needed to take 3-dimentional images to provide a visual demonstration of the microchannels in the threadlike ducts. We conducted high voltage electron microscopic studies for this purpose and obtained corroborating tomograms that exhibit the microchannels. In conclusion, the meridian-like tissues are ducts with microchannels, which implies the circulatory function of the acupuncture meridian network.

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.