siRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo.

Considerable efforts have been directed towards discovering and developing delivery vehicles for RNA therapeutics. While most studies emphasize the efficacy and safety of these delivery vehicles, few reports conduct a comprehensive assessment of their storage stability, a critical property for practical applications. Here, we report a potent and safe lipid nanoparticle with long-term storage stability. Through chemical synthesis and screening of cationic lipids, a formulation has been identified that enables potent knockdown of hepatocyte proteins in mice upon intravenous administration (siRNA ED50 ~0.02 mg/kg). Toxicity studies revealed that a dose of 2mg/kg was well tolerated in rats, the most sensitive rodent model. We identified that a cyclic chemical structure in cationic lipids improved particle stability. The nanoparticles showed over 1.5 year storage stability as a liquid, with over 90% siRNA encapsulation without any changes in particle size. This novel delivery material has promising potential as a drug product that could bring RNA therapeutics to the treatment of liver-related disorders.

Direct measurement of speed of sound in cartilage in situ using ultrasound and magnetic resonance images.

This study verified the accuracy of the speed of sound (SOS) measured by the combination method, which calculates the ratio between the thickness values of cartilage measured by using the magnetic resonance imaging (MRI) and the ultrasonic pulse-echo imaging, and investigated in vivo application of this method. SOS specific to an ultrasound imaging device was used as a reference value to calculate the actual SOS from the ratio of cartilage thicknesses obtained from MR and ultrasound images. The accuracy of the thickness measurement was verified by comparing results obtained using MRI and a non-contact laser, and the accuracy of the calculated SOS was confirmed by comparing results of the pulse-echo and transmission methods in vitro. The difference between laser and MRI measurements was 0.05 ± 0.22 mm. SOS values in a human knee measured by the combination method in the medial and lateral femoral condyles were 1650 ± 79 and 1642 ± 78 m/s, respectively (p < 0.05). The results revealed the feasibility of in situ SOS measurement using the combination method.

Phase-contrast X-ray imaging of the gas diffusion layer of fuel cells.

The understanding of and in situ observation of the transport and distribution of water in carbon-paper gas diffusion layers (GDLs) using non-destructive imaging techniques is critical for achieving high performance in polymer electrolyte fuel cells (PEFCs). To investigate the behavior of water in GDLs of PEFCs, phase-contrast X-ray imaging via X-ray interferometric imaging (XII) and diffraction-enhanced imaging (DEI) were performed using 35 keV X-rays. The XII technique is useful for the radiographic imaging of GDLs and in situ observations of water evolution processes in operating PEFCs. DEI provides a way for tomographic imaging of GDLs in PEFCs. Because high-energy X-rays are applicable to the imaging of both carbon papers and heavy materials, which make up PEFCs, phase-contrast X-ray imaging techniques have proven to be valuable for investigation of GDLs.

Local VEGF administration enhances healing of colonic anastomoses in a rabbit model.

BACKGROUND/AIMS: Many studies report the role of vascular endothelial growth factor (VEGF) in wound healing, but few describe local VEGF administration to the digestive tract. Leakage from colonic anastomoses, including those due to ischemia, represents a major complication causing increased mortality and morbidity. Angiogenesis is crucial to anastomotic healing and restoration of blood supply, and VEGF is a potent angiogenic factor showing improved healing in various models of reconstruction and anastomosis. Here, we examine the effects of local VEGF-A(165) administration on postoperative rabbit colon anastomosis. METHODS: Two colotomies per animal were made in the sinistral colon on opposite sides of the mesentery. Randomly assigned VEGF (10 microg/0.1 ml) or saline (0.1 ml) was injected into the muscularis propria on both sides of each colonic anastomosis before closing the access laparotomy using single-layer sutures. On postoperative days 3, 4 and 7, the bursting pressure of partially healed anastomoses was measured. On postoperative day 4, anastomotic tissues were examined for the following: hydroxyproline; histopathologically for inflammatory infiltrate and tissue organization and immunohistochemically for capillary proliferation and density; vessel density of midzone collaterals around anastomoses by microangiography. RESULTS: Compared to saline, VEGF administration significantly improved bursting pressure (p = 0.014, paired t test) and increased hydroxyproline (p = 0.027, paired t test) on postoperative day 4. Inflammatory cell infiltration and fibroblast proliferation were prominent, and submucosal capillary vascular counts were significantly higher for VEGF. CONCLUSIONS: Administration of VEGF to colonic anastomosis accelerates wound healing and strengthens the anastomosis by increased angiogenesis.

Fat-saturated diffusion-weighted imaging of the rat pelvis using three-dimensional MP-RAGE MR sequence.

In this work we report on the development of a novel technique for fat-saturated three-dimensional (3D) diffusion-weighted (DW) MRI sequence based upon 3D magnetization-prepared rapid gradient-echo (3D-MP-RAGE). In order to saturate fat, two kinds of procedures were competed CHESS-DW-3D-MP-RAGE sequence (CHESS-3D-DWI) and DW-3D-MP-WE-RAGE sequence (WE-3D-DWI) "chemical shift selective: CHESS method vs. water-excitation: WE method". The CHESS-3D-DWI sequence and WE-3D-DWI sequence were compared in terms of their degree of fat suppression. In CHESS-3D-DWI sequence a preparation phase with a "CHESS-90 degrees RF-motion probing gradient: MPG-180 degrees RF-MPG-90 degrees RF" pulse-train was used to sensitize the magnetization to fat-saturated diffusion. In contrast, WE-3D-DWI sequence a RAGE-excitation pulse with a "binominal-pulse 1-1 or 1-2-1" was selected to water-excited (fat-saturated) diffusion imaging. These imaging were done during in vivo studies using an animal experiment. From experimental results obtained with a phantom, the effect of diffusion weighting and the effect of fat-saturation were confirmed. Fat-saturation was much better in the WE-3D-DWI sequence than CHESS-3D-DWI sequence. From rat experimental results, fat-saturated diffusion-weighted image data were obtained. This sequence was useful for in vivo imaging.

Quantitative analysis of amyloid plaques in a mouse model of Alzheimer's disease by phase-contrast X-ray computed tomography.

Densely aggregated beta-amyloid peptides are believed to play a key role in the pathogenesis of Alzheimer's disease. Amyloid plaques are a potential target for molecular imaging to determine the clinical status of Alzheimer's disease. Phase-contrast X-ray imaging combined with computed tomography is a promising technique that can be used to visualize the physical density of structures in biological tissues non-invasively, and without the use of imaging agents. Using brain tissue isolated from a mouse model of Alzheimer's disease, we show that beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques, but not beta-amyloid 40-negative/beta-amyloid 42-positive amyloid plaques, exist as high-density aggregates that can be specifically detected by phase-contrast X-ray computed tomography. The phase-contrast X-ray computed tomography detected beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques in three-dimensions with an extremely high sensitivity comparable to that of histological analysis, and also enabled the load of amyloid plaques to be quantified. Furthermore, the use of phase-contrast X-ray computed tomography reveals that the physical density of beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques increases with age, and that the large volume, high-density, amyloid plaques that are specifically observed in aged Alzheimer's disease mice are closely associated with neuritic dystrophy. These results demonstrate that phase-contrast X-ray computed tomography is a highly sensitive imaging technique for analyzing dense-cored amyloid plaques in postmortem samples, and is beneficial in elucidating amyloid pathophysiology in Alzheimer's disease.

Application of synchrotron X-ray imaging to phase objects in orthopedics.

Novel imaging of the fine structures of the ribs of a pig and a specimen of human osteosarcoma utilizing the spatial coherence of X-rays was successfully performed with an incident X-ray energy of 30 keV at SPring-8, Japan. The image contrast appearing at the periphery of trabecular bone, small calcifications and small fractures is caused by the phase shift of the X-rays at the boundary of these objects that have different X-ray refractive indices. The image is recorded on mammography film without an intensifying screen. Comparison of the image contrast using different sample-to-film distances, Z, such as Z = 5 m and Z approximately 0 m, showed that the former images were always more informative, i.e. better in resolution and/or image contrast when imaging trabecular bone, bone marrow and small fractures in compact bone, and for imaging cartilage. Radiography using synchrotron X-rays for phase objects should be a powerful tool for diagnosis in orthopedics, especially for bone disease.

Preliminary experiment of fluorescent X-ray computed tomography to detect dual agents for biological study.

The simultaneous observation of various information, such as blood flow, tissue metabolism and distribution of receptors, is quite important in order to understand the functional state of biomedical objects. The simultaneous detectability of contrast agents by fluorescent X-ray computed tomography (FXCT) with synchrotron radiation is examined in this study. The system consisted of a silicon (111) double-crystal monochromator, an X-ray slit system, a scanning table, a PIN diode, a highly purified germanium detector and an X-ray charge-coupled device (CCD) camera. The monochromatic X-ray beam energy was adjusted to 37.0 keV and collimated into a pencil beam of 1 x 1 mm. The fluorescent spectra of the K alpha lines for iodine and xenon were detected simultaneously. FXCT could image the distribution of both iodine and xenon agents in a phantom clearly and the contrast ratio was significantly better than that of transmission X-ray computed tomography images.

Design of synchrotron light source and its beamline dedicated to dual-energy x-ray computed tomography.

A synchrotron light source dedicated to medical applications has been designed at National Institute of Radiological Sciences. The storage ring, with circumference of 80 m, is designed for acceleration of 2.3 GeV and a stored current of 420 mA. It is equipped with two multipole wigglers to produce sufficient photon flux in a hard x-ray region required for medical applications. The purposes of the synchrotron light source are clinical performance of medical diagnoses clinically and research and development relating with medical applications. One of the most interesting applications for us is dual-energy x-ray computed tomography (CT). It gives the information about electron density of human tissue. The information plays an important role in advancing heavy-ion radiotherapy of cancers. Electron density can be derived from attenuation coefficients measured by different energy x rays. In this paper, a practical method of the dual-energy x-ray CT with synchrotron radiation is proposed with the theoretical consideration. The primitive experiment using monochromatic x rays emitted from radioisotopes proved the procedure of analysis mentioned here effective to derive electron densities from linear attenuation coefficients for two x rays of a different energy. The beamline dedicated to dual-energy x-ray CT is also proposed. It has a multipole wiggler as a light source and it mainly consists of a dual crystal monochromator and a rotating filter for attenuating photon flux of x rays and two-dimensional detector.

[Clinical laboratory data and reference intervals standardized in Fukuoka].

In Fukuoka whose population is approximately five million inhabitants, surveys on the accuracy of laboratory data have been performed by the Fukuoka Prefecture Medical Association for the last 30 years. We have been attempting to evaluate the data for routine use since 1988, and it has become possible to share laboratory data between all institutions in Fukuoka prefectures. As a result, reference intervals for 23 clinical chemistry analytes were established in 1995, to which were added in 1996 five serum protein constituents that have been utilized for clinical examinations. Methods for documentations and monitorings the data obtained in the prefecture were also established, standardization of the above analytes extended to 97% of the institutions in the prefecture. Results for 14 of the 23 clinical chemistry analytes have become highly reliable and clinically useful as differences between institutions in terms of results have narrowed. Standardization of other analytes is now in progress.

Morphology, fluorescence properties, and their photothermal changes of poly(substituted thiophene) films revealed by near-field fluorescence microspectroscopy.

The morphology and fluorescence spectrum of poly(3-[2-(N-dodecylcarbamoyloxy)ethyl]thiophene-2,5-diyl) film were examined with spatial resolution of 100 nm using near-field fluorescence microspectroscopy. Fluorescence spectra observed at protruding domains were blue-shifted compared with flat areas, and further blue-shift was observed there more appreciably by long-time irradiation via a near-field scanning optical microscope probe. It is considered that the polymer chains at the protruding domains take disordered conformations, in which conjugated lengths are shorter and further disordering can be induced more easily by irradiation compared with those in the flat areas.

Comparison of in-phantom dose distributions for coronary angiography using an x-ray machine and synchrotron radiation.

Coronary cineangiography using synchrotron radiation is anticipated, owing to the high intensity and availability of monoenergy. To investigate allowable dose levels in clinical application, absorbed dose distribution in a tissue substitute phantom for a conventional x-ray machine was measured with thermoluminescent dosimeters at the University of Tsukuba under the practical conditions used for digital angiography. The dose rate at a 0.5-cm depth was 0.145 Gy/s, and the dose per frame was 0.725 mGy for the irradiation period of 5 ms per frame. For synchrotron radiation, the dose distribution measurement was made at a 5-GeV AR (Accumulation Ring) of the High Energy Accelerator Research Organization, in which a polymethylmethacrylate (PMMA) phantom was irradiated with the strongest beam available at the facility, which was 33.32 keV, 5.2 x 6.2 cm2 beam. Using this beam, a 1-mm-diameter coronary artery has been visualized at 1% iodine concentration at the AR. Nonhomogeneous strength distribution in the beam was observed in the vertical direction. The maximum dose rate was 0.556 Gy/s, and it attenuated to 1/3000 at a 30-cm depth in the beam center. At the deep positions, the doses were influenced by the high harmonics, which was confirmed with an EGS4 Monte Carlo calculation. Outside the beam, beam contamination on both sides of the main beam affected the doses. For comparison to the x-ray machine, the measured dose was analytically converted to that needed for a 5.2 x 16 cm2 beam that is used for clinical application. The dose rate at 0.5-cm depth was found to be 0.215 Gy/s, which is 1.48 times larger than that for x-rays. Moreover, the attenuation rate in the phantom was significantly greater than that of the x-ray machine, because of the difference of the energy spectra between the x-rays and synchrotron radiation used.

High-resolution imaging of coronary calcifications by intense low-energy fluoroscopic X-ray obtained from synchrotron radiation.

PURPOSE: To obtain an intense monochromatic low-energy X-ray from synchrotron radiation (SR) and apply it to detect coronary calcifications. METHODS AND RESULTS: The SR beam was reflected with a silicon crystal to be expanded (150 mm in height and 80 mm in width) and to be monochromatized at an energy level of 37 keV. The X-ray was intermittently irradiated to obtain dynamic imaging of 30 images/s. Images were recorded by a digital fluorography system. The low-energy X-ray from SR sharply visualized calcification of coronary arteries, while conventional X-ray could not visualize coronary calcification. CONCLUSION: The intense monochromatic low-energy X-ray from SR is sensitive, has high-resolution for imaging coronary calcification and may serve as a screening method for coronary artery disease.

Visualization, quantification and therapeutic evaluation of angiogenic vessels in cancer by synchrotron microangiography.

The usefulness of a synchrotron microangiography system for depicting, quantitating and therapeutically evaluating angiogenic vessels in cancer is illustrated. In 20 mice transplanted with murine colon cancer, sequential changes in the angiogenic vessels were determined by using synchrotron microangiography, using changes in tumor volume for reference. This system allowed the depiction and quantification of angiogenic vessels in the period from one to four weeks after transplantation. The effects of antiangiogenic therapy were evaluated by using a neutralizing antibody against vascular endothelial growth factor. The neutralizing antibody partially suppressed angiogenesis and tumor growth. Synchrotron microangiography is shown to be useful for the depiction, quantification and evaluation of angiogenic vessels in cancer.

[A study of mental and physical reactions after the Hanshin-Awaji Earthquake: mental and physical conditions of female undergraduate students immediately after, 2 months after, and 9 months after the earthquake].

This study assessed how time brings out changes in one's mental and physical reactions to traumatic experiences. Two surveys were conducted on students of Kobe College, located at one of the areas hardest hit by the Hanshin-Awaji Earthquake in January, 1995. Much of the traumatic reactions decreased in strength through January to March, while feelings of survival guilt did not. Guilt feelings of those whose housing were destroyed increased in March, and kept its level even in October. Although general health conditions of those surveyed in October was normal, several post-traumatic reactions increased in October as compared with March. The rate of recovery from post-traumatic reactions seemed to very according to the depth of one's psychological suffering, to one's financial situation, and to the presence of social support. The factor analysis on the items of mental and physical reactions yielded seven factors. The factor of highest eigenvalue in January was one named "anxiety about after-quake tremors", however, this was replaced by one named "emotional confusions" in March.

Digitized cerebral synchrotron radiation angiography: quantitative evaluation of the canine circle of Willis and its large and small branches.

BACKGROUND AND PURPOSE: Conventional X-ray angiography lacks the sensitivity and spatial resolution needed to detect small amounts of iodinated contrast material and to quantitate diameters of the small vessels in the brain. The purpose of this study was to ascertain whether digitized synchrotron radiation microangiography, with the use of a high-definition TV camera system, can accurately show small cerebral vessels. METHODS: Six anesthetized dogs were exposed to monochromatic synchrotron radiation with an energy level of 33.3 keV optimized for iodine detection while iodinated contrast material was injected into the brachiocephalic and vertebral arteries. The images were detected with a high-definition TV camera system with a spatial resolution of 30 microm. In all, 26 cerebral angiograms of the circle of Willis with its branches were obtained, and the images were digitized at a workstation. RESULTS: The small branches of the circle of Willis were clearly visible on all images. Vasodilatation of the circle of Willis and its large and small branches induced by CO2 inhalation was quantitatively confirmed on the images: for example, the diameter of one small branch was increased from 0.24 +/- 0.04 mm to 0.38 +/- 0.12 mm. Temporal subtraction improved the image quality. CONCLUSION: The synchrotron radiation angiographic system is useful for visualizing large and small vessels deep in the brain as well as for quantitating their diameters.

Branching patterns of intramural coronary vessels determined by microangiography using synchrotron radiation.

The intramural coronary artery (IMCA) with a diameter of 50-500 micrometers is critical for blood supply to the inner layers of heart muscle. We introduced digital measurement to microangiography using monochromatic synchrotron radiation and quantified branching patterns of the IMCA, the epicardial coronary artery (EPCA), and the distal ileal artery (DIA). The pre- and postbranching diameters were measured (95-1,275 micrometers) in seven dogs. A typical arterial segment divided into two nearly equivalent branches, and a regression line of daughter-to-mother diameter plots was almost identical among the EPCA (y = 0.838x - 16.7 in micrometers), IMCA (y = 0.737x - 2.18), and DIA (y = 0.755x + 8.63). However, a considerable difference was present at a segment where a proximal IMCA branched off from an EPCA (y = 0.182x + 90.2). Moreover, a proximal IMCA diameter had no relationship to the branching order from an EPCA. The precision of this method was confirmed by the good correlation of diameter measurements between two independent observers (r = 0.999, y = 1.02x - 1.07). In conclusion, using digital microangiography we demonstrated that the self-similar branching pattern of coronary arteries was discrete at the connection between the IMCA and EPCA.

Dynamic intravenous coronary angiography using 2D monochromatic synchrotron radiation.

A method of examination for coronary artery disease that is less invasive and easier than coronary angiography (CAG) has been sought. We have developed a dynamic intravenous coronary angiography (IVCAG) system using synchrotron radiation (SR) and have used it clinically. Four patients suspected of having angina pectoris underwent IVCAG. An SR beam was reflected asymmetrically with a silicon crystal to produce a wide (150 mm x 80 mm) and monochromatic (37 keV) X-ray beam, with an energy level to achieve high sensitivity to the contrast agent. Following an intravenous injection of contrast agent, irradiation was applied for 4 ms periods at 33 ms intervals for dynamic IVCAG at 30 images s-1. Images were acquired with an image intensifier and recorded with a digital fluorography system. The dynamic images permitted clear visualization of the coronary arteries and permitted evaluation of coronary anatomy. Two patients exhibited no stenotic lesions, one patient had a 90% stenosis in the right coronary artery, and the remaining patient had a 25% stenosis at the site of previous percutaneous transluminal coronary angioplasty in the left anterior descending artery (LAD). The total irradiation doses used for IVCAG were less than those for conventional angiography. Dynamic IVCAG can be readily used for the evaluation of coronary arteries.

Synchrotron microangiography reveals configurational changes and to-and-fro flow in intramyocardial vessels.

In 8 dogs, in situ microangiography using synchrotron radiation visualized penetrating transmural arteries (PTAs) with a diameter of >60 micrometers and allowed quantitation of vessel diameters of >140 micrometers. Myocardial contraction reduced the vascular short-axial diameters to 87 +/- 17% (n = 62, P < 0.001, paired t-test) of the end-diastolic values and increased the longitudinal dimension to 129 +/- 5% (n = 45, P < 0.001). The diameter reduction in the subendocardial PTA segments was significantly more marked than that in the subepicardial PTA segments (60 +/- 12 vs. 88 +/- 12%, n=13, P < 0.001, paired t-test). Intracoronary administration of dobutamine (0.1 micrograms. kg-1. min-1) increased, and in contrast, partial clamping of the coronary artery (ischemia) decreased, the configurational changes. To-and-fro blood flow was clearly observed in PTAs with visual identification of capacitive backflow, resistive forward flow during ischemia on coronary arteriography, and even under baseline conditions in coronary venography. Thus this method advances our understanding of mechanical influences on the coronary circulation.

Dose response of various radiation detectors to synchrotron radiation.

Accurate dosimetry is particularly difficult for low- to medium-energy x-rays as various interaction processes with different dependences on material properties determine the dose distribution in tissue and radiation detectors. Monoenergetic x-rays from synchrotron radiation offer the unique opportunity to study the dose response variation with photon energy of radiation detectors without the compounding effect of the spectral distribution of x-rays from conventional sources. The variation of dose response with photon energies between 10 and 99.6 keV was studied for two TLD materials (LiF:Mg,Ti and LiF:Mg,Cu,P), MOSFET semiconductors, radiographic and radiochromic film. The dose response at synchrotron radiation energies was compared with the one for several superficial/orthovoltage radiation qualities (HVL 1.4 mm Al to 4 mm Cu) and megavoltage photons from a medical linear accelerator. A calibrated parallel plate ionization chamber was taken as the reference dosimeter. The variation of response with x-ray energy was modelled using a two-component model that allows determination of the energy for maximum response as well as its magnitude. MOSFET detectors and the radiographic film were found to overrespond to low-energy x-rays by up to a factor of 7 and 12 respectively, while the radiochromic film underestimated the dose by approximately a factor of 2 at 24 keV. The TLDs showed a slight overresponse with LiF:Mg, Cu, P demonstrating better tissue equivalence than LiF:Mg, Ti (maximum deviation from water less than 25%). The results of the present study demonstrate the usefulness of monoenergetic photons for the study of the energy response of radiation detectors. The variations in energy response observed for the MOSFET detectors and GAF chromic film emphasize the need for a correction for individual dosimeters if accurate dosimetry of low- to medium-energy x-rays is attempted.