Distribution of TiO2 particles in the olfactory bulb of mice after nasal inhalation using microbeam SRXRF mapping techniques

The characterization of different sized TiO₂ (25 nm, 80 nm, and 155 nm) was carried out by transmission electron microscopy (TEM) and the micro-distributions of TiO₂ in the olfactory bulb of mice after nasal inhalation were investigated by microbeam SRXRF mapping techniques. The results show that TiO₂ particles can be translocated to the olfactory bulb through the olfactory nerve system after inhalation. The distributions of Fe, Cu, and Zn in the olfactory bulb were also studied.     

嗅觉记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了C57BL/6J小鼠在嗅觉训练和记忆测试后嗅球蛋白表达的差异, 探讨了与嗅觉记忆相关的蛋白质. C57BL/6J小鼠经嗅觉训练后, 可对相应的气味保持记忆能力, 其嗅球蛋白表达存在明显差异, 5种蛋白与嗅觉记忆形成显著相关. 上述蛋白功能涉及神经发育, 信号转导及细胞骨架和核苷酸代谢, 其中神经发育和信号转导相关蛋白表达上调, 而细胞骨架和核苷酸代谢相关蛋白表达水平降低. 这些与嗅觉记忆形成相关的蛋白深化了对嗅觉记忆机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

In vitro formation and characterization of a perfusable three-dimensional tubular capillary network in microfluidic devices

This paper describes the in vitro formation and characterization of perfusable capillary networks made of human umbilical vein endothelial cells (HUVECs) in microfluidic devices (MFDs). Using this platform, an array of three-dimensional (3D) tubular capillaries of various dimensions (50-150 μm in diameter and 100-1600 μm in length) can be formed reproducibly. To generate connected blood vessels, MFDs were completely filled with fibrin gel and subsequently processed to selectively leave behind gel structures inside the bridge channels. Following gel solidification, HUVECs were coated along the gel walls, on opposite ends of the patterned 3D fibrin gel. After 3-4 days, HUVECs migrating into the fibrin gel from opposite ends fused with each other, spontaneously forming a connected vessel that expressed tight junction proteins (e.g., ZO-1), which are characteristic of post-capillary venules. With ready access to a perfusable capillary network, we demonstrated perfusion of the vessels and imaged red blood cells (RBCs) and beads flowing through them. The results were reproducible (～50% successful perfusable capillaries), consistent, and could be performed in a parallel manner (9 devices per well plate). Additionally, compatibility with high resolution live-cell microscopy and the possibility of incorporating other cell types makes this a unique experimental platform for investigating basic and applied aspects of angiogenesis, anastomosis, and vascular biology.     

Structural aspects of the lymphocyte traffic in rat submandibular lymph node.

Modulation of lymphocyte flow in the lymphatic compartment of the lymph node may serve, in part, to promote lymphocyte sensitization during an antigenic challenge. This study was undertaken to show where this might occur by examining the structural relationships of the intranodal lymphatic pathways, blood vessels, and connective tissue support with respect to lymphocyte and lymph flow. Differently stained plastic resins were injected into the blood vessels and lymphatics of the submandibular lymph node and visualized with a confocal laser scanning microscope. The specimens were corroded to study the three-dimensional cast structures by scanning electron microscopy. Alkali digestion was also used to prepare the reticular fiber network in the lymph node for scanning electron microscopic examination. At the hilus of the node, two to three arteries gave off arterioles running in medullary cords towards the cortex. The medullary cords, the periphery of the deep cortex, and the perifollicular zones had dense capillary networks. In contrast, the center of the follicle and the center of the deep cortex were less highly vascularized. High-endothelial venules were restricted to the perifollicular zone and the periphery of the deep cortex. At the cortico-medullary boundary, they abruptly transformed into medullary venules with a normal endothelium. The marginal sinus of the lymph node was crossed by thick reticular fibers that arose from the inner sheets of the capsule. The lymph pathway went through the marginal sinus, into the trabecular sinus, to the cortical perifollicular sinus, the dense lymphatic sinus around the deep cortex, and finally into the medullary sinus. At present, the exact functional significance of the complex lymph node lymphatic architecture is not clear. However, the highly organized structural organization may play a significant role in regulating and directing lymphocyte flow to facilitate antigen presentation.     

Preparation, characterization and application of pyrene-loaded methoxy poly(ethylene glycol)–poly(lactic acid) copolymer nanoparticles

Pyrene-loaded biodegradable polymer nanoparticles were prepared by incorporating pyrene into the polymer nanoparticles formulated from amphiphilic diblock copolymer, methoxy poly(ethylene glycol)–poly(lactic acid) (MePEG–PLA). Their morphological structure and physical properties were characterized by nuclear magnetic resonance (NMR), dynamic light scattering, fluorescence spectroscopy, transmission electronic microscopy and zeta potential measurements. Further, MePEG–PLA nanoparticles containing pyrene as fluorescent marker were administered intranasally to rats, and the distribution of nanoparticles in the nasal mucosa and the olfactory bulb were visualized by fluorescence microscopy. NMR results confirmed that MePEG–PLA copolymer can form nanoparticles in water, and hydrophilic PEG chains were located on the surface of the nanoparticles. The particle size, zeta potential and pyrene loading efficiency of MePEG–PLA nanoparticles were dependent on the PLA block content in the copolymer. Following nasal administration, the absorption of nanoparticles across the epithelium was rapid, with fluorescence observed in the olfactory bulb at 5 min, and a higher level of fluorescence persisted in the olfactory mucosa than that in the respiratory mucosa. These results show that pyrene could serve as a useful fluorescence probe for incorporation into polymer nanoparticles to study tissue distribution and MePEG–PLA nanoparticles might have a great potential as carriers of hydrophobic drugs.     

Non-Invasive Evaluation of Acute Effects of Tubulin Binding Agents: A Review of Imaging Vascular Disruption in Tumors

Tumor vasculature proliferates rapidly, generally lacks pericyte coverage, and is uniquely fragile making it an attractive therapeutic target. A subset of small-molecule tubulin binding agents cause disaggregation of the endothelial cytoskeleton leading to enhanced vascular permeability generating increased interstitial pressure. The resulting vascular collapse and ischemia cause downstream hypoxia, ultimately leading to cell death and necrosis. Thus, local damage generates massive amplification and tumor destruction. The tumor vasculature is readily accessed and potentially a common target irrespective of disease site in the body. Development of a therapeutic approach and particularly next generation agents benefits from effective non-invasive assays. Imaging technologies offer varying degrees of sophistication and ease of implementation. This review considers technological strengths and weaknesses with examples from our own laboratory. Methods reveal vascular extent and patency, as well as insights into tissue viability, proliferation and necrosis. Spatiotemporal resolution ranges from cellular microscopy to single slice tomography and full three-dimensional views of whole tumors and measurements can be sufficiently rapid to reveal acute changes or long-term outcomes. Since imaging is non-invasive, each tumor may serve as its own control making investigations particularly efficient and rigorous. The concept of tumor vascular disruption was proposed over 30 years ago and it remains an active area of research.     

Functional histology of glioma vasculature by FTIR imaging

Fourier-transform infrared (FTIR) imaging has been used to investigate brain tumor angiogenesis using a mice solid tumor model and bare-gold (∅ 25 nm) or BaSO₄ (∅ 500 nm) nanoparticles (NP) injected into blood vasculature. FTIR images of 20-μm-thick tissue sections were used for chemical histology of healthy and tumor areas. Distribution of BaSO₄-NP (using the 1,218–1,159 cm⁻¹ spectral interval) revealed clearly all details of blood vasculature with morphological abnormalities of tumor capillaries, while Au-NP (using the 1,046–1,002 cm⁻¹ spectral interval) revealed also diffusion properties of leaky blood vessels. Diffusion of Au-NP out of vascular space reached 64 ± 29 μm, showing the fenestration of “leaky” tumor blood vessels, which should allow small NP (<100 nm, as for Au-NP) to diffuse almost freely, while large NP should not (as for BaSO₄-NP in this study). Therefore, we propose to develop FTIR imaging as a convenient tool for functional molecular histology imaging of brain tumor vasculature, both for identifying blood capillaries and for determining the extravascular diffusion space offered by vessel fenestration.     

Tracking the pathway of diesel exhaust particles from the nose to the brain by X-ray florescence analysis

Studies have shown that exposure to nano-sized particles (< 50 nm) result in their translocation to the central nervous system through the olfactory nerve. Translocation commonly occurs via inhalation, ingestion and skin uptake. Little information is available on the specific pathway of cellular localization of nano-sized particles in the olfactory bulb. The nano-sized particles entrance into the postsynaptics cell is of particular interest because the mitral cell projects to the central nucleus of the amygdala and the piriform cortex. Therefore, our objective in this follow-up study has been to determine whether or not the mitral cells project nano-sized particles to the brain.Nano-sized particles in this study were generated using diesel exhaust. Lab mice were exposed for a period of 4 weeks. We employed synchrotron radiation (SPring-8, Japan) to determine the concentration levels of metal in the olfactory neuron pathway. Metal levels were assayed by mapping, using X-ray fluorescence analysis. The major metal components measured in the filter that collected the inhaled diesel exhaust particles were calcium, copper, iron, nickel and zinc. Our studies reveal an increase in the amount of nano-sized particles in the glomerular layer as well as in the neurons in the olfactory epithelium. Higher levels of nickel and iron were found in the olfactory epithelium's lamina propria mucosae in comparison to that in the control group. Higher levels of iron also were observed in the glomerular layer. Our studies do not clarify the specifics of metal adhesion and detachment. This remains to be one of the key issues requiring further clarification.     

Oily drug carriers in cancer chemotherapy. II. Preparation of viscous ethyl oleate for intraarterial infusion therapy and its disposition in rats and hamsters.

Viscous ethyl oleate (VEO) was prepared as an oil drug carrier by the addition of aluminum stearate or ethyl cellulose. Since the rate of shear of VEO containing aluminum stearate was greatly and nonlinearly changed against the shearing stress compared to that containing ethyl cellulose, the latter was used for subsequent microvascular and organ distribution experiments in rats and hamsters. For infusion into the carotid artery in hamsters, neat ethyl oleate (EO, 4cP) or VEOs of various apparent viscosities (40, 80, 120 cP-VEOs) embolized the vascular system in the cheek pouch, although arrival time to the site where the embolization was observed and the embolization period differed depending on the type of oily drug carrier. For infusion into the hepatic artery in rats, however, only 120 cP-VEO embolized the vascular system in the liver. After infusion of the oily drug carrier containing 3H-oleic acid into the artery of hamster cheek pouch and rat liver, 30-50% of the radioactivity was gradually eliminated within 48 h, whereas about 80% of the dose was rapidly eliminated after infusion to rat stomach and kidney. In addition, the amount of 120 cP-VEO remaining in each organ 48 h after infusion was higher than those of EO and 40 and 80 cP-VEOs. Histological observation after infusion in rat liver revealed that 120 cP-VEO slowly migrated from the artery or arteriole to the sinusoidal capillary region. These results suggest that 120 cP-VEO can be used as a drug carrier because of its function of vascular embolization and high retention in a targeted tissue.     

Enricher for fraction of high-pressure liquid chromatography

A device is developed for concentrating a dilute solution without losing the components with boiling points slightly higher than the solvent. The device consists of an evaporator, receptor, and approximately 100 capillaries. A dilute solution is introduced into the evaporator and heated at a lower temperature than the boiling point of the solvent with the addition of a helium gas flow. As a result, mostly only the solvent evaporates, passes through the capillaries, and enters into the receptor. The low-boiling-point components in the solute, with boiling points slightly higher than the solvent, are trapped at the inlet of the capillaries. These components are then recovered by a small amount of solvent supplied from the receptor through the capillaries, with the main components of the solute concentrated at the bottom of the evaporator. A diesel fuel is separated into aliphatic and aromatic fractions by high-pressure liquid chromatography using a silica gel column. These fractions are then analyzed by low-resolution field ionization mass spectrometry, following concentration using the described device. The analytical results show that the final composition of the fractions is almost the same as that of the aliphatic or aromatic hydrocarbons in the original fuel.     

Determination of N-acetylaspartic acid concentration in the mouse brain using HPLC with fluorescence detection

The concentration of brain N-acetylaspartic acid (NAA) in mice was determined by high-performance liquid chromatography (HPLC) using fluorescence detection after pre-column derivatization with 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Six different brain parts, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice, were investigated. The NAA concentration (nmol/mg protein) was highest in the olfactory bulb (58.2 ± 4.0, n = 8) and lowest in the hippocampus (42.8 ± 1.6, n = 8). The proposed HPLC method with fluorescence detection was successfully used to determine the NAA concentration in each investigated brain area.     

Selective-precursor reducing route to cobalt nanocrystals and ferromagnetic property

Cobalt nanocrystals were prepared by controlled chemical route at mild condition through selective-precursor reducing synthesis. Nanorod bundles and three-dimensional (3D) dendritic nanocrystal networks of Co were prepared by selecting different precursors. The samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) technologies. Field-emission scanning electron microscopy (FE-SEM), SEM, and TEM images indicate the nanorod bundles mainly consist of nanorods with the diameter of 70 nm. In 3D dendritic nanocrystal networks there are numerous secondary and sub-secondary branches were grown at right angles on each main stem. Room temperature magnetic measure of the Co samples demonstrates much enhanced ferromagnetic property, which might be attributed to their organization of specific shape. The possible formation mechanism of the cobalt nanocrystals with different morphologies was also discussed.     

外周苯二氮受体示踪剂的合成和评价

合成了用作外周苯二氮受体潜在的选择性配体的N,N-二乙基-2-(4-碘苯基)-6-三氟甲基-咪唑并[1,2-a]吡啶-3-乙酰胺(ITFZOL). 其放射性标记物[125I]ITFZOL通过碘脱锡化反应制备, 放化得率75%~85%, 比活度大于76 GBq/μmol. 小鼠尾静脉注射[125I]ITFZIOL后, 放射性集中分布于肾上腺、肺、肾、心、嗅球和小脑等外周苯二氮受体高密度区域. 预先给与外周苯二氮受体选择性配体PK11195明显减少外周苯二氮受体高密度区域放射性分布, 提示[125I]ITFZOL对外周苯二氮受体具有较高的特异亲和性. 生物活性数据表明, [125I]ITFZO是一种潜在的选择性外周苯二氮受体单光子放射性配体.     

Acute lung injury induced by Staphylococcal enterotoxin B: disruption of terminal vessels as a mechanism of induction of vascular leak

The current hypothesis of alveolar capillary membrane dysfunction fails to completely explain the severe and persistent leak of protein-rich fluid into the pulmonary interstitium, seen in the exudative phase of acute lung injury (ALI). The presence of intact red blood cells in the pulmonary interstitium may suggest mechanical failure of pulmonary arterioles and venules. These studies involved the pathological and ultrastructural evaluation of the pulmonary vasculature in Staphylococcal enterotoxin B (SEB)-induced ALI. Administration of SEB resulted in a significant increase in the protein concentration of bronchoalveolar lavage fluid and vascular leak in SEB-exposed mice compared to vehicle-treated mice. In vivo imaging of mice demonstrated the pulmonary edema and leakage in the lungs of SEB-administered mice. The histopathological studies showed intense clustering of inflammatory cells around the alveolar capillaries with subtle changes in architecture. Electron microscopy studies further confirmed the diffuse damage and disruption in the muscularis layer of the terminal vessels. Cell death in the endothelial cells of the terminal vessels was confirmed with TUNEL staining. In this study, we demonstrated that in addition to failure of the alveolar capillary membrane, disruption of the pulmonary arterioles and venules may explain the persistent and severe interstitial and alveolar edema.     

Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhancement compared to non-fluorinated counterparts. The fluorescence enhancement was attributable to a nanoscale fluorous effect in the Pdots that maintained the molecular planarity and minimized the structure distortion between the excited state and ground state, thus reducing the nonradiative relaxations. By performing through-skull and through-scalp imaging of the brain vasculature of live mice, we quantitatively analyzed the vascular morphology of transgenic brain tumors in terms of the vessel lengths, vessel branches, and vessel symmetry, which showed statistically significant differences from the wild type animals. The bright NIR-II Pdots obtained through fluorination chemistry provide insightful information for precise diagnosis of the malignancy of the brain tumor.     

Confocal microscopy, computer modeling, and quantification of glomerular vascular corrosion casts.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

Analysis of effective molecular diffusion rates for verteporfin in subcutaneous versus orthotopic Dunning prostate tumors

Photosensitizer biodistribution change inside tissue is one of the dominant factors in photodynamic therapy efficacy. In this study, the pharmacokinetics of a benzoporphyrin derivative (BPD), delivered in verteporfin for injection formulation, have been quantified in the rat Dunning prostate tumor MAT-LyLu model, using both subcutaneous and orthotopic sites. Blood plasma sampling indicated that BPD had a bi-exponential metabolic lifetime in vivo, with the two lifetimes being 9.6 min and 8.3 h. The spatial distributions in the tumor were quantified as a function of distance from the perfused blood vessels, using fluorescence histologic images of the tumor. A fluorescent vascular marker was used to obtain locations and shapes of perfused capillaries at a wavelength of emission different from that of BPD and to allow colocalized images to be acquired of vessel and BPD locations. Using the BPD fluorescence images obtained 15 min after intravenous administration, a forward finite-element solution to the diffusion equation was used to predict the drug distribution by matching the fluorescence intensity images observed microscopically. An inverse solver was used to minimize the root mean square error between the image of simulated diffusion and the experimental image, resulting in estimation of the diffusion coefficient of BPD in the tumor models. Effective diffusion coefficients were 0.88 and 1.59 microm2/s for the subcutaneous and orthotopically grown tumors, respectively, indicating that orthotopic tumors have significantly higher vascular extravasation rates as compared with subcutaneous tumors. This analysis supports the hypothesis that leakage rates of the photosensitizer vary considerably. Thus, although varying the time between injection and optical irradiation may be used to vary the targeting between vascular and less vascular areas, the precise time of treatment will depend on the nature of the permeability of the vasculature in the tissue being treated.     

A simplified method for capillary embedment into microfluidic devices - exemplified by sol-gel-based preconcentration

We here describe an alternative method of embedding functionalized capillaries into microdevices fabricated in PDMS. The capillaries have square-shaped outer dimensions and fit into elastic PDMS channel networks of similar dimensions. By modifying the capillary off-chip, the technique makes it possible to integrate a new chip function without risking contamination of already existing chemically patterned areas because of new reagent solutions. Leak-free insertion of these capillaries has earlier been reported, where a thin layer of uncured PDMS bonded the capillary to the microchannel and the lid structure. In this new approach, oxygen plasma is used to bond the square capillary to the PDMS, eliminating the risk of clogging the microsystem with uncured prepolymer. The new embedding technique was exemplified and evaluated by inserting a square capillary piece containing monolithic sol-gel for sample preconcentration application. The assembled microdevice was run with mass spectrometric detection, showing that peptides were preconcentrated without leakage from either the sol-gel itself or around the inserted capillary. Repeated preconcentration runs showed migration times better than 3% for all peptides. We believe that the presented microchip assembling technique greatly simplifies the insertion of functionalized capillary pieces, e.g., an initial preconcentrator to a PDMS device containing other downstream modules.     

Comprehensive two-dimensional gas chromatography in combination with rapid scanning quadrupole mass spectrometry in perfume analysis

Single column gas chromatography (GC) in combination with a flame ionization detector (FID) and/or a mass spectrometer is routinely employed in the determination of perfume profiles. The latter are to be considered medium to highly complex matrices and, as such, can only be partially separated even on long capillaries. Inevitably, several monodimensional peaks are the result of two or more overlapping components, often hindering reliable identification and quantitation. The present investigation is based on the use of a comprehensive GC (GC x GC) method, in vacuum outlet conditions, for the near to complete resolution of a complex perfume sample. A rapid scanning quadrupole mass spectrometry (qMS) system, employed for the assignment of GC x GC peaks, supplied high quality mass spectra. The validity of the three-dimensional (3D) GC x GC-qMS application was measured and compared to that of GC-qMS analysis on the same matrix. Peak identification, in all applications, was achieved through MS spectra library matching and the interactive use of linear retention indices (LRI).