Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Intraoral foreign bodies detected 40 years after a car accident using cone beam computed tomography

Foreign bodies are common findings in the maxillofacial region, most commonly the result of accidents and physical aggression. Among the objects frequently found in the orofacial tissues are fragments of metal, plastic, wood, and glass. Visualization and exact identification of the location of these objects can be challenging but is of major importance prior to surgical removal. The present case report describes the use of cone beam computed tomography to locate, visualize, and surgically remove glass particles in the oral cavity.

Evolution of bombesin conjugates for targeted PET imaging of tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel (64/67)Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC(50): 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle(14) by Met, and deletion of D-Tyr(6), further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6(th), 11(th), and 14(th) position of BN induced only slight influences on affinity to mouse GRPr. [Cu(II)]-CPTA-[βAla(11)] BN(7-14) ([Cu(II)]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [(64/67)Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [(64/67)Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [(64/67)Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

Pathology hinting as the combination of automatic segmentation with a statistical shape model

With improvements in acquisition speed and quality, the amount of medical image data to be screened by clinicians is starting to become challenging in the daily clinical practice. To quickly visualize and find abnormalities in medical images, we propose a new method combining segmentation algorithms with statistical shape models. A statistical shape model built from a healthy population will have a close fit in healthy regions. The model will however not fit to morphological abnormalities often present in the areas of pathologies. Using the residual fitting error of the statistical shape model, pathologies can be visualized very quickly. This idea is applied to finding drusen in the retinal pigment epithelium (RPE) of optical coherence tomography (OCT) volumes. A segmentation technique able to accurately segment drusen in patients with age-related macular degeneration (AMD) is applied. The segmentation is then analyzed with a statistical shape model to visualize potentially pathological areas. An extensive evaluation is performed to validate the segmentation algorithm, as well as the quality and sensitivity of the hinting system. Most of the drusen with a height of 85.5 microm were detected, and all drusen at least 93.6 microm high were detected.

Compensability index for compensation radiotherapy after treatment interruptions

Background The goal of our work was to develop a simple method to evaluate a compensation treatment after unplanned treatment interruptions with respect to their tumour- and normal tissue effect. Methods We developed a software tool in java programming language based on existing recommendations to compensate for treatment interruptions. In order to express and visualize the deviations from the originally planned tumour and normal tissue effects we defined the compensability index. Results The compensability index represents an evaluation of the suitability of compensatory radiotherapy in a single number based on the number of days used for compensation and the preference of preserving the originally planned tumour effect or not exceeding the originally planned normal tissue effect. An automated tool provides a method for quick evaluation of compensation treatments. Conclusions The compensability index calculation may serve as a decision support system based on existing and established recommendations.

Evaluation and quantification of spectral information in tissue by confocal microscopy

A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Phase contrast and time-of-flight magnetic resonance angiography of the intracerebral arteries at 1.5, 3 and 7 T

Time-of-flight (ToF) and phase contrast (PC) magnetic resonance angiographies (MRAs) are noninvasive applications to depict the cerebral arteries. Both approaches can image the cerebral vasculature without the administration of intravenous contrast. Therefore, it is used in routine clinical evaluation of cerebrovascular diseases, e.g., aneurysm and arteriovenous malformations. However, subtle microvascular disease usually cannot be resolved with standard, clinical-field-strength MRA. The purpose of this study was to compare the ability of ToF and PC MRA to visualize the cerebral arteries at increasing field strengths.

Synthesis of Mid-Chain Functionalized Polymers

Polymers with mid-chain alkoxyamine functionality were synthesized by activating monohalogenated polymers in the presence of nitroso or nitrone radical traps. The resulting polymers were either polystyrene (PSt) homopolymers with a mid-chain alkoxyamine or PSt-poly(methyl acrylate) (PMA) diblock copolymers with an alkoxyamine unit at the junction between the segments. Monohalogenated polymers where synthesized by atom transfer radical polymerization (ATRP) and were then reacted to form polymer radicals in the presence of a radical trap, nitrone or nitroso. When only polystyrene radicals were reacted with the radical trap a dimer was formed with an alkoxyamine functionality in the center of the polymer chain. This functionality allowed the polymer chain to be cleaved in order to visualize the extent of the alkoxyamine functionality incorporation into the polymer chains. It was found that near quantitative alkoxyamine mid-chain functionality could be achieved by activating the PStBr in the presence of 10 equivalents of nitrone, 5 equivalents of copper bromide, and 2 equivalents of copper metal. Further reducing the amount of copper metal led to incomplete coupling, while increasing the equivalents beyond 2 generated polymer dimers with less than quantitative mid-chain functionality. Monochlorinated polystyrene (PStCl) precursors gave much poorer coupling results compared to reactions with PStBr, which is consistent with the stronger C-Cl bond resisting activation and the formation of the polystyryl radicals. When poly (methyl acrylate) (PMABr) is reacted with PStBr in the presence of a nitroso group at reduced temperatures (30 oC) block copolymers were selectively formed with an alkoxyamine functionality in the center. This was done by first activating the PSt-Br to form a polymer radical that would react with the radical trap to form a persistent radical on the oxygen. The PMA-Br, once activated, reacted with the radical on the oxygen to form the block copolymer. To test the amount of functionality incorporated, a coupling reaction was performed with no nitroso present, and found that no reaction occurred. This showed that the radical trap is essential for the coupling to occur, and cleavage of the diblock indicated that the alkoxyamine functionality was indeed incorporated into the diblock.

Quantifying The Diffusion And Erosion Of Degradable Hydrogels Using A Microfluidic Technique

Hydrogels are composed of cross-linked networks of hydrophilic polymers that are biocompatible due to their high water content. Mass transfer through hydrogels has been suggested as an effective method of drug delivery, specifically in degradable polymers to minimize lasting effects within the body. Diffusion of small molecules in poly (ethylene glycol) diacrylate (PEG-DA) and dextran methacrylate (dex-MA) hydrogels was characterized in a microfluidic device and by complementary techniques. Microfluidic devices were prepared by crosslinking a formulation of hydrogel and photo-initiator, with and without visible dye, using photolithography to define a central microchannel. Channel sizes within the devices were approximately 600 ¿m to simulate vessels within the body. The microfluidic technique allows for both image and effluent analyses. To visualize the diffusive behavior within the dextran hydrogel, methylene blue and sulforhodamine 101 dyes were used in both elution and uptake experiments. Three analysis techniques for measuring diffusion coefficients were used to quantify the diffusion of solute in the hydrogel, including optical microscopy, characterization of device effluent, and NMR analyses. The optical microscopy technique analyzes images of the dye diffusion captured by a stereomicroscope to generate dye concentration v. position profiles. The data was fit to a diffusion model to determine diffusion coefficients and the dye release profile. In a typical elution experiment, aqueous solution is pumped through the microchannel and dye diffuses out of the hydrogel and into the aqueous phase. During elution, images are taken at regular time intervals and the effluent was collected. Analysis of the device effluent was performed using ultraviolet-visible (UV/Vis) spectroscopy to determine the effluent dye concentration and thus a short-time diffusion coefficient. Nuclear magnetic resonance (NMR) was used to determine a free diffusion coefficient of molecules in hydrogel without the effect of a concentration gradient. Diffusion coefficients for methylene blue and sulforhodamine 101 dyes in dex-MA hydrogel calculated using the three analysis methods all agree well. It was determined that utilizing a combination of the three techniques offers greater insight into molecular diffusion in hydrogels than employing each technique individually. The use of the same microfluidic devices used to measure diffusion is explored in the use of studying the degradation of dex-MA hydrogels. By combining what is known about the degradation rate in regards to the effect of pH and crosslinking and the ability to use a dye solution in contrast to establish the hydrogel boundaries could be a novel approach to studying hydrogel degradation.

Sensitivity and specificity of fluorescence microlymphography for detecting lymphedema of the lower extremity

Fluorescence microlymphography (FML) is used to visualize the lymphatic capillaries. A maximum spread of the fluorescence dye of ≥ 12 mm has been suggested for the diagnosis of lymphedema. However, data on sensitivity and specificity are lacking. The aim of this study was to investigate the accuracy of FML for diagnosing lymphedema in patients with leg swelling. Patients with lower extremity swelling were clinically assessed and separated into lymphedema and non-lymphatic edema groups. FML was studied in all affected legs and the maximum spread of lymphatic capillaries was measured. Test accuracy and receiver operator characteristic (ROC) analysis was performed to assess possible threshold values that predict lymphedema. Between March 2008 and August 2011 a total of 171 patients (184 legs) with a median age of 43.5 (IQR 24, 54) years were assessed. Of those, 94 (51.1%) legs were diagnosed with lymphedema. The sensitivity, specificity, positive and negative likelihood ratio and positive and negative predictive value were 87%, 64%, 2.45, 0.20, 72% and 83% for the 12-mm cut-off level and 79%, 83%, 4.72, 0.26, 83% and 79% for the 14-mm cut-off level, respectively. The area under the ROC curve was 0.82 (95% CI: 0.76, 0.88). Sensitivity was higher in the secondary versus primary lymphedema (95.0% vs 74.3%, p = 0.045). No major adverse events were observed. In conclusion, FML is a simple and safe technique for detecting lymphedema in patients with leg swelling. A cut-off level of ≥ 14-mm maximum spread has a high sensitivity and high specificity of detecting lymphedema and should be chosen.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Interaction of fine particles and nanoparticles with red blood cells visualized with advanced microscopic techniques

So far, little is known about the interaction of nanoparticles with lung cells, the entering of nanoparticles, and their transport through the blood stream to other organs. The entering and localization of different nanoparticles consisting of differing materials and of different charges were studied in human red blood cells. As these cells do not have any phagocytic receptors on their surface, and no actinmyosin system, we chose them as a model for nonphagocytic cells to study how nanoparticles penetrate cell membranes. We combined different microscopic techniques to visualize fine and nanoparticles in red blood cells: (I) fluorescent particles were analyzed by laser scanning microscopy combined with digital image restoration, (II) gold particles were analyzed by conventional transmission electron microscopy and energy filtering transmission electron microscopy, and (III) titanium dioxide particles were analyzed by energy filtering transmission electron microscopy. By using these differing microscopic techniques we were able to visualize and detect particles < or = 0.2 microm and nanoparticles in red blood cells. We found that the surface charge and the material of the particles did not influence their entering. These results suggest that particles may penetrate the red blood cell membrane by a still unknown mechanism different from phagocytosis and endocytosis.

Sonographic visualization and ultrasound-guided block of the third occipital nerve: prospective for a new method to diagnose C2-C3 zygapophysial joint pain

BACKGROUND: Chronic neck pain after whiplash injury is caused by cervical zygapophysial joints in 50% of patients. Diagnostic blocks of nerves supplying the joints are performed using fluoroscopy. The authors' hypothesis was that the third occipital nerve can be visualized and blocked with use of an ultrasound-guided technique. METHODS: In 14 volunteers, the authors placed a needle ultrasound-guided to the third occipital nerve on both sides of the neck. They punctured caudal and perpendicular to the 14-MHz transducer. In 11 volunteers, 0.9 ml of either local anesthetic or normal saline was applied in a randomized, double-blind, crossover manner. Anesthesia was controlled in the corresponding skin area by pinprick and cold testing. The position of the needle was controlled by fluoroscopy. RESULTS: The third occipital nerve could be visualized in all subjects and showed a median diameter of 2.0 mm. Anesthesia was missing after local anesthetic in only one case. There was neither anesthesia nor hyposensitivity after any of the saline injections. The C2-C3 joint, in a transversal plane visualized as a convex density, was identified correctly by ultrasound in 27 of 28 cases, and 23 needles were placed correctly into the target zone. CONCLUSIONS: The third occipital nerve can be visualized and blocked with use of an ultrasound-guided technique. The needles were positioned accurately in 82% of cases as confirmed by fluoroscopy; the nerve was blocked in 90% of cases. Because ultrasound is the only available technique today to visualize this nerve, it seems to be a promising new method for block guidance instead of fluoroscopy.

Distribution Map

Understanding large software systems is a challenging task, and to support it many approaches have been developed. Often, the result of these approaches categorize existing entities into new groups or associates them with mutually exclusive properties. In this paper we present the Distribution Map as a generic technique to visualize and analyze this type of result. Our technique is based on the notion of focus, which shows whether a property is well-encapsulated or cross-cutting, and the notion of spread, which shows whether the property is present in several parts of the system. We present a basic visualization and complement it with measurements that quantify focus and spread. To validate our technique we show evidence of applying it on the result sets of different analysis approaches. As a conclusion we propose that the Distribution Map technique should belong to any reverse engineering toolkit.