Noninvasive assessment of regional lung ventilation is of critical importance in quantifying the severity of disease and evaluating response to therapy in many pulmonary diseases. This work presents for the first time the implementation of a hyperpolarized (HP) gas MRI technique for measuring whole-lung regional fractional ventilation (r) in Yorkshire pigs (n = 5) through the use of a gas mixing and delivery device in supine position. The proposed technique utilizes a series of back-to-back HP gas breaths with images acquired during short end-inspiratory breath-holds. In order to decouple the RF pulse decay effect from ventilatory signal build-up in the airways, regional distribution of flip angle (α) was estimated in the imaged slices by acquiring a series of back-to-back images with no inter-scan time delay during a breath-hold at the tail-end of the ventilation sequence. Analysis was performed to assess the multi-slice ventilation model sensitivity to noise, oxygen and number of flip angle images. The optimal α value was determined based on minimizing the error in r estimation; αopt = 5–6° for the set of acquisition parameters in pigs. The mean r values for the group of pigs were 0.27±0.09, 0.35±0.06, 0.40±0.04 for ventral, middle and dorsal slices, respectively, (excluding conductive airways r > 0.9). A positive gravitational (ventral-dorsal) ventilation gradient effect was present in all animals. The trachea and major conductive airways showed a uniform near-unity r value, with progressively smaller values corresponding to smaller diameter airways, and ultimately leading to lung parenchyma. Results demonstrate the feasibility of measurements of fractional ventilation in large species, and provides a platform to address technical challenges associated with long breathing time scales through the optimization of acquisition parameters in species with a pulmonary physiology very similar to that of human beings.
Pulmonary ventilation; Quantitative lung imaging; Fractional ventilation; Hyperpolarized gas MRI; Mechanical ventilation
Chordomas are rare tumors arising from remnants of the notochord. Because of the challenges in achieving a complete resection, the radioresistant nature of these tumors, and the lack of effective chemotherapeutics, the median survival for patients with chordomas is approximately 6 years. Reproducible preclinical model systems that closely mimic the original patient’s tumor are essential for the development and evaluation of effective therapeutics. Currently, there are only a few established chordoma cell lines and no primary xenograft model. In this study, the authors aimed to develop a primary chordoma xenograft model.
The authors implanted independent tumor samples from 2 patients into athymic nude mice. The resulting xenograft line was characterized by histopathological analysis and immunohistochemical staining. The patient’s tumor and serial passages of the xenograft were genomically analyzed using a 660,000 single-nucleotide polymorphism array.
A serially transplantable xenograft was established from one of the 2 patient samples. Histopathological analysis and immunohistochemical staining for S100 protein, epithelial membrane antigen, and cytokeratin AE1/AE3 of the primary patient sample and the xenografts confirmed that the xenografts were identical to the original chordoma obtained from the patient. Immunohistochemical staining and western blot analysis confirmed the presence of brachyury, a recently described marker of chordomas, in the tumor from the patient and each of the xenografts. Genome-wide variation was assessed between the patient’s tumor and the xenografts and was found to be more than 99.9% concordant.
To the best of their knowledge, the authors have established the first primary chordoma xenograft that will provide a useful preclinical model for this disease and a platform for therapeutic development.
animal model; brachyury; chordoma; xenograft; oncology
Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired.
In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose “analysis pipelines“ that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept.
We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.
Software; Open source; Image analysis; Object-based image analysis
Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static – bone resorptive (R) to moving – nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.
The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3-D fluorescence microscopy images. As example application we present the analysis of drug-induced and S1P1-knock-out-related changes in bone-osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data about T-B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets.
The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
Quantitative measurement of regional lung ventilation is of great significance in assessment of lung function in many obstructive and restrictive pulmonary diseases. A new technique for regional measurement of fractional ventilation using hyperpolarized 3He MRI is proposed, addressing the shortcomings of an earlier approach that limited its use to small animals. The new approach allows for the acquisition of similar quantitative maps over a shortened period and requires substantially less 3He gas. This technique is therefore a better platform for implementation in large species, including humans. The measurements using the two approaches were comparable to a great degree, as verified in a healthy rat lung, and are very reproducible. Preliminary validation is performed in a lung phantom system. Volume dependency of measurements was assessed both in vivo and in vitro. A scheme for selecting an optimum flip angle is proposed. In addition, a dead space modeling approach is proposed to yield more accurate measurements of regional fractional ventilation using either method. Finally, sensitivity of the new technique to model parameters, noise, and number of included images were assessed numerically. As a prelude to application in humans, the technique was implemented in a large animal study successfully.
quantitative pulmonary imaging; regional lung ventilation; fractional ventilation; hyperpolarized gas MRI; mathematical models of lung
RATIONALE AND OBJECTIVES
The use of hyperpolarized 3He MRI as a quantitative lung imaging tool has progressed rapidly in the past decade, mostly in assessment of the airways diseases COPD and asthma. This technique has shown potential to assess both structural and functional information in healthy and diseased lungs. In this study, we apply the regional measurements of structure and function to a bleomycin rat model of interstitial lung disease.
MATERIALS AND METHODS
Male Sprague Dawley rats (300–350 g) were administered intra-tracheal bleomycin. After 3 weeks, apparent diffusion coefficient and fractional ventilation were measured by 3He MRI and pulmonary function testing using a rodent-specific plethysmography chamber. Sensitized and healthy animals were then compared using threshold analysis to assess the potential sensitivity of these techniques to pulmonary abnormalities.
No significant changes were observed in total lung volume and compliance between the two groups. Airway resistance elevated and forced expiratory volume significantly declined in the 3-wk bleomycin rats, and fractional ventilation was significantly decreased compared to control animals (p < 4×10−4). Apparent diffusion coefficient of 3He showed a smaller change, but still a significant decrease in 3-wk bleomycin animals (p < 0.05).
Preliminary results suggest that quantitative 3He MRI can be a sensitive and non-invasive tool to assess changes in an animal interstitial lung disease model. This technique may be useful for longitudinal animal studies and also in the investigation of human interstitial lung diseases.
bleomycin; interstitial lung disease; hyperpolarized 3He MRI; idiopathic pulmonary fibrosis; interstitial lung disease
Intramolecular spin-order transfer is a useful technique for signal enhancement of insensitive and low-concentration molecular species. We present a closed-form, optimized pulse sequence which maximizes the efficiency of transfer between a singlet (para) nuclear pair and a vicinal heteronucleus. Neglecting the decay of coherences while the nuclei are in the transverse plane, the scheme is unity efficient for all combinations of internuclear scalar couplings. Efficiency loss due to T2-like decay is also minimized by keeping the sequence as short as possible. We expect this result to be useful for hyperpolarization experiments in which the spin-order originates in parahydrogen, as well as studies of singlet state decay aimed at longer-term storage of spin-order in hyperpolarized Magnetic Resonance Imaging.
Chemotaxis and chemorepulsion of osteoclast precursors depends on S1P concentrations and expression of the receptors S1PR1 and S1PR2, which act to regulate osteoclast precursor localization.
Sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, controls the dynamic migration of osteoclast (OC) precursors (OPs) between the blood and bone, in part via the S1P receptor 1 (S1PR1) which directs positive chemotaxis toward S1P. We show that OPs also express S1PR2, an S1P receptor which mediates negative chemotaxis (or chemorepulsion). OP-positive chemotaxis is prominent in gradients with low maximal concentrations of S1P, whereas such behavior is minimal in fields with high maximal S1P concentrations. This reverse-directional behavior is caused by S1PR2-mediated chemorepulsion acting to override S1PR1 upgradient motion. S1PR2-deficient mice exhibit moderate osteopetrosis as a result of a decrease in osteoclastic bone resorption, suggesting that S1PR2 contributes to OP localization on the bones mediated by chemorepulsion away from the blood where S1P levels are high. Inhibition of S1PR2 function by the antagonist JTE013 changed the migratory behavior of monocytoid cells, including OPs, and relieved osteoporosis in a mouse model by limiting OP localization and reducing the number of mature OCs attached to the bone surface. Thus, reciprocal regulation of S1P-dependent chemotaxis controls bone remodeling by finely regulating OP localization. This regulatory axis may be promising as a therapeutic target in diseases affecting OC-dependent bone remodeling.
Hyperpolarized 3He (HP 3He) MRI shows promise to assess structural and functional pulmonary parameters in a sensitive, regional and non-invasive way. Structural HP 3He MRI has applied the apparent diffusion coefficient (ADC) for the detection of disease-induced lung microstructure changes at the alveolar level, and HP 3He pulmonary partial pressure of oxygen (pO2) imaging measures the oxygen transfer efficiency between the lung and blood stream. Although both parameters are affected in chronic obstructive pulmonary disease (COPD), a quantitative assessment of the regional correlation of the two parameters has not been reported in the literature. In this work, a single acquisition technique for the simultaneous measurement of ADC and pO2 is presented. This technique is based on the multiple regression method, in which a general linear estimator is used to retrieve the values of ADC and pO2 from a series of measurements. The measurement uncertainties are also analytically derived and used to find an optimal measurement scheme. The technique was first tested on a phantom model, and then on an in-vivo normal pig experiment. A case study was performed on a COPD patient, which showed that in a region-of-interest ADC was 29% higher while oxygen depletion rate was 61% lower than the corresponding global average values.
Hyperpolarized 3He MRI; apparent diffusion coefficient; partial pressure of oxygen; multiple regression method
Improvements in the quantitative assessment of structure, function, and metabolic activity in the lung, combined with improvements in the spatial resolution of those assessments, enhance the diagnosis and evaluation of pulmonary disorders. Radiologic methods are among the most attractive techniques for the comprehensive assessment of the lung, as they allow quantitative assessment of this organ through measurements of a number of structural, functional, and metabolic parameters. Hyperpolarized nuclei magnetic resonance imaging (MRI) has opened up new territories for the quantitative assessment of lung function and structure with an unprecedented spatial resolution and sensitivity. This review article presents a survey of recent developments in the field of pulmonary imaging using hyperpolarized nuclei MRI for quantitative imaging of different aspects of the lung, as well as preclinical applications of these techniques to diagnose and evaluate specific pulmonary diseases. After presenting a brief overview of various hyperpolarization techniques, this survey divides the research activities of the field into four broad areas: lung microstructure, ventilation, oxygenation, and perfusion. Finally, it discusses the challenges currently faced by researchers in this field to translate this rich body of methodology into wider-scale clinical applications.
hyperpolarized gas MRI; hyperpolarized 13C MRI; quantitative lung imaging
To correctly estimate the camera motion parameters and reconstruct the structure of the surrounding tissues from endoscopic image sequences, we need not only to deal with outliers (e.g., mismatches), which may involve more than 50% of the data, but also to accurately distinguish inliers (correct matches) from outliers. In this paper, we propose a new robust estimator, Adaptive Scale Kernel Consensus (ASKC), which can tolerate more than 50 percent outliers while automatically estimating the scale of inliers. With ASKC, we develop a reliable feature tracking algorithm. This, in turn, allows us to develop a complete system for estimating endoscopic camera motion and reconstructing anatomical structures from endoscopic image sequences. Preliminary experiments on endoscopic sinus imagery have achieved promising results.
Endoscopic endonasal skull base surgery (ESBS) requires high accuracy to ensure safe navigation of the critical anatomy at the anterior skull base. Current navigation systems provide approximately 2mm accuracy. This level of registration error is due in part from the indirect nature of tracking used. We propose a method to directly track the position of the endoscope using video data. Our method first reconstructs image feature points from video in 3D, and then registers the reconstructed point cloud to pre-operative data (e.g. CT/MRI). After the initial registration, the system tracks image features and maintains the 2D–3D correspondence of image features and 3D locations. These data are then used to update the current camera pose. We present registration results within 1mm, which matches the accuracy of our validation framework.
Recent advances in computational image processing have made it possible to reconstruct camera motion and scene geometry from a series of monocular images. By applying these methods to endoscopic image sequences, it is possible to create detailed, quantitative anatomic reconstructions. Such anatomic reconstructions have many potential clinical uses. Our objectives in this study are to (1) develop a process flow for reconstruction from endoscopic image sequences and (2) present results supporting the hypothesis that such reconstructions can be computed.
We first outline the overall process flow for endoscopic reconstruction. Then, we present an instantiation of this process flow using recently developed methods in computational vision. We apply these methods to cadaverous specimens for which ground truth endoscopic motion is known.
We are able to produce consistent estimates of endoscopic motion and dense reconstructions of the surrounding anatomy for >65% of 1373 image pairs.
Our study indicates that processing endoscopic images to produce anatomic structure is feasible. Such reconstructions have high potential clinical value for intraoperative navigation, diagnosis, and treatment planning.
Anatomic reconstruction; computer-integrated surgery; computer vision; endoscopy; image processing; navigation; robust statistics; surgery; visual modeling
Osteoclasts (OCs) are bone-resorbing multinuclear giant cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Although many molecules are known to contribute to OC differentiation, RANKL chief among them, the mechanisms controlling the recruitment and homing of OC precursors (OPs) to the bone surface have not been elucidated. Here we report that sphingosine-1-phosphate (S1P) controls the movement of OPs between the blood and their site of differentiation. Cells with the properties of OPs express functional S1P1 receptors and exhibit positive chemotaxis along an S1P gradient in vitro. Intravital two-photon imaging of bone tissues revealed that a potent S1P1 agonist, SEW2871, stimulated motility of OP-containing monocytoid populations in vivo. OC/monocyte (CD11b) lineage-specific conditional S1P1 knockout mice showed osteoporotic changes due to increased OC attachment to bone surface, suggesting a crucial role of the S1P-S1P1 system in recirculation of OPs to blood where S1P levels are high. Furthermore, treatment with the S1P1 agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by facilitating recirculation of OP-containing cell populations and reducing the number of mature OCs attached to the bone surface. This study provides evidence that S1P controls the migratory behavior of OPs, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may be promising as a therapeutic target.
Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.
Rationale and Objectives
Estimation of regional lung function parameters from hyperpolarized gas magnetic resonance images can be very sensitive to presence of noise. Clustering pixels and averaging over the resulting groups is an effective method for reducing the effects of noise in these images, commonly performed by grouping proximal pixels together, thus creating large groups called bins. This method has several drawbacks, primarily that it can group dissimilar pixels together, and it degrades spatial resolution. This study presents an improved approach to simplifying data via principal component analysis (PCA) when noise level prohibits a pixel-by-pixel treatment of data, by clustering them based on similarity to one another rather than spatial proximity. The application to this technique is demonstrated in measurements of regional lung oxygen tension using hyperpolarized 3He MRI.
Materials and Methods
A synthetic data set was generated from an experimental set of oxygen tension measurements by treating the experimentally-derived parameters as “true” values, and then solving backwards to generate “noiseless” images. Artificial noise was added to the synthetic data, and both traditional binning and PCA-based clustering were performed. For both methods, the RMS error between each pixel’s “estimated” and “true” parameters was computed and the resulting effects were compared.
At high signal-to-noise ratios, clustering does not enhance accuracy. Clustering does however improve parameter estimations for moderate SNR values (below 100). For SNR values between 100 and 20, the PCA-based K-means clustering analysis yields greater accuracy than Cartesian binning. In extreme cases (SNR < 5). Cartesian binning can be more accurate.
The reliability of parameters estimation in imaging-based regional functional measurements can be improved in presence of noise by utilizing principal component analysis-based clustering without sacrificing spatial resoltuin as compared to Cartesian binning. Results suggest that this approach has a great potential for robust grouping of pixels in hyperpolarized 3He MRI maps of lung oxygen tension.
Functional lung imaging; principal component analysis; hyperpolarized 3He MRI; parameter estimation
Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K+ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant.
Animals display a variety of skin pigment patterns. How these often intricate patterns are formed, however, is the longstanding question. Zebrafish is the only model organism having a pigment pattern, and thus it provides a unique system in which to investigate the mechanism of pattern formation. The striped pigment pattern of zebrafish comprises two types of pigment cells, melanophores (black chromatophores) and xanthophores (yellow chromatophores), and defects in pigment cell differentiation cause abnormal pigment patterns. However, the mechanism(s) underlying the arrangement of pigmented cells during development is unclear. In this paper, the authors cloned and studied the zebrafish mutant gene jaguar/obelix and identified it as inwardly rectifying potassium channel 7.1 (Kir7.1). Although the development of pigment cells is normal in jaguar/obelix fish, they have abnormally wide body stripes; thus, cell positioning is altered, suggesting that the jaguar/obelix functions in the system that determines pigment patterning. The connection between the Kir7.1 channel and the pigment pattern remains unclear, but the mutant melanophores are defective in intracellular aggregation and dispersion of the melanosome (pigment) controlled by the sympathetic neuron, suggesting that the signaling pathway activated by the neuron is also related to pigment pattern formation.