What can imaging tell us about physiology? Lung growth and regional mechanical strain

The interplay of mechanical forces transduces diverse physico-biochemical processes to influence lung morphogenesis, growth, maturation, remodeling and repair. Because tissue stress is difficult to measure in vivo, mechano-sensitive responses are commonly inferred from global changes in lung volume, shape, or compliance and correlated with structural changes in tissue blocks sampled from postmortem-fixed lungs. Recent advances in noninvasive volumetric imaging technology, nonrigid image registration, and deformation analysis provide valuable tools for the quantitative analysis of in vivo regional anatomy and air and tissue-blood distributions and when combined with transpulmonary pressure measurements, allow characterization of regional mechanical function, e.g., displacement, strain, shear, within and among intact lobes, as well as between the lung and the components of its container-rib cage, diaphragm, and mediastinum-thereby yielding new insights into the inter-related metrics of mechanical stress-strain and growth/remodeling. Here, we review the state-of-the-art imaging applications for mapping asymmetric heterogeneous physical interactions within the thorax and how these interactions permit as well as constrain lung growth, remodeling, and compensation during development and following pneumonectomy to illustrate how advanced imaging could facilitate the understanding of physiology and pathophysiology. Functional imaging promises to facilitate the formulation of realistic computational models of lung growth that integrate mechano-sensitive events over multiple spatial and temporal scales to accurately describe in vivo physiology and pathophysiology. Improved computational models in turn could enhance our ability to predict regional as well as global responses to experimental and therapeutic interventions.     

Detection of Time-Varying Structures by Large Deformation Diffeomorphic Metric Mapping to Aid Reading of High-Resolution CT Images of the Lung

Objectives

To evaluate the accuracy of advanced non-linear registration of serial lung Computed Tomography (CT) images using Large Deformation Diffeomorphic Metric Mapping (LDDMM).

Methods

Fifteen cases of lung cancer with serial lung CT images (interval: 62.2±26.9 days) were used. After affine transformation, three dimensional, non-linear volume registration was conducted using LDDMM with or without cascading elasticity control. Registration accuracy was evaluated by measuring the displacement of landmarks placed on vessel bifurcations for each lung segment. Subtraction images and Jacobian color maps, calculated from the transformation matrix derived from image warping, were generated, which were used to evaluate time-course changes of the tumors.

Results

The average displacement of landmarks was 0.02±0.16 mm and 0.12±0.60 mm for proximal and distal landmarks after LDDMM transformation with cascading elasticity control, which was significantly smaller than 3.11±2.47 mm and 3.99±3.05 mm, respectively, after affine transformation. Emerged or vanished nodules were visualized on subtraction images, and enlarging or shrinking nodules were displayed on Jacobian maps enabled by highly accurate registration of the nodules using LDDMM. However, some residual misalignments were observed, even with non-linear transformation when substantial changes existed between the image pairs.

Conclusions

LDDMM provides accurate registration of serial lung CT images, and temporal subtraction images with Jacobian maps help radiologists to find changes in pulmonary nodules.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

Long-Term In Vivo Imaging of Multiple Organs at the Single Cell Level

Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.     

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.     

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. Download video file.^{(57M, mp4)}     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5-7 h, and a typical in vivo imaging session takes 1-2 h.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases

Summary Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-term culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1m or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellular changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)     

Multimodality registration without a dedicated multimodality scanner

Multimodality scanners that allow the acquisition of both functional and structural image sets on a single system have recently become available for animal research use. Although the resultant registered functional/structural image sets can greatly enhance the interpretability of the functional data, the cost of multimodality systems can be prohibitive, and they are often limited to two modalities, which generally do not include magnetic resonance imaging. Using a thin plastic wrap to immobilize and fix a mouse or other small animal atop a removable bed, we are able to calculate registrations between all combinations of four different small animal imaging scanners (positron emission tomography, single-photon emission computed tomography, magnetic resonance, and computed tomography [CT]) at our disposal, effectively equivalent to a quadruple-modality scanner. A comparison of serially acquired CT images, with intervening acquisitions on other scanners, demonstrates the ability of the proposed procedures to maintain the rigidity of an anesthetized mouse during transport between scanners. Movement of the bony structures of the mouse was estimated to be 0.62 mm. Soft tissue movement was predominantly the result of the filling (or emptying) of the urinary bladder and thus largely constrained to this region. Phantom studies estimate the registration errors for all registration types to be less than 0.5 mm. Functional images using tracers targeted to known structures verify the accuracy of the functional to structural registrations. The procedures are easy to perform and produce robust and accurate results that rival those of dedicated multimodality scanners, but with more flexible registration combinations and while avoiding the expense and redundancy of multimodality systems.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases.

Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-,erm culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1 M or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellualr changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions^1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens^3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network^2,6-12. Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ^12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures^11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal''s environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.     

Organ-specific cellular requirements for in vivo dendritic cell generation

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.     

High-resolution confocal imaging and three-dimensional rendering

Intrinsic opacity and inhomeogeniety of most biological tissues have prevented the efficient light penetration and signal detection for high-resolution confocal imaging of thick tissues. Here, we summarize recent technical advances in high-resolution confocal imaging for visualization of cellular structures and gene expression within intact whole-mount thick tissues. First, we introduce features of the FocusClear technology that render biological tissue transparent and thus improve the light penetration and signal detection. Next, a universal fluorescence staining method that labels all nuclei and membranes is described. We then demonstrate the postrecording image processing techniques for 3D visualization. From these images, regions of interest in the whole-mount brain can be segmented and volume rendered. Together, these technical advances in confocal microscopy allow visualization of structures within whole-mount tissues up to 1mm thick at a resolution similar to that of the observation of single cells in culture. Practical uses and limitations of these techniques are discussed.     

Human induced pluripotent stem cell–derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury

Background aims

Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD.

Cellular imaging of inflammation in atherosclerosis using magnetofluorescent nanomaterials

OBJECTIVE: Magnetofluorescent nanoparticles (MFNPs) offer the ability to image cellular inflammation in vivo. To better understand their cellular targeting and imaging capabilities in atherosclerosis, we investigated prototypical dextran-coated near-infrared fluorescent MFNPs in the apolipoprotein E-deficient (apo E-/-) mouse model. METHODS AND RESULTS: In vitro MFNP uptake was highest in activated murine macrophages (p < .001). Apo E-/- mice (n = 11) were next injected with the MFNP (15 mg/kg iron) or saline. In vivo magnetic resonance imaging (MRI) demonstrated strong plaque enhancement by the MFNPs (p < .001 vs. saline), which was confirmed by multimodality ex vivo MRI and fluorescence reflectance imaging. On fluorescence microscopy, MFNPs were found in cellular-rich areas of atheroma and colocalized with immunofluorescent macrophages over endothelial cells and smooth muscle cells (p < .001). CONCLUSIONS: Here we show that (1) the in vitro and in vivo cellular distribution of atherosclerosis-targeted MFNPs can be quantified by using fluorescence imaging methods; (2) in atherosclerosis, dextranated MFNPs preferentially target macrophages; and (3) MFNP deposition in murine atheroma can be noninvasively detected by in vivo MRI. This study thus provides a foundation for using MFNPs to image genetic and/or pharmacological perturbations of cellular inflammation in experimental atherosclerosis and for the future development of novel targeted nanomaterials for atherosclerosis.