The authors would like to thank Dr. Daniela Pompilio and Roberta Ciccimarra for technical help.

All animal experiments described herein were approved by the intramural animal-welfare committee for animal experimentation of Chiesi Farmaceutici and ERASMUS MC under protocol number: EMC 3349 (138-14-07) complying with the European Directive 2010/63 UE, Italian D.Lgs 26/2014 and the revised &#34;Guide for the Care and Use of Laboratory Animals&#34;23.
NOTE: Prior to use, female inbred C57Bl/6 (7-8 weeks old) mice were acclimatized for at least 7 days to the local vivarium conditions (room temperature: 20-24 &#176;C; relative humidity: 40-70%; 12-h light-dark cycle), having free access to standard rodent chow and softened tap water.
1. Intratracheal Treatment of Mice with Bleomycin
<ol>
	<li>Prepare the equipment for the intra-tracheal instillation12,13,14.</li>
	<li>Put the mice in the anesthetic chamber connected to an isoflurane vaporizer set at 2.5% mixed with oxygen. Check for depth of anesthesia by the lack of a toe-pinch response. The effect of the anesthesia occurred after 3-5 min.</li>
	<li>Position the anesthetized mouse on the intubation platform, hanging it by its incisors placed on the wire.</li>
	<li>Turn on the laryngoscope, take a pair of blunt ended forceps and use either the forceps or laryngoscope&#39;s tip to gently open the mouth.</li>
	<li>Pull out the tongue and hold it to the side with forceps. Place the laryngoscope blade towards the back of the mouth until the opening of the trachea is visualized and keep the laryngoscope in place.</li>
	<li>With the other hand, insert the delivery tube connected to the end of the PE tubing into the trachea, rotating the three-way valve. Deliver 50 &#181;L of bleomycin (0.020 &#181;g/mouse). After instillation, quickly remove the tube from the trachea to prevent suffocation. Hold the mice upright for a few seconds.
	<ol>
		<li>Administer bleomycin intratracheally at day 0 and repeat at day 4 (Figure 1).</li>
	</ol>
	</li>
	<li>Remove the mouse from the intubation platform and monitor the mouse for 30 min (the time needed to fully recover from anesthesia).</li>
	<li>Perform in vivo imaging of the mice lungs by FMT and Micro-CT after double bleomycin instillation at day 7, 14, and 21.</li>
</ol>
2. In vivo Imaging by Fluorescence Molecular Tomography
NOTE: Beforehand, prepare a fresh stock solution of 6 nmol/mL of MMP sensitive fluorescent substrate 680 in saline solution (0.9% sodium chloride), and store protected from the light at 4 &#176;C before use. It is stable for up to 6 months at 4 &#176;C. Allow MMP imaging agent to equilibrate to room temperature before injecting into animals.
<ol>
	<li>Probe injection
	<ol>
		<li>Prepare the equipment for intravenous injection of the MMP probe solution. Pre-warm water at 50 &#176;C to heat the mouse tail.</li>
		<li>To firmly grasp the mouse scruff, take it by the tail and allow it to grip the cage lid. Holding the mice across the animal&#39;s shoulders, put the tail in a warm water beaker, for 4 to maximum 8 s thermal injury to prevent thermal injury to the tail skin.&#160; Check the veins swelling for easier visualization and needle insertion.</li>
		<li>During the injection procedure, put the mice into a plastic restrainer to keep it steady and pull the tail through a special hole in the back. Find the two caudal veins on either side of the tail; not the artery on the underside of the tail.</li>
		<li>Insert the needle (26G) of the 1 mL syringe into the vein. If the needle is correctly placed, injection will be easy and the probe solution will flow into the vessel.</li>
		<li>Inject the MMP probe solution at 10 mL/kg.</li>
		<li>Discard the needle. 
		NOTE: The optimal imaging time point is 24 h after the MMP probe injection because it is the time required by the probe to be activated. If longitudinal studies are performed, the optimal re-injection time is 7 days, which allows for the complete clearance of the agent from the mouse.</li>
	</ol>
	</li>
	<li>FMT imaging 
	NOTE: Before starting, always remove the fur on and around the areas of the animal that are to be imaged, in this case the chest, to avoid scattering and absorbance in tissue.
	<ol>
		<li>To anesthetize the mouse, put it in anesthetic chamber and turn the isoflurane dial to 2.5% for induction and 2% for maintenance.</li>
		<li>Initialize the data acquisition software and open a new study in the database for the experiment.</li>
		<li>Before starting the imaging with FMT, transfer the mouse into the imaging cassette. Place the anesthetized mouse in the middle of imaging cassette to optimally use the field of view of the FMT. Keep the mice flat, secure and gently compressed against both windows of the imaging cassette. Adjust the height by knobs on the cassette, and then slide it into the docking station.</li>
		<li>Click on subject in the scanning window and click preview to see the live image.</li>
		<li>Draw the scan region sufficiently large so that tissue surrounding the anticipated area of fluorescence is captured. Including a total of 25 scan points guarantees the lung region will be totally scanned.</li>
		<li>Once the ROI is drawn, first click add to reconstruction queue and then click Scan to image the mouse. Make sure that the correct laser at 680 nm is selected.</li>
		<li>When the image acquisition is completed, place the mouse back into its cage. Be sure it fully recovers from anesthesia.</li>
		<li>Quantify the picomoles of fluorescence in the Analysis window of the analysis software using the ROI tool and reduce the ROI around 700-800 mm3 on the signal coming from the lung region. Be careful to exclude the liver signal. Copy the ROI on the other subjects to have them similar in dimension and adjust their position in each animal image.</li>
		<li>Export images as jpg files.</li>
	</ol>
	</li>
</ol>
3. In Vivo Imaging by Micro-CT
CAUTION: Before starting, always remove any metal jewelry or metal objects near the imaging area, to avoid scattering of x-rays.
NOTE: Radiation-induced lung fibrosis is a common finding during radiation induced lung injury24. Neither Micro-CT derived indices nor histological findings associated with lung fibrosis were present in saline treated control mice on day 21 subjected to four Micro-CT imaging sessions, indicating that the X-ray dose delivered to the animals during Micro-CT examinations was not sufficient to affect the findings.
<ol>
	<li>Turn on the Micro-CT by pressing the green power button and launch the software to warm up the x-ray source. Use the small bore and the animal bed for mice imaging.</li>
	<li>Create the database by clicking on New database for a new one and build the browser based on number of mice in the experiment, or click connecting to existing database to save the data to a previously created one.</li>
	<li>Before starting the scanning, select the acquisition parameters in the software control window: X-ray tube voltage, 90 kV; CT X-ray tube current, 160 &#181;A; Live X-ray tube current, 80 &#181;A; FOV, 36 mm; No gating technique; Scan technique, High resolution 4 min.</li>
	<li>Anesthetize the mice by inhalation of 3% isoflurane and place them on the bed inserted into the bore with a nose cone providing a constant supply of anesthetic. Immobilize the paws of the mice with tape on the bed to allow the chest to be exposed.</li>
	<li>Slide the instrument door and turn on the Live Mode to see the mouse position in real time by pressing the capture button. Move the animal bed to align the chest with the field of view (FOV) using buttons directly located on the CT instrument. Center the scan on the mouse; if not adjust the position with left and right arrows located on the CT instrument.</li>
	<li>Confirm optimal bed position by rotating the gantry by selecting 90&#176; and clicking Set. Make sure that the region to image is fully inside the FOV.</li>
	<li>Do not apply any gating technique and start the scan by clicking the CT Scan button. Click yes to the message that informs that the x-ray source will be turned on. 
	NOTE: Once the x-rays are turned on, the orange lamp on top of the instrument will be illuminated and the sliding door will be impossible to open for the safety of the operator. Once the scan is finished, a new window of the 2D Viewer software appears showing the transaxial, coronal, and sagittal slice of the reconstruction.</li>
	<li>Check the image quality and be sure not to have blurred images from movements due to the low level of anesthesia. If necessary, repeat the scan.</li>
	<li>Place the animals back into the cage and be sure they fully recover from anesthesia.</li>
</ol>
4. Bronchoalveolar Lavage
<ol>
	<li>Anesthetize animals with 3% isoflurane and sacrifice by bleeding from the abdominal aorta.</li>
	<li>Use scissors to expose the thoracic cage and neck. Then expose the trachea and make a small incision to allow the BAL procedure to be performed with a 21-gauge lavage tube attached to a 1 mL syringe without the needle. Pay attention not to cut through the trachea.</li>
	<li>To perform the BAL, fill the 1 mL syringe without a needle with 0.6 mL of sterile solution [10x Hank&#39;s balanced salt solution (HBSS); 100 mM ethylenediaminetetraacetic acid (EDTA); 1 mM 4-(2-hydroxy-ethyl)-1-piperazineethansulphonic acid (HEPES); distilled water].</li>
	<li>Insert the lavage tube into the incision in the trachea and slowly inject and withdraw the solution 3 times, pausing on the third withdrawal to allow optimum collection of the BALF for subsequent analysis.</li>
</ol>
5. Histology and Histomorphometry
<ol>
	<li>Expose and remove lungs, inflating them with a cannula through the trachea by gentle infusion with 0.6 mL of 10% neutral-buffered formalin and store the samples at room temperature for 24 h.</li>
	<li>Dehydrate samples through different passages in increasing concentrations of alcohol solutions (60% ethanol for 1 h, 70% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 2 h, and 100% ethanol for 2 h) using an automatic tissue processor.</li>
	<li>Place specimens in xylene for 2 h to make them translucent. At the end of dehydration, infiltrate samples in paraffin at 60 &#176;C for 3 h and embed them in the automated processor.</li>
	<li>Obtain 5 &#181;m thick serial sections using a rotary microtome.</li>
	<li>Deparaffinize and rehydrate slides in descending grades of ethanol and stain with Masson&#39;s Trichrome using an automatic tissue processor.</li>
	<li>Manually focus on lung slices, scanning at 20X magnification using a slide scanner and capture digital images of entire lung sections using the viewing software with a resolution of 451 nm/pixel.</li>
	<li>Morphologically assess fibrotic lung injury by semi-quantitative and quantitative parameters as follows:
	<ol>
		<li>Determine the Ashcroft score. Semi-quantitatively grade morphological changes in lung sections according to the scale defined by Ashcroft10 modified by H&#252;bner et al.25 Use the system of 0-8 score to assess all the parenchyma in the lung sections.</li>
		<li>Determine the Collagen content. Quantify the degree of fibrosis10,25 by the microscope image analysis software. Randomly select three ROIs at 10X magnification, per each slide. By standardization of color threshold settings, detect the collagen as green-stained area.</li>
		<li>Determine the Alveolar Air Area. Quantify the alveolar air area as an indirect parameter of fibrosis10,25. By means of the same software and ROIs applied for the fibrosis fraction, detect the air area within the ROIs using a white threshold.</li>
	</ol>
	</li>
</ol>

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<li>Tassali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MR+imaging%2C+targeting+and+characterization+of+pulmonary+fibrosis+using+intra-tracheal+administration+of+gadolinium-based+nanoparticles%5BTitle%5D)+AND+%22Contrast+Media+Mol+Imaging%22%5BJournal%5D)">MR imaging, targeting and characterization of pulmonary fibrosis using intra-tracheal administration of gadolinium-based nanoparticles.</a> Contrast Media Mol Imaging. 11 (5), 396-404 (2016).</li>
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The authors declare that they have no competing interests.

Despite many research groups focusing on developing new drugs to treat IPF, at the moment only two are available for patients. There is an urgent medical need to find more effective therapies7 because only lung transplantationis able to prolong survival of 4-5 years26. The essential prerequisite for translational medicine and development of new drugs is the availability of an animal model that mimics the features of IPF and in which interventional studies are predictive of ...

Idiopathic pulmonary fibrosis (IPF) is chronic lung disease with progressive decrease of lung functions that is unfortunately often fatal within four years of diagnosis1. The major features of IPF are extracellular matrix deposition and fibroblast proliferation, but the pathogenesis is not yet fully understood. The most supported hypothesis is that multiple cycles of lung injuries cause the destruction of alveolar epithelial cells that leads to alteration of the mesenchymal cell cycle proliferation, exaggerated accumulation of fibroblasts and myofibroblasts, and increased matrix production. Mediators involved in these processes such as matrix metalloproteinases (MMPs) have been found dysregulated in fibrosis development either in human IPF or in bleomycin-induced animal models. The uncontrolled MMP production leads to an unbalanced collagen deposition within the lung interstitium and alveolar space, mimicking abnormal wound repair1,2.
One of the main obstacles for drug discovery and development is the availability of accessible mouse models that mimic human pathogenesis and the disease phenotype. Different agents have been used to induce lung fibrosis in animal models: irradiation damage, administration of asbestos and silica, administration of fibrinogenic cytokines and bleomycin3,4; however bleomycin is the most used in mice, rats, guinea pigs, hamsters, rabbits5 or in large animals (non-human primates, horses, dogs and ruminants)6,7. Bleomycin is an antibiotic made by the bacterium Streptomyces verticillus8 and is used as an anti-cancer agent9. Pulmonary fibrosis is a common side effect of the drug and for this reason, it is used in experimental animal models to induce pulmonary fibrosis.
In bleomycin-induced lung fibrosis models, the fibrotic lesions occur 14 - 21 days after bleomycin administration. In the presented work, we used a new protocol to induce lung fibrosis in mice by double bleomycin intratracheal instillation. The bleomycin mouse model is very time consuming because new drugs need to be evaluated on established fibrotic lesions, and tested to distinguish their anti-fibrotic effects from anti-inflammatory effects.
Biochemical determination of collagen content, morphometrical and histological analysis were based on post mortem analysis, limiting the possibility to follow the pathogenesis of the disease in the same animal. Although these parameters were considered a gold standard for fibrosis evaluation, they did not provide any temporal or spatial distribution of the fibrotic lesion and preclude a way to investigate the process of disease progression.10
Recently, non-invasive imaging technologies have been applied to monitor airway remodeling, inflammation, and fibrosis progression in murine models: Magnetic Resonance Imaging (MRI), Micro Computer Tomography (Micro-CT), Fluorescence Molecular Tomography (FMT) and Bioluminescent (BLI)11,12,13,14,15,16,17,18,19,20,21. We propose a non-invasive imaging approach to monitor longitudinally lung fibrosis progression by FMT and Micro-CT at different time-points after a bleomycin challenge22.
Many pathways are involved in the establishment and development of fibrosis, and not much is known. Only a deeper understanding of these processes could translate to more drug targets that may transfer into the clinic. The ability to longitudinally monitor MMP activation by fluorescence molecular tomography coupled to the detection of lung parenchymal changes by Micro-CT might be used in the future to access the clinical response to treatment.

<table><tbody><tr><td>FMT 2500 Fluorescence Tomography System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>MMPsense 680</td><td>Perkin Elmer Inc.</td><td>NEV10126</td><td>Protect from light, store the probe at 4 &amp;deg;C</td></tr><tr><td>TrueQuant software</td><td>Perkin Elmer Inc.</td><td></td><td></td></tr><tr><td>Female inbred C57BL/6</td><td>San Pietro NatisoneHorst, The Netherlands (UD),&amp;nbsp;</td><td></td><td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td></tr><tr><td>Isoflurane</td><td>ESTEVE spa</td><td>571329.8</td><td>Do not inhale</td></tr><tr><td>Automated cell counter</td><td>Dasit XT 1800J</td><td>Experimental Builder</td><td></td></tr><tr><td>Saline Solution, 0.9% Sodium Chloride (NaCl)</td><td>Eurospital</td><td>15A2807</td><td></td></tr><tr><td>Quantum FX Micro-CT scanner&amp;nbsp;</td><td>Perkin Elmer Inc.</td><td></td><td></td></tr><tr><td>Bleomycin sulphate from Streptomyces Verticillus&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;B2434&amp;nbsp;</td><td></td></tr><tr><td>Automatic tissue Processor&amp;nbsp;</td><td>ATP700 Histo-Line Laboratories</td><td>ATP700&amp;nbsp;</td><td></td></tr><tr><td>Embedding system&amp;nbsp;</td><td>EG 1160 Leica Biosystems</td><td>EG 1160</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Slee Cut 6062</td><td></td><td></td></tr><tr><td>Digital slide scanner&amp;nbsp;</td><td>NanoZoomer S60, Hamamatsu Photonics</td><td></td><td></td></tr><tr><td>NIS-AR image analysis software&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Masson&amp;rsquo;s Trichrome Staining</td><td>Histo-Line Laboratories</td><td></td><td></td></tr><tr><td>10% neutral-buffered formalin</td><td>Sigma</td><td>HT5012-1CS</td><td></td></tr><tr><td>Penn-century model DP-4M Dry power insufflator</td><td>Penn-century</td><td>DPM-EXT</td><td></td></tr><tr><td>PE190 micro medical tubing</td><td>2biological instruments snc</td><td>BB31695-PE/8</td><td></td></tr><tr><td>Syringe without needle 5 mL</td><td>Terumo</td><td>SS*05SE1</td><td>Cut the boards of the piston by scissors</td></tr><tr><td>Hamilton 0.10 mL (model 1710)</td><td>Gastight</td><td>81022</td><td></td></tr><tr><td>Discofix 3-way Stopcock</td><td>Braun</td><td>4095111</td><td></td></tr><tr><td>Syringe with needle 1 mL</td><td>Pic solution</td><td>3,071,260,300,320</td><td>Use without needle</td></tr><tr><td>Plastic feeding tubes 18 ga x 50 mm</td><td>2biological instruments snc</td><td>FTP-18-50</td><td>Cut obliquely the tip&amp;nbsp;</td></tr></tbody></table>

a multimodal imaging approach based on micro-ct and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

Spontaneous resolution of the lung fibrosis lesions observed three weeks after single bleomycin administration and moderate structural changes highlight the limits of this model. Only preventive treatment could be performed due to the narrow therapeutic window that does not represent clinical practice17.
Here, we demonstrate that our protocol of double bleomycin intratracheal instillation is able to devel...

Watch this Scientific Journal Video about A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice a

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

a-multimodal-imaging-approach-based-on-micro-ct-fluorescence

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Chiesi Farmaceutici S.p.A.. Beschrijven we een niet-invasieve multimodale aanpak op basis van Micro-CT en fluorescentie moleculaire tomografie voor longitudinale beoordelingvan de muismodel van lung fibrosis geïnduceerd door dubbele intratracheale instillatietest van bleomycine imaging.

Video: A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicine

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Cancer Research

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Bioengineering

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

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