Name: a multimodal imaging approach based on micro-ct and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice
Uploaded: 2018-04-13T12:30:00
Description: 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

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 viva...

<ol>
	<li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+mechanisms+of+pulmonary+fibrosis.">Integrating mechanisms of pulmonary fibrosis.</a> J. Exp. Med. 208 (7), 1339-1350 (2011).</li><li>Wynn, T. A., Ramalingam, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fibrosis:+therapeutic+translation+for+fibrotic+disease.">Mechanisms of fibrosis: therapeutic translation for fibrotic disease.</a> Nat. Med. 18 (7), 1028-1040 (2012).</li><li>Moore, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+fibrotic+lung+disease.">Animal models of fibrotic lung disease.</a> Am J Respir Cell Mol Biol. 49 (2), 167-179 (2013).</li><li>Ackermann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nintedanib+on+the+microvascular+architecture+in+a+lung+fibrosis+model.">Effects of nintedanib on the microvascular architecture in a lung fibrosis model.</a> Angiogenesis. , (2017).</li><li>Moore, B. B., Hogaboam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+pulmonary+fibrosis.">Murine models of pulmonary fibrosis.</a> Am J Physiol Lung Cell Mol Physiol. 294 (2), 152-160 (2008).</li><li>Organ, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+segmental+challenge+model+for+bleomycin-induced+pulmonary+fibrosis+in+sheep.">A novel segmental challenge model for bleomycin-induced pulmonary fibrosis in sheep.</a> Exp Lung Res. 41 (3), 115-134 (2015).</li><li>Organ, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+correlations+in+a+large+animal+model+of+bleomycin-induced+pulmonary+fibrosis.">Structural and functional correlations in a large animal model of bleomycin-induced pulmonary fibrosis.</a> BMC Pulm Med. 15, 81 (2015).</li><li>Shen, B., Du, L., Sanchez, C., Edwards, D. J., Chen, M., Murrell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+the+bleomycin+biosynthetic+gene+cluster+from+Streptomyces+verticillus+ATCC15003.">Cloning and characterization of the bleomycin biosynthetic gene cluster from Streptomyces verticillus ATCC15003.</a> J Nat Prod. 65 (3), 422-431 (2002).</li><li>Yu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Delivery+of+Bleomycin:+A+Comprehensive+Anticancer+Review.">Targeted Delivery of Bleomycin: A Comprehensive Anticancer Review.</a> Curr Cancer Drug Targets. 16 (6), 509-521 (2016).</li><li>Ashcroft, T., Simpson, J. M., Timbrell, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+method+of+estimating+severity+of+pulmonary+fibrosis+on+a+numerical+scale.">Simple method of estimating severity of pulmonary fibrosis on a numerical scale.</a> J Clin Pathol. 41 (4), 467-470 (1988).</li><li>Stellari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+lung+inflammatory+response+to+Pseudomonas+aeruginosa+and+its+modulation+by+azithromycin.">In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.</a> J Transl Med. 13, 251 (2015).</li><li>Stellari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+monitoring+of+lung+inflammation+in+CFTR-deficient+mice.">In vivo monitoring of lung inflammation in CFTR-deficient mice.</a> J Transl Med. 14 (1), 226 (2016).</li><li>Stellari, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transiently+transgenized+mice+with+a+bovine+interleukin+8+(CXCL8)+promoter/luciferase+reporter+construct.">In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct.</a> PloS one. 7 (6), 39716 (2012).</li><li>Stellari, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlightened+Mannhemia+haemolytica+lung+inflammation+in+bovinized+mice.">Enlightened Mannhemia haemolytica lung inflammation in bovinized mice.</a> Vet Res. 45, 8 (2014).</li><li>Tassali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+imaging,+targeting+and+characterization+of+pulmonary+fibrosis+using+intra-tracheal+administration+of+gadolinium-based+nanoparticles.">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><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+asthmatic+inflammation+using+hybrid+fluorescence+molecular+tomography-x-ray+computed+tomography.">Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography-x-ray computed tomography.</a> J Biomed Opt. 21 (1), 15009 (2016).</li><li>Van de Velde, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+micro-CT+provides+biomarkers+of+lung+disease+that+can+be+used+to+assess+the+effect+of+therapy+in+preclinical+mouse+models,+and+reveal+compensatory+changes+in+lung+volume.">Longitudinal micro-CT provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume.</a> Dis Model Mech. 9 (1), 91-98 (2016).</li><li>Stellari, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+Matrix+Metalloproteinase+Gene+Promoter+Activity+Allows+In+Vivo+Real-time+Imaging+of+Bleomycin-Induced+Lung+Fibrosis+in+Transiently+Transgenized+Mice.">Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice.</a> Front Immunol. 8, 199 (2017).</li><li>Hellbach, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+dark-field+radiography+facilitates+the+diagnosis+of+pulmonary+fibrosis+in+a+mouse+model.">X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model.</a> Sci Rep. 7 (1), 340 (2017).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+experimental+lung+fibrosis.">Noninvasive imaging of experimental lung fibrosis.</a> Am J Respir Cell Mol Biol. 53 (1), 8-13 (2015).</li><li>Ruscitti, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+assessment+of+bleomycin-induced+lung+fibrosis+by+Micro-CT+correlates+with+histological+evaluation+in+mice.">Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice.</a> Multidiscip Respir Med. 12, 8 (2017).</li><li>Stellari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+inflammation+and+airway+remodeling+by+fluorescence+molecular+tomography+in+a+chronic+asthma+model.">Monitoring inflammation and airway remodeling by fluorescence molecular tomography in a chronic asthma model.</a> J Transl Med. 13, 336 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals, 8th ed. , (2011).</li><li>Meganck, J. A., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosimetry+in+Micro-computed+Tomography:+a+Review+of+the+Measurement+Methods,+Impacts,+and+Characterization+of+the+Quantum+GX+Imaging+System.">Dosimetry in Micro-computed Tomography: a Review of the Measurement Methods, Impacts, and Characterization of the Quantum GX Imaging System.</a> Mol Imaging Biol. , (2016).</li><li>Hubner, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+quantification+of+pulmonary+fibrosis+in+histological+samples).">Standardized quantification of pulmonary fibrosis in histological samples).</a> BioTechniques. 44 (4), 507-514 (2008).</li><li>King, T. E., Pardo, A., Selman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+fibrosis.">Idiopathic pulmonary fibrosis.</a> Lancet. 378 (9807), 1949-1961 (2011).</li><li>De Langhe, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+lung+fibrosis+and+emphysema+in+mice+using+automated+micro-computed+tomography.">Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography.</a> PloS one. 7 (8), 43123 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:1440px;" width="1441">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">FMT 2500 Fluorescence Tomography System</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;">Experimental Builder</td>
			<td style="width:607px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MMPsense 680</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;">NEV10126</td>
			<td>Protect from light, store the probe at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">TrueQuant software</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Female inbred C57BL/6</td>
			<td>San Pietro NatisoneHorst, The Netherlands (UD),&#160;</td>
			<td style="width:171px;"></td>
			<td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Isoflurane</td>
			<td style="width:335px;">ESTEVE spa</td>
			<td style="width:171px;">571329.8</td>
			<td>Do not inhale</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated cell counter</td>
			<td style="width:335px;">Dasit XT 1800J</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline Solution, 0.9% Sodium Chloride (NaCl)</td>
			<td style="width:335px;">Eurospital</td>
			<td>15A2807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Quantum FX Micro-CT scanner&#160;</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">Bleomycin sulphate from Streptomyces Verticillus&#160;</td>
			<td style="width:335px;">Sigma</td>
			<td>&#160;B2434&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automatic tissue Processor&#160;</td>
			<td style="width:335px;">ATP700 Histo-Line Laboratories</td>
			<td>ATP700&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Embedding system&#160;</td>
			<td style="width:335px;">EG 1160 Leica Biosystems</td>
			<td>EG 1160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rotary microtome&#160;</td>
			<td style="width:335px;">Slee Cut 6062</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Digital slide scanner&#160;</td>
			<td style="width:335px;">NanoZoomer S60, Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">NIS-AR image analysis software&#160;</td>
			<td style="width:335px;">Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Masson&#8217;s Trichrome Staining</td>
			<td style="width:335px;">Histo-Line Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10% neutral-buffered formalin</td>
			<td style="width:335px;">Sigma</td>
			<td>HT5012-1CS</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:328px;">Penn-century model DP-4M Dry power insufflator</td>
			<td style="width:335px;">Penn-century</td>
			<td>DPM-EXT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PE190 micro medical tubing</td>
			<td style="width:335px;">2biological instruments snc</td>
			<td style="width:171px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Syringe without needle 5 mL</td>
			<td style="width:335px;">Terumo</td>
			<td style="width:171px;">SS*05SE1</td>
			<td>Cut the boards of the piston by scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Hamilton 0.10 mL (model 1710)</td>
			<td style="width:335px;">Gastight</td>
			<td>81022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Discofix 3-way Stopcock</td>
			<td style="width:335px;">Braun</td>
			<td>4095111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Syringe with needle 1 mL</td>
			<td style="width:335px;">Pic solution</td>
			<td>3,071,260,300,320</td>
			<td>Use without needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Plastic feeding tubes 18 ga x 50 mm</td>
			<td style="width:335px;">2biological instruments snc</td>
			<td>FTP-18-50</td>
			<td>Cut obliquely the tip&#160;</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 ...

The overall goal of this study on an IPF-like mouse model is to longitudinally monitor lung fibrosis with non-invasive imaging techniques such as FMT and micro-CT. This method can advance what sequence in regarding this specific molecular events occurring in lung fibrosis. The main advantage of this techniques is that anatomical changes of specific molecular events can be monitored noninvasively.The applications of this technologies extend towards therapy of IPF pathology because the fusion of FMT and micro-CT represents the possibility to study disease progression and new therapy evaluation. Start by positioning an anesthetized mouse on the intubation platform hanging it by its incisors placed on the wire. Turn on the laryngoscope, then take a pair of blunt-ended forceps and gently open the mouth.Place the laryngoscope blade towards the back of the mouth until the opening of the trachea is visualized. Use the other hand to insert the delivery tube connected to the end of the PE tubing into the trachea. Rotate the three-way valve to deliver 50 microliters of bleomycin.After installation quickly remove the tube from the trachea to prevent suffocation. Hold the mice upright for a few seconds. For probe injection take the mouse by the tail and allow it to grip the cage lid.Then grasp across the shoulders and hold the mouse by the scruff. Immerse the tail in a beaker of warm water to cause the veins to swell for easier visualization and needle insertion. Find the two caudal veins on either side of the tail, not the artery on the underside of the tail.Insert the 26 gauged needle of the MMP loaded 1 milliliter syringe into the vein and inject the MMP probe solution at 10 milliliters per kilogram of body weight. To begin initialize the date acquisition software and open a new study in the database for the experiment. Before starting the imaging, transfer the shaved and anesthetized mouse to the middle of imaging cassette.Keep the mouse flat, secure, and gently compressed against both windows of the imaging cassette. Then in the scanning window of the software, click on Subject"and then preview to see the live image. Draw the scan region sufficiently large so that the tissues surrounding the anticipated area of fluorescence is captured.Once the ROY is drawn, click Add to Reconstruction Queue"and then click Scan"to image the mouse. When the image acquisition is complete, remove the mouse from the imaging cassette and place back into the home cage. Quantify the picomoles of fluorescence using the ROY tool and reduce the ROY around 700 to 800 cubed millimeters on the signal coming from lung region.Repeat imaging every seven days or as required. To image by micro-CT, first turn on the micro-CT by pressing the green power button and launch the software to warm up the x-ray source. Create the database by clicking on New Database"and build the browser based on the number of mice in the experiment.Before starting the scan, select acquisition parameters in the software control window as follows. Set x-ray tube voltage to 90 kilovolts, the CT x-ray tube current to 160 micro angstroms, live x-ray tube current to 80 micro angstroms, and the field of view to 36 millimeters. Select no Gating Technique"and High Resolution"and four minutes for scan technique.After anesthetizing the mouse by inhalation of 3%isoflurane, place it onto the animal bed inserted into the small bore with a nose cone providing a constant supply of anesthetic. Immobilize the paws with tape on the bed to allow the chest to be exposed. Next side the instrument door closed and turn on live mode to see the mouse position in real time by pressing the capture button.Move the animal bed to align the chest with field of view using the buttons on the CT instrument. Once the mouse is centered, rotate the gantry to confirm optimal bed position by selecting 90 degrees and clicking Set"Then click the CT scan button. Click Yes"to the message that informs that the x-ray source will be turned on.After imaging place the animals back into the cage and monitor until fully recovered from anesthesia. Repeat imaging every seven days or as required. Staining with Masson's trichrome reveals the morphology in the lung tissue.Pictures of the bleomycin treated group showed a pronounced pattern of fibrosis starting from day seven mainly as single fibrotic masses and progressed at day 14 to confluent conglomerates of substituted collagen and remained unaltered until day 21. Repeated micro-CT imaging allowed for quantification and segmentation of airways in fibrosis lung lobes. The airways were divided into a central and distal part.The distal part of the airways is the intrapulmonary tract and splits into the lung lobes as the right cranial lobe, right middle lobe, right caudal lobe, right accessory lobe, and left lung. Quantification of airway radius showed increased radius in the bleomycin treated mice compared to saline controls. Each point represents the mean standard error of the mean of five animals for a total of 30 mice.Total lung fibrosis quantification of vehicle or bleomycin-treated mice at different time points demonstrates that these micro-CT parameters agree with histological findings. Once mastered this technique can be performed for drug screening routinely. After its development this technique paved the way for researcher in the field of lung fibrosis disease to explore efficacies of the repeating treatment in IPF-like animal models.

Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

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 ...