This work was supported by the Italian Cystic Fibrosis Foundation Project FFC#18/2013, FFC#29/2015 and by the Italian Cystic Fibrosis League through the Veneto Branch&#8212;Associazione Veneta Lotta contro la Fibrosi Cistica Onlus.

All animal experiments described were approved by the intramural animal-welfare committee for animal experimentation by the Interdepartmental Centre of Experimental Research Service at the University of Verona and comply 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; Washington, D.C.: National Academy Press, 1996. This protocol and experimentation were approved by the National Institutes of Health (n 273/15). Animals had free access to standard rodent chow and softened tap water and were acclimatized for at least 5 days to the local vivarium conditions (room temperature: 20 - 24 &#176;C; relative humidity: 40 - 70%; light-dark cycle: 12 h) before any treatment.
1. In Vivo Gene Delivery
<ol>
	<li>Use a laminar flow hood to prepare the in vivo delivery reagent/nucleic acid complexes.</li>
	<li>Define the experimental protocol and parameters following the manufacturer&#39;s in vivo instructions.
	<ol>
		<li>Use a total injection volume of 200 &#181;L of complexes per mouse.</li>
		<li>Start with 40 &#181;g of DNA suspended in endotoxin-free water; the optimization range can reach 60 &#181;g. 
		NOTE: The final concentration of nucleic acid in the injection volume must not exceed 0.5 &#181;g/&#181;L, according to the manufacturer&#39;s instructions.</li>
		<li>Use an N/P ratio of 6 - 8 (0.12 - 0.16 &#181;L of delivery reagent per &#181;g of nucleic acid). Calculate the corresponding volume of delivery reagent. 
		NOTE: The complexes should be cationic for effective cell entry. The N/P ratio is defined as the number of nitrogen residues (N) on the in vivo delivery reagent per nucleic acid phosphate (P) and represents the measure of the ionic balance within the complexes.</li>
	</ol>
	</li>
	<li>Dilute the calculated amount (see step 1.2.1) of nucleic acid in 5% glucose (final concentration) using 10% glucose stock solution (provided) and sterile water. Make sure that the dilution volume is half the final injection volume. Vortex gently or mix by pipetting up and down.</li>
	<li>Dilute the calculated amount (see step 1.2.3) of delivery reagent into half the injection volume of 5% glucose (final concentration) using the 10% glucose stock solution (provided) and sterile water. Vortex gently and spin at 13,000 x g for 15 s.</li>
	<li>Add the above diluted delivery reagents to the diluted nucleic acid all at once. Mix them by gentle vortexing and spin down at 13,000 x g for 15 s.</li>
	<li>Incubate the mixture from step 1.5 for 15 min at room temperature. 
	NOTE: From this point on, the complexes are stable for 4 h at room temperature and for up to 7 days when stored at 4 &#176;C.</li>
	<li>Perform tail-vein injections using complexes equilibrated at room temperature. Place the mouse tail into warm water (50 - 53 &#176;C) for 30 s to allow for vein dilation.
	<ol>
		<li>Place the mouse inside the restraining device. Insert a 27- to 30-gauge needle in the tail vein at a 20 - 30&#176; angle and slowly inject 200 &#181;L. Upon completion, remove the needle and apply pressure to the injection site. 
		NOTE: A slight bulge in the tail during the injection indicates incorrect positioning. If this occurs, remove the needle and repeat the process proximal to the previous site.</li>
	</ol>
	</li>
	<li>Visualize gene expression by performing in vivo BLI (see step 2) at 24 and 48 h after the intravenous injection.</li>
</ol>
2. In Vivo BLI
NOTE: Beforehand, prepare a fresh stock solution of 15 mg/mL D-luciferin in DPBS, filter-sterilize it using a 0.22-&#181;m filtering unit, and store it at -20 &#176;C.
<ol>
	<li>Place the mice into a clear plexiglass anesthesia chamber. Make sure that the isoflurane chamber is full. When ready to anesthetize the animals, ensure that the pump (left) and chamber (right) switches are on. Turn the isoflurane dial to 2.5% for induction and 2% for maintenance.&#160;Animals inside the IVIS chamber are kept under 2.5% isofluorane anesthesia during image acquisition.</li>
	<li>After the mice are fully anesthetized, inject 10 mL/kg bodyweight of the D-luciferin solution by an intraperitoneal route 15 min before imaging. 
	NOTE: A kinetic study on D-luciferin should be performed to determine the time of the signal peak after the administration of D-luciferin.</li>
	<li>Open the in vivo imaging system and prepare the imaging chamber by lining it with a piece of black cardstock (discard upon completion). Place the nose cones as needed for correct anesthesia (use one cone per mouse). 
	NOTE: The tube that supplies the anesthesia to the instrument is split so that the same concentration of anesthesia is plumbed to the anesthesia manifolds located inside the imaging system.</li>
	<li>Transfer the mice (up to 5) from the box to the nose cones attached to the manifold in the imaging system and close the door.&#160;Image acquisition lasts 5 min.</li>
	<li>Acquire a BLI using the manufacturer&#39;s software, as follows.
	<ol>
		<li>Initialize the software. In the in vivo imaging system acquisition control panel, put a check mark next to Luminescent. Confirm that the Excitation Filter setting is Block and the Emission Filter setting is Open.</li>
		<li>Click the arrows: for the Luminescent Imaging Mode, select a 5-min exposure time, Binning 8, and F/Stop 1; for the Photograph Imaging Mode, select Binning 4 and F/stop 8.</li>
		<li>From the Field of View dropdown list, select D, 19 cm, and a Subject Height of 1.5 cm. Click Acquire when ready to acquire the image.</li>
	</ol>
	</li>
	<li>When the image acquisition is complete, place the mice back into their cages.</li>
	<li>Quantify the photons emitted from specific regions using the manufacturer&#39;s software.
	<ol>
		<li>Click ROI Tools in the tool palette. In the ROI Tools, select Measurement ROI from the Type dropdown list.</li>
		<li>Click on the Square icon and draw a squared ROI with the proper dimensions to cover the thorax of one animal. Copy and paste the ROI for each animal to obtain ROIs with the same dimensions. In the ROI Tools panel, click on Measure ROI to obtain the measurements of the total intensity in the ROIs.</li>
		<li>Observe the ROI Measurements data for all ROIs created in the images or sequences during a session (one ROI per row). Click Export and select the folder where the file will be saved.</li>
	</ol>
	</li>
</ol>
3. Mouse Challenge with Pro-Inflammatory Stimuli
NOTE: Prior to the mouse challenge with pro-inflammatory stimuli, check the baseline activation by in vivo BLI (see step 2). At least 7 days must pass between in vivo gene delivery and mouse challenge to allow the mild and transient inflammation to disappear.
<ol>
	<li>Prepare the equipment for intratracheal instillation (refer to Figure 1 and Figure 2).
	<ol>
		<li>Connect the 5 ml disposable syringes with the spring (C, E), a 100 &#181;L syringe (B), and a disposable gauge (D) to the 3-way stopcock (A). Place the system on the support (H). Place the system on the support (H).</li>
		<li>Connect the PE190 micro medical tubing (F) to the disposable gauge (D) and to the pen century (G).</li>
		<li>Fill the 5-mL syringe with 800 &#181;L of air and turn the 3-way stopcock. 
		NOTE: Fill the tube with Pseudomonas aeruginosa culture supernatant by aspirating 50 &#181;l in the 100 &#181;l syringe.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55499/55499fig1.jpg" /> 
Figure 1: Schematic Representation of a Single Component of the Intratracheal Device. 
(A) 3-way stopcock, (B) 100-&#181;L Hamilton syringe, (C) disposable 5-mL syringe, (D) disposable gauge, (E) disposable 5-mL syringe with spring, (F) PE190 micro medical tubing, (G) pen century, and (H) support. <a href="http://cloudfront.jove.com/files/ftp_upload/55499/55499fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55499/55499fig2.jpg" /> 
Figure 2: Representation of the Assembled Intratracheal Device. 
The identifications are the same as in Figure 1. <a href="http://cloudfront.jove.com/files/ftp_upload/55499/55499fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Anesthetize the mice using an isoflurane vaporizer chamber set at 2.5% isoflurane mixed with oxygen.</li>
	<li>Monitor the animal to evaluate the effects of the anesthetic after 3 - 5 min. 
	NOTE: To confirm that the mouse is fully anesthetized, monitor the following signs carefully: slowed breathing rate should slow down, lack of arm stretching when picked up by the neck, and a lack of response when the hind limbs are stimulated. Wait for a few additional minutes and check again before proceeding to the next step if these criteria are not met.</li>
	<li>Place the anesthetized mouse on the plexiglass intubation platform, hanging it by its incisors, which are placed on the wire.</li>
	<li>Turn on the laryngoscope with the left hand (for right-handed researchers) and grab a pair of blunt-ended forceps. Use the tip of laryngoscope and the forceps to gently pry open the mouth.</li>
	<li>Pull the tongue out and hold it to the side using the forceps. Guide the laryngoscope blade towards the back of the mouth. Keep the laryngoscope pressed down very gently at a 90&#176; angle until the opening of the trachea is visible. Hold the laryngoscope in place.</li>
	<li>Using the other hand, take the delivery tube connected to the end of the PE tubing and insert it into the trachea. Rotate the three-way valve to deliver the inoculum. Pull the tube out of the trachea as soon as possible. Hold the mouse upright for a few seconds to the allow inoculum to be inhaled into the lungs. 
	NOTE: Mice will suffocate and die if the trachea is blocked for too long.</li>
	<li>Remove the mouse from the platform. The recovery time may vary based on strains; monitor the mouse diligently, ensuring that the animal is fully awake within 30 min after the procedure.</li>
	<li>Intraperitoneally inject 150 mg/kg D-luciferin and image the lungs using an in vivo imaging system 4, 24, and 48 h after the intratracheal instillation of the stimuli. Quantify the photons emitted from specific regions using the manufacturer&#39;s software.</li>
</ol>

<ol>
	<li>Barnes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+approaches+to+asthma-chronic+obstructive+pulmonary+disease+overlap+syndromes.">Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes.</a> J Allergy Clin Immunol. 136 (3), 531-545 (2015).</li><li>Cohen-Cymberknoh, M., Kerem, E., Ferkol, T., Elizur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+inflammation+in+cystic+fibrosis:+molecular+mechanisms+and+clinical+implications.">Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications.</a> Thorax. 68 (12), 1157-1162 (2013).</li><li>Dhooghe, B., Noel, S., Huaux, F., Leal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+inflammation+in+cystic+fibrosis:+pathogenesis+and+novel+therapies.">Lung inflammation in cystic fibrosis: pathogenesis and novel therapies.</a> Clin Biochem. 47 (7-8), 539-546 (2014).</li><li>Durham, A. L., Caramori, G., Chung, K. F., Adcock, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+anti-inflammatory+therapeutics+in+asthma+and+chronic+obstructive+lung+disease.">Targeted anti-inflammatory therapeutics in asthma and chronic obstructive lung disease.</a> Transl Res. 167 (1), 192-203 (2015).</li><li>Sagel, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biomarkers+of+airway+inflammation+in+cystic+fibrosis.">Noninvasive biomarkers of airway inflammation in cystic fibrosis.</a> Curr Opin Pulm Med. 9 (6), 516-521 (2003).</li><li>Starkey, M. R., 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=Murine+models+of+infectious+exacerbations+of+airway+inflammation.">Murine models of infectious exacerbations of airway inflammation.</a> Curr Opin Pharmacol. 13 (3), 337-344 (2013).</li><li>Cacalano, 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=Neutrophil+and+B+cell+expansion+in+mice+that+lack+the+murine+IL-8+receptor+homolog.">Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.</a> Science. 265 (5172), 682-684 (1994).</li><li>Simonet, W. S., 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=Long-term+impaired+neutrophil+migration+in+mice+overexpressing+human+interleukin-8.">Long-term impaired neutrophil migration in mice overexpressing human interleukin-8.</a> J Clin Invest. 94 (3), 1310-1319 (1994).</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), e39716 (2012).</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>De Simone, 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=Host+genetic+background+influences+the+response+to+the+opportunistic+Pseudomonas+aeruginosa+infection+altering+cell-mediated+immunity+and+bacterial+replication.">Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.</a> PLoS One. 9 (9), e106873 (2014).</li></ol>

The authors have nothing to disclose.

In a previous work11, a contrast between bIL-8-Luc-dependent BLI and BAL markers was shown. It relied on the differential degree of sensitivity within mouse strains12. For this reason, the first application of the bIL-8-Luc model to a different mouse strain requires an initial study of the inflammatory response, both in terms of BLI and more standardized inflammatory markers.
Mice transfection causes mild lung inflammation and the activation of b...

Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis, are characterized by airway inflammation. Airway inflammation is characterized by edema, cellular infiltration, T lymphocyte and mast cell activation, increased airway secretions, and excessive collagen deposition. CF is a multisystem disorder, and its major cause of mortality and morbidity is lung bacterial infection with increasing pulmonary exacerbation. The decline in lung function predicts a significantly poorer outcome1,2,3,4.
The inflammation state of the respiratory tract is usually observed through the evaluation of immunological markers recruited during the inflammatory process in material derived from the lower and upper airways, such as sputum, which provides variable results. Bronchoscopies are also performed5. Murine models are valuable tools for investigating the pathogenesis and evolution of diseases characterized by airway inflammation and for which effective treatments or cures have not yet been identified. Animal models of lung infection and inflammation have been used to study asthma and host-pathogen interactions, including the role of chemicals that simulate human conditions (e.g., cigarette smoke exposure, LPS, elastase, ovalbumin, poly I:C, etc., as well as combinations of the above)6. The measurement of inflammation-related parameters requires the sacrifice of the animals, as invasive approaches are required to measure factors such as bacterial load, cytokines in the lungs, and collected bronchoalveolar lavage (BAL) fluid. Also, histological examinations are often required. The possibility of obtaining information on the inflammatory response kinetics requires the use of numerous mice. Therefore, a technique that would allow for obtaining such information without the need to sacrifice the animals is valuable on technical, ethical, economical, and operational bases.
IL-8 is an essential player in the inflammation process, recruiting leukocytes to the inflamed tissue. It represents a molecular read-out for the study of inflammatory pathway activation. MIP-2 and KC may be functional homologs of human IL-8 in mice. Mice express only one potential IL-8 receptor, a homolog of human CXCR27,8, but they are capable of modulating a heterologous IL-8 gene promoter that drives a reporter gene. A lung inflammation murine model has recently been developed after the observation that a bovine IL-8 promoter/luciferase reporter construct can be transactivated in mice. This feature allows for the utilization of bioluminescence imaging (BLI) to monitor the inflammatory response in living animals9.
This model has been adapted to study inflammation triggered by bacterial exoproducts (e.g., LPS or products released by bacterial strains) or TNFalpha10,11. The drug discovery process is focused on the development and optimization of old and new anti-inflammatory molecules that can treat lung diseases, such as CF, asthma, and COPD. These new chemical entities must be quickly and conveniently tested in animal models that can be linked to specific clinical phenotypes in order to facilitate the design of smart clinical trials.

<table><tbody><tr><td>FMT 2500 Fluorescence Tomography System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>IVIS Lumina serie II Pre-clinical &lt;em&gt;In Vivo&lt;/em&gt;; Imaging System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>MMPsense 750 FAST</td><td>Perkin Elmer Inc.</td><td>NEV10001EX</td><td>Protect from light, store the probe at 4 &amp;deg;C</td></tr><tr><td>Female inbred BalbC</td><td>Harlan Laboratories Italy</td><td></td><td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td></tr><tr><td>bIL-8-Luc plasmid</td><td>Department of Medical Veterinary Science, University of Parma, Italy</td><td></td><td>Store the plasmid at -20 &amp;deg;C</td></tr><tr><td>pGL3basic vector</td><td>Promega</td><td>E1751</td><td>Store the vector at -20 &amp;deg;C</td></tr><tr><td>JetPEI DNA transfection reagent</td><td>Polyplus transfection</td><td>201B-001G</td><td>The DNA and JetPEI mix was formulated with a final N/P ratio of 7</td></tr><tr><td>D-luciferin potassium salt 1 g</td><td>Perkin Elmer Inc.</td><td>122796</td><td>Protect from light, store at -20 &amp;deg;C</td></tr><tr><td>Living Image software</td><td>Caliper Life Sciences,</td><td>Experimental Builder</td><td></td></tr><tr><td>Isoflurane</td><td>ESTEVE spa</td><td>571329.8</td><td>Do not inhale</td></tr><tr><td>Bio-Plex Cytokine Assay Kit</td><td>Bio-Rad Laboratories</td><td>M60-009RDPD</td><td>Store the unopened kit at 4 &amp;deg;C</td></tr><tr><td>Automated cell counter</td><td>Dasit XT 1800J</td><td>Experimental Builder</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>Gas anesthesia system XGI-8</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</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 a 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 18ga 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>

an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

The bIL-8-Luc transient transgenic mouse model was used for the in vivo monitoring of lung inflammation in mice challenged with concentrated bacterial supernatant (30x) containing secreted virulence factors. The induced inflammatory response was detectable by in vivo imaging as an increase in the BLI signal. Pro-inflammatory activity was clearly detectable 2.5 h post-instillation, although the BLI signal reached the highest peak between 5 and 24 h and was still detectabl...

Watch this Scientific Journal Video about An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses at JoVE.com

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

an-il-8-transiently-transgenized-mouse-model-for-vivo-long-term

Research

JoVE Journal

Immunology and Infection

7.4K Views.  University of Verona.  De hier beschreven werkwijze maakt het mogelijk om visualisatie van IL-8 promotorafhankelijke ontstekingsactivering in de longen van muizen door middel van niet-invasieve bioluminescentie beeldvorming (BLI). Het zelfde dier kan meerdere keren tot BLI worden onderworpen tot maximaal twee maanden vanaf de tijd van afgifte van het luciferase reporterconstruct. 

Video: An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Neuroscience

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Medicine

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Samenvatting

Meer video's verkennen