We would like to thank Raymond H. Thornton for his insightful and comprehensive early work on this technique.This study was funded by grant support from the National Cancer Institute (NCI 1R37CA250661-01A1), the Children&#39;s Leukemia Research Association, the Hackensack Meridian School of Medicine, and the HUMC Foundation/Tackle Kids Cancer.

All procedures were performed in accordance with animal care guidelines at the Center for Discovery and Innovation (IACUC protocol 290). For the present study, C57BL/6 mice (female, 4-6 weeks old), C57BL/6 mice (female, 6 months old), J:NU female mice, NOD scid gamma (NSG) female mice, and B6;CAG-luc, -GFP mice were used as the young mouse model, aged mouse model, athymic nude model, immunodeficient model, and bioluminescence cell source, respectively. The mice were obtained from a commercial source (see Table of Materials). This procedure will typically require two people (one to remain sterile while performing the injections and another to handle the mice).
1. Animal preparation
<ol>
	<li>Induce anesthesia in the mice using 3%-4% isoflurane gas and maintain anesthesia using 1%-3% isoflurane gas administered&#160;via&#160;a nose cone and precision-calibrated vaporizer (see&#160;Table of Materials).</li>
	<li>Confirm the appropriate anesthetic depth/unconsciousness by unresponsiveness to the hind paw pinch.</li>
	<li>Remove the fur from the anterior chest area of the mice by applying a thin layer of depilatory cream for less than 1 min. Use a wet paper towel to remove the cream completely along with the loose fur. 
	NOTE: Applying too much cream will result in the chest area skin becoming inflamed.</li>
	<li>Place one mouse at a time, supine, on the heated platform of the small animal ultrasound imaging station (see Table of Materials) with the nose cone in place (Figure 1).</li>
	<li>Secure the mouse to the stage with medical adhesive tape at the hind and forelimbs (Figure 1).</li>
	<li>Apply ophthalmic ointment to both eyes to prevent corneal drying.</li>
	<li>Disinfect the fur-free upper thorax skin using a chlorhexidine gluconate applicator (see Table of Materials).</li>
</ol>
2. Preparation of the ultrasound machine and sterile field
<ol>
	<li>Activate the highest frequency linear probe available, typically the probe with the highest spatial resolution for the size of the animal being imaged. Activate the probe by tapping the corresponding button after the startup screen. 
	NOTE: For this application with mice, the probe used is designed specifically for use with mice and small rats (see Table of Materials).</li>
	<li>Optimize the ultrasound settings for imaging and injection following the steps below.
	<ol>
		<li>Adjust the depth of the field of view to an appropriate size for the target animal by adjusting the vertically oriented sliders on the right side of the screen (Figure 2). The maximum depth setting will typically be about 6-8 mm for young mice.</li>
		<li>Adjust the grayscale gain by sliding the button along the horizontal bar on the bottom of the screen (Figure 2). The goal is to start with an image that is just slightly darker than a typical &#34;gray&#34; appearance.</li>
		<li>Adjust the focal zone (blue arrow on the right of the screen, Figure 2) to the anticipated level of the thymus. For young mice, this will be around a depth of 4 mm.</li>
		<li>If image capture is desired, test the functionality of the store image and store clip buttons to ensure the images can be appropriately saved throughout the procedure. Perform this by tapping the Save Clip button on the bottom right of the screen or by tapping the Freeze button, and then tapping Save Image (Figure 2).</li>
	</ol>
	</li>
	<li>Apply a small amount (~1 mL) of ultrasound gel to the transducer surface (see Table of Materials) while it is upright, either resting in the ultrasound machine holder or in the hands of an assistant.</li>
	<li>Prepare a small sterile field next to the heated platform. The optimal positioning for this is usually between the platform and the ultrasound machine.
	<ol>
		<li>Empty these items onto the sterile field: a sterile probe cover, rubber band, sterile gloves, and sterile ultrasound gel (see Table of Materials).</li>
		<li>With the sterile field set up and items in place, put on the sterile gloves.</li>
		<li>Carefully place the sterile probe cover over the ultrasound transducer (as well as over the gel that was initially placed on the probe). Maintain sterility and touch only the sterile cover, nothing else. Slide the sterile rubber band over the sterile probe cover to keep it in place. 
		NOTE: The air foci, regardless of size, can interfere with ultrasound imaging. Hence, it is essential to apply the ultrasound gel between the transducer and sterile probe cover, and on top of the probe cover to ensure an air-free interface between the ultrasound probe and the animal.&#160;</li>
		<li>Place a moderate amount (2-3 mL) of sterile ultrasound gel onto the transducer. 
		&#8203;NOTE: The user is now ready to image an anesthetized mouse.</li>
	</ol>
	</li>
</ol>
3. Imaging and locating the thymus
<ol>
	<li>While maintaining sterility, place the ultrasound gel-topped probe vertically on the disinfected portion of the anterior chest wall of the mouse for initial imaging.
	<ol>
		<li>Take a moment to look at the ultrasound image and optimize it further. Go back to step 2.2 and adjust to get an appearance similar to that of Figure 3.</li>
	</ol>
	</li>
	<li>Scan the anterior chest of the mouse in a transverse plane. Perform this by holding the transducer vertically and moving it up and down from the neck to the abdomen in a paintbrush-like or &#34;sweeping&#34; motion. 
	NOTE: The heart will be the most recognizable structure in the chest due to its rapid motion and &#34;chambered&#34; appearance. Once the heart is localized, this can be used as a reference point to acquire an image of the thymus.</li>
	<li>With the heart centered in the field of view, sweep the transducer slightly toward the neck. Just superior to the heart, the thymus is usually encountered.</li>
	<li>Visualize the thymus as a bilobed, pyramidal, hypoechoic (&#34;dark&#34; or &#34;black&#34; appearing on the screen) structure that is centered in the midline, anterior to the aorta and posterior to the sternum (Figure 3A).</li>
	<li>Make a note of the two round paired black (i.e., &#34;hypoechoic&#34;) structures on either side of the upper chest. 
	&#8203;NOTE: These are the bilateral venae cavae. The aorta is a similar curvilinear hypoechoic structure in the midline between the two venae cavae. These are easily recognizable by their pulsatile motion.</li>
</ol>
4. Injection of the thymus
<ol>
	<li>If needed, apply more (2-3 mL) sterile ultrasound gel to the transducer. 
	NOTE: A relatively large amount of sterile gel on the transducer (compared to the size of the mouse thorax) will act as a &#34;gel pad&#34; around the chest wall of the mouse. This will reduce the number of ultrasound artifacts made by air within the field of view.</li>
	<li>Using the ultrasound probe, locate the widest portion of the thymus, which is usually the ideal target site for injection. Anticipate a horizontal needle trajectory in the chosen location.
	<ol>
		<li>Note where the major blood vessels (SVCs and aorta) are located at this site. Avoid these during the injection.</li>
		<li>The blood vessels will be hypoechoic, pulsatile structures, as described in step 3.7. If unsure, use the color Doppler mode to check for flow within the vessels (Figure 4A). Activate the color Doppler mode by tapping the Color button on the screen.</li>
		<li>If one of the major blood vessels (or the heart) is anticipated to be along the expected needle trajectory, choose a new target area or find a different approach/trajectory.</li>
	</ol>
	</li>
	<li>Hold the transducer in one hand and a 30 G insulin needle (see Table of Materials) with 10 &#181;L of injectate in the other. 
	NOTE: The injectate will vary based on the experimental design. The present study used phosphate-buffered saline, trypan blue, or D-luciferin (0.1 &#181;g/10 &#181;L).</li>
	<li>To begin the injection process, move the transducer laterally so that the thymus is off-center in the ultrasound field of view. Ensure that the other side of the field of view consists of mostly ultrasound gel and nothing else.</li>
	<li>Place the tip of the needle in the gel under the transducer and slowly move the needle until it is visualized adjacent to the skin surface (Figure 4B).</li>
	<li>While continuously imaging the needle under ultrasound, insert the needle into the thymus gland with a percutaneous trajectory, away from blood vessels.
	<ol>
		<li>Use a &#34;cross thymus&#34; horizontal trajectory to place the needle tip in the thymic lobe contralateral to the entry site. This accounts for potential leakage along the needle tract (Figure 5A).</li>
	</ol>
	</li>
	<li>Once the needle tip is inside the desired portion of the thymus, quickly inject the contents (such as 10 &#181;L of trypan blue or D-luciferin, 0.1 &#181;g/10 &#181;L) from the 30 G syringe while using sonographic visualization.
	<ol>
		<li>To stabilize the syringe during needle insertion and injection, hold the syringe between the thumb and third finger and control the syringe plunger with the index finger.</li>
	</ol>
	</li>
	<li>Remove the needle after all the contents have been deposited.</li>
</ol>
5. Post-injection monitoring of animals
<ol>
	<li>Transfer the animal to an empty cage and observe until it regains sufficient consciousness to maintain sternal recumbency. 
	NOTE: Full recovery from anesthesia is expected to occur within 2 min.</li>
	<li>Monitor the animal for an additional 10 min for signs of distress, labored breathing, or bleeding. 
	NOTE: Post-injection pain is not expected, and there is typically no need for post-injection analgesia.</li>
	<li>Once fully recovered and following an uneventful post-injection observation period, return the injected animal to the company of other animals.</li>
</ol>

<ol>
	<li>Chinn, I. K., Blackburn, C. C., Manley, N. R., Sempowski, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+primary+lymphoid+organs+with+aging.">Changes in primary lymphoid organs with aging.</a> Seminars in Immunology. 24 (5), 309-320 (2012).</li><li>Gruver, A. L., Sempowski, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines,+leptin,+and+stress-induced+thymic+atrophy.">Cytokines, leptin, and stress-induced thymic atrophy.</a> Journal of Leukocyte Biology. 84 (4), 915-923 (2008).</li><li>Masopust, D., Sivula, C. P., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice,+dirty+mice,+and+men:+Using+mice+to+understand+human+immunology.">Of mice, dirty mice, and men: Using mice to understand human immunology.</a> Journal of Immunology. 199 (2), 383-388 (2017).</li><li>Mukherjee, P., Roy, S., Ghosh, D., Nandi, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+animal+models+in+biomedical+research:+a+review.">Role of animal models in biomedical research: a review.</a> Laboratory Animals Research. 38 (1), 18 (2022).</li><li>McCaughtry, T. M., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+tolerance:+What+have+we+learned+from+mice.">Central tolerance: What have we learned from mice.</a> Seminars in Immunopathology. 30 (4), 399-409 (2008).</li><li>Zlotoff, D. A., 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=CCR7+and+CCR9+together+recruit+hematopoietic+progenitors+to+the+adult+thymus.">CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus.</a> Blood. 115 (10), 1897-1905 (2010).</li><li>Vukmanovic, S., Grandea, A. G., Faas, S. J., Knowles, B. B., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+of+T-lymphocytes+induced+by+intrathymic+injection+of+a+thymic+epithelial+cell+line.">Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.</a> Nature. 359 (6397), 729-732 (1992).</li><li>Schwarz, B. A., Bhandoola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+hematopoietic+progenitors+with+T+lineage+potential.">Circulating hematopoietic progenitors with T lineage potential.</a> Nature Immunology. 5 (9), 953-960 (2004).</li><li>Marodon, 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=Induction+of+antigen-specific+tolerance+by+intrathymic+injection+of+lentiviral+vectors.">Induction of antigen-specific tolerance by intrathymic injection of lentiviral vectors.</a> Blood. 108 (9), 2972-2978 (2006).</li><li>Adjali, O., 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+correction+of+ZAP-70+immunodeficiency+by+intrathymic+gene+transfer.">In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer.</a> Journal of Clinical Investigation. 115 (8), 2287-2295 (2005).</li><li>Tuckett, A. 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=Image-guided+intrathymic+injection+of+multipotent+stem+cells+supports+life-long+T+cell+immunity+and+facilitates+targeted+immunotherapy.">Image-guided intrathymic injection of multipotent stem cells supports life-long T cell immunity and facilitates targeted immunotherapy.</a> Blood. 123 (18), 2797-2805 (2014).</li><li>Tuckett, A. Z., Thornton, R. H., O'Reilly, R. J., vanden Brink, M. R. M., Zakrzewski, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+injection+of+hematopoietic+progenitor+cells+establishes+functional+T+cell+development+in+a+mouse+model+of+severe+combined+immunodeficiency.">Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency.</a> Journal of Hematology &amp; Oncology. 10 (1), 109 (2017).</li><li>Hogan, B. V., Peter, M. B., Shenoy, H. G., Horgan, K., Hughes, 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=Surgery+induced+immunosuppression.">Surgery induced immunosuppression.</a> Surgeon. 9 (1), 38-43 (2011).</li><li>Blair-Handon, R., Mueller, K., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+intrathymic+injections+in+mice.">An alternative method for intrathymic injections in mice.</a> Laboratory Animals. 39 (8), 248-252 (2010).</li><li>Tuckett, A. Z., Zakrzewski, J. L., Li, D., vanden Brink, M. R., Thornton, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-hand+ultrasound+guidance+permits+safe+and+efficient+minimally+invasive+intrathymic+injections+in+both+young+and+aged+mice.">Free-hand ultrasound guidance permits safe and efficient minimally invasive intrathymic injections in both young and aged mice.</a> Ultrasound in Medicine and Biology. 41 (4), 1105-1111 (2015).</li><li>K&#252;ker, 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=The+value+of+necropsy+reports+for+animal+health+surveillance.">The value of necropsy reports for animal health surveillance.</a> BMC Veterinary Research. 14 (1), 191 (2018).</li><li>Sinclair, C., Bains, I., Yates, A. J., Seddon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+thymocyte+death+underlies+the+CD4:CD8+T-cell+ratio+in+the+adaptive+immune+system.">Asymmetric thymocyte death underlies the CD4:CD8 T-cell ratio in the adaptive immune system.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 2905-2914 (2013).</li><li>Manna, S., Bhandoola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+injection.">Intrathymic injection.</a> Methods in Molecular Biology. 1323, 203-209 (2016).</li><li>de la Cueva, T., Naranjo, A., de la Cueva, E., Rubio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+intrathymic+injection+in+mice.">Refinement of intrathymic injection in mice.</a> Laboratory Animals. 36 (5), 27-32 (2007).</li></ol>

The authors do not have any conflicts of interest to disclose.

An ultrasound-guided free-hand injection is a highly accurate technique for delivering study materials to the thymus in an efficient and aseptic fashion. Following the initial sterilization of the skin at the injection site, sterility is maintained during the procedure owing to the use of sterile gloves, sterile ultrasound probe covers, and sterile ultrasound gel. In contrast to the blind percutaneous approach10,17 or relying on surgical incisions for direct visu...

The thymus has an essential role in T cell development and immunity. T cell deficiency, which can be caused by thymic involution, genetic disorders, infections, and cancer treatments, amongst other factors, leads to high mortality and morbidity1,2. Mouse models are indispensable in both basic and translational immunology research and have been used for decades to study thymic biology and T cell development, as well as to develop treatments for those suffering from thymic dysfunction and T cell deficiency3,4,5.
A central part of thymic investigations has been the intrathymic injection of biological materials such as cells, genes, or proteins in mouse models6,7,8,9,10,11,12. Conventional intrathymic injection methods use thoracotomy followed by intrathymic injection under direct visualization or by &#34;blind&#34; percutaneous injection into the mediastinum. The surgical approach significantly increases the pneumothorax risk, amongst others. Moreover, the elevated stress during this surgery results in immunosuppression, thus potentially compromising immunological data13. Experienced researchers, after some practice, can perform the blind injection technique, but this approach is less accurate and therefore, limits experimental subjects to young mice with a big thymus.
The utilization of ultrasound guidance has been introduced as a precise and minimally invasive alternative to traditional intrathymic injection approaches14. However, this procedure is very time-consuming when using the integrated rail system instead of the free-hand technique. Performing injections with the injection mount requires careful imaging optimization and positioning of the transducer with the help of the various attachments such as the transducer stand and mount, the X, Y, and Z positioning system, as well as proficient operation of the micro-manipulation controls and rail system extensions. A simple alternative technique, ultrasound-guided thymic injection, is presented here performed by a radiologist using a free-hand approach15, which is both a rapid and accurate minimally invasive alternative to the above-described methods. Importantly, the current approach can be performed with any high-resolution ultrasound imaging system without needing an injection mount and integrated rail system. It is especially useful for studies requiring the injection of large numbers of mice11, for experiments involving the injection of both thymic lobes, or for the accurate injection of small thymuses in aged, irradiated, or immunocompromised mice12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquasonic 100 Ultrasound Gel</td>
			<td>Parker Laboratories (Fairfield, NJ, USA)</td>
			<td>01-01</td>
			<td>Sterile Ultrasound Transmission Gel</td>
		</tr>
		<tr>
			<td>B6;CAG-luc, -GFP mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>025854</td>
			<td>Bioluminescence cell source</td>
		</tr>
		<tr>
			<td>BD Insulin Syringes with needle</td>
			<td>Becton Dickinson (Franklin Lakes, NJ, USA)</td>
			<td>328431</td>
			<td>Ultra-fine needle - 12.7 mm, 30 G</td>
		</tr>
		<tr>
			<td>C57BL/6 mouse - aged</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>000664</td>
			<td>age 6 months old; aged model</td>
		</tr>
		<tr>
			<td>C57BL/6 mouse - young</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>000664</td>
			<td>age 4-6 weeks; young model</td>
		</tr>
		<tr>
			<td>Chloraprep One-step 0.67 mL</td>
			<td>CareFusion (El Paso, TX, USA)</td>
			<td>260449</td>
			<td>chlorhexidine gluconate applicator</td>
		</tr>
		<tr>
			<td>Curity Cotton Tipped Applicator</td>
			<td>Cardinal Health (Dublin, OH, USA)</td>
			<td>A5000-2</td>
			<td>Sterile, 6&#34;</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Gold Biotechnology (St Louis, MO, USA)</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein (Melville, NY, USA)</td>
			<td>1182097</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina X5</td>
			<td>PerkinElmer (Melville, NY, USA)</td>
			<td>n/a</td>
			<td>In vivo bioluminescence imaging system</td>
		</tr>
		<tr>
			<td>J:NU mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>007850</td>
			<td>Athymic nude model</td>
		</tr>
		<tr>
			<td>Kendall Hypoallergenic Paper Tape</td>
			<td>Cardinal Health (Dublin, OH, USA)</td>
			<td>1914C</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Surgical Nitrile Gloves</td>
			<td>Kimberly-Clark Professional (Irving, TX, USA)</td>
			<td>56892</td>
			<td>Sterile Gloves</td>
		</tr>
		<tr>
			<td>Nair Hair Remover Lotion</td>
			<td>Church and Dwight (Trenton, NJ, USA)</td>
			<td>n/a</td>
			<td>Depilatory agent</td>
		</tr>
		<tr>
			<td>NOD scid gamma (NSG) mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>005557</td>
			<td>Immunodeficient model</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS), 1x</td>
			<td>Corning (Corning, NY, USA)</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Med Vet International</td>
			<td>PH-PURALUBE-VET</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Sheathes</td>
			<td>Sheathing Technologies (Morgan Hill, CA, USA)</td>
			<td>10040</td>
			<td>Sterile Ultrasound Probe Covers</td>
		</tr>
		<tr>
			<td>Sure-Seal Induction Chamber</td>
			<td>Braintree Scientific (Braintree, MA, USA)</td>
			<td>EZ-17 85</td>
			<td>Anesthesia induction chamber</td>
		</tr>
		<tr>
			<td>Transducer MX550D</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Vevo 3100 imaging probe (25-55 MHz, Centre Transmit: 40 MHz)</td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% solution in PBS</td>
			<td>MP Biomedicals (Solon, OH, USA)</td>
			<td>91691049</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 3100 Imaging System</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Ultrasound imaging system</td>
		</tr>
		<tr>
			<td>Vevo 3100 Lab Software</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Version 3.2.7 for imaging and analysis</td>
		</tr>
		<tr>
			<td>Vevo Compact Dual Anesthesia System</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Tabletop isoflurane-based anesthesia unit</td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Procedural platform</td>
		</tr>
	</tbody>
</table>

a minimally invasive, accurate, and efficient technique for intrathymic injection in mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The successful implementation of this technique relies on a few key steps to be followed. First, reliable identification of the thymus gland itself has to be ensured. In young mice, this is straightforward due to the gland&#39;s large size (Figure 3A). In older mice or immunodeficient mice, it can be more challenging; however, it is still very feasible with modern ultrasound equipment (Figure 3B,C). Second, it is critically important to set the ...

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A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...

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Research

JoVE Journal

Immunology and Infection

2.2K Views.  Hackensack University Medical Center. The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

Video: A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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 tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

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

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

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 successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...