Name: a minimally invasive, accurate, and efficient technique for intrathymic injection in mice
Uploaded: 2022-08-23T14:00:00
Description: Watch this Scientific Journal Video about A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice at JoVE.com

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...

<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>

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 ...

Watch this Scientific Journal Video about A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice at JoVE.com

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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