Name: an immunological model for heterotopic heart and cardiac muscle cell transplantation in rats
Uploaded: 2020-05-08T13:30:00
Description: Watch this Scientific Journal Video about An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats at JoVE.com

We want to thank Britta Trautewig, Corinna L&#246;bbert and Ingrid Meder for their commitment.

All animal experiences have been performed according to the guidelines of the local Ethics Animal Review Board of the regional authorities for consumer protection and food safety of Lower Saxony (LAVES, Oldenburg, Germany) with the approval IDs 12/0768 and 17/2472.
1. Heart explantation and perfusion
NOTE: As graft donors, female or male rats at an age of 7-22 weeks were used.
<ol>
	<li>Anesthetize the donor rat by isoflurane inhalation (induction at 5% and mainte...

<ol>
	<li>Abbott, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Technique+for+Heart+Transplantation+In+the+Rat.">A Technique for Heart Transplantation In the Rat.</a> Archives of Surgery. 89 (4), 645-652 (1964).</li><li>Tomita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+homotransplantation+in+the+rat.">Heart homotransplantation in the rat.</a> Sapporo igaku zasshi. The Sapporo Medical Journal. 30 (4), 165-183 (1966).</li><li>Ono, K., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+of+heart+transplantation+in+rats.">Improved technique of heart transplantation in rats.</a> The Journal of Thoracic and Cardiovascular Surgery. 57 (2), 225-229 (1969).</li><li>Wen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+a+new+working+heterotopic+heart+transplantation+model+in+rats.">A simple technique for a new working heterotopic heart transplantation model in rats.</a> Transplantation Proceedings. 45 (6), 2522-2526 (2013).</li><li>Benke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+abdominal+rat+heart+transplantation+as+a+model+to+investigate+volume+dependency+of+myocardial+remodeling.">Heterotopic abdominal rat heart transplantation as a model to investigate volume dependency of myocardial remodeling.</a> Transplantation. 101 (3), 498-505 (2017).</li><li>Kearns, M. J., 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=Rat+Heterotopic+Abdominal+Heart/Single-lung+Transplantation+in+a+Volume-loaded+Configuration.">Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration.</a> Journal of Visualized Experiments. (99), 52418 (2015).</li><li>Ibrahim, 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=Heterotopic+abdominal+heart+transplantation+in+rats+for+functional+studies+of+ventricular+unloading.">Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading.</a> The Journal of Surgical Research. 179 (1), e31-e39 (2013).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> Journal of Visualized Experiments. (6), 238 (2007).</li><li>Lu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+simplified+volume-loaded+heterotopic+rabbit+heart+transplant+model+with+improved+techniques+and+a+standard+operating+procedure.">A new simplified volume-loaded heterotopic rabbit heart transplant model with improved techniques and a standard operating procedure.</a> Journal of Thoracic Disease. 7 (4), 653-661 (2015).</li><li>Kitahara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+transplantation+of+a+decellularized+and+recellularized+whole+porcine+heart.">Heterotopic transplantation of a decellularized and recellularized whole porcine heart.</a> Interactive Cardiovascular and Thoracic Surgery. 22 (5), 571-579 (2016).</li><li>Kadner, 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=Heterotopic+heart+transplantation:+experimental+development+and+clinical+experience.">Heterotopic heart transplantation: experimental development and clinical experience.</a> European Journal of Cardiothoracic Surgery. 17 (4), 474-481 (2000).</li><li>Zin&#246;cker, 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=Immune+reconstitution+and+graft-versus-host+reactions+in+rat+models+of+allogeneic+hematopoietic+cell+transplantation.">Immune reconstitution and graft-versus-host reactions in rat models of allogeneic hematopoietic cell transplantation.</a> Frontiers in Immunology. 3 (NOV), 1-12 (2012).</li><li>Klempnauer, J., 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=Genetic+control+of+rat+heart+allograft+rejection:+effect+of+different+MHC+and+non-MHC+incompatibilities.">Genetic control of rat heart allograft rejection: effect of different MHC and non-MHC incompatibilities.</a> Immunogenetics. 30, 81-88 (1989).</li><li>Huang, 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=Genetic+manipulations+in+the+rat:+Progress+and+prospects.">Genetic manipulations in the rat: Progress and prospects.</a> Current Opinion in Nephrology and Hypertension. 20 (4), 391-399 (2011).</li><li>Gordon, C. 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=Pulse+doppler+and+M-mode+to+assess+viability+of+cardiac+allografts+using+heterotopic+femoral+heart+transplantation+in+rats.">Pulse doppler and M-mode to assess viability of cardiac allografts using heterotopic femoral heart transplantation in rats.</a> Microsurgery. 27 (4), 240-244 (2007).</li><li>Gordon, C. 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=A+new+modified+technique+for+heterotopic+femoral+heart+transplantation+in+rats.">A new modified technique for heterotopic femoral heart transplantation in rats.</a> The Journal of Surgical Research. 139 (2), 157-163 (2007).</li><li>Ma, Y., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+2+heterotopic+heart+transplant+techniques+in+rats:+cervical+and+abdominal+heart.">Comparison of 2 heterotopic heart transplant techniques in rats: cervical and abdominal heart.</a> Experimental and Clinical Transplantation. 9 (2), 128-133 (2011).</li><li>Bektas, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+donor-specific+blood+transfusions+after+kidney,+heart,+pancreas,+and+skin+transplantation+in+major+histocompatibility+complex-incompatible+rats.">Differential effect of donor-specific blood transfusions after kidney, heart, pancreas, and skin transplantation in major histocompatibility complex-incompatible rats.</a> Transfusion. 37 (2), 226-230 (1997).</li><li>Saiho, K. 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=Long-term+allograft+acceptance+induced+by+single+dose+anti-leukocyte+common+antigen+(RT7)+antibody+in+the+rat.">Long-term allograft acceptance induced by single dose anti-leukocyte common antigen (RT7) antibody in the rat.</a> Transplantation. 71 (8), 1124-1131 (2001).</li><li>Bektas, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+transfers+infectious+immunologic+tolerance+in+MHC-incompatible+heart+transplantation+in+rats.">Blood transfers infectious immunologic tolerance in MHC-incompatible heart transplantation in rats.</a> Journal of Heart and Lung Transplantation. 24 (5), 614-617 (2005).</li><li>J&#228;ger, M. D., 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=Sirolimus+promotes+tolerance+for+donor+and+recipient+antigens+after+MHC+class+II+disparate+bone+marrow+transplantation+in+rats.">Sirolimus promotes tolerance for donor and recipient antigens after MHC class II disparate bone marrow transplantation in rats.</a> Experimental Hematology. 35 (1), 164-170 (2007).</li><li>Timrott, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+allogeneic+bone+marrow+cells+in+view+of+residual+alloreactivity:+Sirolimus+but+not+cyclosporine+evolves+tolerogenic+properties.">Application of allogeneic bone marrow cells in view of residual alloreactivity: Sirolimus but not cyclosporine evolves tolerogenic properties.</a> PLoS ONE. 10 (4), 1-16 (2015).</li><li>Hadamitzky, 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=Memory-updating+abrogates+extinction+of+learned+immunosuppression.">Memory-updating abrogates extinction of learned immunosuppression.</a> Brain, Behavior, and Immunity. 52, 40-48 (2016).</li><li>Beetz, 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=Recipient+natural+killer+cells+alter+the+course+of+rejection+of+allogeneic+heart+grafts+in+rats.">Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats.</a> Plos One. 14 (8), e0220546 (2019).</li><li>Hirschburger, 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=Nicotine+Attenuates+Macrophage+Infiltration+in+Rat+Lung+Allografts.">Nicotine Attenuates Macrophage Infiltration in Rat Lung Allografts.</a> The Journal of Heart and Lung Transplantation. 28 (5), 493-500 (2009).</li><li>Ruzza, 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=Heterotopic+heart+transplantation+in+rats:+improved+anesthetic+and+surgical+technique.">Heterotopic heart transplantation in rats: improved anesthetic and surgical technique.</a> Transplantation Proceedings. 42 (9), 3828-3832 (2010).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+technique+for+heart+transplantation+in+rats:+abdominal+vessel+branch-sparing+and+modified+venotomy.">A simplified technique for heart transplantation in rats: abdominal vessel branch-sparing and modified venotomy.</a> Microsurgery. 26 (6), 470-472 (2006).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+method+for+heterotopic+mouse+heart+transplantion.">A modified method for heterotopic mouse heart transplantion.</a> Journal of Visualized Experiments. (88), (2014).</li><li>Al-Amran, F. G., Shahkolahi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+arterial+anastomosis+heterotopic+heart+transplantation+model.">Total arterial anastomosis heterotopic heart transplantation model.</a> Transplantation Proceedings. 45 (2), 625-629 (2013).</li><li>Hoerstrup, S. P., 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=Modified+technique+for+heterotopic+rat+heart+transplantation+under+cardioplegic+arrest.">Modified technique for heterotopic rat heart transplantation under cardioplegic arrest.</a> Journal of Investigative Surgery. 13 (2), 73-77 (2000).</li><li>Fry, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+vascular+endothelial+changes+associated+with+increased+blood+velocity+gradients.">Acute vascular endothelial changes associated with increased blood velocity gradients.</a> Circulation Research. 22 (2), 165-197 (1968).</li><li>Ahmadi, A. 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=Orthotopic+Rat+Kidney+Transplantation:+A+Novel+and+Simplified+Surgical+Approach.">Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach.</a> Journal of Visualized Experiments. (147), (2019).</li><li>Schmid, C., 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=Successful+heterotopic+heart+transplantation+in+rat.">Successful heterotopic heart transplantation in rat.</a> Microsurgery. 15 (4), 279-281 (1994).</li><li>Shan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+technique+for+heterotopic+heart+transplantation+in+rats.">A modified technique for heterotopic heart transplantation in rats.</a> Journal of Surgical Research. 164 (1), 155-161 (2010).</li><li>Moris, D., 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=Mechanisms+of+liver-induced+tolerance.">Mechanisms of liver-induced tolerance.</a> Current Opinion in Organ Transplantation. 22 (1), 71-78 (2017).</li><li>Bickerstaff, A. 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=Murine+renal+allografts:+spontaneous+acceptance+is+associated+with+regulated+T+cell-mediated+immunity.">Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity.</a> Journal of Immunology. 167 (9), 4821-4827 (2001).</li><li>Mottram, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocardiographic+monitoring+of+cardiac+transplants+in+mice.">Electrocardiographic monitoring of cardiac transplants in mice.</a> Cardiovascular Research. 22 (5), 315-321 (1988).</li><li>Torre-Amione, 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=Decreased+expression+of+tumor+necrosis+factor-&#945;+in+failing+human+myocardium+after+mechanical+circulatory+support:+A+potential+mechanism+for+cardiac+recovery.">Decreased expression of tumor necrosis factor-&#945; in failing human myocardium after mechanical circulatory support: A potential mechanism for cardiac recovery.</a> Circulation. 100 (11), 1189-1193 (1999).</li><li>Goldstein, D. J., 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=Circulatory+resuscitation+with+left+ventricular+assist+device+support+reduces+interleukins+6+and+8+levels.">Circulatory resuscitation with left ventricular assist device support reduces interleukins 6 and 8 levels.</a> The Annals of Thoracic Surgery. 63 (4), 971-974 (1997).</li><li>Tang-Quan, K. 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=Non-volume-loaded+heart+provides+a+more+relevant+heterotopic+transplantation+model.">Non-volume-loaded heart provides a more relevant heterotopic transplantation model.</a> Transplant Immunology. 23 (1-2), 65-70 (2010).</li><li>Lakhal-Naouar, I., 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=Transcutaneous+immunization+using+SLA+or+rLACK+skews+the+immune+response+towards+a+Th1+profile+but+fails+to+protect+BALB/c+mice+against+a+Leishmania+major+challenge.">Transcutaneous immunization using SLA or rLACK skews the immune response towards a Th1 profile but fails to protect BALB/c mice against a Leishmania major challenge.</a> Vaccine. 37 (3), 516-523 (2019).</li><li>Sousa-Batista, A. J., 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=Novel+and+safe+single-dose+treatment+of+cutaneous+leishmaniasis+with+implantable+amphotericin+B-loaded+microparticles.">Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-loaded microparticles.</a> International Journal for Parasitology: Drugs and Drug Resistance. , (2019).</li></ol>

The previously described method of heterotopic cardiac transplantation in rats is mainly based on the description of Ono and Lindsey in 19693. Since then, several modifications have been introduced in various species leading to a wide diversity of this model. Combining several of these modifications and introducing our own experience resulting from over 30 years of performing heterotopic heart transplants in the laboratory, we created a feasible surgical approach, which does not require long train...

Heterotopic heart transplantation is a frequently used experimental model for different investigations regarding transplantation tolerance, acute and chronic allograft rejection, ischemia-reperfusion injury, machine perfusion or cardiac remodelling. Among other advantages, the graft function can be monitored noninvasively by palpation and graft failure does not lead to a vital impairment of the recipient in contrast to other organs, such as kidneys or livers.
In 1964, Abbott et al. initially described heterotopic abdominal heart transplantation in rats1. Later, in 1966, the end-to-side technique for anastomoses was described by Tomita et al.2. The groundwork for the currently used model was reported by Ono and Lindsey in 19693. During the last decades, several modifications have been published to create different types of unloaded, partially loaded or loaded left ventricular heart grafts including combined heterotopic heart-lung transplantation4,5,6. For immunological analyses a non-volume loaded heart graft transplantation is most commonly performed. In this case, the blood flow retrogradely enters the donor ascending aorta and subsequently the coronary arteries. The venous drainage occurs along the coronary sinus into the right atrium and ventricle (Figure 1A-B). Therefore, the left ventricle is excluded from blood flow, apart from marginal amounts of blood from Thebesian veins. This also makes it a useful model for studying the pathophysiological mechanisms during left ventricular assist device therapy7.
Heterotopic heart transplantation has been performed in various species including mice, rabbits, pigs and has even been used as a uni- or biventricular assist device in humans8,9,10,11. The rat still represents a popular experimental animal for transplant models, especially since the graft survival times for different rat strain combinations have been well-defined in the past and a large number of immunological reagents are accessible12,13. Unlike mice, rats are larger making surgery and access to lymphatic tissue for immunological analyses more feasible12. Furthermore, the introduction of commercial cloning technologies in rats in recent years will most likely lead to a recurring interest in experimental rat models14.
In general, heterotopic heart grafts can be attached to the recipient vessels either by performing cervical or abdominal anastomosis. However, a few studies suggest that a femoral anastomosis facilitates improved monitoring due to better access for manual palpation or transfemoral echocardiography and thus allows a more precise detection of graft failure15,16.
It has been shown that there is no difference regarding operation time, complication rate, outcome and graft survival time between both anastomosis techniques17. Clearly, the availability of a sufficient number of draining lymph nodes must be mentioned as a benefit of cervical anastomosis; however, longer training periods are required. In contrast, the abdominal anastomosis is less complicated and equally valuable for immunological investigations, especially when combined with results from a novel method of in-ear injection of allogenic cardiac muscle cells and subsequent cervical lymphadenectomy. A combination of both models offers a broad spectrum of post-interventional immunological analyses.
The following protocol refers to operating in pairs of surgeons in order to reduce ischemia time. However, all experiments can be performed by a single person. The setup of instruments and materials for heart explantation and implantation is displayed in Figure 2A-B.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia device (including isoflurane vaporizer)</td>
			<td>Summit Anesthesia Solutions</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula (27 G)</td>
			<td>BD Microlance</td>
			<td>302200</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer</td>
			<td>Rimadyl 50 mg/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar Tubes (15 mL)</td>
			<td>GreinerBioOne</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (40 &#181;m)</td>
			<td>BD Falcon</td>
			<td>2271680</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type CLSII</td>
			<td>Biochrome</td>
			<td>C2-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresses 5x5 cm</td>
			<td>Fuhrmann</td>
			<td>31501</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresses 7.5x7.5 cm</td>
			<td>Fuhrmann</td>
			<td>31505</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Heinz Herenz Medizinalbedarf</td>
			<td>1032128</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexpathenol (5 %)</td>
			<td>Bayer</td>
			<td>&#34;Bepanthen&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS BioWhittaker</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>B. Braun</td>
			<td>Aesculap BD557R</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>B. Braun</td>
			<td>Aesculap BD313R</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>B. Braun</td>
			<td>Aesculap BD35</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating mat</td>
			<td>Gaymar Industries</td>
			<td>&#34;T/Pump&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic gauze</td>
			<td>Ethicon</td>
			<td>Tabotamp</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-Natrium 25 000 I.E.</td>
			<td>Ratiopharm</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran CP</td>
			<td>CP-Pharma</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Large-pored sieve (stainless steel)</td>
			<td>Forschungswerkst&#228;tten Hannover Medical School</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Astra Zeneca</td>
			<td>2 % Xylocain</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamizol-Natrium</td>
			<td>Ratiopharma</td>
			<td>Novaminsulfon 500 mg/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>B. Braun</td>
			<td>Aesculap BD3361</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>Codman, Johnson &#38; Johnson Medical</td>
			<td>Codmann 80-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>B. Braun</td>
			<td>Aesculap BD336R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>B. Braun</td>
			<td>Aesculap FD241R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>B. Braun</td>
			<td>Aesculap FD101R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>B. Braun</td>
			<td>Aesculap FM471R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>B. Braun</td>
			<td>Aesculap BM221R</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Glutamine (100x)</td>
			<td>PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (18 G)</td>
			<td>B. Braun</td>
			<td>4268334B</td>
			<td></td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (22 G)</td>
			<td>B. Braun</td>
			<td>4268091B</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe pointed scissors</td>
			<td>B. Braun</td>
			<td>Aesculap BC030R</td>
			<td></td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Forschungswerkst&#228;tten Hannover Medical School</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI culture medium</td>
			<td>Lonza</td>
			<td>BE12-702F</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution (NaCl 0.9 %)</td>
			<td>Baxter</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>B. Braun</td>
			<td>Aesculap BC414</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Carl-Zeiss</td>
			<td>OPMI-MDM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (anastomoses)</td>
			<td>Catgut</td>
			<td>Mariderm 8-0 monofil</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (ligature)</td>
			<td>Resorba</td>
			<td>Silk 5-0 polyfil</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (skin, fascia)</td>
			<td>Ethicon</td>
			<td>Mersilene 3-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>B. Braun</td>
			<td>9166017V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>B. Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (20 mL)</td>
			<td>B. Braun</td>
			<td>4606205V</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamp</td>
			<td>B. Braun</td>
			<td>Aesculap FB708R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunological model for heterotopic heart and cardiac muscle cell transplantation in rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

In the past, different immunological issues have been addressed on the basis of the model, which was validated in the work group by more than 500 transplants with a survival rate of more than 95%13,18,19,20,21,22,23,24. Tota...

Watch this Scientific Journal Video about An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats at JoVE.com

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Transplant models are valuable tools for immunological research that often require surgical expertise. Here, we present a feasible approach for heterotopic heart and cardiac muscle cell transplantation in rats. By highlighting and simplifying all of the crucial steps of our model, researchers without a surgical background should be able to master this model in a reasonable amount of time.Both transplant models provide valuable insights into the mechanisms of tolerance and rejection and can be applied in various species. We have a lot of experience in teaching this model to individuals without prior surgical training. After learning the crucial steps, we advice patience and sufficient practice with deceased animals.For donor heart explantation, after confirming a lack of response to pedal reflex in anesthetized seven to 22 week old rat donor animal, apply eye lubricant and use mechanical clippers to remove the abdominal and thoracic fur. Place the rat in the supine position on a heating base and fix the limbs at the base of the operation table with elastic bands. Disinfect the exposed skin with 70%ethanol and perform a median laparotomy.Insert retractors into the incision and use sterilized cotton swabs to mobilize the intestine to the left side of the donor to expose the inferior vena cava. Next, puncture the inferior vena cava to allow intravenous injection of 500 International Units of heparin dissolved in one milliliter of ice-cold isotonic saline solution. Stop the bleeding at the punctured site after needle retraction by light compression using a cotton swab.Incise the diaphragm, perform a lateral thoracotomy to both sides of the donor and pin the thorax wall. Remove the pericardium and the vagal nerve by blunt preparation using two micro-needle holders. For exsanguination, transect the abdominal vessels and insert the blunt branch of a probe pointed scissors into the transverse pericardial sinus.Use a compress to dissect the ascending aorta and pulmonary artery as distally as possible under light caudal traction of the heart. Place a single 5-0 ligature around the superior and inferior vena cava and the pulmonary veins and tighten the ligature step-wise as dorsally as possible. Sever the tissue dorsal to the ligature and extract the heart.To perfuse the explanted heart, use an 18 gauge cannula from an intravenous catheter to flush 30 milliliters of ice-cold, isotonic saline solution supplemented with 1000 International Units of heparin through the ascending aorta and the pulmonary artery before placing the heart in a 15 milliliter tube of saline solution on ice. After preparing the 10 to 14 week old recipient rat as just demonstrated, mobilize the intestines to the upper-left side of the recipient onto a warm, wetted compress. And adjust the surgical microscope or a sufficient alternative.After mobilizing the duodenum and proximal jejunum, respectively, using a five to seven full magnification, use cotton swabs and blunt dissection to expose the abdominal aorta and inferior vena cava. Using two micro needle holders to elevate the abdominal vessels without injuring the lumbar veins, place a Cooley vascular clamp onto the vessels. Use a 30 to 45 degree arched 27 gauge cannula to puncture the abdominal vessels.And use Potts scissors to enlarge the puncture site into a longitudinal incision that matches the size of the lumen of the donor vessels. To remove clots and to prevent postoperative thrombosis, perfuse the vessels with saline and place the graft into the situs. Using two simple interrupted 8-0 monofilament non-resorbable sutures, fix the donor ascending aorta to the recipient abdominal aorta at the cranial and caudal corners of the longitudinal incision.To anastomose the ascending aorta of the donor with the abdominal aorta of the recipient place the graft to the right of the recipient vessels while performing the first half of the anastomosis with a running 8-0 monofilament suture. Then place the graft to the left of the recipient vessels and perform the second half of the anastomosis. Fix the donor pulmonary artery to the inferior vena cava in a similar manner with two corner knots at the cranial and caudal corners.After fixing the donor vessel with knots at each corner, place a stitch from the outside to the inside of the vessel to run an intraluminal anastomosis for the first half of the anastomosis. Before tying down the suture, place another stitch from the inside to the outside. To prevent peripheral embolism after finishing the second half of the anastomosis, flush the anastomosis with saline before tightening the knot of the second half of the anastomosis.Place a hemostatic gauze around both anastomoses and carefully release the Cooley vascular clamp to allow graft reperfusion. Managing any bleeding with light compression and sterilized cotton swabs. After about 60 seconds, the heart should start beating.Carefully replace the intestines and close the abdominal fascia and the skin with separate continuous 3-0 polyfilament running sutures. At the appropriate experimental time point, harvest a donor rat heart as demonstrated and use a sterile scalpel or sterile scissors to shred the heart into three by three millimeter fragments. Digest the pieces in 20 milliliters of culture medium supplemented with 0.5 milligrams per milliliter of collagenase for 30 minutes at 37 degree Celsius.At the end of the digestion, add the tissue to a large-pored sieve, while removing the culture medium. Add five to 10 milliliters of isotonic saline solution and strain the tissue slurry through the sieve. And after centrifuging the cell suspension twice, filter it through a 40 micrometer cell strainer.Rinse the strainer with five to 10 milliliters of isotonic saline solution to collect any remaining cells. And after centrifugation, resuspend the isolated rat cardiac cells in saline solution at a concentration of five times 10 to the fifth cells per milliliter. Load a one milliliters syringe with the cell solution and place the anesthetized recipient animal in a lateral position with the target ear facing up.Fix the ear with a finger and double-sided tape, and use a 27 gauge cannula to subcutaneously inject 20 microliters of cardiac muscle cell solution close to the visual capillary vessels into the recipient's ear. After the appropriate experimental observation period, extract the draining cervical lymph nodes and perform the appropriate downstream analyses of interest. Syngenic transplants survived up to 100 days without signs of graft failure.The specific donor-recipient combination can result in different speeds of heterotopic heart transplant rejection. Cryostat sections of the rejected hearts reveal an increase infiltration of immune effector cells, whereas syngenic grafts are largely free of immune cells. Cervical lymphadenectomy and re-stimulation assays of draining lymph node cells after oral cardiac muscle cell injection reveals distinct strain specific immune responses toward heterotopic cardiac tissue in allogenic transplant recipients, warranting further immunological analyses such as cytokine profiling.Handle the vessels carefully when inserting and extracting the needle. Don't put too much attention on the sutures to prevent stenosis. Don't place too many stitches for each anastomosis.

an-immunological-model-for-heterotopic-heart-cardiac-muscle-cell

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...