Name: optimization of the cuff technique for murine heart transplantation
Uploaded: 2020-06-26T13:00:00
Description: Watch this Scientific Journal Video about Optimization of the Cuff Technique for Murine Heart Transplantation at JoVE.com

This work was supported by the Fujian Provincial Health Education Joint Research Project (WKJ2016-2-20), the National Natural Science Foundation of China (81771271 and 81800664), the National Key R&#38;D Program of China (2018YFA0108304) and the Education and Scientific Research Project for Young and Middle-aged Teachers in Fujian Province (JAT170714), Natural Science Foundation of Hunan Province of China (2019JJ50842) and Huxiang Young Talents of Hunan Province (2019RS2013).

Animals (BALB/c, C57BL/6, male, 8-12 weeks) are housed in a specific pathogen-free facility at the Xiamen University Laboratory Animal Center. C57BL/6 is used as recipient and BALB/c is used as donor. All the procedures are performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines.
NOTE: A set of microsurgical instruments, including a micro scissor, micro straight forceps, micro curved forceps and micro needle holders, are necessary for the operation (Ta...

<ol>
	<li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+transplantation+in+congenic+strains+of+mice.">Heart transplantation in congenic strains of mice.</a> Transplantation Proceedings. 5 (1), 733-735 (1973).</li><li>Que, 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=Prolonged+cold+ischemia+time+in+mouse+heart+transplantation+using+supercooling+preservation.">Prolonged cold ischemia time in mouse heart transplantation using supercooling preservation.</a> Transplantation. , (2019).</li><li>Wang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+murine+cardiac+allograft+arteriopathy+by+long-term+blockade+of+CD40-CD154+interactions.">Suppression of murine cardiac allograft arteriopathy by long-term blockade of CD40-CD154 interactions.</a> Circulation. 105 (13), 1609-1614 (2002).</li><li>Hasegawa, T., Visovatti, S. H., Hyman, M. C., Hayasaki, T., Pinsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+vascularized+murine+cardiac+transplantation+to+study+graft+arteriopathy.">Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy.</a> Nature Protocols. 2 (3), 471-480 (2007).</li><li>Chen, Z. H. <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+of+cervical+heterotopic+heart+transplantation+in+mice.">A technique of cervical heterotopic heart transplantation in mice.</a> Transplantation. 52 (6), 1099-1101 (1991).</li><li>Matsuura, A., Abe, T., Yasuura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+mouse+cervical+heart+transplantation+using+a+cuff+technique.">Simplified mouse cervical heart transplantation using a cuff technique.</a> Transplantation. 51 (4), 896-898 (1991).</li><li>Wang, Q., Liu, Y., Li, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+technique+for+heterotopic+vascularized+cervical+heart+transplantation+in+mice.">Simplified technique for heterotopic vascularized cervical heart transplantation in mice.</a> Microsurgery. 25 (1), 76-79 (2005).</li><li>Li, 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=Surgical+technique+for+lung+retransplantation+in+the+mouse.">Surgical technique for lung retransplantation in the mouse.</a> Journal of Thoracic Disease. 5 (3), 321-325 (2013).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+experience+with+five+hundred+thirty+liver+transplants+in+the+rat.">A surgical experience with five hundred thirty liver transplants in the rat.</a> Surgery. 93 (1), 64-69 (1983).</li><li>Chen, H., Zhang, Y., Zheng, D., Praseedom, R. K., Dong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+kidney+transplantation+in+mice:+technique+using+cuff+for+renal+vein+anastomosis.">Orthotopic kidney transplantation in mice: technique using cuff for renal vein anastomosis.</a> PLoS One. 8 (10), 77278 (2013).</li><li>Miller, M. 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=Spontaneous+restoration+of+transplantation+tolerance+after+acute+rejection.">Spontaneous restoration of transplantation tolerance after acute rejection.</a> Nature Communications. 6, 7566 (2015).</li><li>Lin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Jagged-1+combined+with+blockade+of+CD40+pathway+prolongs+allograft+survival.">Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival.</a> Immunology and Cell Biology. 93 (2), 213-217 (2015).</li><li>Xie, B., 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=Combined+costimulation+blockade+inhibits+accelerated+rejection+mediated+by+alloantigen-primed+memory+T+cells+in+mice.">Combined costimulation blockade inhibits accelerated rejection mediated by alloantigen-primed memory T cells in mice.</a> Immunological Investigations. 38 (7), 639-651 (2009).</li><li>Shao, 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=Combination+of+monoclonal+antibodies+with+DST+inhibits+accelerated+rejection+mediated+by+memory+T+cells+to+induce+long-lived+heart+allograft+acceptance+in+mice.">Combination of monoclonal antibodies with DST inhibits accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.</a> Immunology Letters. 138 (2), 122-128 (2011).</li><li>Dai, 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=Blockade+of+CD27/CD70+pathway+to+reduce+the+generation+of+memory+T+cells+and+markedly+prolong+the+survival+of+heart+allografts+in+presensitized+mice.">Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice.</a> Transplant Immunology. 24 (4), 195-202 (2011).</li><li>Yan, 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=Inhibition+of+accelerated+rejection+mediated+by+alloreactive+CD4(+)+memory+T+cells+and+prolonged+allograft+survival+by+arsenic+trioxide.">Inhibition of accelerated rejection mediated by alloreactive CD4(+) memory T cells and prolonged allograft survival by arsenic trioxide.</a> Immunological Investigations. 42 (5), 438-454 (2013).</li><li>Yan, 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=Inhibiting+accelerated+rejection+mediated+by+alloreactive+CD4(+)+memory+T+cells+and+prolonging+allograft+survival+by+1alpha,25-dihydroxyvitamin+D(3)+in+nude+mice.">Inhibiting accelerated rejection mediated by alloreactive CD4(+) memory T cells and prolonging allograft survival by 1alpha,25-dihydroxyvitamin D(3) in nude mice.</a> Immunology Letters. 149 (1-2), 54-61 (2013).</li><li>Lin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+trioxide+is+a+novel+agent+for+combination+therapy+to+prolong+heart+allograft+survival+in+allo-primed+T+cells+transferred+mice.">Arsenic trioxide is a novel agent for combination therapy to prolong heart allograft survival in allo-primed T cells transferred mice.</a> Transplant Immunology. 25 (4), 194-201 (2011).</li><li>Shao, 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=CD44/CD70+blockade+and+anti-CD154/LFA-1+treatment+synergistically+suppress+accelerated+rejection+and+prolong+cardiac+allograft+survival+in+mice.">CD44/CD70 blockade and anti-CD154/LFA-1 treatment synergistically suppress accelerated rejection and prolong cardiac allograft survival in mice.</a> Scandinavian Journal of Immunology. 74 (5), 430-437 (2011).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+reproducible+cervical+cuff+technique+for+rat-to-mouse+heterotopic+heart+xenotransplantation.">A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.</a> Xenotransplantation. , (2017).</li><li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> Journal of Visualized Experiments. (92), e50753 (2014).</li><li>Blanchard, J. M., Pollak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+perfusion+and+storage+of+heterotopic+heart+transplants+in+mice.">Techniques for perfusion and storage of heterotopic heart transplants in mice.</a> Microsurgery. 6 (3), 169-174 (1985).</li><li>Felix, N. 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=H2-DMalpha(-/-)+mice+show+the+importance+of+major+histocompatibility+complex-bound+peptide+in+cardiac+allograft+rejection.">H2-DMalpha(-/-) mice show the importance of major histocompatibility complex-bound peptide in cardiac allograft rejection.</a> Journal of Experimental Medicine. 192 (1), 31-40 (2000).</li><li>Tomita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+of+heterotopic+cervical+heart+transplantation+in+mice.">Improved technique of heterotopic cervical heart transplantation in mice.</a> Transplantation. 64 (11), 1598-1601 (1997).</li><li>Niimi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+for+heterotopic+cardiac+transplantation+in+mice:+experience+of+3000+operations+by+one+surgeon.">The technique for heterotopic cardiac transplantation in mice: experience of 3000 operations by one surgeon.</a> Journal of Heart and Lung Transplantation. 20 (10), 1123-1128 (2001).</li><li>Wang, K., Zhang, N., Li, H. <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+mouse+heterotopic+heart+graft+retransplantation.">Improved technique of mouse heterotopic heart graft retransplantation.</a> Microsurgery. 26 (3), 200-202 (2006).</li><li>Plenter, R. J., Grazia, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+heterotopic+heart+transplant+technique.">Murine heterotopic heart transplant technique.</a> Journal of Visualized Experiments. (89), (2014).</li><li>Ratschiller, T., 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+Cervical+Heart+Transplantation+in+Mice.">Heterotopic Cervical Heart Transplantation in Mice.</a> Journal of Visualized Experiments. (102), e52907 (2015).</li><li>Zhou, Y., Gu, X., Xiang, J., Qian, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+suture+versus+cuff+anastomosis+in+mouse+cervical+cardiac+transplant.">A comparative study on suture versus cuff anastomosis in mouse cervical cardiac transplant.</a> Experimental and Clinical Transplantation. 8 (3), 245-249 (2010).</li><li>Fukunaga, N., Bissoondath, V., Rao, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submandibular+Gland-preserving+Technique+for+Heterotopic+Cervical+Heart+Transplantation+in+Mice.">Submandibular Gland-preserving Technique for Heterotopic Cervical Heart Transplantation in Mice.</a> Transplantation. 102 (11), 464-465 (2018).</li></ol>

Mouse heart transplants models are important tools for transplant immunology research, as tools and materials for evaluating the immune mechanisms of this model and a large number of gene-modified mice are available. However, microsurgical technical challenges, such as vessels suture and eversion, have limited its widespread use. In the present study, we have investigated certain core technical challenges of murine heart transplantation and have obtained good outcomes. A critical step of the protocol the insertion of an ...

The establishment of mouse heterotopic heart transplantation through end-to-end anastomosis within the abdomen in 1973 was a major milestone in basic transplant immunology research1. This model provided an important and valid tool for analyzing the mechanisms of ischemia reperfusion injury2, immunological rejection, and tolerance3,4. However, the complex and time-consuming nature of the surgery as well as the potential for infections can result in severe perioperative abdominal adhesions and inflammatory reactions, resulting in a low efficiency for heterotopic heart transplantation model.
The cervical heterotopic heart transplantation technique was first described by Chen in 19915. In this model, the recipient&#8217;s external jugular vein is anastomosed to the pulmonary artery of the graft and the carotid artery is anastomosed to the ascending aorta. The main advantages of this method are the convenience of monitoring and the reducing of trauma to the recipient. In the same year, Matsuura described an improved technique, in which the end of the external jugular vein and carotid artery were everted over a Teflon cuff and fixed with a circumferential silk ligature6. Some researchers also fixed the cuff to the right pulmonary artery in the donor heart before inserting the cuff into the external jugular vein of the recipient7. Thus far, the cuff technique has been widely applied in various vascular pedicle transplant models, including those for lung8, liver9, and renal10 transplantation.
To date, there are several difficulties associated with the cuff technique. For example, the carotid artery is difficult to evert over the cuff due to the additional elasticity, resulting in the tissue flipping backwards. Hence, additional practice and a microsurgical dilator may be required to complete this step. In addition, the cervical vessel preparation can take up to 25 minutes.
To resolve these issues, we introduce the inner tube technique, which is based on the cuff technique and includes fixing the cuff on the external jugular vein and carotid artery using an inner tube to helps with the eversion of the vessel wall. In addition, with simple training, the recipient preparation is reduced to 15.5 minutes. This technique reduces the complexity of the operation and does not require additional practice or the use of a vascular dilator. It can be applied in all transplantation immune research, especially for verifying third-party immune tolerance during which the recipient receives two cardiac allografts, one within the abdomen and the other in the neck11. We also recommend co-operation between two skilled surgeons to establish this model, with one surgeon preparing the recipient animal and the other harvesting and implanting the donor heart. Such collaboration can shorten the operation time to 25 minutes. Using this optimized procedure, we have established syngeneic, allogeneic12,13,14,15,16,17,18,19, and xenogeneic mouse heart transplantation models20.
The rationale for the development of the inner tube technique was to reduce the operation time for the establishment of a mouse heart transplant model with a high success rate. Optimization of the cervical heart transplantation model facilitates the acquisition of high success rates in a short period of surgery time compared to the traditional suture and cuff technique21. Further, the cooperation model can further reduce the warm ischemic time of the donor heart compared to surgeries performed with a single operator.

<table>
	<tbody>
		<tr>
			<td>Artery cuff</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. diameter: 0.55 mm,length: 1.0 mm</td>
		</tr>
		<tr>
			<td>Artery inner tube</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. Diameter: 0.28mm</td>
		</tr>
		<tr>
			<td>Micro curved forceps</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA3050</td>
			<td>1/8 arc, 0.3-mm tip without a hook</td>
		</tr>
		<tr>
			<td>Micro needle holders</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA2050</td>
			<td>0.2-mm tip</td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA1050</td>
			<td>Straight, blade length: 10 mm</td>
		</tr>
		<tr>
			<td>Micro straight forceps</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA3060</td>
			<td>0.15-mm tip without a hook</td>
		</tr>
		<tr>
			<td>Scanlan Vascu-Statt Bulldog Clamps</td>
			<td>Scanlan International Inc</td>
			<td>1001-531</td>
			<td>Clamping pressure 20&#8211;25 grams</td>
		</tr>
		<tr>
			<td>Vein cuff</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. diameter: 0.9 mm,length: 1.2 mm</td>
		</tr>
		<tr>
			<td>Vein inner tube</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. Diameter: 0.6 mm</td>
		</tr>
	</tbody>
</table>

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.

Surgical Operation Time
After training, a skilled surgeon can successfully perform the operation within 35 minutes using the inner tube technique, wherein approximately 15.5 minutes are required for recipient preparation, 10.9 minutes are required for donor preparation, and 4.4 minutes are required for donor heart anastomoses. The cold and warm ischemia time (from donor preparation to heart implantation) is reduced to 15.3 minutes compared to the operation using the cuff techn...

Watch this Scientific Journal Video about Optimization of the Cuff Technique for Murine Heart Transplantation at JoVE.com

Optimization of the Cuff Technique for Murine Heart Transplantation

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 ...

Hello, everyone. My name is Dr.Zhongquan Qi.It's widely known that the cuff technique has been used as cardiac transplantation since in 1991. Although there have been great advancements in medical surgery and the new surgical technique, there are still some difficulties based on cuff technique.It's a form of unveiling, where they will show you an optimotic half technique, which can significantly equal the surgical efficiency. The main procedures is inserting a temporarily in that tube into the visor. Proof read the ISO almost half.Inserting the tube technique. A skilled surgeon can perform the operation within 35 minutes with 95%success rate. Then they can employ the Thermal Surgeon Operation Module, which further improves the efficiency of the operation showing in the skeptimic and the operation time will be shortened to about 35 minutes.Thank you. And that's that ties the recipe and miles with pentobarbital. Use atraumatic mechanical clippers to remove the hair at the right later cervical region.Use a sterile cotton tip applicator to wipe the surgical area with iodine antiseptic followed by 70%ethanol. Place the mouse in the supine position on the operation platform. Cover the mouse with sterile gauze.Use an ophthalmic scissor to make a transverse incision from the lower one third NACMEC line to the right shoulder clavicle joint. Isolate the right external jungular vein with micro curved forceps to expose enough length. Cut off the brunches via electro coagulation.And locate the vessel at the disto and using a six 2 0 six suture. Clamp the external junglar vein proximally using a powder clamp. And then translate the vein proximately to the ligature using a micro scissor.Wash the vessel lumen with 100 units per milliliter 02 four Celsius degree heparin salient to remove any residual blood. Pull the external junglar vein through the vena cava using micro straight forceps. Insert the vein inner tube into the lumen as a stent and over the vessel wall over the curve with micro straight forceps.Fix the irritant Faso endothelium at the proximal end of the curve using a circumferential eight to 06 suture. Use micro straight forceps to withdraw the vein inner tube from the vein vessel. Perform blunt dissection with micro forceps to isolate the right carotid artery, adjacent to the inner edge of the sternocleidomastoid.Clamp the right carotid Artery proximally using a babcock clamp. Locate the carotid artery distally using a six to 06 suture. And use a micro scissor to transact the carotid artery proximally to the ligature.Wash the carotid artery with 100 units per milliliter 02 four Celsius heparin salient to remove any residual blood. Pass the carotid artery through the artery curve and insert the artery inner tube into the artery vessel using micro straight forceps. Aim for the vessel over the curve using micro straight forceps.Fix the inverted facile endothelium using a circumferential eight to 06 suture. Withdraw the artery inner tip from the artery vessel with micro straight forceps. Anaesthesia and its infection are the same as the recipient.Make an abdominal mid line incision with an ophthalmic scissor and expose the abdominal cavity. Use micro curve forceps to expose the inferior vena cava. And then intravenously inject 200 microliter of 100 units per milliliter 02 four Celsius degree heparin salient per 20 gram of body weight through the inferior vena cava.Perform thoracotomy with ophthalmic scissors. Cut off the ribs through the palettes Romita axillary light incisions. Flav the anterior chest wall outwards to expose the thoracic cavity.Excise the thymus with micro forceps. Expose the aorta and then profuse 200 microliter of 100 units per milliliter 02 four Celsius degree heparin salient to the coronary artery through the aortic arch. Use a micro scissor to transect the ascending aorta at the beginning of the aortic arch.Transect the pulmonary artery at the beginning of the two main branches with a micro scissor. Locate the superior vena cava and inferior vena cava approximately using a six to 02 suture and use a micro scissor to transect the vein distillate to the ligature. Locate the pulmonary veins together circumferentially using a single six, to 06 suture and cut off the vein branches distally to the ligature using a micro scissor.Remove the heart graft from the surrounding soft tissues. Place the donor heart upside down into the right neck region of the recipient. Input the pulmonary artery of the donor heart to a six to 06 loop with micro straight forceps.Wrap the vessel lumen around the vena cava and then tighten the six to 06 suture loop around the calf to bend the vessel joint. Perform anastomosis of the aorta of the graft and artery curve. Release the clumped jugular vein followed by clumped jugular artery.Keep the vessel joined and twisted and ensure that the blood flow is unobstructed. Set the passing heart graft into the subcutaneous space and then suture the incision. Record the time to normal sinus rhythm and the preservation of normal sinus rhythm for at least five minutes after clamp release to monitor the post operative graft function.Place the recipient alone on the one blanket until the recipient wakes up from anesthesia. Taken together, although our surgery uses cuff techniques that have been reported, we established the inner core technique based on this. Reduce the difficulty in surgery and shorten the anastomosis time by optimization technique.We suggested the double surgery incorporation motto to further improve the efficiency of the operation shown in the schematic. And the operation time will be shortened to about 25 minutes. A fully MHC mismatched BALBC allograft heart can be rejected with eight days after transplantation into C57BL6 recipient mice.Syngeneic heart transplants used in our research survived more than 100 days except one rare case due to a 15%weight loss compared to normal weight before the operation. Histopathology of the allograft on day seven revealed typical features of acute rejection, including a heavy cellular infiltrate with tissue necrosis. Syngeneic grafts are near normal with no evidence of myocyte necrosis or inflammatory cell infiltration.100 days after syngenetic heart transplant, the vascular endothelium of anastomosis site can be collected and stained by immunofluorescent. In this analysis, no averse snaring of vascular wall, thrombosis or thickening of the intima were observed. Electro microscopic imaging revealed that a smooth endothelium and a regular longitudinal crust formation with the endothelial cells arranged neatly and closely with no obvious sediment on the surface.

optimization-of-the-cuff-technique-for-murine-heart-transplantation

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 ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

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 ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.