Name: a modified method for heterotopic mouse heart transplantion
Uploaded: 2014-06-23T19:00:00
Description: Watch this Scientific Journal Video about A Modified Method for Heterotopic Mouse Heart Transplantion at JoVE.com

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia Project Grant 1029205, by the Microsearch Foundation of Australia, and by the Myee Codrington Medical Research Foundation.

Prior to commencement of experiments, obtain approval from the relevant institution&#8217;s Animal Care Ethics Committee for the planned experiments. Maintain mice in the accordance with the requirements of your institution. The following protocol has been approved by Sydney University and Royal Prince Alfred Hospital committees.
Prior to commencing surgery, all instruments should be cleaned thoroughly and sterilized by soaking in 80% ethanol. Some institutions stipulate autoclaving however th...

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	<li>Aramaki, 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=Interleukin-10+but+not+transforming+growth+factor-beta+is+essential+for+generation+and+suppressor+function+of+regulatory+cells+induced+by+intratracheal+delivery+of+alloantigen.">Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen.</a> Transplantation. 79, 568-576 (2005).</li><li>Chen, R. H., Bushell, A., Fuggle, S. V., Wood, K. J., Morris, P. 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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> J. Thorac. Cardiovasc. Surg. 57, 225-229 (1969).</li><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=Primarily+vascularized+allografts+of+heart+in+mice.">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li><li>Larsen, C. 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Mouse heart transplantation is a challenging microsurgical method that requires considerable surgical skill to master. The most challenging aspect is the small diameter of the vessels. In addition, it is necessary to limit the recipient operation time and bleeding. The technique for mouse heart transplantation was first described by Corry et al. in 1973 and subsequently by Mottram et al12. Our modifications include the following points. Firstly, immediate perfusion of the donor heart with col...

Animal models of organ transplantation can provide valuable information for improving treatment of clinical transplant patients. Mouse models are particularly useful for studying the immune mechanism of organ transplant rejection or acceptance due to the wide range of genetically modified mice and reagents specific for mice that are not available for other animal models1-3. A challenge with mouse models of transplantation is the small size of the donors and recipients which requires considerable technical skill to achieve satisfactory outcomes.
A technique was first described for heterotopic transplantation of hearts in rats4 which was subsequently adapted for mouse heart transplantation by Corry et al5. This technique involved preparing the recipient prior to the donor operation and no perfusion of the donor heart, both of which are likely to compromise survival of the transplanted heart or the recipient. The procedure has been widely used as originally described to examine the mechanisms of transplant rejection and tolerance6-8. Others have adapted the original rat heart transplant procedure of Ono and Lindsey for heart transplants in mice9,10. More recently, a technique was published for mouse heart transplantation that overcame some of the problems associated with the method of Corry et al11. The protocol described here incorporates our modifications, based on the method of Mottram et al12, which include: in situ perfusion with cold heparinized saline immediately after thoracotomy and performing the donor operation prior to the recipient operation to minimize recipient operation time. In addition, we use small atraumatic vessel clamps instead of 6-0 silk ties. Although vessel clamps have the disadvantage of taking more space they are easier to control than ties, which should not be too tight or loose and are less straightforward to remove than clamps. Our method uses a single running suture for vessel anastomosis, although in the initial learning stages an alternative is to use stay sutures at the proximal and distal corners to ensure evenness of sutures and thus patency of the anastomosis.

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			<td height="21" style="height:21px;width:293px;">Operating microscope&#160;</td>
			<td style="width:247px;">Leica, Heerbrugg, Switzerland</td>
			<td style="width:124px;">M651</td>
			<td style="width:167px;">10x - 25x magnification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthetic machine&#160;</td>
			<td>Vet Quip Pty Ltd, Sydney, Australia</td>
			<td style="width:124px;">Vett3</td>
			<td>Capable of delivering a mixture of isoflurane and oxygen in air&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Operating board&#160;</td>
			<td style="width:247px;">Hardware store or office supplier</td>
			<td style="width:124px;"></td>
			<td>Dense cork or synthetic capable of taking pins</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:293px;">Heparinised saline (5 U/ml, 4oC)</td>
			<td>Pfizer, USA</td>
			<td style="width:124px;">FW25</td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:293px;">Normal saline (37oC)</td>
			<td style="width:247px;">AstraZeneca Pty Ltd, Australia</td>
			<td style="width:124px;">4538</td>
			<td>In 1ml syringe with 23 gauge needle on warm pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sutures 10- nylon, 5-0 Vicryl</td>
			<td>Ethicon, Inc. NJ, USA</td>
			<td style="width:124px;">2870G/J421H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine (0.05mg/kg in 0.1ml saline)</td>
			<td>Reckitt Benckiser, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampcillin (5mg/kg in 0.1 ml saline)&#160;</td>
			<td>Aspen, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Foam&#160;</td>
			<td>Pharmacia &#38; Upjohn Co. USA</td>
			<td style="width:124px;">801289304</td>
			<td>Cut into small pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High temperature cautery device&#160;</td>
			<td>Medtronic, USA</td>
			<td style="width:124px;">8444000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microsurgery instruments:</td>
			<td colspan="2">Shanghai Medical Instruments Co. Ltd.,&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-needleholders</td>
			<td style="width:247px;">Shanghai, China</td>
			<td style="width:124px;">WT2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro scissors</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WT1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (straight tip)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (curved tip)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-mosquito clamps (1 pair)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-vessel atraumatic clamps (1 pair)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40130/W40150</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Heating Pad/Right Temp</td>
			<td style="width:247px;">Kent Scientific Corporation, Turrington, CT 06790</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified method for heterotopic mouse heart transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

After an initial training period, 227 cases of mouse heterotopic heart transplantation in our group were analyzed. The success rate within the first 24 hr was 90.3% and at 48 hr it was 86.8%. Of the 30 (13.2%) failures within 48 hr, 5 (2.2%) experienced hind limb paralysis and had to be euthanized, 10 (4.4%) had a non-beating heart due to graft ischemia injury and/or thrombosis, while 15 (6.6%) died within 48 hr. Some experimental graft survivals are shown in Table 1 with a variety of strain combinations...

Watch this Scientific Journal Video about A Modified Method for Heterotopic Mouse Heart Transplantion at JoVE.com

A Modified Method for Heterotopic Mouse Heart Transplantion

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

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