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>
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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.

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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 ...

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 ...

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 ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...