This work was supported by 2022 JST basic research (The Japanese Society for Transplantation).

The experimental protocol was approved by the Animal Experimentation Committee at Kyoto University. The study utilized C57BL/6 mice, aged over 10 weeks and weighing between 25 g and 30 g, obtained from a commercial source (see Table of Materials). All animals were anesthetized with 2.5% isoflurane (following institutionally approved protocols), maintained under specific pathogen-free conditions, and all experimental procedures were conducted in compliance with Kyoto University&#39;s Regulations on Animal Experimentation. The appropriate instruments used for the study are listed in the Table of Materials and depicted in Figure 1 and Figure 2.
1. Animal selection
<ol>
	<li>Use mice with a body weight of 25-30 g. 
	&#8203;NOTE: While mouse liver allografts2 are generally accepted across major histocompatibility complex barrier, we opted for the syngeneic MOLT model in this study to focus on the detailed mechanism of cold ischemia reperfusion injury10. Mice weighing less than 25 g are not recommended because it is impossible to insert an inner stent into a thin bile duct. Similarly, mice weighing more than 30 g are not recommended due to the presence of a large amount of intraabdominal fat around the vessels.</li>
</ol>
2. Cuff making
<ol>
	<li>Prepare 20 G and 16 G intravenous catheters for the PV and IHIVC, respectively.</li>
	<li>Using a scalpel, cut the catheter to create the cuff. The main body of the cuff should measure 2 mm in length, with an extension handle of 1 mm (Figure 3A). 
	NOTE: If the handle is too large, it may become challenging to insert the cuff.</li>
	<li>On the cuff&#39;s surface, create a shallow groove for thread fixation using the back of the scalpel blade. 
	NOTE: When applying the back of the scalpel blade to the cuff, ensure that the tip of the blade is at an angle of approximately 60 degrees (as shown in Figure 3B). After making the first groove, secure the scalpel blade and rotate the cuff. Ideally, two grooves are recommended, but a single groove can suffice without issues.</li>
</ol>
3. Cuff attachment
<ol>
	<li>In a plastic rectangular-shaped container, place small ice cubes and position a small metal cup (6 cm in diameter) on top. Fill the cup with organ preservation fluid (see Table of Materials) and place the syngeneic graft within.</li>
	<li>Gently roll the syngeneic liver graft (extracted from another donor mouse) using a cotton swab, ensuring that the porta hepatis faces upwards. Thoroughly remove any blood stored in the lumen of the IVC with organ preservation fluid.</li>
	<li>Thread the PV through the cuff using a thread attached, for example, to the splenic vein (see Figure 4A).</li>
	<li>With the cuff handle at the 12 o&#39;clock position, secure both the handle and cuff with a bulldog clamp (see Table of Materials), positioning it 2 to 3 mm from the PV edge.</li>
	<li>Secure the bulldog clamp further using large vascular forceps and a fixation mold to establish the operative field (see Figure 4B).</li>
	<li>Carefully inspect the PV lumen at approximately 15 to 20x magnification. If necessary, dripping water can enhance visibility of the lumen.</li>
	<li>Gently grasp the PV lumen and externalize it through the cuff.</li>
	<li>Once fully externalized, secure it with a double ligation using 8-0 silk thread along the cuff&#39;s groove (Figure 4C,D). 
	NOTE: Ensure that the ligature is not loose. Also, take care that the ligature point is not too large, as it may interfere with cuff insertion. The ligature point can be positioned in any direction.</li>
	<li>Attach the cuff to the IHIVC using a procedure similar to that used for the PV (Figure 4E). 
	NOTE: When clamping the IHIVC with the cuff, avoid biting into the liver parenchyma. Since the neck is shorter than the PV, carefully determine the clamping position.</li>
	<li>Create a slipknot by passing the thread around the base of the IHIVC to temporarily ligate the IHIVC (Figure 5). This thread will be removed after reperfusion.</li>
</ol>
4. Portal vein reconstruction
<ol>
	<li>Anesthetize the recipient mouse by inducing&#160;anesthesia with isoflurane at 2.5% and reduce it to 0.6% before extracting the native liver as described in published literature2. Dinsinfect the surgical area atleast three times with alternating rounds of iodophor and 70% ethanol before laparotomy.</li>
	<li>Ensure that the hepatic lobe is properly positioned and that the cuffed PV is free from kinking and torsion.</li>
	<li>Gently grasp the recipient&#39;s PV edge using Pean forceps and secure it with a vise. 
	NOTE: In this article, a vise refers to a mechanical apparatus used to secure Pean forceps on the table. For more information on vise, see Table of Materials.</li>
	<li>Apply a vascular clamp to the recipient&#39;s PV and pass an 8-0 silk thread around it.</li>
	<li>Create a 1/4 circumferential section approximately 0.5 mm from the PV edge (Figure 6A). Enlarge the hole while passing saline solution through the lumen using a specialized instrument (a 27 G needle with the tip cut off and bent into an L-shape).</li>
	<li>Use straight forceps to grip the cuff handle and insert it into the lumen.</li>
	<li>Once fully inserted, secure the cuff with an 8-0 silk thread. A single ligation is sufficient for fixation.</li>
	<li>Remove the vascular clamp and reperfuse the liver.</li>
	<li>Carefully release the Pean forceps and complete the portal vein reconstruction. The entire PV reconstruction process should take approximately 5 min.</li>
</ol>
5. Infrahepatic inferior vena cava reconstruction
<ol>
	<li>Position the liver lobe appropriately and move the Pean forceps attached to the recipient IHIVC dorsal to the graft&#39;s right lower lobe, securing it with a vise.</li>
	<li>Eliminate any remaining liver tissue around the recipient&#39;s IVIHC by gently rubbing with a cotton swab.</li>
	<li>Pass an 8-0 silk thread around the recipient&#39;s IVIHC.</li>
	<li>Create a 1/4 circumferential section at approximately 0.5 mm from the recipient&#39;s IVIHC edge (Figure 6B).</li>
	<li>After introducing normal saline solution into the lumen, insert the cuff into the lumen. 
	NOTE: Due to the short neck, the cuff is often inserted more shallowly than in the PV.</li>
	<li>Carefully secure the cuff with an 8-0 silk thread and then remove the vascular clamp and Pean forceps.</li>
</ol>
6. Postoperative care
<ol>
	<li>Utilize a heating pad and warming lamp to facilitate postoperative recovery.</li>
	<li>Continuously monitor blood pressure, heart rate, respiratory rate, body temperature, as well as food and water consumption.</li>
	<li>Adminsiter subcutaneous injection of carprofen (5 mg/kg, every 6 h&#160;or until needed) to alleviate postoperative pain.&#160;Follow local&#160;institutional guidelines. 
	NOTE: If the condition of mice deteriorates, the experiment will be stopped immediately and mice were euthanized by inhalation of carbon dioxide.</li>
	<li>To collect the tissue sample, re-anesthetize the mice and euthanize them by an IACUC approved method.</li>
</ol>

Strategies for Efficient Vascular Anastomosis in Mouse Liver Transplantation

<ol><li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+liver+transplantation+in+the+mouse%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li>
<li>Yokota, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+mouse+liver+transplantation+to+study+liver+biology+and+allograft+tolerance%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.</a> Nat Protoc. 11 (7), 1163-1174 (2016).</li>
<li>Shen, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+responses+in+a+new+mouse+model+of+prolonged+hepatic+cold+ischemia+followed+by+arterialized+orthotopic+liver+transplantation%5BTitle%5D)+AND+%22Liver+Transpl%22%5BJournal%5D)">Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.</a> Liver Transpl. 11 (10), 1273-1281 (2005).</li>
<li>Kageyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemia-reperfusion+injury+in+allogeneic+liver+transplantation%3A+A+role+of+CD4+T+cells+in+early+allograft+injury%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Ischemia-reperfusion injury in allogeneic liver transplantation: A role of CD4 T cells in early allograft injury.</a> Transplantation. 105 (9), 1989-1997 (2021).</li>
<li>Li, T., Hu, Z., Wang, L., Lv, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Details+determining+the+success+in+establishing+a+mouse+orthotopic+liver+transplantation+model%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">Details determining the success in establishing a mouse orthotopic liver transplantation model.</a> World J Gastroenterol. 26 (27), 3889-3898 (2020).</li>
<li>Tian, Y., Rüdiger, H. A., Jochum, W., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+arterialized+and+nonarterialized+orthotopic+liver+transplantation+in+mice%3A+Prowess+or+relevant+model%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: Prowess or relevant model.</a> Transplantation. 74 (9), 1242-1246 (2002).</li>
<li>Steger, U., Sawitzki, B., Gassel, A. M., Gassel, H. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+hepatic+rearterialization+on+reperfusion+injury+and+outcome+after+mouse+liver+transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.</a> Transplantation. 76 (2), 327-332 (2003).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+various+techniques+of+mouse+liver+transplantation%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">A review of various techniques of mouse liver transplantation.</a> Transplant Proc. 45 (6), 2517-2521 (2013).</li>
<li>Kojima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+carcinoembryonic+antigen-related+cell+adhesion+molecule+1-T+cell+immunoglobulin+domain+and+mucin+domain-containing+protein+3+cross+talk+alleviates+liver+transplant+injury+in+mice+and+humans%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">T cell carcinoembryonic antigen-related cell adhesion molecule 1-T cell immunoglobulin domain and mucin domain-containing protein 3 cross talk alleviates liver transplant injury in mice and humans.</a> Gastroenterology. 165 (5), 1233-1248 (2023).</li>
<li>Yokota, S., Yoshida, O., Ono, Y., Geller, D. A., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+transplantation+in+the+mouse%3A+Insights+into+liver+immunobiology%2C+tissue+injury%2C+and+allograft+tolerance%5BTitle%5D)+AND+%22Liver+Transpl%22%5BJournal%5D)">Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.</a> Liver Transpl. 22 (4), 536-546 (2016).</li>
</ol>

No authors have any conflicts of interest to disclose.

Learning vascular reconstruction is the most challenging aspect of achieving successful MOLT. The cuff's quality significantly influences the reconstruction's difficulty, given the small size of mice5. This article provides a detailed protocol for cuff preparation, attachment, and reconstruction.
While there are no major differences from previous reports regarding cuff preparation and connection2,5, some minor points should be considered. First, the cuff handle's thickness should be limited to approximately 1/6 of the total circumference. If it's too thin, the handle can easily bend and become hard to grasp. If it's too thick, it may be challenging to handle in the recipient's abdomen. Identifying your personal preference early on is essential.
Second, visibility of the lumen at the back table is crucial. A 10-0 nylon thread is used, attached to the donor's splenic vein as a landmark to confirm the PV lumen. The nylon thread is placed at 8 o'clock to prevent excessive twisting. The vessel wall is externalized by grasping it at 3 o'clock and 9 o'clock with two forceps and pressing it against the cuff wall at 6 o'clock. Fixation with 8-0 silk thread requires careful movements. Adequate fixation is achieved by securing it in the groove. After attachment, lift the cuff to confirm it doesn't come off.
Third, during reconstruction, it's important not to create too large a hole in the recipient vessel because the hole naturally enlarges as the cuff is inserted. Making a large hole can lead to vessel splitting, the most common cause of failure. Particularly in IHIVC reconstruction, smaller holes should be made since the shorter length of IHIVC makes it easier to pull out than a PV. When passing saline solution through the lumen, support cuff insertion by applying upward force rather than downward.
Common causes of failure and precautions have been discussed in detail. The most crucial points include ensuring clear visualization of the inner vessel lumen and avoiding unnecessary force during cuff insertion to prevent vessel splitting. These technical tips for the cuff method are believed to elevate skills and advance research.

Mouse orthotopic liver transplantation (MOLT) is an effective experimental method first reported in 19911. This experimental model, which utilizes genetically modified mice and various research reagents, has played a pivotal role in investigating warm and cold ischemia and reperfusion injuries. However, the model&#39;s high technical complexity has hindered the development of basic medicine for liver transplantation2. MOLT involves three major steps: (1) retrieval of the liver from the donor mouse, (2) back table surgery, and (3) implantation of the liver into the recipient. Among these recipient procedures, vascular anastomosis poses the greatest challenge. While the suprahepatic inferior vena cava anastomosis is typically completed by hand stitching2, the infrahepatic inferior vena cava (IHIVC) and portal vein (PV) can be reconstructed more efficiently using plastic cuffs in place of hand-sewn sutures.
The anhepatic period signifies the interval between the removal of the recipient&#39;s native liver and the graft implantation. To ensure consistent results, it is imperative to limit the anhepatic time to less than 20 min. Consequently, studies employing this model have been confined to specific institutions3,4,5,6,7,8,9. Among the various stages of MOLT, achieving smooth PV and IHIVC reconstruction is crucial for minimizing anhepatic time and ensuring successful transplantation.
PV and IVC reconstruction is generally carried out using vascular cuffs since the cuff technique simplifies vascular anastomosis compared to hand-sewn sutures2,5,8. The technique involved in vascular cuff preparation and secure attachment of the cuff significantly impacts the complexity of recipient vascular reconstruction. Our objective is to provide detailed visual guidance for numerous technical tips related to the cuff method, thereby reducing the learning curve. These video clips will provide a clear understanding of how to attach the cuff to the vessels and reconstruct the PV and IHIVC during recipient surgery.

<table><tbody><tr><td>16 G intravenous catheter</td><td>TERUMO</td><td>SR-FF2032</td><td>IHIVC cuff</td></tr><tr><td>20 G intravenous catheter</td><td>TERUMO</td><td>SR-FF1651</td><td>PV cuff</td></tr><tr><td>8-0 braid silk</td><td>Natsume Seisakusho</td><td>CR9-80B2</td><td>8-0 silk</td></tr><tr><td>Belzer UW Cold Storage Solution&amp;nbsp;</td><td>Astellas</td><td></td><td>Organ preservation fluid</td></tr><tr><td>Bulldog clamp</td><td>B BRAUN</td><td>FB329R</td><td>Bulldog clamp</td></tr><tr><td>C57BL/6 mice&amp;nbsp;&amp;nbsp;</td><td>Oriental Bio Service</td><td></td><td></td></tr><tr><td>Isoflurane inhalation solution</td><td>Viatris</td><td></td><td>Anesthesic</td></tr><tr><td>Micro Blunted Tips 0.1 mm x 0.06 mm&amp;nbsp;</td><td>F.S.T</td><td>11253-20</td><td>Straight microforceps</td></tr><tr><td>Micro Serrefine Clamp Applicator with Lock</td><td>F.S.T</td><td>18056-14</td><td>Vessel clip applicator</td></tr><tr><td>Micro Serrefines&amp;nbsp;</td><td>F.S.T</td><td>18055-4</td><td>Vessel clip</td></tr><tr><td>No.11 Spare Blades</td><td>FEATHER Safety Razor</td><td>11</td><td>Blades</td></tr><tr><td>Ophthalmic scissor, round handle</td><td>B BRAUN</td><td>FD103R</td><td>Microscissor</td></tr><tr><td>Plastic rectangular-shaped container&amp;nbsp;&amp;nbsp;</td><td>Daiso</td><td></td><td>10 cm long, 15 cm wide and 6 cm high</td></tr><tr><td>SuperGrip Tips</td><td>F.S.T</td><td>00649-11&amp;nbsp;</td><td>Curved microforceps</td></tr><tr><td>SZX7</td><td>Olympus</td><td>SZX7</td><td>Microscope</td></tr><tr><td>Vise</td><td>Niigataseiki</td><td>00505351</td><td>A mechanical tool to secure Pean forceps on the table</td></tr></tbody></table>

vascular reconstruction with the cuff technique in mouse orthotopic liver transplantation

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. However, the technical challenges pose a barrier to utilizing this valuable experimental model and passing on these skills to the next generation. The most challenging aspect of this procedure is vascular reconstruction, including the portal vein (PV), infrahepatic inferior vena cava (IHIVC), and suprahepatic inferior vena cava. The use of plastic cuffs, rather than sutures, allows for smoother PV and IHIVC reconstruction. Vessels are reconstructed by attaching a cuff made from an intravenous catheter to the tip of the graft vessel and interposing the cuff into the recipient vessel. The two most crucial aspects are properly visualizing the inner lumen of the vessel and avoiding the use of excessive force. Our aim is to provide a technical overview of vascular reconstructions using the cuff technique in recipient surgery. These technical tips for the cuff technique are expected to help microsurgeons facilitate vascular reconstruction and advance their research.

PV reconstruction is successful when, upon unclamping the portal vein, there is no tortuosity, and the liver is uniformly perfused. Anhepatic time should be under 20 min, as anhepatic times exceeding 25 min increase the risk of mouse mortality. IHIVC reconstruction is deemed successful if there is no blood regurgitation from the graft.
Storing the graft at cold temperatures for 1 h using organ preservation solution results in a serum alanine aminotransferase level of approximately 2,000 U/L at 6 h after reperfusion (Figure 7A). The survival rate is 100% at 7 days after transplantation (Figure 7B). However, in our model without hepatic artery anastomosis, recipient mice may succumb to intrahepatic biloma-related issues approximately 30 days post-transplantation
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66215/66215fig01.jpg" /> 
Figure 1: Instruments for vascular reconstruction. Straight or curved forceps are used as needed for the situation and the operative field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66215/66215fig02.jpg" /> 
Figure 2: The microscope. The microscope is equipped with an objective lens with 10x magnification and an eyepiece lens with at least 0.8x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66215/66215fig03.jpg" /> 
Figure 3: Preparation of the cuff. (A) The cuff for the PV and IHIVC. (B) A schematic representation of the process of creating a groove on the cuff. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66215/66215fig04.jpg" /> 
Figure 4: Cuff attachment. (A) The thread attached to the PV is passed into the cuff. (B) Instrument setup for cuff attachment. (C) Securing the cuff with 8-0 silk ligations. (D) Schematic illustration of vascular externalization and fixation to the cuff. (E) Completed cuff attachment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66215/66215fig05.jpg" /> 
Figure 5: Temporary ligation of the IHIVC root. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66215/66215fig06.jpg" /> 
Figure 6: Vascular reconstruction. This illustration shows the locations for creating holes for (A) PV and (B) IHIVC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66215/66215fig07.jpg" /> 
Figure 7: Postoperative results. (A) Serum levels of alanine aminotransferase (ALT) at 6 h after reperfusion. CIT, cold ischemic time; Tx, transplantation. ****p &#60; 0.0001. (B) Survival of the recipient after MOLT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66215/66215fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation at JoVE.com

Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

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1.3K Views.  Kyoto University. This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

Video: Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

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 ...

Immunology and Infection

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

Podsumowanie

Przeglądaj więcej filmów

Orthotopic Liver Transplantation in Rats

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Heterotopic Cervical Heart Transplantation in Mice

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

Optimization of the Cuff Technique for Murine Heart Transplantation

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model