Name: using the sleeve technique in a mouse model of aortic transplantation - an instructional video
Uploaded: 2017-10-22T13:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video at JoVE.com

We would like to thank Roma Wieczorek and Peter Kurdybacha for their excellent editing assistance, and Leon Decker and Uli Heuter for their excellent technical assistance.

Procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) at RWTH Aachen University, AZ 84-02.04.2012.A234.
NOTE: The procedure is demonstrated using adult male wild type mice with CD1 background. Keep the mice in a specialized laboratory unit before and after the surgery, assuring proper access to food, specialized veterinary control, and treatment. If the animals are purchased from outside, allow one-week acclimatization before performing surgery.
1. Preparation of the Donor
<ol>
	<li>Use sterile materials and instruments to maintain sterile conditions during surgery to avoid infections.</li>
	<li>Anesthetize each mouse with a mixture of 1.5% by volume isoflurane and 100% oxygen through a face mask. Lay the mouse on a platform in the supine position and tape all its legs to the operating table. Check its reflexes by pinching the hind feet to be sure that the mouse is sufficiently anesthetized. Place ophthalmic ointment on the eyes to prevent drying during the procedure.</li>
	<li>Remove all hair from the abdomen using a depilatory gel or use a shaver. Perform the operation under sterile conditions. Disinfect the abdomen with alternating scrubs of chlorhexidine and sterile water.</li>
	<li>Remove the donor aorta via a midline abdominal incision with scissors or scalpel. Retract the bowel manually to the right. Gently manually reflect the intestines over to the side using powder free gloves.
	<ol>
		<li>Place the bowel on a piece of gauze wetted with saline to keep it moist.</li>
		<li>Dissect away the abdominal aorta very carefully from the surrounding tissue using a blunt dissection with tweezers.</li>
		<li>Separate the segment of the aorta between the renal arteries and its bifurcation from the vena cava with tweezers.</li>
		<li>Secure all the small branches of this segment very carefully using 11-0 monofilament single suture.</li>
		<li>Before removing the aorta, inject 0.5 milliliters (mL) of saline solution containing 50 U of heparin into the inferior vena cava.</li>
		<li>Let the donor animal exsanguinate after the segment of the aorta is removed.</li>
		<li>Rinse the graft fully with saline and then transfer it immediately to a container of ice-cold saline.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Recipient
<ol>
	<li>Anesthetize the recipient animal with a mixture of 1.5% by volume isoflurane and 100% oxygen through a face mask, then remove the hair and disinfect (Section 1). Make a mid-line incision from the xiphoid to the pelvis with a scalpel and retract the abdominal walls. Place ophthalmic ointment on the eyes to prevent dryness during the procedure.</li>
	<li>Wrap the bowel in saline solution moistened gauze and displace very gently to the animal&#39;s right.</li>
	<li>Dissect the infrarenal aorta free between the renal arteries proximally and the bifurcation distally with tweezers.</li>
	<li>Secure all the small branches of this segment very carefully using 11-0 monofilament single suture.</li>
	<li>Clamp the proximal and distal portions of the aorta with a 6-0 single silk suture.</li>
	<li>Divide the aorta in the middle between the clamps and irrigate the cut ends with heparinized saline to flush the lumen open.</li>
	<li>Place the graft in the orthotopic position with the feeding vessel end inserted into the receiving vessel followed by suturing with 11-0 monofilament taking care to avoid any torsion of the aorta by properly aligning the donor and recipient (Figure 1)10.</li>
	<li>Carefully release the ligatures after conducting an inspection of the anastomosis. Release the distal clamp first. This results in low pressure holding the walls together prior to releasing the proximal high-pressure side.</li>
	<li>Perfuse the graft immediately and check for a visible pulse. Gently remove the remnants of the silk. The optimal overlap length between donor and recipient aorta is 1-2 mm.</li>
	<li>Return the abdominal contents to the abdominal cavity and close the whole wound with a running 3-0 polyglycolic acid suture.</li>
	<li>Give the mouse buprenorphine (0.05 mg/kg body weight subcutaneously (SC)) before terminating anesthesia.</li>
	<li>Do not leave an animal unattended until it is fully conscious. Manage pain therapy with buprenophine 0.05 mg/kg body weight administered SC three times a day for three days after the operation as approved by the institutional oversight body.</li>
	<li>For tissue harvesting, anesthetize the recipient mice as described above and flush the vessels with phosphate-buffered saline (PBS) followed by 4% formaldehyde/PBS, (pH = 7.4) by cardiac puncture. Remove the grafts gently. After overnight fixation in 4% formaldehyde/PBS, process specimens further and embed in paraffin.</li>
</ol>

<ol>
	<li>Rowinska, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-+invasive+in+vivo+analysis+of+a+murine+aortic+graft+using+high+resolution+ultrasound+microimaging.">Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging.</a> Eur J Radiol. 81 (2), 244-249 (2012).</li><li>Daugherty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+atherosclerosis.">Mouse models of atherosclerosis.</a> Am J Med Sci. 323 (1), 3-10 (2002).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc .Biol. 24 (3), 429-434 (2004).</li><li>Zernecke, A., Shagdarsuren, E., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+atherosclerosis:+an+update.">Chemokines in atherosclerosis: an update.</a> Arterioscler Thromb Vasc Biol. 28 (11), 1897-1908 (2008).</li><li>Chereshnev, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+heterotopic+aortic+arch+transplantation.">Mouse model of heterotopic aortic arch transplantation.</a> J Surg Res. 111 (2), 171-176 (2003).</li><li>Koulack, J., McAlister, V. C., Giacomantonio, C. A., Bitter-Suermann, H., MacDonald, A. S., Lee, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+aortic+transplant+model+of+chronic+rejection.">Development of a mouse aortic transplant model of chronic rejection.</a> Microsurgery. 16 (2), 110-113 (1995).</li><li>Engelbrecht, G., Kahn, D., Duminy, F., Hickman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+rapid+technique+for+renal+transplantation+in+the+rat.">New rapid technique for renal transplantation in the rat.</a> Microsurgery. 13 (6), 340-344 (1992).</li><li>Baxter, K., Hao, P. M., Howden, B. O., Saunder, A., Jablonski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+of+abdominal+heart+transplantation+in+the+rat.">Modified technique of abdominal heart transplantation in the rat.</a> J Heart Lung Transplant. 17 (11), 1057-1064 (1998).</li><li>Dambrin, C., Calise, D., Pieraggi, M. T., Thiers, J. C., Thomsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice:+a+new+model+of+allograft+arteriosclerosis.">Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.</a> J Heart Lung Transplant. 18 (10), 946-951 (1999).</li><li>Siemionow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+microarterial+anastomoses:+end-to-end+versus+end-in-end+(sleeve)+technique.">Histopathology of microarterial anastomoses: end-to-end versus end-in-end (sleeve) technique.</a> J Hand Surg Am. 15, 619-625 (1990).</li><li>Charo, I. F., Ransohoff, R. 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+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354 (6), 610-621 (2006).</li><li>Sun, 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=Improved+surgical+technique+for+the+establishment+of+a+murine+model+of+aortic+transplantation.">Improved surgical technique for the establishment of a murine model of aortic transplantation.</a> Microsurgery. 18 (6), 368-371 (1998).</li><li>Guo, L., Agarwal, A., George, J. 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The authors declare that they have no competing financial interests.

Prior to this study various other transplantation models in mice were thoroughly analysed3,6,7,10,13,14. The model of aortic transplantation using the sleeve technique with modifications by Dambrin et al. was selected as it matched our criteria and showed a high reliability of the sleeve anastomosis in comparison to conventional end-to-end suture methods1,10.
This technique is favorable in many ways with cross-clamp time considerably reduced minimizing the damage to the aorta during surgery. A low incidence of thrombosis was observed in addition to avoiding a potential mismatch in vessel caliber between the donor and the recipient. The above observations make this technique highly suitable for investigating vascular disease in aortic transplants in mice. 
In a follow-up study in which an ultrasound was performed 8 weeks after transplantation, no significant changes were detected. This confirmed assumptions that any damage to the aorta during surgery would be minimal1. 
The grafting procedure presented in this article guarantees no impairment both in the graft integrity and its function. Therefore, it can be concluded that this experimental transplantation model may serve as a valuable tool for future molecular and pharmacological investigations of degenerative vessel disease in genetically engineered mice.
We believe that the video guide can function as teaching material illustrating the use of this simple arterio-arterial model and that it will contribute to further fruitful debate on many important issues in vascular pathologies. This very rapid anastomosis method may be used to study vascular disease in genetically engineered mice. It may also be used as a modification in the aneurysm model combined with transplantation. 
There are critical points during the procedure. The placing of the suture itself is the most critical step. The surgeon has to take care to avoid any torsion of the aorta by proper alignment of donor and recipient. The clamps are carefully removed after inspection of the anastomosis. The distal clamp should always be released first resulting in low pressure holding the walls together prior to the proximal high-pressure side release. The consequence of not properly following the sequence of release would be bleeding.

As pointed out in a previous study, great attention has been paid to murine aortic transplant models which allow discrimination between specific vascular responses caused by the graft itself and certain systemic factors connected with an arteriogenic environment1,2,3. The major factor that plays a crucial role here is the availability of knockout and transgenic mice. Their involvement in such a model offers the possibility of identifying and determining new pathophysiological pathways associated with the development of degenerative vascular disease, such as atherosclerosis and aneurysm formation4,5.
It is worth noting that during grafting an intrinsic ischemia/reperfusion injury to the vessels intended for the transplant may appear. Therefore, the occurrence of specific problems with graft integrity or an unexpected inflammatory reaction during the post-operative period cannot be excluded possibly precluding pathophysiological changes in degenerative vascular diseases3,4,5,6,7. Sleeve anastomosis is the alternative end-in-end method for arterial anastomosis of vessels with a diameter of less than one millimeter and has been applied successfully in renal and cardiac transplantation in rats which was subsequently adapted to aortic transplantation in mice by Dambrin et al.8,9,10,11.
Aortic damage using the sleeve transplantation technique is minimized with a very low technical failure rate, due to it lasting only 20 min on average. Our previous results have demonstrated excellent functional and structural properties of an isograft in vivo after transplantation using the sleeve technique1. Dambrin et al. describe that after a short learning curve the success rate was over 78%10. Complications such as thrombosis are rare, for example Engelbrecht et al. did not observe thrombosis using the sleeve technique in renal transplantation in the rat8.
The murine aortic transplant model with sleeve anastomoses is a rapid and easy tool to study iso/allograft reactions in the transplanted vessel. This video illustrates the aortic transplantation procedure carried out in our laboratory. This transplantation model may be useful in defining the underlying pathological mechanisms of vascular degenerative disease and may contribute to further evaluation of molecular and pharmacological interventions12.

<table><tbody><tr><td>Halsey Needle Holder</td><td>Fine Science Tools</td><td>12001-13</td><td></td></tr><tr><td>Dumont #5 Forceps -</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Lexer-Baby Scissors</td><td>Fine Science Tools</td><td>14079-10</td><td></td></tr><tr><td>Castroviejo Micro Needle Holder</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>Vannas Scissors</td><td>Aesculap, Germany</td><td>Typ OC498R</td><td></td></tr><tr><td>Castroviejo Suture Forceps</td><td>Geuder, Germany</td><td>19015</td><td></td></tr><tr><td>6-0 silk black (Silk)</td><td>Deknatel, Research Triangle Park NC, USA</td><td>18020-60- FST</td><td></td></tr><tr><td>11-0 monofilament (Ethilon)</td><td>Ethicon, Norderstedt, Germany</td><td>EH7438G</td><td></td></tr><tr><td>3-0 polyglycolic acid suture (Serafit)</td><td>Serag-Wiessner, Naila, Germany</td><td>60203214</td><td></td></tr><tr><td>Isofluorane</td><td>Any genericon</td><td></td><td></td></tr><tr><td>Heparin</td><td>Any genericon</td><td></td><td></td></tr><tr><td>0.9% saline</td><td>Any genericon</td><td></td><td></td></tr><tr><td>Buprenorphine</td><td>Any genericon</td><td></td><td></td></tr><tr><td>Bepanthene eye and nose cream</td><td>Bayer, Germany</td><td></td><td></td></tr><tr><td>Microscop</td><td>Zeiss</td><td>Opmi MDO/S5</td><td></td></tr><tr><td>Vaporiser</td><td>Eickemeyer</td><td>TEC3</td><td></td></tr><tr><td>Ultrasound</td><td>Vevo, Canada</td><td>770,2100</td><td></td></tr></tbody></table>

using the sleeve technique in a mouse model of aortic transplantation - an instructional video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

The mice recovered from anesthesia within 15-30 min with no observed physical impairment, although there was an elevated risk of thrombosis. Ultrasound analysis was used during postoperative follow up. The wild type mice used in the study exhibited no changes in the dimensions of their lumen. Accordingly, neither stenoses nor aneurysmal formations were observed. The transplant animals failed to exhibit plaque development of vessel walls (Figure 2).
Conventional staining procedures such as immunohistology can be used to determine the plaque, pattern of the smooth muscle cells, and accumulation of macrophages. In our study the histological and immunohistochemical staining was performed 6 weeks after grafting to test the integrity of the graft. Histological staining (Hematoxylin and eosin (HE) and immunohistochemical staining (Smooth Muscle Actin (SMA) and Macrophage (MAC2)) (Figure 3) showed us unchanged distribution patterns of smooth muscle cells, intact endothelial cell lining, and no accumulation of cells in the intima. These findings indicate that no significant lesion or cell activation was detected in the grafted vessels (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54915/54915fig1.jpg" /> 
Figure 1: Sleeve technique.&#160;The abdominal aorta was transplanted using the sleeve technique. In this procedure, the donor&#39;s aorta was placed in the orthotopic position with superficial bites present in the feeding vessel. <a href="https://cloudflare.jove.com/files/ftp_upload/54915/54915fig1large.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/54915/54915fig2.jpg" /> 
Figure 2: Intraoperative and ultrasound images.&#160;Examples of the intraoperative (necropsy) view of the grafts 6 weeks after transplantation (A), its three-dimensional ultrasound (B), and B-mode view (C). The postoperative follow up was conducted using ultrasound. The pictures show the patency of the graft with no change in lumen dimensions. Additionally, no stenoses or aneurysmal formations were observed. <a href="https://cloudflare.jove.com/files/ftp_upload/54915/54915fig2large.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/54915/54915fig3.jpg" /> 
Figure 3: Histological and immunohistochemistry images.&#160;The histological and immunohistochemistry representative images of transplanted animals at 6 weeks after transplantation. No significant lesions were observed in transplanted aortas by histology (Hematoxylin and eosin, HE, 100X magnification, Scale bar&#160; = 50 &#181;m), immunohistochemical staining (Smooth Muscle Actin SMA (red), or Macrophage MAC2 (green),&#160;200X magnification, Scale bar = 25 &#181;m). Nuclei were counter-stained by DAPI (blue). <a href="https://cloudflare.jove.com/files/ftp_upload/54915/54915fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video at JoVE.com

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

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Research

JoVE Journal

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8.9K Views.  Ruhr- University Bochum. We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease. 

Video: Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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 ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

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. ...

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 ...

Strategies for Efficient Vascular Anastomosis in Mouse 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. 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.

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.

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

A Simple Cuff Technique for Murine Left Lung Transplantation

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an efficient and highly reliable "cuff technique" of transplantation. This article describes a sophisticated and comprehensive method for orthotopic lung transplantation in a vascularized orthotopic lung model, representing the most physiologic and clinically relevant model of mouse lung transplantation to date. The transplantation process consists of two distinct stages: donor harvest and subsequent implantation into the recipient. The method has been successfully mastered, and with several months of sufficient training, a skilled practitioner can perform the procedure in approximately 90 min from skin-to-skin. Surprisingly, once individuals overcome the initial learning curve, the survival rate during the perioperative period approaches nearly 100%. The mouse model allows for the use of multiple commercially available transgenic and mutant strains of mice, enabling the study of tolerance and rejection. Additionally, the unique features of this model make it a valuable tool for investigating tumor biology and immunology.

The present protocol describes a cuff technique for a mouse left lung transplantation model. This technique has been developed over several years and has performed well, serving effectively in immunological research.

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...