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. F. <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+for+the+study+of+vascular+disease.">Orthotopic aortic transplantation in mice for the study of vascular disease.</a> J Vis Exp. (69), e4338 (2012).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Halsey Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5-45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35&#8232;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

using-sleeve-technique-mouse-model-aortic-transplantation-an

Nanyang Technological University

Research

JoVE Journal

Medicine

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: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

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. ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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 ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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. ...