Name: Orthotopic Hind Limb Transplantation in the Mouse (Video) | JoVE Journal
Uploaded: 2016-02-12T13:00:00.000+00:00
Duration: 7 min 15 s
Description: Learn orthotopic hind limb transplantation in mice using a non-suture cuff technique for super-microvascular anastomosis. This method reduces procedural complexity and increases success rates for studying alloimmune responses in vascularized composite allotransplantation.

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5 minutes

This work was supported by the Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0053. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.
The authors would like to thank Jessica Izzi, D.V.M, Caroline Garrett, D.V.M. and Julie Watson, D.V.M. for their excellent veterinary support during this study.

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH) and were approved by the Johns Hopkins University Animal Care and Use Committee (JHUACUC). The specific procedures were performed under the approved ACUC protocol MO13M108.&#160;
1. Donor Operation
<ol>
	<li>Administer analgesia at the appropriate time point for each pharmacological formulation prior to surgery. As per the approved animal care and use protocol use 0.1 mg/kg BW of buprenorphine subcutaneously 1 hr prior to skin incision.</li>
	<li>Sedate the donor with isoflurane applied through a chamber attached to an isoflurane vaporizer at 4%; maintain sedation and anesthesia at 2% through a nose cone. Perform toe pinch withdrawal reflection to monitor the depth of anesthesia prior to the initiation of the procedure.</li>
	<li>Wear masks, disposable isolation gowns and gloves.</li>
	<li>Shave the surgical area, in particular the hind limb and groin, and prep with 10% Povidone - Iodine.</li>
	<li>Use a sterile field drape, autoclaved instruments and a high magnification microscope (40X).</li>
	<li>Make groin skin incision using scissors proximally to the mid-thigh area and circumferentially connect the incision to demarcate the hind limb from the rest of the mouse body.</li>
	<li>Identify and dissect the femoral artery, vein and nerve. Separate all three structures using forceps and micro-scissors.</li>
	<li>Once the vascular pedicle is dissected divide the vessels at the level of the inguinal ligament using micro scissors.</li>
	<li>Next, continue to divide the individual ventral (gracilis and medial thigh muscles) and dorsal muscle groups 20 proximally at the level of the mid-thigh to separate the graft from the donor animal using scissors.</li>
	<li>Transect the femur and cut at the mid of the femoral shaft using scissors.</li>
	<li>Euthanize animal by isoflurane overdose followed by cervical dislocation. Confirm cessation of heart beat and respiration.</li>
	<li>Flush the limb with 2 ml&#160;heparinized (30 I.E.) cold (4 &#176;C) saline by using a 33 G flushing needle mounted on a syringe (see Materials Table).</li>
	<li>Place one polyimide cuff on the femoral vein and artery, respectively.</li>
	<li>Wrap graft into wet cotton gauze, place in petri dish and store at 4 &#176;C until inset.</li>
</ol>
2. Recipient Operation
<ol>
	<li>Removal of the Hind Limb
	<ol>
		<li>Administer analgesia at the appropriate time point for each pharmacological formulation prior to surgery. As per the approved animal care and use protocol use 0.1 mg/kg BW of Buprenorphine SC 1 hr prior to skin incision.</li>
		<li>Sedate the donor with isoflurane applied through a chamber attached to an isoflurane vaporizer at 4%; maintain sedation and anesthesia at 2% through a nose cone. Perform toe pinch withdrawal reflection to monitor the depth of anesthesia prior to the initiation of the procedure.</li>
		<li>Use veterinary ointment on the eyes of the mouse to prevent dryness while under anesthesia.</li>
		<li>Shave the surgical area, in particular the hind limb and groin and prep with 10% Povidone - Iodine.</li>
		<li>Make groin skin incision using scissors proximally to the mid-thigh area and circumferentially connect the incision to demarcate the hind limb from the rest of the mouse body.</li>
		<li>Identify and dissect the femoral artery, vein and nerve and separate all three structures using forceps and micro-scissors.</li>
		<li>Once the vascular pedicle is dissected, clamp the femoral vessels at the level of the inguinal ligament.</li>
		<li>Cut the vessels distal at the level of the superficial epigastric artery.</li>
		<li>Next, continue to divide the individual ventral (gracilis and medial thigh muscles) and dorsal muscle groups 20 proximally at the level of the mid-thigh to separate the native hind limb from the recipient animals using scissors.</li>
		<li>Transect the femur in the middle of the femoral shaft using scissors.</li>
		<li>Cauterize previously transected thigh muscles to prevent bleeding of the dissection site and thus recipient blood loss.</li>
	</ol>
	</li>
	<li>Implantation
	<ol>
		<li>Minimize fluid loss by irrigating the operative field with warm saline (37 &#176;C) and injecting 0.3 ml warm saline before and after the operation.</li>
		<li>Place the graft in a way that reflects the accurate anatomical position of the native hind limb by aligning the femur bone of the recipient and the graft and connect them using a 20 G spinal needle as an intramedullary rod.</li>
		<li>Coapt the ventral and dorsal muscle groups using absorbable suture material (6-0 Polysorb).</li>
		<li>Connect the femoral vessels using the non-suture cuff technique; in detail, pull the recipient side of the vessel over the cuffs previously mounted on the vessel ends of the graft. Use a 10-0 Nylon suture and perform a circumferential tie to fix the recipient vessel onto the cuff.</li>
		<li>Next release the clamps. At this stage visually verify cuff rotation and optimal positioning to prevent mis-rotation and kinking of the vessels.</li>
		<li>Perform meticulous hemostasis using electro cautery with a particular focus on the muscle recipient donor interface and the bone ends.</li>
		<li>Close the skin using non-absorbable Nylon sutures (6-0 Ethilon).</li>
		<li>Establish normothermic conditions by allowing the animal to recover in its cage under a heating lamp. Continue regular monitoring for at least 4 hr prior to returning it to the housing facility.</li>
		<li>Provide post-operative analgesia with Buprenorphine at a dose of 0.1mg/kg SC every 6-8 hr&#160;for 3 days.</li>
	</ol>
	</li>
</ol>

<ol><li>Khalifian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facial+transplantation%3A+the+first+9+years%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Facial transplantation: the first 9 years.</a> Lancet. , (2014).</li>
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<li>Sucher, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+hind+limb+transplantation%3A+a+new+composite+tissue+allotransplantation+model+using+nonsuture+supermicrosurgery%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Mouse hind limb transplantation: a new composite tissue allotransplantation model using nonsuture supermicrosurgery.</a> Transplantation. 90, 1374-1380 (2010).</li>
<li>Maglione, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+technique+for+heterotopic+vascularized+pancreas+transplantation+in+mice+to+assess+ischemia+reperfusion+injury+and+graft+pancreatitis%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.</a> Surgery. 141, 682-689 (2007).</li>
<li>Sucher, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+hind-limb+transplantation+in+rats%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Orthotopic hind-limb transplantation in rats.</a> J Vis Exp. , (2010).</li>
<li>Zou, Y., Brandacher, G., Margreiter, R., Steurer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cervical+heterotopic+arterialized+liver+transplantation+in+the+mouse%5BTitle%5D)+AND+%22J.+Surg.+Res%22%5BJournal%5D)">Cervical heterotopic arterialized liver transplantation in the mouse.</a> J. Surg. Res. 93, 97-100 (2000).</li>
<li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+mouse+limb+transplantation+model%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Development of a mouse limb transplantation model.</a> Microsurgery. 19, 209-213 (1999).</li>
<li>Schneeberger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Upper-extremity+transplantation+using+a+cell-based+protocol+to+minimize+immunosuppression%5BTitle%5D)+AND+%22Ann.+Surg%22%5BJournal%5D)">Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.</a> Ann. Surg. 257, 345-351 (2013).</li>
<li>Azari, K., Brandacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularized+composite+allotransplantation%5BTitle%5D)+AND+%22Curr+Opin+Organ+Transplant%22%5BJournal%5D)">Vascularized composite allotransplantation.</a> Curr Opin Organ Transplant. 18, 631-632 (2013).</li>
<li>Pomahac, B., Gobble, R. M., Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facial+and+hand+allotransplantation%5BTitle%5D)+AND+%22Cold+Spring+Harb.+Perspect.+Med%22%5BJournal%5D)">Facial and hand allotransplantation.</a> Cold Spring Harb. Perspect. Med. 4, (2014).</li>
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<li>Leto Barone, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gracilis+myocutaneous+free+flap+in+swine%3A+an+advantageous+preclinical+model+for+vascularized+composite+allograft+transplantation+research%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">The gracilis myocutaneous free flap in swine: an advantageous preclinical model for vascularized composite allograft transplantation research.</a> Microsurgery. 33, 51-55 (2013).</li>
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<li>Foster, R. D., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+hindlimb+transplantation+in+the+mouse%5BTitle%5D)+AND+%22J.+Reconstr.+Microsurg%22%5BJournal%5D)">Orthotopic hindlimb transplantation in the mouse.</a> J. Reconstr. Microsurg. 19, 49(2002).</li>
<li>Tung, T. H., Mohanakumar, T., Mackinnon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+Mouse+Model+for+Heterotopic+Limb+and+Composite-Tissue+Transplantation%5BTitle%5D)+AND+%22J.+Reconstr.+Microsurg%22%5BJournal%5D)">Development of a Mouse Model for Heterotopic Limb and Composite-Tissue Transplantation.</a> J. Reconstr. Microsurg. 17, 267-274 (2001).</li>
<li>Zhang, F., Shi, D. Y., Kryger, Z., Moon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+mouse+limb+transplantation+model%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Development of a mouse limb transplantation model.</a> Microsurgery. 19 (5), 209-213 (1999).</li>
<li>Schneeberger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atypical+acute+rejection+after+hand+transplantation%5BTitle%5D)+AND+%22Am.+J.+Transplant%22%5BJournal%5D)">Atypical acute rejection after hand transplantation.</a> Am. J. Transplant. 8, 688-696 (2008).</li>
<li>Mathes, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+mixed+hematopoietic+chimerism+permits+tolerance+of+vascularized+composite+allografts+across+a+full+major+histocompatibility+mismatch+in+swine%5BTitle%5D)+AND+%22Transpl.+Int%22%5BJournal%5D)">Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine.</a> Transpl. Int. 27, 1086-1096 (2014).</li>
<li>Yamada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+CTLA4Ig+for+induction+of+mixed+chimerism+and+renal+allograft+tolerance+in+nonhuman+primates%5BTitle%5D)+AND+%22Am.+J.+Transplant%22%5BJournal%5D)">Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.</a> Am. J. Transplant. 14, 2704-2712 (2014).</li>
<li>Sachs, D. H., Kawai, T., Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+tolerance+through+mixed+chimerism%5BTitle%5D)+AND+%22Cold+Spring+Harb.+Perspect.+Med%22%5BJournal%5D)">Induction of tolerance through mixed chimerism.</a> Cold Spring Harb. Perspect. Med. 4, a015529(2014).</li>
<li>Kawai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. Med. 358, 353-361 (2008).</li>
<li>Kawai, T., Sachs, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tolerance+induction%3A+hematopoietic+chimerism%5BTitle%5D)+AND+%22Curr+Opin+Organ+Transplant%22%5BJournal%5D)">Tolerance induction: hematopoietic chimerism.</a> Curr Opin Organ Transplant. 18, 402-407 (2013).</li>
<li>Kawai, T., Sachs, D. H., Sykes, M., Cosimi, A. .B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. Med. 368, 1850-1852 (2013).</li>
<li>Leventhal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chimerism+and+tolerance+without+GVHD+or+engraftment+syndrome+in+HLA-mismatched+combined+kidney+and+hematopoietic+stem+cell+transplantation%5BTitle%5D)+AND+%22Sci.+Transl.+Med%22%5BJournal%5D)">Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation.</a> Sci. Transl. Med. 4, 124-128 (2012).</li>
<li>Cendales, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Banff+2007+working+classification+of+skin-containing+composite+tissue+allograft+pathology%5BTitle%5D)+AND+%22Am.+J.+Transplant%22%5BJournal%5D)">The Banff 2007 working classification of skin-containing composite tissue allograft pathology.</a> Am. J. Transplant. 8, 1396-1400 (2008).</li>
</ol>

The authors declare that they have no competing financial interest.

Vascularized Composite Allotransplantation, such as upper extremity and face transplantation for reconstruction of devastating tissue defects, has evolved as a valid treatment option for patients not amendable to conventional reconstructive procedures. Technical advances in the field of reconstructive microsurgery as well as a vast experience with potent immunosuppressive and immune modulatory therapies in solid organ transplantation, now enables long-term allograft survival in this unique patient population 3,21</s...

The late nineties heralded the pioneering days of reconstructive transplantation with the first successful hand transplant performed in France in 1998. Since then, the use of VCAs for reconstruction of devastating tissue defects has been successfully employed in a wide spectrum of patients. To date, the world counts 76 recipients of 112 upper extremities as well as 31 faces 1-3. In addition, several other types of VCAs such as abdominal wall 4, larynx 5, trachea 6, vascularized joints 7, and even penis 8 have been performed. Furthermore, the live birth of a baby was recently reported after uterus transplantation 9. This growing world experience is indicative for how reconstructive transplantation has become a valid therapeutic option for patients suffering of significant functional tissue defects not amendable to conventional reconstructive and restorative surgery and treatment. 
While the idea of replacing "like with like" sparked clinical enthusiasm, initial skepticism still prevails with regards to side effects of conventional high-dose immunosuppression required to maintain allografts and their function 10,11. However, as shown by seminal work of Lee et al., these composite grafts are less likely to reject than its individual components, and furthermore, some of the tissue components such as the vascularized bone compartment have fueled optimism as they might exert unique immunological effects onto the balance of immune acceptance and rejection 12. 
Our group pioneered several microsurgical animal models for solid organ transplantation, as well as vascularized composite allotransplantation 13-19. Here we describe a novel surgical procedure using a non-suture cuff technique to perform super micro-vascular anastomosis in an orthotopic mouse hind limb transplantation model. This transplant model provides a useful tool for investigating immune acceptance and rejection mechanisms, as well as the role of individual tissue components, such as the vascularized bone marrow compartment, towards tolerance induction in the immunologically versatile setting of the mouse species. Additionally, the orthotopic placement of the limb opens the possibilities for nerve regeneration and functional outcome studies, which are critically important to the setting of VCA.

<table><tbody><tr><td>Suture, 6-0 Nylon</td><td>MWI</td><td>31849</td><td></td></tr><tr><td>Suture, 6-0 Polysorb</td><td>MWI</td><td>72667</td><td></td></tr><tr><td>Suture, 10-0 Nylon</td><td>Aero Surgical</td><td>TK-107038</td><td></td></tr><tr><td>Polyimide Tubing, Size 25</td><td>Vention Medical</td><td>141-0023</td><td></td></tr><tr><td>Polyimide Tubing, Size 27</td><td>Vention Medical</td><td>141-0015</td><td></td></tr><tr><td>Microvascular Clamps (Single)</td><td>Synovis</td><td>00396</td><td></td></tr><tr><td>Microvascular Clamps (Double)</td><td>Synovis</td><td>00414</td><td></td></tr><tr><td>Micro-Scissors</td><td>Synovis</td><td>SAS-18</td><td></td></tr><tr><td>Micro-Forceps</td><td>Synovis</td><td>FRS-15 RM-8</td><td></td></tr><tr><td>Micro-Dilators</td><td>Synovis</td><td>FRS-15 RM-8d.1</td><td></td></tr><tr><td>Micro-Needledriver</td><td>Synovis</td><td>C-14</td><td></td></tr><tr><td>Micro-Clamp Applicator</td><td>Synovis</td><td>CAF-4</td><td></td></tr><tr><td>Micro-Flushing Needle</td><td>Hamilton</td><td></td><td>10MM, 30&amp;deg;, 33G</td></tr><tr><td>Lactated Ringers Solution</td><td>Fisher Scientific</td><td>NC9968051</td><td></td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>DEA Number required; Obtained from hosptial pharmacy.</td></tr><tr><td>Enrofloxacin; Baytril</td><td>Bayer Health Care</td><td>186599</td><td></td></tr><tr><td>Heparin</td><td></td><td></td><td>Obtained from hosptial pharmacy</td></tr></tbody></table>

orthotopic hind limb transplantation in the mouse

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

Performing vascularized composite allotransplantation in a mouse model using a non-suture cuff technique allows to achieve excellent and long term graft and animal survival as shown in Figure 1. Furthermore, it represents a reliable method of obtaining reproducible outcomes of gradual allograft rejection in vascularized composite allotransplantation as documented by the images shown in Figure 2. H&amp;E histology of tissues obtained from animals undergoin...

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

The overall goal of this instructional video is to introduce a murine model of Orthotopic Hind Limb Transplantation using a non-suture cuff technique for super-microvascular anastomosis. Animal models are paramount to the understanding and treatment of human disease and have paved the way for reconstructive transplantation to become a clinical reality over the past two decades. This particular model can help answer key questions related to the path of physiology of an alloimmune response specific to vascularized composite allografts.The main advantage of the technique is that the non-suture cuff technique significantly reduces the complexity and increases the success rate of this super-microvasular procedure. After confirming the appropriate level of sedation by toe pinch shave the hind limb and the groin of the donor animal. Next disinfect the skin with 10%povidone iodine and place the mouse under a disecting microscope.Using scissors begin the donor limb harvest with a groin skin incision proximal to the mid thigh area. Circumstantially connecting the incision to demarcate the hind limb from the rest of the rest of the body. Identify and dissect the femoral artery, vein, and nerve.Then use forceps and microscissors to separate the three structures. Preventing microtrauma to the vessel wall by using a no touch technique is essential for ensuring the success of the procedure. Once the vascular pedicle has been dissected use the microscissors to divide the vessels at the inguinal ligament.Continue the division of the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the graft from the donor animal. Then, transect the femur cutting at the middle of the femoral shaft. Using a syringe equipped with a 33 gauge needle, flush the harvested limb with two ml of heparinized four degree Celsius saline.Then, place individual polyimide cuffs on the femoral vein and artery. And store the graft in wet cotton gauze in a petri dish at four degrees Celsius until inset. Mounting the pesis onto the polyimide cuffs is the single most critical step of the procedure.As malnotation may impair the bloodflow and improper tish pressmon may expose some antibiotic tissues increasing the likelihood of thrombosis formation. To remove the recipient hind limb, inject 0.3 ML of warm saline intraperitoneally. Make a circumferential incision around the hind limb and identify and dissect the femoral artery, vein, and nerve as just demonstrated.Once the vascular pedicle has been dissected clamp the femoral vessels at the inguinal ligament. And cut the vessels distally at the superficial epigastric artery. Continue to divide the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the native hind limb from the recipient animal.Then transect the femur in the middle of the femoral shaft. Then using a 20 gauge spinal needle as a intramedullary rod and align the femur bones of the recipient and the graft in the native hind limb position. Connect the donor and recipient femur bones.Use absorbable suture material to coapt the ventral and dorsal muscle groups. Performing this alignment accurately and quickly prevents extended blood loss from the bone cavity. To connect the femoral vessels, pull the recipient side of the vessel over the polyimide cuffs.And use a 10-0 nylon suture to fix the recipient vessel onto the cuff with a circumferential tie. When the vessel has been stabilized release the clamps. And visually verify that the cuffs are not misrotated.Visually verify homeostasis and use electrocautery to stop any source of bleeding. Then close the skin with non-absorbable nylon sutures. And monitor the animal until it is fully recovered.Performing vascularized composite allotransplantation in a mouse model using this non-suture cuff technique facilitates the achievement of excellent and long-term graft and animal survival. Moreover, this technique represents a reliable method for obtaining reproduceable outcomes of gradual allograft rejection and a vascularized composite allotransplantation model as documented by the images. Indeed, histological analysis of tissues obtained from animals undergoing rejection further underscores the reproducible dynamics of allograft rejection in this murine model.Once mastered, this technique can be completed in 90 to 120 minutes, if it is performed properly. While attempting this procedure it's important to remember to pay the utmost attention to the handling of the tissue and to avoid any sort of bleeding. After its development, this technique can pave the way for researchers in the field of reconstructive transplantation.To explore basic mechanistic questions related to the alloimmunity of vascularized composite allografts. After watching this video, you should have a good understanding of how to perform all the critical steps of an Orthotopic Hind Limb transplantation in the mouse.

orthotopic-hind-limb-transplantation-in-the-mouse

Research

JoVE Journal

Medicine

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Orthotopic Hind-Limb Transplantation in Rats

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Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

We present a reproducible mouse model of heterotopic hindlimb-to-neck vascularized composite allotransplantation. This supermicrosurgical protocol, involving 0.3-0.5 mm vessel anastomoses, integrates nylon filament guidance and venous cuffing. It provides a reliable platform for translational studies on tolerance, ischemia-reperfusion injury, and immunological mechanisms in vascularized composite allotransplantation.

Heterotopic Hindlimb-to-Neck Transplantation in the Mouse: A Supermicrosurgical Model of Vascularized Composite Allotransplantation

This protocol presents a simplified, end-to-side suture anastomosis technique for murine cervical heart transplantation, providing a robust and reproducible model for studying transplant immunology.

Cervical Heterotopic Mouse Heart Transplantation using End-to-side Anastomosis Technique to Study Transplant Immunology

Learn orthotopic hind limb transplantation in mice using a non-suture cuff technique for super-microvascular anastomosis. This method reduces procedural complexity and increases success rates for studying alloimmune responses in vascularized composite allotransplantation.

Orthotopic Hind Limb Transplantation in the Mouse

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