The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

NOTE: All animals used in this procedure are treated in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research as well as the guidelines set down by the animal oversight committee at Saint Louis University. 
NOTE: All surgical instruments and solutions are sterilized prior to surgery to limit microbial infection of the eye.&#160;It should be noted that while the animals do experience some pain from this procedure, we do not employ analgesics.&#160; The reason for this is because all analgesics are anti-inflammatory and since corneal transplantation responses involve inflammation, the use of anti-inflammatory drugs would compromise our ability to determine what factors are involved in corneal graft failure.
1. Anesthesia
<ol style="margin-left: 40px;">
	<li>Place donor and recipient mice under general anesthesia by IP injections of ketamine (86.98 mg/kg) and xylazine (13.04 mg/kg).</li>
	<li>Keep the recipient mouse under anesthesia throughout the procedure which typically takes 30 min to an hr. Consequently, constantly monitor the mouse for any signs of regaining consciousness.</li>
	<li>Apply puralube ointment to the eye that will not undergo surgery after the animal is anesthetized to prevent dryness.</li>
</ol>
2. Corneal Grafting
<ol style="margin-left: 40px;">
	<li>Obtaining the donor corneal button.
	<ol>
		<li>Once the animal is fully anesthetized, achieve adequate mydriasis by administration of a couple of eye drops of 1% tropicamide and 2.5% phenylephrine hydrochloride.</li>
		<li>Position the head of the donor animal horizontally on a board placed on a sturdy movable support. Fix the head with a strip of tape across the neck to ensure that the eye is in a horizontal position throughout the entire operation.</li>
		<li>Using a 2 mm diameter trephine, whose tip was dyed with methyl blue, outline the central cornea graft site.</li>
		<li>With a sharp blade, penetrate the cornea and inject healon into the anterior chamber to deepen it to reduce the chance of damage to the donor endothelium and&#160;underlying lens.</li>
		<li>Excise the donor graft with vannas scissors and place into a dish containing Hanks&#8217; balanced salt solution until use.</li>
		<li>After the donor graft has been the removed, euthanize the donor mouse via CO2 inhalation.</li>
	</ol>
	</li>
	<li>Preparing the graft bed.
	<ol>
		<li>Repeat the same steps as described in 2.1.1 through 2.1.2 for the recipient.</li>
		<li>Using a 1.5 mm diameter trephine, outline the recipient graft site.</li>
		<li>With a sharp blade, penetrate the cornea and inject healon into the anterior chamber to deepen it to reduce the chance of damage to the underlying lens.</li>
		<li>Remove the outlined central corneal button from the recipient using vannas scissors and discard.</li>
	</ol>
	</li>
	<li>Suturing the graft
	<ol>
		<li>Place the donor cornea over the graft bed in the recipient&#8217;s cornea. Be sure that adequate healon is under the donor cornea to protect the donor endothelial cells from damage by direct contact with lens.</li>
		<li>Using super fine tipped microforceps, place the first bite of 11-0 nylon suture into the donor side, through the donor with 90% depth of full thickness to recipient&#8217;s side, then tie off.</li>
		<li>Once the cornea is anchored in place, perform midcardinal interrupted sutures such that the cornea has 8 to 10 total sutures and the donor cornea is securely lined up with and attached to the recipient corneal graft bed.</li>
	</ol>
	</li>
	<li>Deepening the anterior chamber
	<ol>
		<li>Deepen the anterior chamber by injecting HBSS or air bubble into the anterior chamber and gently check the integrity of the corneal graft for leakage with a cellulose sponge. 
		NOTE: If the anterior chamber cannot be reformed then there is a high probability of cataract which will make future evaluations of the transplanted cornea very difficult and will also potentially lead to donor corneal endothelium dysfunction and thus graft failure.</li>
	</ol>
	</li>
	<li>Final evaluation
	<ol>
		<li>Observe the eye to determine that the pupil is round and the depth of the anterior chamber is normal. 
		NOTE: If the pupil is not round this indicates that during suturing the iris was damaged and thus the graft is considered a technical failure.</li>
		<li>Apply antibiotic ointment to the eye. Optional: Close the eye lid with a 7-0 silk suture.</li>
		<li>Observe mice until they are fully awake and then individually house them for a minimum of two days post-surgery.</li>
	</ol>
	</li>
</ol>
3. Suture Removal
<ol style="margin-left: 40px;">
	<li>In those cases where lid suture is used, anesthetize mice as described above and remove the lid suture at 48 hr.</li>
	<li>Anesthetize mice on day 7 postoperatively. Remove the sutures securing the corneal graft. Once the sutures are removed and the animal has fully awakened, return it to its cage.</li>
</ol>
4. Clinical Evaluation
<ol style="margin-left: 40px;">
	<li>Examine the eye for indications of procedural complications which include, cataract (clouding of the lens), hyphema (blood in the anterior chamber), an anterior chamber that is not of the proper depth, or significant opacity of the cornea. Consider those that demonstrate these complications as &#8220;compromised&#8221; and euthanize them by CO2 inhalation.
	<ol>
		<li>Perform all examinations on unanesthetized mice. Hold the mouse with one hand thus restraining the mouse so that the other hand can proptose the eye to enable a better view of the eye. Once observations are completed, return the animal to its cage.</li>
	</ol>
	</li>
	<li>Have an observer unfamiliar with the treatment groups evaluate transplanted corneas 2 to 3 times a week for signs of corneal graft rejection episodes or corneal graft failure. Use either the surgical microscope or a horizontal slit-lamp biomicroscope for these observations.
	<ol>
		<li>Evaluate every cornea for opacity using a scale of 0 to 5. The scale is defined as follows:
		<ol>
			<li>Assign a score of 0 to those corneas that have no signs of opacity.</li>
			<li>Assign a score of 1 to those corneas that show minimal superficial opacity.</li>
			<li>Assign a score of 2 to corneas that display mild and deeper opacity but the underlying pupil and iris are still discernable.</li>
			<li>Assign a score of 3 to cornea that display stromal opacity wherein the iris cannot be seen in detail with the exception of the pupil margins.</li>
			<li>Assign a score of 4 to cornea that display dense stromal opacity and if no underlying structures can be viewed.</li>
			<li>Assign a score of 5 to cornea that display complete opacity and intensive stromal edema, with pupil and iris totally obscured.</li>
		</ol>
		</li>
		<li>Also evaluate every cornea for the degree of blood vessel infiltration (neovascularization) using a rating scale of 1 to 8. To accomplish this, view the cornea as consisting of 4 equal quadrants and determine the amount of blood vessels in each of these quadrants with a score that will range from 0 (no vessels) to 2 for extensive vascularization of that quadrant. Add the individual scores from each quadrant to calculate the final neovascularization score.</li>
		<li>Classify corneas as acutely rejected if they have a score of 3 for two consecutive observations for time points up to 5 weeks.</li>
		<li>Classify mice whose corneas were clear at 5&#160;weeks but develop opacification at times &#62;45&#160;days post engraftment, with a score of 3 for two consecutive time points, as having undergone late-term corneal allograft rejection. Use Kaplan-Meier survival curves to analyze graft survival.</li>
	</ol>
	</li>
</ol>
5. Manipulation of the Model
<ol style="margin-left: 40px;">
	<li>Preparation of single cells from the spleen.
	<ol>
		<li>To prepare single cells, first euthanize the donor mouse. Then, remove the spleen.</li>
		<li>Place the spleen in a cell strainer and disrupt it with a syringe plunger from a 3 ml syringe.</li>
		<li>Wash cells and resuspend in 10 ml of Hanks Balanced Salt Solution.</li>
		<li>Remove 10 &#956;l of cell suspension and add to 10 &#956;l of 0.4% trypan blue and mix. Add that to hemocytometer and count the cells in the central grid. The number of cells in tube is the cell count x 104 x 2 (dilution factor in trypan blue) x 10 (volume in the tube).</li>
	</ol>
	</li>
	<li>Injection into the anterior chamber.
	<ol>
		<li>Anesthetize mice as previously described.</li>
		<li>Perform injections using a dissecting microscope. For each intracameral injection, use 106 spleen cells in a 0.005 ml volume and a 0.25 ml microliter syringe fitted with a 33 G&#160;needle. 
		NOTE: Other manipulations of the model can be performed by treating animals with reagents that act as either antagonists or agonists to determine the role that a particular factor may play following orthotopic corneal allograft surgery.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

The murine model of corneal transplantation described here enables the investigator to study human corneal allograft rejection in a model that is predictive of what factors are best associated with both rejection15,17,18,20, 26-30 and acceptance21-25 of corneal allografts. Unlike human corneal transplantation, in which patients are given either topical or systemic steroid treatment to either treat or prevent rejection31, this model is typically used to determine those factors that are relevant to allograft rejection in the absence of such therapy. In addition to a model for acute corneal allograft rejection, which typically occurs within 30 to 40 days of transplantation, we also present a late-term model of such rejection that occurs 45&#160;days post-engraftment in mice and mimics many of the features of late-term and chronic rejection32,33 which thus far have not been modeled in animals.
The model&#8217;s strengths are that one can dissect various mechanisms that are responsible for the relatively high rate of corneal allograft acceptance as well as determining which mechanisms are most responsible for corneal allograft failure. This model also allows one to test various therapeutic strategies in an animal which possesses an immune system that is very similar to the human immune system. To that end, the existence of many reagents that react with murine factors as well as transgenic and gene-targeted mice, allow for the evaluation of so many more factors than would be the case with other species. This ability to evaluate so many different factors which are important to both success and failure of corneal allografts is a significant positive in favor of using this murine model versus other animal models in which surgery is easier to perform due to the increased size of the eyes of these species.
While there are several weaknesses of this model, we believe the strengths far outweigh them. One of the most significant, which was alluded to above, is the technical expertise involved in performing corneal transplantation in mice. Due to the small size of the murine eye, this requires someone who is adept at microsurgery, both in performing the transplantation as well as removal of sutures. Consequently, repeated practice is required to master and maintain proficiency at this technique. The second weakness is that while mice are very similar to humans, they are not the same. These animals display a greater tendency towards neovascularization, than do human corneas. Furthermore, this model will not mimic the surgical procedures meant to treat endothelial cell dysfunction like Fuch&#8217;s dystrophy2. Prior to recent year&#8217;s full thickness transplants like the ones used in the described protocol were employed. Presently, endothelial transplants involving only the endothelial portion of the cornea have been employed34,35. Such transplants have not been modeled thus far in mice due to the difficulty of performing this surgery in such small animals.
For late-term rejection (&#62;45&#160;days) two factors unique to this version of the model stand out. The first weakness with this form of the model being that mice must be maintained for a minimum of two to three months in order to study late-term rejection. And secondly, the corneas must first survive potential acute rejection reactions. Such acute rejections occur in 50 to 70% of recipients17,20-24. Thus to study late-term rejection one will engraft mice whose transplants will be rejected acutely and will not survive until 45&#160;days post-engraftment. In order to mitigate this we have found that the rejection rate in male mice is less than that observed for female mice (manuscript in preparation) and thus to study late term rejection it is advised that only male mice be used. We also attempted to prolong corneal allograft survival with steroid treatment, but this did not prove to be particularly useful (personal observations) and thus do not recommend that steroid treatment be used. In spite of these complications and the caveat that likely approximately half or somewhat more of those engrafted with allogeneic corneas will remain clear until after 45&#160;days, this is still a very useful model to study those factors that are relevant to late-term rejection.

Corneal transplantation is one of the most successful and common types of transplantation performed in humans. The reasons why this surgery is performed are a result of injury, infectious disease1, or other forms of non-infectious corneal disease2. Figures from the Eye Bank Association of America indicate that over 46,000 were performed in 2011 (see web site at: restoresight.org/eye_banks/eye_banks.html). An indication of its success is that one year failure rates for allogeneic corneal grafts range from 10 to 15% and at 5 years the success is in excess of 70%3-8. As many studies have shown, the success of corneal allografts is directly related to the fact that the eye is an immunologically privileged site. Factors responsible for the corneas status as an immune privilege site include the lack of both blood and lymph vessels in the cornea, a relative absence of antigen presenting cells, factors produced by the cornea that suppress immune effector funtions9-15, low expression of MHC antigens16, and the expression of FasL17-20.
However, in spite of these factors predisposing these grafts for success, they do undergo rejection3-7. Consequently, understanding those mechanisms that mediate this rejection as well as testing various therapies to prevent rejection is of critical importance. To that end, we describe here a murine model of corneal transplantation that has been in use for over 20 years to study corneal transplantation in a controlled experimental environment. Since transplantation responses involve many different factors working in concert that will ultimate determine whether the transplanted tissue fails or succeeds, it is not possible to understand the importance of those factors in any in vitro model. Consequently, studies using intact animals are required to determine what factors are important to either success or failure of transplanted tissue.
While other species of animals have been used to study corneal transplantation, the murine model has several advantages when compared to using other species. The first is the existence of many strains of mice that express certain transgenes or have been gene-targeted to lack expression of specific immunological factors whose function in transplantation can be better studied. In addition, there are many reagents (both recombinant factors and antibodies that neutralize factors) that are specific for mice and which do not exist for many other species of animals. Because of the existence of these factors, this model has been used extensively to identify relevant factors involved in acute corneal allograft responses15,17,18,20-29. Furthermore, many of the factors involved in corneal transplantation are also known to be functional in transplantation of other tissues.

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

murine corneal transplantation: a model to study the most common form of solid organ transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

The murine model of corneal transplantation has been used for over 20 years to successfully characterize mechanisms of both corneal allograft rejection19-23 and corneal allograft acceptance13,15,16,18, 24-27. This model was used to establish the importance of FasL expression in corneal allograft acceptance, in that animals that lack FasL were not able to accept corneal allografts15. It has also been used to demonstrate that vascular endothelial growth factor receptor 1 morpholino treatment significantly increases corneal graft survival28. In a very recent report this model was used to test whether pretreatment of mice prior to corneal engraftment with corticosteroids held any therapeutic benefit29. The authors showed that such pretreatment did improve the survival rate of corneal allografts29. These reports clearly demonstrate that the primary advantage of this model is that one can study in a live animal those things that are believed to be important to the success of corneal transplants in humans.
Previous studies from this laboratory have reported that one of the chief mechanisms responsible for corneal allograft rejection is the establishment of systemic delayed-type hypersensitivity (DTH) to specific alloantigens expressed by the corneal allograft19,20. We have since used this model to test whether establishment of systemic alloantigen DTH tolerance to alloantigens expressed by the donor corneal allograft improves survival of such grafts. As shown in Figure 1, BALB/c mice tolerized to C57BL/10 (B10) alloantigens did not improve corneal allograft acceptance as measured by mean survival time for these corneal allografts. Thus the mean survival time for rejected corneas was the same whether mice were tolerized or not. We also performed similar studies using skin graft responses as a means of testing the effectiveness of systemic DTH tolerization. These studies demonstrated that when DTH tolerance was established in BALB/c mice towards B10 alloantigens, skin grafts bearing some (B10.D2 and C.B10-H-2b) or all of B10 alloantigens did not display any increased mean survival time (Figure 2). We concluded from these studies that BALB/c mice that had established antigen-specific DTH responses did not derive any measurable benefit from such tolerance for either corneal or skin allografts. These data also indicate that information generated studying a murine model of corneal transplantation will, at times, be directly applicable to other forms of solid tissue transplantation.
<img alt="Figure 1" src="/files/ftp_upload/51830/51830fig1highres.jpg" /> 
Figure 1. BALB/c mice were injected into the anterior chamber with 106 spleen cells from C57BL/10 (B10) mice. Full thickness B10 corneal grafts were then placed on BALB/c mice injected with B10 (n = 10) or mock injected BALB/c mice (n = 11). Mice were observed for 50&#160;days and evaluated for corneal graft rejection. Data reported as mean survival time for those grafts that were rejected for &#8220;tolerized&#8221; mice (B10 injected, 6 mice) or not tolerized (mock injected, 6 mice). These curves did not demonstrate statistical significance (p &#62;0.05).
<img alt="Figure 2" src="/files/ftp_upload/51830/51830fig2highres.jpg" /> 
Figure 2. Injection of spleen cells into the anterior chamber does not impact survival of allogeneic skin grafts. BALB/c mice were injected into the anterior chamber with 106 spleen cells from C57BL/10 mice. After one week these mice were separately engrafted with skin from C57BL/10, n = 10, B10.D2, n = 7, C.B10-H-2b, n = 7 mice and compared to mice that were not injected and engrafted with C57BL/10 skin, n = 10. Results are expressed as the mean survival time + SEM.

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K Views.  Saint Louis University. Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Video: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

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 ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. 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 using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Immunology and Infection

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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.

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 ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

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