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 rel...

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.

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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...

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 ...

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9.6K 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

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 Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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 ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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

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

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

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

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...