Name: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation (Video) | JoVE Journal
Uploaded: 2014-11-17T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Learn murine corneal transplantation for studying acute allograft rejection using a mouse model. This method enables researchers to evaluate graft integrity and test therapeutic strategies.

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5 minutes

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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</ol>

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.

<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

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...

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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JoVE Journal

Medicine

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

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