This work was supported by NIH grants R01 HL109420 to WM and AHA 9320033N to LY.

Mouse allograft and syngeneic graft transplantation model
All animal studies were approved by the institutional animal care and use committees of Yale University. For allograft model, segments of thoracic aorta from male, 4-5 week old WT (C57BL/6J) or IFN-&gamma;R-KO mice were interposed into the abdominal aorta of female recipient, 8-12 week old WT using an end-to-end microsurgical anastomotic technique (see next for details). For syngeneic graft model, segments of thoracic aortae from male, 4-5 week old WT mice were interposed into the abdominal aortae of male, 8-12 week old IFN-&gamma;R1-KO using an end-to-end microsurgical anastomotic technique. At 1 week postoperatively, the animals were inoculated i.v. with Ad5.CMV-mouse IFN-&gamma; or Ad5.CMV-LacZ (Qbiogene) at 1 x 109 PFU. Serum mouse IFN-&gamma; levels were measured by ELISA (eBioscience) at 1 and 5 weeks after adenovirus administration. Certain animals received BrdU (Sigma-Aldrich) at 100 mg/kg s.c. for 2 weeks before sacrifice.
End-to-end microsurgical anastomotic technique (Video will be taken for this part):
1. Donor Procedure
<ol>
	<li>Anesthetize the donor mice with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (10 mg/kg).</li>
	<li>After ensuring adequate anesthesia, place the donor mice under the operating microscope at X8-30 magnification on a tray in a supine position, and use Iodine Prep Pad and Alcohol Prep Pad for chest wall preparation.</li>
	<li>Incise the anterior chest wall through the ribs and diaphragm to expose the heart.</li>
	<li>Open the right atrium, inject 10 ml of heparinized (100 U/ml) saline solution into the left ventricle to flush the mice using a 10-ml syringe with a 25-gauge needle.</li>
	<li>Resect the heart and lungs to expose the entire length of thoracic aorta.</li>
	<li>Dissect the thoracic aorta carefully to minimize the direct trauma, while identify, ligate and divide the lumber branches using 11-0 suture near the aorta.</li>
	<li>Once the thoracic aorta is free from surrounding tissue, excise the whole thoracic aorta for transplantation.</li>
	<li>Flush the cut end of the donor aorta with heparinized (100 U/ml) saline solution, and store it in the same solution on ice (up to 6 hr) until transplantation.</li>
</ol>
2. Recipient Procedure
<ol>
	<li>Inject the recipient mice with i.p. ketoprofen (5 mg/kg) for analgesia, and thenanesthetize the mice with i.p. ketamine (50 mg/kg) and xylazine (10 mg/kg). The recipient mice are deeply anesthetized within 10 minutes for a duration of 60-90 min. During surgery, the level of anesthesia is assessed every 15 min by pinching the anterior abdominal wall or foot using forceps. If the animal reacts to the noxious stimuli at anytime during surgery, an additional dose of ketamine and xylazine (1/4 or 1/3 original dose) will be administered by s.c. or i.m.</li>
	<li>After ensuring adequate anesthesia, shave off the fur in the anterior abdominal wall using an electric shaver, clean the skin using Iodine Prep Pad and Alcohol Prep Pad, cover the eyes using ophthalmic ointment. During surgery, the following aseptic techniques will be used for the operations, including cleaning the operating table with 10% bleach, wearing sterile gloves and using sterilized microsurgery instruments(steam-sterilized instruments for the start of the operation, then hot glass bead-sterilized instruments between multiple operations, etc).</li>
	<li>Place the recipient mice under the operating microscope at a magnification of X8-30 on a tray in a supine position.</li>
	<li>Incise the abdominal wall at the midline from xyphoid to pubis, and spread the abdominal wall apart using a micro-retractor to expose the abdominal cavity.</li>
	<li>Retract the intestines superiorly and wrap with gauze moistened with saline solution. The recipient mice become chilled by loss of heat through externalized intestines. It is important to maintain animal warmth. However, the heating board is not used during the surgery as modest hypothermia is of benefit in preventing spinal injury during occlusion of aortic blood flow as well as there is inefficient heat transfer with absent blood flow to the lower half of the mice body.</li>
	<li>Move the reproductive organs inferiorly, wrap the abdomen areas of recipient mice with saline-solution-moistened gauze, and use it to retract the rectum to right side of the abdomen. The exposed tissues are irrigated periodically with saline solution during transplantation.</li>
	<li>Dissect bluntly a segment of infrarenal aorta between the renal vessel proximally and aortic bifurcation distally and separate from inferior vena cava (IVC) carefully.</li>
	<li>Identify and ligate all of the small branches originating from this segment of abdominal aorta using 11-0 Polyamide Monofilament suture.</li>
	<li>Cross-clamp the isolated segment of abdominal aorta using two vascular clamps proximately 5 mm apart, one at each end.</li>
	<li>Transect the abdominal aorta between the clamps using sharp micro-scissors to create the anastomotic sites.</li>
	<li>Resect a small segment of abdominal aorta (up to 0.4 mm in length) from one cut end of transected recipient aorta to accommodate the donor aortic graft.</li>
	<li>Flush the aortic segments between clamps using heparinized saline solution to remove the residual blood. The aortic segments between clamps and the donor aortic graft are irrigated periodically with heparinized (100 U/ml) saline solution (up to 40 ml/kg) during anastomosis.</li>
	<li>Transect both sides of donor aorta with sharp micro-scissors to create a segment graft of 2.5 mm in length for transplantation.</li>
	<li>Place the donor aortic graft in the orthotopic position, anastomose the donor graft's proximal and distal end to the recipient's proximal and distal cut end of abdominal aorta, respectively, with an end-to-end pattern using 11-0 Polyamide Monofilament suture.</li>
	<li>For the continuous sutures, place the stay sutures at 3 and 9 o'clock positions firstly. Between two stay sutures at each side of graft, anastomose both cut edges in 3-4 stitches using the running sutures, and tie the running sutures to stay sutures with a double knot. Since both layers of closure are continuous, the risk of dehiscence is increased. The donor aortic graft should be in appropriate length to connect the recipient's proximal and distal cut end of abdominal aorta.</li>
	<li>For the interrupted sutures, construct four anastomosis at 3 and 9 o'clock positions in both sides of graft firstly, and anastomose both cut edges in 3-4 stitches between two stay sutures at each side of graft.</li>
	<li>Release the distal clamp to allow the retrograde flow into graft, identify the bleeding sites and make additional stitches within 3 minutes.</li>
	<li>After ascertaining satisfactory hemostasis, release the proximal clamp.</li>
	<li>Confirm the graft patency by presence of vigorous pulsation in the graft and the proximal adjacent segment of native abdominal aorta, in particular in the distal adjacent segment of abdominal aorta and inferior mesenteric artery. Consider thrombosis in anastomotic sites if the pulsation diminish within a few minutes after the restoration of blood flow.</li>
	<li>Return the intestines into the abdominal cavity.</li>
	<li>Suture close the abdominal wall using 5-0 Nylon suture in muscle layer and skin layer, respectively.</li>
	<li>Place the recipient mice into a clean cage on the top of a heating pad, and wait 1-2 hr for the mice to regain consciousness and recover from anesthesia. It is important to maintain animal adequate warmth during recovery. Keep the mice in the warm and dry cage before animal wake from anesthesia.</li>
	<li>Assess the motor function of hind limbs after mice recovery, and again on the second day. A successful transplantation surgery will be confirmed if no dysfunction of hind limbs is observed in recipient mice on the second day.</li>
	<li>Administer the recipient mice with Ketoprofen in drinking water (5 mg/kg/d = 27 ug/ml in drinking water) for 48 hr. If any recipient mice suffer from pain unrelieved by the postoperative analgesics as evidenced by loss of mobility, failure to groom, abnormal bunched posture, etc, they will be euthanized. The animals will be euthanized at predefined end-points after 1-60 days.</li>
</ol>
Graft analysis
Artery grafts in allograft were procured at 4 weeks and grafts in syngeneic graft model were 6 weeks post-operatively (5 weeks after viral infection) and analyzed by standard histological techniques for Elastica-van Gieson (EVG) staining, hematoxylin and eosin (HE) staining and immunofluorescence staining. Pictures were taken using an immunofluorescence microscope system (Zeiss). Cell counting of nuclei surrounded by positive immunostaining was performed under high magnification and averaged from 5 cross-sections for each graft. The graft area measurements of the lumen (within the endothelium), intima (between the endothelium and internal elastic lamina, IEL), media (between the IEL and external elastic lamina, EEL), wall thickness (between the endothelium and external elastic lamina) and whole vessel (within the EEL) were calculated from 5 serial cross-sections, 150 &mu;m apart for each graft, using computer-assisted image analysis and NIH Image 1.60 (<a href="http://rsbweb.nih.gov/nih-image/" target="_blank">http://rsbweb.nih.gov/nih-image</a>).
Statistical analysis
All data are expressed as mean &plusmn; SEM. Two-tailed, paired t tests and a two-way ANOVA analysis were performed using the Prism software program (GraphPad Software). Differences with P&lt;0.05 were considered to indicate statistical significance.

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We have nothing to disclose.

The described protocols are focused on mouse GA models. The procedures can be applied to other graft transplantation models. These models include humanized xenograft (i.e. human coronary artery segments interposed into the infra-renal aortae of immunodeficient mice), and acute rejection mouse GA model (i.e. a mouse aorta from one genetic strain into another genetic strain recipient). Our described mouse models are more to close human GA lesions. The first model involves interposition of a vessel segment...

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree 7. Early stages may cause eccentric and focal stenoses that are more obvious in arteries, thus more closely resembling stenoses seen in conventional atherosclerosis. The lumen loss of the graft vessels results from intimal expansion due to infiltration of host T cells and macrophages and especially to accumulation of extracellular matrix and smooth muscle-like cells originated from graft, host or both 5, 13, 19, that is inadequately compensated by outward vessel remodeling. In cardiac allografts, the most clinically significant lesions are those in the epicardial and intramyocardial coronary arteries. Ultimately, GA of the coronary arteries will cause ischemic heart failure. GA is the major cause of late cardiac graft loss. The stenoses of GA stop at the suture lines, strongly implicating the host response to graft alloantigens in its pathogenesis and leading us to classify GA as a form of chronic rejection 3. However, other forms of arterial injury may increase the risk of GA, either through increasing the net burden of injury or by intensifying and/or modulating the alloimmune response. The endothelial cell (EC) lining of graft arteries is preserved in human GA and the most superficial regions of the intima adjacent to the EC lining is the site most heavily infiltrated by host-derived IFN-&gamma;-producing T cells and macrophages 11; in some patients GA is associated with the development of donor-specific alloantibodies that bind to graft EC 16 but the vessels show little evidence of the fibrinoid necrosis that is characteristic of acute antibody-mediated rejection 11.
The most critical component of GA pathogenesis is the proliferation of smooth muscle -like cells within the intima; if this process can be arrested, GA is unlikely to progress. Previous work from our group had shown that intimas of human coronary artery segments interposed into the infra-renal aortae of immunodeficient mice expand in response to adoptively transferred human T cells allogeneic to the artery donor and that this process could be inhibited by neutralizing human IFN-&gamma; 18. Furthermore exogenous human IFN-&gamma; could cause intimal (and medial) vascular smooth muscle cell (VSMC) proliferation in these arterial grafts in the absence of human T cells 15, 17. (It's important to note that human and mouse IFN-&gamma; do not cross species, ruling out indirect effects on the mouse host in this experimental system.) These humanized mouse models have the benefit of recapitulating human T cell/vascular cell interactions and the intimal lesions are largely composed of human (i.e. graft-derived) cells, as has been observed in clinical specimens, but they do not fully recapitulate the clinical situation because they ignore the role of host macrophages and possibly other cell types involved in clinical transplant lesions. A conventional mouse model of this process could theoretically address this problem, complementing the limitations of the humanized model by involving a complete host immune system and providing the additional advantage of allowing the power of mouse genetic approaches to be applied to GA. The two most widely used mouse models involve heterotopic heart transplantation and orthotopic artery transplantation 1. The lesions that develop in the arteries of heterotopic heart grafts are largely made up of host cells, likely of bone marrow origin, whereas intimal cells of the arteries in human heart grafts are predominantly of graft origin 5, 13, 19. This is a significant distinction that has led us to develop alternative mouse models. Interposition of a mouse aorta from one strain into another mouse strain recipient is even more limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks 19. Consequently, the subsequent changes seen in the interposed vessel segment are solely a response of host cells that have repopulated the decellularized vessel scaffold, creating a highly artifactual situation of limited relevance as a model for the changes in graft vessels that occur in the clinic. We have recently developed two new mouse models to circumvent these problems 21. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; (IFN-&gamma;R-KO) followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. Here, we describe detailed protocols and the advantages of our mouse GA models.

<table border="1" >
<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>C57BL/6J (H-2b)</td>
 <td>Jackson Laboratories 
 (Bar Harbor, ME)</td>
 <td>000664</td>
 <td>Donor (5 weeks) 
 Recipient (8-12weeks)</td>
 </tr>
 <tr>
 <td>Ketamine Hydrochloride Injection </td>
 <td>Hospira Inc.</td>
 <td>NDC 0409-2053</td>
 <td>Storage Solution(50 mg/ml) 
 Working Solution(5 mg/ml)</td>
 </tr>
 <tr>
 <td> Xylazine Sterile Solution</td>
 <td>Lloyd Inc.</td>
 <td>NADA# 139-236</td>
 <td>Storage Solution(100 mg/ml) 
 Working Solution(1 mg/ml)</td>
 </tr>
 <tr>
 <td>Ketoprofen</td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-4396-01</td>
 <td>Storage Solution(100 mg/ml) 
 Working Solution-oral 
 (0.027 mg/ml)</td>
 </tr>
 <tr>
 <td>Heparin Sodium</td>
 <td>Sagent Pharmaceticals</td>
 <td>NDC 25021-400</td>
 <td>Storage Solution(1000 U/ml) 
 Working Solution(100 U/ml)</td>
 </tr>
 <tr>
 <td>Saline solution (Sterile 0.9% Sodium Chloride)</td>
 <td>CareFusion</td>
 <td>AL4109</td>
 <td></td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride Injection</td>
 <td>Hospira Inc.</td>
 <td>NDC 0409-4888-10</td>
 <td>To prepare the anesthetic </td>
 </tr>
 <tr>
 <td>Petrolatum Ophthalmic Ointment </td>
 <td>Dechra Veterinary Products</td>
 <td>NDC 17033-211-38</td>
 <td></td>
 </tr>
 <tr>
 <td>Iodine Prep Pads </td>
 <td>Triad Disposables, Inc.</td>
 <td>NDC 50730-3201-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Alcohol Prep Pads</td>
 <td>McKesson Corp.</td>
 <td>NDC 68599-5805-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Leica</td>
 <td>MZ95</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Scissors</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-5693</td>
 <td></td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>F.S.T</td>
 <td>15009-08</td>
 <td>To transect the aorta of donor or recipient </td>
 </tr>
 <tr>
 <td>Extra Narrow Scissors</td>
 <td>F.S.T</td>
 <td>14088-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle Holder/Forceps</td>
 <td>MICRINS</td>
 <td>MI1542</td>
 <td>To hold the needle</td>
 </tr>
 <tr>
 <td>Fine Forceps</td>
 <td>F.S.T </td>
 <td>11254-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>F.S.T</td>
 <td>11251-35</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Forceps</td>
 <td>F.S.T</td>
 <td>11000-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>F.S.T </td>
 <td>13011-12</td>
 <td></td>
 </tr>
 <tr>
 <td>LANCASTER Eye Speculum</td>
 <td>Zepf Medical Instruments</td>
 <td>42-1209-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Vascular Clip</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-6472</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Clip Applying Forceps With Lock</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-5440</td>
 <td></td>
 </tr>
 <tr>
 <td>Black Polyamide Monofilament Suture</td>
 <td>AROSurgical Instruments Corporation</td>
 <td>Cat #T4A10Q07</td>
 <td>10-0 suture, Needle=70 microns</td>
 </tr>
 <tr>
 <td>Black Monofilament Nylon Suture</td>
 <td>Syneture 
 (Covidien)</td>
 <td>SN-1956</td>
 <td>6-0 suture</td>
 </tr>
 <tr>
 <td>Non-Woven Songes</td>
 <td>McKesson Corp.</td>
 <td>Reorder No. 94442000</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml Syringe</td>
 <td>BD</td>
 <td>REF 309659</td>
 <td></td>
 </tr>
 <tr>
 <td>3 ml Syringe</td>
 <td>BD</td>
 <td>REF 309657</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml Syringe</td>
 <td>BD</td>
 <td>REF 309604</td>
 <td></td>
 </tr>
 <tr>
 <td>18G 1 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305196</td>
 <td></td>
 </tr>
 <tr>
 <td>25G 7/8, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305124</td>
 <td></td>
 </tr>
 <tr>
 <td>27G 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305109</td>
 <td></td>
 </tr>
 <tr>
 <td>30G 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305106</td>
 <td></td>
 </tr>
 <tr>
 <td>Hearting Pad</td>
 <td>Sunbeam</td>
 <td>Z-1228-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Trimmer</td>
 <td>Wahl</td>
 <td>9854-500</td>
 <td></td>
 </tr>
<tr>
<td></td>
<td></td>
<td></td>
<td>Table 2. Specific reagents and equipment.</td>
</tr>
 </tbody>
</table>

mouse models for graft arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

Mouse allograft arteriosclerosis (GA) model: In this model, a male donor aorta is transplanted into female recipient so that the host induces alloreactive T cell-mediated alloimmune responses against a minor Y antigen (the male-specific minor histocompatibility antigen H-Y) expressed on the graft 12, and in turn T cell-produced IFN-&gamma; drives VSMC proliferation 20 as observed in other allograft transplantation models 2, 6, 8-10, 14. A segment of thoracic aorta from a ...

Watch this Scientific Journal Video about Mouse Models for Graft Arteriosclerosis at JoVE.com

Mouse Models for Graft Arteriosclerosis

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Research

JoVE Journal

Medicine

13.6K Views.  Yale University School of Medicine. אנו מתארים פרוטוקולים לarteriosclerois שוחדנו העכבר (GA) דגמים אשר כרוך חציצה של מגזר כלי עכבר לנמען של אותו הזן המולד. על ידי backcrossing שינויים גנטיים נוספים לתורם הכלי, המודל יכול להעריך את ההשפעה של גנים ספציפיים על GA.

Video: Mouse Models for Graft Arteriosclerosis

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

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 ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...