Name: Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis (Video) | JoVE Journal
Uploaded: 2012-05-09T12:00:00.000+00:00
Duration: 5 min 37 s
Description: Explore a humanized rat model for studying intimal hyperplasia after stent deployment using human internal mammary artery transplantation. This translational approach enables assessment of human stent responses in human tissue.

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5 minutes

The authors thank Christiane Pahrmann for her contribution. Special thanks to Ethicon, Norderstedt, Hamburg (Germany) for providing the suture material.
Funding
Sonja Schrepfer has received a research grant from the Deutsche Forschungsgemeinschaft (DFG) (SCHR992/3 1 and SCHR992/4-1).] The work was supported by the ISHLT Shumway Career Development Grant 2010 and the Falk Research Funding (Stanford University).

1. Internal Mammary Artery (IMA) Preparation
<ol>
	<li>The arterial endothelium is denuded by the passage of a 2-french Fogarty arterial embolectomy catheter (Baxter Healthcare, Deerfield, IL, USA). The catheter is pulled through the whole vessel length twice to ensure endothelial damage.</li>
	<li>Use any human stent of 8mm length and 2.5 mm-3 mm in diameter (e.g. Translumina, Yukon stent). CAUTION: The diameter of the stent should not exceed the vessel diameter by more than 10% to avoid pre- and post-stent stenosis. CAUTION: Don't switch the length of the stent within the same study.</li>
	<li>Deploy the stent using the appropriate balloon pressure (which is noted on the stent package) to achieve the desired diameter.</li>
	<li>Store stented IMA in 4 °C RPMI + heparin (500 IE/10 ml) on ice until transplantation.</li>
</ol>
2. Animal Preparation
RNU Nude (Crl:NIH-Foxn1rnu) rats (300-350 g) are housed under conventional conditions in scantainer ventilated cabinets, fed standard rat chow and autoclaved water ad libidum.
<ol>
	<li>Anesthetize rat with isoflurane (2.5-3%) using an induction chamber.</li>
	<li>Shave the abdominal hair and place the rat on its back and place a facemask over its nose and mouth to keep up the anesthesia.</li>
	<li>Disinfect the abdominal area widely using Provo-Iodine, next use 80% ethanol - repeat this step twice. Check reflexes pinching the hind feet to be sure that the rat is sufficient anesthetized.</li>
	<li>Under microscopic view, perform an upper median laparotomy to expose the infrarenal abdominal aorta.</li>
	<li>Place the intestines in a saline moisturized glove. Fold the glove around the intestines to prevent loss of moisture.</li>
	<li>Dissect the aorta from the infrarenal region to the bifurcation, carefully not to cause damage on the branches of the vessels.</li>
	<li>Use micro clamps to stop the aortic blood flow. Place the proximal clamp first, followed by the distal clamp.</li>
	<li>Remove an app. 0.5-0.7 mm aortic segment and flush the remaining aorta with heparin (200 units).</li>
	<li>Take the stented IMA and shorten it to the adequate length and position it into the gap.</li>
	<li>Connect the IMA to the recipient aorta, by running sutures using 8-0 prolene suture (Ethicon, Norderstedt, Germany).</li>
	<li>Carefully open the first cranial clamp and then the caudal clamp.</li>
	<li>There should be a visible pulse in the transplanted IMA and at the distal end of the aorta.</li>
	<li>Place the intestines back into the abdomen.</li>
	<li>Flush the abdomen with pre-warmed sterile saline.</li>
	<li>Close the muscle layer of the abdominal wall using 6-0 prolene running sutures (Ethicon, Norderstedt, Germany).</li>
	<li>While the rat is still in anesthesia, inject 4-5 mg/kg Carprofen subcutaneously.</li>
	<li>Administer post-surgical analgesia as appropriate (e.g. Carprofen or Meloxicam) for 3 days post surgery.</li>
</ol>
3. Representative Results
For histology, the specimens were fixed in 4% formalin, dehydrated in a graded series of alcohol, and infiltrated in a mixture (MMA I) of 80% methylmethacrylate and 20% dibutylphthalate for 1 day, MMA I with 1% dry benzoyl peroxide for 1 day, and MMA I with 3% dry benzoyl peroxide (MMA III) for 1-2 days at 4 °C. Thereafter, the specimens were polymerized in fresh MMA III in glass vials in a water bath on a pre-polymerized base. Slow polymerization was achieved by keeping the vials at 26 °C overnight, increasing the temperature to 28 °C the next morning, and then increasing the temperature gradually by 0.5 °C over 12 h until polymerization occurred. The polymerized blocks were sectioned at 5 μm thickness using a MICROM HM 360 microtome equipped with a tungsten carbide knife.
To identify the origin of proliferating cells (Figure 1), slides were stained with antibodies identifying either the green fluorescent protein (GFP) or rat MHC-I and human smooth muscle cells. For these studies, human IMA was incubated with the reporter gene GFP overnight using lentiviral particles for stable transduction of IMA cells. Dividing daughter cells from human origin could be identified by expressing GFP. After deparaffinization, heat-induced epitope retrieval is performed by heating the slides in antigen retrieval solution using a steamer. The Image-iT FX signal enhancer can be used for the blocking step. Cells of human origin are identified using the mouse monoclonal antibodies against GFP (1:100 diluted in primary antibody diluent (Dako)), and further labeled with goat-anti-mouse IgG, Alexa Fluor 488 (1:1000 diluted in secondary antibody diluent). The smooth muscle cells were marked with the rabbit polycolonal anti-smooth muscle α-actin (1:100 diluted in primary antibody diluent), followed by goat-anti-rabbit IgG, Alexa Fluor 555 (1:1000 diluted in secondary antibody diluents). Each antibody incubation step is performed at 37 °C for 1 hour with three times PBS washing in between. Nuclei are stained with DAPI for 10 minutes. After mounting of the slides using Prolong Gold antifade reagent, samples were analyzed using confocal microscopy.
<img alt="Fluorescence microscopy, GFP/DAPI, SMA/DAPI, rat MHC-I, tissue staining, cell imaging comparison." src="/files/ftp_upload/3663/3663fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. The neointimal cells as human smooth muscle cells. A: Green= anti GFP, labeling cells of human origin; red=anti human smooth muscle cell actin; blue=DAPI, identifying cell nuclei. B: Green= anti rat MHC-I, labeling cells of rat origin; red=anti human smooth muscle cell actin; blue=DAPI, identifying cell nuclei. Proliferating cells are identified as smooth muscle cells and positive for GFP, but negative for the rat MHC-I molecule. Therefore, proliferating cells are human origin.

<ol><li>Deuse, T., Ikeno, F., Robbins, R. C., Schrepfer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+In-Stent+Restenosis%3A+An+Inexpensive%2C+Reliable%2C+and+Rapid+Preclinical+Model%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model.</a> J. Vis. Exp. (31), e1346(2009).</li>
<li>Oyamada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trans-iliac+rat+aorta+stenting%3A+a+novel+high+throughput+preclinical+stent+model+for+restenosis+and+thrombosis%5BTitle%5D)+AND+%22J.+Surg.+Res%22%5BJournal%5D)">Trans-iliac rat aorta stenting: a novel high throughput preclinical stent model for restenosis and thrombosis.</a> J. Surg. Res. 166, e91-e95 (2011).</li>
<li>Chamberlain, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+model+of+in+situ+stenting%5BTitle%5D)+AND+%22Cardiovascular+research%22%5BJournal%5D)">A novel mouse model of in situ stenting.</a> Cardiovascular research. 85, 38-44 (2010).</li>
<li>Deuse, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introducing+the+first+polymer-free+leflunomide+eluting+stent%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">Introducing the first polymer-free leflunomide eluting stent.</a> Atherosclerosis. 200, 126-134 (2008).</li>
<li>Finn, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+healing+after+sirolimus%2C+paclitaxel%2C+and+bare+metal+stent+placement+in+combination+with+peroxisome+proliferator-activator+receptor+gamma+agonists%3A+requirement+for+mTOR%2FAkt2+in+PPARgamma+activation%5BTitle%5D)+AND+%22Circulation+research%22%5BJournal%5D)">Differential healing after sirolimus, paclitaxel, and bare metal stent placement in combination with peroxisome proliferator-activator receptor gamma agonists: requirement for mTOR/Akt2 in PPARgamma activation.</a> Circulation research. 105, 1003-1012 (2009).</li>
<li>Tellez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+bare+metal+stent+implantation+in+homozygous+LDL+receptor+deficient+swine+induces+a+neointimal+formation+pattern+similar+to+humans%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">Coronary bare metal stent implantation in homozygous LDL receptor deficient swine induces a neointimal formation pattern similar to humans.</a> Atherosclerosis. 213, 518-524 (2010).</li>
<li>Suzuki, Y., Yeung, A. C., Ikeno, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pre-clinical+animal+model+in+the+translational+research+of+interventional+cardiology%5BTitle%5D)+AND+%22JACC+Cardiovasc.+Interv%22%5BJournal%5D)">The pre-clinical animal model in the translational research of interventional cardiology.</a> JACC Cardiovasc. Interv. 2, 373-383 (2009).</li>
<li>Holt, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intimal+proliferation+in+an+organ+culture+of+human+internal+mammary+artery%5BTitle%5D)+AND+%22Cardiovascular+research%22%5BJournal%5D)">Intimal proliferation in an organ culture of human internal mammary artery.</a> Cardiovascular research. 26, 1189-1194 (1992).</li>
<li>Swanson, N., Javed, Q., Hogrefe, K., Gershlick, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+internal+mammary+artery+organ+culture+model+of+coronary+stenting%3A+a+novel+investigation+of+smooth+muscle+cell+response+to+drug-eluting+stents%5BTitle%5D)+AND+%22Clin.+Sci.+%28Lond%29%22%5BJournal%5D)">Human internal mammary artery organ culture model of coronary stenting: a novel investigation of smooth muscle cell response to drug-eluting stents.</a> Clin. Sci. (Lond). 103, 347-353 (2002).</li>
</ol>

No conflicts of interest declared.

Although different in vivo research models are existing to investigate the development of intimal hyperplasia after stent placement, these models still facing translational hurdles to overcome. Furthermore, large animal models are expensive and special housing conditions as well as surgical equipment is not available for all laboratories.
Using a human IMA to study the development of human intimal proliferation and in-stent restenosis was studied before ex situ in organ ...

<table><tbody><tr><td>2 French Fogarty catheter</td><td>Baxter Internationl Inc.</td><td>120602F</td><td></td></tr><tr><td>Yukon Stent</td><td>Translumina GmbH, Hechingen, Germany</td><td></td><td>Use the stent of your choice according to your study protocol</td></tr><tr><td>RPMI media</td><td>Biochrom AG</td><td>Nr.F1275</td><td></td></tr><tr><td>heparin</td><td>Baxter Internationl Inc.</td><td>2B0953</td><td></td></tr><tr><td>isoflurane</td><td>Abbott Laboratories</td><td>B506</td><td></td></tr><tr><td>Provo-Iodine</td><td>Betadine Puredue Pharma</td><td>EAN:5995327165830</td><td></td></tr><tr><td>80% ethanol</td><td>Geyer</td><td>ETV 80/0500</td><td></td></tr><tr><td>Micro clamp</td><td>Harvard Apparatus</td><td>PY2-61-0186</td><td></td></tr><tr><td>Sutures 8-0</td><td>Johnson &amp;amp; Johnson</td><td>2808G</td><td></td></tr><tr><td>Sutures 6-0</td><td>Johnson &amp;amp; Johnson</td><td>1698 H</td><td></td></tr><tr><td>Carprofen</td><td>Feizer Vet</td><td>PZN:0110208</td><td></td></tr><tr><td>Metamizol</td><td>Ratiopharm</td><td></td><td></td></tr><tr><td>Target retrieval solution, pH9</td><td>Dako</td><td>S2368</td><td></td></tr><tr><td>Image-iT FX signal enhancer </td><td>Invitrogen</td><td>I36933</td><td></td></tr><tr><td>mouse monoclonal anti-GFP antibody </td><td>BD Biosciences</td><td>632381</td><td></td></tr><tr><td>primary antibody diluent </td><td>Dako</td><td>S3022</td><td></td></tr><tr><td>goat-anti-mouse IgG, Alexa Fluor 488 </td><td>Invitrogen</td><td>A11017</td><td></td></tr><tr><td>secondary antibody diluent </td><td>Dako</td><td>S0809</td><td></td></tr><tr><td>rabbit polycolonal anti-smooth muscle &amp;alpha;-actin </td><td>Abcam</td><td>ab5694</td><td></td></tr><tr><td>goat-anti-rabbit IgG, Alexa Fluor 555 </td><td>Invitrogen</td><td>A21430</td><td></td></tr><tr><td>Prolong Gold antifade reagent</td><td>Invitrogen</td><td>P36930</td><td></td></tr></tbody></table>

human internal mammary artery (ima) transplantation and stenting: a human model to study the development of in-stent restenosis

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

The overall goal of this procedure is to study the development of intimal hyperplasia in a humanized model. This is accomplished by first denuding, a section of human mammary artery or IMA, using a two French fogerty catheter. Next, a stent is inserted into the IMA.Then a segment of rat abdominal aorta is replaced with a stented IMA. Finally, the wound is sutured and the animal is allowed to recover the endothelial damage due to the invasive procedure of balloon dilatation and stenting results in inflammation and the production of inflammatory cytokines. This leads to the proliferation of smooth muscle cells, which progressively occlude the vessel lumen resulting in stenosis.Ultimately, results can be obtained that show the proliferation of human smooth muscle cells through immunofluorescence confocal microscopy. Today we will show you a model for the assessment of wrist stenosis in human arteries in a red host. The advantage of this model of existing techniques like the abdominal wet aortic standing, is that we can assess human stents in human tissue.We first had the idea for this method when we were aiming to find a translational in vivo model for vascular remodeling. To prepare the IMA begin by using a two French Fogarty arterial embolectomy catheter to denude the arterial endothelium by moving the catheter into the vessel to ensure endothelial damage. Insert a human stent of eight millimeters in length and 2.5 to three millimeters in diameter into the vessel.Using the appropriate balloon pressure, deploy the stent to achieve the desired diameter. Store the stented IMA on ice at four degrees Celsius in RPMI and Heparin until transplantation. After anesthetizing, a rat with isoflurane, shave the abdominal hair, place a face mask over its nose and mouth to maintain 2%anesthesia using provo iodine, disinfect the abdominal area widely and follow it with 80%ethanol.Repeat these steps twice. Pinch the hind feet to be sure that the rat is sufficiently sedated. Next, perform an upper median laparotomy to expose the infrarenal abdominal aorta.Then place the intestines in a saline moisturized glove and fold the glove around the intestines to prevent loss of moisture Under a surgical microscope, dissect the aorta from the infrarenal region to the bifurcation, taking care not to cause damage on the branches of the vessels. To stop aortic blood flow, use micro clamps to first clamp the proximal end, then the distal end. Remove an approximately 0.5 to 0.7 millimeter aortic segment and flush the remaining aorta with 200 units of heparin.Next, shorten the stented IMA to an adequate length and position it into the aortic gap. Then using running sutures, connect the IMA to the recipient aorta. Carefully open first the coddle and afterwards the cranial clamp.There should be a visible pulse in the transplanted IMA and at the distal end of the aorta. Place the intestines back into the abdomen and flush it with prewarm sterile saline. Use six aut proline running sutures to close the muscle layer of the abdominal wall, followed by the skin layer.While the rat is still under anesthesia subcutaneously. Inject four to five milligrams per kilogram of carprofen. Monitor the animal daily until removal of the IMA to identify the origin of proliferation.Human IMA was stably transduced to overexpress GFP in the vessel. Histological sections were stained with antibodies against GFP shown here in green and human smooth muscle actin or SMA seen in red. The cell nuclei are in blue.Shown here is emerged confocal image showing the origin of proliferated cells. This 3D video shows the colocalization of SMA in red and GFP in green Following surgery. Other methods like histopathology and the confocal immunofluorescence micro can be performed in order to answer the questions like luminal obliteration, morphology, and the cellularity.This method is a promising model for translational research. Thank you for watching and good luck with your experiments.I.

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Explore a humanized rat model for studying intimal hyperplasia after stent deployment using human internal mammary artery transplantation. This translational approach enables assessment of human stent responses in human tissue.

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

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