The authors thank Christiane Pahrmann for her technical assistance. This study was funded by the Deutsche Stiftung fuer Herzforschung (F/28/14). D.W. was supported by the travel award from the International Society for Heart and Lung Transplantation. T.D. received the Else Kro&#776;ner Excellence Stipend from the Else-Kro&#776;ner-Fresenius-Stiftung (2012_EKES.04). S.S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; DE2133/2-1, T.D.&#160; and SCHR992/3- 1, SCHR992/4-1, S.S.).

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (&#34;Amt f&#252;r Gesundheit und Verbraucherschutz, Hansestadt (Office for Health and Consumer Protection) Hamburg&#34;).
1. Animal Care
<ol>
	<li>Obtain Lewis rats (LEW/Crl) rats and ROSA/luciferase-LEW transgenic rats weighing 300-350 g from the Institute of Laboratory Animals.</li>
	<li>Keep the rats under conventional conditions in ventilated cabinets and feed them standard rat chow and autoclaved water ad libitum.</li>
	<li>Perform a graft transplantation using the ROSA/luciferase-LEW transgenic rats as the donors and the syngenic LEW/Crl rats as the recipients.</li>
</ol>
2. Preparation of the Donor Rat
<ol>
	<li>Use an induction chamber to anaesthetize a rat with isoflurane (2.5-3%).</li>
	<li>Place the rat on its back and maintain the anesthesia with a facemask covering the mouth and nose. Check for sufficient depth of anesthesia by pinching the hind feet and verifying the absence of reflexes. Apply some vet ointment to the eyes to prevent dryness while under anesthesia.&#160; &#160;</li>
	<li>Spread the hind legs and fix their position using tape.</li>
	<li>Shave the inguinal hair with a hair trimmer and disinfect the entire area using povidone-iodine followed by 80% ethanol. Repeat the disinfection step twice. 
	NOTE: The surgical area, gauze, and surgical instruments should be sterilized. Maintain a sterile field throughout the procedure and wear single-use, sterile surgical gloves, masks, and caps.</li>
	<li>Under a microscope, perform a vertical incision along the linea inguinalis. Use two forceps to gently separate the subcutaneous tissues and expose the superficial epigastric vein from its origin on the femoral vein. Carefully isolate the superficial epigastric vein from the surrounding tissues.</li>
	<li>Stop blood flow in the superficial epigastric vein using two micro clamps.</li>
	<li>Harvest an approximately 0.5&#160;to 1&#160;cm segment of the vein by carefully lifting the isolated vein with forceps and cutting through the vessel with microscissors. Leave the micro clamps on the vessel stump to prevent the loss of blood. Place the removed piece of vein on sterile gauze. Carefully place a 30 G&#160;needle inside one end of the harvested vein and flush the vessel with heparin (50 units/ml). 
	NOTE: Handle the vein with care and avoid damage during lifting, cutting, and flushing. Make sure to flush the graft with the proper amount of heparin.</li>
	<li>Keep the vessel segment in 1% lidocaine on ice until transplantation into the recipient rat to prevent a vessel spasm.</li>
	<li>Euthanize the donor rat by increasing the anesthesia to 5% isoflurane. After 2-3 min, open the abdomen along the linea alba, cut through the diaphragm, and remove the heart to stop circulation.</li>
</ol>
3. Preparation of the Recipient Rat
<ol>
	<li>Anesthetize and fix the recipient rat in the same way as the donor rat.</li>
	<li>Shave the medial side of the legs with a hair trimmer and disinfect three times using povidone-iodine and 80% ethanol. &#160;</li>
	<li>Monitor the depth of anesthesia and ensure that it is sufficient by verifying the absence of reflexes when pinching the hind feet.</li>
	<li>Perform a median femoral incision from the knee to the inguinal fold. Under a microscope, use 2 forceps to separate the femoral artery from its surroundings.</li>
	<li>Use micro clamps to stop the flow of blood. Place the proximal clamp first, followed by the distal clamp.</li>
	<li>Cut out a short segment of the clamped femoral artery with microscissors and discard it. Shorten the remaining arterial stump with microscissors, creating a gap that is 1-2 mm larger than the vein graft. Flush the arterial stump with heparin using a 30 G&#160;needle. 
	NOTE: If the adventitia protrudes slightly beyond the vessel stump, use forceps to pull it slightly over the end of the vessel and remove a piece.</li>
	<li>Place the harvested vein from step 2.8 between the arterial stumps and adjust the length so that it fits suitably into the gap. Note the direction of the vein.</li>
	<li>Perform the proximal anastomosis first using a 10-0 prolene suture. Conduct single stitches in the order shown (Figure 1D). Start with a suture on each lateral side before adding three more sutures on the ventral side. Afterwards, place three stitches on the dorsal side of the vessel to complete the anastomosis. &#160;</li>
	<li>Connect the distal vessels with the graft using the same technique as for the proximal anastomosis described in step 3.8. Again, start with a suture on each lateral side, and then place three sutures on the ventral side and the dorsal side.</li>
	<li>Load two 1-mL syringes with fibrin glue component 1 and 2. Carefully lift the graft with forceps and drop approximately 100 &#956;l&#160;of fibrin glue component 1 under the graft, followed by component 2. 
	NOTE: Make sure that components 1 and 2 are applied in a 1:1 ratio.</li>
	<li>Place the graft back in its position and drop an additional 100 &#956;l of components 1 and 2 on top of the graft. Be sure that the glue covers both the graft and the anastomosis in order to prevent anastomotic insufficiency and over-distension of the vein graft. &#160;</li>
	<li>Carefully open the distal clamp, followed by the proximal.</li>
	<li>Confirm a successful surgery by checking for a visible pulse in the transplanted vein and distal artery of the graft.</li>
	<li>Remove excessive glue, which impedes skin closure. Use forceps to lift the cured glue and remove the excess with microscissors. Close the skin layers with 5-0 prolene sutures.</li>
	<li>Inject 4-5 mg/kg Carprofen subcutaneously before allowing the rat to wake up. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Keep the animal in a single cage until it is fully recovered.</li>
	<li>Add Metamizole to the drinking water (50 mg Metamizole per 100 ml) as pain medication for the following 3 days and monitor the animal daily. &#160;</li>
</ol>
4. Duplex Sonography
NOTE: Use duxplex sonography to visualize blood flow non-invasively in rats14.
<ol>
	<li>Anaesthetize a rat in an induction chamber (isoflurane 2%). Place the rat on its back and maintain anesthesia with a facemask covering the nose.</li>
	<li>Use hair clippers and hair removal cream to remove the hair around the area of the thigh.</li>
	<li>Apply ultrasound gel to the thigh. Make sure that there are no air bubbles. Acquire duplex sonography images using an MS 400 transducer (center frequency: 30 MHz) with a frame rate of 230-400 frames/sec. &#160;</li>
</ol>
5. Histopathology
NOTE: Harvest and stain the vessel with Masson&#8217;s trichrome staining for morphometric analysis15.
<ol>
	<li>Fix the harvested vessel in 4% paraformaldehyde overnight and dehydrate it in increasing concentrations of ethanol.&#160; Embed the sample in paraffin and cut it into 5 &#956;m&#160;thick slices using a microtome. 
	NOTE: Paraformaldehyde is toxic and should be handled with special care.</li>
	<li>Deparaffinize the slides before staining them with trichrome staining solution. Dehydrate the stained slides, clear them with xylene, and mount them in mounting medium. After drying the slides, view the samples with a microscope.&#160; &#160;</li>
</ol>
6. Bioluminescence Imaging (BLI)
NOTE: The postoperative graft was tracked over time in vivo by measuring bioluminescent signal16.
<ol>
	<li>Dissolve 1 g of D-Luciferin potassium salt in 22 ml of PBS and inject it intraperitoneally into the rat (375 mg/kg body weight). Wait 15 min for the luciferin to circulate in the animal.</li>
	<li>Place the rat into a real-time bioluminescent quantification system and access the bioluminescence signal.</li>
</ol>

<ol>
	<li>Sabik, J. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+saphenous+vein+graft+patency.">Understanding saphenous vein graft patency.</a> Circulation. 124, 273-275 (2011).</li><li>Goldman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+patency+of+saphenous+vein+and+left+internal+mammary+artery+grafts+after+coronary+artery+bypass+surgery:+results+from+a+Department+of+Veterans+Affairs+Cooperative+Study.">Long-term patency of saphenous vein and left internal mammary artery grafts after coronary artery bypass surgery: results from a Department of Veterans Affairs Cooperative Study.</a> J Am Coll Cardiol. 44, 2149-2156 (2004).</li><li>Fitzgibbon, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+bypass+graft+fate+and+patient+outcome:+angiographic+follow-up+of+5,065+grafts+related+to+survival+and+reoperation+in+1,388+patients+during+25+years.">Coronary bypass graft fate and patient outcome: angiographic follow-up of 5,065 grafts related to survival and reoperation in 1,388 patients during 25 years.</a> J Am Coll Cardiol. 28, 616-626 (1996).</li><li>O'Brien, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+around+and+within+saphenous+vein+bypass+grafts.">Wound healing around and within saphenous vein bypass grafts.</a> J Thorac Cardiovasc Surg. 114, 38-45 (1997).</li><li>El-Kurdi, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovine+femoral+artery+bypass+grafting+using+saphenous+vein:+a+new+model.">Ovine femoral artery bypass grafting using saphenous vein: a new model.</a> J Surg Res. 193, 458-469 (2015).</li><li>Yuda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+receptor+antagonist,+L-158,809,+prevents+intimal+hyperplasia+in+dog+grafted+veins.">Angiotensin II receptor antagonist, L-158,809, prevents intimal hyperplasia in dog grafted veins.</a> Life Sci. 68, 41-48 (2000).</li><li>McCann, R. L., Hagen, P. O., Fuchs, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspirin+and+dipyridamole+decrease+intimal+hyperplasia+in+experimental+vein+grafts.">Aspirin and dipyridamole decrease intimal hyperplasia in experimental vein grafts.</a> Ann Surg. 191, 238-243 (1980).</li><li>Thomas, A. 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The authors have nothing to disclose.

This video demonstrates a rat vein interposition model to investigate vein graft loss and to allow for the exploration of the underlying pathological processes and the testing of new drugs or therapeutic options.
Anesthesia is a crucial aspect of surgical procedures. A continuous inhalative anesthesia system is recommended, as this is a safe and easy method, especially during prolonged operations. This can be of great importance during the training phase, when the operation takes more than 1 hr.
With respect to the surgical procedure, it is critical not to damage the vein graft during harvest and implantation17. Gripping the adventitia of the vessel can prevent damage to the vein graft and avoid subsequent thrombus formation and graft failure. Graft patency can be determined immediately after vein graft construction through the observation of blood flow and of pulsation in the vein graft or distal artery, as well as through duplex sonography.
Another critical aspect of the procedure is to prevent the over-distension of the vein graft. Sudden exposure of the vein graft to the arterial pressure system leads to increased wall tension, subsequent over-distension, and changes in flow pattern. These are sources of thrombosis, anastomotic insufficiency, and early graft failure. Supporting the vein graft with fibrin glue can prevent uncontrolled ballooning and protect the intima and media from mechanical destruction. Absorbable collagen covers are an alternative to fibrin glue and can be used as perivenous covers18.
The most critical step within this protocol is undoubtedly the anastomosis between the vein graft and the artery. Care must be taken not to pierce the two walls of the vessel, as this will lead to the narrowing of the anastomosis, which may result in early failure of the graft. In addition, special attention must be paid to the localization of sutures. Congruency of the suture positions in the artery and vein ensures graft patency and prevents insufficiency. To facilitate this, sutures can be performed in the order shown in Figure 1D.
The degree of technical difficulty can be viewed as a limitation of the technique, because a novice must first become familiar with the microsurgical equipment and the small sizes of the anatomical structures. However, other models used exhibit the same difficulties, and we believe that this video will help novice surgeons to master this technique within a short time.
Numerous small animal models for venous graft failure have been described in the literature. However, most models only provide very small amounts of tissue for analysis13. An advantage of this method is the comparatively large amount of tissue that can be obtained. One graft can be divided into multiple parts and used for different assays, thereby reducing the number of experimental animals required.
Recent advances in zinc-finger nuclease technology enabled the generation of knockout rats19. By selecting suitable knock-out rats, graft patency loss can be studied in different disease conditions. For example, renin knockout rats can be utilized to study hypertension20. These genetic backgrounds can be combined with this animal model to glean information on the mechanisms of vein graft failure in different settings or on the impact of certain genes in the development of myointima hyperplasia.
In conclusion, the model described in this video is reproducible, inexpensive, and easy to perform, and it can be established quickly and reliably. Myointima hyperplasia, which is the main cause of vein graft failure, developed rapidly over four weeks, resulting in progressive luminal obliteration. Successfully tested treatment options in this model can be further confirmed in large animal models.

Coronary artery diseases and their complications are among the leading causes of death worldwide. Current therapeutic strategies focus on re-establishing the blood flow, either by dilating the narrowed vessel or by creating a bypass. Coronary artery bypass grafting (CABG) using vein autografts was first described in 1968 and has been refined over the years. Apart from the revascularization of the left anterior descending coronary artery, saphenous vein conduits are most commonly used1. However, graft patency remains the Achilles heel of saphenous vein grafts (SVG). One year after surgery, graft patency is 85%, dropping to 61% after ten years2,3. Unveiling the pathophysiological mechanisms and causes of SVG patency loss is therefore an important task.
This video demonstrates a rat vein interposition model to investigate vein graft loss. The overall goals of this method are to explore the underlying pathobiological and -physiological processes during disease progression and to develop a suitable model for drug or therapeutic option testing. By transplanting the superficial epigastric vein into the arterial system, this model closely mimics the clinical setting of coronary artery bypass grafting. Surgical trauma, ischemia, and wall stress are important triggers of pathological vascular changes and are imitated in the model described.
Different models and species are available to investigate vein graft patency loss. Large animal models, such as pigs4, sheep5, dogs6, and monkeys7, resemble human vessel and anatomical structures and thus enable complex therapeutic strategies, such as bypass stenting or new surgical techniques, to be tested8. However, special housing, equipment, and staff are required. In addition, high costs and the need for an additional anesthetist during surgery impede their broader application. Small animals, including rats, are easy to handle, do not require special housing, and have manageable costs. Compared to mouse models9,10, rat models have the advantage of better operability and therefore less variability in the outcome. Rats are physiologically and genetically more similar to humans than mice11,12. In addition, most wild-type mice only develop limited myointima13, which make mouse models prone to type II errors. The histology of the main mouse veins, such as the inferior vena cava, only consists of a few cell layers and renders early evaluation difficult13. A further disadvantage is the small amount of tissue available for subsequent analysis after graft recovery.
The model described in this video is reproducible, inexpensive, and easy to perform, and it can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, such as viral vectors for gene therapy, in an economical fashion.

<table border="0" cellpadding="0" cellspacing="0" width="720">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Rat LEW/Crl</td>
			<td style="width:183px;">Charles River</td>
			<td style="width:145px;">Stock number 004</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:236px;">Rat LEW-Tg(Gt(ROSA)26Sor- 1 
			luc)11Jmsk</td>
			<td style="width:183px;">Institute of laboratory animals, Kyoto University, Japan</td>
			<td style="width:145px;">NBPR rat number 0299</td>
			<td style="width:156px;">http://www.anim.med.kyoto-u.ac.jp/NBR/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td align="right">20%</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Forene</td>
			<td>AbbVie</td>
			<td style="width:145px;">PZN 10182054 Art.Nr.: B506</td>
			<td>Isoflurane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">hair removal creme</td>
			<td>Rufin cosmetic</td>
			<td align="right">27618</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Povidone-Iodine</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rimadyl</td>
			<td>Pfizer</td>
			<td style="width:145px;">400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylocain 1%</td>
			<td>AstraZeneca</td>
			<td>PZN: 1137907</td>
			<td>Lidocain</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EVICEL</td>
			<td>J&#38;J Med.Ethicon Biosur</td>
			<td style="width:145px;">PZN 7349697 Art. Nr.:EVK01DE</td>
			<td>fibrin glue</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">NaCl 0,9%</td>
			<td>B.Braun</td>
			<td style="width:145px;">PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:236px;">Vevo 770 high-resolution in vivo micro-imaging system</td>
			<td>VisualSonics</td>
			<td></td>
			<td>duplex sonography</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ecogel 100 ultrasound gel</td>
			<td>Eco-med</td>
			<td>30GB</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">D-Luciferin Firefly, potassium salt</td>
			<td>Biosynth</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xenogen Ivis 200</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>bioluminescence imaging</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Resorcine-Fuchsine Weigert</td>
			<td>Waldeck</td>
			<td>2.00E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td align="right">537020</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylene</td>
			<td>Th. Geyer</td>
			<td align="right">3410</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td align="right">2246</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td align="right">2295</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td align="right">2270</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Slide Rack</td>
			<td>Ted Pella</td>
			<td align="right">21057</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining dish</td>
			<td>Ted Pella</td>
			<td align="right">21075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td align="right">1578675</td>
			<td>Eye ointment</td>
		</tr>
	</tbody>
</table>

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.

The rat vein interposition model is suitable to study the development of myointima hyperplasia and vein graft failure. Animals recover well from the surgery and show excellent physical condition post-operation. Figure 1 shows the key surgical steps. After the skin incision along the linea inguinalis, the epigastric superficial vein and femoral artery are identified (Figure 1A). Harvesting of the graft should be performed carefully, without damaging the graft (Figure 1B), as this can lead to early graft failure and patency loss. After positioning the graft in the recipient animal (Figure 1C), anastomosis stitches are performed in the order shown in Figure 1D. The completed venous interposition graft appears pale (Figure 1E) and should become red and show pulsation after reperfusion (Figure 1F).
The successful integration of the vein into the femoral artery and graft patency after transplantation can be confirmed non-invasively using duplex sonography (Figure 2A). By transplanting the vein of a Luc-positive rat into a syngeneic Luc-negative rat, bioluminescence imaging can be used to monitor graft presence over time (Figure 2B).
Myointima hyperplasia develops progressively in the graft over time. Histological staining with Masson&#8217;s trichrome demonstrates myointima formation inside the internal elastic lamina (Figure 2C). The calculation of luminal obliteration, (dividing the cross-sectional area of the lumen by the area within the internal elastic lamina) revealed a gradual loss of graft patency from day 7 to day 28 post-surgery, thus confirming the reproducible dynamics of myointima hyperplasia in this rat model (Figure 2D).
<img alt="Figure 1" src="/files/ftp_upload/54839/54839fig1.jpg" /> 
Figure 1: Detailed Scheme of the Surgical Procedure. (A)&#160;Anatomy of the inguinal region.&#160;(B)&#160;Harvesting the epigastric superficial vein graft.&#160;(C)&#160;Placing the vein graft between the arterial stumps of the recipient animal.&#160;(D)&#160;Order of anastomosis stitches. (E)&#160;Site after the venous graft construction. (F)&#160;Site after the reperfusion of the venous interposition graft. Black arrows mark the epigastric superficial vein. The Scale bar = 5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54839/54839fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54839/54839fig2.jpg" /> 
Figure 2: Characterization of the Animal Model. (A)&#160;Duplex sonography of a venous interposition graft immediately after transplantation (0d) and 28 days post-operation (28d). (B)&#160;BLI from Luc-positive grafts transplanted into Luc-negative rats at 0 days and 28 days after surgery. (C)&#160;Representative graft cross sections harvested after days 7, 14, 21, and 28 and stained with Masson&#8217;s trichrome. (Upper panel: the Scale bar = 400 &#181;m; Lower panel: the Scale bar = 100 &#181;m)&#160;(D)&#160;Luminal obliteration in percentage is calculated by dividing the new inner lumen from the former lumen. Intergroup differences were assessed by one-way analysis of variance (ANOVA) with Bonferroni&#8217;s post-Hoc test. p &#60;0.01. The error bars are the standard deviation (SD). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54839/54839fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental File 1" src="/files/ftp_upload/54839/54839Sup1.jpg" /> 
Supplemental File 1: Schematic Illustration of the Surgical Procedure. A vertical incision along the linea inguinalis is performed on the donor rat, exposing the inferior epigastric vein. Blood flow is stopped with two micro clamps, and a vein segment 0.5-1.0 cm long is harvested. In the recipient rat, a median femoral incision is performed, exposing the femoral vein, artery, and nerve. After clamping the femoral artery, a vessel segment is removed and replaced by the harvested vein graft. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54839/Vein_interpostition_3.pptx" target="_blank">Please click here to view Supplemental File 1 (or right-click to download).</a>

Watch this Scientific Journal Video about Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency at JoVE.com

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

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. ...

vein-interposition-model-suitable-model-to-study-bypass-graft

Nanyang Technological University

Research

JoVE Journal

Medicine

8.7K Views.  University Heart Center Hamburg. This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats. 

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

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

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. ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...