We thank the entire surgical staff for excellent technical assistance.

The study conforms to the Declaration of Helsinki. The protocol follows the guidelines of an independent institutional ethics committee, and human biomaterials were obtained after informed written consent (ethics committee approval: A 2018-0037).
1. Positioning of the legs
NOTE: Patient inclusion criteria included a history of coronary artery disease with elective/urgent indication for CABG surgery and the need for harvesting of at least one venous bypass graft for complete revascularization. Patients with debilitating chronic disease, emergency operations, status post-deep vein thrombosis, and active wet gangrene were excluded. Pre- and post-operative procedures were comparable with previously described clinical studies12,13. 28 patients undergoing CABG were included for antegrade endoscopic vessel harvesting of 30 great saphenous veins from the lower leg after informed written consent. A cardiac surgeon certified and experienced with the technique (&#62;200 cases) for the upper leg executed the antegrade EVH of the great saphenous veins from the lower leg.
<ol>
	<li>Organization of the surgical theatre
	<ol>
		<li>Before surgery, ensure supine position of the anesthetized patient on the surgical table following institutional standard procedures for CABG surgery.</li>
		<li>Place the vein harvester on the right side of the patient. Place the surgical team and instrumental set-up for cardiac surgery on the left side of the patient. Place the instrumental set-up for EVH near the end of the table (Figure 1, Figure 2; see Table of Materials).</li>
	</ol>
	</li>
	<li>Specific positioning of the legs
	<ol>
		<li>Place two foam rollers (length: 60 cm, diameter: 12 cm) below the extended legs. Place one half-cylindrical foam roller just above the knee in order to avoid overstretched knees and common peroneal nerve lesions. Then, place another full cylindrical foam roller below the Achilles tendon for lifted and outward rotated foot position (Figure 1A-D).</li>
	</ol>
	</li>
</ol>
2. Minimally invasive surgical access to the vein graft
<ol>
	<li>Access site
	<ol>
		<li>Use institutional standard disinfection procedures with Octenidindihydrochlorid followed by standard sterile covering for aseptic surgical conditions.</li>
		<li>Make one longitudinal skin incision (length: 1.5-2 cm) with a curved scalpel (size 10) on the lower leg. Start the incision with the distance of approximately one index finger above the imagined ankle joint and proceed upwards parallel to the medial margin of the tibia bone (Figure 2C).</li>
	</ol>
	</li>
	<li>Bridging vein harvesting technique
	<ol>
		<li>Obtain minimally invasive access to the great saphenous vein with surgical forceps, dissecting surgical scissors, and an electrosurgical pencil, if necessary. Starting from the skin incision, isolate the vessel 4 cm in each direction using a vessel loop, dissecting scissors, small soft tissue retractor, and Langenbeck hook, applying the standard bridging vein harvesting technique (Figure 2C).</li>
		<li>Continuously check vein quality status and surrounding subcutaneous tissue in the working channel. Visualize in order to avoid injury to the saphenous nerve. Avoid harvesting of progressive varicose veins.</li>
	</ol>
	</li>
	<li>Practical hints
	<ol>
		<li>Make sure that one small finger can easily access the working channel (antegrade). Avoid surgical clipping of side branches at this time.</li>
	</ol>
	</li>
</ol>
3. Antegrade EVH with the optical dissector
<ol>
	<li>Insertion of optical dissector
	<ol>
		<li>Assemble the optical dissector by connecting an extended length endoscope (diameter: 7 mm, length: 48 cm) to an optical camera and dissection tip from the endoscopic vessel harvesting system, according to the manufacturer&#39;s instructions. Moisturize the optical dissector with saline containing heparin (i.e., NaCl + Hep: 5.000 per 200 mL).</li>
		<li>Put the inflatable blocker balloon (also provided by the endoscopic vessel harvesting system and moisturized with NaCl + Hep) over the optical dissector. Gently insert the optical dissector (antegrade) and, after, the inflatable blocker balloon into the wound under permanent optical control of the vein (Figure 2D-F).</li>
	</ol>
	</li>
	<li>Dissection of the vein
	<ol>
		<li>Block the inflatable blocker balloon with room air (10 mL). Flood the working channel with CO2 (flow: 5 L/min, pressure: 15 cm H2O) and indicate to the anesthetic medical staff. Make sure that the working channel is extended by the gas pressure.</li>
		<li>Move antegrade until reaching the imagined proximal medial end of tibial diaphysis using the optical dissector, following the manufacturer&#180;s instructions. Gently dissect the main vessel from a majority of subcutaneous tissue until achieving clear identification of side branches.</li>
		<li>For optimal results, dissect the main vessel through antegrade movement of the optical dissector 1) above of, then 2) below, the main vessel. Then, selectively dissect the side branches, with one side of the vein preserving the perivascular tissue as far as possible, followed by the other side (Figure 2G-I).</li>
		<li>Continuously check vein quality status and mechanical stress in the working channel. Visualize in order to avoid injury to the saphenous nerve. Avoid harvesting of progressive varicose veins.</li>
	</ol>
	</li>
</ol>
4. Antegrade EVH with the optical retractor
<ol>
	<li>Insertion of optical retractor
	<ol>
		<li>Remove the optical dissector from the wound and disconnect the dissection tip.</li>
		<li>Adapt the blocker balloon for the optical retractor and block the working channel with a 5 mL syringe. Assemble the optical retractor by connecting the extended length endoscope to the optical camera and retractor device from the endoscopic vessel harvesting system, which is provided with an internal bipolar electrocoagulation device (power output: level 3-4).</li>
		<li>Use anti-fog fluid for the tip of the endoscope (Figure 3A-C). Again, moisturize the optical retractor with NaCl + Hep before antegrade insertion through the blocked balloon.</li>
	</ol>
	</li>
	<li>Isolation of the vein
	<ol>
		<li>Advance the optical retractor antegrade to the end of the working channel. Release the vein from the surrounding subcutaneous tissue with the retractor device and selectively interrupt side branches with the bipolar electrocoagulation device in a retrograde fashion (Figure 3D-F). Here, the bipolar electrocoagulation device must be positioned with the convex ending away from the main vessel.</li>
		<li>Continuously check vein quality status and mechanical stress in the working channel. Visualize in order to avoid injury to the saphenous nerve.</li>
	</ol>
	</li>
</ol>
5. Vein graft retrieval
<ol>
	<li>Finishing of EVH
	<ol>
		<li>Execute a stab incision in the skin with a sharp scalpel (size 11) at the distal end of the dissected vein (regarding venous flow direction). Insert a smooth (anatomical) clamp through the stab incision and clamp the vein under optical control with the optical retractor.</li>
		<li>Gently retrieve the clamped vein through the stab incision and cut it off proximally (regarding venous flow direction). Thereafter, gently remove the optical retractor through the blocked balloon simultaneously relieving the distal portion of the vein (Figure 3G). Deflate the blocker balloon and remove it from the wound.</li>
		<li>Switch off CO2 and indicate it to the anesthetic medical staff. At this time, use surgical clips and interrupt the remaining side branches before retrieval of the vein graft, if necessary.</li>
	</ol>
	</li>
	<li>Finishing of bridging vein harvesting
	<ol>
		<li>Execute a stab incision in the skin with a sharp scalpel (size 11) at the proximal end of the isolated vein approximately 3 cm above imagined ankle joint. Insert an anatomical clamp through the stab incision and retrieve the vein though the skin incision under digital and optical control. Visualize and avoid injury to the saphenous nerve.</li>
		<li>Then, clamp the vein under direct vision and cut off distally (regarding venous flow direction). Thereafter, gently relieve the entire vein graft through the initial minimally-invasive surgical access site and cannulate the proximal end with a 3.0 mm flexible vessel cannula (Figure 3H).</li>
	</ol>
	</li>
</ol>
6. Final preparation of the vein graft
<ol>
	<li>Gently flush the released venous graft with NaCl + Hep (in a 10 mL syringe) alternating with double-clipping of all side branches (Figure 3H). Continuously check vein quality status and repair injuries, if necessary, with polypropylene sutures (7-0 or 8-0). Finally, the vein harvester and primary surgeon must evaluate graft quality of the endoscopic vein, applying the same criteria as is executed for veins harvested by the open technique.</li>
	<li>If necessary, store the vein graft in a NaCl + Hep-moisturized compress at room temperature (RT) for short-term storage. However, avoid longer time periods of storage. Transfer the vein graft into heparinized blood as soon as arterial cannulation for cardiopulmonary bypass is accomplished.</li>
</ol>
7. Wound closure
<ol>
	<li>Ligate the main vessel at both clamped vein ends, each with a 4-0 polyglactin 910 suture. Remove the clamps.</li>
	<li>Insert a 10Fr Redon drain into the wound (Figure 3I). Fixate the Redon drain with 2-0 poly ethylene terephthalate suture at the skin.</li>
	<li>Execute subcutaneous and intracutaneous wound closures at the minimally invasive access site with 2-0 and 4-0 polyglactin 910 sutures, respectively. Close the two small stab incisions at the proximal and distal ends, with one U-suture each, stitched intracutaneously (4-0 polyglactin 910). Drape the wounds with sterile plasters.</li>
	<li>Wrap the leg, except in peripheral artery disease patients.</li>
</ol>

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Manuscript publication was funded by Getinge Group (Germany). Alexander Kaminski is a consultant to Getinge Group and receives speaker honoraria from Getinge Group. All authors declare study conduction and entire scientific analyses were executed independently from industrial partners. All authors declare responsibility for the integrity of the work as a whole and have given final approval to the version to be published. All authors declare that there are no conflicts of interest.

It should be stated that we prefer complete arterial coronary revascularization in our department. There is rising evidence that CABG using bilateral internal mammary artery (IMA) grafts can significantly improve long-term survival of patients14,15,16,17. However, there are valid reasons for a &#34;single IMA plus vein grafts&#34; strategy, especially in patients at advanced ages, patients with...

Open atraumatic &#34;low-touch&#34; and &#34;no-touch&#34; techniques have been developed over the years for harvesting saphenous veins in coronary artery bypass graft (CABG) surgery or peripheral bypass grafting, producing grafts with excellent endothelial integrity and long-term patency. However, wound complications remain a major problem when using the open technique, especially in obese, diabetic, and chronic venous insufficiency patients1,2,3,4. The question arises of how physicians can harvest the saphenous vein with optimal graft quality and reduced risk for wound complications. Endoscopic vein harvesting (EVH) techniques have been proven to be cost-effective, and clinical outcome parameters are comparable with the open technique. However, strategies protecting endothelial integrity, histological structure, and physiological function of vein grafts during EVH are highly appreciated in order to preserve optimal graft quality2. Recent studies have presented superior graft patency after open harvesting compared to endoscopic techniques5. It has also been shown that bridging vein harvest techniques can directly improve vein quality6. Therefore, it is hypothesized that vein graft harvesting may be advanced through synergizing antegrade EVH with minimally invasive bridging vein harvesting, specific leg positioning, and vein isolation in a tensionless working channel.
To date, conventional EVH techniques for harvesting great saphenous veins have used antegrade approaches for the upper leg and retrograde approaches for the lower leg. However, we have experienced limitations of these techniques and hold concerns about graft quality. The great saphenous vein from the knee and upper leg frequently have revealed numerous side branches and occasionally shown dilated vessel diameter, leading to impaired vessel quality and mismatching of conduit and target vessels that can negatively affect long-term graft patency after CABG and re-revascularization rate7,8,9,10,11. In our experience, the retrograde EVH approach for the lower leg has repetitively resulted in prolonged blood stasis inside the vessel (with augmented intravenous blood pressure due to closed venous valves), increased mechanical stress on the tissue, bleeding, thrombus formations, graft damage, and impaired graft quality. Consequently, this standardized protocol was developed for safe antegrade EVH from the lower leg, combining the bridging vein harvest technique for minimally invasive access site with antegrade EVH in a tensionless working channel for adequate vein graft quality.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>disposable scalpel (size 11, Pr&#228;zisa Plus)</td>
			<td>Dahlhausen, Germany</td>
			<td></td>
			<td>a</td>
		</tr>
		<tr>
			<td>small curved smooth (anatomical) clamps </td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>b</td>
		</tr>
		<tr>
			<td>toothed (surgical) forceps</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>c</td>
		</tr>
		<tr>
			<td>surgical scissors </td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>d</td>
		</tr>
		<tr>
			<td>holder for scalpel blade (size 10)</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>e</td>
		</tr>
		<tr>
			<td>fine smoth (anatomical) forcep</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>f</td>
		</tr>
		<tr>
			<td>sponge-holding clamp</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>g</td>
		</tr>
		<tr>
			<td>clipping device</td>
			<td>Fumedica, Switzerland</td>
			<td></td>
			<td>h</td>
		</tr>
		<tr>
			<td>18 Gauge cannula (Sterican)</td>
			<td>B. Braun, Germany</td>
			<td></td>
			<td>i</td>
		</tr>
		<tr>
			<td>light handle</td>
			<td>Simeon Medical, Germany</td>
			<td></td>
			<td>j</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>k</td>
		</tr>
		<tr>
			<td>tissue retractor</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>l</td>
		</tr>
		<tr>
			<td>Redon needle</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>m</td>
		</tr>
		<tr>
			<td>adhesive hook and loop fastener</td>
			<td>M&#246;lnlycke, Germany</td>
			<td></td>
			<td>n</td>
		</tr>
		<tr>
			<td>extended length endoscope </td>
			<td>Karl Storz, Germany</td>
			<td></td>
			<td>o</td>
		</tr>
		<tr>
			<td>optical cable</td>
			<td>Karl Storz, Germany</td>
			<td></td>
			<td>p</td>
		</tr>
		<tr>
			<td>transparent drap camera cover</td>
			<td>ECOLAB Healthcare, Germany</td>
			<td></td>
			<td>q</td>
		</tr>
		<tr>
			<td>connection cable for electrocauterisation</td>
			<td>Maquet, Getinge Group, Germany </td>
			<td></td>
			<td>r</td>
		</tr>
		<tr>
			<td>gas insufflation set</td>
			<td>Dahlhausen, Germany</td>
			<td></td>
			<td>s</td>
		</tr>
		<tr>
			<td>Fred Anti-Fog Solution </td>
			<td>Medtronic, USA</td>
			<td></td>
			<td>t</td>
		</tr>
		<tr>
			<td>bipolar electrocoagulation device </td>
			<td>Maquet, Getinge Group, Germany </td>
			<td></td>
			<td>u</td>
		</tr>
		<tr>
			<td>monitor (WideView)</td>
			<td>Karl Storz, Germany</td>
			<td></td>
			<td>v</td>
		</tr>
		<tr>
			<td>light source (xenon 300)</td>
			<td>Karl Storz, Germany</td>
			<td></td>
			<td>w</td>
		</tr>
		<tr>
			<td>gas insufflation controller (Endoflator)</td>
			<td>Karl Storz, Germany</td>
			<td></td>
			<td>x</td>
		</tr>
		<tr>
			<td>half-cylindrical foam roller</td>
			<td>Almatros, Gebr. Albrecht KG, Germany</td>
			<td></td>
			<td>y</td>
		</tr>
		<tr>
			<td>full-cylindrical foam roller</td>
			<td>Almatros, Gebr. Albrecht KG, Germany</td>
			<td></td>
			<td>z</td>
		</tr>
		<tr>
			<td>bulldog clamp</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>aa</td>
		</tr>
		<tr>
			<td>flexible vessel cannula</td>
			<td>Medtronic, USA</td>
			<td></td>
			<td>ab</td>
		</tr>
		<tr>
			<td>vessel loop (Mediloops)</td>
			<td>Dispomedica, Germany</td>
			<td></td>
			<td>ac</td>
		</tr>
		<tr>
			<td>Heparin-Natrium (5000 U) in 200ml saline</td>
			<td>B. Braun, Germany</td>
			<td></td>
			<td>ad</td>
		</tr>
		<tr>
			<td>Langenbeck hooks</td>
			<td>B. Braun Aesculap, Germany</td>
			<td></td>
			<td>ae</td>
		</tr>
		<tr>
			<td>sutures (polygalctin 910, Vicryl 2-0, 4-0; poly ethylene terephthalate, Ethibond 2-0)</td>
			<td>Ethicon, Johnson &#38; Johnson, USA</td>
			<td></td>
			<td>af</td>
		</tr>
		<tr>
			<td>Endoscopic vessel harvesting system, Vasoview Hemopro II</td>
			<td>Maquet, Getinge Group, Germany </td>
			<td></td>
			<td>ag</td>
		</tr>
		<tr>
			<td>Octenidindihydrochloride, Octeniderm</td>
			<td>Schuelke &#38; Mayr GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synergizing antegrade endoscopic with bridging vein harvesting for improvement of great saphenous vein graft quality from the lower leg

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative wound complications. This standardized protocol for antegrade endoscopic vein harvesting (EVH) from the lower leg has the potential to be introduced to routine coronary artery bypass grafting (CABG). Patients undergoing CABG surgery are positioned on a surgical table with two additional foam rollers below the extended legs, enabling antegrade EVH from the lower leg. Following minimally invasive surgical access through a bridging vein harvest technique, an endoscopic optical dissector is inserted antegrade into the wound. The main vessel and side branches are dissected under continuous optical control of vein quality status and the working channel. After, an endoscopic optical retractor is inserted with an internal bipolar electrocoagulation device for precise, safe, and tissue-protective interruption of side branches. After release of the vein, the vessel is cut off at the proximal and distal ends under optical control, retrieved from the wound, then cannulated and flushed with heparinized saline. Finally, all side branches of the vein graft are double-clipped. Vascular histology is analyzed in a randomized selection of vein samples. After applying this standardized EVH protocol, the learning curve was shown to be steep, and graft quality was sufficient for coronary artery bypass grafting in every case. There was no conversion to surgical harvesting and low risks for tissue damage and bleeding. Leg positioning and synergizing EVH with bridging vein harvesting improved procedural success and vein graft quality. In our hands, antegrade EVH from the lower leg was feasible, demonstrating straightforward graft dissection as well as adequate macroscopic and microscopic graft quality with preserved endothelial integrity. In conclusion, the introduced technique is safe, shows excellent vein autograft quality, and illustrates feasibility for elective and urgent isolated CABG and combined CABG scenarios.

A steep learning curve was demonstrated for an experienced cardiac surgeon performing antegrade EVH of the great saphenous vein from the lower leg (Figure 4). There were no conversions to surgical harvesting. However, there were four cases of vein injury in the beginning of the learning curve. In three of the four cases, major injuries occurred at the distal portion of the vein because of an inadequately narrow working channel when the surgeon isolated the vein above the tibial metaphysis. D...

Watch this Scientific Journal Video about Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg at JoVE.com

Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative ...

synergizing-antegrade-endoscopic-with-bridging-vein-harvesting-for

Research

JoVE Journal

Medicine

8.5K Views.  University of Rostock. Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

Video: Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

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 ...

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. ...

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 ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

Drawing Blood from the Penile Vein

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...