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

Summary

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

Abstract

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.

Introduction

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 years^2,3. 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 models^9,10, 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 mice^11,12. 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.

Protocol

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 ("Amt für Gesundheit und Verbraucherschutz, Hansestadt (Office for Health and Consumer Protection) Hamburg").

1. Animal Care

1. Obtain Lewis rats (LEW/Crl) rats and ROSA/luciferase-LEW transgenic rats weighing 300-350 g from the Institute of Laboratory Animals.
2. Keep the rats under conventional conditions in ventilated cabinets and feed them standard rat chow and autoclaved water ad libitum.
3. Perform a graft transplantation using the ROSA/luciferase-LEW transgenic rats as the donors and the syngenic LEW/Crl rats as the recipients.

2. Preparation of the Donor Rat

1. Use an induction chamber to anaesthetize a rat with isoflurane (2.5-3%).
2. 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.   
3. Spread the hind legs and fix their position using tape.
4. 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.
5. 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.
6. Stop blood flow in the superficial epigastric vein using two micro clamps.
7. Harvest an approximately 0.5 to 1 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 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.
8. Keep the vessel segment in 1% lidocaine on ice until transplantation into the recipient rat to prevent a vessel spasm.
9. 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.

3. Preparation of the Recipient Rat

1. Anesthetize and fix the recipient rat in the same way as the donor rat.
2. Shave the medial side of the legs with a hair trimmer and disinfect three times using povidone-iodine and 80% ethanol.  
3. Monitor the depth of anesthesia and ensure that it is sufficient by verifying the absence of reflexes when pinching the hind feet.
4. 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.
5. Use micro clamps to stop the flow of blood. Place the proximal clamp first, followed by the distal clamp.
6. 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 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.
7. 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.
8. 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.  
9. 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.
10. Load two 1-mL syringes with fibrin glue component 1 and 2. Carefully lift the graft with forceps and drop approximately 100 μl 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.
11. Place the graft back in its position and drop an additional 100 μl 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.  
12. Carefully open the distal clamp, followed by the proximal.
13. Confirm a successful surgery by checking for a visible pulse in the transplanted vein and distal artery of the graft.
14. 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.
15. 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.
16. 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.  

4. Duplex Sonography

NOTE: Use duxplex sonography to visualize blood flow non-invasively in rats¹⁴.

1. Anaesthetize a rat in an induction chamber (isoflurane 2%). Place the rat on its back and maintain anesthesia with a facemask covering the nose.
2. Use hair clippers and hair removal cream to remove the hair around the area of the thigh.
3. 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.  

5. Histopathology

NOTE: Harvest and stain the vessel with Masson’s trichrome staining for morphometric analysis¹⁵.

1. Fix the harvested vessel in 4% paraformaldehyde overnight and dehydrate it in increasing concentrations of ethanol.  Embed the sample in paraffin and cut it into 5 μm thick slices using a microtome.
   NOTE: Paraformaldehyde is toxic and should be handled with special care.
2. 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.   

6. Bioluminescence Imaging (BLI)

NOTE: The postoperative graft was tracked over time in vivo by measuring bioluminescent signal¹⁶.

1. 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.
2. Place the rat into a real-time bioluminescent quantification system and access the bioluminescence signal.

Results

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),...

Discussion

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 h...

Materials

Name	Company	Catalog Number	Comments
Rat LEW/Crl	Charles River	Stock number 004
Rat LEW-Tg(Gt(ROSA)26Sor- 1 luc)11Jmsk	Institute of laboratory animals, Kyoto University, Japan	NBPR rat number 0299	http://www.anim.med.kyoto-u.ac.jp/NBR/
PFA 4%	Electron Microscopy Sciences	#157135S	20%
hair clipper	WAHL	8786-451A ARCO SE
Forene	AbbVie	PZN 10182054 Art.Nr.: B506	Isoflurane
microsurgical clamp	Fine Science Tools	18055-04	Micro-Serrefine - 4 mm
clamp applicator	Fine Science Tools	18056-14
hair removal creme	Rufin cosmetic	27618
Povidone-Iodine	Betadine Purdue Pharma	NDC:67618-152
10-0 Ethilon suture	Ethicon	2814G
5-0 prolene suture	Ethicon	EH7229H
Rimadyl	Pfizer	400684.00.00	Carprofen
Novaminsulfon	Ratiopharm	PZN 03530402	Metamizole
Heparin	Rotexmedica	PZN: 3862340	25.000 I.E./ ml
Xylocain 1%	AstraZeneca	PZN: 1137907	Lidocain
EVICEL	J&J Med.Ethicon Biosur	PZN 7349697 Art. Nr.:EVK01DE	fibrin glue
NaCl 0.9%	B.Braun	PZN 06063042 Art. Nr.: 3570160
Vevo 770 high-resolution in vivo micro-imaging system	VisualSonics		duplex sonography
Ecogel 100 ultrasound gel	Eco-med	30GB
D-Luciferin Firefly, potassium salt	Biosynth	L-8220
PBS pH 7.4	Gibco	10010023
Xenogen Ivis 200	Perkin Elmer		bioluminescence imaging
Weigerts iron hematoxylin Kit	Merck	1.15973.0002	Trichrome staining
Resorcine-Fuchsine Weigert	Waldeck	2.00E-30	Trichrome staining
Acid Fuchsin	Sigma-Aldrich	F8129-25G	Trichrome staining
Ponceau S solution	Serva Electrophoresis	33427	Trichrome staining
Azophloxin	Waldeck	1B-103	Trichrome staining
Molybdatophosphoric acid hydrate	Merck	1.00532.0100	Trichrome staining
Orange G	Waldeck	1B-221	Trichrome staining
Light Green SF	Waldeck	1B-211	Trichrome staining
Vitro-Clud	Langenbrinck	04-0001
Glacial Acetic Acid	Sigma-Aldrich	537020
37% HCl	Sigma-Aldrich	H1758
Xylene	Th. Geyer	3410
Paraffin	Leica biosystems	REF 39602004
Ethanol absolute	Th. Geyer	2246
Ethanol 96%	Th. Geyer	2295
Ethanol 70%	Th. Geyer	2270
Slide Rack	Ted Pella	21057
Staining dish	Ted Pella	21075
Bepanthen Eye and Nose ointment	Bayer	1578675	Eye ointment