In Vivo Laboratory Surgery

• Under high magnification place background material behind the dissected vessel. Place two approximator clips (first proximally then distally) on the artery and cut it completely through with scissors perpendicularly to its axis.
• The lumen should be irrigated in both directions with a solution containing high concentration of heparin.
• The adventitia should be removed in a few millimeter distance from the edges (using forceps and scissors).

• Dilation: this minimizes spasm and enlarges the lumen so that you can see and suture the vessel ends safely. Hold the vessel end by the adventia with a forceps and gently introduce a dilating forceps inside the lumen. Delicately dilate to a maximum of 1.5 times the natural diameter of the vessel.

• Move the two clamps of the approximator closer together, so that there is about a 1 mm distance between the vessel ends.

It is important to include the muscle and also the intima into the stitch (left) (Carrel 1907, Jacobson 1960, Jassinowsky 1889), and not only the muscle (middle) or the adventitia (right).

Frontal surface: (1) stitch at "12 o’clock",
                            (2) stitch at "6 o’clock",
                            (3) place a silastic cannula into the lumen
                            (4) stitch at "3 o’clock",
                            (5) stitch at “1.5 o’clock",
                            (6) stitch at "4.5 o’clock",

Dorsal surface: (7) stitch at 1.5 o’clock",
                            (8) continuous suture which consists of 2 steps:
                                   stitch at "3 o’clock",
                                   stitch at "4.5 o’clock",
                            (9) removal of the silastic cannula, knotting.

• Open the caudal clamp of the approximator first followed by the cranial one.
• Apply compression with a small gauze or cotton ball for 2-3 minutes to stop bleeding.
• If the anastomosis still bleeds, identify the source and place supplementary sutures to stop it.
• Test the patency of the anastomosis and close up the skin.

• Direct observation through the operating microscope: transversal pulsation cranially (dynamic changes in diameter of the vessel) from the anastomosis and that of the caudal segment of the vessel indicate patency of the anastomosis. Longitudinal pulsations reveal obstruction of the anastomosis.
• “Uplift test": Lift the vessel caudally to the anastomosis with forceps, until it collapses. It will pulsate, when lowered, if the anastomosis is patent.
• The “empty-and-refill test": Clamp the artery with a forceps immediately caudal to the anastomosis, and then close the artery again with a second forceps, immediately near the first one. Slide the second forceps over the artery in the direction of the blood flow, keeping it closed and release the first forceps. An almost instant filling of the emptied segment of the artery proves patency of the anastomosis. Any delay, is a sign that the anastomosis is partially blocked (involvement of the adventitia in the lumen or to a throug -stitch). If there is no filling and the vessel remains collapsed the anastomosis has completely failed.
• Opening the anastomosis: after ligating the repaired vessel both cranially and caudally, cut out the anastomosis, transect it longitudinally opening it as a book and evaluate the internal aspects of the sutures underneath the operating microscope.

		The “uplift test"
		The “empty-and-refill test"

Contrary to macrovascular surgical practice, the end-to-end continuous suture is rarely used in microsurgery, especially when arteries are concerned. Still, if correctly performed, this type of anastomosis can produce results that are just as good as anastomosis with separate threads (Mao 1986). Additionally, this technique is considerably faster: there are no knots to tie, no thread ends to cut, and therefore no need to change the instruments in your hands. It is important to use a short thread (3-5 cm) in order to reduce trauma which occurs when the passage of the suture material through the vessel walls is lengthy.
Preparing the artery involves the same steps as the end-to-end suture with separate threads. Apply the microapproximator, section the artery, and prepare the vessel ends. Place the first stay suture "at 3 hr" and tie the knot, leaving one of the thread ends longer so that it will serve to make another knot. Place the second stay suture at 180° in respect to the other, leaving the "short end" also longer. Continue with suturing at the posterior wall, than after knotting it; finish with suturing the anterior wall.
After the first two initial stitches, further 4-5 stitches are usually necessary.

Anastomosis with continuous suture

• In order to avoid any future bleeding of the anastomosis, place the first and the last passes as close to the stay sutures as possible.
• Place the sutures with great accuracy, perpendicular on the margins of the vessel, as you would suture with separate stitches.
• In order to prevent inclusion of the posterior wall into the stitch:
    - lift the margin of the vessel, introduce and open a forceps in the lumen of the vessel or pick up the adventitia;
    - when the tip of the needle is inside the lumen, keep its position parallel to the axis of the vessel;
    - always follow the tip of the needle visually and control its exact location.
• Tie the knot with one of the short ends of the other stay suture.
• The greatest risk in performing this maneuver is the over-tightening of the anastomosis which produces stenosis of your anastomosis. This can be prevented by dilating the anastomosis up to 1.5 times its natural diameter which can be easily performed after the first two stitches.
• Cut the thread ends, irrigate the operating field with saline solution, and open the caudal clamp. After a few seconds open the cranial clamp as well, and remove the microapproximator. Gently compress the anastomosis for about 2-3 minutes. Verify if there are any bleeding sites and perform the patency tests.

• Under a high magnification, place a background material behind the prepared vessel, and place two approximator clips (first proximally then distally). Cut it completely through with scissors perpendicularly to its axis.
• Irrigate and clean not only the lumen, but the entire wound of any blood or tissue traces. Leave some saline solution in the wound so that the vein ends can float in a minimum quantity of liquid.
• Minimally remove the fragments of adventitia, being careful not to cut the venous wall.
• Grab the adventitia with fine forceps and placing the tips of another one dilate the vessel ends gently.
• Bring the approximator clips closer to each other so that there is about a 1 mm distance between the vessel ends (similar to that performed in case of arterial anastomosis). The distance between the edge of the vessel and the penetration point of the needle should be approximately twice the diameter of the needle.
• While inserting the needle, do not involve the posterior wall in the suture. To accomplish this, always support the venous wall with forceps tips carefully inserted into the lumen.
• The distance between the sutures should be double in comparison with the arterial suture, because the pressure of the blood flow is much lower in the vein. Thus, although the diameter of the vein is nearly double the arterial one, you will complete the anastomosis with approximately the same number of stitches.
• After cutting the thread, irrigate the field, remove the microapproximators in the following order: first the proximal one and then the distal one.
• Patency test: After removing the microclamps, the vein should fully dilate and regain its cylindrical shape at the anastomosis site. In case of a distortion or a stricture, it is possible that you involved the posterior wall in the suture, and you should reapply the microapproximators, exploring the anastomosis and correcting the sutures.

• Fully dilated anastomosis, same diameter and blood color distally and proximally to the anastomosis. If the anastomosis is thrombosed, the vein has a larger diameter and the blood is darker distal to the anastomosis.
• "Empty-and-refill test": if we move a caudally places forceps away from the anastomosis and the vessel refills. An almost instant filling of the emptied segment proves the patency of the anastomosis. Any delay, is a sign that the anastomosis is partially blocked (involvement of the adventitia in the lumen or to a through-stitch). If there is no filling and the vessel remains collapsed the anastomosis has completely failed.
• Opening the anastomosis: after ligating the repaired vessel both cranially and caudally, cut out the anastomosis, transect it longitudinally opening it as a book and evaluate the internal aspects of the sutures underneath the operating microscope.

Although end-to-side microsurgical anastomosis is technically more challenging than the end-to-end anastomosis, its importance is undeniable in case of free flap transfers (Godina 1979). In spite of the greater number of delicate maneuvers (such as arteriotomy), the end-to-side anastomosis is often the most suitable solution. It represents a solution for the discrepancies in the vessel diameters and, additionally, it does not interrupt the distal blood flow (Nam 1978).

Carotid or femoral arteries are both suitable. Harvest the longest possible segment of arterial graft by caudal and cranial ligation.

Prepare a 2.5 cm segment of the abdominal aorta as desribed above, and after placing a background material, place 2 clamps on it. Bring the harvested graft into the operating field and place it in a C-shaped position along the aorta, anticipating the future arteriotomy sites. Then remove the adventitia carefully over an area twice the size of the future arteriotomy.

The donor and recipient vessels (on the left), arteriotomy (on the right).

Place a stitch into the area of the future excised vessel part and lift it up. Next lift up the vessel wall while, cutting out an ellipse-shaped piece with a single move with a curved pair of scissors. This excised area can further be enlarged when necessary, while on small-caliber arteries (femoral, carotid) a simple longitudinal incision can be also satisfactory. This incision can be enlarged with a pair of forceps.
After flushing the lumen of the aorta, make sure that the edges of the arteriotomy are neatly cut and free of adventitia. The length of the arteriotomy must be one and a half times the diameter of the vessel that will provide the terminal end of the anastomosis, so that after the suture it will have a trumpet-shaped form.

• Put one end of the graft near the arteriotomy.
• First place the corner knot: by stitching into the graft from the outside towards the inside and the artery from the inside towards the outside (this causes eversion of the vessel wall and knots will be placed outside the vessel) and then place the second corner knot at 180° angle from the first one.

Approximation of the donor segment (left), the first stitch (middle) and suture of the posterior wall (right).

• When suturing the posterior wall of the anastomosis, start the stitches at the corners ending up at the center of the anastomosis. Always enter the graft first and come out through the aorta. This requires a back-hand approach. Usually 3-4 stitches are necessary. The position of threads at the edges is the most important: those must be placed as near as possible to the corner knots.
• At the posterior wall, one should penetrate the vessel wall radially to the center of the arteriotomy and never perpendicular to the vessel.
• If the distance between two adjoining sutures is not equal, it causes bleeding of the anastomotis, which is more persistent than in case of an end-to-end anastomosis.
• It is often useful to place all the central stitches first and knot later.

• Turn over the graft, exposing the anterior wall. Inspect the interior of the anastomosis and check to make sure that the sutures were placed correctly. If needed, remove and replace any and all sutures that have been incorrectly done. If everything is going well, suture the anterior wall of the anastomosis in the same manner.
• When suturing the second graft, you will need to place the first corner knot inside the angle between the graft and the aorta, at the so-called "heel" of the anastomosis. Continue placing the second corner knot at the "toe" of the anastomosis and then suture the anastomosis as specified previously.
• Remove both microclamps. Contrary to the situation of end-to-end anastomosis, if bleeding is significant, it will persist. Only replacement can solve this problem. If we place a clamp on the aorta inside the loop of the bypass, we can force the blood flow through the graft.

When placing a clamp on the aorta inside the loop of the bypass, the blood flow through the graft can forced.

• Prepare both carotid arteries caudally as far as possible and cranially, up to the bifurcation and ligate one of them (this will be the graft). Place a microclamp as far caudally as possible of the same vessel and section and irrigate it the immediately.
• Dilate the free end and remove the adventitia. Bring the donor, sectioned carotid over the trachea, making sure that it reaches the contralateral recicient artery without tension, and estimate the future site of the arteriotomy.
• Remove a portion of the adventitia from the recipient vessel, clamp it cranialy and perform a longitudinal arteriotomy. The length of the incision should be about one and a half times the diameter of the donor carotid at its end, after you have dilated it.
• Place the first corner knot at the "heel" of the anastomosis, that is at the caudal angle of the arteriotomy. Stitch with the needle from the outside towards the inside of the donor carotid and from the inside to the outside of the recipient carotid.
• We can start with either the posterior (Harris 1981) or the anterior wall (Ackland 1989, Yonekawa 1999). Remove the microclamps in the following order: first the cranial one on the receptor artery and after that the caudal one on the same artery. The last microclamp should be removed from the transposed donor artery.

Possible order of the stitches in case of end-to-side anastomosis (when we start with suturing the posterior wall). It is a general rule that stitches should be placed starting from to corner toward the center of the anastomosis.

This is one of the most difficult models of end-to-side anastomosis. Since these vessels are considerably small and their walls are thin, one should try it after being familiar with other types of anastomoses. As desribed above, the femoral vessels should be dissected as distally from the inguinal ligament as possible reaching the difurcation. Ligate both muscle and epigastric branches. Because the adventitia of the vein is extremely thin and it is positioned primarily around two longitudinal lymphatic vessels, the use of moisturized small cotton swabs is recommended.
• After placing a background material underneath the vessels, ligate the femoral artery as caudally as possible and place a vascular clamp cranially.
• Cut the artery, irrigate the segment, dilate its end and remove the adventitia. Position the artery so that it makes a right angle with the vein, while preventing any tension or torsion. You can now approximate the place for the venotomy.
• Place two clamps on the vein and perform the venotomy (stitcs into the wall of the vein where it will be cut and lift it up gently) and cut out a narrow strip with the length of 1.5 times the diameter of the artery.
• Beware that the walls of the vein are extremely thin and transparent and they collapse easily. Wash out the vein lumen until there is no trace of blood in it, and leave some saline solution in the operating field. This will make the walls of the vein float in liquid, they will be easier to identify and less likely to collapse.
• As usual, place the first corner knot cranially, at the "heel" of the anastomosis, and then the second one, at its "toe." Suture posterior then the anterior wall paying special attention to the venous wall that should be well exposed at each passing of the needle. Avoid involvement of the posterior wall into the stitches. If we include too much venous wall in the suture loop, this will inevitably lead to a stenotic anastomosis.
• Remove the clamps in the following order: first the cranial clamp then the caudal one on the vein, and evetually the arterial one.
• The first sign that the anastomosis is working is the appearance of pulsing, red arterial blood in the segment of the vein close to the anastomosis; caudally, through the transparent venous wall you will see the venous blood mixing with the arterial blood. Lift up the vein cranially to the anastomosis until it collapses and see how its lumen fills up rhythmically, with every pulsation.

End-to-side anastomosis with well-executed continuous suture can be as safe as the one with interrupted sutures. Additional benefit is reduced time because knots are not tied, thread ends need not to be cut, instruments are not changed between hands. It is important to use a relatively shorter thread (3-5 cm) to decrease the distress induced by the longer passage of the thread through the vessel wall.
• This technique can be applied in any end-to-side model where we dissected the vessels as detailed above.
• Place the first corner knot at the heel of the anastomosis. Tie the knot leaving the short end somewhat longer so that you will be able to use it later for tying another knot. Do not cut the needle from the thread, leave it attached.
• Take a second thread and suture the toe of the anastomosis at 180° angle from the first corner knot. Leave both ends of the thread longer and then perform continuous stitches. At the first passage of the needle, be assured that you are as close to the corner knot as possible. The safest direction is from the heel towards the toe.
• As you advance, leave the loops of the suture loose: otherwise you will not be able to properly visualize the edges of the vessels.
• A special approach is performing a continuous suture on the posterior wall without rotating it while the intimal surface is facing us (Hou 1987).
• Tightening in two steps: first tighten each loop, starting at the heel, and then pick up the end of the corner knot and the end of the long thread, tractioning until the continuous thread is almost completely tightened. Next, tie up the long end with one of the threads from the corner knot placed at the toe of the anastomosis. You should be very careful: at this point you could cause stricture and stenosis of the anastomosis.

Tightening up the loop after continuous suture

In general, microsurgical exposure and suturing skills should first be honed in the training laboratory, using models with gradually increasing levels of difficulty. Although the peripheral nerve anastomosis is seemingly simple when compared to the more challenging microvascular repairs, it should not be dismissed lightly as it involves specific challenges and particular tricks. Anastomotic techniques can be practiced on rat’s sciatic and femoral nerves. The sciatic nerve is the larger of the two and perhaps a more realistic model. The following basic principles should be observed when performing microsurgical repair of a nerve:
• avoid tension and axis rotation of a nerve;
• resect ends sparingly;
• join the ends in the correct alignment and treat the nerve’s vessels gently.

The nerve should be exposed and cut with a sharp scalpel to avoid crushing injury to and mangling of the axons. It is recommended that a thin blue rubber dam (background) be placed beneath the nerve. The ends are then irrigated and debris carefully trimmed off.

In a healthy nerve the axons are under pressure, and proximally a mushrooming effect will occur as the axons extrude from their nerve sheaths. If the proximal and distal nerve ends were anastomosed in this condition many of the axons would grow erratically and neuroma formation would result.

The protruding exoplasma must be trimmed off to create a straighter path for the regenerating axons. This excision should be performed on both ends of the nerve to the extent that short empty sleeves of nerve sheaths remain, resembling a vein. In case of small diameter nerves like the sciatic and femoral nerve in rats, this exercise is more similar to the human fascicular (perineural) nerve repair than to the epineural approximation.

The first stitch
These stitches involve 8-0 or 9-0 sutures, and are placed under approximately 5x magnification using the operating microscope.

The needle is driven from outside to inside through the epineurium, 1 mm from the cut ends of the nerve in a perpendicular fashion. Note that the needle should penetrate only the epineurium, not the axons. The needle is then driven into the epineurium on the opposite end of the nerve, from inside to outside. The knot is tied with leaving tails approximately 2 cm in length.

Second stitch
The second stitch is placed in the same manner, 180° from the first stitch.

If the bundles are of distinct shapes, then aligning them and creating a match should provide satisfactory results.

The 1-3 additional sutures can be placed in between the first two stitches on the anterior surface. Using the tails of first and second stitches, the nerve can be rotated 180° and another 1-3 sutures can be placed on the posterior surface. The nerve is then rotated back and the long stay sutures are cut.

Evaluation
The completed anastomosis should not be under any tension whatsoever. Enough stitches should be placed to eliminate excessive epineurial gaps. There should be no protruding axons found between stitches.

In preparation for a perineural anastomosis, 5-7 mm of epineurium should be resected from both proximal and distal nerve ends. The nerve bundle is then divided by blunt dissection. Each group of bundles are sutured by their perineurium using 2-3 stitches of 10-0 suture under 5-10x magnification. The nerve does not need to be rotated 180° as in the epineurial closure.

Reconstruction should start from the most distant and deeply located bundles, approximating each of the bundles individually.

If the nerve is to be repaired involves a gap, and it cannot be mobilized so that the repair can be performed tension free, then the only viable solution is either a jump graft or an interposed graft.
The jump graft is applicable if the missing segment does not exceed a few millimeters. It is accomplished using a sleeve of autogenous tissue or synthetic tubing inserted between the proximal and distal ends. This sleeve will prevent neural tissues from migrating randomly between the regenerating nerve ends, thus providing a clear re-growth and distal migration following Valerian regeneration.
An autogenous (interposed) graft involves a suitable piece of peripheral nerve that is fitted between the two ends, filling in the missing length. It is sutured in place, either in with a epineural or perineural fashion, and will provide a scaffold for the regenerating nerve fibers.