This surgical protocol provides a reliable model to study ischemic preconditioning regimen in fasciocutaneous flaps to improve the outcomes of these surgeries. This protocol is easy to learn, reproducible and reliable. The main difference with other protocols is the use of femoral vessels to provoke the ischemic preconditioning, avoiding harming small vessels.
It can eventually lead to optimizing an ischemic preconditioning protocol for free and pedicled flap reconstruction to improve flap survivability and reduce the critical period of the flap vulnerability. This method can contribute to the field of reconstructive surgery and lends itself well to the study of ischemia reperfusion injuries. To begin, place the properly sedated animal in a supine position.
Shave the abdomen from below the inguinal crease to above the level of the xiphoid process. Apply a hair removal cream on the shaved area and wait for five minutes before cleaning it. Using a sterile skin pen and a ruler, first mark the midline of the animal's abdomen and then mark the inguinal crease.
Before making the incision, indicate the position of the desired flap on the animal's body by drawing an oval or a rectangle, having a vertical length of up to six centimeters and a horizontal width of about three centimeters extending cranially from the inguinal crease. Now draw five or six equidistant marks perpendicular to the flap limits. Then using a pair of Ragnell scissors, make a longitudinal incision of three to four centimeters on the inguinal crease.
Be sure to pull skin upward to avoid damaging vessels. Expose and identify the femoral and epigastric vessels using a 4 jewelers microsurgical forceps by opening and closing the forceps to separate the fascia and to gain access to the vessels beneath the inguinal fat pad. Use the inguinal incision to start the flap incision using the Ragnell scissors.
Pay attention to undermining the full thickness of the skin and the connective tissue over the abdominal muscle. As the tip of the flap is initially freed from the surrounding skin, continue the flap procurement by undermining from the distal to the proximal part using the Ragnell scissor tips to separate the flap from the muscle while cauterizing any perforator vessels and dermal plexus vessels around the flap. Once the flap is entirely harvested, aim to encapsulate the entirety of both branches of the superficial inferior epigastric artery or SIEA with the flap by gently lifting the flap upward to visualize the vessels.
Next, separate the fat pads on the inferior aspect on both the medial and lateral sides of the flap. Use the bipolar cautery to cauterize the fat pads close to the border of the incision carefully without harming the superficial inferior epigastric pedicle. Before proceeding for the femoral vessel preparation after completion of the flap harvesting, inject the animal with a single dose of 17.5 international units of sodium heparin via the penile vein.
To allow a better view of the vessels, place the animal within a lone star self-retaining retractor. Now working under a surgical microscope having a 40X magnification, use a couple of 4 microsurgical forceps to dissect the femoral vessels both proximally and distally until the emergence of the superficial epigastric vessels. On the distal femoral vessels, clean the fascia and gently free the nerve from the artery and vein.
Using an 8-0 nylon suture, ligate the distal femoral vessels by circumventing the microsurgical needle holder under the artery and vein, clasping the suture and tying off these vessels right after the emergence of the epigastric vessels, leaving a one millimeter distance after the pedicle origin. On the proximal femoral vessels, repeat the same process of cleaning the connective tissue. However, separate the artery and vein from each other to allow efficient clamping.
To induce intermittent ischemia, place microsurgical clamps separately on each proximal femoral artery and vein. When the ischemic injuries are completed, suture the flap to its original position lining the marks drawn preoperatively. Suture the flap using a running suture with 3-0 nylon at the inguinal crease medially, around the flap, and ending at the inguinal crease laterally.
To verify the blood supply to the flap, inject 0.25 milliliters of 10%sterile fluorescein sodium into the the penile vein using the same technique and tools described for the previous injection. After three minutes, shine a long wave UV lamp at 366 nanometers to reveal the fluorescent areas corresponding to the perfused areas. After closure and verification, spread crushed metronidazole along the sutures to prevent auto-mutilation and spray liquid bandage on the same area.
To remove the flap from the epigastric blood supply at the end of the ischemic preconditioning period, cauterize inferior to the fat pad along the inferior border of the flap. Immediate intravenous fluorescein angiography on postoperative day zero or POD0 showed complete vascularization of the flap by the SIEA alone. The tissue, including the whole flap paddle, was well perfused.
The angiography of the negative control on POD5 and POD10 showed full viability of the flap paddle. All animals in this group where no ligation was performed on the feeding vessels remained healthy. For the positive control group, the absence of green fluorescence immediately after ligation showed no perfusion of the flap, proving the absence of neovascularization.
This was confirmed on POD10 with observed necrosis of 85%of the skin paddle. Interestingly, the distal tip was viable and neovascularized. Statistical analysis of the flap surface viability on POD10 showed 99.5%survivability by the negative control group whereas the positive control group showed 11.25%survivability.
An example experimental group meant to study ischemic preconditioning showed 13.67%survivability demonstrating that the particular ischemic preconditioning protocol was inefficient in this model. The two major steps are flap harvesting and vessel dissection under the microscope. The most important piece of advice is to have good-quality micro instruments.
The next goal is to try different ischemic preconditioning protocols by varying the ischemia durations and rhythms to select the regimen allowing the best flap survivability following early flow disruption. This technique by providing a good model for ischemia fusion injuries can lead to new findings on ischemic preconditioning applied to modern reconstructive surgery and reconstructive transplant.
