The overall goal of the following experiment is to make it possible to create ischemic wounds, not only in normal rabbits, but also in rabbits with reduced resistance such as those which are aged or diabetic. All the currently used techniques for ischemic wound creation are highly invasive, and so far, none of these techniques can be used in diabetic animals. This procedure uses minimally invasive surgery that begins with making three small skin incisions over the three vascular bundles.
The next step is to isolate and protect the central vein and the coddle vascular bundle so that blood circulation to the ear is reduced but not totally eliminated. Finally, the remaining vessels, nerves, and subcutaneous tissues are all severed or ligated via the skin incisions. Ear ischemia is confirmed by the absence of arterial pulse, ear appearance, temperature difference, and subcutaneous oxygen saturation.
This minimally invasive surgery may seem to be difficult to perform, but it is actually very easy. The main advantage of this technique is that it has very little skin disruption. Since we first published this technique in 2007, we have received many questions.
We have since further simplified this procedure. To make it even easier to do without using a microscope, Begin by weighing the selected rabbit, anesthetize the rabbit, using a mixture of ketamine and xylazine. Monitor the vital signs of the rabbit, three vascular bundles that supply the ear can be identified on the dorsal side.
These are the central bundle, which is the largest one with collaterals connecting to other vessels. The coddle bundle in which the artery is usually very small or absent, and the cranial bundle. On the ventral side, the distal skin does not receive blood supply from the proximal vessels.
Instead, vessel bundles on the dorsal ear have numerous penetrating branches through the cartilage to provide blood supply to the distal ventral skin. In some rabbits, a very small subcutaneous vessel also occurs on the ventral side, which supplies blood to no more than one third of the ventral skin. In an open circumferential skin incision, there is essentially no bleeding from a ventral incision.
Before proceeding, make certain the rabbit is not responsive to pain stimuli using a toe pinch and eye reflex, protect the animal's eyes by applying artificial tear.Ointment. Keep the rabbit warm during the surgery with a heating pad. Then using Betadine and alcohol scrub both ears and surrounding skins drape the rabbit, so just the two ears are exposed.
One ear will be rendered ischemic while the other serves as a control. The instruments used for the surgery are a surgical knife with a number 15 blade, two pairs of pickups, one for the skin and another for other soft tissues. Three mosquito clamps, one or two pairs of curved micro scissors, one pair of small scissors for cutting sutures and tough tissues, a needle holder and some stainless steel punches.
Begin the surgery by identifying the three vascular pedicles according to the arteries and veins. Using a number 15 blade. Carefully make one vertical skin incision on the central vascular pedicle.
Isolate the central vein from the surrounding tissues and protect it. This step encircles. The central vein for retraction.
Dissect the central nerve free. It is normally on the coddle side of the vein. The nerve is cut off with scissors.
Dissect the central artery free. It is normally on the cranial side and deeper than the vein, tie off the artery and divide it. Clean up the tissues around the central vein over the ear cartilage.
A second skin incision is made over the coddle pedicle. To preserve the bundle. The vessels are dissected free and encircled for protection.
This is especially useful when the vein is large, as in this rabbit. Make a tunnel between this and the first incision. Cut the subcutaneous tissues over the cartilage.
A third skin incision is made over the cranial bundle. This whole bundle is ligated and separated. The skin over the third bundle is cut open.
This vein is very close to the skin, so do not cut too deeply. The whole vessel bundle is then dissected free, tied off, and separated. Using a knife and scissors, make a subcutaneous tunnel.
The three incisions discontinue all of the subcutaneous tissues, muscles, nerves, and small vascular branches. Use additional sharp and blunt dissections to clean up the base, leaving bare cartilage about five to 10 millimeters wide. Bleeding can be controlled by gauze pressure.
Electrocautery is unnecessary. The skin inside the ear is not cut as it does not carry much blood. The important aspect of this procedure is to ensure the adequate dissection of the subcutaneous tunnel while protecting the vessels.
This should not be such a difficult procedure. After hemostasis, the three skin incisions are closed up using four oh or five oh sutures and checked again to make sure the skin incisions are properly closed and no bleeding is seen For wound drug testing, four. Circular full thickness skin wounds can be created on the ventral side of each ear with a six millimeter stainless steel punch.
The distance between the wounds is at least 20 to 30 millimeters. The skin and perichondrium inside the punch are removed, but the cartilage is not damaged. After the wounds are created on both ears, cover them with gauze temporarily and transfer the rabbit to a procedure room for drug application.
After the wounds are cleaned of any blood clots, apply test and control drugs. The wounds are covered with occlusive dressing to prevent desiccation. When comparing two drugs, apply each drug to both a proximal and distal wound.
This controls for the difference rates of wound healing in the distal and proximal ear postoperatively. The ischemic ear usually becomes cool and cyanotic with a reduced sensation distal to the incision. Mild I edema may present for a few days, but disappears by itself and no treatment is necessary.
The central artery pulse is absent. Ear movement is reduced, but not totally eliminated because some muscles are still attached to the ear base. If the tunnel is made correctly, a clear indentation can be seen between the incisions.
A fentanyl patch is attached to the back skin for relieving possible postoperative pain. The skin temperature is measured on each ear daily with a thermometer. Blood circulation is measured in the ear using a transcutaneous oxygen tension meter or a laser doppler flow meter.
After one week, the sutures of the skin incisions may be removed from 2007. The procedure has been used in more than 70 rabbits. This includes 23 diabetic rabbits and four aged.
The temperature differences between the non-ischemic and ischemic ear ranged from one to 10 degrees Celsius with most rabbits falling within the range of two to six degrees Celsius. The temperature difference was higher in the early days after surgery decreased gradually after day 15, but was still maintained at the end of one month. Six tissue samples were taken from each ear and high energy phosphate contents were measured by HPLC, both a TP and total energy.
Concentrations were higher in the normal ears than in the ischemic ears. Wound healing times were always longer for the ischemic ear than for the non-ischemic ear in non-diabetic and diabetic animals in 15 non-diabetic rabbits. Average healing time was 17.8 days on the non-ischemic ear versus 21.8 days on the ischemic ear.
In 10 12 month diabetic animals, average healing time was 18.3 days on the non-ischemic ear compared to 29.1 days on the ischemic ear. In all animals including diabetic rabbits, the skin incisions healed normally without any complications In rabbits. With 24 month diabetic time, the ventral wounds have a higher tendency to get infected.
This feature makes it closer to human diabetic wounds. A histology sample shows the healing of the subcutaneous tunnel in which muscle is absent and is replaced by fibrous tissues. However, the skin is intact.
While attempting this procedure, it is important to remember to treat the animals in a humane way. Once mastered, this technique can be done in less than one hour. We have used this technique in more than 70 rabbits, including 23 diabetic rabbits without any complications such as bleeding or infection.
It is a very safe procedure following this procedure. Other methods like additional ischemia, nerve damage, and bacterial inoculation can be performed in order to answer additional questions such as vascular ulcers, pressure ulcers, and a diabetic neuropathy.
