The overall goal of this procedure is to induce a consistent survivable and functionally measurable degree of ischemia. Reperfusion induced acute kidney injury in mice by performing unilateral renal pedicle clamping, followed by contralateral nephrectomy ischemia. Reperfusion injury is achieved by making a dorsal incision and exposing the left and right renal pedicle.
That includes the renal artery and vein. Next, a contralateral nephrectomy is performed after liberating the kidney from the surrounding tissue. Then the left renal pedicle is clamped using a vascular clamp and the kidney is covered with the skin.
Finally, after a predetermined time, the clamp is released, the kidney is allowed to reperfuse, and the kidney is pushed back into the retroperitoneal space. Ultimately, functional analysis of kidney injury can be carried out through analysis of serum creatinine levels using an enzymatic cascade assay 24 hours after removal of the contralateral kidney Ischemia. Reperfusion injury is a model that's widely used to study the mechanisms and treatment of acute kidney injury.
The technique itself is relatively straightforward, but there are a number of variables which have resulted in many labs, finding the technique difficult to reproduce with high mortality, and also relatively low throughput with individuals only able to form five to 10 surgeries per day. Dr.Skrinick, who will be presenting the technique to you later on has spent a number of years developing the technique in our lab and has devised a protocol which we have found to be much more reproducible with a low mortality and relatively high throughput. On a good day, NA is able to form 40 to 50 surgeries per day To prepare for surgery.
Autoclave all surgical instruments if they'll be used for multiple mice, treat them using a hot bead sterilizer in between procedures. If using a water bath system, set the heat at 38 degrees Celsius one hour prior to surgery, weigh the mouse, and then administer a subcutaneous injection of 0.5 milliliters of sterile normal saline. After anesthetizing a mouse using an intraperitoneal injection of alaine, ketamine mixture, shave the surgical area and apply ophthalmic ointment to protect the mouse corneas.
Transfer the mouse in a prone position onto a heated surface lined with an absorbent bench pad and tape the legs to the surgical surface using Betadine solution. Scrub from the center of the site towards the periphery, followed by novacan a total of three times. Cover the surgical field with the sterile surgical drape.
Next, palpate the kidney location through the skin. Then cut the dorsal skin along the midline of the mouse with scissors and forceps. Separate the skin and subcutaneous layers over the left and right dorsal sides through the incision.
Make a small incision through the right flank muscle and fascia above the right kidney and exteriorize it carefully dissect the upper and lower poles of the kidney free from surrounding tissue. The adrenal gland is also gently removed from the upper pole of the right kidney along with its blood supply by gently separating the tissue with blunt forceps using a sweeping movement from medial to lateral. This is a little hard to demonstrate by video, but can be understood.
Looking at the anatomy of the adrenal gland with its blood supply illustrated in figure one, then using four oh silk suture, tie a double surgical knot around the hilum. Leave one end of the suture about two inches long and use it to hold up the kidney to visualize the cut site while cutting distal to the knot. Use sterile saline to irrigate the stump.
Then with absorbable suture, close the muscle layer moving to the left side. Make a small incision through the left flank muscle and fascia above the left kidney and exteriorize it with blunt forceps. Carefully hold the kidney while releasing the renal pedicle from the surrounding fat tissue with a second set of forceps.
Taking care not to exert too much pressure on the kidney, which may cause renal injury. If you are including sham operated controls in your experiments, sham operated mice should undergo exactly the same procedures as the injured mice, including releasing the renal pedicle from surrounding fat, but the vascular clamp is not placed on the renal pedicle. Next, while stabilizing the kidney withholding forceps, use a non-traumatic vascular clamp to clamp the left renal pedicle clamping times will vary depending on the mouse strain and severity of injury required with the clamp still in place.
Cover the left kidney with the skin and then use saline soaked sterile gauze to cover the skin incision. Then cover the mouse with a sterile drape to protect the surgical field after the desired length of time, expose the kidney. Check that it has a uniform dusky appearance, and then release the clamp.
The kidney should rapidly and uniformly take on a pink appearance following reperfusion. Gently push the kidney back into the retroperitoneal space. Next, close the muscle layer with absorbable suture and close the skin with clips, administer 0.05 micrograms per gram buprenorphine analgesia, and 0.5 milliliters of sterile normal saline subcutaneously.
Then transfer the mouse onto a heating pad and allow it to wake before transferring it to the animal room up to five. Mice can be placed in each cage after surgery, closely monitor the mouse and administer additional analgesia as needed every eight to 12 hours for pain or discomfort in our hand. Unilateral IR injury with simultaneous contralateral nephrectomy substantially reduces variability of results when compared with bilateral IR injury.
With this procedure, 50 to 60%of mice develop the expected renal insufficiency within 24 hours after injury. In practical terms, this means that there is still significant variability using this model since the remaining mice either develop very little injury or have much more severe injury. For our purposes, we are able to minimize this problem.
Since the treatment groups we use are usually initiated 24 hours or more after the initial injury. In this way, mice can be randomized to different treatment limbs after the 24 hour time point creatinine is obtained. Mice with creatinine values outside of a predetermined range are excluded from further study at this point.
For moderate injury, we usually exclude mice with serum creatinine values outside of a 0.8 to 1.2 milligrams per deciliter range. In addition to performing unilateral IR with simultaneous contralateral nephrectomy, we perform unilateral IR alone. Mice can survive much longer periods of ischemia and tend to develop more severe post-injury fibrosis.
However, because it is difficult to evaluate functional recovery with unilateral IR injury in this model, there is no way to evaluate the severity of injury without harvesting kidney tissues and that may not be possible for longer term studies. For this reason, we have developed a protocol for inducing severe unilateral IR injury and assessing renal functional recovery by performing contralateral nephrectomy eight days after the original injury. Using this approach, 90 to 100%of mice survives the injury, but importantly, the mice developed very consistent renal injury and can be evaluated for functional recovery following acute kidney injury from day nine onwards.
The consistency and predictability of kidney injury is strongly dependent on the consistency and precision of steps performed during surgery. Once master it, this technique can be done in 35 to 40 minutes and can be performed simultaneously on five to 10 miles at a time. We have used this method to operate on as many as 40 to 50 miles per day, so enjoy and good luck.
