The overall goal of this procedure is to deliver biologics directly into the renal artery in order to induce a localized therapeutic effect in the renal microvascular compartment in rats. The main advantage of this technique over existing methods such as systemic administration of anti-inflammatory or antifibrotic drugs is that the intrarenal delivery technique minimizes any potential systemic side effects. The implications of this technique extend to a therapy of a variety of renal pathologies, because the renal microvascular function is involved in a wide spectrum of kidney diseases.
Playing a key role in the progression of kidney damage. To prepare for surgery, begin by autoclaving all surgical instruments. After anesthetizing a rat, according to the text protocol, transfer the animal to a controlled heating pad, to preserve the animal's body temperature at 37 degrees Celsius.
Use 1%to 2%isoflourane in one liter per minute of oxygen to maintain anesthesia. Administer an analgesic subcutaneously, and apply eye ointment to prevent dryness. To compensate for loss of body fluids, administer 10 milliliters per kilogram of 0.9%normal saline subcutaneously.
Then, shave the abdominal area, and use povidone iodine and 70%ethanol pads to clean the skin. To begin surgery, ensure that the depth of sedation is adequate by monitoring physical reflexes such as a withdrawal from a toe pinch. Use a number 10 surgical scalpel blade to perform a laparotomy through a small midline incision.
With cotton swabs, pull the intestine and colon to the right side of the abdomen, and cover them with sterile gauze, soaked in 0.9%normal saline to keep the organs moist. Gently retract upward the spleen, liver, stomach, and pancreas to expose the aorta and the left kidney artery. Next, under a surgical microscope, use blunt dissecting curved forceps with a repeated open close motion along the length of the vessels and sterile cotton swabs, to carefully separate the abdominal aorta above and below the left kidney and the left renal artery from the veins, the fat, and the surrounding connective tissue.
The use of cotton swabs helps avoid damage to nerves and infected blood vessel, in particular to thin-walled veins. Then, place a 4-0 silk suture underneath the aorta, and using microvascular clips, clamp the aorta above and below the renal artery bifurcation. Now, using a 24 gauge intravenous catheter, puncture the aorta at the level of the left kidney artery bifurcation, and carefully advance the catheter into the renal artery.
Because of the small size of the renal artery, the catheter might puncture through the vessel. It is important to work under a surgical microscope and to insert the needle as parallel to the artery as possible. Then, connect a syringe filled with the drug solution or saline to the catheter, and profuse the kidney.
Immediately after profusion, use a microvascular clip to clamp the left renal vein and the left ureter before removing the catheter. Then, place a piece of absorbable hemostat gelatin sponge with a small drop of tissue adhesive over the punctured area of the aorta, and with a cotton swap, gently apply pressure. At the same time, release the clamp from the aorta below the left renal artery bifurcation.
After five minutes, release the clamp from the renal vein and ureter. Carefully release the clamp from the aorta above the left renal artery bifurcation, and allow the kidney to reperfuse. The total renal ischemia should last no longer than seven minutes.
After ensuring that no active bleeding occurs, and closely observing the area for 10 more minutes, use 4-0 absorbable sutures and a continuous pattern to prevent infection, and close the abdominal incision in two layers. Apply topical antibiotic ointment over the incision area to prevent infections. Then, transfer the rat into the observation cage on a warm pad until complete recovery.
After completion of all of the studies, euthanize the animals according to the text protocol. Two different doses of recombinant rat VEGF, or PBS as control, were injected into rats, and then animals were euthanized eight hours post surgery to examine the activation of the VEGF pathway. As seen here, by H&E staining, the surgical procedure did not effect the morphology of the perfused kidney, as compared to the control organ.
In addition, serious red staining did not show an increase in extracellular matrix deposition in response to the ischemic time and the infusion of rrVEGF, as compared to control tissue. Finally, by Western blot analysis, a small but significant increase in the expression of proteins involved in the VEGF pathways was observed, including VEGF, FLK-1, Phospho-AKT and AKT, and Phospho-ERK and ERK. Once mastered, this technique can be done in about 45 minutes if it is performed properly.
While attempting this procedure, it's important to follow institutional and national guidelines for the animal care and use, in particular, to alleviate the pain and discomfort by the administration of analgesics. Following this procedure, other methods, such as Western blotting, or histological staining of the kidney can be performed to determine the efficacy of different therapies as well as activation of specific pathways.
