Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

The overall goal of this procedure is to demonstrate the in vivo insertion of an osmotic pump for local substance delivery to the ischemic hind limbs of mice and the subsequent hind limb vasculature perfusion with microfill for micro computed tomography imaging. This is accomplished by first making an incision in the shoulder and hind limb of the experimental animal to allow tunneling of sheath tubing along the inside of the back of the animal. In the second step, a filled and primed osmotic pump and catheter are inserted through the tubing and the pump is sutured into the back of the animal.

Next, the tubing is removed. The femoral artery is double ligated proximal to the femoral bifurcation, and the incision is sutured. Subsequently, the hindin vasculature is perfused with microfill, a silicone radio pick imaging agent through the infrarenal abdominal aorta catheter.

Ultimately, a 3D image of the vascular cast of the mouse ischemic hind limb made visible with microfill can be imaged. I'm Dr.Richard Dixon, head of the Molecular Cardiology Research Laboratories at the Texas Heart Institute. Our lab has been developing a variety of cell and drug-based therapies for treating cardiovascular diseases and peripheral vascular diseases.

One of the most common models for testing compounds in peripheral vascular disease is the animal rodent hind limb ischemia model. We have developed a number of important advances to this model recently, and we'd like to present those to you in this video presentation. The main advantage of this technique over existing method like daily drug injections, is that this technique reduce the needs for the animal handling and the insurance.

The continues local administration of a substance over the start period. Additionally, micro CT imaging of the microfill casted vessels is advantageous over histological or laser dopper profusion imaging due to the ability of micro CT to render a truly quantitative and volumetric three dimensional analysis that is representative of the vascularity throughout an entire sample. Using sterile technique begin by weighing the empty pump and recording its weight in milligrams.

Next, use a one CC syringe and blunt tipped 27 gauge filling tube to fill the pump slowly with PBS. When the PBS reaches the top of the pump, stop filling. After wiping off any excess solution, weigh the filled pump.

Then use a pair of forceps to break the white flange from the flow. Moderator being careful not to bend or crush the tube cover three to four millimeters of either end of the moderator with a P 50 catheter. Now use a syringe to fill the catheter and the moderator leaving the syringe attached to the distal part of the catheter.

Begin inserting the flow moderator into the pump. When the catheter is flush with the pump surface, remove the syringe from the catheter and incubate the pump in PBS at 37 degrees Celsius overnight. On the day of the surgery, apply eye ointment on a fully sedated prone mouse, and then place a fitted nose cone delivering a continuous flow of isof fluorine onto the animal After injecting anesthetic at the incision site, treat the skin with a povidone iodine solution followed by 70%ethanol.

Then make a four to six millimeter incision in the scapula region to the side of the spinal column. Use fine forceps to separate the subcutaneous membrane. Then after turning the mouse to the supine position, use microsurgical scissors to make a 0.8 to one centimeter incision above the knee towards the medial thigh following the path of the femoral artery.

Then use small sheath tubing to tunnel up the side of the torso from the medial thigh to the incision on the back. Next, after returning the mouse to the prone position, guide the catheter of the prime osmotic pump through the sheath tubing to the thigh, and then position the pump inside the back of the animal. Close the back incision with a six oh prolene suture.

After placing the mouse in the supine position, once again, remove the sheath tubing, leaving the catheter exposed through the incision on the thigh. Secure the leg with surgical tape. Next, use a retractor to open the incision and separate the subcutaneous fat tissue to reveal the vasculature.

Then use fine pointed forceps to gently pierce the membranous femoral sheath. To access the vascular bundle without causing excessive bleeding. Carefully dissect and separate the femoral artery from the femoral vein and nerve near the groin.

Then place two eight oh prolene adjacent sutures to interrupt the femoral artery, two to three millimeters proximal to the femoral bifurcation at the origin of the profunda FMRIs and epigastric arteries. Now cut the catheter to the appropriate length. Place the tip at the anterolateral region of the thigh and secure the tip with a 6.0 prolene suture to the superficial fascia.

To prepare for imaging, place the anesthetized mouse in the supine position and inject 0.2 ccs of 1000 IU heparin sodium subcutaneously to prevent clotting in the animal's vasculature while the mouse is attached to the nose cone. Make an incision large enough to expose the thoracic cavity and the lower abdomen after moving the abdominal organs to the left side of the animal. To expose the infrarenal abdominal aorta, carefully separate the aorta from the inferior vena cava.

Next, ligate the proximal infrarenal abdominal aorta and use the needle tip to make a small incision on the vessel. Then insert a 27 and a half gauge blunt needle attached to a P 10 catheter into the infrarenal abdominal aorta and secure the needle with a six oh suture. After securing the 27 and a half gauge needle, make an incision in the right atrium and administer an overdose of isof fluorine to induce euthanasia and allow for the exit of excess fluid.

Now, attach a heparinized saline filled 20 cc syringe to the needle tip and flush the vasculature for at least five minutes. Removing the nose cone once the flushing begins, The injection of the microfill is the most difficult step of the procedure. In order to ensure success, it is important to make certain that all the blood is rinsed out of the vessels after perfusion with sailing, Now attach a formalin filled 20 cc syringe to the catheter and pressure perfu the vessel for an additional five minutes.

Finally, slowly perfuse the vessel with the microfill for five minutes. Be careful to apply a consistent pressure when administering microfill in order to achieve optimal vessel casting. Optionally, at this point, the skin can be dissected to view the microfill filled vasculature for further on face analysis.

For polymerization of the contrast agent, place the animal in a container filled with 10%NBF and store it at four degrees Celsius after 24 hours. Use dissection tools to remove the skin distal to the lower abdominal aorta. Refresh the NBF and place the animal back into the container at room temperature.

After four days, dissect the contralateral and ischemic hind limbs to facilitate the bone decalcification. Place the specimens in a formic acid solution for 48 hours, and then rinse the specimens under running water for one hour. Finally, return the specimens to 10%NBF and proceed with fron and micro computed tomography imaging.

This first figure shows representative laser doppler images of blood perfusion before and surgical ligation of the femoral artery. In the mouse hind limb ischemia was created in the left hind limb as indicated by the arrows after vascular casting with microfill, A 3D micro computed tomography image of the vascular network shows that microfill can fill the vessels effectively. Discontinuities can occur, however, due to various procedural factors such as air bubbles, a lack of pressure, or the viscosity of the microfill, the points of discontinuity are indicated by the arrows.

After watching this video, you should have a good understanding of how to use an asthmatic pump to stably deliver a substance to the hind limb of a mouse after ischemia surgery.