Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

This protocol can be used to answer questions about drug delivery and immune interactions within the organs included in our circuit. This technique allows for the perfusion of organs that are not possible to perfuse by traditional ex-vivo methods, while avoiding the main clearance organs. Before beginning the surgery, set a circulating water bath and all of the water-jacketed components, to 37 degrees Celsius.

Confirm that the tubing is cleaned, and use a bubble trap within a moist chamber as the perfusate reservoir to limit the perfusate volume. To perform the catheterization procedure, confirm a lack of response to pedal reflex, in an eight to 10 week old male anesthetized bulb see mouse. And place the mouse in the supine position on a styrofoam board, with the head at six o'clock.

Immobilize the animals limbs with tape, and wipe the abdomen with isopropyl alcohol. Make a T-shaped incision in the abdomen, using electro coagulation to stop any bleeding around the edge of the incision. Push the stomach, jejunum and colon to the right side of the abdomen, to reveal the abdominal aorta, vena cava and common iliac and iliolumbar arteries and veins.

Under a dissection microscope, locate and ligate the ovarian and iliolumbar arteries and veins with 4-O silk sutures. Loop two 4-O silk sutures under the abdominal aorta and inferior vena cava about one centimeter above the iliac artery and vein, one millimeter apart. Then make a loose nod in the suture closest to the iliac vessels.

Use a needle holder to horizontally align and stretch both the inferior vena cava and the abdominal aorta. And, keeping the vessels stretched, use a 24-gauge winged shield IV catheter to puncture the abdominal aorta. As soon as the needle penetrates about one millimeter into the vessel, depress the button to retract the needle core, and insert the catheter about five millimeters into the vessel.

Insert a catheter five millimeters into the inferior vena cava, in the same manner. And tie both sutures around the catheterized vessels. Then apply instant glue to immobilize the catheters to the erector spin a, and replace the abdominal organs.

To set up the perfusion system, place the mouse on a silicon pad in the water-jacketed moist chamber, and immobilize the catheter wings to the pad with 19-gauge needles. Fill the arterial catheter inlet, with pre-warmed perfusion buffer. Use hemostatic forceps, and a screw-on connector to attach the catheter inlet to the tubing, and immobilize the inlet perfusion tubing with tape.

Adjust the peristaltic flow rate to 0.6 milliliters per minute, and keep the perfusion outflow open-ended for five to 10 minutes, to wash the blood out through the venous catheter. Some clots will be present in the outlet catheter, flush out the clots with perfusate buffer, and use a screw on connector to attach to the end of the venous catheter to the outlet tubing to close the circuit. Then cover the moist chamber with a pre-warmed lid for up to two hours of perfusion, periodically checking on the level of perfusate.

Here, confocal images after perfusion with ringer solution containing Hoechst 33342 and DyLight-649 lectin, are shown. The muscle, bone marrow, testes, bladder, prostate and foot skin typically demonstrate an efficient nuclear and vascular staining. Hematoxylin and eosin staining of these tissues after three hours of normal thermic perfusion, reveals a successful flushing of the organs, with no apparent tissue injury.

This model can be used to understand drug uptake pathways, in the physiological irrelevant environment. For example, to understand the role of serum components in drug accumulation. We're using the system, to understand the mechanism of liposome accumulation in the skin.