Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

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The protocol combines a mouse model of abdominal aortic aneurysm resembling established human disease and repetitive treatment via intravenous catheter. To address the clinical need to block aneurysm progression. The protocol provides a complete workflow for testing drugs to reduce aneurysm progression in mice including comprehensive information on the expansion of aortic diameter volume and morphology.

Plus 3D ultrasound. 3D ultrasound may be adapted to measure the infrarenal aorta where aneurysms develop in less taste based mouse models. The jugular vein catheterization can be utilized for any set of requiring intravenous injections.

After anesthetizing 12 to 14 weeks old APOE deficient mail mouse shave the small area on the upper left side of the back, over the shoulder blade, apply 10%povidone iodine solution to disinfect the shaved area. Make a one centimeter transversal incision in the skin of the upper back with a scalpel between the mid spinal and left scapular line. Holding the skin up with forceps and using blunt curved scissors.

Make a subcutaneous pocket by pushing toward the left hind limb. Open the scissors and pull the open scissors out of the cut to widen the pocket. Gently insert the pump into the pocket with the flow moderator toward the tail to minimize potential interference of angiotensin two released by the incision site.

Then close the wound with 4.0 absorbable interrupted sutures. Using the vascular access system. Prepare the catheter by cutting the three French side to the desired length and pushing the catheter over the 22 gauge metal connector of the vascular access system or VAS with at least three millimeter overlap.

Place the aluminum cap on the button to protect the port. Shave the fur from the right side of the neck on the ventral side and the right side of the upper back of an anesthetized mouse. Then apply the povidone iodine solution to disinfect the shaved area.

For jugular vein preparation, make a 0.5 centimeter transversal super clavicular skin incision at the right side of the neck over the right clavicle. Use blunt microsurgical tweezers to separate the connective tissue and fat exposing the external jugular vein. Avoid tearing small blood vessels in the fat.

Isolate five millimeters of the vessel close to the pectoral muscles. Then blunt dissect tissue under the vein using bent micro tweezers and pass through two to three of the 6.0 silk ligatures. Tuck in the ligatures and add a drop of saline to the site.

For button implantation flip the mouse over and place it in the prone position. Apply the povidone iodine solution to disinfect the shaved area. Make a one centimeter sagittal incision on the upper back with a scalpel between the mid spinal and right scapular lines.

Next, use blunt curved scissors to make a circular pocket slightly larger than the size of the VAS around the incision site by blunt dissection. Use the blunt curved scissors to tunnel cranially over the right shoulder toward the ventral incision at the neck by slightly opening the scissors. Then pulling the open scissors out and repeating the action as it is pushed further in.

Once the tunnel has reached the ventral incision pass through surgical clamps from the ventral to the dorsal incision. Attach three French end of the catheter to the clamp and pull the catheter through the tunnel so that it is out of the ventral neck incision and the VAS is in place at the dorsal incision. Then insert the VAS surgical felt disc subcutaneously at the incision on the back.

Unclamp the catheter check for patency using the fork end of the handling tool to remove the protective aluminum cap and use the magnetic end to hold the button and inject saline or PBS with a one milliliter syringe attached to the corresponding injector until the liquid leaks from the one French end. Push the button caudally in the pocket and close the skin over the felt disc of the VAs under the flange of the VAS with two four oh interrupted sutures cranially. For vein catheterization, flip the mouse back to the supine position and add a drop of saline to the cut side.

Separate the ligatures and tie the first ligature around the catheter and the jugular vein with two to three knots as far cranially as possible to ligate the vein and anchor the catheter to the outside. Move the second ligature as close as possible to the pectoral muscles. Using micro scissors cut a diagonal angle to create a sharp end so that three to five millimeters of the catheter will be in the vein.

Then pierce a hole in the vein using a 27 gauge needle attached to a one milliliter syringe filled with saline by pulling on the secured cranial ligature and pushing the needle parallel to the vein. Next, insert the catheter into the vein in the same manner by pulling on the secured cranial ligature and sliding the catheter into the vein using the bent tweezers. Push the catheter until it is aligned with the vein.

Tie the second ligature over the catheter inserted region with two to three knots and check that there is no blood leakage. Cut off the excess end of both ligatures with micro scissors and add a drop of saline. Then close the skin with 4.0 absorbable interrupted sutures.

Inject the mouse with the desired volume of inhibitor or PBS saline as demonstrated earlier. Maintain positive pressure on the plunger while disconnecting the syringe from the VAS. Monitor the mice post recovery.

Using the ultrasound software adjust the settings to gain of 30 DB image depth to 9.0 millimeter and width to 8.08 millimeter. Next, add a drop of electro gel to each of the four electrocardiograms or ECG electrodes on the stage and tape the mouse extremities to them. Spread warm ultrasound gel on the mouse's abdomen and lower the transmitter to put it in contact with the abdomen.

Identify the aorta as a circular, fast pulsating vessel. Locate the left renal artery and survey the area manually up to 12 millimeter cranially to ensure no interference in the area of interest. Return to the left renal artery.

Then set the probe at six millimeters cranially from the artery. For 3D ultrasound acquisition set the respiratory gating to 25%delay and a window of 50%and the ECG trigger to 50 milliseconds. From the 3D options set the scan distance to 11.96 millimeters with a step size of 0.076 millimeters.

After acquiring 157 frames, scroll through to check for image quality before saving the image. For 2D diameter acquisition turn off the respiratory gating and ECG trigger and manually locate the area with the largest diameter in the 12 millimeter stretch of the super renal aorta. Press B mode on the screen and acquire a B mode image.

Additionally, for the EKV image without moving the transducer, acquire an EKV image with the system's standard settings at the same site. The development and progression of the super renal aneurysms were monitored by ultrasound at baseline day eight and day 27. A trichrome stain of the day 27 aorta illustrates the morphology of the formed aneurysm with wall dissection and intramural thrombus.

The incidence rate of abdominal aortic aneurysm or AAA at day eight and aortic ruptures within the first nine days showed that 9%of mice failed to form a AAA. However, 35%of the mice experienced aortic ruptures before catheter implantation, thus resulting in a total of 56%of the remaining mice with established AAA disease being minimal to stratification into treatment groups. An inverse relation was observed between the initial expansion and further disease progression of initially fast forming versus moderately growing aneurysms in PBS control treated mice.

The exemplary ultrasound results revealed that GSK 4 84 treatment inhibited AAA progression while the aneurysms continued to enlarge and control mice. Catheter patency is essential as catheter disconnection from the vein or vascular access button results in drug delivery into the subcutaneous space with unknown drug concentrations at the aneurysm site. When mice are sacrificed at the end of the experiment, the water and other tissues can be histologically investigated and blood analysis conducted to characterize on and off target drug effects.

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Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

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