Echocardiography-guided Injection for Targeted and Reliable Intramyocardial Stem Cell Delivery in a Rat Model of Myocardial Infarction

To begin, use a sterile applicator to apply ophthalmic gel onto both eyes of an anesthetized rat to prevent dryness. After complete hair removal, disinfect the injection site by alternating an appropriate surgical scrub agent with 70%ethanol. Transfer the rat from the heating pad onto the animal platform of the ultrasound imaging system.

Apply a small amount of electrode gel to attach the rat's paws to the platform electrodes, enhancing signal quality. Then insert the rectal temperature probe to monitor body temperature continuously. Now, acquire the desired baseline or pre-injection images, including parasternal long axis and short axis views in both B mode and M mode, as well as four chamber images for anatomical and functional assessment.

To prepare the injection system, attach a 22 gauge sterile guide needle to a one milliliter syringe. Mount the syringe with the guide needle onto the injection clamp and secure it in place. Next, align the ultrasound transducer with the injection mount to ensure the guide needle is visible in the imaging field.

Adjust the transducer position using the transducer mount and holding clamp. Without moving the rat, rotate the animal platform until the notch on the transducer points toward the rat's right shoulder. To fine-tune the imaging, adjust the micromanipulator screws on the animal platform rail.

Keep the transducer steady to maintain alignment with the needle. Now, using parasternal long axis B mode and M mode imaging, visualize the infarcted area. Evaluate the extent of infarction, regional wall motion, and areas of wall thinning to determine injection feasibility.

Identify the peri-infarct zone and choose a hypokinetic area next to the infarct core that has an end diastolic wall thickness greater than one millimeter. Next, using the rail system, advance the injection mount towards the animal platform. Then adjust the injection mount's micromanipulator screws to fine-align the needle tip with the exact center of the transducer field.

Now, to slowly advance the guide needle and puncture the skin, turn the inject micromanipulator screw on the injection mount and confirm the needle tip is visible in the ultrasound field. Then activate the needle guide feature in the ultrasound software to confirm the planned trajectory of the guide needle. Advance the guide needle toward the selected injection site, stopping with the bevel one to two millimeters from the left ventricular anterior wall.

Once the needle is in position, instruct one operator to stabilize the guide needle at its base to maintain constant ultrasound visualization. Ask another operator to loosen the syringe clamp, carefully remove the syringe, and replace it with the syringe containing the injectate. Next, attach an 88 millimeter long 29 gauge needle to the syringe containing the injectate.

Secure the syringe onto the injection mount. Manually insert the needle through the stationary guide needle, making minor adjustments with the micromanipulator screws along the x-axis if required. Continue to advance the needle manually until the bevel becomes visible in the thoracic cavity on ultrasound.

Once the 29 gauge needle is visible on ultrasound, advance it into the myocardium of the left ventricular anterior wall using the micromanipulator screws for precise control. Ensure the full length of the needle bevel is embedded in the myocardium. Slowly inject the injectate into the myocardium.

Confirm a successful injection by observing a bright, dense echogenic spot at the injection site that moves with the wall motion of the left ventricular anterior wall. Finally, remove the syringe and needles from the injection mount to prevent accidental needle sticks. Carefully lift the rat off the animal platform and place it on a heating pad for recovery.

Bioluminescence imaging revealed partial leakage of the injected stem cells due to premature needle withdrawal indicated by diffuse luminescent signal in the thoracic cavity. A successful intramyocardial injection was confirmed by a concentrated luminescent signal at the left ventricular mid-apex region. Ex vivo imaging of sectioned hearts showed the strongest bioluminescent signal in the mid-apex slice with weaker signals in the apex and mid-ventricle sections.

Postmortem imaging of the heart showed a visible purple hydrogel adjacent to the infarct zone, confirming targeted delivery. Fluorescence imaging of heart tissue showed that the red tracer was confined to the peri-infarct zone, confirming localized delivery. Sirius red staining confirmed that the tracer was delivered to a region of intermediate wall thickness between the infarct core and healthy myocardium.

Color Doppler imaging confirmed that the needle was correctly placed inside the myocardium with no visible blood flow before injection. During injection, the anterior wall remained intact with no external blood flow detected. After needle withdrawal, the retained injectate was visible within the myocardium and no hemorrhage was observed.