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

Summary

This article details an optimized protocol for echocardiography-guided intramyocardial injections in rat models of myocardial infarction using a 29 G x 88 mm needle. This technique ensures robust, precise, and reproducible delivery of therapeutic agents directly into the peri-infarct zone.

Abstract

Echocardiography-guided intramyocardial injection (EGI) is a minimally invasive technique for delivering stem cell therapies in preclinical myocardial infarction (MI) models. Compared to traditional open-chest approaches, EGI offers improved clinical translatability, reduced invasiveness, and minimized physiological impact on the animal. While EGI is well established in murine models, its application in rats remains limited due to anatomical and technical challenges. In particular, thinning of the left ventricular anterior wall (LVAW) in infarcted and peri-infarct regions complicates safe and accurate myocardial delivery, as wall thickness can fall below the needle bevel size of commonly used 27 G or 28 G needles, increasing the risk of ventricular perforation or failed delivery. To address this limitation, we optimized a protocol for EGI in rat MI models using 29 G Spinocan needles. The smaller-diameter, longer needle enables precise targeting of thin myocardial tissue, minimizing damage and enhancing injection accuracy, independent of LVAW thickness. This technique is compatible with standard transthoracic echocardiography platforms and eliminates the need for thoracotomy, allowing longitudinal studies in the same animal. Our refined method enables robust, reproducible delivery of therapeutic agents into viable myocardium adjacent to the infarct zone, where regenerative therapies are most effective. By improving safety and targeting precision, this approach increases the translational relevance of preclinical cardiac research and supports the development of standardized protocols across laboratories.

Introduction

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, with myocardial infarction (MI) being a significant contributor to both acute and chronic cardiac conditions¹. Despite advances in pharmacological and interventional management, the regenerative capacity of the adult human heart is limited, often resulting in adverse remodeling and progression to heart failure^2,3. Consequently, stem cell-based therapies have gained attention as potential strategies to repair damaged myocardium, preserve cardiac function, and improve clinical outcomes⁴.

Robust preclinical models are essential for evaluating the safety, efficacy, and delivery strategies of these therapies. Among small animal models, rats offer several advantages, including manageable heart size, well-characterized infarction techniques, and translationally relevant cardiac remodeling responses⁵. Conventionally, intramyocardial administration of stem cells in rat MI models is achieved via thoracotomy, allowing direct visualization of the injection site⁶. However, this approach is invasive, introduces significant procedural risks, and impedes repeated interventions in longitudinal studies. Moreover, it lacks alignment with clinical delivery modalities, such as catheter-based or percutaneous injection⁷.

Echocardiography-guided intramyocardial injection (EGI) is a minimally invasive alternative that enables targeted delivery of therapeutic agents under real-time imaging guidance. While well established in murine models, the use of EGI in rat models has been limited. EGI is more extensively developed in mice than rats primarily because most cardiovascular research has historically focused on mice as the dominant preclinical model⁸. Additionally, the smaller size and heart anatomy of mice allowed for the optimization of ultrasound-guided procedures⁹. In contrast, rats have a thicker chest wall and greater respiratory motion, complicating stable, high-resolution imaging during the procedures¹⁰.

Advances in high-frequency ultrasound technology have made EGI in rats feasible, enhancing the clinical relevance of their use. Yet, the thinning of the left ventricular anterior wall (LVAW) in infarcted regions remains a major challenge for EGI in rodents. In rats, wall thickness often decreases to less than 1 mm, whereas standard 27 G or 28 G needles have bevel lengths of 1.25-1.5 mm, increasing the risk of ventricular perforation or poorly directed cell delivery. To address this limitation, we refined the EGI technique in rats by utilizing 29 G x 88 mm Spinocan needles. These needles feature a bevel length of 1 mm, allowing precise, atraumatic delivery of cells into the thinned myocardium of the infarct and peri-infarct regions. The procedure is performed using high-resolution transthoracic echocardiography, enabling visualization of both anatomical landmarks and needle trajectory in real time.

This refined EGI protocol provides a minimally invasive, reproducible method for targeted delivery of therapeutic agents into the peri-infarct myocardium in rat models of both acute and chronic MI, independent of LVAW thickness. It facilitates injection into viable border zones-critical sites for achieving therapeutic benefit-while significantly reducing surgical burden and recovery time compared to open-chest approaches. Moreover, it supports longitudinal studies involving repeated injections and follow-up imaging, thereby reducing animal numbers in accordance with the 3Rs (Replacement, Reduction, Refinement) principle¹¹. The protocol is adaptable for the delivery of pharmacological agents, gene therapies, biomaterials, and various stem cell types. By standardizing EGI in rat models and aligning with clinical delivery modalities, this approach enhances reproducibility across laboratories and strengthens the translational relevance of preclinical cardiac research.

Protocol

All animal experiments were approved by the Local Ethical Committee of UHasselt (Ethical Commission for Animal Experimentation, UHasselt, Diepenbeek, Belgium, ID202308 and ID202497) and conducted in accordance with EU Directive 2010/63/EU.

1. Equipment setup

1. Turn on the ultrasound imaging platform, the integrated warmed platform, the physiology monitoring unit, and the gel warmer.
   NOTE: To enable clear and sufficiently detailed visualization of the left ventricular anterior wall during the EGI procedure, a high-frequency ultrasound system equipped with a ≥ 15 MHz linear array transducer providing approximately 75 µm axial resolution is recommended.
2. Turn on the heating pad to maintain the body temperature of the rat during chest hair removal.
3. Select the appropriate transducer and initialize the 3D motor to ensure full range of motion of the transducer.
   NOTE: In addition to using the micromanipulator of the animal's platform railing system, the 3D motor allows for highly precise transducer adjustments, which can facilitate the alignment with the injection needle when needed. If necessary, the procedure can also be performed without the 3D motor.
4. Ensure proper system alignment before imaging. Set the micromanipulator of screws controlling the animal platform to a neutral position (i.e., centered within the full range of motion). Center the animal platform itself in the middle of the railing system. Position the transducer mounting system so that the transducer is aligned directly above the center of the animal platform.

2. Animal preparation and anesthesia

1. Weigh the rat and calculate the required volume of buprenorphine (0.04 mg/kg) to administer for pain relief.
2. Place the rat in an induction chamber connected to an anesthesia machine. Induce anesthesia with 2.5% isoflurane supplemented with oxygen at a flow rate of 2 L/min.
3. Once anesthesia is induced, transfer the rat onto a heating pad to maintain the body temperature at 37 0.5 °C. Maintain adequate anesthesia by positioning the rat's snout into a nose cone connected to the anesthesia system, delivering a constant flow of 1-3% isoflurane supplemented with 1-1.5 L/min oxygen, adjusted as needed to ensure appropriate depth of anesthesia.
4. Shave the rat's chest and remove residual hair with depilatory cream. After hair removal, disinfect the injection site by alternating an appropriate surgical scrub agent (e.g., povidone-iodine) with 70% ethanol, repeated 3x. Apply ophthalmic gel to both eyes to prevent dryness.
5. Administer the precalculated dose of buprenorphine intramuscularly into the hind leg to minimize the discomfort related to the EGI.
6. Transfer the rat from the heating pad to the animal platform of the ultrasound imaging system. Ensure consistent anesthesia delivery through the platform's integrated nose cone, maintaining a steady flow of 1-3% isoflurane in 1-1.5 L/min oxygen. To ensure adequate anesthesia, perform a toe pinch to check the absence of the pain reflex. Secure the rat's paws onto the platform electrodes, applying a small amount of electrode gel to ensure optimal signal quality. Insert the rectal temperature probe for continuous body temperature monitoring.
   NOTE: Continuous monitoring of physiological parameters is essential throughout imaging and injection procedures. Heart rate (HR), respiratory rate (RR), and body temperature of the rat must remain within acceptable ranges to avoid anesthesia-related complications. As a general guide, HR should be in the range of 250-400 bpm, whereas RR should be at least 30 breaths per minute. If HR or RR drop below these thresholds, immediately decrease the isoflurane concentration (e.g., from 2-3% down to 1-2%) and verify that the animal remains properly positioned and does not become hypothermic. Adjustments should be made gradually, with close monitoring until parameters return to target levels.
7. At this stage, acquire any desired baseline or pre-injection images. In general, parasternal long axis (PSLAX) and short axis (SAX) images in both B-mode and M-mode, along with four-chamber images, are recommended for baseline functional and anatomical assessment.
   NOTE: General imaging can be performed with the system set up as shown in Figure 1A. For the injection procedure, the animal platform will need to be repositioned and the transducer adjusted accordingly. While PSLAX B-mode imaging is used for guidance during injection, these images may not be optimal for post-hoc functional analyses due to the necessity of precise needle alignment.

3. Echocardiography-guided intramyocardial injection procedure

NOTE: From this point onward, two operators are required to successfully perform the injection. Operator A is responsible for manipulating the animal platform and monitoring the live echocardiography image, while Operator B should position themselves behind the injection mount to accurately align the needle and transducer.

1. Prepare the injection system by attaching a 22 G sterile guide needle to a 1 mL syringe. Place the syringe with the guide needle onto the injection mount and secure the injection clamp.
   NOTE: The 29 G x 88 mm needle is too flexible to puncture skin and muscle layers directly; the guide needle is necessary to facilitate the injection.
2. Align the transducer with the injection mount to ensure the needle can be visualized. Adjust the transducer position using the transducer mount and holding clamp, and, if needed, rotate the injection mount along its rail to achieve appropriate alignment.
3. Without moving the rat, rotate the animal platform so that the notch of the transducer points to the rat's right shoulder (Figure 1B-D). This setup allows the acquisition of a clear PSLAX B-mode image of the left ventricle. Verify that the guide needle remains within the transducer's field of motion. If misaligned, adjust the transducer position via the 3D motor system rather than altering the injection mount.
4. Once correct needle-transducer alignment is achieved, fine-tune the imaging by using the micromanipulator screws of the animal platform rail. Maintain the transducer position to preserve alignment with the needle. Additional rotation or translation of the animal platform might be necessary to optimize image quality.
5. Visualize the infarcted area. Evaluate the infarct extent, regional wall motion, and wall thinning, using both PSLAX B-mode and M-mode imaging to assess the feasibility of injection site. Apply a 16-segment wall motion scoring approach to identify akinetic or severely hypokinetic regions, which represent the infarct core.
6. Identify the peri-infarct zone and select the target injection site. Choose a hypokinetic area adjacent to the infarct core with an end-diastolic wall thickness greater than 1 mm, which allows complete insertion of the 29 G needle bevel at a shallow angle.
7. Using the rail system, advance the injection mount towards the animal platform. Fine-align the needle with the exact middle of the transducer using the micromanipulator screws on the injection mount.
   NOTE: Precise alignment is critical. If the needle is not centered to the transducer, it will not be visible during the injection.
8. Advance the guide needle using the inject micromanipulator screw on the injection mount until the skin is punctured. Confirm that the needle tip is visible in the ultrasound image. If the needle is not visible, retract the needle carefully using the 'inject' micromanipulator screw and repeat step 3.7 to realign.
9. Activate needle guide feature in the ultrasound software to confirm the needle trajectory. Advance the guide needle towards the pre-determined target region, positioning the needle bevel approximately 1-2 mm from the left ventricular anterior wall (LVAW) (Figure 2A).
   NOTE: Avoid puncturing the myocardium with the guide needle to prevent excessive bleeding or cardiac injury.
10. Once the needle is positioned, have Operator A firmly stabilize the guide needle at its base ensuring constant visualization of the needle on the ultrasound screen. Simultaneously, ask Operator B to loosen the clamp securing the syringe, remove the syringe, and prepare the injectate syringe. Instruct Operator A to keep the guide needle stationary.
11. Attach the 29 G x 88 mm needle on the syringe containing the injectate (Operator B) and secure the syringe onto the injection mount. Manually guide the 29 G x 88 mm needle through the stationary guide needle, making minor adjustments with the micromanipulator screws in the x-axis plane (left or right of the probe) if necessary. Advance manually until the needle bevel becomes visible in the thoracic cavity on ultrasound.
    NOTE: Due to the flexibility of the 29 G needle, it may bend during insertion into the thoracic cavity. To minimize this, Operator A can stabilize the external portion of the needle shaft while operator B advances the needle into the thoracic cavity.
12. Once the 29 G x 88 mm needle passes through the guide needle and becomes visible on ultrasound, advance it into the myocardium of the LVAW using the micromanipulator screws for added precision and safety. Confirm that the needle bevel is located entirely within the myocardium (Figure 2B).
    NOTE: Due to the relatively long bevel of the 29 G needle compared to the myocardial wall thickness, it is critical to ensure that the entire bevel is embedded within the myocardium. Use the 'Freeze image' function to verify placement if needed. Although ultrasound depth calibration may aid in precision, in this protocol, we verified depth by selecting injection sites with an end-diastolic wall thickness >1 mm and visually confirming the needle tip approaching-but not breaching-the endocardial border (Figure 2B).
13. Inject the injectate slowly into the myocardium (Operator B). Successful intramyocardial injection is confirmed visually by a bright/dense echogenic spot at the injection site, which should move synchronously with the LVAW (Figure 2C). To minimize backflow of the injectate, wait for approximately 10 s after completing the injection before retracting the needle.
14. First, remove the syringe with the needles from the injection mount to minimize the risk of needle stick injury. Next, carefully remove the rat from the animal platform and allow it to recover on a heating pad. Monitor vital parameters continuously until full recovery from anesthesia.

Results

Validation of injection accuracy by in- and ex vivo bioluminescence imaging (BLI)
To evaluate the success and efficiency of the EGI, stem cells were first transduced to express firefly luciferase (Fluc) via a lentiviral vector, enabling in vivo tracking by bioluminescence imaging (BLI). BLI confirmed precise intramyocardial delivery in 86% of cases (n = 14).

Discussion

As preclinical research focusing on regenerative therapies to restore the heart after myocardial infarctions advances, hurdles regarding optimal therapeutic agent delivery remain¹². Minimally invasive EGI techniques are increasingly favored over open-chest approaches because they better mimic clinical percutaneous delivery, improve animal welfare, and permit repeated administrations in longitudinal studies^8,13. Yet, detailed, standardized ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
21?MHz MX250 transducer	FUJIFILM VisualSonics, Inc.	/	High-frequency ultrasound transducer compatible with the Vevo 3100 system
3M Transpore Surgical Tape	3M	1527	To secure the paws of the animal to the animal platform
Aquasonic  100 Ultrasound Transmission Gel	Parker Laboratories BV	01-08	Ensures proper acoustic coupling between transducer and animal skin for optimal image quality
Bupaq 0.3 mg/mL Solution for Injection for Dogs and Cats	Richter Pharma AG	57446/4008	Opioid analgesic (buprenorphine) administered for pain management
ISOFLUTEK 1000	Alivira	/	Isoflurane-based inhalation anesthetic
Multi-bottle gel warmer 230 V	Parker Laboratories BV	83-03-20 CE (LCD)	Warms ultrasound gel to body temperature to prevent hypothermia of animal discomfort
Signagel Electrode Gel	Parker Laboratories BV	15-25	Applied to ECG electrodes to improve signal conduction for physiological monitoring
SPINOCAN 29 G X 88 MM	Bbraun	4501900	Fine-gauge needle for precise intramyocardial injection
Terumo 3-part syringe with pre-connected hypodermic needle	Terumo Europe N.V.	MDSS01S2516E	Used to mount and secure the 22 G guide needle and the 29 G injection needle for controlled delivery into the myocardium.
THM150 Physiological Monitoring Unit	FUJIFILM VisualSonics, Inc.	/	Monitors vital signs such as heart rate, respiratory rate, and body temperature of the animal during the procedure
Veet Minima Pure	Reckitt Benckiser	/	Depilatory creme
Vevo 3100	FUJIFILM VisualSonics, Inc.	/	High-resolution ultrasound system
Vidisic carbomerum 980 2mg/g ophthalmic gel	Bausch + Lomb	/	Applied to eyes of anesthetized animal to prevent corneal drying