The overall goal of this procedure is to safely and accurately deliver a volume of injectate into the left ventricular myocardium of the rabbit heart. And to do this by taking advantage of percutaneous contrast echocardiography guidance. This is accomplished by first anesthetizing the animal, and then preparing the chest for the procedure.
Next, after placing the rabbit on a thermal blanket, and verifying that an ECG signal is correctly displayed, use a clinical ultrasound system to obtain an appropriate acoustic window, and optimize the image in a wide field of view. Then, after loading the syringe with marked cells mixed with a suitable contrast agent, and under ultrasound guidance, carefully align the needle opposite to the transducer and position it closely to the skin of the left hemithorax, before slowing advancing the needle to the target region of the myocardium. Finally the injectate is safely delivered as demonstrated by transmural hyperechogenicity at the site of injection in the echocardiographic image.
Ultimately, the confirmation ex vivo of successful delivery of injectate is demonstrated by the presence of dye in the myocardium, whereas cross-sectional immunohistochemical analysis of myocardial tissue sections is used to show the successful delivery of a cell marker and dye into the target myocardium. The purpose of this video is to demonstrate how to perform this technique safely and accurately, for our possibility, on today's rabbit, using a clincal echocardiography system. The main advantages of this technique are the minimal and basic nature of the machine, and the ability to use contrast echocardiographic guidance for precise visualization of the target sites of injection, thus reducing to a minimum the risk of accidental interchamber delivery.
Before proceeding with intramyocardial injection, start by injecting a combination of ketamine and medetomidine to anesthetize the animal. As demonstrated, the rabbit remains calm when the head is covered with a surgical blanket. Once the animal is anesthetized, use a hair clipper to remove the hair from the skin of the thorax, starting below the neck, and then moving to the subxiphoid region.
Shave additional regions of one to three centimeters of the internal face of the right forelimb as well as the medial tibial regions of both right and left hindlimbs. Then, apply a suitable conducting gel to the electrodes and position these in the shaved regions of the limbs. Fix the electrodes with surgical tape to prevent the loss of contact whilst moving the animal during the procedure.
Next, place the animal on a thermal blanket, verify that a correct ECG signal is displayed on the screen of the system, maintain the animal in a decubitus supine position and allow the animal to breathe spontaneously whilst administering oxygen by face mask throughout the whole procedure. Thoroughly clean and disinfect the shaved skin of the chest with a chlorhexidine-based solution. To maintain good skin contact with the transducer, and to reduce artifacts and improve overall imaging quality, apply a sufficient amount of ultrasound gel to the transducer.
Then, manually place the transducer at the fourth to the sixth intercostal space two to three centimeters away from the right parasternal line, and at an angle of incidence of the ultrasound beam of approximately 90 degrees, relative to the chest wall. Make sure the transducer orientation mark is facing forward to facilitate its alignment with the needle in subsequent steps. You should obtain a parasternal short axis view of the heart at the level of the papillary muscles.
And it is important to have a wide field of view by increasing the depth with the appropriate control in the system. If necessary, adjust the location of the transducer relative to the intercostal space, as well as its anteroposterior and dorsoventral angle to optimize the image, and then identify in this view the right ventrical, RV, left ventrical, LV, interventricular septum, IVS, posterior wall, PW, as well as antero and posteromedial papillary muscles. Pay particular attention to obtain a symmetric image in this view, as well as appropriate differentiation of endocardial and epicardial contorts, and if necessary, adjust through image optimization controls such as gain control.
Once the correct echocardiographic view is obtained, maintain this position throughout the rest of the procedure whilst a second operator performs the intramyocardial injection. For this, use a pre-loaded syringe with a needle attached which should be placed close to the skin of the left hemithorax in a symmetric mirroring position with respect to the transducer. Then, manually align the needle with the transducer orientation mark at an angle of approximately 90 degrees and slowly advance the needle through the skin and into the chest cavity.
The percutaneous needle insertion in this position and orientation allows the visualization of the needle in the plane of the ultrasound beam, thus allowing realtime monitoring, and when necessary, adjustment of the location of the needle relative to the target region of the myocardium, as shown here. Note in this image, the presence of the needle in the left ventricular chamber, shown by the arrowheads. This can easily be repositioned by gently retracting and repositioning the needle to the desired location.
With the tip at the target location, slowly deliver the injectate over a period of 20 to 30 seconds, while slowly and gently retracting the needle to increase the extent of myocardium treated. Perform subtle changes in the angle of incidence of the needle as necessary to complete injections at four intramyocardial injection sites. One in the intraventricular septum, and three in the left ventricular free wall, and deliver a volume of 250 microliters of injectate per injection site.
After completing the procedure, continue monitoring the rabbit's biserial window echocardiographic scans, as well as ECG tracings to verify the absence of complications secondary to the procedure, until the animal is fully awake from anesthesia. The use of 24 hour Holter ECG monitoring is advisable to help determine the effects of intramyocardial injections on the heart rhythm. Using the protocol described here, once the intramyocardial positioning of the tip of the needle was confirmed by echocardiography, and the injection initiated, transmural hyperechogenicity was observed during the delivery of India ink to the target region in this parasternal short axis view, white arrow heads.
Hyperechogenicity was also seen shortly after the injection as shown in this parasternal long axis view of the heart where an echo bright image can clearly be seen at the interventricular septum white arrow heads. When the intramyocardial injection was immediately followed by euthanasia, deposits of India ink were readily visible on external examination of the heart, as shown in this image. Black arrow heads.
Similarly, heart tissue sections at the papillary muscles level revealed transmural deposits of India ink in the free wall of the left ventrical, black arrow heads, demonstrating an efficient and effective delivery of injectate into the myocardium. In this same image, a deposit of India ink was also visible at the interventricular septum. White arrow.
Whilst the procedure was generally well-tolerated, it was very common to observe transient acceleration or deceleration of heart rate, associated with isolated premature ventricular contractions, PVC, both during as well as shortly after the procedure. Indeed, monitoring with 24 hour Holter ECG in animals that received intramyocardial injection with India ink revealed that arrhythmias were very common and that their relative frequency could be affected by the underlying physiological status of the animal. This image shows an example of isolated PVC, the most frequently observed arrhythmia, detected during Holter monitoring.
A comparison between a group of rabbits with normal phenotype, and a group of rabbits that received intravenous administration of Doxorubicin, or DOX, a cardiotoxic and cancer drug, for eight weeks. Prior to intramyocardial injection with India ink indicated that isolated PVCs were significantly more frequent in the DOX group. Other arrhythmias detected by Holter ECG monitoring in both groups were PVC couplets, PVC triplets, and non-sustained ventricular tachycardia.
Significantly, whilst the mean heart rate in the normal and DOX groups were comparable, the standard deviation of the normal to normal, R to R intervals, or SDNN, a measure of nonlinear dynamics related to heart health status, exhibited a marked reduction in the doxorubicin-treated group. Prior to the procedure, the successful transfection of enhanced green fluorescent protein, eGFP positive marked cells, was confirmed by a fluorescence microscopy during in vitro cell culture conditions. Then, upon intramyocardial injection, which was immediately followed by euthanasia, the successful delivery of eGFP positive marked cells into the myocardium was demonstrated via immunohistochemical analysis of heart tissue sections.
Thus, as shown in this image, abundant marked cells were present within the myocardium. Black arrow heads, interspersed with India ink deposits. Deposits of India ink and abundant eGFP positive cells, white arrow heads, were still present when euthanasia was delayed by 24 hours after the intramyocardial injection.
Of note, an acute inflammatory response was observed at this time point, with neutrophils infiltrating the myocardium, red arrows, suggesting an acute cellular rejection response to the xenogeneic cells that were transplanted. Acute inflammation was also observed 24 hours after intramyocardial injection with India ink alone. However, in this instance, the predominant cells infiltrating the myocardium were macrophages.
White arrow heads. After having watched this video, you should have a good understanding of how to perform a percutaneous intramyocardial injection, guided by contrast echocardiography in a rabbit heart. Once mastered, this technique can be performed in 25 minutes after stasis.
