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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Abstract

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Introduction

Cell and gene therapies are exciting and ever developing strategies for regenerating/repairing the injured myocardium in HFrEF. A few studies have compared the effectiveness (e.g., cell retention rate) of the different routes of cell delivery, which have consistently demonstrated the superiority of IMI over intracoronary or intravenous routes1,2,3,4,5. Thus, it is not surprising that a large proportion of studies on translational models of stem cell therapy of the injured myocardium, deliver the injectate via IMI performed under direct view in an open chest procedure6,7. However, this approach has several limitations, including the invasive nature of the procedure, which carries the risk of peri-procedural mortality (often under-reported)8. In addition, an IMI under direct view does not eliminate the possibility for inadvertent injection into the ventricular cavity. In clinical practice an IMI during open chest surgery could be an appropriate method for therapeutic cell delivery, e.g., during coronary artery bypass graft surgery (CABG); however, this approach may not be appropriate for cell delivery in global cardiomyopathy of non-ischemic origin (e.g., HFrEF secondary to anthracycline-induced cardiomyopathy (AICM)).

There is no doubt that ischemic heart disease (IHD) is the most common cause of HFrEF (~ 66%)9,10; however, non-ischemic cardiomyopathy, including AICM, still affects a significant proportion of patients with HFrEF (33%)9. Indeed, recent advances in clinical oncology have resulted in more than 10 million survivors of cancer in the USA alone11, with estimates of a similar number in Europe, consistent with an overall trend towards improved survival of cancer patients12,13. Thus, exploring the benefits of novel therapies such as stem cell transplant for non-ischemic cardiomyopathy, as well as the trialing of an effective and minimally invasive route of stem cell delivery is of utmost importance, given the increasing number of patients affected by cardiotoxicity secondary to anticancer drugs.

Of note, hypothesis testing studies using stem cell therapy aiming to repair/regenerate the injured myocardium frequently involves the use of small rodents (e.g., mice and rats). These models often require expensive high frequency ultrasound systems for evaluation of myocardial function, usually equipped with linear array transducers which have some inherent associated limitations (e.g., reverberation)14. However, other models such as rabbits, representing a large preclinical model, have some advantages for hypothesis testing of stem cell therapies in HFrEF. Thus, in contrast to rats and mice, rabbits maintain a Ca+2 transport system and cellular electrophysiology that resembles that of humans and other large animals (e.g., dogs and pigs)15,16,17,18,19. Another advantage, is their amenability for cardiac ultrasound imaging using relatively inexpensive and widely available clinical echocardiography systems equipped with relatively high frequency phase array transducers, e.g., 12 MHz, such as those frequently used in neonatal and pediatric cardiology. These systems allow excellent echocardiographic imaging with state of the art technology, and they take advantage of the superiority of harmonic imaging20. Furthermore, extensive hypothesis testing of the potential of cardiac regenerative therapies (e.g., stem cell therapy), their safety, efficacy, cardiomyogenic potential, as well as evaluation of the fate of the injectate once delivered into the myocardium, is mandatory before they can be considered for human use, and they require the use of large preclinical animal models, such as the rabbit17,19. Here, we describe a minimally invasive technique for cell delivery via percutaneous contrast-echocardiography guided IMI using a clinical echocardiography system, which is aimed at stem cell transplant-based therapy for non-ischemic cardiomyopathy20. We also describe the benefits of India Ink (InI, also known as China Ink) as an ultrasound contrast agent and in situ tracer of the injectate in the rabbit heart.

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Protocol

The experiments described herein were approved by the Ethical Research Committee of the University of Murcia, Spain, and were performed in accordance with Directive 2010/63/EU of the European Commission. The steps described were performed under standard operating protocols that were part of the plan of work and have not been performed solely for the purpose of filming the accompanying video to this paper.

1. Preparation of Cells and Mammalian Expression Vector

NOTE: Here, we briefly describe a protocol for preparation and transfection of a cell line (human embryonic kidney 293 (HEK-293)); however, appropriate cell specific protocols for the cell type of interest should be optimized (e.g., stem cells).

  1. Maintain HEK-293 cells in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% sodium pyruvate, 2 mM Glutamine, 1% penicillin/streptomycin, and incubated at 37 °C in a humidified atmosphere containing 5% CO2. Once cells are sub-confluent, split at a ratio of 1:3.
  2. Start splitting the cells by aspirating the media, then wash once with sterile phosphate buffered saline (PBS), remove excess PBS and then incubate with 2.5 mL of 0.5x Trypsin-Ethylenediaminetetraacetic acid (EDTA) (5 min, 37 °C).
    1. Add one volume of DMEM media (supplemented as described above) to stop the reaction, and then detach the cells by slowly and gently aspirating up and down with an electronic pipette.
  3. Then, transfer the cell suspension to an appropriate container (e.g., 15-50 mL conical centrifuge tube). Centrifuge in a swing bucket (100 x g), discard the supernatant and wash the pellet twice with sterile PBS.
  4. Resuspend the pellet in fresh DMEM media and seed at an appropriate cell density (e.g., 1 x 106 cells in a 75 cm2 flask) in fresh culture flasks or large dishes according to local laboratory practices.
    1. Replace existing media with fresh media every 2 days.
      NOTE: The expression vector was derived from pIRES1hyg and used according to the manufacturer's instructions as previously described21. p(EGFP) IRES1hyg was generated by subcloning EGFP cDNA as a BamHI + NotI insert from pEGFP-N1 into the BamHI + NotI digested pIRES1hyg21.
  5. 1 day before transfection, seed HEK-293 cells at a density of 0.5 x 105 cells/cm2 in 12- or 24-well tissue culture plates.
  6. On the day of transfection, prepare DNA-lipid transfection reagent complexes in separate 1.5 mL microcentrifuge tubes (1 tube/well).
    1. Start by adding 250 µL of reduced serum medium into each tube, and then add 4 µg DNA (mix gently).
    2. Subsequently, add 250 µL from a previously prepared mix of 10 µL lipid transfection reagent and 250 µL of reduced serum medium, to each tube (mix again gently).
    3. Finally, proceed with transfections, by incubating HEK-293 cells with DNA lipid transfection reagent complexes for 4 h, and then replace with DMEM medium supplemented as described above (step 1.1), and incubate for another 48 h. Perform drug selection of stable transfectants with 100 µg/mL hygromycin B.
  7. Detach HEK-293 cells with trypsin as described above (step 1.2). After washing the cells in sterile PBS, resuspend in an appropriate vehicle (e.g., 10% v/v InI in PBS), to achieve a final cell concentration of 5 x 106/mL.

2. Preparation of the Rabbit

NOTE: The positioning of the rabbit and the transducer for IMI is not optimal to evaluate morphology and function of the heart of the animal. Thus, it is advisable to perform a complete echocardiographic examination20 prior to the IMI (see below), and at subsequent time points as defined by the experimental design. This will aim to evaluate the baseline anatomical and functional characteristics of the heart in the animal that will receive an injection, and also evaluate the effects, of IMI in the function of the heart.

  1. Perform this examination in a blinded fashion and following the guidelines of the Echocardiography Committee of the American College of Veterinary Internal Medicine and the American Society of Echocardiography/European Association for Cardiovascular Imaging22,23,24.
  2. Obtain a simultaneous 1-lead electrocardiogram (ECG) tracing throughout the whole echocardiographic study.
  3. Anesthetize the rabbit with ketamine 10 mg/kg, combined with medetomidine 200 µg/kg, intramuscular (I.M.) injection.
  4. Verify the level of anesthesia after 10-20 min following administration of anesthesia.
  5. Using a hair clipper remove the hair of the chest widely (e.g., below the neck to the sub-xiphoid region) (Figure 1A).
  6. Shave additional regions of 1-2 cm2 in the internal face of the right forelimb (mediocubital region) and of both hind limbs (mediotibial region) (Figure 1B).
  7. Place adhesive ECG electrodes on the shaved regions of the limbs to synchronously monitor the heart rhythm during the procedure (Figure 1C).
  8. Place the animal in the decubitus supine position on a thermal blanket, with the limbs outstretched using surgical tape attached to the table (Figure 1C).
    1. Make sure the ears are flexed backwards behind the head/back of the rabbit, and at a position that is lower than its forelimbs, since this helps to maintain correct positioning of the thorax of the animal throughout the procedure.
  9. Allow the animal to breathe spontaneously whilst administering oxygen by face mask throughout the whole procedure (100%, 2-3 L/min).

figure-protocol-6279
Figure 1. Preparation of the rabbit for IMI. (A) Clip hair from thorax; (B) Clip hair from limbs; (C) Attach electrodes and position the rabbit with legs outstretched on a thermal blanket. Please click here to view a larger version of this figure.

3. Percutaneous Contrast Echocardiography-guided IMI Technique in the Rabbit

  1. Clean and disinfect the skin of the thorax with a chlorhexidine-based solution.
    1. Use an aseptic technique throughout the whole procedure, according to current best practice.
    2. Whenever possible, and if reasonably practicable, perform a fully sterile procedure, including, but not limited to, the use of sterile material such as gowns, gloves, surgical wound drapes, sterile dressing material for the table, as well as a sterile ultrasound transducer cover and sterile ultrasound gel. This will reduce to a minimum the risk of introducing pathogens to the animal receiving IMI, and is standard practice in the clinical setting (e.g., during cardiac puncture).
      NOTE: It is recommended to always proceed in line with local and national regulations of animal research applicable to the local institution and country of practice.
  2. Apply ultrasound transmission gel to the chest and/or to the transducer, and with the transducer cord around the experimenter's neck, perform a quick window scan of the animal's heart, which is often helpful to visualize anatomy and to plan for the IMI.
  3. Place the transducer manually at the 4th-6th intercostal space, 2-3 cm away from the right parasternal line with an angle of incidence of ~ 90° with respect to the right side of the thoracic wall (Figure 2A).
  4. Adjust the location of the transducer relative to the intercostal space as well as its anteroposterior and dorsoventral angle to optimize a modified short axis view at the level of the papillary muscles. Identify in this view the right ventricle (RV), left ventricle (LV), interventricular septum (IVS), posterior wall (PW), as well as anterolateral (AL) and posteromedial (PM) papillary muscles (Figure 2B).
    1. Have a wide field of view by significantly increasing the depth using the appropriate control on the system (e.g., button, dial).
    2. Pay particular attention to obtaining a symmetrical image in this view, as well as appropriate differentiation of endocardial and epicardial contours, and, if necessary, adjust through image optimization controls (e.g., gain).
  5. Once the optimal echocardiographic view is obtained (Figure 2B), maintain this position throughout the rest of the procedure, whilst a second operator performs the IMI (see below).
    1. Whilst holding the transducer, avoid concealing the transducer orientation mark, which should always be facing forward, thus allowing its alignment with the needle in subsequent steps (Figure 2A, C).
  6. With a 24-G needle attached to a 1 mL syringe, place the needle 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 ~ 90° (Figure 2C), and slowly advance the needle through the skin and into the chest cavity.
    NOTE: The percutaneous needle insertion in this position and orientation facilitates the visualization of the needle in the plane of the ultrasound beam (Figure 2D, E), thus allowing real time monitoring and, when necessary, adjustment of the location of the needle relative to the target region of the myocardium (Figure 2G, H).
  7. With the tip at the target location, slowly deliver the injectate (up to 0.25 mL per injection site) within 10-30 s (Figure 2E), whilst slowly and gently retracting the needle during injection to increase the extent of the myocardium treated.
    1. Use InI 10% (v/v) diluted in PBS for standardization of the technique, and as an in situ tracer whilst acquiring competency, as well as to confirm successful targeting of all four IMI sites within the myocardium by gross pathology and histopathology (see Representative Results). Once competency is achieved, InI could be substituted by a suitable commercial ultrasound contrast agent if desired.
      NOTE: Delivery of 10% (v/v) InI diluted in PBS either with or without cells into the myocardium results in transmural hyperechogenicity (i.e., echo bright appearance) at the target site of injection (Figure 2E, F). Transient deceleration or acceleration of the heart rate, associated with premature ventricular contractions (e.g., isolated, couplets and triplets) are frequently observed even from the first contact of the needle with the epicardium as well as during and/or shortly after IMI. However, no life-threatening arrhythmias are developed, and acute adverse effects are rarely observed using up to 0.25 mL (1.25 x 106 cells) of injectate per IMI injection site (see Representative Results and Discussion).
  8. Perform subtle changes in the angle of incidence of the needle as necessary to complete injections of 0.25 mL to each of the four target IMI sites (three in the left ventricular free wall (LVFW) and one in the IVS).
  9. After completing the percutaneous contrast echocardiography-guided IMI procedure, evaluate heart rhythm (e.g., through serial ECG trace and/or 24 h Holter ECG), and perform serial window echocardiographic scans to verify the absence of complications, until the animal is fully recovered from anesthesia, and only then transfer to a light cycle room.
    1. Here, we use 24 h Holter ECG to monitor the effects of IMI with InI on heart rhythm for 24 h. For this, we compared a group of 6 rabbits with normal phenotype (Normal Group) and a group of 6 rabbits that received intravenous administration of Doxorubicin (a cardiotoxic anthracycline drug, commonly used for the treatment of cancer; DOX group) at a dose of 2 mg/kg/week for 8 weeks, and then both cohorts received IMI with InI.

figure-protocol-13181
Figure 2. Percutaneous contrast echocardiography-guided intramyocardial injection in the rabbit. (A) Placement of the transducer in the right hemithorax at an angle of ~ 90°. (B) Representative image of a parasternal short axis view (PSSX) of the heart at the level of papillary muscles in the rabbit. (C) Alignment of the needle at an angle of ~ 90° relative to the transducer orientation mark. (D) Location of the needle at the target site in a PSSX view of the heart (note that the needle is easily visualized in the plane of the ultrasound beam). (E and F) Demonstration of hyperechogenicity at the target site upon intramyocardial injection with India Ink (arrowheads highlight the transmural hyperechogenicity). (G) Accidental location of the needle in the LV chamber (arrowheads highlight the needle shaft). (H) Repositioning of the needle to the LV free wall (arrowheads highlight the needle shaft). RV = right ventricle; LV = left ventricle; IVS = interventricular septum; PW = posterior wall; AL = anterolateral papillary muscle; PM = posteromedial papillary muscle. Please click here to view a larger version of this figure.

4. Post IMI Analyses

  1. Perform histopathological analysis of heart tissue samples from rabbits.
    1. Fix tissue for 24 h in 10% formaldehyde, followed by dehydration with increasing ethanol concentrations as follows:
      • 1x in 70% (60 min)
      • 1x in 95% ethanol/5% methanol (60 min)
      • 1x in 100% (60 min)
      • 1x in 100% (90 min)
      • 1x in 100% (120 min)
      NOTE: All the above incubations are conducted at room temperature (RT). Then, substitute twice with 100% xylene (1 h, RT), and finally embed in paraffin in two steps (60 min, 58 °C)25.
    2. Perform 4-5 µm tissue sections with a microtome25. Mount sections on slides.
    3. Perform staining with hematoxylin-eosin and Masson's trichrome methods25,26,27.
  2. In tissue sections from transplanted hearts, perform immunohistochemistry to detect EGFP(+) HEK-293 cells (e.g., using the avidin-biotin complex (ABC) method), briefly:
    1. De-wax 4-5 µm thick heart sections in 100% xylene (10 min, at RT). Rehydrate tissue by washing with decreasing ethanol concentration solutions (2x in 100% (2 min); 2x in 95% (2 min); 1x in 70% (2 min); 1x in 50% (2 min); 1x in 30% (2 min); 1x ddH2O (2 min)) at RT.
    2. Perform endogenous peroxidase inhibition by covering the sections with 100 µL of 3% H2O2 diluted in methanol (prepared with 5 mL of 30% stock solution of H2O2, and adding methanol up to a total volume of 50 mL) (incubate 30 min, RT), and then wash by immersion with Tris buffered saline (TBS; pH 7.6).
    3. Perform antigen unmasking via enzymatic treatment, by covering the sections with 100 µL of 0.1% Pronase (prepared with 0.01 g Pronase diluted in 10 mL TBS) (incubate 12 min, RT), then wash with TBS (5 min, RT).
    4. Incubate in blocking solution (normal goat serum at 10% in TBS) using 100 µL per slide (30 min, RT), and wash with TBS (5 min, RT).
    5. Incubate with chicken anti-green florescent protein (GFP) as primary antibody (1:500 in TBS) (1 h, 30 °C), and wash with TBS (5 min, RT).
    6. Incubate with secondary antibody biotinylated goat-anti-chicken IgG (1:250 in TBS) (1 h, 30 °C), and then wash with TBS (5 min, RT).
    7. Incubate with avidin-biotin complex (20 min, 30 °C), and then label using 3,30-diaminobenzidine tetrahydrochloride (DAB) (RT, 5-10 min).
    8. Finally, dehydrate by washing in increasing ethanol concentrations (1x in 30% (2 min); 1x in 50% (2 min); 1x in 70% (2 min); 2x in 95% (2 min); 2x in 100% (2 min)) at RT, counterstain sections using the hematoxylin-eosin method25, and then mount cover slides. Include positive, negative as well as isotype controls.
      NOTE: The brief protocol described above is not intended for general immunohistochemistry use; optimization for the tissue of interest and conditions is necessary.

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Results

Percutaneous Contrast Echocardiography-guided IMI with InI:

Using the protocol described above, and once the optimal positioning of the tip of the needle was confirmed by echocardiography and the injection initiated, transmural hyperechogenicity was observed during the delivery of InI (10% v/v in PBS) (Figure 2E), as well as shortly after the IMI to the target region (

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Discussion

The primary goal was to develop a minimally-invasive technique that could be used for the delivery of stem cells into the myocardium of rabbits (a large sized preclinical animal model)17,18, whilst taking advantage of the use of a relatively inexpensive imaging system readily available in many clinical and research centers. Here, we show that, using a clinical echocardiography system, and aided by InI, a widely available agent, with both in situ tracing ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Sheila Monfort, Brenda Martínez, Carlos Micó, Alberto Muñoz, and Manuel Molina for excellent support provided during the collection of data, and Carlos Bueno for providing the EGFP(+) HEK-293 cells. This work was supported in part by: Fundación Séneca, Agencia de Ciencia y Tecnología, Región de Murcia, Spain (JT) (Grant number: 11935/PI/09); Red de Terapia Celular, ISCIII-Sub. Gral. Redes, VI PN de I+D+I 2008-2011 (Grant no. RD12/0019/0001) (JMM), Co-financed with Structural funding of the European Union (FEDER) (JMM); and, the University of Reading, United Kingdom (AG, GB) (Central Funding). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Materials

NameCompanyCatalog NumberComments
HD11 XE Ultrasound SystemPhilips10670267Echocardiography system.
S12-4PhilipsB01YgG4-12 MHz phase array transducer
Ultrasound Transmision Gel (Aquasone)Parket laboratories IncN 01-08
Vasovet 24GBraunREF 381212 over-the-needle catheter
Omnifix-F 1 ml syringeBraun9161406V
Imalgene (Ketamine)MerialRN 9767Veterinary prescription is necessary
Domtor (Medetomidine)EsteveCN 570686.3Veterinary prescription is necessary
Heating Pad
Faber-Castel TG1Faber-Castel16 33 99India (China) Ink
Holter SyneflashEla medicalSF0003044S24 h Holter ECG system.
Electrodes Blue Sensor®Ambu (NUMED)VLC-00-SHolter ECG electrodes.
MicrotomeLeica BiosystemsRM2155
MicroscopeOlimpusCO11
ABC Vector EliteVector LaboratoriesPK-6200Avidin Biotin Complex Kit.
Chicken anti-GFP antibodyInvitrogenA10262Primary antibody.
Biotinylated goat-anti-chicken IgG AntibodyVector LaboratoriesBA-9010Secondary Antibody.
3,30-diaminobenzidine tetrahydrochloride (DAB)DAKO (Agilent)S3000
Fluorescence MicroscopeCarl Zeiss
MicroImaging		Zeiss AX10 Axioskop
Holter ECGElamedicalSyneflash SF0003044S
Dulbecco’s modified Eagle medium (DMEM) Fisher Scientific11965084
10% fetal calf serum (FCS)Fisher Scientific11573397
0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA)Fisher Scientific25300054
Lipofectamine 2000 (Lipid transfection reagent)Fisher Scientific11668019
Reduced serum medium (Opti-MEM)Fisher Scientific31985070
Hygromycin BCalbiochem (MERCK)400051
Xylene (histological)Fisher ScientificX3S-4
Hydrogen Peroxide Solution (H2O2)SigmaH1009
PronaseFisher Scientific53-702-250KU

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