This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.
A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes – but is not limited to – hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.
Close to 360,000 individuals in the United States1 and many more worldwide2 suffer an episode of sudden cardiac arrest every year. Attempts to restore life require not only that cardiac activity be reestablished but that damage to vital organs be prevented, minimized, or reversed. Current cardiopulmonary resuscitation techniques yield an initial resuscitation rate of approximately 30%; however, survival to hospital discharge is only 5%1. Myocardial dysfunction, neurological dysfunction, systemic inflammation, intercurrent illnesses, or a combination thereof occurring post-resuscitation account for the large proportion of patients who die in spite of initial return of circulation. Thus, greater understanding of the underlying pathophysiology and novel resuscitation approaches are urgently needed to increase the rate of initial resuscitation and subsequent survival with intact organ function.
Animal models of cardiac arrest play a critical role in the development of new resuscitation therapies by providing insights on the pathophysiology of cardiac arrest and resuscitation and offering practical means to conceptualize and test new interventions before they can be tested in humans3. The rat model of closed chest cardiopulmonary resuscitation (CPR) described here has played an important role. The model was developed in 1988 by Irene von Planta – a research fellow at the time – and her collaborators4 in the laboratory of late Professor Max Harry Weil M.D., Ph.D. at the University of Health Sciences (renamed Rosalind Franklin University of Medicine and Science in 2004) and has been extensively used in the field of resuscitation predominantly by fellows of Professor Weil and their trainees.
The model simulates an episode of sudden cardiac arrest with resuscitation attempted by conventional CPR techniques and thus includes induction of ventricular fibrillation (VF) by delivering an electrical current to the right ventricular endocardium and provision of closed chest CPR by a pneumatically driven piston device while concomitantly delivering positive pressure ventilation with oxygen-enriched gas. Termination of VF is accomplished by transthoracic delivery of electrical shocks. The rat model strikes a balance between models developed in large animals (e.g., swine) and models developed in smaller animals (e.g., mice) allowing exploration of new research concepts in a well-standardized, reproducible, and efficient manner with access to a robust inventory of pertinent measurements. The model is particularly useful in early stages of research to explore new concepts and examine the effects of confounders before conducting studies in larger animal models that are more costly, but of greater translational impact.
A Medline search for all peer-reviewed articles reporting a similar rat model having VF as the mechanism of cardiac arrest and some form of closed chest resuscitation revealed a total of 69 additional original studies using the model since it was first published in 19884. The research areas include pathophysiological aspects of resuscitation5-17, factors influencing outcomes18-30, the role of pharmacological interventions examining vasopressor agents31-43, buffer agents44, inotropic agents45, agents aimed at myocardial or cerebral protection46-70, and also the effects of mesenchymal stem cells71-73.
The model and protocol described in this article is currently being used at the Resuscitation Institute. Yet, there are multiple opportunities to “customize” the model based on the capabilities available to individual investigators and the goals of the studies.
NOTE: The protocol was approved by the Institutional Animal Care and Use Committee at Rosalind Franklin University of Medicine and Science. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council.
1. Experimental Setup and Anesthesia
2. Vascular Cannulations
2.1) Left femoral artery for advancing the T-type thermocouple catheter into the descending thoracic aorta
2.2) Left femoral vein for advancing the PE25 catheter into the right atrium
2.3) Right femoral artery for advancing the PE25 catheter into the descending thoracic aorta
2.4) Right external jugular vein for advancing the 3F polyurethane pediatric venous catheter into the right atrium
3. Tracheal Intubation
3.1) Tracheal exposure
3.2) Tracheal intubation
4. Confirmation of Baseline Stability
5. Experimental Protocol
5.1) Induction of ventricular fibrillation (VF)
5.2) Chest compressions and positive pressure ventilation
NOTE: The chest compressor featured in this publication is a custom-made pneumatically driven and electronically controlled piston device. The ventilator is a commercially available device.
5.3) Defibrillation
5.4) Post-resuscitation
The rat model described here was recently used to compare the effects of two inhibitors of the sarcolemmal sodium-hydrogen exchanger isoform 1 (NHE-1) on myocardial and hemodynamic function during chest compression and post-resuscitation61. It was previously reported that NHE-1 inhibitors attenuate myocardial reperfusion injury by limiting sodium-induced cytosolic and mitochondrial calcium overload, and thereby help preserve left ventricular distensibility during chest compression and attenuate post-resuscitation myocardial dysfunction12. In this study, the NHE-1 inhibitor cariporide (1 mg/kg), which had been extensively investigated in the past, was compared with the newer compound AVE4454B (1 mg/kg) and vehicle control in three groups of 10 rats each, all subjected to 10 min of untreated VF followed by 8 min of chest compression before delivering electrical shocks. Either compound or vehicle control was randomized for administration into the right atrium immediately before starting chest compression with the investigators blind to the assignment. The effects of the NHE-1 inhibitors were analyzed individually and combined (i.e., against control). As shown in Figure 6, NHE-1 inhibition enabled attaining a predefined aortic diastolic pressure (between 26 mm Hg and 28 mm Hg) with less depth of compression consistent with preservation of left ventricular distensibility. When the coronary perfusion pressure was indexed to the depth of compression (CPP/Depth ratio) – an index of left ventricular distensibility – only rats treated with cariporide attained statistical significance. Post-resuscitation, both compounds ameliorated myocardial dysfunction and this effect was associated with greater survival as shown in Figure 7. It was concluded based on this study that cariporide is more effective than AVE4454B for resuscitation from cardiac arrest in this rat model.
Figure 1: Rat Instrumentation. Schematic rendition of the rat model of VF and closed-chest resuscitation illustrating the various instrumentations and devices used in the model to induce VF and perform cardiac resuscitation. AC = alternating current, ECG = electrocardiogram.
Figure 2: Representative Induction of Ventricular Fibrillation. Experiment depicting the ECG and the aortic pressure at baseline 6 min before inducing VF, at the start of the 60 Hz alternating current delivery to induce VF, and after turning the current off 3 min later. The current delivery typically masks the VF waveform superimposing a 60 Hz waveform, which is no longer seen after turning off the current, documenting sustained VF.
Figure 3: Defibrillation Protocol. Algorithm used to guide when to deliver electrical shocks and when to resume chest compression (CC) based on the electrical cardiac rhythm and the mean aortic pressure (MAP) level. VF = ventricular fibrillation, SHOCK = delivery of electrical shocks. The possible resuscitation outcomes include: (1) ROSC, return of spontaneous circulation defined as a MAP ≥40 mm Hg lasting >5 min; (2) ROCA, return of cardiac activity defined as an organized rhythm with an aortic pulse pressure ≥5 mm Hg but MAP <40 mm Hg; (3) refractory VF, defined as the persistency of VF upon completion of the 5th cycle; (4) PEA, pulseless electrical activity defined as an organized cardiac electrical activity with an aortic pulse pressure <5 mm Hg; and (5) asystole, defined as the absence of electrical and mechanical cardiac activity.
Figure 4: Representative Defibrillation Protocol. Experiment depicting the ECG, the aortic pressure, and the piston displacement (Depth) at the end of chest compression and one additional cycle. Shown are the effects of chest compression (CC) on the aortic pressure while the heart is in VF followed by a pause in chest compression to deliver the initial electrical shock. The shock terminated VF but resulted in weak cardiac activity unable to sustain a mean aortic pressure ≥25 mm Hg prompting resumption of chest compression, this time yielding a pulsatile mean aortic pressure >25 mm Hg which rapidly increased to >40 mm Hg consistent with return of spontaneous circulation (ROSC).
Figure 5: Experimental Timeline. Timeline of a typical acute rat experiment showing interventions and measurements. Ao = aortic, BG = blood gases, Co-Ox = co-oximetry, ECG = electrocardiogram, FiO2 = fraction of inspired oxygen, Lac = lactate, RA = right atrium.
Figure 6: Effect of NHE-1 Inhibitors on CPR Efficiency. The depth of chest compression (Depth) and the ratio between coronary perfusion pressure and depth of compression (CPP/Depth) comparing the control solution (C) with AVE4454B (AVE) and cariporide (CRP) before chest compression. NHEI = AVE and CRP groups combined. Line graphs depict Depth and CPP/Depth throughout chest compression comparing NHEI (o) with controls (●). Numbers in brackets denote rats remaining in ventricular fibrillation. The bar graphs depict the same variables at the last min of chest compression. Values are means ± SEM. †p <0.01, ‡p <0.001 vs control by Student’s t-test; p <0.01, p <0.001 vs control by one-way ANOVA using Holm-Sidak’s test for multiple comparisons; p <0.05 vs control by one-way ANOVA using Dunn’s test for multiple comparisons (This figure has been modified from Radhakrishnan et al.61).
Figure 7: Effect of NHE-1 Inhibitors on Survival. Kaplan-Meier curves in rats that received cariporide (CRP), AVE4454B (AVE), or vehicle control solution. Shown on the left are survival curves for all rats and on the right only those that had return of spontaneous circulation (ROSC). Upper graphs depict survival for the individual interventions and bottom graphs survival for the AVE and CRP groups combined (NHEI). p <0.01 vs control by Gehan-Breslow analysis using Holm-Sidak’s test for multiple comparisons; †p = 0.01 vs control by Gehan-Breslow analysis (This figure has been modified from Radhakrishnan et al.61).
Variables | Baseline | Post-Resuscitation | ||
-5 min | 60 min | 120 min | 180 min | |
Temperature (°C) | 36.9 ± 0.3 [12] | 36.9 ± 0.4 [6] | 36.7 ± 0.3 [6] | 37.0 ± 0.6 [5] |
HR (min-1) | 379 ± 30 | 334 ± 27 | 346 ± 21 | 370 ± 35 |
Cardiac Output (ml/min) | 87 ± 13 | 48 ± 11 | 33 ± 11 | 30 ± 10 |
Cardiac Index (ml/kg∙ min-1) | 175 ± 28 | 93 ± 22 | 65 ± 20 | 58 ± 19 |
Ao Sysolic Pressure (mmHg) | 162 ± 15 | 108 ± 19 | 107 ± 24 | 102 ± 20 |
Ao Diastolic Pressure (mmHg) | 130 ± 13 | 84 ± 13 | 86 ± 21 | 82 ± 16 |
Ao Mean Pressure (mmHg) | 141 ± 13 | 92 ± 15 | 93 ± 22 | 89 ± 17 |
RA Mean Pressure (mmHg) | 0 ± 1 | 2 ± 1 | 2 ± 2 | 1 ± 2 |
End-tidal CO2 (mmHg) | 37 ± 10 | 34 ± 14 | 24 ± 16 | 24 ± 17 |
pH, Aorta (unit) | 7.40 ± 0.04 | 7.28 ± 0.11 | 7.36 ± 0.10 | 7.34 ± 0.08 |
Lactate, Aorta (mmol/L) | 0.56 ± 0.32 | 5.68 ± 2.64 | 3.24 ± 1.63 | 3.38 ± 2.15 |
PO2, Aorta (mmHg) | 84 ± 8 | 178 ± 18 | 206 ± 9 | 206 ± 25 |
PCO2, Aorta (mmHg) | 40 ± 6 | 30 ± 11 | 29 ± 9 | 24 ± 10 |
Table 1: Representative Hemodynamic and Metabolic Values. Baseline values were obtained in 12 male retired breeder Sprague-Dawley rats after completion of surgical instrumentation and before induction of ventricular fibrillation. Subsequent values were obtained at 60, 120, and 180 minutes post-resuscitation. Numbers in brackets denote rats that remained alive in the post-resuscitation interval. Data are shown as mean ± SD. Ao = aortic, HR = heart rate, RA = right atrial.
Critical Steps in the protocol
There are critical steps in the protocol. When mastered, the preparation and protocol proceed as succinctly described below. The surgical preparation is expeditious, advancing catheters rapidly through small incisions triggering minimal or no vessel spasm and positioning the catheter tips as intended, followed by successful tracheal intubation after a single or a few attempt(s); thus, completing the preparation in ≈ 90 min from the initial pentobarbital dose to induction of VF with baseline measurements within reference values (Table 1). VF is electrically induced in every instance leading to spontaneously sustained VF after 3 min of uninterrupted electrical stimulation in >95% of the instances. During chest compression, an aortic diastolic pressure ≥24 mm Hg and end-tidal CO2 ≥10 mm Hg is generated without exceeding a compression depth of 17 mm depth and without injuring intrathoracic organs. Implementation of a defibrillation protocol (e.g., as shown in Figure 3) occurs with ease and with <5 sec interruptions in chest compression. Finally, return of spontaneous circulation occurs in >60% of the experiments using the present protocol or similar ones leading to post-resuscitation myocardial dysfunction with a 240 min survival >40% and metabolic abnormalities indicative of the systemic oxygen deficit that occurs during cardiac arrest and reverses in the post-resuscitation phase in survivors, as shown on Table 1.
Modifications and troubleshooting
The model is highly versatile, allowing for relatively simple adaptations to meet specific research objectives. Recently, use of PE25 size tubing was preferred over PE50 size tubing, which has been used in the past by other investigators, and found it easier to advance into proper position without compromising the fidelity of the pressure measurements. The left ventricle can been catheterized from a carotid artery to assess left ventricular function34,61 or to inject microspheres for measuring regional organ blood flow6,55. The trachea may be cannulated directly via tracheostomy instead of the oral – more challenging – technique featured in this article, especially in acute experiments without recovery from anesthesia. Other approaches to induce VF have been described including transcutaneous electrical epicardium stimulation74, current delivery to the entrance of the superior vena cava into the heart75, and electrical stimulation of the esophagus using a pacing electrode76. The method of chest compression may be varied by starting compression at the maximal depth, using lateral restraints, compressing at other rates and duty cycles, and also by using manual technique instead of a piston device. Ventilation can also be varied; the original description used a ventilatory rate of 100 min-1 synchronized 1:2 to compressions whereas the present model uses a ventilatory rate of 25 min-1 unsynchronized to compressions; consistent with the reduced ventilatory demands of CPR77 and current clinical recommendations against pausing for compressions after having established a secured airway. Ventilation can also be passive and promoted by chest compression provided the airway is patent20 or obviated while administering oxygen directly into the trachea25. If an experiment requires removal of large amounts of blood relative to the animal’s blood volume [BV(ml) = 0.06 x body weight (g) + 0.77]78; e.g., for blood collection for determining organ blood flow with microspheres6,55 or for repetitive measurement of blood analytes, blood may be transfused from a donor rat from the same colony6,55. Current analytical techniques, however, allow determination of multiple analytes in small samples and administration of equivalent amounts of normal saline or another accepted intravascular solution compensates for small blood losses. The model can also be used to study asphyxia as the mechanism of arrest9, which is typically accomplished by inducing neuromuscular blockade and occluding the airway.
Limitations of the technique
The model lacks underlying coronary artery disease and it is technically difficult to acutely induce coronary artery occlusion; conditions most commonly associated with sudden cardiac arrest in humans. The need to maintain the current to induce VF is not ideal and raises concerns of potential injury to the myocardium. Indeed minor thermal injury at the site of current delivery was recognized in the original study, and noted that it could be minimized by reducing the current to the minimum requirement during the 3 min interval needed to induced self-sustained VF4. In addition, the electrical current unintendedly triggers skeletal muscle contraction, which could contribute to lactic acid production. The calcium cycling physiology of the rat heart compared to other mammals is less dependent on the sodium-calcium exchanger79, and interpretation of related therapies should consider this aspect of the rat cardiac physiology. The rate of compression and ventilation exceeds that used in humans precluding direct extrapolation of related findings. The effects of anesthesia80 including cell protective effects81 should be considered when interpreting findings, although it is not clear that pentobarbital obfuscate findings compared to inhalant anesthetics which have cardioprotective effects81. Most of the studies reported in the literature have been conducted in male rats intended to minimize possible experimental confounders stemming from different timing within the estrous cycle. Further work is required to assess the effects of gender on resuscitation physiology and outcomes. Another important limitation is the reduced availability of genetically engineered rats relative to mice having to resort to customized genetic engineering or targeted gene manipulation of adult animals through introduction of genetic material (e.g., viral vectors and antisense oligonucleotides).
Significance of the technique with respect to existing/alternative methods
The model is best suited to explore new concepts, new interventions, and to challenge existing paradigms as part of a larger translational strategy that eventually includes focused studies in larger animal models, such as swine, before human trials. Studies in smaller animals (e.g., mice) are complicated by difficulties in inducing VF, limited surgical instrumentation, and the small blood volume that precludes repetitive blood analysis.
Future applications or directions after mastering this technique
The rat model was originally developed to simulate various aspects of human CPR after sudden cardiac arrest. As highlighted in the introduction, the model has been used by investigators to address several aspects of cardiac resuscitation, including its physiology, conventional determinant of outcomes, and mostly the effects of established and novel therapeutic interventions as referenced in this article. The Resuscitation Institute expects the reader to be inspired and use the model to address the many questions in resuscitation research that need further exploration given the disappointing outcomes with current resuscitation methods.
The authors have nothing to disclose.
The authors would like to acknowledge Dr. Wanchun Tang MD, MCCM, FCCP, FAHA and Jena Cahoon of the Weil Institute of Critical Care Medicine in Rancho Mirage, CA. for their contributions to the resuscitation protocol outline and for having helped train the rodent surgeon (LL). The preparation of this article was in part supported by a gift in memory of US Navy Retired SKC Robert W. Ply by Ms. Monica Ply for research in heart disease and Parkinson’s disease and by a discretionary fund from the Department of Medicine at Rosalind Franklin University of Medicine and Science.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Sodium pentobarbital | Sigma Aldrich | P3761 | http://www.sigmaaldrich.com/catalog/product/sigma/p3761?lang=en®ion=US |
Rectal thermistor | BIOPAC Systems, INC | TSD202A | http://www.biopac.com/fast-response-thermistor |
Needle electrode biopolar concentric 25 mm TP | BIOPAC Systems, INC | EL451 | http://www.biopac.com/needle-electrode-concentric-25mm |
PE25 polyethylene tubing | Solomon Scientific | BPE-T25 | http://www.solsci.com/products/polyethylene-pe-tubing |
26GA female luer stub adapter | Access Technologies | LSA-26 | http://www.norfolkaccess.com/needles.html |
Stopcocks with luer connections; 3-way; male lock, non-sterile | Cole-Parmer | UX-30600-02 | http://www.coleparmer.com/Product/Large_bore_3_way _male_lock_stopcocks _10_pack_Non_sterile/EW-30600-23 |
TruWave disposable pressure transducer | Edwards Lifesciences | PX600I | http://www.edwards.com/products/pressuremonitoring/Pages/truwavemodels.aspx?truwave=1 |
Type-T thermocouple | Physitemp Instruments | IT-18 | http://www.physitemp.com/products/probesandwire/flexprobes.html |
Central venous pediatric catheter | Cook Medical | C-PUM-301J | https://www.cookmedical.com/product/-/catalog/display?ds=cc_pum1lp_webds |
Abbocath-T subclavian I.V. catheter (14g x 5 1/2") | Hospira | 453527 | http://www.hospira.com/products_and_services/iv_sets/045350427 |
Novametrix Medical Systems, Infrared CO2 monitor | Soma Technology, Inc. | 7100 CO2SMO | http://www.somatechnology.com/MedicalProducts/novametrix_respironics_co2smo_ 7100.asp |
Harvard Model 683 small animal ventilator | Harvard Apparatus | 555282 | http://www.harvardapparatus.com/webapp/wcs/stores/servlet/haisku2_10001_11051_44453_-1_ HAI_ProductDetail_N_37322_37323 |
Double-flexible tipped wire guides | Cook Medical | C-DOC-15-40-0-2 | https://www.cookmedical.com/product/-/catalog/display?ds=cc_doc_webds |
High accuracy AC LVDT displacement sensor | Omega Engineering | LD320-25 | http://www.omega.com/pptst/LD320.html |
HeartStart XL defibrillator/monitor | Phillips Medical Systems | M4735A | http://www.healthcare.philips.com/main/products/resuscitation/products/xl/ |
Graefe micro dissection forceps 4 inches | Roboz | RS-5135 | http://shopping.roboz.com/Surgical-Instrument-Online-Shopping?search=RS-5135 |
Graefe micro dissection forceps 4 inches with teeth | Roboz | RS-5157 | http://shopping.roboz.com/Surgical-Instrument-Online-Shopping?search=RS-5157 |
Extra fine micro dissection scissors 4 inches | Roboz | RS-5882 | http://shopping.roboz.com/micro-scissors-micro-forceps-groups/micro-dissecting-scissors/Micro-Dissecting-Scissors-4-Straight-Sharp-Sharp |
Heiss tissue retractor | Fine Science Tools | 17011-10 | http://www.finescience.com/Special-Pages/Products.aspx?ProductId=321&CategoryId=134& lang=en-US |
Crile curve tip hemostats | Fine Science Tools | 13005-14 | http://www.finescience.com/Special-Pages/Products.aspx?ProductId=372 |
Visistat skin stapler | Teleflex Incorporated | 528135 | http://www.teleflexsurgicalcatalog.com/weck/products/9936 |
Braided silk suture, 3-0 | Harvard Apparatus | 517706 | http://www.harvardapparatus.com/webapp/wcs/stores/servlet/haisku2_10001_11051_43051_-1_ HAI_ProductDetail_N_37916_37936 |
Betadine solution | Butler Schein | 3660 | https://www.henryscheinvet.com/ |
Sterile saline, 250 ml bags | Fisher | 50-700-069 | http://www.fishersci.com/ecomm/servlet/itemdetail?catnum=50700069&storeId=10652 |
Heparin sodium injection, USP | Fresenius Kabi | 504201 | http://fkusa-products-catalog.com/files/assets/basic-html/page25.html |
Loxicom (meloxicam) | Butler Schein | 045-321 | https://www.henryscheinvet.com/ |
Thermodilution cardiac output computer for small animals | N/A | N/A | Custom-developed at the Resuscitation Institute using National Instruments hardware and LabVIEW software |
Analog-to-digital data acquisition and analysis system | N/A | N/A | Custom-developed at the Resuscitation Institute using National Instruments hardware and LabVIEW software |
Pneumatically-driven and electronically controlled piston device for chest compression in small animals | N/A | N/A | Custom-developed at the Weil Institute of Critical Care Medicine |
60 Hz alternating current generator | N/A | N/A | Custom-developed at the Weil Institute of Critical Care Medicine |