Aby wyświetlić tę treść, wymagana jest subskrypcja JoVE. Zaloguj się lub rozpocznij bezpłatny okres próbny.
Method Article
The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.
The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.
Myocardial stunning is defined as reversible reduced contractile activity despite restoration of adequate blood flow following a brief period of ischemia or prolonged periods of ischemic insults with cardioprotection1,2,3,4,5. The method presented is specifically used to model clinically relevant ischemic insults which can result in reversible impairments in contractile function (i.e., ischemic insults associated with cardiac surgery utilizing cardioplegic arrest, brief periods of ischemia, angina, etc.). In contrast to severe ischemia studies (myocardial infarction, necrosis) this protocol was developed to evaluate myocardial functional recovery and cardioprotection without tissue injury, remodeling, and cell death. The majority of the paper discusses a standard cardioplegic arrest protocol with elements similar to a cardiac surgery using hypothermia and intermittent cardioplegia delivery.
Myocardial protection during the majority of cardiac surgeries relies on cardioplegia and cardiopulmonary bypass. Although cardioplegia (CP) solutions and strategies vary widely (blood, crystalloid, cold, warm etc.) the most common elements are 1) hyperkalemia and/or other strategies to arrest the heart in diastole, thereby limiting energy utilization resulting from myocardial contraction, and 2) hypothermia to slow metabolism and help maintain ATP and other energy reserves while arrested. Current cardioplegia solutions provide protection to the heart against ischemic insults that would otherwise prove lethal. However, cardioprotective strategies during surgical ischemic insults are not perfect, and the resultant mild ischemic injury can result in reversible cardiac contractile dysfunction despite adequate blood flow (myocardial stunning), acidosis, cardiomyocyte damage, and vascular effects including reduced coronary perfusion and vasospasm.
This protocol differs from standard isolated heart ischemia models evaluating myocardial infarction and severe ischemia in that it evaluates milder ischemic insults which can result in impaired cardiac function following brief ischemia or ischemic insults associated with cardioplegic arrest. (for review on Langendorff perfusion techniques and I/R studies see6–8). For general guidelines and a thorough analysis of experimental parameters associated with mouse isolated perfused hearts see Sutherland et al. 20039 The technique presented here details the necessary equipment, reagents, steps, strategies and tips to reliably induce stunning in mouse hearts. Minor modifications are necessary to apply the technique to rats.
Briefly isolated mouse hearts are Langendorff perfused for approximately 30 min with physiologic Krebs-Henseleit buffer (KHB), followed by cold protected cardiac arrest via delivery of a hyperkalemic hypothermic cardioplegia solution. Following arrest, cardiac functional recovery is monitored during rewarming and reperfusion of the heart with KHB. Changes in the degree of recovery of cardiac contractile function can be evaluated to assess cardioprotective agents and different cardioprotection strategies.
NOTE: All Procedures were approved by the Lifespan Institutional Animal Care and Use Committee and all animals and procedures according to the National research Council Guide for Care and Use of Laboratory Animals10.
1. Balloon Fabrication and Left Ventricular Pressure Monitoring Circuit
2. Preparation of the Isolated Heart Perfusion System
3. Mouse Surgery
3.1) Mouse Anesthesia and Handling
3.2) Remove the Heart
3.3) Clean the Heart
4. Mounting the Heart, Starting Perfusion, and Placing the Balloon
4.1) Mounting and Cannulating the Heart
4.2) Establishing Perfusion and LVP Measurement
4.3) Basal Measurement
5. Initiating Cardioplegic Arrest
6. Reperfusion
7. Collecting the Tissue
Figure 1 presents typical results from one mouse experiment. LVP (red line), dP/dt (green line), and temperature (purple line) were continusouly recorded over ~3 hr. Letters indicate a - baseline measurement, b, c, d, e - delivery of cardioplegia solution, f - start of reperfusion, g, h, i, j - measurement of cornary flow during reperfusion. Note, depressed LVDP and dP/dt upon reperfusion compared to baseline. Figure 1B includes the data from A recorded over ~2 sec. Note, the decrease in...
The preceding protocol details methods to evaluate myocardial stunning secondary to global ischemia associated with cardioplegic arrest. In our hands this protocol produces an approximate ~40% reduction in cardiac function (LVDP, +/- dP/dt) with minimal changes in heart rate at the 30 min post-reperfusion time point. As the heart is reperfused and rewarmed all parameters of cardiac function are reduced at initial time points with a greatly reduced heart rate before stabilizing between 20 and 30 min. Coronary flow is gene...
The authors have nothing to disclose.
Name | Company | Catalog Number | Comments |
Cardioplegia Solution (St Thomas II) | Symbol / Concentrations (mM) | ||
Sodium Chloride | NaCl; 110 | ||
Potassium chloride | KCl; 16 | ||
Calcium Chloride | CaCL2; 1.5 | ||
Magnesium Chloride | MgCL2; 16 | ||
Sodium Bicarbonate | NaHCO3; 10 | ||
Krebs-Heinslet Buffer | |||
Sodium Chloride | NaCl; 118 | ||
Potassium Chloride | KCl; 4.8 | ||
Magnesium Sulfate | MgSO4; 1.7 | ||
Sodium Bicarbonate | NaHCO3; 24.9 | ||
Potassium Phosphate (monobasic) | KH2PO4; 1.2 | ||
Calcium Chloride | CaCL2; 1.4 | ||
Sodium Pyruvate | Na pyruvate; 2 | ||
Glucose | C6H12O6; 6 | ||
Balloon reagents | |||
Corn Syrup | |||
Spaghetti | |||
Silicon Dispersion Gel | |||
styrofoam block | |||
lab oven/incubator ( 50C) | |||
Langendorff Perfusion equipment | |||
Isolated perfused heart sytem (IH-SR (Hugo-Sachs) or equivalent) | |||
Data acquisition system (DSI, ADinstruments or equivalent) | |||
Heated water circulator | |||
Cooling water circulator | |||
Perfusion pump capable of 2-30 ml/min | |||
Inline perfusion filters - 1 um glass fiber | |||
Pressure sensors and amplifiers for LVP and perfusion pressure | |||
Small graduated cylinder (~10 mL) | |||
Small temperature probe and thermometer (Werner or equivalent) | |||
perfusion resevoir (1L) | |||
cardioplegia resevoir (~200 mL) | |||
gas bubbler | |||
95/5 O2/CO2 mix | |||
Surgical tools and reagents | |||
Metzenbaum and Potz surgical scissors | |||
two Dumont size 5 forceps | |||
ketamine | |||
xylazine | |||
heparin | |||
small clamp with soft sides to hold aorta (i.e. terminal clamp with taped ends) | |||
Silk 2-0 and 4-0 sutures |
Zapytaj o uprawnienia na użycie tekstu lub obrazów z tego artykułu JoVE
Zapytaj o uprawnieniaThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. Wszelkie prawa zastrzeżone