Radnoti Working Heart - Langendorff Application Notes

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Radnoti Working Heart - Langendorff Application Notes

  1. 1. Radnoti Glass Technology The Radnoti Working Heart System Isolated Perfused Heart Presented by: Radnoti LLCwww.radnoti.com 800-428-1416 2010
  2. 2. Radnoti Glass Technology Introduction Isolated Perfused Heart The Isolated Perfused Heart System, as originated by Oscar Langendorff more than a century ago, has become a predominant technique in pharmacological and physiological research. The technique allows the examination of cardiac contractile strength (inotropic effects), heart rate (chronotropic effects) and vascular effects without the complications of an intact animal model. From its simple beginning the technique and equipment has evolved to include both constant pressure and constant flow models in a working heart mode as well as both recirculating and non recirculating modes. The Radnoti Isolated Perfused Heart System has the capacity to function in any of these configurations allowing flexibility in experimental research and design. This presentations intention is a quick reference towards the use of the Radnoti Isolated Working Heart System.www.radnoti.com 800-428-1416
  3. 3. Radnoti Glass Technology Typical Values Isolated Perfused Heart These values are obtained from a variety of sources and are displayed to demonstrate the approximate ranges of these values. Values are for adult animals. In vivo heart rate and blood pressure are taken at rest. Cation values are from serum. Left ventricular volume (LVV) is given for a balloon inserted into the left ventricle. CF (coronary flow) is given for a saline solution at 50-60 mmHg Rate Bpm BP mm/Hg Na,mM K,mM Ca,mM Mg,mM LVVml heart Heart Cat 110-140 125/70 163 4.4 1.3 0.7 0.7-2.4 2-3 Rat 330-360 129/91 140 5.7 2.6 1.1 0.1-0.2 8-10 Guinea Pig 280-300 120/170 145 7.4 2.6 1.2 0.1-0.2 5-8 R.pipens 37-60 31/21 Carp 40-78 43www.radnoti.com Rabbit 205-220 110/73 155 4.6 3.5 1.6 0.4-0.7 2-5 800-428-1416
  4. 4. Radnoti Glass Technology Ready, Set, Go Isolated Perfused Heart Once the experimental protocol has been decided and the instrumentation is selected and placed on the system, the system can be primed and readied for the first experimental set up.www.radnoti.com 800-428-1416
  5. 5. Radnoti Glass Technology Pumps Overview Isolated Perfused Heart Perfusate or Buffer Delivery: PERISTALTIC PUMP PERISTALTIC: The Peristaltic Pump is used to transport the buffer solution from the reservoir through the system and to the heart. It is important that the buffer delivery pump offer a range of flow well within the flow demand of the system. The pump should be operating at a mid range of its speed capability to insure a long pump life. The Peristaltic Pump provided with the Radnoti 120101BEZ Isolated Working Heart System is the 170100A Peristaltic Pump with Two 170110 easy load pump heads: Water Jacket Temperature Control: THERMAL CIRCULATING PUMP THERMAL CIRCULATOR: The Thermal Circulator is used to warm and maintain temperature of the system by warming the water and circulating throughout the water jacket of the system. The thermal circulator must have sufficient pump strength to move the water through the system and overcome the hydrostatic pressure head created by the elevated components of the system. In addition, the tank volume must be of sufficient size to minimize the effect of the returning fluids’ temperature variation. The combination of these two features will insure an accurate and stable temperature control throughout the system. The thermal circulator provided with the Radnoti 120101BEZ Isolated Working Heart system is the 170051A Thermal Circulating Water Bathwww.radnoti.com 800-428-1416
  6. 6. Radnoti Glass Technology Water Jacket Diagram Isolated Perfused Heart When connecting the Water Jacketed tubing it is very important to have the flow of the water go from the bottom of the component to the top. Make sure that you remove all air bubbles from the components.www.radnoti.com 800-428-1416
  7. 7. Radnoti Glass Technology Priming the System 2mm dia. air bubble Isolated Perfused Heart – close stopcock – control with syringe When bubble trap is 2/3 full close vent stopcock. When bubble trap is 2/3 full – close vent stopcock. Redirect aortic stop cock to prime compliance loopwww.radnoti.com Start Pump Buffer can be sent to waste 800-428-1416 or returned to the buffer Fill Buffer Reservoir reservoir.
  8. 8. Radnoti Glass Technology Anesthesia and Cardiac Removal Isolated Perfused Heart Preparation of the Donor: The anesthetic(s) used will depend on: the donor, potential problems with side effects in the experimental protocol, the extent of the surgical procedures and the regulations of your Animal Care and Use Committee. The most common are barbiturates, such as nembutal or thiopental, ketamine/xylazine, ethyl ether and common volatile surgical anesthetics. The latter two can present potential personnel hazards due to fire or intoxication. Carbon dioxide and euthanasia solutions should not be used due to the danger of an anesthetic overdose causing severe or prolonged cardiac impairment or hypoxia. Unless there is an overriding experimental concern, the donor should be heparinized prior to surgery to reduce the formation of emboli in the vasculature.www.radnoti.com 800-428-1416
  9. 9. Radnoti Glass Technology Excision of the Heart Isolated Perfused Heart Cardiac removal may be performed by a simple medical incision (median sternotomy) with the blunt end of a pair of blunt-sharp pointed scissors to open the thoracic cavity. This is followed by exposure of the heart, opening of the pericardium, support of the organ, and removal of the heart by cutting across the arch of the aorta and the vena cava. Care should be taken not to cut the aorta so short as to impair mounting on the cannula. The heart may be placed in a beaker of chilled, heparinized perfusate to arrest the beating of the heart.www.radnoti.com 800-428-1416
  10. 10. Radnoti Glass Technology Working Heart Mode Isolated Perfused Heart Fluid is delivered in retrograde NOTE: Before any working heart experiment can be initiated the heart must be stabilized in Langendorff mode. Aortic valve forced shut The Langendorff heart preparation involves the cannulation of the aorta. This buffer solution is then delivered retrograde via the aorta either at a constant flow rate (delivered by the peristaltic pump) or a constant hydrostatic pressure (usually in the range of 60-100mmHg). In both instances the aortic valves are forced shut and the perfusion fluid is directed into the coronary ostia thus perfusing the entire ventricular mass of the heart and draining into the right atrium via the coronary sinus. Perfusion fluid is directed into the coronary ostiawww.radnoti.com 800-428-1416 Perfused ventricular mass
  11. 11. Radnoti Glass Technology Cannulation and Stabilization Isolated Perfused Heart Langendorff (Retrograde) The heart is removed from the beaker of chilled, Heparinized perfusate is used to arrest the beating of the heart and the organ then mounted on the aortic cannula as perfusate is flowing from the cannula. Some researchers prefer to use two pair of tweezers to position the aorta onto the cannula. Care must be taken to avoid puncturing the aorta. The heart can be held on Correct the cannula with a blood vessel clamp such as Dieffenbach serafine while tying the heart onto the aortic cannula with sutures. In the case of the flanged cannula, the aorta is slid down the cannula so that the tie is against the flange. The most critical part of the preparation is the delay in time from the removal of perfusion in the donor, to the Left Coronary reperfusion of the heart. Since this normally highly metabolically active organ has only the oxygen and substrate contained in the vessels at the time of removal to sustain itself, this Ostia time should be kept to a minimum (<30-60 seconds). Incorrectwww.radnoti.com 800-428-1416 Aortic Valve
  12. 12. Radnoti Glass Technology Perfusion of the Heart Isolated Perfused Heart Once mounted on the cannula and reperfusion has begun, the heart should begin to beat strongly within seconds. If a constant flow system is used, should be carefully monitored to avoid under perfusion or over perfusion. Perfusion rates are about 3-15ml/g heart weight for constant flow systems using Tyrodes, etc., and for both constant pressure and constant flow systems the initial pressure should be about 50-60mm Hg for most mammalian hearts, dependent on the donor, heart rate (pacing), oxygen delivery and work output. Physiologically normal perfusion pressures of 80-100mm Hg as in blood-perfused hearts are not used in saline-perfused hearts due to enhanced edema and potential valve damage. The heart will stabilize rapidly and most experiments can begin within 10-15 minutes after the preparation has been mounted and the various monitoring systems attached. The heart should be functional for several hours, although it is prudent to reduce the experimental time as much as possible. Preparations will suffer edema if uncompensated by a plasma expander concomitant with protein loss from the heart.www.radnoti.com 800-428-1416
  13. 13. Radnoti Glass Technology Working Heart Preparation Isolated Perfused Heart The Working heart preparation: When changing to Working Heart mode, the heart is perfused through the left atria. Perfusate enters the left ventricle and is ejected Left Coronary Ostia out of the aorta. The afterload hydrostatic pressure forces a portion of Mitral Valve the perfusate into the coronary bed via the coronary ostia with the coronary perfusate collecting into the right atria from the Thebesian veins. Click on Heart to Animate Left Atrium Aorta Aortic Valve Left Ventriclewww.radnoti.com 800-428-1416 Coronary Bed
  14. 14. Radnoti Glass Technology Working Heart in Detail Isolated Perfused Heart There is just one additional step following the establishment of perfusion (without the insertion of an LVP balloon). Which is cannulation and secure tying off (without any leaks) of the left atrium via one of the orifices of the pulmonary veins. The dimensions and relative positions of the aortic and atrial cannulae are critical to a successful preparation. The Radnoti Heart Chamber will allow you to adjust the distance between cannula by rotating the cams on the heart chamber lid. Vertical position may be adjusted by the loosening of the locking cap on the cannula, setting the position, and then re-securing.www.radnoti.com 800-428-1416
  15. 15. Radnoti Glass Technology Working Heart in Detail cont. Isolated Perfused Heart Once aortic and left atrial cannulation are accomplished, the aortic cannula stopcock is switched over and perfusion initiated by the left atrium whilst simultaneously opening up the aortic outflow line . In this way, oxygenated perfusion fluid from a constant pressure head left atrial perfusion reservoir (which is continuously filled by a roller pump from the Radnoti Buffer Reservoir flows under gravity into the left atrial cannula. The preload of the preparation is determined by the height of the overflow from the atrial perfusion bubble trap above the heart. This is usually set around 7cm for isolated rat hearts but can be varied to suit other preparations or to allow construction of "Starling Curves" relating preload to cardiac function.www.radnoti.com 800-428-1416
  16. 16. Radnoti Glass Technology Working Heart in Detail cont. Isolated Perfused Heart It is important to stress that, in vivo, cardiac output is equal to the venous return from the lungs to the left atrium. In the isolated Working Heart the venous return is represented by the flow from the left atrial cannula. An important point, in using a working heart apparatus is that the left atrial perfusion line must be capable of delivering perfusion fluid at a rate sufficient to support the maximum cardiac output of a working heart at any particular preload. If the left atrial cannula is too small it will artificially limit the cardiac output of the preparation. The problem is compounded by the pulsatile nature of atrial filling. This means that the atria only fill during about half of the cardiac cycle. To ensure that this problem does not arise and that left ventricular filling is not limited by inadequate left atrial inflow it is essential to check that the left atrial perfusion line can deliver a flow rate of at least twice the expected maximal cardiac output. This is easily checked by running the apparatus without a heart attached and measuring the flow from the left atria line. A rate of at least 150ml/min is recommended for a 1g heart. Having flowed from the left atrial cannula into the left atrium, the perfusion fluid is ejected via the mitral valve into the left ventricle from where it is ejected through the aortic cannula against a hydrostatic pressure set via the compliance loop. The after load is determined by the height of the compliance reservoir above the aortic cannula. The compliance bubble trap contains a 2mm diameter air bubble and mimics normal vascular elasticity. It is an essential component of the perfusion circuit greatly increasing the chances of successful working heart function. In the course of left ventricular ejection, a portion of the perfusion fluid is forced into the coronary ostia and thereby perfuses the coronary vessels of the heart.www.radnoti.com 800-428-1416
  17. 17. Radnoti Glass Technology Working Heart in Detail cont. Isolated Perfused Heart The coronary effluent exits from the right heart into the heart chamber where it may be sampled for assay or returned (via a roller pump) to the supply reservoir for reoxygenation. Depending on the species and experimental design, filling pressures are usually in the range of 7cmH2O and after loads in the range 60-100 cmH2O or more. Under these conditions and using the heart from a 250g rat, coronary flows of up to 25 ml/minute and aortic flows of 50-80 ml/minute can be expected. These can be measured by timed collection into graduated cylinders or by optional flow meters. Summation of coronary and aortic flow gives the cardiac output. Hearts may be paced or allowed to beat spontaneously under which circumstances heart rate may be derived from a pressure recording which is usually via a side arm of the aortic cannula. Because of the large volumes of perfusion fluid pumped by the heart, the working preparation usually operates in the recirculating mode and for this reason it is essential to have an in-line filter (1µm porosity) in the circuit to remove any particulate contaminants which may originate from the heart, connecting tubing, glassware or perfusion solutions.www.radnoti.com 800-428-1416
  18. 18. Radnoti Glass Technology Switching the system to Working Heart Mode Isolated Perfused Heart The Isolated Perfused Heart System, as originated by Oscar Recirculate Langendorff more than a century ago, has become a mainstay of pharmacological and physiological research. The system allows the examination of cardiac inotropic, Or waste chronotropic and vascular effects without the complications of an intact animal model. The original design has evolved to encompass both constant pressure and constant flow models in both recirculating and non-recirculating modes, as well as "working heart“.. The Radnoti Model 120101BEZ Perfused Heart System has the capacity to function in any of these configurations, allowing the researcher to have maximal flexibility in experimental design. This capability is enhanced through the use of solid state monitoring and recording technology Switch Aortic and Atrial combined with precision fabrication resulting in a stopcocks convenient, easy to use and easy to maintain package.www.radnoti.com 800-428-1416
  19. 19. Radnoti Glass Technology After Load Pressure Isolated Perfused Heart Having flowed from the left atrial cannula into the left atrium, the perfusion fluid is ejected via the mitral valve into the left ventricle from where it is ejected through the aortic cannula against a after load hydrostatic pressure via the compliance bubble trap chamber. The afterload is determined by the height of the compliance loop reservoir the heart. In the compliance loop, the bubble trap compliance chamber is pre-filled with perfusate to the point where it contains approximately a 2mm diameter bubble of air for the working heart. The trapped air bubble mimics normal vascular elasticity. The air bubble may be adjusted by placing a syringe at the top vent outlet of the bubble trap compliance chamber. Once the size has been set, close the stopcock to trap the bubble. Keeping the compliance air bubble relatively close to the heart is an essential component of the perfusion circuit and greatly improves successful long term function of the heart.www.radnoti.com 800-428-1416
  20. 20. Radnoti Glass Technology After Load Pressure cont. Isolated Perfused Heart In the course of left ventricular ejection, a portion of the perfusion fluid is forced into the coronary ostia and thereby perfuses the coronary vessels of the heart. The coronary effluent exits from the right heart into the heart chamber Left Coronary Ostia from whence it may be sampled for assay or returned (via a roller pump) to the buffer reservoir for reoxygenation. Mitral Valve Depending on the species and experimental design, filling pressures are usually in the range of 7 cmH2O and after loads in the range 60-100 cmH2O or more. Under these conditions and using the heart from a 250g rat, coronary flows of up to 25 ml/minute and aortic flows of 50-80 ml/minute can be expected. These can be measured by timed collection into measuring cylinders or by in-line float or Aorta electromagnetic flow meters. Summation of coronary and aortic flow gives the cardiac output. Aortic Valve Left Ventriclewww.radnoti.com 800-428-1416 Coronary Bed
  21. 21. Radnoti Glass Technology Recommended Reading Isolated Perfused Heart If the experimenter is not conversant with cardiovascular pharmacology and physiology there are a number of excellent texts for familiarization. Besides the medical physiology and pharmacology standards, there are a number of specialized texts. Pharmacologic Analysis of Drug-receptor Interaction by Terrence P. Kenakin (Raven Press, NY) is compact with practical emphasis on isolated tissues and organs in pharmacological research.www.radnoti.com 800-428-1416
  22. 22. Radnoti Glass Technology Disclaimer Isolated Perfused Heart These procedures and devices are intended for research and experimentation. All statements, technical information and recommendations herein are based on tests and sources we believe to be reliable, but the accuracy or completeness thereof is not guaranteed. Before using, user shall determine the suitability of the product for its intended use, and user assumes all risk and liability whatsoever in connection therewith. Neither seller nor manufacturer shall be liable in tort or in contract for any loss or damage, direct, incidental, or consequential arising out of the use or the inability to use the product. No statement or recommendation contained herein shall have any force or effect unless in an agreement signed by officers of seller and manufacturer.www.radnoti.com 800-428-1416

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