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Working Heart Langendorff Instrumentation Radnoti LLC

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					                                          Radnoti Glass Technology
    Isolated Perfused Heart


                              The Radnoti Working Heart System




                                                             Presented by:
                                             Radnoti Glass Technology, Inc.




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                                                                              2007
                                                  Radnoti Glass Technology
    Isolated Perfused Heart


                              Introduction


                              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 to instrumentation of the Radnoti Isolated Working Heart System.




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    Isolated Perfused Heart


                              Experimental Options
                              There are a great number of physiological parameters that can be measured in the Isolated
                              Perfused Heart preparation.

                              A pressure measurement while in constant flow mode will show the resistance of the heart,
                              indicating vasodilatation or vasoconstriction.

                              A left ventricular pressure measurement made using a combination of pressure transducer,
                              flexible balloon catheter and latex balloon can serve as an indicator of contractile force.
                              Electrocardiograms (ECGs) are readily obtained using surface electrodes of monopolar, bipolar
                              construction or needle electrodes that pierce the heart muscle and are of interest in studies
                              involving arrhythmias.

                              Oxygen consumption can be determined with dual oxygen electrodes, one placed in the perfusate
                              stream entering the heart, the other monitoring the effluent leaving the coronary sinus. This
                              effluent can be removed through the use of a peristaltic pump and then transferred to the second
                              oxygen electrode.

                              Similarly, ion selective electrodes can be placed in the effluent or perfusate stream or
                              oxygenation chamber of the Radnoti Isolated Perfused Heart apparatus thus permitting
                              measurement of pH and other cations and anions.

                              Radiolabelled compounds can be used for metabolic studies, as well as the release or uptake of
                              various ions or substrates.
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                              Optical studies measuring intracellular constituents have been performed on the fluorescence of
                              endogenous or exogenous fluorescent compounds.
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    Isolated Perfused Heart


                              Instrumentation of the System
                                                                                                 Perfusion Pressure and Ejection
                                                                                                 Pressure
                                                                                                 159905
                                 LVP
                                 159907 IVP transducer & Balloon or 159905 Pressure Transducer   Perfusion pressure on Aortic Cannula
                                 with 170424 flexible balloon catheter and balloon               (Langendorff constant flow)

                                                                                                 Ejection Pressure on stopcock of Aortic
                                                                                                 Cannula feed to Compliance loop
                                                                                                 (Working Heart)




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    Isolated Perfused Heart


                              Instrumentation of the System cont.




                                    ECG
                                    #140155 ECG electrodes and ML132 Bioamp


                                                                              Flow-
                                                                              Perfusion
                                                                              IUF1000 Flow Meter mounted at
                                                                              feed to atrial cannula
                                                                              Ejection
                                                                              IUF1000 Flow Meter mounted
                                                                              inline on compliance loop



                                                Pacing
                                                #140157 Pacing electrodes
                                                GR-SD9 Stimulator




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                              Instrumentation of the System cont.




                                                           O2
                                                           MI-730 Dip-type O2 microelectrode
                                                           O2-ADPT Oxygen Adapter




                                                                                                       pH
                                                                                                       MI-410 Micro-combination pH microelectrode
                                                                                                       MV-ADPT Millivolt Adapter




                                                 Flexible Balloon Catheter
                                                 #170423 The balloon catheter is
                                                 for ventricular insertion. It is a
                                                 simple, reliable way to measure
                                                 left ventricular isovolumetric                Temperature
www.radnoti.com                                  pressure.                                     BAT-12 Microprobe Thermometer
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                              Typical Values


                              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        43



www.radnoti.com                             Rabbit         205-220      110/73         155          4.6       3.5       1.6       0.4-0.7           2-5
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    Isolated Perfused Heart


                              Pumps Overview
                              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:

                              View Instruction Manual

                              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 Bath

                              View Instruction Manual



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                              Thermal Circulator Quick Setup
                              Installation
                              Locate the unit on a sturdy work area. Ambient temperatures should be inside the range of +50°F to +104°F (+10°C to +40°C).
                              The maximum operating relative humidity is 80%. Never place the unit in a location where excessive heat, moisture, or
                              corrosive materials are present. Make sure the voltage of the power source meets the specified voltage, ±10%. The pump
                              connections are located at the rear of the pump box and are labeled              and            These connections are angled
                              upward so the recirculating fluid will drain back into the reservoir when the hoses are disconnected. Both connections are
                              capped with stainless steel serrated plugs. The pump lines have ¼" MPT for mating with standard plumbing fittings. For your
                              Convenience, stainless steel adapters ¼" FPT to 3/8" O.D. serrated fitting are provided. The bath work area has a high and
                              low level marker to guide filling. The markers are 1 inch horizontal slits located in the center of the stainless steel baffle
                              separating the work area and the pump assembly. The correct fluid level falls between these two markers. The unit will not
                              start if the fluid level is below the lower slit.
                              Operation
                              Before starting the unit, double-check all electrical and plumbing connections. Make sure the bath is properly filled with fluid.
                              To start the unit, press           To turn the unit off press     again. The LED           indicates the status of the heater. It
                              illuminates to indicate the heater is on.
                              Temperature Adjustment
                              To display the temperature set point simply press on the controller. The          indicator will illuminate and the display will flash the
                              current set point value. To adjust the temperature set point, press the arrow buttons until the desired temperature set point is
www.radnoti.com               indicated. Press          again to confirm the change. The display will rapidly flash the new value for a short time and then return to the
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                              recirculating fluid temperature. Please refer to the manual for periodic maintenance.
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    Isolated Perfused Heart


                              Peristaltic Pump Quick Setup

                                      The L/S EASY-LOAD Pump Head is compatible with most MASTERFLEX® L/S® drives with a standard tang
                                      interface. Mount the Pump Heads using the 2 long screws provided. The pump heads line up with each other
                                      by the tangs and are held together with the long thumb screws.




                                     Be sure the pump drive is turned off. To open the pump
                                     head rotate lever to the left.
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                              Peristaltic Pump Quick Setup cont.



                                                        1. With the lever released, load tubing of the
                              NOTE: Tubing              correct size. Center the tubing between the
                              retainers usually do      retainers. Make sure that the tubing is in the
                              not need to be            tubing retainer slot on each side of the pump
                              readjusted when           head.
                              changing tubing of the
                              same type
                              and size.

                              NOTE: There are a
                              few combinations of                            2. Rotate the lever to the
                              tubing and materials                           right to close.
                              that may require a
                              further adjustment of
                              the tubing retainers if
                              the tubing creeps.
                              Open pump head,
                              move tubing retainer
                              down one (1) notch,
                              then close pump           3. To set the tubing retainer to the
                              head. Turn pump on.       tubing, release the retainer from the
                              If tubing creep           locked position by first pushing in
                              persists, repeat          slightly towards the body, then
                              procedure.                downward firmly against the tubing.
www.radnoti.com                                         Set retainer on both sides.
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    Isolated Perfused Heart


                              Water Jacket Diagram


                              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.




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    Isolated Perfused Heart




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    Isolated Perfused Heart


                              Cardiac Pacing
                              The experimenter must decide whether the heart will be paced or
                              allowed to beat spontaneously. Pacing is used to maintain a
                              standard contractile response and metabolic demand. Spontaneous
                              beating may permit the experimenter to measure changes in heart
                              rate and rhythm that will occur with various drugs or manipulations.

                              To pace a heart, the stimulus rate must exceed the natural cardiac
                              pacemaker rate. Often the sinoatrial node is crushed or the right
                              atrium excised to eliminate the contribution of the primary intrinsic
                              pacemaker. Pacing voltage is determined as a set percentage
                              (normally 110-150%) above the voltage required to capture (pace)
                              the heart and should not have to exceed 3-5 v, with a duration of 0.1-
                              1 msec. Hearts may be paced using either surface or plunge
                              electrodes inserted into the cardiac tissue by running Teflon-coated
                              wires into needles, exposing the tips of the wires and bending the
                              wires over the tips of the needles. The needles are then pushed into
                              the heart and withdrawn, leaving the wire embedded in the tissue.

                              Should tissue damage be an issue, it is recommended that the
                              Radnoti Pacing Electrode be used.

                              Pacing may also be used to induce arrhythmias in attempts to
                              measure changes in fibrillation threshold.
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    Isolated Perfused Heart


                              Left Ventricular Pressure

                              (In Langendorff modes only)

                              A saline-filled balloon catheter inserted into the left ventricle is often used to
                              measure isovolumetric work.

                              Balloons should be slightly larger than the maximum expanded
                              volume of the ventricle to avoid effects of measuring the resistance of
                              the balloon to stretch.

                              The balloon is secured to a flexible balloon catheter, which is then
                              connected to a pressure transducer. The balloon may be inserted by
                              passage through the left atrium or by passing the catheter through the wall
                              of the left ventricle for pressure measurements. In this case, a one-way
                              valve must be placed in the aortic cannula if the intraventricular pressure
                              exceeds the perfusion pressure.




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    Isolated Perfused Heart


                              Working Heart Contractile Function

                              In the Working Heart Model, contractile function can be assessed by the initial
                              ejection pressure at the aorta. The concomitant ability to pump against an
                              afterload and/or reach a set ejection pressure with a preload is set by adjusting the
                              height of the compliance loop reservoir.

                              Pressure-volume work is determined by the total volume of fluid ejected by the          Afterload pressure
                              ventricle over time. In any of these cases, the experimenter should determine the
                              appropriate amount of resting force or pressure required to maintain the heart on
                              the ascending limb of the Starling Curve and avoid overstretching the heart
                              muscle.

                              Other useful functions derived from contractile measurements include the first
                              derivative, dP/dt, a determinant of the rate of change of developed pressure and
                              the integral of pressure as an index of work. Heart rate can be monitored from
                              force measurements or monitored independently with an ECG amplifier.




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                              Starling Curve Defined
                              The Frank-Starling law of the heart (also known as Starling's law or the Frank-Starling mechanism)
                              states that the more the ventricle is filled with blood during diastole (end-diastolic volume), the
                              greater the volume of ejected blood will be during the resulting systolic contraction (stroke volume).
                              There is an optimum end-diastolic volume, after which cardiac performance declines (see below).
                              This means that the force of contractions will increase as the heart is filled with more blood and is a
                              direct consequence of the effect of an increasing load on a single muscle fiber. In particular, such
                              increased load stretches the myocardium further and enhances the affinity of troponin C for
                              calcium, hence increasing the contractile force. The force that any single muscle fiber generates is
                              proportional to the initial sarcomere length (known as preload), and the stretch on the individual
                              fibers is related to the end-diastolic volume of the ventricle. In the human heart, maximal force is
                              generated with an initial sarcomere length of 2.2 micrometers, a length which is rarely exceeded in
                              the normal heart. Initial lengths larger or smaller than this optimal value will drop the force of the
                              muscle owing to less overlap of the thin and thick filaments for larger values and more overlap of the
                              thin filaments for smaller values. This can be seen most dramatically in the case of a premature
                              ventricular contraction. The premature ventricular contraction causes early emptying of the left
                              ventricle (LV) into the aorta. Since the next ventricular contraction will come at its regular time, the
                              filling time for the LV increases, causing an increased LV end diastolic volume. Because of the
                              Frank-Starling law, the next ventricular contraction will be more forceful, causing the ejection of the
                              larger than normal volume of blood, and bringing the LV end-systolic volume back to baseline.
                              For example, during vasoconstriction the end diastolic volume increases. Increasing preload will
                              increase stroke volume. The heart will pump what it receives. The above is true of healthy
                              myocardium. In the failing heart with an over-dilated ventricle, cardiac performance is compromised
www.radnoti.com               as described by Laplace's law.
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                              LVP Max Developed Pressure and Preload
                                                                                                         Trouble Shooting
    Isolated Perfused Heart


                              (Balloon Method, Langendorff Only).

                              LVP measurements are greatly affected by balloon size selection
                              and pre-load based on a Starling curve.

                              Select the appropriate balloon size.

                              It is imperative that the balloon size selected not be too small for the
                              donor heart. An indication of too small balloon size is that diastolic
                              minimum pressure is too high in order to achieve maximum developed
                              pressure.




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                              LVP Max Developed Pressure and Preload
                                                                                                        Trouble Shooting
    Isolated Perfused Heart


                              (Balloon Method, Langendorff only) cont.

                              Performing the starling curve to determine optimum preload to left
                              ventricular balloon.

                              The preload on the balloon should be increased gradually while
                              monitoring the developed pressure. An increase increment of 2mmHg
                              followed by a review of maximum developed pressure and systolic
                              pressure. Continue with the process until such time as an optimum
                              developed pressure is achieved while maintaining a physiological normal
                              systolic pressure.




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                                                                                                                    Trouble Shooting
    Isolated Perfused Heart


                              The Frank Starling Law of the Heart
                              The Frank-Starling Law of the Heart (also known as Starling's law or the Frank-Starling mechanism) states that the more the
                              ventricle is filled with blood during diastole (end-diastolic volume), the greater the volume of ejected blood will be during the
                              resulting systolic contraction (stroke volume).
                              This means that the force of contractions will increase as the heart is filled with more blood and is a direct consequence of the
                              effect of an increasing load on a single muscle fiber. In particular, such increased load stretches further the myocardium and
                              enhances the affinity of troponin C for Calcium, hence increasing the contractile force. The force that any single muscle fiber
                              generates is proportional to the initial sarcomere length (known as preload), and the stretch on the individual fibers is related to the
                              end-diastolic volume of the ventricle. In the human heart, maximal force is generated with an initial sarcomere length of 2.2
                              micrometers, (a length which is rarely exceeded in the normal heart). Initial lengths larger or smaller than this optimal value will
                              drop the force of the muscle owing to: less overlap of the thin and thick filaments for larger values, and more overlap of the thin
                              filaments for smaller values. This can be seen most dramatically in the case of a premature ventricular contraction. The premature
                              ventricular contraction causes early emptying of the left ventricle (LV) into the aorta. Since the next ventricular contraction will
                              come at its regular time, the filling time for the LV increases, causing an increased LV end diastolic volume. Because of the
                              Frank-Starling Law, the next ventricular contraction will be more forceful, causing the ejection of the larger than normal volume of
                              blood, and bringing the LV end-systolic volume back to baseline. For example, during vasoconstriction the end diastolic volume
                              increases, increasing preload. This will increase stroke volume. The heart will pump what it receives. The above is true of healthy
                              myocardium. In the failing heart, the more the myocardium is dilated, the weaker it can pump, as it then reverts to Laplace's Law.
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                              Radnoti Glass Technology
    Isolated Perfused Heart




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    Isolated Perfused Heart




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    Isolated Perfused Heart


                              Recommended Reading


                              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.




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    Isolated Perfused Heart


                              Disclaimer



                              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.




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