this is an experiment for assessment of cardiac contractility using langendorff apparatus connected to a powerlab.

1. How to assess cardiac
contractility using langendorff
apparatus connected to a
powerlab.
Dina Hamdy Merzeban
Physiology Lecturer
Fayoum University
dhm00@Fayoum.edu.eg

2. Aim of the work
• How to isolate heart.
• How to calibrate powerlab from millivolt recording to mmHg.
• How to assess contractility using langendorff apparatus
connected to a powerlab.

3. Introduction

4. • As isolated/ hanging heart models lack fresh blood
circulation, hormonal and autonomic nervous
responses and otherwise very complex in-vivo factors
are decoupled which helps to perform a variety of
basic analyses of fundamental cardiac properties
unmasking the potential direct action of studied
compounds

5. Types of isolated heart models:
• Langendorff: (retrograde perfusion).
• Working Heart: (antegrade perfusion).

6. APPLICATIONS OF LANGENDORFF SET-UP USING
PRESSURE CATHETER MEASUREMENTS
• Assessment of contractility (inotropy).
• Assessment of HR (chronotropy).
• Coronary vasculature dilations.
• Arrhythmogenic, anti-arrhythmic, anti-fibrillatory effects .
• Gradual determination of hypoxic damage.
• Calcium antagonism.

7. Fig 1: langendorff apparatus.

8. Langendorff apparatus:
• Principle : is to maintain the heart perfused at constant
temperature.
• Langendorff apparatus consists of two tube systems, the first
system contains perfusate (Kreb-Henseleit KH buffer) that states in
a reservoir and ends in the aotic cannula passing through a pump
to maintain constant flow to the isolated heart (non recirculating
constant flow Langendorff apparatus)
• A second tube system that jacket the first system with water
maitained at 37 ºC using a heater unit that the fluid recirculates to
it.
• After aortic cannulation of the heart, it is placed in water jacketed
heart chamber maintained at 37 ºC

9. Types of flow in langendorff
CONSTANT PRESSURE LANGENDORFF
• Pressure is held constant and changes in coronary resistance are
detected as changes in blood flow.
• Constant pressure is achieved by elevating the aortic bubble trap
chamber as 1 mmHg = 13.6 mm of water column therefore
0.816 m would be needed to create 60 mmHg.
CONSTANT FLOW LANGENDORFF:
• Flow is held constant at fixed flow rate via peristaltic pump and
changes in coronary resistance are detected as changes in
pressure.

10. Excision of the heart and its
cannulation (rat):
• Male Wistar rats weighing 350-450 g were used. They were
anaesthetized using ketamine hydrochloride (25 mg/kg,
intraperitonial) and heparinized by intraperitoneal injection of
Heparin(1000IU). A left thoracotomy was performed and
hearts were rapidly exposed, excised by cutting across the arch
of the aorta (leaving enough space for mounting on the
cannula) along with all other vascular structures in the area. The
heart is removed into ice-cold heparinized phosphate buffered
saline (4°C) to arrest beating. The ascending aorta is then
mounted on the aortic cannula (Louch et.al., 2011).
•

11. • Mounted heart should be perfused by flow of up to 15
ml/min/g of heart (Bell et.al.,2011).

12. Introduction of fluid-filled balloon into
the LV :

13. Caliberation of powerlab:

14. Experimental preparation
1. Frog Pithing Procedure
2. Dissecting out the Frog Sciatic Nerve:

15. Frog’s sciatic nerve in nerve bath

16. Powerlab 4/30

17. Proper Connections to the Powerlab

18. Experimental procedure
3. Setup and calibration of equipment:

19. •the nerve bath above, stimulating
electrode in black wire,
•two stimulating electrodes in white
color,
• red is anode +,
•black is cathode -,
•green is ground

20. LabChart 7 welcome center

21. The test connection data shown in the Scope
view.

22. Using the Scope view to show
increasing CAP amplitude.

23. Waveform cursor on the
maximum CAP amplitude

24. CAP amplitude versus stimulus intensity (data obtained from
the present experiment).
Proximal CAP amplitude
Stimulus
amplitude
Proximal CAP
amplitude
Stimulus amplitude
Δ(mV) mV Δ(mV) mV
2.1109 20 42.0859 400
4.2641 40 63.125 500
6.3141 60 103.875 1000
8.3594 80 253.8125 2000
10.6297 100 382.9375 3000
12.8141 120 510.5313 4000
14.5984 140 767.1563 6000
16.8875 160 774.7 7000
19.025 180 882.5 8000
21.0953 200 1003 9000
23.1406 220 1051.7 9500
25.2953 240 1117.2 10000
27.4719 260 1113.8 10500
29.5641 280 1122.2 11000
31.8297 300 1129.2 11500
33.4781 320 1130.4 12000
35.7344 340 1131.4 12500
38.0953 360 1131 13000
40.2172 380 1131 13500
Threshold stimulus voltage: 20 mV
Maximum CAP amplitude: 11500 mV

25. CAP amplitude versus stimulus intensity (as
obtained from the threshold test)
0
2000
4000
6000
8000
10000
12000
14000
16000
020040060080010001200
stimulusamplitude
CAP amplitude
CAP amplitude versus stimulus intensity
Proximal CAP amplitude Δ(mV)
Stimulus amplitude mV

26. Determination of nerve conduction
velocity
• Using a ruler, measure the distance in millimeters between
the black negative leads of each of the two recording
electrodes.
• Read the value for time differential (Δt) from the Cursor
display in the LabChart application window in milliseconds.

27. Measure the distance in millimeters between the
black negative leads of each of the two recording
electrodes.

28. Zoom window in overlay mode showing analysis
procedure for calculating conduction velocity.
Waveform Cursor information is displayed at the
top of the window.
(overlay in zoom view not activated!!!)

30. Calculation of conduction velocity
The conduction velocity of nerve is normally reported in meters per second. It is
more easily recorded as millimeters per millisecond, which yields the same result.
Distance between recording
electrodes:
10 Mm
Time interval between CAP1
and CAP2:
0.02 Ms
Conduction velocity 500 m/s

31. Methods for Calculation of
conduction velocity
• Conduction velocity is systematically related to fiber
diameter
• Velocity (m/s) = Diameter (µmeters) x 2.5
• Two methods for Calculation of conduction velocity
• Difference method
• Absolute method

32. The difference method:
• The stimulus duration is set to 0.2 ms, and two pairs
of recording leads are connected

33. • A maximal CAP is elicited, and the latencies (in
ms) to the peaks of the responses are measured.
• This latency signifies the time it took for average
fibers in the nerve to transmit their APs from the
stimulating electrodes to the first recording electrode

34. Zoom window in overlay mode showing analysis
procedure for calculating conduction velocity.
Waveform Cursor information is displayed at the
top of the window.
(overlay in zoom view not activated!!!)

35. • Now subtract the latency determined proximally from the latency
determined distally,
• a value for the time it took for the CAP to travel from proximal
electrode R1 to distal electrode R7.
• Conduction velocity (difference method)
• = (d2-d1)/(latencydistal - latencyproximal)
• =(20mm-10mm)/ (0.08msec- 0.05 msec)= 10/0.03= 333m/sec

36. The absolute method:
• The conduction velocity can also be calculated by the
absolute method, which means that the velocity is
calculated using a single latency and distance
measurement.

37. Calculation using proximal electrode
only

38. Measure from the cathode (the stimulating
electrode nearest the recording electrodes) to the
first recording electrode = 0.5 cm
the stimulating electrode
nearest the recording
electrodes
The first recording
electrode

39. • Measure from the cathode (the stimulating electrode nearest
the recording electrodes) to the first recording electrode R1.
• Latency = latency at electrode R1 (ms)
d = distance (mm) from stimulating electrode to recording
electrode R1
• Velocity = d / latency (m/s) or (mm/ms)
• One can also calculate the velocity for distal electrode R7: the same
formula applies except that the distance d and the latency are of
course longer.

40. Bi-Directionality of Nerve
Conduction

41. Characteristics of the CAP:
• The peak amplitude of the CAP .
• The latency of the onset of the CAP.
• The latency of the peak of the CAP
• The duration of the CAP
• The threshold stimulus voltage
• The maximal stimulus voltage

42. Stimulator Settings

43. Stimulator Settings• Stimulus Duration: 0.2 ms
• Stimulus Voltage: 0.05 V
• Delay: 1 ms
• Rate: 1 Hz
• Mode: Repeat

44. The peak amplitude of the CAP:
is the voltage value of the peak of the CAP
response.
The peak amplitude of the CAP is 63 mV (as revealed in the
present experiment).

45. the latency of the onset of CAP is 0.00002 sec (=0.02 msec) (as revealed in
the present experiment).
The latency of the onset of the CAP:
is the time from the onset of the stimulus
artifact to the onset of the CAP.

46. The latency of the peak of the CAP:
is the time from the onset of the stimulus
artifact to the peak of the CAP.
The latency of the peak of the CAP is 0.00004 sec (=0.04msec) (as
revealed from the present experiment).

47. The duration of the CAP:
is the time from the beginning of the positive
phase to the end of the negative phase of the
CAP.
the duration of the CAP is 0.00029 sec (=0.29 msec) (as revealed from
the present experiment).

48. The threshold stimulus voltage:
is determined by raising and lowering the stimulus voltage
a little to find the voltage at which the CAP is just
discernible.
The threshold stimulus voltage is 20 mv and the CAP peak is 2.09 mV
(as revealed from the present experiment table 1).

49. The maximal stimulus voltage:
is the point at which a further increase in
stimulus voltage produces no further increase in
the CAP amplitude.
The maximal stimulus voltage is 11.5 V and the CAP peak is 1149
mV (as revealed from the present experiment table 1)

50. The significance of measuring the latency
to the beginning of the CAP, versus
measuring it to the peak of the CAP:
• The latency of the beginning of the CAP reflects
how long it takes for the fastest fibers to conduct
action potentials from the stimulus source to the
recording electrodes.
• When the latency is measured to the peak of the
CAP, we obtain the latency of an average fiber in
the nerve.

51. The significance of the threshold
voltage
• the threshold voltage, measured from the stimulator, is the
voltage needed to generate at least one AP from a fiber in
the sciatic nerve bundle.
• (Remember when we speak of CAP thresholds, we are
dealing with a whole nerve and not an individual fibre.)
• As the resolution of the screen is poor, it is hard to
measure an accurate threshold. The true thresholds are in
reality much lower.