Langendroff apparatus - Standard operating procedure

How to use langendorff apparatus and it's applications, advantages & limitations.

1. LANGENDORFF APPARATUS By- Dr. Chitranshi Hyanki JR 2 Department of Pharmacology and Therapeutics KGMU, Lucknow

2. Contents Introduction Instruments used in Langendorff’s Apparatus Set up Modes of perfusate Perfusion solution Retrograde perfusion of heart Measurements Advantages Limitations

3. Introduction In 1897, Oscar Langendorff established the isolated perfused mammalian heart preparation which was considered to be the breakthrough in cardiovascular research. Principle- The heart is perfused in retrograde direction from the aorta at constant pressure or constant flow with oxygenated saline solutions.

4. Used to perfuse all sizes of heart – mice, rats and guinea pigs. Parameters usually measured are- Contractile force Coronary flow and cardiac rhythm

5. Langendorff’s apparatus ADINSTRUMENTS.com

6. Modified langendorff apparatus set up

7. The instruments used in langendorff’s apparatus set up - Power lab

8. It has eight channel data acquisition system. Features include- Filtering (hardware and software) Analogue outputs Trigger input USB connection to computers.

9. Bridge amplifier Software-controlled, single-channel bridge amplifier. Suitable for measuring the pressure. Seamlessly interfaces with the power lab system & pressure tansducers .

10. Pressure transducer Connects to bridge amplifier. Measures perfusion and left ventricular pressure.

11. Animal bio amplifier Single-channel bio-amplifier. Measures cardiac electrical activity from the isolated perfused heart.

12. Peristaltic pump Low-noise peristaltic pump. Provides laminar flow of perfusate through the system. Eliminates the need for elevated reservoirs.

13. STH Pump controller Controls the peristaltic pump. Provides an analogue output corresponding to calibrated flow rates. Provides a pressure/flow feedback system to the peristaltic pump when switching between constant flow & constant pressure modes.

14. Thermostat heater Manages temperature of the perfusate & heart Keeps it at 37°C.

15. T-type temperature pod Software-controlled pod used to measure perfusate temperature.

16. Labchart software ADINSTRUMENTS.com

17. Langendorff System is supplied with LabChart Pro software Also includes LabChart Modules LabChart Modules add advanced analysis functions to LabChart software Like specific applications to fast track analysis

18. Animals Albino Rats (300 gm and at least of 1 yr of age) New Zealand Rabbits (1.5-3 kg and 3 years of age) Guinea pig (300-450 gm and 2-3 years of age) Ambient temperature (23°C ±2°C) under a 12:12 hr light and dark cycle Free access to tap water and food ad libitum. The animals are acclimatized to the laboratory conditions for at least 1 week prior to experimentation

19. Method Animal is pretreated with the heparin (55-110 μg / kg, i.p or s.c [for rabbit] or heparin (500–1000 IU/kg, i.v in rabbit and rat and i.p in guinea pig). Animal is anaesthetised – thorax opened immediately - heart exposed. Heart is dissected out – transferred Cold Krebs solution (rat and guinea pig heart) or McEwens solution (rabbit heart)

20. The ascending aorta is separated from pulmonary artery Dissect the heart with at least 1 cm aorta intact Heart is gently squeezed several times Trim away any excess connective and lung tissue from the heart

21. Modes of perfusate Two modes Perfusion at- Constant pressure is given preference for studies of ischemia otherwise Constant flow technique is preferred.

22. Perfusion solution Perfusion solutions available for use can be- 1) asanguinous solution ( krebs henseleit buffer [ khb ]) 2) washed blood cells 3) whole blood. All media have to be aerated. Typically 95% O2 +5% CO2

23. Perfusion temperature Perfusion carried out close to the body temperature of the species under study. For rats, it is 35.9-37.5°c The thermostatically controlled water jacketed system provides the required temperature.

24. Precautions to be taken - In preparation for the experiments solid food should be withdrawn from the animals 24 h prior to the experiment. No limitation in water supply to the animal. Before starting the experiment, the perfusion solution has to be equilibrated with the appropriate gas & is to be warmed. All tubing to the aortic cannula has to be freed from air bubbles which might enter the coronary circulation & cause ischemia.

25. Prior to insertion of cannula in aorta of isolated heart the stopcock of the perfusing system should be opened so that there is already some flow through the aortic cannula - 1) This helps to avoid air bubbles entering the coronary arteries 2) This is the most critical step of the entire procedure 3) The cannula should not be inserted too deeply into the aorta 4) If too deep it may obstruct the orifices of the coronary arteries

26. Retrograde perfusion of heart Perfusion of isolated heart - reservoir containing oxygenated perfusion fluid.  Cannula inserted and fixed in the ascending aorta.  Reservoir opened, the perfusate is forced to flow into the aorta.  Closes the leaflets of aortic valve  Prevents perfusate from entering left ventricles.

27. The perfusion fluid goes to the coronary arteries that come off the side of the aorta  During diastole entire perfusate solution enters the coronary arteries via the ostia  Perfuses the entire ventricular mass of the heart  Right atrium via the coronary sinus  Driven out via the right ventricle & the pulmonary artery

28. Uses The preparation measures contractility of tissue in vitro. Analysis of contractile function & responses to ischemic insult studied. Studying the effect of pro-arrhythmic & anti-arrhythmic mechanism of various drugs. Testing the actions of drugs on ventricular fibrillation induced by regional or global myocardial ischemia. Contractility of tissue in vitro Heart rate

29. Drugs Catecholamines 5-HT, Histamine Acetylcholine Bradykinin Vasopressin Angiotensin Sodium Nitrite

30. Advantages Long viability duration, simple preparation, low cost, reliable data collection in quality & quantity. Permits the measurement of inotropic, chronotropic & coronary vascular effects, without the interference of hormones or nervous system. The effects of drugs directly on the heart can also be studied A range of coronary vasoactive drugs can be examined.

31. Limitations Requires high skill & care while harvesting the heart from animal Harvesting of heart to cannulation within an appropriate time frame to ensure good results The cannulation step is important in terms of the viability of heart Time from excision of heart to mount on cannula should be less 5 mins time to avoid damage to the heart. The major disadvantage is that 5-10% deterioration per hour in contractility & chronotropic function is seen.

32. References Bell RM, Mocanu MM, Yellon DM. Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion. Vol. 50, Journal of Molecular and Cellular Cardiology. 2011. p. 940–50. Gupta SK. Drug Screening Methods. 3rd edition, New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2019. Langendorff Isolated Heart Perfusion Technique | ADInstruments [Internet].  https:// www.adinstruments.com /research/animal/cardiovascular/isolated-heart-systems/ langendorff Langendorff Apparatus with Thermostat Controller | ADInstruments Dhein S. The Langendorff Heart.

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