Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Podsumowanie

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Streszczenie

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

Wprowadzenie

Elucidation of the events underlying the cardiac response to both ischemia and reperfusion are essential in improving the treatment of myocardial infarction¹ and cardiac surgical procedures that require aortic cross-clamping². While in vivo models of ischemia reperfusion injury allow very useful endpoint analysis, they are not as effective for studying the functional effects of ischemia reperfusion injury acutely in real time. Additionally, in vivo ischemia reperfusion models generally produce significant variability in infarct size, and direct delivery of drug to the heart at the time of reperfusion is challenging. The utilization of a Langendorff isolated heart system for studying ischemia reperfusion injury allows for real-time functional assessment of pharmacological treatments, uniform area of infarcted tissue, and instantaneous delivery of drug directly to the myocardium.

First described by Oscar Langendorff in 1895³, the Langendorff isolated heart is a robust model for studying ischemia reperfusion injury, having been used in ischemia reperfusion research for the last 40 years^4,5. Here, some modifications are made to optimize the isolated heart for functional analysis. In situ cannulation of the aorta while the heart is beating ensures that the heart does not experience ischemic preconditioning, which would alter the results of ischemia reperfusion trials⁶. To facilitate this, a tracheotomy is performed, allowing ventilation and ensuring physiological stability of the rat during surgery. The heart is then attached to a glass water-jacketed spiral column through which Krebs Henseleit buffer is delivered via retrograde perfusion directly into the aorta. A saline-filled balloon is inserted into the left ventricle and attached to a pressure transducer, which allows for real time measurement of pressures from within the ventricle and calculation of multiple functional parameters. At the conclusion of the experiment, the heart is flushed with cold saline to arrest contraction and flash frozen in liquid nitrogen to enable downstream analysis of DNA, RNA and protein levels. Thus modified, the Langendorff perfused heart serves as an effective system for direct monitoring of the physiological effect of pharmacological interventions at any time acutely during the ischemia reperfusion injury.

Protokół

All procedures listed here have been approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. The experiments described here are acute, non-survival experiments. As such, there is no use of eye ointment and a sterile operating suite is not required. Euthanasia is achieved by exsanguination during harvesting of the heart.

1. Experimental Preparation

1. Set up either a constant pressure or constant flow Langendorff Perfusion Apparatus⁴.
2. Prepare 4 L of modified Krebs Henseleit Buffer (in mM: 112 NaCl, 5 KCl, 1.2 MgSO₄, 1 K₂HPO₄, 1.25 CaCl₂, 25 NaHCO₃, 11 D-glucose, 0.2 octanoic acid, pH = 7.4).
   1. Make 10x stock buffers.
      1. Dissolve NaCl, KCl, MgSO₄, and K₂HPO₄ in 3 L ultrapure water.
      2. Dissolve CaCl₂ in 500 ml ultrapure water with 40 ml of 1 N HCl.
      3. Slowly add CaCl₂ solution to the buffer made in 1.2.1.1. Add enough ultrapure water to total 4 L. Allow to stir for 1 hr before filtering through a 0.8 µm filter. Store at 4 °C.
      4. Dissolve NaHCO₃ in 4 L ultrapure water. Allow to stir for 1 hr before filtering through at 0.8 µm filter. Store at 4 °C
   2. Combine 400 ml 10x Krebs Henseleit buffer with 400 ml 10x NaHCO₃ buffer. Add enough ultrapure water to total 4 L. Dissolve glucose and octanoic acid while stirring. Filter through 0.8 µm filter.
3. Prepare system for heart.
   1. Add 2 L of filtered buffer to a 4 L bottle placed 38 in above the stopcock attached to the aortic cannula. This will deliver a perfusion pressure of 70-80 mmHg to the heart, which is the recommended perfusion pressure⁴.
   2. Insert gas dispersion bubbler into the buffer in the bottle.
   3. Run the buffer through the system, being sure to eliminate any bubbles from inside the tubing.
   4. Turn on gas (95% O₂/5% CO₂) at least 30 min before heart is to be placed on the system. Use a gas flow probe and blood gas analyzer to verify oxygen saturation.
4. Prepare surgical supplies and balloon.
   1. For harvesting of heart, prepare one pair of 6.75” surgical scissors, one pair of 4.5” scissors, two pairs of hemostats, two sets of caramalt forceps, two syringes with 27 G needles, 2 pieces of 0-guage silk suture (about 15 cm in length), one 1/16 inch barbed cannula, and one tracheal cannula.
   2. To assemble tracheal cannula, glue a short piece of 1/16 inch diameter plastic tubing to a Y-connector.
   3. To assemble balloon apparatus: attach a gavage needle to a piece of 1/16 inch tubing, then attach tubing to pressure transducer housing. A small syringe attached to the other side of the pressure transducer housing will allow inflation and deflation of the balloon.
      1. Cut a square of plastic wrap and place tip of a gavage needle in the center. Wrap the plastic wrap around the gavage needle.
      2. Fill balloon with saline and tie off with two sutures. Inflate balloon to ensure there are no leaks. The volume of saline within the inflated balloon should be roughly 200 µl, but can vary depending on the size of the rat.
      3. Alternatively, use a latex finger cot in place of the plastic wrap. Place gavage needle within finger cot, fill with saline and tie off.

2. Harvest the heart

1. Restrain a Sprague Dawley rat using a decapicone or other manual restraint device. Weigh rat.
2. Administer ketamine/xylazine mixture (0.85 mg/kg ketamine and 0.15 mg/kg xylazine) via intraperitoneal injection to anesthetize rat.  This dose of anesthetic will provide sufficient anesthesia for 20-40 min, but the procedure should be completed as quickly as possible after anesthesia is confirmed.
3. Confirm proper degree of anesthesia by testing toe pinch reflex.
4. Tracheotomy
   1. Remove pelt from mid-forepaw to base of jaw, using hemostats to elevate pelt and scissors to make incision. Keep the pelt elevated to separate it from the underlying muscle, allowing one to cut along the pelt and remove it.
   2. Using hemostats, lift glandular tissue and make a horizontal incision in fascia inferior to the glandular tissue. Use hemostats to spread incision open, taking care not to nick the jugular veins.
   3. Using hemostats, pinch muscle overlying trachea, and using scissors make a transverse incision in the muscle just below the tip of the hemostats. Using hemostats, spread tissue apart to reveal trachea.
   4. Carefully insert hemostats under trachea, clearing fascia while progressing by gently advancing and retracting hemostats.
   5. Once hemostats are successfully placed behind the trachea, pinch 0-0 silk suture with hemostats and hemisect the trachea with a small, sharp pair of scissors.
   6. Insert tracheal cannula into the hemisected trachea, pull suture behind the trachea, and tie the suture around the cannulated trachea, anchoring the suture to the Y-groin of the cannula with a surgeons knot.
   7. Using a 27 G needle, inject 1,000 U/kg heparin into the jugular vein and allow to circulate for 30 sec.
5. Thoracotomy
   1. Remove pelt from mid-abdomen to mid-forepaw region by pinching with hemostats and cutting with scissors.
   2. Make a ¾ inch transverse incision into the abdominal muscle wall, just below the diaphragm.
   3. Make a vertical incision up the abdominal wall and chest wall, cutting along the sternum.
   4. Cut the diaphragm back bilaterally, then spread ribcage to reveal heart, clamp ribcage with hemostats to ensure it remains spread.
   5. Using hemostats, gently remove thymus, exposing the ascending aorta.
   6. Tease hemostats through aortic loop, advancing and retracting hemostats and gently spreading fascia to allow tip of hemostats through.
   7. Use hemostats to pull 0-0 silk suture behind ascending aorta, tie suture into a loose half-square knot.
   8. Turn stopcock to start flow of perfusion buffer, hemisect aorta and immediately insert cannula into aortic lumen. Rapidly cinch a half-square knot and complete the knot, tightening thoroughly.
   9. Use scissors to dissect heart away from the great vessels, remove heart from chest and attach to perfusion column.
   10. Trim away excess lung tissue and allow heart to equilibrate on column for about 15 min before insertion of balloon. Use a normal flow rate of buffer through the heart of 10-20 ml/min.

3. Langendorff Perfusion and Ischemia Reperfusion Injury

1. Placement of LV pressure balloon
   1. Deflate balloon while rolling into a cone shape and turn stopcock to ensure balloon remains deflated.
   2. Excise left atrium with a small pair of scissors to expose access to mitral valve.
   3. Insert balloon through left atrium and mitral valve into left ventricle.
   4. Start pressure monitoring software (LabChart Pro).
   5. Inflate balloon by opening stopcock and gently increasing amount of saline in balloon until diastolic pressure reading from micromanometer exceeds zero.
   6. To check length-tension response, inflate balloon slowly to a diastolic pressure of 75 mmHg, deflate slowly, repeat once.
   7. Set diastolic pressure to 10 mmHg by modulating the amount of saline in the balloon. Turn stopcock to seal balloon at this level of inflation, and maintain closed pressure system between LV balloon and pressure transducer.
   8. Lower heart into heated chamber and cover chamber with plastic.
2. Administration of Ischemia Reperfusion Injury
   1. Plug the bottom of the heated chamber and allow chamber to fill with effused buffer.
   2. When buffer has submerged heart, turn stopcock to stop the flow of buffer into the heart, inducing global ischemia. Ischemia time can vary depending on the goals of the experiment.
   3. After 30 min of ischemia, turn stopcock to restore flow of buffer into heart, initiating reperfusion.
   4. Allow reperfusion of heart for 1 hr before terminating the experiment. Use variable reperfusion times, depending on the goals of the experiment.
3. Termination of Experiment and Harvesting of Tissue
   1. Using side port of stopcock, administer 10 ml of phosphate buffered saline at 4 °C into the heart at a rate of 10 ml/min to arrest the heart.
   2. Remove heart from perfusion column, trim away remaining atrial tissue.
   3. Place heart into a stainless steel tissue slicing matrix, cover with Parafilm, and place at -80 °C for 8 min, or until the heart has a marshmallow-like consistency.
   4. Remove block from freezer and insert razorblades into slicing matrix to slice heart into 2 mm slices. Tissue blocks can also be purchased with slice sizes other than 2 mm.
   5. Remove slices one at a time and drop into liquid nitrogen to flash freeze for biochemical studies. Retain the third ventricular slice from the apex for infarct staining.
   6. Remove tissue from liquid nitrogen and store at -80 °C until use in biochemical experiments.
4. Infarct Staining
   1. Incubate the third ventricular slice in 10 ml of 1% w/v triphenyltetrazolium chloride (TTC) at 37 °C for 15 min.
      Note: Incubation in TTC can be performed for 15 or 30 min with no change in effectiveness¹⁵.
   2. Transfer tissue slice from TTC to 10% buffered formalin and allow to incubate at room temperature overnight. For optimal results, hearts should be taken out of formalin and imaged within 24 hr⁴.
   3. Photograph stained slice and use software such as ImageJ to estimate infarct size according to manufacturer’s protocol. Take photographs as soon as possible after fixation, as the dye will fade over time.

Wyniki

The left ventricular balloon apparatus allows for real-time monitoring of the pressure developed by the contracting left ventricle (Figure 1). As described previously⁷, this pressure trace can be used to calculate many of the parameters of ventricular function. These calculations can be made in the baseline phase as well as the reperfusion phase, averaged over multiple traces within each group, and compared in order to determine whether the pharmacological intervention resulted in cardiac prec...

Dyskusje

The isolated perfused rat heart can be successfully used to study the effect of pharmacological intervention on cardiac preconditioning in ischemia reperfusion injury⁹. However, there are some essential steps to the procedure that must be standardized in order to ensure reproducible results. Maintaining a temperature of 37.4 °C within the system is critical, as even mild hypothermia and hyperthermia can cause cardiac preconditioning^10,11. The overall time that elapses from injection of anesthet...

Materiały

Name	Company	Catalog Number	Comments
Sodium Chloride	Sigma Aldrich	S3014
Potassium Chloride	Sigma Aldrich	P9541
Magnesium Sulfate	Sigma Aldrich	203726
Potassium Phosphate Dibasic	Sigma Aldrich	RES20765-A7
Calcium Chloride Dihydrate	Sigma Aldrich	C8106
Sodium Bicarbonate	Sigma Aldrich	S5761
D-Glucose	Sigma Aldrich	G8270
Octanoic Acid	Sigma Aldrich	C2875
2,3,5-triphenyltetrazolium chloride	Sigma Aldrich	T8877
Medical Pressure Transducer	MEMSCAP	SP844
Masterflex Peristaltic Pump	Cole Parmer	EW-07521-40
Masterflex Easy Load Pump Head	Cole Parmer	EW-07518-10
Heated circulating water bath	Lauda	M3
Tubing Flow Module	Transonic	TS410
Modular Research Console	Transonic	T402
Inline flow sensor	Transonic	ME2PXN
PowerLab Data Acquisition Device	AD Instruments	PL3508
LabChart data acquisition software	AD Instruments	MLU60/8