Experiment 10
Perfusion of the toad heart
【objective】 With the method of the heart in vitro, to observe the influence of the sodium, potassium, calcium three kinds of ions and certain nerval factors to the heart activity. 【introduction】 The autorhythmicity activity of the heart needs to have an appropr iate physics and chemistry environment. Once the suitable environment is disturbed or destroyed, the heart activity can be affected. Heart is controlled by the autonomic nerve. When the sympathetic nerve is excite, its end releases the noradrenalin to make the cardiac muscle shrinkage force to strengthen, the conduction to speed up, the heart rate to speed up, and the cardiac output to increase; when the vagus nerve is excite, its end releases acetylcholine to cause the cardiac muscle shrinkage force to weaken, the heart rate to reduce speed and the cardiac output reduction. When the toad heart is in vitro, it is filled with the ringer’s solution which its characteristic of the physics and c hemistry is approximate the blood plasma, the heart may maintain the rhythmicity contraction and the relaxation in certain time. If the fluid is changed, its frequency and the scope also change.
【equipment and drug 】 BL-420 animal experiment system ，tension transducer ， operating apparatus， frog heart canula ， frog leg clips， frog heart clip ， beaker，thread，ringer’s solution，0.65% NaCl，1% CaC12，l% KCl，1:10,000 noradrenalin，l:10,000 acetylcholine
【object】 Toad 【method and procedure 】 1 .Destroy the brain and the spinal cord, expose the heart and cut the pericardium membrane. Carefully indentity the structure of the heart.
Fig 1
the structure of the heart
2. separately pass a thread under the aortas and the left aorta. Tie up the left aorta, the left hand holds the line to fix the aorta, cut a small slanting mouth with the small scissors in the conus arteriosus root, then put the the tube which is filled with the ringer’s sol ution into the conus arteriosus .when insert to the conus arteriosus, back o ff slightly. When the ventricle contracts, insert downward along the ventricle behind direction, by the aorta petal insertion ventricle cavity.
When the tube is inserted the ventricle, the liquid level shifts with the activity of the heart. (if it isn’t so, you may revolve 90 degrees in order to avoid inserting orifice incline to paste on ventricle wall, and block orifice). If you determine to do it rightly, the line is gripped tightly and fixed on the side pipe. 3. cut off the organization which is around the heart and take out the heart from the chest, when you cut the vein, you cut them far away the vinous sinus to avoid damaging it. Aspirate the fluid and wash it with the ringer’s s olution to avoid stopping the frog heart ca nula because of the blood coagulation. At last, fix it on the iron framing with the tes t-tube holder. 4. adjust instrument Fix the tensity transducer on the bracket of the iron, the frog heart clip which is joined with the tension transducer will be clamped on the apex cordis in the ventricle relaxing period. The line with the tention transducer should be identical straight line with the tube of the middle axle, in the namely is vertical with the ground. Turn on the computer, activate the icon of “BL-420 biological data acquisition and analysis system” on the desktop→click model→ circulation→perfusion of the toad heart
【observe item】 (1) descripe the normal curve of the heart. The scope of the curve represents the strong and the weak of the heart; the density of the curve represents the speed of the heart rate; the disciplines of the curve represents the rhythmicity of the heart; the baseline of the curve represents the degree of the ventricle relaxation.
Fig 2. The curve of the natural heart (2) 0.65% nacl influence: take out the whole perfusation and put the 0.65% nacl into the t ube, observe the curve change of the heart. After the effect is obvious, aspirate the fluid and wash it with the ringer’s solution until the curve restores normally. (3) 1% cac1 2 influence: add 1-2 drop 1% cac1 2 in the tube, observe the curve change of the heart. After the effect is obvious, aspirate the fl uid and wash it with the ringer’s solution until the curve restores normally. (4) l%kcl influence: add 1-2 drop l%kcl in the tube, ob serve the curve change of the heart. After the effect is obvious, aspirate the fluid and wash it with the ringer’s solution until t he curve restores normally.
① Ringer’s solution
② Na+
③ Ca2+
④ K+
fig 3. The curve of the three kinds of irons (5) 1:10,000 adrenalin influence: add 1-2 drop l:10,000 adrenalin in the tube, observe the curve change of the heart. After the effect is obvious, a spirate the fluid and wash it with the ringer’s solution until the curve restores normally. (6) l:10,000 acetylcholin influence: add 1 drop of l:10,000 acetylcholin in the tube, observe the curve change of the heart. After the effect is obvious, aspirate the fluid and wash it with the ringer’s solution until the curve restores normally.
【result】 Order
1
Observation project
The curve change of the heart
Ringer’s solution
2
0.65% NaCl
3
1% CaCl2
4
1% KCl
5
1:10000 NE
6
1:10000 Ach
【requiring and attention 】 1. when the function of each chemistry medicine is obvious, you should aspirate the liquid and change the ringer’s solution several times in the frog heart canula, in order to avoid injurying the cardiac muscle. The next experimental project can be carried on when the palpitation restores normally. 2. after the reagent is added each time, you may mix evenly gently to enable it to play the r ole rapidly. When the function of the medicine is not obvious, you may increase the dose of the medicine. 3. don’t mix up the straws. One inserts the ringer’s solution, the other inserts t he fluid in the frog heart canula. In order to avoid influencing the experiment results, the straws in the different
reagents may not be confused. 4. when the fluid is changed each time, the liquid level of the frog heart canula should be maintained the same altitude. 5 .add the ringer’s solution to the surface o f heart to make it moist as necessary. 6 .you should make a mark when you add the regment or change the fluid each time. 7 .when you fix the tension transducer, you should incline it slightly downward, in order to avoid the current of water which trickles from the heart into the tension transducer.
【questions】 1. analyse the reason of each e xperiment. 2. tests must maintain the constant lever of the perfusion. Why?
