This experimental method was obtained from the official website of the Fourth Military Medical University
Operation method
Experiments on chemical factors affecting heart activity
Principle
Normal physiological activities of cells depend on the stability of the environment in which they are located. The maintenance of normal cardiac function also requires an appropriate physicochemical environment, such as the concentration of K+ and Ca2+ in tissue fluid, appropriate pH and temperature. The in vivo heart is also subject to the dual innervation of sympathetic and parasympathetic nerves. When the sympathetic nerve is excited, its endings release norepinephrine (NE), which acts on the corresponding receptors in cardiomyocytes to increase the heart rate, accelerate the conduction, and enhance the myocardial contraction; and when the vagus nerve is excited, its endings release acetylcholine (Ach), which slows down the heart rate, slows down conduction, and reduces the contraction force of the heart muscle. After the heart of the toad was isolated, the heart was perfused with Ren's fluid, whose physicochemical properties were similar to those of toad plasma, and within a certain period of time, its rhythmic contraction and diastole could be maintained. Changing the composition of its perfusate affects the functional activity of the heart. In disease states, the contractility of the heart is sometimes markedly diminished, and cardiac glycosides such as digitalis improve the contractility of the myocardium and, therefore, have the effect of improving cardiac function.
Materials and Instruments
Frogs Toads Move I. Material preparation Caveat 1. Do not injure the venous sinus when preparing the frog heart specimen. 2. The fluid level in the frog heart cannula should be maintained at the same level every time the fluid is changed. 3. Add Ren's solution on the surface of the heart at any time to keep it moist. 4. The droppers of various reagents should be differentiated and used exclusively, and should not be mixed, so as not to affect the experimental results. 5. When adding reagents, first add l ~ 2 drops, mixed with a dropper, if the effect is not obvious can be added appropriately, but must be added drop by drop, and closely observe the results of the experiments after the addition of the drug dose. 6. The above experimental items, once the role of the cannula should be immediately sucked out of the liquid and normal Ren's solution for washing, so as to avoid myocardial damage, and repeated washing several times so that the heart beat curve back to normal before the next experiment. 7. Each experiment should be controlled before and after. For more product details, please visit Aladdin Scientific website.
Ren's solution Calcium-free Ren's solution Calcium chloride solution Potassium chloride 1% solution Norepinephrine Acetylcholine Lactic acid solution Sodium bicarbonate solution Digitalis Ren's solution
Two channel physiological recorder Microcomputerized biosignal acquisition and processing system Tension transducer Frog surgical instruments Glass frog heart cannula Frog heart clip Bi-concave clip Iron stent Wire Thin pipette Beaker
1. Preparation of isolated frog heart specimens
2. Connection of the experimental device The frog heart cannula was fixed on the iron stand with test tube clamp and double concave clamp. During the diastolic period of the ventricle, the tip of the heart was clamped with the frog heart clamp and the wire on the frog heart clamp was connected to the tension transducer, and the connecting wires should be kept perpendicular and appropriately loosened and tightened. Then connect the tension transducer to the microcomputer physiological signal acquisition equipment (Figure 1). 
II. Observation projects
1. Trace the normal frog heart beat curve, paying attention to the heart rate and the degree of contraction and diastole of the ventricles. Record a segment of the normal frog heart beat curve to use as a control.
2. Replace all the Ren's solution in the frog heart cannula with equal amount of calcium-free Ren's solution, observe the heartbeat changes, and record a section of heartbeat curve.
3. Aspirate out the calcium-free Ren's solution, use the normal Ren's solution to change several times, and wait until the heartbeat curve returns to normal, add 1-2 drops of 3% calcium chloride solution into the perfusate, observe the heartbeat changes, and record a section of the heartbeat curve.
4. Aspirate out the perfusion fluid containing calcium chloride, use Ren's solution to change and wash repeatedly, wait until the curve returns to normal, add 1-2 drops of 1% potassium chloride solution into the perfusion fluid, observe the heartbeat changes, and record a section of the heartbeat curve.
5. Aspirate the perfusate containing potassium chloride and replace it with Ren's solution repeatedly. After the curve returns to normal, add 1~2 drops of 1:10,000 epinephrine solution to the perfusion solution, observe the heartbeat changes, and record a segment of the heartbeat curve.
6. Aspirate the perfusate containing epinephrine and replace it repeatedly with fresh Ren's solution. After the curve returns to normal, add 1 to 2 drops of 1:100,000 acetylcholine solution to the perfusate, observe the heartbeat changes, and record a section of the heartbeat curve.
7. Add 1 drop of 10% digitalis Ren's solution in the frog heart cannula, observe for 1~2 min, if there is no obvious change in the contraction amplitude, add another 1~2 drops until there is obvious cardiotonic effect, after the cardiotonic effect is obvious, then add 10% digitalis Ren's solution drop by drop until there is obvious intoxication, observe the heartbeat changes, and record a section of the heart beat curve.