Download Section 3 ：Physiology Experiments Experiment 1 Recording of the

Section 3 ：Physiology Experiments
Experiment 1
Recording of the electrical signal of toad’s sciatic nerve
I. Preparation of the sciatic nerve --gastrocnemius muscle sample
[PURPOSE]
To study and master the preparation method of toad’s sciatic nerve- gastrocnemius muscle
sample.
[PRINCIPLE]
The life and physiological functions of amphibian are similar to that of mammals. Toad is a
kind of amphibian and its tissue samples are easy to obtain and the experimental conditions are
controlled easily. So in many animal experiments of physiology, the sciatic nerve-gastrocnemius
muscle sample is often used to observe the excitability of nerves and muscles, rules of stimulus and
response and the properties of muscle contraction.
[OBJECTS]
Toads or Frogs
[MATERIALS]
Frog board, glass board, big scissors, surgical scissors, forceps, metal probe, glass separating
needles, frog pins, burette, culture dishes, Ringer's solution.
[METHODS]
1. Destroying the toad’s brain and spinal cord
Take a toad and wash it with water. Hold the toad with one hand, press the front part of the
head with forefinger in order to expose the great occipital foramen. Hold the toad’s limbs and
abdomen with the rest fingers.
Then destroy the brain and spinal cord completely by pithing with a probe.
Hold the probe with the other hand, find the site of the great occipital foramen along the middle line
of the head and neck, prick into the great occipital foramen with the probe, then put the probe
forward to the brain, stirring it in order to destroy it. After that, withdraw the probe backward but do
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not out of the skin, then thrust it into the spinal cord, moving it around to destroy the spinal cord
completely.
If the movements and any reflexes of toad's four limbs disappear, that means brain and spinal
cord have been destroyed completely. Otherwise, the brain and spinal cord should be destroyed
again.
Fig. 3-1 Destroying the brain and spinal cord of the Toad
2. Preparation the lower limb sample
Traverse the spinal column with big scissors at the site of 1 cm away from the sacroiliac joint.
Hold the toad’s lower limbs, cut the skin on the two sides of abdomen. Be careful not to bring
the skin in contact with tools used on the sciatic nerve since the skin carries neurotoxins that will
kill the sciatic nerve. Then cut away all upper trunk and the internal organs. Also cut away the skin
around anus.
Clamp the spinal column with round head forceps (don’t touch the sciatic nerve), remove the
skin off the legs. Soak the sample in Ringer's solution and wash all tools you’ve just used and your
hands.
Separate the two parts of the thigh along the middle line of spinal column and pelvis. One half
is used to make a sciatic nerve - gastrocnemius muscle sample and the other part soaked into
Ringer's solution.
3. Preparation the sciatic nerve - gastrocnemius muscle sample
Fix the toad sample on the frog board with pins, separate the two major muscles of the thigh to
expose the white sciatic nerve, free the sciatic nerve till knee joint with glass needle along the spinal
column. Gently raise the nerve (without pinching or pulling it).
Retain a small part of vertebra linking with the nerve and cut off all the branches of the sciatic
nerve.
Separate the gastrocnemius muscle’s till the heel tendon and ligate it with thread. Cut away the
bones of the crus and muscles on thigh. Keep 1~2 cm thighbone used to fix the sample to the hole of
a electrode board. Put the sample in Ringer's solution for several minutes in order to recover of
excitability of the nerve.
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Fig.3-2 Preparing the sciatic nerve- gastrocnemius muscle sample
[POINTS for ATTENTION]
1. Be careful not let neurotoxins of the toad’s skin contaminate the nerve sample. In case
the neurotoxins spurt into your eyes, wash your eyes with water immediately.
2. Don’t clamp the nerve with forceps and pulling it.
3. Be careful and don’t damage the nerve trunk when cut away some branches.
4. To moisten the sample with Ringer's solution frequently to keep the activity of the nerve.
II. Generation of the action potential of nerve trunk
[PURPOSE]
To study the recording method of the action potential(AP) of nerve trunk. Also to know how to
analyze the AP and understand further the excitability of the nerve.
[PRINCIPLE]
AP is the basis of communications in the body. Since for the most part the nervous system, it is
the primary director of homeostasis, and it is the AP that is the signal the nervous system uses in
communications, thus understanding AP’s function is one of the primary goals of physiology.
It is very difficult to penetrate a cell with a microelectrode and record the transmembrane
action potential under laboratory conditions. However, the electrical activity of nerve can be
monitored by measuring the potential changes produced by action potentials on the extracellular
surface of the tissue, such as the surface of sciatic nerve trunk. The sciatic nerve trunk is made up of
hundreds of descending (signals from the CNS to the periphery) and ascending nerve fibers (signals
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from the periphery to the CNS). The descending fibers of the sciatic nerve innervate the muscles and
other effectors of the leg.
A resting nerve is typically polarized with a resting potential of approximately -70 mV. If both
leads of a voltmeter, separated by a distance (d), are placed on the surface of the nerve trunk, no
difference in potential between these two locations will be recorded while the nerve is at
rest(Fig.3-3A).
An action potential (indicated by the lightly shaded area in the Fig.3-3B) is initiated at the left
end of the nerve. The sodium influx in the region makes this surface negative with respect to the
surrounding regions. However, the regions under the two recording electrodes are still at rest and
there is no difference in potential between them.
A
B
Fig.3-3 Generation of the action potential of nerve trunk
When the action potential conducts into the region under the first recording electrode, the
surface of the membrane becomes negative, while the surface under the second recording electrode
is still positive. There is a potential difference.
As the action potential conducts along the nerve, the difference in potential will be zero when
the action potential is between the two electrodes, negative when the action potential is under the
second recording electrode, and zero again when the action potential has conducted past the second
recording electrode.
Because there are both positive and negative phases to this response, it is referred to as a
biphasic action potential.
[OBJECTS]
Toads or Frogs
[MATERIALS]
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Computer, biological and physiological experiments system, specimen box of nerve, a set of
surgical instruments for frog, culture dishes, burette, basin for waste, Ringer's solution.
[METHODS]
1. Preparation of sciatic nerve trunk sample of toad
(1) Destroy the brain and spinal cord of toad completely.
(2) Remove the skin off the leg.
(3) Wash the surgical instruments and your hands.
(4) Cut the sample into two parts along the middle line of spinal column and pelvis. Put one
part on the frog board for next step and the other part into Ringer's solution.
(5) Make the sciatic nerve trunk sample: Fix one leg on the frog board. Separate the sciatic
nerve along the spinal column. Tie the end of nerve with a thread near to the vertebra and
cut it away from the vertebra. Divide the muscle and tissue with nerve with glass needles,
separate the sciatic nerve from the spinal column to ankle joint making the nerve enough
longer. Cut all the branches of the nerve trunk. Tie the other end of the nerve at the ankle
joint and cut it away. Put the sciatic nerve sample in Ringer's solution for several minutes,
then do next steps of the experiments.
2. Setting up the equipment
(1) Connect stimulating electrodes and recording electrodes to the computer. Put the nerve
sample on the electrodes and make sure that the thicker end near the nerve centre are put on the
stimulating electrodes and the other end on the recording electrodes.
(2) Turn on the computer and begin to do the experiment.
Enter the menu of Biolap420 system→ experimental item(click)→the muscle and nerve
experiment(click)→the generation of the action potential of nerve trunk(click).
3. Observation and measurement
(1) Biphasic AP: observe the biphasic AP wave carefully and the phenomenon that the AP
amplitude of nerve trunk changes with the stimulus intensity to a certain extent. Then find
out the maximal stimulus intensity (the minimal effective stimulus intensity that cause the
excitation of all the never fibers).
(2) Monophasic AP: crush the nerve between two recording electrodes by forceps or drop
procaine on it, then the monophasic AP will be observed on the screen of computer.
[NOTICES]
1. Be careful not to damage the nerves when you are separating the nerve trunk.
2. The nerve trunk sample should be as long as possible and be immersed frequently with
Ringer's solution.
3. Make sure that the nerve trunk is touched to the stimulating electrode, ground electrode and
recording electrode.
4. The distance between the two recording electrodes should be much longer.
III. Conduction velocity determination of the action potential in the
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sciatic nerve.
[PURPOSE]
To understand the concept of conduction velocity of nerve excitation and learn the determining
method of it.
[PRINCIPLE]
Action potential has a definite conduction velocity. Conduction velocity is an important aspect
of cellular physiology. Different nerve fiber has different conduction velocity which depends on
many factors such as the diameter, mylenation (or the lack thereof) and the environmental
temperature and so on. Fiber Aα is the main element to the frog sciatic nerve s and its velocity is
about 35~40m/s. By measuring the distance (D) and time (t) of the nerve that the nerve impulse
travel the stimulating and recording sites, conduction velocity can be calculated from the equation
V=D/t.
[OBJECTS]
Toads or Frogs
[MATERIALS]
Computer, biological and physiological experiments system, box of nerve specimen, a set of
surgical instruments for frog, culture dish, burette, basin for waste, Ringer's solution.
[METHODS]
1. Preparation for sciatic nerve sample of toad
The method of preparing the nerve sample is the same as the preceding experiment.
2. Setting up the equipment
Connect the two pair of the recording electrodes on box of nerve specimen, also to the first
and second channels of the computer.
3. Starting the experimental item
(1) Put the nerve sample on the electrodes and make sure that the thicker end(near the nerve
center)is on the stimulating electrodes and the other end on the recording ones.
(2) Enter the Biolap420 system of the computer→muscle and nerve experiment(click) →the
determination of conduction velocity of nerve trunk excitation(click). After a dialogue
window appeared, fill in the value of the distance between the two recording electrodes,
then click the key “enter” to start a stimulus. Immediately two AP of the first and second
channel appear respectively. At the same time the data of conduction velocity also appears
automatically.
4. You can also calculate the conduction velocity.
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Record the maximal two APs led by two pair of recording electrodes R1R2 and R3 R4. Time
difference (t) is the time between the peaks of the two APs. Measure and record the distance (D)
between two recording sites R1 and R3. Then the conduction velocity can be calculated by the
equation:
Conduction Velocity (V) = distance(D)/time(t) (m/s)
[POINTS for ATTENTION]
1. The nerve trunk sample should be longer enough.
2. D value should be bigger enough and the distance between stimulating electrodes and R1
should not be too short. It’s better longer than 2 cm.
IV. Refractory period determination of the action potential in the
sciatic nerve.
[PURPOSE]
To measure absolute and relative refractory period in toad sciatic nerve by double pulses
method.
[PRINCIPLE]
When the excitable tissue is excited by a stimulus, its excitability has a regular periodical
change and goes through the absolute refractory period, the relative refractory period, the
supernormal period and the subnormal period, then returns to normal condition. Measurement of the
threshold can be used to observe the excitability of tissue. In order to measure the periodical change
of the nerve excitability, a conditional stimulus is n to the nerve firstly, then a testing stimulus is
used at different phases of the previous excitation and check the threshold of the nerve responding
to the testing stimulus as well as the AP amplitude to determine the periodical change excitability of
excitable tissue.
[OBJECTS]
Toads or Frogs
[MATERIALS]
Computer, biological and physiological experiments system, box of nerve specimen, a set of
surgical instruments for frog, culture dish, burette, basin for waste, Ringer's solution.
[METHODS]
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1. Preparation for sciatic nerve sample of toad
The method of preparing the nerve sample is the same as the previous experiment.
2. Setting up the equipment
(1) Connect stimulating and recording electrodes with computer, also to the box of nerve
specimen respectively. Put the sample on the electrodes and make sure that the thicker end
(near to nerve center) is on the stimulating electrodes and the other end on the recording ones.
Pay attention to not let the nerve fold or twist.
(2) Enter the experimental item
Turn on the computer and enter the Biolap420 system, → the experimental item(click)→
the muscle and nerve experiment(click) →measurement of refractory period on toad sciatic
nerve(click).
3. Measurement of refractory period on toad sciatic nerve
(1) Give a stimulus intense enough and generate a biphasic AP. Regulate the interval and
generate two separate biphasic AP.
(2) Slowly reduce the stimulus interval until the second AP’s amplitude just starts to be reduced
when compared to the first AP. This interval is the Relative Refractory period in ms.
(3) Continue to slowly reduce the interval until the second AP completely disappears. This
interval is the Absolute Refractory Period in ms.
(4) Repeat steps 1-3 at least three times.
（Aiping Li）
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Experiment 2
Muscle contraction experiments with toad’s sciatic nervegastrocnemius muscle sample
I. Influences of Different Stimulus Intensity on the Muscle Contraction
[PURPOSE]
1. To observe the influences the different stimulus intensity on muscle contraction by
stimulating the sciatic nerve-gastrocnemius muscle sample;
2. To understand the concepts of stimulus, reaction and excitability.
[PRINCIPLE]
The characteristics that the living tissues produce the reaction on the stimulus are called the
excitability. Muscle, nerve and gland are called excitable tissues. Different tissue and cell shows
different excitability. Nerve excitability represents as action potential (AP)and muscle represents as
contraction. So we can judge if the muscle’s excitability by observing it’s contraction.
The threshold intensity is the standard to judge the tissue excitability. As far as the muscle
tissue is concerned, the minimum stimulus intensity which causes the muscle contraction is called
the threshold intensity (the stimulus of this intensity is called the threshold stimulus; the stimulus
much lower in intensity than the threshold intensity is called the sub-threshold stimulus; the
stimulus much greater in intensity than threshold intensity is called the supra-threshold stimulus).
When the stimulus intensity increases gradually, the amplitude of the muscle contraction increases
correspondingly, too. When the stimulus intensity rises to certain level, the amplitude of the muscle
contraction reaches the greatest value. The stimulus intensity increases continually after this, but the
amplitude of the muscle contraction no longer increases. The minimum stimulus intensity which
causes the muscle to have the maximal contractive amplitude is called the optimal intensity, and the
stimulus with this intensity is called the maximal stimulus
Some basic vital activities and the physiological functions of the toad are similar to that of the
mammal. Because the living conditions of the frog tissue in vitro are very simple and easy to control,
the sciatic nerve-gastrocnemius muscle sample is usually used to observe the excitability, the course
of excitement and the contraction characteristics of muscles in physiology experiment.
[OBJECTS]
Toads
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[MATERIALS]
BL-410 computer teaching system of physiological and pharmacological experiment, operating
table for frog, frog board, dropper, the zinc copper bend, Ringer’s solution.
[METHODS]
1. Preparation of sciatic nerve-gastrocnemius muscle sample（Fig3-4）
Fig3-4 Preparation of the sciatic nerve-gastrocnemius muscle sample
The procedure of the Preparation of sciatic nerve-gastrocnemius muscle sample see previous
Experiment 1.
(1) Destroy brain and spinal cord.
(2) Cut away the upper part of it’s body and internal organs, shell it’s skin.
(3) Separate sciatic nerve
Separate the sciatic nerve along the spiral cord, tie the end of it and cut away. Separate the
sciatic nerve from the sciatic nerve ditch, cut all the branches to the knee joint.
(4) Get the sample
Cut all the muscle at knee joint’s crape the thighbone with a big scissors, cut the upper
thighbone(remain 2/3 of it). Separate the gastrocnemius muscle’s tendon and ligate it with
thread. Cut away the fibula muscle and separate it, and cut away the other part below the
knee joint.
（5） Check the excitability of the sample
Touch the sciatic nerve with the zinc copper bend which immerses the Ringer’s solution,
and observe the gastrocnemius contraction. If the gastrocnemius contracts, it indicates that
the sample has excitability. Dip the sample in Ringer’s solution for about 10 minutes and
await recover of excitation.
2.
Fixation of the sample and connection of the equipment
(1) Fix the thighbone of the sample in a hole near the electrode, put the gastrocnemius muscle
above the thighbone, connect ligation thread with the tension transducer, the to the
computer. Put the sciatic nerve on the electrode. (Fig3-5);
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Fig 3-5 schematic diagram of experimental connnection
(2) Join up the output of electronic stimulator and electrode;
(3) Open the computer experimental system. Enter the menu of Biolap420 → item of
experiment → muscle-nerve experiment(click) → the relationship of stimulus and
response(click). When the stimulus intensity is increase gradually; the muscle contraction
will appear. The stimulus at this moment is called the threshold stimulus. Write down the
intensity at this moment. The stimulus lower than threshold intensity is called the
sub-threshold stimulus. Continue to increase the intensity of stimulus, the amplitude of the
muscle contraction increases correspondingly, too. Observe the maximal stimulus. When
the stimulus intensity rises to a certain intensity, the amplitude of the muscle contraction
reaches the greatest value. The stimulus intensity increases continually after this, but the
amplitude of the muscle contraction increases no longer. The minimum stimulus intensity
that causes the nerve to have the maximal amplitude is called the maximal stimulus.
(4) After the experiment, identify the threshold stimulus, the sub-threshold stimulus, the
supra-threshold stimulus and the maximal stimulus.
3.
Observation items
Observe the relationship between intensity and contraction, record the muscle-shrink-curve,
work out the threshold stimulus, the maximal stimulus .
[POINTS for ATTENTION]
1. When you separating the nerve , prevent metal apparatus from touching the nerve .
2. Soaked the sample with Ringer solution in order to keep the activity.
3. Make sure that all the equipments were connected well.
Ⅱ. Influences of Different Stimulation Frequencies on the Muscle
Contraction
[PURPOSE]
To observe the single twitch, the incomplete tetanus and the complete tetanus of the
gastrocnemius muscle.
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[PRINCIPLE]
The forms of muscle contraction is not only depend on the stimulus intensity, but also the
stimulus frequency. When the interval between two stimuli is longer than the period of single
muscle contraction and relaxation, the contraction is called single twitch. After the stimulus
frequencies are changed, the muscle has the different contraction forms. If the interval is longer,
there are a series of signal twitches. If the stimulus frequencies are increased gradually, the stimulus
interval becomes shorter. If the diastole of the previous contraction cycle has not finished, and the
next contraction appears again, then the toothed curve will appear. The toothed curve shows that the
muscle contraction form is the incomplete tetanus. If the stimulus frequencies are increased
continually, the interval time of stimulus is much shorter than the systole, and the muscle contract
persistently . This muscle contraction form is the complete tetanus.
[OBJECTS]
Toads
[MATERIALS]
BL-410 computer teaching system of physiological and pharmacological experiment, operating
table for frog, frog board, dropper, the zinc copper bend ,Ringer’s solution, the iron bracket, the
glass needles, the threads, the stimulation electrode, the tension transducer.
[METHODS]
1. Preparation of sciatic nerve-gastrocnemius muscle sample Put the sample into the culture
dish of the Ringer’s solution for 10 min to keep its excitability;
2. Open the computer experimental system. Enter the menu of Biolap420 → item of
experiment →muscle-nerve experiment(click) →the classic experiment(click) to observe the signal
twitch, the incomplete tetanus and the complete tetanus(Fig 3-6)
Fig 3-6 Influences of the muscle contraction through different stimulation frequencies
3. Observation items
Observe the relationship between frequency and contraction, record single twitch, incomplete
tetanus and complete tetanus.
[POINTS for ATTENTION]
1.Use the supra-threshold stimulus, and keep the stimulus intensity unchangeable in the
experiment;
2. Don’t stimulate the muscle consistently;
3. Add the Ringer’s solution frequently on the sample.
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[GUIDANCE]
1. Preview
(1) Master the concept of excitability.
(2) Master the mechanism of the chemical transmission of excitation at nerve-muscle joint
(3) Understand the principle of contraction.
(4) Understand the response of contraction and analysis its mechanism.
2. Manipulation
(1) Master the method of preparation of sciatic nerve- gastrocnemius muscle. Do not damage
the joint of nerve and muscle. Do not touching the nerve with the hand and other metal
tools except glass needle when separating the sample; Do not pull the nerve excessively .
(2) stop stimulus in time in order to avoid fatiguing of nerve.
3. Reports
(1) Get the clear curves of the muscle contraction.
(2) Discuss the relationship between stimulus intensity, frequency and muscle contraction,
and work out the principle of contraction.
（Liang Zhu）
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Experiment 3
Premature Contraction and Compensatory Pause of Toad Cardiac
Muscle
[PURPOSE]
1. To study the methods of recording the beating curve of the frog’s heart in vivo;
2. To prove the characteristics of especially long effective refractory period of the cardiac
muscle by observing the premature systole and the compensatory pause and try to get a further
knowledge of the periodicity regulation of cardiac muscle’s excitability.
[PRINCIPLE]
The specialized excitatory and conductive system of the heart has the autorhythmicity, but
autorhythmicity differs in various positions of the heart. The autorhythmicity of the sinoatrial node
(also called the sinus node in Amphibia) is the highest, and controls the rhythmic heartbeat, so it is
called the normal pacemaker. The pacemakers elsewhere except the sinus node are called the ectopic
pacemakers.Their autorhythmicity is lower than sinoatrial node .
Once cardiac muscle excites, its excitability will change periodically. And its effective
refractory period is comparatively much longer, almost equals to the whole systole and pre-diastole.
During this period, any stronger stimulus can not cause the cardiac muscle excite and contract. But
during the later period of diastole, a suprathreshold stimulus before the normal rhythmicity
excitation arriving at ventricle can cause the heart excitation and contract . It is called premature
contraction. Premature contraction has also its own refractory period. So if the normal rhythmicity
excitation transmitted by sinus venous is in the effective refractory period of premature contraction,
the heart can’t be excited and contracted. and ventricle is in diastole. It is not until the next normal
rhythmicity excitation arrives that the ventricle recovers to normal rhythmicity contract. This long
diastole after the premature systole is called the compensatory pause
[OBJECTS]
Toads
[MATERIALS]
BL-410 computer experimental teaching system, stimulate electrode, tension transducer, iron
bracket, double cave clamp, operational plate for toad, frog board, toad heart clip, beaker, dropper,
suture, probe, the glass needles, the burettes, the threads, Ringer’s solution
[METHODS]
1. Destroy the toad’s brain and spinal cord by probe, and put the toad on frog board. Open the
thoracic cavity, and cut pericardium to expose the intact, in-situ heart.
2. Know about the structure of Toad’s heart (Fig.3-7):
(1) Anterior view: the ventricle, the atrium (left & right), the arterial cone, the aorta (left &
right), the atrio-ventricular groove (A-V groove, between the atrium & the ventricle)
(2) Dorsal view: the sinus (conjoin with the atrium), the sinus-atrium groove (S-A groove,
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between the atrium & the sinus, the lune-shaped white list)
Fig. 3-7 Sketch map of the toad heart
2. Clip apex of the heart for 1 mm in the diastole with a toad heart clip connected with tension
transducer. Fix the stimulating electrode at iron bracket with ventricle between the two poles
of electrode, and then connect the heart to recording equipment (fig 3-8).
Fig. 3-8 The connection of recording equipment
3. Open BL-410 computer experimental teaching system Enter the menu of Biolap420→ item
of experiment →circulation experiment(click) →Premature Contraction and Compensatory
Pause (click).
4. Observation Items
(1) Record a period of normal heart beating curves, to recognize the curve of systole and
diastole.
(2) Stimulate the ventricle in the systole by the single supra-threshold stimulus, observe the
curve of the contraction of the ventricle.
(3) During the earlier, middle, later period of diastole, Stimulate the ventricle by the single
suprathreshold stimulation, observe the premature contraction and compensatory pause.
(some forms of the results are shown in Fig 3-9)
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Fig 3-9 The cardiac contraction curve of premature contraction and compensatory pause on
toad. PC. premature contraction CP. compensatory pause a, b, c stimulation in absolute refractory
period, d, stimulation in relative refractory period
[GUIDANCE]
1. Preview
(1) Preview the physiological characteristics of cardiac muscle.
(2) Preview the operation methods of BL-410 computer experiment teaching system.
2. Manipulation
Stimulating electrode should be contact to ventricle.
3. Points for attention
(1) Thoroughly destroy the brain and spinal cord.
(2) Add Ringer’s solution frequently during the experiment to keep the heart’s activity during
the experiment.
(3) Don’t stimulate the heart constantly.
(4) The thread should be erect between the heart and the tension transducer.
(5) The stimulating electrode should keep in touch with the ventricle in both the systole and the
diastole.
(6) The stimulus intensity which stimulates the abdomen muscles to bring the contraction
should be checked before the stimulation electrode is connected with the heart.
4. Report
Mark, Clip and paste the experiment record curve. Each student should have one copy of the
curve and try to account for the premature contraction and compensatory pause.
（Liang Zhu）
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Experiment 4
Effects of chemical substances on the activity of the isolated toad heart
[PURPOSE]
To observe the effects of three ions (sodium, potassium and calcium) and two chemical
substances (acetylcholine and adrenaline) on the activity of the toad heart in vitro.
[PRINCIPLE]
1. The cardiac muscle has autorhythmicity. For human body, the pacemaker of the heart is SA
node.
2. The vein sinus, which is the pacemaker of the heart in the amphibious animal, such as toad
or frog, can automatically produce the rhythmic excitation.
3. The isolated toad heart in a simulated physiological environment, despite out of the nerve
control, can also keep the rhythmic activity and produce rhythmic constriction in a time. Therefore,
the normal rhythmic activity of the isolated heart depends on the stability of the internal
environment.
4. Ringer’s solution is the simulated physiological environment. Therefore, which is the
internal environment of the isolated heart.
Tab.1 The main composition of Ringer’s solution
Composition
Percentage
NaCl
0.65%
KCl
0.014%
CaCl2
0.012%
5. Alteration of the component in the Ringer’s solution, such as no Ca2+, high Ca2+, high K+,
can change the activity of the isolated heart.
[OBJECTS]
Toads or Frogs
[MATERIALS]
1. Equipments BL-410 computer teaching system of physiological and pharmacological
experiment, tension transducer, operational instruments of frog, toad heart clip, pulley, tube clip,
iron bracket, double cave clamp, toad heart cannula, operational plate for toad, dropper, beaker,
suture thread.
2. Solutions Ringer’s solution, 0.65% NaCl, 2% CaCl2, 1% KCl, 1:10000 adrenaline and
1:10000 acetylcholine.
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[METHODS]
1. Preparation of the isolated heart
(1) Take a toad, after destroying its brain and spinal cord, fix it on the toad plate on back. Open
the thoracic cavity, and cut pericardium to expose the heart.
(2) Put two lines under the two aortas. One, made a knot at the superior extremity of the left
aorta, is used to tow when intubation; the other, above arterial cone, is joined a slipknot to ligate and
fix the cannula of the heart.
(3)The left hand holds the ligature above the left aorta, shears a reversed “V” shaped incision
with ophthalmological scissors at root of the left aorta and above the slipknot (doesn’t cut off the
aorta); the right hand inserts a proper cannula of toad heart that contains Ringer’s solution into the
artery cone from the cut position. When the top of the cannula reaches the arterial cone, withdraw it
a little, and turn it to the direction of the ventricle central (left rear). The left hand holds up the tissue
around atrioventricular groove with a forceps, the little finger or the ring finger of right hand pushes
the ventricle gently. Insert the cannula into the ventricle at the systole. Avoid overexerting or
intubating deeply. If the cannula has already entered the ventricle, the surface of Ringer’s solution in
the cannula would fluctuate up and down following the relaxation and contraction of the ventricle.
Take out the blood in the cannula with a dropper, and wash it 1-2 times with fresh Ringer’s solution.
Ligate the prepared slipknot tightly, and fix it on the side hook of the cannula to prevent the cannula
to slip out of the ventricle. Cut off two aortas.
(4) Hold up the cannula gently to raise heart, make a ligation at the boundary of the venous
sinus and the vena cava. In order to avoid harming the venous sinus, the ligation should be made
as low as possible. Cut off the vena cava under the ligation; dissociate the heart from the toad.
(5) Exchange the liquid in the cannula with fresh Ringer’s solution repeatly avoiding the
blood coagulation. At this time an isolated toad heart has already been made successfully and can be
used for experiment.
2. Apparatus installment
Link the BL-410 computer experimental teaching system according to fig. 3-10.
Fig3-10 The apparatus installment of the isolated toad heart
At first， clamp the canula with a wooden tube clip, and then the wooden tube clip is fixed on
the iron bracket by a double cave clamp. Clamp the tip of the heart at ventricle diastole with a toad
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heart clip that is linked with a long line in the other end, and then link the line to the adaptive bridge
in the tension transducer (also can be linked to a transducer through a pulley). Regulate the position
of the pulley or the double cave clamp in order that the tensility of line is appropriate.
3. Observation items
(1) Record the normal curve of the relaxation and constriction of the isolated heart. Pay
attention to the frequency and the amplitude of the heartbeat.
(2) After the Ringer’s solution in cannula is all replaced by 0.65% NaCl, observe the change of
the heartbeat.
(3) Take out the 0.65% NaCl, wash it several times repeatedly with fresh Ringer’s solution.
When the heartbeat recovers to normal, add 2% CaCl2 1~2 drops to the Ringer’s solution in the
cannula, and then observe the change of the heartbeat.
(4)Take out the Ringer’s solution including CaCl2, wash it several times repeatedly with fresh
Ringer’s solution. After the curve recovers to normal, add 1%KCl 1~2 drops in the Ringer’s solution
in the cannula, observe the change of the heartbeat.
(5)Take out the Ringer’s solution including KCl, wash it several times repeatedly with fresh
Ringer’s solution. After the curve recovers to normal, then add 1:10000 adrenaline 1~2 drops in the
Ringer’s solution in the cannula, observe the change of the heartbeat.
(6)Take out the Ringer’s solution including adrenaline, wash it several times repeatedly with
fresh Ringer’s solution. After the curve recovers to normal, add 1:10000 acetylcholine 1~2 drops in
the Ringer’s solution in the cannula, observe the change of the heartbeat.
[GUIDANCE]
1.Preview
(1)Review the associated theory of nerve innervation of the heart and the regulation on the heart
activity.
(2) Review the four physiological characteristics of the cardiac muscle and the factors influencing
the heart activity.t
2.Manipulation
The difficult point in the experiment is how to insert the cannula into the ventricle. Insert the
cannula at ventricular systole, because the aortic valve is open at this time. Moreover, when ligating
between the venous sinus and the vena cava, don’t ligate the venous sinus(pacemaker position) .
When one experimenter is ligating, the other experimenter can press down the line knot with a
forceps, then ligate it tightly.
3.Points for attention
(1) When isolating the heart, don’t harm the venous sinus, take out the toad heart with the venous
sinus together.
(2) The droppers to take the fresh Ringer’s solution and to take the liquid in the cannula should be
separated in case it affects the observation of the experiment phenomenon and analysis of the
results.
(3) Keep the stable height(2cm) of the liquid in cannula.
(4) When observing each item, wash the cannula with fresh Ringer’s solution in time after the effect
becomes obvious lest harming the cardiac muscle and recovering the heartbeat hardly. Don’t turn to
the next item until the heartbeat recovers to normal.
19
(5) As altering the component in the infuse liquid, mark it in time
4.Report
(1)Attach the recorded curve on the experimental report and describe the experiment phenomenon.
(2)Analyze the effects of Na+, Ca+, K+, adrenaline and acetylcholine on the heartbeat according to
the result. What is the mechanism ?
(Yumei Zhang)
20
Experiment 5
The human body experiments
Ⅰ. Measurement of the arterial blood pressure of the human body
[PURPOSE]
The aims of this experiment are to comprehend the principles of measuring of the arterial blood
pressure by using a sphygmomanometer with a cuff and master the methods. The systolic pressure,
diastolic pressure and pulse pressure will be measured at the brachial artery.
[PRINCIPLE]
The arterial blood pressure is formed by the wall pressure which the flowing blood exerts on
the walls of blood vessels. Up to now the most simplest and useful indirect method for measuring
the blood pressure in human is still using a sphygmomanometer with a cuff.
The principle of the method is that to place a cuff over the branchial artery around the upper
arm, and a stethoscope is placed over the antecubital artery. Firstly occlude the underlying artery
and interrupt pulse transmission via inflating air into the cuff to a level above the expected systolic
pressure. Then through the slowly deflating, the cuff pressure is lowered gradually, the occluded
brachial artery opens more and more with each pulse to allow blood to spurt through at high velocity.
Turbulence is generated and the arterial wall oscillates, producing characteristic changes in sound
“Korotkoff sounds”. The sounds are heard in auscultation from no noise to sharp tapping sounds, the
pulse of the radial artery could be palpated. And the value of the blood pressure is read at the same
time on the dial. The maximum blood pressure is measured in the arterial system during cardiac
ejection at the cardiac cycle, the arterial pressure falls progressively. The sound continues to be
heard during the blood goes interruptedly through the artery beneath the cuff, the artery remains
distended with blood as soon as the pressure in the cuff is equal or lower slightly than the diastolic
pressure, no sounds are heard in the lower artery as the factors forming an eddy current no exist.
The minimal blood pressure at the point of silence read on the dial is the diastolic blood pressure.
The difference between systolic and diastolic pressure is the pulse pressure.
The arterial blood pressure of human body,which is regulated by both neural regulation and
humoral regulation, is stable under natural status. But it is dynamic and many factors such as body
position, movement, respiration and temperature can affect it.
[OBJECTS]
21
Humans
[MATERIALS]
Stethoscope, sphygmomanometer.
[METHODS]
1. The volunteer takes off a sleeve and calmly sits down for more than five minutes.
2. Lay the forearm on the table with palm upwards, keeping the height of forearm equals to the
position of heart. A cuff is winded over the forearm with proper tightness. The low border of
the cuff should be 2 cm above the elbow joint at least.
Fig3-11 Methods of the blood pressure measurement
3. Palpate the pulse point at the brachial artery in the elbow fossa, then put the chest piece of the
stethoscope there. Attention: don’t press the chest piece strongly.
4. Use the rubber ball to pump gas into the cuff with hand as one hand flip slightly the chest
piece of the stethoscope, The mercury circle in the glass dial rises gradually. When no sound
can be heard from the stethoscope, stop pumping gas (generally near to 180 mmHg). Thereby
occluding the underlying artery and interrupting pulse transmission. Whereat loosen the nut in
the balloon, and the pressure cuff is then slowly deflated. Listen to the sound carefully the
column of mercury declines slowly .When suddenly hearing the sharp sound likes “tapping,
tapping”(Korotkoff sounds), please pay attention to the height of the mercury column. Record
the scale beside the height, it expresses the systolic pressure (SP).
5. Continue deflating the gas slowly and the sound occurs a series of change at the same time:
first from low to high, then from high to low suddenly, and at last disappears completely.
When the sound changes from strong to weak suddenly, the scale of mercury circle of
sphygmomanometer represents the diastolic pressure (DP). Record the value.
6. Calculate the pulse pressure (PP): The difference between the systolic pressure the and
diastolic pressure is the pulse pressure.
[POINTS for ATTENTION]
1. Keep the room in silence in order to auscultate.
2. The tightness of the cuff winded should be proper.
3. The artery pressure is usually measured 2 or 3 times repeatly, the interval of measurement is
two or three minutes. The cuff pressure must be returned to zero when you want to measure
again. The value usually take the approximate values in three times measurement.
4. The forearm and the right atrium is at the same level, and the cuff of the sphygmomanometer
22
should be winded above the elbow fossa.
5. The chest piece of stethoscope is put on the pulse position of the brachial artery. Don’t presse
it heavily and also can’t don’t presse it under the cuff. The chest piece may be touched to the
skin too loosely to hear nothing.
6. When the blood pressure exceeds the normal scope, let the volunteer have a rest for 10
minutes and measure again.
7. Use the sphygmomanometer correctly: when the cuff begins to be inflated, turn on the switch
which is at the root of the mercury column, and turn off the switch once the measurement of
the blood pressure is over.
[APPENDIX]
1.The Korotkoff sound diagram in measurement of blood pressure in normal individual with
auscultatory method.
Fig.3-12 The Korotkoff sound diagram
Ⅱ. ABO Blood Group System and its Identification
[PURPOSE]
To learn the method of erythrocyte’s blood grouping identification , and grasp the principle and
significance of ABO blood group system identification by the observation of erythrocyte
agglutination.
[PRINCIPLE]
Blood has been classified, according to its antigenic activity, as types O, A, B and AB. The
principle of ABO blood group system identification is mainly determined by specific antigen which
is on the surface of the membrane of erythrocyte. The antigen (or be called agglutinogen) is
determined by the inherited properties of red cells. Blood-group antigens belong to glycoproteins
and glycolipids. There are three types of blood-group antigens: O, A, and B. They differ only
23
slightly in the composition of carbohydrates (see the figure 3-13.). Antibody or agglutinin, which is
in the serum, can cause agglutination with the antagonistic antigen or agglutinogen and result in
erythrocyte haemolysis. Thus, the blood group identification still should be done clinically for safe
blood transfusion.
Fig 3-13 Specific blood group antigenantigen
on locating in the erythrocyte membrane
Generally, when an antigen is present in the blood, the antagonistic antibody is absent.For
example, type O blood possesses “no antigen” and both antibodies A and B are present, type AB
blood possesses both antigen A and B consequently no antibodies are present and so forth.
[OBJECTS]
Humans
[MATERIALS]
Standard antibody A and antibody B, single-use blood taking needles, slides, 75% alcohol,
small bamboo needles, medical cottons, microscope.
[METHODS]
1. Divided a clean slide along the midline with a pen;
mark the left as “A” and the right as “B” to indicate
antibody A and B respectively. Put a drop of antibody A in the middle of the left side of the slide;
and put a drop of antibody B in the middle of the right side of the slide.
2.Rub ear lobe or finger tip for 2 or 3 minutes with the cleaning hands, making these sites getting
red.
3. Disinfect the skin of ear lobe or finger tip with cottons in 75% alcohol, and then make it bleeding
by sting into the skin for about 2 mm with a single-used blood taking needle. Scrape a drop of blood
with one side of a bamboo needle and add it into the serum A；Dip a drop of blood with another side
of this bamboo needle and add it into the serum B. Mix up the blood and serum by shaking the slide
with hands .
24
Fig.3-14 Blood typing of ABO blood types
4. Put the slide in static under room temperature for 10 minutes and observe whether there is a
agglutination with naked eyes. Shake the slide again if the agglutination can not be observed
clearly.Observe whether or not the agglutination happens under a microscope after 30 minutes, and
identify blood types of subjects according to the agglutination. If you have questions about the
result, you may detect it again .
5.Determine blood group (see Fig.3-14).
[POINTS for ATTENTION]
1.Don’t dip blood too much to avoid the mass formation in the serum.
2.Use different side of on bamboo needle to add blood into the serum. Avoid the cross
contamination.
Ⅲ．Human ECG recordings
[PURPOSE]
The purpose of this experiment is to study a method of the electrocardiogram ( ECG) recording
and comprehend the physiological significance of all waves and its intervals.
[PRINCIPLE]
The cardiac muscles excite before the contraction. Electric potentials will show a serial of
changes during the conduction of excitation in the heart according to some procedure. The change
of electric potential spreads from the heart into the adjacent tissues surrounding the heart, a small
proportion of the current spreads all the way to the surface of the body. If electrodes are placed on
the body skin according to some rules, electrical potentials generated by the current can be recorded.
The recording is known as an electrocardiogram.
[OBJECTS]
25
Humans
[MATERIALS]
Electrocardiogram machine, electric paste, compasses, magnifier.
[METHODS]
1. Operating procedures of the recording electrocardiogram
⑴ After connecting to the power line, ground wire, lead lines, turn on the power switch ,and
warm up the machine for three to five minutes.
⑵ The volunteer should lie on the examining table and relax muscles.The knobs are placed on
the wrists, ankles and the front of chest, then link the lead lines. The conduct electric paste is
coated to those positions where the electrode will be placed in order to reduce the skin electric
resistance. The way of connecting the lead lines is red-right hand, yellow-left hand, green-left
foot, black-right foot (groundwire) and white-chest.
Fig. 3-15 Mode chart of electrocardiogram
⑶ Regulate the enlargement factor of electrocardiograph ,then record the Electrocardiogram,
ECG.Ⅰ,Ⅱ,Ⅲ,aVR,aVL,aVF,V1, V3,V5 are chosen by “lead choice” respectively, record the
ECG of all the leads one by one, generally only doⅡin this experiment. Take down
electrocardiograph paper for analysis.
2. Analysis of ECG
⑴ Measure wave amplitude and interphase
① Wave amplitude: If the standard voltage is 1mV/10mm, then 1 mm in ECG equals to 0.1 mV.
The amplitude of the positive wave is from the upper edge of the base line to the top of the
wave. The amplitude of the negative wave is from the nether edge of the base line to the
lowest point of the wave.
② Interval: When recording the ECG, generally the paper speed is 25 mm/s and 50 mm/s.The
paper speed of 25 mm/s is often selected, then each small pane is 0.04 s.
⑵ Identify the normal waves and interphase in ECG:P wave, QRS complex, T wave and P-R
interval,Q-T interval, and analyse these items.
① Measure heart rate: measure the P-P interval or R-R interval between two sequential cardiac
cycles, then calculate the heart rate with the formula .If the intervals of cardiac cycle is
obvious different, calculate the average value with five intervals of cardiac cycle.
Heart rate = 60 /P-P interval(s) or R-R interval(s)
② Analysis of cardiac rhythm: the analysis of cardiac rhythm include judgements of the main
26
cardiac rhythm, identification of cardiac rhythm and whether or not appearance premature
systole and ectopic rhythm.
The recording of Sinus rhythm in ECG is positive P waves in lead Ⅱ,negative
in lead aVR and P-R interval is up 0.12s.You can diagnose arrhythmia if the
difference between the longest P-P interval and the shortest P-P interval is over 0.12 s. The
normal heart rate in adult is 60~90/min.
[POINTS for ATTENTION]
1.
2.
3.
Metal substance must be removed from the volunteer before recording ECG.
Muscles in the body should be relaxed in order to eliminate the interference with the muscle
electricity.
The machine must be connected rightly with the ground.
(Hua Piao)
27
Experiment 6
Digestion Experiment of Rabbit
[PURPOSE]
To observe the motion types of mammalian gastrointestinal tract under anaesthesia and their
regulations by the nervous and humoral systems.
[PRINCIPLE]
The gastrointestinal smooth muscles contract with spontaneous rhythm. Peristalsis is the basic
form of gastrointestinal motility. Moreover, adverse peristalsis, migrating myoelectric complex
(MMC) and segmentation contraction all are other patterns of movement and serve different
functions. In vivo, the motility of gastrointestinal tract is regulated by nervous and humoral factors.
[OBJECTS]
Rabbits
[MATERIALS]
Operating instruments of mammals, protective electrode, Tyrode’s solution, normal saline, 25%
urethan, atropine, neostigmine, 1:10 000 adrenalin solution, 1:10000 acetylcholine solution.
[METHODS]
1. Anaesthesia
(1) Grasping and weighing the rabbit.
(2) 25% urethan solution (4ml/kg) was injected into the vein in the edge of the rabbit’s ear.
2. Operation
(1) Operation on cervix: After fixed on the operating table on back, the rabbit was subjected to
a skin incision on midline of its neck with its hair sheared. The unilateral vagus nerve was
separated and prepared for the next step.
(2) Operation on abdomen: The rabbit was undergone laparotomy along linea alba abdominis
with its abdomenal hair sheared and its stomach and gut were exposed. The two brims of
the abdomenal incision were nipped and lift up to form a furry bag by two pairs of dentate
hemostats, which were fixed on the support of the C-shape clamp.
3．Observation of gastrointestinal motility under various conditions
Firstly the gastric peristalsis and tensity; intestinal peristalsis and segmentation
contraction in physiological states should be observed..
28
After the following treatments, the gastric peristalsis and tensity; intestinal peristalsis and
segmentation contraction should be observed again.
(12) Stimulation of vagus nerve.
(2) Adding 2-3 drops of 1:10 000 adrenalin on surface of the intestine.
(3) Adding 2-3 drops of 1:10 000 acetylcholine on surface of the intestine.
(4) Adding 0.2~0.3 ml atropin on surface of the intestine.
(5) Adding 0.2~0.3 ml neostigmine on surface of the intestine.
[POINTS for ATTENTION]
1. The skin in the rabbit’s back can be grasped when it is captured, but not its ears.
2. Slow injection was necessary during anaesthesia to avoid dying of the rabbit and the status
of the rabbit should be observed closely including cornea reflex, respiration（slow and
rhythmic）, and the reaction to pain.
3. The stomach and the gut should keep wet using warm normal saline during the experiment
to avoid that the celiac temperature decreased and the gastrointestinal motility was affected.
4. In observation items, the first 4?steps can be operated on the same segment of the intestine
except the last one.
（Lili Guan & Yuan Zou）
29
Experiment 7
Respiration Experiment of Rabbit
[PURPOSE]
To observe the effects of some factors on respiratory movement.
[PRINCIPLE]
Respiratory movement depends upon cyclical inspiratory muscle excitation by the nerves to
the diaphragm and intercostals muscles. This neural activity is triggered by the inspiratory neurons
in medulla. Ventilation occurs reflexly via chemoreceptors or medullary inspiratory neurons by
various stimuli.
[OBJECTS]
Rabbits
[MATERIALS]
A computer, bioenginery experimental system, 25% urethan, tensile transducer, protective
electrode, tracheal cannula, operating instruments of mammals, rubber tube (50~100cm), ballonet, a
bottle filled with soda lime, gauze, beaker, thread.
[METHODS]
1. Anaesthesia
(1) Grasping and weighing the rabbit.
(2) 25% urethan solution (4ml/kg) was injected into the vein in the edge of the rabbit’s ear.
2. Operation:
(1) Endotracheal intubation: The rabbit was fixed on the operation table on back and
subjected to a skin incision on midline of its neck with its hair sheared. The trachea was
detached from nearby constitutions and thread was placed under the trachea. An inverse
“T” shaped incision was made on the trachea 1cm far from the cricoid cartilage and the
trachea cannula (“Y”shape) was inserted into the airway fixed with the thread. (Note: to
keep the incline of the cannula orifice downward and overturn it when inserted in). The
blood and secretion within the trachea should be cleaned out. The operating field should
be covered with warm gauze soaked by normal saline.
(2) The bilateral vagus nerves were separated and prepared for the next step with silk thread
put under each nerve.
30
3. Experimental equipments
(1) The tensile transducer was tied around the rabbit’s chest with appropriate tension and its
output was connected with the input of the computer.
(2) Turn on the computer and enter the menu of Biolap420 → item of experiment (click) →
Respiratory experiment(click) → Regulation of respiratory movement (click).
4. Observation items
(1) Record a normal respiratory curve as control.
(2) Effect of high CO2 on respiratory movement: One side of the trachea cannula was
connected with the CO2 ballonet with the other side blocked. Then the screw clip on the
ballonet was opened to make some CO2 influx into trachea. Observe and record the
changes of respiratory movement.
(3) Effect of decreased external O2 tension on respiratory movement: One side of the trachea
cannula was connected with the air ballonet through the bottle filled with soda lime, and
the other side of the airflow was blocked. The oxygen in the bottle became less and less
along with the rabbit’s respiration because the carbon dioxide was absorbed by soda lime.
Observe and record the changes of respiratory movement.
(4) Effect of anatomical dead space enlargement on respiratory movement: One side of the
trachea cannula was connected with a rubber tube (50~100cm) to increase the anatomical
dead space of pulmonary ventilation, and the other side was blocked. Observe and record
the alteration of respiratory movement.
(5) Effect of blocked airflow entirely (asphyxia) on respiratory movement: Both sides of the
trachea cannula were blocked promptly. Then the clips were undone immediately when
the rabbit’s respiration was strengthened and otherwise the rabbit would die of asphyxia.
(6) Effect of stimulating vagus nerve on respiratory movement: The unilateral or bilateral
vagus nerve was ligated and cut off, and the changes of respiratory movement would be
observed. Then the central end of vagus nerve was stimulated and lasted 5-10s, and the
changes of respiratory movement would be observed.
[POINTS for ATTENTION]
Observe the respiration of the rabbit closely and clean out the blood and secretion within
the trachea in time to avoid asphyxia.
（Lili Guan & Yuan Zou）
31
Experiment 8
Regulation of artery blood pressure
[PURPOSE]
To measure directly the arterial blood pressure (BP) and observed the effects of neural and
humoral factors on artery blood pressure.
[PRINCIPLE]
The intra-cardiac sympathetic nerve and vagus nerve are both dominating nerves of heart. By
changing heart rate, ventricular contractility and excitation conduction, stimulation of the cardiac
sympathetic nerve through the neuron’s release of the neurotransmitter－noradrenalin which
combine with β– adrenalin receptor on the myocytes’ membrane, contributes to positive inotropic
action, increasing cardiac output, but stimulation of the cardiac vagus nerve contributes to decrease
cardiac output.
Autonomic nerves dominating blood vessels mostly belong to sympathetic vasoconstrictor
nerves. When they are excited, vascular smooth muscles contract, blood vessel caliber shrinks and
peripheral resistance increase. At the same time, cardiac output increases due to the contraction of
capacitance vessels and the increase of venous return.
Blood pressure is a complex trait that is determined by the interaction of multiple genetic and
environmental factors. The arterial BP depends on two fundamental hemodynamic variable factors
including cardiac output (CO ) and total peripheral resistance (TPR.). The changes of the CO are
important in BP modulation, and the CO is determined by myocontractility, heart rate and blood
volume (venous return), and TPR is exquisitely sensitive to small changes of the vessels radius.
Several neural and humoral mechanisms modulate changes of CO and TPR.
In addition to neural regulation, cardiovascular activities are affected by humoral
factors released from adrenal medulla and etc. Adrenalin can activateβ–receptors to
increase cardiac output. Its effects on vessels depend on dominating receptor located in
vessel walls. Noradrenalin mainly activates alpha-receptors to increase peripheral
resistance and elevate arterial blood pressure. Noradrenalin injected intravenously elevates
blood pressure and induces bradycardia.
[OBJECTS]
Rabbits
[MATERIALS]
A computer, bioenginery experimental system, pressure transducer, operating instruments of
mammals, artery clip, biconcave clamp, tracheal cannula, syringe, protective electrode, iron stand,
32
artery cannula. Normal saline, 25% urethan solution, 5% sodium citrate solution, 1:10 000
noradrenalin solution.
[METHODS]
1. Operation
(1) Anaesthesia: 25% urethan solution (4ml/kg) was injected into the vein in the edge of an
ear for anesthesia and the rabbit was fixed on operating table on back..
(2) Separation of common carotid arteries, vagus nerve and aortic nerve: The left and right
common carotid arteries parallel the trachea on both sides of it. A bunch of nerves go
along with the common carotid artery including vagus nerve, sympathetic nerve and
aortic nerve, composing the arterial sheath. After arterial sheath was separated, the three
strips of nerves should be distinguished carefully. As shown in Figure 3-16, all of the
nerves within the sheath parallel artery and among which, the vagus nerve is the thickest,
the sympathetic nerve is thinner, and the aortic nerve is the thinnest (just as hair) that
usually adheres to the sympathetic nerve. (Commonly, the aortic nerve and the
sympathetic nerve were separated firstly, then the vagus nerve and the left and right
common carotid arteries.) . Each nerve should be detached out for 2-3 cm long but the
carotid arteries should be separated as long as possible. Various threads were put under
each nerve and artery respectively for further use. The left common carotid artery was
used to measure blood pressure and the right nerves were used to be stimulated
respectively in this experiment.
Fig.3-16 Distribution of trachea, blood vessels and nerves in cervix of rabbit
1. trachea; 2.sympathetic nerve; 3.common carotid artery; 4.vagus nerve; 5.aortic nerve
(3)
Artery intubatton: The common carotid artery was ligated with a silk thread at the
separated segment far from heart, and was subjected to cross-clamp with an arterial clip at
the separated segment near the heart to block the local blood stream temporarily. The
vessel segment between the knot and the artery clip should be separated as long as
possible. Then a thread was placed under the separated artery with a slipknot for further
33
step. At the artery segment 0.5 cm distance from the knot, an inclined cut towards the
heart was made with an ophthalmologic scissors. Then the arterial cannula filled with 5%
sodium citrate was inserted into the cut towards the heart and fixed with the silk thread
prepared, and the surplus thread was ligated on the side of the cannula to avoid slippage.
Be attention to keep the incline of the cannula adown and overturn it after being inserted
into avoiding infarct. It should keep certain distance between the cannula and the artery
clip to avoid vessel damage in that friction between artery clip and the top of the cannula.
Arterial cannula should be fixed and paralleled the common carotid artery in order to
avoid blocking blood stream.
2. Experimental equipments
The output of pressure transducer was connected with the input of the computer. Turn on
the computer and enter the menu of Biolap 98→item of experiment (click)→cardiovascular
experiment(click)→regulation of arterial blood pressure (click).
3. Observation items
(1) Normal curve of the blood pressure and heart beat. The normal curve of blood pressure
consists of three-level waves. The second level wave often is seen on blood pressure
curve and the third level wave sometimes.
1) First level wave (Heart beat wave): It was induced by fluctuations of blood pressure due to
the systole and diastole in accordance with the heart rate.
2) Second level wave (Respiratory wave): The fluctuations of blood pressure along with
respiratory cycle in accordance with the respiratory rhythm.
3) Third level wave: It possibly results from the tonic periodical change of vasomotor centers.
(2) Observe the changes of blood pressure when following events happened.
1) Common carotid artery was cross-clamped for 10 seconds;
2) Aortic nerve was stimulated by a protective electrode;
3) 0.3ml 1:10 000 noradrenalin solution was injected intravenously;
4) Unilateral vagus nerve was ligated and cut off (at the distal end of knot)，and the peripheral
end of the separated vagus nerve was stimulated.
[POINTS for ATTENTION]
1. The narcotic should be injected slowly and the respiratory change should be noticed carefully
during anaesthesia.
2. Press hemostasia with gauzes is recommended if the seeping of blood is localized.
Hemostatic forceps should be used to stop bleeding by clamping vessels or hemorrhagic
spots.
3. During the experiment, arterial cannula should parallel carotid arterial trunk to avoid damage
of the artery.
4. The next item doesn’t begin until the blood pressure recovers to normal.
5. The gas vacuole should be evacuated in the arterial cannula that is connected with the pressure
transducer when 5% sodium citrate is injected into.
（Lili Guan & Yuan Zou）
34
Experiment 9
Factors influencing urine formation
[PURPOSE]
To observe some factors influencing urine formation the change the urinary output and
analyze their mechanisms, further understand the renal excretory function.
[PRINCIPLE]
The kidneys have excretory, regulatory, metabolic and endocrine function and are largely
responsible for the control of the body fluid equilibrium.
The excretory product of the renal system is urine which contains metabolic end products,
notably the nitrogenous waste products, urea and creatinine. The processes of urine formation
include glomerular filtration, reabsorption and secretion of the tubules. All factors affecting the
above three processes would influence the formation of urine and thus the amount of urine.
Clinically, the changes in urinary output (e.g. oliguria or polyuria) are usually an early manifestation
of renal excretory dysfunction.
[OBJECTS]
Rabbit
[MATERIALS]
A computer, bioenginery experimental system, surgical instruments of mammals, bladder
cannula, syringes (50ml, 10ml and 1ml), protective electrode, test tube, test tube rack, silk thread,
gauze, Normal saline, 25% urethane, 20% glucose, 1:10000 noradrenalin, heparin, furosemide,
qualitative test paper for glucose in urine.
[METHODS]
1. Operation
(1) 25% urethan solution (4ml/kg) was injected into the vein in the edge of an ear for anesthesia
and the rabbit was fixed on the operating table in supine body position.
(2) Operation in cervix: The right vagus nerve was separated and a silk thread was put under it
preparing for the next step.
(3) Operation in abdomen: First, ligate external orifice of urethra with a silk thread in order to
prevent the loss of urine when the bladder was stimulated during the experiment. Second,
shear the ventral hair of rabbit underwent laparotomy, and a 2-3 cm incision was made above
the public symphysis along linea alba abdominis and the bladder was moved out. Then a
35
longitudinal incision was made on the apex of the bladder in which few blood vessels
distributed. The bladder cannula was inserted into it immediately and fixed with the silk
thread. Then the urine can be collected with a test tube. The operating field should be covered
with warm gauze soaked by normal saline.
2. Observation item
After the urine outflow was steady, record the urine volume for 5 minutes as control. Then
observe changes of the urine volume of rabbit subjected to the following treatments.
(1) The effects of normal saline on the volume of urine: 20ml normal saline (38℃) was injected
into the vein in the rabbit’s ear within 1 minutes, and the volume of urine for 5 minutes was
recorded.
(2) The effects of noradrenalin on the volume of urine: 1:10000 noradrenalin (0.3ml) was injected
into the vein in the rabbit’s ear, then the volume of urine for 5 minutes was recorded.
(3) The effects of glucose on the volume of urine: 2 drops of urine was collected to test glucose in
urine as control with a qualitative test paper. Then 20% glucose (1.5ml) was injected into the
vein in the rabbit’s ear, and the volume of urine for 5 minutes was observed. At the same time,
2 drops of urine was collected to test glucose in urine qualitatively every 1 or 2 minutes.
(4) The effects on the volume of urine when stimulating the peripheral end of the vagus nerve.
After the right vagus nerve was ligated and cut off, the peripheral end of it was stimulated for
20s~30s with moderate intensity of current, and the volume of urine for 5 minutes was
recorded.
(5) The effects of diuretic agent on the volume of urine: The rabbit was administered with
furosemide(5mg/kg) intravenously through the vein in its ear. After 5 minutes, the volume of
urine for 5 minutes was recorded.
[POINTS for ATTENTION]
1. The vein in the edge of the rabbit’s ear should be carefully injected because many times of
intravenous injection were applied in this experiment.
2. The operation should be done gently to avoid traumatic anuresis. Too long incision in
abdomen is not recommended. Don’t injure the internal organ when the peritoneum is cut
open.
(Qiying Yao)
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Experiment 10
Experiment of center nervous system
I. Analysis of the reflex arc
[PURPOSE]
To understand the relationship between the integrity of a reflex arc and the reflex activity through
certain spinal motor reflexes.
[PRINCIPLE]
Reflex is a rapid (and unconscious) response to changes in the internal or external
environment which maintains homeostasis. Reflex arc is its structural foundation. A typical reflex
arc is composed of sensor, afferent nerve fiber, reflex centre, efferent nerve fiber and effector. The
completion of a reflex activity relies on the integrity of the reflex arc. The reflex would be
interrupted if any part of the reflex arc was broken.
[OBJECTS]
Toads or Frogs
[MATERIALS]
1cm2
Frog operating instruments, iron bracket, hemostat, muscle clamp, cup, filter paper (about 1×
) , two stimulating electrodes, beakers, 0.5%、1% sulfuric acid, gauze.
[METHODS]
1. Preparation of spinal toad
Put a scissors into the buccal cavity of the toad horizontally. Cut down its cranio-brain along
posterior border of drum membrane on two sides but remain its submaxilary. (In addition, spinal
toad can be prepared by a probe sticking into cranial cavity from great occipital foramen to destroy
brain tissue.) Fix the toad on the frog board on abdomen. Cut open the skin lengthways at the back
of the right hind limb. Find the sciatic nerve in the fossa between the biceps femoris muscle and the
semimembranosus muscle. Put a thread under the nerve for further use. After the operation, clamp
the toad’s submaxilary with a muscle clamp, and hang it on the bracket.
2. Observing items
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(1) Stick a piece of filter paper with 1% sulfuric acid on the abdomen skin and then observe
the response of the toad. After that, immerse the toad into the water in a beaker to wash up
and rub them to dry with a piece of gauze.
(2) Dip the toes of the toad hind limbs in 0.5% sulfuric acid in a watch-glass respectively (the
scope immerged into sulfuric acid must be same of these two times and just limit to the toe
tine). Then observe the response of the toad.
(3) Cut a circular nick on the skin along the toe joint of left hind limb, strip its limb skin under
the nick (make sure that the skin of toe tine must be striped completely), then dip the toe in
1% sulphuric acid (pay attention that the other toes must not be dipped into the sulphuric
acid), and observe the response of the hind limb.
(4) Ligate the right sciatic nerve, cut off the nerve between two ligations, then repeat the step (2)
and step (3).
(5) Destroy the spinal cord with a metal probe, and repeat the step (2) and step (3).
[POINTS for ATTENTION]
1. The place where the cranio-brain is cut should be chosen properly. If it is too high, some
brain tissues may be remained and independent activity may appear. If it is too low, the upper
spinal cord may be injured and the reflex may be disappeared.
2. After stimulation, sulfuric acid should be washed up immediately with water to prevent skin
injury. And then wipe the water to prevent diluting the sulfuric acid.
3. The toe dipped into the sulphuric acid should be only one, and the depth dipped in should be
same and not too much every time.
II. Observation of decerebellar animals
[PURPOSE]
To observe the symptom caused by cerebella injury of mice, in order to know it’s influence on
motor function such as muscle tonus and equilibrium of the trunk etc.
[PRINCIPLE]
Cerebellum is an important center for controlling posture and balance and for coordinating
and learning movements. It receives the information from motor organ, balance organ and motor
area of cerebral cortex, and sends information through thalamus to cortex motor area, through
nucleus ruber and hyperoliva nucleus to cerebellum, or through nucleus ruber and reticular
formation of brain stem to spinal cord. These compose a complicated reflex loop and control
complex body motion. Cerebellum injury will lead to dyskinesia, mainly represent body unbalance,
muscle tone facilitation or inhibition and ataxia.
[OBJECTS]
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Mice
[MATERIALS]
Mammalian surgical equipment, mouse operating-table, number 9 injector, cotton, 200 ml
beaker and aether.
[METHODS]
1. Anesthesia
Observe the posture, muscle tone and motor behaving of the mouse before anaesthesia.
Then put the mouse into a beaker containing a block of aether cotton with a cover. When the mouse
breathes deeply and slowly and could not move voluntarily, take it out and fix it on the
operating-table on abdomen.
2. Surgery and observation
(1) Wipe out the vertical fair, cut open the skins of the head along the median to ear back. Fix the
head with your left hand, scrape off the muscle and periosteum. We can see the cerebellum
through the skull.
(2) Identify every suturas (sutura coronalis, sutura arrowy and sutura lambdoidea) on the skull.
Pierce one side of os parietale with a needle uprightly (Fig.3-17):
Fig3-17: Area of destroy of mouse’s cerebellum
piercing position is 1mm under sutura lambdoidea, 2mm aside sutura arrowy): firstly a
shallow injure-pierce about 2mm, turn the needle slightly, destroy the tissue around, then take
out the needle and hemostasia by a block of cotton. After mouse awake, observe the change of
posture and muscle tone. Secondly a deep injure-pierce into about 3mm, stir the tissues around
within the cerebellum to destroy it, then take out the needle and hemostasia by a block of
cotton. Observation methods and items are the same as above.
[POINTS for ATTENTION]
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1. Pay attention to the animal’s breath when anaesthesia to avoid death induced by super
anaesthesia. If animals awaked, we can supply aether by a block of cotton again.
2. The probes should not stab extremely and only destroy one side of cerebellum, do not damage
the mesencephalon.
III. Semi-crosscut of the mouse’s spinal cord
[PURPOSE]
To verify the conduction function of spinal cord by comparing body’s motor function and
sensory function.
[PRINCIPLE]
Spinal cord is not only the base reflex center but also a pathway between body receptor, body
effector and many central neurons in the brain. In order to prove the conductive function of spinal
cord, cross cut half of the spinal cord transversely and compare the motor function and sensory
function between the two sides of the animal’s body under the cutting level.
[OBJECTS]
Mice
[MATERIALS]
Mammal surgical instruments, board, two elastics, 200ml beaker, tampons, a needle, forceps,
aether.
[METHODS]
1. Observe the activity of four limbs when the mouse moving about on the table. Sting the
mouse’s toe of hind limbs and observe their reaction. Stimulate two hind limbs with the
freezing cotton stick and observe their reaction.
2. Anaesthesia the mouse with aether and fix it on the operating-table on abdomen. Feel out the
floating rib of the mouse with your thumb and index finger, wipe off the back hair and cut
open the skins along the median. Cut off the tendon beside the processus spinosus and the
tendon between the vertebras clinging to the processus spinosus of 1~3 lumbar. Separate the
muscles on them by tampons and the forceps to exposure the vertebras. Cut out the
processus spinosus and the arcus vertebrae of one lumbar to exposure the white spinal cord
about 2 mm. In sign of spinal postvein on the post center of the spinal, cut out the spinal
transversely from center to one side by a needle, and cover the wound with a NS cotton ball.
Observe the following items when the mouse become awake:
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(1) Compare the skin color between the two side soles.
(2) Observe the posture of mouse’s four limbs and compare the forelimbs’ posture with the
backlimbs’ posture.
(3) Have the mouse walked on the table and observe if the mouse is paralysed on it’s hind limb.
(4) Sting the mouse’s hind limb respectively with a needle and observe it’s reaction.
[POINTS for ATTENTION]
1. Pay attention to the animal’s breath when anaesthesia to avoid death induced by super
anaesthesia
2. Make sure not to injure the spinal cord during cutting off the vertebrae.
3. Make sure it is the spinal cord before cutting. Don’t injure the post spinal artery at the back
median of the spinal cord when cut it to avoid over bleeding.
(Qiying Yao)
41