Download EXPERIMENTS ON FROG CARDIOVASCULAR AND

Experiments on frog cardiovascular and respiratory system
7
EXPERIMENTS ON FROG CARDIOVASCULAR AND RESPIRATORY SYSTEM
Dr. Metz Júlia, Dr. Orbán-Kis Károly, Dr. Szilágyi Tibor
PURPOSE
The purpose of this laboratory practice is to investigate the cardiovascular and respiratory
function in the frog. Because of the similarities in the function of the systems, in vivo and in
vitro experimentation on frog organs is often used for understanding human physiology.
The aim of the practice is:
► to demonstrate ciliary movement in the respiratory mucosa,
► to visualize the capillaries of the nail bed,
► to demonstrate arterial, venous and capillary circulation on frog tongue,
► to register the mechanical and electrical activity of the frog heart, to demonstrate
the thermal effect, the effect of inotropic and chronotropic substances and of the
change of ionic concentrations on the activity of the frog heart,
► to determine the excitation threshold and to demonstrate the refractory period.
Animals used for demonstration and experimental purpose are healthy.
Nevertheless avoid direct contact with their blood, faeces or secretion. Do not
touch animals except when specifically told by the demonstrator and always
wear gloves. Do not pet the animals, do not feed the animals. Further details
regarding laboratory safety rules as well as ethical considerations of animal
experimentation are provided at the end of this Handbook.
THEORETICAL BACKGROUND
Ciliary movement
The mucosa lining the upper and lower airways provides a barrier against injury from inhaled
toxics, bacteria, and other biogenic agents (organic dusts, pollen, mold spores, etc.). The
respiratory epithelium is coated with mucus, which lubricates, insulates, and humidifies the
epithelium and protects it by entrapping bacteria and other particles for removal by
mucociliary clearance. The respiratory epithelium is composed of ciliated cells, having 200
cilia on each cell; the cilia are continuously moving with a frequency of 10-20 beats/sec. Thus
mucus is moved towards the pharynx: in the upper airways the direction of the movement is
downwards, in the lower airways is upwards. This continuous movement shifts the mucus
with the entrapped particles slowly, with a speed of 1cm/min towards the pharynx where it is
swallowed or eliminated.
A characteristic of the frog anatomy is that the ciliated epithelium is also present in the upper
third of the esophagus.
Microcirculation
Microcirculation involves several types of vessels: arterioles, venules and capillaries. These
can be visualized in man at the nail bed, in frog at the tongue and at the membrane which
joins the toes (interdigital membrane). Visualization of these vessels in human allows the
counting of the capillary arches, measuring the diameter of the vessels and determining the
speed of blood flow using Doppler method. These can be used in the diagnoses of diseases
such as Raynaud's diseseas, scleroderma, etc.
Examining the vessels of the frog tongue the different types of vessels can be identified: in
arterioles, the blood flow is towards the branches, in venules the blood flows from the
branches to the larger vessels. In arterioles the speed of the blood flow is higher and the
vessels pulsate. In larger vessels the laminar flow of the blood can be observed: the blood
8
Physiology laboratory exercises
flows in parallel layers; in the center of the vessel the flow has higher speed than near the
walls of the vessel. (Figure no. 1)
Figure no. 1. Laminar flow in the arterioles (arrows in the left represent the
speed of the layers).
The electrical and mechanical activity of the heart
The action potential is the electrical response of the excitable cell to a stimulus that has
higher intensity than the threshold value. During the action potential the myocardium is in the
refractory period: the myocardial cell dos not respond to any stimuli regardless of their
intensity. The action potential is followed by the contraction of the myocardial cells after a
short period necessary for the electromechanical coupling. The action potential and the
muscle contraction-relaxation have similar duration (Figure no. 2). Because of the relatively
long refractory period the mechanical phenomena cannot be summated, thus the
myocardium cannot present tetanical contraction. The sequence of contraction and
relaxation is essential for the pumping function (ejection and inflow) of the heart.
Figure no. 2. The electrical and mechanical activity of the heart (AP – action
potential, MCG – mechanocardiogram, ARP – absolute refractory period, Δt
– the period necessary for the electromechanical coupling).
Modulation of the activity of the heart
► Thermal effect: Warming up the venous sinus increases the frequency of the
contractions, warming up the ventricle increases the amplitude of its contractions.
Chilling the venous sinus and the ventricle decreases the frequency and the
Experiments on frog cardiovascular and respiratory system
9
amplitude of the contractions. Contraction and excitability depend on enzymatic
systems and conformational change of certain proteins (ionic channels), which are
influenced by temperature.
► Chemical mediators: Adrenalin increases the frequency and amplitude of the
contractions acting on β1 adrenergic receptors. Acetylcholine decreases the
frequency and amplitude of the contractions through its action on muscarinic (M2)
cholinergic receptors. Because of the existence of these receptors on the myocardial
cells applying the mediators of the vegetative nervous system on the denervated
heart reproduces the effects of the sympathetic and parasympathetic stimulation.
► The concentration of ions in the extracellular space: Increasing the concentration
of extracellular Ca2+ increases the strength of the contraction while the diastolic
relaxation becomes shortened, finally the heart stops in systole (contraction) due to
the accumulation of Ca2+ in the extracellular space ("calcium rigor"). Increasing the
concentration of extracellular K+ blocks in a non-selective manner the external pores
of the Ca2+ and Na+, thus decreases the amplitude of the contractions, until the heart
stops in diastole (relaxation) (Figure no. 3).
Figure no. 3. The effect of increased concentration of extracellular ions on
the mechanical activity of the frog heart.
► The effect of removing the pericardium: Opening the pericardial sac allows the
chambers of the heart to move/expand more forcefully, which leads to increased
sensibility of the Frank-Starling mechanism and to higher stroke volume. Removing
the pericardial sac leads to lack of vagal effect thus increasing the heart rate.
Method for simultaneous recording of the electrical and mechanical activity
Three metal electrodes are used to simultaneously record the electrical and mechanical
activity of the frog heart. One electrode is inserted in the base of the heart, the second (a
metal hook) is fixed on the apex of the heart, and the third one is the grounding electrode.
The electrical activity is recorded using a bipolar method and differential amplification. The
electric signal is amplified and digitalized (Figure no. 4 and Figure no. 5).
Figure no. 4. Scheme of the equipment used to record the electrical and
mechanical activity of the frog heart.
10
Physiology laboratory exercises
The mechanical activity is recorded with a mechano-electrical transducer (Figure no. 5):
elongation of the spring is proportionate to the force of the contraction. The iron core of
inductance coil is connected to the apex of the heart through a metal hook, thus
contractions of the heart will modify the position of the iron core and the inductance of
coil proportionately.
the
the
the
the
Figure no. 5. Recording electrode placement and the scheme of the
mechano-electrical transducer
The metal hook inserted in the apex of the heart penetrates both the extracellular and
intracellular space; thereby a lesion potential is recorded (Figure no. 6). This qualitatively
resembles the action potential which is recorded with microelectrodes (using an intracellular
electrode); but the signal is distorted by an extracellular shunting current that has to be
diminished.
Figure no. 6. Methods used for recording the lesion potential from the frog
heart (left) and the action potential (right).
This can be achieved by increasing the resistance of the extracellular space: by stretching
the wire, the heart can be elongated, thus its electrical resistance increases (see equations).
V
R
L
R  
S
I
where I – current, V – potential, R – electrical resistance, L – length, S – section area, ρ –
specific resistance.
Experiments on frog cardiovascular and respiratory system
11
Frog anatomy and physiology
Frogs are often used in experiments because their cardiovascular and respiratory systems
present several functional similarities with human ones, and their organs have lower
sensibility to ischemia and hypoxia.
The frog heart shows particular characteristics in contrast to human heart. The frog heart
consists of four compartments (Figure no. 7): venous sinus, atria, ventricle, aortic bulb, which
contract successively.
Figure no. 7. The chambers of the frog heart: on the left the heart is in the
anatomical position, on the right the apex is turned upwards.
The excitation is generated in the venous sinus and propagates from one compartment to
other, but every compartment has its own pacemaker, too. Under physiological conditions,
the activity of the heart is governed by the pacemaker situated in the venous sinus, which
generates excitation with the highest frequency, around 60 beats per minute (the principle by
which the highest frequency pacemaker governs the rhythm of the heart is called overdrive
suppression). The atrial pacemaker generates excitation with a frequency of around 50 beats
per minute; the ventricular pacemaker has a frequency of around 25 beats per minute and
the pacemaker of the aortic bulb 10 beats per minute. Between the compartments there is a
junctional zone where the propagation of the electrical signal is slowed down. At these levels
the electrical signal delays thus allowing a better inflow of blood to the downstream
compartment and a more efficient hemodynamics. In the human heart the venous sinus is
included in the wall of the atria; there is a sino-atrial pacemaker node that is surrounded by
the first junctional zone (corresponding to the junction between the venous sinus and the
atria), site of the sino-atrial blocks.
Stannius ligatures
Stannius ligatures are experimentally used to study the intracardiac propagation of excitation.
When surrounding the jonctional zone with a piece of thread and tightening the knot, the
propagation of the electrical impulses from one compartment to the other is cut off (Figure
no. 8). The first Stannius ligature is placed at the junction between the venous sinus and the
atria. After stretching the thread the venous sinus continues to contract with its initial
frequency (approx. 60 beats per minute) dictated by the physiological pacemaker, localized
in the venous sinus (the Remak ganglion, equivalent to the sinusal node in human), but the
atria and the ventricle stop in diastole. After a few minutes they start to contract again, but
with a lower frequency, approx. 50 beats per minute, this is dictated by the pacemaker
localized in the atria (the Ludwig ganglion, equivalent to the atrio-ventricular node in human).
The second Stannius ligature is placed at the limit between the atria and the ventricle,
preserving the first ligature. After stretching the thread, the venous sinus contracts with its
12
Physiology laboratory exercises
initial frequency, the atria contracts with the frequency acquired after the first ligature; the
ventricle, usually after a longer period (used to determine by the experimenter the refractory
periods) starts to contract with an even lower frequency, approx. 25 beats per minute,
asynchronously to the sinusal rhythm. Thus the human third degree atrio-ventricular block is
modeled. The ventricular rhythm is dictated by the Bidder ganglion situated in this
compartment.
Figure no. 8.The frog heart's scheme from lateral view and the position of
the Stannius ligatures.
Determination of the excitation threshold, the refractory periods and the extrasystole.
Action potentials are triggered when the membrane potential is depolarized up to the
threshold of excitation. The minimal current amplitude necessary to reach the threshold can
be determined using the ventricle without spontaneous activity after placing the second
Stannius ligature. The stimulus intensity is gradually increased until a response is generated.
Even if the stimulus strength is increased more the same response is obtained, this is known
as the all-or-nothing law.
The all-or-none law is the principle that the strength by which an excitable cell
responds to a stimulus is not dependent on the strength of the stimulus. If the
stimulus strength is above threshold, the nerve or muscle fiber will give a
complete response or otherwise no response at all.
To determine the refractory period two consequent stimuli are applied having at least the
intensity necessary to initiate action potentials. Then the interval between the stimuli is
decreased till the second action potential disappears. Thus the absolute refractory period is
determined.
If the ventricle started to contract the action potential is recorded and used as a trigger to
apply the electric stimulus. Stimulating the heart during the first part of an action potential
does not produce any response; this demonstrates the existence of the absolute refractory
period. At the end of the action potential refractory period ends as well and the myocardium
responds with a supplementary contraction called premature ventricular contraction
(extrasystole). This is followed by a longer break, called compensatory pause; this occurs
because the next stimulus arising from the ventricular node finds the myocardium in the
refractory period following the extrasystole.
Experiments on frog cardiovascular and respiratory system
13
PRACTICAL EXECUTION
Examination of the human microcirculation
Blood circulation in humans can be visualized using a capillary microscope. The capillary
microscope is a dissecting microscope; the examined object is lighted up from above. Blue or
green light is used. The nail bed of the ring finger is oiled with one drop of glycerin. The
finger is placed on the microscope stage so that the border between the nail and the skin of
the finger is situated in the center of the visual field. The examination is started using low
power (ocular of 10x, objective of 0.63x), gradually increasing the magnification. At a
magnification of 40x the capillary loops are visible (Figure no. 9).
Figure no. 9. Vessels of the nail bed.
Examination of the blood flow of the frog's tongue
The frog is weighted and is wrapped in a cloth. The frog is injected intraperitoneally with 20%
urethane with a dose of 1 ml/50 g. The onset of anesthesia is indicated by the loss of the
righting (flip) response (the frog does not rights itself when turned on its back) and the loss of
the corneal reflex (the frog does not blink when the cornea is touched with a blunt object).
The frog is placed on a horizontal board. The tongue is dragged out and fixed with needles
on a cork board with a hole on the middle. The board is then placed on the microscope stage
(Figure no. 10).
Figure no. 10. Examination of the blood flow of the frog's tongue.
14
Physiology laboratory exercises
Urethane [ethyl carbamate (NH2COOCH2CH3)] is a common compound
found in very small quantities in alcoholic beverages, soy sauce and bread. At
larger doses, urethane has anesthetic properties.
Urethane is a commonly used anesthetic for animal experiments. It is popular
because a single injection (typically 1.0 to 1.5 g/kg) provides immobility for
surgical procedures of long duration (several hours) and because there is a
general impression that it produces minimal changes in circulation and
respiration, preserves reflex responses, and minimally interferes with the
physiological relevance of data collected in anesthetized animals.
The blood circulation is examined with a low power objective (10x). In case of a prolonged
examination the tongue is regularly moistened with Ringer's solution.
Ringer's solution for frog (or amphibians in general) contains sodium chloride,
potassium chloride, sodium bicarbonate and glucose (the main ions of the
extracellular space in physiological concentrations, energy source and buffer
system); is used to maintain the vitality of the tissues during experiments.
This solution was invented by Sidney Ringer (British physiologist and
clinician, 1836-1910). Variants of this solution adapted to the physiology of
different species are used up to these days in experiments and clinical
practice.
Demonstration of ciliary movement
The anesthetized frog is wrapped in a cloth. One blade of the scissor is introduced in the oral
cavity and the head is removed cutting above the level of eyes with a single fast stroke
(Figure no. 11).
Figure no. 11. Fast decapitation of the anesthetized frog.
The frog is placed on a dissecting board and hereafter the cut head of the frog is used.
Ringer's solution with suspended charcoal particles is dropped on the mucosa of the hard
palate. After a few minutes the charcoal particles are visibly shifted towards the pharynx.
Examination of ciliary movement using an optical microscope
Scraping is collected from the mucosa of the hard palate by abrasion with a blade; placed on
a microscope slide and moistened with a drop of Ringer's solution on room temperature.
When examining the slide at a low-power microscope (objective of 10x) the uniform
movement of the cilia can be observed.
Frog dissection, identification of the inner organs, examination of the heart
Next the frog is dissected. The frog is placed and stretched out in supine position on a
dissection board. A V-shaped incision is made through the skin and abdominal wall
Experiments on frog cardiovascular and respiratory system
15
musculature. Then the incision is extended towards the clavicles (collar bones) which are
also cut and with a transverse section the complete anterior thoracic wall is removed. The
frog's organs can be seen and identified. The abdominal and thoracic cavities are not
separated; the frog does not have a diaphragm. The heart is beating in the pericardial sac,
the main arteries and veins can be identified. If the lungs contain air, the acinar structure can
be observed.
Count the heart beats while the heart is in its anatomical position!
The pericardium (a thin white membrane) is clipped with a small forceps and cut carefully cut
without injuring the heart.
Count the heart beats after the removal of the pericardium!
The heart is examined in its anatomical position and with the apex turned upwards, after
sectioning the posterior ligament of the heart.
Identify the chambers of the heart and note the successive contraction of the
chambers! Next observe the thermal effect; by dropping cold, then warm
Ringer's solution is on the heart. Determine the ventricular frequency after
applying the solutions.
Than the heart is removed and placed in a Petri dish containing Ringer's solution.
Count the ventricular beats. Add adrenaline in the Petri dish and note the
change in ventricular frequency. If the heart stopped beating after the
removal, apply mechanical stimulation with a pointless needle.
Examination of the ciliary movement on the frog esophagus
After the removal of the heart the esophagus, identify the stomach and intestines, observing
the intestinal motility. Identify the liver, gall-bladder and spleen. Lifting the intestines the
kidneys with the suprarenal glands, the urinary bladder and the genital organs can be seen.
The upper third of the esophagus is removed and is pulled on a glass pipette kept in
horizontal position. After a few minutes a change in the position of the esophagus can be
observed due to the ciliary movement.
Recording the electrical and mechanical activity of the frog's heart
The anesthetized, decapitated and despinalized frog is stretched out in supine position on a
dissection board. The anterior thoracic wall is dissected and removed as described earlier. At
this point the heart can be seen. The pericardial sac is opened. The electrodes are fixed; the
metal hook is inserted in the apex of the heart. The heart is lifted by stretching the wire of the
mechano-electrical transducer. The electrical and mechanical activity of the heart in basal
conditions is recorded using a computer. Then warm (40°C) and ice-cold Ringer's solution is
dropped on the heart, followed by chemical mediators (adrenaline, acetylcholine) and
electrolytes (Ca2+, K+) noting the effects. Then the Stannius ligatures are applied and their
effect on the heart is observed. The stimulating electrode is attached to the heart and first the
current intensity necessary to reach the threshold potential (and initiate the action potential),
16
Physiology laboratory exercises
and then the refractory period is determined. When the ventricle starts contracting the
extrasystole and the compensatory pause can be demonstrated.