General Physiology and Excitable Tissue – Dental Medicine
Muscle twitch
Rat phrenic nerve – hemidiaphragm simulation
Introduction
The Virtual Rat is a simulation of the rat
phrenic nerve-hemidiaphragm preparation - a
robust in vitro preparation which has been widely
used in the study the actions of neuromuscular
blocking and reversal agents, and other drugs
which affect neuromuscular transmission.
The hemidiaphraghm is a large, focally
innervated, respiratory skeletal muscle, composed
of fast-type muscle fibres. Neurotransmission is
mediated by nicotinic cholinoceptors. Electrical
stimulation of the phrenic nerve evokes fast,
shorting-lasting muscle twitches.
The preparation
A rat (0.5 kg) is killed by anaesthetising it with 100% CO2 (a rapid and fairly painless
procedure). The rib cage is opened and the phrenic nerve and triangular-shaped hemidiaphragm is
removed and placed into an organ bath containing Kreb's solution, bubbled with 95%O2/5%CO2, and
maintained at 32C in a surrounding water bath. As shown in the diagram, the base of the muscle is
attached to a support and the tendon attached to a tension transducer. The nerve is looped through a
stimulation electrode and another electrode is placed directly upon the muscle. Drugs are applied
directly into the bath. When required, drugs can be washed out, by flushing it out with new Kreb's
solution.
Computer simulation
To start the simulation, select the icon:
by double-clicking it or in the menu Start –>
At the beginning you could write your name.
všechny programy –> Twitch V2.1.8.
Program control:
Basic control of the program is in the menu bar at the top of the window.
File:
 New Rat – Start experiment with new rat, all settings will be reset.
 Load Rat – Load saved experiment.
 Save Rat – Save current experiment.
 Print – Print out actual window.
 Exit – Leave program.
Drugs:
To apply a drug, choose one from the list of drugs. The dose of the drug can be selected from
the select menu. Drugs can be applied in various doses and in any combination during experiment.
Concentration is expressed in mol per liter (M) and corresponds to just added (not total)
concentration. Total concentration of the drug appears below the graphical record of the contraction.
The drugs accumulate in solution. When you want to replace a working solution with a new
Krebs solution, select Wash. Effect of some drugs lasts sometimes even after washing out.
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General Physiology and Excitable Tissue – Dental Medicine
Ions:
Concentrations of calcium and magnesium in solution can be changed in this menu. Calcium
is responsible for acetylcholine release from the nerve endings. Calcium influx enables moving of
vesicles with acetylcholine to cell membrane and their exocytosis. Leak and entry of calcium from
and to the muscle cell is very limited. Thus muscle contraction is almost independent on extracellular
concentration of calcium. Contraction can be affected only after long-lasting changes in calcium
concentration.
Magnesium competitively blocks the calcium entry to the nerve cell. Thus magnesium affects
the amount of acetylcholine release. High concentration of magnesium blocks a neuromuscular
transmission.
Wash:
When you change the solution in the bath, all drugs and ionic changes will be removed. You
can choose between normal Krebs solution and Krebs solution without calcium. Effect of some drugs
lasts sometimes even after washing out.
Experiment control:
In the left lower part of the window there are control elements of the experiment. They are
divided into two parts: Controls a Stimulator.
The experiment is started up by pushing the button Start. Button Stop disrupts the
experiment, which can continue after pushing Start button. After disruption of the experiment you can
browse through the whole record using scrollbar. The actual time and magnitude of the contraction in
a point of the mouse cursor are showed in the field below Start and Stop buttons .
The muscle can be stimulated in either of two ways. DIRECTLY – by injecting a small
current into the muscle fiber via the recording electrode. The amplitude and duration of the stimulus
current can be set within the simulation. INDIRECTLY - by stimulating the phrenic nerve via
platinum wire electrode. You can also turn off the stimulation.
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General Physiology and Excitable Tissue – Dental Medicine
Applying drugs
The following drugs can be applied to the preparation:
Tubocurarine
Non-depolarizing competitive cholinergic antagonist, reversible blocker of the nicotinic
receptors. After the block of the nicotinic receptors, the sodium channels could not operate and nerve
evoked depolarization of the subsynaptic membrane decrease. If this depolarization is too small, the
action potential on the muscle fiber does not appear. Tubocurarine and acetylcholine bonds on the
nicotinic receptors are reversible and competitive (i.e. dependent on drugs concentration).
Recommended lower dose is 0.2 μM.
Neostigmine
Competitive inhibitor of acetylcholinesterase (ACHE), that causes reversible blockade of this
enzyme. ACHE decreases concentration of acetylcholine in the synaptic gap and dissociates it into
cholin and acetic acid. ACHE blockade prolongs acetylcholine effect on the subsynaptic membrane.
Recommended lower dose is 0.2 μM.
4-aminopyridin
Blocker of potassium channel, which prolongs action potential duration. Recommended lower
dose is 20 μM.
Atropine
Competitive antagonist of muscarin receptors.
Suxamethonium
Short-time depolarizing myorelaxant. It binds to nicotinic receptors, where it effects like longtermed agonist. This bond causes prolonged depolarization of the membrane that is way the action
potential does not appear. Recommended lower dose is 5 μM.
Tetrodotoxin
Blocker of sodium channels. Tetrodotoxin decreases or prevents depolarization of the nerve
and muscle. Recommended lower dose is 0.2 μM.
PROTOCOL
Describe effect of each drug on the action potential during the direct and the indirect
stimulation and explain your observations. (c = concentration of drug in the solution):
Indirect stimulation
Direct stimulation
Tubocurarine
c=
Neostigmine
c=
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General Physiology and Excitable Tissue – Dental Medicine
4-aminopyridine
c=
Atropine
c=
Suxamethonium
c=
Tetrodotoxin
c=
Magnesium
c=
Plot the graph of the dependence of the contraction magnitude on the drug concentration in the
solution with tubocurarine (minimal concentration 0.5 μM and increment 0.2 μM) or with
suxamethonium (minimal concentration 5 μM and increment 2 μM). Maximum concentration used
corresponds to the dose at which the muscle does not response to stimulation. Use only indirect
stimulation.
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contraction force (gms)
General Physiology and Excitable Tissue – Dental Medicine
concentration (mol/l)
Which drugs and in which mechanism can compensate low level of calcium concentration (0.5 mM)?
(Wash the bath with Krebs solution without calcium and then add 0.5 mM of calcium.)
How and why you should compensate hypermagnesemia (6 mM) without affecting potassium
channels or acetylcholinesterase?
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General Physiology and Excitable Tissue – Dental Medicine
How can be completely restored the contraction after application of tetrodotoxin (0,5 μM) or
suxamethonium (20 μM)?
What is the difference in muscle response between the direct and the indirect stimulation, when the
bath solution is without calcium? Explain it.
Electromyography – Muscle Fatigue
Introduction
Mechanical work, in the physical sense, refers
to the application of a force that results in the movement
of an object. Skeletal muscle performs mechanical work
when the muscle contracts and an object is moved, as in
lifting a weight. To lift a weight, your muscles must
exert a force great enough to overcome the weight. If
you exert less force, then the weight does not move
(Fig.1).
Fig. 1
Physiologically, skeletal muscle is stimulated to contract when the brain or spinal cord
activates motor units of the muscle.Motor units are defined as a motoneuron and all of the
muscle fibers that the motoneuron innervates. An action potential (AP) in a human motoneuron
always causes an action potential in all of the muscle fibers of the motor unit. As a matter of fact,
humans generally do not send just one AP at a time down a motoneuron. Instead, a train of APs is
sent — enough to induce tetany (the sustained fusion of individual muscle twitches) in the
muscle fibers of the motor unit. [A discussion of motor units and their control was presented in
Lesson 1.]
Most human skeletal muscles are composed of hundreds of motor units (Fig. 2). When a skeletal
muscle is called on to perform mechanical work, the number of motor units in the muscle activated by
the brain is proportional to the amount of work to be done by the muscle; the greater the amount of
work to be done, the greater the number of motor units activated. Thus, more motor units are
simultaneously active when a skeletal muscle lifts 20 kilograms than when the same muscle lifts 5
kilograms.
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General Physiology and Excitable Tissue – Dental Medicine
Fig. 2.Example of Motor Units
The brain determines the number of active motor units required for a muscle to perform a given
task by utilizing sensory information from stretch receptors in the muscle and associated tendons and
joint capsules. For example, when lifting a bucket of water from the ground, the brain first activates
several motor units in the requisite skeletal muscles. If sensory information returning from the
muscles indicates the muscles are contracting but not developing adequate power to lift the bucket,
the brain activates additional motor units until the sensory information indicates the bucket is being
lifted. The sequential activation of motor units to perform a designated task is called motor unit
recruitment.
Once you have lifted a light object, the brain recruits approximately the same number of motor
units to keep the object up, but cycles between different motor units. The muscle fibers consume
stored energy available in the muscle, and generate a force by contracting. As the muscle fibers
deplete this fuel source, more energy must be created in order to continue contracting. By recruiting
different motor units, motor units can relax and replenish their fuel sources.
Skeletal muscles performing acute maximum work or chronic submaximum work of a repetitive
nature will eventually fatigue. Fatigue is defined as a decrease in the muscle’s ability to generate
force due to a reversible exhaustion of energy resources. If the muscle uses its energy sources faster
than they can be generated by cellular metabolism, fatigue occurs. During contraction, skeletal muscle
cells convert chemical energy into thermal and mechanical energy, and, in the process, produce
chemical waste products, mainly lactic acid, whose accumulation leads to acidosis. Some accumulated
waste products also stimulate pain receptors in surrounding connective tissue and induce cramping of
skeletal muscle. Pain and cramp of the working muscle are, in addition to a decrease in contraction
force, characteristic signs of the muscle fatigue. Normally the waste products are removed from the
muscle by the circulatory system as the blood brings nutrients to the muscle for energy
transformation. If certain waste products (metabolites) are not removed at an adequate rate, they will
accumulate and chemically interfere with the contractile process, thereby hastening the onset of
fatigue.
The muscle work is divided into two basic groups – static work and dynamic work. The
dynamic work is characterized by a regular change of contraction and relaxation. During the
dynamic work, the blood flow in the working muscle varies, it decreases during contraction and
increases during relaxation. Thus the waste products are removed from the active muscle during
relaxation. The static work is characterized by an increase in muscle tone without a change in muscle
length. The working muscle is contracted and the blood flow is permanently decreased during the
static work. This implies that the signs of the muscle fatigue appear earlier in the static work then in
the dynamic one.
In this lesson, you will examine motor unit recruitment and skeletal muscle fatigue by
combining electromyography with dynamometry.When a motor unit is activated, the component
muscle fibers generate and conduct their own electrical impulses, which cause the fibers to contract.
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General Physiology and Excitable Tissue – Dental Medicine
Although the electrical impulse generated and conducted by each fiber is very weak (less than 100
volts), many fibers conducting simultaneously induce voltage differences in the overlying skin
which are large enough to be detected by a pair of surface electrodes.
The detection, amplification, and recording of changes in skin voltage produced by underlying
skeletal muscle contraction is called electromyography, and the recording thus obtained is called an
electromyogram (EMG). Power is defined as the amount of work done per unit of time.
Dynamometry means the measurement of power (dyno = power, meter = measure), and the graphic
record derived from the use of a dynamometer is called a dynagram. In this lesson, the power of
contraction of clench muscles will be determined by a hand dynamometer equipped with an
electronic transducer for recording.
The BIOPAC system will simultaneously record three bands of information:
1) The force you exert on the transducer,
2) The electrical signal produced by the muscle during contraction, and
3) The integrated waveform, which is an indication of the activity levels of the muscle.
Experimental Objectives
1)
2)
To record the force produced by clench muscles, EMG, and integrated EMG when inducing
fatigue.
To compare the time to fatigue during static and dynamic work.
Materials










BIOPAC electrode lead set (SS2L)
BIOPAC disposable vinyl electrodes (EL503), 6 electrodes per subject
BIOPAC electrode gel (GEL1) and abrasive pad (ELPAD)
or
Skin cleanser or alcohol prep
BIOPAC SS25 Hand Dynamometer
BIOPAC Headphones (OUT1)
Computer system
Biopac Student Lab software v3.6.7 PC or v3.0.7 Mac or greater
BIOPAC acquisition unit (MP30)
BIOPAC wall transformer (AC100A)
BIOPAC serial cable (CBLSERA) or USB cable (USB1W) if using a USB port.
Experimental Methods
Overview
 As you complete the Experimental Methods (Set Up, Calibration, and Recording) and the
Analysis, you may need to use the following tools and/or display options. The window display
shown below is only a reference sample — it does not represent any lesson specific data. The
sample screen shows 3 channels of data and four channel measurement boxes, but your screen
display may vary between lessons and at different points within the same lesson.
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General Physiology and Excitable Tissue – Dental Medicine
channel measurement boxes
(channel #
)
measurement type result)
marker
marker tools
channel boxes
(Data analysis mode only)
vertical scales
marker label
vertical (amplitude)
scroll bar
channel labels
selection tool
horizontal (time) scroll bar
horizontal scale
zoom tool
I-Beam cursor
 The symbols explained below are used throughout Experimental Methods and Analysis.
Key to Symbols

If you encounter a problem or need further explanation of a concept, refer to the
Orientation Chapter for more details.

The data collected in the associated step needs to be recorded in the Data Report (in the
section indicated by the alpha character). You can record the data individually by hand
or choose Edit > Journal > Paste measurements to paste the data to your journal for
future reference.

Most markers and labels are automatic. Markers appear at the top of the window as
inverted triangles. This symbol is used to indicate that you need to insert a marker and
key in a marker label similar to the text in quotes. You can insert and label the marker
during or after acquisition. On a Mac, press “ESC” and on a PC, press “F9.”
 Each section is presented in a two-column format, as described below.
FAST TRACK Steps
Detailed Explanation of Steps
This side of the lesson (left, shaded
This side of the lesson contains more detailed information to
column) is the “FAST TRACK” through
clarify the steps and/or concepts in the FAST TRACK, and may
the lesson, which contains a basic
include reference diagrams, illustrations, and screen shots.
explanation of each step.
A.
SET UP
FAST TRACK SET UP
1. Turn your computer ON.
2. Make sure the BIOPAC MP30 unit is
OFF.
Detailed Explanation of Steps for SET UP
The desktop should appear on the monitor. If it does not appear,
ask the laboratory instructor for assistance. 

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General Physiology and Excitable Tissue – Dental Medicine
3. Plug the equipment in as follows:
Hand dynamometer (SS25L) — CH 1
Electrode lead (SS2L) — CH 3
Headphones (OUT1) — back of unit
Headphones (BIOPAC OUT1)
Plugs into back of MP30 unit
BIOPAC SS2L
plugs into CHannel 3
BIOPAC SS25L
plugs into CHannel 1
4. Turn ON the BIOPAC MP30 unit.
5. Attach three electrodes to the
forearm (Fig. 4).
Set up continues...
Fig. 3 Equipment Setup

For the first recording segment, select the Subject’s dominant
forearm (generally the right forearm if the subject is righthanded, or the left forearm if the subject is left-handed) and
attach the electrodes onto the forearm as shown; this will be
Forearm 1.
Use the Subject’s “Other arm” for the second recording
segment; this will be Forearm 2.
Attach three electrodes to the forearm as shown in Fig. 4.
Fig. 4 Electrode placement
Note: For optimal electrode adhesion, the electrodes should be
placed on the skin at least 5 minutes before the start of the
Calibration procedure.
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General Physiology and Excitable Tissue – Dental Medicine
6. Attach the electrode lead set
(SS2L) to the electrodes,
following the color code (Fig.
5).
IMPORTANT
Make sure the electrode lead
colors match Fig. 5.
White Lead
(-)
Red Lead
(+)
Black Lead
(Ground)
Fig. 5 Electrode lead attachment
Each of the pinch connectors on the end of the electrode cable
needs to be attached to a specific electrode. The electrode
cables are each a different color. Follow Fig. 5 to ensure that
you connect each cable to the proper electrode.
The pinch connectors work like a small clothespin, but will
only latch onto the nipple of the electrode from one side of the
connector.

7. Start the BIOPAC Student Lab
Program.
8. Choose lesson “L02-EMG-2” and click 
OK.
No two people can have the same filename, so use a unique
9. Type in a unique filename.
identifier, such as the subject’s nickname or student ID#. 
This ends the Set Up procedure.
10. Click OK.
END OF SET UP
B. CALIBRATION
The Calibration procedure establishes the hardware’s internal parameters (such as gain, offset, and
scaling) and is critical for optimum performance. Pay close attention to the Calibration procedure.
FAST TRACK CALIBRATION
Detailed Explanation of Steps for Calibration
A pop-up window will tell you to remove any grip force from
1. Click on Calibrate.
the hand dynamometer. To do so, set the hand dynamometer
down.
2. Set the hand dynamometer down and Remove your hands from the transducer to ensure there is no
force on the transducer. This establishes a zero-force calibration
click OK.
before you continue the calibration sequence.
3. Grasp the BIOPAC hand dynamometer For Forearm 1, clench with the hand of your dominant forearm.
with your hand as close to the dynagrip For Forearm 2, clench with your other hand.
crossbar as possible without actually
touching the crossbar (Fig. 6).
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General Physiology and Excitable Tissue – Dental Medicine
Dynagrip crossbar
IMPORTANT
You need to hold the
dynamometer in the same
position for all measurements
from each arm, so note your
hand position with respect to
the crossbar for the first
segment and try to repeat it for
the other segments.
4. Follow the instructions on the two
successive pop-up windows and click
on OK when ready for each.
5. Wait about two seconds, then clench
the hand dynamometer as hard as
possible, then release.
6. Wait for Calibration to stop.
7. Check the Calibration data.
Hand close to
bracket but not
touching.
Fig. 6
Pop-up windows will guide you through the initial calibration
sequence. After you click OK on the third pop-up window, the
Calibration recording will begin.
The program needs a reading of your maximum clench to
perform an auto-calibration.
The Calibration procedure will last eight seconds and stop
automatically, so let it run its course.
At the end of the eight-second Calibration recording, the screen
should resemble Fig.7.
 If correct, proceed to the data
Recording Section.
 If incorrect, Redo Calibration.
Fig. 7
If your calibration recording did not begin with a zero baseline
(Subject clenched before waiting two seconds), you need to
repeat calibration to obtain a reading similar to Fig. 7.
END OF CALIBRATION
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General Physiology and Excitable Tissue – Dental Medicine
C. RECORDING LESSON DATA
FAST TRACK RECORDING
1. Prepare for the recording.
SEGMENT 1
2. Click on Record.
3. Clench the hand dynamometer with
your maximum force. Note this force
and try to maintain it.
4. When the maximum clench force
displayed on the screen has decreased
by more than 50%, click on Suspend.
5. Review the data on the screen.
 If correct, go Step 7.
 If incorrect, go to Step 6.
6. Click on Redo if your data was
incorrect, and repeat Steps 3-5.
SEGMENT 2
7. Click on Resume.
8. Repeatedly and periodically clench the
Detailed Explanation of Steps for Recording Lesson Data
You will record two segments on each forearm:
a. Segment 1 records Fatigue during the static work.
b. Segment 2 records Fatigue during the dynamic work
In order to work efficiently, read this entire section so you will
know what to do before recording.
Check the last line of the journal and note the total amount of
time available for the recording. Stop each recording segment as
soon as possible so you don’t waste recording time (time is
memory) .
After you click on record, the screen will change to display only
the hand dynamometer channel, and a grid using your assigned
increment as a division scale will appear so that you can visually
review the force level. You will begin to record data.
Note the maximum clench force so you can determine when the
force has decreased by 50% (the maximum force may scroll out
of view). Try to maintain the maximum clench force (the
forearm will fatigue and the force will decrease).
The time to fatigue to 50% of maximal clench force will vary
greatly among individuals.
When you click on Suspend, the recording should halt, giving
you time to review the data for segment one.
If all went well, your data should look similar to Fig. 8.
Fig. 8 Fatigue during the static work
Note that the peak found immediately following the start of
Segment 2 represents the maximal clench force. This example
shows the point of fatigue to 50% maximal clench force
captured on the same screen, but your maximum force may
scroll out of view. You may use the horizontal (time) scroll bar
to view your entire recording.
The data would be incorrect if:
a) You didn’t record to the point of 50% maximal clench
force.
b) The Suspend button was pressed prematurely.
c) The instructions were not followed.
Click “Redo” and have the Subject rest so the arm muscles
recover and the fatigue data will be meaningful. When ready
repeat Steps 3-5. Note that once you press Redo, the data you
have just recorded will be erased.
A marker labeled “Continued clench at maximum force” will
automatically be inserted when you click Resume and the
recording will continue from the point it left off.
Repeat a cycle of Clench-Release. One cycle should last about 1
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General Physiology and Excitable Tissue – Dental Medicine
hand dynamometer with your maximum
force and then relax.
9. When the maximum clench force
displayed on the screen has decreased
by more than 50% of the initial value,
click on Suspend
10. Review the data on the screen.
 If correct, go to Step 13.
11. If incorrect, go to Step 12.
12. Click on Redo if your data was
incorrect, and repeat Steps 8-10.
13. Click Forearm 2.
or, if you’ve recorded both arms,
Go to Step 16.
14. To record Forearm 2, attach electrodes
(per Set Up Step 5 and Step 6) to the
Subject’s other forearm.
15. Complete the entire Calibration section
and the Recording section up to this
point for the Forearm 2.
16. Click Done.
17. Remove the electrodes from your
forearm.
second.
The time to fatigue to 50% of maximal clench force will vary
greatly among individuals.
When you click on Suspend, the recording should halt, giving
you time to review the data for segment two.
If all went well, your data should look similar to Fig. 9.
Fig. 9 Fatigue during the dynamic work
The data would be incorrect if:
a) You didn’t record to the point of 50% maximal clench force.
b) The Suspend button was pressed prematurely.
c) The instructions were not followed.
Click “Redo” and have the Subject rest so the arm muscles
recover and the fatigue data will be meaningful. When ready
repeat Steps 8-10. Note that once you press Redo, the data you
have just recorded will be erased.
When you click on Forearm 2, the program returns you to the
Calibration sequence.
Refer to the Set Up section for proper placement of electrodes
and electrode leads.
Complete the entire Calibration sequence (outlined in the
preceding section) and then follow the entire Recording
procedure.
A pop-up window with four options will appear. Make your
choice, and continue as directed.
If choosing the “Record from another subject” option:
a) Attach electrodes per Set Up Step 5 and continue the
entire lesson from Set Up Step 8.
b) Each person will need to use a unique file name.
Remove the electrode cable pinch connectors, and peel off the
electrodes. Throw out the electrodes (BIOPAC electrodes are
not reusable). Wash the electrode gel residue from the skin,
using soap and water. The electrodes may leave a slight ring on
the skin for a few hours, which is quite normal.
END OF RECORDING
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General Physiology and Excitable Tissue – Dental Medicine
DATA ANALYSIS
FAST TRACK DATA ANALYSIS
Detailed Explanation of Data Analysis Steps
1. Enter the Review Saved Data mode and Enter Review Saved Data from the Lessons menu. 
choose the correct file.
For the first part of the analysis, choose the data file from the
Subject’s first arm (Forearm 1, saved with file name extension
“1-L02”).
For the second part of the analysis, choose the data file from the
subject’s second arm (Forearm 2, saved with file name extension
“2-L02”).
Note Channel Number (CH)
designations:
Channel Displays
CH 1
Force
CH 3
Raw EMG
CH 40
Integrated EMG
Fig. 10
2. Setup your display window for optimal The first data segment was the recording before the first marker.
viewing of the first data segment.
See Fig. 11.
The following tools help you adjust the data window: 
Autoscale horizontal Horizontal(Time) Scroll Bar
Autoscale waveforms Vertical (Amplitude) Scroll Bar
Zoom Tool
Zoom Previous
3. Set up the measurement boxes as
The following is a brief description of the specific
follows:
measurements not previously covered. 
Channel Measurement
value: displays the amplitude value for the channel at the
CH 1
value
point selected by the I-beam cursor. If a single point is
CH 40
delta T
selected, the value is for that point, if an area is selected,
the value is the endpoint of the selected area.
delta T: displays the amount of time in the selected
segment (the difference in time between the endpoints of
the selected area).
4. Using the I-Beam cursor, select a point
of maximal clench force immediately
following the start of Segment 1.
A
Fig. 11
The point selected should represent the maximal clench force at
the start of Segment 1 (continuous maximal clench), as shown in
Fig. 11.
You will need this number to complete Step 7.
5. Calculate 50% of the maximum clench
force from Step 4.
A
6. Find the point of 50% maximum clench Make an eyeball approximation of the point that is 50% down
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General Physiology and Excitable Tissue – Dental Medicine
force by using the I-beam cursor and
leave the cursor at this point.
from the maximal clench point. Then, use the I-beam cursor to
click on points near this region, noting the value displayed in the
measurement box, until you are on a point within 5% of the
maximal clench force. Leave the cursor at this point.
7. Select the area from the point of 50%
One way to select the area is as follows:
clench force back to the point of
The cursor should be flashing on the point of 50% maximal
maximal clench force by using the Iclench force. Hold down the mouse button and drag to the left of
beam cursor and dragging. (Fig. 12).
this point until you reach the point of maximal clench force, then
Note the time to fatigue (CH 40 delta T) release the mouse button.
measurement.
A
8. Scroll to Segment 2 and set up your
display for optimal viewing.
Fig. 12 showing area max-50%
Segment 2 begins at the second append marker.
9. Set up the measurement boxes as
The following is a brief description of the specific
follows:
measurements not previously covered. 
Channel Measurement
max: finds the maximum amplitude value within the area
CH 1
max
selected by the I-Beam cursor (including the endpoints).
CH 40
delta T
10. Using the I-Beam cursor, select an area
of the first maximal clench force
immediately following the start of
Segment 2.
B
Fig. 13
The area selected should represent the maximal clench force at
the start of Segment 2 (continuous maximal clench), as shown in
Fig. 13.
You will need this number to complete Step 13.
11. Calculate 50% of the maximum clench
force from Step 10.
B
12. Find the point of 50% maximum clench Make an eyeball approximation of the point that is 50% down
force by using the I-beam cursor and
from the maximal clench point. Then, use the I-beam cursor to
leave the cursor at this point.
click on points near this region, noting the value displayed in the
measurement box, until you are on a point within 5% of the
maximal clench force. Leave the cursor at this point.
13. Select the area from the point of 50%
One way to select the area is as follows:
clench force back to the point of
The cursor should be flashing on the point of 50% maximal
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General Physiology and Excitable Tissue – Dental Medicine
maximal clench force by using the Iclench force. Hold down the mouse button and drag to the left of
beam cursor and dragging. (Fig. 14).
this point until you reach the point of maximal clench force, then
Note the time to fatigue (CH 40 delta T) release the mouse button.
measurement.
B
Fig. 14 showing area max-50%
14. Repeat the entire Analysis section
starting with Step 1 with the data file
for Forearm 2.
15. Exit the program.

END OF DATA ANALYSIS
PROTOCOL
I.
Data and Calculations
Subject Profile
Name
Age
Gender: Male / Female
Height
Weight
Dominant forearm: Right / Left
Muscle Fatigue
A. Complete Table 1 using Segment 1 data from each arm.
Table 1 Segment 1 data – Static work
Forearm 1 (Dominant)
Maximum
50% of max
Time to
Maximum
Clench Force
clench force
fatigue
Clench Force
calculate
CH 1 value
CH 40 delta T* CH 1 value
Forearm 2
50% of max
clench force
calculate
Time to
fatigue
CH 40 delta T*
Forearm 2
50% of max
clench force
calculate
Time to
fatigue
CH 40 delta T*
B. Complete Table 2 using Segment 2 data from each arm.
Table 2 Segment 2 data – Dynamic work
Forearm 1 (Dominant)
Maximum
50% of max
Time to
Maximum
Clench Force
clench force
Fatigue
Clench Force
calculate
CH 1 max
CH 40 delta T* CH 1 max
*Note: You do not need to indicate the delta T (time to fatigue) polarity. The polarity of the delta T
measurement reflects the direction the "I-beam" cursor was dragged to select the data. Data
selected left to right will have a positive ("+") polarity, while data selected right to left will have
a negative ("-") polarity.
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General Physiology and Excitable Tissue – Dental Medicine
Questions
C. Compare the initial contraction force during the static and dynamic works.
Forearm 1 (dominant)
Forearm 2
D. Does the time to fatigue vary between the static and dynamic works? If yes, explain why.
Forearm 1 (dominant)
Forearm 2
E. Define Fatigue
F. As you fatigue, the force exerted by your muscles decreases. What physiological
processes explain the decline in strength?
G. Define Motor unit
H. Define Motor unit recruitment
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General Physiology and Excitable Tissue – Dental Medicine
Determination of Blood Volume using T1824 (Evans Blue)
The volume of any fluid compartment in the body can be measured by administration of a
substance into that compartment, allowing it to spread throughout the compartment, and then by
measuring the extent to which the substance had become diluted. The concentration of the injected
substance is analyzed chemically, or by other means.
Ideal properties of a substance used for measurements of the body fluid volumes are:
1. It must be non-toxic.
2. It should diffuse quickly and uniformly.
3. It should be eliminated and metabolized slowly.
4. It should not have any influence on the distribution of water and other substances in the body.
5. It should be easy to determine.
A substance used for measuring blood volume must be capable of spreading throughout the
blood with ease, and it must remain in the circulatory system long enough for measurements to be
made. The plasma proteins remain in the circulatory system and any foreign substance that combines
with them is kept in the blood stream. A number of dyes, generally known as "vital dyes", have the
ability to combine with proteins.
The dye the most universally used for measuring the plasma volume is T-1824, also called
Evans blue. In making determinations of the plasma volume, a known quantity of the dye is injected,
and it immediately combines with the proteins and spreads throughout the circulatory system within
approximately five minutes. A sample of the blood is then taken and the red blood cells are removed
from the plasma by centrifugation. By spectrophotometric analysis, one can determine the exact
quantity of the dye in the sample of the plasma. From the quantity of the dye detected in each milliliter
of the plasma and the quantity of the dye injected, the plasma volume is calculated using this formula:
Volume in ml =
Quantity of dye injected
Concentration of dye per ml of plasma
This method does not measure the total blood volume because the dye does not enter the red
blood cells. However, the blood volume can be calculated from the plasma volume and the haematocrit
using the following formula:
Blood volume = Plasma volume 
100
100 - Haematocrit
Procedure:
1. Anaesthetize the first adult rat, at first lightly with ether and then with urethan injected
intraperitoneally (1.5 g/kg of the body weight).
2. Put the anaesthetized rat on the operation table. Incise the skin on the neck along the midline
and reflect the skin laterally on each side. Make a vena jugularis externa preparation with
surgical forceps.
3. Aspirate the solution of the Evans blue into the insulin syringe in the amount proportional to
the rat's weight as indicated in the Table 1. Carefully inject the solution intravenously.
4. After 5 minutes decapitate the rat with scissors and collect its blood into a heparinized beaker.
5. Prepare the second rat in the same way (steps 1, 2)
6. Aspirate into the glass 2ml syringe the Evans blue solution in the amount indicated in the
Table 1 and replenish the syringe up to the volume of 2 ml with hypertonic glucose (20%).
Inject the solution carefully intravenously into vena jugularis externa.
7. After 5 minutes decapitate the rat and collect its blood in the heparinized beaker in the same
way as in the first case (step 4)
8. Prepare the third rat in the same way (steps 1, 2)
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General Physiology and Excitable Tissue – Dental Medicine
9. After vena jugularis externa preparation inject the NaCl solution in the same amount as if it
were Evans blue according to the Table 1.
10. After 5 minutes decapitate the rat and collect its blood in the beaker in the same way as in
step 4.
11. Take samples of the blood from the first and second rats into two glass capillaries, seal them
on the end and centrifuge them in the hematocrit centrifuge for 2 minutes at 16 000 r.p.m.
Determine the haematocrit value.
12. Transfer the blood from each of the three beakers into centrifuge test tubes and centrifuge
them 5 minutes at 16 000 r.p.m. in the laboratory centrifuge.
13. Aspirate the plasmas with Pasteur's pipettes and transfer them to spectrophotometer cuvettes.
The plasma from the third rat serves as the blank. Read the absorbances of the first and
second samples against the blank on the spectrophotometer.
14. Determine the concentrations of the Evans blue using the calibration curve (Figure 1) and
calculate the plasma volume and the blood volume of the first and second rats according to
the formulas above.
15. Calculate the blood volume per kg of weight in both rats.
16. Compare both values.
17. Describe consequences of the injection of the hypertonic solution.
Table 1.
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General Physiology and Excitable Tissue – Dental Medicine
1,1
1,0
0,9
Absorbance
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Concentration (mg/l)
Absorbance calibration curve
PROTOCOL
1.
Weight of the rat in kg
m1 =
2.
m2 =
Quantity of dye injected in mg
Q1 =
3.
Q2 =
Haematocrit
H1 =
4.
H2 =
Determination of Evans blue concentration (mg/l) using the calibration curve
C1 =
5.
C2 =
Plasma volume
V
P1

Q
C
1
V
P2
1
C
Q
6.

Q
C
2
2
Evans blue concentration in the plasma (mg/l)
Quantity of the dye injected (mg)
Blood volume
V
B1
 V P1 
1
1 H
V
21
B2
 V P2 
1
1 H
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General Physiology and Excitable Tissue – Dental Medicine
VP
H
7.
Plasma volume
Haematocrit
The blood volume per kg of weight of the rat
V
m
B1
1

V
m
B2

2
Conclusion:
Compare the blood volumes in both animals and explain eventual differences.
22