Physiological Science lab: Frog Skeletal Muscle

7/25/2019 Physiological Science lab: Frog Skeletal Muscle

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Frog Skeletal MuscleIn this experiment, you will investigate the physiological properties of skeletal muscle using the isolated

frog gastrocnemius. You will explore the single twitch, the graded response, the relationship between

muscle length and tension, muscle tetanus, and muscle fatigue. These experiments illustrate thecollective understanding of muscle physiology gained from over 400 years of research.

BackgroundThe frog muscle preparation you will use in the laboratory is the earliest isolated tissue preparation. Thefirst experiments on muscle physiology appear to have been performed between 1661 and 1665 by Jan

Swammerdam, who demonstrated that an isolated frog muscle could be made to contract when thesciatic nerve was irritated with a metal object. Later, Luigi Galvani (1737-1798) demonstrated that frog

muscle responded to electrical currents. This experiment focuses on the mechanical properties of

skeletal muscle. The invention of the kymograph (a rotating drum powered by a clockwork motor) in thelate 1840s, attributed to either Carlo Matteucci (1811-1868) or Carl Ludwig (1816-1895), revolutionized

experimental physiology for it enabled events such as muscle contractions to be recorded and analyzed

for the first time. Today, the computer has taken the place of the kymograph but physiology students ofthe late 1800's would recognize these experiments. These demonstrate some of the important functional

characteristics of skeletal muscle.

The basic unit of a muscle is the muscle cell, or fiber. Whole muscles are made up of bundles of these

fibers. Unlike cardiac muscle cells, there are no gap junctions between adjacent cells. This means each

fiber behaves independently. A single muscle fiber has a very regular structure, defined by myofibrils(Figure 1). Each myofibril consists of an arrangement of the contractile proteins actin and myosin, which

are able to slide past each other in the presence of calcium ions (Ca2+) and ATP.

Figure 1. Skeletal Muscle Structure

A single motor neuron, and all the muscle fibers that it innervates, is known as a motor unit (Figure 2).

Skeletal muscle is similar to nerve tissue in that the fiber responds to a stimulus in an all-or-none fashion.This response is called a twitch. One motor neuron supplies a number of muscle fibers to constitute a

motor unit. Motor units vary greatly in size, from just a few muscle fibers innervated by a single neuron

(small motor unit) up to thousands (large motor unit). The smaller the motor unit, the finer the controlof movement in that muscle; thus, the muscles controlling the movements of the fingers and eyes have

small motor units whereas those controlling the large limb muscles may have very large motor units.

However, most muscle consists of a range of motor unit sizes.


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Figure 2. Motor Unit

Depending on the intensity and frequency of stimulation, greater numbers of fibers are activated. Thestrength of a muscle contraction, therefore, can be increased in two ways: by increasing the number of

active motor units (termed recruitment) and by stimulating existing active motor units more frequently.The absolute force that a muscle can generate is dependent on the total number of muscle fibers. Somuscles with large cross-sectional areas are able to generate larger forces than those with small cross-

sectional areas.

Motor nerves release the neurotransmitter acetylcholine from their terminals, called motor end plates.

The acetylcholine released into the junctional cleft binds to receptors on the muscle membrane that are

directly coupled to cation-selective ion channels (Figure 3). Opening of these channels depolarizes themuscle fiber and leads to the release of intracellular calcium from the sarcoplasmic reticulum, a variant of

smooth endoplasmic reticulum. The increased cytosolic calcium sets in motion the biochemical eventsthat underlie contraction. The acetylcholine is rapidly hydrolyzed by acetylcholine esterase on the

skeletal muscle membrane in this region and thus does not accumulate in the junctional cleft.

Figure 3. Neuromuscular Junction


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Skeletal muscle can be studied under isometric (constant length) or isotonic (constant load) conditions.Here the force is measured isometrically. Action potentials in skeletal muscle, like those in nerve, last for

only a few milliseconds. In contrast the mechanical response of the muscle the muscle twitch last

significantly longer (Figure 4).

Figure 4. Temporal Relationship between Muscle Action Potential and Consequent Contraction

A second stimulus arriving before the muscle has relaxed again causes a second twitch on top of the first

so that greater peak tension is developed. This is called summation. With increasing frequency ofstimulation, there is less and less time for the muscle fiber to relax between stimuli, and eventually the

contractions fuse and a smooth powerful contraction tetanus is seen. Normally skeletal muscles areactivated by volleys of action potentials and operate in a state of fused contractions.

Figure 5. Effect of Frequency on Repeated Stimulation

The strength of muscle contraction is also influenced by the degree of stretch of the muscle. When

considering the force of the response to stimulation, it is necessary to separate out the passive and active

forces. The passive forces reflect the contributions of elastic elements in the muscle, both extracellularlyand within the fibers themselves. The active force is generated by the contractile machinery when thefibers are stimulated (Figure 6).

Experimentally, what is measured at different degrees of stretch is the total force during nerve

stimulation and contraction and passive force in the absence of nerve stimulation. The difference

between the two is the active force at any muscle length.


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Figure 6. Skeletal Muscle Length-Tension Relationship

Skeletal muscle contraction requires metabolic energy. A depletion of energy stores results in fatigue.Some muscle fibers are more resistant to fatigue than others; these have a greater capacity for oxidative

metabolism. Note that in the intact animal, fatigue occurs primarily because the motor drive from the

brain is reduced, rather than as a result of an appreciable depletion of the muscle energy reserves.


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Required Equipment LabChart 7 software

PowerLab Data Acquisition Unit

Bridge Pod

Force Transducer

Small weight between 550 grams Ring Stand

Manipulator/Micropositioner and clamps

Strong thread

Petri dish

Pasteur pipette

One frog (Rana pipiensor Xenopus laevis)

Normal Frog Ringers solution

Cold (10 oC) Frog Ringers solution

Small millimeter ruler

Tape

Medium-sized beaker

Dissection tools:

o

Glass fingerbowlo

Sharp scissors or scalpel

o Bone shearso Blunt probeo

Dissection tray with wax or pad

o Dissection pins Muscle Stimulation Equipment: You need one of the lists below (consult your instructor for more

information)o LIST 1

! Stimulator Cable (BNC to Alligator Clips)

! Muscle Holdero LIST 2

! Animal Nerve Stimulating Electrode!

Ring Stand clamp for the electrode! Femur clamp


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Procedure

Equipment Setup and Calibration

1. Make sure the PowerLab is turned off and the USB cable is connected to the computer.

2.

Connect the Force Transducer cable to the back of the Bridge Pod. Connect the Bridge Pod to Input1 on the front panel of the PowerLab (Figure 7). Connect the Stimulating Electrodes to the output on

the front panel of the PowerLab. Follow the color scheme in Figure 7.

Figure 7. Equipment Setup for PowerLab 26T

3. Securely mount the Force Transducer and Manipulator/Micropositioner on the Ring Stand as shown inFigure 8.

Figure 8. Ring Stand, Manipulator/Micropositioner, and Force Transducer Setup

4. Tape a small millimeter ruler to the side of the Manipulator/Micropositioner to use as a reference

when adjusting the height.


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5. Ask your instructor whether you are using a Muscle Holder or femur clamp to secure the muscle.Proceed to the proper setup after you calibrate the Force Transducer and dissect the frog.

6. Turn on the PowerLab.

Calibrating the Force Transducer

Raw output from the Force Transducer is in millivolts (mV). It needs to be calibrated to give the moremeaningful units of Newtons (N). The Force Transducer also has some residual offset voltage that needs

to be corrected for.

1. Launch LabChart and open the settings file Frog Muscle Settings from the Experimentstab in the

Welcome Center. It will be located in the folder for this experiment.

2. Select Bridge Podfrom the Channel 1 Channel Function pop-up menu. Leave the Force Transducer

undisturbed. Observe the signal (Figure 9) in the dialog. Zero this signal by turning the knob on thefront of the Bridge Pod. Close the Bridge Pod pop-up menu.

Figure 9. Bridge Pod Dialog

3. Start recording. Record for five seconds, and then hang a known weight (between 550 grams,

provided by your instructor) from the Force Transducer. Record for a further five seconds, and Stop.

4. Click-and-drag to selectall the data. Select Units Conversionfrom the Channel 1 Channel

Function pop-up menu (Figure 10).


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Figure 10. Units Conversion Dialog

5. Select a small area when no weight was added, and click the arrow next to Point 1.

6.

Select a small area when the weight was added, and click the arrow next to Point 2.

7. Enter the desired unit value in Newtons for each weight. Use the equation below:

Frog Dissection

1. Obtain a double-pithed frog from your instructor.

2. Use sharp scissors or a scalpel to cut the skin of the frog around its abdomen. Peel the skin down

and off the legs of the frog (Figure 11).

Figure 11. How to Remove the Skin

Note:Keep the tissue moist at all times with normal Ringers solution.


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3. Remove the leg from the frog by severing at the hip joint. Carefully dissect the gastrocnemiusmuscle away from the tibiofibula bone, but leave it attached to the knee and heel.

4. While the muscle is still attached, pass a 15 centimeter piece of strong thread under the Achillestendon at the heel of the frog. Tie this thread securely to the tendon (Figure 12). You will use this

thread to attach the muscle to the Force Transducer.

Figure 12. Thread Positioned under the Achilles Tendon

5. Sever the Achilles tendon below the attached thread.

6. Using bone shears or strong scissors, cut the tibiofibula bone below the knee, and cut the femurbone above the knee. If you are using the Muscle Holder, cut the femur close to the knee joint,

leaving little femur remaining. If you are using the femur clamp, cut the femur close to the hip joint,

leaving as much femur bone as possible for clamping (Figure 13).

Figure 13. Locations to Cut the Leg Bones

Note:Keep the muscle in a Petri dish of cold Frog Ringers solution until you are ready to mount it in the

Muscle Holder or femur clamp.


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Equipment Setup if Using a Muscle Holder

1. Attach the Muscle Holder to the Ring Stand.

2. Fix the muscle in the Muscle Holder by positioning the knee joint just below the constriction in the

Perspex molding, as shown in Figure 14. Connect the Alligator Clips to the two innerconnectors at

the top of the Muscle Holder.

Figure 14. Muscle Holder

3. Secure the thread attached to the Achilles tendon of the muscle through the hole in the metal tab of

the Force Transducer. Make sure there is some slack in the thread.

4. Raise the Manipulator/Micropositioner using the adjustment knob so that the muscle is vertical but

not under tension. The thread should not be loose but should have some slack in it. Make surethere is room to increase the height of the Force Transducer by at least 10 mm (Figure 15).

Figure 15. Equipment Setup with Muscle Holder


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5. Rotate the base of the Muscle Holder and insert an appropriately-sized beaker to collect wastesolution. Rinse the muscle with regular Ringers solution to keep it moist.

6. Make sure the silver wires on the Muscle Holder are still in contact with the muscle. Check thateverything is connected correctly.

Equipment Setup if Using a Femur Clamp

1. Attach the femur clamp to the Ring Stand.

2. Fix the muscle in the femur clamp with the knee joint side of the muscle in the clamp.

3. Secure the thread attached to the Achilles tendon of the muscle through the hole in the metal tab ofthe Force Transducer. Make sure there is some slack in the thread.

4. Raise the Manipulator/Micropositioner using the adjustment knob so that the muscle is vertical butnot under tension. The thread should not be loose but should have some slack in it. Make sure

there is room to increase the height of the Force Transducer by at least 10 mm (Figure 16).

5. Connect the stimulating electrode to a Ring Stand clamp to keep it in place. Touch the electrode tipto the belly of the muscle. (It is referred to as a bipolar stimulator in Figure 16 because there aretwo electrodes on the tip.)

Figure 16. Equipment Setup with Femur Clamp

6.

Insert an appropriately-sized beaker to collect waste solution under the femur clamp. Rinse the

muscle with regular Ringers solution to keep it moist.

7. Make sure the stimulating electrode is still in contact with the muscle. Check that everything is

connected correctly. Turn on the PowerLab.


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Exercise 1: Twitch Recruitment

In this exercise, you will examine the effects of stimulus amplitude (strength) on contractile force as the

muscle is given a series of stimuli of increasing amplitude.

Note:It is essential you watch what is happening to the muscle. You will be asked to describe your

observations in the Data Notebook.

1. LabChart should be open. If not, open the settings file Frog Muscle Settings.

2. Make sure the muscle is moist and is in contact with the electrodes.

3. Zero the Bridge Pod. Use the same procedure as before. You do not need to calibrate the data.

Note:For this exercise, you will be running a macro to apply a series of increasing stimuli. A macro is a

recorded set of commands and operations that can be executed with a single command.

4. Go to the Macromenu and select Recruitment to start the macro. Alternatively, you can press F2.

LabChart will start recording, increase the stimulus on its own, and stop recording. (Do not click

Start before playing the macro.)

5. When the macro is finished, save your data, but do not close the file.

6. Scroll through your data andAutoscale, if necessary. Start at the end of the data and move toward

the beginning. Determine the minimum voltage required to elicit the maximum contraction; this is

the maximum excitation voltage.

7. Multiply this voltage by 1.5. This new value is the supramaximal stimulus voltage. Record thesevalues in Table 2 of the Data Notebook. The supramaximal stimulus voltage will be used in each of

the following exercises.

8. Wait at least 30 seconds before moving on to the next exercise. Keep the muscle moist with Ringers

solution.

Exercise 2: Effect of Stretch on Contractile Force

In this exercise, you will examine the effect of stretch (also called preload) on muscle contractile force, by

raising the Manipulator/Micropositioner.

1. Make sure the muscle is moist and the electrodes are still positioned correctly.

2. Zero the Bridge Pod as before. You do not need to calibrate the data.

3. Note the position of the Manipulator/Micropositioner using the ruler. This is the reference point.

Record this value in Table 3 of the Data Notebook.

4. Click on the data at the end of the last data block, and go to the Commandsmenu and selectAdd

comment. Type exercise 2 and clickAdd.

5. Go to the Setupmenu and select Stimulator Panel. In the Pulse Heighttext box, enter the

value (in volts) for the supramaximal stimulus voltage calculated in Exercise 1. Do not adjust any

other settings. You can close the panel or keep it open.


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Note:For this exercise, you will be running another macro to set up the Stimulator and record the data.Do not start recording before playing the macro.

6. Go to the Macromenu and select Muscle Tension to start the macro. Alternatively, you can pressF3. LabChart will start recording and will prompt you with messages. Follow the on-screen

instructions to record data.

7. After each recording, there will be a prompt to raise the Manipulator/Micropositioner by one

millimeter. Before doing so, wait 30 seconds to allow the muscle to recover. Turn the adjustmentknob at the top to raise the instrument. Do not try to reposition the entire unit on the Ring Stand.

During the exercise, the Manipulator/Micropositioner will increase a total of 10 millimeters.

8. At the end of the macro, immediately return the Manipulator/Micropositioner to its original position

and release the tension on the muscle by turning the adjustment knob.

9. Save your data. Do not close the file.

10.Wait at least two minutes before moving on to the next exercise to give the muscle time to recover.

Make sure you keep it moist with Ringers solution.

Exercise 3: Muscle Summation

In this exercise, you will stimulate the muscle with twin pulses at different pulse intervals and willobserve their effect on muscle contractions.


2. Zero the Bridge Pod as before. Do not calibrate the data.

3. Click on the data at the end of the last data block, and go to the Commandsmenu and selectAddcomment. Type exercise 3 and clickAdd.

Note:For this exercise, you will be running a macro to set up the Stimulator and record the data. Donot click Start before playing the macro.

4. Go to the Macromenu and select Summation to start the macro. Alternatively, you can press F4.LabChart will start recording and will prompt you with messages.

5. Follow the on-screen instructions. The PowerLab will stimulate the muscle with twin pulses 400 ms,

200 ms, 100 ms, 50 ms, and 20 ms apart. Each recording will appear in a separate block.

6. When the macro has finished, add a commentto each block of data with the stimulus interval as

indicated above. Click on the data at the beginning of a data block, and go to the Commandsmenu

and selectAdd comment. Type the stimulus interval, select Insert at selection and clickAdd.

7.

Save your data. Do not close the file.

8. Wait at least 30 seconds before moving on to the next exercise. Make sure you keep the musclemoist with Ringers solution.

Exercise 4: Muscle Tetanus

In this exercise, you will examine the muscles response to repetitive stimulation at five different

frequencies, each for a 1 second duration.


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2. Zero the Bridge Pod as before, but do not calibrate the data.



Note:For this exercise, you will be running a different macro to set up the Stimulator and record the

data. Do not click Start before playing the macro.

4. Go to the Macromenu and select Tetanus to start the macro. Alternatively, you can press F5.LabChart will start recording and will prompt you with messages.

5. Follow the on-screen instructions. The PowerLab will stimulate the muscle for one second withrepetitive pulses at intervals of 400 ms, 200 ms, 100 ms, 50 ms, and then 20 ms. Each recording willappear in a separate block.

6. When the macro has finished, add a commentto each block of data with the stimulus interval as

indicated above. Click on the data at the beginning of a data block, and go to the Commandsmenu

and selectAdd comment. Type the stimulus interval and select the comment to be inserted at theselection.


8. Wait at least 30 seconds before moving on to the next exercise. Make sure you keep the musclemoist with Ringers solution.

Exercise 5: Muscle Fatigue

In this exercise, you will examine muscle fatigue, evoked by prolonged stimulation of the muscle at a

high frequency (50 Hz for 30 seconds).

1.

Make sure the muscle is moist and the electrodes are still positioned correctly.

2. Zero the Bridge Pod as before, but do not calibrate the data.



Note:For this exercise, you will be running a final macro to stimulate the muscle repetitively. Do not

click Start before playing the macro.

4. Go to the Macromenu and select Fatigue to start the macro. Alternatively, you can press F6.

LabChart will start recording and will prompt you with messages.

5.

Follow the on-screen instructions. The PowerLab will stimulate the muscle with a stimulus interval of20 ms for 30 seconds. The recording duration of the macro is 45 seconds.



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Analysis

Exercise 1: Twitch Recruitment

1. Examine the data in the Chart View. Autoscale, if necessary. Up to 20 contractions should be seen

in the Force channel, each in a separate data block.

2. Place the Markeron the baseline of the waveform in the Force channel.

3. Place the Waveform Cursorat the top of the last contraction peak.

4. Record the peak height for each of the peaks in Table 1 of the Data Notebook, starting at the lastpoint and working backwards to the beginning of the trace. Do not move the Marker, only the

Waveform Cursor. Fill out Table 1 starting at the bottom.

Note:The PowerLab will have stimulated the muscle 20 times, but not all of the stimuli may have elicited

a twitch. If there are fewer than 20 contractions, enter a zero in Table 1 for those stimulus intensitieswithout a twitch.

Exercise 2: Effect of Stretch on Contractile Force1. Examine the data in the Chart View, andAutoscale, if necessary.

2. If the Markeris still in the Chart View, click in the lower left corner to return it to its Marker box.

3. Using the Waveform Cursor, measure the baseline value. This is the preload force.

4. Determine the raw twitch force with the Waveform Cursorby placing the cursor on the maximumvalue in the data block. Record this value in Table 3 of the Data Notebook.

5. Repeat steps 3 and 4 for the other data blocks in this exercise.

6.

Calculate the net twitch force by subtracting the preload (baseline) value from the raw twitch forcevalue.

7. Record these values in Table 3 of the Data Notebook.

Exercise 3: Muscle Summation

1. Examine the data in the Chart View, andAutoscale, if necessary. There will be five blocks of

recorded data.

2. For each data block, determine the maximum contractile force of the first peak and second peakusing the Markerand Waveform Cursor. Position the Markeron the baseline immediately before

the first contraction. Use the Waveform Cursorto measure the maximum heights of the two

contractions.


Note:When the peaks merge, put a single measurement in the second response column of Table 4.


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Exercise 4: Muscle Tetanus

1. Examine the data in the Chart View, andAutoscale, if necessary. There should be five blocks of

data.

2. Determine the maximum contractile force using the Markerand Waveform Cursor, as done for

Exercise 3. Follow the steps above.


Exercise 5: Muscle Fatigue

1. Examine the data in the Chart View, andAutoscale, if necessary.

2. Place the Markeron the waveform baseline in the Force channel immediately prior to stimulation.

3. Use the Waveform Cursorto determine the maximum contractile force. Record this value in Table6 of the Data Notebook.

4. Record the time of maximum stimulation in Table 6 of the Data Notebook.

5. Determine the contractile force at the following times:

t = 1 s

t = 5 s

t = 10 s

t = 15 s t = 20 s

t = 25 s

t = 30 s

6. Record each of these values in Table 6 of the Data Notebook.


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Data Notebook

Exercise 1 Observations

a) Describe the contractile force of the muscle when the stimulus strength was increased.

Table 1. Effect of Stimulus Intensity on Contractile Force

Stimulus Amplitude (V) Contractile Force (N)

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.80

0.85

0.90

0.95

1.00


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Table 2. Supramaximal Stimulus Voltage

Minimum Voltage Required

For Maximal Contraction (V)

Supramaximal Stimulus

Voltage (V)

Table 3. Effect of Preload on Contractile Force

Reference position of micropostitioner (mm) =

Preload(N)

Raw TwitchForce (N)

Net TwitchForce (N)

Block 1

(Reference Point___ mm)

Block 2(___ mm)

Block 3

(___ mm)Block 4

(___ mm)

Block 5(___ mm)

Block 6

(___ mm)

Block 7

(___ mm)

Block 8(___ mm)

Block 9

(___ mm)Block 10

(___ mm)

Table 4. Effect of Stimulus Frequency on Contractile Force

Stimulus Interval

(ms)

Force of First

Peak (N)

Force of Second

Peak (N)

400

200

100

50

20


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Table 5. Muscle Tetanus

Stimulus Interval

(ms)

Contractile

Force (N)

400

200

100

50

20

Table 6. Muscle Fatigue

Time at Maximum Force(s, post-stimulation)

Maximum Force(N)

Time Since Stimulation

(s)

Contractile Force

(N)

1

5

10

15

20

25

30


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Study Questions

1. In light of the all or none law of muscle contraction, how can you explain twitch

recruitment (also called the graded response)?

2. What effect does stretching the muscle have on contractile strength? Is this effectlinear? What preload force resulted in the highest contractile force?

3. What effect does varying the stimulation frequency have on contractile force? Whichstimulus interval caused the greatest contractile force?


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4. Define tetanus. At which stimulus interval did you observe tetanus?

5. At what time point did your muscle begin to fatigue? Calculate the percent decrease incontractile force by comparing the force at the end of the experiment with themaximum contractile force.

6. In your own words, explain a possible mechanism for why the muscle was unable to

maintain a prolonged contraction in Exercise 5.

Physiological Science lab: Frog Skeletal Muscle

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