DMT 720MO Bedienungsanleitung
1TISSUE BATH MYOGRAPH SYSTEM - 720MO - USER GUIDE
USER GUIDE
TISSUE BATH 720MO
USER GUIDE
3TISSUE BATH MYOGRAPH SYSTEM - 720MO - USER GUIDE
CONTENTS
Chapter 1 - tissue bath overview..............................................................................................................................................................................................3
Chapter 2 - setting up the tissue bath ..................................................................................................................................................................................4
2.1 Changing and adjusting the mounting supports .............................................................................................................................4
2.1.1 Changing the mounting supports (gure 2.1): ............................................................................................................................4
2.1.2 Fine adjusting the pins for larger vessels (gure 2.2 and 2.3) ...................................................................................................5
2.2 Calibration of the force transducer ..................................................................................................................................................5
Chapter 3 - Experimental set-up ..............................................................................................................................................................................................6
3.1 Mounting protocol ?? ........................................................................................................................................................................6
3.2 Normalization ....................................................................................................................................................................................7
3.2.1 Principles of the normalization procedure ...................................................................................................................................7
3.3 Standard start....................................................................................................................................................................................8
3.3.1 Principles of the standard start procedure ..................................................................................................................................8
3.4 Endothelium function........................................................................................................................................................................9
3.4.1 Principles of checking endothelium function...............................................................................................................................9
3.5 In vitro experiment 1: Noradrenaline contractile response............................................................................................................9
3.5.1 Background ....................................................................................................................................................................................9
3.6 In vitro experiment 2: Acetylcholine relaxation curve .................................................................................................................. 10
3.6.1 Background ................................................................................................................................................................................. 10
3.5.2 Protocol........................................................................................................................................................................................ 10
3.6.2 Protocol........................................................................................................................................................................................ 11
Chapter 4 - CleAning and maintenance ............................................................................................................................................................................. 12
4.1 Cleaning the Tissue Bath ............................................................................................................................................................... 12
4.2 Maintenance of the force transducer ........................................................................................................................................... 13
3.3.1 Checking the force transducer................................................................................................................................................... 13
4.3.1 Force transducer replacement................................................................................................................................................... 14
4.4 Maintenance of the linear slides................................................................................................................................................... 15
Appendix 1 - Buer recipes ..................................................................................................................................................................................................... 16
Appendix 2 - Normalization theory ...................................................................................................................................................................................... 18
Appendix 3 - Reading a millimetre micrometer............................................................................................................................................................... 20
Notes................................................................................................................................................................................................................................................ 21
4CHAPTER 1
CHAPTER 1 TISSUE BATH OVERVIEW
Micrometer
Pin support connected to micrometer
Force transducer pin
Pin support connected to
force transducer
Figure 1.1 Tissue Bath with close-up of chamber
Transducer house
5TISSUE BATH MYOGRAPH SYSTEM - 720MO - USER GUIDE
CHAPTER 2 SETTING UP THE TISSUE BATH
2.1 Changing and adjusting the mounting supports
Each chamber can accommodate mounting supports for larger segments (>500 µm). Because the mounting supports can be
changed easily, experiments can be performed with different vessels of varying internal diameter from 500 µm up to 10 mm.
Continuous use and repeated greasing of the transducer arm holes will cause some misalignment of the mounting supports. The
mounting supports will need occasional adjustments. Changing and adjustment of the mounting supports is performed using
the following step-by-step procedure.
OBS
THE TRANSDUCERS ARE FRAGILE AND SENSITIVE TO MECHANICAL STRAIN. BE VERY CAREFUL WHEN CHANGING OR AD
JUSTING THE MOUNTING SUPPORTS!
2.1.1 Changing the mounting supports (gure 2.1):
1. Use the micrometer to separate the supports as far apart as possible.
2. Use the small screwdriver provided to gently loosen screw “D” on the support attached on the transducer side. Screw “D” is
the screw on the support closest to the transducer.
3. Gently pull the support away from the transducer pin.
4. Loosen screw “B” on the micrometer side with the appropriate Allen key.
5. Pull the pin support away.
NOTE
NUMBER THE MOUNTING SUPPORTS WITH THE NUMBER OF THE CHAMBER THEY WERE REMOVED FROM USING A
PERMANENT MARKER. STORE THE MOUNTING SUPPORTS IN THE PROVIDED PLASTIC CASE. NUMBERING THE MOUNTING
SUPPORTS WILL SAVE TIME WHEN THE MOUNTING SUPPORTS ARE CHANGED AGAIN, MINIMIZING THE AMOUNT OF
ADJUSTMENTS NEEDED AFTER EACH CHANGE.
A B C D
Figure 2.1 Illustration of the screws for changing supports and adjustment of the pins
Micrometer
6CHAPTER 2
Tissue holding pins from top view
Tissue holding pins from side view
Figure 2.3 - Illustrations of properly aligned tissue holding pins (depicted on the far left) and
incorrectly aligned pins (depicted in the middle and far right).
Figure 2.2 - Illustration of the screws for ne adjustment of the pins
A B E D C
2.1.2 Fine adjusting the pins for larger vessels (gure 2.2 and 2.3)
1. Loosen screw “A” to move the micrometer-side arm holder sideways
2. Loosen screw “B” to move the micrometer-side pin toward or away from the transducer.
3. Loosen screw “C” to align the transducer-side tissue holding pin horizontally.
4. Loosen screws “D” and “E” to align the heights of the tissue holding pins vertically.
2.2 Calibration of the force transducer
As a part of the general maintenance of the Tissue Bath, DMT recommends that the Tissue Bath is force calibrated at least once a
month. The Tissue Bath should also be force calibrated every time the Multi Interface has been moved. Although lab benches are
all supposedly perfectly horizontal, small differences in lab bench pitch can affect the calibration of the system. The Tissue Bath
should also be calibrated if the system has been idle for longer than a month. A step-by-step procedure is explained in chapter
3.5.1.2 in Multi Myograph System - User Manual.
7TISSUE BATH MYOGRAPH SYSTEM - 720MO - USER GUIDE
CHAPTER 3 EXPERIMENTAL SETUP
This chapter contains experimental set-up for the Tissue Bath. For dissection of a vessel, please see Procedures for investiga-
tions of small vessels using a small vessel Myograph by M.J. Mulvany.
3.1 Mounting protocol
Below is a short description of how to mount a tissue ring preparation on the mounting pins of the 720MO system. DMT deliver
the 720MO system with 200µm pins allowing the user to mount ring preparation with an internal diameter from 450µm and up.
For really large tissue ring preparation with the ability to make high force contractions the 200µm pins may be too thin. DMT can
delivery mounting pins with the following thickness:
L-shaped mounting pins (Diameter): 200µm, 250µm, 300µm and 400µm.
1. Fill the chamber with a given buffer (5-7ml) (see appendix 1 for Buffer recipes). DO NOT FILL THE CHAMBER TO THE EDGE
OF THE CHAMBER.
2. Make sure that the tissue holding pins are properly aligned.
3. Use the micrometer to get the pins close together as possible without touching each other.
4. Slide your tissue ring preparation onto the two mounting pins.
5. Slowly move the pins apart using the micrometer until the force reading is increasing.
6. The tissue is now mounted and ready for a normalization protocol (see chapter 3.2) nding the optimal pre-load tension for
the mounted tissue
7. Place the micrometer at the position giving the optimal pre-load tension on the mounted tissue.
8. Continue with a standard start (see chapter 3.3).
9. The mounted tissue is now ready for experiments.
Properly aligned pins, seen from side and above
8
Figure 3.9 Illustration of the exponential
curve tting and determination of IC100
Figure 3.8 Illustration of the stepwise
normalization procedure
3.2 Normalization
The importance of normalizing the preparation is three-fold:
1. Experiments with elastic preparations like vessels can only have meaning if they are performed under conditions where the
size is clearly dened.
2. Clearly dened conditions are required in pharmacological experiments as the sensitivity of preparations to agonists and
antagonists is dependent on the amount of stretch.
3. The active response of a preparation is dependent on the extent of stretch, which makes it important to set the preparation
to an internal circumference giving maximal response.
The aim of the normalization procedure is to stretch the segment to a so-called normalized internal circumference (IC1): dened
as a set fraction of the internal circumference (IC100) that a fully relaxed segment would have at a specied transmural pressure.
For small rat arteries the target transmural pressure is typically 100 mmHg = 13.3 kPa.
3.2.1 Principles of the normalization procedure
In practice the normalization is performed by distending the segment stepwise and measuring sets of micrometer and force read-
ings (gure 3.8). These data are converted into values of internal circumference (μm) and wall tension T (mN/mm) respectively.
Plotting wall tension against internal circumference reveals an exponential curve and by applying the isobar curve corresponding
to 100 mmHg, IC100 is calculated from the point of intersection using the Laplace relation (gure 3.9). IC1 is calculated from IC100
by multiplying a factor giving an internal circumference at which the active force production as well as the sensitivity to agonists
of the segment is maximal. For rat mesenteric arteries the factor is 0.9 but both this factor as well as the transmural pressure has
to be optimized for each particular segment. The normalized internal diameter is calculated by dividing IC1 with .
Appendix 2 contains a complete description of the mathematical rationale and calculations of the normalization procedure.
3.3 Standard start
The purpose of performing a standard start is to:
1. Re-activate the mechanical and functional properties of the vessel segment.
2. Check that responses to different types of stimuli are normal in appearance and thereby ensuring that the functionality of
the vessel segment has not been damaged during the dissection or mounting procedures.
3. Ensure that the tension development gives an effective active pressure that is above the chosen accepted value (usually
13.3 kPa = 100 mmHg).
The standard start is performed after the vessel segment has been heated, equilibrated and normalized. The present procedure
is suitable for rat mesenteric arteries. Another procedure may be needed for other animal species and tissue or vessel types.
CHAPTER 3
9TISSUE BATH MYOGRAPH SYSTEM - 720MO - USER GUIDE
-- Stimulus 1 & 2 --
KPSS + 10 M NA
Stimulate for 3 minutes
-- Wash out --
4 x with PSS
Wait 5 minutes
-- Stimulus 3 --
PSS + 10 M NA
Stimulate for 3 minutes
-- Wash out --
4 x with PSS
Wait 5 minutes
-- Stimulus 4 --
KPSS
Stimulate for 3 minutes
-- Wash out --
4 x with PSS
Wait 5 minutes
-- Stimulus 5 --
KPSS + 10 M NA
Stimulate for 3 minutes
-- Wash out --
4 x with PSS
Ready for experiment
Repeat 1 x
3.4 Endothelium function
The reasons for checking endothelium function may include:
1. To check whether the relaxing function of the endothelium is intact. The procedure is performed to make sure that the en-
dothelium is not damaged during the dissection or mounting procedure.
2. If an experiment requires removal of the endothelium this procedure is useful to check whether the endothelial cells were
successfully removed.
The procedure can be performed after the vessel segment has been heated, equilibrated and normalized. Preferably the proce-
dure should be done after performing a standard start to make sure that the vessel segment is viable.
The present procedure is for use with rat mesenteric arteries. Another procedure may be needed for other animal species and
tissue or vessel types.
3.4.1 Principles of checking endothelium function
Stimulating a vessel segment with acetylcholine causes a release of nitric oxide (NO, also known as EDRF) from the endothelium
cells and subsequent relaxation of the vascular smooth muscle cells. If the endothelium is undamaged by the dissection and
mounting procedures, then a substantial relaxation will occur. With complete removal or damaged endothelium, a partial relaxa-
tion or no relaxation to acetylcholine is observed.
It is important to note that the amount of NO or EDRF in a vessel is often dependent upon its size. In certain vessels, endothelium-
derived hyperpolarizing factor (EDHF) can contribute more or less than EDRF, and in other vessels the same stimulation with ACh
can promote release of endothelium-derived contracting factor (EDCF). Therefore, it is important to check the existing literature
in order to determine the expected response in your particular vessel with the given concentration of agonist.
3.3.1 Principles of the standard start procedure
The standard start procedure consists of a series of ve stimuli and washout periods. The rst two stimuli are performed using
a mixture of KPSS and 10 μM noradrenaline to give a maximum contractile response. The third stimulus is performed using a
mixture of PSS and 10 μM noradrenaline to give a maximum pure agonist mediated (α-adrenoceptor) contraction. The fourth
stimulus is performed using KPSS to give a depolarising contractile response (this stimulus also includes a component from neu-
rally released noradrenaline). The nal stimulus is performed using a mixture of PSS and 10 μM noradrenaline. All solutions are
preheated to 37oC and aerated with a mixture of 95% O2 and 5% CO2 before use. Instructions for making the necessary solutions
are described in appendix 1.
10
3.5 In vitro experiment 1: Noradrenaline contractile response
The purpose of the present protocol is to determine the sensitivity of rat mesenteric small arteries to the vasoconstrictor
noradrenaline/norepinephrine with a cumulative concentration-response curve.
3.5.1 Background
Noradrenaline (norepinephrine) causes contraction of mesenteric small arteries through activation of α-adrenoceptors whereas
noradrenaline activation of β-adrenoceptors causes vasodilatation. As the purpose is to determine the contraction sensitivity to
noradrenaline, the vasodilatory effect of noradrenaline is eliminated throughout the experiment by the constant presence of the
β-adrenoceptor antagonist, propranolol.
Rat mesenteric arteries are densely innervated by sympathetic nerves, which have a highly efcient reuptake mechanism that
removes noradrenaline from the neuromuscular junction. The reuptake mechanism will create a concentration gradient between
the solution around the vessel segment and the receptors on the smooth muscle. To correctly determine the sensitivity to
noradrenaline it is necessary to eliminate this concentration gradient by performing the experiment in the presence of cocaine
to block the noradrenaline reuptake.
To determine the sensitivity to noradrenaline the vessel segment is exposed to increasing concentrations of noradrenaline.
Each concentration is applied until a steady response has been reached and then the next concentration is applied. When the
vessel segment is fully contracted or does not response more upon increasing the noradrenaline concentration, the experiment
is ended.
3.5.2 Protocol
Prepare the following stock solutions:
Noradrenaline: 10-4, 10-3, 10-2 M
Propranolol: 10-3 M
Cocaine: 10-3 M
1. Mount and normalize the vessels as described in chapter 3.1 and 3.2.
2. Perform a standard start as described in chapter 3.3.
3. Incubate the vessel segment in 1 μM propranolol (add 5 µL of 10-3 M to 5 mL PSS in chamber) and 3 μM cocaine (add 15
µL of 10-3 M to 5 mL PSS in chamber) for at least 10 minutes.
4. Add increasing concentrations of noradrenaline into the bath (use the table below as a guideline). Wait for a stable contractile
response or a standard time such as 2 minutes between each application.
[NA] in chamber (µM)* Volume of stock solution to add to chamber
0.1 5 μL of 10-4 M
0.3 1 μL of 10-3 M
0.5 1 μL of 10-3 M
12.5 μL of 10-3 M
1.3 1.5 μL of 10-3 M
1.5 1 μL of 10-3 M
37.5 μL of 10-3 M
51 μL of 10-2 M
10 2.5 μL of 10-2 M
*In calculating the [NA] in the chamber, the applied volume of noradrenaline is ignored.
CHAPTER 3
Andere Handbücher für 720MO
1
Inhaltsverzeichnis
Andere DMT Badezimmerarmatur Handbücher
