DMT 630MA User manual

USER GUIDE
MYOGRAPH SYSTEM - 630MA

AUTOMATED MULTI WIRE
MYOGRAPH SYSTEM

CONTENTS
CHAPTER 1 - WIRE MYOGRAPH OVERVIEW 4
CHAPTER 2 - THE MULTI WIRE MYOGRAPH UNIT 5
2.1 Changing and adjusting the mounting supports 5
2.2 Calibration of the force transducer 8
CHAPTER 3 - EXPERIMENTAL SET-UP 9
3.1 Mounting protocol for small arteries 9
3.2 Mounting protocol for larger arteries 15
3.3 Normalization 17
3.4 Standard start 24
3.5 Endothelium function 25
3.6 In vitro experiment 1: Noradrenaline contractile response 26
3.7 In vitro experiment 2: Acetylcholine relaxation curve 28
CHAPTER 4 - CLEANING AND MAINTENANCE 30
4.1 Cleaning the 630MA Myograph system 30
4.2 Maintenance of the force transducer 32
4.3 Maintenance of the linear slides 36
APPENDIX 1 - BUFFER RECIPES 37
Physiological Saline Solution (PSS) 37
High potassium Physiological Saline Solution (KPSS) 39
APPENDIX 2 - NORMALIZATION THEORY 40
Mathematical calculations 40
APPENDIX 3 - READING A MILLIMETRE MICROMETER 42

4
CHAPTER 1 - WIRE MYOGRAPH OVERVIEW
Figure 1.1 Wire Myograph with close-up of chamber
Micropositioner
Allen screws for ne
alignment of the
myograph jaws
Connection to Myo-Interface
Force transducer pin
Supports
Myograph jaw connected to force transducer
Myograph jaw connected to micropositioner
Figure 2.3 Chamber cover
For drug application
Temperature probe
Funnel

5
CHAPTER 2 - THE MULTI WIRE MYOGRAPH UNIT
2.1 CHANGING AND ADJUSTING THE MOUNTING SUPPORTS
NOTE: The transducers are fragile and sensitive to mechanical strain. Be very careful
when changing or adjusting the mounting supports!
Each chamber can accommodate mounting
supports for either small vessels (>30µm) or
larger segments (>500µm). Because the mounting
supports can be changed easily, experiments can
be performed with dierent vessels of varying
internal diameter. Continuous use and repeated
greasing of the transducer pin holes will cause
some misalignment of the mounting supports.
The mounting supports, therefore, whether
they are the jaws for wires or the pins, will need
occasional adjustments.
Changing and adjustment of the supports is
performed using the following step-by-step
procedure.
2.1.1 CHANGING THE MOUNTING SUPPORTS (FIGURE 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 using the small screwdriver.
Screw D is the screw on the transducer-side
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 tting allen key.
5. Pull the support away. Note: Number the
supports with the chamber number they
were removed from using some kind of
permanent marker. Store the supports in
the provided plastic case. Numbering the
supports will save time when the sup ports
are changed again, limiting the amount of
adjustments needed after each change.

6
A B D C
2.1.2 COARSE ADJUSTING THE JAWS FOR SMALL VESSELS (FIGURE 2.1)
1. Loosen screw A to move the micrometer-side
jaw toward or away from the micrometer.
2. Loosen screw B to move transducer-side jaw
toward or away from the transducer.
3. Loosen screw C to vertically alight the
transducer-side jaw. Screw C is the screw on
the transducer-side support that is furthest
away from the transducer.
NOTE: Number the supports with the number of the chamber they were removed from
using some kind of permanent marker. Store the supports in the provided plastic case.
Numbering the supports will save time when the supports are changed again, limiting
the amount of adjustments needed after each change.
Figure 2.1 Illustration of the screws for changing supports and coarse adjustment of the jaws
Micrometer
Transducer house

7
Figure 2.3 - Illustrations of properly aligned jaws (depicted on the far left) and incorrectly aligned jaws
(depicted in the middle and far right).
Jaws from top view
Jaws from side view
2.1.3 FINE-ADJUSTING THE JAWS FOR SMALL VESSELS (FIGURE 2.2 AND FIGURE 2.3)
1. TighteningScrew“D”willmovethemicrometer
side jaw downward and to the left.
2. Tightening both screws “D” and “B” will move
the micrometer side jaw straight down
3. Tightening both screws “C” and “A” will move
the micrometer side jaw straight up.
Figure 2.2 Fine adjustments of the jaws in the Wire Myograph chamber
A B
C D

8
Figure 2.4 - Fine adjustments of the pins in the Myograph chamber
Pins from top view
Figure 2.5 - Illustrations of properly aligned pins (depicted on the far left) and incorrectly
aligned pins (depicted in the middle and far right).
2.1.4 FINE-ADJUSTING THE PINS FOR LARGER VESSELS (FIGURE 2.4 AND FIGURE 2.5)
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
pin horizontally.
4. Loosen screws D and E to align the heights of
the pins vertically.
Micrometer
Transducer house
A B
C D
2.2 CALIBRATION OF THE FORCE TRANSDUCER
As a part of the general maintenance of the Wire
Myograph, DMT recommends that the Wire
Myograph is force calibrated at least once a
month. The Wire Myograph should also be force
calibrated every time the interface has been
moved. Although lab benches are all supposedly
perfectly horizontal, small dierences in lab bench
pitch can aect the calibration of the system. The
Wire Myograph 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
in Multi Myograph System User Manual.
Pins from side view

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CHAPTER 3 - EXPERIMENTAL SET-UP
This chapter contains experimental set-up for the
Wire Myograph. For dissection of a vessel, please
see Procedures for investigations of small vessels
using a small vessel Myograph by M.J. Mulvany.
3.1 MOUNTING PROTOCOL FOR SMALL ARTERIES
The procedure involves attaching the mounting
wires to jaws which are in turn mounted on the
force transducer. This force transducer is capable
of measuring with a sensitivity of about 0.01 mN
(1 mg), but can be damaged if the applied force
exceeds about 1600 mN (160 g). Therefore, care
must be taken to avoid pressing the jaws too hard
together. A movement of ~20 µm after they have
touched is sucient to hold the wires clamped.
3.1.1 MOUNTING STEP ONE
• Cut lengths of 40 μm wire ~2.2 cm long.
Mount one wire on left-hand jaw of the Wire
Myograph as follows.
• Holding wire at far end, place center of wire
between jaws and screw jaws together so
that the wire is clamped (gure 3.1 A).
• Bend the far end of the wire towards the left,
and wrap it around under xing screw, so the
wire is wound clockwise: tightening the screw
will then tighten the wire. This procedure
should result in the wire being clamped
between the jaws and with near end of wire
pointing towards operator (gure 3.1 B-C).
• Fill the Wire Myograph chamber with PSS
(at room temperature). See appendix 1 for
example of buer recipes.
WARNING: Do NOT close the jaws
too hard against each other.
Figure 3.1 A, B and C Mounting step 1
A B C

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3.1.2 MOUNTING STEP TWO
• Using forceps to hold the handle segment,
transfer excised vessel from Petri dish to the
Wire Myograph chamber. Hold the vessel as
close to the proximal end as possible and try
to mount the vessel onto the wire.
• If the lumen is shut, try one of the following
possibilities:
1. Use the wire to gently push the lumen
open (blood streaming out is a good sign)
2. Hold excised vessel about 3 mm from
the cut end with one set of forceps and
use the other forceps to squeeze the blood
remaining in lumen out through the cut
end.
• Pull the proximal end of the excised vessel
segment along the wire such that the vessel
segment acts as its own feeder to be feed
into the wire into the vessel (gure 3.2 A-C).
Be careful not to stretch the vessel segment
if the end of the wire catches the vessel wall.
Figure 3.2 A, B and C Mounting step 2
A B C

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3.1.3 MOUNTING STEP THREE
• Once the vessel segment is threaded onto
the wire, catch the free end of the wire (near-
est you) with the forceps and move the jaws
apart.
• While controlling the movement of the wire
with the forceps, use the other forceps to
gently pull the vessel segment along the wire
until the area of interest is situated in the gap
between the jaws. The near end of the vessel
segment shall lie about 0.1 mm inside the jaw
gap to insure no point of contact (gure 3.3
A).
• Still controlling the free wire end with the for-
ceps, move the jaws together to clamp the
wire and in one movement secure the wire
under the near xing screw on the left-hand
jaw. Again in a clockwise direction so that
tightening the screw also tightens the wire
(gure 3.3 B).
Figure 3.3 A and B Mounting step 3
3.1.4 MOUNTING STEP THREE
• Using forceps, gently rub the vessel segment
on the far side of the jaw to separate any
excess vessel segment from the area of
interest clamped in the gap between the
jaws (gure 3.4 A). Make sure that the vessel
segment is separated as close as possible to
the jaws (gure 3.4 B). The excessive vessel
segment is nally dissected free and removed
from the wire (gure 3.4 C).
ABC
Figure 3.4 A, B and C Mounting step 4
A B

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3.1.5 MOUNTING STEP FIVE
• Move the jaws apart (gure 3.5 A). Take a
second wire holding it about one third down
from the far end using a forceps. Align the
wire parallel with the vessel segment such
that the wire can be passed into the far end
of the lumen. Gently feed the wire through
the lumen of the vessel segment in one
movement using the rst mounted wire
as a guide (gure 3.5 B-C). Hold the wire at
a point at least 10 mm from the vessel to
prevent the vessel being stretched during
the maneuverer. Be careful not to touch the
lumen of the vessel with the end of the wire
and when pushing the wire end through the
near end of the lumen. Once the wire has
successfully passed through the lumen of the
vessel segment, place the wire in a position,
which ensures sucient length for the wire
to be secured both at the near and far xing
screws on the right-hand jaw.
Figure 3.5 A, B and C Mounting step 5
3.1.6 MOUNTING STEP SIX
• Carefully move the jaws together while
ensuring that the second mounted wire lies
underneath the rst one secured on the left-
hand jaw (gure 3.6 A). The procedure clamps
the second wire to prevent it from damaging
the vessel segment when securing the wire
to the right-hand jaw (connected to the
transducer). Secure the near end of the wire
in a clockwise direction under the far xing
screw on the right-hand jaw (gure 3.6 B).
Figure 3.6 A and B Mounting step 6
A B C
A B

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3.1.7 MOUNTING STEP SEVEN
• Secure the far end of the wire under the near
xing screw on the right-hand jaw. Again the
wire is passed clockwise around the screw
stretching the wire as the screw is tightened
(gure 3.7 A-B). Move the jaws apart to
slightly stretch the vessel segment. Make
sure that the vessel on the far side of the jaws
does not extend beyond the jaws, as even a
small extension will aect the normalization
procedure. In case of excess of vessel on
the far side of the jaws then move the jaws
together again and remove excessive tissue
using a forceps as described in mounting step
four. A better method for the skilled operator
is to move the jaws slightly apart and use
scissors to make a small slit in the vessel wall
where the vessel is clamped.
Figure 3.7 A and B Mounting step 7
3.1.8 MOUNTING STEP EIGHT
• Adjust the two wires in a way that the two
wires will ‘bump’ into each other and not will
go beneath or on top of each other as shown
here below by using the small screwdriver or
forces to move the wires on the two opposing
Jaws a bit up or down.
• Use a dissection microscope at this step while
moving the jaws together using the manual
micropositioner until the two wires are as
close to each other as possible but without
touching.
AB

14
Correct position of the two wires (Blue color) on the Jaws. This is a side
view of the two wires on the two jaws.
In-correct position of the two wires (Blue color). Here one wire is lower
positioned than the other. Correct the wires before continuing. This is a
side view of the two wires on the two jaws.
In-correct position of the two wires (Blue color). Here one wire is completely
above the other wire. Correct the wires as shown in rst example before
continuing. This is a side view of the two wires on the two .
• The artery is now mounted correctly and
can moved back onto the 630MA interface
with the heat turned on. When the buer
in the chamber have reached 37°C, replace
the buer in the chamber with fresh well
gassed warm buer. Wait 5 min and start the
Automated Normalization as described in
Chapter 3.3.5

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3.2 MOUNTING PROTOCOL FOR LARGER ARTERIES
For mounting of larger arteries with an internal
diameter larger than 500µm and up to 11mm
replace the mounting Jaws with Pins (see chapter
2.1). With the 630MA system a box with 4 sets of
200µm pins is delivered. For really large arteries
pins with 250µm, 300µm or 400µm pins can be
purchased from DMT.
3.2.1 MOUNTING STEP ONE
• First make sure that the mounting pins is placed correctly across each other in the chambers as
shown below.
Pins from top view
Pins from side view

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3.2.2 MOUNTING STEP TWO
• Using the manual micropositioner move
the two mounting pins as close together as
possible without touching each other.
• Take the isolated artery/tissue section and
slide it over the two pins as shown in the
gures below.
The artery is now mounted correctly and the
heat of the 630MA interface can be turned ON.
When the buer in the chamber have reached
37C, replace the buer in the chamber with fresh
well gassed warm buer. Wait 5 min and start
the Automated Normalization as described in
Chapter 3.3.5.
NOTE: For large arteries in could be necessary to add a small tension (1-2mN) to the
mounted artery on the pins to avoid that the artery falls of the pins during heating and
replacement of the buer. Remember to place the pins as close to each other as possible
without touching just before starting the automated normalization.

17
3.3 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 dened.
2. Clearly dened 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): dened as a set fraction of the internal circumference (IC100) that a fully relaxed
segment would have at a specied transmural pressure. For small rat arteries the target transmural
pressure is typically 100 mmHg = 13.3 kPa.

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3.3.1 PRINCIPLES OF THE NORMALIZATION PROCEDURE
In practice the normalization is performed by
distending the segment stepwise and measuring
sets of micrometer and force readings (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).
IC1is calculated from IC100 by multiplying a factor,
the Norm 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 Norm 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 .
3.3.2 THE AUTOMATED NORMALIZATION PROCEDURE ON THE 630MA SYSTEM
Make sure the wires/pins do not touch and are
close together without touching
1. Zero the force of the Chamber going to be
normalized automatically.
2. Go into the Norm. Set-up menu.
NORMALIZATIONSETUP
SELECT
SELECT
SELECT
SELECTCHAMBER1:

ENTER
SELECT
SELECTCHAMBER2:
SELECTCHAMBER3:
SELECTCHAMBER4:


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NORMALIZATIONSETUP
SELECT
SELECT
SELECT
SELECTCHAMBER1:

ENTER
SELECT
SELECTCHAMBER2:
SELECTCHAMBER3:
SELECTCHAMBER4:

3. Select the appropriate chamber.
CH1NORM.PARAMETERS1
SELECT
SELECT
SELECT
Norm.Time:60Sec.
Wirediameter:40um
ReleaseCH1:1000um
ENTER
SELECT
NEXT

SELECT
SELECT
SELECT
Norm.Pressure:13.3KPa
Norm.Factor:0.9
Eyepicececal.:0.40mm/div.

ENTERBACK
CH1NORM.PARAMETERS2

4. Enter the values for the Norm. Time, Wire/Pin diameter.
DMT have the following wire and Pin products:
Steel Wire : 40µm in diameter
Tungsten wires: 25µm, 15µm and 10µm in diameter
Pins: 200µm, 250µm, 300µm and 400µm
in diameter
5. Press NEXT to go into the PARAMETERS 2 menu
Myograph chamber tubing.

20
6. Enter the Norm. Pressure, Norm Factor and
Eyepiece cal values. It is very important that
the Norm Pressure and Norm factor is right
for the type of artery mounted in the chamber.
There can be a huge dierence between
artery type and species e.g for rat mesenteric
arteries the Norm Pressure is 13.3kPa and the
Norm Factor is 0.9 but for mouse mesenteric
arteries the Norm Pressure is 13.3kPa and
the Norm Factor 1.1. To nd the Norm factor
for your specic artery and specie please
read DMT Normalization Guide.
7. Go into the Mounting Artery
8. Select the appropriate Chamber
MOUNTONCHAMBERNO.
SELECT
SELECT
SELECT
SELECTCHAMBER1

ENTER
SELECT
SELECTCHAMBER2
SELECTCHAMBER3
SELECTCHAMBER4

IMPORTANT: The Norm Factor has to be found for the type of vessel and species BEFORE
using the automated normalization.