DMT 320A Manual de usuario
USER GUIDE, VOL.2.1
SINGLE WIRE MYOGRAPH SYSTEM - 320A
SINGLE WIRE MYOGRAPH 320A
USER GUIDE
CONTENTS
Chapter 1 - Single Wire Myograph overview........................................................................................................................................................................3
Chapter 2 - Setting up the Single Wire Myograph .............................................................................................................................................................4
2.1 Adjustment of supports.....................................................................................................................................................................4
2.2 Force transducer calibration.............................................................................................................................................................5
Chapter 3 - Experimental set-up ..............................................................................................................................................................................................6
3.1 Mounting protocol for small arteries................................................................................................................................................6
3.1.1 Mounting step one ......................................................................................................................................................................6
3.1.2 Mounting step two ......................................................................................................................................................................7
3.1.3 Mounting step three ...................................................................................................................................................................7
3.1.4 Mounting step four......................................................................................................................................................................8
3.1.5 Mounting step ve.......................................................................................................................................................................8
3.1.6 Mounting step six........................................................................................................................................................................9
3.1.7 Mounting step seven...................................................................................................................................................................9
3.2 Normalization ....................................................................................................................................................................................9
3.2.1 Principles of the normalization procedure ............................................................................................................................. 10
3.3 Standard start................................................................................................................................................................................. 10
3.3.1 Principles of the standard start procedure ............................................................................................................................ 10
3.4 Endothelium function..................................................................................................................................................................... 11
3.4.1 Principles of checking endothelium function......................................................................................................................... 11
3.5 In vitro experiment 1: Noradrenaline contractile response......................................................................................................... 12
3.5.1 Background .............................................................................................................................................................................. 12
3.5.2 Protocol..................................................................................................................................................................................... 12
3.6 In vitro experiment 2: Acetylcholine relaxation curve .................................................................................................................. 13
3.6.1 Background .............................................................................................................................................................................. 13
3.6.2 Protocol..................................................................................................................................................................................... 13
Chapter 4 - Cleaning and Maintenance .............................................................................................................................................................................. 14
4.1 Cleaning the Single Wire Myograph .............................................................................................................................................. 14
4.2 Maintenance of the force transducer ........................................................................................................................................... 15
4.2.1 Checking force transducer ...................................................................................................................................................... 15
4.2.2 Force transducer replacement................................................................................................................................................ 16
4.4 Maintenhance of the linear slide .................................................................................................................................................. 17
4.3 Changing the Single Wire Myograph window glass...................................................................................................................... 17
Appendix 1 - Buer recipes ..................................................................................................................................................................................................... 18
Appendix 2 - Normalization theory ...................................................................................................................................................................................... 20
Appendix 3 - Reading a millimetre micrometer............................................................................................................................................................... 22
Notes................................................................................................................................................................................................................................................ 23
4CHAPTER 1
CHAPTER 1 SINGLE WIRE MYOGRAPH OVERVIEW
Port for connection to Wire Interface
Myograph jaw connected
to force transducer
Myograph jaw connected
to micrometer
Micrometer
Suction pipe for connection
to vacuum pump
Pipe for gas supply Pipe for lling the chamber
(using the 40 mm funnel)
Force transducer pin
Figure 1.1 Single Wire Myograph with close-up of chamber
Transducer house
Allen screws for ne alignment of the jaws
(2 on each side)
Window at the bottom of the Myograph chamber
for imaging (under the wire jaws)
5SINGLE WIRE MYOGRAPH SYSTEM - 320A - USER GUIDE
B
A E
CD
Figure 2.1 Illustration of screws for adjusting supports and horizontal adjustment
CHAPTER 2 SETTING UP THE SINGLE WIRE MYOGRAPH
2.1 Adjustment of supports
A successful mounting of any kind of tubular tissue segment in the Single Wire Myograph is to a high extent dependent on per-
fectly matching supports. The supports are matched prior to the shipment but daily use of the Single Wire Myograph and greasing
of the transducer pinhole will over time create a need for an adjustment of the supports.
NOTE
THE TRANSDUCERS ARE FRAGILE AND SENSITIVE TO MECHANICAL STRAIN. BE VERY CAUTIOUS NOT TO PUT STRAIN ON THE
TRANSDUCER WHEN CHANGING OR ADJUSTING THE MOUNTING SUPPORTS. IN ADDITION, VERY LITTLE FORCE SHOULD BE
APPLIED TO THE SCREWS IN ORDER TO AVOID BREAKING THE THREADS.
Adjustment of the supports is performed using the following step-by-step procedure. The procedure is illustrated in gure 2.1.
1. Carefully loosen screw “E” on the top of the support connected to the force transducer. Align the horizontal support and
carefully tighten the screw again.
2. Loosen screw “D” on the top of the support connected to the linear slide. Align the horizontal support matching the force
transducer connected support as carefully as possible and gently tighten the screw again.
3 Loosen screw “C” on the linear slide to roughly match the linear slide support to the force transducer support in the horizon-
tal plane. Tighten the screw before proceeding with step 4.
4. The plate “B” on which the linear slide is mounted is balanced on top of a small stainless steel ball making it possible to
nely adjust the linear slide support in all vertical and horizontal planes using the four Allen screws “A”. Use the four Allen
screws to make the nal horizontal (see arrow in gure 2.1) and vertical (see arrow in gure 2.2) adjustments to match the
linear slide support to the force transducer support. The correct matching of the supports is illustrated in gure 2.3.
IMPORTANT
AVOID CONTINUOUSLY TIGHTENING THE ALLEN SCREWS DURING THE FINAL ADJUSTMENTS: LOOSEN THE ALLEN SCREW
PLACED DIAGONALLY TO THE ALLEN SCREW BEING TIGHTENED, OTHERWISE THERE IS A HIGH RISK OF DAMAGING THE
SINGLE WIRE MYOGRAPH FRAME.
6
Figure 2.2 Illustration of vertical adjustment
Figure 2.3 Illustration of correctly aligned supports for small vessels (left) and
incorrectly aligned supports (middle and right)
CHAPTER 2
2.2 Force transducer calibration
DMT recommends that the Single Wire Myograph is force calibrated at least once every month. DMT also recommends that the
Single Wire Myograph is force calibrated every time the system has been moved or has not been used for a long period of time.
See the force calibration procedure in chapter 3.7.1 in Wire Myograph Systems - User Manual.
7SINGLE WIRE MYOGRAPH SYSTEM - 320A - USER GUIDE
CHAPTER 3 EXPERIMENTAL SETUP
This chapter contains experimental set-up for the Single 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 trans-
ducer is capable of measuring with a sensitivity of about 0.01 mN (1 mg), but can be damaged if the applied force exceeds about
1 N (100 g). Therefore care must be taken to avoid pressing the jaws too hard together. A movement of ~20 µm after they have
touched is sufficient 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 Single Wire Myograph as follows.
• Holding wire at far end, place centre of wire between jaws and screw jaws together so that the wire is clamped (gure 3.1 A).
NOTE
DO NOT CLOSE THE JAWS TOO HARD AGAINST EACH OTHER.
• Bend the far end of the wire towards the left, and wrap it around under xing screw, so the wire is wound clockwise: tighten-
ing 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 Single Wire Myograph chamber with PSS (at room temperature). See appendix 1 for example of buffer recipes.
Figure 3.1 A, B and C Mounting step 1
ABC
8
3.1.2 Mounting step two
• Using forceps to hold the handle segment, transfer excised vessel from Petri dish to Single 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.
3.1.3 Mounting step three
• Once the vessel segment is threaded onto the wire, catch the free end of the wire (nearest 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 forceps, 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
ABC
B
Figure 3.2 A, B and C Mounting step 2
CHAPTER 3
9SINGLE WIRE MYOGRAPH SYSTEM - 320A - USER GUIDE
Figure 3.5 A, B and C Mounting step 5
ABC
ABC
Figure 3.4 A, B and C Mounting step 4
3.1.5 Mounting step ve
• Move the jaws apart (gure 3.5 A). Take a second wire holding it about one third down from the far end using 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 manoeuvre. 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 sufcient length for the wire to be secured both at the near and far xing screws on the right-hand jaw.
3.1.4 Mounting step four
• 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 Single Wire
Myograph chamber (gure 3.4 C).
10
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 direc-
tion under the far xing screw on the right-hand jaw (gure 3.6 B).
Figure 3.6 A and B Mounting step 6
AB
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
seg- ment. Make sure that the vessel on the far side of the jaws does not extend beyond the jaws, as even a small extension
will affect the normalisation 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 forceps as described in mounting step four. A better method for the skilled opera-
tor is to move the jaws slightly apart and use scissors to make a small slit in the vessel wall where the vessel is clamped.
B
A
Figure 3.7 A and B Mounting step 7
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.
CHAPTER 3
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