DMT 620M User manual

USER GUIDE, VOL.2.0
WIRE MYOGRAPH SYSTEM - 620M

User Guide
Version 1.0
WIRE MYOGRAPH  620M
USER GUIDE

3
CONTENTS
Chapter 1 - Wire Myograph overview .................................................................................................................................................................................... 3
Chapter 2 - setting up the wire myograph - 620M............................................................................................................................................................ 4
2.1 Changing and adjusting the mounting supports ...................................................................................................................................................... 4
2.1.1 Changing the mounting supports (gure 2.1)......................................................................................................................................................... 4
2.1.2 Coarse adjusting the jaws for small vessels (gure 2.1)........................................................................................................................................ 4
2.1.3 Fine adjusting the jaws for small vessels (gure 2.2 and gure 2.3)................................................................................................................. 5
2.1.4 Fine adjusting the pins for larger vessels (gure 2.4 and gure 2.5):............................................................................................................... 6
2.2 Calibration of the force transducer.................................................................................................................................................................................. 6
Chapter 3 - Experimental set-up ............................................................................................................................................................................................. 7
3.1 Mounting protocol for small arteries .............................................................................................................................................................................. 7
3.1.1 Mounting step one............................................................................................................................................................................................................. 7
3.1.2 Mounting step two............................................................................................................................................................................................................. 8
3.1.3 Mounting step three.......................................................................................................................................................................................................... 8
3.1.4 Mounting step four............................................................................................................................................................................................................ 9
3.1.5 Mounting step ve............................................................................................................................................................................................................. 9
3.1.6 Mounting step six.............................................................................................................................................................................................................10
3.1.7 Mounting step seven.......................................................................................................................................................................................................10
3.2 Normalization........................................................................................................................................................................................................................10
3.2.1 Principles of the normalization procedure..............................................................................................................................................................11
3.3 Standard start ........................................................................................................................................................................................................................11
3.3.1 Principles of the standard start procedure..............................................................................................................................................................11
3.4 Endothelium function ........................................................................................................................................................................................................12
3.4.1 Principles of checking endothelium function........................................................................................................................................................12
3.5 In vitro experiment 1: Noradrenaline contractile response..................................................................................................................................13
3.5.1 Background.........................................................................................................................................................................................................................13
3.5.2 Protocol ................................................................................................................................................................................................................................13
3.6 In vitro experiment 2: Acetylcholine relaxation curve............................................................................................................................................14
3.6.1 Background.........................................................................................................................................................................................................................14
3.6.2 Protocol ................................................................................................................................................................................................................................14
Chapter 4 - Cleaning and maintenance ..............................................................................................................................................................................15
4.1 Cleaning the Wire Myograph ..........................................................................................................................................................................................15
4.2 Maintenance of the force transducer............................................................................................................................................................................16
4.2.1 Checking the force transducer.....................................................................................................................................................................................16
4.2.2 Force Transducer Replacement....................................................................................................................................................................................17
4.3 Maintenance of the linear slides.....................................................................................................................................................................................18
Appendix 1 - Buer recipes .....................................................................................................................................................................................................19
Appendix 2 - Normalization theory ......................................................................................................................................................................................21
Appendix 3 - Reading a millimetre micrometer...............................................................................................................................................................23

4WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE
Connection to Interface
Allen screws for ne alignment
of the Wire Myograph jaws
Micrometer
Jaw connected
to Micrometer
Jaw connected
to force transducer
Force transducer pin
Supports
Figure 1.1 Wire Myograph with close-up of chamber
CHAPTER 1
Figure 1.2 Chamber cover
Access for drug application
Access or funnel
Access for temperature probe
CHAPTER 1  WIRE MYOGRAPH OVERVIEW

5
CHAPTER 2  SETTING UP THE WIRE MYOGRAPH  620M
2.1 Changing and adjusting the mounting supports
Each chamber can accommodate mounting supports for either small vessels (>50µm) or larger segments (>500µm).The mount-
ing supports can be changed easily and experiments can be performed with dierent vessels of varying internal diameter.
Continuous use and repeated greasing of the transducer arm holes will cause some misalignment of the mounting supports.
Therefore whether it is mounting supports for jaws or for pins they 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 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 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.
2.1.2 Coarse adjusting the jaws for small vessels (gure 2.1)
1. Loosen screw“A” to move the micrometer side jaw toward or away from the micrometer.
6. Loosen screw“D” 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.
A B C D
Figure 2.1 Illustration of the screws for changing supports and coarse adjustment of the jaws
Micrometer
Transducer

6WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE
2.1.3 Fine adjusting the jaws for small vessels (gure 2.2 and gure 2.3)
1. Tightening Screw“D” will move the micrometer 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.
CHAPTER 2
Figure 2.2 Fine adjustments of the jaws in the Wire Myograph chamber
Figure 2.3 - Illustrations of properly aligned jaws (depicted on the far left) and incorrectly aligned jaws
(depicted in the middle and far right).
A B
C D
Jaws from top view
Jaws from side view

7
2.1.4 Fine adjusting the pins for larger vessels (gure 2.4 and gure 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.
Figure 2.4 - Fine adjustments of the pins in the Myograph chamber
A B E D C
Pins from top view
Pins from side 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).
2.2 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

8WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE
CHAPTER 3  EXPERIMENTAL SETUP
This chapter contains experimental set-up for the Wire Myograph. For dissection of a vessel, please see Procedures for investi-
gations 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 m wire ~2.2 cm long. Mount one wire on left-hand jaw of the 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 Wire Myograph chamber with PSS (at room temperature). See appendix 1 for example of buer recipes.
Figure 3.1 A, B and C Mounting step 1
ABC
CHAPTER 3

9
3.1.2 Mounting step two
• Using forceps to hold the handle segment, transfer excised vessel from Petri dish to the Auto Dual 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

10 WIRE MYOGRAPH SYSTEM - 620M - 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 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 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 sucient 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 wire (gure
3.4 C).
CHAPTER 3