Pressure Myograph System - 110P
Contents
1. LLL R IIa aaa aaa S aaa R saw Rr sus m as 34 PROLOG ON TOR nuc M ec T EO EO 34 sae gie WC OS NINE TTE EEUU of Appendix 1 Fuse replacement oen crx wx iv x wx ici i ON ERE ORDER RR EN nc Rd BR b Anl Rc lk oic a Bil ER RR RIED 39 Appendix 2 System Specifications 111 ee ee nnns a nhau use a a aaa a IIa RR RR IIIS S DN RRR RS SN RR RES ennnen nnmnnn suas c ausa n rra 40 NOTOS 41 4 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL SAFETY The Pressure Myograph System has been designed for use only in teaching and research applications It is not intended for clinical or critical life care use and should never be used for these purposes nor for the prevention diagnosis curing treatment or alleviation of disease injury or handicap e Do not open the unit the internal electronics pose a risk of electric shock Do not use this apparatus near water e To reduce the risk of fire or electric shock do not expose this apparatus to rain or moisture Objects filled with liquids should not be placed on the apparatus e Do not block any ventilation openings Install in accordance with the manufacturer s instructions e Do not install near any he2. PRESSURE MYOGRAPH SYSTEM MODEL 110P User Manual Version 3 4 2 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL PRESSURE MYOGRAPH SYSTEM MODEL 110P DMT reserves the right to alter specifications as required This document was as far as possible accurate at the time of printing Changes may have been made to the software and hardware it describes since then New information may be supplied separately This documentation is provided with the DMT Pressure Myograph System Model 110P Document Number 110P 001A All rights reserved No part of this manual may be reproduced or transmitted in any form or by any means without the written permission of Danish Myo Technology A S Every attempt is made to ensure accurate information misprints construction and specification changes can occur Danish Myo Technology A S reserves the right to alter change content as required and without any notice Copyright Danish Myo Technology A S DANISA MYO T ECHANOL OGS CONTENTS SANE OV 5 E 6 6 Certificate OF CODTOIIHIEV o cocco nervt CN ERMN CB E E MUN NIKE NK 7 Unpacking the Pressure Myograph System cccsssscssnsscsnssseeesseeeaseeenssenenssecenaseeoassenensseessseoeasseoaseeoaseeeesseeneaseenasssennsssonnasensasas 8 Chapte
3. 1 3 2 Pressure Calibration Procedure on page 25 During the pressure calibration monitor the three relative pressure reading values in the calibration menu on the Myo Interface e If the value is O or above 6500 for one of the pressure transducers then the pressure transducer is broken and needs to be changed e fthe reading is between 1 499 or 5000 6500 then contact DMT for further instructions If the message OFF is displayed in either of the pressure reading lines in the Main Menu on the Myo Interface then the pressure transducer is broken and needs to be replaced In case of any other problems related to the pressure transducer please contact DMT for further instruction and advice 4 2 Inverted Microscope Pressure Myograph System set ups require high flexibility in image analysis DMT recommend to use inverted microscopes with high quality objectives and contrast features DMT recommend the DMT Microscope Zeiss Axiovert 40 Nikon TS 100F and Motic AE2000 4 3 The Pressure Regulator The Pressure Regulator controls the flow and pressure applied to the vessel mounted in the pressure myograph chamber via settings in the DMT VAS program or the Myo Interface This Chapter is intended as an overview of the Pressure Regulator to un derstand how it works to control flow and pressure settings 4 3 1 Control of Flow and Pressure Settings The Pressure Regulator consists of two individual circuits an air circuit and a buffer circuit a
4. 15 30 C 250 ml Built in Safety and reduction Optional accessories FlowMeter range 15 ul min to 1500 ul min Enable pH meter on the interface range temp correction Peristaltic pump pH O 14 0 C 50 C 2 5 50 rpm for superfusion of the chamber 40 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL NOTES NOTES 41 DMT A S Skejby Science Center Skejbyparken 152 DK 8200 Aarhus N Denmark Tel 45 87 41 11 00 FEE 115 AL LIL OL www dmt dk sales dmt dk support dmt dk DMT Asia Ltd Rm 2402B Great Eagle Centre 23 Harbour Road Wanchai Hong Kong S A R P R China Tel 852 6621 8337 Fax 852 3020 7554 www dmt asia com sales dmt asia com support dmt asia com DMT Asia China office Rm 28C No 8 Dong Fang Road Lu Jia Zui Financial District Shanghai 200120 P R China TEA O ZL LSS Fax 86 0 21 5877 0063 www dmt asia com sales dmt asia com supportedmt asia com DMT USA Inc 201 East Liberty Street Suite 6 Ann Arbor MI 48104 USA Tel 1 770 612 8014 Fax 1 678 302 7013 www dmt usa com sales dmt usa com support dmt usa com
5. 3 Valve 4 i Air UT 3 No Flow Valve Needle Valve Calibration Valve Buffer Flow Air Flow Figure 4 10 Pressure Regulator Diagram CHAPTER4 29 4 4 Pressure Myograph Maintenance and Cleaning The Pressure Myograph System Model 110P is a very delicate and sophisticated piece of research equipment In order to keep it working at its best DMT recommend that the following sections are read carefully and that the instructions are followed at all times 4 4 1 Pressure Myograph Chamber Pipes To prevent the pipes from being blocked by buffer salt deposits after an experiment use the chamber cover to remove the clean ing solutions Afterwards remove the cover from the myograph chamber and turn on the vacuum pump and vacuum valve for about 10 seconds Wait to turn off the oxygen supply until turning off the vacuum pump Wipe off any buffer remaining on the outside of the pipes using a piece of paper 4 4 2 Force Transducer The force transducer is one of the most delicate and fragile components of the Pressure Myograph System Therefore careful handling of the force transducer is of most importance to prevent it being damaged The left glass cannula in the pressure myograph is connected to the transducer pin coming through a small hole in the transducer house located on top of the pressure myograph unit as illustrated in figure 4 11 below To prevent the buffer from running into the transduc
6. Pressure Regulator to the Pressure Myograph Unit use the silicone tube internal diameter 1mm delivered with the Pressure Myograph System Connect the perfusion inlet at P1 on the Pressure Myograph Unit see figure 2 1 on previous page to the P1 outlet on the cap of the buffer bottle see figure 2 1 on previous page Connect the perfusion outlet at P2 on the Pressure Myograph Unit see figure 2 1 on previous page and figure 2 2 above to the P2 inlet in the Pressure Regulator Unit see figure 2 1 on previous page NOTE TO FILL THE TUBING AND AVOID AIR BUBBLES MOUNT THE RESISTANCE TUBE SHUNT AS DESCRIBED IN CHAPTER 4 1 3 2 PRESSURE CALIBRATION PROCEDURE ON PAGE 25 FILL THE SCHOTT BOTTLE WITH 250 ML BUFFER AND RUN THE FOLLOWING FLUSH PROGRAMME USING THE MYO INTERFACE OR DMT VAS 1 150mm Hg P2 7Omm Hg Enter the pressure values in the Pressure Menu and press F2 in the Main Menu of the Myo Interface to start the flush pro gramme Run the programme until no air bubbles are visible in the perfusion circuit NOTE Il NEVER FILL THE BUFFER BOTTLE WITH MORE THAN 250 ML OF BUFFER AS HIGHER VOLUMES MAKES PRECISE CONTROL OF THE PRESSURE MORE DIFFICULT IMPORTANT NEVER RUN THE FLUSH PROGRAMME WITH A VESSEL MOUNTED IN THE PRESSURE MYOGRAPH CHAMBER AS THIS MAY CAUSE SERIOUS DAMAGE TO THE VESSEL 14 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 2 2 4 2 Extra Perfusion In Outlet The extra perfusion
7. VAS Manual Core amp Analysis Functions and or the Quick Guide for DMT VAS and IC Capture for installing the USB camera CHAPTER2 13 2 2 3 The first Weight and Pressure Calibrations Prior to the shipment of the Pressure Myograph System it has gone through two days of continuous testing including final weight and pressure calibrations However in order to ensure that the myograph is working at highest performance DMT recommends that new weight and pressure calibrations are performed before starting to use the Pressure Myograph System The weight and pressure calibration procedures are described in detail respectively in 4 1 3 1 Weight Calibration Procedure on page 24 and 4 1 3 2 Pressure Calibration Procedure on page 25 2 2 4 External Pressure Myograph Unit Connections This section describes how to connect the perfusion circuit the superfusion circuit oxygen supply pressure supply and suction device Perfusion outlet Suction pipe to vacuum Superfusion outlet Perfusion inlet e Extra perfusion Superfusion Pipe for oxy Access hole for reagents Extra perfusion inlet outlet inlet gen supply buffer temperature or pH probe Figure 2 2 External Pressure Myograph connection 2 2 4 1 Connecting Perfusion Flow The perfusion flow is controlled by the Pressure Regulator via the Myo Interface or DMT VAS How to control the perfusion flow is described in detail in chapter 3 1 4 Pressure Menu on page 18 To connect the
8. both the Myo Interface and the Pressure Regulator 4 Pressure Regulator Oxygen Supply Connection Connect the Main Pressure Inlet on the back panel of the Pressure Regulator to an adjustable oxygen supply using the Atlas Copco Cablair hose delivered with the Pressure Myograph System IMPORTANT MAKE SURE THAT THE HOSE IS SECURELY FASTENED TO THE MAIN PRESSURE INLET USING THE HOSE CLIP THE PRESSURE FROM THE OXYGEN SUPPLY MUST NOT EXCEED 1 0 bar 5 Microscope Camera PC Connection Connect the USB cable from the USB Camera to your PC 6 Myo Interface External Data Acquisition System Connection optional If you have NOT obtained the DMT VAS then the four BNC ports on the Myo Interface front enables connection of an external data acquisition system for recording the inlet pressure the outlet pressure the longitudinal force the actual temperature and the pH value of the superfusion or perfusion buffer NOTE ONLY FOUR OF THE FIVE DATA CHANNELS CAN BE RECORDED SIMULTANEOUSLY 4 Turn on the Power Turn the main power to the Myo Interface and the Pressure Regulator on at the power switch Turn on the computer and the Pressure Myograph System is now ready to be used with the DMT VAS NOTE A DETAILED GUIDANCE IN HOW TO CONNECT THE PRESSURE MYOGRAPH SYSTEM FOR AN EXPERIMENTAL SETUP IS DE SCRIBED IN CHAPTER 3 THE PRESSURE MYO INTERFACE ON PAGE 16 2 2 2 Installation of DMT VAS and USB Camera See the user manual DMT
9. contract Wash the KPSS until baseline diameter is reached Repeat the KPSS contraction and washes An agonist such norepinephrine can be added 10 8M final in the bath to cause a contraction However the vessel should also have developed some myogenic tone The amount of tone will depend on the vessel used Once the vessel is contracted Ach 10 5M final in the bath can be added to assess endothelial function Wash the vessel 5 to 6 times over 20 to 30 minutes Vessel is now ready for experiments PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 5 2 Buffer recipes 5 2 1 Physiological Saline Solution PSS 1x PSS 1000ml Solution 1 Chemical NaCl KCl MgSO 7H O KH PO Solution 2 Chemical CaCl 2H O Solution 3 Chemical NaHCO EDTA Glucose MW g mol 58 44 74 56 246 48 136 09 MW g mol 147 02 MW g mol 84 01 372 24 198 77 Conc mmol l 118 99 4 69 1 17 1 18 Conc mmol l 2 50 Conc mmol l 25 00 0 03 5 50 Conc g l 6 954 0 350 0 289 0 161 Conc g l 0 368 Conc g l 2 100 0 010 1 091 1 Dissolve the chemicals in approximately 100ml double distilled H O as three individual solutions as described in the table above Gently heat solution 3 to dissolve the EDTA 2 Solution 1 is added to a graduated bottle and the bottle is filled with double distilled H O to a final volume of 500ml 3 Solution 3 is added to the graduated bottle which
10. external you are at the top of a menu use f to change to the previous di data acquisition system optional main menu e Myo Interface Display F Keys Have different but specific functions Numeric Keys dependent on the active main menu Enter data or setting values Enter Key Confirm entered numeric values or change between available choices INFO Key Push this key in any menu to display all specific F key functions ESC Key Delete incorrectly entered numeric values In some submenus used to return to main menu Figure 1 3 Pressure Myo Interface Front 115 230 V 50 60 Hz Automatic Voltage Selector 9 9 ON OFF Switch 9 Power Connector UG E CES REGULATOR RS232 pH MYOGRAPH Connection Port for control of Connection Port for Pressure Pressure Regulator Myograph Unit RS 232 Port for Serial to USB connection to R Connection Port for pH Meter optional GE Booten dni DU EIDEM i n n e 12V DC Port Figure 1 4 Pressure Myo Interface Back 10 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 1 3 Pressure Regulator Pressure and Perfusion Buffer Inlet and Outlet Pipes Pressure Transducer Calibration Valve Pressure Valve Activity Indicators Figure 1 5 Pressure Regulator Front Main Pressure Inlet e 115 230 V 50 60 Hz Automatic Voltage Selector Connection Port for the Pressure Myo Interface Inter
11. in and outlet on the Pressure Myograph Unit 3 way valve makes it possible to add special agents or reagents to the perfusion circuit during an experiment The small handle on top of the valves controls the flow directions through the 3 way valves at P1 and P2 as described in chapter 4 1 2 3 Way Valve Adjustments on page 23 2 2 4 3 Connecting Superfusion Flow Optional Creating a superfusion flow in the Pressure Myograph Chamber requires an external peristaltic pump which is not part of the standard Pressure Myograph System Contact DMT for further information about recommended perfusion pump models speci fications and prices The superfusion circuit is connected to the Pressure Myograph Unit as illustrated in Figure 2 1 Example of the Complete Pres sure Myograph System Model 110P on page 12 2 2 4 4 Connecting Flowmeter Model 161FM Optional The DMT Flowmeter Model 161FM is optional for flow measurements in the range of 1 1500yl min The Flowmeter is easily combined with any DMT Pressure Myograph System as illustrated in Figure 2 1 Example of the Complete Pressure Myograph System Model 110P on page 12 Use of the FlowMeter makes it possible to control intravascular pressure pressure gradient and flow conditions with high ac curacy which enables the performance of in vitro studies on vessel segments under conditions very close to those found in vivo Application of the FlowMeter also enables measurement of flow rate
12. page 22 The recommended size for the new glass cannulas is 16 mm for the glass cannula con nected to P1 right side and 14 mm for the glass cannula connected to P2 left side 4 1 5 Checking the Force Transducer The myograph force transducer is a strain gauge connected in a Wheatstone bridge The force transducer is placed in a separate compartment on top of the pressure myograph unit The separate compartment provides some mechanical protection for the force transducer but the transducer is still very vulnerable to applied forces exceeding 1 newton 100 gram or fluid running into the transducer compartment due to insufficient greasing of the transducer pinhole This chapter describes how to check the force transducer for any kind of damage 4 1 5 1 Simple Force Transducer Check Unstable readings 1 If the force reading on the Myo Interface appears unstable then first check that the Myo Interface and the Myograph Unit are properly connected through the 25 pin grey cable 2 f the force reading still appears unstable then perform a new weight calibration of the fore transducer as described in 4 1 3 1 Weight Calibration Procedure on page 24 During the weight calibration monitor the relative force reading values in the calibration menu on the Myo Interface e fthe value is O or above 6500 then the force transducer is broken and needs to be changed e If the reading is between 1 499 or 5000 6500 then contact Danish Myo Technology fo
13. program 3 1 Myo Interface Manual Figure 3 1 below is an overview of the menus in the Pressure Myo Interface The menus are divided into main menus and sub menus In the following sections all menus are described in detail Myo Interface Model 110P Version 8 2 F1 Recorder Menu Rec 1 gt Press 3 gt Rec 1 lo TO Rec 1 hi 200 Rec 2 gt Press 2 Rec 2 lo TO Rec 2 hi 2 200 Rec 3 gt Force Rec 3 lo 5 0 Rec 3 hi OO Rec 4 gt Temp Rec 4 lo TO Rec 4 hi E P1 Inlet P2 Outlet Status Flow mm Hg sec SetupMenu Calibrate Press Resovoir e eG Option 208 Main Menu P 64 4 mm Hg po 55 2 mn he Force 10 15 mN Temp 210265 Set Temp 37 0 C Heat 2 On H a FOO p Info Change menu Cal pressure Info Change menu Start pres Ctrl Info Change menu Start pres Ctrl Zero force Condensed menu F4 Near 2 T2 Far 2 7 mN Setup ACEI 615 Calibrate Menus Heat OFF Set 10 pH 7 00 Setup F4 F4 F4 Options Menus Calibrate Pressure Calibrate Force Calibrate pH OFFSET AmA NNA Apply 50 mm Hg Apply no Force Offset buffer 7 00 No 7 Comm Menu Serial mode RS485 Myograph a A No 10 pH Menu Offset buffer 7 00 Slope buffer gt 400 Temp comp Auto Manual temp Figure 3 1 Menus in the 110P Myo Interface 16 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL Push F3 whe
14. vessel on the P2 cannula 13 Gently mount the vessel on the P2 cannula gently without pulling it off the P1 cannula Secure the vessel as described in step 7 14 Once the vessel is secured on the P2 cannula open the P1 cannula and gently push more buffer into the mounted vessel The vessel should slightly fill and bulge from the small pressure head Close the P1 2 way valve to maintain a slightly pres surized vessel 15 Transfer the mounting chamber to the microscope and attach all the tubing 16 Fill the tubing leading from the P1 reservoir bottle marked P1 at the top of the bottle with the buffer that has been pre warmed and pre bubbled The easiest way to do this is attach a syringe to the P1 3way valve as illustrated and draw buffer from the bottle The 3 way valve should be positioned the same way A P1 Connected to P1 Reservoir Bottle Connected to Syringe draw buffer from here 17 Once the buffer is drawn through the tubing turn the 3 way valve as shown 18 Disconnect the syringe and move it to the P2 side of the chamber but DO NOT EMPTY THE SYRINGE Attach to the 3 way valve as shown Connected to port of Pressure Regulator marked P2 Connected to Syringe filled with buffer CHAPTERS 35 19 Use the buffer that was drawn from the bottle to charge the remainder of the system The buffer should be pushed gently from the 3 way valve all the way to the waste bottle When the buffer starts dripp
15. BEEN INSTALLED ALONG WITH DRIVERS FOR THE DIGITAL CCD USB CAMERA FOLLOW THE PROCEDURES IN SECTION 2 2 1 BELOW TO SET UP THE PRESSURE MYOGRAPH SYSTEM If you have NOT purchased a computer from DMT follow the procedures in the user manual DMT VAS Manual Core amp Analysis Functions to install DMT VAS on your own computer 2 2 1 Setting up the complete Pressure Myograph System This section describes how to connect the cables in the Pressure Myograph System as illustrated in figure 2 1 on previous page Before connecting any of the myograph equipment ensure that the USB Camera is connected to your PC and the DMT VAS is installed on your PC as described in the DMT VAS Manual NOTE BEFORE PROCEEDING WITH THE CONNECTION PROCEDURE MAKE SURE THAT THE MYO INTERFACE THE PRESSURE REG ULATOR THE MICROSCOPE AND THE PC ARE SWITCHED OFF 1 Myograph Unit Myo Interface Connection Connect the Pressure Myograph Unit to the Myo Interface by using the grey 25 pin connection cable The end of the cable with the temperature probe is used with the Pressure Myograph Unit 2 Myo Interface PC Connection Connect the Myo Interface to the computer with the serial to USB cable on the back panel of the Myo Interface to the USB port on the computer 3 Myo Interface Pressure Regulator Connection Connect the Myo Interface to the Pressure Regulator by using the grey 15 pin connection cable Connect the power cord to the power inlet on the back panel of
16. T BE VERY CAREFUL NOT TO DAMAGE THE GLASS CANNULAS DURING THE CLEANING PROCEDURE AFTER CLEANING ALWAYS CHECK THAT THE GREASING AROUND THE TRANSDUCER PIN IS SUFFICIENT TO KEEP OUT THE BUFFER SOLUTION FROM THE TRANSDUCER COMPARTMENT In cases of red or brown discolorations appearing on the chamber sides the following cleaning procedure will work in most cases 1 Incubate the myograph chamber for 30 minutes with 20ul of a 2mM T 1210 Tetrakis 2 pyridylmethyl ethylenediamine solu tion dissolved in double distilled water 2 Use a swabestick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period 3 Wash the myograph chamber several times with double distilled water 4 Incubate the myograph chamber with 96 ethanol for 10 minutes while continuing the mechanical cleaning with a swab stick 5 Remove the ethanol solution and wash a few times with double distilled water Incubate the myograph chamber with an 8 acetic acid solution for 10 minutes and continue the mechanical cleaning with a swab stick 6 Wash the myograph chamber several times with double distilled water CHAPTER4 33 CHAPTER 5 EXPERIMENTAL SETUP STEP BY STEP 5 1 Mounting protocol for small arteries This section shortly describes the basic technique of mounting small vessels in the Pressure Myograph Figure 5 1 Mouting of vessel in the Pressure Myograph Chamber Preparation 1 Prepare your PSS 60mM KPSS can
17. afterwards is filled with additional double distilled H O to a final volume of about 850ml 4 Aerate the solution with carbogen 9576 O 5 CO for about 20 minutes 5 Solution 2 is added and the graduated bottle is filled with additional double distilled H O to reach the final volume of 1000ml Continue the carbogen bubbling until the pH of the buffer solution reaches 6 4 25x Concentrated PSS 1000ml Solution 1 Chemical NaCl KCl CaCl 2H O Solution 2 Chemical MgSO 7H O KH2PO Solution 3 Chemical EDTA MW g mol 58 44 74 56 147 02 MW g mol 246 48 136 09 MW g mol 372 24 Conc mmol l 118 99 4 69 2 50 Conc mmol l 147 1 18 Conc mmol l 0 03 Conc g l 173 850 8 750 9 200 Conc g l 1225 4 025 Conc g l 0 250 CHAPTER5 37 1 Dissolve the chemicals for solution 1 in about 800ml double distilled H O in a 1000ml graduated bottle Dissolve the chemicals for solutions 2 and 3 in 75ml double distilled H O in individually cylinders Gently heat solution 3 to dissolve the EDTA 2 Solution 2 and 3 is added to solution 1 and the graduated bottle is filled with additional double distilled H O to reach a final volume of 1000 0ml Before use 3 Dilute the 25 x PSS stock solution 1 25 using double distilled 4 Add 1 091 g l Glucose 2 100 g l NaHCO Aerate the solution with carbogen 95760 5 CO for at least 20 minutes If necessary wait furth
18. al features of the Pressure Myograph System This includes how to use the DMT Microscope and how to use adjust and separate the Pressure Myograph Unit 4 1 The Pressure Myograph Unit The pressure myograph unit can be placed on the DMT microscope as illustrated in figure 4 1 A below or an inverted microscope as illustrated in figure 4 1 B below Press the black lever to lock the Pressure Myograph Unit on the DMT Microscope Figure 4 1 A B DMT Microscope setup and Zeiss inverted Microscope setup CHAPTER4 21 4 1 1 Adjustment of Glass Cannulas Adjustment of the glass cannulas is divided into a pre experimental alignment and general adjustments done during mounting of a blood vessel or when running an experiment General Adjustments General adjustments are performed using the two micropositioners on top of the Pressure Myograph Unit The vertical micropo sitioner figure 4 2 B below is used to adjust the vertical position of the right glass cannula and thereby the vertical position of the mounted blood vessel The horizontal micropositioner fig 4 2 C below is used to define the length between the two glass cannulas and thereby the horizontal stretch of the mounted blood vessel A B C e e Figure 4 2 Adjustment of glass cannulas Pre Experimental Alignment To make the general adjustments as easy as possible during an experiment it is important to make an alignment of the glass cannulas prio
19. as a parameter as well as shear stress Reynolds number and vascular resistance due to changes in vessel structure and function in various physiological and pharmacological studies The FlowMeter has to be added as a hardware in the DMT VAS program see DMT VAS Manual Besides calculating the flow rate MyoFlow enables real time calculations of total flow and current drop volume In addition MyoFlow features a real time flow graph showing flow rate development over time 2 2 4 5 Connecting Oxygen Supply Connect the oxygen supply pipe on the Pressure Myograph Chamber cover to an adjustable oxygen supply 2 2 4 6 Connecting Suction Device Connect the large pipe on the Pressure Myograph Chamber cover to a vacuum pump via a suction bottle and a vacuum valve as illustrated in Figure 2 1 Example of the Complete Pressure Myograph System Model 110P on page 12 The internal diameter of the silicone tube used for this connection must be 2 mm a 2 mm tube is not included in the myograph system CHAPTER2 15 CHAPTER 3 THE PRESSURE MYO INTERFACE The DMT VAS control the Pressure Myo Interface However the user NOT having DMT VAS the following chapter will go through how to use the Myo Interface without DMT VAS This chapter is a complete manual for the Pressure Myo Interface The chapter contains a step by step description of how to navi gate in the menus and how to use the special features of the Pressure Myograph System like the sequencer
20. at sources such as radiators heat registers stoves or other apparatus that produce heat e Only use attachments and accessories specified by the manufacturer e Unplug this apparatus during lightning storms or when unused for long periods of time e This apparatus must be grounded e Use a three wire grounding type cord similar to the one supplied with the product e Do not defeat the safety purpose of the polarized or grounding type plug A polarized plug has two flat blades one being wider than the other A grounding type plug has two blades and a third round grounding pin The wide blade or the third prong is provided for your safety If the provided plug does not fit into your outlet consult an electrician for replacement of the obsolete outlet e Be advised that different operating voltages require the use of different types of line cord and attachment plugs Check the voltage in your area and use the correct type See the table below Voltage Line plug according to standard 110 125 V UL817 and CSA C22 2 No 42 220 230 V CEE 7 page VII SR section 107 2 D1 IEC 83 page C4 240 V BS 1363 of 1984 Specification for 13A fused plugs and switched and unswitched socket outlets Protect the power cord from being walked on or pinched particularly at power plugs and the point where they connect to the apparatus Refer all servicing to qualified service personnel Servicing is required when the apparatus has been damaged in any way su
21. be calibrated Pressure force or pH Reservoir Temperature setting for perfusion buffer reservoir Option The option line allows access to submenus using special codes To change any of the settings in the Setup Menu move the line to be changed to the top line of the display Press Enter to change between calibration of pressure transducers force trans ducer or pH in the calibrate line Use the numeric keys to change the reservoir temperature setting and press Enter to confirm Enter the specific access code and press Enter to go to the submenu The Setup Menu has the following functions Info Press the Info key and the display shows the F key options in the Setup Menu Press Info again to return to the Setup Menu or one of the F keys to proceed with one of these options F1 Press F1 to proceed to the Main Menu F4 Pressure transducer calibration Choose Press in the calibrate line and press F4 to enter the window shown to the right Apply a pressure of 50mm Hg on the Pressure Myograph using the Big Ben pressure manometer When the relative readings at the bottom line of the win dow appears stable press F3 to proceed Apply a pressure of 125mm Hg on the Pressure Myograph using the Big Ben pressure manometer When the relative readings at the bottom line of the win dow appears stable press F3 to proceed Apply a pressure of 200mm Hg on the Pressure Myograph using the Big Ben pressur
22. be used to test contractions and 16mM KPSS can be used to cause relaxations usually 16mM KPSS only works in small arteries for relaxation 2 Make at least 4 loops with nylon suture to use for securing the vessel to the mounting cannulas Double loops are best and will prevent the loops from slipping loose once tightened 3 Pre heat PSS to 37 C making sure the PSS is bubbled with carbogen 5 CO2 9596 O2 or PraxAir 5 CO2 21 O2 bal ance N2 for at least 20 to 30 minutes 4 f desired rinse the mounting chamber and all tubing that sees buffer with ddH20 5 Fill the P1 reservoir bottle in the pressure regulator with pre warmed PSS DO NOT FILL MORE THAN 250mL Keep the tub ing from the bottle attached to port on top of the bottle marked P1 higher than the bottle otherwise capillary action will draw buffer from the bottle and will leak all over the bench Mounting 1 Isolate small vessel of interest If you want to learn more about dissection of mesenteric arteries please request for the Dissection amp Mounting video 2 Clean the vessel as best as possible of perivascular fat and tissue 3 Place the small artery into a small container of cold or room temperature PSS 4 Place 10mL of cold or room temperature PSS in the mounting chamber 5 Have a syringe with buffer attached to the P1 3 way valve but the P1 and P2 3 way valves should be closed to the vessel 6 Position the sutures appropriately on both cannulas so that they ar
23. ch as the power supply cord or plug is damaged liquid has spilled onto or objects have fallen into the apparatus the apparatus has been exposed to rain or moisture does not operate normally or has been dropped SAFETY 5 EMC EMI This equipment has been tested and found to comply with the limits for a Class B Digital device pursuant to part 15 of the FCC rules These limits are designed to provide reasonable protection against harmful interference in residential installations This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by monitoring the interference while turning the equipment off and on the user is encouraged to correct the interference by one or more of the following measures e Reorient or relocate the receiving antenna e Increase the separation between the equipment and receiver e Connect the equipment into an outlet on a circuit different to that which the receiver is connected to e Consult the dealer or an experienced radio TV technician for help APPROVALS Complies with the EMC standards EMC 89 336 EEC EN 50 081 1 and EN 50 082 1 FCC part 15 Class B CISPR 22 Class B Certi
24. e Force DONE Push Esc 9 After calibrating the force transducer carefully remove weight balance and calibra Force Zero 20315 tion bridge The Pressure Myograph System is now ready for use longitudinal force Forco eal posa measurements 4 1 3 2 Pressure Calibration Procedure The setup to perform the pressure transducer calibration procedure is illustrated in figure 4 7 below Carefully follow the proce dure described below to pressure calibrate the Pressure Myograph System Figure 4 7 Illustration of set up for pressure transducer calibration 1 Connect the Pressure Myograph Unit to the Pressure Regulator as to perform a standard pressure myograph experiment as illustrated in figure 4 7 above 2 Connect the Big Ben Pressure Manometer to the 3 way valve at P1 on the Pressure Myograph Unit as illustrated in figure 4 7 above 3 Inside the myograph chamber on the right side is a small stainless steel pipe which via a small silicone tube is connected to the right glass cannula see figure 4 8 B on next page Carefully disconnect the silicone tube from the stainless steel pipe 4 Carefully disconnect the silicone tube between the outlet connecting pipe and the outlet pressure transducer connecting pipe see figure 4 8 A on next page 5 Connect the enclosed resistance tube shunt to the stainless steel pipe inside the myograph chamber and the outlet pres sure transducer connection pipe as i
25. e manometer When the relative readings at the bottom line of the win dow appears stable press F3 to proceed The calibration is now finished The readings in the two bottom lines are the cal ibration parameters stored in the internal memory of the Myo Interface Press ESC to return to the Setup Menu For a more detailed instruction to the calibration procedure see chapter 4 1 3 Force and Pressure Calibration on page 23 Setup Menu Calibrate Press Resovoir 2 OG Option 0 Info Change menu Cal pressure F4 Calibrate Pressure Apply 50 mm Hg Push F3 when stable Pi 3614 3593 F3 Calibrate Pressure Apply 125 mm Hg Push F3 when stable Pal 4252 P2 4222 F3 Calibrate Pressure Apply 200 mm Hg Push F3 when stable P1 4956 P2 4933 F3 Calibrate Pressure DONE Push Esc 36148 42527 49565 35334 42223 49332 CHAPTER 3 19 Force transducer calibration F4 Calibrate Force Chose Force in the calibrate line and press F4 to enter the window shown to the right Apply no Force Push F3 when stable Force 1938 Make sure that no force is applied on the force transducer and when the relative reading at the bottom line of the window appears stable press F3 to proceed F3 Carefully place the 2 gram weight on the load pan and when the relative reading at the UP bottom line of the window appears stable press F3 to proceed Push F3 when stable Force 2245 The calibration
26. e ready for use when the vessel is mounted Place 2 loops on each cannula in case one breaks or fails f Carefully transfer the vessel from the small container to the mounting chamber Using a large bore pipette tip and pipette or an eye dropper carefully suck up the isolated vessel and transfer it to the chamber This will minimize handling and potential for damaging the vessel 8 Continue to mount the vessel on the glass cannulas Start with the P1 side Once the P1 side of the vessel is mounted on 34 the cannula secure the vessel to the cannula by tightening the loops around the vessel onto the cannula PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 9 Make sure the vessel is mounted in the correct direction Flow through the vessel is directional so proximal and distal ends for flow will need to be noted somehow Mount the proximal end to flow on the P1 cannula 10 Once secured gently rinse the vessel of any blood Before washing the lumen gently push some buffer from the syringe through the 3 way valve to the port that is not connected to the vessel so as to bleed out any air that might have been trapped in the valve 11 Open the valve to the vessel and gently push buffer through to wash out any blood in the lumen This will also help open the distal end if it is pinched shut Close the P1 3 way valve so as to keep the vessel from collapsing 12 Position the P1 cannula with attached vessel so as to facilitate mounting of the
27. ed Psp uM P1 3614 2 3533 10 Apply a pressure of 125mm Hg on the pressure myograph using the Big Ben pressure manometer When the relative readings at the bottom line of the window Ae us IDr ressure appears stable press F3 to proceed p Push F3 when stable P14252 P2 4222 11 Apply a pressure of 200mm Hg on the pressure myograph using the Big Ben pres sure manometer When the relative readings at the bottom line of the window ap F3 pears stable press F3 to proceed Calibrate Pressure Apply 200 mm Hg Push F3 when stable 12 The calibration is now finished The readings in the two bottom lines are the calibra P14956 P2 4933 tion parameters stored in the internal memory of the Myo Interface Press ESC to return to the Setup Menu F3 Calibrate Pressure u T DONE Push Esc 13 Carefully remove the resistance tube shunt and reconnect the tubing to the glass 36148 42527 49565 cannulas Open the calibration valve on the Pressure Regulator by turning it coun 35334 42223 49332 ter clockwise IMPORTANT MAKE SURE THAT THE CALIBRATION VALVE IS COMPLETELY OPENED AFTER THE PRESSURE CALIBRATION A HALF CLOSED CALIBRATION VALVE WILL MAKE IT VERY DIFFICULT TO CONTROL THE PRESSURE AND MAKE THE PRESSURE READINGS UNRELIABLE 4 1 4 Replacement of Glass Cannulas The mounted glass cannulas are easily replaced with new ones by carefully loosening the two screws F and G in Figure 4 2 Adjustment of glass cannulas on
28. emoved works the best 3 Fill the syringe with air 4 Toclean the cannulas attach the syringe with needle at the point in the chamber indicated at figure 4 12 below 5 Make sure the cannulas are not aligned If they are still aligned pushing the air into the cannulas will only cause the buffer or water to shoot into the cannula straight across from it 6 Push the air from the syringe through the cannula This should push out any buffer in the cannula f Remove the syringe and fill with double distilled water Detach tubing here and insert end of blunted 18 gauge needle attached to syringe into tubing to clean P2 can nula Detach tubing here and insert end of blunted 18 gauge needle attached to syringe into tubing to clean P1 can nula Figure 4 12 By attaching the syringe at these point the P1 abd P2 pressure transducer is bypassed and water or air can be pushed through the cannula as hard as the user likes 32 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 8 Re attach as in step 4 above 9 Push water through the cannula to wash 10 Remove the syringe remove the water from the syringe and reattach the syringe as in step 4 11 Push air through the cannula to dry 12 Repeat steps 3 through 11 on the P2 cannula Attach the syringe at the point in the chamber as indicated in figure 4 12 on previous page Using this cleaning procedure will minimize damage to the pressure transducers will prevent clogs
29. er for the pH of the buffer to reach pH 7 4 38 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL APPENDIX 1 FUSE REPLACEMENT The main fuse of the myograph system is placed inside the power inlet on the Myo Interface If the fuse blows it is easily changed using the following procedure IMPORTANT IF THE FUSE NEEDS TO BE CHANGED MAKE SURE THAT THE REPLACEMENT FUSE IS EQUAL TO THE ONE BLOWN Specifications T1 6A 250V 6 3 x 32mm Use a small screwdriver to open the voltage selector block 9 Red Fuse Block e APPENDIX 1 39 APPENDIX 2 SYSTEM SPECIFICATIONS Technical specifications Vessel size Vessel alignment Mounting supports Chamber Chamber volumes Chamber material Chamber cover Force range Force resolution Pressure range Pressure resolution Pressure calibration Heating Temp range Temp resolution Temp probe Weight calibration Analogue output Digital output Voltage Ambient temperature Fluid capacity Gas superfusion ports Valves 260 um Manually X Y amp Z settings Glass pipettes Single Max 10 ml 5 ml typical Acid resistant stainless steel With pipes for suction gassing superfusion 50 mN 0 1 mN 0 250 mmHg requires external pressure source app 1 ATO 0 1 mmHg Manual Built in Ambient 50 C 0 1 C Included Semi automatic O 1 0V full scale 12 bit Serial interface RS232 RS485 100 to 240 VAC auto 50 60 Hz
30. er house the hole is filled with high vacuum grease As a part of daily maintenance it is very important to inspect the greasing of the transducer hole before starting any experiment Insufficient greasing causes damage and malfunction of the force transducer IMPORTANT DMT RECOMMENDS THAT THE HIGH VACUUM GREASE SEALING UP THE TRANSDUCER HOLE BE CHANGED AT LEAST ONCE A WEEK SEE FIGURE 4 11 BELOW DMT TAKES NO RESPONSIBILITIES FOR THE USE OF ANY OTHER KINDS OF HIGH VACUUM GREASE THAN THE ONE TO BE PURCHASED FROM DMT DMT TAKES NO RESPONSIBILITIES FOR ANY KIND OF DAMAGE APPLIED TO THE FORCE TRANSDUCERS QU Transducer hole 98 d a 4 r M Figure 4 11 Transducer pin hole sealed up with high vacuum grease 4 4 3 Pressure Transducer and Glass Cannulas To prevent the pressure transducers and the tubing inside the pressure myograph unit from being blocked by buffer salt deposits after an experiment DMT recommends that the system be flushed using the following procedure 1 Disconnect the Pressure Regulator from the Pressure Myograph Unit Keep the Pressure Myograph Unit connected to the Myo Interface to enable continuous monitoring of the pressure readings from P1 and P2 2 Fill a small syringe with 896 acetic acid and flush both pressure transducers and glass cannulas by connecting the syringe needle to the 3 way valve for the individua
31. f the transducer arm is placed freely behind the glass cannula fixation plate which means it does not touch the fixation plate Figure 4 5 Weight Calibration Set up Figure 4 6 Illustration of how to fit the balance just behind the glass cannula fixation plate 24 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 5 Goto the Setup Menu on the Myo Interface and choose Force to weight calibrate the force transducer Press F4 to start calibration SetupMenu Calibrate 2 Pes Resovoir IW C 6 Make sure that absolutely no force is applied on the force transducer by checking Option that the tip of the transducer arm is not touching the glass cannula fixation plate Also check that the relative force reading in the display is stable Press F3 to pro ceed with calibrating F4 Calibrate Force Apply no Force f Carefully place the 2 g weight on the pan The force applied on the force transducer ipo E E orce should mimic the stretch created by the contraction of a mounted ring preparation Wait until the relative force reading is stable Press F3 to finish the calibration Calibrate Force Place 2 g on the pan 8 Press Ese and go to the Main Menu on the Myo Interface The force reading on dern the Myo Interface should now be very close to 9 81 mN If the force reading is differ Force 2245 ent from 9 81 mN then try to calibrate the fore transducer once again starting with step 3 F3 Calibrat
32. face Pressure inlet e ON OFF Switch Inlet Pressure Manometer Power Connector Figure 1 6 Pressure Regulator Back CHAPTER 1 11 CHAPTER 2 SETTING UP 2 1 Pressure Myograph System Model 110P pH Meter optional Myo Interface i Rear Panel i a Peristaltic Pump j optional Connection cable with temperature probe PC Data Acquisition and Software D op N S e C I I I I I I I I I I I I I l FlowMeter 161FM DMT Microscope Not included Superfusion Buffer I I I I I I I I I I I I I I I I I I Oxygen Supply Pressure and Oxygen Supply Peewee ee ee omm omm mm mm mm m mm mm mm um omm Vaccum Pump optional I I I I I I I I I I I I I I I I I 4 I I I I optional I I I I I I I I I I I I I I I I I I I I I I I Sucction Bottle Outlet Superfusion buffer Inlet Superfusion buffer Inflow Perfusion buffer Outflow Perfusion buffer Pressure Regulator Rear Panel Figure 2 1 Example of the Complete Pressure Myograph System Model 110P Figure 2 1 above is a diagram of complete set up for the Pressure Myograph System 110P The diagrams include optional equip ment such as a peristaltic pump to enable superfusion flow and pH probes for
33. fied with the safety standards 60 065 IEC 60065 Complies with the safety standards UL6500 CSA E65 6 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL CERTIFICATE OF CONFORMITY DMT A S Skejbyparken 152 8200 Aarhus N Denmark hereby declares its responsibility that the following product Pressure Myograph System Model 110P is covered by this certificate and marked with CE label conforms with the following standards EN 60 065 IEC 65 Safety requirements for mains operated Electronic and related apparatus for household and similar general use EN 50 081 1 Electromagnetic compatibility Generic emission standard Part 1 Residential commercial and light industry EN 50 082 1 Electromagnetic compatibility Generic immunity standard Part 1 Residential commercial and light industry With reference to regulations in the following directives 73 23 EEC 89 336 EEC CERTIFICATE OF CONFORMITY 7 UNPACKING THE PRESSURE MYOGRAPH SYSTEM Please take a few minutes to carefully inspect your new Pressure Myograph System for damage which may have occurred during handling and shipping If you suspect any kind of damage please contact us immediately and the matter will be pursued as soon as possible If the packing material appears damaged please retain it until a possible claim has been settled We recommend that you store the packing material for any possible future transport of the Pressure Myograph System In case of transpo
34. figure 4 3 and figure 4 4 below Figure 4 3 Three way valve flow settings at P1 Figure 4 4 Three way valve flow settings at P2 4 1 3 Force and Pressure Calibration This section contains a complete explanation of how to perform the force weight calibration and pressure calibrations As a part of the general maintenance of the myograph DMT recommends that the myograph is force and pressure calibrated at least once every month DMT also recommends that the myograph is force and pressure calibrated every time the system has been moved or has not been used for a long period of time CHAPTER4 23 4 1 3 1 Weight Calibration Procedure The section contains a complete step by step description of how to weight calibrate the force transducer 1 Fill the myograph chamber with double distilled water and move the glass cannulas apart 2 Place the calibration bridge balance and weight on the myograph unit allowing it to be pre warmed together with the myo graph unit Turn on the heating in the Main Menu on the Myo Interface 3 After approximately 20 30 minutes the whole system will have reached the target temperature normally 379 C Place the warmed calibration bridge and balance on the myograph unit as illustrated in figure 4 5 below 4 Make sure that the tip of the transducer arm on the balance is placed behind the glass cannula fixation plate as illustrated in figure 4 6 below Carefully move the calibration bridge until the tip o
35. from occurring in the pressure regulator and will minimize clogs in the cannulas IMPORTANT ACETIC ACID MAY BE USED MORE LIBERALLY IN CLEANING THE CHAMBER AND GLASS CANNULAS IT IS IMPORTANT TO THOROUGHLY WASH THE ACETIC ACID FROM THE CANNULAS OTHERWISE PRECIPITATE WILL FORM IN THE CANNULAS AND CLOG THEM REQUIRING THAT THE CANNULAS BE CHANGED MORE OFTEN If the chamber still have salt build up then continue with the following procedure 1 Fill the myograph chamber to the edge with an 8 acetic acid solution and allow it to stand for a few minutes to dissolve calcium deposits and other salt build up Use a swab stick to mechanically clean all chamber surfaces 2 Remove the acetic acid and wash the myograph chamber and glass cannulas several times with double distilled water 3 If any kind of hydrophobic reagent have been used which might be difficult to remove using step 1 and 2 then try incubating the chamber and glass cannulas with 96 ethanol or a weak detergent solution e g Treepol 4 To remove more resistant or toxic chemicals incubate the myograph chamber and glass cannulas with 1M HCI for up to 1 hour In exceptional cases incubate the chamber and supports with a up to 3M HNO solution for about 15 minutes 5 Wash the myograph chamber and glass cannulas several times with double distilled water IMPORTANT NOTES BE VERY CAREFUL USING STEP 3 AND 4 REPEATEDLY AS STRONG REAGENTS CAN CAUSE EXTREME DAMAGE TO THE MYO GRAPH UNI
36. ing When the relative pH output in the bottom line is stable press F3 to proceed The calibration is now finished The parameters are stored in the internal memory of the Myo Interface Press ESC to return to the Setup Menu pH submenu optional To open the special pH submenu enter the code 10 in the option line in the Setup Menu Offset buffer pH value of offset buffer z Piu Slope buffer pH value of slope buffer IE bd ion ope Temp comp AUTO or MANUAL temperature compensation en comp zo CAPS Manual temp Manual temperature setting Manual temp 37 C The pH submenu contains the settings of the pH calibration procedure Offset buffer and Slope buffer are the pH values of the buffers used to calibrate the pH probe pH measurements are temperature dependent and in the temperature compensation line it is possible to choose between an AUTO or MANUAL temperature compensation In the AUTO mode the temperature probe is used to measure the temperature of the calibration buffers The temperature is then automatically used in the pH calibration In the MANUAL mode the temperature of the calibration buffers is measured with a thermometer and entered manually in the manual temperature line Press F1 to return to the Setup Menu 20 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL CHAPTER 4 PRESSURE MYOGRAPH MANUAL Chapter 4 contains a complete explanation to all mechanic
37. ing gradi ent The Pressure Menu has the following functions Info Press the Info key and the display shows the F key options in the Pressure Menu Press Info again to return to the Pressure Menu or one of the F keys to pro ceed with one of these options F1 Press F1 to proceed to the Sequencer Menu F2 Press F2 to start the pressure control programme 3 1 5 Recorder Menu The Pressure Myo Interface has four analogue output ports at the front panel for con nection to a data recording system Each of these output ports can be programmed to represent inlet pressure reading outlet pressure reading longitudinal force reading temperature or pH meter The default settings are Rec 1 P1 Inlet pressure output Rec 2 P2 Outlet pressure output Rec 3 Longitudinal force output Rec 4 Temperature output The full scale output on the recorder is one volt In the Recorder Menu it is possible to change the value that corresponds to O V lo and 1 V hi 18 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL Pressure Menu P1 Inlet 90 mm Hg P2 Outlet 80 mm Hg Status Flow ON mm Hg sec 0 Info Change menu Start pres Ctrl Recorder Menu Rec 1 gt Press 1 Rec 1 lo 3 10 Rec 1 hi 200 Rec 2 Rec 2 lo Rec 2 hi Rec 3 Rec 3 lo Rec 3 hi Rec 4 Rec 4 lo Rec 4 hi 3 1 6 Setup Menu The Setup Menu displays Calibrate Choose which sensor to
38. ing in the waste bottle stop pushing buffer BE CAREFUL WHEN CHARGING THIS PART OF THE SYSTEM EXCESSIVE PRESSURE CAN DAMAGE THE INTERNAL 3 HIDDEN TRANSDUCER IN THE PRESSURE REGULATOR 20 Close the 3 way valve as shown below 21 The system is charged and ready Equilibration 1 Turn on the heat which should be preset to 37 C 2 3 T 8 9 Start bubbling the chamber with oxygen Set P1 to 2OmmHg and P2 to 5mmHg This will cause a flow gradient and perfuse the vessel with some of the newly charged buffer Make sure that the charged lines DO NOT have air bubbles otherwise endothelium will be stripped from the lumen Perfuse for 2 to 3 minutes Set P1 and P2 pressure to 10mmHg Increase pressure by 10mmHg every 5 minutes until target pressure is reached For cerebral arteries from rats this may be 60 to 70 mmHg For mesenteric resistance arteries this would be G0mmHg After the first 20 minutes after starting the heat and pressurization remove the old PSS and replace it with new PSS that should be pre warmed and oxygenated Continue to pressurize the vessel After 40 minutes from the start of heat and pressurization do another wash as in step 6 Once 60 minutes have passed the Standard Start or Wake Up Protocol can be initiated Wake up or standard start protocol 1 2 3 36 Once the vessel is equilibrated remove the old PSS and add 60mM KPSS to cause a contraction Allow the vessel to
39. is now finished The readings in the two bottom lines are the calibration parameters stored in the internal memory of the Myo Interface Press ESC to return to the Setup Menu F3 Calibrate Force DONE Push Esc For a more detailed instruction to the calibration procedure see 4 1 3 Force and Pres i eee en sure Calibration on page 23 pH calibration Optional Choose pH in the calibrate line and press F4 to enter the window shown to the right FA The first line shows the value of the offset buffer first buffer solution which is always Calibrate pH OFFSET 7 00 Offset buffer 7 00 j t Temp auto 22 4 c pH output SEE um VI The second line shows the temperature of the buffer solution The temperature is an important parameter in the calibration formula and is obtained automatically by placing the myograph temperature probe in the buffer solution SE SIS The third line shows the output from the pH probe as raw data from the A D converter Slope buffer 4 00 Temp auto c DD OE pH output a 177mV Place the pH meter electrode and temperature probe in the offset buffer solution and turn on stirring When the relative pH output in the bottom line is stable press F3 to proceed F3 Calibrate pH OFFSET i ff a 22 In the first line is now shown the value of the slope buffer second buffer solution which ee p is always 4 00 Place the pH and temperature probes in the slope buffer solution and turn on stirr
40. l pressure transducers and glass cannulas using a small piece of silicone tube 30 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL IMPORTANT BE VERY CAREFUL NOT TO APPLY A TOO HIGH PRESSURE WHEN FLUSHING AS THIS MAY DAMAGE THE PRESSURE TRANS DUCERS KEEP AN EYE ON THE PRESSURE TRANSDUCER READINGS ON THE MYO INTERFACE WHEN FLUSHING AND MAKE SURE THAT THE PRESSURE DOES NOT EXCEED 150 mmHg S Flush the pressure transducers and glass cannulas as described in step 2 Repeat the procedure two to three times 4 4 4 Pressure System Cleaning L 2 3 10 11 12 Tta 14 15 16 17 18 19 20 After completing an experiment remove the vessel mounted on the cannulas Remove buffer from the tissue chamber Turn off heat Dump any buffers in the bottles both P1 and P2 bottles P1 bottle is the bottle that starts with buffer P2 bottle is the waste bottle If looking at the pressure regulator from the front P1 bottle is on the right and P2 bottle is on the left Put the bottles back in place and tighten the caps Make sure all the tubing is connected to the chamber and bottles Make sure that the system is NOT in No Flow turn off No Flow if this function was used If using glass cannulas for small vessels then insert the calibration shunt that came with your system to bypass the can nulas in the same fashion you would use the shunt to calibrate your pressure transducers If looking at the cap of
41. llustrated in figure 4 9 on next page CHAPTER4 25 Figure 4 8 Disconnection of silicone tubes B P Figure 4 9 Mounting of Resistance tube shunt 6 Close the inlet from the pressure regulator to P1 by turning the handle on the 3 way valve towards the normal inlet way The inlet to P1 now comes from the pressure manometer See Figure 4 3 Three way valve flow settings at P1 on page 23 and Figure 4 7 Illustration of set up for pressure transducer calibration on page 25 f Close the calibration valve on the front of the Pressure Regulator by turning it clockwise IMPORTANT MAKE SURE THAT THE CALIBRATION VALVE IS COMPLETELY CLOSED IF THE CALIBRATION VALVE IS NOT COMPLETELY CLOSED IT WILL RESULT IN INCORRECT RESULTS AND UNRELIABLE PRESSURE READINGS AN SETTINGS DURING AN EX PERIMENT 8 Turn on the Pressure Regulator and the Myo Interface Use F1 to go to the Setup Menu in the Myo Interface Bring the calibrate line to the top of the display by using the numeric keys and press Enter to choose between calibrating pressure force or pH Choose calibrate pressure in the calibrate line and press F4 to enter the calibration menu 26 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL 9 Apply a pressure of 50mm Hg on the pressure myograph using the Big Ben pres sure manometer When the relative readings at the bottom line of the window ap Calibrate Pressure pears stable press F3 to proce
42. measuring pH in the superfusion buffer Also the diagram include the DMT Flowmeter Model 161FM The Flowmeter enables flow measurements in the range of 1 500ul min Figure 2 1 above is intended as an overview for the set up of the Pressure Myograph Systems followed by a detailed step by step description in the remaining sections of Chapter 2 The Pressure Myograph System will work with a wide range of different microscopes DMT Microscope Zeiss Axiovert 40 Nikon TS 100F Motic AE2000 or your own inverted microscope of choice NOTE THE MICROSCOPE IS ESSENTIAL FOR THE DIMENSIONAL ANALYSIS OF THE MOUNTED VESSELS The DMT Microscope is a standard inverted microscope with a built in digital CCD USB camera The DMT Microscope is very use ful and recommended in set ups requiring standard image analysis The Zeiss Axiovert 40 is an inverted microscope which provides the highest degree of flexibility in image analysis The Zeiss Ax iovert 40 and Motic AE 2000 enables the use of a wide range of different objectives and sophisticated fluorescence techniques Contact DMT Sales Department for further product information and prices on any optional equipment for the different available microscopes 12 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL Waste Bottle 2 2 Setting up Step by Step NOTE IF YOU HAVE PURCHASED A COMPUTER FROM DMT IN CONJUNCTION WITH YOUR PRESSURE MYOGRAPH SYSTEM THEN THE DMT VESSEL ACQUISITION SUITE DMT VAS HAS ALREADY
43. n stable P1 3614 P2 3533 F3 Calibrate Pressure Apply 125 mm Hg Push F3 when stable 1 4252 P2 4222 F3 Calibrate Pressure Apply 200 mm Hg Push F3 when stable P1 4956 P2 4933 F3 Calibrate Pressure DONE Push Esc 36148 42527 49565 35334 42223 49332 Push F3 when stable Force 1938 F3 Calibrate Force Place 2 g on the pan Push F3 when stable Force 2245 F3 Calibrate Force DONE Push Esc Force Zero 20315 Force gain 668 3 Temp auto 22 4 c pH output 2 F3 Calibrate pH Slope Slope buffer 4 00 Temp auto 22 4 c pH output 177mV F3 Calibrate pH OFFSET Offset 2208 Slpoe 26 4 3 1 1 General Key Functions 1 1 Scroll up and down in the menus Being in the top line of a menu use f to change to the previous main menus Info Push this key in any menu to display all specific F key functions Numeric keys Enter data or setting values Enter Confirm entered numeric values or change between available choices Esc Delete incorrectly entered numeric values In some submenus used to return to main menu F1 Change to the next menu F2 F4 Have different but specific functions dependent of the active main menu 3 1 2 Turn on Message Turning on the Pressure Myo Interface a message appears as shown to the right Use the T and keys to adjust the display contrast setting Press F1 for proceeding to the Main Menu 3 1 3 Main Menu The Main Menu displays P1 Inlet pressure readi
44. ng in mm Hg P2 Outlet pressure reading in mm Hg Force Longitudinal force reading in mN Temp Actual temperature probe reading in C Set temp Heating temperature setting in C Heat Actual heating status ON OFF pH Actual pH reading Optional Use the f and keys to see all values as the display is only capable of showing four lines at the time Temperature setting To change the temperature setting move the Set Temp line to the top of the display Use the numeric keys to enter a new temperature setting and press Enter to confirm Heating control To turn on heating move the Heat line to the top of the display and use Enter to switch between on and off The Main Menu has the following functions Info Press the Info key and the display shows the F key options in the Main Menu Press Info again to return to the Main Menu or one of the F keys to proceed with one of these options F1 Press F1 to proceed to the Pressure Menu F2 Press F2 to start the pressure control programme The settings of the pressure control programme are adjusted in the Pressure Menu F3 Press F3 to zero the longitudinal force reading F4 Press F4 for the display to show a condensed Main Menu showing pressure inlet reading P1 pressure inlet setting P1 set pressure outlet reading P2 pressure outlet setting P2 set force reading F pH reading Temperature read ing T and temperature setting T se
45. nual FlowMeter Model 161FM MyoFlow FlowMeter software including drivers CD ROM e Peristaltic Pump for Superfusion Flow Optional e Alita Watson Marlow400 VS2 10R Midi 2 5 bOrpm Computer Optional 8 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL CHAPTER 1 SYSTEM OVERVIEW 1 1 Pressure Myograph Unit Horizontal Adjustment Screw e Micropositioner for Vertical Adjustment of Glass Cannulas Force Transducer Outlet Pressure Transducer 25 Pin Serial Port for Communication with Connection Pipe Myo Interface Inlet Valve Outlet Valve e Outlet Pressure Transducer Inlet Pressure Transducer Micropositioner for Longitudinal Adjustment of Glass Cannulas Figure 1 1 The Pressure Myograph Unit Fixation Plate for Left Glass Cannula Outlet Connecting Pipe e e 9 y e e 9 9 9 e e 9 9 9 9 9 eS Silicone Tube connecting Outlet Connecting Silicone Tube connecting Perfusion Inlet Pipe and Right Pipe with Left Glass Cannula TES Glass Cannula Left Glass Cannula g e Right Glass Cannula 9 Myo Chamber Window Figure 1 2 The Pressure Myograph chamber close up CHAPTER1 9 1 2 Pressure Myo Interface Up and Down Arrow Keys Scroll up and down in the menus If Analogue Recorder Output Connection to
46. r 1 System OVOrIVIOW ossnxpndntui ianaddnnie opone nip OG DOR nina 9 DVS SS UNS MVO IPA Mb epson Doro RI EON E dUadS 9 T Pressure NP OS nte eb CRAB EE III DNI NRI Sivas YA DIN 10 1 5 Pressure FM ica etcetera ee eh CEU S CUN DI E M MNA Dad TI Chapter UD c 12 2 1 Pressure Myograph System Model 110P d ddl OVER DNE 12 Fe E OEE AA 13 Chapter 3 The Pressure Myo Interface 1 c eiie ecce mnnn IRR RR IIS a S suus Rr sas a na 16 MVO MEN Ce MaN Ua esses a REN 16 Chapter 4 Pressure Myograph Manual rece erre nnne nana naa III RR RR RARE RAE RARE S RRR S Sys R au RS Saa 21 A1 The Pressure Myograph UMIE mcr T 21 A VETE MITO CONG a E E E O E E 28 2 3 Ine Te SO A E A A Conde A TNA E E 28 4 4 Pressure Myograph Maintenance and Cleaning ha 30 Chapter 5 Experimental setup step by step
47. r further instructions If the message OFF is displayed in the force reading line in the Main Menu on the Myo Interface then the force transducer is broken and needs to be replaced In case of any other problems related to the force transducer please contact DMT for further instruction and advice CHAPTER4 27 4 1 6 Checking the Pressure Transducers The Pressure Myograph Unit contains of two pressure transducers P1 and P2 based on Wheatstone bridge circuit The trans ducers are placed inside the pressure myograph unit to protect them from mechanical damage The pressure transducers are very vulnerable to applied pressures exceeding 300mm Hg a pressure which is easily exceeded if trying to flush the transducers using a syringe IMPORTANT DMT RECOMMENDS THAT THE MYO INTERFACE IS CONNECTED AND TURNED ON WHEN FLUSHING THE PRESSURE TRANS DUCERS TUBING AND GLASS CANNULAS USING A SYRINGE THIS ENABLES AN EXACT CONTROL OF THE PRESSURE AP PLIED ON THE PRESSURE TRANSDUCERS WHICH WILL PREVENT THEM FROM BEING DAMAGED THE APPLIED FLUSHING PRESSURE SHOULD NOT EXCEED 150 mmHg 4 1 6 1 Simple Pressure Transducer Check 1 If the pressure reading on the Myo Interface appears unstable then first check that the Myo Interface and the Pressure Myograph Unit are properly connected through the 25 pin grey cable 2 Ifthe pressure reading still appears unstable then perform a new pressure calibration of the pressure transducers as de scribed in 4
48. r to the mounting of the blood vessel Left Glass Cannula Horizontal positioning of the left glass cannula is adjusted by carefully loosening screw D figure 4 2 above Vertical positioning of the left glass cannula is adjusted by carefully loosening screw E figure 4 2 above Longitudinal positioning of the left glass cannula is adjusted by gently loosening of screw F figure 4 2 above IMPORTANT BE VERY CAREFUL NOT TO APPLY TOO MUCH FORCE gt 100 GRAM ON THE FORCE TRANSDUCER PIN WHEN ADJUSTING THE LEFT GLASS CANNULA THE FORCE TRANSDUCER HAS A MECHANICAL PROTECTION IN ITS LONGITUDINAL DIRECTION OF MOVEMENT BUT IS VERY VULNERABLE TO FORCE APPLIED ON THE FORCE TRANSDUCER PIN 22 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL Right Glass Cannula Horizontal positioning of the right glass cannula is adjusted by carefully loosening screw A figure 4 2 on previous page Vertical positioning of the right glass cannula is adjusted with the micropositioner C figure 4 2 on previous page Longitudinal position ing of the right glass cannula is adjusted by gently loosening of screw G figure 4 2 on previous page NOTE BE CAREFUL NOT TO DAMAGE THE TIP OF THE GLASS CANNULAS DURING THE GENERAL ADJUSTMENT 4 1 2 3 Way Valve Adjustments The 3 way valves on each side of the Pressure Myograph Unit at P1 amp P2 have three different settings to control the in or outlet flow to the pressure transducers The flow settings are illustrated in
49. rt and the original packing material is unavailable please contact DMT Sales Department for advice and packing instruc tions After unpacking your new Pressure Myograph System please use the following list to check that the system is complete Pressure Myograph Unit e Force calibration kit Including bridge balance and 2 gram weight Resistance tube shunt Calibration tube Two glass cannulas e Chamber cover e Pressure Myo Interface e Pressure Myograph Unit connection cable with a temperature probe e Power cord the shape of the AC plug varies by country be sure that the plug has the right shape for your location e Serial Cables for PC connection or serial connection to other DMT Pressure Myograph Systems e Pressure Regulator e Power cord the shape of the AC plug varies by country be sure that the plug has the right shape for your location e Atlas Copco Cablair hose for main pressure supply e Two 500ml Schott Duran Bottles incl special designed screw caps Riester Big Ben Pressure Manometer e Accessories e Allen key e Spare screws e Small screwdriver e Nylon suture e Tube of high vacuum grease e Tube of grease for linear slides e Silicone tubing internal diameter 1mm e Hardware Software amp Manuals e USB digital Camera CD with user manual Pressure Myograph System Model 110P CD with DMT Vessel Acquisition Suite Software DMT VAS optional e FlowMeter Model 161FM Optional User ma
50. s illustrated in figure 4 10 on next page The Pressure Regulator transfers the pressure and flow settings from the DMT VAS program or the Myo Interface by indi vidual control of the air pressure in the two buffer bottles IMPORTANT NEVER FILL THE BOTTLE WITH MORE THAN 250 ML BUFFER AS HIGHER VOLUMES MAKES PRECISE CONTROL OF THE PRESSURE MORE DIFFICULT 4 3 2 No Flow Setting Activating the No Flow setting in the Myo Interface closes the No Flow Valve in the diagram shown in figure 4 10 on next page Closing the No Flow Valve serves to prevent any buffer back flow through the mounted vessel Activating the No Flow setting turns on the No Flow indicator lamp on front of the Pressure Regulator Activating the No Flow setting in the Myo Interface deactivates the P2 pressure transducer and the P1 pressure transducer takes control of the pressure applied on the mounted vessel At the same time the P3 pressure transducer is activated controlling the air pressure in the left bottle with bufffer in figure 4 10 on next page Activating P3 serves to prevent a huge pressure to build up in the left bottle with buffer which may damage the mounted vessel when re establishing the flow 28 PRESSURE MYOGRAPH SYSTEM MODEL 110P USER MANUAL Air In Max 1 atm Inlet to P1 on Pressure Myograph unit Reduction Valve Manometer 0 5 bar Outlet from P2 on Pressure Myograph Unit Relief Valve 0 8 bar pm A Valve 2 Valve
51. t Myo Interface Model 110P Version 3 3 Main Menu Set Temp Heat pH Info Change menu Start pres Ctrl Zero force Condensed menu F4 P1 141 P1 set 140 P2 set 130 pH 7 65 T set 37 CHAPTER 3 17 3 1 4 Pressure Menu The Pressure Menu displays P1 Inlet Inlet pressure setting in mm Hg P2 Outlet Outlet pressure setting in mm Hg Status Activation of pressure control ON OFF Flow Activation of flow control ON OFF mm Hg sec Setting of pressure changing gradient The Pressure Menu enables control and adjustment of pressures and flow P1 Inlet and P2 Outlet settings control the inlet and outlet pressure of the perfusion buffer The pressure control is activated or deactivated in the Status line by choosing ON OFF IMPORTANT PLEASE NOTE THAT THE P1 INLET PRESSURE CANNOT BE SMALLER THAN THE P2 OUTLET PRESSURE IF A P1 VALUE SMALLER THAN THE P2 VALUE IS ENTERED THE P1 VALUE WILL AUTOMATICALLY BE SET TO A VALUE EQUAL P2 FURTHERMORE IF A P2 VALUE LARGER THAN THE P1 VALUE IS ENTERED THEN THE P2 VALUE WILL AU TOMATICALLY BE SET TO A VALUE EQUAL P1 A SETTING WHERE P1 P2 ACTIVATES THE NO FLOW FUNCTION IN THE PRESSURE REGULATOR AS DESCRIBED IN 4 3 2 NO FLOW SETTING ON PAGE 28 THE FUNCTION WORKS TO PREVENT BACKFLOW The perfusion flow can be activated or deactivated in the Flow line ON OFF In the mm Hg sec line it is possible to adjust and control the pressure chang
52. t these steps and make sure that the system is thoroughly washed with double distilled water again to remove the acetic acid An acetic acid wash is not needed after each use Use this spar ingly If only buffer has been used in the perfusion line then there is no need to ever use acetic acid to clean the tubing internal buffer lines of the chamber and inside the regulator IMPORTANT I BE VERY CAREFUL NOT TO DAMAGE PRESSURE TRANSDUCER P3 WHEN FLUSHING THE BUFFER CIRCUIT THERE ARE NO READINGS FROM P3 VISIBLE ON THE MYO INTERFACE DANISH MYO TECHNOLOGY THEREFORE RECOMMENDS THAT THE PRESSURE MYOGRAPH UNIT ALWAYS BE FLUSHED FIRST IN THIS WAY IT IS POSSIBLE TO GET A FEELING OF HOW MUCH FLUSHING PRESSURE THAT IS EQUAL TO 150 mmHg IMPORTANT II NEVER FLUSH THE AIR CIRCUITS PORT NO 2 3 AND 4 ON THE PRESSURE REGULATOR WITH ANY KIND OF FLUID 4 4 5 Cleaning Pressure Myograph Chamber and cannulas DMT STRONGLY RECOMMENDS THAT THE MYOGRAPH CHAMBER AND SURROUNDINGS BE CLEANED AFTER EACH EXPERI MENT After a normal experiment use the following procedure to clean the myograph chamber and supports The chamber and cannulas can be cleaned at the same time while the systems is being flushed if the flow program is used as described in step 15 above 1 clean the chamber fill the chamber with double distilled water and wash at least 3 times 2 Toclean the cannulas one can use a syringe and needle An 18 guage needle with the sharp bevel r
53. the P1 bottle the frits at the top where the tubing is connected should be clearly marked with P1 4 and 5 Detach the tubing from the top of the P1 bottle marked 5 Attach the Big Ben manometer at the frit marked 5 Using the Big Ben manometer push the residual buffer out of the system Using the Big Ben manometer will allow you to pressurize the system by knowing exactly how much pressure is being used without having to worry about blowing out the P1 and P2 pressure transducers More importantly this will protect the hidden P3 transducer in the pressure regulator DO NOT EXCEED 250 mmHg IF YOU EXCEED 250 mmHg YOU WILL DAMAGE ALL 3 PRESSURE TRANDUCERS Once the buffer has been cleared from the system and into the waste bottle fill the P1 bottle with double distilled water DO NOT FILL WITH MORE THAN 250 mL OF WATER Re attach the P1 bottle top and make sure it is tight Using the Big Ben manometer that should still be attached to the 5 frit on top of the bottle pressurize the bottle using the manometer DO NOT EXCEED 250 mmHg IF YOU EXCEED 250 mmHg YOU WILL DAMAGE ALL 3 PRESSURE TRANDUC ERS Water should be pushed from the bottle through the system Do this for at least 5 minutes As an alternative re attach the tubing to the 5 frit on top of the P1 bottle Make sure all the other tubing is connected in the system so that if flow is induced the water from the P1 bottle will flow from the P1 bottle to the P2
54. waste bottle freely Induce flow by setting P1 on the interface to 200 mmHg and setting P2 to 50 mmHg This should cause the water in the P1 bottle to flow through the system to wash the tubing and internal parts of the chamber and pressure regulator If this method is used to clean the system then run this flow program for at least 10 minutes Once the system has been flushed with water empty the water from the P1 bottle Re attach the cap to the P1 bottle and using the Big Ben Manometer push the water out from the system by attaching the Big Ben to frit 5 on the P1 bottle Again DO NOT EXCEED 250 mmHg IF YOU EXCEED 250 mmHg YOU WILL DAMAGE ALL 3 PRESSURE TRANDUCERS Once the system is emptied dry the system by continuing to push air through the lines Push air through for at least 5 minutes Once air has been pushed through the system detach the Big Ben manometer from the P1 bottle and reattach the appro priate tubing CHAPTER4 31 21 Detach the tubing from the regulator NOT THE BOTTLES where it is marked P2 This frit on the regulator is right next to the frit marked 1 22 Connect the Big Ben manometer here and push air through to make sure the inside of the regulator is dried If buffer or water stays in this part of the regulator it can cause problems that are not covered by warranty and this step will guarantee that this part of the regulator stays clear 23 f an 8 acetic acid rinse is performed then repea
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