Home

User Manual

1. 2 1 The complete Myograph 610M system 1 eese eee nnnm 13 2 2 SOUS UD SLOP OV STOI see ee ee ne een 13 2 3 The first weight calibration nee sera 14 Chapter 3 The Myo Interface u 2 222000200nn0nnn0nnnunnnunnnnnnnnnnnnnnnnnnnnnnn 15 3 1 Turning on the HO nea C Cud EG YO o saw ATO RF WLR I OW nu G 15 3 2 Main menus and UDD UO een sense 15 Chapter 4 The Multi wire myograph unit eeu 19 4 1 Change and adjustment of mounting supports 222u2002000000000nnnnnnnnnnnnnnnnnnnnnnnnn 19 4 2 Force transducer Cali DTU OM aenivcscsnincescstecaneasncsanreoswncivatantenndnenainiecnndiustsanieowuntccwsanseous 21 4 3 Checking force transducer nenne 24 4 4 Force transducer replacement 0 cccssccccesscssescceesecsesccnesccnesecnueccnenecnessscsnscnssecesaseons 24 iod esie didici e EO FT EF REY NN FER FN EC HYN TF 25 Chapter 5 Dissection mounting and normalization 28 5 1 Dissection protocol for small mesenteric arteries eeeeeeeeeeeeen 28 5 2 Mounting protocol Tor small arteries ana nee on gt INV zii Ad TETTE 35 Chapter 6 Experimental protocols cernere 36 eit 1a 015148 LE Ui FED te cco esteem chee ace sete een A Y RE RR RR FY IN 36 In vitro experiment 1 Noradrenaline contractile response eene 38 In vitro experiment 2 Acetylcholine
2. ing 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 and supports with an 8 acetic acid solution for 10 minutes and continue the mechanical cleaning with a swab stick 6 Wash the myograph chamber and supports several times with double distilled water Important e In exceptional cases it may be necessary to unmount the supports and clean them and the myograph chamber separately to ensure that all surfaces are cleaned User manual 610M Chapter 4 27 Chapter 5 Dissection mounting and normalization This chapter contains some practical protocols and procedures describing how to quickly get started using the Multi Wire Myograph 610M system While the chapter mostly addresses users who are not yet familiar with the wire myograph technique the information may also be useful for the experienced myograph user 5 1 Dissection protocol for small mesenteric arteries The Multi Wire Myograph technique is versatile in that a large variety of physiologi cal and pharmacological studies of ring preparations from different species can be performed The wire myograph is predominantly used for investigation of small blood vessels and as an example this chapter describes the dissection of rat mesenteric arteries 1 A laboratory rat is euthanized in accordance to the local national law and regula tions A midline laparotomy is performed to e
3. 03 mN Force 3 1 87 mN Force 4 0 23 mN NOW MOUNT MYO NO 1 Force 2 1 03mN Force 3 1 87mN Force 4 0 23mN gt Zero 1 Zero 2 F1 Slct Zero 3 F2 Zero Zero 4 Chapter 3 15 16 After selecting F2 the selected transducer will be zeroed and the display will automatically return to the force menu Re enter the zero menu using the arrow down button choose the next transducer with the F1 button and zero with F2 Repeat this pro cedure until all transducers have been zeroed To simultaneously zero all channels enter the zero menu and press the right arrow gt and F2 at same time Note there should be no force applied to the transducers when zeroing Heat menu In this menu the heating and temperature settings are control led The F1 button turns the heat ON and F2 switches it OFF The a temperature the myograph heats to Set temp is decreased jen with the button and increased with Act Temp is the actual temperature measured by the external temperature probe when connected to the interface If there is no probe connected Act Temp OFF will be displayed Note Whenever the system is turned on by the power switch on the rear panel the heating is automatically off until turned on by the user When the heating is on the set temperature will be reached in 20 minutes Recorder menu The 610M Myo Interface has four 1 V full scale analog outputs s i gt A AOU which pro
4. 2 L DMT recommends a 2 L thick walled glass bottle to collect the used solution from the myographs and act as a suction trap to prevent fluid entering the vacuum pump Vacuum pump DMT recommends a membrane vacuum pump having a volume of at least 6 L minute Dissection petri dish DMT Item PD 2000 DMT recommends a 9 cm glass Petri dish coated with a 5 mm Sylgard polymer layer User manual 610M Appendix 4 45 A4 2 Myograph 610M System accessories This section contains a list of special accessories available for the multi wire myo graph system Model 610M ADInstruments PowerLab data acquisition system Including LabChart data acquisition and analysis software e DMT Normalisation module DMT Item MLSO65 LabChart add on Module software pH Meter Including pH electrode Plastic mounting jaws DMT Item PJ 345610 With built in platinum electrodes for electrophysiological experiments 25 um tungsten wire DMT Item TW 345610 For mounting of ring preparations with internal diameter down to 60 um Standard PC system DMT Item ff 80150 e CS 200 4 Channel Current Stimulators DMT Item ff CS 200 Combined pulse and train generator A5 3 Myograph 610M System spare parts This section contains a complete list of standard spare parts available for the myo graph 610M For parts not listed in this appendix or for special parts which may need to be custom made please contact DMT for further information Force
5. 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 230 V CEE 7 page VII SR section 107 2 CEE 7 page VII SR section 107 2 D1 IEC 83 page C4 83 page C4 V BS 1363 of 1984 USE aa IE 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 such 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 User manual 610M Safety 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 agains
6. distilled H O to reach a final volume of 1000 mL Before use 3 Dilute the 25 x PSS stock solution 1 25 with double distilled H O 4 Add 1 091 g L Glucose 2 100 g L NaHCO o Aerate the solution with carbogen 95 O 5 CO for at least 20 minutes If necessary wait further for the pH of the buffer to reach pH 7 4 High Potassium physiological saline solution KPSS To make 1 L of KPSS Use the recipe for regular PSS but replace the desired concentration of NaCl with KCI For example to make 60 mM KPSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 64 86 3 79 KCI 74 56 58 82 4 39 MgSO 7H O 246 48 1 17 0 29 KH PO 136 09 1 18 0 16 Calcium free physiological saline solution Ca free PSS To make 1 L of Ca free PSS Use the recipe for regular PSS but omit the CaCl and add EGIA to buffer the residual Ca in solution The concentration of EGTA in the PSS should be 1 5 mM to ensure sufficient buffering User manual 610M Chapter 6 41 42 Appendix 1 Terms of warranty Warranty DMT A S warrants to the original user that myograph systems manufactured by DMT A S will be free from defects in materials and workmanship for a period of three years after the date of delivery DMT A S will repair or replace any defective part subject to the conditions limitations and exclusions Exclusions Force and pressure transducers separately or part of myograph systems manufac tured by DMT A S are dis
7. iugbof1DM O 05 JJ 4 J iubof1DM O 8 UU 754e19M Lo 8 awof0 M ord 25yLof M In calculating the NA in the myograph chamber the mE volume of no radrenaline is ignored Rat mesenteric arteries do not show spontaneous tone in the wire myograph which is why it is necessary to first induce a contraction to be able to observe the relaxa tion to acetylcholine In this protocol the contraction is induced by noradrenaline The required concentration of noradrenaline needs to be optimised since a too low concentration makes it impossible to evaluate the relaxation On the other hand it may be difficult to relax super maximally contracted arteries which may lead to an un derestimation of the sensitivity to acetylcholine Therefore it is recommended to apply a concentration of noradrenaline inducing 60 70 of maximal contraction response In practice this concentration is found by performing a noradrenaline concentration response curve as described in the previous section The vessel segment is exposed to the noradrenaline concentration and when the response has stabilised increasing concentrations of acetylcholine are added to relax the vessel Each concentration is applied until a steady response has been reached and then the next concentration is applied When the vessel segment is either fully relaxed or does not relax more upon increasing the acetylcholine concentration the exper
8. line respectively T 4 Scroll up and down in the menus The active line in the menu is indicated by a gt symbol displayed in the left side of the display Force menu The force menu provides an online reading of the force values in millinewtons mN Removing and replacing a chamber unit When a chamber unit is unplugged from the Myo Interface the force display does not show a force value An example display is depicted to the right where myograph unit 1 is unplugged To replace the unit firstly press the arrow displayed in the active line The display now changes to Now mount myo No 1 Put the chamber unit in place on the interface always place chamber unit one at No 1 on the interface unit two on No 2 and so forth and plug the chamber cable into the corresponding input on the interface s rear panel If the unit is not plugged into the interface within 10 seconds then the arrow key should be pressed again the interface will display the previous message Once the unit is connected the active line will again display a force reading Zero menu This menu is used to zero the output of the transducers The F1 button changes which transducer is active in the list Slct stands for select Pressing the F2 button will zero the selected trans ducer indicated by gt User manual 610M Multi Myograph System Model 610M Version 2 2 Force 1 Force 2 Force 3 Force 4 PUSH BEFORE MOUNT Force 2 1
9. microscope stage and focus on it Fit one of the division lines on the stage micrometer to one of the division lines of the reticule scale very precisely While keeping the stage micrometer absolutely fixed on the microscope stage find another position on both scales where the division lines also fit precisely Read the position of the two fit points on both scales and fill in the values in the Ocular Calibration Sheet Repeat the procedure twice DMT Multi wire myograph system Model 610M Ocular calibration sheet A m Stage micrometer Reticular scale Calculations 1 Calculate the length between the two positions on the stage micrometer by sub tracting the value of position 1 from the value of position 2 Multiply the length in divisions with the length of each division to get the length in mm 2 Calculate the length between the two positions on the reticule scale by subtracting the value of position 1 from the value of position 2 3 Calculate the mean length value of both the stage micrometer and the reticular scale 4 Calculate the conversion factor 5 Mean Stage Micrometer Length mm Mean Reticule Scale div User manual 610M Appendix 6 49 50 Appendix 7 How to read a millimeter micrometer Sleeve scale Thimble scale Figure A7 1 Overview of the micrometer parts actual reading 20000 um 20 mm Sleeve scale The micrometer sleeve scale has a total length of 25 mm divided into fifty equal parts
10. submenu is found under Setup submenu 3 By default the valve delay is set to 1 second the available delay range is 1 to 99 seconds The valve delay is the time the valve is open once you have released the valve button on the front panel The delay time is changed using the and buttons Force range submenu The Force Range submenu is found in Setup submenu 7 By default the force range is set at 200 mN The available ranges are 200 400 800 and 1600 mN The force range is changed using the and buttons Serial port submenu For digital storage of data the interface has an integrated RS232 port and two RS485 serial ports The RS232 serial port is used when a single system is connected to a computer and the RS485 serial ports are used for multiple connections up to four systems linked together allowing 16 force channels and four temperature readings By default the system is set to use RS232 and functions for the serial settings are found in Setup submenu 10 To switch between the two outputs use the and buttons Using the enclosed serial cable with the mounted adapter the 9 pin plug is connected to the RS232 plug of the interface and the 25 pin plug connected to the serial port COM port on the computer RS232 Serial Port This serial communication protocol must be used when only one system is connected to one PC RS485 Serial Port This serial communication protocol must be used when multi ple
11. transducer DMT Item FT 61 80 Roll of 40 um stainless steel wire DMT Item ff SSW 123456 High vacuum grease and grease for linear slides DMT Item HVG 1000 Stainless steel jaws DMT Item SSJ 345610 Mounting support pins DMT Item MO 200 Calibration kit DMT Item CK 6127800 e Chamber cover DMT Item CC 61700 e 40 mm funnels DMT Item F 2000 46 DMT Multi wire myograph system Model 610M Appendix 5 Fuse replacement The main fuse of the myograph system is placed inside the power inlet on the Myo Interface When a fuse blows and needs to be changed it is imperative that the replacement fuse is equal to the one blown The 610M system uses T1 6A 250 V 6 3 x 32 mm DMT recommends that both fuses in the fuse block are changed at the same time as it can be difficult to determine which fuse is blown To replace the fuses Use a small screwdriver to open the voltage selector block Remove the red fuse block Remove the existing fuses Insert the new fuses Replace the fuse block back into the voltage selector block Note ensure that the correct voltage for your country is displayed Se IE Voltage selector block User manual 610M Appendix 5 47 Appendix 6 Calibration of ocular reticule Principles of ocular calibration The purpose of calibrating the eyepiece reticule is to determine a conversion factor 0 allowing the microscope to be used for measuring vessel segment lengths mounted i
12. um wires IC 205 6 um Using the Laplace relation the effective pressure P is calculated for each pair of readings The effective pressure is an estimate of the internal pressure which is nec essary to extend the vessel to the measured internal circumference T ce The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure The target value needs to be optimized for the indi vidual tissue preparation optimal active force as determined by the length tension relationship for that tissue For rat mesenteric arteries the target transmural pres sure is normally 100 mmHg 13 3 kPa 100 mmHg IC T 100 mmHg 2T User manual 610M Appendix 8 51 52 An exponential curve is fitted to the internal circumference pressure data as illus trated in fig 6 14 in Chapter 6 Now the isobar corresponding to 100 mmHg is used to calculate the IC value from the point of interception between the function of the exponential curve and the function of the 100 mmHg isobar The normalized internal circumference IC is calculated by multiplying the internal circumference corresponding to 100 mmHg IC by a factor k The factor is for rat mesenteric arteries 0 9 Again this value should be optimized for the particular tissue preparation being used by a length tension curve IC k IC The normalized internal lumen diameter is then calculated by 42 Jc 1 TU The mi
13. 00 300 um 6 Use high quality forceps and ocular dissection scissors to dissect the vessel segment of interest Start cutting through the mesenteric membrane along both sides of the vessel about 1 2 mm from the vessel To avoid accidentally cutting the artery always cut along the length of the vessels and never perpendicular to them fig 6 2A B f Dissect away as much adipose tissue as needed around the vessels to distin guish between the artery and vein The artery can easily be identified by the fol lowing characteristics fig 6 3 DMT Multi wire myograph system Model 610M Second Branch Figure 6 1 Branching of the mesenteric arteries Figure 6 2 Removal of adipose tissue around the area of interest Figure 6 3 Distinguishing between artery and vein User manual 610M Chapter 5 29 30 e The branch points of arteries are V shaped whereas those of veins are more U shaped The arterial wall contains a thick layer of smooth muscle cells compared to the vein wall which only contains a single or a few layers of smooth muscle cells The histological difference is clearly visible in the stereomicroscope e If you still have difficulty and the vein and artery still contain some blood then try to move the blood forward by very gently squeezing the vessels with a for ceps In the artery the blood will run back quickly whereas in the vein the blood will run back very slowly if it even does so Note it is impor
14. 00030 USA China Tel 45 87 411100 Tel 86 O 21 64869685 Tel 1 770 612 8014 Fax 45 87 411101 Fax 86 0 21 64280591 Fax 1 678 302 7013 www dmt dk www dmt asia com www dmt usa com sales dmt dk sales dmt asia com sales dmt usa com support dmt dk support dmt asia com support dmt usa com User manual 610M Trademarks Introduction Until the mid 1970s most of the details about the mechanical morphological and pharmacological properties of vascular smooth muscle was obtained from studies on relatively large vessels At that time rat tail arteries were the smallest vessels to be investigated in detail due to limitations in the available in vitro techniques For example studies measuring the contraction force were routinely performed with only one of the mounting wires secured Furthermore relatively large wires LOO 200 um were used which precluded the use of small vessels and the vessel segment had to be directly manipulated with the dissecting equipment causing inevitable mechanical trauma Investigations of smaller vessels were therefore limited to in vivo perfusion experiments and histological examinations In 1976 Professors Mulvany and Halpern described for the first time a new technique that made it possible to investigate highly isometric responses from vessels with internal diameters as small as 100 um The mounting procedure was refined twofold both ends of each mounting wire were secured under tension without any d
15. 90000000000909090000000000090090000000090000000000090000900090000000909000000000009000000900000000909 0 s H E 2 999900000090000000900000000000000000000090000000000000000000002000000000000000000000005000000000000000000009000000009000009099009 H 00900900900900900000000000090000000000000000000000000000090090000000090000009000009090009009000090009000009000909009090090900909009090000000000000000090009 optional Suction bottle optional Figure 2 1 The complete multi wire myograph system model 610M The dotted lines repre sent optional connections to the system 2 2 Setting up step by step The chapter contains a complete step by step description of how to set up a complete Myograph 610M System as illustrated in fig 2 1 1 Myo Interface PC connection Data acquisition is possible either by connecting the Myo Interface directly to a PC or through a PowerLab data acquisition and analysis system optional I Direct PC connection Connect the Myo Interface to one of the COM ports on the PC using a serial cable Cable not included ll PowerLab optional Follow the DMT Quick Guide instructions delivered with the PowerLab sys tem to install the PowerLab driver and LabChart software on the computer Follow the instructions provided in the DMT QuickStart Guide Data Acquisi tion Multi Wire Myograph System 610M and PowerLab LabChart 2 Gas supply The gas supply into the chamber is a plastic tube which is co
16. Each part of division above the horizontal line represents 1 mm where each fifth line is marked by a longer line and a number which designates the length in mm Each part of division below the horizontal line is placed between each 1 mm mark scale above the horizontal line and represents 0 5 mm Thimble scale The thimble is divided into fifty egual parts and one complete rotation of the thimble is indicated by the smallest division on the sleeve which equals 0 5 mm Each divi son on the thimble scale is 10 um If the thimble scale falls between two lines then a number between O and 10 um must be approximated Example 1 1 Note that the thimble has stopped at a point beyond 10 on the sleeve indicat ing 10000 um 10 mm 2 Note that there is no mark completely visible between the 10 mm mark and the thimble 3 Read the value on the thimble corresponding to the intersection with the hori zontal line on the sleeve A _ Reading on sleeve 10000 um unida B _ No additional mark visible O um C Thimble reading 380 um 0 a Total reading 10380 um Figure A7 2 Example 1 reading 10380 um Example 2 1 Note that the thimble has stopped at a point beyond 16 on the sleeve indicat ing 16000 um 16 mm 2 Note that this time a mark is visible between the 16 mm mark and the thimble indication 500 um 3 Read the value on the thimble corresponding to the intersection with the horizon tal line on the sleeve A Readi
17. Multi Wire Myograph System Model 610M User Manual Version 2 2 DANISH NO T FT U OGS DANISA MO T amp CHANOL OGS S DMT Multi wire myograph system Model 610M Multi Wire Myograph System Model 610M User manual Trademarks PowerLab and LabChart are registered trademarks of ADInstruments Pty Ltd The names of specific recording units such as PowerLab 4 25 are trademarks of ADInstruments Pty Ltd Pentium is a registered trademark of the Intel Corporation Windows Windows 95 Windows 98 Windows ME Windows NT Windows 2000 and Windows XP are registered trademarks of Microsoft Corporation All other trademarks are the properties of their respective owners 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 multi wire myograph system Model 610M v 2 2 Document Number 610M UG2 2A No part of this document may be reproduced by any means without the prior written permission of DMT Copyright 2008 DMT A S M DANISH NO TECHNOL LC OG DMT A S DMT Asia DMT USA Inc Skejbyparken 152 Everwin Gardens 1201 Peachtree Street DK 8200 Aarhus N Rm 502 Block B 400 Colony Square Suite 200 630 Denmark 521 Wanping Nan Lu Atlanta GA 30361 Shanghai 2
18. ales dmt asia com support dmt asia com DMT USA Inc 1201 Peachtree Street 400 Colony Square Suite 200 630 Atlanta GA 30361 USA Tel 1 770 612 8014 Fax 1 678 302 7013 www dmt usa com sales dmt usa com support dmt usa com
19. anel Gas regulator needle valve ON OFF switch 2 Gas input Power connector Suction input H RS 485 Port for serial connection to PC 4 Recorder outputs e e RS 282 Port for serial connection to PC Temperature probe User manual 610M Chapter 1 11 12 1 3 Multi wire myograph unit Allen screws for fine alignment e Connection to Myo Interface o of the myograph jaws Micropositioner e Force transducer pin eo e i Myograph jaw connected to force transducer e Myograph jaw connected to micropositioner e MN Supports ee eeeeeennnnns i rc TRW EYN T ER Mounting support jaws for small vessels Mounting support pins for larger vessels Ek i DMT Multi wire myograph system Model 610M Chapter 2 Setting up 2 1 The complete Myograph 610M system Myo Interface front panel Power supply PowerLab data acquisition system ee 3 ki optional DMT CS200 Pulse Train Stimulator optional PC data acquisition and analysis software optional Power supply Connection to oxygen supply O BNC Cables yd o o 007 E1 3 M t yd gt T H poc Naoosdfeoeccccccccccccccocccccccseccccccccccccccoccsccsosesccsocccccsocccsesocccseoccccccocccccccccscon p d Vacuum um Q09990909090000000090009009
20. claimed from any warranty Limitations This warranty shall not apply to equipment subjected to accidental damage improper use alteration or deterioration Warranty on third party products will be as determined by their respective manufac turer DMT A S shall not be liable for consequential incidental special or other direct or indirect damages resulting from economic loss or property damage sustained by you or any end user from the use of the products sold or services rendered hereunder Warranty Returns A Return Material Authorisation RMA number is required for all returns This number should be clearly indicated on all returned myograph systems Products damaged due to improper or inadequate packaging when returned for RMA purposes are not granted warranty coverage DMT Multi wire myograph system Model 610M Appendix 2 Service check For successful studies of small blood vessels or other small tubular tissue it is im perative that the myograph is performing optimally To make sure that our customers are always dealing with first class myographs DMT offers a Myograph Service Check at a very favourable price The Myograph Service Check includes a complete separation of the system for in spection of all mechanical and electronic parts The myograph is then reassembled adjusted and finally all electronic and mechanical parts are tested Please note that the service does not include or cover replacement of t
21. cover 4 Chamber covers The chamber covers are used to keep the temperature and other conditions gas tension pH of the solution surrounding the mounted segment as accurate as possible There are holes in the covers which serve multiple functions fig 2 3 and slots for the myograph supports and suction gas tubes 2 3 The first weight calibration Prior to the shipment of the Multi Wire Myograph 610M System it has gone through two days of continuous testing including a final weight calibration However in order to ensure that the myograph is working at highest performance DMT recommends that a new weight calibration is performed before starting to use the myograph system The weight calibration procedure is described in detail in Chapter 4 DMT Multi wire myograph system Model 610M Chapter 3 The Myo Interface 3 1 Turning on the Myo Interface When the 610M Myo Interface is switched on the start up mes sage depicted to the right is shown After a few seconds during which the system autocalibrates the A D converters pressing one of the arrow buttons will display the force menu 3 2 Main menus and submenus General navigation The various menus are selected the data changed and values entered with the F1 F2 and the arrow buttons The F1 and F2 button functions differ according to the current menu while the arrow buttons typically have the following functions Increase and decrease the data in the active
22. crometer reading X at which the internal circumference of the normalized ves sel is set to is calculated by IC IC 2 DMT Multi wire myograph system Model 610M Appendix 9 System specifications Tissue size Chamber Chamber material Chamber volume Chamber suction Chamber gassing Chamber cover Force range Force resolution Micrometers Weight calibration Heating Temp range Temp resolution Temp probe Output reading Analog outputs Digital output Voltage gt 60 um gt 450 um up to 10 mm Four individual chambers Acid resistant stainless steel Max 8 mL Manual or automatic time controlled user defined Individually controlled per chamber by needle valves Supplied with connections for gassing User selectable at 200 400 800 or 1600 mN 0 01 mN Manually operated Semi automatic Built in Ambient temp 50 C 0 1 C External Force mN 1 0V F S Serial interface RS232 RS485 115 VAC 10 50 60Hz 230 VAC 10 50 60Hz User manual 610M Appendix 9 53 54 Notes DMT Multi wire myograph system Model 610M ri MOD TE n r T CO5L_ oa DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Tel 45 87 41 11 00 Fax 45 87 41 11 01 www dmt dk sales dmt dk support dmt dk DMT Asia Everwin Gardens Rm 502 Block B 521 Wanping Nan Lu Shanghai 200030 China Tel 86 O 21 64869685 Fax 86 O 21 64280591 www dmt asia com s
23. d that you store the packing material for any possible future transport of the Multi Wire Myograph System In case of transport and the original packing material is unavailable please contact the DMT Sales Department for advice and packing instructions After unpacking your new Multi Wire Myograph System please use the following list to check that the system is complete e 1 Myo Interface unit e 4 chamber units with mounted stainless steel jaws e 4 set mounting support pins 200 um e 4chamber covers e 1external temperature probe e 1 power cord e 1 calibration kit including bridge balance and 2 gram weight e 4 plastic funnels e 1 roll of 40 um stainless steel wire e 1tube of high vacuum grease e 1 tube of grease for linear slides e 5 spare screws for mounting of jaws e 4 Allen keys e 1 small screwdriver e 1 Multi Wire Myograph System 610 M User Manual e 1 manual by Professor M J Mulvany Procedures for Investigation of Small Vessels Using Small Vessel Myograph 1 CD Dissection and mounting of small vessels in wire myographs The shape of the AC plug varies by country be sure that the plug has the right shape for your location DMT Multi wire myograph system Model 610M Chapter 1 System overview cc c 1 1 Myo Interface front panel Power indicator Valve buttons Myo Interface display AU SEW FU FFF nicer X Function buttons 1 2 Myo Interface rear p
24. e excessive vessel segment is finally dis sected free and removed from the myograph cham ber fig 6 9B C C Figure 6 9 A Band C Mounting step 4 Mounting step five e Move the jaws apart fig 6 10A Take a second wire holding it about one third down from the far end us ing 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 first mounted wire as a guide fig 6 10B 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 push ing 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 sufficient length for the wire to be secured both at the near and far fixing screws on the right hand jaw Figure 6 10 A Band C Mounting step 5 User manual 610M Chapter 5 Mounting step six e Carefully move the jaws together while en suring that the second mounted wire lies underneath the first one secured on the left hand jaw fig 6 11A The procedure clamps the second wire to prevent it from damaging the vessel segment when secur ing the wire to the right hand jaw connect ed to the transducer Secure the near end
25. her the wire jaw or the pin 4 Once the system has warmed up to the set temperature check by placing the temperature probe into the chamber go to the Calibration menu on the Myo Interface In the Calibration menu ensure Calibrate 1 is active the symbol is displayed to the left use the F1 button to scroll through the list and press F2 to initiate calibration of force transducer 1 DMT Multi wire myograph system Model 610M Figure 4 6 Weight calibration kit shown in place on an isolated myograph unit 5 Ensure that the tip of the transducer arm is not touching either the wire or the jaw or the mounting support pin if used It is imperative that the force trans ducer is not subjected to any force at this stage When the relative force reading in the display is stable press F2 to proceed with the calibration 6 Carefully place the 2 g weight on the pan as illustrated in fig 4 7 The force ap plied on the force transducer as the tip of the arm pushes against the mounted wire or Support pin should mimic the stretch created by the contraction of a mounted ring preparation Wait until the relative force reading is stable then press F2 to finish the calibration Force transducer 1 is now weight calibrated to an output of 9 81 mN The display will automatically return to the Force menu Note If the force reading is unstable or gt 0 1 mN different from 9 81 mN repeat the weight calibration If the instability continues refe
26. ial cells were successfully removed The procedure can be performed after the vessel segment has been heated equili brated and normalised Preferably the procedure should be done after performing a standard start to make sure that the vessel segment is viable The present procedure is for use with rat mesenteric arteries Another procedure may be needed for other animal species and tissue or vessel types Principles of checking endothelium function Stimulating a vessel segment with acetylcholine causes a release of nitric oxide NO also known as EDRF from the endothelium cells and subsequent relaxation of the vascular smooth muscle cells If the endothelium is undamaged by the dissection and mounting procedures then a substantial relaxation will occur With complete removal or damaged endothelium a partial relaxation or no relaxation to acetylcholine is observed It is important to note that the amount of NO or EDRF in a vessel is often dependent upon its size In certain vessels endothelium derived hyperpolarizing factor EDHF can contribute more or less than EDRF and in other vessels the same stimulation with ACh can promote release of endothelium derived contracting factor EDCF Therefore it is important to check the existing literature in order to determine the expected response in your particular vessel with the given concentration of agonist Procedure for Checking Endothelium Function Step 1 PSS 3 uM NA Stimu
27. illed H O as three 40 individual solutions as described in the table above Gently heat solution 3 to dissolve the EDTA Solution 1 is added to a graduated bottle and the bottle is filled with double distilled H O to a final volume of 500 mL Solution 3 is added to the graduated bottle which afterwards is filled with additional double distilled H O to a final volume of about 850 mL Aerate the solution with carbogen 95 O 5 CO for about 20 minutes Solution 2 is added and the graduated bottle is filled with additional double distilled H O to reach the final volume of 1000 mL Continue the carbogen bubbling until the pH of the buffer solution reaches 7 4 DMT Multi wire myograph system Model 610M 25x concentrated PSS To make 1 L concentrated PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 173 85 KCI 14 56 4 69 8 75 CaCl 2H 0 147 02 2 50 9 20 Solution 2 Chemical MW g mol Conc mmol L Conc g L MgSO 7H O 246 48 1 17 1 23 KH2PO 136 09 1 18 4 02 Solution 3 Chemical MW g mol Conc mmol L Conc g L EDTA 3 2 24 0 03 0 25 1 Dissolve the chemicals for solution 1 in about 800 mL double distilled H O in a 1000 mL graduated bottle Dissolve the chemicals for solutions 2 and 3 in 75 mL 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
28. imal 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 fig 6 A C Be careful not to stretch the vessel segment if the end of the wire catches the vessel wall Z Figure 6 7 A Band C Mounting step 2 Mounting step three e 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 e While controlling the movement of the wire with the forceps use the other forceps to gently pull the ves sel 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 O 1 mm inside the jaw gap to insure no point of contact fig 6 8A Still controlling the free wire end with the forceps move the jaws together to clamp the wire and in B one movement secure the wire under the near fixing screw on the left hand jaw Again in a clockwise di Figure 6 8 A and B rection so that tightening the screw also tightens the Mounting step 3 wire fig 6 8B 32 DMT Multi wire myograph system Model 610M 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 fig 6 9A Make sure that the vessel segment is separated as close as possible to the jaws Th
29. iment is ended Protocol Prepare the following stock solutions e Acetylcholine 10 10 10 M e Noradrenaline 107M 1 Mount and normalise the vessels as described in Chapter 5 2 Perform a standard start and check the vessel segment for endothelium func tion as described at the start of this chapter 3 Add noradrenaline to obtain a response around 60 of maximum determined from the previous noradrenaline concentration response curve When the con tractile response is stable add increasing concentrations of acetylcholine to the chamber using Table 6 2 as a guideline Wait for a stable contractile response or a standard time such as two Minutes between each application User manual 610M Chapter 6 39 ACh in chamber uM Volume of stock solution to add to chamber lt M 1 1 uL of 107M 2 5 uL of 10 In calculating the ACh in the myograph chamber the applied volume of ACh is ignored 10 6 1 Buffer recipes Physiological saline solution PSS To make 1 L of PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 6 95 KCI 14 56 4 69 0 35 MgSO 7H O 246 48 1 17 0 29 KH PO 136 09 1 18 0 16 Solution 2 Chemical MW g mol Conc mmol L Conc g L CaCl 2H O 147 02 2 50 0 37 Solution 3 Chemical MW g mol Conc mmol L Conc g L NaHCO 84 01 25 00 2 10 EDTA 31224 0 03 0 01 Glucose 198 77 5 50 1 09 1 Dissolve the chemicals in approximately 100 mL double dist
30. irect manipulation of the vessel segment Segments of small vessels could now be atraumatically mounted as ring preparations in a myograph for recording of highly isometric force measurements During the late 1970s some improvements were made to the myograph and in 1981 a new dual myograph that allowed simultaneous testing of two vessels was introduced In parallel the technique became widely acknowledged resulting in a growing interest in the myograph systems In 1986 the growing demand resulted in the foundation of the private company J P Trading with the purpose of making the myograph systems commercially available worldwide At the same time J P Trading initiated a comprehensive improvement programme for the existing myograph systems as well as a development programme of new myograph systems in close co operation with Professor M J Mulvany and the University of Aarhus During the late 1980s and through the 1990s several improvements were applied to the myograph systems such as a new mechanical design a more robust transducer and new electronic systems In addition new systems were introduced like the Automatic Dual Myograph 510A the Dual Myograph 410A the Multi Myograph 610M and the Confocal Myograph 120CW In 2000 J P Trading changed its company structure and became known as DMT Today DMT is one of the world s leading designers and manufacturers of wire myographs pressure myographs culture myographs and organ tissue baths Dri
31. justment 6 Loosen screw A fig 4 1 to move the associated support back and forward f Loosen screw B fig 4 1 to move the associated support back or forward 8 Loosen screw C fig 4 1 to vertically align the associated support Fine adjustment small vessel supports jaws 9 Tightening screw A fig 4 2 will move the attached jaw downwards and to the left 10 Tightening both screw A fig 4 2 and screw D fig 4 2 will move the nearest mounting head downwards only 11 Tightening both screw B fig 4 2 and screw C fig 4 2 will move the nearest head upward User manual 610M Chapter 4 19 EB Transducer house Micrometer e mn gur Pe gt Figure 4 2 Myograph unit screws for fine adjustment Dr See V x x Figure 4 3 Illustration of correctly aligned supports for small vessels left and incorrectly aligned supports middle and right Fine adjustment large vessel supports pins 12 Loosen screw A fig 4 4 to align the support vertically 13 Loosen screw B fig 4 4 to move the support back or forward 14 Loosen screw C fig 4 4 to move the support horizontally CL SE HER AB n 6 Figure 4 4 Myograph unit adjustment for mounting support pins 20 DMT Multi wire myograph system Model 610M 4 2 Force transducer calibration As a part of the general maintenance of the myograph DMT recommends that the myograph is weight calibrated at least once ever
32. late for 3 minutes Step 2 Add 10 pl 5 x 10 M ACh Wait 2 minutes Step 3 Wash out 4 x with PSS Ready for experiment User manual 610M Chapter 6 37 38 In vitro experiment 1 Noradrenaline contractile response The purpose of the present protocol is to determine the sensitivity of rat mesenteric small arteries to the vasoconstrictor noradrenaline norepinephrine with a cumulative concentration response curve Background Noradrenaline norepinephrine causes contraction of mesenteric small arteries through activation of a adrenoceptors whereas noradrenaline activation of B adrenoceptors causes vasodilatation As the purpose is to determine the contraction sensitivity to noradrenaline the vasodilatory effect of noradrenaline is eliminated throughout the experiment by the constant presence of the B adrenoceptor antagonist propranolol Rat mesenteric arteries are densely innervated by sympathetic nerves which have a highly efficient reuptake mechanism that removes noradrenaline from the neuromuscular junction The reuptake mechanism will create a concentration gradient between the solution around the vessel segment and the receptors on the smooth muscle To correctly determine the sensitivity to noradrenaline it is necessary to eliminate this concentration gradient by performing the experiment in the presence of cocaine to block the noradrenaline reuptake To determine the sensitivity to noradrenaline the vessel seg
33. making it possible to calculate the force acting on the force transducer Leroi To g sind weigt FT fac m as 8 sind ong sind As the length of the pan arm is 2 cm the length of the transducer arm is 4 cm the weight is 2 g both angles are 90 and the acceleration of gravity is 9 81 ms the force acting on the force transducer is 2 0ms 28 9 51 ms Sin90 4 cm sin90 59 01 gramem s As 1N is equal 1 kg m s F is equal to F 7 9 81 mN Weight calibration procedure 1 Move the jaws pins apart in the myograph chambers Mount a wire on the trans ducer side of the myograph jaws in all chambers Place the units on the inter face plug in the cables and fill the chambers with double distilled water This is not required if the mounting support pins are used instead of the jaws 2 Turn on the heating in the Heat main menu of the Myo Interface The system will typically reach the target set temperature by default 37 C after 20 minutes 3 Place the calibration bridge balance and weight on myograph unit 1 as illustrat ed in fig 4 6 It is important that the calibration kit is pre warmed together with the myograph unit Make sure that the tip of the transducer arm on the balance is placed in the gap between the wire pin and the jaw as illustrated in fig 4 7 Carefully move the calibration bridge until the tip of the transducer arm is placed freely in the gap which means it does not touch eit
34. ment is exposed to increasing concentrations of noradrenaline Each concentration is applied until a steady response has been reached and then the next concentration is applied When the vessel segment is fully contracted or does not response more upon increasing the noradrenaline concentration the experiment is ended Protocol Prepare the following stock solutions e Noradrenaline 10 103 102M e Propranolol 10 M e Cocaine 10 M 1 Mount and normalise the vessels as described in Chapter 5 2 Perform a standard start as described at start of this chapter 3 Incubate the vessel segment in 1 uM propranolol add 5 uL of 10 Mto 5 mL PSS in chamber and 3 uM cocaine add 15 uL of 10 M to 5 mL PSS in chamber for at least 10 minutes 4 Add increasing concentrations of noradrenaline into the bath use Table 6 1 as a guideline Wait for a stable contractile response or a standard time such as 2 minutes between each application In vitro experiment 2 Acetylcholine relaxation curve The purpose of the present protocol is to determine the sensitivity of the endothelium dependent vasodilator acetylcholine in noradrenaline pre contracted rat mesenteric small arteries Background Acetylcholine causes relaxation of rat mesenteric small arteries by activating of mus carinic M3 receptors at the endothelial cell layer leading to release of endothelium derived relaxing factors DMT Multi wire myograph system Model 610M 03 J J
35. n the wire myograph Several types of eyepiece reticules are available for such a purpose The most simple and yet very useful type is a horizontal scale as illustrated in fig A6 1 amp 70 l 9 0 d m X 40 50 0 im Figure A6 1 Horizontal eyepiece reticule scale The basic principle is to use the eyepiece reticule typically consisting of 50 100 divi sions to measure the length of an object in terms of reticular divisions spanned by the object Having the conversion factor specific for the eyepiece reticule and magnifi cation used the length of the object in millimetres is easily calculated All reticules need to be calibrated in order to determine the conversion factor char acteristic for that specific eyepiece reticule and the magnification used For such purpose a stage micrometer is needed A stage micrometer is simply a microscope glass slide with a scale engraved on the surface A typical micrometer scale is 2 00 mm long engraved with divisions of 0 01 mm equalling 10 um per division However micrometer glass slides with less fine divisions are also useful for calibrating the ster eomicroscope to be used with the myograph Ocular calibration procedure 1 Decide which microscope magnification is to be used for the segment length measurements Use the largest possible fixed magnification where the eyepiece reticule scale still covers the whole gap of the myograph jaws 2 Place the stage micrometer on the
36. ng on sleeve 16000 um B One additional mark visible 500 um C Thimble reading 280 um Total reading 16780 um Figure A7 3 Example 2 reading 16780 um DMT Multi wire myograph system Model 610M Appendix 8 Normalization theory The importance of making a normalization before initiating an experiment with any tubular tissue segment is described in Chapter 6 In this appendix the mathematical rationale and calculations underlying the normalization procedure are described in detail Mathematical calculations Let X Y be the pair of values representing the micrometer reading and force read ing respectively characterising each step in the normalization procedure Y is the force reading at the start position of the normalization procedure where the wires are just separated and the force reading is approximately zero Then given that tension on the vessel is equal to force divided by wall length the wall tension at the i th mi crometer reading is calculated by ee MER 25 a a where is the microscope eyepiece reticule calibration factor in mm per division and a and a are the vessel end points when measuring the length of the mounted vessel segment The internal circumference of the mounted vessel at the i th reading is calculated by IC IC 2A AJ where IC is the internal circumference of the mounted vessel when the wires are just separated and is given by I 2x d where d is the wire diameter For 40
37. nsufficient to cover damage or loss of the myograph system In most cases an additional insurance coverage is needed DMT Multi wire myograph system Model 610M Appendix 4 Myograph accessories and spare parts This appendix contains a complete register of equipment needed to set up a Multi Wire Myograph System In addition the appendix contains a list of special Myograph 610M accessories and spare parts Besides the main focus on development and manufacturing DMT has specialised in offering our costumers first class laboratory equipment needed for a Multi Wire Myograph set up at very competitive prices Please contact DMT Sales Department for further product information and prices A4 1 General Myograph equipment This section contains a complete checklist of laboratory equipment needed when set ting up a Multi Wire Myograph system Dissection stereo microscope Including ocular micrometer and stage microm eter DMT recommends the Zeiss Stemi 2000 Stereo Microscope Mounting forceps DMT Item DF 3000 DMT recommends Dumont Medical No 5 tip 0 40 mm x 0 06 mm e Dissection scissors DMT Item ff DS 1000 DMT recommends Geuder G 19745 8 cm straight trabeculum Pipettes DMT recommends CappAero u pipettes Light source DMT recommends Schott Cold Light Source either Model KL 200 or Model KL 1500 LCD Water bath including heater DMT recommends Julabo 5 L open bath circulator with plexiglass bath tank Glass bottle
38. ntractile response The third stimulus is performed using a mixture of PSS and 10 uM noradrenaline to give a maximum pure agonist mediated a adrenoceptor contraction The fourth stimulus is performed using KPSS to give a depolarising contractile response this stimulus also includes a component from neurally released noradrenaline The final stimulus is performed using a mixture of PSS and 10 uM noradrenaline All solutions are preheated to 37 C and aerated with a mixture of 95 O and 5 CO before use Instructions for making the necessary solutions are described at the end of this chapter Repeat 1 x Stimulus 1 amp 2 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 3 Wash out PSS 10 uM NA 4x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 4 Wash out KPSS 4x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 5 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Ready for experiment DMT Multi wire myograph system Model 610M Endothelium function The reasons for checking endothelium function may include 1 To check whether the relaxing function of the endothelium is intact The procedure is performed to make sure that the endothelium is not damaged during the dissec tion or mounting procedure 2 If an experiment requires removal of the endothelium this procedure is useful to check whether the endothel
39. nveniently attached User manual 610M Chapter 2 13 14 to the stainless steel suction pipe The tube must be placed in the chamber in order to have the solution aerated described below Connect the main gas sup ply to the gas input tube on the rear panel of the interface The needle valves are used to regulate the level of bubbling to each chamber Each needle valve has a lock device attached Note To ensure longevity the needle valves should be turned regularly complete ly in and out a few times and greased to prevent them from becoming stiff use the linear grease provided with the system 3 Suction connection The system has an inbuilt aeration manifold with separate valves that allow each chamber to be drained individually After connecting your suction source to the interface the appropriate chamber will be drained by pressing the associated numeric button The suction pipes are inserted into the chambers by gently pull ing the pipe up turning it 90 counter clockwise and releasing it into the chamber fig 2 2 Note When draining the chambers using the automatic suction function continu ing to press the button for an additional 3 5 seconds after the chamber appears to be empty will ensure no leftovers from the previous solution are retained in the tubing and valves PE RATEN HMM e Funnel D e Pe e For drug application e m Mw enn e Temperature probe X Figure 2 2 Suction connection Figure 2 3 Chamber
40. of the wire in a clockwise direction under the far fixing screw on the right hand jaw fig 6 11B Figure 6 11 A and B Mounting step 6 Mounting step seven e Secure the far end of the wire under the near fixing screw on the right hand jaw Again the wire is passed clockwise around the screw stretching the wire as the screw is tightened fig 6 12A B Move the jaws apart to slightly stretch the ves sel segment Make sure that the vessel on the far side of the jaws does not extend beyond the jaws as even a small extension will 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 a forceps as described in mounting step four A better method for the skilled operator is to move the jaws slightly apart and use scissors to make a small slit in the vessel wall where the vessel is clamped Figure 6 12 A and B Mounting step 7 B DMT Multi wire myograph system Model 610M 5 3 Normalization The importance of normalizing the preparation is three fold 1 Experiments with elastic preparations like vessels can only have meaning if they are performed under conditions where the size is clearly defined 2 Clearly defined 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 dependen
41. oid pressing the jaws too hard together A movement of 20 um after they have touched is sufficient to hold the wires clamped Mounting step one e Cut lengths of 40 um wire 2 2cm long Mount one wire on left hand jaw of myograph as follows e Holding wire at far end place centre of wire between Jaws and screw jaws together so that the wire is clamped fig 6 6A Note do not close the jaws too hard against each other See above e Bend the far end of the wire towards the left and wrap it around under fixing screw so the wire is wound clockwise tightening the screw will then tighten the wire This procedure should result in the wire being clamped between the jaws and with near end of wire pointing towards operator fig 6 6B C e Fill the myograph chamber with PSS at room tem perature UJ Figure 6 6 A Band C Mounting step 1 User manual 610M Chapter 5 31 Mounting step two e Using forceps to hold the handle segment transfer excised vessel from Petri dish to myograph chamber Hold the vessel as close to the proximal end as possi ble and try to mount the vessel onto the wire If the lumen is shut try one of the following possibilities e Use the wire to gently push the lumen open blood streaming out is a good sign e 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 e Pull the prox
42. ption of the mathematical rationale and cal culations of the normalization procedure ma DEM IR lem NB o o WN Force mN mm Wall Tension T mN mm 100 200 300 ao 500 600 700 800 900 Internal Circumference gum Time min Figure 6 13 Illustration of the stepwise Figure 6 14 Illustration of the expo normalization procedure nential curve fitting and determina tion of IC oo User manual 610M Chapter 5 35 Chapter 6 Experimental protocols Standard start The purpose of performing a standard start is to 1 Re activate the mechanical and functional properties of the vessel segment 2 Check that responses to different types of stimuli are normal in appearance and thereby ensuring that the functionality of the vessel segment has not been dam aged during the dissection or mounting procedures 3 Ensure that the tension development gives an effective active pressure that is above the chosen accepted value usually 13 3 kPa 100 mmHg The standard start is performed after the vessel segment has been heated equili brated and normalised The present procedure is suitable for rat mesenteric arter ies Another procedure may be needed for other animal species and tissue or vessel types Principles of the standard start procedure The standard start procedure consists of a series of five stimuli and washout periods The first two stimuli are performed using a mixture of KPSS and 10 uM noradrena line to give a maximum co
43. r to section 4 3 6 Carefully remove the weight balance and calibration bridge from myograph unit 1 and move to myograph unit 2 Repeat steps 4 6 for force transducer 2 3 and 4 Figure 4 7 Illustration of how to fit the balance between the wire and the gap in the support User manual 610M Chapter 4 23 4 3 Checking force transducer The myograph force transducer is a strain gauge connected in a Wheatstone bridge The force transducer for each myograph unit is located in a separate compartment transducer house see Chapter 1 3 While this provides some mechanical protection the force transducers are 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 If the force reading continues to be unstable in spite of a recent weight calibration then repeat the weight calibration and note down the relative force reading values shown in the Calibration menu on the Myo Interface e f the value is O or above 6500 then the force transducer is broken and needs to be changed e f the value is between 1 499 or 3001 6250 then contact DMT for further instructions Important If at any time the message OFF is displayed in the Force menu on the Myo Inter face this indicates that the force transducer is broken and must be replaced In this instance or in case of other problems related to the force transducer plea
44. ransducers or other parts Please contact DMT for information about prices User manual 610M Appendix 2 43 44 Appendix 3 Shipping instructions If the myograph system needs to be sending back for service or repair please read the following shipping instructions very carefully Before you start packing the myograph system please remember that you are dealing with very delicate equipment and therefore care must be taken DMT recommends that each part of the myograph system be wrapped individually i e with bubble wrap and placed together in a large box preferable the box you once received the Myo graph system in Place the wrapped items in the middle of the box and fill out the surroundings with chips of expanded polystyrene Important Ensure before closing the box that none of the enclosed items are loose as transport by road or air from time to time can be quite rough Address the box to DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Make sure that all four sides of the box are marked fragile or similar Make an indi cation on the top of the box that it contains goods returned for repair service Customers outside the EC must further enclose a pro forma invoice stating that the box contains goods being returned for repair or service If arranging transportation through a courier please keep in mind the high value of the myograph system and that a standard insurance provided by the courier in most cases is i
45. re 4 9 Transducer pin hole to be sealed up with high vacuum grease red ar rows indicate points where grease should be checked and replaced User manual 610M Chapter 4 25 Important e DMT recommends with frequent use of the myograph that the high vacuum grease sealing up the transducer hole is checked and if necessary replaced once a week e DMT takes no responsibilities for the use of any other kinds of high vacuum grease than the one available from DMT e DMT takes no responsibilities for any kind of damage applied to the force trans ducer Linear slides Check the linear slides under the black covers for grease at least once a week In case of insufficient lubrication grease the slides with the Grease for Linear Slides enclosed with the myograph Apply the linear slide grease at the places marked by the arrows in fig 4 10 Figure 4 10 Greasing points on the linear slides Myograph cleaning DMT strongly recommends that the myograph chamber and surroundings are cleaned after each experiment At the end of the experiment use the following procedure to clean the myograph chamber and supports 1 Fill the myograph chamber to the edge with an 8 acetic acid solution and allow it to work 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 supports several times with double distilled
46. re Myograph System Model 610M is a delicate and sophisticated piece of research equipment DMT recommend that the following sections are read carefully and that the instructions are followed at all times Myograph chamber pipes To prevent the pipes from being blocked by buffer salt deposits after an experiment remove the chamber cover from the myograph and turn on the vacuum pump and vacuum valve for about 10 seconds continuously press the valve button down Turn off the vacuum pump and then the oxygen supply Remove any buffer remaining on the outside of the pipes with absorbant paper Force transducer The force transducer is the most delicate and fragile component of the myograph system and it should therefore be handled with the utmost care One of the jaws in each myograph is connected to the transducer pin The transducer pin is attached to the myograph mounting supports and is located outside of the chamber as illustrated in fig 4 9 Despite there being no direct contact to the salt solution in the chamber there is a risk for evaporating solution to deposit calcium in the pinhole The hole running into the transducer house the hole is therefore filled with high vacuum grease As a part of daily maintenance it is very important to inspect the greasing of the trans ducer hole before starting any experiment Insufficient greasing permits salts and fluid to enter thereby causing damage and malfunction of the force transducer Figu
47. relaxation curve eese 38 Appendix 1 Terms of warranty 1 ee eeee eee nena nnn nnu nana 42 Appendix 2 Service check eeeeeeee eere eene enean nnn anna aun 43 Appendix 3 Shipping instructions ecce e eere nnn AA Appendix 4 Myograph accessories and spare parts 45 A4 1 General myograph equipment 11 eese ener nnne nnn nnne 45 A4 2 Myograph 610M system accessories eese eee ener nn nente 46 A5 3 Myograph 610M system spare parts eeeeseeeeeeeeee nennen nnne nnnm nnns 46 Appendix 5 Fuse replacement 2 2202200n00nn00n0nnnunnunnnnnnnunnnnn nun nnnn nun 47 Appendix 6 Calibration of ocular reticule eese 48 Appendix 7 How to read a millimeter micrometer 50 Appendix 8 Normalization theory 220 2200200n00nn0nnnnnnunn nun nun n nun nun 51 Appendix 9 System specifications eene ener 53 DMT Multi wire myograph system Model 610M About this manual This manual contains a complete list of procedures describing how to install main tain and get started using the multi wire myograph system model 610M version 2 2 Chapter 1 provides an overview of the construction and basic fea
48. se contact DMT for further instruction and advice 4 4 Force transducer replacement If the force transducer is broken and needs to be changed please follow this step by step replacement procedure carefully 1 Disconnect the myograph unit from the Myo Interface grey cable 2 Turn the myograph unit upside down and remove the bottom plate by loosen ing the two screws A B as illustrated in fig 4 8 Carefully disconnect the force transducer plug and remove the old transducer Note how the plug is connected to the old force transducer to prevent incorrect connection of the new force transducer 3 Remove any remaining grease from the transducer pin left inside of the trans Figure 4 8 The two screws holding the transducer house in place DMT Multi wire myograph system Model 610M ducer compartment of the myograph unit Also clean the hole leading from the transducer compartment to the myograph chamber 4 Plug the replacement transducer in Replace the bottom plate and tighten the two Allen screws 6 Place some high vacuum grease Supplied with the system around the transduc er pin in the myograph chamber see fig 4 9 Make sure that the hole is com pletely sealed so that absolutely no buffer solution is able to enter the transducer compartment and damage the force transducer Ol Important The new force transducer must be weight calibrated prior to running an experiment 4 5 Myograph maintenance The Multi Wi
49. systems are linked together In order to use this feature an external or internal signal converter that is half duplex and tog gles RTS signals are required The converter if external must be placed between the computer and the first myograph Note For further information please contact DMT Linking multiple systems Connect a serial cable to the computer and to the signal convert er Connect a second serial cable to the other end of the signal converter and the other end of the cable to the RS485 plug on the myograph system that will be 1 Connect a serial cable to the other RS485 plug on the interface Other end of cable must be connected to system 2 Repeat this if 3 or 4 systems are linked together Important When using the serial connection the systems must always be linked as follows system 1 to 2 2 to 3 and 3 to 4 Valve delay 1sec Force Range 200mN gt Serial port RS232 gt Serial port RS485 gt Serial port RS485 Myograph no 1 User manual 610M Chapter 3 17 18 When all systems are linked together in the above order enter submenu 10 Press to change serial port to RS485 Use F1 to move the active line marker gt down and select the system number equivalent to the way in which the serial cables have been connected Exit the serial port menu by pressing f DMT Multi wire myograph system Model 610M Chapter 4 The multi wire myograph unit This chapter contains a comple
50. t 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 61326 2 6 2005 EN 61000 3 2 Certified with the safety standards Directive 2006 95 EC EN 61010 1 2001 EN 61010 1 Corr 1 2003 EN 61010 1 Corr 1 2003 EN 61010 2 101 2003 DMT Multi wire myograph system Model 610M Certificate of Conformity DMT A S Skejbyparken 152 8200 Aarhus N Denmark hereby declares its responsibility that the following product Multi Wire Myograph System Model 610M Version 2 2 is covered by this certificate and marked with CE label conforms with the following s
51. t on the extent of stretch which makes it important to set the preparation to an internal circumference giving maximal response The aim of the normalization procedure is to stretch the segment to a so called normalized internal circumference IC defined as a set fraction of the internal cir cumference IC that a fully relaxed segment would have at a specified transmural pressure For small rat arteries the target transmural pressure is typically LOO mmHg 13 3 kPa Principles of the normalization procedure In practice the normalization is performed by distending the segment stepwise and measuring sets of micrometer and force readings fig 6 13 step 1 4 These data are converted into values of internal circumference um and wall tension T mN mm respectively Plotting wall tension against internal circumference reveals an exponential curve and by applying the isobar curve corresponding to 100 mmHg IC oo is calculated from the point of intersection using the Laplace relation fig 6 14 IC is calculated from IC oo by multiplying a factor giving an internal circumference at which the active force production as well as the sensitivity to agonists of the segment is maximal For rat mesenteric arteries the factor is O 9 but both this factor as well as the transmural pressure has to be optimized for each particular segment The normalized internal diameter is calculated by dividing IC with m Appendix 8 contains a complete descri
52. tandards EN 61010 1 2001 Safety requirements for electrical equipment for EN61010 1 Corr 1 2003 measurement control and laboratory use EN 61010 1 Corr 1 2003 Part 1 General requirements EN 61010 2 101 2003 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 101 Particular requirements for in vitro diagnostic IVD medical equipment EN 61326 2 6 2005 Electrical equipment for measurement control and laboratory use EMC requirements Part 2 6 Particular requirements In vitro diagnostic IVD medical equipment With reference to regulations in the following direc tives 2006 95 EC 89 336 EEC User manual 610M Certificate of Conformity Contents ACS OE KS une ent anne greta aa aa REA a aaa RRES 3 sea oe act A EENORGETER 5 ENG EN anne en anne HEFIN EE RHAN FFF SF FFF 6 ABDFOVAIIS PEIEE S HN rer 6 Certificate of Conformity 22 2000200n200nnnnnnnnnnnnnnnnnnnnnn nun nun nun nun nnnnnnEnn 7 About this MANUAL ni enianesan nungen YNN ER nn ERE ERE RES MUNERE SEN SN YR 9 Unpacking the myograph system 1 eco eere rne enn nun nun nun 10 Chapter 1 System overview 1er eee nun nun nn nun ALAR RE nnau 11 T Myo Interf ce front pane ee een 11 1 2 Myo Interface rear Panel een dU US RY 11 1 3 Multi wire myograph unit ee NN ee naar TE PWT HYF 12 Chapter 2 Setting Up Rena 13
53. tant that you per form this on vessels other than those you will use as this procedure damages the vessels 8 Dissect away the vein using scissors to cut the adipose and connective tissue between the artery and vein One method is to cut the vein in one position and af terwards gently to pull the vein away from the artery In this way a fine membrane of connective tissue becomes visible between the adipose tissue and the artery Carefully cut the fine membrane to remove the vein and adipose tissue while avoiding any direct contact between the scissor and artery fig 6 4 A B Figure 6 4 Removal of vein Figure 6 5 A B and C Cutting free the artery to be studied DMT Multi wire myograph system Model 610M 9 Clean the artery by removing any remaining adipose or connective tissue Gently pull away adipose or connective tissue to make the connective tissue membrane become visible Cut the membrane to remove the tissue 10 Cut the distal end of the artery section to be investigated Afterwards cut the proximal end while ensuring that the vessel segment has the correct length fig 6 5A C 5 2 Mounting protocol for small arteries The procedure involves attaching the mounting wires to jaws which are in turn mount ed on the force transducer This force transducer is capable of measuring with a sensitivity of about 0 01 mN 1 mg but can be damaged if the applied force exceeds about 1 N 100 g Therefore care must be taken to av
54. te explanation of how to adjust calibrate and maintain the myograph 610M units to ensure the equipment performs to the highest standard 4 1 Change and adjustment of mounting supports The chamber units can accommodate mounting supports for either small vessels gt 80 um or larger segments gt 500 um As the mounting supports are readily changed it is easy to perform experiments with different vessel sizes Furthermore the mounted supports will require adjustment with time due to continuous use of the myograph system and repeated greasing of the transducer pinhole 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 sup ports Changing supports 1 Screw the supports as far apart as possible with the micrometer 2 Using the small screwdriver provided gently loosen screw A fig 4 1 on the sup port attached to the transducer side Avoid exerting any force on the screw or transducer pin Gently pull the support away from the transducer pin Loosen screw B fig 4 1 on the micrometer side 5 Pull the support away Note store the removed supports for each unit together and a number indicating which unit they came from this will save time adjusting the supports when they are mounted again T gt W CB A Figure 4 1 Myograph unit screws for changing supports and coarse adjustment Coarse horizontal and vertical ad
55. tures of the Myo In terface and the multi wire myograph unit Chapter 2 describes step by step how to set up a complete 610M wire myograph system including accessories Chapter 3 is a complete manual to the 610M Myo Interface version 2 2 The chapter contains a step by step description of how to navigate in the menus and how to use the special features of a 610M myograph Chapter 4 contains procedures describing general as well as daily maintenance of the myograph unit e g adjustment of supports weight calibration of the force trans ducer and cleaning instructions Chapter 5 describes how to get started using the wire myograph system This in cludes a complete dissection and mounting procedure for small mesenteric arteries and a few basic pharmacological experiments Appendices contain additional information about normalization theory ocular calibra tion myograph service shipping instructions system specifications equipment lists accessories and spare parts and fuse replacement User manual 610M About this manual 10 Unpacking the myograph system Please take a few minutes to carefully inspect your new Multi Wire Myograph System for damage which may have occurred during handling and shipping If you suspect any kind of damage please contact DMT 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 recommen
56. ven by our global customer base our number one goal is to develop and manufacture first class research equipment within the fields of physiology and pharmacology DMT Multi wire myograph system Model 610M Safety The 610M multi wire 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 e 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 heat 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
57. vide the force signal in volts for connection to an exter Force 2 OUT nal data acquisition system By default the interface is set at 20 dub mN where 20 mN corresponds to 1 V As the force value will vary according to the maximum response from the preparation used the maximum force value can be changed by firstly making the channel of interest active and then increasing or decreasing the value using the or buttons Irrespective of which maximum force value is entered the analog output is fixed to 1 V at max Calibration menu The calibration menu permits calibration of the transducers using mm the calibration kit provided with the 610M system The F1 but E ton changes which transducer is active in the list Slct stands for Calibrate 4 select Pressing the F2 button will begin the calibration process for the force transducer selected indicated by Calibration of the transducers is described in full in Chapter x Setup menu The Setup menu provides access to submenus which are used to change the gain setting serial port connection and link if multi ple systems are connected To enter the desired submenu use the or buttons and when the appropriate number is shown the submenu is activated by pressing F2 Whenever any changes have been made one must return to the Setup menu using 7 for the changes to take effect DMT Multi wire myograph system Model 610M Valve delay submenu The valve delay
58. 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 supports with 96 ethanol or a weak detergent solution 4 To remove more resistant or toxic chemicals incubate the myograph chamber and supports with 1 M HCI for up to 1 hour In exceptional cases incubate the chamber and supports with an up to 3 M HNO solution for about 15 minutes 5 Wash the myograph chamber and supports several times with double distilled water DMT Multi wire myograph system Model 610M Important Notes Be very careful using step 3 and 4 repeatedly as strong reagents may damage the myograph unit e After cleaning ALWAYS check that the greasing around the transducer pin is suf ficient to keep the buffer solution away from the transducer compartment In cases of red or brown discolorations appearing on the chamber sides or on the supports the following cleaning procedure will work in most cases 1 Incubate the myograph chamber and supports for 30 minutes with 20 ul of a 2 mM T 1210 Tetrakis 2 pyridylmethyl ethylenediamine solution dissolved in double distilled water 2 Use a cotton swab stick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period 3 Wash the myograph chamber and supports several times with double distilled water 4 Incubate the myograph chamber with 96 ethanol for 10 minutes while continu
59. xpose the mesenteric bed 2 Use scissors to remove about 10 cm of intestine together with its feeding vascu lature including part of the superior mesenteric artery Be careful not to damage the vasculature during this procedure The proximal end of the intestine section must be about 10 cm from pylorus Make a cut in the proximal end of the intes tine for later identification 3 Place the excised intestine section in a Petri dish about 9 cm in diameter coated with a 5 mm thick layer of Sylgard at the bottom to hold the fixing pins Immedi ately fill the Petri dish with cold PSS which has been well aerated with carbogen The dissection is performed without further oxygenation of the PSS The cold tem perature ensures the artery is relaxed during the mounting procedure 4 Pin down the proximal end of the intestine section on the left hand side of the Petri dish without stretching the vessels Pin down the remaining of the intestine section in an anti clockwise direction In this configuration proximal end at the left side distal end at the right side and running anti clockwise from proximal to distal side the feeding vasculature is on the far side of the intestine and the veins are usually uppermost 5 Select the vessel segment to be investigated fig 6 1 First time myograph users are recommended to start dissecting and mounting vessel segments from the first or second branch from the superior mesenteric artery internal diameter 2
60. y month DMT also recommends that the myograph is weight calibrated every time the system has been moved or has not been used for a long period of time Principles of weight calibration Weight calibrating the force transducer is based on simple physics the net torque acting on a balance when applying a certain amount of weight The magnitude of the torque T about a point of rotation P is defined by a tT r F sing where r is the distance from the point of rotation to the point on the object where the force F is acting with the angle of 8 Applying the physics to the weight calibration setup is illustrated in Fig 4 5 Oen Transducer Arm TA Figure 4 5 Theoretical principle of the weight calibration Applying the weight on the pan arm creates a net torque acting at the center of grav ity resulting in a force F acting on the force transducer The following two equations describe the forces working in the weight calibration system User manual 610M Chapter 4 21 22 i eT m ser 8 SING Zy 21 6 SING where r is the length of the pan arm F is the force acting on the pan arm when applying the weight F is equal acceleration of gravity times the mass of the weight r is the length of the transducer arm and F is the force acting on the force trans ducer The net torque acting at center of gravity is constant for the weight calibration setup which makes equation 1 and 2 equal

User Manual

Contents

Download Pdf Manuals

Related Search

Related Contents