User Guide 420A
Contents
1. na 14 Cleaning Me Dual Wire WIV ORT OD ssa Edo Paus chua ehem dran cimus ducc ductum daas ac psu SGH aab duca A AD ad cta su ERU REB P 14 4 2 Maintenance of the force transducer CUR RU ua Ri Reach cR kr ew ds QD Reine 15 LN SU RETE T I ee 18 2 2 2 Force transducer nuc DEN Rune 16 4 3 Changing the Dual Wire Myograph window 2 ena aua Rua auk ua sua E cidunt 17 4 4 Maintenance of the linear 511966 RS XH URDU EEG Pica Eo PRICE EN ERR Rt ce iR e 17 Appendix 1 Buffer FeCIDOeS cessa remm a Exe Cuni Ea Ex ES Ein KEA ENEAN 18 Appendix 2 Normalization theory ioskxccenkxuEEXETREEMUEN EUER SERYXRDEENEAKEESKNMENMUP NGRKNKESKNUON EUR FEEKERNXEKBNRUREVEXEFEG MSN RUN REUNKODEEUANKFD YR nnmnnn 20 Appendix Reading millimetre icri e pnr iex nob i c all c cR c rer n 22 NOTOS eR TP P 23 2 WIRE MYOGRAPH SYSTEM 420A USER GUIDE CHAPTER 1 DUAL WIRE MYOGRAPH OVERVIEW Port for connection to Wire Interface Allen screws for fine alignment of the e Myograph jaws 4 on each side T2. 0229 M DAI e Hener ae Micrometer d rio 06 EH dI Ln Bal Myograph jaw connected 12 11 CJ Myograph jaw connected to force transducer UM re 49 Window at the bottom of the Myograph chamber for imaging under the wire jaws Myograph chamber separator Figure 1 1 Dual Wire Myograph with close up of the chamber CHAPTER 1 3 CHAPTER 2 SETTING UP THE DUAL WIRE 2 1 Adjustment of supports A successful mounting of any kind of tubular tissue segment in the Dual Wire Myograph is to a high extent dependent on perfectly matching supports The supports are matched prior to the shipment but daily use of the Dual Wire Myograph and greasing of the transducer pinhole will over time create a need for an adjustment of the supports NOTE THE TRANSDUCERS ARE FRAGILE AND SENSITIVE TO MECHANICAL STRAIN BE VERY CAUTIOUS NOT TO PUT STRAIN ON THE TRANSDUCER WHEN CHANGING OR ADJUSTING THE MOUNTING SUPPORTS IN ADDITION VERY LITTLE FORCE SHOULD BE APPLIED TO THE SCREWS IN ORDER TO AVOID BREAKING THE THREADS Adjustment of the supports is performed using the following step by step procedure The procedure is illustrated in figure 2 1 1 Carefully loosen screw D on the top of the support connected to the force transducer Align the horizontal Support and carefully tighten the screw again Loosen screw E on the top of the support connec
3. Carefully disconnect the plug on the force transducer 5 Loosen and remove the two Allen screws and washers as illustrated in figure 4 2 and carefully remove the old trans ducer 6 Remove any remaining grease from the transducer pin left inside of the transducer compartment of the Dual Wire Myo graph Clean the hole leading from the transducer compartment to the Dual Wire Myograph chamber f Gently place the new force transducer into position Place the Allen screws and washers in their positions ready to be tight ened 8 Before tightening the Allen screws be sure that the transducer pin is placed directly in the center of the chamber pinhole when viewing from the side of the Dual Wire Myograph Figure 4 2 Illustration of how to replace the force transducer 16 WIRE MYOGRAPH SYSTEM 420A USER GUIDE 9 Tighten the two Allen screws and replace the bottom plate Tighten the four screws on the bottom plate 10 Place some high vacuum grease around the transducer pin in the chamber Make sure that the hole is completely sealed so absolutely no buffer solution is able to enter the transducer compartment which will damage the force transducer 11 Mount the support on the transducer pin and adjust the supports as described in chapter 2 1 IMPORTANT BEFORE MAKING ANY EXPERIMENT ON THE DUAL WIRE MYOGRAPH REMEMBER TO PERFORM A NEW FORCE CALIBRATION 4 3 Changing the Dual Wire Myograph window glass The glass in the Dua
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5. Figure 4 3 Greasing points on the linear slides CHAPTER4 17 APPENDIX 1 BUFFER RECIPES Physiological Saline Solution PSS 1x PSS Chemical Mol Wt mM g 0 5L gL g 2L g 4L 58 45 130 3 799 7 598 15 20 30 39 KCI 74 557 4 7 0 175 0 35 0 70 1 40 KH PO 136 09 1 18 0 08 0 16 0 64 MgSO 7H O 246 498 1 17 0 145 0 29 0 58 1 16 NaHCO 84 01 14 9 0 625 1 25 2 50 5 00 Glucose 180 16 5 5 0 5 1 00 2 00 4 00 EDTA 380 0 026 0 005 0 01 0 02 0 04 CaCl 110 99 1 16 O 8mL 1 6mL 3 2 ML 6 4mL 1 0 M solution 1 Make a 1 0M solution of CaCl 110 99 in double distilled H O Filter sterilize the calcium solution through a 0 22 um filter The sterilized solution can be stored in the refrigerator for up to 3 months 2 Dissolve all the chemicals except the CaCl in approximately 80 of the desired final volume of double distilled H O while being constantly stirred For example if 1 litre of PSS is to be made then dissolve all the chemicals in 800mL of double distilled H O 3 Add the appropriate volume of 1 0M CaCl for the total volume of PSS being made for example 1 6mL of 1 0M CaCl for 1 litre of buffer Continue to stir the PSS while the CaCl is being added 4 Bring the solution up to the final volume with double distilled H O Continue to stir the solution until the EDTA is fully dissolved This takes about 15 minutes at room temperature 5 Aerate the solution with carbogen 95 5 for about 20 mi
6. Myograph is a very delicate and sophisticated piece of research equipment DMT recommends that the following sections are read carefully and that the instructions are followed at all times As a part of the general maintenance of the Dual Wire Myograph DMT recommends that the Dual Wire Myograph is force cali brated at least once a month The Dual Wire Myograph should also be force calibrated every time the Wire Interface has been moved Although lab benches are all Supposedly perfectly horizontal small differences in lab bench pitch can affect the calibra tion of the system The Dual Wire Myograph also should be calibrated if the system has been idle for longer than a month A step by step procedure is explained in chapter 3 7 1 in Wire Myograph System User Manual 4 1 Cleaning the Dual Wire Myograph DMT STRONGLY RECOMMENDS THAT THE DUAL WIRE MYOGRAPH AND SURROUNDINGS WILL BE CLEANED AFTER EACH EXPERIMENT PLEASE REMEMBER TO TAKE OUT THE CHAMBER DIVIDER 1 Fill the chamber to the edge with an 8 acetic acid solution and allow it to stand for a few minutes to dissolve calcium de posits and other salt build up Use a swab stick to mechanically clean all the surfaces of the Dual Wire Myograph 2 Remove the acetic acid and wash the chamber and supports several times with double distilled water 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 supp
7. NT DMT RECOMMENDS USE OF THE HIGH VACUUM GREASE ONCE A WEEK TO SEAL UP THE TRANSDUCER HOLE BY FRE QUENTLY USE 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 TRANSDUCER Figure 4 1 Transducer pin hole to be sealed up with high vacuum grease seen in the transducer house left and inside the chamber right 4 2 1 Checking force transducer The force transducer is a strain gauge connected in a Wheatstone bridge The force transducers are placed in separate compartment The separate compartments provide some mechanical protection for the force transducer but the transducers are still very vulnerable to applied forces exceeding 1 newton 100 g or fluid running into the transducer compartment due to insufficient greasing of the transducer pinhole If the force reading on the Wire Interface appears unstable then first check that the Wire Interface and the Dual Wire Myograph are properly connected through the 25 pin grey cable If the force reading still appears unstable then perform a new force cali bration of the force transducer During the force calibration monitor the relative force reading values in the Calibration Menu on the Wire Interface The normal operating values for the force transducer during calibration should be between 3000 and 3500 e lf the value is O a single digit or a three
8. Stimulus 1 amp 2 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus Wash out PSS 10 uM 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 4 Wash out KPSS 4 x 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 3 4 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 en dothelium is not damaged during the dissection or mounting procedure 2 f an experiment requires removal of the endothelium this procedure is useful to check whether the endothelial cells were successfully removed The procedure can be performed after the vessel segment has been heated equilibrated and normalized Preferably the proce dure 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 3 4 1 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 en
9. apart While controlling the movement of the wire with the forceps use the other forceps to gently pull the vessel segment along the wire until the area of interest is situated in the gap between the jaws The near end of the vessel segment shall lie about 0 1 mm inside the jaw gap to insure no point of contact figure 3 3 A Still controlling the free wire end with the forceps move the jaws together to clamp the wire and in one movement secure the wire under the near fixing screw on the left hand jaw Again in a clockwise direction so that tightening the screw also tightens the wire figure 3 3 B Figure 3 3 A and Mounting step 3 CHAPTERS 7 3 1 4 Mounting step four e 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 figure 3 4 A Make sure that the vessel segment is Separated as close as possible to the jaws figure 3 4 B The excessive vessel segment is finally dissected free and removed from the Dual Wire Myograph chamber figure 3 4 C ba 94 94 Figure 3 4 B and Mounting step 4 3 1 5 Mounting step five e Move the jaws apart figure 3 5 A Take a second wire holding it about one third down from the far end using forceps Align the wire parallel with the vessel segment such that the wire can be passed into the far end of the lumen Gently feed the wire through the lumen of the vessel s
10. by IC IC ot 1 0 2 APPENDIX2 21 APPENDIX 3 READING A MILLIMETRE MICROMETER Sleeve scale Thimble scale Figure A3 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 50 equal parts Each part of a division above the horizontal line represents 1 mm where each 5th line is marked by a longer line and a number designating the length in mm Each 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 5O equal parts and one complete rotation of the thimble is indicated by the smallest division on the sleeve which equals 0 5 mm Each division on the thimble scale is 10 um If the thimble scale falls between two lines then 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 indicating 10000 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 horizontal line on the sleeve A Reading on sleeve 10000 um B Noadditional mark visible um C Thimble reading 380 um Total reading 10380 Figure A2 2 Example 1 reading 10380 um Example 2 1 Note that the thimble has stopped at a point beyon
11. ciples of the normalization procedure In practice the normalization is performed by distending the segment stepwise and measuring sets of micrometer and force read ings figure 3 8 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 5 calculated from the point of intersection using the Laplace relation figure 3 9 IC is calculated from IC 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 Appendix 2 contains a complete description of the mathematical rationale and calculations of the normalization procedure Force mN mm Wal Tension mN mm Time min d a lee PIER oed ub T Figure 3 8 lllustration of the stepwise Figure 3 9 Illustration of the exponential normalization procedure curve fitting and determination of IC po 3 3 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 differ
12. d 16 on the sleeve indicating 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 horizontal line on the sleeve A Reading on sleeve 16000 um B One additional mark visible 500 um C Thimble reading 280 um Total reading 16780 ym Figure A2 3 Example 2 reading 16780 um 22 WIRE MYOGRAPH SYSTEM 420A USER GUIDE NOTES NOTES 23 DMT A S Skejby Science Center Skejbyparken 152 DK 8200 Aarhus N Tel 45 87 41 11 00 Fax 45 87 41 11 01 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 6021 5425 1330 Fax 86 0 21 5877 0063 www dmt asia com sales dmt asia com supportedmt asia com DMT USA Inc 525 Avis Drive Suite 10 Ann Arbor MI 48108 USA Tel 1 770 612 8014 Fax 1 678 302 7013 www dmt usa com sales dmt usa com support dmt usa com
13. d surfaces during the last 15 minutes of the incubation period 3 Wash the chamber and supports several times with double distilled water 4 ncubate the chamber with 9690 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 chamber and supports with an 8 acetic acid solution for 10 minutes and continue the mechanical cleaning with a swab stick 6 Wash the chamber and supports several times with double distilled water IMPORTANT IN EXCEPTIONAL CASES IT MAY BE NECESSARY TO UNMOUNT THE SUPPORTS AND CLEAN THEM AND THE CHAMBER SEPARATELY TO ENSURE THAT ALL SURFACES ARE CLEANED 14 WIRE MYOGRAPH SYSTEM 420A USER GUIDE 4 2 Maintenance of force transducer The force transducer is the most delicate and fragile component of the Dual Wire Myograph Careful handling is very important Two of the jaws is connected to the transducer pins on each side in the chamber The transducer pins enters the chamber through a pinhole in the chamber wall located below the surface level of the buffer See figure 4 1 To prevent the buffer from running into the transducer 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 will cause damage and malfunction of the force transducer IMPORTA
14. digit number the force transducer is broken and needs to be replaced e the value is less than 2000 or greater than 4500 the force transducer is broken and needs to be replaced e the force reading s appear yellow cannot be reset to zero or the transducer cannot be recalibrated the force transducer is broken and needs to be replaced If any other problems related to the force transducer are encountered please contact DMT for advice or further instructions IMPORTANT IF THE MESSAGE OFF IS DISPLAYED IN EITHER OF THE FORCE READING LINES IN THE MAIN MENU ON THE WIRE 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 CHAPTER 4 15 4 2 2 Force transducer replacement In case that the force transducer is broken and needs to be changed please follow this step by step replacement procedure carefully 1 Disconnect the Dual Wire Myograph from the Wire Interface grey cable 2 Carefully remove the support connected to the transducer pin by loosening the screw D on top of the support as illustrated in figure 2 1 3 Turn the Dual Wire Myograph up side down and remove the bottom plate by loosening the four screws A as illustrated in figure 4 2 IMPORTANT NOTICE HOW THE PLUG IS CONNECTED TO THE FORCE TRANSDUCER SO NO MISTAKE IS MADE WHEN CONNECTING THE PLUG TO THE NEW FORCE TRANSDUCER 4
15. dothelium is undamaged by the dissection and mounting procedures then a substantial relaxation will occur With complete removal or damaged endothelium a partial relaxa tion or no relaxation to acetylcholine is observed Itis 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 CHAPTER 11 3 5 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 3 5 1 Background Noradrenaline norepinephrine causes contraction of mesenteric small arteries through activation of o 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 d
16. e 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 3 7 A and B Mounting step 7 3 2 Normalization The importance of normalizing the preparation is three fold 1 Experiments with elastic preparations like vessels can only have meaning if they are performed under conditions where the size is clearly 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 The active response of a preparation is dependent on the extent of stretch which makes it important to set the preparation to an internal circumference giving maximal response CHAPTER3 9 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 circumference that a fully relaxed segment would have at a specified transmural pressure For small rat arteries the target transmural pressure is typically 100 mmHg 13 3 kPa 3 2 1 Prin
17. egment in one movement using the first mounted wire as a guide figure 3 5 B C Hold the wire at a point at least 10 mm from the vessel to prevent the vessel being stretched during the manoeuvre Be careful not to touch the lumen of the vessel with the end of the wire and when pushing the wire end through the near end of the lumen Once the wire has successfully passed through the lumen of the vessel segment place the wire in a position which ensures sufficient length for the wire to be secured both at the near and far fixing screws on the right hand jaw Figure 3 5 Mounting step 5 8 WIRE MYOGRAPH SYSTEM 420 USER GUIDE 3 1 6 Mounting step six e Carefully move the jaws together while ensuring that the second mounted wire lies underneath the first one secured on the left hand jaw figure 3 6 A The procedure clamps the second wire to prevent it from damaging the vessel segment when securing the wire to the right hand jaw connected to the transducer Secure the near end of the wire in a clockwise direc tion under the far fixing screw on the right hand jaw figure 3 6 B Figure 3 6 A and B Mounting step 6 3 1 7 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 figure 3 7 A B Move the jaws apart to slightly stretch the vessel seg ment Make sure that th
18. ensely 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 segment 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 3 5 2 Protocol Prepare the following stock solutions Noradrenaline 105 103 10 M Propranolol 10 M Cocaine 10 M 1 Mount and normalize the vessels as described in chapter 3 1 and 3 2 2 Perform a standard start as described in chapter 3 3 3 Incubate the vessel segment in 1 uM propranolol add 5 uL of 103 M to 5 mL PSS in chamber and 3 uM cocaine 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 the table below as a guideline Wait for a stable contractile response or a sta
19. ent types of stimuli are normal in appearance and thereby ensuring that the functionality of the vessel segment has not been damaged 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 equilibrated and normalized The present procedure is suitable for rat mesenteric arteries Another procedure may be needed for other animal species and tissue or vessel types 3 3 1 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 noradrenaline to give a maximum contractile response The third stimulus is performed using a mixture of PSS and 10 uM noradrenaline to give a maximum pure agonist mediated o adrenoceptor contraction The fourth stimulus is performed using KPSS to give a depolarising contractile response this stimulus also includes a component from neu rally 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 9576 O and 5 CO before use Instructions for making the necessary solutions are described in appendix 1 10 WIRE MYOGRAPH SYSTEM 420A USER GUIDE Repeat 1 x
20. fixing screw so the wire is wound clockwise tighten ing the screw will then tighten the wire This procedure should result in the wire being clamped between the jaws and with near end of wire pointing towards operator figure 3 1 B C e Fill the Dual Wire Myograph chamber with PSS at room temperature See appendix 1 for example of buffer recipes Figure 3 1 A B and C Mounting step 1 6 WIRE MYOGRAPH SYSTEM 420A USER GUIDE 3 1 2 Mounting step two Using forceps to hold the handle segment transfer excised vessel from petri dish to Dual Wire Myograph chamber Hold the vessel as close to the proximal end as possible and try to mount the vessel onto the wire If the lumen is shut try one of the following possibilities 1 Use the wire to gently push the lumen open blood streaming out is a good sign 2 Hold excised vessel about 3 mm from the cut end with one set of forceps and use the other forceps to squeeze the blood remaining in lumen out through the cut end Pull the proximal end of the excised vessel segment along the wire such that the vessel segment acts as its own feeder to be feed into the wire into the vessel figure 3 2 A C Be careful not to stretch the vessel segment if the end of the wire catches the vessel wall Figure 3 2 A B and C Mounting step 2 3 1 3 Mounting step three Once the vessel segment is threaded onto the wire catch the free end of the wire nearest you with the forceps and move the jaws
21. h carbogen 9576 0 576 CO for about 20 minutes APPENDIX 1 19 APPENDIX 2 NORMALIZATION THEORY The importance of making a normalization before initiating an experiment with any tubular tissue segment is described in chapter 3 2 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 see appendix 3 and force reading respectively character izing 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 micrometer reading is calculated by Y Y T 90 28 e a where 6 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 IO PEZ e where IC is the internal circumference of the mounted vessel when the wires are just separated and is given by IC 2 m d where d is the wire diameter For 40 wires IC 205 6 um Using the Laplace relation the effective pressure is calc
22. l Wire Myograph chamber window is fixed in place and kept waterproof by a thin layer of high vacuum grease on the circular edge between the glass and the chamber base The following procedure describes how to change the window glass 1 Screw the jaws as far apart as possible and carefully remove the jaw from the transducer pin side it should not be neces sary to remove the jaw on the micrometer side 2 Loosen the glass from the chamber by gently pushing up on the glass from below the window with a blunt tool 3 Remove the old grease and clean the area thoroughly with 96 ethanol 4 Carefully apply a small continuous amount of high vacuum grease around the edge of the window Using forceps place the new window glass in place Push down gently around the edges to create a seal between the glass the grease and the chamber base 5 Check that the new window forms a tight seal by filling the chamber with distilled water If there is a leak repeat the replace ment procedure 6 Finally replace the jaw on the transducer pin and adjust the supports according to the instructions provided in chapter 2 1 4 4 Maintenance of the linear slides Check the linear slides under the black covers for grease at least once a week In case of insufficient lubrication the micrometer will not move as effortlessly as it Should grease the slides with the original enclosed grease for linear slides at the points marked by the arrows in figure 4 3
23. ndard time such as 2 minutes between each application NA in chamber uM Volume of stock solution to add to chamber ea es sO OM sorte 12 WIRE MYOGRAPH SYSTEM 420A USER GUIDE 3 6 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 3 6 1 Background Acetylcholine causes relaxation of rat mesenteric small arteries by activating of muscarinic M3 receptors at the endothelial cell layer leading to release of endothelium derived relaxing factors Rat mesenteric arteries do not show spontaneous tone in the wire myograph which is why it is necessary to first induce a con traction to be able to observe the relaxation to acetylcholine In this protocol the contraction is induced by noradrenaline The required concentration of noradrenaline needs to be optimized 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 underestima tion 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 previ
24. nutes 25x Concentrated PSS Chemical Mol Wt mM g 0 5L gL g 2L g 4L 58 45 3250 94 98 189 96 379 92 759 84 KCI 74 557 117 5 4 375 8 75 17 5 35 0 KH PO 136 09 29 5 2 0 4 0 8 0 16 0 MgSO 7H O 246 498 29 25 3 625 1 25 14 5 29 0 NaHCO 84 01 14 9 0 625 1 25 2 50 5 00 Glucose 180 16 5 0 0 5 1 00 2 00 4 00 EDTA 380 0 65 0 125 0 25 0 50 1 0 CaCl 110 99 40 20mL 40mL SOmL 160mL 1 0 M solution 18 WIRE MYOGRAPH SYSTEM 420A USER GUIDE Make 1 0M solution of CaCl 110 99 in double distilled H O Filter sterilize the calcium solution through a 0 22 um filter The sterilized solution can be stored in the refrigerator for up to 3 months Dissolve all the chemicals except the CaCl in approximately 80 of the desired final volume of double distilled H O while being constantly stirred For example if 1 litre of PSS is to be made then dissolve all the chemicals in 800mL of double distilled H O 2 Add the appropriate volume of 1 0M CaCl for the total volume of PSS being made for example 1 6mL of 1 0M CaCl for 1 litre of buffer Continue to stir the PSS while the CaCl is being added Bring the solution up to the final volume with double distilled H O Continue to stir the solution until the EDTA is fully dis solved This takes about 15 minutes at room temperature Before use Dilute the 25 x PSS stock solution 1 25 using double distilled H O Add 091 g L Glucose 100 g L NaHCO Aerate the solution wi
25. orts with 9690 ethanol or a weak detergent solution 4 Toremove more resistant or toxic chemicals incubate the 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 HNO3 solution for about 15 minutes 5 Wash the chamber and supports several times with double distilled water To prevent the pipes from being blocked by buffer salt deposits after an experiment remove the chamber cover from the myo graph 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 tissue IMPORTANT NOTES BE VERY CAREFUL USING STEP 3 AND 4 REPEATEDLY AS STRONG REAGENTS CAN CAUSE EXTREME DAMAGE TO THE DUAL WIRE MYOGRAPH BE VERY CAREFUL NOT TO EXERT ANY FORCE ON THE WIRE JAWS 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 SEE FIGURE 4 1 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 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 swabestick to mechanically clean all the affecte
26. ous section The vessel segment is exposed to the noradrenaline concentration and when the response has stabilised increasing concentra tions 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 experiment is ended 3 6 2 Protocol Prepare the following stock solutions Acetylcholine 10 103 10 M Noradrenaline 10 M 1 Mount and normalize the vessels as described in chapter 3 1 and 3 2 2 Perform a standard start and check the vessel segment for endothelium function as described in chapter 3 3 and 3 4 3 Add noradrenaline to obtain a response around 6096 of maximum determined from the previous noradrenaline concen tration response curve When the contractile response is stable add increasing concentrations of acetylcholine to the chamber using the table below as a guideline Wait for a stable contractile response or a standard time such as two minutes between each application ea iMWiM 1 2 5 uL of 103 M 1 3 1 5 uL of 103 M 1 5 1 uL of 10 M 7 5 uL of 10 M 1 uL of 102 M 2 5 uL of 10 M In calculating the ACh in the Dual Wire Myograph chamber the applied volume of ACh is ignored CHAPTER 3 13 CHAPTER 4 CLEANING AND MAINTENANCE The Daul Wire
27. quise A A L O A Ed oS das A E E EA E E E A ONE 9 ca NONN ZUON sanra oi o m 9 GN Re EIC V TETTE 10 3 3 1 Principles of the standard start procedure E n bx Date imei od nare Duce e 10 3 2 1 Principles of the normalization scs ous cui virt nca Riu ERES URSI ERR Ia EQ RARE YII A OA v FC Ck Ra ue dis 10 3 4 Endotheium CO DM mme en 11 3 4 1 Principles or CHECKING endothelium function ofits dus Sada nb un Eur ificnigue Dao 11 3 5 In vitro experiment 1 Noradrenaline contractile reSPONSE ccccssssscesececcsssseetececcccsseeeeenccccsseesencceceseeesenececesseerseececesseesesecees 12 uM ON ENERO 12 DL FOO OT E O E N A E OE E EAO E E E QUEE EU R VUE If EE 42 3 6 In vitro experiment 2 Acetylcholine relaxation CUrve cccccssssecesececcsssseetececcecseseseeecccussseeeeeccccasseesenccecesseerenececesseerenececesssetenesees 15 Nc sere teeth 15 OZ POLO C Ol RENE TNT IT A m 15 Chapter 4 Cleaning and Maintenance randi OR Ra CR EN A OK RUN RD ED ebd FH DN arr END
28. rce calibration DMT recommends that the Dual Wire Myograph is force calibrated at least once every month DMT also recommends that the Dual Wire Myograph is force calibrated every time the system has been moved or has not been used for a long period of time See the force calibration procedure in chapter 3 7 1 in Wire Myograph Systems User Manual CHAPTER2 5 CHAPTER 3 EXPERIMENTAL SET UP This chapter contains experimental set up for the Dual Wire Myograph For dissection of a vessel please see Procedures for investigations of small vessels using a small vessel Myograph by M J Mulvany 3 1 Mounting protocol for small arteries The procedure involves attaching the mounting wires to jaws which are in turn mounted on the force transducer This force trans ducer is capable of measuring with a sensitivity of about 0 01 mN 1 mg but can be damaged if the applied force exceeds about 1 N 100 g Therefore care must be taken to avoid pressing the jaws too hard together A movement of 20 um after they have touched is sufficient to hold the wires clamped 3 1 1 Mounting step one e Cut lengths of 40 um wire 2 2 cm long Mount one wire on left hand jaw of the Dual Wire 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 figure 3 1 A NOTE DO NOT CLOSE THE JAWS TOO HARD AGAINST EACH OTHER e Bend the far end of the wire towards the left and wrap it around under
29. ted to the linear slide micrometer Align the horizontal support matching the force transducer connected support as carefully as possible and gently tighten the screw again 2 Loosen screw C on the linear slide to roughly match the linear slide support to the force transducer support in the horizon tal plane Tighten the screw before proceeding with step 4 3 The plate on which the linear slide is mounted is balanced on top of a small stainless steel ball making it possible to finely adjust the linear slide support in all vertical and horizontal planes using the four Allen screws Use the four Allen screws to make the final horizontal See arrow in figure 2 1 and vertical See arrow in figure 2 2 adjustments to match the linear slide support to the force transducer support The correct matching of the supports is illustrated in figure 2 3 IMPORTANT AVOID CONTINUOUSLY TIGHTENING THE ALLEN SCREWS DURING THE FINAL ADJUSTMENTS LOOSEN THE ALLEN SCREW PLACED DIAGONALLY TO THE ALLEN SCREW BEING TIGHTENED OTHERWISE THERE 15 A HIGH RISK OF DAMAGING THE DUAL WIRE MYOGRAPH FRAME gt O gt Figure 2 1 Illustration of screws for adjusting supports and horizontal adjustment 4 WIRE MYOGRAPH SYSTEM 420A USER GUIDE Figure 2 2 Illustration of vertical adjustment Figure 2 3 lllustration of correctly aligned supports for small vessels left and incorrectly aligned supports middle and right 2 2 Fo
30. th carbogen 95760 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 55 1x 60mM KPSS Chemical Mol Wt mM g 0 5L g L g 2L g 4L 58 45 14 7 2 18 4 37 8 73 17 46 KCI 74 557 60 2 24 4 47 8 95 17 89 136 09 1 18 0 08 0 16 0 32 0 64 MgSO 7H O 246 498 1 17 0 145 0 29 0 58 1 16 NaHCO 84 01 14 9 0 625 1 00 2 00 5 00 Glucose 180 16 bib 0 5 1 00 2 00 4 00 EDTA 380 0 026 0 005 0 01 0 02 0 04 CaCl 110 99 1 6 0 8mL 1 6mL 32ml 6 4mL 1 0 M solution T Make a 1 0M solution of CaCl 110 99 in double distilled H O Filter sterilize the calcium solution through a 0 22 um filter The sterilized solution can be stored in the refrigerator for up to 3 months Dissolve all the chemicals except the CaCl in approximately 80 of the desired final volume of double distilled H O while being constantly stirred For example if 1 litre of PSS is to be made then dissolve all the chemicals in 800mL of double distilled H O 2 Add the appropriate volume of 1 0M CaCl for the total volume of PSS being made for example 1 6mL of 1 0M CaCl for 1 litre of buffer Continue to stir the PSS while the CaCl is being added Bring the solution up to the final volume with double distilled H O Continue to stir the solution until the EDTA is fully dis solved This takes about 15 minutes at room temperature Aerate the solution wit
31. ulated for each pair of readings The effective pressure is an estimate of the internal pressure which is necessary to extend the vessel to the measured internal circumference T amp The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure The target value needs to be optimized for the individual tissue preparation optimal active force as determined by the length tension rela tionship for that tissue For rat mesenteric arteries the target transmural pressure is normally 100 mmHg 13 3 kPa IC T 100 7 100 mmHg T An exponential curve is fitted to the internal circumference pressure data as illustrated in figure 3 9 Now the isobar correspond ing to 100 mmHg is used to calculate the value from the point of interception between the function of the exponential curve and the function of the 100 mmHg isobar The normalised internal circumference IC is calculated by multiplying the internal circumference corresponding to LOO mmHg op 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 20 WIRE MYOGRAPH SYSTEM 420A USER GUIDE The normalized internal lumen diameter is then calculated by HE 1 TU The micrometer reading X at which the internal circumference of the normalized vessel is set to is calculated
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