User Guide for 360CW
Contents
1. 12 a OUN PRICE RR O 12 2 0 POO O a dI A E E E A A 12 3 6 In vitro experiment 2 Acetylcholine relaxation csssscceeccssssseeeeeccccesseeseeccccesseesseccccesseeseeececesseesssececesssasseececasesassescsoasess 13 OLB O RR s tnn 15 ie FO LOC Ol eR 25 Chapter 4 Cleaning and Maintenance EU ED UK ENFIN RE EET E GU NEA KE 14 4 1 Cleaning the Confocal Wire cite crt cla eae neste uera cl dns Qu VR Irem di 14 4 2 Maintenance of the force coe kr ew rt e Ri 18 mo ACEC te TONGS halls OIC CI E SURE 15 4 2 2 Force transducer FeO CS TSIM ER m ERE rene ndr diem cv 16 4 3 Changing Confocal Wire Myograph window amp 1 5 16 4 4 Maintenance of the linear 1652. lor aiat UR xs TC C oko yicQ 16 Appendix 1 Buffer FOCI DC RECETTE IE Aana KaK Aaaa KaKa na iniaa 17 Appendix 2 Normalization theory ssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn OON XN NRI FE KM REX RU XD UK 19 Appendix 3 Reading a millimetre micrometer PERRO A SEEN ua Ug HE RE EE ERE ERREUR E RYE CERE VERE E ERKKENCNRUIK ER EX NEA TEES KR EH EE EE ER 21 2 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE CHAPTER 1 CONFOCAL WIRE MYOGRAPH OVERVIEW Port for connection with Wire Interface Vertical micrometer screw Horizontal micrometer Allen screws for fine alignment of the wire jaws 2 on each side force transducer Force transducer pin Window at the bottom of the Myograph chamber for imaging under the wire jaws Figure 1 1 Confocal Wire Myograph with close up of the chamber for adding drugs suction connection NNNM Oxygen supply Spare connection for chamber superfusion 6 Figure 1 2 Chamber cover CHAPTER 1 3 CHAPTER 2 SETTING UP THE CONFOCAL WIRE MYOGRAPH 2 1 Adjustment of supports A successful mounting of any kind of tubular tissue segment in the Confocal Wire Myograph is very dependent on perfectly match ing supports The supports are matched prior to the shipment but daily use of
3. um um C Thimble reading 280 um Total reading 16780 um Figure A2 3 Example 2 reading 16780 um APPENDIX 3 21 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
4. 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 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 minutes 25x Concentrated PSS Chemical Mol Wt mM g 0 5L g L g 2L g 4L 58 45 3250 94 98 189 96 379 92 759 84 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 29 14 5 29 0 NaHCO 84 01 14 9 0 625 1 25 250 5 00 Gluco
5. 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 16 535 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 O 8mL 1 6mL 3 2mL 6 4mL 1 0 M solution d 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 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 with carbogen 9576 0 576 CO for about 20 minutes 18 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 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 a
6. an experiment use the chamber cover to remove the clean ing solutions Afterwards remove the cover from the chamber and turn on the vacuum pump 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 tissue IMPORTANT NOTES BE VERY CAREFUL USING STEP 3 AND 4 REPEATEDLY AS STRONG REAGENTS CAN CAUSE EXTREME DAMAGE TO THE 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 3 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 myograph chamber for 30 minutes with 20yl of a 2 mM T 1210 Tetrakis 2 pyridylmethyl ethylenediamine solution dissolved in double distilled water 2 Use a swabestick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period 3 Wash the chamber 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 with an 896 acetic acid solution for 10 minutes a
7. the Confocal 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 below 1 Carefully loosen screw A on the top of the support connected to the force transducer Align the horizontal Support and carefully tighten the screw again 2 Loosen screw B on the top of the support connected to the linear slide Align the horizontal support matching the force transducer connected support as carefully as possible and gently tighten the screw again 3 Loosen screw C on the linear slide to roughly match the linear slide Support to the force transducer support in the horizon tal plane Tighten the screw before proceeding with step 4 4 The plate 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 D Use the four Allen screws to make the final horizontal and vertical adjustments to match the linear slide Support to t
8. working distance objectives 2 4 Force calibration DMT recommends that the Confocal Wire Myograph is force calibrated at least once every month DMT also recommends that the 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 Confocal 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 difficulty of mounting ring preparations in any Wire Myograph mostly depends on the lumen size The Wire Myograph tech nique is usable for ring preparations with internal diameter gt 100 using 40 stainless steel wires 25 um tungsten wires are available for ring preparations with an internal diameter as low as 60 um For large diameter vessels mounting pins 200 um and larger are an available option The Confocal Wire Myograph reverse mounting jaws are shaped with pegs at the bottom to position the vessel as close as possi ble to the objective of the inverted confocal microscope The special jaws with a vessel mounted are illustrated in figure 3 1 The pegs make the mounting of the vessel a little more difficult than in the other Wire Myographs Another differenc
9. DANISH MYO TECHNOL Ost CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE CONTENTS Chapter 1 Confocal Wire Myograph overview ccssssssscsenssseccensseencensseccennnseecennaseecoonssseneensseeneeasseneenssseeeensaseeeonsssesoensssssnensssensens 3 Chapter 2 Setting up the Confocal Wire Myograph sccsssssccssnsseeccennsseeceenseeeneessseeeeeasseneenssseeensssecensseeeeonsseeseonsssensensseseonns h AL PACS US IC O SUDDO caasas e OS me 4 22 SOC CII SIC SITUE rrr 5 2 X a a A E E T E E A E AA 5 AAFO dU r Cal O ea E AE r GUI QUE 5 Chapter 3 Experimental set up ssssnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn nnmnnn nnmnnn nnna nnmnnn nanmanna 6 2L Protocol TON tS ccc rrerre a E E A 6 OE O REINO A E A AOE A E E n 10 3 2 1 Principles of the normalization DFOCS AUN IM iet tor vd on Rv innt 10 3 3 I PET TO DET 10 3 3 1 Principles or the standard start procedure caisse iet nnde Rb Rn red t E ec t n DR Dn OE eir 11 cvi cenas IE NS UU TT m 11 34 T Principles or checking endothelium aerate Gasp uu RR RO ouf me cii d tuit dui 11 3 5 In vitro experiment 1 Noradrenaline contractile
10. arget 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 gt 100 mmHg T An exponential curve is fitted to the internal circumference pressure data as illustrated in figure 3 3 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 by a factor 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 APPENDIX 2 19 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 by IC IC EF 1 O 2 20 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE APPENDIX 3 READING A MILLIMETRE MICROMETER Sleeve scale Thimble scale Figure A2 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 equa
11. d the chamber base Daily use of the Confoacl Wire Myograph objectives that require water or other immersion solvents will mean that the glass needs to be replaced daily prior to mounting The following procedure describes how to change the Confoacl Wire Myograph window glass 1 Carefully loosen the glass from the chamber edge using small forceps or similar tool 2 Clean the edge at the bottom of the chamber to remove any remaining old grease 3 Carefully apply a small amount of high vacuum grease around the edge using a wood stick or similar equipment Take a new window glass using forceps and gently push it into place Before starting a new experiment make sure that the complete edge is sealed up with high vacuum grease to keep the Myograph window waterproof 4 4 Maintenance of the linear slides Check the linear slides underneath the black cover for grease at least once a week In case of insufficient lubrication grease the Slides with the original enclosed grease for linear slides at the places marked by the arrows in figure 4 2 below NOTE ONLY USE THE GREASE MARKED WITH GREASE FOR LINEAR SLIDES Figure 4 2 Greasing points on the linear slides 16 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 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
12. e from the other Wire Myographs is that the mounting supports for the jaws are longer and thus less rigid Slight pressure on the jaw can result in a significant stress upon the mounting supports On the transducer side this results in stress upon the transducer pin which could damage the transducer Furthermore there is a risk you will push the jaws out of alignment For these reasons the demands upon your carefulness and awareness are increased in these procedures These instructions hopefully will help you to succeed with the mounting procedure Experience with mounting in other Wire Myographs is an advantage and will ease the process Figure 3 1 lllustartion of mounted vessel in Confocal Wire Myograph special designed jaws 1 A vessel size 150 500 is dissected as described in Procedures for investigations of small vessels using a small vessel Myograph by M J Mulvany 3 Before mounting it is important that the cover slip diameter 19 mm the thickness may need to be optimized to your optics at the bottom of the Confoacl Wire Myograph is clean It is recommended that the glass be changed every day To ease this process the top part of the Confoacl Wire Myograph with the heads can be tilted up and out of the way this also permits easy access for cleaning To remove the old glass press up gently from below Remove the old grease and fix the new glass with a little grease high vacuum grease It is important to remove any air bubbles betw
13. e the Confoacl Wire Myograph to the confocal microscope and attach bubbling to the bath Connect the Confocal Wire Myo graph to the Wire Interface and turn the heating on Zeroing of the micrometer X can now be done wires moved together until they are just touching and a normalization performed as described in the next section After the artery is normalized contractility tested and loading with the fluorescent dye completed move the heads downwards with the vertical micrometer to position the artery just above the cover slip Once you can see the jaws are close to the cover slip follow the force reading on the Myo Interface It is important to now advance slowly and carefully too much downwards force and you will put unnecessary strain on the transducer and risk cracking the cover slip When the artery is touching the bottom of the chamber the force reading with increase At this point move the artery up slightly The artery is now positioned for imaging and the objective should be able to focus on the vessel Note that movement artifacts can occur when the artery is stimulated with agonists or depolarized with potassium This factor can be somewhat avoided by the use of ratiometric dyes Contraction can be inhibited by wortmannin which covalently binds to the myosin light chain kinase Alternatively the artery can be hyperpolarized with potassium channel openers thereby preventing the opening of voltage sensitive calcium channels and contract
14. een the glass and the chamber to ensure a good water tight seal 4 is also important that the mounting heads are carefully adjusted and aligned so that there are no disturbances in the force recording when the heads are close together The alignment procedure is described in chaper 2 1 For the mounting procedure the following equipment is used a stereomicroscope magnification 4 40x and a fibre light source two pairs of fine forceps a pair of fine scissors trabecular type a small screwdriver and wire 5 have as much space as possible move the supports up with the vertical micrometer screw before you start the mounting procedure 6 Place the Confoacl Wire Myograph in front of you with the micrometer to your right 6 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE f Close heads partly 8 Hold a wire with the forceps one third from the top Place the wire between the heads underneath the top left peg and over the lower right peg 9 When the wire is in position the heads are moved together and the top end of the wire is bent up and fixed by the top left screw Remember always to place the wires clockwise under the screw heads so that tightening of the screws will tighten the wires too 10 The protruding wire should be straight and long enough to reach the lower left screw Excess wire can be cut off cold PSS 11 Fill the chamber with cold PSS and transfer the vessel to the Confoacl Wire M
15. 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 If 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 endothelium 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 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 t
16. gment 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 107 10 104 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 iWioM es imi9M E 13 CHAPTER 4 CLEANING AND MAINTENANCE The Confocal Wire Myograph is a very delicate and sophisticated piece of research equipment In order to keep it working at its best DMT recommend that the following sections are read carefully and that the instructions are followed at all times As a part of the general maintenance of the Confocal Wire Myograph DMT recommends that the Confocal Wire Myograph is force calibrated at least once a month The Confocal Wire Myograph should also be force calibrated every time the Wire Interface has been moved Although lab benche
17. he force transducer support The correct matching of the supports is illustrated in figure 2 2 IMPORTANT BE CAREFUL TIGHTEN THE ALLEN SCREWS WHEN DOING THE FINAL ADJUSTMENTS ALWAYS REMEMBER TO LOOSEN THE ALLEN SCREW PLACED DIAGONAL TO THE ALLEN SCREW BEING TIGHTENED OTHERWISE THERE IS A HIGH RISK OF DAMAGING THE CONFOCAL WIRE MYOGRAPH FRAME A Screw on support connected to the force transducer TILIPIDI C Screw on the linear slide TED D Four Allen screws B Screw on support connected to the linear slide Figure 2 1 Illustration of how to adjust the supports 4 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE Figure 2 2 Illustration of correctly aligned supports of the Confocal Wire Myograph special designed jaws view from the side left and view from the top right 2 2 Special designed jaws The Confocal Wire Myograph is supplied with jaws for inverted scopes but can be used with normal stainless steel jaws to mount small vessels on an upright system For experimental work requiring electrical field stimulation a set of plastic jaws with attached platinum electrodes is available 2 3 Chamber The chamber has a conical shape and can thus be used with a small amount of buffer liquid The chamber can be customized to operate with volumes of 3 ml or less The conical milling on the bottom outside of the chamber allows objectives on inverted microscopes to be raised to the thin glass window to accommodate very low
18. he 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 3 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 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 c
19. ion CHAPTERS 9 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 3 The active response of a preparation is dependent on the extent of stretch which makes it important to set the preparation to an internal circumference giving maximal response The aim of the normalization procedure is to stretch the segment to a so called normalized internal circumference IC o defined as a set fraction of the internal circumference IC 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 Principles 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 2 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 Lap
20. l parts Each part of a division above the horizontal line represents 1 mm where each 5 line is marked by a longer line 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 50 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 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 indicating 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 horizontal line on the sleeve A Reading on sleeve 10000 um B No additional mark visible O um C Thimble reading 380 um Total reading 10380 um Figure 2 2 Example 1 reading 10380 um Example 2 1 Note that the thimble has stopped at a point beyond 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 B One additional mark visible
21. lace relation figure 3 3 IC is calculated from IC o 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 T mN mm 100 200 3 40 500 600 700 800 900 Time min Internal Circumference pm Figure 3 2 Illustration of the stepwise Figure 3 3 Illustration of the exponential normalization procedure curve fitting and determination of IC 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 different 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 f
22. nd 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 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 lt 20 a where 9 is microscope eyepiece reticule calibration factor in mm per division and a and 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 IU SETA FUA I 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 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 necessary to extend the vessel to the measured internal circumference T The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure The t
23. nd continue the mechanical cleaning with a swab stick 6 Wash the chamber several times with double distilled water IMPORTANT NOTES IN EXCEPTIONAL CASES IT MAY BE NECESSARY TO REMOVE THE SUPPORTS FOR INDIVIDUAL CLEANING TO MAKE SURE THAT ALL SURFACES ARE CLEAN 14 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 4 2 Maintenance of force transducer The force transducer is the most delicate and fragile component of the myograph system Therefore careful handling is important One of the jaws is connected to the transducer pin To prevent the buffer from running into the transducer house the hole is filled with high vacuum grease see figure 4 1 below As part of the daily maintenance it is very important to inspect the greasing of the transducer hole before starting any experiment Insufficient greasing causes damage and malfunction of the force transducer IMPORTANT DMT RECOMMENDS THAT THE HIGH VACUUM GREASE SEALING THE TRANSDUCER PINHOLE IS CHECKED AND SEALED AT LEAST ONCE A WEEK ESPECIALLY IF THE MYOGRAPH SYSTEM IS USED FREQUENTLY 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 4 2 1 Checking the force transducer The force transducer is a strain gauge connected in a Wheatstone bridge The f
24. ocaine 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 10 10 107M 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 10 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 Addincreasing concentrations of noradrenaline into the bath use the table below as a guideline Wait for a stable contractile response or a standard time such as 2 minutes between each application NA in chamber uM Volume of stock solution to add to chamber In calculating the NA in the Confocal Wire Myograph chamber the applied volume of noradrenaline is ignored M M M lt lt 12 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 3 6 In vitro experiment 2 Acetylcholine relaxation curve The purpose of the present protocol is to dete
25. or rat mesenteric arteries Another procedure may be needed for other animal species and tissue or vessel types 10 CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 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 a 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 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 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 4 x with PSS Stimulate for 3 minutes Ready for experiment 3 4 Endothelium function The reasons
26. orce transducer is placed in a separate compart ment on top of the Wire Myograph The separate compartment provides some mechanical protection for the force transducer but the transducer is still very vulnerable to applied forces exceeding 1 newton 100 gram or fluid running into the transducer compartment due to insufficient greasing of the transducer pinhole This section describes how to check the force transducer for any kind of damage Simple force transducer check 1 If the force reading on the Wire Interface appears unstable then first check that the Wire Interface and the Confocal Wire Myograph are properly connected through the 25 pin grey cable 2 the force reading still appears unstable then perform a new force calibration of the force transduce During the force calibration monitor the relative force reading values in the Calibration Menu on the Wire Interface e the value is or above 6500 then the force transducer is broken and needs to be changed e lf the reading is between 1 499 or 3001 6250 then contact DMT for further instructions CHAPTER4 15 4 2 2 Force transducer replacement In case that the force transducer is broken and needs to be changed please contact DMT for further instructions 4 3 Changing Confocal Wire Myograph window glass The glass in the Confoacl 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 an
27. rmine 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 previous 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 se
28. s are all Supposedly perfectly horizontal small differences in lab bench pitch can affect the calibration of the system The Confocal 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 Confocal Wire Myograph DMT STRONGLY RECOMMENDS THAT THE CONFOCAL WIRE MYOGRAPH AND SURROUNDINGS BE CLEANED AFTER EACH EXPERIMENT After an experiment use the following procedure to clean the chamber and supports 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 Confocal Wire Myograph 2 Remove the acetic acid and wash the chamber and jaws 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 jaws with 9690 ethanol or a weak detergent solution 4 To remove more resistant or toxic chemicals incubate the chamber and glass cannulas with 1M HCI for up to 1 hour In exceptional cases incubate the chamber and supports with a up to 3M HNO solution for about 15 minutes 5 Wash the chamber and supports several times with double distilled water To prevent the pipes from being blocked by buffer salt deposits after
29. se 180 16 5 5 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 160mL 1 0 M solution APPENDIX 1 17 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 with 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 gL 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 KH PO
30. the way until it passes the lower right peg and enough wire is through to reach the lower right screw The jaws are screwed closely together The top end of the wire with the 90 angle should point upwards between the jaws and the wire should run under neath the right side pegs To ease the coming steps which maybe the most critical ones for a viable preparation the myograph is turned 90 degrees so the vessel runs from right to left CONFOCAL WIRE MYOGRAPH SYSTEM 360CW USER GUIDE 20 The end of the second wire is bent towards the top right screw screws are still named as if the Confoacl Wire Myograph was not turned 90 and fixed below the screw head Take care that the vessel is not stretched or moved in the longitudinal direction You might prevent this by holding the other end of the wire with your second pair of forceps 21 The lower end of the wire now needs to be fixed to the lower right screw Take care that the second wire is mounted parallel to the first wire and of course on the outside of it Remember again that the wire should be straight and tight 22 Bylooking in the microscope and at the same time watching the force signal the height of the left wire is adjusted to allow it to move freely when the wires are moved together or away from each other The height of the wire is adjusted by adjusting the Allen keys on the micrometer side Mov
31. yograph chamber See appendix 1 for example of buffer recipes 12 Holding the vessel close to the proximal end with the forceps the vessel is mounted onto the wire 13 The vessel can now be cut off to give it the right length to fit the gap between the shortest pegs 2 mm Add a little bit more the width of one peg Expla nation follows in step 15 CHAPTERS 7 14 15 The jaws are now moved widely apart and the wire is released so the vessel can pass the pegs without touching them and thereby be damaged The vessel is pulled gently up the wire and the wire is positioned underneath the top left peg NOTE WHEN THE PROXIMAL END OF THE VESSEL 15 FIXED BETWEEN THE WIRE AND THE PEG THIS PREVENTS THE VESSEL SLIPPING DOWN AND BECOMING COMPRESSED WHEN THE SECOND WIRE IS INSERTED THE VESSEL SHOULD NOT BE FIXED AT THE DISTAL END A FIXATION AT THE DISTAL END WOULD HINDER THE VESSEL TO MOVE FREELY IN THE LONGITUDINAL DIRECTION 16 17 18 19 8 The wire is fixed with the lower left screw and at the same time is pulled to run tight from the screws along the head and underneath the left pegs The vessel should run parallel to the front of the head A long wire 25 mm is bent one third from the top in a 90 angle This wire is guided into the vessel along the first wire from the top end of the vessel When the wire is through the whole length of the vessel it is cautiously pulled through the rest of
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