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USER GUIDE, VOL.2.0

W I R E M Y O G R A P H S Y S T E M - 6 2 0 M

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Summary of Contents for 620M

Page 1: ...USER GUIDE VOL 2 0 W I R E M Y O G R A P H S Y S T E M 6 2 0 M ...

Page 2: ...User Guide Version 1 0 WIRE MYOGRAPH 620M USER GUIDE ...

Page 3: ...unting step five 9 3 1 6 Mounting step six 10 3 1 7 Mounting step seven 10 3 2 Normalization 10 3 2 1 Principles of the normalization procedure 11 3 3 Standard start 11 3 3 1 Principles of the standard start procedure 11 3 4 Endothelium function 12 3 4 1 Principles of checking endothelium function 12 3 5 In vitro experiment 1 Noradrenaline contractile response 13 3 5 1 Background 13 3 5 2 Protocol...

Page 4: ...Myograph jaws Micrometer Jaw connected to Micrometer Jaw connected to force transducer Force transducer pin Supports Figure 1 1 Wire Myograph with close up of chamber CHAPTER 1 Figure 1 2 Chamber cover Access for drug application Access or funnel Access for temperature probe CHAPTER 1 WIRE MYOGRAPH OVERVIEW ...

Page 5: ...ed to gently loosen screw D on the support attached on the transducer side using the small screwdriver Screw D is the screw on the transducer side support closest to the transducer 3 Gently pull the support away from the transducer pin 4 Loosen screw B on the micrometer side with the appropriate fitting Allen key 5 Pull the support away NOTE NUMBER THE SUPPORTS WITH THE NUMBER OF THE CHAMBER THEY ...

Page 6: ...h screws D and B will move the micrometer side jaw straight down 3 Tightening both screws C and A will move the micrometer side jaw straight up CHAPTER 2 Figure 2 2 Fine adjustments of the jaws in the Wire Myograph chamber Figure 2 3 Illustrations of properly aligned jaws depicted on the far left and incorrectly aligned jaws depicted in the middle and far right A B C D Jaws from top view Jaws from...

Page 7: ...Figure 2 5 Illustrations of properly aligned pins depicted on the far left and incorrectly aligned pins depicted in the middle and far right 2 2 Calibration of the force transducer As a part of the general maintenance of the Wire Myograph DMT recommends that the Wire Myograph is force calibrated at least once a month The Wire Myograph should also be force calibrated every time the interface has be...

Page 8: ...ovement of 20 µm after they have touched is sufficient to hold the wires clamped 3 1 1 Mounting step one Cut lengths of 40 μm wire 2 2 cm long Mount one wire on left hand jaw of the Wire Myograph as follows Holding wire at far end place centre of wire between jaws and screw jaws together so that the wire is clamped figure 3 1 A NOTE DO NOT CLOSE THE JAWS TOO HARD AGAINST EACH OTHER Bend the far en...

Page 9: ... Be careful not to stretch the vessel segment if the end of the wire catches the vessel wall 3 1 3 Mounting step three Once the vessel segment is threaded onto the wire catch the free end of the wire nearest you with the forceps and move the jaws apart While controlling the movement of the wire with the forceps use the other forceps to gently pull the vessel segment along the wire until the area o...

Page 10: ...retched 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 3 1 4 M...

Page 11: ... 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 th...

Page 12: ... diameter is calculated by dividing IC1 with 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 ...

Page 13: ...o 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...

Page 14: ... 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 noradrenal...

Page 15: ...e 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 segment is either fully relaxed or does not relax more upon increasing the ...

Page 16: ...or buffer remaining in the chamber or on the tubing using absorbent paper IMPORTANT NOTES BE VERY CAREFUL USING HCL OR HNO3 BECAUSE THESE ACIDS MAY CAUSE EXTREME DAMAGE TO THE STAINLESS STEEL CHAMBERS AND SUPPORTS DO NOT USE BLEACH TO CLEAN THE CHAMBERS REPEATED USE OF CHLORINATED SOLUTIONS SUCH AS BLEACH AND HCL WILL CAUSE DAMAGE TO THE STAINLESS STEEL PARTS OF YOUR WIRE MYOGRAPH SYSTEM AVOID USI...

Page 17: ...ection for the force transducers they are still very vulnerable to applied forces exceeding 1 Newton 100 grams or fluid running into the transducer compartment due to insufficient greasing of the transducer pinhole If the force readings on the Multi Interface appear unstable or noisy then first check that the Wire Myographs are connected properly to the Multi Interface and that the chambers are pl...

Page 18: ...ducer inside a new transducer house 5 Place a small amount of vacuum grease clear or whitish grease around the bottom of the transducer housing to seal when set back in place see figure 4 4 6 Carefully realign the transducer housing with the new transducer on the Wire Myograph and reinsert the Allen screws through the bottom of the Wire Myograph 7 Tighten the screws and place some vacuum grease ar...

Page 19: ...rease at least once a week In case of insufficient lubrication grease the slides with the Grease for Linear Slides included with your system See figure 4 5 below Figure 4 4 Inside the transducer housing and close up of transducer pin The orange arrows in the dashed frame indicates the place that the vacuum grease needs to be applied to prevent water and buffer from damaging the transducer ...

Page 20: ...uble distilled H2 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 H2 O 3 Add the appropriate volume of 1 0M CaCl2 for the total volume of PSS being made for example 1 6mL of 1 0M CaCl2 for 1 litre of buffer Continue to stir the PSS while the CaCl2 is being added 4 Bring the solution up to the final volume wit...

Page 21: ...hysiological Saline Solution KPSS 1x 60mM KPSS Chemical Mol Wt mM g 0 5L g L g 2L g 4L NaCl 58 45 74 7 2 18 4 37 8 73 17 46 KCl 74 557 60 2 24 4 47 8 95 17 89 KH2 PO4 136 09 1 18 0 08 0 16 0 32 0 64 MgSO4 7H2 O 246 498 1 17 0 145 0 29 0 58 1 16 NaHCO3 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 CaCl2 110 99 1 6 0 8mL 1 6mL 3 2mL 6 4mL 1...

Page 22: ...sel when the wires are just separated and is given by IC0 2 d where d is the wire diameter For 40 µm wires IC0 205 6 µm Using the Laplace relation the effective pressure Pi 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 Pi Ti ICi 2 The stepwise distension is contin...

Page 23: ...23 The normalized internal lumen diameter is then calculated by IC1 d1 The micrometer reading X1 at which the internal circumference of the normalized vessel is set to is calculated by IC1 IC0 2 X1 X0 ...

Page 24: ...Note that the thimble has stopped at a point beyond 10 on the sleeve indicating 10000 µm 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 B No additional mark visible C Thimble reading Total reading Example 2 1 Note that the thimbl...

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