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DMT 630MA, User Manual

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26

3.6 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.

3.6.1 BACKGROUND

Noradrenaline (norepinephrine) causes 
contraction of mesenteric small arteries 
through  activation  of  α-adrenoceptors  whereas 
noradrenaline  activation  of  β-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 
β-adrenoceptor antagonist, propranolol.

Rat mesenteric arteries are densely innervated 
by sympathetic nerves, which have a highly 
efficient  re-uptake  mechanism  that  removes 
noradrenaline from the neuromuscular 
junction.  The  re-uptake  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 re-uptake.

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.

Summary of Contents for 630MA

Page 1: ...USER GUIDE MYOGRAPH SYSTEM 630MA ...

Page 2: ...AUTOMATED MULTI WIRE MYOGRAPH SYSTEM ...

Page 3: ...3 5 Endothelium function 25 3 6 In vitro experiment 1 Noradrenaline contractile response 26 3 7 In vitro experiment 2 Acetylcholine relaxation curve 28 CHAPTER 4 CLEANING AND MAINTENANCE 30 4 1 Cleaning the 630MA Myograph system 30 4 2 Maintenance of the force transducer 32 4 3 Maintenance of the linear slides 36 APPENDIX 1 BUFFER RECIPES 37 Physiological Saline Solution PSS 37 High potassium Phys...

Page 4: ...r Micropositioner Allen screws for fine alignment of the myograph jaws Connection to Myo Interface Force transducer pin Supports Myograph jaw connected to force transducer Myograph jaw connected to micropositioner Figure 2 3 Chamber cover For drug application Temperature probe Funnel ...

Page 5: ...djustments Changing and adjustment of the supports is performed using the following step by step procedure 2 1 1 CHANGING THE MOUNTING SUPPORTS FIGURE 2 1 1 Use the micrometer to separate the supports as far apart as possible 2 Use the small screwdriver provided to gently loosenscrewDonthesupportattachedonthe transducer side using the small screwdriver Screw D is the screw on the transducer side s...

Page 6: ... is the screw on the transducer side support that is furthest away from the transducer NOTE Number the supports with the number of the chamber they were removed from using some kind of permanent marker Store the supports in the provided plastic case Numbering the supports will save time when the supports are changed again limiting the amount of adjustments needed after each change Figure 2 1 Illus...

Page 7: ... 1 3 FINE ADJUSTING THE JAWS FOR SMALL VESSELS FIGURE 2 2 AND FIGURE 2 3 1 TighteningScrew D willmovethemicrometer side jaw downward and to the left 2 Tightening both 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 Figure 2 2 Fine adjustments of the jaws in the Wire Myograph chamber A B C D ...

Page 8: ...ly 4 Loosen screws D and E to align the heights of the pins vertically Micrometer Transducer house A B C D 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 been moved Although lab benches are ...

Page 9: ... 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 center of wire between jaws and screw jaws together so that the wire is clamped figure 3 1 A Bend the far end of the wire towards the left and wrap it around under fixing screw...

Page 10: ...h 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 carefu...

Page 11: ... with the for ceps 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 B Mounting step 3 3 1 4 MOUNTING STEP THREE Using forceps gently rub the vessel segment on the far side of the jaw to separate any excess vess...

Page 12: ...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 A B and C Mounting step 5 3 1 6 MOUNTING STEP SIX Carefully move the jaws together while ensuring that the second mount...

Page 13: ...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 1 8 MOUNTING STEP EIGHT Adjust the two wires in a way that the two wires will bump into each other and not will go beneath or o...

Page 14: ...s In correctpositionofthetwowires Bluecolor Hereonewireiscompletely above the other wire Correct the wires as shown in first example before continuing This is a side view of the two wires on the two The artery is now mounted correctly and can moved back onto the 630MA interface with the heat turned on When the buffer in the chamber have reached 37 C replace the buffer in the chamber with fresh wel...

Page 15: ... Jaws with Pins see chapter 2 1 With the 630MA system a box with 4 sets of 200µm pins is delivered For really large arteries pins with 250µm 300µm or 400µm pins can be purchased from DMT 3 2 1 MOUNTING STEP ONE First make sure that the mounting pins is placed correctly across each other in the chambers as shown below Pins from top view Pins from side view ...

Page 16: ...rned ON When the buffer in the chamber have reached 37C replace the buffer in the chamber with fresh well gassed warm buffer Wait 5 min and start the Automated Normalization as described in Chapter 3 3 5 NOTE For large arteries in could be necessary to add a small tension 1 2mN to the mounted artery on the pins to avoid that the artery falls of the pins during heating and replacement of the buffer...

Page 17: ...nists 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 IC1 defined as a set fraction of the internal circumference IC10...

Page 18: ... multiplying a factor the Norm 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 Norm Factor is 0 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 IC1 with 3 3 ...

Page 19: ...ease CH1 1000 um ENTER SELECT NEXT SELECT SELECT SELECT Norm Pressure 13 3 KPa Norm Factor 0 9 Eyepicece cal 0 40 mm div ENTER BACK CH1 NORM PARAMETERS 2 4 Enter the values for the Norm Time Wire Pin diameter DMT have the following wire and Pin products Steel Wire 40µm in diameter Tungsten wires 25µm 15µm and 10µm in diameter Pins 200µm 250µm 300µm and 400µm in diameter 5 Press NEXT to go into the...

Page 20: ... Norm Factor is 0 9 but for mouse mesenteric arteries the Norm Pressure is 13 3kPa and the Norm Factor 1 1 To find the Norm factor for your specific artery and specie please read DMT Normalization Guide 7 Go into the Mounting Artery 8 Select the appropriate Chamber MOUNT ON CHAMBER NO SELECT SELECT SELECT SELECT CHAMBER 1 ENTER SELECT SELECT CHAMBER 2 SELECT CHAMBER 3 SELECT CHAMBER 4 IMPORTANT Th...

Page 21: ...0 mN ENTER START NORM Segment Length 0 4 mm 11 Enter the Eyepiece values of the artery mounted in the given chamber When the a1 and a2 values have been entered the 630MA will calculate the artery Segment Length based on the a1 a2 and Eyepiece Cal values Check if the segment length is approx correct The gap in the jaws are 2 00 mm shown with red arrows in the figure to the right 12 Enter the Norm F...

Page 22: ...is successful a parameter screen is shown and the mounted artery is ready for use with the optimal pre load tension Norm Pressure 13 3 kPa Norm Factor IC1 IC100 0 9 Norm Time 60 sec Norm Force 1 5 mN Wire diameter 40um Eyepiece cal 0 40mm div Eyepiece a1 0 5 Eyepiece a2 2 5 Segement Length 0 800 mm r 0 98445 Xo 0 00 um Yo 37 44mN Normalized lumen Dia 397 26 um Motor pos 458 80 um CH1 NORM RESULTS ...

Page 23: ... of the next artery segment on chamber 2 CH1 NORMALIZATION Step no 15 Time 0 Xo 0 00 um Yo 48 13 mN Force 53 14 mN GO BACK To many steps to normalize GO BACK and check parameter NOTE It is ONLY possible to perform the automated normalization on one chamber at a time 19 Therefore the working process should be Mount chamber 1 and start the automated normalization Mount chamber 2 and start the automa...

Page 24: ...fore use Instructions for making the necessary solutions are described in appendix 1 Stimulus 1 2 KPSS 10 μM NA Stimulate for 3 minutes Wash out 4 x with PSS Wait 5 minutes Stimulus 3 PSS 10 μM NA Stimulate for 3 minutes Wash out 4 x with PSS Wait 5 minutes Stimulus 4 KPSS Stimulate for 3 minutes Wash out 4 x with PSS Wait 5 minutes Stimulus 5 KPSS 10 μM NA Stimulate for 3 minutes Wash out 4 x wit...

Page 25: ...r vessel types 3 5 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 dam...

Page 26: ...at mesenteric arteries are densely innervated by sympathetic nerves which have a highly efficient re uptake mechanism that removes noradrenaline from the neuromuscular junction The re uptake 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 e...

Page 27: ... 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 µM Volume of stock solution to add to chamber 0 1 5 μL of 10 4 M 0 3 1 μL of 10 3 M 0 5 1 μL of 10 3 M 1 2 5 μL of 10 3 M 1 3 1 5 μL of 10 3 M 1 5 1 μL of 10 3 M 3 7 5 μL of 10 3 M 5 1 μL of 10 2 M 10 2 5 μL of 10 2 M In calculating the NA in the Wire Myogr...

Page 28: ...aline 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 underestimation 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 conce...

Page 29: ...f 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 3 7 2 PROTOCOL Prepare the following stock solutions Acetylcholine 10 4 10 3 10 2 M Noradrenaline 10 2 M ACh in chamber µM Volume of stock solution to add to chamber 0 1 5 μL of 10 4 M 0 3 1 μL of 10 3 M 0 5 1 μL of 10 3 M 1 2 5 ...

Page 30: ...lled water 6 If acids such as 1M HCl and 3M HNO3 are used to clean the chambers make sure ALL surfaces are thoroughly dried after copious washes with double distilled water Any residual acid will cause corrosion of the stainless steel jaws and pins CHAPTER 4 CLEANING AND MAINTENANCE 4 1 CLEANING THE 630MA MYOGRAPH SYSTEM At the end of each experiment use the following procedure to clean the Wire M...

Page 31: ...b stick 12 Wash the chamber and supports several times with double distilled water 13 Dry the surfaces using absorbent paper i e Kim Wipes or cotton tip applicators If red or brown discolorations appear on the chamber sides or on the supports the following cleaning procedure will work in most cases IMPORTANT Be very careful using HCl or HNO3 because these acids may cause extreme damage to the stai...

Page 32: ...ducers As a part of daily maintenance inspect the grease around the transducer pin extending from the transducer housing pinhole before starting any experiment see figure 4 1 Insufficient grease in this area will allow buffer and water to enter the transducer housing and causing damage to the force transducer IMPORTANT DMT recommends that the high vacuum grease sealing the transducer pinhole is ch...

Page 33: ...d by ground of the system using the ground Connection on the back side of the 630MA system During the new calibration monitor the relative force reading values in the Force Calibration sub menu on the Multi Interface The normal operating values for the force transducer during calibration should be between 3000 and 3500 If the value is 0 a single digit or a three digit number the force transducer i...

Page 34: ...ansducer connector marked with the blue box in figure 4 4 5 Lift carefully the broken transducer and discard it Figure 4 4 6 The replacement transducer will be shipped with the new transducer inside a new transducer house 7 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 8 Drag the transducer connector through...

Page 35: ... 4 3 The 8 screws that secure the transducer house to the chamber 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 36: ...the black covers for grease 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 5 Myograph unit screws for changing supports and coarse adjustment of the jaws ...

Page 37: ...erilized solution can be stored in the refrigerator for up to 3 months 2 Dissolve all the chemicals except the CaCl2 in approximately 80 of the desired final volume of double distilled H2 O while being constantly stirred For example if 1 liter 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 PS...

Page 38: ...approximately 80 of the desired final volume of double distilled H2 O while being constantly stirred For example if 1 liter 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 liter of buffer Continue to stir the PSS while the CaCl2 is being add...

Page 39: ...lized solution can be stored in the refrigerator for up to 3 months 2 Dissolve all the chemicals except the CaCl2 in approximately 80 of the desired final volume of double distilled H2 O while being constantly stirred For example if 1 liter 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 b...

Page 40: ...ely 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 where δ is the microscope eyepiece reticule calibration factor in mm per division and a measuring the length of the mounted vessel segment The internal circumference of the mounted vessel at the i th reading is calculated by ICi IC0 2 Xi X0 where ...

Page 41: ...g to 100 mmHg is used to calculate the IC100 value from the point of interception between the function of the exponential curve and the function of the 100 mmHg isobar The normalized internal circumference IC1 is calculated by multiplying the internal circumference corresponding to 100 mmHg IC100 by a factor k The factor is for rat mesenteric arteries 0 9 Again this value should be optimized for t...

Page 42: ...ch 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 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 t...

Page 43: ...ontal line on the sleeve A Reading on sleeve B One additional mark visible C Thimble reading Total reading Figure A2 3 Example 2 reading 16780 µm 16000 µm 500 µm 280 µm 16780 µm Example 1 1 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 thim...

Page 44: ...Danish Myo Technology A S E mail sales dmt dk Tel 45 87 41 11 00 Fax 45 87 41 11 01 DMT USA Inc E mail sales dmt usa com Tel 1 734 707 0250 Fax 1 678 302 7013 ...

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