1. welcome

2. Wu Liping 2004-6-4

3. Background   The technique was first developed for investigating small vessels with internal diameters down to around 100 μm by Mulcany & Halpern, in 1976,1977   Multi Myograph System – 610M is suited to vessels with internal diameters of 100-400 μm   At least 50% of the precapillary pressure drop occurs in the vessels with internal diameters than 100 μm ( proximal resistance vessels)

4. General Principles   Opening order: powerlab myograph software   Closing order: software myograph powerlab   Line connection: one-to-one correspondingly

5. General Principles   Buttons’ function: : main options (zero/calibrate/temperature, etc.) : number changing (decrease/increase) F1 : channel selecting F2 : detailed operation

6. General Principles   Zero before experiments: F = 9.81 m N 2 g weight Calibration bridge tip wire

7. solutions MW PSS 2.5 K-PSS 2.5 g/molmmol/lg/lmmol/lg/l NaCl58.441196.95400 KCl74.564.70.35123.79.223 CaCl 2 ● 2H 2 O 147.022.50.3682.50.368 MgSO 4 ● 7H 2 O 246.681.170.2891.170.289 NaHCO 3 84.01252.1252.1 KH 2 PO 4 136.11.180.1611.180.161 EDTA372.240.0270.0100.0270.010 Glucose198.775.51.0915.51.091 NAK: KPSS+NE 10 μ M NAPSS: PSS+NE 10 μM

8. dissection   The dissection of mesenteric small ateries from rats at autopsy mid-line laparotomy to exteriorize mesenteric bed Use scissors to remove 10cm of intestine together with its feeding vasculature, including part of the superior mesenteric artery. Proximal end of intestinal section should be 10cm from pylorus Example Place excised section in Petri dish containing PSS. Petri dish contains a 5 mm thick layer of Sylgaard to hold fixing pins.

9. dissection Pin down the proximal end of the intestine on the left hand side of the dish, and pin out the remainder of the intestine in an anticlockwise direction Dissect out the test segment (normally the third branch) together with a piece of the proximal branch Using ocular dissection scissors, cut through the mesenteric membrane along either side of vessel, about 1-2mm from the vessel

10. dissection Dissect away the vein (distinguish from the pattern of the branch points) Clean artery of connective tissue by holding the connective tissue with forceps and pulling gently away from the artery Cut distal end of vessel to be investigated and cut unwanted arcade 2mm from feeder artery, which are to be used as a “handle” to move the vessel Cut proximal end of feeder artery

11. mounting   Caution! The procedure involves attaching the mounting wires to jaws which are in turn mounted on the force transducer ( sensitivity- 0.01 mN (1mg) ; upper limit- 1N (100g) )   So ! Do not press the jaws too hard together: a movement of 50mm after they have touched is sufficient to hold the wires clamped

12. mounting   Attaching the first mounting wire Clamp the wire between two jaws Wrap the far end of the wire around under fixing screw clockwise and tighten the screw Fill myograph with PSS at room temperature jaw screw

13. mounting   Mounting of the vessel Hold the “handle” segment of the excised arcade and try to mount the vessel onto the proximal end of wire. Pull the proximal end of excised arcade along wire until the vessel to be investigated is situated between the mounting jaws Screw jaws together to clamp the wire. Wrap the near end of wire around under near fixing screw again in a clockwise direction

14. mounting Using forceps, gently rub vessel on the far side of the jaw and sever the vessel so that the portion of the excised arcade on the far side of the jaw can be pulled away Screw jaws apart, take a second wire and align it parallel with the vessel, passing into the far end of the lumen Screw jaws together. Ensure that the second wire moves under the first. Wrap the ends of wire like the former Screw jaws apart so that the wires should be levelled Connect oxygen line to chamber and start heating

15. Normalization T : the myograph vessel supports had been moved together (the mounting wires just touched) B : the vessel supports were moved slightly apart, so that the record at B shows zero tension 1, 2, 3, and 4 : the vessel was slowly stretched, so that the records show the forces 1 min after each stretch. At 4, the vessel supports were again moved together to release some of the resting tension N : the vessel internal circumference was set to IC 1 (the internal circumference at which the active force production of the vessel is maximal)

16. Normalization IC 100 : the vessel’s internal circumference when it is fully relaxed and under a transmural pressure of 100mmHg P i : effective pressure, p i = wall tension/(IC/2  ) T : wall tension, T = pi*(IC/2  ) IC 1 : 0.9* IC 100 Normalized lumen diameter is taken as I 1 = IC 1 / 

17. Measurement of responses   The response of a vessel to an agonist is normally presented in terms of the change in wall force above the resting force. F resting = alpha*(reading resting -reading baseline ) F agonist = alpha*(reading agonist -reading baseline ) Alpha is the force transducer calibration (mN/recorder division) i.e. the force response ΔF=F agonist – F resting ΔF = alpha*(reading agonist -reading resting )

18. removing Endothelium   Purpose: To remove the endothelium from an artery, trying to eliminate endothelial effects on the rest of the vessel To rule out the influence of the endothelium on a certain mechanism To confirm the role of the endothelium in the mechanism

19. removing Endothelium   Procedure: First, fix the vessel and check the endothelium function Set the vessel to a small tension (0.5mN) Use a coarse pair of forceps to hold a human hair (clean, round, straight, thick), with a large microscope- magnification to look closely at the artery Enter the tip of hair into the vessel lumen, and work down through the hole inner surface of the vessel rubbing forwards and backwards

20. removing Endothelium   Procedure (cont.): Change to fresh solution in the chamber, and let the vessel equilibrate for 5-10 min Stretch the vessel to its normalized micrometer setting and let it relax for 5 min Check the endothelium-function again

21. Checking endothelium-function   Purpose: To check whether the relaxing function of the endothelium is intact after mounting a vessel To check the success of an endothelium-removal procedure which you have applied to a vessel

22. Checking endothelium-function   Principle: By stimulating a vessel with Acetylcholine (Ach.), the vessel will relax, following the release of EDRF (Endothelium Derived Relaxing Factor) from the endothelium. The procedure can be performed at any time after the heating and equilibration of the vessel.

23. Checking endothelium-function   Protocol 1 : Quick check-single dose Stimulate 4 min with NA 3 PSS (10ml ） Add 10  l stock B to the chamber to give 5*10 (-6 ） M Ach Wait 2 min Wash out 4 times with PSS 2.5

24. Checking endothelium-function   Protocol 2 : Ach-concentration response curve (Ach-CRC) Stimulate 4 min with NA 3 PSS (10ml ） Add 20  l stock E to the chamber to give 10 (-8 ） M Ach After 2 min, add 18  l stock D to the chamber to give 10 (-7 ） M Ach Repeat 3) with stock C and B to give 10 (-6) and 10 (-5 ） M Ach After 2 min, add 90  l stock A to the chamber, to give 10 (-4 ） M Ach Wait 2 min Wash out 4 times with PSS 2.5

25. Checking endothelium-function   Solutions: NA3PSS=PSS2.5+noradrenaline 3 mM Stock A=Acetylcholine (Ach) 10(-2)M (stock solution in freezer) Stock B= Ach 5* 10(-3)M. Made by mixing stock A and water 1:1 Stock C= Ach 5* 10(-4)M. Made by diluting stock B 10- fold with water Stock D= Ach 5* 10(-5)M. Made by diluting stock C 10- fold with water Stock E= Ach 5* 10(-6)M. Made by diluting stock D 10- fold with water

26. That’s all, thank you