The goal of this webinar was to provide an overview of the essential tools and techniques required for successful isolation of blood vessels and study in pressurized arteriograph systems. Presenters review dissection and mounting techniques, equipment requirements, proper experimental design, and discuss the difference between pressurized cannulation systems and wire myograph/tissue bath systems. Dr. Scott Earley shares strategies for skills development, in particular starting research with 2nd order mesenteric artery sample and how to generate vessel constrictions and dilations. In addition, he discusses an advanced application involving the assessment of myogenic reactivity in a "blind-sac" vessel preparation using a cerebral parenchymal arteriole. Key Topics: - equipment needs and proper operation - how to design an experiment to assess blood vessel function in-vitro - techniques for vessel dissection, mounting, and assessing viability - how to interpret experimental results - studying myogenic responses using a cerebral parenchymal arteriole

1. Getting Started with
In-Vitro Blood Vessel
Research

2. Getting Started with In-Vitro Blood Vessel Research
1. History of the Pressurized Arteriograph (Gerry Herrera)
2. Anatomy of an Arteriograph System (Gerry Herrera)
3. Introduction to Research Applications and Methodology (Scott Earley)
4. Getting Started: (Scott Earley)
• KCl and PE induced constriction
• ACh-induced dilation
• Myogenic constriction
5. Tips & Troubleshooting (Scott Earley)
6. Q&A Session (Scott Earley & Gerry Herrera)

3. InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.

4. Getting Started with In-Vitro
Blood Vessel Research
Gerry Herrera, PhD
President,
Catamount Research &
Development Inc.,
Living Systems Instrumentation
Copyright 2015 InsideScientific & Living Systems Instrumentation. All Rights Reserved.

5. The Halpern/Mulvany Wire Myograph
Pioneering The Way…

6. The Halpern/Mulvany Wire Myograph
Pioneering The Way…

7. The Halpern/Mulvany Wire Myograph
Pioneering The Way…

8. The First Arteriograph for In Vitro Vessel Research

9. The First Arteriograph for In Vitro Vessel Research

10. Automating Data Collection: Edge Detection

11. Automating Data Collection: Edge Detection

12. Automating Data Collection: Edge Detection

13. Anatomy of a Pressurized Arteriograph System
To be modified…
Constant Pressure
• The vast majority of
applications are well-served by
constant pressure, no
intraluminal flow setup
• Distal end of vessel is occluded
• Key components include a
pressure source, vessel
chamber, means for
temperature control, and data
acquisition hardware/software

14. Perfused Resistance Vessels
Advanced Application:
• It is also possible to measure vascular
responses at either constant or
variable intraluminal flow rate

15. Anatomy of a Pressurized Arteriograph System
To be modified…
Constant Pressure
& Flow
• Setup for intravascular flow is a bit
more complex
• Distal end of vessel remains open
• In addition to the basic items for
regulating intravascular pressure,
you will also need a pump to control
intraluminal solution flow and a way
to monitor pressure on both sides of
the cannulated vessel

16. Shown with optional thermistor (THRS)
• Vessel is mounted
between the cannulae
and tied in place with
nylon thread (provided
with the chamber)
• Blood is flushed out of
the vessel
• Superfusion of desired
buffer into chamber and
into vessel
Parts of a Pressure Arteriograph
Click Here for
Chamber Information

17. Additional Information on Equipment Required for
Setting up a Pressurized Arteriograph System
• Vessel Chambers
• Specialty Chambers
• Myographs
• Pressure Control
• Flow Control
• Video Analyzers
• Temperature Control
• Aeration & Oxygenation
• pH Measurements
• Dissection Tools
• Microscopes
• Data Acquisition

18. Getting Started with In-Vitro
Blood Vessel Research
Scott Earley, PhD
Associate Professor of
Pharmacology,
University of Nevada School of
Medicine
Copyright 2015 InsideScientific & Scott Earley. All Rights Reserved.

19. What kinds of studies can we do using Pressurized
Arteriograph Systems?
1. Smooth Muscle Cell
Contractility
2. Endothelial Function
3. Vessel Structure:
Wall Thickness & Remodeling

20. 1. Basic Equipment and Procedures
2. Example Experiments:
• KCl- and PE-induced constriction of mouse mesenteric arteries
• ACh-induced dilation of mouse mesenteric arteries
• Myogenic constriction - mouse cerebral parenchymal
arterioles
3. Tips and Troubleshooting
What are we going to cover today?

21. How To Get Started

22. • MOPS buffered Saline
• Physiological Saline
Solution (PSS)
• High K+ PSS
• Ca2+-free PSS
• Prepare all solutions the
day of the experiment
Prepare the Solutions

23. Physiological Solutions
Recipes for solutions that Dr. Earley uses can be found in his
publications. Here, we provide a few saline formulations that can be
useful specifically for cases in which you cannot bubble your saline
solution – for example, when you need to keep the bath solution stagnant
without superfusion to minimize amounts of expensive drugs being added.
In most other cases, you should use a bicarbonate buffered saline in which
you bubble the saline with at CO2 mixture to get pH control. There are
many recipes for bicarbonate buffered saline in the literature.

24. Physiological Solutions
The following saline formulations are buffered by HEPES, including the HEPES-bicarbonate recipe.
These solutions do not require bubbling.
• HEPES-PSS Solution: This is a general purpose saline.
• High K+ (40 mM) HEPES: This is a saline in which K+ concentration has been increased to ~40 mM by
equimolar substitution with Na+. Use this for eliciting K+-induced vasoconstrictions. Some labs increase
K+ to 60 mM. If you do this, make sure to use equimolar substitution of K+ and Na+ to avoid osmolalility
problems.
• 0 Ca2+ HEPES: This is a saline used to monitor the passive responses of the vessel. Ca2+ has been
removed and a Ca2+ chelator (EGTA) has been added.
• HEPES-Bicarbonate Solution: This is a general purpose saline. Many labs prefer to have bicarbonate
present in the saline to maintain cellular bicarbonate transporter function. If you use this solution as a
standard solution, you can also prepare High K+ and 0 Ca2+ versions.

25. HEPES-PSS Solution
Compound M.W. mM g/L
NaCl 58.44 141.9 8.29
KCl 74.55 4.7 0.35
MgSO4 (7H2O) 246.28 1.7 0.42
EDTA 292.25 0.5 0.15
CaCl2 (2H2O) 147.02 2.8 0.41
HEPES 238.30 10.0 2.38
KH2PO4 136.09 1.2 0.16
Glucose 180.16 5.0 0.90
Titrate to pH 7.4 with 10 N NaOH. Prepare fresh daily.
(This buffer does not require bubbling)

26. High K+ (40 mM) HEPES
Compound M.W. mM g/L
NaCl 58.44 95.8 5.60
KCl 74.55 38.0 2.84
MgSO4 (7H2O) 246.28 1.7 0.42
EDTA 292.25 0.5 0.15
CaCl2 (2H2O) 147.02 2.8 0.41
HEPES 238.30 10.0 2.38
KH2PO4 136.09 1.2 0.16
Glucose 180.16 5.0 0.90
Use this equimolar substitution of KCl and NaCl. All other reagent amounts stay the same. Titrate to pH 7.4 with 10 N NaOH .
Prepare fresh daily.
(This buffer does not require bubbling)

27. 0 Ca2+ HEPES
Compound M.W. mM g/L
NaCl 58.44 141.9 8.29
KCl 74.55 4.7 0.35
MgSO4 (7H2O) 246.28 1.7 0.42
EDTA 292.25 0.5 0.15
EGTA 380.40 2.0 0.75
HEPES 238.30 10 2.38
KH2PO4 136.09 1.2 0.16
Glucose 180.16 5.0 0.90
This formula is Identical to HEPES-PSS, except CaCl2 is replaced by EGTA. Titrate to pH 7.4 with 10 N NaOH . Prepare fresh daily.
(This buffer does not require bubbling)

28. HEPES-Bicarbonate Solution (This buffer does not require bubbling)
Compound M.W. mM g/L
NaCl 58.44 130.00 7.60
KCl 74.55 4.00 0.30
MgSO4 (7H2O) 246.28 1.20 0.30
NaHCO3 84.00 4.00 0.34
CaCl2 (2H2O) 147.02 1.80 0.27
HEPES 238.30 10.0 2.38
KH2PO4 136.09 1.18 0.16
Glucose 180.16 6.00 1.08
EDTA 292.25 0.03 0.009
Osmolality before adding glucose is approximately 300 mOsm/L. Titrate to pH 7.4 with 10 N NaOH . Prepare fresh daily.

29. Isolating the Vessels
• Microscope
• Dissection dishes
• Dissection tools

30. Mesentery Brain
Isolating the Vessels

31. • Mesentery pinned out
and fat removed
• Veins and Arteries
Isolating the Vessels

32. • We weren’t able to come
up with any images for
this – Gerry, do you have
anything?
• ….
• ….
• ….
Making Vessel Ties
Mounting Vessels in the Pressure Myograph
Making Vessel Ties:
• Use multi-filament braided nylon fibers
• Cut to desired length (~5-10mm)
• Tease individual filaments apart –
use 1 filament per tie
• Make several ties and store them in a
covered petri-dish using double-sided
tape
• Pick up tie from end attached to tape,
NOT from the free end

33. • We weren’t able to come
up with any images for
this – Gerry, do you have
anything?
• ….
• ….
• ….
Attach proximal end
& flush out blood…
Attach distal end.
Mounting Vessels in the Pressure Myograph

34. Transfer Chamber to
Recording System
• Place chamber on the
microscope stage
• Connect inlet and outlet
superfusion
• Connect pressure
control system

35. Keeping the Vessels Alive and Happy
Intraluminal
Pressure
Temperature Flow Rate pH
Follow links for additional information on suggested equipment

36. Collecting Data
• Edge Detection
• Data Acquisition
System

37. The Complete System

38. Example Experiments

39. Example 1: KCl-Induced Constriction
• Expose the vessel to elevated
extracellular KCl (60 mM) – High
K+ PSS.
• Collapses the K+ gradient,
resulting in depolarization of the
smooth muscle cell plasma
membrane.
• Ca2+ influx through L-type Ca2+
channels causes vascular smooth
muscle contraction and
vasoconstriction.
K+
K+ K+
K+ K+K+
Ca2+
SMC

40. Vessel superfused with High K+ PSS:
Baseline Diameter High K+ PSS

41. Example 2: PE-Concentration Response Curve
• Phenylephrine (PE) binds to
the type-1 adrenergic
receptor (α1 receptor) on the
smooth muscle cell plasma
membrane.
• This causes an increase in
intracellular Ca2+, resulting in
smooth muscle cell
contraction and
vasoconstriction.
PE
Ca2+
SMC

42. 10-9 M PE 10-8 M PE 10-7 M PE 10-6 M PE 10--5 M PE
Vessel superfused with increasing concentrations of PE:

43. Effects of increasing PE concentration on vessel diameter:
• Recording of
luminal diameter
versus time as
increasing
concentrations of
PE are delivered
• When doing dose
response be sure to
allow the recording
to stabilize

44. Plot the Data…
10-9 10-8 10-7 10-6 10-5 10-4 10-3
0
20
40
60
[PE]
%Constriction
n=5
% Constriction
=
(Baseline Diameter – Diameter with PE)
Baseline Diameter

45. Example 3: ACh-Induced Relaxation (Endothelium Viability)
• Acetylcholine (ACh) binds to
muscarinic type-2 (M2) receptors
on the endothelial cell plasma
membrane.
• This causes the release of NO and
EDHF.
• These factors diffuse to the
underlying vascular smooth muscle
cells to decrease intracellular Ca2+
and cause dilation.
• Gold standard for intact endothelial
function.
Endothelial
cell
SMC
ACh
Ca2+
NO
EDHF

46. Baseline Diameter Pre-Constrict: 10-6 M PE 10-4 M ACh + 10-6 M PE
Example 3: ACh-Induced Relaxation (Endothelium Viability)

47. ACh
Example 3: ACh-Induced Relaxation (Endothelium Viability)
• Vessel was pre-constricted
with PE
• ACh was applied at the peak
of the steady-state PE-
constriction

48. Damaging the endothelium
Perfuse Vessel With AirBaseline Diameter

49. Following Endothelial Cell Damage:
Baseline diameter Pre-constrict 10-6 M PE 10-4 M ACh + 10-6 M PE

50. Plot the Data…
10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
0
50
100
150
[ACh]
%Vasodilation
Endothelium Intact
Endothelium Damaged
n=5
%Vasodilation
=
% Reversal of Preconstriction
=
[(Baseline – PE+ACh)/Baseline] –
[(Baseline – PE/Baseline)]
[(Baseline – PE)/Baseline]

51. Example 4: Myogenic Response – Cerebral Parenchymal Arterioles
• Record diameter during step-
wise increases in intraluminal
pressure
• Repeat while superfusing
vessels with Ca2+-free PSS to
determine the passive
diameter
• %Myogenic tone = [(Passive
Diameter – Active
Diameter)/Passive Diameter]
P (mmHg)
passive
active

52. • The learning curve – do the procedure in examples
1-3 (KCl-induced constriction, PE-concentration response curve,
ACh-induced dilation) using second order mesenteric arteries until
you get reproducible results.
• Work fast- remove organs and isolate vessels as quickly as possible
• Make solutions fresh each day.
• Stretching the vessel in the initial prep
• pH/O2/temperature
Tips & Pointers

53. Thank You!
For additional information on Pressure Arteriograph
Systems, Wire Myographs, and solutions for
studying vessel function in-vitro visit:
http://www.livingsys.com/index.html