1. Determinants of Muscle Glucose Delivery (MGU)
Skeletal muscle blood flow
Endothelial permeability to glucose
Capillary recruitment
An increase in the number of perfused capillaries is known as capillary recruitment
10% of capillaries are perfused in resting skeletal muscles
Capillary density is a function of metabolic requirements

2. Project Objective
To design a technique to measure capillary recruitment in vivo in the skeletal muscle microvasculature of mice for the purpose of investigating basal and insulin-stimulated MGU.

3. Need for Technique
Size of capillaries (~8mm) limits study of recruitment
No commercial in vivo technique available to measure capillary recruitment
Current technique (fluorescent microspheres) is insufficient
Time consuming and difficult
Results are inconsistent
Indirect, not real time
To assess muscle glucose delivery

4. Protocol Summary Catheterized mouse anesthetized with Metaphane.
Superficial vastus lateralis muscle exposed on right leg.
Mouse mounted on confocal microscope using stereotaxic magnetic plate.
1.1 mg/mL Rhodamine B-Dextran T70 in saline infused at mL/min for 30min.
Norepinephrine (NE) infused at mL/min for 30min.
Sodium nitroprusside (SNP) infused at mL/min for 30min.
Observations made and images collected.
Assessment of capillary recruitment.

5. Choosing Optimal Materials
Materials Needed
Materials Chosen
Reason
Fluorophore
Rhodamine B
1. Excitation/emission do not significantly overlap w/muscle absorption band.
2. High quantum yield (Q~0.5)
Conjugate
Dextran T70
1. Allows capillary confinement
2. High soluability
3. Little quenching
Anesthesia
Metaphane
1. Easily administered inhalant
2. Minor effect on blood flow
Vasoconstrictor
Norepinephrine (NE)
1. Constrictor to muscle
2. Available in lab
Vasodilator
Sodium Nitroprusside (SNP)
1. Dilator to muscle
Microscope
Confocal
1. Allows depth of ~150mm
2. Available at Vanderbilt
3. Allows slicing and 3D-reconstruction

6. Confocal Microscopy Hardware: Zeiss LSM410 Confocal Laser
Scanning Inverted Microscope with He/Ne laser
Software: 3D reconstruction and topographical analysis software
Operating Parameters:
Excitation wavelength: 543nm (green)
XY-Resolution: mm
Z-Resolution: 4.1mm
Depth: 100mm
Magnification: 20x
Image size: 512 x 512 pixels
Operation:
1. Pinhole aperture blocks out-of-focus planes
2. Sample scanned in multiple planes (“slices”)
3. 3D image developed from “slices”.

7. Stereotaxic Magnetic Plate

8. Orientation of Mouse on the Mouse Plate

9. Infusion Set-up and Timeline

10. Red Blood Cell Velocity
100mm
100mm
1.8 s
Velocity vs. Time 5 10 15 20 25 30 35
0.5 1
1.5
Time (s)
Velocity (mm/s)

11. Recruitment Assessment
NE Vasoconstriction
100mm
SNP Vasodilation
100mm
500
1000
1500
2000 50
100
150
200
250
Pixel Frequency
Intensity Histogram
Intensity (Arbitrary Units)
500
1000
1500
2000 50
100
150
200
250
Pixel Frequency
Intensity Histogram
Intensity (Arbitrary Units)

12. Accomplishments Imaging technique identified for in vivo measurement
Technique for in vivo muscle exposure designed
Anesthesia that is least affecting to blood flow identified
Optimal fluorophore identified
Mouse plate designed for imaging
Vasodilation/vasoconstriction times quantified
Blood velocity quantified
Complete technique to measure capillary recruitment designed
Capillary recruitment measured in vivo