Transcript Design of an in vivo Technique to Measure Capillary

Technique for Measuring
Capillary Recruitment
Stephanie Beatrous
Snehal Patel
Samar Sharma
Advisor:
David Wasserman, Ph.D.
Muscle Glucose Uptake

Muscle Glucose Uptake (MGU) is a linear
sequence of three processes.
–
–
–
Glucose delivery from blood to the interstitial
space
Glucose transport from interstitial space to the
intercellular environment.
Glucose phosphorylation by Hexokinase II
Significance of MGU
Glucose plays a role in cell function by
serving as an energy source.
 Each of the three processes in MGU
determine whether cellular needs for energy
source are met.
 As MGU is controlled by many
physiological subsystems, alterations could
lead to pathology, such as diabetes.

Muscle Glucose Delivery
Skeletal muscle blood flow
 Capillary recruitment
 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

Regulation of Microcirculation
QM= Blood Flow to Muscle
QC= Blood Flow per Capillary
NC= Number of Perfused Capillaries
VC= Blood Velocity
AC= Capillary Cross Sectional Area
QM=QCNC=VCACNC


Blood flow to the muscles is directly proportional
to the number of perfused capillaries (NC)
Surface area for glucose exchange is proportional
to NC, so a higher blood velocity will not be as
efficient
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.
Protocol Summary







1.1 ug/mL saline solution of Rhodamine B-Dextran T70
infused at 1.375 uL/min until equilibrium reached
Catheterized mouse anesthetized using Methophane
Surgery performed, vasus lateralus muscle exposed on
right leg
Mouse mounted on confocal microscope
Norepinephrine injected to vasoconstrict
50 uL sodium nitroproside (SNP) injected to vasodilate
Observations made and images collected
Orienting the Mouse on Plate
Approach Technique
Fluorescence Microscopy
Infusion Set-up
Technique Timeline
Results







Sequence of .tiff images
.tiff images compiled into .avi videos
Illustrates microvasculature
Evidence of capillary blood flow
Erythrocyte movement imaged
Data can be used to find blood velocity
Can determine relative percentage of perfused
capillaries
Capillary Images
Design of Plate
Future Plans
Formalize protocol
 Quantify the results

–
–

Calculate blood velocity
Measure recruitment
Test mouse plate
Project Significance
Factors that determine insulin resistance or
insulin sensitivity in vivo may be identified
 Sites involved in the regulation of insulin
action in vivo may be determined
 Therapies can be designed to target the sites
of dysfunction
 Insulin resistance treated more effectively
