1. Stephanie Beatrous Snehal Patel Samar Sharma Advisor: David Wasserman, Ph.D. Technique for Measuring Capillary Recruitment

2. Muscle Glucose Uptake n 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

3. Significance of MGU n Glucose plays a role in cell function by serving as an energy source. n Each of the three processes in MGU determine whether cellular needs for energy source are met. n As MGU is controlled by many physiological subsystems, alterations could lead to pathology, such as diabetes.

4. Muscle Glucose Delivery n Skeletal muscle blood flow n Capillary recruitment n Endothelial permeability to glucose

5. Capillary Recruitment n An increase in the number of perfused capillaries is known as capillary recruitment n 10% of capillaries are perfused in resting skeletal muscles n Capillary density is a function of metabolic requirements

6. Regulation of Microcirculation Q M = Blood Flow to Muscle Q C = Blood Flow per Capillary N C = Number of Perfused Capillaries V C = Blood Velocity AC= Capillary Cross Sectional Area Q M =Q C N C =V C A C N C n Blood flow to the muscles is directly proportional to the number of perfused capillaries (N C ) n Surface area for glucose exchange is proportional to N C, so a higher blood velocity will not be as efficient

7. Project Objective n 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.

8. Protocol Summary n 1.1 ug/mL saline solution of Rhodamine B-Dextran T70 infused at 1.375 uL/min until equilibrium reached n Catheterized mouse anesthetized using Methophane n Surgery performed, vasus lateralus muscle exposed on right leg n Mouse mounted on confocal microscope n Norepinephrine injected to vasoconstrict n 50 uL sodium nitroproside (SNP) injected to vasodilate n Observations made and images collected

9. Orienting the Mouse on Plate

10. Approach Technique Fluorescence Microscopy

11. Infusion Set-up

12. Technique Timeline

13. Results n Sequence of.tiff images n.tiff images compiled into.avi videos n Illustrates microvasculature n Evidence of capillary blood flow n Erythrocyte movement imaged n Data can be used to find blood velocity n Can determine relative percentage of perfused capillaries

14. Capillary Images

15. Design of Plate

16. Future Plans n Formalize protocol n Quantify the results – Calculate blood velocity – Measure recruitment n Test mouse plate

17. Project Significance n Factors that determine insulin resistance or insulin sensitivity in vivo may be identified n Sites involved in the regulation of insulin action in vivo may be determined n Therapies can be designed to target the sites of dysfunction n Insulin resistance treated more effectively