1. Stephanie Beatrous Snehal Patel Samar Sharma Advised by: David Wasserman, Ph.D. Measurement of Capillary Recruitment

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

3. 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

4. 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. 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

7. 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.

8. Diabetes n Diabetes is fundamentally a disorder of MGU. – 16million Americans – 98billion dollars in costs – 18% of deaths (>25yrs old). – 7th leading cause of death in 1993 – pharminfo.com

9. Jablonski Diagram

10. Methods n 1.1 ug/mL saline solution of TX Red-Dextran T70 infused at 1.375 uL/min n Dye solution equilibrated in mouse after 100 min n Catheterized mouse anesthetized using Nambitol n Surgery performed, vasus lateralus muscle exposed on right leg n Mouse mounted on confocal microscope n 50 uL sodium nitroproside (SNP) injected to vasodilate n Observations made and images collected

11. Results n “Honeycomb” structure imaged n Capillary flow evident in structure

12. Capillary Images

13. Past Work n Research of current imaging techniques – Optical Coherence Tomography (OCT) – Intravital Microscopy – Microspheres – Functional Magnetic Resonance Imaging (fMRI) – Fluorescent Microscopy – Two-photon Excitation Microscopy – Whole-Field Microscopy – Widefield Imaging – Confocal Microscopy

14. Current Status n Research almost complete – Need to continue assessing feasibility of methods n Most Promising method identified – Fluorescent Microscopy

15. Future Plans n Test Fluorescent Microscopy on 2/16/01 using the confocal microscope and implemented technique. n Adapt this technique to study MGU. n If adaptation unsuccessful, use Optical Coherence Tomography (OCT)

16. Future Plans (cont.) n Test variations in the technique n Refine protocol for measuring capillary recruitment n Obtain images and assess quality of results