Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Medical Biophysics

Collaborative Specialization

Scientific Computing

Supervisor

Ellis, Christopher G.

2nd Supervisor

Fraser, Graham M.

Co-Supervisor

Abstract

The purpose of this thesis was to develop tools for studying oxygen-dependent regulation of red blood cell (RBC) flow distribution in the microcirculation. At the microvascular level, arterioles dictate the distribution of oxygen (O2) carrying RBCs to downstream capillaries, a process which needs to be tightly regulated and coupled to O2 off loading from capillaries to the tissue. To investigate potential regulatory mechanisms, an O2 exchange platform was developed to manipulate the RBC hemoglobin O2 saturation (SO2) at the muscle surface while limiting the changes in SO2 to only a single capillary network. Decreasing SO2 in a single capillary network resulted in an increase in supply rate, while increasing SO2 caused a decrease in supply rate. This finding is consistent with our hypothesis that ATP released in capillaries in response to low SO2 is responsible for vasodilation of upstream arterioles to regulate blood flow. To determine whether the dynamics of ATP was fast enough to enable RBC signalling in capillaries, an in vitro microfluidic system was developed to generate a rapid decrease in RBC SO2. The feasibility of this experimental design was first tested computationally using a mathematical model that consisted of blood flow, oxygen and ATP transport as well as a model for hemoglobin binding, ATP release, ATP/luciferin/luciferase reaction and digital camera light detection. The model demonstrated that the concept was theoretically feasible and yielded important insights such as the signal sensitivity to flow rate. The model further revealed that measured light intensity levels would not be directly related to ATP concentrations, thus, care must be taken when interpreting the data. It was determined that the microfluidic device would be fabricated using soft lithography techniques that resulted in a device that differed significantly from our original theoretical design since all of the layers would be oxygen permeable except for a glass coverslip with a small opening for gas exchange between the liquid and gas channel. To optimize the geometric design of this microfluidic device, to maximize the desaturation the RBCs, a finite element model was developed. Based on this design a device was constructed. To test whether the design generated a rapid decrease in RBC SO2, a low hematocrit high SO2 RBC suspension was perfused through the device exposed to 95% N2 and 5% CO2 in the gas channel. Finally, to overcome challenges with existing approaches for measuring SO2 in the device, a novel image analysis technique using digital inpainting was developed. The inpainting approach demonstrated a rapid change in RBC SO2 at the entrance to the window, thus the microfluidic device is ready to be used to measure the dynamics of O2-dependent ATP release from RBCs. The new inpainting algorithm was also applied to in vivo video sequences where it was shown to provide more accurate SO2 measurements and to work under conditions where existing approaches fail. In summary, this thesis provides a set of in vivo, in vitro and computational tools that can be used to study the mechanisms of SO2-dependent regulation of the microvascular blood flow.

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