Application of a Novel Microfluidic Device for Studying Microvascular Blood Flow Regulation In Vivo

Objective Validate the efficacy of a novel liquid microfluidic device (LMFD) to manipulate microvascular blood flow by maintaining a fixed solute concentration, temperature, and physiologic gas conditions in a microscale region of tissue while simultaneously allowing for visualization and quantification of vascular geometry and hemodynamics. Methods LMFD flow channels were moulded in polydimethylsiloxane (PDMS) using soft lithography techniques. A diffusive exchange window was created by patterning a 600×300 μm laser cut micro‐outlet into a glass coverslip. Cured PDMS flow channels were plasma bound to both the coverslip containing the micro‐outlet, and a glass slide to produce a complete LMFD. Ten Sprague‐Dawley rats (149–197g) were anesthetized with sodium pentobarbital; indwelling cannulas were placed into the left common carotid and right jugular vein for systemic monitoring, anaesthetic administration and fluid resuscitation; animals were then tracheotomized and mechanically ventilated. The extensor digitorum longus muscle of the lower hind‐limb was carefully dissected, externalized, and reflected across the LMFD secured in the stage of an inverted Olympus IX73 microscope. The muscle was allowed to equilibrate for 30 minutes while the LMFD was perfused with buffered Krebs solution at 37□. Logarithmic doses (10 −8 M to 10 −3 M) of adenosine triphosphate (ATP), acetylcholine (Ach), and phenylephrine (PE) were sequentially administered to the muscle via perfusion through the LMFD. The LMFD was perfused with each drug concentration for 5 minutes prior to data collection. Video of microvascular blood flow was recorded from multiple focal planes within the volume directly overlying the micro‐outlet. Recordings were analyzed offline to measure velocity, hematocrit, and supply rate. All animal protocols were approved by Memorial University's Institutional Animal Care Committee. Results ATP significantly increased red blood cell supply rate (RBC SR) to 36.1 and 36.5 cell/s at concentrations of 10 −4 and 10 −3 M respectively (p < 0.01) compared to the baseline level of 14.4 cell/s. Increasing concentrations of PE caused a graded change in RBC SR with significant decreases to 9.1, 3.5, and 0.8 cells/s at 10 −5 , 10 −4 , 10 −3 M respectively (p < 0.02). Changes in perfusion were observed only in areas directly overlying the micro‐outlet and within 200–300 μm of the window. Functional images generated at the highest drug concentrations showed that microvascular perfusion was unaffected in regions at greater distances from the micro‐outlet. Evan's Blue dye was used to confirm there were no leaks around the exchange window and that only tissue overlying the micro‐outlet was directly exposed to solutions perfusing the LMFD. Conclusions Our novel LMFD allows for a controlled, continuous delivery of dissolved substances to highly constrained regions of microvasculature while simultaneously allowing visualization and measurement of blood flow within discrete vessels and networks. Blood flow changes can be directly observed in the region of interest while the surrounding vasculature is unaffected thus preserving integrated regulation to the tissue as a whole. Support or Funding Information Project funded through a NSERC Discovery grant to GM Fraser. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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