Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and K_ATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.     

EFFECTS OF MEAL SIZE ON OTOLITH RECOVERY FROM FECAL SAMPLES OF GRAY AND HARBOR SEAL PUPS

Recovered otoliths from pinniped feces provide valuable information on diet composition and prey size. We studied the effect of meal size on otolith recovery from the feces of one harbor and eight gray seal pups. Each of 11 experiments comprised a half-ration meal, a period of fecal collection, a 1.5-or double-ration meal again followed by a period of fecal collection. A significantly lower percentage of Atlantic herring otoliths were recovered from half-ration meals (25%± 12.5% in the harbor seal, 8.6%± 6.9% in eight gray seals) than from 1.5- or double-ration meals (62.5%± 3.1 % in the harbor seal, 32.8%± 23.5% in gray seals). Meal size also significantly affected the percentage of Atlantic cod otoliths recovered from gray seal feces (65.0%± 26.3% from half ration, 98.3%± 2.9% from 1.5 ration). For both size meals, recovered cod otoliths were more significantly eroded than herring otoliths. The development of correction factors to account for the effects of digestion will need to consider the distribution of meal sizes of free-ranging pinnipeds.     

Divergent compensatory growth responses within species: linked to contrasting migrations in salmon?

Animals often exhibit accelerated or “compensatory” growth (CG) after periods of environmentally induced growth depression, raising important questions about how they cope with environmental variability. We tested an underexplored hypothesis regarding the evolutionary consequences of CG; namely, that natural populations differ in CG responses. Common-garden experiments were used to compare subadult growth following food restriction between groups (control, treatment) of two Atlantic salmon (Salmo salar) populations and their first-generation (F₁) hybrids. The populations are found at similar latitudes but characterized by differences in migration distance. We predicted that long-distance migrants would better maintain growth trajectories following food restriction than short-distance migrants because they: (1) require larger body sizes to offset energetic costs of migration and (2) face greater time constraints for growth as they must leave non-breeding areas earlier to return to breeding areas. Long-distance migrants grew faster, achieved quicker CG (relative to controls), and their overall body morphology was more streamlined (a trait known to improve swimming efficiency) than slower growing short-distance migrants. F₁ hybrids were generally intermediate in “normal” growth, CG, and body morphology. We concluded that CG responses may differ considerably among populations and that the conditions generating them are likely interconnected with selection on a suite of other traits.     

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo . The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo . The device can be sterilized for cell culture using common methods without loss of function. The device''s applicability to studying the in vitro wound healing response will be demonstrated.Open in a separate windowClick here to view.^{(69M, flv)}     

Brain slice stimulation using a microfluidic network and standard perfusion chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.