Spreading depolarization suppression from inter-astrocytic gap junction blockade assessed with multimodal imaging and a novel wavefront detection scheme.

Spreading depolarizations (SDs) are an enigmatic and ubiquitous co-morbidity of neural dysfunction. SDs are propagating waves of local field depolarization and increased extracellular potassium. They increase the metabolic demand on brain tissue, resulting in changes in tissue blood flow, and are associated with adverse neurological consequences including stroke, epilepsy, neurotrauma, and migraine. Their occurrence is associated with poor patient prognosis through mechanisms which are only partially understood. Here we show in vivo that two (structurally dissimilar) drugs, which suppress astroglial gap junctional communication, can acutely suppress SDs. We found that mefloquine hydrochloride (MQH), administered IP, slowed the propagation of the SD potassium waveform and intermittently led to its suppression. The hemodynamic response was similarly delayed and intermittently suppressed. Furthermore, in instances where SD led to transient tissue swelling, MQH reduced observable tissue displacement. Administration of meclofenamic acid (MFA) IP was found to reduce blood flow, both proximal and distal, to the site of SD induction, preceding a large reduction in the amplitude of the SD-associated potassium wave. We introduce a novel image processing scheme for SD wavefront localization under low-contrast imaging conditions permitting full-field wavefront velocity mapping and wavefront parametrization. We found that MQH administration delayed SD wavefront's optical correlates. These two clinically used drugs, both gap junctional blockers found to distinctly suppress SDs, may be of therapeutic benefit in the various brain disorders associated with recurrent SDs.

Novel in vitro microfluidic platform for osteocyte mechanotransduction studies.

Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-µm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.