Chlorpyrifos Disrupts Acetylcholine Metabolism Across Model Blood-Brain Barrier.

Despite the significant progress in both scientific understanding and regulations, the safety of agricultural pesticides continues to be called into question. The need for complementary analytics to identify dysregulation events associated with chemical exposure and leverage this information to predict biological responses remains. Here, we present a platform that combines a model organ-on-chip neurovascular unit (NVU) with targeted mass spectrometry (MS) and electrochemical analysis to assess the impact of organophosphate (OP) exposure on blood-brain barrier (BBB) function. Using the NVU to simulate exposure, an escalating dose of the organophosphate chlorpyrifos (CPF) was administered. With up to 10 µM, neither CPF nor its metabolites were detected across the BBB (limit of quantitation 0.1 µM). At 30 µM CPF and above, targeted MS detected the main urinary metabolite, trichloropyridinol (TCP), across the BBB (0.025 µM) and no other metabolites. In the vascular chamber where CPF was directly applied, two primary metabolites of CPF, TCP and diethylthiophosphate (DETP), were both detected (0.1-5.7 µM). In a second experiment, a constant dose of 10 µM CPF was administered to the NVU, and though neither CPF nor its metabolites were detected across the BBB after 24 h, electrochemical analysis detected increases in acetylcholine levels on both sides of the BBB (up to 24.8 ± 3.4 µM) and these levels remained high over the course of treatment. In the vascular chamber where CPF was directly applied, only TCP was detected (ranging from 0.06 µM at 2 h to 0.19 µM at 24 h). These results provide chemical evidence of the substantial disruption induced by this widely used commercial pesticide. This work reinforces previously observed OP metabolism and mechanisms of impact, validates the use of the NVU for OP toxicology testing, and provides a model platform for analyzing these organotypic systems.

A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.

There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 µL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (µCA) for detecting glutamate in real time. Using the chamber of the µCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the µCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.

Fibrotic Encapsulation Is the Dominant Source of Continuous Glucose Monitor Delays.

Continuous glucose monitor (CGM) readings are delayed relative to blood glucose, and this delay is usually attributed to the latency of interstitial glucose levels. However, CGM-independent data suggest rapid equilibration of interstitial glucose. This study sought to determine the loci of CGM delays. Electrical current was measured directly from CGM electrodes to define sensor kinetics in the absence of smoothing algorithms. CGMs were implanted in mice, and sensor versus blood glucose responses were measured after an intravenous glucose challenge. Dispersion of a fluorescent glucose analog (2-NBDG) into the CGM microenvironment was observed in vivo using intravital microscopy. Tissue deposited on the sensor and nonimplanted subcutaneous adipose tissue was then collected for histological analysis. The time to half-maximum CGM response in vitro was 35 ± 2 s. In vivo, CGMs took 24 ± 7 min to reach maximum current versus 2 ± 1 min to maximum blood glucose (P = 0.0017). 2-NBDG took 21 ± 7 min to reach maximum fluorescence at the sensor versus 6 ± 6 min in adipose tissue (P = 0.0011). Collagen content was closely correlated with 2-NBDG latency (R = 0.96, P = 0.0004). Diffusion of glucose into the tissue deposited on a CGM is substantially delayed relative to interstitial fluid. A CGM that resists fibrous encapsulation would better approximate real-time deviations in blood glucose.