Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

PURPOSE: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2] (2.5%; [O2]2.5) and room air ([O2]A). METHODS: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2]2.5 and [O2]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. RESULTS: HCEnCs grown at [O2]A and [O2]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2]2.5 than at [O2]A. HIF-1α was undetectable. Cells displayed greater viability at [O2]2.5 than at [O2]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. CONCLUSIONS: Primary HCEnCs show unique metabolic characteristics at physiologic [O2]. The effect of [O2] for optimization of HCEnC culture conditions should be considered. TRANSLATIONAL RELEVANCE: With the advance of cell-based therapeutics for corneal endothelial diseases, [O2] should be considered an important variable in the optimization of HCEnC culture conditions.

Disrupted Glutamatergic Transmission in Prefrontal Cortex Contributes to Behavioral Abnormality in an Animal Model of ADHD.

Spontaneously hypertensive rats (SHR) are the most widely used animal model for the study of attention deficit hyperactivity disorder (ADHD). Here we sought to reveal the neuronal circuits and molecular basis of ADHD and its potential treatment using SHR. Combined electrophysiological, biochemical, pharmacological, chemicogenetic, and behavioral approaches were utilized. We found that AMPAR-mediated synaptic transmission in pyramidal neurons of prefrontal cortex (PFC) was diminished in SHR, which was correlated with the decreased surface expression of AMPAR subunits. Administration of methylphenidate (a psychostimulant drug used to treat ADHD), which blocks dopamine transporters and norepinephrine transporters, ameliorated the behavioral deficits of adolescent SHR and restored AMPAR-mediated synaptic function. Activation of PFC pyramidal neurons with a CaMKII-driven Gq-coupled designer receptor exclusively activated by designer drug also led to the elevation of AMPAR function and the normalization of ADHD-like behaviors in SHR. These results suggest that the disrupted function of AMPARs in PFC may underlie the behavioral deficits in adolescent SHR and enhancing PFC activity could be a treatment strategy for ADHD.