Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice

Abstract

Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. METHODS. We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. RESULTS. Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP. CONCLUSIONS. Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Targeting YAP mechanosignaling to ameliorate stiffness-induced Schlemm’s canal cell pathobiology

“…The temperature-controlled perfusion chamber (35°C) was filled with prewarmed DPBS with added 5.5 mM D-glucose (DBG), submerging the eyes. To facilitate drug exchange (42, 43), two microneedles per eye were used: one needle connected to the syringe pump and used for the drug exchange and the other needle for the perfusion. The exchange needles were filled with DPBS and the perfusion needles were filled with filtered DBG containing verteporfin (10 µM) or vehicle control (DMSO).…”

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms

“…Importantly, anterior chamber exchange allows precise control over the drug concentration which would otherwise be diluted without an exchange due to the presence of drug-free fluid within the anterior chamber. Insertion of two needles in the small mouse eye is technically challenging but has been demonstrated 114 ; however, drugs are also often infused with a single needle. Researchers must be aware that drug introduced into the mouse eye with a single needle will not be delivered immediately to the TM and, even when it reaches the TM, will be delivered at a diluted concentration that changes over time and is less than the infused concentration.…”

Ferroptosis in Glaucoma: A Promising Avenue for Therapy

“…[ 23 ] As both a component and co‐factor, iron is vital for the normal functioning of mitochondria, particularly in the energy‐intensive retinal ganglion cells (RGCs). Furthermore, the traditionally considered passive process of trabecular meshwork‐mediated aqueous humor drainage has been revealed to be energy‐dependent, [ 24 ] and its impairment is relevant in the context of ferroptosis. Regarding ferroptosis in glaucoma, much attention has been given to mechanisms involving mitochondrial function and oxidative stress.…”

“…Contrary to the conventional belief that aqueous humor outflow is a passive, non‐energy‐consuming process, recent studies have challenged this view, demonstrating it to be an active process dependent on cell metabolism. [ 24 ] Therefore, mitochondrial dysfunction in TMCs critically impacts outflow and leads to uncontrollable IOP, a hallmark of progressive glaucoma. In terms of ion content, intracellular copper overload can alter mitochondrial permeability through the opening of the mitochondrial permeability transition pore (MPTP), inducing mitochondrial swelling and reactive oxygen species (ROS) generation.…”